1

Miniaturised glucose-oxygen biofuel cell Yoko Kikuchi

A thesis submitted for

the degree of Doctor of Philosophy of Imperial College London

Department of Bioengineering

Imperial College London

SW7 2AZ

2

I hereby declare that the work presented in this thesis is my own

Yoko Kikuchi

3

ABSTRACT

Miniaturized glucose-oxygen biofuel cells are useful to power implantable

medical devices such as biosensors They are small more biocompatible and run

continuously on glucose and oxygen providing cleaner energy at neutral environment

A typical glucose-oxygen biofuel cell consists of an anode with glucose oxidase (GOx)

and a cathode with various oxygen reducing catalysts This thesis describes

experimental investigations of the major issues of such systems viz complex electrode

fabrication enzyme instability and inefficient oxygen reduction Electrodes were built

using the simple and scaleable bulk modification method where all the material was

simply mixed and bound together into composites with epoxy resin For the anodes the

composite made of 10 GOx with 77 TTF-TCNQ was found optimal The GOx

electrodes were modified with various enzyme stabilisers (PEI DTT PEG GLC FAD

and mixture of PEIDTT and PEIFAD) and 2 of PEI-DTT (11 ww) was most

effective in the presence of O2 Its maximum output current density was 18 x 10-2 plusmn 99

x 10-3 Am-2 It also showed the resistant against O2 electron deprivation and enzyme

inhibition Its KMwas 5 mM For the cathodes various oxygen reducing catalysts

(metalised carbon anthroquinone modified carbon laccase and bilirubin oxidase) were

incorporated into graphite composite and the electrodes were pretreated in different

media in order to enhance their catalytic activity None showed four-electron O2

reduction NaOH-pretreated cobalt (II) salophen composite electrodes showed

two-electron O2 reduction and were most catalytic Its standard catalytic rate constant

was 12 x 10-5 plusmn 12 x 10-6 ms-1 Of the catalysts examined metal complex composites

gave the best results for oxygen-reducing cathodes and their pretreatment led to the

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synergetic effect because it increased the concentration of catalytic surface oxygen

groups and enhanced oxygen reduction

5

ありがとう Thank you Terima kashi ขอบคณ คะ Merci 고마워 謝謝 Danke

やったね

copy鳥

山

明

集

英

社

copy Akira Toriyama Shueisha Inc

6

ACKNOWLEDGEMENTS

I would like to express deepest gratitude to my supervisor Dr Danny OrsquoHare

for his guidance on and patience for my research Frankly speaking the project on

glucose-oxygen fuel cells was very challenging and I came to realise the hard reality of

creating new clean energy despite of the world huge demand for such energy in a time

of global warming I could not help but feel that the research was turning into a

wild-goose chase however there was much to learn and gain from the experience here

For that and the people I met during this time I feel deeply grateful for the opportunity

given and I am convinced that this will enlighten my life

I would like to specially thank Dr Pei Ling Leow and Mr Parinya Seelanan for

their sincere support understanding and encouragement Without their help I would not

have been able to achieve my PhD I would like to thank to the current and past

members of Biosensor group Ms Nitin Bagha Dr Christopher Bell Dr Eleni Bitziou

Mr Jin-Young Kim Mr Wei-Chih Lin Dr Beinn Muir Mr Raphael Trouillon Dr

Martyn Boutelle Mr Yao-Min Chang Dr Delphine Feuerstein Ms Michelle Rogers

Dr Costas Anastassiou Dr Arun Arora Dr Martin Arundell Dr Catherine Dobson Dr

Melanie Fennan Dr Severin Harvey Dr Alex Lindsay Dr Bhavik Patel Dr Hong

Zhao Dr Emma Corcoles and Dr Parastoo Hashemi for their support during research

and sharing the wonderful time for many occasions I would like to thank Mr David

Roche for his sincere devotion to our work and being a great lab company I would like

to give my warm thanks to Dr Andrew Bond Dr Stephanie Cremers Ms Christina

Hood Dr Asimina Kazakidi Dr Peter Knox Dr Bettina Nier Ms Eileen Su Mr

Che-Fai Yeong Mr Krause Oliver and Dr Clifford Talbot for appropriate support fun

7

and encouragement I would also like to thank Dr Kate Hobson Mr Martin Holloway

Mr Ken Keating Mr Alan Nyant Mr Richard Oxenham Ms Paula Smith Ms Dima

Cvetkova Dr Anna Thomas-Betts and Mr Nick Watkins for their academic and

laboratory support Finally I would like to say thank you to all my family for their trust

and unconditional support my teacher Prof Fukuyama my friends Dr Pieta

Nasanen-Gilmore Jingzi Kofi Mariama Masa Maya Miki Pedro Sandra Sascha and

Wei Fong for being always close supportive and encouraging even when we were apart

8

CONTENTS

ABSTRACT 3

ACKNOWLEDGEMENTS 6

CONTENTS 8

LIST OF ABBREVIATIONS 15

LIST OF FIGURES 20

LIST OF TABLES 26

LIST OF EQUATIONS 27

1 INTRODUCTION 31

11 INTRODUCTION TO FUEL CELLS 31

111 History of the fuel cells 31 112 Principles of the fuel cells 33 113 Electric power from the fuel cells 35

12 CATALYSTS 37

121 Faradayrsquos second law ndash Current and reaction rate37 122 Transition state theory Eyring equation and reaction rate 38 123 Overcoming the activation free energy 40 124 Variations in catalysts42

13 ELECTRODE POTENTIAL AND REACTION RATE 44

131 Fermi level electrode potential and electron transfer 44 132 Electrode potential equilibrium potential and Nernst equation47

9

133 Electrode potential and activation free energy 50 134 Potential shift and standard rate constant 52 135 Butler-Volmer equation overpotential and current 53 136 Butler-Volmer equation vs Tafel equation 55

14 OVERPOTENTIAL AND FUEL CELLS 57

141 Four overpotentials that decrease the EMF57 142 Internal current 58 143 Activation overpotential58 144 Diffusion overpotential 59 145 Resistance overpotential60 146 Total overpotential and cell voltage 62

15 BIOFUEL CELLS 63

151 Microbial and enzymatic fuel cells 63 152 Enzyme fuel cells and mediators64

16 MINIATURISED MEDICAL POWER SOURCES 66

161 Realisation of miniaturised fuel cells and medical power supply 66 162 Miniaturised potential medical power sources67

17 OUTLINE OF THE WORK 69

171 Introduction to miniaturised glucose-oxygen fuel cells 69 172 Outline71

2 ANODE INTRODUCTION 73

21 GLUCOSE OXIDATION ENZYMES 73

22 DESIGNING ENZYME ELECTRODES 77

221 Physical adsorption 78 222 Reconstituting apo-enzyme78 223 Tethered Osmium-complexes linked to conductive hydrogel 80 224 Entrapment in the conductive polymer 81 225 Bulk modification83

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties 84 226 Enzyme covalent attachment and mass loading 88

23 PROTEIN INACTIVATION AND STABILISATION 90

10

24 PROTEIN DESTABILISATION 92

241 Deamidation 92 242 Oxidation93 243 Proteolysis and racemisation94 244 Physical instabilities95

25 ENZYME STABILISERS 95

251 Polyehyleneimine (PEI) 96 252 Dithiothreitol (DTT)97 253 Polyethylene glycol (PEG)98 254 Polyols99 255 FAD100

26 ENZYME KINETICS AND ELECTROCHEMISTRY 101

261 Michaelis-Menten kinetics 101 262 Electrochemistry and Michaelis-Menten kinetics 105 263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics108

2631 Lineweaver-Burk linear regression 108 2632 Eadie-Hofstee linear regression 109 2633 Electrochemical Hanes-Woolf linear regression 110

264 Cornish-Bowden-Wharton method 112 265 Nonlinear regression methods113

3 ANODE MATERIAL AND METHOD115

31 ELECTRODE FABRICATION 115

311 TTF-TCNQ synthesis115 312 Electrode template fabrication 115 313 Electrode composite fabrication117

3131 Composite for conductance study 117 3132 Bulk modified GOx enzyme electrode composite 118 3133 GOx enzyme electrode composite with enzyme stabilisers 119

314 Electrode wiring and insulation120

32 CONDUCTIVITY EXPERIMENT 121

33 POLISHING COMPOSITE ELECTRODES 122

34 GLUCOSE AMPEROMETRY 123

341 Glucose solution123

11

342 Electrochemical system123 343 Glucose amperometry with GOx electrodes 124

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA 125

4 ANODE RESULTS AND DISCUSSION 127

41 AIMS OF ANODE STUDIES 127

42 CONDUCTANCE OF THE COMPOSITE 128

421 Conductance of various conductive materials128 422 Influence of enzyme concentration on conductivity 130

43 CATALYTIC ACTIVITY OF GOX COMPOSITE ELECTRODES 132

431 Influence of GOx concentration on the catalytic activity of GOx electrodes132 432 Influence of O2 on the catalytic activity of GOx electrodes134

44 THE EFFICACY OF GOX STABILISERS 139

441 Introduction139 442 PEI140 443 DTT142 444 PEG 143 445 GLC144 446 FAD146 447 PEI and DTT mixture147 448 PEI and FAD mixture149 449 Most effective stabiliser content for GOx anodes 150

45 CONCLUSION TO ANODE STUDY 152

5 CATHODE INTRODUCTION 155

51 OXYGEN REDUCTION REACTION (ORR) 155

52 REACTIVITY OF OXYGEN 157

53 ORR CATALYSTS 159

531 Noble metal ORR catalysts 160 532 Macrocycle and Schiff-base complex ORR catalysts 165

12

533 Multicopper enzymes as ORR catalyst 168 5331 BOD 171 5332 Lac 173 5333 CueO 173

534 Anthraquinone ORR catalyst 174

54 CARBON PROPERTIES 177

541 Graphite180 542 Glassy carbon (GC)181 543 Activated carbon (AC) 182 544 Carbon fibre (CF)182 545 Carbon nanotubes (CNTs)183 546 Boron-doped diamond (BDD)184

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON 185

551 Electrochemical properties of carbon185 552 Electrochemical pretreatments (ECPs) of the carbon185

56 ELECTROCHEMICAL TECHNIQUES FOR ORR 187

561 Cyclic voltammetry (CV)188 562 Non-faradic current and electric double layer capacitance 189 563 Diagnosis of redox reactions in cyclic voltammetry 190 564 Coupled reactions in cyclic voltammetry194 565 Voltammograms for the surface-confined redox groups 195 566 Hydrodynamic voltammetry (HDV) and Levich equation197 567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the charge

transfer rate constant 200 568 Finding the standard rate constant (kdeg)205 569 Summary of the electrochemical techniques206

57 SUMMARY OF THE CHAPTER 207

6 CATHODE MATERIAL AND METHOD 208

61 ELECTRODE FABRICATION 208

611 Graphite PtC and RhC electrodes208 612 Anthraquinone(AQ)-carbon composite electrodes208

6121 Derivatisation of graphite with AQ 208 6122 Fabrication of AQ-carbon composite electrode 209

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes209

13

6131 Information on synthesis of metallosalophen 209 6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes 210

62 COMPOSITE RDE CUTTING 210

63 ELECTRODE POLISHING 210

631 Composite RDE polishing210 632 Glassy carbon electrode polishing211

64 ENZYME RDES 212

641 Membrane preparation 212 642 Membrane enzyme electrodes212

65 ELECTROCHEMICAL EXPERIMENTS 213

651 Electrochemical system213 652 Electrode pretreatments214

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes 214 6522 Pretreatments of AQ-carbon PtC and RhC 215

653 Electrochemical experiments 215 654 Data analysis 216

7 CATHODE RESULTS AND DISCUSSION 218

71 AIMS OF THE CATHODE STUDIES 218

72 INFLUENCE AND EFFICACY OF ECPS 219

721 Glassy carbon electrodes (GCEs)219 7211 Untreated GCEs 219 7212 Influence of electrochemical pretreatments on GCE 220 7213 Effect of GCE pretreatment on ORR 227

722 Graphite composite electrodes 230 7221 Untreated graphite composite electrodes 230 7222 Influence of electrochemical pretreatments on graphite composite electrodes 232 7223 The effect of graphite composite electrode pretreatment on ORR 235

723 Platinum on carbon composite electrodes (PtC) 240 724 Rhodium on carbon composite electrodes (RhC)240

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR 241 725 Co(salophen) composite electrodes243

7251 Untreated Co(salophen) composite electrodes 243 7252 Influence of electrochemical pretreatments on Co(salophen) composite 244 7253 The effect of Co(salophen) composite electrode pretreatment on ORR 246

726 Fe(salophen) composite electrodes 248 7261 Untreated Fe(salophen) composite electrodes 248 7262 Influence of electrochemical pretreatments on Fe(salophen) composite 250

14

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR 252 727 Anthraquinone (AQ) composite electrodes253 728 Lac and BOD enzyme membrane electrodes 255

73 MOST EFFICIENT ORR CATALYSTS 257

731 Best five electrodes for nα 258 732 Best five electrodes for kdeg 259 733 Best five electrodes for Epc2(HDV) 260 734 Best three candidates for ORR catalytic electrodes 260

74 CONCLUSION TO CATHODE STUDY 262

8 CONCLUSION AND FURTHER STUDIES 264

9 BIBLIOGRAPHY 268

15

LIST OF ABBREVIATIONS

Α Transfer coefficient

A Electrode surface area

ABTS 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

AC Activated carbon

AFC Alkaline fuel cell

AQ Anthraquinone

Asn Asparagine

Asp Aspartic acid

BDD Boron-doped diamond

BOD Bilirubin oxidase

C Reactant concentration

C O (0t) Electrode surface oxidant concentration

C R (0t) Electrode surface reactant concentration

C(0t) Electrode surface concentration

C Bulk concentration

Cd Double layer capacitance

CF Carbon fibre

CNH Carbon nanohorn

CNT Carbon nanotubes

Co(salen) N-N-bis(salicylaldehyde)-ethylenediimino cobalt(II)

Co(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminocobalt (II)

CO Bulk oxidant concentration

CR Bulk reactant concentration

CueO Copper efflux oxidase

CV Cyclic voltammetry

Cys Cysteine

(DEAE)-dextran Diethylaminoethyl-dextran

DET Direct electron transfer

DHG Dehydrogenase

DMFC Direct methanol fuel cell

DTE Dithioerythriotol

DTT Dithiothreitol

[E0] Initial enzyme concentration

16

E Arbitrary electrode potential Enzyme

Edeg Standard electrode potential

Edeg Formal potential

Ea Anode potential

EC Electrochemical-chemical

Ec Cathode potential

Ecell Cell voltage

ECP Electrochemical pretreatment

Ee Electrode equilibrium potential

EF Fermi potential

EMF Electro motive force

Ep2 Half-peak potential

Epa Anodic peak potential

Epc Cathodic peak potential

Epc2(HDV) Half-wave reduction potential in HDV

ES Enzyme-substrate complex

Eλ Switching potential

∆E Potential difference Potential shift

∆Edeg Standard peak separation

δEp Peak width

εF Fermi energy

F Faraday constant

FAD Flavin adenine dinucleotide

Fe(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminoiron (II)

GC Glassy carbon

GCE Glassy carbon electrode

GHP 1-(6-D-gluconamidohexyl) pyrrole

GLC Glycerol

Gln Glutamine

GOx Glucose oxidase

GOx(O) Oxidised GOx

GOx(R) Reduced GOx

GSH Gluthatione

∆G Gibbs free energy shift

∆Gdeg Standard Gibbs free energy change

∆GdegDagger Standard activation free energy

17

∆GDagger Gibbs free energy of activation

∆GadegDagger Anode standard activation free energy

∆GaDagger Anode activation free energy

∆GodegDagger Oxidation standard activation free energy

∆GoDagger Oxidation activation free energy

∆GRdegDagger Reduction standard activation free energy

∆GRDagger Reduction activation free energy

h Planck constant

HDA Hydrodynamic amperometry

HDV Hydrodynamic voltammetry

His Histidine

HOMO Highest occupied molecular orbital

∆Hdeg Standard enthalpy change

I Electric current Net current

I0 Exchange current

Ia Anodic current

Ic Cathodic current

Id Disc limiting current

IK Kinetic current

IL Limiting current Diffusion-limited current

Im Maximum steady state limiting current

Ip Peak current

Ipa Anodic peak current

Ipc Cathodic peak current

I-V Current-Voltage

J Current density

Jm Maximum limiting current density

k Rate constant

K Equilibrium constant

k(E) Rate constant at the applied potential E

kdeg Standard heterogeneous rate constant

kdegb Backward reaction standard rate constant

kdegf Forward reaction standard rate constant

KDagger The equilibrium constant between the activated complexes and the reactants

k1 Rate constant for ES formation from E and S

k-1 Rate constant for ES dissociation into E + S

18

k2 Rate constant for P formation from ES

kB Boltzmann constant

kb Backward reaction rate constant

kf Forward reaction rate constant

kf(E) Rate constant for forward reactions at the applied potential E

KM Michaelis enzyme dissociation constant

Lac Laccase

LDH Lactate dehydrogenase

LMCT Ligand-to-metal charge transfer

LPG Liquefied petroleum gas

LUMO Lowest unoccupied molecular orbital

MCO Multicopper oxidase

MDH Methanol dehydrogenase

Me2SQ Dimethylmercaptobenzoquinone

MEMS Microelectromechanical systems

MeSQ Methylmercapttobenzoquinone

MET Mediated electron transfer

Met Methionine

mPQQ-GDH Membrane-bound PQQ dehydrogenase

MWNT Multi-walled nanotube

n Overall number of electrons transferred

na The number of electrons transferred up to the rate limiting step

NAD Nicotinamide adenine dinucleotide

NAD-GDH Nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

OCV Open circuit voltage

ORR Oxygen reduction reaction

P Electric power Product

PAFC Phosphoric acid fuel cell

PBS Phosphate buffer saline

PC Pyrolytic carbon

PDMS Polydimethylsiloxane

PEFC Polymer electrolyte fuel cell

PEG Polyethylene glycol

PEI Polyethyleneimine

P-I Power-Current

POx Pyranose oxidase

19

PQ Phenanthrenequinone

PQQ pyrrolo-quinoline quinone

PQQ-GDH Quinoprotein glucose dehydrogenase

Pro Proline

PtC Platinum on carbon

Q Charge

RDE Rotating disc electrode

RhC Rhodium on carbon

RLS Rate limiting step

rpm Rotaion per minute

S Substrate

SEN Single-enzyme nanoparticle

sPQQ-GDH Soluble PQQ dehydrogenase

SQ Mercaptobenzoquinone

SWNT Single-walled nanotube

∆Sdeg Standard entropy change

t Time

T1 Type I (blue Cu in Lac)

T2 Type II ( trinuclear Cu in Lac)

T3 Type III (trinuclear Cu in Lac)

TCNQ 7788-Tetracyanoquinodimethane

TEM Transmission electron microscopy

Trp Tryptophan

TTF Tetrathiafulvalene

TTF-TCNQ Tetrathiafulvalene 7788-tetracyanoquinodimethane

Tyr Tyrosine

V Voltage Maximum limiting catalytic velocity

VHT Vacuum heat treatment

η Overpotential

ν Vibrational frequency Kinematic viscosity

υ Reaction rate Scan rate Enzyme catalytic rate

ω Angular velocity

20

LIST OF FIGURES

Fig No Page Description

Fig 1 32 Historical fuel cells

Fig 2 33 Photographs of modern fuel cells

DMFC Cogeneration system Hydrogen car Glucose-Oxygen fuel cell

Fig 3 34 The schema of glucose-oxygen fuel cell

Fig 4 41 Transition state diagram with the reaction coordinate

Fig 5 45 Fermi level and electron transfer

Fig 6 46 Fermi level and electron transfer at an arbitrary electrode potential

Fig 7 48 Standard free energy change along the electrode potential at

(a) E = Edeg (b) E gt Edeg and (c) E lt Edeg

Fig 8 51 The shift in ∆GdegDagger accompanied by ∆E

Fig 9 57 Tafel plot

Fig 10 59 Activation overpotential vs current

Fig 11 60 Diffusion overpotential vs current

Fig 12 61 Resistance overpotential vs current

Fig 13 62 V-I curve for a typical low temperature fuel cell

Fig 14 65 Mediated electron transfer reaction by the enzyme electrode

Fig 15 70 The schema for a glucose-oxidising anode

Fig 16 75 The structure and redox reactions of FAD NAD and PQQ

Fig 17 79 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode

Fig 18 81 GOx wired to conductive hydrogel complexed with Os

Fig 19 83 Monomer structures of conductive polymers

1-(6-D-gluconamidohexyl) pyrrole and Mercapto-p-benzoquinone

Fig 20 85 TTF-TCNQ structure and its charge transfer

Fig 21 86 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 20 mVs-1

Fig 22 89 The procedure for the enzyme mass-loaded biocatalytic nanofibre

Fig 23 90 The fabrication procedure of single-enzyme nanoparticles

Fig 24 92 The chemical structures of the side groups of Gln and Asn

Fig 25 93 The chemical structures of the side groups of Cys Met Tyr Trp and His

Fig 26 95 The chemical structures of Asp and Pro

Fig 27 96 The structure of PEI

Fig 28 98 The structures of DTT from a linear to a cyclic disulfide form

Fig 29 98 The structures of PEG

21

Fig 30 100 The structure of glycerol

Fig 31 104 A typical Michaelis-Menten hyperbolic curve of the enzyme

Fig 32 107 Electrochemical Michaelis-Menten hyperbolic curve of the enzyme electrode

Fig 33 109 Electrochemical Lineweaver-Burk linear regression of the enzyme electrode

Fig 34 110 Electrochemical Eadie-Hofstee linear regression of the enzyme electrode

Fig 35 111 Electrochemical Hanes-Woolf linear regression of the enzyme electrode

Fig 36 113 Electrochemical Cornish-Bowden-Wharton plot of the enzyme electrode

Fig 37 116 The photographs of PDMS electrode templates

Fig 38 118 The photographs of empty and filled PDMS templates

Fig 39 119 The schema for bulk modification method

Fig 40 121 The photographs of insulated composite electrodes

Fig 41 122 The photographs of measuring electrode resistance

Fig 42 125 The photograph of glucose amperometry with a multichannel potentiostat

Fig 43 126 The analytical output by SigmaPlot Enzyme Kinetics

Fig 44 129 Conductance of various electrode composite

Fig 45 131 Conductance of GOx composite with varying GOx content

Fig 46 133 Difference in the glucose-response amperometric curve by the enzyme electrodes

containing various GOx content 5 10 and 15 in the absence of O2

Fig 47 134 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the absence of O2

Fig 48 135 (i) Difference in the glucose-response amperometric curve by 10 GOx composite

electrodes in the absence and presence of O2

(ii) The re-plotted amperometric curves in the presence of N2 air and O2

Fig 49 136 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the presence N2 O2 and air

Fig 50 140 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEI in the presence and absence of O2

Fig 51 142 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 DTT in the presence and absence of O2

Fig 52 145 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEG in the presence and absence of O2

Fig 53 146 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 GLC in the presence and absence of O2

Fig 54 147 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 FAD in the presence and absence of O2

22

Fig 55 143 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-DTT

(051 11 1505 and 22) in the presence and absence of O2

Fig 56 148 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051 11

1505 and 22)

Fig 57 149 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-FAD (21

22 and 24 ) in the presence and absence of O2

Fig 58 151 Jm KM in the absence and presence of O2 for various stabilisers

PEIDTT (11 0515 221505 ) and GLC 1

Fig 59 156 Oxygen reduction pathways 2 and 4 electron reduction pathways

Fig 60 157 Degradation of superoxide anion radical in aqueous solutions

Fig 61 158 An energy level diagram for ground state dioxygen molecule

Fig 62 159 Diagrams for the state of π orbitals in the ground state oxygen and peroxide anion

radical

Fig 63 162 Three models of oxygen adsorption Griffith Pauling and Bridge models

Fig 64 163 Models for oxygen adsorption on metals and ORR pathways

Fig 65 164 A volcano-shaped trend between ORR rate and oxygen binding energy of various

transition metals

Fig 66 165 Examples of transition metallomacrocycles Heme and copper phthalocyanine

Fig 67 166 Schiff base complexes

Co(II) salen Co(II) salophen and Co(III) salen conductive polymer

Fig 68 167 The molecular structure of dicobalt face-to-face diporphyrin [Co2(FTF4)]

Fig 69 168 ORR pathways by Co2(FTF4)

Fig 70 170 The structure of laccase from Trametes versicolor

Fig 71 171 Redox reaction cycle of MCOs with O2

Fig 72 172 Common mediators for BOD ABTS and Os complex

Fig 73 174 Two-electron ORR by anthraquinone

Fig 74 176 The structures of anthraquinone and its derivative Fast red AL salt

Fig 75 177 HDV of ORR at various quinone electrodes at pH 7

Unmodified GC AQGC and PQGC

Fig 76 178 The carbon structures graphite and diamond

Fig 77 179 The carbon sections basal and edge planes

Fig 78 181 A TEM image of graphene bilayer at 106 Aring resolution

Fig 79 182 A TEM image and a schema of GC

Fig 80 183 Computer graphic models of various CNTs Bundled SWNTs MWNT and CNHs

23

Fig 81 184 A TEM image of diamond

Fig 82 187 The CVs showing the influence of ECP on GCE at pH 7 in the presence and absence

of O2 The CVs for both pretreated and unpretreated GCEs are shown

Fig 83 188 The triangular waveform for CV and a typical voltammetric curves for BQ

Fig 84 189 The CV showing the double layer capacitance at graphite electrode surface at pH 7

Fig 85 194 A voltammogram examples of a quasi-reversible reaction at various scan rates

Fig 86 195 Voltammograms of two-electron transfer in the EE systems with different ∆Edegrsquo

Fig 87 196 A voltammogram of the reversible redox reaction by surface-confined groups

Fig 88 198 A diagram of hydrodynamic electrochemical method using a RDE

Fig 89 200 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH 74

Fig 90 201 Levich plot examples for reversible and quasi-reversible systems

Fig 91 203 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in oxygenated

001 M PBS at pH 74 at pretreated GCE

Fig 92 204 Koutecky-Levich plot for O2 reduction at a pretreated GCE in oxygenated 001 M PBS at

pH 74 with applied potential ndash 04 06 and 08 V

Fig 93 206 Log kf(E) plot for O2 reduction at various pretreated GCEs

Fig 94 207 The protocol for finding kinetic and thermodynamic parameters

Fig 95 211 The photographs of fabricated composite RDEs

Fig 96 213 The photograph of RDE experiment setting with a rotor and gas inlet

Fig 97 220 CVs of untreated GCEs at pH 74 in 001 M PBS in the absence and presence of O2

at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 98 221 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of O2

between + 09 and - 09 V vs AgAgCl at 005 Vs-1

(i) CVs for all the pretreated GCEs

(ii) CVs excludes PBS pretreatments

Fig 99 223 The bar graphs for the capacitance of GCEs in 015 M NaCl buffered with 001 M

phosphate to pH 74

Fig 100 225 Overlaid CVs during various pretreatments for GCEs involving

01 M H2SO4 alone 001 M PBS at pH 74 alone and both pretreatments

Fig 101 227 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M oxygenated

PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 102 229 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Fig 103 231 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M PBS in

the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

24

Fig 104 233 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M N2-sparged PBS

between + 09 and - 09 V vs AgAgCl

Fig 105 236 Overlaid CVs of ORR at various pretreated graphite composite electrodes at pH 74

in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 106 237 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at pH 74

in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various scan rates

50 100 200 and 500 mVs-1

Fig 107 238 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

Fig 108 239 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

Fig 109 241 CVs of untreated and H2SO4-pretreated RhC composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

Fig 110 242 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M PBS in

the absence and presence O2 at 005 Vs-1 between + 09 and - 09 V vs AgAgCl

Fig 111 243 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M PBS in the

absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 112 245 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 113 247 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 114 248 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite electrodes

Fig 115 249 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

Fig 116 251 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 117 253 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 118 254 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M PBS in

theabsence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 119 256 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated PBS at

005 Vs-1 between 0 and -09 V vs AgAgCl

Fig 120 257 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Fig 121 258 The bar graphs of the best five nα

25

Fig 122 259 The bar graphs of the best five kdeg

Fig 123 260 The bar graphs of the best five Epc2(HDV)

Fig 124 266 A photograph of the fuel cell stack made with GOx anodes and Co(salophen)

cathodes

26

LIST OF TABLES

Table No Page Description

Table 1 35 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Table 2 87 Electrochemical behaviour of TTF-TCNQ at pH 74

Table 3 104 Michaelis-Menten equation at various [S]

Table 4 117 Composite composition table

Table 5 119 Blended stabiliser composition

Table 6 139 Four possible causes of glucose amperometric current reduction

Table 7 152 Comparison of maximum output current and KM with some published data

Table 8 159 Bond orders bond lengths and bond energies of some dioxygen species

Table 9 180 The electric conductivity of various carbon forms and sections

Table 10 186 Various ECP examples

Table 11 192 CV diagnosis for reversible system at 25 ˚C

Table 12 193 CV diagnosis for irreversible system at 25 ˚C

Table 13 196 CV diagnosis for reversible system by surface-confined groups at 25 ˚C

Table 14 214 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Table 15 216 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC

Table 16 217 Parameter values for Koutecky-Levich equation

O2 diffusion coefficient O2 concentration and PBS kinematic viscosity

Table 17 261 Three candidate electrodes for high ORR catalysis

Table 18 261 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

27

LIST OF EQUATIONS

Eq No Page Description

Eq 1 35 Anode glucose oxidation reaction

Eq 2 35 Cathode oxygen reduction reaction

Eq 3 35 Overall glucose-oxygen fuel cell reaction

Eq 4 36 Electric power equation

Eq 5 36 Electric current equation in electrochemistry

Eq 6 38 Faradayrsquos second law of electrolysis Charge equation

Eq 7 38 Faradayrsquos second law of electrolysis Reaction equation

Eq 8 39 Transition state theory equation Rate constant equation

Eq 9 39 Transition state theory equation Equilibrium constant

Eq 10 39 Transition state theory equation Atomic vibration frequency equation

Eq 11 40 Transition state theory equation Erying equation

Eq 12 42 Gibbs free energy shift equation in thermodynamic terms

Eq 13 42 Gibbs free energy shift equation in electrochemical terms

Eq 14 49 A typical redox reaction

Eq 15 50 Nernst equation

Eq 16 50 Electrode potential shift

Eq 17 50 Free energy shift in electrochemical terms

Eq 18 52 Anodic oxidation activation free energy shift in terms of potential shift

Eq 19 52 Cathodic reduction activation free energy shift in terms of potential shift

Eq 20 53 Forward reduction rate constant equation in terms of kdeg and potential shift

Eq 21 53 Backward oxidation rate constant equation in terms of kdeg and potential shift

Eq 22 54 Butler-Volmer equation

Eq 23 54 Overpotential equation

Eq 24 54 Exchange current equation

Eq 25 55 Butler-Volmer equation in terms of electrode equilibrium constant

Eq 26 55 Butler-Volmer equation in terms of overpotential

Eq 27 56 Butler-Volmer equation for small overpotential

Eq 28 56 Tafel equation for large overpotential

Eq 29 60 Diffusion overpotential equation

Eq 30 61 Ohmrsquos law

Eq 31 63 Cell voltage equation

Eq 32 63 Gibbs free energy available from the fuel cell

28

Eq 33 73 Glucose oxidation reaction by GOx

Eq 34 73 Glucose oxidation reaction by NAD-DHG

Eq 35 87 TTF-TCNQ reaction At equilibrium state

Eq 36 87 TTF-TCNQ reaction TCNQ reduction at lt + 015 V vs AgAgCl

Eq 37 87 TTF-TCNQ reaction TCNQ lattice reduction at lt - 015 V vs AgAgCl

Eq 38 87 TTF-TCNQ reaction Anodic TCNQ salt formation at gt + 031 V vs AgAgCl

Eq 39 87 TTF-TCNQ reaction TTF lattice oxidation at gt + 035 V vs AgAgCl

Eq 40 87 TTF-TCNQ reaction TTF further oxidation at gt + 038 V vs AgAgCl

Eq 41 101 Enzyme kinetics A typical enzyme reaction

Eq 42 102 Enzyme kinetics Enzyme-substrate complex formation at steady state

Eq 43 102 Enzyme kinetics Rate of enzyme-substrate complex formation in terms of k-1 and k2

Eq 44 102 Enzyme kinetics

Enzyme-substrate complex equation in terms of [E0] k1 k-1 k2 and [S]

Eq 45 102 Enzyme kinetics Enzyme catalytic reaction

Eq 46 102 Enzyme kinetics Enzyme catalytic reaction equation

Eq 47 102 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of enzyme-substrate complex

Eq 48 103 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of [E0] k1 k-1 k2 and [S]

Eq 49 103 Enzyme kinetics Michaelis-Menten constant definition

Eq 50 103 Michaelis-Menten equation

Eq 51 104 Michaelis-Menten equation when [S] ge KM

Eq 52 104 Michaelis-Menten equation when [S] = KM

Eq 53 104 Michaelis-Menten equation when [S] le KM

Eq 54 106 Electrochemical Michaelis-Menten equation

Eq 55 106 Electrochemical Michaelis-Menten equation for current density

Eq 56 108 Electrochemical Lineweaver-Burk linear regression equation

Eq 57 109 Electrochemical Eadie-Hofstee linear regression equation

Eq 58 111 Electrochemical Hanes-Woolf linear regression equation

Eq 59 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for Jm

Eq 60 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for KM

Eq 61 128 Enzyme electrode specificity constant

Eq 62 139 The causes of anodic current reduction A typical GOx-O2 reaction

Eq 63 139 The causes of anodic current reduction TTF reduction by GOx

Eq 64 139 The causes of anodic current reduction GOx allosteric inhibition

Eq 65 139 The causes of anodic current reduction TTF oxidation by O2

29

Eq 66 139 The causes of anodic current reduction TTF oxidation by GOx

Eq 67 161 O2 Dissociative mechanism Simultaneous oxygen adsorption and molecular fission

Eq 68 161 O2 Dissociative mechanism Surface hydroxide formation

Eq 69 161 O2 Dissociative mechanism Oxygen reduction to water

Eq 70 161 O2 Associative mechanism Oxygen adsorption

Eq 71 161 O2 Associative mechanism Peroxide formation

Eq 72 161 O2 Associative mechanism Peroxide reduction

Eq 73 161 O2 Associative mechanism Surface oxygen reduction to hydroxide

Eq 74 161 O2 Associative mechanism Surface hydroxide reduction to water

Eq 75 175 Anthraquinone O2 reduction Semiquinone anion radical formation

Eq 76 175 Anthraquinone O2 reduction O2 reduction by a semiquinone anion radical

Eq 77 175 Anthraquinone O2 reduction Spontaneous superoxide reduction to O2

Eq 78 175 Anthraquinone O2 reduction

Spontaneous superoxide reduction to hydroperoxide

Eq 79 190 Electric double layer capacitance per unit area

Eq 80 192 CV diagnosis ndash Reversible system Ip vs υfrac12

Eq 81 192 CV diagnosis ndash Reversible system Ep

Eq 82 192 CV diagnosis ndash Reversible system ∆Ep

Eq 83 192 CV diagnosis ndash Reversible system δEp

Eq 84 192 CV diagnosis ndash Reversible system Reversal parameter

Eq 85 193 CV diagnosis ndash Irreversible system Ip vs υ

Eq 86 193 CV diagnosis ndash Irreversible system Ep a function of kdeg α and υ

Eq 87 193 CV diagnosis ndash Irreversible system ∆Ep

Eq 88 193 CV diagnosis ndash Irreversible system δEp

Eq 89 195 CV diagnosis ndash EE system Peak separation of EE system

Eq 90 196 CV diagnosis ndash Surface-confined groups Ip vs υ

Eq 91 196 CV diagnosis ndash Surface-confined groups Ep

Eq 92 196 CV diagnosis ndash Surface-confined groups ∆Ep2

Eq 93 198 Diffusion layer thickness in terms of ω

Eq 94 199 Levich equation in terms of ω

Eq 95 199 Levich equation in terms of diffusion layer thickness

Eq 96 202 Koutecky-Levich equation

Eq 97 204 The rate constant for arbitrary potential E

Eq 98 205 k(E) in terms of kdeg and η for a forward reaction

Eq 99 205 kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

Eq 100 238 Surface semiquinone radical formation

30

Eq 101 238 ORR by surface semiquinone radical

31

1 INTRODUCTION

11 INTRODUCTION TO FUEL CELLS

111 History of the fuel cells

Fuel cells are electrochemical devices which directly convert fuel into

electricity via chemical reactions The invention dates back to 19th century when a

British physicist Sir William Grove and a German-Swiss chemist Christian Schoumlnbein

discovered the reverse reaction of water electrolysis The current output by this system

was however very small Therefore the discovery stayed in the shadow of the steam and

internal combustion engines and it needed another century to gain its attention

In the 1930s a British engineer Francis Bacon started working towards

alkaline fuel cells (AFCs) and in the 1950s General Electric developed polymer

electrolyte fuel cells (PEFCs) Those fuel cells were loaded on the spaceships in the

1960s by NASA because they were compact provided clean power with large energy

density and generated drinkable water as a by-product [1-5] Since then fuel cells have

been continuously used for space and marine technologies [3] In the 1970s studies on

fuel cells were further expanded for consumer applications [1 3 5 6] The first

air-breathing fuel cell hybrid car was built by Kordesch [1 7] and phosphoric acid fuel

cells (PAFCs) were developed and introduced to offices and hospitals [2 3 5]

32

Fig 1 Some historical fuel cell applications (i) Alkaline fuel cell for Apollo 11 [8] (ii)

Kordeschrsquos Austin A40 city hybrid car [7]

Since the 1990s fuel cells have been drawing keen attention as flexible renewable

energy in conjunction with the depletion of fossil fuel global warming and the demand

in cleaner energy [6 9] Great effort has been made to miniaturise fuel cells for

individual users such as mobile devices vehicles and households [1 3 5 6] The recent

advance in such technologies has led to direct methanol fuel cells (DMFCs) for

mobile-phone handsets [10 11] cogeneration systems to supply household power [1 5]

hydrogen fuel cell cars [12-15] and bio-batteries running on glucose and air oxygen for

portable music players [16-18] Glucose-oxygen biofuel cells have been also further

miniaturised for medical implantable purposes [18 19]

33

Fig 2 Modern fuel cell technologies (i) Toshibarsquos world smallest DMFC [20] (ii)

TokyoGasrsquos cogeneration system Enefarm (iii) BMWrsquos Hydrogen 7 (iV)

Sonyrsquos Bio Battery generating electricity from sugar [16]

112 Principles of the fuel cells

A fuel cell unit simply consists of an anode a cathode electrolyte and a

conducting wire A conducting wire connects an anode and a cathode to form an

external circuit This allows coupling of electrochemical reactions taking place at each

electrode and provides a conductive path for electrons between them Electrolyte

allows charged species to move between two electrodes but blocks out the entry of

electrons This completes the electric circuit and permits electric conduction At the

anode fuel is oxidised by an oxidation reaction and this supplies electrons to the

cathode through the external circuit The number of electrons generated depends on the

34

type of fuels reactions and environment in which the reactions take place In the anode

reaction ions are also released to balance overall charge They move to the cathode

though the electrolyte At the cathode ions from the electrolyte and electrons from the

circuit meet and react together with an oxidiser supplied The completion of the coupled

reactions generate electricity and products [3 4 21] Fig 3 shows a glucose-oxygen

fuel cell as an example

Fig 3 A glucose-oxygen fuel cell

At the anode glucose is oxidised into gluconolactone This gives out two electrons and

two protons At the cathode oxygen gas receives them and is reduced to water Each

reaction has a unique redox potential called standard electrode potential Edeg and

electromotive force (EMF) is the potential difference between such electrode potentials

It is also called open circuit voltage (OCV) This is the theoretical size of potential

difference available from the fuel cell [3 4] The coupled reactions for glucose-oxygen

fuel cell is shown in Table 1 Each redox potential was measured against silver

35

silver-chloride reference electrode (AgAgCl) at pH 7 25degC therefore shown in formal

potential (Edegrsquo)

Table 1 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Anode Edegrsquo = - 042 V (Eq 1)

(via FADFADH2 redox [22 23])

Cathode [24] Edegrsquo = 062 V (Eq 2)

Overall ∆Edegrsquo = 104 V (Eq 3)

The reaction potential at each electrode is measured separately as half cell potential

against the universal reference electrode such as AgAgCl because it is impossible to

directly measure both inner potentials simultaneously

113 Electric power from the fuel cells

Electric power (P) is the amount of work can be handled by electric current per

unit time It is defined as in Eq 33 The size of power is determined by the size of

electric current (I) and voltage (V) since P is proportional to I and P

36

Electric power

VIP times= middotmiddotmiddotmiddotmiddot Eq 4

P = Electric power W

I = Electric current A

V = Voltage V

Electric current is the amount of charge (Q) flows per unit time Voltage is the electric

potential energy per unit charge and synonymous with potential difference (∆E) EMF

or OCV Increase in I andor V increases P V is however constrained by the type of

electrode reactions as discussed previously I depends on (i) reaction rate (ii) the

electrode surface and (iii) reactant concentration as shown in Eq 5

Electric current in electrochemistry

nFAkCnFAI == υ middotmiddotmiddotmiddotmiddot Eq 5

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

A = Electrode surface area m-2

υ = Reaction rate (1st or pseudo 1st order) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

It is indispensable to have large rate constant to increase output current This demands

lowering activation energy for a chemical transition during the reaction and can be

achieved by use of the catalysts

37

12 CATALYSTS

121 Faradayrsquos second law ndash Current and reaction rate

Catalysts accelerate specific chemical reactions without altering the change in

overall standard Gibbs free energy In other words they change the reaction kinetics but

not the thermodynamics They only provide an alternative lower-energy pathway to

shorten the reaction time without changing themselves before and after the reaction

[25-27] In fuel cells catalysts are used to increase the current output by accelerating the

electrode reaction The relationship between current and rate of reaction is defined by

Faradayrsquos second law of electrolysis (Eq 5 - Eq 7) This expresses the size of electric

charge generated in terms of the number of moles of reactant consumed or product

produced

38

Faradayrsquos second law of electrolysis

nFNQ = middotmiddotmiddotmiddotmiddot Eq 6

AtNCktimes

=times=υ middotmiddotmiddotmiddotmiddot Eq 7

Q = Charge C

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

N = The number of moles of reactant or product mol

υ = Reaction rate (1st or pseudo 1st ordedr) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

t = Time s

A = Electrode surface area m-2

Faradayrsquos second law of electrolysis states that an increase in reaction rate increases the

current output To increase the reaction rate intrinsically its Gibbs free energy of

activation (∆GDagger) must be lowered The relationship between k and ∆GDagger is stated in

Erying equation [28]

122 Transition state theory Eyring equation and reaction rate

When a reaction proceeds it must go through a critical state called transition

state of maximum potential energy also called the activated complex Activated

complexes are unstable molecules existing for only a few molecular vibrations before

decaying into the products Transition state theory assumes that the reactants and the

39

activated complexes are in quasi-equilibrium and the reaction rate is constrained by the

rate at which the activated complex decays into the products This implies that the

reaction rate depends on the equilibrium constant between the activated complexes and

the reactants (KDagger) and the atomic vibrational frequency (ν) as shown in Eq 9 ν is

defined by statistical mechanics in terms of Boltzmann constant (kB) Planck constant

(h) and the temperature (Eq 10) [28]

Transition state theory and reaction rate [28]

[ ]RTexpDagger Dagger

Dagger GC

CK ∆minus== middotmiddotmiddotmiddotmiddot Eq 8

Dagger Kk ν= middotmiddotmiddotmiddotmiddot Eq 9

hTkB=ν middotmiddotmiddotmiddotmiddot Eq 10

KDagger = Equilibrium constant between the reactants and the activated

complexes

s-1

CDagger = Activated complex concentration molm-3

C = Reactant concentration molm-3

∆GDagger = Gibbs free energy of activation Jmol-1

R = Gas constant = 8314 Jmol-1K-1

T = Thermodynamic temperature K

k = Rate constant (1st or pseudo 1st reaction) s-1

ν = Atomic vibrational frequency s-1

kB = Boltzmann constant = 13807 x 10-23 JK-1

h = Planck constant = 662621 x 10-34 Js

40

Based on this the Erying equation expresses the relationship between the reaction rate

and Gibbs activation free energy as shown in Eq 11 [25 28 29]

Erying equation TG

B

hTkk

RDagger ∆minus

= κ middotmiddotmiddotmiddotmiddot Eq 11

where κ = transmission coefficient

This tells that to increase the rate constant reaction temperature must be increased or

the activation free energy must be lowered

123 Overcoming the activation free energy

Activation free energy is a potential energy gap between the reactant and

activated complex At the transition state intra-molecular reorganisation takes place

through the bond destruction and formation This demands the energy and slows the

kinetics The reaction does not proceed unless this energy barrier is overcome Hence

energy input in someway or lowering activation free energy is required for the reaction

to proceed andor accelerate the reaction Heat energy can provide atomic and molecular

kinetic energy so that the system can overcome the energy barrier as indicated in Eq 11

[4 21 29] Applying electric potential also has the similar effect It provides the electric

energy to overcome the energy barrier The rate constant therefore can be altered

extrinsically by temperature andor electrode potential [21 29 30] Alternatively

catalysts can reduce ∆GDagger so that the reaction rate can be increased intrinsically They

enable or accelerate a reaction by providing alternative lower-energy reaction pathway

41

This does not change the reaction path or the standard enthalpy change (∆Hdeg) of the

system however lowers the activation free energy for this path so that the reactants can

be converted more quickly to the products as shown in Fig 4

Fig 4 Energy transition for catalytic reaction through the transition state

Fig 4 shows the energy state transition during a reaction with and without a catalyst

The reaction coordinate represents the reaction progress along the reaction pathway

∆Hdeg is a state function [31] therefore determined only by the initial and final state of

the system but independent of the reaction path ∆Hdeg is divided into standard Gibbs free

energy change (∆Gdeg) and the term of standard entropy change (∆Sdeg) as Eq 12 shows [4

21 23 26 32]

The key insight therefore from transition state theory is that for a catalyst to function it

42

must spontaneously bind the transition state

Gibbs free energy change

deg∆minusdeg∆=deg∆ STHG middotmiddotmiddotmiddotmiddot Eq 12

deg∆minus= EnF middotmiddotmiddotmiddotmiddot Eq 13

∆Gdeg is the standard free energy difference which became available for work through

the reaction and must be lt 0 for a spontaneous reaction ∆Gdeg can be related to the

standard electrical potential difference as Eq 13 shows [4 21 29 30 33 34] It is the

maximum EMF theoretically available from the system ∆Sdeg is the difference in the

degree of disorder in the system The entropy term (T∆Sdeg) is the energy not available for

work and temperature dependent This and Eq 13 imply that ∆Gdeg is also altered by

temperature as shown in Fig 4 [35]

124 Variations in catalysts

Given the general applicability of transition state theory it is not surprising that

catalysts have extensive variation and applications the reaction phase can be

homogeneous or heterogeneous catalysts can be inorganic organic or biological

Inorganic catalysts are metals such as transition or noble metals or complexes

on metal ions in various oxidation states Elemental metal catalysts typically catalyse

reactions through surface adsorption Metal particles have extensive variation in

physical properties such as particle size shape and surface area They often come in

dispersed on the porous support material for improved catalytic performance due to

43

increased surface area [36] and cost efficiency [1] Composition and structure of the

particles thickness of support material and condition of metal deposition all affect

reactions [27 37-39] A complex is a compound in which a ligand is loosely bonded to

a metal centre through coordination bonds An unsaturated complex has a dissociated

ligand and catalysis takes place when a substrate binds to that in the single orientation

[27 40] Complexes also have important roles in biological system [41-43] Organic

catalysts are non-metal non-biological catalysts They are often functionalised solid

surfaces for surface catalysis [27] In electrochemistry such catalysts can be chemically

bonded to conductive material [44] Simple electrochemical pretreatment can generate

or attach chemical groups onto the electrode surface and they mediate electron transfer

[45-47] Biological catalysts are generally called enzymes Enzymes are biological

polymers mostly made of protein and usually have high reaction and substrate

specificity They often have cofactors which are not proteinous but essential for their

catalytic activities Enzymes are involved in virtually all biological activities [23 25 27

48] and in a few industrial applications due to their substrate specificity [27]

Catalysts are by definition not supposed to be consumed in their reactions

however they could lose their catalytic power Impurities fouling and poisoning are

common deactivating factors for the metal catalysts Competing reactions also convert

them into inactive forms Particle migration and agglomeration reduce their catalytic

surface area hence their overall activity [27] Biological catalysts are very sensitive to

the environment Slight changes in pH and temperature can deactivate enzymes Their

stability is also affected by presence of metal ions surfactants oxidative stress and

organic solvent molecules [49] Metal catalysts can be possibly regenerated by

redispersion [27] however it is very difficult to recover biological catalytic functions

44

Therefore enzyme stabilisers are often added to extend their life Protein engineering

also can confer advantageous properties to the enzymes Amino acid sequences leading

to favourable protein folding functions or stability are determined in silico and they are

introduced to existing enzymes by genetic engineering such as site-directed mutagenesis

Protein engineering of serine protease subtilisin is a classic example [50]

13 ELECTRODE POTENTIAL AND REACTION RATE

131 Fermi level electrode potential and electron transfer

In the sect 123 the relationship between the electric potential and activation free

energy was briefly discussed As heat energy can provide the kinetic energy to atoms

and molecules for a reaction to proceed the electric energy also can provide kinetic

energy to electrons for electron transfer to proceed This leads to the redox reaction at

the electrode surface

Each substance has a set of discrete energy levels Each energy level can

accommodate up to two electrons Electrons fill these energy levels in ascending order

from the lower level This can divide these energy levels into two groups electron-filled

lower energy level and empty higher energy level without electrons In between the

critical energy level the called Fermi level lies It is designated as εF (Fermi energy) or

EF (Fermi potential) The Fermi level contains electrons of highest energy A substance

can transfer only these electrons to the external system as a donor while as an acceptor

it can accommodate external electrons only within the empty energy level which is

above its Fermi level (Fig 5)

45

Fig 5 Fermi level and electron transfer

The energy state of electrons however varies by substance therefore electrons at Fermi

level so-called highest occupied molecular orbital (HOMO) of a donor may need to

gain various kinetic energy to jump into the lowest empty energy level so-called lowest

unoccupied molecular orbital (LUMO) of an acceptor for electron transfer (Fig 6)

46

Fig 6 Electrode potential and electron transfer

As soon as the energy of electrons at Fermi level of a substance or an electrode meets

the energy level of LUMO of the other redox molecule electron transfer proceeds as

long as there is room to accommodate electrons in the acceptor This is the cathode

reaction The potential can be swept back by applying positive potential to the electrode

This lowers the energy of LUMO of the electrode so that an electron in HOMO of the

donor can be transferred back to the electrode This is the anode reaction If the electron

transfer is fast in both directions compared with other reaction steps it is a

thermodynamically reversible system and the standard electrode potential (Edeg) for this

redox reaction lies somewhere between and close to those two electrode potentials

which have enabled electron transfer At Edeg both anode and cathode reactions have the

same reaction rate hence they are at equilibrium [33] and there is no net current [21 30]

47

132 Electrode potential equilibrium potential and Nernst equation

Activation free energy of an electrode reaction can be altered by applying

potential The extent of its alteration and the direction of the reaction depend on the size

and direction of potential applied Fig 7 shows the difference in the standard activation

free energy (∆GdegDagger) for redox reactions at various electrode potentials [30]

48

Fig 7 Standard free energy and electrode potential [30]

(a) At E = Edeg (b) E gt Edeg and (c) E lt Edeg

More positive potential lowers the activation free energy for oxidation hence shifts the

reaction in favour of oxidation and vice versa This implies that applying extra potential

to the electrode perturbs the equilibrium and to restore it the electrode equilibrium

49

potential (Ee) must be shifted This phenomenon is clearly stated in Nernst equation (Eq

15 on p 50) as a relationship between Ee and the equilibrium constant (K) at the

electrode surface At equilibrium both oxidation and reduction reactions take place at

the same rate and there is no net current therefore the surface concentration (C(0t))

approximates bulk concentration (C) Nernst equation shows that Ee is always under

the influence of such concentration but can be established provided the reaction is fast

enough to restore the equilibrium [21 30] In the dilute solutions of the electroactive

species Edeg is substituted with the formal potential (Edegrsquo) which considers the influence

of inter-solute and solvent interactions [21 29]

Redox reaction

middotmiddotmiddotmiddotmiddot Eq 14

O = Oxidant

ne- = The number of electrons transferred

R = Reactant

kf = Forward reaction rate constant (1st or pseudo 1st reaction) s-1

kb = Backward reaction rate constant (1st or pseudo 1st reaction) s-1

50

Nernst equation

lowast

lowast

+deg=R

Oe

CC

nFRTEE ln middotmiddotmiddotmiddotmiddot Eq 15

Ee = Equilibrium potential V

Edegrsquo = Formal potential V

R = Gas constant = 8314 Jmol-1K-1

T = Absolute temperature K

n = The number of electron transferred

F = Faraday constant = 96487 Cmol-1

CO = Bulk oxidant concentration molm-3

CR = Bulk reductant concentration molm-3

133 Electrode potential and activation free energy

When E ne Edegrsquo the difference in these potentials is expressed as the electrode

potential shift (∆E) as in Eq 16 and its free energy shift (∆G) as in Eq 17

Electrode potential shift

degminus=∆ EEE middotmiddotmiddotmiddotmiddot Eq 16

Free energy shift

)( degminus=∆=∆ EEnFEnFG middotmiddotmiddotmiddotmiddot Eq 17

When ∆E is applied positively it lowers the anode oxidation activation free energy

(∆GaDagger) by fraction of the total energy change (∆G) as shown in Fig 8 on p 51 The

fraction for the oxidation reaction is defined as (1-α) The transfer coefficient (α) is a

51

kinetic parameter which states the degree of symmetry in the potential energy barrier

for redox reactions The symmetry in energy barrier is present when the slopes of two

potential energy curves are the same and it is defined as α = 05 α is dependent on

redox potential and could vary within the range between 0 and 1 In most cases α = 05

and appears to be constant when overpotential is small [21 30]

Fig 8 The shift of ∆E and ∆GdegDagger [21 30]

With α the shift in electrode activation free energy can be expressed in terms of

potential shift for both oxidation and reduction reactions as shown in Eq 18 and Eq 19

52

∆GDagger in terms of (E-Edegrsquo)

Anode Oxidation )()1(DaggerDagger degminusminusminus∆=∆ EEnFGG aa αo middotmiddotmiddotmiddotmiddot Eq 18

Cathode Reduction )(DaggerDagger degminus+∆=∆ EEnFGG cc αo middotmiddotmiddotmiddotmiddot Eq 19

∆GoDagger = Anode oxidation activation free energy Jmol-1

∆GodegDagger = Anode oxidation standard activation free energy Jmol-1

α = Transfer coefficient molm-3

n = The number of electrons transferred Jmol-1

F = Faraday constant = 96485 Cmol-1

E = Arbitrary electrode potential V

Edegrsquo = Formal potential V

∆GRDagger = Cathode reduction activation free energy Jmol-1

∆GRdegDagger = Cathode reduction standard activation free energy Jmol-1

134 Potential shift and standard rate constant

By substituting the activation free energy in Eyring equation (Eq 11) with the

shift in electrode activation free energy in Eq 18 and Eq 19 it is possible to express k

in terms of potential shift and standard rate constant (kdeg) (Eq 20 and Eq 21) kdeg is the

rate constant at Edegrsquo[21 30]

53

k in terms of (E-Edegrsquo) and kdeg

Forward reduction cathode rate constant (kf) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bf

Daggercexpκ

⎥⎦⎤

⎢⎣⎡ degminusminus

⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )(expexp

Dagger

EERT

nFRT

GhTk cB ακ

o

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= )(exp EERT

nFk fα middotmiddotmiddotmiddotmiddot Eq 20

Backward oxidation anode rate constant (kb) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bb

Daggeraexpκ

⎥⎦⎤

⎢⎣⎡ degminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )()1(expexp

Dagger

EERT

nFRT

GhTk aB ακ

o

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= )()1(exp EE

RTnFkb

α middotmiddotmiddotmiddotmiddot Eq 21

kf and kb = Forward and backward reaction rate constant s-1

kdegf and kdegb = Forward and backward reaction standard rate constant s-1

135 Butler-Volmer equation overpotential and current

At the equilibrium potential E = Ee kf = kb hence anodic current (Ia) =

cathodic current (Ic) and therefore the net current (I =Ia ndash Ic) is macroscopically zero (I =

0) However both forward and backward reactions still occur microscopically at the

electrode surface The absolute value of this electric current is called exchange current

(I0) and is not directly measurable When CO = CR at E = Ee C O (0t) = C R (0t) and kdegf

= kdegb = kdeg hence the net current any potential E can be defined as below by

Butler-Volmer equation (Eq 22)

54

Butler-Volmer equation

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡ degminus

minusminus⎥⎦

⎤⎢⎣⎡ degminusminus

deg= )()1(exp)(exp )0()0( EERT

nFCEERT

nFCnFAkI tRtOαα middotmiddotmiddotmiddotmiddot Eq 22

The Butler-Volmer equation states the relationship between the electric current

and the potential shift This can be further rephrased in terms of overpotential (η)

through defining I0 using Nernst equation (Eq 15) η is the potential required to drive a

reaction in a favourable direction from its equilibrium state and defined as n Eq 23

Overpotential (η)

eEE minus=η middotmiddotmiddotmiddotmiddot Eq 23

At E = Ee both anode and cathode exchange currents are the same therefore both

exchange currents can be defined together as I0 (see below) and I0 can be expressed in

terms within Nernst equation by substituting (Ee-Edegrsquo) as in Eq 24

Exchange current

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= lowast )(exp0 EERT

nFCnFAkI eO

α

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= lowast )()1(exp EE

RTnFCnFAk e

Rα

αα )()( 1RO CCnFAk minus= o middotmiddotmiddotmiddotmiddot Eq 24

Now the Butler-Volmer equation (Eq 22) can be expressed in terms of η (= E ndash Ee) (Eq

25) by expressing it with I0 and the with re-arranged Nernst equation

55

⎥⎦⎤

⎢⎣⎡ degminus=lowast

lowast

)(exp EERTnF

CC e

R

O The term

)0(

CC t in Eq 25 indicates the influence of mass

transport For purely kinetically controlled region the electrode reaction is independent

of mass transport effects therefore C(0t) asymp C and Bulter-Volmer equation approximates

to Eq 26

Butler-Volmer equation and η

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminusminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminus

minus= lowast

lowast

lowast

minusminus

lowast

lowast

lowast

αααα

R

O

O

tO

R

O

R

tR

CCEE

RTnF

CC

CCEE

RTnF

CC

II )(exp)()1(exp )0()1(

)0(0

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= lowastlowast ηαηαRT

nFC

CRT

nFC

CI

O

tO

R

tR exp)1(exp )0()0(0

middotmiddotmiddotmiddotmiddot Eq 25

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= ηαηαRT

nFRT

nFI exp)1(exp0 middotmiddotmiddotmiddotmiddot Eq 26

The Butler-Volmer equation shows the dependence of current and hence the reaction

rate on the overpotential This implies that small kdeg can be overcome by overpotential

The kinetically-limited region of the electrode reaction is however very small because

the rate constant sharply increases exponentially with overpotential and soon the

reaction became diffusion limited [21 30] At this state the surface equilibrium is

totally perturbed and the overpotential is used to drive the diffusion for the one-way

reaction

136 Butler-Volmer equation vs Tafel equation

At small η lt RTnF Butler-Volmer equation further approximates to a linear

equation as shown in Eq 27

56

Small η Butler-Volmer equation

η⎟⎠⎞

⎜⎝⎛=

RTFnII 0

middotmiddotmiddotmiddotmiddot Eq 27

At large |η| gt RTnF the rate of the backward reaction is negligible hence one

of the exponential terms in Butler-Volmer equation becomes negligible and the

equations are solved for η for both anode and cathode reactions as below

Large η Tafel equations

InFRTI

nFRT lnln 0 plusmn= mη in the form of ||ln Iba +=η middotmiddotmiddotmiddotmiddot Eq 28

Anode oxidation InF

RTInF

RT ln)1(

ln)1( 0 αα

ηminus

+minus

=

Cathode reduction InF

RTInF

RT lnln 0 ααη minus=

In the system which follows the Tafel equations I0 (and hence kdeg ) is very small and the

electrode reaction does not proceed unless large η is supplied In this potential range the

reaction is already totally irreversible [4 21 30] An irreversible slow reaction is

graphically presented in Tafel plot in which ln (I) is plotted against η as shown in Fig

9

57

Fig 9 Tafel plot [4 21 30]

I0 is deduced from the y-intercept of the extrapolated slope Once |I| has become too

large and reached to the mass transport limit at large |η| the slope deviates from the

linear trend [30]

14 OVERPOTENTIAL AND FUEL CELLS

141 Four overpotentials that decrease the EMF

As discussed so far overpotential is extra potential required to drive a reaction

In the fuel cells this can only be taken from the potential generated within the system

causing potential drop and reduces EMF The overpotential in the system is attributed to

the four causes [1 4] (i) internal current (ii) activation overpotential (iii) diffusion

overpotential and (iv) resistance overpotential

58

142 Internal current

Internal current is a reverse current caused by crossover and leakage current

Crossover happens when the fuel flows to the opposite electrode through the electrolyte

Leakage current occurs when the current flows through the electrolyte rather than the

external circuit Internal current therefore happens without a load and is observed as the

negative offset from the theoretical value of EMF This is noted especially in

low-temperature fuel cells such as direct methanol fuel cells (DMFCs) [4 51] In such

systems The inner current can be several 10 Am-2 and potential drop could be more

than 03 V [4] In order to prevent fuel mixture and crossover a separator is used to

partition the cell Alternatively it is also possible to confer high specificity to electrodes

as seen in glucose - oxygen fuel cells Such systems do not require

compartmentalisation and this is advantageous for miniaturisation [52]

143 Activation overpotential

Activation overpotential is a potential loss due to the slow reaction kinetics as

discussed in sect 135 This is unavoidable to some extent and is typically the biggest

problem in most fuel cells involving O2 cathode reduction because this reaction is

kinetically hindered Finding or inventing catalysts with high kdeg is the key solution for

this A sluggish reaction exhibits a sharp rise in activation overpotential for a slight

increase in low current After the steep increase the current increase becomes linear and

constant as shown in Fig 10 until the reaction reaches the limit of mass transport at the

higher overpotential [4 30 34]

59

Fig 10 Activation overpotential against current [4]

144 Diffusion overpotential

Diffusion overpotential is a potential loss due to concentration gradient This

tries to drive flux of electroactive species toward and from the electrode surface It

happens when the reaction kinetics is faster than mass transport [4 21 30] In fuel cells

diffusion overpotential is caused by fuel depletion due to the inefficient fuel convection

or supply [21 34] This problem is observed in reformers because the fuel refining and

conversion such as liquefied petroleum gas (LPG) to hydrogen gas take place outside

the system and it makes difficult to respond to the sudden fuel demand from the system

[4] It also happens when fuel diffusion is interfered with by bad electrode geometry

electrode fouling and adsorption [21 27] Diffusion overpotential is defined as Eq 29

60

Diffusion overpotential equation

⎟⎟⎠

⎞⎜⎜⎝

⎛minus=

Ldiffusion I

InFRT 1lnη middotmiddotmiddotmiddotmiddot Eq 29

ηdiffusion = Diffusion overpotential V

IL = Limiting current A

Limiting current (IL) is generated when a reaction takes place at the rate limited by mass

transport Diffusion overpotential sharply increases when reaction kinetics have reached

to the maximum and very large current are drawn as shown in Fig 11 [21 30 34 53]

Fig 11 Diffusion overpotential against current [4]

145 Resistance overpotential

Resistance overpotential arises within electrodes separators connectors circuit

or electrolyte [4 34] It is called Ohmic resistance or IR drop because it follows Ohmrsquos

61

law (Eq 30) where resistance overpotential is linearly proportional to the electric

current as shown in Fig 12 [4 30]

Ohmrsquos law

IRV = middotmiddotmiddotmiddotmiddot Eq 30

V = Voltage consumed due to the resistance V

I = Electric current drawn due to the resistance A

R = Resistance Ω

| |

Fig 12 Resistance overpotential against current [4]

Causes of internal resistance are scattered through the system therefore there are many

possible ways to resolve this One can possibly reduce it by using more conductive

material The difficulty lies in finding the conductive material which can be modified as

well as carry out the functions as one desires under the required conditions [53]

Resistance can be reduced by reducing the distance between two electrodes [54]

however this might cause short circuit and fuel leakage This becomes a serious issue in

62

use of solid electrolyte such as polymer electrolyte because both performance efficiency

and mechanical stability of electrolyte must be achieved [53] Excess in inert supporting

electrolyte minimises solution resistance and matrix effect It sustains a thin electric

double layer uniform ionic strength and buffering effect against pH change [21 30]

However electrolyte could become a problem when electrode material dissolves into

electrolytic solution and reacts there because it might change nature of electrolyte [1

21]

146 Total overpotential and cell voltage

Total overpotential is the sum of all four overpotentials Current ndash Voltage (IV)

polarisation curve visualises the tendency of each potential drop as shown in Fig 13 [1

4]

Fig 13 V-I curve for a typical low temperature fuel cell [4]

63

The cell voltage (Ecell) is determined by subtracting the cathode potential (Ec)

from the anode potential (Ea) (Eq 31) The actual cell voltage is found by subtracting

the total overpotential from the theoretical cell voltage The energy available from this

cell voltage can be calculated as in Eq 32

Cell voltage

cacell EEE minus= middotmiddotmiddotmiddotmiddot Eq 31

totalltheoreticacellactualcell EE ηminus= )()(

cellcell nFEG minus=∆ middotmiddotmiddotmiddotmiddot Eq 32

15 BIOFUEL CELLS

151 Microbial and enzymatic fuel cells

Biofuel cells are fuel cells which use biocatalysts for fuel reactions

Biocatalysts can be either the whole living cells or isolated enzymes The former is

called a microbial fuel cell and the latter is called an enzymatic fuel cell [55-57] They

often function under ambient conditions and allow wider choice of fuels Microbial fuel

cells use common carbohydrates such as glucose and lactate [55] but also have

potentials in extracting power from waste and sediments [34 55 57 58] Harnessing

such microbial life activities has some advantages They can complete fuel oxidation

within the cell It was reported that a microorganism Rhodoferax ferrireducen has over

80 glucose conversion rate [58] Microbial fuel cells also have long-term stability

[56-58] of up to five years [55] However their output current and power densities are

64

small due to high internal resistance [55-57] Enzymatic fuel cells in contrast have

higher current and power densities but short life incomplete reactions require

mediators to shuttle electron between the enzyme catalytic centre and the electrode and

become deactivated easily [55 56 58] Addition of mediators and their immobilisation

with enzyme complicates fuel cells design further However they can be more

advantageous due to their substrate specificity Both anode and cathode to reside in the

same compartment of mixed fuels without crossover This enables the miniaturisation of

fuel cells [52 59 60] and is particularly useful for medical implantation Stability and

lifetime of the enzyme fuel cells can be improved by appropriate enzyme

immobilisation fixing up the suitable conditions for their function [55] and adding

some enzyme stabilisers Addition of the right mediators and increased enzyme loading

will also increase output current and power density [55 59]

152 Enzyme fuel cells and mediators

Enzyme reactions are available for wide range of substrates yet each enzyme

has very high specificity for its substrates Many also function at neutral atmospheric

conditions therefore miniaturised enzyme fuel cells are of interest for in vivo

application Many redox reactions however take place at the catalytic centre which is

deeply buried in the protein body This proteinous content is an electronic insulator and

interrupts the electrical communication between the redox centre and the electrode It is

therefore difficult for many enzymes to establish efficient electron transfer without

mediators as shown in Fig 14 This is called mediated electron transfer (MET)

65

Fig 14 Mediated electron transfer reaction [55 61]

It is possible for some enzymes such as multicopper oxidases (MCOs) to perform direct

electron transfer (DET) without mediators however mediators ensure good electrical

communication and cell performance However the presence of mediators as well as the

enzyme specificity could complicate the fuel cell composition and its reaction path due

to the reaction cascade [55 59 62 63] Palmore built a methanol - oxygen enzyme fuel

cells and this involved four enzymes two mediators and six reactions to complete the

full reaction path [64] It is more advantageous to use enzymatic fuel cells for very

specific applications [59] such as medical implantation where there are environment

constraints but where applications demand only small power output through simple

reactions

Mediators are usually small redox active species which can be organic or

metal complexes In the enzymatic fuel cells they should be carefully selected because

improper mediators can cause unwanted overpotential or can destabilise the system

They must have (i) small overpotential (ii) fast reversible electron transfer [55 56 59

60 63 65] and (iii) stability [62 65] However there should be small but reasonable

66

potential gap between the enzyme and the mediator to drive electron transfer [60 66]

The gap of 50 mV is optimal [19 64] because of the small potential loss yet less

competing with enzyme reactions It is also possible to mediate electron transfer by

building a conductive nanowire between the electrode and the redox centre [60 63 67]

or a connection by plugging metal nanoparticles into the enzyme [60 67]

16 MINIATURISED MEDICAL POWER SOURCES

161 Realisation of miniaturised fuel cells and medical power supply

Medical application is one of the obvious targets for miniaturised biofuel cells

Glucose-oxygen biofuel cells which continuously run on glucose and oxygen were

thought to be ideal for implantable medical devices because both oxygen and glucose

are abundant in the body [63] They were initially intended to power cardiac assist

devices and pacemakers The studies on such systems have been made for more than 40

years however none has been made into practical use because their power density

stability operation life and biocompatibility were inadequate [19 67-69] Their output

power ranges between microW ndash mWcm-2 and they operate only for a few days to a few

weeks Most applications need a few W power for a longer duration [18 60 67] A

pacemaker requires 1microW [56] for more than 8 ndash 10 years with 996 reliability Such

power has been supplied by lithium (Li) iodine battery [19 56 70] which is

characterised by extended operational life [68] Li is fairly stable light-weight metal

and has high energy density of 386 kAhkg-1 Its standard potential is highly reducing ndash

323 V vs AgAgCl These properties enable Li batteries to power most devices

67

including pacemakers laptops and vehicles [19 21] However there are some problems

in Li batteries They are still bulky because they need to store their own fuel and it has

to be sealed with the metal case [19 68] They occupy half of the space in the

pacemaker volume [68 71] They use corrosive [72] and irritating materials There is

potential of explosion [68] leakage and the necessity of exchange Li also has potential

resource constraint due to the geopolitical issues as seen for oil and platinum More than

80 of its production is based in South America and China [73] With increased public

concern over using greener safer energy and saving resources as well as the global

political intervention in that it is natural to consider about using alternative

technologies to power implantable medical devices where it is possible

162 Miniaturised potential medical power sources

There are numerous implantable electrical medical devices with different

power requirements These include cardiac pacemakers defibrillators neurostimulators

for pain control drug delivery device hearing aids [70] and biosensors [74]

Implantable autonomous body sensors for physiological parameters such as glucose

concentration local pH flow and pressure [19] require power as low as 1 microW [56 70

74] and the operation period of 1 year at least [74] Such power can be supplied by

various implantable energy harvesting devices using body glucose [19] motion

vibration [75] and temperature differences [75] Heller developed the smallest

glucose-oxygen biofuel cells made of carbon fibres with the dimension of 17-microm

diameter and 2-cm length Anode glucose oxidase and cathode laccase were wired to

conductive hydrogel backbone with a flexible osmium (Os) mediator complex Its

68

output was 048 mWcm-2 with 06 V at 37 degC and it lasted for 1 week [19 67]

Kinetic energy of motion and vibration can be converted into electricity with

electrostatic transduction using microelectromechanical systems (MEMS) This

technology allows integration of various microelectronic devices on the same substrate

[76] NTT Corporation developed a system generating power from body motion and

temperature It consists of a thermoelectric and a comb-shaped Au-Si vibrational device

on a monolithic Si substrate The voltage produced by a thermoelectric device is

boosted by capacitance which was generated by the vibrational device then its charge

is stored in the capacitor [75] Another vibrational device was reported to have 20 x 20 x

2 mm volume and output 6 microW and 200 V at 10 Hz low frequency and 39 ms-2

acceleration The principal difficulty in using MEMS is to adjust and synchronise

flexibly the resonance frequency of vibrational device to the human body frequency

which is low and has constant fluctuation in order to increase energy conversion

efficiency The typical vibration has the frequency ranging from 1 to 100 Hz and

acceleration from 1 - 10 ms-2 [76]

Thermoelectric devices generate power from temperature difference Each

module consists of a number of thermocouples on Si substrate Thermocouples are

made of different material so that it can generate voltage from the heat flow following

temperature gradient This is called Seebeck effect and its size depends on the material

and number of thermocouples used and the spacing within the device [75 76] Typical

ambient temperature difference is less than 10K [74 76] and its power is 1microWcm-2 [74]

Seiko Instrument developed a wristwatch called Seiko Thermic which was powered by

human body temperature in 1998 [77 78] It consists of n-type Bismuth-Tellurium

(BiTe) and p-type Bismuth-Antimony-Tellurium on rods reinforced by Ni on oxidised

69

silicon wafer It generated microWcm-2 power at extremely low 0047 conversion

efficiency With a booster circuit it powered a wristwatch operating at 1microW and 15 V

It could however operate only when ambient temperatures were below 27 degC [78] and

so could not be used in tropical regions

Heller also developed an implantable miniature Nafion-coated zinc ndash silver

chloride (ZnAgCl) battery The anode was made of zinc fibre coated with Nafion It

was 76-microm diameter 2-cm length and 75 x 10-2 cm2 surface area The cathode was

made of AgCl wire covered with chloride-permeable membrane On discharge anode

zinc formed Zn3(PO4)2middot4H2O hopeite complex with Nafion growing non-porous

lamellae on the surface The lamellale had a unique property which was permeable to

zinc ions but impermeable to O2 therefore the battery became corrosion free It output

was 13 microAcm-2 at 1 V and operated for three weeks under physiological condition The

advantage of this battery was its size which was 100-fold smaller than the smallest

conventional batteries [79] The smallest silver oxide button buttery has the volume 30

mm3 [19 80] with energy density 04 mWh mm-3 at 15 V The smallest Li-ion buttery

has the volume 190 mm3 [80] with energy density 025 mWh mm-3 at 4 V [21]

17 OUTLINE OF THE WORK

171 Introduction to miniaturised glucose-oxygen fuel cells

A miniaturised glucose-oxygen biofuel cell runs on glucose and oxygen This

would be ideal to power implantable autonomous low-power body sensors since both

glucose and oxygen are abundantly present in the body The range in their physiological

70

concentration is between 4 ndash 8 mM for glucose and 01 ndash 02 mM for oxygen [56 67]

The aim of this study was to build miniaturised glucose-oxygen fuel cells with reliable

enzyme anodes and powerful oxygen-reducing cathodes using straightforward and

scaleable technology The general structure and mechanism of glucose-oxygen fuel cells

were described in sect 112

In this study both anode and cathode reactions took place at the room

temperature and physiological pH in the presence of catalysts Glucose oxidation was

catalysed by the enzyme glucose oxidase (GOx) from Aspergillus niger and its electron

transfer and conduction were facilitated by an conducting organic salt called

tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) (Fig 15)

Fig 15 The schema for a glucose-oxidising anode

GOx and TTF-TCNQ were mixed into a composite employing a feasible electrode

fabrication method called bulk modification method Various enzyme stabilisers were

added to the composite at different concentration to improve its performance The

influence of various enzyme stabilisers on anode performance was studied in the

71

presence of both N2 and O2 and the most ideal anode composition was determined

Cathodes were also fabricated in the similar manner using graphite as conductive

material and mixing with various oxygen reducing catalysts except for the enzyme

membrane cathodes The cathode composites were pretreated in various media to

enhance their activity For enzyme membrane electrodes glassy carbon electrodes were

used instead of graphite composite and enzyme solution was loaded onto the surface

and trapped with membrane Catalytic power and mechanism of each cathode were

studied with various electrochemical methods and the most catalytic cathode was

determined

172 Outline

Chapter 1 is a general introduction about fuel cells It explains the history and

basic concept of fuel cells and outlines the study

Chapter 2 explains about the enzyme anodes The introduction part refers to

glucose oxidising enzymes enzyme immobilisation TTF-TCNQ enzyme inactivation

enzyme stabilisers and enzyme kinetics The material and method section reports the

protocol for the enzyme electrode fabrication electrode conduction study and

amperometry study for enzyme electrodes Result and discussion section mentions

enzyme electrode conduction enzyme kinetic studies based on amperometry

mechanism of enzyme stabilisation in each stabiliser and ideal electrode composition

Chapter 3 discusses oxygen-reducing cathodes The introduction part reviews

the mechanism of the oxygen reduction reaction oxygen reactivity oxygen reduction

catalysts carbon properties electrochemical pretreatments electrochemical techniques

72

and how to determine the catalytic efficiency of cathodes The material and method

section describes the protocol for the oxygen-reducing electrode fabrication preparation

and electrochemical pretreatment electrochemical studies electrochemical studies for

qualitative analysis and to obtain the kinetic parameters and analysis to determine the

efficiency of catalytic cathodes The result and discussion section describes the

voltammetric behaviour of each pretreated cathode influence of pretreatment and

catalytic efficiency The most catalytic cathode was determined at the end

Chapter 4 refers to the overall conclusion and further studies

73

2 ANODE INTRODUCTION

21 GLUCOSE OXIDATION ENZYMES

Glucose-oxygen fuel cells extract power by oxidising glucose at the anode The

anode contains glucose-oxidising enzymes There are several enzymes available for

glucose oxidation (i) β-D glucose oxidase (GOx) (EC 1134) [56 81] (ii) pyranose

oxidase (POx) (EC 11310) [56 81] (iii) nicotinamide adenine dinucleotide-dependent

glucose dehydrogenase (NAD-GDH) (EC 11147) [18 56] and (iv) quinoprotein

glucose dehydrogenase (PQQ-GDH) (EC 1152) [56 81] These enzymes are

classified into the family of oxidoreductases which catalyse either transfer of (i)

hydride (H-) (ii) oxygen atoms or (ii) electrons from one molecule to another Oxidases

transfer electrons from substrates to O2 and form H2O or H2O2 as products

Dehydrogenases transfer electrons from substrates to coenzymes without involving O2

(Eq 33 and Eq 34)

Glucose oxidation by GOx and NAD-DHG

GOx middotmiddotmiddotmiddotmiddot Eq 33

NAD-DHG middotmiddotmiddotmiddotmiddot Eq 34

To function catalytically enzymes require non-peptide inorganic or organic cofactors or

prosthetic groups Non-peptide organic cofactors are also called coenzymes Enzymes

lacking such groups are called apoenzymes and hardly functional Cofactors are

generally loosely bound to the apoenzymes and freely diffusible but on catalysis they

74

bind to the enzymes as the second substrates Prosthetic groups are always tightly bound

to the apoenzymes through covalent or non-covalent bonding [22 23 48] GOx and

POx are fungal flavoprotein enzymes containing flavin adenine dinucleotide (FAD)

cofactors FAD in GOx is tightly bound to the apoenzyme but not covalently bonded

hence still dissociable [67 82-84] NAD-GDH contains dissociable NAD+ [65] while

PQQ-GDH contains pyrrolo-quinoline quinone (PQQ) redox cofactor [56 65] which is

moderately bound to the apoenzyme [65 67] The structure of each cofactor is shown in

Fig 16

75

N

N

NH

N O

O

R

N

N

NH

HN O

O

R

NH

N

NH

HN O

O

R

H+ + e- H+ + e-

2H+ +2e-

N+

NH2

O

R

H

N

NH2

OH

R

HH

N

NH

O

O

OHO

OH

O

OH

O

N

NH

O

HO

OHO

OH

O

OH

O

N

NH

OH

HO

OHO

OH

O

OH

O

H+ + e- H+ + e-

FAD FADH2

NAD NADH

PQQ PQQH2

Fig 16 The structure of FAD NAD and PQQ and their redox reactions

Moieties involved in the redox reactions are circled in gray [23 65]

Cofactors also stabilise the enzyme structure Irreversible thermal GOx denaturisation

involves the perturbation of FAD biding site causing FAD dissociation although 70 of

its secondary structure is retained Chemical denaturisation untangles the dimmer

structure of GOx [65 81 83 85] and dissociates monomers and FAD [86 87] Excess

FAD does not prevent or regain function after thermal inactivation [87] However FAD

can rapidly bind to apoenzymes at 25 degC pH 61 and stabilise the tertiary structure

through conformational change and give resistance to proteolysis [82]

76

The choice of enzyme is based on the reaction involved enzyme availability

stability enzymatic properties physical properties and operational environment In the

absence of O2 or when there is concern about system degradation due to the competition

of electrons between O2 and artificial electron acceptors or due to the by-product H2O2

GDHs are more preferred to GOx [23 60 65-67 88] However the use of GDHs poses

different disadvantages Membrane-bound PQQ dehydrogenase (mPQQ-GDH) has very

high glucose specificity however it is difficult to solubilise and purify hence not for

commercial use [88] Soluble PQQ dehydrogenase (sPQQ-GDH) has been used in

commercial glucose sensors [67 89] however it suffers from low thermostability and

very low glucose specificity [88] Moreover PQQ-GDHs add complication to the

system because metal ions such as calcium ions (Ca2+) and magnesium ions (Mg2+) is

required to activate them [89 90] In the case of NAD-GDH NAD must be provided

because it is a dissociable cofactor but must bind first to the apoenzyme to form a

catalytic complex for glucose oxidation In addition to this complication NAD was also

reported to be more prone to causing electrodes poisoning without a quinone acceptor

[65] NAD forms adduct with phosphate in the buffer its decomposition releases free

adenine and electrochemically oxidised adenine adsorbs onto the electrode surface

causing electrode poisoning [91]

The redox potential of enzyme is also important because it is the driving force

for electron transfer and the power although it is difficult to match both catalytic

efficiency and electrochemical efficiency It is also difficult to determine the redox

potential exactly because the redox potential of cofactors is influenced by

microenvironment such as where they are embedded in the enzyme as well as

77

macroenvironment such as pH and temperature For example the redox potential of

FAD itself is ndash 042 V vs AgAgCl at pH 7 25 degC [22 23] however that of

flavoproteins could range from ndash 069 to ndash 005 V [65] and FAD-containing GOx has

the redox potential approximately ndash 03 V [67 92] It was also reported glucose

gluconolactone redox potential was ndash 056 V [93] PQQ itself has - 013V in absence of

Ca2+ and ndash 012 V in presence of 1M CaCl2 [94] while sPQQ-GDH has the redox

potential approximately - 019 V in presence of Ca2+ [66 67] Furthermore the enzyme

redox potential leads to a complex issue when mediators which assist electron transfer

between the enzyme redox centre and the electrode are involved because the improper

combination of the enzyme and mediator causes inefficient electron transfer and hence

potential drop

For enzyme availability stability and simplicity in handling GOx has been

most popular glucose-oxidising enzyme It has optimal pH 55 and temperature 35 ndash

40 degC [93] but is stable between pH 4 ndash 7 [95] and up to 50 - 55 degC [82 87] GOx has

higher substrate specificity and is most active toward β-D-glucose oxidation [56 86 88]

and very stable therefore its use has prevailed among various sensor applications [60

67]

22 DESIGNING ENZYME ELECTRODES

Construction of enzyme electrodes is complex because it simultaneously

incorporates enzymes mediators conductive materials and stabilisers ensuring that all

of them work harmoniously under restricted performance conditions There are several

78

immobilisation techniques available These include physical adsorption cross-linking

covalent bonding and entrapment in gels or membranes [96]

221 Physical adsorption

Physical adsorption is a simplest immobilisation method Electrodes are dipped

in the solution containing enzymes and mediators or such solution can be applied to the

electrode surface In this method however the enzyme tends to leach due to the weak

interaction between the electrode and enzyme [55] In order to avoid the loss of

electrode material the electrode surface is often covered or coated with material One

classic method is that the solution on the electrode surface is trapped with a

semipermeable dialysis membrane [61 65 92] This allows enzymes free from the

possible denaturisation caused by immobilisation on the solid material Enzymes inside

the dialysis membrane also can be fixed onto the cellulose acetate membrane or the

enzyme layer can be cross-linked with bifunctional reagents such as glutaraldehyde

[61]

222 Reconstituting apo-enzyme

Willner constructed enzyme electrodes by reconstituting apo-enzyme on the

gold (Au) electrodes surface with quinone mediators The electrode consisted of three

layers which were made of PQQ synthetic FAD and apo-GOx [97] PQQ were

mediating groups and attached onto Au electrode by cystamine PQQ attached on Au

was then covalently linked to synthetic FAD called N6-(2-aminoethyl)-FAD Covalently

79

linked FAD self-assembles with apo-GOx so that holo-GOx assembly is formed on the

top of the PQQ mediating layer attached to the Au surface The reconstitution procedure

is shown in Fig 17

Fig 17 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode [97]

The output current density was 300 microAcm-2 at pH 7 35 degC and it dropped by 5 in the

presence of O2 although the decrease was negligible at 01 mM physiological O2

concentration [98] The reconstitution of apo-enzymes is a flexible system potentially

applicable to enzyme electrodes [97] The fabrication process is cumbersome however

the enzymes are neatly aligned and electrically connected in the correct orientation

therefore electrocommunication between the enzymes and electrodes can be

maximised

80

223 Tethered Osmium-complexes linked to conductive hydrogel

It has been reported that embedding enzymes in the conductive hydrogel

complexed with tethered osmium (Os) mediator led to high output current [19 60 66

67] The conductive hydrogel is made of cross-linked polyvinylpyridine or

polyvinylimidazole They are soluble as monomers however they gain their

water-swelling property in cross-linked polymer This is ideal for enzyme mobility and

electrical conductivity Electron transfer is realised by the Os complex which is tethered

to the conductive hydrogel back bone The tether is made of 10 ndash 15 atoms [19 67] in

order to allow flexile movement of mediators Tethered Os complexes sweep the large

volume made of multiple layers of hydrogel form electrostatic adducts with enzymes

[19] and facilitate collide-and-hop electron conduction [19 99] Polymer tether and Os

complexes can be flexibly tailored for different enzymes and different operational

environments [19] One of its great advantages is that the redox potential for electron

transfer can be adjusted by changing the ligand structure of Os complex [19 60 97]

Heller constructed such electrodes made of carbon fibers whose dimension was 7 microm

diameter 2 cm length 08 mm2 surface area and 26 x 10-3 mm3 volume GOx was

wired to poly(4-vinylpyridine) conductive hydrogel backbone by the tethered complex

of tris-dialkylated NNrsquo-biimidazole Os2+3+ (Fig 18)

81

Fig 18 GOx wired to conductive hydrogel complexed with Os [19 67]

The redox potential of its redox centre was ndash 02 V vs AgAgCl The glucose oxidation

took place at ndash 036V and its output current density was 11 mAcm-2 [19] Disadvantage

of the tethered enzyme is short life It was stable for one week in the physiological

buffer then 5 current drop per day was observed When it was implanted in grape sap

50 current drop occurred within 20 hours In calf serum 50 current dropped

occurred in 2 hours with electrode fouling [19]

224 Entrapment in the conductive polymer

Entrapping enzymes in conductive polymer such as polypyrrole polyaniline

and polythiophene [65] is a popular method for enzyme electrodes [96] This is made by

82

electropolymerisation in which potential is applied to the solution containing enzymes

and monomors for certain duration It is a simple method but can be complicated if the

conditions for polymer growth are not compatible with enzyme entrapment [99]

Enzyme leakage occurs when enzymes are not trapped securely but it can be reduced

when derivatised polymers are used [96 100] Their conductivity is affected by

monomer structure [100] acidity and electrolyte [101] It can be degraded by products

of enzymatic reactions [65] Fujii fabricated GOx electrodes which were stable for

more than four months though a gradual sensitivity drop was observed GOx was

entrapped in saccharide-containing polypyrrole made of 1-(6-D-gluconamidohexyl)

pyrrole (GHP) (Fig 19(i)) on Pt electrodes It was reported that entrapped GOx was

secured by hydrogen bonding present between sugar gluconyl groups and enzymes

Enzyme leaching was also reduced by improved polymerisation with extended

conductive hydrocarbon chain [100] The output current density was 75 microAcm-2 at 055

V pH 74 in the presence of 56 mM glucose and 175 microA cm-2 under glucose saturated

condition [100] Arai entrapped various enzymes in polymer of mercaptobenzoquinone

(SQ) (Fig 19(ii)) and its methylated derivatives methylmercapttobenzoquinone

(MeSQ) and dimethylmercaptobenzoquinone (Me2SQ) on Au electrode surface

83

OH

H

H

OH

OH

H

OH

H

CH2OHN(CH2)6NHCHOH

(i)O

O

SH

Fig 19 Monomer structures of conductive polymers

(i) 1-(6-D-gluconamidohexyl) pyrrole [100]

(ii) Mercapto-p-benzoquinone [101]

Quinones have been widely used as good mediators [21 60 61 65] Poly(SQ) is a

conductive polymer functionalised with quinone mediating groups and can be easily

attached to Au electrode due to the presence of thiol group (-SH) [100] It was revealed

that more methylated SQ polymer led to better performance The maximum current

density for SQ- MeSQ- and Me2SQ-GOx anodes was 006 522 and 544 microAcm-2 and

their electrode enzyme dissociation constant KM was 13 18 and 20 mM respectively

[102 103] FAD cofactors have been also electropolymerised to entrap lactate

dehydrogenase and lactate oxidase It was reported that polymerised FAD extended the

shelf and operational life of electrodes to more than one year and also reduced O2

influence [104]

225 Bulk modification

Another popular electrode fabrication involves bulk modification of solid

composite This is simply made by mixing electrode material and inert binding reagent

84

such as epoxy resin Typical composite electrodes consist of 70 conductive material

by weight [105] Bulk-modified electrodes are robust stable inexpensive and easy to

modify build and reuse [105-107] therefore often used for enzyme electrodes [108]

For GOx electrodes tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ)

has been used as both mediating and conducting material

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties

TTF-TCNQ is a black crystalline charge transfer complex having both

mediating and conducting properties Its handling is also easy and often used for GOx

electrodes [65] Its conductivity is 102 ndash 103 Scm-1 [109 110] and comparable to that of

graphite [65 105] TTF-TCNQ complex consists of TTF electron donor and TCNQ

electron acceptor and they are arranged in a segregated stack structure TTF-TCNQ has

metal-like electric conductivity and this derives from intra-stack and inter-stack

molecular interactions Within the stack π-orbitals of adjacent molecules overlap This

builds the major conductive path in the direction of stack [61 65 110 111] Between

the stacks there is partial charge transfer between different chemical groups and this

gives the metallic feature in which charge carriers are mobile among the stacks

[110-112] as shown in Fig 20

85

S

S S

S

N

C

C

N

C

C

N

N

S

S S

S

7 6

N

C

C

N

C

C

N

N

-

TTF - electron donor

TCNQ - electron acceptor

6 - localised 6 - delocalised

+ e-

+ e-

Fig 20 TTF-TCNQ structure and its charge transfer [61]

TTF-TCNQ is a good electrode material because it has low background current and is

efficient at electron transfer [65] is stable [65] insoluble in water and not toxic [113]

However it exhibits complex electrochemistry (Fig 21) and its behaviours have not yet

been fully elucidated

86

vs AgAgCl

TCNQTCNQ e⎯⎯rarr⎯minus minusbull+

+minus+ ⎯⎯rarr⎯minus 2e TTFTTF

minus+minusbull⎯⎯rarr⎯

minus 2e TCNQTCNQ

+minus⎯⎯rarr⎯minus

TTFTTF e

Fig 21 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 01M potassium

phosphate 01 M KCl at 20 mVs-1 [92] Modified

The range of its effective redox potential varies according to the authors [65 92 114

115] Following the Lennoxrsquos study its potential extremes should lie within the range

between - 015 V and + 031 V vs AgAgCl (Table 2) because beyond these potential

ranges irreversible lattice reduction or oxidation takes place Even within the range

crystal dissolution salt formation with buffer ions and its deposition are still present and

could change the electrochemical profile TTF-TCNQ stability is affected by many

factors such as potential applied buffer component and concentration ion solubility

mass transport and analyte [92]

87

Table 2 Electrochemical behaviour of TTF-TCNQ at pH 74

Equilibrium TCNQTTFTCNQ-TTF +⎯rarr⎯ middotmiddotmiddotmiddotmiddot Eq 35

TCNQ reduction + 015 V MTCNQTCNQTCNQ Me ⎯⎯rarr⎯⎯⎯rarr⎯+minus +minusbull+ middotmiddotmiddotmiddotmiddot Eq 36

Lattice reduction - 015 V minus+minusbull ⎯⎯rarr⎯minus 2e TCNQTCNQ middotmiddotmiddotmiddotmiddot Eq 37

Anode salt formation + 031 V MTCNQTCNQ M⎯⎯rarr⎯++minusbull middotmiddotmiddotmiddotmiddot Eq 38

Lattice oxidation + 035 V +minus⎯⎯rarr⎯minus

TTFTTF e middotmiddotmiddotmiddotmiddot Eq 39

Further oxidation + 038 V +minus+ ⎯⎯rarr⎯minus 2e TTFTTF middotmiddotmiddotmiddotmiddot Eq 40

In the presence of a redox couple it also exhibits different electron transfer mechanism

When Edegrsquo of the redox couple is Edegrsquo gt 0 V TTF-TCNQ shows metal-like behaviour

facilitating direct electron transfer (DET) while it presents mediated electron transfer

(MET) for the redox couple of Edegrsquo lt 0 V Lennox made a study on TTF-TCNQ GOx

electrodes and reported that large anodic current was observed at ndash 015 V He also

reported that stable current was obtained when constant potential + 02 V was applied

for 24 hours at pH 7 in the presence of glucose He concluded that hydrophobic protein

core solubilised TCNQ and it formed enzyme-mediator complex GOx oxidation could

be catalysed at low potential because it was still in the range of the stable redox

potential He also conducted experiments with GOx and methanol dehydrogenase

(MDH) modified TTF-TCNQ electrodes in the presence of O2 The performance was

interfered by O2 in both electrodes against his expectation because DHG is in general

resistant to O2 He gave a conclusion that reduced mediators were oxidised by O2 before

electrooxidation took place [92]

88

226 Enzyme covalent attachment and mass loading

Enzymes can be covalently attached to the electrode however it becomes

complicated when mediators and stabilisers are must be also attached for practical fuel

cell applications This is however an attractive technique once ideal means of

co-immobilisation are established because enzymes can be retained firmly on the

electrode surface Enzymes can be covalently linked to oxidised or aminated surface

through amide bonds [116 117] Covalent linkage often lowers enzyme activity because

it partially alters the enzyme structure and confines the enzyme conformation [55]

however Kim succeeded in fabricating stable biocatalytic nanofibre by loading enzyme

aggregate to the enzyme monolayer which was covalently attached to the nanofibres

(Fig 22) Nanofibres with ester groups were synthesised from polystyrene and

poly(styrene-co-maleic anhydride) using electrospinning Seed enzyme was covalently

attached to the chemically modified nanofibre to form an enzyme monolayer on the

nanofibre This enzyme-attached nanofibre was further modified with the mixture of

glutaraldehyde and enzymes so that enzyme mass load was achieved by loading enzyme

aggregate to the enzyme monolayer covalently attached to the fibre Minter reported

that enzyme immobilisation with cross-linking increased the enzyme stability but

limited the enzyme conformation and thus reduced the activity However the activity

reported was nine times higher than that with an enzyme monolayer only Moreover the

activity was retained more than one month under rigorous shaking [118] The activity of

each enzyme particle might have been reduced however mass loading increased the

reactant flux hence increased the overall activity

89

Fig 22 The procedure for the enzyme mass-loaded biocatalytic nanofibre [118]

He also fabricated single-enzyme nanoparticles (SENs) in which each enzyme particle

was encapsulated within a hybrid organic and inorganic network The surface amino

groups of the enzyme were converted into surface vinyl groups and then polymerised

The polymer was then cross-linked to form the shell around the enzyme particle (Fig

23)

90

Fig 23 Fabrication procedure of single-enzyme nanoparticles [55]

It was reported that high stability was achieved after polymer cross-linking The enzyme

activity was approximately half of the free enzyme however there was no mass

transport interference [55] due to their porous structure Km in SENs was nearly

equivalent that of the free enzyme but the storage stability went beyond five months

[119] Eight-fold increase on storage stability after GOx covalently linked to

poly(phosphorothioate) was also reported [120]

23 PROTEIN INACTIVATION AND STABILISATION

Enzyme inactivation is one of the biggest problems in enzyme electrodes

Enzymes are excellent catalysts in their native environment however they are only

designed to work optimally under specific conditions therefore very sensitive to the

slight change in their environment and easily undergo inactivation The issue is

persistent in the enzyme electrodes because their fabrication introduces the enzymes to

the unusual environment yet enzyme catalytic activity must be maintained to obtain

sufficient power output and operational life The problem can be alleviated by (i)

91

protein engineering [56 121] (ii) environmental engineering [56 60 63 67] and (iii)

addition of appropriate enzyme stabilisers [120 122] Protein engineering involves the

alteration of enzyme structure to gain desired functions stability or resistance However

it is very challenging to improve both stability and activity since protein stability is

conferred by its rigidity while its activity depends on the conformational flexibility

Alteration of protein properties is realised by genetic engineering such as site-directed

mutangenesis [123] or directed evolution [49] Environmental engineering is closely

linked to the enzyme immobilisation It aims to achieve the same goals as in protein

engineering but extrinsically The environment which rigidifies the protein structure

such as cross-linking or inclusion in electrode composite tends to increase the enzyme

stability and prolong its life In contrast the environment which more favours over

flexible protein conformation such as hydrogel and membrane trap exhibits higher

enzyme activity but shorter life However it was reported that enzyme cross-linking and

the presence of hydrophilic macromolecules improved both stability and activity while

electrostatic interaction improved selectivity [124] Addition of stabilisers is more

targeted smaller scale environmental engineering Intrinsic conformational stability of

the protein cannot be improved [120] however it can target and eliminate specific

inactivating factors increases the local specific protein stability and prevents or slows

its unfolding There are two factors to improve for enzymes to achieve ideal operation

(i) activity by hydration and (ii) increased lifetime by increased protein rigidity which

might involve the removal of bond-destabilising factors or amplifying the bond strength

within the enzymes In fact both must be probably achieved simultaneously for gaining

the optimal state because they interrelate to each other in terms of protein dynamics As

mentioned earlier protein hydration improves enzyme activity but also allows its

92

regeneration hence affects the protein internal dynamics Protein dehydration distorted

the protein structure [125]

24 PROTEIN DESTABILISATION

There are many protein destabilising factors apart from dehydration and these

can be classified into chemical and physical instability Chemical instability happens

due to chemical modification such as deamidation oxidation proteolysis and

racemisation

241 Deamidation

Deamidation is the hydrolysis of amide link in the amino acid side groups such

as glutamine (Gln) and asparagine (Asn) (Fig 24)

Glutamine (Gln) Asparagine (Asn)

Fig 24 Side groups of Gln and Asn

It is favoured by increase in pH temperature and ionic strength The buffer

concentration influences the rate of deamidation at pH 7 ndash 12 Urea also accelerates

deamidation It often implies the protein aging and then modifies the protein function

93

242 Oxidation

Oxidation takes place at the side chains of cystine (Cys) methionine (Met)

tyrosine (Ty) tryptophan (Trp) and histidine (His)

Cysteine (Cys) Methionine (Met)

Tyrosine (Tyr) Tryptophan (Trp) Histidine (His)

Fig 25 Side groups of Cys Met Tyr Trp and His

Atmospheric oxygen can oxidise Met residues H2O2 modifies indole thiol (SH) [123

126] disulfide (SS) imidazole phenol and thioether (RSR) groups at neutral or slightly

alkaline conditions At acidic condition it oxidises Met into Met sulfoxide (R-S=O-R)

Under extremely oxidising condition Met sulfoxide is further oxidised into Met sulfone

(R-S(=O)2-R) Met oxidation leads to loss of biological activity however it does not

abolish the enzyme activity Therefore if oxidised Met is reduced back by a reductant

such as thiols it will restore the full biological activity Met reactivity depends on its

position within the protein Cys plays an important role in protein folding because it

forms intermolecular disulfide bonds however very susceptible to oxidation It can

undergo autooxidation in the absence of oxidants It is oxidised in several stages in

94

which thiol R-SH group is oxidised to (i) sulfenic acid (R-S-OH) (ii) disulfide

(R-S-S-R) (iii) sulfinic acid (R-S=O-OH) and (iv) sulfonic acid (R-S(=O)2-OH) [123

127] Oxidised thiol groups can be reduced back however they can be also reduced

back in the wrong way This leads to protein misfolding and loss of activity There are

many factors that influence the rate of oxidation They are (i) temperature (ii) pH (iii)

buffer medium (iv) metal catalysts (v) O2 tension (vi) steric condition (vii) neighbouring

groups (viii) photooxidation The presence of metal ions such as Fe2+ and Cu2+

accelerates oxidation by air [123] and the presence of O2 also might accelerate thiol

oxidation with other oxide such as nitric oxide radical [127] Metal-catalysed oxidation

involves a site-specific metal binding to Trp His and Met This leads to severer form of

oxidation causing protein aggregation Peroxide-catalysed oxidation on the other hand

is milder and tends to affect Cys and Trp but does not form aggregates [126] Increase in

pH increases pKa hence accelerates dissociation of ndashSH All of Cys Met Typ Tyr and

His are susceptible to photooxidatoin however His photooxidation is fatal leading to

enzymatic inactivity [123]

243 Proteolysis and racemisation

Proteolysis and racemisation takes place at aspartic acid (Asp) residue The

bond between Asp and proline (Pro) is especially susceptible to hydrolysis Asp also

easily undergoes base-catalysed isomerisation to isoAsp through a ring formation [123]

95

Aspartic acid (Asp) Proline (Pro)

Fig 26 Side group of Asp and the amino acid structure of Pro

244 Physical instabilities

Physical instability includes denaturation adsorption aggregation and

precipitation and [123] This refers to loss of tertiary structure and does not involve

chemical modification It is defined as Gibbs free energy change between the native and

unfolded state of protein Physical instability is induced by change in temperature

extreme pH organic solvent denaturant [123] and solvation [120 125]

25 ENZYME STABILISERS

By clarifying the possible major causes of instability the choice of optimal

approach and stabilisers can be made Considering the case of glucose-oxygen fuel cells

the stabilisers should be selected on the basis of following points (i) conformational

stability for longer operation and shelf life (ii) hydrophilic environment for optimal

enzyme functions (iii) optimal interaction between enzyme and substrate and enzyme

and environment Conformational stability is to secure protein folding and avoid

denaturation This is achieved by thiol group protection reduction of oxidised thiol

96

groups removal of oxidising species an increase in internal hydrophobic interaction

and environmental hydrophilic and electrostatic interaction Optimal enzyme activities

can be achieved by an increase in hydrogen bonding and electrostatic interactions

251 Polyehyleneimine (PEI)

Polyethyleneimine (PEI) has been reported as an efficient antioxidant as well

as chelating agent It is also cationic (Fig 27) [120] therefore provides electrostatic

stabilisation with anionic protein [126] such as GOx [67 85] Anti-microbial activity

has been also reported [126] It is not effective against thermal inactivation [128]

H+

N+H3N+H3

N+H3H3N+

H2+ H+

H+

H+

H2

+

H2+

Fig 27 Structure of PEI [129]

PEI is available in different molecular weights [120] and smaller molecules are more

effective in binding and flexibility [130] However its optimal loading concentration

varies [120 128 131] PEI protects thiol groups efficiently [120] from both metal- and

peroxide- catalysed oxidation despite of their different oxidation mechanisms [126] PEI

also forms a complex with the enzyme and coats it with polycations This occludes the

97

oxidants from the protein surface [126 130] Complex formation might be involving

hydrogen bonding between carboxylic acid groups in the enzyme and nitrogen atoms in

PEI [130] This possibly confers stability in dried state [128] It was reported that PEI

increased the maximum limiting catalytic velocity (V) for GOx [131] and lactate

dehydrogenase (LDH) [128] However it also increased Km for LDH and shifted its

optimal pH slightly toward acidic [128]

252 Dithiothreitol (DTT)

Dithiothreitol (DTT) is another small water-soluble [132] thiol protecting

reagent [132 133] with the reducing potential at ndash 033 V at pH 7 It has only little

odour [132] and has low toxicity [134] Its epimer is dithioerythriotol (DTE) Both are

excellent reducing reagents with two thiol groups (Fig 28) [132] however DTT might

be marginally better in reducing power [135] It reduces back disulfide quantitatively

[127 132] at lower concentration than other thiol-containing reducing reagents such as

Cys and gluthatione (GSH) But it cannot restore some of the lost catalytic activity

[136] because it only reduces disulfides but not other thiol oxides Therefore its

efficacy depends on (i) the oxidation mechanism involved in the protein inactivation

(ii) the velocity at which the inactivation proceeds and (iii) how thiol-contaning amino

acids are present in the protein DTT is not effective against mild oxidation caused by

low concentration of H2O2 or isolated Cys residues [127] It is probably because that

DTT reduces thiol oxide by converting itself from a linear into a cyclic disulfide form

which is called trans-45-dihydroxy-o-dithiane with (Fig 28) [132]

98

Fig 28 Structures of DTT from a linear to a cyclic disulfide form [132]

On the other hand arsenite was effective in reducing only mildly oxidated thiol groups

but not disulfides [127] It is however toxic [137] and not suitable for practical use

Neither of DTT or arsenite was effective if thiol oxidation is outcompeting [127]

253 Polyethylene glycol (PEG)

Polyethylene glycol (PEG) (Fig 29) has been reported as an enzyme stabiliser

however also has mixed reception and its stabilisation mechanism has not yet been

clearly understood

Fig 29 Structures of PEG

OrsquoFagain reported that PEG increased storage and thermo stability [120] while

99

Andersson reported the increase in LDH activity with PEG 10000 Mw but was

followed by a decrease after 10-day storage and then eventually became worse than

those without stabilisers The stabilisation was also found to be concentration dependent

[128] As for protein stabilisation mechanism it was proposed that PEG preferentially

hydrates the protein and stabilises its native structure in the solution [138-140]

According to Arakawa this causes the exclusion of PEG from protein surface due to the

unfavourable interaction between them hence increases water density around the

protein This results in the water shell around the protein surface and increases the

hydrogen bonds [138 139] Better hydration favoured larger PEG Mw due to the larger

steric exclusion [138] on the other hand lower PEG Mw was also reported to be better

because of its better solubility and viscosity [139] Optimal concentration is unknown

however one proposal is that higher concentration is more advantageous to stabilise the

denatured protein because it can penetrate and stabilise the protein hydrophobic core

exposed during denaturation [138] In contrast Lee reported that this penetration affects

the hydrophobic core of active enzyme and decreased the activity and denatured the

protein [139] Both however concluded that a fine balance between preferential

hydration and hydrophobic interaction is important and PEG concentration must be up

to 30 [138 139] It was also reported that PEG stabilised the protein of low water

content better because it prevented the water evaporation while polyols tended to strip

water from the protein dehydrated microenvironment [139]

254 Polyols

Polyols such as glycerol (GLC) (Fig 30) were reported to increase shelf life

100

[141] thermostability [120] and also provide preferential solvation as seen in PEG

[141-143] Higher GLC concentration resulted in better protein activity [143 144] and

reduced protein aggregation due to the tighter protein packing [144] However observed

hydration was lower than expected for high GLC concentration therefore Gekko

proposed that the contribution of higher GLC concentration did not derive form the

increase in steric exclusion Instead GLC might penetrate the solvation layer of protein

and stabilise the structured water network around the protein [143] Andersson also

compared the influence on protein activity after the storage among several candidate

stabilisers diethylaminoethyl-dextran (DEAE)-dextran Mw 500000 sorbitol and PEI

DEAE-dextran increased the activity both before and after the storage while sorbitol

decreased it However PEI was only found to be superior to all [128]

OH

OHHO

Fig 30 Structure of glycerol

255 FAD

FAD (Fig 16 p 75) is also another possible stabiliser as well as mediator

because it can regenerate denatured aged enzymes which have lost its cofactors [82

108] However it has not been clarified well and Bartlett questioned its efficacy due to

the contradictory results he obtained [145] It was reported that addition of FAD

increased the electrode sensitivity by 70 especially in the concentration range below

101

3mM and activity by 8 times although it also increased Km by 2 times [108]

26 ENZYME KINETICS AND ELECTROCHEMISTRY

261 Michaelis-Menten kinetics

A simple enzyme reaction can be expressed in terms of equilibrium between

the free enzyme (E) substrate (S) enzyme-substrate complex (ES) and product release

(P) The enzyme is regenerated on completion of the catalytic cycle

General enzyme reaction mechanism

middotmiddotmiddotmiddotmiddot Eq 41

E = Enzyme concentration mol

S = Substrate concentration mol

ES = Enzyme-substrate complex concentration mol

P = Product concentration mol

k1 = Rate constant for ES formation from E and S mol-1s-1

k-1 = Rate constant for ES dissociation into E + S s-1

k2 = Rate constant for P formation from ES s-1

The rate of ES formation is defined as

102

][][]])[[]([][2101 ESkESkSESEk

dtESd

minusminusminus= minus middotmiddotmiddotmiddotmiddot Eq 42

Where [E0] is the initial enzyme concentration

When the reaction is at steady state 0][=

dtESd

])[(][][]])[[]([ 212101 ESkkESkESkSESEk +=+=minus minusminus middotmiddotmiddotmiddotmiddot Eq 43

Re-arranging the above equation for [ES] as below

][]][[

][121

01

SkkkSEk

ES++

=minus

middotmiddotmiddotmiddotmiddot Eq 44

When the rate of product formation is defined as follows

middotmiddotmiddotmiddotmiddot Eq 45

][2 ESktP=

∆∆

=υ middotmiddotmiddotmiddotmiddot Eq 46

Where υ is the catalytic rate

When all the enzyme active sites are occupied such a condition is called saturated and

defined as [E0] = [ES] The maximum catalytic rate (V) is therefore available as below

Maximum catalytic rate V

][][ 202 ESkEkV == middotmiddotmiddotmiddotmiddot Eq 47

103

Express υ in terms of [S] by inserting Eq 44 into Eq 46

][

]][[

1

21

02

Sk

kkSEk

++

=minus

υ middotmiddotmiddotmiddotmiddot Eq 48

Michaelis-Menten constant (KM) is a dissociation constant about ES and defined as

Michaelis-Menten constant

1

21

kkk

K M+

= minus middotmiddotmiddotmiddotmiddot Eq 49

Michaelis-Menten equation (Eq 50) is obtained when υ is expressed in terms of V and

KM by inserting Eq 47 and Eq 49 into Eq 48

Michaelis-Menten equation

][][SK

SV

M +=υ middotmiddotmiddotmiddotmiddot Eq 50

This is a hyperbola equation to lead the enzyme catalytic rate Most enzymes typically

show this hyperbolic curve (Fig 31) when they are free from allosteric controls or

enzyme inactivation

104

0E+00

1E-09

2E-09

3E-09

4E-09

5E-09

0E+00 2E-03 4E-03 6E-03 8E-03 1E-02 1E-02 1E-02 2E-02

[S] M

Fig 31 A typical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

As shown in Fig 31 Michaelis-Menten plot has different kinetic profiles at high and

low [S] Mathematical expression of its transition over [S] is shown in Table 3

Table 3 Michaelis-Menten equation at various [S]

[S] ge KM Vcongυ middotmiddotmiddotmiddotmiddot Eq 51

[S] = KM 2V

=υ middotmiddotmiddotmiddotmiddot Eq 52

[S] le KM MM K

SEkK

SV ]][[][ 02=congυ middotmiddotmiddotmiddotmiddot Eq 53

At high saturated substrate concentration the catalytic rate becomes zero-order

therefore Michaelis-Menten curve becomes plateau When [S] = KM the rate becomes

105

half of the maximum catalytic rate At low substrate concentration the catalytic rate is

linearly dependent on [S] therefore the kinetics becomes first-order about [S]

262 Electrochemistry and Michaelis-Menten kinetics

In electrochemistry enzyme activity is studied by amperometry This involves

measuring measuring the current generated by the enzyme electrode reaction at a fixed

potential over an arbitrary period It therefore shows the transition in current response

over time For enzyme electrodes substrate concentration is changed over time so that

the current response for a different substrate concentration can be measured over a long

period With intact enzyme and appropriate heterogeneous electron transfer present

most enzyme electrodes follow Michaelis-Menten kinetics (Fig 32) in the amperometry

in the absence of allosteric control or enzyme inactivation Therefore the amperometry

data can be analysed in the similar way to that of biochemical data and for this

Michaelis-Menten equation is expressed in electrochemical terms [146] In

electrochemical enzyme kinetics enzyme catalytic rate (υ) is obtained in terms of

electric current therefore V is substituted with maximum steady state limiting current

(Im) or its current density (Jm)

106

Electrochemical Michaelis-Menten equation

][][

SKSI

IM

m

+times

= middotmiddotmiddotmiddotmiddot Eq 54

][][

SKSJ

JAI

M

m

+times

== middotmiddotmiddotmiddotmiddot Eq 55

I = Current A

Im = Max steady-state limiting current A

[S] = Substrate concentration mol

KM = Michaelis-Menten constant mol

A = Electrode surface area m-2

J = Current density Am-2

Jm = Max steady-state limiting current density Am-2

There is often an offset at zero substrate concentration due to competing reactions and

trace contaminants and this must be subtracted before analysis

107

V

0E+00

2E-04

4E-04

6E-04

8E-04

1E-03

0 2 4 6 8 10 12 14 16

frac12 Jm

KM

Jm Am-2

[S] mM

Jm

Fig 32 Electrochemical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

KM and Jm (or V in biochemical enzyme kinetics) are important parameters to analyse

both enzyme catalytic power and stability Electrochemical Michaelis-Menten equation

and curves can be used for rough estimation of KM and Jm however it often leads to

inaccurate result because measuring initial rate for high substrate concentration is very

difficult in this method hence very prone to errors Therefore Michaelis-Menten

equation is linearised using one of the following four methods (i) Lineweaver-Burk (ii)

Eadie-Hofstee (iii) Hanes-Woolf and (iv) Cornish-Bowden-Wharton equations

Michaelis-Menten plot is however good for intuitive diagnosis of general enzyme

behaviours

108

263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics

2631 Lineweaver-Burk linear regression

Lineweaver-Burk linear regression is one of the classic regression methods

taking double reciprocal of Michaelis-Menten equation (Eq 56)

Electrochemical Lineweaver-Burk linear regression

mm

M

JSJK

J1

][11

+times= middotmiddotmiddotmiddotmiddot Eq 56

It plots 1J against 1[S] as shown in Fig 33 The value of each parameter is found by

extrapolating the line The reciprocal of Jm is found at y-intercept and KM can be derived

from either x-intercept or the slope once Jm is found

109

-50E+02

00E+00

50E+02

10E+03

15E+03

20E+03

-06 -05 -04 -03 -02 -01 00 01 02 03 04

1[S] mM-1

1J A-1m

2

Fig 33 Electrochemical Lineweaver-Burk linear regression

Based on the real data Refer to sect 343 p 124

Lineweaver-Burk linear regression is however largely influenced by the errors in j at

low substrate concentrations [22 147] Its reciprocal could vary by a factor of 80 over

the range of substrate concentration varying one-third to three times the KM [148]

Hence it is not an ideal method for the analysis

2632 Eadie-Hofstee linear regression

Eadie-Hofstee linear regression plots J against J[S] This plot is strongly

affected by the errors in υ because υ is present in both coordinates (Eq 57)

Electrochemical Eadie-Hofstee linear regression

mM JSJKJ +timesminus=

][ middotmiddotmiddotmiddotmiddot Eq 57

110

Again the plot must be extrapolated to find Jm at y-intercept KM can be derived from

the slope (Fig 34)

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

0E+00 5E-05 1E-04 2E-04 2E-04 3E-04

J [S] AmM-1

J Am

-2

Jm

-Km

Fig 34 Electrochemical Eadie-Hofstee linear regression Based on the real data

Refer to sect 343 p 124

Eadie-Hofstee linear regression will give very strict evaluation [148] and might be used

to detect the deviations from Michaelis-Menten behaviour only if the data is precise and

accurate [147]

2633 Electrochemical Hanes-Woolf linear regression

Hanes-Woolf linear regression plots [S]J against [S] KM is derived from the

x-intercept and Jm is found from the slope

111

Electrochemical Hanes-Woolf linear regression

m

M

m JKS

JJS

+times= ][1][ middotmiddotmiddotmiddotmiddot Eq 58

This is a better method compared to the previous two regression methods because all

the parameter values can be derived from the reasonable range of [S] and they donrsquot

heavily rely on the extrapolation [147] The data is normalised by [S] therefore does not

cause many errors

-20E+08

00E+00

20E+08

40E+08

60E+08

80E+08

10E+09

12E+09

14E+09

-40E+00 00E+00 40E+00 80E+00 12E+01 16E+01

[S] mM[S] J m

MA

-1m2

Fig 35 Electrochemical Hanes-Woolf linear regression Based on the real data

Refer to sect 343 p 124

However either of these three linear regressions might not be adequate for the enzyme

electrode analysis [65] because the data reflect not only the single-substrate

Michaelis-Menten kinetics but also include the influence from other factors such as

enzyme immobilisation mediator reactions and electrode composition as well as

112

general experimental errors Their contributions to the overall kinetics therefore must be

considered [65 149] although the enzyme electrode reactions can approximate the

Michaelis-Menten model when one substrate is a variable and other variables are fixed

over an experiment [147 149] Under such a complex system it is not ideal to use those

graphical linear regression methods because they assume that the results follow

Michaelis-Menten model and that all the experimental errors follow normal distribution

[150] without giving any measure in the precision [148] Hence it is more suitable to

use a non-parametric direct linear plot or nonlinear regression methods because they

respect the influence and errors from various sources without any bias

264 Cornish-Bowden-Wharton method

Cornish-Bowden-Wharton method is distribution-free non-parametric direct

linear plot In this method observations are plotted as lines in parameter space rather

than points in observation space [151] It then takes the median of Jm and KM from every

possible non-duplicate pair of observed [S] and υ (Eq 59 and Eq 60) as the best-fit

estimate

Electrochemical Cornish- Bowden-Wharton non-parametric method

])[][(])[]([

1221

122121 SJSJ

SSJJJm minusminus

= middotmiddotmiddotmiddotmiddot Eq 59

])[][()](][[

1221

122121 SJSJ

JJSSKM minusminus

= middotmiddotmiddotmiddotmiddot Eq 60

It is a simple method without complex calculation insensitive to outliers and no

113

weighting is required [147 152]

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

14E-03

16E-03

18E-03

20E-03

-5 -3 -1 1 3 5

KM mM

Jm Am-2Median Jm

Median KM

Fig 36 Electrochemical Cornish-Bowden-Wharton plot Based on the real data

Refer to sect 343 p 124

265 Nonlinear regression methods

Nonlinear regression methods manipulate the parameters to minimise the sum

of squared difference between the measured and expected values [150] They fit the

integrated equation to the enzyme progress curve and calculate the approximate location

of the minimum for the sum of squared difference at a particular point in the slope It

then recalculates the new minimum location to improve the prediction until there is no

further improvement This is called gradient method based on Gauss-Newton algorithm

or non-linear least square method [150 153] This is computationally intensive due to

114

the recursive operations however advanced computational technology has made it

possible The parameters obtained may not be perfectly correct [150] The method still

assumes errors normally distributed [153] and Cornish-Bowden has criticized about the

ambiguous statistical assumption of such computer programmes [154] However

non-linear regression methods are insensitive to outliers [150 153] have better estimate

accuracy compared to conventional linear regression methods and the estimations

mostly fit Michaelis-Menten model Graphical methods arose at a time when nonlinear

regression methods were computationally not available however it is now possible

thanks for the great technological advance and more recommended to use in analysis to

yield better precision and reliability [153]

115

3 ANODE MATERIAL AND METHOD

31 ELECTRODE FABRICATION

311 TTF-TCNQ synthesis

Tetrathiafulvalene ge980 (TTF) and 7788-Tetracyanoquinodimethane

ge980 (TCNQ) were purchased from Sigma Aldrich Spectroscopic grade acetonitrile

and diethyl ether were purchased from VWR International TTF and TCNQ were

weighted to 1 g each and dissolved separately in 50 ml of acetonitrile TTF-acetonitrile

solution had yellow colour while TCNQ-acetonitrile solution was green Both were

heated on the hot plate under the fume hood On the full dissolution two solutions were

quickly mixed to form black solid crystals The mixture was immediately taken off the

hot plate and cooled down at the room temperature for a few hours The crystals were

collected on Buchner funnel and washed under vacuum twice with acetonitrile twice

with diethyl ether and then twice again with acetonitrile The washed crystals were left

to dry for overnight with a cover The dried crystals were collected and kept in the

sealed container for future use [61]

312 Electrode template fabrication

Sylgard 184 Silicone Elastomer Kit were purchased from Dow Corning

Corporation for polydimethylsiloxane (PDMS) Base and hardener were mixed at 101

(vv) for use CY1301 and aradur hardener HY 1300 were purchased from Robnor

116

resins to make epoxy resin They were mixed at 31 (vv) for use

Silicone electrode templates (Fig 16(i)) were made to shape electrode discs

Each template had round holes in line Two types of templates template A and B were

made Template A had holes with the dimension 2-mm diameter x 1-mm thickness

while template B had holes with the dimension with 5-mm diameter x 2-mm thickness

In order to make PDMS templates for electrode discs different templates were made to

shape the holes in PDMS (Fig 16(ii)) Discs were made with epoxy resin and were

fixed onto the plastic Petri dish PDMS was poured into disk-mounted Petri dish

degassed for 20 minutes and then cured at 60 degC Cured PDMS electrode templates

were taken out from the epoxy resin-Petri dish templates and then cleaned with

analytical grade ethanol for use

Fig 37 PDMS electrode templates (i) PDMS template and (ii) their epoxy resin-Petri

dish template

117

313 Electrode composite fabrication

3131 Composite for conductance study

Graphite at microcrystal grade (2-15 micron 999995 purity) was purchased

from Alfa Aesar GmbH amp CoKG Glassy carbon (GC) powder was kindly donated from

Dr Zhao Carbon nanofibre (CNF) was also kindly donated from Loughborough

University Composite electrodes were built from the mixture of the carbon conductive

or TTF-TCNQ and epoxy resin Each composite mixture had different composition ratio

[108] as below in Table 4

Composite Ratio I Ratio II Ratio III Ratio IV Ratio V

Graphite Epoxy resin 73 64 55

GC Epoxy resin 64 73

CNF Epoxy resin 37 46 55 64 73

TTF-TCNQ Epoxy resin 64 73 82

Table 4 Composite composition

TTF-TCNQ was mixed with epoxy resin at 64 73 and 82 Graphite was mixed at 73

64 and 55 GC powder was mixed at 64 and 73 CNT was mixed at 37 46 55 64

and 73 (ww) Three to five copies were fabricated for each electrode type Composite

mixture was packed into the template A (Fig 38) and cured at room temperature for 72

hours [106]

118

Fig 38 PDMS template (i) packed with electrode material and (ii) empty one

3132 Bulk modified GOx enzyme electrode composite

Glucose oxidase (GOx) [Type II-S 15000-50000 unitsg] from Aspergillus

niger was purchased from Sigma Aldrich Conductive composite paste was first made

with TTF-TCNQ and epoxy resin at 73 as in sect 3131 GOx was then incorporated into

the conductive composite mixture at varying mixing ratio 5 10 and 15 (ww) by

bulk-modification method (Fig 39) 10 GOx composite electrodes were also made

with graphite Enzyme composite mixture was packed into the template B and cured at

4 degC for 96 hours

119

R

RR R

R

RR R

RRR

Conducting particles

Insulating binder

Chemical modification

Add biological receptor R

Cast extrude or print into suitable form

R

R

RR

R

R

R

R

R

R

RR

Fig 39 Schema for bulk modification method

3133 GOx enzyme electrode composite with enzyme stabilisers

Poly(ethyleneimine) (PEI) solution [50 (wv) in H2O] DL-dithiothreitol

(DTT) Poly(ethylene glycol) (PEG) [average mol wt 200] glycerol (GLC) [ge99] and

flavin adenine dinucleotide disodium (FAD) salt hydrate (ge95) were purchased from

Sigma Aldrich Each stabiliser was mixed with GOx at different fraction 0 1 2 and

4 PEI was also blended with DTT or FAD for synergetic effects as below in Table 5

Blend type Ratio I Ratio II Ratio III Ratio VI

PEIDTT 0515 1010 1505 2020

PEIFAD 21 22 24

Table 5 Blended stabiliser composition

120

PEI was mixed with DTT at the ratio of 0515 11 1505 and 22 PEI was also

mixed with FAD at 21 22 and 24 Each enzyme-stabiliser mixture was weighted to

10 of the whole composite It was then mixed with the conductive composite mixture

made earlier The conductive composite mixture was made of TTF-TCNQ and epoxy

resin at 73 The final composite mixture was packed into the template B and cured at

4 degC for 96 hours

314 Electrode wiring and insulation

Silver wire (temper annealed 025-mm diameter) was purchased from Advent

Portex polyethylene tubing (028 x 0165 mm) purchased from Scientific Laboratory

Supplies Silver epoxy was purchased from ITW chemotronics Silver epoxy came in

two parts A and B and they were mixed at 11 (vv) for use Silver wire was cut for

5-cm length and its tip was cleaned with analytical grade ethanol dried and coved with

insulating polyethylene tubes Cured electrode composite discs were popped out of the

PDMS template An insulated silver wire was fixed onto to each electrode disc with

conductive silver epoxy and it was left at room temperature for 24 hours to cure silver

epoxy After silver epoxy had been cured the wired electrodes were coated and

insulated with degassed epoxy resin and cured at room temperature for 3 days for

non-enzyme composite electrodes and 4degC for 4 days for enzyme composite electrodes

7-9 days were taken to build the complete composite electrodes (Fig 40) The enzyme

electrodes were stored at 4degC when not in use

121

Fig 40 Insulated composite electrodes (i) non-enzyme (ii) GOx electrodes

32 CONDUCTIVITY EXPERIMENT

Copper foil with electrically conductive acrylic adhesive was purchased from

RS Components Digital multimeter from Metra was used to measure resistance

Electrode surface was covered with adhesive copper foil evenly and both ends were

clamped with crocodile clips (Fig 41(i)) to measure electrode resistance of the

composite as in Fig 41(ii)

122

Fig 41 Measuring electrode resistance (i) connecting the electrodes to the

multimeter (ii) measuring the electrode resistance with the multimeter

33 POLISHING COMPOSITE ELECTRODES

MicroPolish II aluminium oxide abrasive powder (particle range 1 03 and

005 microm) silicone carbide (SiC) grinding paper for metallography wet or dry (grit 600

1200 and 2400) and MicroCloth Oslash73 mm for MiniMet for fine polishing were

purchased from Buehler Self adhesive cloth for intermediate polishing was purchased

from Kemet Electrodes were polished immediately before the experiment until

mirror-like flat surface was obtained This erases the electrode history and gives

assigned electrode surface area [105] Composite electrodes were hand polished

successively with SiC grinding paper in 400 1200 and 2400 grit During polishing

deionised water of 18 MΩ was added occasionally to the polishing surface This

prevents damaging electrode surface with frictional heat It was followed by successive

polishing with alumina oxide slurry with particle size 1 03 and 005 microm Slurry was

123

sonicated to dissolve agglomerates before use [155] Kemet polishing pad was used for

rougher polishing with 1 and 03 microm powder Felt-like MicroCloth was used for

polishing with 005microm Electrodes were sonicated in the deionised water briefly after

polishing at each grit or grade

34 GLUCOSE AMPEROMETRY

341 Glucose solution

Phosphate buffer saline (PBS) powder for 001 M at pH 74 containing 0138

M NaCl and 00027 M KCl [156] was purchased from Sigma Aldrich PBS was kept in

the fridge when not in use [157] 1M glucose solution was made by mixing

D-(+)-Glucose with 001 M PBS and left for 12 hours at 4degC for mutarotation It is

mandatory because GOx has stereochemical specificity and only active toward

β-D-glucose while glucose solution consists of two enantiomers α- and β-D glucose

and their concentration changes over time until they reach the equilibrium at 3862

Experiments should be carried out in the equilibrated solution for accuracy

342 Electrochemical system

A single-channel CHI 650A and multi-channel CHI 1030 potentiostats were

used for glucose amperometry with three-electrode system in which the

electrochemical cell contained (a) working electrode(s) an AgCl reference electrode

and a homemade Pt counter electrode The AgCl reference electrode was purchased

124

from IJ Cambria AgCl reference electrode was saturated with 3M KCl Pt counter

electrode was made of Pt wire and Pt mesh was fixed at its tip to give large surface area

Pt mesh at its tip

343 Glucose amperometry with GOx electrodes

Glucose amperometry was conducted by adding a known volume of 1M

glucose aliquot into 100 ml of 001 M PBS electrolyte solution containing GOx anodes

with convection applied Convection was however ceased on during current

measurement to avoid the noise The electrolytic solution was gassed with either N2 O2

or compressed air for 15 minutes before the experiment O2 concentration is 123 mM in

fully oxygenated PBS [158] and 026 mM in air [112] Gas was continuously supplied

through Dreschelrsquos gas washing bottle containing PBS during experiment The response

of GOx anodes to glucose was obtained as steady state current The amperometry was

carried on until no further current increase was observed against glucose added Each

experiment took more than 8 - 12 hours The sample size ranged from two to three in

most cases

125

Fig 42 Amperometry setting with CHI 1030 multichannel potentiostat

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA

Normality of enzyme function was confirmed by plotting Michaelis-Menten

curve based on the amperometry data obtained The capacitive current before glucose

addition was subtracted from the data Enzyme kinetic parameters the maximum

current density (Jm) and the enzyme dissociation Michaelis constant (KM) were

automatically estimated with standard error using an analytical software called

SigmaPlot Enzyme Kinetics version 13 The estimation is based on non-linear

regression analysis about Michaelis-Menten kinetics as shown in Fig 43

126

Glucose M

0 1e-2 2e-2 3e-2 4e-2 5e-2

J A

m-2

00

20e-3

40e-3

60e-3

80e-3

Vmax = 0008445 Km = 0005842

Fig 43 Analytical output by SigmaPlot Enzyme Kinetics Based on the real data

Influence of enzyme immobilisation electrode composition O2 and stabilisers on GOx

electrode performance was studied The most efficient GOx anodes was determined on

the basis of the parameters obtained

127

4 ANODE RESULTS AND DISCUSSION

41 AIMS OF ANODE STUDIES

This section describes the fundamental study on the enzyme composite

electrodes and the influence of enzyme stabilisers on the performance and stability of

GOx electrodes Firstly the difference in conductivity as a function of various

conductive materials and the component fraction were studied They did not contain

enzyme The most suitable composite was then selected and used to build the enzyme

electrodes Secondly GOx electrodes were built at various GOx fractions in the

composite The best component fraction was determined from the data obtained in

amperometry Amperometry was conducted to obtain two enzyme kinetic parameters

the maximum current density (Jm) and the enzyme dissociation Michaelis constant (KM)

Jm indicates the size of catalytic velocity KM indicates the extent of enzyme-substrate

dissociation Smaller KM values indicate better enzyme-substrate affinity and enzyme

stability With the component ratio selected enzyme electrodes were then constructed

using various types and concentrations of enzyme stabiliser Amperometry was again

conducted to study the influence of enzyme stabilisers on the GOx anode performance

and stability The catalytic efficiency was determined by the fraction M

mK

J This is

based on the concept of specificity constant M

catK

k as defined below

128

Enzyme electrode specificity constant

MMM

cat

KEV

Kk

Kk

sdot==

][2 middotmiddotmiddotmiddotmiddot Eq 61

where kd = k2 Rate constant for P formation from ES

The specificity constant is a common measure of the relative catalytic rate at low

substrate concentration [22] M

mK

J therefore can be used as a comprehensive

indicator for both catalytic velocity and enzyme stability under the fixed enzyme

concentration In this study GOx concentration was fixed to approximately 10 weight

of the total composite According to the M

mK

J obtained the five most efficient and

stable GOx electrodes were determined The possible enzyme stabilisation mechanisms

of each enzyme stabiliser were also discussed together in the light of published

hypothesises

42 CONDUCTANCE OF THE COMPOSITE

421 Conductance of various conductive materials

Various electrodes were built with different conductive materials graphite GC

CNF and TTF-TCNQ at different component fraction The electrode dimension was

fixed to 314 mm2 x 1 mm Their conductance was measured and presented in a box plot

as shown in Fig 44 The sample size ranged from 3 to 6 The median is indicated by a

short line within the box

129

00E+00

50E-02

10E-01

15E-01

20E-01

25E-01

30E-01

35E-01

40E-01

45E-01C

ondu

ctan

ce

S

Lower quartile S 103E-03 549E-03 219E-01 111E-01 142E-01 599E-04 390E-03 115E-02 972E-03

Max S 281E-03 338E-02 268E-01 312E-01 415E-01 810E-04 113E-02 467E-02 262E-02

Min S 739E-04 467E-03 200E-01 933E-02 855E-02 418E-04 296E-03 264E-03 587E-03

Upper quartile S 206E-03 282E-02 253E-01 220E-01 253E-01 795E-04 807E-03 232E-02 199E-02

Median S 131E-03 847E-03 238E-01 129E-01 216E-01 780E-04 484E-03 155E-02 136E-02

TTF-TCNQ64

TTF-TCNQ73

Graphite 55 Graphite 64 Graphite 73 CNF 37 CNF 46 CNF 55 GC 73

Fig 44 Conductance of various electrode composite n = 3 ndash 6

Graphite composites showed higher conductance than other composites It is explained

by graphite having the highest intrinsic conductivity ranging between 103 ndash 104 Scm-1

[105 159] although being compounded in a composite lowered the conductivity by 4 ndash

5 order of magnitude compared to that of raw material The conduction did not show a

linear relationship with the electrode composition ratio for graphite composite because

the graphite composite 55 had larger conductance of 024 S than that of 64 and 73 It

also showed the smallest deviation than the other graphite composites while the

deviation increased with increase in the graphite content One possible cause of this is

the presence of patchy conductive paths due to the uneven distribution of conductive

islands within the composite [105 106] The conduction of the composite is therefore

often locally biased unpredictable and also leads to low reproducibility TTF-TCNQ

composite at 73 showed the conductance of 85 mS smaller than the graphite

composited 55 approximately by 30 times Conductivity of TTF-TCNQ is 102 ndash 103

130

Scm-1 [109 110] and smaller than that of graphite by one order of magnitude so this is

consistent with the intrinsic conductivity of the conducting paticles

There were also different interactions observed among conductive materials

with insulating materials Mixing epoxy resin with graphite was easier than mixing with

other conductive materials Some even did not cure at the same mixing ratios The

carbon nanofibre (CNF) composite mixture did not cure when it exceeded more than

50 weight content With 40 CNF content however it showed the conductance of 48

mS It was not comparable to graphite composite despite its similar conductivity of 103

S [105] however it was similar to that of TTF-TCNQ 73 composite 50 CNF content

even showed larger conductance of 155 mS than 70 TTF-TCNQ CNFs have been

reported to have mediating function [117] therefore with this high conduction property

at low content CNF might be very useful to enzyme composite and even superior to

TTF-TCNQ

422 Influence of enzyme concentration on conductivity

Conductance of the GOx composite was measured The conductive composite

consisted of either TTF-TCNQ 73 or graphite composite 73 GOx content was varied

by 5 10 and 15 for TTF-TCNQ composite and 10 only for graphite composite

The electrode dimension was fixed to 196 mm2 x 2 mm The sample numbers ranged

from 2 ndash 4 The study revealed that the presence of enzyme interfered with conduction

in GOx concentration dependent manner for TTF-TCNQ composite as shown in Fig 45

131

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01C

ondu

ctan

ce

S

Low er quartile S 131E-02 924E-03 124E-05 165E-01

Max S 484E-02 143E-02 368E-05 593E-01

Min S 120E-02 754E-03 390E-06 122E-01

Upper quartile S 441E-02 126E-02 288E-05 400E-01

Median S 281E-02 109E-02 208E-05 207E-01

TTF-TCNQGOx 5 TTF-TCNQGOx 10 TTF-TCNQGOx 15 GraphiteGOx 10

Fig 45 Conductance of GOx composite with various GOx content n= 2 - 4

This was due to insulating property of the protein although higher enzyme loading is

preferred for better catalytic performance as discussed in sect 226 on p 88 The issue

may be circumvented by fixing the enzyme onto the electrode surface It is however

more technically tedious and not as feasible for mass manufacture as bulk modification

It is also difficult to modify conductive salt for covalent attachment of the enzyme This

is because the conductive property of conducting salt is very sensitive to its chemical

structure and slight modification can lead to insulating properties [61] A similar

phenomenon has been also observed in CNT functionalisation [117]

Graphite-GOx composite showed larger conductance than TTF-TCNQ-GOx

composite by 20 times at the same component fraction This was due to the better

conductive property of graphite However graphite does not have mediating property

and the use of graphite as conductive material for enzyme electrodes requires the

132

loading of mediator separately The key to designing enzyme electrodes is in the balance

and compromise among the conductivity mediation enzyme activity mechanical and

chemical stability and difficulty in fabrication process A good candidate material to

make up for the TTF-TCNQ lower conductivity is CNT It has been reported that CNF

has both good conductivity and mediating functions [117] and might be suitable for bulk

modification More safety study on use of CNF is however necessary [117 160]

43 CATALYTIC ACTIVITY OF GOx COMPOSITE ELECTRODES

431 Influence of GOx concentration on the catalytic activity of GOx electrodes

Amperometry was conducted for GOx composite electrodes in N2 O2 and air

for various GOx content The conductive composite consisted of either TTF-TCNQ 73

or graphite composite 73 GOx content was varied by 5 10 and 15 for

TTF-TCNQ composite and 10 only for graphite composite The sample size was 3 for

each electrode type Control experiments were also performed with enzyme-free

composite electrodes and there was no glucose response observed There was also no

stable current observed for 10 GOx-graphite electrodes despite of its high

conductivity reported in sect 422 while Michaelis-Menten curve was observed in any

TTF-TCNQ-GOx composite electrodes This confirmed the necessity of mediating

function for enzyme anodes and its presence in TTF-TCNQ

All the TTF-TCNQ-GOx electrodes showed the Michaelis-Menten current

response curves however differed in the current as shown in Fig 46

133

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

35E-02

00E+00 10E-02 20E-02 30E-02 40E-02 50E-02

Glucose M

J A

m-2

GOx 5GOx 10GOx 15

Fig 46 Difference in the glucose-response amperometric curve of enzyme

electrodes for various GOx content 5 10 and 15 in the presence N2

The 15 GOx composite showed the smallest glucose response current due to the

interfered conduction by high protein content while 5 GOx composite showed smaller

response than 10 composite due to the enzyme deficiency Their Jm only counted for

26 and 41 of that of 10 GOx composite (Fig 47(i)) 10 GOx electrode was

therefore most catalytic KM for 5 and 10 GOx were 71 and 73 mM and little

different (Fig 47(ii)) This implies that the size of catalytic activity can be flexibly

altered by only slight change in KM

134

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

Jm

Am

-2

Jm Am-2 729E-03 180E-02 466E-03

GOx 5 GOx 10 GOx 15

00E+00

20E-03

40E-03

60E-03

80E-03

10E-02

12E-02

14E-02

Km

M

KM M 710E-03 729E-03 187E-03

GOx 5 GOx 10 GOx 15

Fig 47 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 n = 1 - 3

The results also tell that an increase in GOx loading could enhance the catalysis hence

output current unless the increase is not excessive and gt 15 at least The loading limit

should lie somewhere between 10 and 15 for this composite Similar electrodes

were also built by Alegret with 75 epoxy resin 10 TTT-TCNQ 10 graphite and

5 GOx [115] Their Jm was around 7 mAm-2 and little different from that of 5 GOx

with 70 TTF-TCNQ composite It suggests that less TTF-TCNQ content might be

sufficient as long as the conductivity is sustained by more conductive material such as

graphite

432 Influence of O2 on the catalytic activity of GOx electrodes

O2 and air suppressed the current output by GOx composite electrodes and

delayed the response to the glucose as shown in Fig 48 A current increase was

observed immediately after glucose addition for N2 but not for O2 and air They showed

135

sigmoidal current curves with a glucose response lag

(i)

(ii)

-50E-03

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

00E+00 50E-03 10E-02 15E-02 20E-02 25E-02 30E-02 35E-02 40E-02 45E-02

Glucose M

J A

m-2

GOx 10 N2GOx 10 O2GOx 10 Air

Fig 48 Difference in the glucose-response amperometric curve of 10 GOx

composite electrodes in the presence (i) N2 and O2 and (ii) re-plotted

amperometric curves for N2 air and O2

136

As expected O2 and air decreased Jm and increased KM as shown in Fig 49

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

GOx 5 GOx 10 GOx 15

Jm

Am

-2

N2O2Air

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

70E-02

80E-02

GOx 5 GOx 10 GOx 15

Km

M

N2O2Air

Fig 49 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 O2 and air n = 1 - 3

Both O2 and air decreased the current of 15 GOx composite electrodes Jm dropped to

approximately 20 of that for N2 KM was increased by 10 and 2 times for O2 and air

respectively The glucose response lag was 8 mM for O2 but none for air Air showed

milder suppression toward current output than O2 and this was expected because O2

counts for only 20 of the air [31] and should cause less influence on GOx activity

Similar phenomena were observed for 10 GOx composite electrodes For O2 and air

Jm was 13 and 40 of that of N2 and decreased to 23 x 10-3 and 67 x 10-3 from 18 x

10-2 Am-2 respectively KM was 73 for N2 and 10 mM for both O2 and air although there

was a response lag of 8 and 1 mM respectively Little difference in KM was observed

among all cases and this implies that KM itself hence enzyme stability or substrate

affinity is little affected by O2 but slight change in KM has large influence on the

catalytic activity This implies that change in catalytic activity induced by O2 is

137

reversible 5 GOx composite electrodes showed slightly unexpected result KM was

increased by approximately 4 times in both O2 and air Jm also decreased however Jm

for air was smaller than O2 by 36 despite of less O2 content There was a response lag

by 10 mM for O2 and 2 mM for air

The overall conclusions from these results is that O2 reduces GOx catalytic rate

andor electron transfer rate and hence reduces the current output but the effect is

reversible because it hardly alters KM value implying that O2 does not affect the enzyme

stability or its affinity to the substrate This phenomenon is typical in non-competitive

inhibition for enzyme kinetics Non-competitive inhibition occurs when an inhibitor

binds to the enzyme-substrate complex and slows its catalysis It therefore changes

catalytic velocity but not KM In the glucose amperometry this implies two possibilities

as a cause of Jm reduction (i) allosteric binding of O2 to GOx-glucose complex or (ii)

interference in electron transfer after the catalysis In the former case O2 does not

inhibit glucose binding to GOx but allosterically interacts with its complex and hinders

its catalysis hence it reduces catalytic current output In the latter case either glucose

binding or GOx catalysis is not affected by O2 but competition for electrons produced

by GOx catalysis occurs between the natural and artificial mediators namely O2 and

TTF-TCNQ and this reduces the electron flow to the electrode therefore it is reflected

as the reduction of catalytic current In addition to this the presence of glucose response

lag also suggests the presence of electron competition [161-163] It was reported that O2

reacts with reduced GOx faster than any other mediators with the second order rate

reaction of 15 x l06 M-1s-l [112 161 163] It is therefore quite possible that O2 swiftly

deprives electrons from reduced GOx andor reduced mediators Reduced GOx reacts

with O2 much faster than with TTF-TCNQ because it was observed that O2 had

138

diminished glucose response current at low glucose concentration Most GOx hence

reacts with O2 first then oxidised TTF-TCNQ once most O2 has been converted into

H2O2 This is compatible with the hypothesis suggested by Hindle [161] Even after the

lag the reaction could go on with residual O2 because KM did not recover fully to the N2

level although a familiar Michaelis-Menten hyperbolic curve was regained This simply

indicates that O2 concentration became smaller than TTF-TCNQ to oxidise GOx The

glucose response lag also decreased with an increase in GOx loading and was smaller

for air than O2 This implies the presence of competition because the lag depends on the

concentration of reduced GOx and electron thieves The large KM increase in the 5

GOx composite electrodes was probably due to the deficiency in the electron transfer

rate to suppress O2 activity How O2 interacts with its environment is unknown however

O2 reduces the catalytic output current due to the following possible mechanisms (i)

electron competition between O2 and TTF+ (ii) non-competitive enzyme inhibition by

allosteric binding of O2 to GOx (iii) direct interaction between O2 and reduced TTF or

(iv) backward reaction between reduced TTF and oxidised GOx

139

Table 6 Four possible causes of glucose amperometric current reduction

(i) middotmiddotmiddotmiddotmiddot Eq 62

middotmiddotmiddotmiddotmiddot Eq 63

(ii) middotmiddotmiddotmiddotmiddot Eq 64

(iii) middotmiddotmiddotmiddotmiddot Eq 65

(iv) middotmiddotmiddotmiddotmiddot Eq 66

Diagnosis can be made by doing experiments with different concentration of O2 and

mediators

44 THE EFFICACY OF GOX STABILISERS

441 Introduction

Various GOx stabilisers PEI DTT PEG GLC and FAD were mixed with GOx

composite at the concentration varying among 1 2 and 4 PEI was also mixed with

DTT or FAD and their synergetic effects were studied All the GOx electrodes were

built on the basis of TTF-TCNQ 73 conductive composite with 10 weight

GOx-stabiliser mixture Control composite electrodes were built with 2 stabilisers but

without GOx Glucose amperometry was conducted as before in the both presence and

absence of O2 Two kinetic parameters Jm and KM were obtained through non-linear

regression analysis The standard kinetic parameter values were set to those obtained in

140

the absence of O2 for GOx electrodes containing no stabiliser They are Jm = 18 x 10-2 plusmn

73 x 10-3 Am-2 and KM = 73 plusmn 57 mM respectively The glucose response lag

observed for this electrode was 10 mM Comprehensive catalytic efficiency was

determined by the size of the fraction M

mK

J under the influence of high O2 and the

most catalytic GOx composite electrodes were chosen on the basis of M

mK

J The

sample size ranged between 2 and 3 None of control experiments showed any glucose

response

442 PEI

Kinetic parameters for various PEI concentrations are shown below in Fig 50

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser PEI 1 PEI 2 PEI 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEI 1 PEI 2 PEI 4

KM

M

N2 O2

Fig 50 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEI in the presence and absence of O2 n = 1 - 3

141

In the absence of O2 PEI lowered Jm without largely affecting KM compared to the

standard value This might imply the presence of allosteric non-competitive inhibition

of glucose catalysis It is certainly possible because PEI can lock in the enzyme

conformation with hydrogen and electrostatic bonding [120] or form a complex with

GOx [126 130] This interferes in its catalysis although it also might confer the

resistance against oxidative stress as reported [126] In the presence of O2 two out of

three electrodes of PEI 1 and 4 did not show Michaelis-Menten curves The data

presented here is therefore based on a single sample This may mean that the electrodes

at those PEI concentrations are in fact prone to the catalytic suppression by O2

Moreover 4 PEI data presented here looks unreliable due to the simultaneous increase

in Jm and KM by 3 ndash 4 times compared to those obtained in the presence of N2 It may

need more data to confirm the phenomenon and be premature to conclude it is however

probably more reasonable to think that PEI 4 is not suitable for GOx anodes At other

PEI concentrations electrodes showed more or less similar kinetic parameter values to

those without stabilisers since Jm decreased and KM increased in the presence of O2

There was no O2-scavenging function observed because their glucose response lag and

KM resembled those without stabilisers Among them 2 PEI containing electrodes

sustained 50 of catalytic activity obtained in N2 and showed marginally better Jm than

stabiliser-free electrodes in the presence of O2 It might therefore have some resistance

against O2 It can be however concluded that PEI is not suitable as an enzyme stabiliser

and not effective enough to enhance the catalytic current output because their Jm was

already smaller by 60 at minimum in N2 compared to the standard value and little

improvement in catalytic performance was observed in the presence of O2

142

443 DTT

Kinetic parameters for various DTT concentrations are shown below in Fig 51

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

Jm

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

KM

M

N2 O2

Fig 51 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 DTT in the presence and absence of O2 n = 3

All DTT containing electrodes showed smaller Jm and KM than the standard values in

the absence of O2 This might imply the possible presence of uncompetitive inhibition

by DTT although the size of inhibition was not in concentration dependent manner In

uncompetitive enzyme inhibition inhibitors bind both free enzyme and

enzyme-substrate complexes hence decreases both catalytic velocity and KM [22] In the

presence of O2 all Jm KM and glucose response lag were larger than those of

stabiliser-free electrodes The increase in glucose response lag was probably due to both

electron competition and uncompetitive inhibition while the inhibition by DTT itself

was less influential on catalytic velocity than non-competitive inhibition by O2 itself

143

and this resulted in larger Jm despite of their larger KM The truth of O2 enzyme

inhibition might be reversible Met oxidation as discussed in sect 242 on p 93 because

atmospheric O2 could oxidise Met residue [123] and DTT might be capable of reducing

it back although DTT has been reported to be effective only in reducing disulphide [127

132] The increase in Jm in the presence of O2 compared to N2 was probably due to the

reduced content of DTT working toward stabilising enzyme structure This freed up

some GOx portion from rigid conformation

444 PEG

Kinetic parameters for various PEG concentrations are shown below in Fig 52

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEG 1 PEG 2 PEG 4

J m

Am

-2

N2 O2

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

No Stabiliser PEG 1 PEG 2 PEG 4

KM

M

N2 O2

Fig 52 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEG in the presence and absence of O2 n = 2 - 3

PEG decreased Jm to 10 of the standard value at maximum in the absence of O2

144

although 4 PEG electrodes showed much larger Jm than the other PEG electrodes

unexpectedly 4 PEG however showed glucose response lag gt 10 mM even in the

absence of O2 The outcome of low Jm is compatible with the result reported by Lee

[139] while among PEI electrodes Jm however increased with PEG content and this

agreed with the results presented by Arakawa [138] The hypothesis by both authors

therefore might be correct but stabilisation mechanism might differ at different

concentrations Since KM was lowered at low PEG content compared to the standard

value PEG might have penetrated the protein core and stabilised the enzyme structure

[164] however it also lowered GOx activity because the protein conformation has been

locked in At 4 PEG concentration PEG still penetrated the protein core but residual

PEG also brought the preferential hydration and this allowed more flexible enzyme

conformation and increased the catalytic activity The residual PEG however might also

have an adverse effect on electron transfer rates at low glucose concentration In the

presence of O2 KM was increased compared to when in N2 and larger than that of

stabiliser-free electrodes while Jm only marginally better compared to that except for 4

PEG It is altogether not clear about the interaction between GOx and PEG however

only high PEG content effectively enhances the catalytic current output

445 GLC

Kinetic parameters for various GLC concentrations are shown below in Fig 53

145

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser GLC 1 GLC 2 GLC 4

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

20E-02

No Stabiliser GLC 1 GLC 2 GLC 4

KM

M

N2 O2

Fig 53 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 GLC in the presence and absence of O2 n = 3

In the absence of N2 GLC showed smaller Jm and KM compared to the standard values

This indicates the presence of uncompetitive inhibition and the decrease in Jm showed

the reciprocal dependency on GLC concentration It is also quite possible that

concentration-dependent inhibition was due to the tighter protein packing by GLC as

previously reported [144] This might contribute more to non-competitive inhibition

though GLC-containing electrodes showed more resistance against O2 because their Jm

was larger than that of stabiliser-free electrodes and again it showed reciprocal

dependence on GLC concentration In both N2 and O2 cases Jm decreased with increase

in GLC content KM also showed the dependence on GLC concentration in the presence

of O2 and increased with GLC concentration Glucose response lag was also decreased

in all cases Among all 1 GLC was most effective because it lowered KM for both N2

and O2 and increased Jm for O2 although it decreased Jm by 40 in N2 It also decreased

glucose response lag from 8 to 5 mM and this might indicate that glycerol reduced and

scavenged O2

146

446 FAD

Kinetic parameters for various FAD concentrations are shown below in Fig 54

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

No Stabiliser FAD 1 FAD 2 FAD 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser FAD 1 FAD 2 FAD 4

KM

M

N2 O2

Fig 54 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 FAD in the presence and absence of O2 n = 1 - 3

FAD may be reasonably effective as an enzyme stabiliser at concentration gt 1 because

its Jm was larger in both absence and presence of O2 compared to that of stabiliser-free

electrodes It also slightly decreased glucose response lag therefore it might be able to

react with oxidising species as previously reported [108] It is however only effective at

higher concentration because two out of three 1 FAD electrodes did not show

Michaelis-Menten curves in the absence of O2 and none worked in its presence although

4 FAD was not most effective either The relationship between increase and decrease

in Jm and KM in N2 is unknown Improvement in electron transfer was not particularly

observed because higher FAD content decreased the current output although FAD has a

147

function as a mediator Mechanism of GOx stabilisation by FAD is not clear as Bartlett

reported [145] however 2 FAD was shown to be most effective among all and the only

one which showed M

mK

J gt 1 for both N2 and O2

447 PEI and DTT mixture

Kinetic parameters for various PEI-DTT concentrations are shown below in

Fig 55

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

KM

M

N2 O2

Fig 55 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

PEI-DTT (051 11 1505 and 22) in the presence and absence of O2 n =

1 - 2

PEI-DTT stabiliser mix showed the most dramatic effect and promising as a stabiliser

formulation In the presence of O2 it had larger Jm and smaller or equivalent KM than

those found in stabiliser-free electrodes Glucose response lag was also decreased from

148

8 to 3 mM and this implies the presence of O2 scavengers which were new synergetic

effects achieved and not observed in use of either stabilisers as discussed in sect 442 and

443 In the absence of O2 most PEI-DTT showed smaller Jm and marginally smaller

KM than those of the standard values except for some as it can be seen Fig 55 This

probably implies the presence of non-competitive-inhibition like effect due to the

stabilisation of enzyme structure by PEI-DTT Since it was difficult to determine the

comprehensively most effective mixture and the relative relationship in the increase and

the decrease in the two kinetic parameter values were not obvious M

mK

J was found

and compared for each PEI-DTT mixture as shown below in Fig 56

00

05

10

15

20

25

30

35

40

45

50

1505 11 0515 22

PEIDTT ()

Jm

KM

(Am

-2M

-1)

N2O2

Fig 56 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051

11 1505 and 22)

It is clear that DTT must exceed 1 to achieve in the fraction to achieve efficient O2

resistance Since the equivalent glucose response lag was observed in all mixtures this

implies that at least 1 DTT is required for simultaneous reduction of Met amino acid

149

residue On the other hand gt 2 PEI seems required for high catalytic efficiency in N2

or equal weight PEIDTT mixing is preferable to increase catalytic efficiency in both N2

and O2 Since PEIDTT at 22 data presented here is based on a single sample the study

on more samples is required to confirm the results however PEIDTT mixture with

DTT gt 1 is effective to improve catalytic efficiency and output current

448 PEI and FAD mixture

Kinetic parameters for various PEI-FAD concentrations are shown below in

Fig 57

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

J m

Am

-2

N2 O2

00E+00

20E-02

40E-02

60E-02

80E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

KM

M

N2 O2

Fig 57 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with PEI-FAD (21 22 and 24 ) in the presence and absence of O2 n = 2

Firstly the result clearly showed total 6 stabiliser content hindered catalysis because it

showed extremely low Jm compared to other PEIFAD containing electrodes It only

counts for 15 and 9 of Jm obtained for other PEIDTT concentration in presence and

150

absence of O2 respectively Secondly there was no large difference in the performance

between PEIFAD at 21 and 22 hence PEIFAD 21 was most effective of all This

might involve non-competitive inhibition because Jm was smaller by 70 while KM was

smaller by only 20 compared to the standard values in the absence of O2 In the

presence of O2 it showed larger Jm by 7 times however also increased KM by 3 times

compared to those of stabiliser-free electrodes and did not decrease the glucose response

lag

449 Most effective stabiliser content for GOx anodes

To determine the size of comprehensive catalytic efficiency M

mK

J was

found for all the stabilisers and its value was compared for O2 for catalytic efficiency in

the presence of O2 Among all five most efficient catalysts were selected and presented

below in Fig 58

151

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

J m

Am

-2

N2 Am-2O2 Am-2

N2 Am-2 180E-02 125E-02 827E-03 292E-02 710E-03 104E-02O2 Am-2 168E-02 181E-02 151E-02 267E-02 122E-02 157E-02

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505 GLC 1

00E+00

50E-03

10E-02

15E-02

KM

M

N2 MO2 M

N2 M 729E-03 671E-03 576E-03 647E-03 938E-03 328E-03

O2 M 104E-02 471E-03 433E-03 801E-03 658E-03 886E-03

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505

GLC 1

Fig 58 Jm KM in the absence and presence of O2 for various stabilisers PEIDTT

(11 0515 221505 ) and GLC 1

PEIDTT mixture was found to be most effect as GOx stabiliser as discussed in sect 447

For Jm PEIDTT 22 showed the largest in both absence and presence of O2 and this

was followed by PEIDTT 11 For KM PEIDTT 0515 showed the smallest

comprehensively for both N2 and O2 and this followed by PEIDTT 11 PEIDTT

22 and GLC 2 showed relatively large KM in the presence of O2 It can be therefore

concluded that PEIDTT 11 is the most effective in stabilising GOx has largest

resistance against O2 and increases the catalytic output current It showed Jm = 13 x 10-2

plusmn 19 x 10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2

with KM = 47 plusmn 42 mM for O2 The current output obtained is much lower than some

reported results by Willner Heller and Sasaki [18 19 97] by 2 ndash 3 order of magnitudes

their electrodes however involved more complex fabrication methods or system as

discussed sect 22 on p 77 On the other hand it was slightly better than the result

reported by Merkoci who composed a similar composite with 5 GOx and was similar

to the results reported by Arai who built GOx electrodes by more complex methods KM

152

obtained is also smaller by 2 ndash 4 times than some of results by Arai [103]

Max current output Am-2 KM mM Method involved Author

125 x 10-3 ndash 181 x 10-2 47 ndash 67 10 GOx composite Data botained

55 x 10-3 Not available 5 GOx composite Merkoci [115]

30 Not available Apo-GOx reconstituted Willner [97]

11 x 10 Not available Os-complex hydrogel Heller [19]

18 Not available Saccharide-modified polypyrrole Fujii [100]

54 x 10-2 Not available QS conductive polymer Arai [103]

41 x 10 13 - 20 Poly(acrylic) acid hydrogel modified

carbon fibre GDH electrodes

Sakai [18]

Table 7 Comparison of maximum output current and KM with some published data

45 CONCLUSION TO ANODE STUDY

Bulk modified enzyme electrodes are good because they are mechanically very

stable and their fabrication is straightforward and scaleable requiring only mixing

electrode components conductive material mediators enzymes and stabiliser with

binding material TTF-TCNQ is useful material for GOx electrodes because it is

conductive as well as mediating The electrode component ratio is important for its

function and conductivity High binder and protein content hinder the conduction It was

found that 73 weight ratio of TTF-TCNQepoxy resin with 10 GOx loading optimal

The balance among the conductivity mediation enzyme activity mechanical and

153

chemical stability and difficulty in fabrication process is key to the successful

fabrication

O2 is known to be the natural mediator of GOx In the glucose-oxygen fuel

cells GOx anode must be resistant to O2 because O2 is an electron acceptor at the

cathode O2 lowers the current output by competing for electron transfer between GOx

and artificial mediators as well as non-competitive inhibition Various GOx stabilisers

were examined to alleviate such O2 influence

PEI DTT PEG GLC FAD PEIDTT and PEIFAD mixtures were studied as

candidate stabilisers to enhance catalytic activity and the current output PEI was not

effective It stabilised the structure but made conformation more rigid and did not

increase the output current in both the absence and presence of O2 DTT was reasonably

effective It showed uncompetitive inhibition in N2 but stabilised the enzyme structure

It therefore increased Jm and KM in the presence of O2 but increase in KM was probably

due to alleviating inhibition by O2 and loosening up the enzyme conformation PEG was

not effective It probably stabilised the enzyme structure but not show much

improvement for O2 competition It might also deprive electrons from reduced GOx

GLC was effective The effect was concentration dependent Lower concentration was

more effective It also scavenged O2 FAD was reasonably effective but only at higher

concentration PEI-FAD was also reasonably effective It decreased Jm but Km was

marginally equivalent to that of stabiliser-free electrodes for N2 But it increased Jm for

O2 Among all PEIDTT at 11 mixture was found to be most effective because it

stabilised the GOx and scavenged O2 Such powerful influence was not observed in the

use of either stabilisers hence it is a synergetic effect It showed Jm = 13 x 10-2 plusmn 19 x

154

10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2 with KM

= 47 plusmn 42 mM for O2 Although it lowered Jm by 30 in the absence of O2 compared

to the standard value of 18 x 10-2 plusmn 73 x 10-3 Am-2 it also decreased KM in both

absence and presence of O2 and increased Jm by 8 times compared to that of

stabiliser-free electrodes PEIDTT 11 is therefore most effective in stabilising GOx

has largest resistance against O2 and increases the catalytic output current

155

5 CATHODE INTRODUCTION

51 OXYGEN REDUCTION REACTION (ORR)

The oxygen reduction reaction (ORR) takes place at the cathode as oxygen

accepts electrons from the anode Oxygen is an ideal oxidiser for the cathode because it

is readily available and its reaction product water is harmless [62] However ORR has

been a long-time bottleneck especially for low-temperature fuel cells because it causes

large potential loss [3 4 62 165-167] due to a strong double bond in the dioxygen

molecule Its difficult bond dissociation causes slow irreversible kinetics [24 165 166]

so a catalyst is required for this reaction to proceed at useful rates [62 165] ORR is a

multi-electron reaction which involves various reduction pathways and different

oxygen intermediates (Fig 16) In aqueous solution either the direct four- or

two-electron ORR pathway may proceed [165 166] The direct four-electron transfer

pathway reduces O2 into H2O in acid and neutral mediaor OH- in alkaline media This

occurs via several reduction steps [24 165 166] which may involve adsorbed peroxide

intermediates These peroxide intermediates never diffuse away in the solution in this

pathway [166] The two-electron transfer pathway reduces O2 into peroxide species as

final products These peroxide species either diffuse away are reduced further or

decomposed into H2O or OH- [24 165 166] This is not favourable because it leads to

lower energy conversion efficiency and its product hydrogen peroxide may degrade the

catalysts and the fuel cells [165 167] Four- and two-electron transfers also may occur

in series or in parallel [165]

156

Fig 59 Oxygen reduction pathways [165 166]

Reduction kinetics and mechanisms depend on the type of catalysts electrode material

electrode surface condition solution pH temperature contaminant and reactant

concentration [24 165 166] Rate-limiting step also varies by type of the catalysts

[166] However the first electron transfer to dioxygen leading to the formation of

superoxide anion radical (O2־) is the rate limiting step [168] O2־ is very rapidly

protonated in water and converted to hydroperoxyl radical (HO2)

157

Fig 60 Degradation of superoxide anion radical in aqueous solutions [24]

In aqueous solution O2־ is unstable and rapidly converted to O2 and H2O2 via a

proton-driven disproportionation process (Fig 60) [24]

52 REACTIVITY OF OXYGEN

A dioxygen molecule is a homodiatomic molecule consisting of two oxygen

atoms double bonded It has two parallel unpaired electrons in degenerate anti-bonding

π designated as π orbitals therefore it is diradical and paramagnetic (Fig 61) [169]

158

Fig 61 An energy level diagram for ground state dioxygen molecule [169]

When one electron is transferred to a dioxygen molecule the electron is placed in π

orbital and it turns into a reactive O2[24] ־ reducing the bond order from 2 to 15

Oxygen reduction changes the molecular symmetry or stretches the double bond

causing bond disruption [165 166] Electron paring in one of the π orbitals also

changes their orbital energy state and lifts the degeneracy as in Fig 62 [170]

Hereogeneous catalysis involves chemisorption of the oxygen and therefore some

transfer of electron density from the oxygen to the catalyst This facilitates the rate

determining initial electron transfer Oxygen reduction catalysts are reviewed in the

next section

159

(O2) 3Σg- O2־

Fig 62 Diagrams for the state of π orbitals in the ground state oxygen (3Σg-) (left)

and peroxide anion radical (right) [24]

O2 species Bond Order Bond length pm Bond energy kJmol-1

O2 2 1207 493

O2+ 2frac12 1116 643

O2- 1frac12 135 395

O22- 1 149 -

Table 8 Bond orders bond lengths and bond energies of some dioxygen species [169]

53 ORR CATALYSTS

Catalysts are required to reduce the overpotential and improve the efficiency of

160

ORR [3 4 62 165-167] It was reported that 80 of the whole potential loss can be

attributed to cathode ORR overpotential [171] The choice of ORR catalysts varies by

applications [36 171] and direct four-electron transfer pathway is most preferred for

ORR at the lowest possible overpotential in every fuel cell application however its

kinetics and reduction pathway are limited by the type of catalysts and experimental

conditions [24 165 166] Miniaturised glucose-oxygen fuel cells operate in the neutral

aqueous solution at ambient or body temperature with relatively low electrolyte and

oxygen concentrations therefore they require powerful ORR catalysts Platinum (Pt)

has been most ideal however its availability and cost are major limiting factors Many

studies have been made on other noble metals [172 173] transition metal macrocyclic

compounds [174] multicopper oxidases [62 175] and quinones [165] as alternatives

ORR catalysts to Pt These ORR catalysts are reviewed below

531 Noble metal ORR catalysts

Noble and transition metals are well-known ORR catalysts There are two

possible ORR mechanisms for metal surface dissociative and associative

161

Dissociative mechanism

middotmiddotmiddotmiddotmiddot Eq 67

middotmiddotmiddotmiddotmiddot Eq 68

middotmiddotmiddotmiddotmiddot Eq 69

( indicates the catalyst surface)

Associative mechanism

middotmiddotmiddotmiddotmiddot Eq 70

middotmiddotmiddotmiddotmiddot Eq 71

middotmiddotmiddotmiddotmiddot Eq 72

middotmiddotmiddotmiddotmiddot Eq 73

middotmiddotmiddotmiddotmiddot Eq 74

In dissociative mechanism the O2 double bond is ruptured rapidly on its adsorption on

the metal surface [165 166 172] This involves a strong interaction between oxygen

and metal atoms [166] and does not generate H2O2 [165] Electron and proton transfer

take place simultaneously and this is the rate-limiting step [166 171] In the associative

mechanism O2 does not dissociate before proton transfer Adsorbed O2 stays on the

metal surface [165 172] and two-electron and proton transfer [166 173] take place via

the formation of superoxide or peroxide intermediate [165 166] This reaction is

thermodynamically easier than the dissociative pathway and its final product is H2O2 or

162

H2O if both pathways occur in series [165] O2 adsorption on the metal surface is an

inner sphere interaction which involves complex formation with the metal surface [30]

There are three ways in which O2 adsorbs (i) Griffiths (ii) Pauling and (iii) bridge

model (Fig 63)

Griffiths model Side-on Pauling model End-on Bridge model

OO

M

Fig 63 Three models of oxygen adsorption [165 166 173]

The Griffiths model is called lsquoside-onrsquo type O2 functions as a bidentate ligand against a

single metal atom in this complex and its π orbitals laterally interact with empty metal

dz2 orbitals causing ligand-to-metal charge transfer (LMCT) πrarrdz

2 At the same time

electrons partially filling metal dxz or dyz orbitals are back donated to oxygen π orbitals

[166 171] Therefore a strong metal-oxygen interaction is present in this model This

effectively disrupts the O=O bond [166] and facilitates the electron transfer from the

metal ion by changing its oxidation number The Pauling model is called the lsquoend-onrsquo

type [165 166 173] O2 is a monodentate ligand in this model and only one of the

oxygen atoms in the dioxygen molecule can be bonded to the metal surface An oxygen

π orbital interacts with metal dz2 orbitals and this allows either the direct or peroxide

ORR pathway after partial charge transfer [166] The bridge model resembles Griffiths

model in the way the metal atom and oxygen interact however it requires two adjacent

163

adsorption sites so that O2 can ligate two different metal atoms The bridge-model

interaction is easily interfered with by the surface contamination and the oxide layer

because the surface species block the adsorption sites for ORR catalysis [173]

Fig 64 Models for oxygen adsorption and ORR pathways [166]

Both the Griffiths and bridge models follow dissociative mechanism (pathway I and III

respectively Fig 64) due to the lateral interaction leading to the dissociation of O2

double bond The Pauling model on the other hand follows the associative mechanism

(pathway II Fig 64) This is further divided into two other pathways IIa and IIb

according to its end product H2O2 or H2O (Fig 64) respectively [166] O2 adsorption

and the ORR mechanism therefore depend on (i) the extent of oxygen-metal

interaction and (ii) the availability of oxygen adsorption sites A volcano-shaped trend

between ORR rate and oxygen-metal interaction has been plotted by Norskov as in Fig

65 [172]

164

Fig 65 A volcano-shaped trend between ORR rate and energy required for oxygen

binding on various transition metal surface [172]

Many have reported platinum (Pt) as the most ideal metal catalyst due to its high

catalytic ORR activity [165 166 171 172] Fig 65 shows that Pt and Pd (palladium)

have largest ORR rate yet medium oxygen adsorption energy and iridium (Ir) and

rhodium (Rd) follow them On the other hand metals with either higher or lower

oxygen adsorption energy result in lower ORR rate This is because metals with high

oxygen adsorption energy such as gold (Au) imposes too large energy barrier for the

formation of a stable oxygen-metal bond while too small adsorption energy as in nickel

(Ni) leads to excessively stable oxygen-metal interaction and slows the proton transfer

Therefore associative pathway is dominant at Au surface while oxygen dissociation is

deactivated at Ni surface but instead this increases the surface oxide coverage [172

173] Pt performs best because it has moderate oxygen-metal surface interaction [165]

Pt alloys with other transition metals such as Ni Co and Fe have been also reported to

increase the catalytic activity [36 167 171 172] because alloying dilutes the Pt-O

165

interaction and alters the overall oxygen-metal surface interaction [171 172] This is a

useful property to add because Pt is known to form a surface oxide layer leading to both

associative and dissociative catalytic pathways [165 171] This surface state is

potential-dependent and stays persistenly at less than 08 V vs AgAgCl [172]

532 Macrocycle and Schiff-base complex ORR catalysts

Macrocyclic transition metal complexes have been widely investigated as

potential ORR catalyst both for fuel cells and as catalysts to activate dioxygen for

synthetic purposes They have been widely reviewed by [165 176] Macrocyclic

complexes consist of (i) transition metals such as Fe Co Ni or Cu (ii) chelating atoms

N4 N2O2 N2S2 O4 and S4 and conjugate π systems such as porphyrin and

phthalocyanine [165] Examples of transition metal macrocycles are shown in Fig 66

Fig 66 Examples of transition metallomacrocycles Heme (left) [177] and copper

phthalocyanine (right) [178]

166

Schiff base complexes such as metallosalens and metallosalophens are other

well-known chelating ORR catalysts [174 179] Transition metal macrocycles and

Schiff bases can be also polymerised into conductive films [180] (Fig 67)

Fig 67 Schiff base complexes

Co(II) salen Co(II) salophen and

Co(II) salen modified poly(34-ethylenedioxythiophene) [174 180]

Interaction between the oxygen molecule and the metal complex is similar to that with

the noble metals and involves partial charge transfer between the filled metal dz orbitals

and the π orbital of O2 as described in sect 531 The catalytic activity of the metal

complexes depends on the type of central metal atom ligand and the size of the

π-electon systems The reducing ability of the ligand is important because the central

metal atom must transfer considerable electron density to oxygen atoms during the

167

catalysis [170] The thermodynamically most stable metal-oxygen complexes are

bionuclear complexes such as dicobalt face-to-face 4-atom-bridged diporphyrin

(Co2(FTF4)) (Fig 68) [165-167]

Fig 68 Molecular structure of dicobalt face-to-face diporphyrin [166]

Mononuclear Co complexes take the associative pathway [165] forming superoxo

cobaltic complexs (Co-O-O־) [181 170] while their dimers or binuclear complexes can

take either direct four-electron or the parallel pathways (Fig 69) [165 167] because

they can lock the oxygen moiety in rigid orientation [170] Co2(FTF4) favours

four-electron transfer and its ORR pathway depends on the distance between two

cofacial metal atoms [165-167] Spacing distance can be altered by inserting a spacer

molecule [165 166]

168

Fig 69 ORR pathway by Co2(FTF4) [166]

However Co2(FTF4) is not stable enough for practical use [165 166] Practical ORR

catalysts for fuel cell application must be stable inexpensive and feasible to synthesise

533 Multicopper enzymes as ORR catalyst

Multicopper oxidases (MCOs) are copper-containing enzymes such as bilirubin

oxidase (BOD) laccase (Lac) and copper efflux oxidase (CueO) Oxygen is widely used

as an electron acceptor in nature because it is ubiquitous and oxidation is a good mean

to obtain energy as in combustion Millions of years of evolution have therefore

provided enzymes with good catalytic activity for oxygen reduction Although oxygen

169

reduction in biological system is complex due to involving various substrates and

catalytic steps as well as it tends to be incomplete and leads to hydrogen peroxide or

superoxide formation as the product the enzymes catalyse the rate-determining initial

electron transfer For application in biofuel cells it is essential that the enzymes are

widely available and possess good stability activity in immobilised form and ideally

four-electron ORR catalysis MCOs have been prevalent in biofuel cell applications

because they are active at neutral pH catalyse four-electron ORR and bear

immobilisation Fungal laccase and bilirubin oxidase have been most widely studied

MCOs [56 59 60]

Most MCOs have four Cu atoms which are designated as type I (T1) type II (T2) and

a pair of type III (T3) T2 and T3 together form a trinuclear Cu centre while T1-Cu

called blue copper [175 182 183] is located away from the trinuclear Cu centre but

adjacent to the hydrophobic substrate-binding site Fig 70 shows the location of the

trinuclear Cu centre (copper coloured) and the blue copper (in blue) Oxygen binding

and ORR take place at the trinuclear Cu centre [175] while T1-Cu receives electrons

from the substrate and mediates electron transfer between substrate and the trinuclear

Cu centre [175 184]

170

Fig 70 Laccase from Trametes versicolor [185]

Enzyme catalytic efficiency depends on the redox potential [175] of T1-Cu It has been

reported that fungal laccase has the highest redox potential between 053 ndash 058 V vs

AgAgCl [62 183] T1-Cu also allows DET due to being located close to the surface

[183 186 187]

MCO reaction cycle starts from substrate oxidation to form a fully reduced state MCO

is initially at native form or so-called intermediate II Native form is characterised by

doubly OH--bridged structure (Fig 71(ii)) In the absence of substrate the native form

decays to the resting form which is at fully oxidised state (Fig 71(i)) After

four-electron reduction of enzyme the fully reduced MCO (Fig 71(iii)) reacts with O2

The first reduction step involves two-electron transfer from each of T3-Cu+ to O2 This

forms peroxide intermediate or intermediate I (Fig 71(iv)) and lowers the energy

barrier to disrupt O=O It is followed by swift protonation of adsorbed peroxide at

T3-Cu site This protonation reaction is induced by the charge transfer from T1- and

171

T2-Cu+ This leads to the complete reduction of oxygen to water and the enzyme native

form is retrieved

(i)

(ii)

(iii)

(iv)

Fig 71 Redox reaction cycle of MCOs with O2 [175]

All MCOs have this common ORR mechanisms despite the different origin structure

biological functions substrate reducing power and optimal environment

5331 BOD

BOD is an anionic monomer [182] most active at neutral pH [183 188] and

172

capable of both DET and MET The mediators must have their redox potential close or

slightly lower than that of BOD 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

(ABTS) (Fig 72(i)) [62 97] Os-pyridine complexes linked to conductive polymer (Fig

72(ii)) [66 189] and cyanometals [18 56] have been used as its mediators [56 185]

ABTS is a divalent anion which works as co-substrate of MCOs and its redox potential

is 067 V vs AgAgCl [56] Os-pyridine complex with conductive polymer follows the

similar idea for the wired GOx anodes discussed in sect 223 on p 80

4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine) has been used

especially for the cathode and its redox potential is 055 V vs AgAgCl

Fig 72 Common mediators for BOD (i) ABTS [56 62]

(ii) Os[4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine)] [190]

173

Tsujimura showed that ORR catalytic constant was 6-times greater in the presence of

Os-pyridine complex than in DET [66 188] Reduction starts around 05 V in most

cases [66 97 183 188] The size of current density varies roughly between 05 - 1

mAcm-2 [56 97 188] and 95 mAcm-2 was achieved by Heller [189]

5332 Lac

Fungal laccase is another anionic monomer [183] It is less expensive than

BOD and has broad substrate specificity including xenobiotics [175] although it has

optimum pH around 5 and activity hindered by chloride [19 182] Its T1-Cu redox

potential ranges between 053 ndash 058 V vs AgAgCl [62 183] Highest redox potential

066 V and current density 057 mAcm-2 were achieved by the use of anthracene

plugging molecule which provides strong binding between Lac and pyrolytic graphite

edge electrode as well as long-term activity 57 of the initial current was retained after

eight weeks [185]

5333 CueO

CueO is a less common enzyme in biofuel cells however it is active for a wide

range of pH between 2 - 8 [186] and also it has better catalytic activity than other

MCOs because it has five Cu atoms This extra Cu atom is believed to have mediating

function and 4 mAcm-2 has been achieved at pH 5 [191] However it has lower redox

potential around 03 V [186 191] Protein engineering has been introduced to increase

T1-Cu redox potential [186]

174

534 Anthraquinone ORR catalyst

Anthraquinone (AQ) is known to catalyse two-electron transfer in ORR (Fig

73) [44 165 192-194] and it is used in industries to produce hydrogen peroxide [165

192]

Fig 73 Two-electron ORR by anthraquinone [193]

ORR by anthraquinone is an electrochemical-chemical (EC) process [165 192] in

which quinone must be first electrochemically reduced to an active species

semiquinone anion radical (Q־) The active semiquinone radical can then react with

dioxygen so that it converts the dioxygen into O2־ via forming of the intermediate

superoxo-C-O(O2) species This is the rate-limiting step O2־ produced by a

semiquinone radical either disproportionates (Eq 77) or it is further reduced into HO2-

(Eq 78) [192]

175

Oxygen reduction reaction by anthraquinone

ltlt Rate limiting step gtgt

middotmiddotmiddotmiddotmiddot Eq 75

middotmiddotmiddotmiddotmiddot Eq 76

ltlt Following spontaneous reactions gtgt

middotmiddotmiddotmiddotmiddot Eq 77

middotmiddotmiddotmiddotmiddot Eq 78

(Q indicates quinone on the surface)

Derivatised anthraquinone such as Fast red (Fig 74) can be attached to the carbon either

physically or covalently Grafted quinones are very stable [192 194 195] and resistant

against continuous potential cycling [194] metal ions and surfactant despite of some

fouling present [44]

176

O

O

N N Cl

frac12 ZnCl2

1

2

4

6

5

3

10

9 7

8

12

14

13

11

O

O

(i) (ii)

Fig 74 The structures of (i) anthraquinone [196] and (ii) Fast red AL salt [44 197]

However its ORR is heavily affected by pH [194] because its catalytic efficiency

depends on the concentration of surface Q־ species [192 194] The concentration

varies by different pH and the catalytic efficiency drops at both high and low pH due to

the depletion of Q־ When pH falls below its dissociation constant Q־ is not

reproducible anymore and ORR is inhibited This phenomenon appears at as high as pH

7 and the ORR vanishes at pH lt 7 [194] AQ-grafted GC (AQGC) shows only sluggish

kinetics at pH lt 10 Its voltammogram also becomes complex due to the presence of

native quinone groups originated from the carbon surface [192] ORR mediated by the

native quinone groups is observed as a prewave and this varies by different quinone

derivatives such as phenanthrenequinone (PQ) (Fig 75) electrode composition and pH

[194]

177

Fig 75 Hydrodynamic voltammogram for ORR at various quinone electrodes with various

rotation speeds [194] (i) unmodified GC (ii) AQGC and (iii) PQGC at pH 7

54 CARBON PROPERTIES

Carbon is widely used for electrochemical applications because it is

inexpensive mechanically stable [105] and sufficiently chemically inert yet reactive

due to the presence of surface defects It has wide potential window between 1 and -13

V [21 30] Carbon is available in various solid forms such as graphite glassy carbon

(GC) pyrolytic carbon (PC) activated carbon (AC) carbon fibres (CFs) carbon

nanotubes (CNT) and diamond It is easy to renew the surface and allows facile

chemical modification [195] However it is also prone to adsorption and surface

oxidation simply by air and water exposure and its electron transfer is under the

constant influence of the electrode surface condition and history [198] This leads to the

deviation and the low reproducibility in carbon electrochemical experiments [105]

178

Physical properties of carbon vary by its form and section The carbon consists of either

(i) layers of the conductive planar graphene sheets or (ii) the insulating

tetrahedral-puckered forms as seen in the diamond (Fig 76) Conductivity in diamond

is gained by doping with boron [199 200]

Fig 76 Carbon structures (i) graphite (ii) diamond [201]

The electrical conductivity is anisotropic and closely correlates with the carbon

structure and section The carbon section is classified into (i) basal and (ii) edge planes

The basal plane extends along the graphene plane while the edge plane is perpendicular

to this

179

Fig 77 Carbon section (i) basal plane (ii) edge plane [105]

The basal plane has larger conductivity due to being parallel to the vast graphene

network while the edge plane is superior in electrochemical reactivity due to being

richer in reactive graphene sheet termini and carbon defects The ratio between the basal

and edge planes therefore affects the conductivity and electrochemical properties of

the carbon and this varies by the carbon form and its treatment [45 105 195 202 203]

The electrical conductivities for various carbon forms and sections is shown in Table 9

180

The type of carbon Electrical conductivity Scm-1

Graphite [105 159] 1 x 103 - 104

Highly ordered PG basal plane 250 x 104

Highly ordered PG edge plane 588

PG basal plane 400 x 103

PG edge plane 333

Polycrystalline graphite 100 x 103

GC [105 159 203] 01 - 250 x 102

Activated carbon [204] 1 x 10

Carbon fibre 133 x 103

Boron-doped diamond [199 205] 01 - 1 x 102

Multi-wall carbon nanotube [206] 1 ndash 2 x 103

Pt 909 x 104

Cu 588 x 105

Table 9 The electric conductivity of various carbon forms and sections Data from

[105] unless defined specifically

541 Graphite

Graphite is black-coloured material with pores and high gas permeability It is

available in many physical forms as powders moulded structures and thin sheets

therefore it is used in many commercial applications [203] Powdered graphite is

convenient to make carbon paste and composite electrodes [105] Graphite consists of

181

layers of graphene sheets each of which is a huge π-conjugate system π-conjugate

system is made of hexagonal rings of sp2 hybridised carbon atoms [105 195] (Fig 76(i)

and Fig 78) and allows free electron movement within graphene This gives carbon

metallic properties such as electric and thermal conductivity

Fig 78 A direct image of a graphene bilayer by transmission electron microscopy

(TEM) at resolution of 106 Aring and with a 2-Aring scale bar [207]

Each graphene sheet is structurally stable in horizontal direction but fragile in vertical

direction because each layer is only held by van der Waalsrsquo force Therefore the

graphene sheets easily slide and collapse [31]

542 Glassy carbon (GC)

Glassy carbon (GC) is a non-graphitised carbon made of randomly interwoven

sp2-carbon fibres which are tangled and heavily cross-linked (Fig 79) It contains many

closed voids [203] and fullerene-like elements [208] It is impermeable to gases [203

182

208] chemically more inert [208] and mechanically stronger but electrically more

resistive than other carbons [203]

Fig 79 GC (i) High resolution TEM image (ii) A schema of GC [208]

543 Activated carbon (AC)

Activated carbon (AC) is thermally and chemically activated carbon It is

highly porous and has large internal surface area This is an excellent adsorbent for

solvent recovery and gas refining [203 208]

544 Carbon fibre (CF)

Carbon fibres (CFs) have small diameter ranging between 5 to 30 microm They are

lightweight have lower fabrication cost and are superior in mechanical properties

therefore they have been used for sporting automotive and military equipments They

are available in many forms such as tow mat felt cloth and thread They consist of a

random arrangement of flat or crumpled graphite sheets but their structure and

183

molecular composition depend on the type of precursors [203 208]

545 Carbon nanotubes (CNTs)

Carbon nanotubes (CNTs) are made rolled-up sheets of sp2-graphene in nano to

micro-meter range They are available in various formats single-walled nanotube

(SWNT) multi-walled nanotube (MWNT) carbon nanohorn (CNH) (Fig 80) and

various forms powder arrays and posts [117]

Fig 80 Various CNTs (i) Bundled SWNTs (ii) MWNT (iii) CNHs [209]

CNTs have been drawing keen attention due to their wide surface area conductive or

semiconductive properties light weight mechanical stability and facile

functionalisation [117 195 202 210] They have been used as support for the dispersed

metal catalysts [3] and electrode materials [117 202] The drawbacks of CNTs are the

high presence of impurities alteration of their properties on acid cleaning [202 211]

and unknown toxicity [117 160 202]

184

546 Boron-doped diamond (BDD)

Diamond is different from other carbons because it is transparent and not

electrically conductive It has a cubic lattice structure made of closely-packed repetitive

units of sp3-hybridised carbon atoms as seen in Fig 81

Fig 81 A TEM image of diamond [212]

Each unit consists of four σ-bonded sp3-carbon atoms in tetrahedral coordination as in

Fig 76(ii) Each bond is localised and this gives structural stability but does not conduct

electricity By doping it with boron diamond can become semiconductive Such

diamond is called boron-doped diamond (BDD) and boron functions as an electron

acceptor BDD is mechanical more stable chemically more inert sensitive to analyte

and has wide potential window lower back ground current and stable electrochemical

responses [195 199 205]

185

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON

551 Electrochemical properties of carbon

The edge plane of carbon which contains graphene termini and carbon defects

is chemically reactive and accounts for carbon electrochemistry while the vast network

of graphene sheets is responsible for its electrical conductivity The termini and defects

include some sp3 hydrocarbons and they are oxidised into various oxygen functional

groups such as carboxyl quinoyl hydroxyl lactone groups ethers cyclic esters and

lactone-like groups [195] These electrode functional groups facilitate electron transfer

provide bonding between the electrode and the catalysts or enzymes or simply cause

electrode capacitance or resistance Various carbon pretreatments such as

electrochemical pretreatment (ECP) vacuum heat treatment (VHT) carbon fracturing

and polishing laser irradiation [195 213-215] are used to attach active surface

functional groups and increase the rate of electron transfer in various electrochemical

reactions [198 216]

552 Electrochemical pretreatments (ECPs) of the carbon

Electrochemical pretreatments (ECPs) provide feasible electrode surface

modification Various ECPs have been used to introduce carbon-oxygen surface

functional groups Most common method is potential cycling in the acid or basic media

or the combination of anodisation and cathodisation as shown in Table 10

186

Table 10 ECPs examples

Potential cycling at 02 Vs-1 between 0 and 02 V in 01 M H2SO4 [105]

Anodisation at 18 V in pH 7 phosphate buffer for 5 minutes followed by 1-minute cathodisation at

ndash 15 V [216 217]

Potential cycling between 06 and 2 V at 5 Vs-1 for 15 to 30 minutes in 05 M NaOH [218] or their

combination [219]

ECPs introduce the electrodes various new properties They (i) attach electrochemically

active functional groups and increase the oxygencarbon ratio [105] (ii) introduce

carbon defects and active sites [105 218] (iii) increase the surface area [105 198 215

216 218] (iv) form a surface film and (v) increase the background current [105] and

double layer capacitance [105 215 216] Most notable effect of EPCs is the increase in

electron transfer rate in many electrochemical reactions This is observed as a decreases

in peak separation and an increase in peak current in voltammetry as seen in Fig 82

[105 216 218 220] As for ORR however it was reported that surface quinone groups

attached by EPC or originated from the carbon itself reduce ORR overpotential but do

not mediate ORR as in quinone groups physically or covalently attached [216 220]

187

Fig 82 The influence of ECP on GCE and its voltammogram at 01 Vs-1 in pH 7

phosphate buffer in the presence of (i) N2 and (ii) O2 The voltammograms

of both pretreated and unpretreated GCEs are shown [216] The

presentation has been altered for convenience

56 ELECTROCHEMICAL TECHNIQUES FOR ORR

ORR has been studied by two electrochemistry techniques cyclic voltammetry

(CV) and hydrodynamic voltammetry (HDV) using the rotating disc electrode (RDE)

These techniques provide overlapping but complementary information CV is a dynamic

technique and allows calculation of the number of electrons up to and including the rate

determining step (nα) With numerical simulation and data fitting CV can be used to

estimate the standard heterogeneous rate constant (kdeg) RDE is a steady state technique

and can give the overall number of electrons transferred (n) The well-established

Koutecky-Levich plot (see sect 567 on p 200) provides a robust method for determining

kdeg provided the standard electrode potential (Edeg) for the reaction is known

188

561 Cyclic voltammetry (CV)

Cyclic voltammetry (CV) is widely used for initial exploration and

characterisation of a redox active system It is of limited electroanalytical value

however kinetic parameters can be obtained from the current-voltage (I-V) plot In

cyclic voltammetry potential is swept linearly with a triangular waveform (Fig 83(i))

and applied cyclically The resulting time-dependent current is typically plotted versus

applied potential (Fig 83(ii)) [30 221] The potential range and scan rate are decided in

advance according to the type of analytes reactions and materials of interest

Fig 83 Schemas for (i) a Triangular waveform (ii) Typical cyclic voltammogram for

benzoquinone (BQ) CV was taken at 005 Vs-1 between + 025 and ndash 05 V vs

AgAgCl for 1 mM BQ in 1M KCl at graphite composite electrode Switching

potential (Eλ) is ndash 05 V and time taken to reach Eλ (t) is 15 seconds Based on

the real data

189

562 Non-faradic current and electric double layer capacitance

Transient techniques such as CV always contain two types of electric current

non-faradaic and faradaic current The non-faradaic charging current is due to the

electric double layer which is formed at the electrode-solution interface This occurs

because such an interface behaves like a capacitor as counter ions accumulate at the

interface in order to balance the potential at the electrode This does not account for the

charge transfer reactions involved however provides the information about the

electrode surface [30] and also can affect the rate of electrode process and distort the

voltammogram Cd can be estimated by electrochemical impedance spectroscopy or

deduced by averaging Cd over the potential range as in Fig 84 and Eq 33 [21 30]

Fig 84 The double layer capacitance (Cd) observed in CV for deoxygenated pH 74 at

the acid-pretreated graphite electrode See Eq 33 for the calculation and

symbols Based on the real data

190

Electric double layer capacitance per unit area

AII

AEQareaunitperC ccca

d sdotminus

=sdot

=υ2

)()( middotmiddotmiddotmiddotmiddot Eq 79

Cd = Double layer capacitance per unit area microFcm-2

Q = Charge C

E = Potential V

A = Electrode surface area cm-2

Ica = Anodic non-faradic current A

Icc = Cathodic non-faradic current A

υ = Scan rate Vs-1

Cd needs to be either negligible or subtracted before interpretation of faradaic current

which accounts for the charge transfer phenomena in redox reactions In order to

minimise the influence of Cd on the voltammogram the scan must be taken at slow scan

rate and the electrolyte concentration must be always kept reasonably high [30]

563 Diagnosis of redox reactions in cyclic voltammetry

A typical voltammogram contains current peaks as seen in Fig 83(ii) and these

are due to the redox reactions in the system Diagnosis of the redox system using CV is

based on (i) peak separation (ii) peak current height (iii) peak shape or peak width (iv)

the number of peaks and (v) the dependency of peak shape on the scan rate The

analysis of such peaks involves the following four parameters anodic peak current (Ipa)

cathode peak current (Ipc) anodic peak potential (Epa) and cathode peak potential (Epc)

This reveals the type of electrochemical reactions involved which are either (i)

191

reversible (ii) quasi reversible or (iii) irreversible When the peak current is proportional

to υfrac12 the redox reaction is diffusion-limited in the solution while a linear dependence

shows that the redox active species is confined to the electrode surface

In the reversible system charge transfer between the redox species and the electrode is

rapid [221] therefore it has fast kinetics and the reaction is diffusion-limited The

electrode potential follows the Nernst equation (Eq 15 on p 50) because the electrode

potential is dependent on the concentration equilibrium at the electrode surface and the

peak potential is independent of the scan rate The features on the reversible system are

listed in Table 11 The peak current (Ip) is linearly proportional to υ12 as the redox

reaction is diffusion-limited The peak separation (∆Ep) is the difference between Epa

and Epc and it is 59 mV at 25 ˚C for the single-charge transfer [29 30] The peak width

(δEp) is the difference between Ep and half-peak potential (Ep2) Ep2 is the potential

where the peak current is half It is 565 mV for a single charge transfer The ratio

between Ipc and Ipa is called a reversal parameter [105] It is unity at any scan rate in the

reversible system

192

Table 11 Reversible system at 25 ˚C [30]

2121235 )10692( υlowasttimes= CADnI p middotmiddotmiddotmiddotmiddot Eq 80

mVn

E p528

= independent of υ middotmiddotmiddotmiddotmiddot Eq 81

mVn

EEE pcpap59)( =minus=∆ middotmiddotmiddotmiddotmiddot Eq 82

mVn

EEE ppp556

2 =minus=δ middotmiddotmiddotmiddotmiddot Eq 83

1=pa

pc

II middotmiddotmiddotmiddotmiddot Eq 84

Ip = Peak current A

n = The number of charges transferred

A = Electrode surface area m-2

D = Analyte diffusion coefficient m-2s-1

ν = Solution kinematic viscosity m-2s-1

Ep = Peak potential V

∆Ep = Peak separation V

Epa = Anodic peak potential V

Epc = Cathodic peak potential V

δEp = Peak width V

Ep2 = Half-wave potential V

Ipa = Anodic peak current A

Ipc = Cathodic peak current A

In a totally irreversible system charge transfer between the redox species and the

electrode takes place only slowly [221] The electrode must be activated by applying

193

large potential to drive a charge transfer reaction at a reasonable rate Ep becomes

dependent on scan rate and this increases ∆Ep and skews the voltammogram [30]

However Ip is still proportional to υfrac12 because at high overpotential the redox reaction

is diffusion-limited [29]

Table 12 Irreversible system at 25 ˚C [222]

2121215 )()10992( υα αlowasttimes= CADnnI p middotmiddotmiddotmiddotmiddot Eq 85

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛

deg+minusdeg=

2121

lnln780RT

Fnk

DFn

RTEE pυα

αα

α

middotmiddotmiddotmiddotmiddot Eq 86

mVn

E pαα

59gt∆ middotmiddotmiddotmiddotmiddot Eq 87

mVn

E pαα

δ 747= middotmiddotmiddotmiddotmiddot Eq 88

α = Transfer coefficient

nα = The number of charge transferred up to the limiting step

( Please refer to Table 11 for other terms and symbols)

A quasi-reversible system is controlled by both kinetics and diffusion Its

electrochemical reversibility is relative to the scan rate [21 30] The peak potential

depends on scan rate for rapid scans and the peak shape and peak parameters become

dependent on kinetic parameters such as α and Λ where 21

1

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛

deg=Λ

minus υαα

RTFDD

k

RO

[30]

Since Λ is a function of k˚υ-frac12 where kdeg is the standard rate constant the voltammogram

hence kdeg is affected by the scan rate as seen in Fig 85

194

Fig 85 Voltammograms of a quasi-reversible reaction at various scan rates [21]

564 Coupled reactions in cyclic voltammetry

Redox reactions are sometimes coupled with different electrochemical or

chemical reactions Voltammogram with altering scan rates can indicate the presence of

such reactions A redox reaction coupled with another redox reaction is called EE

mechanism while with homogeneous chemical reaction it is called EC mechanism If

the coupled reaction is catalytic then it is called ECrsquo mechanism [21]

Many ORR catalysts show coupled reactions with ECrsquo mechanism ORR is a

quasi-reversible reaction because the reaction is limited by oxygen diffusion yet O2

adsorption and reduction heavily relies on overpotential or catalytic power of catalysts

Catalysed reactions show distinctive voltammetric features There is only a single-sided

large current but with no reverse current because they catalyse a reaction only in a

single direction The reversal parameter therefore becomes non-unity [30]

195

ORR catalysts sometimes contain multiple reducible centres or an atom with various

oxidation states as in seen transition metal They might exhibit a multi-peak

voltammogram under non-catalytic condition The shape of voltammogram depends on

the difference in the standard potential (∆E˚rsquo) between two redox centres or states (Eq

89) as seen in Fig 86 [21 30]

21 ooo EEE minus=∆ middotmiddotmiddotmiddotmiddot Eq 89

Fig 86 Voltammograms of two-electron transfer EE systems of different ∆Edegrsquo [21]

565 Voltammograms for the surface-confined redox groups

Catalysts encapsulated in or attached to the electrode provide catalytic groups

or moieties on the electrode surface Such surface-confined redox species show different

voltammograms from solution redox species because their redox reactions do not

196

involve diffusional redox species They exhibit a characteristic voltammogram of

perfect symmetry with Epa = Epc as seen in Fig 87 and their peak current is linearly

proportional to υ rather than υfrac12 as in Eq 90 [21 30] The features of surface-confined

groups are listed in Table 13

Fig 87 A voltammogram of the reversible redox reaction by surface-confined groups [21]

Table 13 Reversible system by surface-confined groups at 25 ˚C [30]

RTAFn

I p 4

22 Γ=

υ middotmiddotmiddotmiddotmiddot Eq 90

oadsp EE = middotmiddotmiddotmiddotmiddot Eq 91

mVn

E p690

2 =∆ middotmiddotmiddotmiddotmiddot Eq 92

Where Γ isiInitial concentration of the absorbed species

However such an ideal voltammogram is seldom observed because the density of redox

species is so high in the surface-confined environment compared to in the solution and a

197

voltammogram is also influenced by the interaction among those packed redox species

A multi-electron system of the surface-confined groups is also not this case [21]

566 Hydrodynamic voltammetry (HDV) and Levich equation

Hydrodynamic methods purposely introduce convective flow to the system

using RDEs This allows the study of precise kinetics because the diffusion is under

precise control Unknown influence by mass transfer and double layer capacitance on

the electrode reaction is eliminated and steady state is quickly achieved [29 30] In

RDE method laminar flow is applied perpendicular to the electrode surface by spinning

a disc electrode at arbitrary rotation velocity (Fig 88) The rotation rate is expressed in

either rpm (rotation per minute) or angular velocity (ω) ω is defined as ω = 2πf where f

is frequency With disc rotation diffusion becomes a function of electrode rotation rate

as in Eq 93 However the limit of disc rotation rate must be set in order to avoid the

thick hydrodynamic boundary layer and turbulent flows to maintain steady state

Allowable range is between 100 rpm lt f lt 10000 rpm or 10 s-1 lt ω lt 1000 s-1 [30]

198

ω

Fig 88 RDE is rotated at ω and laminar flows applied to the electrode surface

Diffusion layer thickness and disc rotation rate [30]

612131611 νωδ minus= D middotmiddotmiddotmiddotmiddot Eq 93

δ = Diffusion layer thickness cm

D = Analyte diffusion coefficient m2s-1

ω = RDE angular velocity s-1

ν = Solution kinematic viscosity m2s-1

Under such a condition disc limiting current (Id) becomes a function of disc rotation

rate as described in Levich equation (Fig 85) [30] This allows finding the total number

of charge transferred (n)

199

Levich equation [30]

612132620 CnFADI Lminus= νω middotmiddotmiddotmiddotmiddot Eq 94

δ

nFAC= middotmiddotmiddotmiddotmiddot Eq 95

IL = Levich current A

n = Total number of charge transferred

F = Faraday constant Cmol-1

A = Electrode surface area m2

C = Bulk analyte concentration molm-3

( Please refer to Eq 93 for other terms and symbols)

Due to the steady state condition the current response in HDV becomes sigmoidal [223]

as seen in Fig 89

200

Fig 89 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH

74 Based on the real data

567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the

charge transfer rate constant

Levich equation (Eq 94) shows that Id is proportional to ωfrac12 therefore also a

function of diffusion layer thickness (Eq 95) This is true for reversible kinetics

because the electrode reaction is limited by diffusion which is a function of ωfrac12 and

therefore Id also becomes dependent on ωfrac12 at any potential However in

quasi-reversible system Id depends on diffusion but also affected by its kinetics [223]

Therefore when Id reaches the kinetic limitation for charge transfer the slope gradually

deviates from its Levich current as seen in Fig 90 [30]

201

Fig 90 Levich plots for reversible (blue) and quasi-reversible (red) systems [30]

In addition its charge transfer kinetics is altered by the potential applied As Tafel plot

(Fig 9 on p 57) presents the current is exponentially dependent on the overpotential

The electrode reaction is kinetically limited at low overpotential then gradually

becomes diffusion-limited as overpotential increases [223] This implies that Id in

quasi-reversible system is always under the influence of potential applied because the

potential alters its kinetics

When Id becomes completely free from the mass transport influence such current is

called kinetic current (IK) because Id now purely depends on only its kinetics Arbitrary

Id then defined as the sum of diffusion-limited IL and kinetically-limited IK The

double-reciprocal equation of this summation is defined as Koutecky-Levich equation

as in Eq 96

202

Koutecky-Levich equation [30]

LKd III111

+=

216132

11620

11ων

times⎟⎟⎠

⎞⎜⎜⎝

⎛+= minus nCFADI K

middotmiddotmiddotmiddotmiddot Eq 96

Id = Disc limiting current A

IK = Kinetic current A

IL = Levich current A

( Please refer to Eq 93 and Eq 94 for other terms and symbols)

By extrapolating the Koutecky-Levich plot IK can be easily estimated [30] from the

reciprocal of its y-intersect In order to plot Koutecky-Levich equation and find IK a

sequence of arbitrary Id is recorded with altering disc rotation velocity at arbitrary

potential This technique is called hydrodynamic amperometry (HDA) and it is shown

in Fig 91

203

Fig 91 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in

oxygenated 001 M PBS at pH 74 at pretreated GCE Based on the real data

Based on the data obtained by HDA Koutecky-Levich plot is plotted as in Fig 92

204

0E+00

5E-02

1E-01

2E-01

2E-01

3E-01

-002 000 002 004 006 008 010 012 014 016 018

ωˉfrac12 (sˉ

frac12)

1 IK (A-1)

- 04 V

- 06 V- 08 V

05 x 10-1

10 x 10-1

15 x 10-1

0

20 x 10-1

25 x 10-1

-1

I d(A

-1)

Fig 92 Koutecky-Levich plot for O2 reduction at pretreated GCE in oxygenated 001 M

PBS at pH 74 with applied potential ndash 04 06 and 08 V Based on the real data

With Ik from Koutecky-Levich plot and nα from CV the rate constant for arbitrary

potential (k(E)) can be calculated using Eq 97

The rate constant for arbitrary potential E [30]

lowast=nFAC

Ik K

E )( middotmiddotmiddotmiddotmiddot Eq 97

k(E) = Rate constant for the applied potential E ms-1

( Please refer to Eq 94 Eq 95 and Eq 96 for other terms and symbols)

205

568 Finding the standard rate constant (kdeg)

Finding the standard rate constant (kdeg) for ORR of each catalyst is the ultimate

aim of this long tedious cathode analysis because kdeg tells the pure catalytic power

independent of reactant diffusion and overpotential (η) k(E) can be expressed in terms of

kdeg and η as in Eq 98 therefore it is possible to estimate kdeg with known η and k(E)

k(E) in terms of kdeg and η for a forward reaction [30 181]

⎥⎦⎤

⎢⎣⎡ minusminus

deg= )(exp 0)( EE

RTnFkk Ef

α

⎥⎦⎤

⎢⎣⎡ minus

deg= ηαRT

nFk )1(exp middotmiddotmiddotmiddotmiddot Eq 98

kf(E) = Rate constant for a reduction reaction at the applied potential E ms-1

kdeg = Standard rate constant ms-1

η = Overpotential V

( Please refer to Table 11 and Table 12 for other terms and symbols)

η can be found by subtracting Edegrsquo of the reaction involved from the applied potential in

HDA n obtained by HDV is used to specify the reaction involved hence its Edegrsquo In

order to estimate kdeg Eq 98 is first converted into logarithmic expression as in Eq 99

kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

ηαRT

nFkk Efminus

minusdeg= loglog )( middotmiddotmiddotmiddotmiddot Eq 99

206

Log(k(E)) is plotted against a range of η and by extrapolating the plot log(kdeg) is obtained

from its y-intersect where η = 0 hence kdeg can be calculated (Fig 93) [181]

-12

-10

-8

-6

-4

-2

0

-10 -08 -06 -04 -02 00 02 04

η V

log kf(E)

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS NaOH PBS NaOH H2SO4 PBS

NaOH H2SO4

NaOH H2SO4 PBSH2SO4 PBS

H2SO4

NaOH PBS

log kdeg

η V

Fig 93 Log kf(E) plot for O2 reduction at various pretreated GCEs Based on the real data

569 Summary of the electrochemical techniques

CV and hydrodynamic methods are complementary techniques for qualitative

examination of redox reactions and extraction of thermodynamic and kinetic parameters

The protocol for calculating kdeg from hydrodynamic methods and nα from CV is shown

in Fig 94

207

Fig 94 The protocol for finding kinetic and thermodynamic parameters

57 SUMMARY OF THE CHAPTER

The chemistry and electrochemistry of O2 has been reviewed The mechanisms of most

widely investigated catalysts noble metals macrocyclic complexes and enzymes have

been reviewed and their suitability for application in biofuel cells has been discussed

The key physico-chemical parameters which characterise these reactions have been

identified and methods for their determination have been described

208

6 CATHODE MATERIAL AND METHOD

61 ELECTRODE FABRICATION

611 Graphite PtC and RhC electrodes

Microcrystalline graphite of grade (2-15 micron 999995 purity) was

purchased from Alfa Aesar GmbH amp CoKG 5 wt-loaded rhodium on activated

charcoal (RhC) and platinum on activated carbon (PtC) were purchased from Sigma

Aldrich CY1301 and Aradur hardener HY 1300 were purchased from Robnor resins

and they were mixed at 31 (vv) for use RDE templates were specially designed and

made on site They were made of Teflon with the dimension 4-mm ID 20-mm OD

and 15-mm height The depth of composite-holding cavity for each RDE was 25 mm

The composite was made by mixing graphite RhC or PtC with epoxy resin at 73 by

hand Composite mixture was packed into five RDE templates and cured at room

temperature for 72 hours [106]

612 Anthraquinone(AQ)-carbon composite electrodes

6121 Derivatisation of graphite with AQ

Fast red AL salt with 25 dye content and hypophosphorous acid at 50 wt

in H2O were purchased from Sigma Aldrich HPLC grade acetonitrile was purchased

from VWR international 5 mM of Fast Red AL salt solution was prepared in 50 mM

209

hypophosphorous acid 10 ml of the Fast Red solution was mixed with 2 g of graphite

The mixture was left at 5 degC for 30 minutes with regular stirring at every 10 minutes It

was finally washed thoroughly with deionised water followed by vacuum suction in

acetonitrile flow The washed derivatised AQ-carbon was left to dry overnight It was

kept in the sealed container for future use [44]

6122 Fabrication of AQ-carbon composite electrode

AQ-graphite was mixed with epoxy resin at 64 by hand The composite was

packed into five RDE templates and cured at room temperature for 72 hours

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes

6131 Information on synthesis of metallosalophen

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminocobalt (II)] and

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminoiron (II)] denoted as Co(salophen)

and Fe(salophen) respectively were kindly donated by Dr Peter Cragg (School of

Pharmacy Brighton University) They were synthesised by dissolving metal acetate in

the ligand solution which was made by condensation of 21 molar ratio of

salicylaldehyde and diethylenediamine in ethanol or methanol [224 225]

210

6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes

Analytical grade dichloromethane was purchased from VWR international In

the presence of small quantity of dichloromethane 5 wt of Co(salophen) or

Fe(salophen) was mixed and homogenised with graphite [181] After the

dichloromethane evaporated the metallosalophen-graphite mixture was further mixed

with epoxy resin at 64 [219] by hand The composite was packed into five RDE

templates and cured at room temperature for 72 hours

62 COMPOSITE RDE CUTTING

The electrode discs of all the RDE composite electrodes were cut into 1

mm-thick sections using a Buehler IsoMet Low diamond saw with a 04 mm-thick

diamond blade

63 ELECTRODE POLISHING

631 Composite RDE polishing

Composite RDEs were polished successively with SiC grinding paper of 400

1200 and 2400 grit followed by with alumina oxide slurry of particle size 1 03 and

005 microm as in sect 33 on p 122 Electrode polishing was done just before the experiment

in order to avoid air oxidation [195]

211

Fig 95 Fabricated composite RDEs

632 Glassy carbon electrode polishing

Glassy carbon electrodes (GCE) (5-mm disc diameter) were purchased from

Pine Instruments Activated carbon (AC) was purchased from Chemviron Carbon GC

cleaning solvent mixture was made by mixing AC and HPLC grade acetonitrile in a

ratio of 13 (vv) The mixture was covered and left at least for 30 minutes then filtered

before use [226] GCEs were successively polished with alumina oxide powder slurry of

1- 03- and 005-microm particles on MicroCloth pad Polished GCEs were sonicated in

filtered ACacetonitrile mixture for 10 ndash 15 minutes to remove alumina powder debris

and contaminants [226] The GCEs were then sonicated in deoxygenated deionised

water Air contact of the electrode surface was strictly avoided [195]

212

64 ENZYME RDEs

641 Membrane preparation

Cellulose acetate dialysis tubing visking 3632 was purchased from Medicell

International Ltd Sodium carbonate (Na2CO3) was obtained from Sigma Aldrich

Dialysis membrane was cut into 10x10-mm2 pieces which were boiled in 1 wv

aqueous Na2CO3 solution for 10 minutes The membrane pieces were rinsed thoroughly

with deionised water and stored [61] in PBS in a sealed container at 4degC until its use

642 Membrane enzyme electrodes

Laccase (Lac) from Trametes versicolor gt20 unitsmg-1 and lyophilised

bilirubin oxidase (BOD) from Myrothecium verrucaria 15 ndash 65 units mg-1 were

purchased from Sigma Aldrich 4 mgml-1 of Lac and BOD solution was made with PBS

respectively BOD immediately dissolved in PBS while a vortex mixer was needed to

dissolve Lac In order to attach enzyme on the GCE surface the tip of polished cleaned

GCEs was dipped and left in the enzyme solution for two hours with gentle stirring The

electrodes were then lightly washed with deionised water Pretreated dialysis membrane

was carefully placed onto the surface of the enzyme-modified GCE and fixed with

rubber O-ring

213

65 ELECTROCHEMICAL EXPERIMENTS

651 Electrochemical system

CHI 650A with single-channel and CHI 1030A with multi-channel

potentiostats were used for electrode pretreatment and electrochemical experiments CV

HDV and HAD An adjustable rotor from Pine Instrument was used to rotate a RDE

Three-electrode system (sect 342 on p 123) was employed with an AgCl reference and a

Pt counter electrodes

Fig 96 RDE experiment setting with a rotor and gas inlet

214

652 Electrode pretreatments

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes

Graphite GC Co(salophen) and Fe(salophen) electrodes were pretreated in

seven different ways in Table 14 Untreated electrodes were also prepared as controls

All the pretreatments were made in series

Treatment type 1st pretreatment 2nd pretreatment 3rd pretreatment

Type I Untreated -

Type II Acidic 01 M H2SO4

Type III Basic 01 M NaOH

Type IV Neutral 001 M PBS

Type V Base followed by Acid 01 M NaOH 01 M H2SO4

Type VI Acid followed by Neutral 01 M H2SO4 001 M PBS

Type VII Base followed by Neutral 01 M NaOH 001 M PBS

Type VIII Base Acid then Neutral 01 M H2SO4 01 M NaOH 001 M PBS

Table 14 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Concentrated sulphuric acid at 90 - 98 and sodium hydroxide tablets were purchased

from VWR international 001 M PBS was purchased from Sigma Aldrich All the

pretreatments were based on potential cycling in 100-ml electrochemical cell except

untreated ones Acidic pretreatment was performed at 5 Vs-1 between 15 V and ndash 05 V

215

in 01 M H2SO4 [105 219] for 30 minutes Basic pretreatment was performed at 5 Vs-1

between 06 V and 2 V in 01 M NaOH [218 219] for 30 minutes Neutral pretreatment

was done at 5 Vs-1 between 15 V and ndash 05 V in deoxygenated 001 M PBS pH 74

[219 220] Constant N2 flow was applied to PBS through the pretreatment In addition

pretreatments were performed in various combinations as in Table 14 Type V Base

followed by Acid Type VI Acid followed by Neutral Type VII Base followed by

Neutral and Type VIII Base followed by Acid then Neutral [219] The influence of

pretreatments on electrode surface and ORR was studied by CV HDV and HDA

6522 Pretreatments of AQ-carbon PtC and RhC

AQ-carbon PtC and RhC composite electrodes were pretreated only in 01 M

H2SO4 at 5 Vs-1 between 15 V and ndash 05 V in for 30 minutes Untreated electrodes were

also prepared as controls

653 Electrochemical experiments

All experiments were carried out under both N2 and O2 saturation 100 ml of

001 M PBS electrolyte solution was sparged with either N2 or O2 for 15 minutes before

the experiment Gas was continuously supplied through a Dreschel gas washing bottle

containing PBS throughout the experiment

CV was performed at 002 005 01 02 and 05 Vs-1 between + 09 V and ndash

09 V HDV was performed at fixed scan rate of 005 Vs-1 sweeping the potential from

+ 09 V to ndash 09 V with disc rotation velocity altered through 900 1600 and 2116 rpm

216

HDA was performed at a range of fixed potentials - 02 - 04 - 06 and ndash 08 V at disc

rotation velocity of 400 625 900 1156 1225 1600 2116 and 2500 rpm All

experiments were repeated twice or three times The sample size ranges from one to

three The median values were taken for the analysis Obtaining a set of data for each

electrode sample took from three weeks to more than one month

654 Data analysis

CV data was used to calculate nα and reaction reversibility HDV data was used

to calculate n using Levich equation (Eq 30 on p 61) Edegrsquo for ORR involved at pH 7

25 degC was specified by n value as in Table 15

n Redox type Redox potential vs AgAgCl

1 Edegrsquo(O2H2O2) 0082 V

2 Edegrsquo(O2H2O) 0616 V

Table 15 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC [24]

η was calculated by subtracting Edegrsquo (ORR) from the potential applied in HDA HDA

data was used to estimate kf(E) at each applied potential using Koutecky-Levich plot (Eq

30 on p 61 and Fig 92 on p 204) N2 data was subtracted from O2 data as background

Other parameters required for Koutecky-Levich plot were set as in Table 16 All

parameters assume the condition of 001 M PBS at 24 degC

217

Parameter Value Reference

O2 diffusion coefficient 23 x 10-9 m2s-1 [227]

O2 concentration 123 molm-3 [158]

Kinematic viscosity (ν) 884 x 10-7 m2s-1 [30]

Table 16 Parameter values for Koutecky-Levich equation

kdeg was estimated by plotting log(kf(E)) vs η (Eq 99 and Fig 93 on p 206) kdeg for

various and pretreated cathodes were compared The best catalyst shows the largest kdeg

The procedure for finding kdeg is summarised in (Fig 94 on p 207)

218

7 CATHODE RESULTS AND DISCUSSION

71 AIMS OF THE CATHODE STUDIES

This section describes the electrochemical characterisation of various modified

electrodes which were considered as candidate cathodes for the biofuel cells Various

carbons were used as electrode materials in all cases The electrochemical behaviour of

carbon depends strongly on history and also can be radically altered by pre-treatment

The effects of electrochemical pretreatments (ECPs) on the kinetic parameters will be

described for both glassy carbon and graphite composites The cathode modifiers

considered in this section include noble metals (Pt Rh) inorganic Schiff bases

(Co(salophen) Fe(salophen)) an organic modifier (anthroquinone) and the enzymes

bilirubin oxidase (BOD) and laccase (Lac) In each case of non-biological catalysts the

effects of ECPs were also quantified using cyclic voltammetry (CV) and hydrodynamic

methods with rotating disc electrodes (RDEs)

CV allowed the investigation of

Capacitance from the double layer region

Qualitative description of the mechanism such as number of discrete reaction

steps reversibility and surface confinement

nα the number of electrons transferred up to and including the rate limiting step

(RLS)

219

while hydrodynamic methods were used to quantify

n the total number of electrons transferred

kdeg the heterogeneous standard rate constant

Epc2(HDV) the half-wave reduction potential in HDV

The data was taken under both N2 and O2 to allow characterisation of the effects of

electrochemical pretreatment and its effects on the kinetics of O2 reduction The best

candidate catalytic cathode was determined according to kdeg nα and Epc2(HDV)

72 INFLUENCE AND EFFICACY OF ECPS

721 Glassy carbon electrodes (GCEs)

7211 Untreated GCEs

Voltammetry curves of untreated GCEs at pH 74 in the presence of N2 and O2

are shown in Fig 97

220

Fig 97 CVs of untreated GCEs at pH 74 in 001 M PBS in the presence of (i) N2 (ii)

O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Untreated GCEs did not show any peak in the absence of O2 (Fig 97(i)) indicating that

there was no electrochemically active redox species on the electrode surface and in the

solution CV was in the presence of O2 (Fig 97(ii)) showed a peak at ndash 08 V but

without a reverse anodic current This indicates the presence of catalytic activity

however it occurred at only high overpotential and is therefore not efficient for ORR

This result is consistent with previous work [215 216]

7212 Influence of electrochemical pretreatments on GCE

GCEs were pretreated in various media in order to improve ORR efficiency It

has been reported that ECPs activate the electrode surface by introducing

electrochemically active functional groups and that this improves electrochemical

(i) N2

-3E-05

-2E-05

-1E-05

0E+00

1E-05

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

221

reversibility and [105 218] and the electron transfer rate of inner-sphere catalysis [228]

Fig 82 shows the voltammetry curves in the absence of O2 for various pretreated GCEs

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-60E-05

-50E-05

-40E-05

-30E-05

-20E-05

-10E-05

00E+00

10E-05

20E-05

30E-05

40E-05

50E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH

Fig 98 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of

O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1 Arrows indicate the

peak position (i) includes CVs for all the pretreated GCEs and (ii) excludes

PBS pretreatments for convenience

222

As the figure shows any pretreatment resulted in an increase in capacitance and

introduced characteristic voltammetric peaks which were not observed in the untreated

GCEs In all cases the peak current was linearly proportional to υ indicating that the

pretreatments led to surface-confined redox active functional groups most likely to be

quinone groups That ECPs give rise to surface quinones is well established [105 218

220 229 230] The Epc shifted with υ and was separated from Epa hence redox

reactions by these surface quinone groups are quasi-reversible The effect of

pretreatments however varied by the type of medium used in pretreatments

Above all PBS-related pretreatments gave qualitatively different results which

were characterised by a great increase in average capacitance calculated from CVs in

001 M PBS at pH 74 at 50 100 and 200 mVs-1 in the double layer region of the CV at

04 V Summary results are shown in Fig 82(i) and Fig 99

223

GCE Capacitance vs Pretreatment

0100200300400500600700800900

1000

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS

NaOH PBS

NaOH H2SO4

PBS

Pretreatment

Capa

cita

nce

microF

cm-2

Fig 99 The capacitance of GCEs in 015 M NaCl buffered with 001 M phosphate to

pH 74 Error bars represent the standard deviation (n = 3)

The average capacitance of the untreated GCEs was 76 microFcm-1 and is within the typical

ranges reported hitherto [221] while PBS pretreatment increased the capacitance to 700

- 800 microFcm-1 A similar phenomenon was reported by Nagaoka and by Swain [215

216] There are two possible reasons for this (i) PBS treatment caused the

microfractures of GC and increased the electrode area and therefore double layer [105

215 216 218] andor (ii) attached the electrochemically inactive charged surface group

such as phenol and carboxylate [216 229]

In the pretreatment involving NaOH the carbon dissolution was observed This

is harsher pretreatment so erodes the electrode and makes the electrode surface more

porous [216 218] NaOH also forms unstable oxide film and eventually removes

graphite oxide [229] and surface impurities [218] However the resultant capacitance

224

was unexpectedly smaller than that of sole PBS pretreatment Therefore NaOH

pretreatment increased the capacitance by increasing the exposed electrode area [218

231] while PBS pretreatment increased capacitance by attaching redox inactive charged

surface groups The largest capacitance after the double pretreatment involving NaOH

and PBS was therefore derived from the layer of charged groups attached on the

electrode surface area made larger by NaOH pretreatment

H2SO4 pretreatment on the other hand resulted in a smallest capacitance

increase The electrode behaviour was also slightly altered from that of other

PBS-related pretreatments when the pretreatments involved H2SO4 together The

capacitance became smaller as shown in Fig 99 and the peak positions were shifted to

more positive potential side (Fig 82(i)) To investigate the causes of this phenomena

the voltammograms during such pretreatments were studied as Fig 100 below

225

Fig 100 Overlaid CVs during various pretreatments for GCEs involving 01 M H2SO4

alone (red) 001 M PBS at pH 74 alone (green) and both pretreatments (pink)

It is clearly shown that either pretreatment resulted in distinctive peaks for the

attachment of active surface quinone groups However the peak potentials were

different when in sole H2SO4 or PBS-related pretreatments This is because as proposed

by Nagaoka those quinone groups were present in the different chemical environment

leading to different behaviours [216] H2SO4 pretreatment attached fewer inactive

surface oxygen groups while PBS pretreatment attached more Therefore H2SO4

pretreatment led to the smaller capacitance change than PBS pretreatment This implies

the presence of different electrode surface conditions hence quinones present in such

different environment behave differently showing different peak positions The

behaviours of surface quinone groups depends on the conditions of the pretreatment

involved [229 230 232] This fact can also explain the phenomena caused by the

226

double pretreatment with H2SO4 and PBS as shown in Fig 82(i) More quinone groups

had been attached by H2SO4 pretreatment in advance therefore only smaller area than

usual became available for PBS pretreatment to stably attach their charged oxygen

surface groups This is the most likely causes of the reduced capacitance and different

peak positions in such a double pretreatment compared to other PBS-related

pretreatments Furthermore quinone peak positions should be close to those found for

the H2SO4 pretreatment alone because quinone groups attached by preceding H2SO4

pretreatment dominate the electrode surface and should be have more influence on the

electrochemistry However they also resulted in larger capacitance because the

subsequent PBS pretreatment attached inactive oxygen surface group which led to an

increase in fixed charge density This is a typical phenomenon of synergetic effects as

already seen in the double pretreatment with NaOH and PBS

On the other hand voltammetric curves for the double pretreatment of NaOH

and H2SO4 did resemble to those by sole NaOH pretreatment Similar phenomena were

observed between the double pretreatment with NaOH and PBS and the triple

pretreatment with NaOH H2SO4 and PBS It seems that preceding NaOH pretreatment

overwhelmed the subsequent H2SO4 pretreatment although some influence by H2SO4

can be observed in terms of capacitance as shown in Fig 99 This implies that the

preceding NaOH pretreatment made most of the electrode area not available for

subsequent H2SO4 pretreatment However if NaOH pretreatment creates micropores in

the GC protons from subsequent H2SO4 pretreatment can still penetrate such

micropores These micropores are accessible for protons but not most ions [216]

227

7213 Effect of GCE pretreatment on ORR

Voltammograms for ORR at various pretreated GCEs at pH 74 are shown in

Fig 101 below

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO4

01M NaOH01M NaOH + 01M H2SO4

001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS

01M NaOH + 01 M H2SO4 + 001M PBS

Fig 101 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

In the figure any pretreated GCE shows larger cathode peaks between ndash 03 and ndash 05 V

without reverse anodic current They were due to O2 reduction by surface quinone

groups whose peaks were observed at ndash 01 V in Fig 82(i) in the absence of O2 This

indicates that ORR at any pretreated GCEs is regulated by ECrsquo mechanism [30] Ipc was

linearly proportional to radicυ in all cases indicating the diffusion-limited charge transfer

228

However untreated GCE was catalytic only at high overpotential since Epc2(HDV) = ndash 08

V This is compatible to the observation made by Tammeveski [233] It was seldom

catalytic since n = 102 plusmn 011 and nα = 059 plusmn 004 This single-electron ORR implies

the presence of O2־ disproportionation from which little power can be extracted [233]

kdeg was could not be calculated because the standard Koutecky-Levich plot could not be

obtained since it did not show a normal linear slope In contrrast all the other

pretreatments improved ORR as seen in Fig 101 They showed large cathodic peaks at

smaller overpotentials PBS-related pretreatments in particular increased the catalytic

current by gt 15 times and positively shifted Epc2(HDV) by gt 300 mV relative to that of

untreated GCEs Similar phenomena were reported elsewhere [220 234] All the GCE

pretreatments resulted in an increase in both nα and n The increase in kdeg accompanied

by an increase in nα was also observed as a general tendency as shown in Fig 102

229

0

2

4

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

kordm

ms

-1

nα 059 107 094 106 168 278 174 178

kordm ms-1 000E+00 715E-09 529E-09 631E-08 131E-07 279E-07 654E-08 113E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

- nα

kdeg ms-1

Untreated H2SO4 NaOHNaOHH2SO4

H2SO4PBS

NaOHPBS

NaOHH2SO4PBS

Fig 102 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Above all the H2SO4-PBS double pretreatment was most effective giving kdeg = 279 x

10-7 plusmn 507 x 10-9 ms-1 and Epc2(HDV) = - 039 plusmn 002 V However its nα was calculated

to be 278 while n = 185 This is an unusual result It might imply the presence of serial

ORR pathway in which H2O2 is further reduced or it is simply impossible to obtain

correct parameters by using Eq 88 on p 193 for ORR at pretreated GCEs due to their

excessively narrow peak width Pretreatment of GCE was proved to increase kdeg of ORR

but how this improves its catalysis has been uncertain for a long time between two

hypotheses whether they mediate electron transfer or they alter the adsorption of O2

and O2[233 232 220] ־ The latter or both is more likely because as Fig 102 shows

PBS-related pretreatments increased the capacitance which was derived from redox

inactive surface oxygen groups yet made significant contribution to an increase in kdeg It

230

is therefore natural to deduce that the largest kdeg and nα were the consequence of

synergetic effect derived from different surface oxygen groups attached by H2SO4 and

PBS

722 Graphite composite electrodes

7221 Untreated graphite composite electrodes

Graphite composite electrodes are different from GCEs They are made of

graphite particles which are more porous and permeable to the solution and gas This

practically results in larger electrode surface area therefore becomes more subject to

facile adsorption and surface modification This leads to the different surface conditions

and electrode behaviours from GCEs as proposed by Sljukic [232] CVs of untreated

graphite composite electrodes at pH 74 in the presence of N2 and O2 are shown in Fig

103 together with those of untreated GCEs

231

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

Fig 103 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl Small peaks on graphite electrodes are indicated by arrows

Different electrode behaviour is noticeable especially in nitrogenated solution because

untreated graphite composite electrodes showed small peaks at + 100 mV and - 100 mV

as indicated by arrows No peak was present fo untreated GCE This indicates the

presence of some reactive surface quinone groups on graphite composite without

pretreatment This is because graphite composite electrodes have more reactive edge

plane graphite exposed and the porous electrode structure allows larger electrode area to

be exposed There was however little difference in averaged capacitance between

graphite composite and GCEs This indicates that an increase in capacitance does not

necessarily correlate to an increase in the surface area as proposed by Regisser [235]

Voltsmmograms for ORR at untreated GC and graphite composite electrodes resembles

each other though for the latter the reduction peak is around 50 mV more positive It can

232

be seen that graphite composites are slightly more catalytic toward ORR presumably

because of the intrinsic quinone groups derived from the graphite electrode surface

7222 Influence of electrochemical pretreatments on graphite composite electrodes

Cvs for the graphite composite electrodes differed from those of GCEs Peaks

were observed in all graphite cases including untreated ones as shown in Fig 104

233

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

(ii)

01M H2SO401M NaOH01M NaOH + 01M H2SO4Untreated

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBSPBS

(iii)01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS001M PBS

Fig 104 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M

nitrogen-sparged PBS between + 09 and - 09 V vs AgAgCl (i) All

pretreatments at 005 Vs-1 (ii) Non-PBS-related pretreatment and (iii)

PBS-related-pretreatment CVs at 002 Vs-1

The capacitance became larger after pretreatments Any multi-pretreatment involving

NaOH and H2SO4 resulted in the largest capacitance which was 20 times larger than

234

that of untreated graphite composite electrodes The capacitance increase was also

larger than that of any pretreated GCEs except for PBS-pretreated ones PBS

pretreatment led to the smallest capacitance increase in graphite composite electrodes in

contrast to the GCEs Instead H2SO4 pretreatment led to the largest capacitance

increase Moreover this outcome was found to correspond to a specific proportional

relation between Ip and υ Ip of electrodes involving any pretreatment in PBS NaOH or

their combinstions were linearly proportional to radicυ This can only arise from on or more

diffusion limited step or steps in the redox reactions of the surface groups most likely

proton diffusion though a porus film or to microscopic porous environments Surface

oxygen groups have previously been reported to involve involve proton uptake and this

leads to a broad peak [216 230] H2SO4-pretreated electrodes showed Ip proportional to

υ and the redox reaction is independent of proton diffusion

These phenomena show that different pretreatments provide different chemical

environment for active quinone groups leading to different quinone behaviours and

voltammetry peaks [229 230 232] In the multi-pretreatment with NaOH and H2SO4

synergetic effects became obvious Its redox reaction was diffusion-limited and showed

voltammograms with two partially resolved sets of peaks yet its peak still partially

overlaps with that of diffusion-independent H2SO4-pretreated electrodes as seen in Fig

104(i) When this double-pretreatment was further followed by PBS pretreatment the

peak derived from quinones attached by H2SO4 disappeared This implies that

subsequent PBS pretreatment suppressed the activity of proton-independent quinones

and the effect by H2SO4 The similar phenomenon was observed when electrodes were

pretreated doubly by H2SO4 and PBS (Fig 104(ii)) Every case presented

235

quasi-reversible redox systems because peak potentials shifted with scan rates Small

multiple peaks were present in slower scan rates merged into one broad peak at faster

scan rate as the contribution of capacitance charging and increased iR drop obscured

voltammetic detail

7223 The effect of graphite composite electrode pretreatment on ORR

ORR at pretreated graphite composite electrodes was found to be less effective

than for pretreated GCEs Only NaOH-related pretreatments were effective (Fig 105)

enough to achieve two-electron ORR Untreated graphite composite electrodes were

catalytic only at high overpotential despite the presence of surface quinone groups PBS

pretreatment was hardly effective in contrast to the results obtained for GCEs

Electrochemical pre=treatment in H2SO4 and its double pretreatment with PBS reduced

Epc2(HDV) by 40 ndash 90 mV however the electrodes showed poor catalytic performance nα

lt 05 and n lt 2 consistent with the initial electron transfer still being rate determining

236

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 105 Overlaid CVs of ORR at various pretreated graphite composite electrodes

at pH 74 in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005

Vs-1

Pretreatment of the graphite composites with just H2SO4 showed a single merged peak

at slower scan rate while they showed an addition prewave as indicated by arrows in

Fig 106 at faster scan rate

237

-16E-03

-14E-03

-12E-03

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

-1 -08 -06 -04 -02 0 02

E V

J Acm-2

50 mV100 mV200 mV500 mV

Fig 106 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at

pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various

scan rates 50 100 200 and 500 mVs-1

Coupled with the fact of n asymp 1 this prewave is due to the rate-limiting slow formation of

semiquinone radicals [233] A semiquinone radical is formed when one electron is

transferred to a quinone group and its formation is necessary to mediate ORR as shown

in Eq 100

238

middotmiddotmiddotmiddotmiddot Eq 100

[233] middotmiddotmiddotmiddotmiddot Eq 101

Q = Surface quinone group

O2־ = Semiquinone radical

Any NaOH-related pretreatment on the other hand showed a prewave around ndash 035 V

at slower scan rate as shown in Fig 107 but this merged into a broad peak at faster scan

rate

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

01M NaOH01M NaOH + 01M H2SO4 01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 107 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

239

Ipc was linearly proportional to radicυ except for the triple pretreatment which was difficult

to define due to the ambiguous peak positions over different scan rates The prewave

was due to the slow reduction of O2 into O2־ (Eq 101) rather than semiquinone radical

formation (Eq 100) as seen in H2SO4-pretreated electrodes They had n asymp 2 and nα asymp 1

hence they catalysed two-electron ORR Those pretreatments positively shifted

Epc2(HDV) by 173 plusmn 26 mV on average from ndash 730 mV at the untreated graphite

composite electrodes The largest catalytic activity was achieved by doubly pretreated

electrodes with NaOH and H2SO4 with kdeg = 382 x 10-7 plusmn 32 x 10-8 ms-1 (Fig 108)

0

2

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

35E-07

40E-07

45E-07

kordm

ms

-1

nα 042 035 080 114 045 040 101 072

kordm ms-1 000E+00 000E+00 233E-07 382E-07 000E+00 000E+00 132E-07 172E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBSUntreated H2SO4 NaOH

NaOHH2SO4

H2SO4

PBS NaOHPBS

NaOHH2SO4

PBS

- - - - nα

kdeg ms-1

Fig 108 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

240

723 Platinum on carbon composite electrodes (PtC)

Noble metals are popular ORR catalysts [172 173] with Pt showing the highest

catalytic power [165 166 171 172] It was reported that Pt has a 98 O2-to-H2O

conversion rate [174] However for PtC composite electrodes the voltammetry was

dominated by showed solution resistance both in the presence and absence of O2 and

therefore were not either catalytic or conductive H2SO4 pretreatment was not at all

effective This is probably due to the large electronic resistance of activated carbon itself

[204] as well as intrinsic resistance of composite conductors

724 Rhodium on carbon composite electrodes (RhC)

Rhodium is a platinum group metal and has been reported as a good ORR

catalysis after Pt and Ir [172] Voltammograms for untreated RhC composite electrodes

only showed the capacitance but no redox peaks in nitrogenated solution The

capacitance decreased by 80 for every ten-fold increase in the scan rate Non-faradic

current is normally linearly proportional to υ however it exponentially decreased with υ

in the untreated RhC composite electrodes It did not show any peak in the presence of

O2 and was not catalytic toward ORR (Fig 110(i)) H2SO4-pretreated RhC composite

also did not show any voltammetric peaks Its capacitance was roughly x30 larger than

that of untreated electrodes and again exponentially decreased with an increase in υ

ORR catalysis was however present (Fig 110(ii)) therefore it is reasonable to deduce

that H2SO4 attached the surface quinone groups but the large capacitance charging

prevented voltammetic observation of quinones

241

(i) Untreated

-16E-03

-12E-03

-80E-04

-40E-04

00E+00

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

N2O2

(ii) H2SO4

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

10E-03

-1 -08 -06 -04 -02 0 02 04 06 08 1

E VJ

Ac

m-2

N2O2

Fig 109 CVs of (i) untreated and (ii) H2SO4-pretreated RhC composite electrodes at

pH 74 in 001 M PBS in the presence of N2 and O2 at 005 Vs-1 between + 09

and -09 V vs AgAgCl

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR

Untreated RhC composite electrodes did not show ORR catalysis however a

steep decrease in cathodic current was observed without any peak as the potential

decreases This steep current decrease was probably caused by O2 adsorption on the Rh

particle surface H2SO4-treated RhC on the other hand was found to be catalytic with n

= 177 and nα = 068 plusmn 011 A reduction prewave was observed at around ndash 05 V and

this merged into a broad peak at faster scan rates This prewave was due to ORR by

surface quinone groups attached by H2SO4 because it overlaps with the peak observed

in H2SO4-treated graphite composite electrodes as shown in (Fig 110) This prewave

242

therefore indicates the slow first electron transfer to form O2־

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

-1 -05 0 05 1

E V

J Acm-2

RhC H2SO4

Graphite H2SO4

Fig 110 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

O2־ was probably further reduced to H2O2 by Rh particles because n = 177 plusmn 011 The

catalytic activity was much larger compared with H2SO4-pretreated graphite composite

electrodes as shown in Fig 110 with kdeg =257 x 10-7 plusmn 230 x 10-8 ms-1

243

725 Co(salophen) composite electrodes

7251 Untreated Co(salophen) composite electrodes

Untreated Co(salophen) electrode showed couples of distinctive peaks Epc(1) =

053 Epc(2) = -024 Epa(3) = -016 and Epa(4) = 016 V as shown in Fig 111(i) These

peaks correspond to stepwise cobalt redox reactions Co3+2+ and Co2+1+ respectively

The peak potentials hardly changed over different scan rates consistent with previously

published results [181] Similar peaks have been observed in deoxygenated organic

solvents [174 181] as well as for Co(salen) [181]

(i) N2

-2E-05

-1E-05

0E+00

1E-05

2E-05

3E-05

4E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

2E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fig 111 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

244

In the presence of O2 it showed a reduction peak at ndash 04 V as shown in Fig 111(ii)

ORR potential was shifted by approximately 300 mV relative to that of untreated

graphite composite This result reasonably compatible to the result reported by Ortiz

[181] At this potential O2 forms an adduct with Co2+(salophen) and then is reduced to

O2־ while Co2+ undergoes one-electron oxidation to Co3+ [170] To be catalytic Co3+

formed here must be reduced back to Co2+ to reduce O2־ further into H2O2 [181] or a

binuclear complex must be formed with another Co2+ [170] Untreated Co(salophen)

composite electrodes were however not strongly catalytic due to n = 138 plusmn 009 and nα

= 055 plusmn 012 Ip was linearly proportional to radicυ and the peak potentials Epc(2) and Epa(3)

varied only by plusmn 8 over different five scan rates This indicates nearly reversible

multi-electron transfer redox system of Co(salophen) composite electrodes

7252 Influence of electrochemical pretreatments on Co(salophen) composite

There was a particular redox pattern found in voltammetry waves according to

the type of pretreatments (Fig 112)

245

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 112 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in

001 M nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

NaOH-pretreatment showed similar voltammetric faradaic features as those in untreated

electrodes (Fig 112) but increased the capacitance by six times relative to that of

untreated electrodes The size of capacitance was 382 microFcm-1 and approximately equal

to that of NaOH-pretreated graphite composite electrodes This implies that NaOH

attached electrochemically inactive surface oxygen groups and the attachment of active

surface quinone groups were not clearly confirmed

PBS-pretreatment on the other hand attached quinone groups because Epa asymp 04 V at

which similar peaks were observed in PBS-pretreated graphite composite (Fig 104(iii)

p 233) Their cathodic peaks were however broader because they merged into peaks

246

for Co-redox processes The effect of PBS pretreatment was much smaller compared to

that of H2SO4-related pretreatment in the deoxygenated solution Single NaOH

treatment and any PBS-related pretreatment showed Ip linearly proportionally to radicυ

while any H2SO4-related pretreatment but without involving PBS-pretreatment showed

a proportional relation with υ This indicates that H2SO4 and PBS or NaOH

pretreatments lead to different surface redox mechanism Peaks due to H2SO4-related

pretreatments were prominent and are indicated by a red circle in Fig 112

7253 The effect of Co(salophen) composite electrode pretreatment on ORR

Untreated electrodes were not catalytic Parameters for H2SO4-pretreated

Co(salophen) composite found to have n asymp 1 and nα le 1 although they look catalytic in

Fig 113 reducing its ORR overpotential by 400 ndash 500 mV compared to GCEs This

inconsistent result arose because it was impossible to find the correct parameter using

the conventional calculation using Eq 88 on p 193 and need more sophisticated

method such as a simulation probramme The other pretreatments were on the other

hand found strikingly effective toward ORR as shown in Fig 113

247

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

40E-04

60E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 113 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Such pretreated Co(salophen) electrodes showed Epc asymp - 03 V having reduced the

overpotential against unpretreated Co(salophen) by 100 mV and 300 mV against

untreated graphite composite electrodes ORR was synergistically catalysed by Co2+ and

surface semiquinone radicals [181 233] They showed n asymp 2 and nα gt 07 hence they

catalysed two-electron ORR Ipc was linearly proportional to radicυ hence the redox

reactions were diffusion limited except for the double pretreatment with NaOH and

H2SO4 Large catalytic activity was achieved by sole NaOH and by the double

pretreatment with NaOH and H2SO4 or the combination of H2SO4 and PBS Among

them sole NaOH pretreatment was revealed to be most effective as shown in Fig 114

and showed kdeg = 119 x 10-5 plusmn 118 x 10-6 nα = 083 plusmn 014 and Epc2(HDV) = 037 V

248

0

1

2

nα

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

kordm

ms

-1

nα 055 028 083 074 068 091 071 078

kordm ms-1 000E+00 000E+00 119E-05 747E-06 271E-06 684E-06 296E-06 587E-06

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

Fig 114 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite

electrodes

One possible reason for this largest catalytic activity by NaOH pretreatment is that

NaOH might attach more reductive surface OH groups [218] which became involved in

reducing back Co3+ into Co2+ during ORR [181]

726 Fe(salophen) composite electrodes

7261 Untreated Fe(salophen) composite electrodes

Untreated Fe(salophen) electrodes showed a couple of distinctive peaks Epa = -

024 plusmn 001 V and Epc = - 036 plusmn 001 V These peaks are due to Fe3+2+ redox reaction as

249

shown in Fig 115(i) The voltammetry curve and redox peaks are reasonably similar to

those of Fe(salen) reported [224]

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

(ii) O2

-6E-04

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

Fig 115 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74

in 001 M PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and

- 09 V vs AgAgCl

However the redox system is not perfectly reversible here because ∆Ep slightly

increases with an increase in the scan rate and its Ip was linearly proportional to radicυ It is

therefore a quasi-reversible system Untreated Fe(salophen) electrodes was not as

catalytic as Co(salophen) electrodes Epc was ndash 079 plusmn 007 V and little different from

that of untreated graphite composite electrode as shown in Fig 115(ii) although Ipc was

slightly bigger The low ORR catalytic activity of Fe(salophen) was due to the low ORR

rate of Fe itself (Fig 65 on p 164) [172]

250

7262 Influence of electrochemical pretreatments on Fe(salophen) composite

The effect of pretreatments to Fe(salophen) was striking They resulted in

additional voltammetric features due to the Fe redox peaks at Epc (Fe) = - 03 V and Epa

(Fe) = - 02 V and by attaching the surface quinone groups at Epc (Q) = 015 V and Epa (Q) =

02 V as shown in Fig 116(i)

251

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

60E-05

80E-05

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

Untreated01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS001M PBS

-1E-04

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

1E-04

1E-04

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

Fig 116 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1 (i) All

pretreatments (ii) Non-PBS-related pretreatments and (iii)

PBS-related-pretreatments CVs

Non-PBS-related pretreatments (Fig 116(ii)) showed redox peaks clearly however the

252

two cathodic peaks merged into a broad peak at faster scan rate than 100 mVs-1

indicating quasi-reversible surface quinone redox reactions Only a broad merged

reduction peak was observed for all PBS-related pretreatments (Fig 116(iii)) at all scan

rates

Any PBS-related pretreatments resulted led to a broad cathodic peak (from + 01 V to

approximately ndash 03 V) made up of both Fesalphen reduction and reduction of surface

confined quinones arising from the electrochemical pretreatment PBS pretreatment

always greatly increases the surface area leading to micro fissures and consequently a

greater variety of microenvironments for electrochemically generated quinone

functionality

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR

Most were found to be only weakly catalytic (Fig 117) since either n or nα or

both of them were lt 1 Only double-pretreatment with NaOH and H2SO4 led to

sufficient ORR catalysis with n = 185 plusmn 017 nα = 079 plusmn 023 kdeg = 112 x 10-7 plusmn 771 x

10-9 ms-1 and Epc2(HDV) = - 057 V The double pretreatment showed a prewave at ndash 035

V and it disappeared at faster scan rate indicating a slow single-electron transfer to O2

Ipc was linearly proportional to radicυ hence the catalysis was diffusion limited

253

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS

Fig 117 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

727 Anthraquinone (AQ) composite electrodes

Anthraquinone-grafted graphite electrodes were pretreated with H2SO4 Both

untreated and H2SO4-pretreated electrodes showed couples of peaks (Fig 118(i)) one of

which Epc(AQ) was compatible with Em = - 049 reported by McCreery [44] These

peaks were therefore derived from anthraquinone chemically attached to the graphite

254

(i) N2

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

-1 -05 0 05 1

E V

J A

cm

-2

Untreated FRE01M H2SO4 FREUntreated Graphite

(ii) O2

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

-1 -05 0 05 1

E VJ

Ac

m-2

Untreated FRE01M H2SO4 FREUntreated Graphite

Fig 118 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl CVs of untreated graphite electrodes are also shown together

The other couple of peaks Epc(SQ) and Epa(SQ) was observed at - 003 plusmn 003 V and +

020 plusmn 003 V respectively They were due to quinone groups on the carbon surface

H2SO4-pretreatment did not alter the voltammogram pattern but only increased the

capacitance by x3 and enhanced the peak current Both electrodes showed Ip linearly

proportional to radicυ hence the redox reactions involve proton diffusion ORR by those

electrodes is shown in Fig 118(ii) together with the voltammogram of graphite

composite electrodes Both untreated and pretreated AQ-grafted composite electrodes

positively shifted Epc by 60 and 230 mV respectively relative to that of untreated

graphite composite electrodes However their voltammetric curves resembled those for

untreated graphite composite electrodes Besides this their Ip dropped by 20 relative

255

to untreated graphite composite electrodes and showed n asymp 1 and nα asymp 05 Therefore

either was not catalytic in this study This outcome does not correspond to the finding

by Manisankar who confirmed the presence of ORR catalysis at AQ-carbon paste

electrodes at pH 7 with a reduction prewave at ndash 04 and a peak at ndash 07 V [193] To the

contrary this result fits to the data presented by Schiffrin who observed ORR

suppression at AQGC electrodes at pH lt 7 He reported that voltammetry curves for

ORR at GC and GCAQ became similar at this pH hence implied the possibility of

disproportionation of semiquinone intermediates [194]

728 Lac and BOD enzyme membrane electrodes

In the absence of O2 neither Lac nor BOD showed any peaks in their cyclic

voltammograms However they showed ORR catalytic activity even in the absence of

mediator (Fig 119) DET is therefore possible as already confirmed by many [183

186-188] Carbon surface groups [195] also might mediate electron transfer [188]

256

-14E-04

-12E-04

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

-1 -09 -08 -07 -06 -05 -04 -03 -02 -01 0

E V

J Acm -2

LacBOD

Fig 119 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated

PBS at 005 Vs-1 between 0 and -09 V vs AgAgCl

Ipc was linearly proportional to radicυ therefore ORR was diffusion limited Epc were -054

plusmn 002 and ndash 067 plusmn 006 V at Lac and BOD respectively They reduced ORR

overpotential by 120 and 250 mV relative to that of GCEs however the Epc shifted with

υ hence they were quasi-reversible It is known that MCOs catalyse four-electron ORR

[175 184 236] however n = 005 and nα asymp 1 in this study This is probably due to the

difficulty in electrochemical measurement of enzyme catalysis It is however possible

to find nα because the rate-limiting step is the single electron transfer between the

electrode and the blue copper atom of the enzyme Assuming this kdeg was calculated for

four-electron ORR at the enzyme electrodes and the result is shown in Fig 120

257

0

1

2

nα

00E+00

40E-09

80E-09

12E-08

16E-08

20E-08

kordm

ms

-1

nα 077 081

kordm ms-1 134E-08 168E-09

Lac Bod

Fig 120 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Lac had kdeg = 134 x 10-8 plusmn 251 x 10-9 while BOD had kdeg = 168 x 10-9 plusmn 271 x 10-10

Lac electrodes showed better ORR efficiency despite of non-optimal pH condition Lac

is most active at pH 5 while BOD at pH 7 The amount of enzyme loaded on the

electrode surface also might have affected the catalytic activity It was however difficult

to control the loading because the enzyme was attached by dip coating Using mediators

could have also improved the ORR efficiency Many authors used mediators such as

ABTS [62 66] and Os-pyridine complexes linked to conductive polymer [97 237] to

assist the electron transfer between the electrode and enzyme

73 MOST EFFICIENT ORR CATALYSTS

The kinetic and thermodynamic parameters (kdeg nα Epc2(HDV)) were organised

258

by magnitude and are summarised in figures 25-27 and tables 1 amp 2 below For each

parameter the best five candidates were selected to present the effects of pretreatment

and their relation with each parameter The best three were chosen from them on the

basis of the parameter size and the number of top-ranked parameters they were

occupying kdeg was given the highest priority for selection process

731 Best five electrodes for nα

Pretreated GCEs were found to have prominent nα (Fig 121)

00

05

10

15

20

25

30

Electrode type

n α

nα 278 178 174 168 114

GC H2SO4-PBS GC NaOH-H2SO4-PBS

GC NaOH-PBS GC PBS GP NaOH-H2SO4

Fig 121 The best five electrodes for nα

The GCEs doubly pretreated with H2SO4 and PBS was found best with nα = 278 Its kdeg

was 279 x 10-7 ms-1 and also biggest among pretreated GCEs Correlation between nα

259

and kdeg has not been confirmed since it is possible for the intial electron transfer to be

accelerated whilst still remaining the RDS

732 Best five electrodes for kdeg

Top six kdegs were dominated by pretreated Co(salophen) composite electrodes

(Fig 122)

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

Electrode type

kdeg

ms

-1

kordm ms-1 119E-05 747E-06 684E-06 587E-06 296E-06

CoS NaOH CoS NaOH-H2SO4

CoS H2SO4-PBS CoS NaOH-H2SO4-PBS

CoS NaOH-PBS

Fig 122 The best five electrodes for kdeg

PBS pretreatment was found to suppress kdeg in Co(salophen) composite electrodes Clear

correlation between kdeg and nα has not yet confirmed however it was found that

favourable kdeg tends to lower overpotential

260

733 Best five electrodes for Epc2(HDV)

Although it is not an exact correspondence some correlation was observed

between lower overpotential and larger kdeg or lower overpotential and larger nα but not

between kdegand nα A range of pretreated Co(salophen) composite electrodes which

showed largest kdeg and a pretreated GCE which showed the largest nα were found to be

dominating low overpotential (Fig 123)

-040

-039

-038

-037

-036

-035

Electrode type

E pc

2(H

DV)

V

Epc2(HDV) -037 -037 -039 -039 -040

CoS H2SO4-PBS CoS NaOH CoS NaOH-H2SO4

GC H2SO4-PBS CoS NaOH-PBS

Fig 123 The best five electrodes for Epc2(HDV)

734 Best three candidates for ORR catalytic electrodes

Pretreated Co(salophen) composite electrodes were the best candidates for

ORR catalysis because of largest kdeg and lowest overpotential Top three catalytic

261

cathodes were listed in Table 17

kordm ms-1 Epc2(HDV) V nα

Co(salophen) NaOH 119E-05 -037 083

Co(salophen) NaOH-H2SO4 747E-06 -039 074

Co(salophen) H2SO4-PBS 684E-06 -037 091

Average 875E-06 -037 083

SD 278E-06 001 009

Table 17 Three candidates for catalytic cathodes for ORR

NaOH-pretreated Co(salophen) was found to be most efficient ORR catalytic cathode

because it has the largest kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 Epc2(HDV) = - 037 V and

was the second smallest of all kdeg of the three candidate Co(salophen) electrodes were

compared with those of catalytic metals and shown in Table 18

Electrode type kdeg ms-1 Experiment condition

Co(salophen) NaOH 119 x 10-5 In 001M PBS pH 74

Co(salophen) NaOH ndashH2SO4 747 x 10-6 In 001M PBS pH 74

Co(salophen) H2SO4-PBS 684 x 10-6 In 001M PBS pH 74

Au 4 x 10-3 In 005 M H2SO4 [173]

Au 3 x 10-5 In 01 M KOH

Ag 8 x 10-3 In 01 M KOH

Pt 4 x 10-3 In 01 M KOH

Table 18 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

262

NaOH-pretreated Co(salophen) composite electrode showed good catalysis at neutral

pH It could manage to achieve as efficient ORR as Au in acidic solution

74 CONCLUSION TO CATHODE STUDY

The large overpotential of ORR has been known to degrade the fuel cell

performance [3 4 62 165-167] Finding efficient ORR catalysts therefore has been a

subject of intensive research In this study various catalytic cathodes were fabricated

using inorganic organic and biological catalysts and they were electrochemically

pretreated in different media to improve the catalytic activity Studies were conducted

using various electrochemical methods (i) CV (ii) HDV and (iii) HDA The data

obtained was analysed both qualitatively and quantitatively The ORR catalytic

efficiency was calculated according to the three parameters (i) kdeg (ii) nα and (iii)

Epc2(HDV) and based on that most catalytic cathode was determined

None of the cathodes was catalytic enough without pretreatment or unless at high

overpotential In many cases electrochemical pretreatments were found to attach

various surface oxygen groups activated the electrodes and led to two-electron ORR

catalysis Their influence on electrochemistry varies by the electrode type and media

used for pretreatment It was found that PBS-related pretreatments were effective to

GCEs When the pretreatment is combined with H2SO4 this might lead to serial ORR

where H2O2 produced is further reduced to water NaOH-related pretreatments were

effective for graphite composite electrodes PtC composite electrodes were not active

due to the high resistance within the activated carbon The performance by RhC was

263

only marginally improved by H2SO4 pretreatment Any NaOH- or PBS- related

pretreatments dramatically increased kdeg of Co(salophen) composite electrodes and

reduced the overpotential by 100 mV Fe(salophen) composite electrodes were not

sufficiently catalytic without a double pretreatment with NaOH and H2SO4

Anthraquinone-grafted graphite composite electrodes were not catalytic at neutral pH

even after pretreatment Multi-copper oxidases were catalytic at DET however its RLS

was electron transfer between the electrode and the enzyme hence appropriate

mediators should be used to achieve higher catalysis

Many of pretreated Co(salophen) composite electrodes were strongly catalytic toward

ORR Among them NaOH-pretreated Co(slophen) electrodes were found to be most

catalytic at neutral pH with kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 nα = 083 and Epc2(HDV) =

- 037 V Its catalytic efficiency was close to that of Au in the acid solution

264

8 CONCLUSION AND FURTHER STUDIES

Miniaturised glucose-oxygen fuel cells are ideal to power small implantable

medical devices Typical specification include at least 1 microW power output and 1-year

operational life Therefore fuel cells must have efficient catalysis and electron transfer

good conductivity chemical and mechanical stability as well as biocompatibility The

electrodes must be feasible to build and scaleable In this study electrodes were built

using bulk modification method because it was simple to construct and scale-up and

they were easy to modify and mechanically stable The anodes were built with 10

GOx with 77 TTF-TCNQ composite and the cathodes were built with graphite

composite containing ORR catalysts using bulk modification method TTF-TCNQ was

used because it was conductive as well as mediating and necessary for electron transfer

of the GOx anodes Problems were enzyme stability and ORR catalytic efficiency

Enzyme mass loading is known to be effective to improve catalytic efficiency and

operational life however this also decreases conductivity of the composite electrodes

The key to optimising enzyme electrodes is in the balance and compromise among the

conductivity mediation enzyme activity mechanical and chemical stability and

difficulty in the fabrication process CNF composites showed reasonable conductivity at

30 content and this might allow higher enzyme loading with mediators without losing

conductivity CNT is also a candidate material because it has comparable or larger

conductivity than TTF-TCNQ and can operate without added mediator species Ideally

enzyme electrodes should be functional with minimum components simple to build

renew and store in dry so that they become easily portable Moreover when they are

further miniaturised this will allow the medical application under harsher conditions so

265

that powering disposable implantable sensors in economically-deprived regions might

become possible Many physiological enzymes however easily lose catalytic activity

outside the optimal condition Incorporation of enzyme stabilisers makes the enzyme

robust and long lasting as well as scavenging reactive species In the glucose-oxygen

fuel cells O2 has been known to reduce the catalytic output current of GOx anodes

PEI-DTT stabiliser mixtures were found to be good for this because they conferred

stability on GOx against oxidative stress prevented O2 electron deprivation and

improved the catalytic current output It was however a smaller effect than when

conductive hydrogel was used Conductive hydrogel can be a good candidate if it

becomes resistant against degradation ORR at the cathode has posed a long-term

problem in fuel cells because ORR is inefficient and consumes potential generated

within the fuel cell Pt has been used as an ORR catalyst but it is expensive and is

becoming scarce Use of the fuel cell in the physiological condition also imposes further

operational limitations Pretreated metal complexes such as Co salophen were found to

be good candidates for ORR-catalytic cathodes Surrounding complexes with

electron-rich environment such as conductive film might improve ORR further Once

candidate anode and cathode are identified and built a biofuel cell unit can be made To

increase the power each cell is connected together and made into a stack The prototype

was made as below in Fig 124 by connecting twenty cell units

266

Fig 124 Fuel cell stack made with various GOx anodes and Co(salophen) cathodes

Each cell contained various GOx anodes and pretreated Co(salophen) electrode in 10 ml

of oxygenated 01 M glucose 001 M PBS solution pH 74 The output voltage was

found to be 02 V and it did not change for one week The fuel cell must be studied

further with an adjustable resistor for voltage-current (V-I) and power-current (P-I)

curves so that OCV can be determined properly The passive type GOx fuel cell could

output 15 mWcm-2 at 03 V [18]

In vivo studies are also necessary for implantable purposes to examine the catalytic

efficiency electrode fouling and biocompatibility This can be studied systematically by

changing the type and concentration of interfering substances such as ascorbate

acetoaminophen and urate [18] Heller tested miniturised GOx-BOD fuel cells tested in

the buffer grape and calf serum It showed only 5 power decrease in buffer however

50 drop within 20 hours in grape and 50 drop within 2 hours in calf serum [19] To

avoid electrode fouling the electrode surface can be coated with a membrane such as

267

Nafion However this imposes another problem because it can interfere with the

reactant flux to the electrode surface In the real physiological system this might cause

a serious problem because glucose and O2 concentration is much lower than in vitro

Furthermore in the physiological conditions active transport of the reactant is not

available and this will cause local concentration polarisation It seems that there are

many obstacles lying in the medical application of miniaturised glucose-oxygen fuel

cells although they have widespread potentials applications

268

9 BIBLIOGRAPHY

1 Carrette L KA Friedrich and U Stimming Fuel cells Principles types fuels and applications

Chemphyschem 2000 1(4) p 162-193

2 Gregoire Padro CE and JO Keller Hydrogen Energy in Kirk-Othmer Encyclopedia of Chemical

Technology 2005 John Wiley amp Sons Inc p 837-866

3 Ohta K T Kazama and M Nishizawa All about fuel cells in Newton 2006 p 31-63

4 Tanabe S Mastering basics of the fuel cells 2009 Denkishoin

5 Tokyo-Gas Fuel cell academy 2009 2901 [cited Available from httpwwwtokyo-gascojppefc

6 Cropper MAJ S Geiger and DM Jollie Fuel cells a survey of current developments Journal of Power

Sources 2004 131(1-2) p 57-61

7 Kordesch K et al Intermittent use of a low-cost alkaline fuel cell-hybrid system for electric vehicles

Journal of Power Sources 1999 80(1-2) p 190-197

8 UTC-Power The fuel cell for Apollo 11

9 Connor S Warning Oil supplies are running out fast in The Independent 2009

10 Mori Y Mobile fuel cell providing new advantages for mobile electronic appliances Toshiba review 2007

62(6) p 44-47

11 Mori H Electronic components and materials Toshiba review 2009 64(3) p 62-63

12 Imperial-College-London Racing Green - Vehicles 2009 [cited Available from

httpwwwunionicacukrccracinggreenvehicleshtml

13 Cleave V Formula Zero kart race could drive fuel cell technology New Scientist 2008 [cited

Available from

httpwwwnewscientistcomarticledn14596-formula-zero-kart-race-could-drive-fuel-cell-technologyhtml

14 HyNor Hydrogen highway opens in Norway 2009 [cited Available from

httpwwwhynornohydrogen-highway-opens-in-norway

15 BMW Hydrogen 7 - The future is closer than you think 2009 [cited Available from

httpwwwbmwcomcomeninsightstechnologyefficient_dynamicsphase_2clean_energybmw_hydroge

n_7html

httpwwwbmwcojpjpjabrandtechnologycleanenergywallpaperhtml

16 Sony Side view - Sony developes Bio Battery generating electricity from sugar 2007 Sony

17 Burton N Sugar-powered electronics in RSC Chemical technology news 2008

18 Sakai H A high-power glucoseoxygen biofuel cell operating under quiescent conditions Energy amp

Environmental Science 2009 2(1) p 133-138

19 Heller A Miniature biofuel cells Physical Chemistry Chemical Physics 2004 6(2) p 209-216

20 Toshiba Toshiba announces worlds smallest direct methanol fuel cell with energy output of 100 milliwatts

269

2004 [cited Available from httpwwwtoshibacojpaboutpress2004_06pr2401htm

21 Osakai T K Kanoh and S Kuwabata Basic Electrochemistry 2007 Kagakudojin Ltd Kyoto Japan

22 Wilson K and J Walker Practical Biochemistry 1992 Cambridge UK Cambridge University Press

23 Murray RK et al Harpers Biochemistry 25 ed 2000 Stamford Connecticut Appleton amp Lange

24 Sawyer DT A Sobkowiak and JL Roberts Jr Electrochemistry for chemists Revised ed 1995

Wiley-Interscience

25 IUPAC IUPAC GOLD BOOK 2009 [cited Available from httpgoldbookiupacorgindexhtml

26 Kitamura T and K Fujisawa English-Japanese The students dictionary of physics 1995 Tokyo ALC

Inc

27 Gates BC Catalysis in Kirk-Othmer Encyclopedia of Chemical Technology 2002 John Wiley amp Sons p

200-254

28 Taguchi S and E Nakano Kinetic Study of Bromination of Malonic Acid Methyl Malonic Acid

and Ethyl Malonic Acid An Undergraduate Physical Chemistry Laboratory Experimentfor

Understanding of the Infuluence of Thermodynamic Parameter on Chemical Kinetics The Chemical

Education Jounal 1999 3(1) p 1-20

29 Pletcher D A First course in electrode processes 1991 The Electrochemical Consultancy (Romsey) Ltd

23 - 26 135 - 145

30 Bard AJ and LR Faulker Electrochemical Methods Fundamentals and Applications 2000 NY USA

John Wiley and Sons Inc

31 Daintith J A concise dictionary of chemistry 2 ed 1990 Oxford UK Oxford University Press

32 Tinoco IJ et al Physical Chemistry 2002 Prentice Hall p 157

33 Watanabe K and S Nakabayashi Introduction to electrochemistry The chemistry of electron transfer 13

ed 2005 Tokyo Japan The Chemical Society of Japan

34 Hamelers B et al Microbial fuel cells Methodology and technology Environmental science amp

technology 2006 40(17) p 5181-5192

35 Koike T Chapter 10 Free energy in Physical chemistry I amp II Lecture handouts for dept of pharmacy

2010 University of Hiroshima p 57-62

36 Lopes T E Gonzalez and E Antolini An overview of platinum-based catalysts as methanol-resistant

oxygen reduction materials for direct methanol fuel cells Journal of alloys and compounds 2008 461(1-2)

p 253-262

37 Vork FTA and E Barendrecht The reduction of dioxygen at polypyrrole-modified electrodes with

incorporated Pt particles Electrochimica Acta 1990 35(1) p 135-139

38 Marino W B Scharifker and A Leone ELECTRODEPOSITION AND

ELECTROCHEMICAL-BEHAVIOR OF PALLADIUM PARTICLES AT POLYANILINE AND

POLYPYRROLE FILMS Journal of the Electrochemical Society 1992 139(2) p 438-443

39 Tsakova V How to affect number size and location of metal particles deposited in conducting polymer

270

layers Journal of Solid State Electrochemistry 2008 12(11) p 1421-1434

40 Lemest Y et al Dioxygen Fixation by a Mixed-Valence Dicobalt Cofacial Porphyrin Journal of the

Chemical Society-Chemical Communications 1983(22) p 1286-1287

41 Kim E et al Superoxo micro-peroxo and micro-oxo complexes from hemeO2 and heme-CuO2 reactivity

Copper ligand influences in cytochrome c oxidase models 2003 p 3623-3628

42 Fukuzumi S et al Mechanism of Four-Electron Reduction of Dioxygen to Water by Ferrocene

Derivatives in the Presence of Perchloric Acid in Benzonitrile Catalyzed by Cofacial Dicobalt Porphyrins

Journal of the American Chemical Society 2004 126(33) p 10441-10449

43 Solomon EI et al O2 and N2O Activation by Bi- Tri- and Tetranuclear Cu Clusters in Biology

Accounts of Chemical Research 2007 40(7) p 581-591

44 Wildgoose GG et al Anthraquinone-derivatised carbon powder reagentless voltammetric pH electrodes

Talanta 2003 60(5) p 887-893

45 McCreery R Carbon electrodes Structural effects on electron transfer kinetics in Electroanalytical

Chemistry A series advances 1990 Marcel Dekker Inc p 221-374

46 Kneten K R McCreery and C McDermott ELECTRON-TRANSFER KINETICS OF AQUATED

FE+3+2 EU+3+2 AND V+3+2 AT CARBON ELECTRODES - INNER-SPHERE CATALYSIS BY

SURFACE OXIDES Journal of the Electrochemical Society 1993 140(9) p 2593-2599

47 Engstrom RC ELECTROCHEMICAL PRETREATMENT OF GLASSY-CARBON ELECTRODES

Analytical Chemistry 1982 54(13) p 2310-2314

48 Lawrence E Hendersons dictionary of biological terms 10 ed 1989 UK Longman group UK Ltd

49 Eijsink V Directed evolution of enzyme stability Biomolecular engineering 2005 22(1-3) p 21-30

50 Pantoliano M et al PROTEIN ENGINEERING OF SUBTILISIN BPN - ENHANCED STABILIZATION

THROUGH THE INTRODUCTION OF 2 CYSTEINES TO FORM A DISULFIDE BOND Biochemistry

1987 26(8) p 2077-2082

51 Bagotzky VS NV Osetrova and AM Skundin Fuel cells State-of-the-art and major scientific and

engineering problems Russian Journal of Electrochemistry 2003 39(9) p 919-934

52 Heller A Integrated medical feedback systems for drug delivery Aiche Journal 2005 51(4) p

1054-1066

53 Nakamura M et al High-performance ultrathin solid oxide fuel cells for low-temperature operation

Journal of the Electrochemical Society 2007 154(1) p B20-B24

54 Lapedes DN McGraw-Hill Dictionary of Physics and Mathematics in McGraw-Hill 1978 McGraw-Hill

Inc

55 Svoboda V et al Enzyme catalysed biofuel cells Energy amp Environmental Science 2008 1(3) p

320-337

56 Barton SC J Gallaway and P Atanassov Enzymatic biofuel cells for Implantable and microscale devices

Chemical Reviews 2004 104(10) p 4867-4886

271

57 Schroder U Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency

Physical chemistry chemical physics 2007 9(21) p 2619-2629

58 Chaudhuri SK and DR Lovley Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells Nature Biotechnology 2003 21(10) p 1229-1232

59 Vincent K F Armstrong and J Cracknell Enzymes as working or inspirational electrocatalysts for fuel

cells and electrolysis Chemical reviews 2008 108(7) p 2439-2461

60 Tsujimura S and K Kano Recent development of enzyme-based biofuel cells GS Yuasa technical report

2008 5(2) p 1-6

61 Cass AEG Biosensors A Practical Approach 1990 Oxford University Press Oxford UK p 49-53

62 Palmore GTR and HH Kim Electro-enzymatic reduction of dioxygen to water in the cathode

compartment of a biofuel cell Journal of Electroanalytical Chemistry 1999 464(1) p 110-117

63 Bullen RA et al Biofuel cells and their development Biosensors amp Bioelectronics 2006 21(11) p

2015-2045

64 Bertschy H et al A methanoldioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as

catalysts application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at

low overpotentials Journal of electroanalytical chemistry and interfacial electrochemistry 1998 443(1) p

155-161

65 Bartlett PN P Tebbutt and RG Whitaker Kinetic aspects of the use of modified electrodes and

mediators in bioelectrochemistry Progress in reaction kinetics 1991 16(2) p 55-155

66 Kano K T Ikeda and S Tsujimura GlucoseO-2 biofuel cell operating at physiological conditions Denki

kagaku oyobi kōgyō butsuri kagaku 2002 70(12) p 940-942

67 Heller A Electrochemical glucose sensors and their applications in diabetes management Chemical

Reviews 2008 108(7) p 2482-2505

68 Ilankumaran V et al Trends in cardiac pacemaker batteries Indian pacing and electrophysiology journal

2004 4(4) p 201-12

69 Schlindwein W and R Linford Medical applications of solid state lonics Solid State Ionics 2006

177(19-25) p 1559-1565

70 Latham R R Linford and W Schlindwein Biomedical applications of batteries Solid State Ionics 2004

172(1-4) p 7-11

71 Soykan O Power sources for implantable medical devices Business Briefing Medical Device

Manufacturing and Technology London 2002

72 Heller A Potentially implantable miniature batteries Analytical and Bioanalytical Chemistry 2006

385(3) p 469-473

73 Taniguchi M Achilles heel of the next-generation electric vehicles - Lithium World map of the resrouce

wars 2008 [cited 2009 Available from

httpbusinessnikkeibpcojparticlemanage20080407152450P=1

272

74 Yeatman EM Advances In Power Sources For Wireless Sensor Nodes International Workshop on Body

Sensor Networks 2004

75 Sato N et al Novel MEMS power generator with integrated thermoelectric and vibrational devices in

Solid-State Sensors Actuators and Microsystems 2005 Digest of Technical Papers TRANSDUCERS 05

The 13th International Conference on 2005

76 Hudak NS and GG Amatucci Small-scale energy harvesting through thermoelectric vibration and

radiofrequency power conversion Journal of Applied Physics 2008 103(10)

77 Seiko-Instruments SEIKO Worlds First 2009 [cited Available from

httpwwwseikowatchescomheritageworlds_firsthtml

78 Kishi M et al Micro thermoelectric modules and their application to wristwatches as an energy source in

Thermoelectrics 1999 Eighteenth International Conference on 1999

79 Lee J et al Ionic conduction in Zn-3(PO4)(2)()4H(2)O enables efficient discharge of the zinc anode in

serum Journal of the American Chemical Society 2005 127(42) p 14590-14591

80 Maxell Button battery table 2009 [cited Available from

httpwwwmaxellcojpproductsmaterialsbutton_batteryhtml

81 Wilson R and APF Turner Glucose-Oxidase - an Ideal Enzyme Biosensors amp Bioelectronics 1992 7(3)

p 165-185

82 Swoboda BEP The relationship between molecular conformation and the binding of flavin-adenine

dinucleotide in glucose oxidase Biochimica et Biophysica Acta (BBA) - Protein Structure 1969 175(2) p

365-379

83 Wohlfahrt G et al 18 and 19 angstrom resolution structures of the Penicillium amagasakiense and

Aspergillus niger glucose oxidases as a basis for modelling substrate complexes Acta Crystallographica

Section D-Biological Crystallography 1999 55 p 969-977

84 Patel RN Biocatalysis in the pharmaceutical and biotechnology industries 1st ed 2006 CRC Press

85 Gouda M Reversible denaturation behavior of immobilized glucose oxidase Applied Biochemistry and

Biotechnology 2002 102 p 471-480

86 Hecht HJ et al Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 3 Angstrom

Resolution Journal of Molecular Biology 1993 229(1) p 153-172

87 Zubrik A et al Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the

transition to the denatured state with residual structure The Journal of biological chemistry 2004 279(46)

p 47601-47609

88 Kojima S et al Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose

biosensor Bioscience biotechnology and biochemistry 2006 70(3) p 654-659

89 Duine J The PQQ story Journal of bioscience and bioengineering 1999 88(3) p 231-236

90 Kyoto-University KEGG - Kyoto Encyclopedia of Genes and Genomes 2009 Kyoto University

91 Delbem MF WJ Baader and SHP Serrano Mechanism of 34-dihydroxybenzaldehyde

273

electropolymerization at carbon paste electrodes catalytic detection of NADH 2002 scielo p 741-747

92 Zhao S et al Electrochemistry of Organic Conducting Salt Electrodes - a Unified Mechanistic

Description Journal of Physical Chemistry 1992 96(13) p 5641-5652

93 Lemuel BW Jr CC Liu and LN Niren Electrochemical measurements with glucose oxidase

immobilized in polyacrylamide gel Constant current voltametry 1971 p 629-639

94 Sato A et al Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Biochemical Journal

2001 357 p 893-898

95 Sigma-Aldrich Glucose Oxidase from Aspergillus niger Type II-S 15000-50000 unitsg solid (without

added oxygen) 2009 [cited

96 Cosnier S Biosensors based on electropolymerized films new trends Analytical and Bioanalytical

Chemistry 2003 377(3) p 507-520

97 Willner I et al Integrated Enzyme-Based Biofuel Cells-A Review Fuel Cells 2009 9(1) p 7-24

98 Heleg-Shabtai V et al Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers

assembled onto Au-electrodes Journal of the American Chemical Society 1996 118(42) p 10321-10322

99 Georganopoulou DG et al Development and comparison of biosensors for in-vivo applications Faraday

Discussions 2000 p 291-303

100 Fujii S et al Properties of glucose sensors prepared by the electropolymerization of pyrrole containing a

gluconyl group Analytical sciences 1999 15(12) p 1175-1176

101 Hoshi T Deposition of electron mediators by electrochemical polymerization Electrochemistry 2003

71(5) p 346-347

102 Arai G M Masuda and I Yasumori Direct Electrical Response between Glucose-Oxidase and

Poly(Mercapto-P-Benzoquinone) Films Bulletin of the Chemical Society of Japan 1994 67(11) p

2962-2966

103 Arai G K Shoji and I Yasumori Electrochemical characteristics of glucose oxidase immobilized in

poly(quinone) redox polymers Journal of Electroanalytical Chemistry 2006 591(1) p 1-6

104 Leonida MD et al Polymeric FAD used as enzyme-friendly mediator in lactate detection Analytical and

Bioanalytical Chemistry 2003 376(6) p 832-837

105 Kissinger PT and WR Heineman Laboratory techniques in electroanalytical chemistry 1996 Marcel

Dekker Inc NY USA p 294-332

106 OHare D and H Zhao Characterisation and modeling of conducting composite electrodes The journal of

physical chemistry C 2008 112(25) p 9351-9357

107 Wang J et al Metal-Dispersed Carbon Paste Electrodes Analytical Chemistry 1992 64(11) p

1285-1288

108 Khurana MK CP Winlove and D OHare Detection mechanism of metallized carbon epoxy oxidase

enzyme based sensors Electroanalysis 2003 15(12) p 1023-1030

109 Sasaki M et al Fabrication and Electrical Characterization of

274

Tetrathiafulvalene-tetracyanoquinodimethane Molecular Wires Japanese Journal of Applied Physics 2003

42 p 2488-2491

110 Iizuka M et al Highly oriented TTF-TCNQ crystal growth using an electric-field induced evaporation

technique Molecular crystals and liquid crystals science and technology Section A Molecular crystals and

liquid crystals 2000 349 p 367-370

111 Saito I and K Yoshida Development of new features on organic conductive material TCI mail 2005

127

112 Cass AEG et al FERROCENE-MEDIATED ENZYME ELECTRODE FOR AMPEROMETRIC

DETERMINATION OF GLUCOSE Analytical Chemistry 1984 56(4) p 667-671

113 Kulys J V Simkeviciene and IJ Higgins Concerning the Toxicity of 2 Compounds Used as Mediators in

Biosensor Devices - 7788-Tetracyanoquinodimethane (Tcnq) and Tetrathiafulvalene (Ttf) Biosensors amp

Bioelectronics 1992 7(7) p 495-501

114 Malinauskas A et al Conductive organic complex salt TTF-TCNQ as a mediator for biosensors An

overview Electroanalysis 2007 19(24) p 2491-2498

115 Llopis X et al Integration of a glucose biosensor based on an epoxy-graphite-TTF center dot

TCNQ-GOD biocomposite into a FIA system Sensors and Actuators B-Chemical 2005 107(2) p 742-748

116 Bourgeois J D Thomas and C Bourdillon COVALENT LINKAGE OF GLUCOSE-OXIDASE ON

MODIFIED GLASSY-CARBON ELECTRODES - KINETIC PHENOMENA Journal of the American

Chemical Society 1980 102(12) p 4231-4235

117 Yun Y et al Nanotube electrodes and biosensors Nano Today 2007 2(6) p 30-37

118 Kim BC et al Preparation of biocatalytic nanofibres with high activity and stability via enzyme

aggregate coating on polymer nanofibres Nanotechnology 2005 16(7) p S382-S388

119 Kim J and JW Grate Single-Enzyme Nanoparticles Armored by a Nanometer-Scale OrganicInorganic

Network 2003 p 1219-1222

120 OFagain C Enzyme stabilization - recent experimental progress Enzyme and Microbial Technology

2003 33(2-3) p 137-149

121 Sillery E et al Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a

platinum catalyst Chemical communications 2002(8) p 866-867

122 Gouda M et al Enhancement of stability of immobilized glucose oxidase by modification of free thiols

generated by reducing disulfide bonds and using additives Biosensors amp bioelectronics 2004 19(6) p

621-625

123 Manning M STABILITY OF PROTEIN PHARMACEUTICALS Pharmaceutical Research 1989 6(11) p

903-918

124 Cao L Immobilised enzymes science or art Current opinion in chemical biology 2005 9(2) p 217-226

125 Daniel R The role of dynamics in enzyme activity Annual review of biophysics and biomolecular

structure 2003 32 p 69-92

275

126 Andersson MM JD Breccia and R Hatti-Kaul Stabilizing effect of chemical additives against oxidation

of lactate dehydrogenase Biotechnology and Applied Biochemistry 2000 32 p 145-153

127 Souza JM and R Radi Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Archives of Biochemistry and Biophysics 1998 360(2) p 187-194

128 Andersson MA and R Hatti-Kaul Protein stabilising effect of polyethyleneimine Journal of

Biotechnology 1999 72(1-2) p 21-31

129 Sigma-Aldrich Poly(ethylenimine) 2009 [cited Available from

httpwwwsigmaaldrichcomstructureimages27mfcd00084427gif

130 Andersson MM R Hatti-Kaul and W Brown Dynamic and static light scattering and fluorescence

studies of the interactions between lactate dehydrogenase and poly(ethyleneimine) Journal of Physical

Chemistry B 2000 104(15) p 3660-3667

131 Wang J L Chen and J Liu Critical comparison of metallized and mediator-based carbon paste glucose

biosensors Electroanalysis 1997 9(4) p 298-301

132 Cleland WW Dithiothreitol a New Protective Reagent for SH Groups Biochemistry 1964 3(4) p

480-482

133 Atanasov P E Wilkins and Q Yang An integrated needle-type biosensor for intravascular glucose and

lactate monitoring Electroanalysis 1998 10(11) p 752-757

134 Sigma-Aldrich DL-Dithiothreitol 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=43815|SIGMAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

135 Ghisalba O and A Brunella Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate

reductase as biocatalyst in the preparative synthesis of 2 -deoxyribonucleoside-5 -triphosphates Journal

of molecular catalysis B Enzymatic 2000 10(1-3) p 215-222

136 Gollhofer D EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM

ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Enzyme and Microbial Technology 1995 17(3) p 235-240

137 Sigma-Aldrich Arsenite standard solution - 005 M in H2O 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=70039|FLUKAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

138 Arakawa T MECHANISM OF POLY(ETHYLENE GLYCOL) INTERACTION WITH PROTEINS

Biochemistry 1985 24(24) p 6756-6762

139 Lee C Improvement of protein stability in protein microarrays Biotechnology Letters 2002 24(10) p

839-844

140 Lee L THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE GLYCOLS)

Biochemistry 1987 26(24) p 7813-7819

141 Costa SA et al Studies of stabilization of native catalase using additives Enzyme and Microbial

276

Technology 2002 30(3) p 387-391

142 Lee J and L Lee THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE

GLYCOLS) Biochemistry 1987 26(24) p 7813-7819

143 Gekko K and SN Timasheff Mechanism of protein stabilization by glycerol preferential hydration in

glycerol-water mixtures Biochemistry 1981 20(16) p 4667-4676

144 Leandro P Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes

produced in a prokaryotic expression system Molecular genetics and metabolism 2001 73(2) p 173-178

145 Bartlett PN VQ Bradford and RG Whitaker Enzyme Electrode Studies of Glucose-Oxidase Modified

with a Redox Mediator Talanta 1991 38(1) p 57-63

146 Joseph S et al Drug metabolism biosensors electrochemical reactivities of cytochrome P450(cam)

immobilised in synthetic vesicular systems Journal of Pharmaceutical and Biomedical Analysis 1998

17(6-7) p 1101-1110

147 Cornish-Bowden A Fundamentals of enzyme kinetics 3rd ed 2004 London Portland Press Ltd

148 Wilkinson G STATISTICAL ESTIMATIONS IN ENZYME KINETICS The Biochemical journal 1961

80(2) p 324

149 Ikeda T and K Kano Fundamentals and practices of mediated bioelectrocatalysis Analytical sciences

2000 16(10) p 1013-1021

150 Duggleby RG and LP Daniel [3] Analysis of enzyme progress curves by nonlinear regression in

Methods in Enzymology 1995 Academic Press p 61-90

151 Eisenthal R and A Cornish-Bowden DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR

ESTIMATING ENZYME KINETIC-PARAMETERS The Biochemical journal 1974 139(3) p 715-720

152 Baici A Enzyme kinetics the velocity of reactions Biochem J 2006 p 1-3

153 Yamazaki A et al Improvement of precision in pharmacological studies using the non-linear regression

method

estimation of enzyme inhibition constants as an example Folia Pharmacologica Japonica 2008 131(3) p 195-203

154 Cornish-Bowden A Teaching Enzyme Kinetics and Mechanism in the 21st Century in 3rd Symposium

on ESCES 2008 Rudensheim Germany

155 Weisshaar DE and T Kuwana Considerations for Polishing Glassy-Carbon to a Scratch-Free Finish

Analytical Chemistry 1985 57(1) p 378-379

156 Stedman Stedmans medical dictionary 25th ed 2002 Lippincott Williams amp Wilkins

157 Tamura T Buffer preparation for biology Buffer preparation for biology ed 6 2008 Tokyo Yodosha

Ltd

158 Battino R and HL Clever The Solubility of Gases in Liquids 1966 p 395-463

159 Pandolfo A and A Hollenkamp Carbon properties and their role in supercapacitors Journal of power

sources 2006 157(1) p 11-27

160 Maynard AD Nanotechnology assessing the risks Nano Today 2006 1(2) p 22-33

277

161 Hindle A E Hall and N Martens AN ASSESSMENT OF MEDIATORS AS OXIDANTS FOR

GLUCOSE-OXIDASE IN THE PRESENCE OF OXYGEN Biosensors amp bioelectronics 1995 10(3-4) p

393-403

162 Mo J et al Comparison of oxygren-rich and mediator-based glucose-oxidase carbon-paste electrodes

Analytica Chimica Acta 2001 441(2) p 183-189

163 Turner A and G Palleschi AMPEROMETRIC TETRATHIAFULVALENE-MEDIATED LACTATE

ELECTRODE USING LACTATE OXIDASE ABSORBED ON CARBON FOIL Analytica Chimica Acta

1990 234(2) p 459-463

164 Mizutani F et al Glucose-Sensing Electrode Based on Carbon Paste Containing Ferrocene and

Polyethylene Glycol-Modified Enzyme Bulletin of the Chemical Society of Japan 1991 64(9) p

2849-2851

165 Zhang J PEM Fuel Cell Electrocatalysts and Catalyst Layers Fundamentals and Applications 2008

London Springer-Verlag

166 Kinoshita K Electrochemical oxygen technology 1992 John Willey amp Sons Inc

167 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of Power

Sources 2005 152 p 1-15

168 Sawyer DT Oxygen Chemistry International series of monographs on chemistry 1991 Oxford

University Press

169 Winter M Chemical Bonding Oxford Chemistry Primers 1997 New York Oxford University Press Inc

170 McLendon G and AE Martell Inorganic Oxygen Carriers as Models for Biological-Systems

Coordination Chemistry Reviews 1976 19(1) p 1-39

171 Toda T et al Enhancement of the electroreduction of oxygen on Pt alloys with Fe Ni and Co Journal of

the Electrochemical Society 1999 146(10) p 3750-3756

172 Norskov JK et al Origin of the overpotential for oxygen reduction at a fuel-cell cathode Journal of

Physical Chemistry B 2004 108(46) p 17886-17892

173 Fischer P and J Heitbaum MECHANISTIC ASPECTS OF CATHODIC OXYGEN REDUCTION Journal

of Electroanalytical Chemistry 1980 112(2) p 231-238

174 Okada T et al Oxygen reduction characteristics on pyrolytic graphite electrodes modified with

electro-polymerized salen compounds Chemistry Letters 1998(8) p 841-842

175 Sakurai T and K Kataoka Basic and applied features of multicopper oxidases CueO bilirubin oxidase

and laccase 2007 p 220-229

176 Sanders J Supramolecular catalysis in transition Chemistry a European journal 1998 4(8) p

1378-1383

177 Hart H LE Craine and DJ Hart Organic Chemistry A Short Course 10 ed 1998 Houghton Miffin

Company USA

178 Hayatsu H CELLULOSE BEARING COVALENTLY LINKED COPPER PHTHALOCYANINE

278

TRISULPHONATE AS AN ADSORBENT SELECTIVE FOR POLYCYCLIC COMPOUNDS AND ITS USE

IN STUDIES OF ENVIRONMENTAL MUTAGENS AND CARCINOGENS Journal of chromatography A

1992 597(1-2) p 37-56

179 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of power

sources 2005 152(1) p 1-15

180 Kingsborough RP and TM Swager Electrocatalytic Conducting Polymers Oxygen Reduction by a

Polythiophene-Cobalt Salen Hybrid Chemistry of Materials 2000 12(4) p 872-874

181 Ortiz B and SM Park Electrochemical and spectroelectrochemical studies of cobalt salen and salophen

as oxygen reduction catalysts Bulletin of the Korean Chemical Society 2000 21(4) p 405-411

182 Mano N et al An oxygen cathode operating in a physiological solution Journal of the American

Chemical Society 2002 124(22) p 6480-6486

183 Shleev S et al Direct electron transfer between copper-containing proteins and electrodes Biosensors

and Bioelectronics 2005 20(12) p 2517-2554

184 Solomon EI et al Oxygen binding activation and reduction to water by copper proteins Angewandte

Chemie-International Edition 2001 40(24) p 4570-4590

185 Heath R F Armstrong and C Blanford A stable electrode for high-potential electrocatalytic O-2

reduction based on rational attachment of a blue copper oxidase to a graphite surface Chemical

communications 2007(17) p 1710-1712

186 Miura Y et al Direct Electrochemistry of CueO and Its Mutants at Residues to and Near Type I Cu for

Oxygen-Reducing Biocathode Fuel cells 2009 9(1) p 70-78

187 Tarasevich MR VA Bogdanovskaya and LN Kuznetsova Bioelectrocatalytic reduction of oxygen in

the presence of laccase adsorbed on carbon electrodes Russian Journal of Electrochemistry 2001 37(8) p

833-837

188 Tsujimura S et al Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase

at carbon electrodes Electrochemistry 2004 72(6) p 437-439

189 Fernandez JL et al Optimization of wired enzyme O-2-electroreduction catalyst compositions by

scanning electrochemical microscopy Angewandte Chemie-International Edition 2004 43(46) p

6355-6357

190 Soukharev V N Mano and A Heller A four-electron O-2-electroreduction biocatalyst superior to

platinum and a biofuel cell operating at 088 V Journal of the American Chemical Society 2004 126(27)

p 8368-8369

191 Miura Y et al Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on

a highly oriented pyrolytic graphite electrode Chemistry Letters 2007 36(1) p 132-133

192 Seinberg JM et al Spontaneous modification of glassy carbon surface with anthraquinone from the

solutions of its diazonium derivative An oxygen reduction study Journal of Electroanalytical Chemistry

2008 624(1-2) p 151-160

279

193 Manisankar P and A Gomathi Electrocatalytic reduction of dioxygen at the surface of carbon paste

electrodes modified with 910-anthraquinone derivatives and dyes Electroanalysis 2005 17(12) p

1051-1057

194 Jurmann G DJ Schiffrin and K Tammeveski The pH-dependence of oxygen reduction on

quinone-modified glassy carbon electrodes Electrochimica Acta 2007 53(2) p 390-399

195 Abiman P R Compton and G Wildgoose Characterising chemical functionality on carbon surfaces

Journal of materials chemistry 2009 19(28) p 4875-4886

196 Thorogood CA et al Differentiating between ortho- and para-Quinone Surface Groups on Graphite

Glassy Carbon and Carbon Nanotubes Using Organic and Inorganic Voltammetric and X-ray

Photoelectron Spectroscopy Labels Chemistry of Materials 2007 19(20) p 4964-4974

197 Sigma-Aldrich Fast red AL salt 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildoN4=380660|SIALampN5=SEARCH_CONCAT_PN

O|BRAND_KEYampF=SPEC

198 Sljukic B C Banks and R Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

199 Granger M et al Polycrystalline diamond electrodes basic properties and applications as amperometric

detectors in flow injection analysis and liquid chromatography Analytica Chimica Acta 1999 397(1-3) p

145-161

200 Compton R J Foord and F Marken Electroanalysis at diamond-like and doped-diamond electrodes

Electroanalysis 2003 15(17) p 1349-1363

201 Arima S Do Science - Why diamond is so hard in Asahicom 2006

202 Dumitrescu I P Unwin and J Macpherson Electrochemistry at carbon nanotubes perspective and issues

Chemical communications 2009(45) p 6886-6901

203 Kinoshita K Carbon Electrochemical and Physicochemical Properties 1988 Wiley-Interscience

204 Kastening B et al Electronic properties and double layer of activated carbon Electrochimica Acta 1997

42(18) p 2789-2799

205 Swain G and R Ramesham THE ELECTROCHEMICAL ACTIVITY OF BORON-DOPED

POLYCRYSTALLINE DIAMOND THIN-FILM ELECTRODES Analytical chemistry 1993 65(4) p

345-351

206 Kaneto K et al Electrical conductivities of multi-wall carbon nano tubes Synthetic metals 1999

103(1-3) p 2543-2546

207 Meyer J et al Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes Nano

letters 2008 8(11) p 3582-3586

208 Harris P New perspectives on the structure of graphitic carbons Critical Reviews in Solid State and

Materials Sciences 2005 30(4) p 235-253

209 Akasaka T Chemical illustration - Carbon materials 2007 Hokkaido Japan

280

210 Iijima S M Yudasaka and F Nihey Carbon nanotube technology NEC Technical Journal 2007 2(1) p

52-56

211 Day T N Wilson and J Macpherson Electrochemical and conductivity measurements of single-wall

carbon nanotube network electrodes Journal of the American Chemical Society 2004 126(51) p

16724-16725

212 Fujita H A TEM image of the atomic structure of the diamond surface 2010 Britannica - The Online

Encyclopedia Osaka Japan

213 McCreery RL et al Control of reactivity at carbon electrode surfaces Colloids and Surfaces A

Physicochemical and Engineering Aspects 1994 93 p 211-219

214 Rice R C Allred and R McCreery FAST HETEROGENEOUS ELECTRON-TRANSFER RATES FOR

GLASSY-CARBON ELECTRODES WITHOUT POLISHING OR ACTIVATION PROCEDURES Journal of

electroanalytical chemistry and interfacial electrochemistry 1989 263(1) p 163-169

215 Swain Electrochemical formation of high surface area carbon fibers Analytical chemistry 1991 63(5) p

517-519

216 Nagaoka T and T Yoshino Surface-Properties of Electrochemically Pretreated Glassy-Carbon Analytical

Chemistry 1986 58(6) p 1037-1042

217 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 2002 58(9) p 1953-1955

218 Motta N and AR Guadalupe Activated Carbon-Paste Electrodes for Biosensors Analytical Chemistry

1994 66(4) p 566-571

219 Leung E et al A novel in vivo nitric oxide sensor Chemical Communications 1996(1) p 23-24

220 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 1986 58(9) p 1953-1955

221 Kissinger PT and WR Heineman Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy

Current Separations 1989 9 p 15-18

222 Wang J Analytical Electrochemistry 3 ed 2006 Wiley-VCH

223 BAS Measurement of electron transfer kinetics using rotating disk voltammetry Electrocehmistry

Principles methods and applications 1995 2(43)

224 Miomandre F et al Electrochemical behaviour of iron(III) salen and poly(iron-salen) Application to the

electrocatalytic reduction of hydrogen peroxide and oxygen Journal of electroanalytical chemistry and

interfacial electrochemistry 2001 516(1-2) p 66-72

225 Brighton-University Inorganic Chemistry Practical C170 Salen lab script 2009 Dept of Pharmacy and

Biomolecular Sciences Brighton University Brighton

226 Ranganathan S TC Kuo and RL McCreery Facile Preparation of Active Glassy Carbon Electrodes

with Activated Carbon and Organic Solvents 1999 p 3574-3580

227 Ohare D CP Winlove and KH Parker Electrochemical method for direct measurement of oxygen

281

concentration and diffusivity in the intervertebral disc electrochemical characterization and tissue-sensor

interactions J Biomed Eng 1991 13(4) p 304-312

228 Chen PH and RL McCreery Control of electron transfer kinetics at glassy carbon electrodes by specific

surface modification Analytical Chemistry 1996 68(22) p 3958-3965

229 Beilby AL TA Sasaki and HM Stern Electrochemical Pretreatment of Carbon Electrodes as a

Function of Potential Ph and Time Analytical Chemistry 1995 67(5) p 976-980

230 Dai H and K Shiu Voltammetric studies of electrochemical pretreatment of rotating-disc glassy carbon

electrodes in phosphate buffer Journal of electroanalytical chemistry and interfacial electrochemistry 1996

419(1) p 7-14

231 Musameh M N Lawrence and J Wang Electrochemical activation of carbon nanotubes

Electrochemistry communications 2005 7(1) p 14-18

232 Sljukic B CE Banks and RG Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

233 Tammeveski K et al Surface redox catalysis for O-2 reduction on quinone-modified glassy carbon

electrodes Journal of Electroanalytical Chemistry 2001 515(1-2) p 101-112

234 Yang HH and RL McCreery Elucidation of the mechanism of dioxygen reduction on metal-free carbon

electrodes Journal of the Electrochemical Society 2000 147(9) p 3420-3428

235 Regisser F et al Randomly oriented graphite electrode 1 Effect of electrochemical pretreatment on the

electrochemical behavior and chemical composition of the electrode Journal of electroanalytical chemistry

and interfacial electrochemistry 1996 415(1-2) p 47-54

236 Shleev S et al Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic

graphite electrode Electrochemistry Communications 2004 6(9) p 934-939

237 Mano N F Mao and A Heller A miniature biofuel cell operating in a physiological buffer Journal of the

American Chemical Society 2002 124(44) p 12962-12963

2

I hereby declare that the work presented in this thesis is my own

Yoko Kikuchi

3

ABSTRACT

Miniaturized glucose-oxygen biofuel cells are useful to power implantable

medical devices such as biosensors They are small more biocompatible and run

continuously on glucose and oxygen providing cleaner energy at neutral environment

A typical glucose-oxygen biofuel cell consists of an anode with glucose oxidase (GOx)

and a cathode with various oxygen reducing catalysts This thesis describes

experimental investigations of the major issues of such systems viz complex electrode

fabrication enzyme instability and inefficient oxygen reduction Electrodes were built

using the simple and scaleable bulk modification method where all the material was

simply mixed and bound together into composites with epoxy resin For the anodes the

composite made of 10 GOx with 77 TTF-TCNQ was found optimal The GOx

electrodes were modified with various enzyme stabilisers (PEI DTT PEG GLC FAD

and mixture of PEIDTT and PEIFAD) and 2 of PEI-DTT (11 ww) was most

effective in the presence of O2 Its maximum output current density was 18 x 10-2 plusmn 99

x 10-3 Am-2 It also showed the resistant against O2 electron deprivation and enzyme

inhibition Its KMwas 5 mM For the cathodes various oxygen reducing catalysts

(metalised carbon anthroquinone modified carbon laccase and bilirubin oxidase) were

incorporated into graphite composite and the electrodes were pretreated in different

media in order to enhance their catalytic activity None showed four-electron O2

reduction NaOH-pretreated cobalt (II) salophen composite electrodes showed

two-electron O2 reduction and were most catalytic Its standard catalytic rate constant

was 12 x 10-5 plusmn 12 x 10-6 ms-1 Of the catalysts examined metal complex composites

gave the best results for oxygen-reducing cathodes and their pretreatment led to the

4

synergetic effect because it increased the concentration of catalytic surface oxygen

groups and enhanced oxygen reduction

5

ありがとう Thank you Terima kashi ขอบคณ คะ Merci 고마워 謝謝 Danke

やったね

copy鳥

山

明

集

英

社

copy Akira Toriyama Shueisha Inc

6

ACKNOWLEDGEMENTS

I would like to express deepest gratitude to my supervisor Dr Danny OrsquoHare

for his guidance on and patience for my research Frankly speaking the project on

glucose-oxygen fuel cells was very challenging and I came to realise the hard reality of

creating new clean energy despite of the world huge demand for such energy in a time

of global warming I could not help but feel that the research was turning into a

wild-goose chase however there was much to learn and gain from the experience here

For that and the people I met during this time I feel deeply grateful for the opportunity

given and I am convinced that this will enlighten my life

I would like to specially thank Dr Pei Ling Leow and Mr Parinya Seelanan for

their sincere support understanding and encouragement Without their help I would not

have been able to achieve my PhD I would like to thank to the current and past

members of Biosensor group Ms Nitin Bagha Dr Christopher Bell Dr Eleni Bitziou

Mr Jin-Young Kim Mr Wei-Chih Lin Dr Beinn Muir Mr Raphael Trouillon Dr

Martyn Boutelle Mr Yao-Min Chang Dr Delphine Feuerstein Ms Michelle Rogers

Dr Costas Anastassiou Dr Arun Arora Dr Martin Arundell Dr Catherine Dobson Dr

Melanie Fennan Dr Severin Harvey Dr Alex Lindsay Dr Bhavik Patel Dr Hong

Zhao Dr Emma Corcoles and Dr Parastoo Hashemi for their support during research

and sharing the wonderful time for many occasions I would like to thank Mr David

Roche for his sincere devotion to our work and being a great lab company I would like

to give my warm thanks to Dr Andrew Bond Dr Stephanie Cremers Ms Christina

Hood Dr Asimina Kazakidi Dr Peter Knox Dr Bettina Nier Ms Eileen Su Mr

Che-Fai Yeong Mr Krause Oliver and Dr Clifford Talbot for appropriate support fun

7

and encouragement I would also like to thank Dr Kate Hobson Mr Martin Holloway

Mr Ken Keating Mr Alan Nyant Mr Richard Oxenham Ms Paula Smith Ms Dima

Cvetkova Dr Anna Thomas-Betts and Mr Nick Watkins for their academic and

laboratory support Finally I would like to say thank you to all my family for their trust

and unconditional support my teacher Prof Fukuyama my friends Dr Pieta

Nasanen-Gilmore Jingzi Kofi Mariama Masa Maya Miki Pedro Sandra Sascha and

Wei Fong for being always close supportive and encouraging even when we were apart

8

CONTENTS

ABSTRACT 3

ACKNOWLEDGEMENTS 6

CONTENTS 8

LIST OF ABBREVIATIONS 15

LIST OF FIGURES 20

LIST OF TABLES 26

LIST OF EQUATIONS 27

1 INTRODUCTION 31

11 INTRODUCTION TO FUEL CELLS 31

111 History of the fuel cells 31 112 Principles of the fuel cells 33 113 Electric power from the fuel cells 35

12 CATALYSTS 37

121 Faradayrsquos second law ndash Current and reaction rate37 122 Transition state theory Eyring equation and reaction rate 38 123 Overcoming the activation free energy 40 124 Variations in catalysts42

13 ELECTRODE POTENTIAL AND REACTION RATE 44

131 Fermi level electrode potential and electron transfer 44 132 Electrode potential equilibrium potential and Nernst equation47

9

133 Electrode potential and activation free energy 50 134 Potential shift and standard rate constant 52 135 Butler-Volmer equation overpotential and current 53 136 Butler-Volmer equation vs Tafel equation 55

14 OVERPOTENTIAL AND FUEL CELLS 57

141 Four overpotentials that decrease the EMF57 142 Internal current 58 143 Activation overpotential58 144 Diffusion overpotential 59 145 Resistance overpotential60 146 Total overpotential and cell voltage 62

15 BIOFUEL CELLS 63

151 Microbial and enzymatic fuel cells 63 152 Enzyme fuel cells and mediators64

16 MINIATURISED MEDICAL POWER SOURCES 66

161 Realisation of miniaturised fuel cells and medical power supply 66 162 Miniaturised potential medical power sources67

17 OUTLINE OF THE WORK 69

171 Introduction to miniaturised glucose-oxygen fuel cells 69 172 Outline71

2 ANODE INTRODUCTION 73

21 GLUCOSE OXIDATION ENZYMES 73

22 DESIGNING ENZYME ELECTRODES 77

221 Physical adsorption 78 222 Reconstituting apo-enzyme78 223 Tethered Osmium-complexes linked to conductive hydrogel 80 224 Entrapment in the conductive polymer 81 225 Bulk modification83

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties 84 226 Enzyme covalent attachment and mass loading 88

23 PROTEIN INACTIVATION AND STABILISATION 90

10

24 PROTEIN DESTABILISATION 92

241 Deamidation 92 242 Oxidation93 243 Proteolysis and racemisation94 244 Physical instabilities95

25 ENZYME STABILISERS 95

251 Polyehyleneimine (PEI) 96 252 Dithiothreitol (DTT)97 253 Polyethylene glycol (PEG)98 254 Polyols99 255 FAD100

26 ENZYME KINETICS AND ELECTROCHEMISTRY 101

261 Michaelis-Menten kinetics 101 262 Electrochemistry and Michaelis-Menten kinetics 105 263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics108

2631 Lineweaver-Burk linear regression 108 2632 Eadie-Hofstee linear regression 109 2633 Electrochemical Hanes-Woolf linear regression 110

264 Cornish-Bowden-Wharton method 112 265 Nonlinear regression methods113

3 ANODE MATERIAL AND METHOD115

31 ELECTRODE FABRICATION 115

311 TTF-TCNQ synthesis115 312 Electrode template fabrication 115 313 Electrode composite fabrication117

3131 Composite for conductance study 117 3132 Bulk modified GOx enzyme electrode composite 118 3133 GOx enzyme electrode composite with enzyme stabilisers 119

314 Electrode wiring and insulation120

32 CONDUCTIVITY EXPERIMENT 121

33 POLISHING COMPOSITE ELECTRODES 122

34 GLUCOSE AMPEROMETRY 123

341 Glucose solution123

11

342 Electrochemical system123 343 Glucose amperometry with GOx electrodes 124

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA 125

4 ANODE RESULTS AND DISCUSSION 127

41 AIMS OF ANODE STUDIES 127

42 CONDUCTANCE OF THE COMPOSITE 128

421 Conductance of various conductive materials128 422 Influence of enzyme concentration on conductivity 130

43 CATALYTIC ACTIVITY OF GOX COMPOSITE ELECTRODES 132

431 Influence of GOx concentration on the catalytic activity of GOx electrodes132 432 Influence of O2 on the catalytic activity of GOx electrodes134

44 THE EFFICACY OF GOX STABILISERS 139

441 Introduction139 442 PEI140 443 DTT142 444 PEG 143 445 GLC144 446 FAD146 447 PEI and DTT mixture147 448 PEI and FAD mixture149 449 Most effective stabiliser content for GOx anodes 150

45 CONCLUSION TO ANODE STUDY 152

5 CATHODE INTRODUCTION 155

51 OXYGEN REDUCTION REACTION (ORR) 155

52 REACTIVITY OF OXYGEN 157

53 ORR CATALYSTS 159

531 Noble metal ORR catalysts 160 532 Macrocycle and Schiff-base complex ORR catalysts 165

12

533 Multicopper enzymes as ORR catalyst 168 5331 BOD 171 5332 Lac 173 5333 CueO 173

534 Anthraquinone ORR catalyst 174

54 CARBON PROPERTIES 177

541 Graphite180 542 Glassy carbon (GC)181 543 Activated carbon (AC) 182 544 Carbon fibre (CF)182 545 Carbon nanotubes (CNTs)183 546 Boron-doped diamond (BDD)184

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON 185

551 Electrochemical properties of carbon185 552 Electrochemical pretreatments (ECPs) of the carbon185

56 ELECTROCHEMICAL TECHNIQUES FOR ORR 187

561 Cyclic voltammetry (CV)188 562 Non-faradic current and electric double layer capacitance 189 563 Diagnosis of redox reactions in cyclic voltammetry 190 564 Coupled reactions in cyclic voltammetry194 565 Voltammograms for the surface-confined redox groups 195 566 Hydrodynamic voltammetry (HDV) and Levich equation197 567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the charge

transfer rate constant 200 568 Finding the standard rate constant (kdeg)205 569 Summary of the electrochemical techniques206

57 SUMMARY OF THE CHAPTER 207

6 CATHODE MATERIAL AND METHOD 208

61 ELECTRODE FABRICATION 208

611 Graphite PtC and RhC electrodes208 612 Anthraquinone(AQ)-carbon composite electrodes208

6121 Derivatisation of graphite with AQ 208 6122 Fabrication of AQ-carbon composite electrode 209

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes209

13

6131 Information on synthesis of metallosalophen 209 6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes 210

62 COMPOSITE RDE CUTTING 210

63 ELECTRODE POLISHING 210

631 Composite RDE polishing210 632 Glassy carbon electrode polishing211

64 ENZYME RDES 212

641 Membrane preparation 212 642 Membrane enzyme electrodes212

65 ELECTROCHEMICAL EXPERIMENTS 213

651 Electrochemical system213 652 Electrode pretreatments214

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes 214 6522 Pretreatments of AQ-carbon PtC and RhC 215

653 Electrochemical experiments 215 654 Data analysis 216

7 CATHODE RESULTS AND DISCUSSION 218

71 AIMS OF THE CATHODE STUDIES 218

72 INFLUENCE AND EFFICACY OF ECPS 219

721 Glassy carbon electrodes (GCEs)219 7211 Untreated GCEs 219 7212 Influence of electrochemical pretreatments on GCE 220 7213 Effect of GCE pretreatment on ORR 227

722 Graphite composite electrodes 230 7221 Untreated graphite composite electrodes 230 7222 Influence of electrochemical pretreatments on graphite composite electrodes 232 7223 The effect of graphite composite electrode pretreatment on ORR 235

723 Platinum on carbon composite electrodes (PtC) 240 724 Rhodium on carbon composite electrodes (RhC)240

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR 241 725 Co(salophen) composite electrodes243

7251 Untreated Co(salophen) composite electrodes 243 7252 Influence of electrochemical pretreatments on Co(salophen) composite 244 7253 The effect of Co(salophen) composite electrode pretreatment on ORR 246

726 Fe(salophen) composite electrodes 248 7261 Untreated Fe(salophen) composite electrodes 248 7262 Influence of electrochemical pretreatments on Fe(salophen) composite 250

14

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR 252 727 Anthraquinone (AQ) composite electrodes253 728 Lac and BOD enzyme membrane electrodes 255

73 MOST EFFICIENT ORR CATALYSTS 257

731 Best five electrodes for nα 258 732 Best five electrodes for kdeg 259 733 Best five electrodes for Epc2(HDV) 260 734 Best three candidates for ORR catalytic electrodes 260

74 CONCLUSION TO CATHODE STUDY 262

8 CONCLUSION AND FURTHER STUDIES 264

9 BIBLIOGRAPHY 268

15

LIST OF ABBREVIATIONS

Α Transfer coefficient

A Electrode surface area

ABTS 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

AC Activated carbon

AFC Alkaline fuel cell

AQ Anthraquinone

Asn Asparagine

Asp Aspartic acid

BDD Boron-doped diamond

BOD Bilirubin oxidase

C Reactant concentration

C O (0t) Electrode surface oxidant concentration

C R (0t) Electrode surface reactant concentration

C(0t) Electrode surface concentration

C Bulk concentration

Cd Double layer capacitance

CF Carbon fibre

CNH Carbon nanohorn

CNT Carbon nanotubes

Co(salen) N-N-bis(salicylaldehyde)-ethylenediimino cobalt(II)

Co(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminocobalt (II)

CO Bulk oxidant concentration

CR Bulk reactant concentration

CueO Copper efflux oxidase

CV Cyclic voltammetry

Cys Cysteine

(DEAE)-dextran Diethylaminoethyl-dextran

DET Direct electron transfer

DHG Dehydrogenase

DMFC Direct methanol fuel cell

DTE Dithioerythriotol

DTT Dithiothreitol

[E0] Initial enzyme concentration

16

E Arbitrary electrode potential Enzyme

Edeg Standard electrode potential

Edeg Formal potential

Ea Anode potential

EC Electrochemical-chemical

Ec Cathode potential

Ecell Cell voltage

ECP Electrochemical pretreatment

Ee Electrode equilibrium potential

EF Fermi potential

EMF Electro motive force

Ep2 Half-peak potential

Epa Anodic peak potential

Epc Cathodic peak potential

Epc2(HDV) Half-wave reduction potential in HDV

ES Enzyme-substrate complex

Eλ Switching potential

∆E Potential difference Potential shift

∆Edeg Standard peak separation

δEp Peak width

εF Fermi energy

F Faraday constant

FAD Flavin adenine dinucleotide

Fe(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminoiron (II)

GC Glassy carbon

GCE Glassy carbon electrode

GHP 1-(6-D-gluconamidohexyl) pyrrole

GLC Glycerol

Gln Glutamine

GOx Glucose oxidase

GOx(O) Oxidised GOx

GOx(R) Reduced GOx

GSH Gluthatione

∆G Gibbs free energy shift

∆Gdeg Standard Gibbs free energy change

∆GdegDagger Standard activation free energy

17

∆GDagger Gibbs free energy of activation

∆GadegDagger Anode standard activation free energy

∆GaDagger Anode activation free energy

∆GodegDagger Oxidation standard activation free energy

∆GoDagger Oxidation activation free energy

∆GRdegDagger Reduction standard activation free energy

∆GRDagger Reduction activation free energy

h Planck constant

HDA Hydrodynamic amperometry

HDV Hydrodynamic voltammetry

His Histidine

HOMO Highest occupied molecular orbital

∆Hdeg Standard enthalpy change

I Electric current Net current

I0 Exchange current

Ia Anodic current

Ic Cathodic current

Id Disc limiting current

IK Kinetic current

IL Limiting current Diffusion-limited current

Im Maximum steady state limiting current

Ip Peak current

Ipa Anodic peak current

Ipc Cathodic peak current

I-V Current-Voltage

J Current density

Jm Maximum limiting current density

k Rate constant

K Equilibrium constant

k(E) Rate constant at the applied potential E

kdeg Standard heterogeneous rate constant

kdegb Backward reaction standard rate constant

kdegf Forward reaction standard rate constant

KDagger The equilibrium constant between the activated complexes and the reactants

k1 Rate constant for ES formation from E and S

k-1 Rate constant for ES dissociation into E + S

18

k2 Rate constant for P formation from ES

kB Boltzmann constant

kb Backward reaction rate constant

kf Forward reaction rate constant

kf(E) Rate constant for forward reactions at the applied potential E

KM Michaelis enzyme dissociation constant

Lac Laccase

LDH Lactate dehydrogenase

LMCT Ligand-to-metal charge transfer

LPG Liquefied petroleum gas

LUMO Lowest unoccupied molecular orbital

MCO Multicopper oxidase

MDH Methanol dehydrogenase

Me2SQ Dimethylmercaptobenzoquinone

MEMS Microelectromechanical systems

MeSQ Methylmercapttobenzoquinone

MET Mediated electron transfer

Met Methionine

mPQQ-GDH Membrane-bound PQQ dehydrogenase

MWNT Multi-walled nanotube

n Overall number of electrons transferred

na The number of electrons transferred up to the rate limiting step

NAD Nicotinamide adenine dinucleotide

NAD-GDH Nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

OCV Open circuit voltage

ORR Oxygen reduction reaction

P Electric power Product

PAFC Phosphoric acid fuel cell

PBS Phosphate buffer saline

PC Pyrolytic carbon

PDMS Polydimethylsiloxane

PEFC Polymer electrolyte fuel cell

PEG Polyethylene glycol

PEI Polyethyleneimine

P-I Power-Current

POx Pyranose oxidase

19

PQ Phenanthrenequinone

PQQ pyrrolo-quinoline quinone

PQQ-GDH Quinoprotein glucose dehydrogenase

Pro Proline

PtC Platinum on carbon

Q Charge

RDE Rotating disc electrode

RhC Rhodium on carbon

RLS Rate limiting step

rpm Rotaion per minute

S Substrate

SEN Single-enzyme nanoparticle

sPQQ-GDH Soluble PQQ dehydrogenase

SQ Mercaptobenzoquinone

SWNT Single-walled nanotube

∆Sdeg Standard entropy change

t Time

T1 Type I (blue Cu in Lac)

T2 Type II ( trinuclear Cu in Lac)

T3 Type III (trinuclear Cu in Lac)

TCNQ 7788-Tetracyanoquinodimethane

TEM Transmission electron microscopy

Trp Tryptophan

TTF Tetrathiafulvalene

TTF-TCNQ Tetrathiafulvalene 7788-tetracyanoquinodimethane

Tyr Tyrosine

V Voltage Maximum limiting catalytic velocity

VHT Vacuum heat treatment

η Overpotential

ν Vibrational frequency Kinematic viscosity

υ Reaction rate Scan rate Enzyme catalytic rate

ω Angular velocity

20

LIST OF FIGURES

Fig No Page Description

Fig 1 32 Historical fuel cells

Fig 2 33 Photographs of modern fuel cells

DMFC Cogeneration system Hydrogen car Glucose-Oxygen fuel cell

Fig 3 34 The schema of glucose-oxygen fuel cell

Fig 4 41 Transition state diagram with the reaction coordinate

Fig 5 45 Fermi level and electron transfer

Fig 6 46 Fermi level and electron transfer at an arbitrary electrode potential

Fig 7 48 Standard free energy change along the electrode potential at

(a) E = Edeg (b) E gt Edeg and (c) E lt Edeg

Fig 8 51 The shift in ∆GdegDagger accompanied by ∆E

Fig 9 57 Tafel plot

Fig 10 59 Activation overpotential vs current

Fig 11 60 Diffusion overpotential vs current

Fig 12 61 Resistance overpotential vs current

Fig 13 62 V-I curve for a typical low temperature fuel cell

Fig 14 65 Mediated electron transfer reaction by the enzyme electrode

Fig 15 70 The schema for a glucose-oxidising anode

Fig 16 75 The structure and redox reactions of FAD NAD and PQQ

Fig 17 79 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode

Fig 18 81 GOx wired to conductive hydrogel complexed with Os

Fig 19 83 Monomer structures of conductive polymers

1-(6-D-gluconamidohexyl) pyrrole and Mercapto-p-benzoquinone

Fig 20 85 TTF-TCNQ structure and its charge transfer

Fig 21 86 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 20 mVs-1

Fig 22 89 The procedure for the enzyme mass-loaded biocatalytic nanofibre

Fig 23 90 The fabrication procedure of single-enzyme nanoparticles

Fig 24 92 The chemical structures of the side groups of Gln and Asn

Fig 25 93 The chemical structures of the side groups of Cys Met Tyr Trp and His

Fig 26 95 The chemical structures of Asp and Pro

Fig 27 96 The structure of PEI

Fig 28 98 The structures of DTT from a linear to a cyclic disulfide form

Fig 29 98 The structures of PEG

21

Fig 30 100 The structure of glycerol

Fig 31 104 A typical Michaelis-Menten hyperbolic curve of the enzyme

Fig 32 107 Electrochemical Michaelis-Menten hyperbolic curve of the enzyme electrode

Fig 33 109 Electrochemical Lineweaver-Burk linear regression of the enzyme electrode

Fig 34 110 Electrochemical Eadie-Hofstee linear regression of the enzyme electrode

Fig 35 111 Electrochemical Hanes-Woolf linear regression of the enzyme electrode

Fig 36 113 Electrochemical Cornish-Bowden-Wharton plot of the enzyme electrode

Fig 37 116 The photographs of PDMS electrode templates

Fig 38 118 The photographs of empty and filled PDMS templates

Fig 39 119 The schema for bulk modification method

Fig 40 121 The photographs of insulated composite electrodes

Fig 41 122 The photographs of measuring electrode resistance

Fig 42 125 The photograph of glucose amperometry with a multichannel potentiostat

Fig 43 126 The analytical output by SigmaPlot Enzyme Kinetics

Fig 44 129 Conductance of various electrode composite

Fig 45 131 Conductance of GOx composite with varying GOx content

Fig 46 133 Difference in the glucose-response amperometric curve by the enzyme electrodes

containing various GOx content 5 10 and 15 in the absence of O2

Fig 47 134 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the absence of O2

Fig 48 135 (i) Difference in the glucose-response amperometric curve by 10 GOx composite

electrodes in the absence and presence of O2

(ii) The re-plotted amperometric curves in the presence of N2 air and O2

Fig 49 136 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the presence N2 O2 and air

Fig 50 140 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEI in the presence and absence of O2

Fig 51 142 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 DTT in the presence and absence of O2

Fig 52 145 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEG in the presence and absence of O2

Fig 53 146 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 GLC in the presence and absence of O2

Fig 54 147 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 FAD in the presence and absence of O2

22

Fig 55 143 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-DTT

(051 11 1505 and 22) in the presence and absence of O2

Fig 56 148 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051 11

1505 and 22)

Fig 57 149 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-FAD (21

22 and 24 ) in the presence and absence of O2

Fig 58 151 Jm KM in the absence and presence of O2 for various stabilisers

PEIDTT (11 0515 221505 ) and GLC 1

Fig 59 156 Oxygen reduction pathways 2 and 4 electron reduction pathways

Fig 60 157 Degradation of superoxide anion radical in aqueous solutions

Fig 61 158 An energy level diagram for ground state dioxygen molecule

Fig 62 159 Diagrams for the state of π orbitals in the ground state oxygen and peroxide anion

radical

Fig 63 162 Three models of oxygen adsorption Griffith Pauling and Bridge models

Fig 64 163 Models for oxygen adsorption on metals and ORR pathways

Fig 65 164 A volcano-shaped trend between ORR rate and oxygen binding energy of various

transition metals

Fig 66 165 Examples of transition metallomacrocycles Heme and copper phthalocyanine

Fig 67 166 Schiff base complexes

Co(II) salen Co(II) salophen and Co(III) salen conductive polymer

Fig 68 167 The molecular structure of dicobalt face-to-face diporphyrin [Co2(FTF4)]

Fig 69 168 ORR pathways by Co2(FTF4)

Fig 70 170 The structure of laccase from Trametes versicolor

Fig 71 171 Redox reaction cycle of MCOs with O2

Fig 72 172 Common mediators for BOD ABTS and Os complex

Fig 73 174 Two-electron ORR by anthraquinone

Fig 74 176 The structures of anthraquinone and its derivative Fast red AL salt

Fig 75 177 HDV of ORR at various quinone electrodes at pH 7

Unmodified GC AQGC and PQGC

Fig 76 178 The carbon structures graphite and diamond

Fig 77 179 The carbon sections basal and edge planes

Fig 78 181 A TEM image of graphene bilayer at 106 Aring resolution

Fig 79 182 A TEM image and a schema of GC

Fig 80 183 Computer graphic models of various CNTs Bundled SWNTs MWNT and CNHs

23

Fig 81 184 A TEM image of diamond

Fig 82 187 The CVs showing the influence of ECP on GCE at pH 7 in the presence and absence

of O2 The CVs for both pretreated and unpretreated GCEs are shown

Fig 83 188 The triangular waveform for CV and a typical voltammetric curves for BQ

Fig 84 189 The CV showing the double layer capacitance at graphite electrode surface at pH 7

Fig 85 194 A voltammogram examples of a quasi-reversible reaction at various scan rates

Fig 86 195 Voltammograms of two-electron transfer in the EE systems with different ∆Edegrsquo

Fig 87 196 A voltammogram of the reversible redox reaction by surface-confined groups

Fig 88 198 A diagram of hydrodynamic electrochemical method using a RDE

Fig 89 200 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH 74

Fig 90 201 Levich plot examples for reversible and quasi-reversible systems

Fig 91 203 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in oxygenated

001 M PBS at pH 74 at pretreated GCE

Fig 92 204 Koutecky-Levich plot for O2 reduction at a pretreated GCE in oxygenated 001 M PBS at

pH 74 with applied potential ndash 04 06 and 08 V

Fig 93 206 Log kf(E) plot for O2 reduction at various pretreated GCEs

Fig 94 207 The protocol for finding kinetic and thermodynamic parameters

Fig 95 211 The photographs of fabricated composite RDEs

Fig 96 213 The photograph of RDE experiment setting with a rotor and gas inlet

Fig 97 220 CVs of untreated GCEs at pH 74 in 001 M PBS in the absence and presence of O2

at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 98 221 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of O2

between + 09 and - 09 V vs AgAgCl at 005 Vs-1

(i) CVs for all the pretreated GCEs

(ii) CVs excludes PBS pretreatments

Fig 99 223 The bar graphs for the capacitance of GCEs in 015 M NaCl buffered with 001 M

phosphate to pH 74

Fig 100 225 Overlaid CVs during various pretreatments for GCEs involving

01 M H2SO4 alone 001 M PBS at pH 74 alone and both pretreatments

Fig 101 227 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M oxygenated

PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 102 229 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Fig 103 231 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M PBS in

the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

24

Fig 104 233 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M N2-sparged PBS

between + 09 and - 09 V vs AgAgCl

Fig 105 236 Overlaid CVs of ORR at various pretreated graphite composite electrodes at pH 74

in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 106 237 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at pH 74

in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various scan rates

50 100 200 and 500 mVs-1

Fig 107 238 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

Fig 108 239 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

Fig 109 241 CVs of untreated and H2SO4-pretreated RhC composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

Fig 110 242 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M PBS in

the absence and presence O2 at 005 Vs-1 between + 09 and - 09 V vs AgAgCl

Fig 111 243 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M PBS in the

absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 112 245 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 113 247 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 114 248 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite electrodes

Fig 115 249 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

Fig 116 251 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 117 253 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 118 254 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M PBS in

theabsence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 119 256 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated PBS at

005 Vs-1 between 0 and -09 V vs AgAgCl

Fig 120 257 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Fig 121 258 The bar graphs of the best five nα

25

Fig 122 259 The bar graphs of the best five kdeg

Fig 123 260 The bar graphs of the best five Epc2(HDV)

Fig 124 266 A photograph of the fuel cell stack made with GOx anodes and Co(salophen)

cathodes

26

LIST OF TABLES

Table No Page Description

Table 1 35 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Table 2 87 Electrochemical behaviour of TTF-TCNQ at pH 74

Table 3 104 Michaelis-Menten equation at various [S]

Table 4 117 Composite composition table

Table 5 119 Blended stabiliser composition

Table 6 139 Four possible causes of glucose amperometric current reduction

Table 7 152 Comparison of maximum output current and KM with some published data

Table 8 159 Bond orders bond lengths and bond energies of some dioxygen species

Table 9 180 The electric conductivity of various carbon forms and sections

Table 10 186 Various ECP examples

Table 11 192 CV diagnosis for reversible system at 25 ˚C

Table 12 193 CV diagnosis for irreversible system at 25 ˚C

Table 13 196 CV diagnosis for reversible system by surface-confined groups at 25 ˚C

Table 14 214 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Table 15 216 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC

Table 16 217 Parameter values for Koutecky-Levich equation

O2 diffusion coefficient O2 concentration and PBS kinematic viscosity

Table 17 261 Three candidate electrodes for high ORR catalysis

Table 18 261 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

27

LIST OF EQUATIONS

Eq No Page Description

Eq 1 35 Anode glucose oxidation reaction

Eq 2 35 Cathode oxygen reduction reaction

Eq 3 35 Overall glucose-oxygen fuel cell reaction

Eq 4 36 Electric power equation

Eq 5 36 Electric current equation in electrochemistry

Eq 6 38 Faradayrsquos second law of electrolysis Charge equation

Eq 7 38 Faradayrsquos second law of electrolysis Reaction equation

Eq 8 39 Transition state theory equation Rate constant equation

Eq 9 39 Transition state theory equation Equilibrium constant

Eq 10 39 Transition state theory equation Atomic vibration frequency equation

Eq 11 40 Transition state theory equation Erying equation

Eq 12 42 Gibbs free energy shift equation in thermodynamic terms

Eq 13 42 Gibbs free energy shift equation in electrochemical terms

Eq 14 49 A typical redox reaction

Eq 15 50 Nernst equation

Eq 16 50 Electrode potential shift

Eq 17 50 Free energy shift in electrochemical terms

Eq 18 52 Anodic oxidation activation free energy shift in terms of potential shift

Eq 19 52 Cathodic reduction activation free energy shift in terms of potential shift

Eq 20 53 Forward reduction rate constant equation in terms of kdeg and potential shift

Eq 21 53 Backward oxidation rate constant equation in terms of kdeg and potential shift

Eq 22 54 Butler-Volmer equation

Eq 23 54 Overpotential equation

Eq 24 54 Exchange current equation

Eq 25 55 Butler-Volmer equation in terms of electrode equilibrium constant

Eq 26 55 Butler-Volmer equation in terms of overpotential

Eq 27 56 Butler-Volmer equation for small overpotential

Eq 28 56 Tafel equation for large overpotential

Eq 29 60 Diffusion overpotential equation

Eq 30 61 Ohmrsquos law

Eq 31 63 Cell voltage equation

Eq 32 63 Gibbs free energy available from the fuel cell

28

Eq 33 73 Glucose oxidation reaction by GOx

Eq 34 73 Glucose oxidation reaction by NAD-DHG

Eq 35 87 TTF-TCNQ reaction At equilibrium state

Eq 36 87 TTF-TCNQ reaction TCNQ reduction at lt + 015 V vs AgAgCl

Eq 37 87 TTF-TCNQ reaction TCNQ lattice reduction at lt - 015 V vs AgAgCl

Eq 38 87 TTF-TCNQ reaction Anodic TCNQ salt formation at gt + 031 V vs AgAgCl

Eq 39 87 TTF-TCNQ reaction TTF lattice oxidation at gt + 035 V vs AgAgCl

Eq 40 87 TTF-TCNQ reaction TTF further oxidation at gt + 038 V vs AgAgCl

Eq 41 101 Enzyme kinetics A typical enzyme reaction

Eq 42 102 Enzyme kinetics Enzyme-substrate complex formation at steady state

Eq 43 102 Enzyme kinetics Rate of enzyme-substrate complex formation in terms of k-1 and k2

Eq 44 102 Enzyme kinetics

Enzyme-substrate complex equation in terms of [E0] k1 k-1 k2 and [S]

Eq 45 102 Enzyme kinetics Enzyme catalytic reaction

Eq 46 102 Enzyme kinetics Enzyme catalytic reaction equation

Eq 47 102 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of enzyme-substrate complex

Eq 48 103 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of [E0] k1 k-1 k2 and [S]

Eq 49 103 Enzyme kinetics Michaelis-Menten constant definition

Eq 50 103 Michaelis-Menten equation

Eq 51 104 Michaelis-Menten equation when [S] ge KM

Eq 52 104 Michaelis-Menten equation when [S] = KM

Eq 53 104 Michaelis-Menten equation when [S] le KM

Eq 54 106 Electrochemical Michaelis-Menten equation

Eq 55 106 Electrochemical Michaelis-Menten equation for current density

Eq 56 108 Electrochemical Lineweaver-Burk linear regression equation

Eq 57 109 Electrochemical Eadie-Hofstee linear regression equation

Eq 58 111 Electrochemical Hanes-Woolf linear regression equation

Eq 59 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for Jm

Eq 60 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for KM

Eq 61 128 Enzyme electrode specificity constant

Eq 62 139 The causes of anodic current reduction A typical GOx-O2 reaction

Eq 63 139 The causes of anodic current reduction TTF reduction by GOx

Eq 64 139 The causes of anodic current reduction GOx allosteric inhibition

Eq 65 139 The causes of anodic current reduction TTF oxidation by O2

29

Eq 66 139 The causes of anodic current reduction TTF oxidation by GOx

Eq 67 161 O2 Dissociative mechanism Simultaneous oxygen adsorption and molecular fission

Eq 68 161 O2 Dissociative mechanism Surface hydroxide formation

Eq 69 161 O2 Dissociative mechanism Oxygen reduction to water

Eq 70 161 O2 Associative mechanism Oxygen adsorption

Eq 71 161 O2 Associative mechanism Peroxide formation

Eq 72 161 O2 Associative mechanism Peroxide reduction

Eq 73 161 O2 Associative mechanism Surface oxygen reduction to hydroxide

Eq 74 161 O2 Associative mechanism Surface hydroxide reduction to water

Eq 75 175 Anthraquinone O2 reduction Semiquinone anion radical formation

Eq 76 175 Anthraquinone O2 reduction O2 reduction by a semiquinone anion radical

Eq 77 175 Anthraquinone O2 reduction Spontaneous superoxide reduction to O2

Eq 78 175 Anthraquinone O2 reduction

Spontaneous superoxide reduction to hydroperoxide

Eq 79 190 Electric double layer capacitance per unit area

Eq 80 192 CV diagnosis ndash Reversible system Ip vs υfrac12

Eq 81 192 CV diagnosis ndash Reversible system Ep

Eq 82 192 CV diagnosis ndash Reversible system ∆Ep

Eq 83 192 CV diagnosis ndash Reversible system δEp

Eq 84 192 CV diagnosis ndash Reversible system Reversal parameter

Eq 85 193 CV diagnosis ndash Irreversible system Ip vs υ

Eq 86 193 CV diagnosis ndash Irreversible system Ep a function of kdeg α and υ

Eq 87 193 CV diagnosis ndash Irreversible system ∆Ep

Eq 88 193 CV diagnosis ndash Irreversible system δEp

Eq 89 195 CV diagnosis ndash EE system Peak separation of EE system

Eq 90 196 CV diagnosis ndash Surface-confined groups Ip vs υ

Eq 91 196 CV diagnosis ndash Surface-confined groups Ep

Eq 92 196 CV diagnosis ndash Surface-confined groups ∆Ep2

Eq 93 198 Diffusion layer thickness in terms of ω

Eq 94 199 Levich equation in terms of ω

Eq 95 199 Levich equation in terms of diffusion layer thickness

Eq 96 202 Koutecky-Levich equation

Eq 97 204 The rate constant for arbitrary potential E

Eq 98 205 k(E) in terms of kdeg and η for a forward reaction

Eq 99 205 kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

Eq 100 238 Surface semiquinone radical formation

30

Eq 101 238 ORR by surface semiquinone radical

31

1 INTRODUCTION

11 INTRODUCTION TO FUEL CELLS

111 History of the fuel cells

Fuel cells are electrochemical devices which directly convert fuel into

electricity via chemical reactions The invention dates back to 19th century when a

British physicist Sir William Grove and a German-Swiss chemist Christian Schoumlnbein

discovered the reverse reaction of water electrolysis The current output by this system

was however very small Therefore the discovery stayed in the shadow of the steam and

internal combustion engines and it needed another century to gain its attention

In the 1930s a British engineer Francis Bacon started working towards

alkaline fuel cells (AFCs) and in the 1950s General Electric developed polymer

electrolyte fuel cells (PEFCs) Those fuel cells were loaded on the spaceships in the

1960s by NASA because they were compact provided clean power with large energy

density and generated drinkable water as a by-product [1-5] Since then fuel cells have

been continuously used for space and marine technologies [3] In the 1970s studies on

fuel cells were further expanded for consumer applications [1 3 5 6] The first

air-breathing fuel cell hybrid car was built by Kordesch [1 7] and phosphoric acid fuel

cells (PAFCs) were developed and introduced to offices and hospitals [2 3 5]

32

Fig 1 Some historical fuel cell applications (i) Alkaline fuel cell for Apollo 11 [8] (ii)

Kordeschrsquos Austin A40 city hybrid car [7]

Since the 1990s fuel cells have been drawing keen attention as flexible renewable

energy in conjunction with the depletion of fossil fuel global warming and the demand

in cleaner energy [6 9] Great effort has been made to miniaturise fuel cells for

individual users such as mobile devices vehicles and households [1 3 5 6] The recent

advance in such technologies has led to direct methanol fuel cells (DMFCs) for

mobile-phone handsets [10 11] cogeneration systems to supply household power [1 5]

hydrogen fuel cell cars [12-15] and bio-batteries running on glucose and air oxygen for

portable music players [16-18] Glucose-oxygen biofuel cells have been also further

miniaturised for medical implantable purposes [18 19]

33

Fig 2 Modern fuel cell technologies (i) Toshibarsquos world smallest DMFC [20] (ii)

TokyoGasrsquos cogeneration system Enefarm (iii) BMWrsquos Hydrogen 7 (iV)

Sonyrsquos Bio Battery generating electricity from sugar [16]

112 Principles of the fuel cells

A fuel cell unit simply consists of an anode a cathode electrolyte and a

conducting wire A conducting wire connects an anode and a cathode to form an

external circuit This allows coupling of electrochemical reactions taking place at each

electrode and provides a conductive path for electrons between them Electrolyte

allows charged species to move between two electrodes but blocks out the entry of

electrons This completes the electric circuit and permits electric conduction At the

anode fuel is oxidised by an oxidation reaction and this supplies electrons to the

cathode through the external circuit The number of electrons generated depends on the

34

type of fuels reactions and environment in which the reactions take place In the anode

reaction ions are also released to balance overall charge They move to the cathode

though the electrolyte At the cathode ions from the electrolyte and electrons from the

circuit meet and react together with an oxidiser supplied The completion of the coupled

reactions generate electricity and products [3 4 21] Fig 3 shows a glucose-oxygen

fuel cell as an example

Fig 3 A glucose-oxygen fuel cell

At the anode glucose is oxidised into gluconolactone This gives out two electrons and

two protons At the cathode oxygen gas receives them and is reduced to water Each

reaction has a unique redox potential called standard electrode potential Edeg and

electromotive force (EMF) is the potential difference between such electrode potentials

It is also called open circuit voltage (OCV) This is the theoretical size of potential

difference available from the fuel cell [3 4] The coupled reactions for glucose-oxygen

fuel cell is shown in Table 1 Each redox potential was measured against silver

35

silver-chloride reference electrode (AgAgCl) at pH 7 25degC therefore shown in formal

potential (Edegrsquo)

Table 1 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Anode Edegrsquo = - 042 V (Eq 1)

(via FADFADH2 redox [22 23])

Cathode [24] Edegrsquo = 062 V (Eq 2)

Overall ∆Edegrsquo = 104 V (Eq 3)

The reaction potential at each electrode is measured separately as half cell potential

against the universal reference electrode such as AgAgCl because it is impossible to

directly measure both inner potentials simultaneously

113 Electric power from the fuel cells

Electric power (P) is the amount of work can be handled by electric current per

unit time It is defined as in Eq 33 The size of power is determined by the size of

electric current (I) and voltage (V) since P is proportional to I and P

36

Electric power

VIP times= middotmiddotmiddotmiddotmiddot Eq 4

P = Electric power W

I = Electric current A

V = Voltage V

Electric current is the amount of charge (Q) flows per unit time Voltage is the electric

potential energy per unit charge and synonymous with potential difference (∆E) EMF

or OCV Increase in I andor V increases P V is however constrained by the type of

electrode reactions as discussed previously I depends on (i) reaction rate (ii) the

electrode surface and (iii) reactant concentration as shown in Eq 5

Electric current in electrochemistry

nFAkCnFAI == υ middotmiddotmiddotmiddotmiddot Eq 5

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

A = Electrode surface area m-2

υ = Reaction rate (1st or pseudo 1st order) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

It is indispensable to have large rate constant to increase output current This demands

lowering activation energy for a chemical transition during the reaction and can be

achieved by use of the catalysts

37

12 CATALYSTS

121 Faradayrsquos second law ndash Current and reaction rate

Catalysts accelerate specific chemical reactions without altering the change in

overall standard Gibbs free energy In other words they change the reaction kinetics but

not the thermodynamics They only provide an alternative lower-energy pathway to

shorten the reaction time without changing themselves before and after the reaction

[25-27] In fuel cells catalysts are used to increase the current output by accelerating the

electrode reaction The relationship between current and rate of reaction is defined by

Faradayrsquos second law of electrolysis (Eq 5 - Eq 7) This expresses the size of electric

charge generated in terms of the number of moles of reactant consumed or product

produced

38

Faradayrsquos second law of electrolysis

nFNQ = middotmiddotmiddotmiddotmiddot Eq 6

AtNCktimes

=times=υ middotmiddotmiddotmiddotmiddot Eq 7

Q = Charge C

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

N = The number of moles of reactant or product mol

υ = Reaction rate (1st or pseudo 1st ordedr) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

t = Time s

A = Electrode surface area m-2

Faradayrsquos second law of electrolysis states that an increase in reaction rate increases the

current output To increase the reaction rate intrinsically its Gibbs free energy of

activation (∆GDagger) must be lowered The relationship between k and ∆GDagger is stated in

Erying equation [28]

122 Transition state theory Eyring equation and reaction rate

When a reaction proceeds it must go through a critical state called transition

state of maximum potential energy also called the activated complex Activated

complexes are unstable molecules existing for only a few molecular vibrations before

decaying into the products Transition state theory assumes that the reactants and the

39

activated complexes are in quasi-equilibrium and the reaction rate is constrained by the

rate at which the activated complex decays into the products This implies that the

reaction rate depends on the equilibrium constant between the activated complexes and

the reactants (KDagger) and the atomic vibrational frequency (ν) as shown in Eq 9 ν is

defined by statistical mechanics in terms of Boltzmann constant (kB) Planck constant

(h) and the temperature (Eq 10) [28]

Transition state theory and reaction rate [28]

[ ]RTexpDagger Dagger

Dagger GC

CK ∆minus== middotmiddotmiddotmiddotmiddot Eq 8

Dagger Kk ν= middotmiddotmiddotmiddotmiddot Eq 9

hTkB=ν middotmiddotmiddotmiddotmiddot Eq 10

KDagger = Equilibrium constant between the reactants and the activated

complexes

s-1

CDagger = Activated complex concentration molm-3

C = Reactant concentration molm-3

∆GDagger = Gibbs free energy of activation Jmol-1

R = Gas constant = 8314 Jmol-1K-1

T = Thermodynamic temperature K

k = Rate constant (1st or pseudo 1st reaction) s-1

ν = Atomic vibrational frequency s-1

kB = Boltzmann constant = 13807 x 10-23 JK-1

h = Planck constant = 662621 x 10-34 Js

40

Based on this the Erying equation expresses the relationship between the reaction rate

and Gibbs activation free energy as shown in Eq 11 [25 28 29]

Erying equation TG

B

hTkk

RDagger ∆minus

= κ middotmiddotmiddotmiddotmiddot Eq 11

where κ = transmission coefficient

This tells that to increase the rate constant reaction temperature must be increased or

the activation free energy must be lowered

123 Overcoming the activation free energy

Activation free energy is a potential energy gap between the reactant and

activated complex At the transition state intra-molecular reorganisation takes place

through the bond destruction and formation This demands the energy and slows the

kinetics The reaction does not proceed unless this energy barrier is overcome Hence

energy input in someway or lowering activation free energy is required for the reaction

to proceed andor accelerate the reaction Heat energy can provide atomic and molecular

kinetic energy so that the system can overcome the energy barrier as indicated in Eq 11

[4 21 29] Applying electric potential also has the similar effect It provides the electric

energy to overcome the energy barrier The rate constant therefore can be altered

extrinsically by temperature andor electrode potential [21 29 30] Alternatively

catalysts can reduce ∆GDagger so that the reaction rate can be increased intrinsically They

enable or accelerate a reaction by providing alternative lower-energy reaction pathway

41

This does not change the reaction path or the standard enthalpy change (∆Hdeg) of the

system however lowers the activation free energy for this path so that the reactants can

be converted more quickly to the products as shown in Fig 4

Fig 4 Energy transition for catalytic reaction through the transition state

Fig 4 shows the energy state transition during a reaction with and without a catalyst

The reaction coordinate represents the reaction progress along the reaction pathway

∆Hdeg is a state function [31] therefore determined only by the initial and final state of

the system but independent of the reaction path ∆Hdeg is divided into standard Gibbs free

energy change (∆Gdeg) and the term of standard entropy change (∆Sdeg) as Eq 12 shows [4

21 23 26 32]

The key insight therefore from transition state theory is that for a catalyst to function it

42

must spontaneously bind the transition state

Gibbs free energy change

deg∆minusdeg∆=deg∆ STHG middotmiddotmiddotmiddotmiddot Eq 12

deg∆minus= EnF middotmiddotmiddotmiddotmiddot Eq 13

∆Gdeg is the standard free energy difference which became available for work through

the reaction and must be lt 0 for a spontaneous reaction ∆Gdeg can be related to the

standard electrical potential difference as Eq 13 shows [4 21 29 30 33 34] It is the

maximum EMF theoretically available from the system ∆Sdeg is the difference in the

degree of disorder in the system The entropy term (T∆Sdeg) is the energy not available for

work and temperature dependent This and Eq 13 imply that ∆Gdeg is also altered by

temperature as shown in Fig 4 [35]

124 Variations in catalysts

Given the general applicability of transition state theory it is not surprising that

catalysts have extensive variation and applications the reaction phase can be

homogeneous or heterogeneous catalysts can be inorganic organic or biological

Inorganic catalysts are metals such as transition or noble metals or complexes

on metal ions in various oxidation states Elemental metal catalysts typically catalyse

reactions through surface adsorption Metal particles have extensive variation in

physical properties such as particle size shape and surface area They often come in

dispersed on the porous support material for improved catalytic performance due to

43

increased surface area [36] and cost efficiency [1] Composition and structure of the

particles thickness of support material and condition of metal deposition all affect

reactions [27 37-39] A complex is a compound in which a ligand is loosely bonded to

a metal centre through coordination bonds An unsaturated complex has a dissociated

ligand and catalysis takes place when a substrate binds to that in the single orientation

[27 40] Complexes also have important roles in biological system [41-43] Organic

catalysts are non-metal non-biological catalysts They are often functionalised solid

surfaces for surface catalysis [27] In electrochemistry such catalysts can be chemically

bonded to conductive material [44] Simple electrochemical pretreatment can generate

or attach chemical groups onto the electrode surface and they mediate electron transfer

[45-47] Biological catalysts are generally called enzymes Enzymes are biological

polymers mostly made of protein and usually have high reaction and substrate

specificity They often have cofactors which are not proteinous but essential for their

catalytic activities Enzymes are involved in virtually all biological activities [23 25 27

48] and in a few industrial applications due to their substrate specificity [27]

Catalysts are by definition not supposed to be consumed in their reactions

however they could lose their catalytic power Impurities fouling and poisoning are

common deactivating factors for the metal catalysts Competing reactions also convert

them into inactive forms Particle migration and agglomeration reduce their catalytic

surface area hence their overall activity [27] Biological catalysts are very sensitive to

the environment Slight changes in pH and temperature can deactivate enzymes Their

stability is also affected by presence of metal ions surfactants oxidative stress and

organic solvent molecules [49] Metal catalysts can be possibly regenerated by

redispersion [27] however it is very difficult to recover biological catalytic functions

44

Therefore enzyme stabilisers are often added to extend their life Protein engineering

also can confer advantageous properties to the enzymes Amino acid sequences leading

to favourable protein folding functions or stability are determined in silico and they are

introduced to existing enzymes by genetic engineering such as site-directed mutagenesis

Protein engineering of serine protease subtilisin is a classic example [50]

13 ELECTRODE POTENTIAL AND REACTION RATE

131 Fermi level electrode potential and electron transfer

In the sect 123 the relationship between the electric potential and activation free

energy was briefly discussed As heat energy can provide the kinetic energy to atoms

and molecules for a reaction to proceed the electric energy also can provide kinetic

energy to electrons for electron transfer to proceed This leads to the redox reaction at

the electrode surface

Each substance has a set of discrete energy levels Each energy level can

accommodate up to two electrons Electrons fill these energy levels in ascending order

from the lower level This can divide these energy levels into two groups electron-filled

lower energy level and empty higher energy level without electrons In between the

critical energy level the called Fermi level lies It is designated as εF (Fermi energy) or

EF (Fermi potential) The Fermi level contains electrons of highest energy A substance

can transfer only these electrons to the external system as a donor while as an acceptor

it can accommodate external electrons only within the empty energy level which is

above its Fermi level (Fig 5)

45

Fig 5 Fermi level and electron transfer

The energy state of electrons however varies by substance therefore electrons at Fermi

level so-called highest occupied molecular orbital (HOMO) of a donor may need to

gain various kinetic energy to jump into the lowest empty energy level so-called lowest

unoccupied molecular orbital (LUMO) of an acceptor for electron transfer (Fig 6)

46

Fig 6 Electrode potential and electron transfer

As soon as the energy of electrons at Fermi level of a substance or an electrode meets

the energy level of LUMO of the other redox molecule electron transfer proceeds as

long as there is room to accommodate electrons in the acceptor This is the cathode

reaction The potential can be swept back by applying positive potential to the electrode

This lowers the energy of LUMO of the electrode so that an electron in HOMO of the

donor can be transferred back to the electrode This is the anode reaction If the electron

transfer is fast in both directions compared with other reaction steps it is a

thermodynamically reversible system and the standard electrode potential (Edeg) for this

redox reaction lies somewhere between and close to those two electrode potentials

which have enabled electron transfer At Edeg both anode and cathode reactions have the

same reaction rate hence they are at equilibrium [33] and there is no net current [21 30]

47

132 Electrode potential equilibrium potential and Nernst equation

Activation free energy of an electrode reaction can be altered by applying

potential The extent of its alteration and the direction of the reaction depend on the size

and direction of potential applied Fig 7 shows the difference in the standard activation

free energy (∆GdegDagger) for redox reactions at various electrode potentials [30]

48

Fig 7 Standard free energy and electrode potential [30]

(a) At E = Edeg (b) E gt Edeg and (c) E lt Edeg

More positive potential lowers the activation free energy for oxidation hence shifts the

reaction in favour of oxidation and vice versa This implies that applying extra potential

to the electrode perturbs the equilibrium and to restore it the electrode equilibrium

49

potential (Ee) must be shifted This phenomenon is clearly stated in Nernst equation (Eq

15 on p 50) as a relationship between Ee and the equilibrium constant (K) at the

electrode surface At equilibrium both oxidation and reduction reactions take place at

the same rate and there is no net current therefore the surface concentration (C(0t))

approximates bulk concentration (C) Nernst equation shows that Ee is always under

the influence of such concentration but can be established provided the reaction is fast

enough to restore the equilibrium [21 30] In the dilute solutions of the electroactive

species Edeg is substituted with the formal potential (Edegrsquo) which considers the influence

of inter-solute and solvent interactions [21 29]

Redox reaction

middotmiddotmiddotmiddotmiddot Eq 14

O = Oxidant

ne- = The number of electrons transferred

R = Reactant

kf = Forward reaction rate constant (1st or pseudo 1st reaction) s-1

kb = Backward reaction rate constant (1st or pseudo 1st reaction) s-1

50

Nernst equation

lowast

lowast

+deg=R

Oe

CC

nFRTEE ln middotmiddotmiddotmiddotmiddot Eq 15

Ee = Equilibrium potential V

Edegrsquo = Formal potential V

R = Gas constant = 8314 Jmol-1K-1

T = Absolute temperature K

n = The number of electron transferred

F = Faraday constant = 96487 Cmol-1

CO = Bulk oxidant concentration molm-3

CR = Bulk reductant concentration molm-3

133 Electrode potential and activation free energy

When E ne Edegrsquo the difference in these potentials is expressed as the electrode

potential shift (∆E) as in Eq 16 and its free energy shift (∆G) as in Eq 17

Electrode potential shift

degminus=∆ EEE middotmiddotmiddotmiddotmiddot Eq 16

Free energy shift

)( degminus=∆=∆ EEnFEnFG middotmiddotmiddotmiddotmiddot Eq 17

When ∆E is applied positively it lowers the anode oxidation activation free energy

(∆GaDagger) by fraction of the total energy change (∆G) as shown in Fig 8 on p 51 The

fraction for the oxidation reaction is defined as (1-α) The transfer coefficient (α) is a

51

kinetic parameter which states the degree of symmetry in the potential energy barrier

for redox reactions The symmetry in energy barrier is present when the slopes of two

potential energy curves are the same and it is defined as α = 05 α is dependent on

redox potential and could vary within the range between 0 and 1 In most cases α = 05

and appears to be constant when overpotential is small [21 30]

Fig 8 The shift of ∆E and ∆GdegDagger [21 30]

With α the shift in electrode activation free energy can be expressed in terms of

potential shift for both oxidation and reduction reactions as shown in Eq 18 and Eq 19

52

∆GDagger in terms of (E-Edegrsquo)

Anode Oxidation )()1(DaggerDagger degminusminusminus∆=∆ EEnFGG aa αo middotmiddotmiddotmiddotmiddot Eq 18

Cathode Reduction )(DaggerDagger degminus+∆=∆ EEnFGG cc αo middotmiddotmiddotmiddotmiddot Eq 19

∆GoDagger = Anode oxidation activation free energy Jmol-1

∆GodegDagger = Anode oxidation standard activation free energy Jmol-1

α = Transfer coefficient molm-3

n = The number of electrons transferred Jmol-1

F = Faraday constant = 96485 Cmol-1

E = Arbitrary electrode potential V

Edegrsquo = Formal potential V

∆GRDagger = Cathode reduction activation free energy Jmol-1

∆GRdegDagger = Cathode reduction standard activation free energy Jmol-1

134 Potential shift and standard rate constant

By substituting the activation free energy in Eyring equation (Eq 11) with the

shift in electrode activation free energy in Eq 18 and Eq 19 it is possible to express k

in terms of potential shift and standard rate constant (kdeg) (Eq 20 and Eq 21) kdeg is the

rate constant at Edegrsquo[21 30]

53

k in terms of (E-Edegrsquo) and kdeg

Forward reduction cathode rate constant (kf) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bf

Daggercexpκ

⎥⎦⎤

⎢⎣⎡ degminusminus

⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )(expexp

Dagger

EERT

nFRT

GhTk cB ακ

o

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= )(exp EERT

nFk fα middotmiddotmiddotmiddotmiddot Eq 20

Backward oxidation anode rate constant (kb) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bb

Daggeraexpκ

⎥⎦⎤

⎢⎣⎡ degminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )()1(expexp

Dagger

EERT

nFRT

GhTk aB ακ

o

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= )()1(exp EE

RTnFkb

α middotmiddotmiddotmiddotmiddot Eq 21

kf and kb = Forward and backward reaction rate constant s-1

kdegf and kdegb = Forward and backward reaction standard rate constant s-1

135 Butler-Volmer equation overpotential and current

At the equilibrium potential E = Ee kf = kb hence anodic current (Ia) =

cathodic current (Ic) and therefore the net current (I =Ia ndash Ic) is macroscopically zero (I =

0) However both forward and backward reactions still occur microscopically at the

electrode surface The absolute value of this electric current is called exchange current

(I0) and is not directly measurable When CO = CR at E = Ee C O (0t) = C R (0t) and kdegf

= kdegb = kdeg hence the net current any potential E can be defined as below by

Butler-Volmer equation (Eq 22)

54

Butler-Volmer equation

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡ degminus

minusminus⎥⎦

⎤⎢⎣⎡ degminusminus

deg= )()1(exp)(exp )0()0( EERT

nFCEERT

nFCnFAkI tRtOαα middotmiddotmiddotmiddotmiddot Eq 22

The Butler-Volmer equation states the relationship between the electric current

and the potential shift This can be further rephrased in terms of overpotential (η)

through defining I0 using Nernst equation (Eq 15) η is the potential required to drive a

reaction in a favourable direction from its equilibrium state and defined as n Eq 23

Overpotential (η)

eEE minus=η middotmiddotmiddotmiddotmiddot Eq 23

At E = Ee both anode and cathode exchange currents are the same therefore both

exchange currents can be defined together as I0 (see below) and I0 can be expressed in

terms within Nernst equation by substituting (Ee-Edegrsquo) as in Eq 24

Exchange current

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= lowast )(exp0 EERT

nFCnFAkI eO

α

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= lowast )()1(exp EE

RTnFCnFAk e

Rα

αα )()( 1RO CCnFAk minus= o middotmiddotmiddotmiddotmiddot Eq 24

Now the Butler-Volmer equation (Eq 22) can be expressed in terms of η (= E ndash Ee) (Eq

25) by expressing it with I0 and the with re-arranged Nernst equation

55

⎥⎦⎤

⎢⎣⎡ degminus=lowast

lowast

)(exp EERTnF

CC e

R

O The term

)0(

CC t in Eq 25 indicates the influence of mass

transport For purely kinetically controlled region the electrode reaction is independent

of mass transport effects therefore C(0t) asymp C and Bulter-Volmer equation approximates

to Eq 26

Butler-Volmer equation and η

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminusminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminus

minus= lowast

lowast

lowast

minusminus

lowast

lowast

lowast

αααα

R

O

O

tO

R

O

R

tR

CCEE

RTnF

CC

CCEE

RTnF

CC

II )(exp)()1(exp )0()1(

)0(0

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= lowastlowast ηαηαRT

nFC

CRT

nFC

CI

O

tO

R

tR exp)1(exp )0()0(0

middotmiddotmiddotmiddotmiddot Eq 25

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= ηαηαRT

nFRT

nFI exp)1(exp0 middotmiddotmiddotmiddotmiddot Eq 26

The Butler-Volmer equation shows the dependence of current and hence the reaction

rate on the overpotential This implies that small kdeg can be overcome by overpotential

The kinetically-limited region of the electrode reaction is however very small because

the rate constant sharply increases exponentially with overpotential and soon the

reaction became diffusion limited [21 30] At this state the surface equilibrium is

totally perturbed and the overpotential is used to drive the diffusion for the one-way

reaction

136 Butler-Volmer equation vs Tafel equation

At small η lt RTnF Butler-Volmer equation further approximates to a linear

equation as shown in Eq 27

56

Small η Butler-Volmer equation

η⎟⎠⎞

⎜⎝⎛=

RTFnII 0

middotmiddotmiddotmiddotmiddot Eq 27

At large |η| gt RTnF the rate of the backward reaction is negligible hence one

of the exponential terms in Butler-Volmer equation becomes negligible and the

equations are solved for η for both anode and cathode reactions as below

Large η Tafel equations

InFRTI

nFRT lnln 0 plusmn= mη in the form of ||ln Iba +=η middotmiddotmiddotmiddotmiddot Eq 28

Anode oxidation InF

RTInF

RT ln)1(

ln)1( 0 αα

ηminus

+minus

=

Cathode reduction InF

RTInF

RT lnln 0 ααη minus=

In the system which follows the Tafel equations I0 (and hence kdeg ) is very small and the

electrode reaction does not proceed unless large η is supplied In this potential range the

reaction is already totally irreversible [4 21 30] An irreversible slow reaction is

graphically presented in Tafel plot in which ln (I) is plotted against η as shown in Fig

9

57

Fig 9 Tafel plot [4 21 30]

I0 is deduced from the y-intercept of the extrapolated slope Once |I| has become too

large and reached to the mass transport limit at large |η| the slope deviates from the

linear trend [30]

14 OVERPOTENTIAL AND FUEL CELLS

141 Four overpotentials that decrease the EMF

As discussed so far overpotential is extra potential required to drive a reaction

In the fuel cells this can only be taken from the potential generated within the system

causing potential drop and reduces EMF The overpotential in the system is attributed to

the four causes [1 4] (i) internal current (ii) activation overpotential (iii) diffusion

overpotential and (iv) resistance overpotential

58

142 Internal current

Internal current is a reverse current caused by crossover and leakage current

Crossover happens when the fuel flows to the opposite electrode through the electrolyte

Leakage current occurs when the current flows through the electrolyte rather than the

external circuit Internal current therefore happens without a load and is observed as the

negative offset from the theoretical value of EMF This is noted especially in

low-temperature fuel cells such as direct methanol fuel cells (DMFCs) [4 51] In such

systems The inner current can be several 10 Am-2 and potential drop could be more

than 03 V [4] In order to prevent fuel mixture and crossover a separator is used to

partition the cell Alternatively it is also possible to confer high specificity to electrodes

as seen in glucose - oxygen fuel cells Such systems do not require

compartmentalisation and this is advantageous for miniaturisation [52]

143 Activation overpotential

Activation overpotential is a potential loss due to the slow reaction kinetics as

discussed in sect 135 This is unavoidable to some extent and is typically the biggest

problem in most fuel cells involving O2 cathode reduction because this reaction is

kinetically hindered Finding or inventing catalysts with high kdeg is the key solution for

this A sluggish reaction exhibits a sharp rise in activation overpotential for a slight

increase in low current After the steep increase the current increase becomes linear and

constant as shown in Fig 10 until the reaction reaches the limit of mass transport at the

higher overpotential [4 30 34]

59

Fig 10 Activation overpotential against current [4]

144 Diffusion overpotential

Diffusion overpotential is a potential loss due to concentration gradient This

tries to drive flux of electroactive species toward and from the electrode surface It

happens when the reaction kinetics is faster than mass transport [4 21 30] In fuel cells

diffusion overpotential is caused by fuel depletion due to the inefficient fuel convection

or supply [21 34] This problem is observed in reformers because the fuel refining and

conversion such as liquefied petroleum gas (LPG) to hydrogen gas take place outside

the system and it makes difficult to respond to the sudden fuel demand from the system

[4] It also happens when fuel diffusion is interfered with by bad electrode geometry

electrode fouling and adsorption [21 27] Diffusion overpotential is defined as Eq 29

60

Diffusion overpotential equation

⎟⎟⎠

⎞⎜⎜⎝

⎛minus=

Ldiffusion I

InFRT 1lnη middotmiddotmiddotmiddotmiddot Eq 29

ηdiffusion = Diffusion overpotential V

IL = Limiting current A

Limiting current (IL) is generated when a reaction takes place at the rate limited by mass

transport Diffusion overpotential sharply increases when reaction kinetics have reached

to the maximum and very large current are drawn as shown in Fig 11 [21 30 34 53]

Fig 11 Diffusion overpotential against current [4]

145 Resistance overpotential

Resistance overpotential arises within electrodes separators connectors circuit

or electrolyte [4 34] It is called Ohmic resistance or IR drop because it follows Ohmrsquos

61

law (Eq 30) where resistance overpotential is linearly proportional to the electric

current as shown in Fig 12 [4 30]

Ohmrsquos law

IRV = middotmiddotmiddotmiddotmiddot Eq 30

V = Voltage consumed due to the resistance V

I = Electric current drawn due to the resistance A

R = Resistance Ω

| |

Fig 12 Resistance overpotential against current [4]

Causes of internal resistance are scattered through the system therefore there are many

possible ways to resolve this One can possibly reduce it by using more conductive

material The difficulty lies in finding the conductive material which can be modified as

well as carry out the functions as one desires under the required conditions [53]

Resistance can be reduced by reducing the distance between two electrodes [54]

however this might cause short circuit and fuel leakage This becomes a serious issue in

62

use of solid electrolyte such as polymer electrolyte because both performance efficiency

and mechanical stability of electrolyte must be achieved [53] Excess in inert supporting

electrolyte minimises solution resistance and matrix effect It sustains a thin electric

double layer uniform ionic strength and buffering effect against pH change [21 30]

However electrolyte could become a problem when electrode material dissolves into

electrolytic solution and reacts there because it might change nature of electrolyte [1

21]

146 Total overpotential and cell voltage

Total overpotential is the sum of all four overpotentials Current ndash Voltage (IV)

polarisation curve visualises the tendency of each potential drop as shown in Fig 13 [1

4]

Fig 13 V-I curve for a typical low temperature fuel cell [4]

63

The cell voltage (Ecell) is determined by subtracting the cathode potential (Ec)

from the anode potential (Ea) (Eq 31) The actual cell voltage is found by subtracting

the total overpotential from the theoretical cell voltage The energy available from this

cell voltage can be calculated as in Eq 32

Cell voltage

cacell EEE minus= middotmiddotmiddotmiddotmiddot Eq 31

totalltheoreticacellactualcell EE ηminus= )()(

cellcell nFEG minus=∆ middotmiddotmiddotmiddotmiddot Eq 32

15 BIOFUEL CELLS

151 Microbial and enzymatic fuel cells

Biofuel cells are fuel cells which use biocatalysts for fuel reactions

Biocatalysts can be either the whole living cells or isolated enzymes The former is

called a microbial fuel cell and the latter is called an enzymatic fuel cell [55-57] They

often function under ambient conditions and allow wider choice of fuels Microbial fuel

cells use common carbohydrates such as glucose and lactate [55] but also have

potentials in extracting power from waste and sediments [34 55 57 58] Harnessing

such microbial life activities has some advantages They can complete fuel oxidation

within the cell It was reported that a microorganism Rhodoferax ferrireducen has over

80 glucose conversion rate [58] Microbial fuel cells also have long-term stability

[56-58] of up to five years [55] However their output current and power densities are

64

small due to high internal resistance [55-57] Enzymatic fuel cells in contrast have

higher current and power densities but short life incomplete reactions require

mediators to shuttle electron between the enzyme catalytic centre and the electrode and

become deactivated easily [55 56 58] Addition of mediators and their immobilisation

with enzyme complicates fuel cells design further However they can be more

advantageous due to their substrate specificity Both anode and cathode to reside in the

same compartment of mixed fuels without crossover This enables the miniaturisation of

fuel cells [52 59 60] and is particularly useful for medical implantation Stability and

lifetime of the enzyme fuel cells can be improved by appropriate enzyme

immobilisation fixing up the suitable conditions for their function [55] and adding

some enzyme stabilisers Addition of the right mediators and increased enzyme loading

will also increase output current and power density [55 59]

152 Enzyme fuel cells and mediators

Enzyme reactions are available for wide range of substrates yet each enzyme

has very high specificity for its substrates Many also function at neutral atmospheric

conditions therefore miniaturised enzyme fuel cells are of interest for in vivo

application Many redox reactions however take place at the catalytic centre which is

deeply buried in the protein body This proteinous content is an electronic insulator and

interrupts the electrical communication between the redox centre and the electrode It is

therefore difficult for many enzymes to establish efficient electron transfer without

mediators as shown in Fig 14 This is called mediated electron transfer (MET)

65

Fig 14 Mediated electron transfer reaction [55 61]

It is possible for some enzymes such as multicopper oxidases (MCOs) to perform direct

electron transfer (DET) without mediators however mediators ensure good electrical

communication and cell performance However the presence of mediators as well as the

enzyme specificity could complicate the fuel cell composition and its reaction path due

to the reaction cascade [55 59 62 63] Palmore built a methanol - oxygen enzyme fuel

cells and this involved four enzymes two mediators and six reactions to complete the

full reaction path [64] It is more advantageous to use enzymatic fuel cells for very

specific applications [59] such as medical implantation where there are environment

constraints but where applications demand only small power output through simple

reactions

Mediators are usually small redox active species which can be organic or

metal complexes In the enzymatic fuel cells they should be carefully selected because

improper mediators can cause unwanted overpotential or can destabilise the system

They must have (i) small overpotential (ii) fast reversible electron transfer [55 56 59

60 63 65] and (iii) stability [62 65] However there should be small but reasonable

66

potential gap between the enzyme and the mediator to drive electron transfer [60 66]

The gap of 50 mV is optimal [19 64] because of the small potential loss yet less

competing with enzyme reactions It is also possible to mediate electron transfer by

building a conductive nanowire between the electrode and the redox centre [60 63 67]

or a connection by plugging metal nanoparticles into the enzyme [60 67]

16 MINIATURISED MEDICAL POWER SOURCES

161 Realisation of miniaturised fuel cells and medical power supply

Medical application is one of the obvious targets for miniaturised biofuel cells

Glucose-oxygen biofuel cells which continuously run on glucose and oxygen were

thought to be ideal for implantable medical devices because both oxygen and glucose

are abundant in the body [63] They were initially intended to power cardiac assist

devices and pacemakers The studies on such systems have been made for more than 40

years however none has been made into practical use because their power density

stability operation life and biocompatibility were inadequate [19 67-69] Their output

power ranges between microW ndash mWcm-2 and they operate only for a few days to a few

weeks Most applications need a few W power for a longer duration [18 60 67] A

pacemaker requires 1microW [56] for more than 8 ndash 10 years with 996 reliability Such

power has been supplied by lithium (Li) iodine battery [19 56 70] which is

characterised by extended operational life [68] Li is fairly stable light-weight metal

and has high energy density of 386 kAhkg-1 Its standard potential is highly reducing ndash

323 V vs AgAgCl These properties enable Li batteries to power most devices

67

including pacemakers laptops and vehicles [19 21] However there are some problems

in Li batteries They are still bulky because they need to store their own fuel and it has

to be sealed with the metal case [19 68] They occupy half of the space in the

pacemaker volume [68 71] They use corrosive [72] and irritating materials There is

potential of explosion [68] leakage and the necessity of exchange Li also has potential

resource constraint due to the geopolitical issues as seen for oil and platinum More than

80 of its production is based in South America and China [73] With increased public

concern over using greener safer energy and saving resources as well as the global

political intervention in that it is natural to consider about using alternative

technologies to power implantable medical devices where it is possible

162 Miniaturised potential medical power sources

There are numerous implantable electrical medical devices with different

power requirements These include cardiac pacemakers defibrillators neurostimulators

for pain control drug delivery device hearing aids [70] and biosensors [74]

Implantable autonomous body sensors for physiological parameters such as glucose

concentration local pH flow and pressure [19] require power as low as 1 microW [56 70

74] and the operation period of 1 year at least [74] Such power can be supplied by

various implantable energy harvesting devices using body glucose [19] motion

vibration [75] and temperature differences [75] Heller developed the smallest

glucose-oxygen biofuel cells made of carbon fibres with the dimension of 17-microm

diameter and 2-cm length Anode glucose oxidase and cathode laccase were wired to

conductive hydrogel backbone with a flexible osmium (Os) mediator complex Its

68

output was 048 mWcm-2 with 06 V at 37 degC and it lasted for 1 week [19 67]

Kinetic energy of motion and vibration can be converted into electricity with

electrostatic transduction using microelectromechanical systems (MEMS) This

technology allows integration of various microelectronic devices on the same substrate

[76] NTT Corporation developed a system generating power from body motion and

temperature It consists of a thermoelectric and a comb-shaped Au-Si vibrational device

on a monolithic Si substrate The voltage produced by a thermoelectric device is

boosted by capacitance which was generated by the vibrational device then its charge

is stored in the capacitor [75] Another vibrational device was reported to have 20 x 20 x

2 mm volume and output 6 microW and 200 V at 10 Hz low frequency and 39 ms-2

acceleration The principal difficulty in using MEMS is to adjust and synchronise

flexibly the resonance frequency of vibrational device to the human body frequency

which is low and has constant fluctuation in order to increase energy conversion

efficiency The typical vibration has the frequency ranging from 1 to 100 Hz and

acceleration from 1 - 10 ms-2 [76]

Thermoelectric devices generate power from temperature difference Each

module consists of a number of thermocouples on Si substrate Thermocouples are

made of different material so that it can generate voltage from the heat flow following

temperature gradient This is called Seebeck effect and its size depends on the material

and number of thermocouples used and the spacing within the device [75 76] Typical

ambient temperature difference is less than 10K [74 76] and its power is 1microWcm-2 [74]

Seiko Instrument developed a wristwatch called Seiko Thermic which was powered by

human body temperature in 1998 [77 78] It consists of n-type Bismuth-Tellurium

(BiTe) and p-type Bismuth-Antimony-Tellurium on rods reinforced by Ni on oxidised

69

silicon wafer It generated microWcm-2 power at extremely low 0047 conversion

efficiency With a booster circuit it powered a wristwatch operating at 1microW and 15 V

It could however operate only when ambient temperatures were below 27 degC [78] and

so could not be used in tropical regions

Heller also developed an implantable miniature Nafion-coated zinc ndash silver

chloride (ZnAgCl) battery The anode was made of zinc fibre coated with Nafion It

was 76-microm diameter 2-cm length and 75 x 10-2 cm2 surface area The cathode was

made of AgCl wire covered with chloride-permeable membrane On discharge anode

zinc formed Zn3(PO4)2middot4H2O hopeite complex with Nafion growing non-porous

lamellae on the surface The lamellale had a unique property which was permeable to

zinc ions but impermeable to O2 therefore the battery became corrosion free It output

was 13 microAcm-2 at 1 V and operated for three weeks under physiological condition The

advantage of this battery was its size which was 100-fold smaller than the smallest

conventional batteries [79] The smallest silver oxide button buttery has the volume 30

mm3 [19 80] with energy density 04 mWh mm-3 at 15 V The smallest Li-ion buttery

has the volume 190 mm3 [80] with energy density 025 mWh mm-3 at 4 V [21]

17 OUTLINE OF THE WORK

171 Introduction to miniaturised glucose-oxygen fuel cells

A miniaturised glucose-oxygen biofuel cell runs on glucose and oxygen This

would be ideal to power implantable autonomous low-power body sensors since both

glucose and oxygen are abundantly present in the body The range in their physiological

70

concentration is between 4 ndash 8 mM for glucose and 01 ndash 02 mM for oxygen [56 67]

The aim of this study was to build miniaturised glucose-oxygen fuel cells with reliable

enzyme anodes and powerful oxygen-reducing cathodes using straightforward and

scaleable technology The general structure and mechanism of glucose-oxygen fuel cells

were described in sect 112

In this study both anode and cathode reactions took place at the room

temperature and physiological pH in the presence of catalysts Glucose oxidation was

catalysed by the enzyme glucose oxidase (GOx) from Aspergillus niger and its electron

transfer and conduction were facilitated by an conducting organic salt called

tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) (Fig 15)

Fig 15 The schema for a glucose-oxidising anode

GOx and TTF-TCNQ were mixed into a composite employing a feasible electrode

fabrication method called bulk modification method Various enzyme stabilisers were

added to the composite at different concentration to improve its performance The

influence of various enzyme stabilisers on anode performance was studied in the

71

presence of both N2 and O2 and the most ideal anode composition was determined

Cathodes were also fabricated in the similar manner using graphite as conductive

material and mixing with various oxygen reducing catalysts except for the enzyme

membrane cathodes The cathode composites were pretreated in various media to

enhance their activity For enzyme membrane electrodes glassy carbon electrodes were

used instead of graphite composite and enzyme solution was loaded onto the surface

and trapped with membrane Catalytic power and mechanism of each cathode were

studied with various electrochemical methods and the most catalytic cathode was

determined

172 Outline

Chapter 1 is a general introduction about fuel cells It explains the history and

basic concept of fuel cells and outlines the study

Chapter 2 explains about the enzyme anodes The introduction part refers to

glucose oxidising enzymes enzyme immobilisation TTF-TCNQ enzyme inactivation

enzyme stabilisers and enzyme kinetics The material and method section reports the

protocol for the enzyme electrode fabrication electrode conduction study and

amperometry study for enzyme electrodes Result and discussion section mentions

enzyme electrode conduction enzyme kinetic studies based on amperometry

mechanism of enzyme stabilisation in each stabiliser and ideal electrode composition

Chapter 3 discusses oxygen-reducing cathodes The introduction part reviews

the mechanism of the oxygen reduction reaction oxygen reactivity oxygen reduction

catalysts carbon properties electrochemical pretreatments electrochemical techniques

72

and how to determine the catalytic efficiency of cathodes The material and method

section describes the protocol for the oxygen-reducing electrode fabrication preparation

and electrochemical pretreatment electrochemical studies electrochemical studies for

qualitative analysis and to obtain the kinetic parameters and analysis to determine the

efficiency of catalytic cathodes The result and discussion section describes the

voltammetric behaviour of each pretreated cathode influence of pretreatment and

catalytic efficiency The most catalytic cathode was determined at the end

Chapter 4 refers to the overall conclusion and further studies

73

2 ANODE INTRODUCTION

21 GLUCOSE OXIDATION ENZYMES

Glucose-oxygen fuel cells extract power by oxidising glucose at the anode The

anode contains glucose-oxidising enzymes There are several enzymes available for

glucose oxidation (i) β-D glucose oxidase (GOx) (EC 1134) [56 81] (ii) pyranose

oxidase (POx) (EC 11310) [56 81] (iii) nicotinamide adenine dinucleotide-dependent

glucose dehydrogenase (NAD-GDH) (EC 11147) [18 56] and (iv) quinoprotein

glucose dehydrogenase (PQQ-GDH) (EC 1152) [56 81] These enzymes are

classified into the family of oxidoreductases which catalyse either transfer of (i)

hydride (H-) (ii) oxygen atoms or (ii) electrons from one molecule to another Oxidases

transfer electrons from substrates to O2 and form H2O or H2O2 as products

Dehydrogenases transfer electrons from substrates to coenzymes without involving O2

(Eq 33 and Eq 34)

Glucose oxidation by GOx and NAD-DHG

GOx middotmiddotmiddotmiddotmiddot Eq 33

NAD-DHG middotmiddotmiddotmiddotmiddot Eq 34

To function catalytically enzymes require non-peptide inorganic or organic cofactors or

prosthetic groups Non-peptide organic cofactors are also called coenzymes Enzymes

lacking such groups are called apoenzymes and hardly functional Cofactors are

generally loosely bound to the apoenzymes and freely diffusible but on catalysis they

74

bind to the enzymes as the second substrates Prosthetic groups are always tightly bound

to the apoenzymes through covalent or non-covalent bonding [22 23 48] GOx and

POx are fungal flavoprotein enzymes containing flavin adenine dinucleotide (FAD)

cofactors FAD in GOx is tightly bound to the apoenzyme but not covalently bonded

hence still dissociable [67 82-84] NAD-GDH contains dissociable NAD+ [65] while

PQQ-GDH contains pyrrolo-quinoline quinone (PQQ) redox cofactor [56 65] which is

moderately bound to the apoenzyme [65 67] The structure of each cofactor is shown in

Fig 16

75

N

N

NH

N O

O

R

N

N

NH

HN O

O

R

NH

N

NH

HN O

O

R

H+ + e- H+ + e-

2H+ +2e-

N+

NH2

O

R

H

N

NH2

OH

R

HH

N

NH

O

O

OHO

OH

O

OH

O

N

NH

O

HO

OHO

OH

O

OH

O

N

NH

OH

HO

OHO

OH

O

OH

O

H+ + e- H+ + e-

FAD FADH2

NAD NADH

PQQ PQQH2

Fig 16 The structure of FAD NAD and PQQ and their redox reactions

Moieties involved in the redox reactions are circled in gray [23 65]

Cofactors also stabilise the enzyme structure Irreversible thermal GOx denaturisation

involves the perturbation of FAD biding site causing FAD dissociation although 70 of

its secondary structure is retained Chemical denaturisation untangles the dimmer

structure of GOx [65 81 83 85] and dissociates monomers and FAD [86 87] Excess

FAD does not prevent or regain function after thermal inactivation [87] However FAD

can rapidly bind to apoenzymes at 25 degC pH 61 and stabilise the tertiary structure

through conformational change and give resistance to proteolysis [82]

76

The choice of enzyme is based on the reaction involved enzyme availability

stability enzymatic properties physical properties and operational environment In the

absence of O2 or when there is concern about system degradation due to the competition

of electrons between O2 and artificial electron acceptors or due to the by-product H2O2

GDHs are more preferred to GOx [23 60 65-67 88] However the use of GDHs poses

different disadvantages Membrane-bound PQQ dehydrogenase (mPQQ-GDH) has very

high glucose specificity however it is difficult to solubilise and purify hence not for

commercial use [88] Soluble PQQ dehydrogenase (sPQQ-GDH) has been used in

commercial glucose sensors [67 89] however it suffers from low thermostability and

very low glucose specificity [88] Moreover PQQ-GDHs add complication to the

system because metal ions such as calcium ions (Ca2+) and magnesium ions (Mg2+) is

required to activate them [89 90] In the case of NAD-GDH NAD must be provided

because it is a dissociable cofactor but must bind first to the apoenzyme to form a

catalytic complex for glucose oxidation In addition to this complication NAD was also

reported to be more prone to causing electrodes poisoning without a quinone acceptor

[65] NAD forms adduct with phosphate in the buffer its decomposition releases free

adenine and electrochemically oxidised adenine adsorbs onto the electrode surface

causing electrode poisoning [91]

The redox potential of enzyme is also important because it is the driving force

for electron transfer and the power although it is difficult to match both catalytic

efficiency and electrochemical efficiency It is also difficult to determine the redox

potential exactly because the redox potential of cofactors is influenced by

microenvironment such as where they are embedded in the enzyme as well as

77

macroenvironment such as pH and temperature For example the redox potential of

FAD itself is ndash 042 V vs AgAgCl at pH 7 25 degC [22 23] however that of

flavoproteins could range from ndash 069 to ndash 005 V [65] and FAD-containing GOx has

the redox potential approximately ndash 03 V [67 92] It was also reported glucose

gluconolactone redox potential was ndash 056 V [93] PQQ itself has - 013V in absence of

Ca2+ and ndash 012 V in presence of 1M CaCl2 [94] while sPQQ-GDH has the redox

potential approximately - 019 V in presence of Ca2+ [66 67] Furthermore the enzyme

redox potential leads to a complex issue when mediators which assist electron transfer

between the enzyme redox centre and the electrode are involved because the improper

combination of the enzyme and mediator causes inefficient electron transfer and hence

potential drop

For enzyme availability stability and simplicity in handling GOx has been

most popular glucose-oxidising enzyme It has optimal pH 55 and temperature 35 ndash

40 degC [93] but is stable between pH 4 ndash 7 [95] and up to 50 - 55 degC [82 87] GOx has

higher substrate specificity and is most active toward β-D-glucose oxidation [56 86 88]

and very stable therefore its use has prevailed among various sensor applications [60

67]

22 DESIGNING ENZYME ELECTRODES

Construction of enzyme electrodes is complex because it simultaneously

incorporates enzymes mediators conductive materials and stabilisers ensuring that all

of them work harmoniously under restricted performance conditions There are several

78

immobilisation techniques available These include physical adsorption cross-linking

covalent bonding and entrapment in gels or membranes [96]

221 Physical adsorption

Physical adsorption is a simplest immobilisation method Electrodes are dipped

in the solution containing enzymes and mediators or such solution can be applied to the

electrode surface In this method however the enzyme tends to leach due to the weak

interaction between the electrode and enzyme [55] In order to avoid the loss of

electrode material the electrode surface is often covered or coated with material One

classic method is that the solution on the electrode surface is trapped with a

semipermeable dialysis membrane [61 65 92] This allows enzymes free from the

possible denaturisation caused by immobilisation on the solid material Enzymes inside

the dialysis membrane also can be fixed onto the cellulose acetate membrane or the

enzyme layer can be cross-linked with bifunctional reagents such as glutaraldehyde

[61]

222 Reconstituting apo-enzyme

Willner constructed enzyme electrodes by reconstituting apo-enzyme on the

gold (Au) electrodes surface with quinone mediators The electrode consisted of three

layers which were made of PQQ synthetic FAD and apo-GOx [97] PQQ were

mediating groups and attached onto Au electrode by cystamine PQQ attached on Au

was then covalently linked to synthetic FAD called N6-(2-aminoethyl)-FAD Covalently

79

linked FAD self-assembles with apo-GOx so that holo-GOx assembly is formed on the

top of the PQQ mediating layer attached to the Au surface The reconstitution procedure

is shown in Fig 17

Fig 17 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode [97]

The output current density was 300 microAcm-2 at pH 7 35 degC and it dropped by 5 in the

presence of O2 although the decrease was negligible at 01 mM physiological O2

concentration [98] The reconstitution of apo-enzymes is a flexible system potentially

applicable to enzyme electrodes [97] The fabrication process is cumbersome however

the enzymes are neatly aligned and electrically connected in the correct orientation

therefore electrocommunication between the enzymes and electrodes can be

maximised

80

223 Tethered Osmium-complexes linked to conductive hydrogel

It has been reported that embedding enzymes in the conductive hydrogel

complexed with tethered osmium (Os) mediator led to high output current [19 60 66

67] The conductive hydrogel is made of cross-linked polyvinylpyridine or

polyvinylimidazole They are soluble as monomers however they gain their

water-swelling property in cross-linked polymer This is ideal for enzyme mobility and

electrical conductivity Electron transfer is realised by the Os complex which is tethered

to the conductive hydrogel back bone The tether is made of 10 ndash 15 atoms [19 67] in

order to allow flexile movement of mediators Tethered Os complexes sweep the large

volume made of multiple layers of hydrogel form electrostatic adducts with enzymes

[19] and facilitate collide-and-hop electron conduction [19 99] Polymer tether and Os

complexes can be flexibly tailored for different enzymes and different operational

environments [19] One of its great advantages is that the redox potential for electron

transfer can be adjusted by changing the ligand structure of Os complex [19 60 97]

Heller constructed such electrodes made of carbon fibers whose dimension was 7 microm

diameter 2 cm length 08 mm2 surface area and 26 x 10-3 mm3 volume GOx was

wired to poly(4-vinylpyridine) conductive hydrogel backbone by the tethered complex

of tris-dialkylated NNrsquo-biimidazole Os2+3+ (Fig 18)

81

Fig 18 GOx wired to conductive hydrogel complexed with Os [19 67]

The redox potential of its redox centre was ndash 02 V vs AgAgCl The glucose oxidation

took place at ndash 036V and its output current density was 11 mAcm-2 [19] Disadvantage

of the tethered enzyme is short life It was stable for one week in the physiological

buffer then 5 current drop per day was observed When it was implanted in grape sap

50 current drop occurred within 20 hours In calf serum 50 current dropped

occurred in 2 hours with electrode fouling [19]

224 Entrapment in the conductive polymer

Entrapping enzymes in conductive polymer such as polypyrrole polyaniline

and polythiophene [65] is a popular method for enzyme electrodes [96] This is made by

82

electropolymerisation in which potential is applied to the solution containing enzymes

and monomors for certain duration It is a simple method but can be complicated if the

conditions for polymer growth are not compatible with enzyme entrapment [99]

Enzyme leakage occurs when enzymes are not trapped securely but it can be reduced

when derivatised polymers are used [96 100] Their conductivity is affected by

monomer structure [100] acidity and electrolyte [101] It can be degraded by products

of enzymatic reactions [65] Fujii fabricated GOx electrodes which were stable for

more than four months though a gradual sensitivity drop was observed GOx was

entrapped in saccharide-containing polypyrrole made of 1-(6-D-gluconamidohexyl)

pyrrole (GHP) (Fig 19(i)) on Pt electrodes It was reported that entrapped GOx was

secured by hydrogen bonding present between sugar gluconyl groups and enzymes

Enzyme leaching was also reduced by improved polymerisation with extended

conductive hydrocarbon chain [100] The output current density was 75 microAcm-2 at 055

V pH 74 in the presence of 56 mM glucose and 175 microA cm-2 under glucose saturated

condition [100] Arai entrapped various enzymes in polymer of mercaptobenzoquinone

(SQ) (Fig 19(ii)) and its methylated derivatives methylmercapttobenzoquinone

(MeSQ) and dimethylmercaptobenzoquinone (Me2SQ) on Au electrode surface

83

OH

H

H

OH

OH

H

OH

H

CH2OHN(CH2)6NHCHOH

(i)O

O

SH

Fig 19 Monomer structures of conductive polymers

(i) 1-(6-D-gluconamidohexyl) pyrrole [100]

(ii) Mercapto-p-benzoquinone [101]

Quinones have been widely used as good mediators [21 60 61 65] Poly(SQ) is a

conductive polymer functionalised with quinone mediating groups and can be easily

attached to Au electrode due to the presence of thiol group (-SH) [100] It was revealed

that more methylated SQ polymer led to better performance The maximum current

density for SQ- MeSQ- and Me2SQ-GOx anodes was 006 522 and 544 microAcm-2 and

their electrode enzyme dissociation constant KM was 13 18 and 20 mM respectively

[102 103] FAD cofactors have been also electropolymerised to entrap lactate

dehydrogenase and lactate oxidase It was reported that polymerised FAD extended the

shelf and operational life of electrodes to more than one year and also reduced O2

influence [104]

225 Bulk modification

Another popular electrode fabrication involves bulk modification of solid

composite This is simply made by mixing electrode material and inert binding reagent

84

such as epoxy resin Typical composite electrodes consist of 70 conductive material

by weight [105] Bulk-modified electrodes are robust stable inexpensive and easy to

modify build and reuse [105-107] therefore often used for enzyme electrodes [108]

For GOx electrodes tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ)

has been used as both mediating and conducting material

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties

TTF-TCNQ is a black crystalline charge transfer complex having both

mediating and conducting properties Its handling is also easy and often used for GOx

electrodes [65] Its conductivity is 102 ndash 103 Scm-1 [109 110] and comparable to that of

graphite [65 105] TTF-TCNQ complex consists of TTF electron donor and TCNQ

electron acceptor and they are arranged in a segregated stack structure TTF-TCNQ has

metal-like electric conductivity and this derives from intra-stack and inter-stack

molecular interactions Within the stack π-orbitals of adjacent molecules overlap This

builds the major conductive path in the direction of stack [61 65 110 111] Between

the stacks there is partial charge transfer between different chemical groups and this

gives the metallic feature in which charge carriers are mobile among the stacks

[110-112] as shown in Fig 20

85

S

S S

S

N

C

C

N

C

C

N

N

S

S S

S

7 6

N

C

C

N

C

C

N

N

-

TTF - electron donor

TCNQ - electron acceptor

6 - localised 6 - delocalised

+ e-

+ e-

Fig 20 TTF-TCNQ structure and its charge transfer [61]

TTF-TCNQ is a good electrode material because it has low background current and is

efficient at electron transfer [65] is stable [65] insoluble in water and not toxic [113]

However it exhibits complex electrochemistry (Fig 21) and its behaviours have not yet

been fully elucidated

86

vs AgAgCl

TCNQTCNQ e⎯⎯rarr⎯minus minusbull+

+minus+ ⎯⎯rarr⎯minus 2e TTFTTF

minus+minusbull⎯⎯rarr⎯

minus 2e TCNQTCNQ

+minus⎯⎯rarr⎯minus

TTFTTF e

Fig 21 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 01M potassium

phosphate 01 M KCl at 20 mVs-1 [92] Modified

The range of its effective redox potential varies according to the authors [65 92 114

115] Following the Lennoxrsquos study its potential extremes should lie within the range

between - 015 V and + 031 V vs AgAgCl (Table 2) because beyond these potential

ranges irreversible lattice reduction or oxidation takes place Even within the range

crystal dissolution salt formation with buffer ions and its deposition are still present and

could change the electrochemical profile TTF-TCNQ stability is affected by many

factors such as potential applied buffer component and concentration ion solubility

mass transport and analyte [92]

87

Table 2 Electrochemical behaviour of TTF-TCNQ at pH 74

Equilibrium TCNQTTFTCNQ-TTF +⎯rarr⎯ middotmiddotmiddotmiddotmiddot Eq 35

TCNQ reduction + 015 V MTCNQTCNQTCNQ Me ⎯⎯rarr⎯⎯⎯rarr⎯+minus +minusbull+ middotmiddotmiddotmiddotmiddot Eq 36

Lattice reduction - 015 V minus+minusbull ⎯⎯rarr⎯minus 2e TCNQTCNQ middotmiddotmiddotmiddotmiddot Eq 37

Anode salt formation + 031 V MTCNQTCNQ M⎯⎯rarr⎯++minusbull middotmiddotmiddotmiddotmiddot Eq 38

Lattice oxidation + 035 V +minus⎯⎯rarr⎯minus

TTFTTF e middotmiddotmiddotmiddotmiddot Eq 39

Further oxidation + 038 V +minus+ ⎯⎯rarr⎯minus 2e TTFTTF middotmiddotmiddotmiddotmiddot Eq 40

In the presence of a redox couple it also exhibits different electron transfer mechanism

When Edegrsquo of the redox couple is Edegrsquo gt 0 V TTF-TCNQ shows metal-like behaviour

facilitating direct electron transfer (DET) while it presents mediated electron transfer

(MET) for the redox couple of Edegrsquo lt 0 V Lennox made a study on TTF-TCNQ GOx

electrodes and reported that large anodic current was observed at ndash 015 V He also

reported that stable current was obtained when constant potential + 02 V was applied

for 24 hours at pH 7 in the presence of glucose He concluded that hydrophobic protein

core solubilised TCNQ and it formed enzyme-mediator complex GOx oxidation could

be catalysed at low potential because it was still in the range of the stable redox

potential He also conducted experiments with GOx and methanol dehydrogenase

(MDH) modified TTF-TCNQ electrodes in the presence of O2 The performance was

interfered by O2 in both electrodes against his expectation because DHG is in general

resistant to O2 He gave a conclusion that reduced mediators were oxidised by O2 before

electrooxidation took place [92]

88

226 Enzyme covalent attachment and mass loading

Enzymes can be covalently attached to the electrode however it becomes

complicated when mediators and stabilisers are must be also attached for practical fuel

cell applications This is however an attractive technique once ideal means of

co-immobilisation are established because enzymes can be retained firmly on the

electrode surface Enzymes can be covalently linked to oxidised or aminated surface

through amide bonds [116 117] Covalent linkage often lowers enzyme activity because

it partially alters the enzyme structure and confines the enzyme conformation [55]

however Kim succeeded in fabricating stable biocatalytic nanofibre by loading enzyme

aggregate to the enzyme monolayer which was covalently attached to the nanofibres

(Fig 22) Nanofibres with ester groups were synthesised from polystyrene and

poly(styrene-co-maleic anhydride) using electrospinning Seed enzyme was covalently

attached to the chemically modified nanofibre to form an enzyme monolayer on the

nanofibre This enzyme-attached nanofibre was further modified with the mixture of

glutaraldehyde and enzymes so that enzyme mass load was achieved by loading enzyme

aggregate to the enzyme monolayer covalently attached to the fibre Minter reported

that enzyme immobilisation with cross-linking increased the enzyme stability but

limited the enzyme conformation and thus reduced the activity However the activity

reported was nine times higher than that with an enzyme monolayer only Moreover the

activity was retained more than one month under rigorous shaking [118] The activity of

each enzyme particle might have been reduced however mass loading increased the

reactant flux hence increased the overall activity

89

Fig 22 The procedure for the enzyme mass-loaded biocatalytic nanofibre [118]

He also fabricated single-enzyme nanoparticles (SENs) in which each enzyme particle

was encapsulated within a hybrid organic and inorganic network The surface amino

groups of the enzyme were converted into surface vinyl groups and then polymerised

The polymer was then cross-linked to form the shell around the enzyme particle (Fig

23)

90

Fig 23 Fabrication procedure of single-enzyme nanoparticles [55]

It was reported that high stability was achieved after polymer cross-linking The enzyme

activity was approximately half of the free enzyme however there was no mass

transport interference [55] due to their porous structure Km in SENs was nearly

equivalent that of the free enzyme but the storage stability went beyond five months

[119] Eight-fold increase on storage stability after GOx covalently linked to

poly(phosphorothioate) was also reported [120]

23 PROTEIN INACTIVATION AND STABILISATION

Enzyme inactivation is one of the biggest problems in enzyme electrodes

Enzymes are excellent catalysts in their native environment however they are only

designed to work optimally under specific conditions therefore very sensitive to the

slight change in their environment and easily undergo inactivation The issue is

persistent in the enzyme electrodes because their fabrication introduces the enzymes to

the unusual environment yet enzyme catalytic activity must be maintained to obtain

sufficient power output and operational life The problem can be alleviated by (i)

91

protein engineering [56 121] (ii) environmental engineering [56 60 63 67] and (iii)

addition of appropriate enzyme stabilisers [120 122] Protein engineering involves the

alteration of enzyme structure to gain desired functions stability or resistance However

it is very challenging to improve both stability and activity since protein stability is

conferred by its rigidity while its activity depends on the conformational flexibility

Alteration of protein properties is realised by genetic engineering such as site-directed

mutangenesis [123] or directed evolution [49] Environmental engineering is closely

linked to the enzyme immobilisation It aims to achieve the same goals as in protein

engineering but extrinsically The environment which rigidifies the protein structure

such as cross-linking or inclusion in electrode composite tends to increase the enzyme

stability and prolong its life In contrast the environment which more favours over

flexible protein conformation such as hydrogel and membrane trap exhibits higher

enzyme activity but shorter life However it was reported that enzyme cross-linking and

the presence of hydrophilic macromolecules improved both stability and activity while

electrostatic interaction improved selectivity [124] Addition of stabilisers is more

targeted smaller scale environmental engineering Intrinsic conformational stability of

the protein cannot be improved [120] however it can target and eliminate specific

inactivating factors increases the local specific protein stability and prevents or slows

its unfolding There are two factors to improve for enzymes to achieve ideal operation

(i) activity by hydration and (ii) increased lifetime by increased protein rigidity which

might involve the removal of bond-destabilising factors or amplifying the bond strength

within the enzymes In fact both must be probably achieved simultaneously for gaining

the optimal state because they interrelate to each other in terms of protein dynamics As

mentioned earlier protein hydration improves enzyme activity but also allows its

92

regeneration hence affects the protein internal dynamics Protein dehydration distorted

the protein structure [125]

24 PROTEIN DESTABILISATION

There are many protein destabilising factors apart from dehydration and these

can be classified into chemical and physical instability Chemical instability happens

due to chemical modification such as deamidation oxidation proteolysis and

racemisation

241 Deamidation

Deamidation is the hydrolysis of amide link in the amino acid side groups such

as glutamine (Gln) and asparagine (Asn) (Fig 24)

Glutamine (Gln) Asparagine (Asn)

Fig 24 Side groups of Gln and Asn

It is favoured by increase in pH temperature and ionic strength The buffer

concentration influences the rate of deamidation at pH 7 ndash 12 Urea also accelerates

deamidation It often implies the protein aging and then modifies the protein function

93

242 Oxidation

Oxidation takes place at the side chains of cystine (Cys) methionine (Met)

tyrosine (Ty) tryptophan (Trp) and histidine (His)

Cysteine (Cys) Methionine (Met)

Tyrosine (Tyr) Tryptophan (Trp) Histidine (His)

Fig 25 Side groups of Cys Met Tyr Trp and His

Atmospheric oxygen can oxidise Met residues H2O2 modifies indole thiol (SH) [123

126] disulfide (SS) imidazole phenol and thioether (RSR) groups at neutral or slightly

alkaline conditions At acidic condition it oxidises Met into Met sulfoxide (R-S=O-R)

Under extremely oxidising condition Met sulfoxide is further oxidised into Met sulfone

(R-S(=O)2-R) Met oxidation leads to loss of biological activity however it does not

abolish the enzyme activity Therefore if oxidised Met is reduced back by a reductant

such as thiols it will restore the full biological activity Met reactivity depends on its

position within the protein Cys plays an important role in protein folding because it

forms intermolecular disulfide bonds however very susceptible to oxidation It can

undergo autooxidation in the absence of oxidants It is oxidised in several stages in

94

which thiol R-SH group is oxidised to (i) sulfenic acid (R-S-OH) (ii) disulfide

(R-S-S-R) (iii) sulfinic acid (R-S=O-OH) and (iv) sulfonic acid (R-S(=O)2-OH) [123

127] Oxidised thiol groups can be reduced back however they can be also reduced

back in the wrong way This leads to protein misfolding and loss of activity There are

many factors that influence the rate of oxidation They are (i) temperature (ii) pH (iii)

buffer medium (iv) metal catalysts (v) O2 tension (vi) steric condition (vii) neighbouring

groups (viii) photooxidation The presence of metal ions such as Fe2+ and Cu2+

accelerates oxidation by air [123] and the presence of O2 also might accelerate thiol

oxidation with other oxide such as nitric oxide radical [127] Metal-catalysed oxidation

involves a site-specific metal binding to Trp His and Met This leads to severer form of

oxidation causing protein aggregation Peroxide-catalysed oxidation on the other hand

is milder and tends to affect Cys and Trp but does not form aggregates [126] Increase in

pH increases pKa hence accelerates dissociation of ndashSH All of Cys Met Typ Tyr and

His are susceptible to photooxidatoin however His photooxidation is fatal leading to

enzymatic inactivity [123]

243 Proteolysis and racemisation

Proteolysis and racemisation takes place at aspartic acid (Asp) residue The

bond between Asp and proline (Pro) is especially susceptible to hydrolysis Asp also

easily undergoes base-catalysed isomerisation to isoAsp through a ring formation [123]

95

Aspartic acid (Asp) Proline (Pro)

Fig 26 Side group of Asp and the amino acid structure of Pro

244 Physical instabilities

Physical instability includes denaturation adsorption aggregation and

precipitation and [123] This refers to loss of tertiary structure and does not involve

chemical modification It is defined as Gibbs free energy change between the native and

unfolded state of protein Physical instability is induced by change in temperature

extreme pH organic solvent denaturant [123] and solvation [120 125]

25 ENZYME STABILISERS

By clarifying the possible major causes of instability the choice of optimal

approach and stabilisers can be made Considering the case of glucose-oxygen fuel cells

the stabilisers should be selected on the basis of following points (i) conformational

stability for longer operation and shelf life (ii) hydrophilic environment for optimal

enzyme functions (iii) optimal interaction between enzyme and substrate and enzyme

and environment Conformational stability is to secure protein folding and avoid

denaturation This is achieved by thiol group protection reduction of oxidised thiol

96

groups removal of oxidising species an increase in internal hydrophobic interaction

and environmental hydrophilic and electrostatic interaction Optimal enzyme activities

can be achieved by an increase in hydrogen bonding and electrostatic interactions

251 Polyehyleneimine (PEI)

Polyethyleneimine (PEI) has been reported as an efficient antioxidant as well

as chelating agent It is also cationic (Fig 27) [120] therefore provides electrostatic

stabilisation with anionic protein [126] such as GOx [67 85] Anti-microbial activity

has been also reported [126] It is not effective against thermal inactivation [128]

H+

N+H3N+H3

N+H3H3N+

H2+ H+

H+

H+

H2

+

H2+

Fig 27 Structure of PEI [129]

PEI is available in different molecular weights [120] and smaller molecules are more

effective in binding and flexibility [130] However its optimal loading concentration

varies [120 128 131] PEI protects thiol groups efficiently [120] from both metal- and

peroxide- catalysed oxidation despite of their different oxidation mechanisms [126] PEI

also forms a complex with the enzyme and coats it with polycations This occludes the

97

oxidants from the protein surface [126 130] Complex formation might be involving

hydrogen bonding between carboxylic acid groups in the enzyme and nitrogen atoms in

PEI [130] This possibly confers stability in dried state [128] It was reported that PEI

increased the maximum limiting catalytic velocity (V) for GOx [131] and lactate

dehydrogenase (LDH) [128] However it also increased Km for LDH and shifted its

optimal pH slightly toward acidic [128]

252 Dithiothreitol (DTT)

Dithiothreitol (DTT) is another small water-soluble [132] thiol protecting

reagent [132 133] with the reducing potential at ndash 033 V at pH 7 It has only little

odour [132] and has low toxicity [134] Its epimer is dithioerythriotol (DTE) Both are

excellent reducing reagents with two thiol groups (Fig 28) [132] however DTT might

be marginally better in reducing power [135] It reduces back disulfide quantitatively

[127 132] at lower concentration than other thiol-containing reducing reagents such as

Cys and gluthatione (GSH) But it cannot restore some of the lost catalytic activity

[136] because it only reduces disulfides but not other thiol oxides Therefore its

efficacy depends on (i) the oxidation mechanism involved in the protein inactivation

(ii) the velocity at which the inactivation proceeds and (iii) how thiol-contaning amino

acids are present in the protein DTT is not effective against mild oxidation caused by

low concentration of H2O2 or isolated Cys residues [127] It is probably because that

DTT reduces thiol oxide by converting itself from a linear into a cyclic disulfide form

which is called trans-45-dihydroxy-o-dithiane with (Fig 28) [132]

98

Fig 28 Structures of DTT from a linear to a cyclic disulfide form [132]

On the other hand arsenite was effective in reducing only mildly oxidated thiol groups

but not disulfides [127] It is however toxic [137] and not suitable for practical use

Neither of DTT or arsenite was effective if thiol oxidation is outcompeting [127]

253 Polyethylene glycol (PEG)

Polyethylene glycol (PEG) (Fig 29) has been reported as an enzyme stabiliser

however also has mixed reception and its stabilisation mechanism has not yet been

clearly understood

Fig 29 Structures of PEG

OrsquoFagain reported that PEG increased storage and thermo stability [120] while

99

Andersson reported the increase in LDH activity with PEG 10000 Mw but was

followed by a decrease after 10-day storage and then eventually became worse than

those without stabilisers The stabilisation was also found to be concentration dependent

[128] As for protein stabilisation mechanism it was proposed that PEG preferentially

hydrates the protein and stabilises its native structure in the solution [138-140]

According to Arakawa this causes the exclusion of PEG from protein surface due to the

unfavourable interaction between them hence increases water density around the

protein This results in the water shell around the protein surface and increases the

hydrogen bonds [138 139] Better hydration favoured larger PEG Mw due to the larger

steric exclusion [138] on the other hand lower PEG Mw was also reported to be better

because of its better solubility and viscosity [139] Optimal concentration is unknown

however one proposal is that higher concentration is more advantageous to stabilise the

denatured protein because it can penetrate and stabilise the protein hydrophobic core

exposed during denaturation [138] In contrast Lee reported that this penetration affects

the hydrophobic core of active enzyme and decreased the activity and denatured the

protein [139] Both however concluded that a fine balance between preferential

hydration and hydrophobic interaction is important and PEG concentration must be up

to 30 [138 139] It was also reported that PEG stabilised the protein of low water

content better because it prevented the water evaporation while polyols tended to strip

water from the protein dehydrated microenvironment [139]

254 Polyols

Polyols such as glycerol (GLC) (Fig 30) were reported to increase shelf life

100

[141] thermostability [120] and also provide preferential solvation as seen in PEG

[141-143] Higher GLC concentration resulted in better protein activity [143 144] and

reduced protein aggregation due to the tighter protein packing [144] However observed

hydration was lower than expected for high GLC concentration therefore Gekko

proposed that the contribution of higher GLC concentration did not derive form the

increase in steric exclusion Instead GLC might penetrate the solvation layer of protein

and stabilise the structured water network around the protein [143] Andersson also

compared the influence on protein activity after the storage among several candidate

stabilisers diethylaminoethyl-dextran (DEAE)-dextran Mw 500000 sorbitol and PEI

DEAE-dextran increased the activity both before and after the storage while sorbitol

decreased it However PEI was only found to be superior to all [128]

OH

OHHO

Fig 30 Structure of glycerol

255 FAD

FAD (Fig 16 p 75) is also another possible stabiliser as well as mediator

because it can regenerate denatured aged enzymes which have lost its cofactors [82

108] However it has not been clarified well and Bartlett questioned its efficacy due to

the contradictory results he obtained [145] It was reported that addition of FAD

increased the electrode sensitivity by 70 especially in the concentration range below

101

3mM and activity by 8 times although it also increased Km by 2 times [108]

26 ENZYME KINETICS AND ELECTROCHEMISTRY

261 Michaelis-Menten kinetics

A simple enzyme reaction can be expressed in terms of equilibrium between

the free enzyme (E) substrate (S) enzyme-substrate complex (ES) and product release

(P) The enzyme is regenerated on completion of the catalytic cycle

General enzyme reaction mechanism

middotmiddotmiddotmiddotmiddot Eq 41

E = Enzyme concentration mol

S = Substrate concentration mol

ES = Enzyme-substrate complex concentration mol

P = Product concentration mol

k1 = Rate constant for ES formation from E and S mol-1s-1

k-1 = Rate constant for ES dissociation into E + S s-1

k2 = Rate constant for P formation from ES s-1

The rate of ES formation is defined as

102

][][]])[[]([][2101 ESkESkSESEk

dtESd

minusminusminus= minus middotmiddotmiddotmiddotmiddot Eq 42

Where [E0] is the initial enzyme concentration

When the reaction is at steady state 0][=

dtESd

])[(][][]])[[]([ 212101 ESkkESkESkSESEk +=+=minus minusminus middotmiddotmiddotmiddotmiddot Eq 43

Re-arranging the above equation for [ES] as below

][]][[

][121

01

SkkkSEk

ES++

=minus

middotmiddotmiddotmiddotmiddot Eq 44

When the rate of product formation is defined as follows

middotmiddotmiddotmiddotmiddot Eq 45

][2 ESktP=

∆∆

=υ middotmiddotmiddotmiddotmiddot Eq 46

Where υ is the catalytic rate

When all the enzyme active sites are occupied such a condition is called saturated and

defined as [E0] = [ES] The maximum catalytic rate (V) is therefore available as below

Maximum catalytic rate V

][][ 202 ESkEkV == middotmiddotmiddotmiddotmiddot Eq 47

103

Express υ in terms of [S] by inserting Eq 44 into Eq 46

][

]][[

1

21

02

Sk

kkSEk

++

=minus

υ middotmiddotmiddotmiddotmiddot Eq 48

Michaelis-Menten constant (KM) is a dissociation constant about ES and defined as

Michaelis-Menten constant

1

21

kkk

K M+

= minus middotmiddotmiddotmiddotmiddot Eq 49

Michaelis-Menten equation (Eq 50) is obtained when υ is expressed in terms of V and

KM by inserting Eq 47 and Eq 49 into Eq 48

Michaelis-Menten equation

][][SK

SV

M +=υ middotmiddotmiddotmiddotmiddot Eq 50

This is a hyperbola equation to lead the enzyme catalytic rate Most enzymes typically

show this hyperbolic curve (Fig 31) when they are free from allosteric controls or

enzyme inactivation

104

0E+00

1E-09

2E-09

3E-09

4E-09

5E-09

0E+00 2E-03 4E-03 6E-03 8E-03 1E-02 1E-02 1E-02 2E-02

[S] M

Fig 31 A typical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

As shown in Fig 31 Michaelis-Menten plot has different kinetic profiles at high and

low [S] Mathematical expression of its transition over [S] is shown in Table 3

Table 3 Michaelis-Menten equation at various [S]

[S] ge KM Vcongυ middotmiddotmiddotmiddotmiddot Eq 51

[S] = KM 2V

=υ middotmiddotmiddotmiddotmiddot Eq 52

[S] le KM MM K

SEkK

SV ]][[][ 02=congυ middotmiddotmiddotmiddotmiddot Eq 53

At high saturated substrate concentration the catalytic rate becomes zero-order

therefore Michaelis-Menten curve becomes plateau When [S] = KM the rate becomes

105

half of the maximum catalytic rate At low substrate concentration the catalytic rate is

linearly dependent on [S] therefore the kinetics becomes first-order about [S]

262 Electrochemistry and Michaelis-Menten kinetics

In electrochemistry enzyme activity is studied by amperometry This involves

measuring measuring the current generated by the enzyme electrode reaction at a fixed

potential over an arbitrary period It therefore shows the transition in current response

over time For enzyme electrodes substrate concentration is changed over time so that

the current response for a different substrate concentration can be measured over a long

period With intact enzyme and appropriate heterogeneous electron transfer present

most enzyme electrodes follow Michaelis-Menten kinetics (Fig 32) in the amperometry

in the absence of allosteric control or enzyme inactivation Therefore the amperometry

data can be analysed in the similar way to that of biochemical data and for this

Michaelis-Menten equation is expressed in electrochemical terms [146] In

electrochemical enzyme kinetics enzyme catalytic rate (υ) is obtained in terms of

electric current therefore V is substituted with maximum steady state limiting current

(Im) or its current density (Jm)

106

Electrochemical Michaelis-Menten equation

][][

SKSI

IM

m

+times

= middotmiddotmiddotmiddotmiddot Eq 54

][][

SKSJ

JAI

M

m

+times

== middotmiddotmiddotmiddotmiddot Eq 55

I = Current A

Im = Max steady-state limiting current A

[S] = Substrate concentration mol

KM = Michaelis-Menten constant mol

A = Electrode surface area m-2

J = Current density Am-2

Jm = Max steady-state limiting current density Am-2

There is often an offset at zero substrate concentration due to competing reactions and

trace contaminants and this must be subtracted before analysis

107

V

0E+00

2E-04

4E-04

6E-04

8E-04

1E-03

0 2 4 6 8 10 12 14 16

frac12 Jm

KM

Jm Am-2

[S] mM

Jm

Fig 32 Electrochemical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

KM and Jm (or V in biochemical enzyme kinetics) are important parameters to analyse

both enzyme catalytic power and stability Electrochemical Michaelis-Menten equation

and curves can be used for rough estimation of KM and Jm however it often leads to

inaccurate result because measuring initial rate for high substrate concentration is very

difficult in this method hence very prone to errors Therefore Michaelis-Menten

equation is linearised using one of the following four methods (i) Lineweaver-Burk (ii)

Eadie-Hofstee (iii) Hanes-Woolf and (iv) Cornish-Bowden-Wharton equations

Michaelis-Menten plot is however good for intuitive diagnosis of general enzyme

behaviours

108

263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics

2631 Lineweaver-Burk linear regression

Lineweaver-Burk linear regression is one of the classic regression methods

taking double reciprocal of Michaelis-Menten equation (Eq 56)

Electrochemical Lineweaver-Burk linear regression

mm

M

JSJK

J1

][11

+times= middotmiddotmiddotmiddotmiddot Eq 56

It plots 1J against 1[S] as shown in Fig 33 The value of each parameter is found by

extrapolating the line The reciprocal of Jm is found at y-intercept and KM can be derived

from either x-intercept or the slope once Jm is found

109

-50E+02

00E+00

50E+02

10E+03

15E+03

20E+03

-06 -05 -04 -03 -02 -01 00 01 02 03 04

1[S] mM-1

1J A-1m

2

Fig 33 Electrochemical Lineweaver-Burk linear regression

Based on the real data Refer to sect 343 p 124

Lineweaver-Burk linear regression is however largely influenced by the errors in j at

low substrate concentrations [22 147] Its reciprocal could vary by a factor of 80 over

the range of substrate concentration varying one-third to three times the KM [148]

Hence it is not an ideal method for the analysis

2632 Eadie-Hofstee linear regression

Eadie-Hofstee linear regression plots J against J[S] This plot is strongly

affected by the errors in υ because υ is present in both coordinates (Eq 57)

Electrochemical Eadie-Hofstee linear regression

mM JSJKJ +timesminus=

][ middotmiddotmiddotmiddotmiddot Eq 57

110

Again the plot must be extrapolated to find Jm at y-intercept KM can be derived from

the slope (Fig 34)

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

0E+00 5E-05 1E-04 2E-04 2E-04 3E-04

J [S] AmM-1

J Am

-2

Jm

-Km

Fig 34 Electrochemical Eadie-Hofstee linear regression Based on the real data

Refer to sect 343 p 124

Eadie-Hofstee linear regression will give very strict evaluation [148] and might be used

to detect the deviations from Michaelis-Menten behaviour only if the data is precise and

accurate [147]

2633 Electrochemical Hanes-Woolf linear regression

Hanes-Woolf linear regression plots [S]J against [S] KM is derived from the

x-intercept and Jm is found from the slope

111

Electrochemical Hanes-Woolf linear regression

m

M

m JKS

JJS

+times= ][1][ middotmiddotmiddotmiddotmiddot Eq 58

This is a better method compared to the previous two regression methods because all

the parameter values can be derived from the reasonable range of [S] and they donrsquot

heavily rely on the extrapolation [147] The data is normalised by [S] therefore does not

cause many errors

-20E+08

00E+00

20E+08

40E+08

60E+08

80E+08

10E+09

12E+09

14E+09

-40E+00 00E+00 40E+00 80E+00 12E+01 16E+01

[S] mM[S] J m

MA

-1m2

Fig 35 Electrochemical Hanes-Woolf linear regression Based on the real data

Refer to sect 343 p 124

However either of these three linear regressions might not be adequate for the enzyme

electrode analysis [65] because the data reflect not only the single-substrate

Michaelis-Menten kinetics but also include the influence from other factors such as

enzyme immobilisation mediator reactions and electrode composition as well as

112

general experimental errors Their contributions to the overall kinetics therefore must be

considered [65 149] although the enzyme electrode reactions can approximate the

Michaelis-Menten model when one substrate is a variable and other variables are fixed

over an experiment [147 149] Under such a complex system it is not ideal to use those

graphical linear regression methods because they assume that the results follow

Michaelis-Menten model and that all the experimental errors follow normal distribution

[150] without giving any measure in the precision [148] Hence it is more suitable to

use a non-parametric direct linear plot or nonlinear regression methods because they

respect the influence and errors from various sources without any bias

264 Cornish-Bowden-Wharton method

Cornish-Bowden-Wharton method is distribution-free non-parametric direct

linear plot In this method observations are plotted as lines in parameter space rather

than points in observation space [151] It then takes the median of Jm and KM from every

possible non-duplicate pair of observed [S] and υ (Eq 59 and Eq 60) as the best-fit

estimate

Electrochemical Cornish- Bowden-Wharton non-parametric method

])[][(])[]([

1221

122121 SJSJ

SSJJJm minusminus

= middotmiddotmiddotmiddotmiddot Eq 59

])[][()](][[

1221

122121 SJSJ

JJSSKM minusminus

= middotmiddotmiddotmiddotmiddot Eq 60

It is a simple method without complex calculation insensitive to outliers and no

113

weighting is required [147 152]

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

14E-03

16E-03

18E-03

20E-03

-5 -3 -1 1 3 5

KM mM

Jm Am-2Median Jm

Median KM

Fig 36 Electrochemical Cornish-Bowden-Wharton plot Based on the real data

Refer to sect 343 p 124

265 Nonlinear regression methods

Nonlinear regression methods manipulate the parameters to minimise the sum

of squared difference between the measured and expected values [150] They fit the

integrated equation to the enzyme progress curve and calculate the approximate location

of the minimum for the sum of squared difference at a particular point in the slope It

then recalculates the new minimum location to improve the prediction until there is no

further improvement This is called gradient method based on Gauss-Newton algorithm

or non-linear least square method [150 153] This is computationally intensive due to

114

the recursive operations however advanced computational technology has made it

possible The parameters obtained may not be perfectly correct [150] The method still

assumes errors normally distributed [153] and Cornish-Bowden has criticized about the

ambiguous statistical assumption of such computer programmes [154] However

non-linear regression methods are insensitive to outliers [150 153] have better estimate

accuracy compared to conventional linear regression methods and the estimations

mostly fit Michaelis-Menten model Graphical methods arose at a time when nonlinear

regression methods were computationally not available however it is now possible

thanks for the great technological advance and more recommended to use in analysis to

yield better precision and reliability [153]

115

3 ANODE MATERIAL AND METHOD

31 ELECTRODE FABRICATION

311 TTF-TCNQ synthesis

Tetrathiafulvalene ge980 (TTF) and 7788-Tetracyanoquinodimethane

ge980 (TCNQ) were purchased from Sigma Aldrich Spectroscopic grade acetonitrile

and diethyl ether were purchased from VWR International TTF and TCNQ were

weighted to 1 g each and dissolved separately in 50 ml of acetonitrile TTF-acetonitrile

solution had yellow colour while TCNQ-acetonitrile solution was green Both were

heated on the hot plate under the fume hood On the full dissolution two solutions were

quickly mixed to form black solid crystals The mixture was immediately taken off the

hot plate and cooled down at the room temperature for a few hours The crystals were

collected on Buchner funnel and washed under vacuum twice with acetonitrile twice

with diethyl ether and then twice again with acetonitrile The washed crystals were left

to dry for overnight with a cover The dried crystals were collected and kept in the

sealed container for future use [61]

312 Electrode template fabrication

Sylgard 184 Silicone Elastomer Kit were purchased from Dow Corning

Corporation for polydimethylsiloxane (PDMS) Base and hardener were mixed at 101

(vv) for use CY1301 and aradur hardener HY 1300 were purchased from Robnor

116

resins to make epoxy resin They were mixed at 31 (vv) for use

Silicone electrode templates (Fig 16(i)) were made to shape electrode discs

Each template had round holes in line Two types of templates template A and B were

made Template A had holes with the dimension 2-mm diameter x 1-mm thickness

while template B had holes with the dimension with 5-mm diameter x 2-mm thickness

In order to make PDMS templates for electrode discs different templates were made to

shape the holes in PDMS (Fig 16(ii)) Discs were made with epoxy resin and were

fixed onto the plastic Petri dish PDMS was poured into disk-mounted Petri dish

degassed for 20 minutes and then cured at 60 degC Cured PDMS electrode templates

were taken out from the epoxy resin-Petri dish templates and then cleaned with

analytical grade ethanol for use

Fig 37 PDMS electrode templates (i) PDMS template and (ii) their epoxy resin-Petri

dish template

117

313 Electrode composite fabrication

3131 Composite for conductance study

Graphite at microcrystal grade (2-15 micron 999995 purity) was purchased

from Alfa Aesar GmbH amp CoKG Glassy carbon (GC) powder was kindly donated from

Dr Zhao Carbon nanofibre (CNF) was also kindly donated from Loughborough

University Composite electrodes were built from the mixture of the carbon conductive

or TTF-TCNQ and epoxy resin Each composite mixture had different composition ratio

[108] as below in Table 4

Composite Ratio I Ratio II Ratio III Ratio IV Ratio V

Graphite Epoxy resin 73 64 55

GC Epoxy resin 64 73

CNF Epoxy resin 37 46 55 64 73

TTF-TCNQ Epoxy resin 64 73 82

Table 4 Composite composition

TTF-TCNQ was mixed with epoxy resin at 64 73 and 82 Graphite was mixed at 73

64 and 55 GC powder was mixed at 64 and 73 CNT was mixed at 37 46 55 64

and 73 (ww) Three to five copies were fabricated for each electrode type Composite

mixture was packed into the template A (Fig 38) and cured at room temperature for 72

hours [106]

118

Fig 38 PDMS template (i) packed with electrode material and (ii) empty one

3132 Bulk modified GOx enzyme electrode composite

Glucose oxidase (GOx) [Type II-S 15000-50000 unitsg] from Aspergillus

niger was purchased from Sigma Aldrich Conductive composite paste was first made

with TTF-TCNQ and epoxy resin at 73 as in sect 3131 GOx was then incorporated into

the conductive composite mixture at varying mixing ratio 5 10 and 15 (ww) by

bulk-modification method (Fig 39) 10 GOx composite electrodes were also made

with graphite Enzyme composite mixture was packed into the template B and cured at

4 degC for 96 hours

119

R

RR R

R

RR R

RRR

Conducting particles

Insulating binder

Chemical modification

Add biological receptor R

Cast extrude or print into suitable form

R

R

RR

R

R

R

R

R

R

RR

Fig 39 Schema for bulk modification method

3133 GOx enzyme electrode composite with enzyme stabilisers

Poly(ethyleneimine) (PEI) solution [50 (wv) in H2O] DL-dithiothreitol

(DTT) Poly(ethylene glycol) (PEG) [average mol wt 200] glycerol (GLC) [ge99] and

flavin adenine dinucleotide disodium (FAD) salt hydrate (ge95) were purchased from

Sigma Aldrich Each stabiliser was mixed with GOx at different fraction 0 1 2 and

4 PEI was also blended with DTT or FAD for synergetic effects as below in Table 5

Blend type Ratio I Ratio II Ratio III Ratio VI

PEIDTT 0515 1010 1505 2020

PEIFAD 21 22 24

Table 5 Blended stabiliser composition

120

PEI was mixed with DTT at the ratio of 0515 11 1505 and 22 PEI was also

mixed with FAD at 21 22 and 24 Each enzyme-stabiliser mixture was weighted to

10 of the whole composite It was then mixed with the conductive composite mixture

made earlier The conductive composite mixture was made of TTF-TCNQ and epoxy

resin at 73 The final composite mixture was packed into the template B and cured at

4 degC for 96 hours

314 Electrode wiring and insulation

Silver wire (temper annealed 025-mm diameter) was purchased from Advent

Portex polyethylene tubing (028 x 0165 mm) purchased from Scientific Laboratory

Supplies Silver epoxy was purchased from ITW chemotronics Silver epoxy came in

two parts A and B and they were mixed at 11 (vv) for use Silver wire was cut for

5-cm length and its tip was cleaned with analytical grade ethanol dried and coved with

insulating polyethylene tubes Cured electrode composite discs were popped out of the

PDMS template An insulated silver wire was fixed onto to each electrode disc with

conductive silver epoxy and it was left at room temperature for 24 hours to cure silver

epoxy After silver epoxy had been cured the wired electrodes were coated and

insulated with degassed epoxy resin and cured at room temperature for 3 days for

non-enzyme composite electrodes and 4degC for 4 days for enzyme composite electrodes

7-9 days were taken to build the complete composite electrodes (Fig 40) The enzyme

electrodes were stored at 4degC when not in use

121

Fig 40 Insulated composite electrodes (i) non-enzyme (ii) GOx electrodes

32 CONDUCTIVITY EXPERIMENT

Copper foil with electrically conductive acrylic adhesive was purchased from

RS Components Digital multimeter from Metra was used to measure resistance

Electrode surface was covered with adhesive copper foil evenly and both ends were

clamped with crocodile clips (Fig 41(i)) to measure electrode resistance of the

composite as in Fig 41(ii)

122

Fig 41 Measuring electrode resistance (i) connecting the electrodes to the

multimeter (ii) measuring the electrode resistance with the multimeter

33 POLISHING COMPOSITE ELECTRODES

MicroPolish II aluminium oxide abrasive powder (particle range 1 03 and

005 microm) silicone carbide (SiC) grinding paper for metallography wet or dry (grit 600

1200 and 2400) and MicroCloth Oslash73 mm for MiniMet for fine polishing were

purchased from Buehler Self adhesive cloth for intermediate polishing was purchased

from Kemet Electrodes were polished immediately before the experiment until

mirror-like flat surface was obtained This erases the electrode history and gives

assigned electrode surface area [105] Composite electrodes were hand polished

successively with SiC grinding paper in 400 1200 and 2400 grit During polishing

deionised water of 18 MΩ was added occasionally to the polishing surface This

prevents damaging electrode surface with frictional heat It was followed by successive

polishing with alumina oxide slurry with particle size 1 03 and 005 microm Slurry was

123

sonicated to dissolve agglomerates before use [155] Kemet polishing pad was used for

rougher polishing with 1 and 03 microm powder Felt-like MicroCloth was used for

polishing with 005microm Electrodes were sonicated in the deionised water briefly after

polishing at each grit or grade

34 GLUCOSE AMPEROMETRY

341 Glucose solution

Phosphate buffer saline (PBS) powder for 001 M at pH 74 containing 0138

M NaCl and 00027 M KCl [156] was purchased from Sigma Aldrich PBS was kept in

the fridge when not in use [157] 1M glucose solution was made by mixing

D-(+)-Glucose with 001 M PBS and left for 12 hours at 4degC for mutarotation It is

mandatory because GOx has stereochemical specificity and only active toward

β-D-glucose while glucose solution consists of two enantiomers α- and β-D glucose

and their concentration changes over time until they reach the equilibrium at 3862

Experiments should be carried out in the equilibrated solution for accuracy

342 Electrochemical system

A single-channel CHI 650A and multi-channel CHI 1030 potentiostats were

used for glucose amperometry with three-electrode system in which the

electrochemical cell contained (a) working electrode(s) an AgCl reference electrode

and a homemade Pt counter electrode The AgCl reference electrode was purchased

124

from IJ Cambria AgCl reference electrode was saturated with 3M KCl Pt counter

electrode was made of Pt wire and Pt mesh was fixed at its tip to give large surface area

Pt mesh at its tip

343 Glucose amperometry with GOx electrodes

Glucose amperometry was conducted by adding a known volume of 1M

glucose aliquot into 100 ml of 001 M PBS electrolyte solution containing GOx anodes

with convection applied Convection was however ceased on during current

measurement to avoid the noise The electrolytic solution was gassed with either N2 O2

or compressed air for 15 minutes before the experiment O2 concentration is 123 mM in

fully oxygenated PBS [158] and 026 mM in air [112] Gas was continuously supplied

through Dreschelrsquos gas washing bottle containing PBS during experiment The response

of GOx anodes to glucose was obtained as steady state current The amperometry was

carried on until no further current increase was observed against glucose added Each

experiment took more than 8 - 12 hours The sample size ranged from two to three in

most cases

125

Fig 42 Amperometry setting with CHI 1030 multichannel potentiostat

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA

Normality of enzyme function was confirmed by plotting Michaelis-Menten

curve based on the amperometry data obtained The capacitive current before glucose

addition was subtracted from the data Enzyme kinetic parameters the maximum

current density (Jm) and the enzyme dissociation Michaelis constant (KM) were

automatically estimated with standard error using an analytical software called

SigmaPlot Enzyme Kinetics version 13 The estimation is based on non-linear

regression analysis about Michaelis-Menten kinetics as shown in Fig 43

126

Glucose M

0 1e-2 2e-2 3e-2 4e-2 5e-2

J A

m-2

00

20e-3

40e-3

60e-3

80e-3

Vmax = 0008445 Km = 0005842

Fig 43 Analytical output by SigmaPlot Enzyme Kinetics Based on the real data

Influence of enzyme immobilisation electrode composition O2 and stabilisers on GOx

electrode performance was studied The most efficient GOx anodes was determined on

the basis of the parameters obtained

127

4 ANODE RESULTS AND DISCUSSION

41 AIMS OF ANODE STUDIES

This section describes the fundamental study on the enzyme composite

electrodes and the influence of enzyme stabilisers on the performance and stability of

GOx electrodes Firstly the difference in conductivity as a function of various

conductive materials and the component fraction were studied They did not contain

enzyme The most suitable composite was then selected and used to build the enzyme

electrodes Secondly GOx electrodes were built at various GOx fractions in the

composite The best component fraction was determined from the data obtained in

amperometry Amperometry was conducted to obtain two enzyme kinetic parameters

the maximum current density (Jm) and the enzyme dissociation Michaelis constant (KM)

Jm indicates the size of catalytic velocity KM indicates the extent of enzyme-substrate

dissociation Smaller KM values indicate better enzyme-substrate affinity and enzyme

stability With the component ratio selected enzyme electrodes were then constructed

using various types and concentrations of enzyme stabiliser Amperometry was again

conducted to study the influence of enzyme stabilisers on the GOx anode performance

and stability The catalytic efficiency was determined by the fraction M

mK

J This is

based on the concept of specificity constant M

catK

k as defined below

128

Enzyme electrode specificity constant

MMM

cat

KEV

Kk

Kk

sdot==

][2 middotmiddotmiddotmiddotmiddot Eq 61

where kd = k2 Rate constant for P formation from ES

The specificity constant is a common measure of the relative catalytic rate at low

substrate concentration [22] M

mK

J therefore can be used as a comprehensive

indicator for both catalytic velocity and enzyme stability under the fixed enzyme

concentration In this study GOx concentration was fixed to approximately 10 weight

of the total composite According to the M

mK

J obtained the five most efficient and

stable GOx electrodes were determined The possible enzyme stabilisation mechanisms

of each enzyme stabiliser were also discussed together in the light of published

hypothesises

42 CONDUCTANCE OF THE COMPOSITE

421 Conductance of various conductive materials

Various electrodes were built with different conductive materials graphite GC

CNF and TTF-TCNQ at different component fraction The electrode dimension was

fixed to 314 mm2 x 1 mm Their conductance was measured and presented in a box plot

as shown in Fig 44 The sample size ranged from 3 to 6 The median is indicated by a

short line within the box

129

00E+00

50E-02

10E-01

15E-01

20E-01

25E-01

30E-01

35E-01

40E-01

45E-01C

ondu

ctan

ce

S

Lower quartile S 103E-03 549E-03 219E-01 111E-01 142E-01 599E-04 390E-03 115E-02 972E-03

Max S 281E-03 338E-02 268E-01 312E-01 415E-01 810E-04 113E-02 467E-02 262E-02

Min S 739E-04 467E-03 200E-01 933E-02 855E-02 418E-04 296E-03 264E-03 587E-03

Upper quartile S 206E-03 282E-02 253E-01 220E-01 253E-01 795E-04 807E-03 232E-02 199E-02

Median S 131E-03 847E-03 238E-01 129E-01 216E-01 780E-04 484E-03 155E-02 136E-02

TTF-TCNQ64

TTF-TCNQ73

Graphite 55 Graphite 64 Graphite 73 CNF 37 CNF 46 CNF 55 GC 73

Fig 44 Conductance of various electrode composite n = 3 ndash 6

Graphite composites showed higher conductance than other composites It is explained

by graphite having the highest intrinsic conductivity ranging between 103 ndash 104 Scm-1

[105 159] although being compounded in a composite lowered the conductivity by 4 ndash

5 order of magnitude compared to that of raw material The conduction did not show a

linear relationship with the electrode composition ratio for graphite composite because

the graphite composite 55 had larger conductance of 024 S than that of 64 and 73 It

also showed the smallest deviation than the other graphite composites while the

deviation increased with increase in the graphite content One possible cause of this is

the presence of patchy conductive paths due to the uneven distribution of conductive

islands within the composite [105 106] The conduction of the composite is therefore

often locally biased unpredictable and also leads to low reproducibility TTF-TCNQ

composite at 73 showed the conductance of 85 mS smaller than the graphite

composited 55 approximately by 30 times Conductivity of TTF-TCNQ is 102 ndash 103

130

Scm-1 [109 110] and smaller than that of graphite by one order of magnitude so this is

consistent with the intrinsic conductivity of the conducting paticles

There were also different interactions observed among conductive materials

with insulating materials Mixing epoxy resin with graphite was easier than mixing with

other conductive materials Some even did not cure at the same mixing ratios The

carbon nanofibre (CNF) composite mixture did not cure when it exceeded more than

50 weight content With 40 CNF content however it showed the conductance of 48

mS It was not comparable to graphite composite despite its similar conductivity of 103

S [105] however it was similar to that of TTF-TCNQ 73 composite 50 CNF content

even showed larger conductance of 155 mS than 70 TTF-TCNQ CNFs have been

reported to have mediating function [117] therefore with this high conduction property

at low content CNF might be very useful to enzyme composite and even superior to

TTF-TCNQ

422 Influence of enzyme concentration on conductivity

Conductance of the GOx composite was measured The conductive composite

consisted of either TTF-TCNQ 73 or graphite composite 73 GOx content was varied

by 5 10 and 15 for TTF-TCNQ composite and 10 only for graphite composite

The electrode dimension was fixed to 196 mm2 x 2 mm The sample numbers ranged

from 2 ndash 4 The study revealed that the presence of enzyme interfered with conduction

in GOx concentration dependent manner for TTF-TCNQ composite as shown in Fig 45

131

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01C

ondu

ctan

ce

S

Low er quartile S 131E-02 924E-03 124E-05 165E-01

Max S 484E-02 143E-02 368E-05 593E-01

Min S 120E-02 754E-03 390E-06 122E-01

Upper quartile S 441E-02 126E-02 288E-05 400E-01

Median S 281E-02 109E-02 208E-05 207E-01

TTF-TCNQGOx 5 TTF-TCNQGOx 10 TTF-TCNQGOx 15 GraphiteGOx 10

Fig 45 Conductance of GOx composite with various GOx content n= 2 - 4

This was due to insulating property of the protein although higher enzyme loading is

preferred for better catalytic performance as discussed in sect 226 on p 88 The issue

may be circumvented by fixing the enzyme onto the electrode surface It is however

more technically tedious and not as feasible for mass manufacture as bulk modification

It is also difficult to modify conductive salt for covalent attachment of the enzyme This

is because the conductive property of conducting salt is very sensitive to its chemical

structure and slight modification can lead to insulating properties [61] A similar

phenomenon has been also observed in CNT functionalisation [117]

Graphite-GOx composite showed larger conductance than TTF-TCNQ-GOx

composite by 20 times at the same component fraction This was due to the better

conductive property of graphite However graphite does not have mediating property

and the use of graphite as conductive material for enzyme electrodes requires the

132

loading of mediator separately The key to designing enzyme electrodes is in the balance

and compromise among the conductivity mediation enzyme activity mechanical and

chemical stability and difficulty in fabrication process A good candidate material to

make up for the TTF-TCNQ lower conductivity is CNT It has been reported that CNF

has both good conductivity and mediating functions [117] and might be suitable for bulk

modification More safety study on use of CNF is however necessary [117 160]

43 CATALYTIC ACTIVITY OF GOx COMPOSITE ELECTRODES

431 Influence of GOx concentration on the catalytic activity of GOx electrodes

Amperometry was conducted for GOx composite electrodes in N2 O2 and air

for various GOx content The conductive composite consisted of either TTF-TCNQ 73

or graphite composite 73 GOx content was varied by 5 10 and 15 for

TTF-TCNQ composite and 10 only for graphite composite The sample size was 3 for

each electrode type Control experiments were also performed with enzyme-free

composite electrodes and there was no glucose response observed There was also no

stable current observed for 10 GOx-graphite electrodes despite of its high

conductivity reported in sect 422 while Michaelis-Menten curve was observed in any

TTF-TCNQ-GOx composite electrodes This confirmed the necessity of mediating

function for enzyme anodes and its presence in TTF-TCNQ

All the TTF-TCNQ-GOx electrodes showed the Michaelis-Menten current

response curves however differed in the current as shown in Fig 46

133

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

35E-02

00E+00 10E-02 20E-02 30E-02 40E-02 50E-02

Glucose M

J A

m-2

GOx 5GOx 10GOx 15

Fig 46 Difference in the glucose-response amperometric curve of enzyme

electrodes for various GOx content 5 10 and 15 in the presence N2

The 15 GOx composite showed the smallest glucose response current due to the

interfered conduction by high protein content while 5 GOx composite showed smaller

response than 10 composite due to the enzyme deficiency Their Jm only counted for

26 and 41 of that of 10 GOx composite (Fig 47(i)) 10 GOx electrode was

therefore most catalytic KM for 5 and 10 GOx were 71 and 73 mM and little

different (Fig 47(ii)) This implies that the size of catalytic activity can be flexibly

altered by only slight change in KM

134

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

Jm

Am

-2

Jm Am-2 729E-03 180E-02 466E-03

GOx 5 GOx 10 GOx 15

00E+00

20E-03

40E-03

60E-03

80E-03

10E-02

12E-02

14E-02

Km

M

KM M 710E-03 729E-03 187E-03

GOx 5 GOx 10 GOx 15

Fig 47 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 n = 1 - 3

The results also tell that an increase in GOx loading could enhance the catalysis hence

output current unless the increase is not excessive and gt 15 at least The loading limit

should lie somewhere between 10 and 15 for this composite Similar electrodes

were also built by Alegret with 75 epoxy resin 10 TTT-TCNQ 10 graphite and

5 GOx [115] Their Jm was around 7 mAm-2 and little different from that of 5 GOx

with 70 TTF-TCNQ composite It suggests that less TTF-TCNQ content might be

sufficient as long as the conductivity is sustained by more conductive material such as

graphite

432 Influence of O2 on the catalytic activity of GOx electrodes

O2 and air suppressed the current output by GOx composite electrodes and

delayed the response to the glucose as shown in Fig 48 A current increase was

observed immediately after glucose addition for N2 but not for O2 and air They showed

135

sigmoidal current curves with a glucose response lag

(i)

(ii)

-50E-03

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

00E+00 50E-03 10E-02 15E-02 20E-02 25E-02 30E-02 35E-02 40E-02 45E-02

Glucose M

J A

m-2

GOx 10 N2GOx 10 O2GOx 10 Air

Fig 48 Difference in the glucose-response amperometric curve of 10 GOx

composite electrodes in the presence (i) N2 and O2 and (ii) re-plotted

amperometric curves for N2 air and O2

136

As expected O2 and air decreased Jm and increased KM as shown in Fig 49

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

GOx 5 GOx 10 GOx 15

Jm

Am

-2

N2O2Air

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

70E-02

80E-02

GOx 5 GOx 10 GOx 15

Km

M

N2O2Air

Fig 49 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 O2 and air n = 1 - 3

Both O2 and air decreased the current of 15 GOx composite electrodes Jm dropped to

approximately 20 of that for N2 KM was increased by 10 and 2 times for O2 and air

respectively The glucose response lag was 8 mM for O2 but none for air Air showed

milder suppression toward current output than O2 and this was expected because O2

counts for only 20 of the air [31] and should cause less influence on GOx activity

Similar phenomena were observed for 10 GOx composite electrodes For O2 and air

Jm was 13 and 40 of that of N2 and decreased to 23 x 10-3 and 67 x 10-3 from 18 x

10-2 Am-2 respectively KM was 73 for N2 and 10 mM for both O2 and air although there

was a response lag of 8 and 1 mM respectively Little difference in KM was observed

among all cases and this implies that KM itself hence enzyme stability or substrate

affinity is little affected by O2 but slight change in KM has large influence on the

catalytic activity This implies that change in catalytic activity induced by O2 is

137

reversible 5 GOx composite electrodes showed slightly unexpected result KM was

increased by approximately 4 times in both O2 and air Jm also decreased however Jm

for air was smaller than O2 by 36 despite of less O2 content There was a response lag

by 10 mM for O2 and 2 mM for air

The overall conclusions from these results is that O2 reduces GOx catalytic rate

andor electron transfer rate and hence reduces the current output but the effect is

reversible because it hardly alters KM value implying that O2 does not affect the enzyme

stability or its affinity to the substrate This phenomenon is typical in non-competitive

inhibition for enzyme kinetics Non-competitive inhibition occurs when an inhibitor

binds to the enzyme-substrate complex and slows its catalysis It therefore changes

catalytic velocity but not KM In the glucose amperometry this implies two possibilities

as a cause of Jm reduction (i) allosteric binding of O2 to GOx-glucose complex or (ii)

interference in electron transfer after the catalysis In the former case O2 does not

inhibit glucose binding to GOx but allosterically interacts with its complex and hinders

its catalysis hence it reduces catalytic current output In the latter case either glucose

binding or GOx catalysis is not affected by O2 but competition for electrons produced

by GOx catalysis occurs between the natural and artificial mediators namely O2 and

TTF-TCNQ and this reduces the electron flow to the electrode therefore it is reflected

as the reduction of catalytic current In addition to this the presence of glucose response

lag also suggests the presence of electron competition [161-163] It was reported that O2

reacts with reduced GOx faster than any other mediators with the second order rate

reaction of 15 x l06 M-1s-l [112 161 163] It is therefore quite possible that O2 swiftly

deprives electrons from reduced GOx andor reduced mediators Reduced GOx reacts

with O2 much faster than with TTF-TCNQ because it was observed that O2 had

138

diminished glucose response current at low glucose concentration Most GOx hence

reacts with O2 first then oxidised TTF-TCNQ once most O2 has been converted into

H2O2 This is compatible with the hypothesis suggested by Hindle [161] Even after the

lag the reaction could go on with residual O2 because KM did not recover fully to the N2

level although a familiar Michaelis-Menten hyperbolic curve was regained This simply

indicates that O2 concentration became smaller than TTF-TCNQ to oxidise GOx The

glucose response lag also decreased with an increase in GOx loading and was smaller

for air than O2 This implies the presence of competition because the lag depends on the

concentration of reduced GOx and electron thieves The large KM increase in the 5

GOx composite electrodes was probably due to the deficiency in the electron transfer

rate to suppress O2 activity How O2 interacts with its environment is unknown however

O2 reduces the catalytic output current due to the following possible mechanisms (i)

electron competition between O2 and TTF+ (ii) non-competitive enzyme inhibition by

allosteric binding of O2 to GOx (iii) direct interaction between O2 and reduced TTF or

(iv) backward reaction between reduced TTF and oxidised GOx

139

Table 6 Four possible causes of glucose amperometric current reduction

(i) middotmiddotmiddotmiddotmiddot Eq 62

middotmiddotmiddotmiddotmiddot Eq 63

(ii) middotmiddotmiddotmiddotmiddot Eq 64

(iii) middotmiddotmiddotmiddotmiddot Eq 65

(iv) middotmiddotmiddotmiddotmiddot Eq 66

Diagnosis can be made by doing experiments with different concentration of O2 and

mediators

44 THE EFFICACY OF GOX STABILISERS

441 Introduction

Various GOx stabilisers PEI DTT PEG GLC and FAD were mixed with GOx

composite at the concentration varying among 1 2 and 4 PEI was also mixed with

DTT or FAD and their synergetic effects were studied All the GOx electrodes were

built on the basis of TTF-TCNQ 73 conductive composite with 10 weight

GOx-stabiliser mixture Control composite electrodes were built with 2 stabilisers but

without GOx Glucose amperometry was conducted as before in the both presence and

absence of O2 Two kinetic parameters Jm and KM were obtained through non-linear

regression analysis The standard kinetic parameter values were set to those obtained in

140

the absence of O2 for GOx electrodes containing no stabiliser They are Jm = 18 x 10-2 plusmn

73 x 10-3 Am-2 and KM = 73 plusmn 57 mM respectively The glucose response lag

observed for this electrode was 10 mM Comprehensive catalytic efficiency was

determined by the size of the fraction M

mK

J under the influence of high O2 and the

most catalytic GOx composite electrodes were chosen on the basis of M

mK

J The

sample size ranged between 2 and 3 None of control experiments showed any glucose

response

442 PEI

Kinetic parameters for various PEI concentrations are shown below in Fig 50

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser PEI 1 PEI 2 PEI 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEI 1 PEI 2 PEI 4

KM

M

N2 O2

Fig 50 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEI in the presence and absence of O2 n = 1 - 3

141

In the absence of O2 PEI lowered Jm without largely affecting KM compared to the

standard value This might imply the presence of allosteric non-competitive inhibition

of glucose catalysis It is certainly possible because PEI can lock in the enzyme

conformation with hydrogen and electrostatic bonding [120] or form a complex with

GOx [126 130] This interferes in its catalysis although it also might confer the

resistance against oxidative stress as reported [126] In the presence of O2 two out of

three electrodes of PEI 1 and 4 did not show Michaelis-Menten curves The data

presented here is therefore based on a single sample This may mean that the electrodes

at those PEI concentrations are in fact prone to the catalytic suppression by O2

Moreover 4 PEI data presented here looks unreliable due to the simultaneous increase

in Jm and KM by 3 ndash 4 times compared to those obtained in the presence of N2 It may

need more data to confirm the phenomenon and be premature to conclude it is however

probably more reasonable to think that PEI 4 is not suitable for GOx anodes At other

PEI concentrations electrodes showed more or less similar kinetic parameter values to

those without stabilisers since Jm decreased and KM increased in the presence of O2

There was no O2-scavenging function observed because their glucose response lag and

KM resembled those without stabilisers Among them 2 PEI containing electrodes

sustained 50 of catalytic activity obtained in N2 and showed marginally better Jm than

stabiliser-free electrodes in the presence of O2 It might therefore have some resistance

against O2 It can be however concluded that PEI is not suitable as an enzyme stabiliser

and not effective enough to enhance the catalytic current output because their Jm was

already smaller by 60 at minimum in N2 compared to the standard value and little

improvement in catalytic performance was observed in the presence of O2

142

443 DTT

Kinetic parameters for various DTT concentrations are shown below in Fig 51

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

Jm

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

KM

M

N2 O2

Fig 51 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 DTT in the presence and absence of O2 n = 3

All DTT containing electrodes showed smaller Jm and KM than the standard values in

the absence of O2 This might imply the possible presence of uncompetitive inhibition

by DTT although the size of inhibition was not in concentration dependent manner In

uncompetitive enzyme inhibition inhibitors bind both free enzyme and

enzyme-substrate complexes hence decreases both catalytic velocity and KM [22] In the

presence of O2 all Jm KM and glucose response lag were larger than those of

stabiliser-free electrodes The increase in glucose response lag was probably due to both

electron competition and uncompetitive inhibition while the inhibition by DTT itself

was less influential on catalytic velocity than non-competitive inhibition by O2 itself

143

and this resulted in larger Jm despite of their larger KM The truth of O2 enzyme

inhibition might be reversible Met oxidation as discussed in sect 242 on p 93 because

atmospheric O2 could oxidise Met residue [123] and DTT might be capable of reducing

it back although DTT has been reported to be effective only in reducing disulphide [127

132] The increase in Jm in the presence of O2 compared to N2 was probably due to the

reduced content of DTT working toward stabilising enzyme structure This freed up

some GOx portion from rigid conformation

444 PEG

Kinetic parameters for various PEG concentrations are shown below in Fig 52

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEG 1 PEG 2 PEG 4

J m

Am

-2

N2 O2

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

No Stabiliser PEG 1 PEG 2 PEG 4

KM

M

N2 O2

Fig 52 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEG in the presence and absence of O2 n = 2 - 3

PEG decreased Jm to 10 of the standard value at maximum in the absence of O2

144

although 4 PEG electrodes showed much larger Jm than the other PEG electrodes

unexpectedly 4 PEG however showed glucose response lag gt 10 mM even in the

absence of O2 The outcome of low Jm is compatible with the result reported by Lee

[139] while among PEI electrodes Jm however increased with PEG content and this

agreed with the results presented by Arakawa [138] The hypothesis by both authors

therefore might be correct but stabilisation mechanism might differ at different

concentrations Since KM was lowered at low PEG content compared to the standard

value PEG might have penetrated the protein core and stabilised the enzyme structure

[164] however it also lowered GOx activity because the protein conformation has been

locked in At 4 PEG concentration PEG still penetrated the protein core but residual

PEG also brought the preferential hydration and this allowed more flexible enzyme

conformation and increased the catalytic activity The residual PEG however might also

have an adverse effect on electron transfer rates at low glucose concentration In the

presence of O2 KM was increased compared to when in N2 and larger than that of

stabiliser-free electrodes while Jm only marginally better compared to that except for 4

PEG It is altogether not clear about the interaction between GOx and PEG however

only high PEG content effectively enhances the catalytic current output

445 GLC

Kinetic parameters for various GLC concentrations are shown below in Fig 53

145

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser GLC 1 GLC 2 GLC 4

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

20E-02

No Stabiliser GLC 1 GLC 2 GLC 4

KM

M

N2 O2

Fig 53 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 GLC in the presence and absence of O2 n = 3

In the absence of N2 GLC showed smaller Jm and KM compared to the standard values

This indicates the presence of uncompetitive inhibition and the decrease in Jm showed

the reciprocal dependency on GLC concentration It is also quite possible that

concentration-dependent inhibition was due to the tighter protein packing by GLC as

previously reported [144] This might contribute more to non-competitive inhibition

though GLC-containing electrodes showed more resistance against O2 because their Jm

was larger than that of stabiliser-free electrodes and again it showed reciprocal

dependence on GLC concentration In both N2 and O2 cases Jm decreased with increase

in GLC content KM also showed the dependence on GLC concentration in the presence

of O2 and increased with GLC concentration Glucose response lag was also decreased

in all cases Among all 1 GLC was most effective because it lowered KM for both N2

and O2 and increased Jm for O2 although it decreased Jm by 40 in N2 It also decreased

glucose response lag from 8 to 5 mM and this might indicate that glycerol reduced and

scavenged O2

146

446 FAD

Kinetic parameters for various FAD concentrations are shown below in Fig 54

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

No Stabiliser FAD 1 FAD 2 FAD 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser FAD 1 FAD 2 FAD 4

KM

M

N2 O2

Fig 54 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 FAD in the presence and absence of O2 n = 1 - 3

FAD may be reasonably effective as an enzyme stabiliser at concentration gt 1 because

its Jm was larger in both absence and presence of O2 compared to that of stabiliser-free

electrodes It also slightly decreased glucose response lag therefore it might be able to

react with oxidising species as previously reported [108] It is however only effective at

higher concentration because two out of three 1 FAD electrodes did not show

Michaelis-Menten curves in the absence of O2 and none worked in its presence although

4 FAD was not most effective either The relationship between increase and decrease

in Jm and KM in N2 is unknown Improvement in electron transfer was not particularly

observed because higher FAD content decreased the current output although FAD has a

147

function as a mediator Mechanism of GOx stabilisation by FAD is not clear as Bartlett

reported [145] however 2 FAD was shown to be most effective among all and the only

one which showed M

mK

J gt 1 for both N2 and O2

447 PEI and DTT mixture

Kinetic parameters for various PEI-DTT concentrations are shown below in

Fig 55

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

KM

M

N2 O2

Fig 55 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

PEI-DTT (051 11 1505 and 22) in the presence and absence of O2 n =

1 - 2

PEI-DTT stabiliser mix showed the most dramatic effect and promising as a stabiliser

formulation In the presence of O2 it had larger Jm and smaller or equivalent KM than

those found in stabiliser-free electrodes Glucose response lag was also decreased from

148

8 to 3 mM and this implies the presence of O2 scavengers which were new synergetic

effects achieved and not observed in use of either stabilisers as discussed in sect 442 and

443 In the absence of O2 most PEI-DTT showed smaller Jm and marginally smaller

KM than those of the standard values except for some as it can be seen Fig 55 This

probably implies the presence of non-competitive-inhibition like effect due to the

stabilisation of enzyme structure by PEI-DTT Since it was difficult to determine the

comprehensively most effective mixture and the relative relationship in the increase and

the decrease in the two kinetic parameter values were not obvious M

mK

J was found

and compared for each PEI-DTT mixture as shown below in Fig 56

00

05

10

15

20

25

30

35

40

45

50

1505 11 0515 22

PEIDTT ()

Jm

KM

(Am

-2M

-1)

N2O2

Fig 56 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051

11 1505 and 22)

It is clear that DTT must exceed 1 to achieve in the fraction to achieve efficient O2

resistance Since the equivalent glucose response lag was observed in all mixtures this

implies that at least 1 DTT is required for simultaneous reduction of Met amino acid

149

residue On the other hand gt 2 PEI seems required for high catalytic efficiency in N2

or equal weight PEIDTT mixing is preferable to increase catalytic efficiency in both N2

and O2 Since PEIDTT at 22 data presented here is based on a single sample the study

on more samples is required to confirm the results however PEIDTT mixture with

DTT gt 1 is effective to improve catalytic efficiency and output current

448 PEI and FAD mixture

Kinetic parameters for various PEI-FAD concentrations are shown below in

Fig 57

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

J m

Am

-2

N2 O2

00E+00

20E-02

40E-02

60E-02

80E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

KM

M

N2 O2

Fig 57 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with PEI-FAD (21 22 and 24 ) in the presence and absence of O2 n = 2

Firstly the result clearly showed total 6 stabiliser content hindered catalysis because it

showed extremely low Jm compared to other PEIFAD containing electrodes It only

counts for 15 and 9 of Jm obtained for other PEIDTT concentration in presence and

150

absence of O2 respectively Secondly there was no large difference in the performance

between PEIFAD at 21 and 22 hence PEIFAD 21 was most effective of all This

might involve non-competitive inhibition because Jm was smaller by 70 while KM was

smaller by only 20 compared to the standard values in the absence of O2 In the

presence of O2 it showed larger Jm by 7 times however also increased KM by 3 times

compared to those of stabiliser-free electrodes and did not decrease the glucose response

lag

449 Most effective stabiliser content for GOx anodes

To determine the size of comprehensive catalytic efficiency M

mK

J was

found for all the stabilisers and its value was compared for O2 for catalytic efficiency in

the presence of O2 Among all five most efficient catalysts were selected and presented

below in Fig 58

151

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

J m

Am

-2

N2 Am-2O2 Am-2

N2 Am-2 180E-02 125E-02 827E-03 292E-02 710E-03 104E-02O2 Am-2 168E-02 181E-02 151E-02 267E-02 122E-02 157E-02

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505 GLC 1

00E+00

50E-03

10E-02

15E-02

KM

M

N2 MO2 M

N2 M 729E-03 671E-03 576E-03 647E-03 938E-03 328E-03

O2 M 104E-02 471E-03 433E-03 801E-03 658E-03 886E-03

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505

GLC 1

Fig 58 Jm KM in the absence and presence of O2 for various stabilisers PEIDTT

(11 0515 221505 ) and GLC 1

PEIDTT mixture was found to be most effect as GOx stabiliser as discussed in sect 447

For Jm PEIDTT 22 showed the largest in both absence and presence of O2 and this

was followed by PEIDTT 11 For KM PEIDTT 0515 showed the smallest

comprehensively for both N2 and O2 and this followed by PEIDTT 11 PEIDTT

22 and GLC 2 showed relatively large KM in the presence of O2 It can be therefore

concluded that PEIDTT 11 is the most effective in stabilising GOx has largest

resistance against O2 and increases the catalytic output current It showed Jm = 13 x 10-2

plusmn 19 x 10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2

with KM = 47 plusmn 42 mM for O2 The current output obtained is much lower than some

reported results by Willner Heller and Sasaki [18 19 97] by 2 ndash 3 order of magnitudes

their electrodes however involved more complex fabrication methods or system as

discussed sect 22 on p 77 On the other hand it was slightly better than the result

reported by Merkoci who composed a similar composite with 5 GOx and was similar

to the results reported by Arai who built GOx electrodes by more complex methods KM

152

obtained is also smaller by 2 ndash 4 times than some of results by Arai [103]

Max current output Am-2 KM mM Method involved Author

125 x 10-3 ndash 181 x 10-2 47 ndash 67 10 GOx composite Data botained

55 x 10-3 Not available 5 GOx composite Merkoci [115]

30 Not available Apo-GOx reconstituted Willner [97]

11 x 10 Not available Os-complex hydrogel Heller [19]

18 Not available Saccharide-modified polypyrrole Fujii [100]

54 x 10-2 Not available QS conductive polymer Arai [103]

41 x 10 13 - 20 Poly(acrylic) acid hydrogel modified

carbon fibre GDH electrodes

Sakai [18]

Table 7 Comparison of maximum output current and KM with some published data

45 CONCLUSION TO ANODE STUDY

Bulk modified enzyme electrodes are good because they are mechanically very

stable and their fabrication is straightforward and scaleable requiring only mixing

electrode components conductive material mediators enzymes and stabiliser with

binding material TTF-TCNQ is useful material for GOx electrodes because it is

conductive as well as mediating The electrode component ratio is important for its

function and conductivity High binder and protein content hinder the conduction It was

found that 73 weight ratio of TTF-TCNQepoxy resin with 10 GOx loading optimal

The balance among the conductivity mediation enzyme activity mechanical and

153

chemical stability and difficulty in fabrication process is key to the successful

fabrication

O2 is known to be the natural mediator of GOx In the glucose-oxygen fuel

cells GOx anode must be resistant to O2 because O2 is an electron acceptor at the

cathode O2 lowers the current output by competing for electron transfer between GOx

and artificial mediators as well as non-competitive inhibition Various GOx stabilisers

were examined to alleviate such O2 influence

PEI DTT PEG GLC FAD PEIDTT and PEIFAD mixtures were studied as

candidate stabilisers to enhance catalytic activity and the current output PEI was not

effective It stabilised the structure but made conformation more rigid and did not

increase the output current in both the absence and presence of O2 DTT was reasonably

effective It showed uncompetitive inhibition in N2 but stabilised the enzyme structure

It therefore increased Jm and KM in the presence of O2 but increase in KM was probably

due to alleviating inhibition by O2 and loosening up the enzyme conformation PEG was

not effective It probably stabilised the enzyme structure but not show much

improvement for O2 competition It might also deprive electrons from reduced GOx

GLC was effective The effect was concentration dependent Lower concentration was

more effective It also scavenged O2 FAD was reasonably effective but only at higher

concentration PEI-FAD was also reasonably effective It decreased Jm but Km was

marginally equivalent to that of stabiliser-free electrodes for N2 But it increased Jm for

O2 Among all PEIDTT at 11 mixture was found to be most effective because it

stabilised the GOx and scavenged O2 Such powerful influence was not observed in the

use of either stabilisers hence it is a synergetic effect It showed Jm = 13 x 10-2 plusmn 19 x

154

10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2 with KM

= 47 plusmn 42 mM for O2 Although it lowered Jm by 30 in the absence of O2 compared

to the standard value of 18 x 10-2 plusmn 73 x 10-3 Am-2 it also decreased KM in both

absence and presence of O2 and increased Jm by 8 times compared to that of

stabiliser-free electrodes PEIDTT 11 is therefore most effective in stabilising GOx

has largest resistance against O2 and increases the catalytic output current

155

5 CATHODE INTRODUCTION

51 OXYGEN REDUCTION REACTION (ORR)

The oxygen reduction reaction (ORR) takes place at the cathode as oxygen

accepts electrons from the anode Oxygen is an ideal oxidiser for the cathode because it

is readily available and its reaction product water is harmless [62] However ORR has

been a long-time bottleneck especially for low-temperature fuel cells because it causes

large potential loss [3 4 62 165-167] due to a strong double bond in the dioxygen

molecule Its difficult bond dissociation causes slow irreversible kinetics [24 165 166]

so a catalyst is required for this reaction to proceed at useful rates [62 165] ORR is a

multi-electron reaction which involves various reduction pathways and different

oxygen intermediates (Fig 16) In aqueous solution either the direct four- or

two-electron ORR pathway may proceed [165 166] The direct four-electron transfer

pathway reduces O2 into H2O in acid and neutral mediaor OH- in alkaline media This

occurs via several reduction steps [24 165 166] which may involve adsorbed peroxide

intermediates These peroxide intermediates never diffuse away in the solution in this

pathway [166] The two-electron transfer pathway reduces O2 into peroxide species as

final products These peroxide species either diffuse away are reduced further or

decomposed into H2O or OH- [24 165 166] This is not favourable because it leads to

lower energy conversion efficiency and its product hydrogen peroxide may degrade the

catalysts and the fuel cells [165 167] Four- and two-electron transfers also may occur

in series or in parallel [165]

156

Fig 59 Oxygen reduction pathways [165 166]

Reduction kinetics and mechanisms depend on the type of catalysts electrode material

electrode surface condition solution pH temperature contaminant and reactant

concentration [24 165 166] Rate-limiting step also varies by type of the catalysts

[166] However the first electron transfer to dioxygen leading to the formation of

superoxide anion radical (O2־) is the rate limiting step [168] O2־ is very rapidly

protonated in water and converted to hydroperoxyl radical (HO2)

157

Fig 60 Degradation of superoxide anion radical in aqueous solutions [24]

In aqueous solution O2־ is unstable and rapidly converted to O2 and H2O2 via a

proton-driven disproportionation process (Fig 60) [24]

52 REACTIVITY OF OXYGEN

A dioxygen molecule is a homodiatomic molecule consisting of two oxygen

atoms double bonded It has two parallel unpaired electrons in degenerate anti-bonding

π designated as π orbitals therefore it is diradical and paramagnetic (Fig 61) [169]

158

Fig 61 An energy level diagram for ground state dioxygen molecule [169]

When one electron is transferred to a dioxygen molecule the electron is placed in π

orbital and it turns into a reactive O2[24] ־ reducing the bond order from 2 to 15

Oxygen reduction changes the molecular symmetry or stretches the double bond

causing bond disruption [165 166] Electron paring in one of the π orbitals also

changes their orbital energy state and lifts the degeneracy as in Fig 62 [170]

Hereogeneous catalysis involves chemisorption of the oxygen and therefore some

transfer of electron density from the oxygen to the catalyst This facilitates the rate

determining initial electron transfer Oxygen reduction catalysts are reviewed in the

next section

159

(O2) 3Σg- O2־

Fig 62 Diagrams for the state of π orbitals in the ground state oxygen (3Σg-) (left)

and peroxide anion radical (right) [24]

O2 species Bond Order Bond length pm Bond energy kJmol-1

O2 2 1207 493

O2+ 2frac12 1116 643

O2- 1frac12 135 395

O22- 1 149 -

Table 8 Bond orders bond lengths and bond energies of some dioxygen species [169]

53 ORR CATALYSTS

Catalysts are required to reduce the overpotential and improve the efficiency of

160

ORR [3 4 62 165-167] It was reported that 80 of the whole potential loss can be

attributed to cathode ORR overpotential [171] The choice of ORR catalysts varies by

applications [36 171] and direct four-electron transfer pathway is most preferred for

ORR at the lowest possible overpotential in every fuel cell application however its

kinetics and reduction pathway are limited by the type of catalysts and experimental

conditions [24 165 166] Miniaturised glucose-oxygen fuel cells operate in the neutral

aqueous solution at ambient or body temperature with relatively low electrolyte and

oxygen concentrations therefore they require powerful ORR catalysts Platinum (Pt)

has been most ideal however its availability and cost are major limiting factors Many

studies have been made on other noble metals [172 173] transition metal macrocyclic

compounds [174] multicopper oxidases [62 175] and quinones [165] as alternatives

ORR catalysts to Pt These ORR catalysts are reviewed below

531 Noble metal ORR catalysts

Noble and transition metals are well-known ORR catalysts There are two

possible ORR mechanisms for metal surface dissociative and associative

161

Dissociative mechanism

middotmiddotmiddotmiddotmiddot Eq 67

middotmiddotmiddotmiddotmiddot Eq 68

middotmiddotmiddotmiddotmiddot Eq 69

( indicates the catalyst surface)

Associative mechanism

middotmiddotmiddotmiddotmiddot Eq 70

middotmiddotmiddotmiddotmiddot Eq 71

middotmiddotmiddotmiddotmiddot Eq 72

middotmiddotmiddotmiddotmiddot Eq 73

middotmiddotmiddotmiddotmiddot Eq 74

In dissociative mechanism the O2 double bond is ruptured rapidly on its adsorption on

the metal surface [165 166 172] This involves a strong interaction between oxygen

and metal atoms [166] and does not generate H2O2 [165] Electron and proton transfer

take place simultaneously and this is the rate-limiting step [166 171] In the associative

mechanism O2 does not dissociate before proton transfer Adsorbed O2 stays on the

metal surface [165 172] and two-electron and proton transfer [166 173] take place via

the formation of superoxide or peroxide intermediate [165 166] This reaction is

thermodynamically easier than the dissociative pathway and its final product is H2O2 or

162

H2O if both pathways occur in series [165] O2 adsorption on the metal surface is an

inner sphere interaction which involves complex formation with the metal surface [30]

There are three ways in which O2 adsorbs (i) Griffiths (ii) Pauling and (iii) bridge

model (Fig 63)

Griffiths model Side-on Pauling model End-on Bridge model

OO

M

Fig 63 Three models of oxygen adsorption [165 166 173]

The Griffiths model is called lsquoside-onrsquo type O2 functions as a bidentate ligand against a

single metal atom in this complex and its π orbitals laterally interact with empty metal

dz2 orbitals causing ligand-to-metal charge transfer (LMCT) πrarrdz

2 At the same time

electrons partially filling metal dxz or dyz orbitals are back donated to oxygen π orbitals

[166 171] Therefore a strong metal-oxygen interaction is present in this model This

effectively disrupts the O=O bond [166] and facilitates the electron transfer from the

metal ion by changing its oxidation number The Pauling model is called the lsquoend-onrsquo

type [165 166 173] O2 is a monodentate ligand in this model and only one of the

oxygen atoms in the dioxygen molecule can be bonded to the metal surface An oxygen

π orbital interacts with metal dz2 orbitals and this allows either the direct or peroxide

ORR pathway after partial charge transfer [166] The bridge model resembles Griffiths

model in the way the metal atom and oxygen interact however it requires two adjacent

163

adsorption sites so that O2 can ligate two different metal atoms The bridge-model

interaction is easily interfered with by the surface contamination and the oxide layer

because the surface species block the adsorption sites for ORR catalysis [173]

Fig 64 Models for oxygen adsorption and ORR pathways [166]

Both the Griffiths and bridge models follow dissociative mechanism (pathway I and III

respectively Fig 64) due to the lateral interaction leading to the dissociation of O2

double bond The Pauling model on the other hand follows the associative mechanism

(pathway II Fig 64) This is further divided into two other pathways IIa and IIb

according to its end product H2O2 or H2O (Fig 64) respectively [166] O2 adsorption

and the ORR mechanism therefore depend on (i) the extent of oxygen-metal

interaction and (ii) the availability of oxygen adsorption sites A volcano-shaped trend

between ORR rate and oxygen-metal interaction has been plotted by Norskov as in Fig

65 [172]

164

Fig 65 A volcano-shaped trend between ORR rate and energy required for oxygen

binding on various transition metal surface [172]

Many have reported platinum (Pt) as the most ideal metal catalyst due to its high

catalytic ORR activity [165 166 171 172] Fig 65 shows that Pt and Pd (palladium)

have largest ORR rate yet medium oxygen adsorption energy and iridium (Ir) and

rhodium (Rd) follow them On the other hand metals with either higher or lower

oxygen adsorption energy result in lower ORR rate This is because metals with high

oxygen adsorption energy such as gold (Au) imposes too large energy barrier for the

formation of a stable oxygen-metal bond while too small adsorption energy as in nickel

(Ni) leads to excessively stable oxygen-metal interaction and slows the proton transfer

Therefore associative pathway is dominant at Au surface while oxygen dissociation is

deactivated at Ni surface but instead this increases the surface oxide coverage [172

173] Pt performs best because it has moderate oxygen-metal surface interaction [165]

Pt alloys with other transition metals such as Ni Co and Fe have been also reported to

increase the catalytic activity [36 167 171 172] because alloying dilutes the Pt-O

165

interaction and alters the overall oxygen-metal surface interaction [171 172] This is a

useful property to add because Pt is known to form a surface oxide layer leading to both

associative and dissociative catalytic pathways [165 171] This surface state is

potential-dependent and stays persistenly at less than 08 V vs AgAgCl [172]

532 Macrocycle and Schiff-base complex ORR catalysts

Macrocyclic transition metal complexes have been widely investigated as

potential ORR catalyst both for fuel cells and as catalysts to activate dioxygen for

synthetic purposes They have been widely reviewed by [165 176] Macrocyclic

complexes consist of (i) transition metals such as Fe Co Ni or Cu (ii) chelating atoms

N4 N2O2 N2S2 O4 and S4 and conjugate π systems such as porphyrin and

phthalocyanine [165] Examples of transition metal macrocycles are shown in Fig 66

Fig 66 Examples of transition metallomacrocycles Heme (left) [177] and copper

phthalocyanine (right) [178]

166

Schiff base complexes such as metallosalens and metallosalophens are other

well-known chelating ORR catalysts [174 179] Transition metal macrocycles and

Schiff bases can be also polymerised into conductive films [180] (Fig 67)

Fig 67 Schiff base complexes

Co(II) salen Co(II) salophen and

Co(II) salen modified poly(34-ethylenedioxythiophene) [174 180]

Interaction between the oxygen molecule and the metal complex is similar to that with

the noble metals and involves partial charge transfer between the filled metal dz orbitals

and the π orbital of O2 as described in sect 531 The catalytic activity of the metal

complexes depends on the type of central metal atom ligand and the size of the

π-electon systems The reducing ability of the ligand is important because the central

metal atom must transfer considerable electron density to oxygen atoms during the

167

catalysis [170] The thermodynamically most stable metal-oxygen complexes are

bionuclear complexes such as dicobalt face-to-face 4-atom-bridged diporphyrin

(Co2(FTF4)) (Fig 68) [165-167]

Fig 68 Molecular structure of dicobalt face-to-face diporphyrin [166]

Mononuclear Co complexes take the associative pathway [165] forming superoxo

cobaltic complexs (Co-O-O־) [181 170] while their dimers or binuclear complexes can

take either direct four-electron or the parallel pathways (Fig 69) [165 167] because

they can lock the oxygen moiety in rigid orientation [170] Co2(FTF4) favours

four-electron transfer and its ORR pathway depends on the distance between two

cofacial metal atoms [165-167] Spacing distance can be altered by inserting a spacer

molecule [165 166]

168

Fig 69 ORR pathway by Co2(FTF4) [166]

However Co2(FTF4) is not stable enough for practical use [165 166] Practical ORR

catalysts for fuel cell application must be stable inexpensive and feasible to synthesise

533 Multicopper enzymes as ORR catalyst

Multicopper oxidases (MCOs) are copper-containing enzymes such as bilirubin

oxidase (BOD) laccase (Lac) and copper efflux oxidase (CueO) Oxygen is widely used

as an electron acceptor in nature because it is ubiquitous and oxidation is a good mean

to obtain energy as in combustion Millions of years of evolution have therefore

provided enzymes with good catalytic activity for oxygen reduction Although oxygen

169

reduction in biological system is complex due to involving various substrates and

catalytic steps as well as it tends to be incomplete and leads to hydrogen peroxide or

superoxide formation as the product the enzymes catalyse the rate-determining initial

electron transfer For application in biofuel cells it is essential that the enzymes are

widely available and possess good stability activity in immobilised form and ideally

four-electron ORR catalysis MCOs have been prevalent in biofuel cell applications

because they are active at neutral pH catalyse four-electron ORR and bear

immobilisation Fungal laccase and bilirubin oxidase have been most widely studied

MCOs [56 59 60]

Most MCOs have four Cu atoms which are designated as type I (T1) type II (T2) and

a pair of type III (T3) T2 and T3 together form a trinuclear Cu centre while T1-Cu

called blue copper [175 182 183] is located away from the trinuclear Cu centre but

adjacent to the hydrophobic substrate-binding site Fig 70 shows the location of the

trinuclear Cu centre (copper coloured) and the blue copper (in blue) Oxygen binding

and ORR take place at the trinuclear Cu centre [175] while T1-Cu receives electrons

from the substrate and mediates electron transfer between substrate and the trinuclear

Cu centre [175 184]

170

Fig 70 Laccase from Trametes versicolor [185]

Enzyme catalytic efficiency depends on the redox potential [175] of T1-Cu It has been

reported that fungal laccase has the highest redox potential between 053 ndash 058 V vs

AgAgCl [62 183] T1-Cu also allows DET due to being located close to the surface

[183 186 187]

MCO reaction cycle starts from substrate oxidation to form a fully reduced state MCO

is initially at native form or so-called intermediate II Native form is characterised by

doubly OH--bridged structure (Fig 71(ii)) In the absence of substrate the native form

decays to the resting form which is at fully oxidised state (Fig 71(i)) After

four-electron reduction of enzyme the fully reduced MCO (Fig 71(iii)) reacts with O2

The first reduction step involves two-electron transfer from each of T3-Cu+ to O2 This

forms peroxide intermediate or intermediate I (Fig 71(iv)) and lowers the energy

barrier to disrupt O=O It is followed by swift protonation of adsorbed peroxide at

T3-Cu site This protonation reaction is induced by the charge transfer from T1- and

171

T2-Cu+ This leads to the complete reduction of oxygen to water and the enzyme native

form is retrieved

(i)

(ii)

(iii)

(iv)

Fig 71 Redox reaction cycle of MCOs with O2 [175]

All MCOs have this common ORR mechanisms despite the different origin structure

biological functions substrate reducing power and optimal environment

5331 BOD

BOD is an anionic monomer [182] most active at neutral pH [183 188] and

172

capable of both DET and MET The mediators must have their redox potential close or

slightly lower than that of BOD 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

(ABTS) (Fig 72(i)) [62 97] Os-pyridine complexes linked to conductive polymer (Fig

72(ii)) [66 189] and cyanometals [18 56] have been used as its mediators [56 185]

ABTS is a divalent anion which works as co-substrate of MCOs and its redox potential

is 067 V vs AgAgCl [56] Os-pyridine complex with conductive polymer follows the

similar idea for the wired GOx anodes discussed in sect 223 on p 80

4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine) has been used

especially for the cathode and its redox potential is 055 V vs AgAgCl

Fig 72 Common mediators for BOD (i) ABTS [56 62]

(ii) Os[4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine)] [190]

173

Tsujimura showed that ORR catalytic constant was 6-times greater in the presence of

Os-pyridine complex than in DET [66 188] Reduction starts around 05 V in most

cases [66 97 183 188] The size of current density varies roughly between 05 - 1

mAcm-2 [56 97 188] and 95 mAcm-2 was achieved by Heller [189]

5332 Lac

Fungal laccase is another anionic monomer [183] It is less expensive than

BOD and has broad substrate specificity including xenobiotics [175] although it has

optimum pH around 5 and activity hindered by chloride [19 182] Its T1-Cu redox

potential ranges between 053 ndash 058 V vs AgAgCl [62 183] Highest redox potential

066 V and current density 057 mAcm-2 were achieved by the use of anthracene

plugging molecule which provides strong binding between Lac and pyrolytic graphite

edge electrode as well as long-term activity 57 of the initial current was retained after

eight weeks [185]

5333 CueO

CueO is a less common enzyme in biofuel cells however it is active for a wide

range of pH between 2 - 8 [186] and also it has better catalytic activity than other

MCOs because it has five Cu atoms This extra Cu atom is believed to have mediating

function and 4 mAcm-2 has been achieved at pH 5 [191] However it has lower redox

potential around 03 V [186 191] Protein engineering has been introduced to increase

T1-Cu redox potential [186]

174

534 Anthraquinone ORR catalyst

Anthraquinone (AQ) is known to catalyse two-electron transfer in ORR (Fig

73) [44 165 192-194] and it is used in industries to produce hydrogen peroxide [165

192]

Fig 73 Two-electron ORR by anthraquinone [193]

ORR by anthraquinone is an electrochemical-chemical (EC) process [165 192] in

which quinone must be first electrochemically reduced to an active species

semiquinone anion radical (Q־) The active semiquinone radical can then react with

dioxygen so that it converts the dioxygen into O2־ via forming of the intermediate

superoxo-C-O(O2) species This is the rate-limiting step O2־ produced by a

semiquinone radical either disproportionates (Eq 77) or it is further reduced into HO2-

(Eq 78) [192]

175

Oxygen reduction reaction by anthraquinone

ltlt Rate limiting step gtgt

middotmiddotmiddotmiddotmiddot Eq 75

middotmiddotmiddotmiddotmiddot Eq 76

ltlt Following spontaneous reactions gtgt

middotmiddotmiddotmiddotmiddot Eq 77

middotmiddotmiddotmiddotmiddot Eq 78

(Q indicates quinone on the surface)

Derivatised anthraquinone such as Fast red (Fig 74) can be attached to the carbon either

physically or covalently Grafted quinones are very stable [192 194 195] and resistant

against continuous potential cycling [194] metal ions and surfactant despite of some

fouling present [44]

176

O

O

N N Cl

frac12 ZnCl2

1

2

4

6

5

3

10

9 7

8

12

14

13

11

O

O

(i) (ii)

Fig 74 The structures of (i) anthraquinone [196] and (ii) Fast red AL salt [44 197]

However its ORR is heavily affected by pH [194] because its catalytic efficiency

depends on the concentration of surface Q־ species [192 194] The concentration

varies by different pH and the catalytic efficiency drops at both high and low pH due to

the depletion of Q־ When pH falls below its dissociation constant Q־ is not

reproducible anymore and ORR is inhibited This phenomenon appears at as high as pH

7 and the ORR vanishes at pH lt 7 [194] AQ-grafted GC (AQGC) shows only sluggish

kinetics at pH lt 10 Its voltammogram also becomes complex due to the presence of

native quinone groups originated from the carbon surface [192] ORR mediated by the

native quinone groups is observed as a prewave and this varies by different quinone

derivatives such as phenanthrenequinone (PQ) (Fig 75) electrode composition and pH

[194]

177

Fig 75 Hydrodynamic voltammogram for ORR at various quinone electrodes with various

rotation speeds [194] (i) unmodified GC (ii) AQGC and (iii) PQGC at pH 7

54 CARBON PROPERTIES

Carbon is widely used for electrochemical applications because it is

inexpensive mechanically stable [105] and sufficiently chemically inert yet reactive

due to the presence of surface defects It has wide potential window between 1 and -13

V [21 30] Carbon is available in various solid forms such as graphite glassy carbon

(GC) pyrolytic carbon (PC) activated carbon (AC) carbon fibres (CFs) carbon

nanotubes (CNT) and diamond It is easy to renew the surface and allows facile

chemical modification [195] However it is also prone to adsorption and surface

oxidation simply by air and water exposure and its electron transfer is under the

constant influence of the electrode surface condition and history [198] This leads to the

deviation and the low reproducibility in carbon electrochemical experiments [105]

178

Physical properties of carbon vary by its form and section The carbon consists of either

(i) layers of the conductive planar graphene sheets or (ii) the insulating

tetrahedral-puckered forms as seen in the diamond (Fig 76) Conductivity in diamond

is gained by doping with boron [199 200]

Fig 76 Carbon structures (i) graphite (ii) diamond [201]

The electrical conductivity is anisotropic and closely correlates with the carbon

structure and section The carbon section is classified into (i) basal and (ii) edge planes

The basal plane extends along the graphene plane while the edge plane is perpendicular

to this

179

Fig 77 Carbon section (i) basal plane (ii) edge plane [105]

The basal plane has larger conductivity due to being parallel to the vast graphene

network while the edge plane is superior in electrochemical reactivity due to being

richer in reactive graphene sheet termini and carbon defects The ratio between the basal

and edge planes therefore affects the conductivity and electrochemical properties of

the carbon and this varies by the carbon form and its treatment [45 105 195 202 203]

The electrical conductivities for various carbon forms and sections is shown in Table 9

180

The type of carbon Electrical conductivity Scm-1

Graphite [105 159] 1 x 103 - 104

Highly ordered PG basal plane 250 x 104

Highly ordered PG edge plane 588

PG basal plane 400 x 103

PG edge plane 333

Polycrystalline graphite 100 x 103

GC [105 159 203] 01 - 250 x 102

Activated carbon [204] 1 x 10

Carbon fibre 133 x 103

Boron-doped diamond [199 205] 01 - 1 x 102

Multi-wall carbon nanotube [206] 1 ndash 2 x 103

Pt 909 x 104

Cu 588 x 105

Table 9 The electric conductivity of various carbon forms and sections Data from

[105] unless defined specifically

541 Graphite

Graphite is black-coloured material with pores and high gas permeability It is

available in many physical forms as powders moulded structures and thin sheets

therefore it is used in many commercial applications [203] Powdered graphite is

convenient to make carbon paste and composite electrodes [105] Graphite consists of

181

layers of graphene sheets each of which is a huge π-conjugate system π-conjugate

system is made of hexagonal rings of sp2 hybridised carbon atoms [105 195] (Fig 76(i)

and Fig 78) and allows free electron movement within graphene This gives carbon

metallic properties such as electric and thermal conductivity

Fig 78 A direct image of a graphene bilayer by transmission electron microscopy

(TEM) at resolution of 106 Aring and with a 2-Aring scale bar [207]

Each graphene sheet is structurally stable in horizontal direction but fragile in vertical

direction because each layer is only held by van der Waalsrsquo force Therefore the

graphene sheets easily slide and collapse [31]

542 Glassy carbon (GC)

Glassy carbon (GC) is a non-graphitised carbon made of randomly interwoven

sp2-carbon fibres which are tangled and heavily cross-linked (Fig 79) It contains many

closed voids [203] and fullerene-like elements [208] It is impermeable to gases [203

182

208] chemically more inert [208] and mechanically stronger but electrically more

resistive than other carbons [203]

Fig 79 GC (i) High resolution TEM image (ii) A schema of GC [208]

543 Activated carbon (AC)

Activated carbon (AC) is thermally and chemically activated carbon It is

highly porous and has large internal surface area This is an excellent adsorbent for

solvent recovery and gas refining [203 208]

544 Carbon fibre (CF)

Carbon fibres (CFs) have small diameter ranging between 5 to 30 microm They are

lightweight have lower fabrication cost and are superior in mechanical properties

therefore they have been used for sporting automotive and military equipments They

are available in many forms such as tow mat felt cloth and thread They consist of a

random arrangement of flat or crumpled graphite sheets but their structure and

183

molecular composition depend on the type of precursors [203 208]

545 Carbon nanotubes (CNTs)

Carbon nanotubes (CNTs) are made rolled-up sheets of sp2-graphene in nano to

micro-meter range They are available in various formats single-walled nanotube

(SWNT) multi-walled nanotube (MWNT) carbon nanohorn (CNH) (Fig 80) and

various forms powder arrays and posts [117]

Fig 80 Various CNTs (i) Bundled SWNTs (ii) MWNT (iii) CNHs [209]

CNTs have been drawing keen attention due to their wide surface area conductive or

semiconductive properties light weight mechanical stability and facile

functionalisation [117 195 202 210] They have been used as support for the dispersed

metal catalysts [3] and electrode materials [117 202] The drawbacks of CNTs are the

high presence of impurities alteration of their properties on acid cleaning [202 211]

and unknown toxicity [117 160 202]

184

546 Boron-doped diamond (BDD)

Diamond is different from other carbons because it is transparent and not

electrically conductive It has a cubic lattice structure made of closely-packed repetitive

units of sp3-hybridised carbon atoms as seen in Fig 81

Fig 81 A TEM image of diamond [212]

Each unit consists of four σ-bonded sp3-carbon atoms in tetrahedral coordination as in

Fig 76(ii) Each bond is localised and this gives structural stability but does not conduct

electricity By doping it with boron diamond can become semiconductive Such

diamond is called boron-doped diamond (BDD) and boron functions as an electron

acceptor BDD is mechanical more stable chemically more inert sensitive to analyte

and has wide potential window lower back ground current and stable electrochemical

responses [195 199 205]

185

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON

551 Electrochemical properties of carbon

The edge plane of carbon which contains graphene termini and carbon defects

is chemically reactive and accounts for carbon electrochemistry while the vast network

of graphene sheets is responsible for its electrical conductivity The termini and defects

include some sp3 hydrocarbons and they are oxidised into various oxygen functional

groups such as carboxyl quinoyl hydroxyl lactone groups ethers cyclic esters and

lactone-like groups [195] These electrode functional groups facilitate electron transfer

provide bonding between the electrode and the catalysts or enzymes or simply cause

electrode capacitance or resistance Various carbon pretreatments such as

electrochemical pretreatment (ECP) vacuum heat treatment (VHT) carbon fracturing

and polishing laser irradiation [195 213-215] are used to attach active surface

functional groups and increase the rate of electron transfer in various electrochemical

reactions [198 216]

552 Electrochemical pretreatments (ECPs) of the carbon

Electrochemical pretreatments (ECPs) provide feasible electrode surface

modification Various ECPs have been used to introduce carbon-oxygen surface

functional groups Most common method is potential cycling in the acid or basic media

or the combination of anodisation and cathodisation as shown in Table 10

186

Table 10 ECPs examples

Potential cycling at 02 Vs-1 between 0 and 02 V in 01 M H2SO4 [105]

Anodisation at 18 V in pH 7 phosphate buffer for 5 minutes followed by 1-minute cathodisation at

ndash 15 V [216 217]

Potential cycling between 06 and 2 V at 5 Vs-1 for 15 to 30 minutes in 05 M NaOH [218] or their

combination [219]

ECPs introduce the electrodes various new properties They (i) attach electrochemically

active functional groups and increase the oxygencarbon ratio [105] (ii) introduce

carbon defects and active sites [105 218] (iii) increase the surface area [105 198 215

216 218] (iv) form a surface film and (v) increase the background current [105] and

double layer capacitance [105 215 216] Most notable effect of EPCs is the increase in

electron transfer rate in many electrochemical reactions This is observed as a decreases

in peak separation and an increase in peak current in voltammetry as seen in Fig 82

[105 216 218 220] As for ORR however it was reported that surface quinone groups

attached by EPC or originated from the carbon itself reduce ORR overpotential but do

not mediate ORR as in quinone groups physically or covalently attached [216 220]

187

Fig 82 The influence of ECP on GCE and its voltammogram at 01 Vs-1 in pH 7

phosphate buffer in the presence of (i) N2 and (ii) O2 The voltammograms

of both pretreated and unpretreated GCEs are shown [216] The

presentation has been altered for convenience

56 ELECTROCHEMICAL TECHNIQUES FOR ORR

ORR has been studied by two electrochemistry techniques cyclic voltammetry

(CV) and hydrodynamic voltammetry (HDV) using the rotating disc electrode (RDE)

These techniques provide overlapping but complementary information CV is a dynamic

technique and allows calculation of the number of electrons up to and including the rate

determining step (nα) With numerical simulation and data fitting CV can be used to

estimate the standard heterogeneous rate constant (kdeg) RDE is a steady state technique

and can give the overall number of electrons transferred (n) The well-established

Koutecky-Levich plot (see sect 567 on p 200) provides a robust method for determining

kdeg provided the standard electrode potential (Edeg) for the reaction is known

188

561 Cyclic voltammetry (CV)

Cyclic voltammetry (CV) is widely used for initial exploration and

characterisation of a redox active system It is of limited electroanalytical value

however kinetic parameters can be obtained from the current-voltage (I-V) plot In

cyclic voltammetry potential is swept linearly with a triangular waveform (Fig 83(i))

and applied cyclically The resulting time-dependent current is typically plotted versus

applied potential (Fig 83(ii)) [30 221] The potential range and scan rate are decided in

advance according to the type of analytes reactions and materials of interest

Fig 83 Schemas for (i) a Triangular waveform (ii) Typical cyclic voltammogram for

benzoquinone (BQ) CV was taken at 005 Vs-1 between + 025 and ndash 05 V vs

AgAgCl for 1 mM BQ in 1M KCl at graphite composite electrode Switching

potential (Eλ) is ndash 05 V and time taken to reach Eλ (t) is 15 seconds Based on

the real data

189

562 Non-faradic current and electric double layer capacitance

Transient techniques such as CV always contain two types of electric current

non-faradaic and faradaic current The non-faradaic charging current is due to the

electric double layer which is formed at the electrode-solution interface This occurs

because such an interface behaves like a capacitor as counter ions accumulate at the

interface in order to balance the potential at the electrode This does not account for the

charge transfer reactions involved however provides the information about the

electrode surface [30] and also can affect the rate of electrode process and distort the

voltammogram Cd can be estimated by electrochemical impedance spectroscopy or

deduced by averaging Cd over the potential range as in Fig 84 and Eq 33 [21 30]

Fig 84 The double layer capacitance (Cd) observed in CV for deoxygenated pH 74 at

the acid-pretreated graphite electrode See Eq 33 for the calculation and

symbols Based on the real data

190

Electric double layer capacitance per unit area

AII

AEQareaunitperC ccca

d sdotminus

=sdot

=υ2

)()( middotmiddotmiddotmiddotmiddot Eq 79

Cd = Double layer capacitance per unit area microFcm-2

Q = Charge C

E = Potential V

A = Electrode surface area cm-2

Ica = Anodic non-faradic current A

Icc = Cathodic non-faradic current A

υ = Scan rate Vs-1

Cd needs to be either negligible or subtracted before interpretation of faradaic current

which accounts for the charge transfer phenomena in redox reactions In order to

minimise the influence of Cd on the voltammogram the scan must be taken at slow scan

rate and the electrolyte concentration must be always kept reasonably high [30]

563 Diagnosis of redox reactions in cyclic voltammetry

A typical voltammogram contains current peaks as seen in Fig 83(ii) and these

are due to the redox reactions in the system Diagnosis of the redox system using CV is

based on (i) peak separation (ii) peak current height (iii) peak shape or peak width (iv)

the number of peaks and (v) the dependency of peak shape on the scan rate The

analysis of such peaks involves the following four parameters anodic peak current (Ipa)

cathode peak current (Ipc) anodic peak potential (Epa) and cathode peak potential (Epc)

This reveals the type of electrochemical reactions involved which are either (i)

191

reversible (ii) quasi reversible or (iii) irreversible When the peak current is proportional

to υfrac12 the redox reaction is diffusion-limited in the solution while a linear dependence

shows that the redox active species is confined to the electrode surface

In the reversible system charge transfer between the redox species and the electrode is

rapid [221] therefore it has fast kinetics and the reaction is diffusion-limited The

electrode potential follows the Nernst equation (Eq 15 on p 50) because the electrode

potential is dependent on the concentration equilibrium at the electrode surface and the

peak potential is independent of the scan rate The features on the reversible system are

listed in Table 11 The peak current (Ip) is linearly proportional to υ12 as the redox

reaction is diffusion-limited The peak separation (∆Ep) is the difference between Epa

and Epc and it is 59 mV at 25 ˚C for the single-charge transfer [29 30] The peak width

(δEp) is the difference between Ep and half-peak potential (Ep2) Ep2 is the potential

where the peak current is half It is 565 mV for a single charge transfer The ratio

between Ipc and Ipa is called a reversal parameter [105] It is unity at any scan rate in the

reversible system

192

Table 11 Reversible system at 25 ˚C [30]

2121235 )10692( υlowasttimes= CADnI p middotmiddotmiddotmiddotmiddot Eq 80

mVn

E p528

= independent of υ middotmiddotmiddotmiddotmiddot Eq 81

mVn

EEE pcpap59)( =minus=∆ middotmiddotmiddotmiddotmiddot Eq 82

mVn

EEE ppp556

2 =minus=δ middotmiddotmiddotmiddotmiddot Eq 83

1=pa

pc

II middotmiddotmiddotmiddotmiddot Eq 84

Ip = Peak current A

n = The number of charges transferred

A = Electrode surface area m-2

D = Analyte diffusion coefficient m-2s-1

ν = Solution kinematic viscosity m-2s-1

Ep = Peak potential V

∆Ep = Peak separation V

Epa = Anodic peak potential V

Epc = Cathodic peak potential V

δEp = Peak width V

Ep2 = Half-wave potential V

Ipa = Anodic peak current A

Ipc = Cathodic peak current A

In a totally irreversible system charge transfer between the redox species and the

electrode takes place only slowly [221] The electrode must be activated by applying

193

large potential to drive a charge transfer reaction at a reasonable rate Ep becomes

dependent on scan rate and this increases ∆Ep and skews the voltammogram [30]

However Ip is still proportional to υfrac12 because at high overpotential the redox reaction

is diffusion-limited [29]

Table 12 Irreversible system at 25 ˚C [222]

2121215 )()10992( υα αlowasttimes= CADnnI p middotmiddotmiddotmiddotmiddot Eq 85

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛

deg+minusdeg=

2121

lnln780RT

Fnk

DFn

RTEE pυα

αα

α

middotmiddotmiddotmiddotmiddot Eq 86

mVn

E pαα

59gt∆ middotmiddotmiddotmiddotmiddot Eq 87

mVn

E pαα

δ 747= middotmiddotmiddotmiddotmiddot Eq 88

α = Transfer coefficient

nα = The number of charge transferred up to the limiting step

( Please refer to Table 11 for other terms and symbols)

A quasi-reversible system is controlled by both kinetics and diffusion Its

electrochemical reversibility is relative to the scan rate [21 30] The peak potential

depends on scan rate for rapid scans and the peak shape and peak parameters become

dependent on kinetic parameters such as α and Λ where 21

1

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛

deg=Λ

minus υαα

RTFDD

k

RO

[30]

Since Λ is a function of k˚υ-frac12 where kdeg is the standard rate constant the voltammogram

hence kdeg is affected by the scan rate as seen in Fig 85

194

Fig 85 Voltammograms of a quasi-reversible reaction at various scan rates [21]

564 Coupled reactions in cyclic voltammetry

Redox reactions are sometimes coupled with different electrochemical or

chemical reactions Voltammogram with altering scan rates can indicate the presence of

such reactions A redox reaction coupled with another redox reaction is called EE

mechanism while with homogeneous chemical reaction it is called EC mechanism If

the coupled reaction is catalytic then it is called ECrsquo mechanism [21]

Many ORR catalysts show coupled reactions with ECrsquo mechanism ORR is a

quasi-reversible reaction because the reaction is limited by oxygen diffusion yet O2

adsorption and reduction heavily relies on overpotential or catalytic power of catalysts

Catalysed reactions show distinctive voltammetric features There is only a single-sided

large current but with no reverse current because they catalyse a reaction only in a

single direction The reversal parameter therefore becomes non-unity [30]

195

ORR catalysts sometimes contain multiple reducible centres or an atom with various

oxidation states as in seen transition metal They might exhibit a multi-peak

voltammogram under non-catalytic condition The shape of voltammogram depends on

the difference in the standard potential (∆E˚rsquo) between two redox centres or states (Eq

89) as seen in Fig 86 [21 30]

21 ooo EEE minus=∆ middotmiddotmiddotmiddotmiddot Eq 89

Fig 86 Voltammograms of two-electron transfer EE systems of different ∆Edegrsquo [21]

565 Voltammograms for the surface-confined redox groups

Catalysts encapsulated in or attached to the electrode provide catalytic groups

or moieties on the electrode surface Such surface-confined redox species show different

voltammograms from solution redox species because their redox reactions do not

196

involve diffusional redox species They exhibit a characteristic voltammogram of

perfect symmetry with Epa = Epc as seen in Fig 87 and their peak current is linearly

proportional to υ rather than υfrac12 as in Eq 90 [21 30] The features of surface-confined

groups are listed in Table 13

Fig 87 A voltammogram of the reversible redox reaction by surface-confined groups [21]

Table 13 Reversible system by surface-confined groups at 25 ˚C [30]

RTAFn

I p 4

22 Γ=

υ middotmiddotmiddotmiddotmiddot Eq 90

oadsp EE = middotmiddotmiddotmiddotmiddot Eq 91

mVn

E p690

2 =∆ middotmiddotmiddotmiddotmiddot Eq 92

Where Γ isiInitial concentration of the absorbed species

However such an ideal voltammogram is seldom observed because the density of redox

species is so high in the surface-confined environment compared to in the solution and a

197

voltammogram is also influenced by the interaction among those packed redox species

A multi-electron system of the surface-confined groups is also not this case [21]

566 Hydrodynamic voltammetry (HDV) and Levich equation

Hydrodynamic methods purposely introduce convective flow to the system

using RDEs This allows the study of precise kinetics because the diffusion is under

precise control Unknown influence by mass transfer and double layer capacitance on

the electrode reaction is eliminated and steady state is quickly achieved [29 30] In

RDE method laminar flow is applied perpendicular to the electrode surface by spinning

a disc electrode at arbitrary rotation velocity (Fig 88) The rotation rate is expressed in

either rpm (rotation per minute) or angular velocity (ω) ω is defined as ω = 2πf where f

is frequency With disc rotation diffusion becomes a function of electrode rotation rate

as in Eq 93 However the limit of disc rotation rate must be set in order to avoid the

thick hydrodynamic boundary layer and turbulent flows to maintain steady state

Allowable range is between 100 rpm lt f lt 10000 rpm or 10 s-1 lt ω lt 1000 s-1 [30]

198

ω

Fig 88 RDE is rotated at ω and laminar flows applied to the electrode surface

Diffusion layer thickness and disc rotation rate [30]

612131611 νωδ minus= D middotmiddotmiddotmiddotmiddot Eq 93

δ = Diffusion layer thickness cm

D = Analyte diffusion coefficient m2s-1

ω = RDE angular velocity s-1

ν = Solution kinematic viscosity m2s-1

Under such a condition disc limiting current (Id) becomes a function of disc rotation

rate as described in Levich equation (Fig 85) [30] This allows finding the total number

of charge transferred (n)

199

Levich equation [30]

612132620 CnFADI Lminus= νω middotmiddotmiddotmiddotmiddot Eq 94

δ

nFAC= middotmiddotmiddotmiddotmiddot Eq 95

IL = Levich current A

n = Total number of charge transferred

F = Faraday constant Cmol-1

A = Electrode surface area m2

C = Bulk analyte concentration molm-3

( Please refer to Eq 93 for other terms and symbols)

Due to the steady state condition the current response in HDV becomes sigmoidal [223]

as seen in Fig 89

200

Fig 89 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH

74 Based on the real data

567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the

charge transfer rate constant

Levich equation (Eq 94) shows that Id is proportional to ωfrac12 therefore also a

function of diffusion layer thickness (Eq 95) This is true for reversible kinetics

because the electrode reaction is limited by diffusion which is a function of ωfrac12 and

therefore Id also becomes dependent on ωfrac12 at any potential However in

quasi-reversible system Id depends on diffusion but also affected by its kinetics [223]

Therefore when Id reaches the kinetic limitation for charge transfer the slope gradually

deviates from its Levich current as seen in Fig 90 [30]

201

Fig 90 Levich plots for reversible (blue) and quasi-reversible (red) systems [30]

In addition its charge transfer kinetics is altered by the potential applied As Tafel plot

(Fig 9 on p 57) presents the current is exponentially dependent on the overpotential

The electrode reaction is kinetically limited at low overpotential then gradually

becomes diffusion-limited as overpotential increases [223] This implies that Id in

quasi-reversible system is always under the influence of potential applied because the

potential alters its kinetics

When Id becomes completely free from the mass transport influence such current is

called kinetic current (IK) because Id now purely depends on only its kinetics Arbitrary

Id then defined as the sum of diffusion-limited IL and kinetically-limited IK The

double-reciprocal equation of this summation is defined as Koutecky-Levich equation

as in Eq 96

202

Koutecky-Levich equation [30]

LKd III111

+=

216132

11620

11ων

times⎟⎟⎠

⎞⎜⎜⎝

⎛+= minus nCFADI K

middotmiddotmiddotmiddotmiddot Eq 96

Id = Disc limiting current A

IK = Kinetic current A

IL = Levich current A

( Please refer to Eq 93 and Eq 94 for other terms and symbols)

By extrapolating the Koutecky-Levich plot IK can be easily estimated [30] from the

reciprocal of its y-intersect In order to plot Koutecky-Levich equation and find IK a

sequence of arbitrary Id is recorded with altering disc rotation velocity at arbitrary

potential This technique is called hydrodynamic amperometry (HDA) and it is shown

in Fig 91

203

Fig 91 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in

oxygenated 001 M PBS at pH 74 at pretreated GCE Based on the real data

Based on the data obtained by HDA Koutecky-Levich plot is plotted as in Fig 92

204

0E+00

5E-02

1E-01

2E-01

2E-01

3E-01

-002 000 002 004 006 008 010 012 014 016 018

ωˉfrac12 (sˉ

frac12)

1 IK (A-1)

- 04 V

- 06 V- 08 V

05 x 10-1

10 x 10-1

15 x 10-1

0

20 x 10-1

25 x 10-1

-1

I d(A

-1)

Fig 92 Koutecky-Levich plot for O2 reduction at pretreated GCE in oxygenated 001 M

PBS at pH 74 with applied potential ndash 04 06 and 08 V Based on the real data

With Ik from Koutecky-Levich plot and nα from CV the rate constant for arbitrary

potential (k(E)) can be calculated using Eq 97

The rate constant for arbitrary potential E [30]

lowast=nFAC

Ik K

E )( middotmiddotmiddotmiddotmiddot Eq 97

k(E) = Rate constant for the applied potential E ms-1

( Please refer to Eq 94 Eq 95 and Eq 96 for other terms and symbols)

205

568 Finding the standard rate constant (kdeg)

Finding the standard rate constant (kdeg) for ORR of each catalyst is the ultimate

aim of this long tedious cathode analysis because kdeg tells the pure catalytic power

independent of reactant diffusion and overpotential (η) k(E) can be expressed in terms of

kdeg and η as in Eq 98 therefore it is possible to estimate kdeg with known η and k(E)

k(E) in terms of kdeg and η for a forward reaction [30 181]

⎥⎦⎤

⎢⎣⎡ minusminus

deg= )(exp 0)( EE

RTnFkk Ef

α

⎥⎦⎤

⎢⎣⎡ minus

deg= ηαRT

nFk )1(exp middotmiddotmiddotmiddotmiddot Eq 98

kf(E) = Rate constant for a reduction reaction at the applied potential E ms-1

kdeg = Standard rate constant ms-1

η = Overpotential V

( Please refer to Table 11 and Table 12 for other terms and symbols)

η can be found by subtracting Edegrsquo of the reaction involved from the applied potential in

HDA n obtained by HDV is used to specify the reaction involved hence its Edegrsquo In

order to estimate kdeg Eq 98 is first converted into logarithmic expression as in Eq 99

kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

ηαRT

nFkk Efminus

minusdeg= loglog )( middotmiddotmiddotmiddotmiddot Eq 99

206

Log(k(E)) is plotted against a range of η and by extrapolating the plot log(kdeg) is obtained

from its y-intersect where η = 0 hence kdeg can be calculated (Fig 93) [181]

-12

-10

-8

-6

-4

-2

0

-10 -08 -06 -04 -02 00 02 04

η V

log kf(E)

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS NaOH PBS NaOH H2SO4 PBS

NaOH H2SO4

NaOH H2SO4 PBSH2SO4 PBS

H2SO4

NaOH PBS

log kdeg

η V

Fig 93 Log kf(E) plot for O2 reduction at various pretreated GCEs Based on the real data

569 Summary of the electrochemical techniques

CV and hydrodynamic methods are complementary techniques for qualitative

examination of redox reactions and extraction of thermodynamic and kinetic parameters

The protocol for calculating kdeg from hydrodynamic methods and nα from CV is shown

in Fig 94

207

Fig 94 The protocol for finding kinetic and thermodynamic parameters

57 SUMMARY OF THE CHAPTER

The chemistry and electrochemistry of O2 has been reviewed The mechanisms of most

widely investigated catalysts noble metals macrocyclic complexes and enzymes have

been reviewed and their suitability for application in biofuel cells has been discussed

The key physico-chemical parameters which characterise these reactions have been

identified and methods for their determination have been described

208

6 CATHODE MATERIAL AND METHOD

61 ELECTRODE FABRICATION

611 Graphite PtC and RhC electrodes

Microcrystalline graphite of grade (2-15 micron 999995 purity) was

purchased from Alfa Aesar GmbH amp CoKG 5 wt-loaded rhodium on activated

charcoal (RhC) and platinum on activated carbon (PtC) were purchased from Sigma

Aldrich CY1301 and Aradur hardener HY 1300 were purchased from Robnor resins

and they were mixed at 31 (vv) for use RDE templates were specially designed and

made on site They were made of Teflon with the dimension 4-mm ID 20-mm OD

and 15-mm height The depth of composite-holding cavity for each RDE was 25 mm

The composite was made by mixing graphite RhC or PtC with epoxy resin at 73 by

hand Composite mixture was packed into five RDE templates and cured at room

temperature for 72 hours [106]

612 Anthraquinone(AQ)-carbon composite electrodes

6121 Derivatisation of graphite with AQ

Fast red AL salt with 25 dye content and hypophosphorous acid at 50 wt

in H2O were purchased from Sigma Aldrich HPLC grade acetonitrile was purchased

from VWR international 5 mM of Fast Red AL salt solution was prepared in 50 mM

209

hypophosphorous acid 10 ml of the Fast Red solution was mixed with 2 g of graphite

The mixture was left at 5 degC for 30 minutes with regular stirring at every 10 minutes It

was finally washed thoroughly with deionised water followed by vacuum suction in

acetonitrile flow The washed derivatised AQ-carbon was left to dry overnight It was

kept in the sealed container for future use [44]

6122 Fabrication of AQ-carbon composite electrode

AQ-graphite was mixed with epoxy resin at 64 by hand The composite was

packed into five RDE templates and cured at room temperature for 72 hours

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes

6131 Information on synthesis of metallosalophen

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminocobalt (II)] and

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminoiron (II)] denoted as Co(salophen)

and Fe(salophen) respectively were kindly donated by Dr Peter Cragg (School of

Pharmacy Brighton University) They were synthesised by dissolving metal acetate in

the ligand solution which was made by condensation of 21 molar ratio of

salicylaldehyde and diethylenediamine in ethanol or methanol [224 225]

210

6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes

Analytical grade dichloromethane was purchased from VWR international In

the presence of small quantity of dichloromethane 5 wt of Co(salophen) or

Fe(salophen) was mixed and homogenised with graphite [181] After the

dichloromethane evaporated the metallosalophen-graphite mixture was further mixed

with epoxy resin at 64 [219] by hand The composite was packed into five RDE

templates and cured at room temperature for 72 hours

62 COMPOSITE RDE CUTTING

The electrode discs of all the RDE composite electrodes were cut into 1

mm-thick sections using a Buehler IsoMet Low diamond saw with a 04 mm-thick

diamond blade

63 ELECTRODE POLISHING

631 Composite RDE polishing

Composite RDEs were polished successively with SiC grinding paper of 400

1200 and 2400 grit followed by with alumina oxide slurry of particle size 1 03 and

005 microm as in sect 33 on p 122 Electrode polishing was done just before the experiment

in order to avoid air oxidation [195]

211

Fig 95 Fabricated composite RDEs

632 Glassy carbon electrode polishing

Glassy carbon electrodes (GCE) (5-mm disc diameter) were purchased from

Pine Instruments Activated carbon (AC) was purchased from Chemviron Carbon GC

cleaning solvent mixture was made by mixing AC and HPLC grade acetonitrile in a

ratio of 13 (vv) The mixture was covered and left at least for 30 minutes then filtered

before use [226] GCEs were successively polished with alumina oxide powder slurry of

1- 03- and 005-microm particles on MicroCloth pad Polished GCEs were sonicated in

filtered ACacetonitrile mixture for 10 ndash 15 minutes to remove alumina powder debris

and contaminants [226] The GCEs were then sonicated in deoxygenated deionised

water Air contact of the electrode surface was strictly avoided [195]

212

64 ENZYME RDEs

641 Membrane preparation

Cellulose acetate dialysis tubing visking 3632 was purchased from Medicell

International Ltd Sodium carbonate (Na2CO3) was obtained from Sigma Aldrich

Dialysis membrane was cut into 10x10-mm2 pieces which were boiled in 1 wv

aqueous Na2CO3 solution for 10 minutes The membrane pieces were rinsed thoroughly

with deionised water and stored [61] in PBS in a sealed container at 4degC until its use

642 Membrane enzyme electrodes

Laccase (Lac) from Trametes versicolor gt20 unitsmg-1 and lyophilised

bilirubin oxidase (BOD) from Myrothecium verrucaria 15 ndash 65 units mg-1 were

purchased from Sigma Aldrich 4 mgml-1 of Lac and BOD solution was made with PBS

respectively BOD immediately dissolved in PBS while a vortex mixer was needed to

dissolve Lac In order to attach enzyme on the GCE surface the tip of polished cleaned

GCEs was dipped and left in the enzyme solution for two hours with gentle stirring The

electrodes were then lightly washed with deionised water Pretreated dialysis membrane

was carefully placed onto the surface of the enzyme-modified GCE and fixed with

rubber O-ring

213

65 ELECTROCHEMICAL EXPERIMENTS

651 Electrochemical system

CHI 650A with single-channel and CHI 1030A with multi-channel

potentiostats were used for electrode pretreatment and electrochemical experiments CV

HDV and HAD An adjustable rotor from Pine Instrument was used to rotate a RDE

Three-electrode system (sect 342 on p 123) was employed with an AgCl reference and a

Pt counter electrodes

Fig 96 RDE experiment setting with a rotor and gas inlet

214

652 Electrode pretreatments

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes

Graphite GC Co(salophen) and Fe(salophen) electrodes were pretreated in

seven different ways in Table 14 Untreated electrodes were also prepared as controls

All the pretreatments were made in series

Treatment type 1st pretreatment 2nd pretreatment 3rd pretreatment

Type I Untreated -

Type II Acidic 01 M H2SO4

Type III Basic 01 M NaOH

Type IV Neutral 001 M PBS

Type V Base followed by Acid 01 M NaOH 01 M H2SO4

Type VI Acid followed by Neutral 01 M H2SO4 001 M PBS

Type VII Base followed by Neutral 01 M NaOH 001 M PBS

Type VIII Base Acid then Neutral 01 M H2SO4 01 M NaOH 001 M PBS

Table 14 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Concentrated sulphuric acid at 90 - 98 and sodium hydroxide tablets were purchased

from VWR international 001 M PBS was purchased from Sigma Aldrich All the

pretreatments were based on potential cycling in 100-ml electrochemical cell except

untreated ones Acidic pretreatment was performed at 5 Vs-1 between 15 V and ndash 05 V

215

in 01 M H2SO4 [105 219] for 30 minutes Basic pretreatment was performed at 5 Vs-1

between 06 V and 2 V in 01 M NaOH [218 219] for 30 minutes Neutral pretreatment

was done at 5 Vs-1 between 15 V and ndash 05 V in deoxygenated 001 M PBS pH 74

[219 220] Constant N2 flow was applied to PBS through the pretreatment In addition

pretreatments were performed in various combinations as in Table 14 Type V Base

followed by Acid Type VI Acid followed by Neutral Type VII Base followed by

Neutral and Type VIII Base followed by Acid then Neutral [219] The influence of

pretreatments on electrode surface and ORR was studied by CV HDV and HDA

6522 Pretreatments of AQ-carbon PtC and RhC

AQ-carbon PtC and RhC composite electrodes were pretreated only in 01 M

H2SO4 at 5 Vs-1 between 15 V and ndash 05 V in for 30 minutes Untreated electrodes were

also prepared as controls

653 Electrochemical experiments

All experiments were carried out under both N2 and O2 saturation 100 ml of

001 M PBS electrolyte solution was sparged with either N2 or O2 for 15 minutes before

the experiment Gas was continuously supplied through a Dreschel gas washing bottle

containing PBS throughout the experiment

CV was performed at 002 005 01 02 and 05 Vs-1 between + 09 V and ndash

09 V HDV was performed at fixed scan rate of 005 Vs-1 sweeping the potential from

+ 09 V to ndash 09 V with disc rotation velocity altered through 900 1600 and 2116 rpm

216

HDA was performed at a range of fixed potentials - 02 - 04 - 06 and ndash 08 V at disc

rotation velocity of 400 625 900 1156 1225 1600 2116 and 2500 rpm All

experiments were repeated twice or three times The sample size ranges from one to

three The median values were taken for the analysis Obtaining a set of data for each

electrode sample took from three weeks to more than one month

654 Data analysis

CV data was used to calculate nα and reaction reversibility HDV data was used

to calculate n using Levich equation (Eq 30 on p 61) Edegrsquo for ORR involved at pH 7

25 degC was specified by n value as in Table 15

n Redox type Redox potential vs AgAgCl

1 Edegrsquo(O2H2O2) 0082 V

2 Edegrsquo(O2H2O) 0616 V

Table 15 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC [24]

η was calculated by subtracting Edegrsquo (ORR) from the potential applied in HDA HDA

data was used to estimate kf(E) at each applied potential using Koutecky-Levich plot (Eq

30 on p 61 and Fig 92 on p 204) N2 data was subtracted from O2 data as background

Other parameters required for Koutecky-Levich plot were set as in Table 16 All

parameters assume the condition of 001 M PBS at 24 degC

217

Parameter Value Reference

O2 diffusion coefficient 23 x 10-9 m2s-1 [227]

O2 concentration 123 molm-3 [158]

Kinematic viscosity (ν) 884 x 10-7 m2s-1 [30]

Table 16 Parameter values for Koutecky-Levich equation

kdeg was estimated by plotting log(kf(E)) vs η (Eq 99 and Fig 93 on p 206) kdeg for

various and pretreated cathodes were compared The best catalyst shows the largest kdeg

The procedure for finding kdeg is summarised in (Fig 94 on p 207)

218

7 CATHODE RESULTS AND DISCUSSION

71 AIMS OF THE CATHODE STUDIES

This section describes the electrochemical characterisation of various modified

electrodes which were considered as candidate cathodes for the biofuel cells Various

carbons were used as electrode materials in all cases The electrochemical behaviour of

carbon depends strongly on history and also can be radically altered by pre-treatment

The effects of electrochemical pretreatments (ECPs) on the kinetic parameters will be

described for both glassy carbon and graphite composites The cathode modifiers

considered in this section include noble metals (Pt Rh) inorganic Schiff bases

(Co(salophen) Fe(salophen)) an organic modifier (anthroquinone) and the enzymes

bilirubin oxidase (BOD) and laccase (Lac) In each case of non-biological catalysts the

effects of ECPs were also quantified using cyclic voltammetry (CV) and hydrodynamic

methods with rotating disc electrodes (RDEs)

CV allowed the investigation of

Capacitance from the double layer region

Qualitative description of the mechanism such as number of discrete reaction

steps reversibility and surface confinement

nα the number of electrons transferred up to and including the rate limiting step

(RLS)

219

while hydrodynamic methods were used to quantify

n the total number of electrons transferred

kdeg the heterogeneous standard rate constant

Epc2(HDV) the half-wave reduction potential in HDV

The data was taken under both N2 and O2 to allow characterisation of the effects of

electrochemical pretreatment and its effects on the kinetics of O2 reduction The best

candidate catalytic cathode was determined according to kdeg nα and Epc2(HDV)

72 INFLUENCE AND EFFICACY OF ECPS

721 Glassy carbon electrodes (GCEs)

7211 Untreated GCEs

Voltammetry curves of untreated GCEs at pH 74 in the presence of N2 and O2

are shown in Fig 97

220

Fig 97 CVs of untreated GCEs at pH 74 in 001 M PBS in the presence of (i) N2 (ii)

O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Untreated GCEs did not show any peak in the absence of O2 (Fig 97(i)) indicating that

there was no electrochemically active redox species on the electrode surface and in the

solution CV was in the presence of O2 (Fig 97(ii)) showed a peak at ndash 08 V but

without a reverse anodic current This indicates the presence of catalytic activity

however it occurred at only high overpotential and is therefore not efficient for ORR

This result is consistent with previous work [215 216]

7212 Influence of electrochemical pretreatments on GCE

GCEs were pretreated in various media in order to improve ORR efficiency It

has been reported that ECPs activate the electrode surface by introducing

electrochemically active functional groups and that this improves electrochemical

(i) N2

-3E-05

-2E-05

-1E-05

0E+00

1E-05

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

221

reversibility and [105 218] and the electron transfer rate of inner-sphere catalysis [228]

Fig 82 shows the voltammetry curves in the absence of O2 for various pretreated GCEs

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-60E-05

-50E-05

-40E-05

-30E-05

-20E-05

-10E-05

00E+00

10E-05

20E-05

30E-05

40E-05

50E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH

Fig 98 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of

O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1 Arrows indicate the

peak position (i) includes CVs for all the pretreated GCEs and (ii) excludes

PBS pretreatments for convenience

222

As the figure shows any pretreatment resulted in an increase in capacitance and

introduced characteristic voltammetric peaks which were not observed in the untreated

GCEs In all cases the peak current was linearly proportional to υ indicating that the

pretreatments led to surface-confined redox active functional groups most likely to be

quinone groups That ECPs give rise to surface quinones is well established [105 218

220 229 230] The Epc shifted with υ and was separated from Epa hence redox

reactions by these surface quinone groups are quasi-reversible The effect of

pretreatments however varied by the type of medium used in pretreatments

Above all PBS-related pretreatments gave qualitatively different results which

were characterised by a great increase in average capacitance calculated from CVs in

001 M PBS at pH 74 at 50 100 and 200 mVs-1 in the double layer region of the CV at

04 V Summary results are shown in Fig 82(i) and Fig 99

223

GCE Capacitance vs Pretreatment

0100200300400500600700800900

1000

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS

NaOH PBS

NaOH H2SO4

PBS

Pretreatment

Capa

cita

nce

microF

cm-2

Fig 99 The capacitance of GCEs in 015 M NaCl buffered with 001 M phosphate to

pH 74 Error bars represent the standard deviation (n = 3)

The average capacitance of the untreated GCEs was 76 microFcm-1 and is within the typical

ranges reported hitherto [221] while PBS pretreatment increased the capacitance to 700

- 800 microFcm-1 A similar phenomenon was reported by Nagaoka and by Swain [215

216] There are two possible reasons for this (i) PBS treatment caused the

microfractures of GC and increased the electrode area and therefore double layer [105

215 216 218] andor (ii) attached the electrochemically inactive charged surface group

such as phenol and carboxylate [216 229]

In the pretreatment involving NaOH the carbon dissolution was observed This

is harsher pretreatment so erodes the electrode and makes the electrode surface more

porous [216 218] NaOH also forms unstable oxide film and eventually removes

graphite oxide [229] and surface impurities [218] However the resultant capacitance

224

was unexpectedly smaller than that of sole PBS pretreatment Therefore NaOH

pretreatment increased the capacitance by increasing the exposed electrode area [218

231] while PBS pretreatment increased capacitance by attaching redox inactive charged

surface groups The largest capacitance after the double pretreatment involving NaOH

and PBS was therefore derived from the layer of charged groups attached on the

electrode surface area made larger by NaOH pretreatment

H2SO4 pretreatment on the other hand resulted in a smallest capacitance

increase The electrode behaviour was also slightly altered from that of other

PBS-related pretreatments when the pretreatments involved H2SO4 together The

capacitance became smaller as shown in Fig 99 and the peak positions were shifted to

more positive potential side (Fig 82(i)) To investigate the causes of this phenomena

the voltammograms during such pretreatments were studied as Fig 100 below

225

Fig 100 Overlaid CVs during various pretreatments for GCEs involving 01 M H2SO4

alone (red) 001 M PBS at pH 74 alone (green) and both pretreatments (pink)

It is clearly shown that either pretreatment resulted in distinctive peaks for the

attachment of active surface quinone groups However the peak potentials were

different when in sole H2SO4 or PBS-related pretreatments This is because as proposed

by Nagaoka those quinone groups were present in the different chemical environment

leading to different behaviours [216] H2SO4 pretreatment attached fewer inactive

surface oxygen groups while PBS pretreatment attached more Therefore H2SO4

pretreatment led to the smaller capacitance change than PBS pretreatment This implies

the presence of different electrode surface conditions hence quinones present in such

different environment behave differently showing different peak positions The

behaviours of surface quinone groups depends on the conditions of the pretreatment

involved [229 230 232] This fact can also explain the phenomena caused by the

226

double pretreatment with H2SO4 and PBS as shown in Fig 82(i) More quinone groups

had been attached by H2SO4 pretreatment in advance therefore only smaller area than

usual became available for PBS pretreatment to stably attach their charged oxygen

surface groups This is the most likely causes of the reduced capacitance and different

peak positions in such a double pretreatment compared to other PBS-related

pretreatments Furthermore quinone peak positions should be close to those found for

the H2SO4 pretreatment alone because quinone groups attached by preceding H2SO4

pretreatment dominate the electrode surface and should be have more influence on the

electrochemistry However they also resulted in larger capacitance because the

subsequent PBS pretreatment attached inactive oxygen surface group which led to an

increase in fixed charge density This is a typical phenomenon of synergetic effects as

already seen in the double pretreatment with NaOH and PBS

On the other hand voltammetric curves for the double pretreatment of NaOH

and H2SO4 did resemble to those by sole NaOH pretreatment Similar phenomena were

observed between the double pretreatment with NaOH and PBS and the triple

pretreatment with NaOH H2SO4 and PBS It seems that preceding NaOH pretreatment

overwhelmed the subsequent H2SO4 pretreatment although some influence by H2SO4

can be observed in terms of capacitance as shown in Fig 99 This implies that the

preceding NaOH pretreatment made most of the electrode area not available for

subsequent H2SO4 pretreatment However if NaOH pretreatment creates micropores in

the GC protons from subsequent H2SO4 pretreatment can still penetrate such

micropores These micropores are accessible for protons but not most ions [216]

227

7213 Effect of GCE pretreatment on ORR

Voltammograms for ORR at various pretreated GCEs at pH 74 are shown in

Fig 101 below

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO4

01M NaOH01M NaOH + 01M H2SO4

001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS

01M NaOH + 01 M H2SO4 + 001M PBS

Fig 101 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

In the figure any pretreated GCE shows larger cathode peaks between ndash 03 and ndash 05 V

without reverse anodic current They were due to O2 reduction by surface quinone

groups whose peaks were observed at ndash 01 V in Fig 82(i) in the absence of O2 This

indicates that ORR at any pretreated GCEs is regulated by ECrsquo mechanism [30] Ipc was

linearly proportional to radicυ in all cases indicating the diffusion-limited charge transfer

228

However untreated GCE was catalytic only at high overpotential since Epc2(HDV) = ndash 08

V This is compatible to the observation made by Tammeveski [233] It was seldom

catalytic since n = 102 plusmn 011 and nα = 059 plusmn 004 This single-electron ORR implies

the presence of O2־ disproportionation from which little power can be extracted [233]

kdeg was could not be calculated because the standard Koutecky-Levich plot could not be

obtained since it did not show a normal linear slope In contrrast all the other

pretreatments improved ORR as seen in Fig 101 They showed large cathodic peaks at

smaller overpotentials PBS-related pretreatments in particular increased the catalytic

current by gt 15 times and positively shifted Epc2(HDV) by gt 300 mV relative to that of

untreated GCEs Similar phenomena were reported elsewhere [220 234] All the GCE

pretreatments resulted in an increase in both nα and n The increase in kdeg accompanied

by an increase in nα was also observed as a general tendency as shown in Fig 102

229

0

2

4

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

kordm

ms

-1

nα 059 107 094 106 168 278 174 178

kordm ms-1 000E+00 715E-09 529E-09 631E-08 131E-07 279E-07 654E-08 113E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

- nα

kdeg ms-1

Untreated H2SO4 NaOHNaOHH2SO4

H2SO4PBS

NaOHPBS

NaOHH2SO4PBS

Fig 102 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Above all the H2SO4-PBS double pretreatment was most effective giving kdeg = 279 x

10-7 plusmn 507 x 10-9 ms-1 and Epc2(HDV) = - 039 plusmn 002 V However its nα was calculated

to be 278 while n = 185 This is an unusual result It might imply the presence of serial

ORR pathway in which H2O2 is further reduced or it is simply impossible to obtain

correct parameters by using Eq 88 on p 193 for ORR at pretreated GCEs due to their

excessively narrow peak width Pretreatment of GCE was proved to increase kdeg of ORR

but how this improves its catalysis has been uncertain for a long time between two

hypotheses whether they mediate electron transfer or they alter the adsorption of O2

and O2[233 232 220] ־ The latter or both is more likely because as Fig 102 shows

PBS-related pretreatments increased the capacitance which was derived from redox

inactive surface oxygen groups yet made significant contribution to an increase in kdeg It

230

is therefore natural to deduce that the largest kdeg and nα were the consequence of

synergetic effect derived from different surface oxygen groups attached by H2SO4 and

PBS

722 Graphite composite electrodes

7221 Untreated graphite composite electrodes

Graphite composite electrodes are different from GCEs They are made of

graphite particles which are more porous and permeable to the solution and gas This

practically results in larger electrode surface area therefore becomes more subject to

facile adsorption and surface modification This leads to the different surface conditions

and electrode behaviours from GCEs as proposed by Sljukic [232] CVs of untreated

graphite composite electrodes at pH 74 in the presence of N2 and O2 are shown in Fig

103 together with those of untreated GCEs

231

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

Fig 103 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl Small peaks on graphite electrodes are indicated by arrows

Different electrode behaviour is noticeable especially in nitrogenated solution because

untreated graphite composite electrodes showed small peaks at + 100 mV and - 100 mV

as indicated by arrows No peak was present fo untreated GCE This indicates the

presence of some reactive surface quinone groups on graphite composite without

pretreatment This is because graphite composite electrodes have more reactive edge

plane graphite exposed and the porous electrode structure allows larger electrode area to

be exposed There was however little difference in averaged capacitance between

graphite composite and GCEs This indicates that an increase in capacitance does not

necessarily correlate to an increase in the surface area as proposed by Regisser [235]

Voltsmmograms for ORR at untreated GC and graphite composite electrodes resembles

each other though for the latter the reduction peak is around 50 mV more positive It can

232

be seen that graphite composites are slightly more catalytic toward ORR presumably

because of the intrinsic quinone groups derived from the graphite electrode surface

7222 Influence of electrochemical pretreatments on graphite composite electrodes

Cvs for the graphite composite electrodes differed from those of GCEs Peaks

were observed in all graphite cases including untreated ones as shown in Fig 104

233

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

(ii)

01M H2SO401M NaOH01M NaOH + 01M H2SO4Untreated

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBSPBS

(iii)01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS001M PBS

Fig 104 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M

nitrogen-sparged PBS between + 09 and - 09 V vs AgAgCl (i) All

pretreatments at 005 Vs-1 (ii) Non-PBS-related pretreatment and (iii)

PBS-related-pretreatment CVs at 002 Vs-1

The capacitance became larger after pretreatments Any multi-pretreatment involving

NaOH and H2SO4 resulted in the largest capacitance which was 20 times larger than

234

that of untreated graphite composite electrodes The capacitance increase was also

larger than that of any pretreated GCEs except for PBS-pretreated ones PBS

pretreatment led to the smallest capacitance increase in graphite composite electrodes in

contrast to the GCEs Instead H2SO4 pretreatment led to the largest capacitance

increase Moreover this outcome was found to correspond to a specific proportional

relation between Ip and υ Ip of electrodes involving any pretreatment in PBS NaOH or

their combinstions were linearly proportional to radicυ This can only arise from on or more

diffusion limited step or steps in the redox reactions of the surface groups most likely

proton diffusion though a porus film or to microscopic porous environments Surface

oxygen groups have previously been reported to involve involve proton uptake and this

leads to a broad peak [216 230] H2SO4-pretreated electrodes showed Ip proportional to

υ and the redox reaction is independent of proton diffusion

These phenomena show that different pretreatments provide different chemical

environment for active quinone groups leading to different quinone behaviours and

voltammetry peaks [229 230 232] In the multi-pretreatment with NaOH and H2SO4

synergetic effects became obvious Its redox reaction was diffusion-limited and showed

voltammograms with two partially resolved sets of peaks yet its peak still partially

overlaps with that of diffusion-independent H2SO4-pretreated electrodes as seen in Fig

104(i) When this double-pretreatment was further followed by PBS pretreatment the

peak derived from quinones attached by H2SO4 disappeared This implies that

subsequent PBS pretreatment suppressed the activity of proton-independent quinones

and the effect by H2SO4 The similar phenomenon was observed when electrodes were

pretreated doubly by H2SO4 and PBS (Fig 104(ii)) Every case presented

235

quasi-reversible redox systems because peak potentials shifted with scan rates Small

multiple peaks were present in slower scan rates merged into one broad peak at faster

scan rate as the contribution of capacitance charging and increased iR drop obscured

voltammetic detail

7223 The effect of graphite composite electrode pretreatment on ORR

ORR at pretreated graphite composite electrodes was found to be less effective

than for pretreated GCEs Only NaOH-related pretreatments were effective (Fig 105)

enough to achieve two-electron ORR Untreated graphite composite electrodes were

catalytic only at high overpotential despite the presence of surface quinone groups PBS

pretreatment was hardly effective in contrast to the results obtained for GCEs

Electrochemical pre=treatment in H2SO4 and its double pretreatment with PBS reduced

Epc2(HDV) by 40 ndash 90 mV however the electrodes showed poor catalytic performance nα

lt 05 and n lt 2 consistent with the initial electron transfer still being rate determining

236

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 105 Overlaid CVs of ORR at various pretreated graphite composite electrodes

at pH 74 in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005

Vs-1

Pretreatment of the graphite composites with just H2SO4 showed a single merged peak

at slower scan rate while they showed an addition prewave as indicated by arrows in

Fig 106 at faster scan rate

237

-16E-03

-14E-03

-12E-03

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

-1 -08 -06 -04 -02 0 02

E V

J Acm-2

50 mV100 mV200 mV500 mV

Fig 106 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at

pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various

scan rates 50 100 200 and 500 mVs-1

Coupled with the fact of n asymp 1 this prewave is due to the rate-limiting slow formation of

semiquinone radicals [233] A semiquinone radical is formed when one electron is

transferred to a quinone group and its formation is necessary to mediate ORR as shown

in Eq 100

238

middotmiddotmiddotmiddotmiddot Eq 100

[233] middotmiddotmiddotmiddotmiddot Eq 101

Q = Surface quinone group

O2־ = Semiquinone radical

Any NaOH-related pretreatment on the other hand showed a prewave around ndash 035 V

at slower scan rate as shown in Fig 107 but this merged into a broad peak at faster scan

rate

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

01M NaOH01M NaOH + 01M H2SO4 01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 107 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

239

Ipc was linearly proportional to radicυ except for the triple pretreatment which was difficult

to define due to the ambiguous peak positions over different scan rates The prewave

was due to the slow reduction of O2 into O2־ (Eq 101) rather than semiquinone radical

formation (Eq 100) as seen in H2SO4-pretreated electrodes They had n asymp 2 and nα asymp 1

hence they catalysed two-electron ORR Those pretreatments positively shifted

Epc2(HDV) by 173 plusmn 26 mV on average from ndash 730 mV at the untreated graphite

composite electrodes The largest catalytic activity was achieved by doubly pretreated

electrodes with NaOH and H2SO4 with kdeg = 382 x 10-7 plusmn 32 x 10-8 ms-1 (Fig 108)

0

2

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

35E-07

40E-07

45E-07

kordm

ms

-1

nα 042 035 080 114 045 040 101 072

kordm ms-1 000E+00 000E+00 233E-07 382E-07 000E+00 000E+00 132E-07 172E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBSUntreated H2SO4 NaOH

NaOHH2SO4

H2SO4

PBS NaOHPBS

NaOHH2SO4

PBS

- - - - nα

kdeg ms-1

Fig 108 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

240

723 Platinum on carbon composite electrodes (PtC)

Noble metals are popular ORR catalysts [172 173] with Pt showing the highest

catalytic power [165 166 171 172] It was reported that Pt has a 98 O2-to-H2O

conversion rate [174] However for PtC composite electrodes the voltammetry was

dominated by showed solution resistance both in the presence and absence of O2 and

therefore were not either catalytic or conductive H2SO4 pretreatment was not at all

effective This is probably due to the large electronic resistance of activated carbon itself

[204] as well as intrinsic resistance of composite conductors

724 Rhodium on carbon composite electrodes (RhC)

Rhodium is a platinum group metal and has been reported as a good ORR

catalysis after Pt and Ir [172] Voltammograms for untreated RhC composite electrodes

only showed the capacitance but no redox peaks in nitrogenated solution The

capacitance decreased by 80 for every ten-fold increase in the scan rate Non-faradic

current is normally linearly proportional to υ however it exponentially decreased with υ

in the untreated RhC composite electrodes It did not show any peak in the presence of

O2 and was not catalytic toward ORR (Fig 110(i)) H2SO4-pretreated RhC composite

also did not show any voltammetric peaks Its capacitance was roughly x30 larger than

that of untreated electrodes and again exponentially decreased with an increase in υ

ORR catalysis was however present (Fig 110(ii)) therefore it is reasonable to deduce

that H2SO4 attached the surface quinone groups but the large capacitance charging

prevented voltammetic observation of quinones

241

(i) Untreated

-16E-03

-12E-03

-80E-04

-40E-04

00E+00

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

N2O2

(ii) H2SO4

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

10E-03

-1 -08 -06 -04 -02 0 02 04 06 08 1

E VJ

Ac

m-2

N2O2

Fig 109 CVs of (i) untreated and (ii) H2SO4-pretreated RhC composite electrodes at

pH 74 in 001 M PBS in the presence of N2 and O2 at 005 Vs-1 between + 09

and -09 V vs AgAgCl

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR

Untreated RhC composite electrodes did not show ORR catalysis however a

steep decrease in cathodic current was observed without any peak as the potential

decreases This steep current decrease was probably caused by O2 adsorption on the Rh

particle surface H2SO4-treated RhC on the other hand was found to be catalytic with n

= 177 and nα = 068 plusmn 011 A reduction prewave was observed at around ndash 05 V and

this merged into a broad peak at faster scan rates This prewave was due to ORR by

surface quinone groups attached by H2SO4 because it overlaps with the peak observed

in H2SO4-treated graphite composite electrodes as shown in (Fig 110) This prewave

242

therefore indicates the slow first electron transfer to form O2־

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

-1 -05 0 05 1

E V

J Acm-2

RhC H2SO4

Graphite H2SO4

Fig 110 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

O2־ was probably further reduced to H2O2 by Rh particles because n = 177 plusmn 011 The

catalytic activity was much larger compared with H2SO4-pretreated graphite composite

electrodes as shown in Fig 110 with kdeg =257 x 10-7 plusmn 230 x 10-8 ms-1

243

725 Co(salophen) composite electrodes

7251 Untreated Co(salophen) composite electrodes

Untreated Co(salophen) electrode showed couples of distinctive peaks Epc(1) =

053 Epc(2) = -024 Epa(3) = -016 and Epa(4) = 016 V as shown in Fig 111(i) These

peaks correspond to stepwise cobalt redox reactions Co3+2+ and Co2+1+ respectively

The peak potentials hardly changed over different scan rates consistent with previously

published results [181] Similar peaks have been observed in deoxygenated organic

solvents [174 181] as well as for Co(salen) [181]

(i) N2

-2E-05

-1E-05

0E+00

1E-05

2E-05

3E-05

4E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

2E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fig 111 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

244

In the presence of O2 it showed a reduction peak at ndash 04 V as shown in Fig 111(ii)

ORR potential was shifted by approximately 300 mV relative to that of untreated

graphite composite This result reasonably compatible to the result reported by Ortiz

[181] At this potential O2 forms an adduct with Co2+(salophen) and then is reduced to

O2־ while Co2+ undergoes one-electron oxidation to Co3+ [170] To be catalytic Co3+

formed here must be reduced back to Co2+ to reduce O2־ further into H2O2 [181] or a

binuclear complex must be formed with another Co2+ [170] Untreated Co(salophen)

composite electrodes were however not strongly catalytic due to n = 138 plusmn 009 and nα

= 055 plusmn 012 Ip was linearly proportional to radicυ and the peak potentials Epc(2) and Epa(3)

varied only by plusmn 8 over different five scan rates This indicates nearly reversible

multi-electron transfer redox system of Co(salophen) composite electrodes

7252 Influence of electrochemical pretreatments on Co(salophen) composite

There was a particular redox pattern found in voltammetry waves according to

the type of pretreatments (Fig 112)

245

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 112 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in

001 M nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

NaOH-pretreatment showed similar voltammetric faradaic features as those in untreated

electrodes (Fig 112) but increased the capacitance by six times relative to that of

untreated electrodes The size of capacitance was 382 microFcm-1 and approximately equal

to that of NaOH-pretreated graphite composite electrodes This implies that NaOH

attached electrochemically inactive surface oxygen groups and the attachment of active

surface quinone groups were not clearly confirmed

PBS-pretreatment on the other hand attached quinone groups because Epa asymp 04 V at

which similar peaks were observed in PBS-pretreated graphite composite (Fig 104(iii)

p 233) Their cathodic peaks were however broader because they merged into peaks

246

for Co-redox processes The effect of PBS pretreatment was much smaller compared to

that of H2SO4-related pretreatment in the deoxygenated solution Single NaOH

treatment and any PBS-related pretreatment showed Ip linearly proportionally to radicυ

while any H2SO4-related pretreatment but without involving PBS-pretreatment showed

a proportional relation with υ This indicates that H2SO4 and PBS or NaOH

pretreatments lead to different surface redox mechanism Peaks due to H2SO4-related

pretreatments were prominent and are indicated by a red circle in Fig 112

7253 The effect of Co(salophen) composite electrode pretreatment on ORR

Untreated electrodes were not catalytic Parameters for H2SO4-pretreated

Co(salophen) composite found to have n asymp 1 and nα le 1 although they look catalytic in

Fig 113 reducing its ORR overpotential by 400 ndash 500 mV compared to GCEs This

inconsistent result arose because it was impossible to find the correct parameter using

the conventional calculation using Eq 88 on p 193 and need more sophisticated

method such as a simulation probramme The other pretreatments were on the other

hand found strikingly effective toward ORR as shown in Fig 113

247

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

40E-04

60E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 113 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Such pretreated Co(salophen) electrodes showed Epc asymp - 03 V having reduced the

overpotential against unpretreated Co(salophen) by 100 mV and 300 mV against

untreated graphite composite electrodes ORR was synergistically catalysed by Co2+ and

surface semiquinone radicals [181 233] They showed n asymp 2 and nα gt 07 hence they

catalysed two-electron ORR Ipc was linearly proportional to radicυ hence the redox

reactions were diffusion limited except for the double pretreatment with NaOH and

H2SO4 Large catalytic activity was achieved by sole NaOH and by the double

pretreatment with NaOH and H2SO4 or the combination of H2SO4 and PBS Among

them sole NaOH pretreatment was revealed to be most effective as shown in Fig 114

and showed kdeg = 119 x 10-5 plusmn 118 x 10-6 nα = 083 plusmn 014 and Epc2(HDV) = 037 V

248

0

1

2

nα

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

kordm

ms

-1

nα 055 028 083 074 068 091 071 078

kordm ms-1 000E+00 000E+00 119E-05 747E-06 271E-06 684E-06 296E-06 587E-06

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

Fig 114 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite

electrodes

One possible reason for this largest catalytic activity by NaOH pretreatment is that

NaOH might attach more reductive surface OH groups [218] which became involved in

reducing back Co3+ into Co2+ during ORR [181]

726 Fe(salophen) composite electrodes

7261 Untreated Fe(salophen) composite electrodes

Untreated Fe(salophen) electrodes showed a couple of distinctive peaks Epa = -

024 plusmn 001 V and Epc = - 036 plusmn 001 V These peaks are due to Fe3+2+ redox reaction as

249

shown in Fig 115(i) The voltammetry curve and redox peaks are reasonably similar to

those of Fe(salen) reported [224]

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

(ii) O2

-6E-04

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

Fig 115 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74

in 001 M PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and

- 09 V vs AgAgCl

However the redox system is not perfectly reversible here because ∆Ep slightly

increases with an increase in the scan rate and its Ip was linearly proportional to radicυ It is

therefore a quasi-reversible system Untreated Fe(salophen) electrodes was not as

catalytic as Co(salophen) electrodes Epc was ndash 079 plusmn 007 V and little different from

that of untreated graphite composite electrode as shown in Fig 115(ii) although Ipc was

slightly bigger The low ORR catalytic activity of Fe(salophen) was due to the low ORR

rate of Fe itself (Fig 65 on p 164) [172]

250

7262 Influence of electrochemical pretreatments on Fe(salophen) composite

The effect of pretreatments to Fe(salophen) was striking They resulted in

additional voltammetric features due to the Fe redox peaks at Epc (Fe) = - 03 V and Epa

(Fe) = - 02 V and by attaching the surface quinone groups at Epc (Q) = 015 V and Epa (Q) =

02 V as shown in Fig 116(i)

251

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

60E-05

80E-05

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

Untreated01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS001M PBS

-1E-04

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

1E-04

1E-04

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

Fig 116 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1 (i) All

pretreatments (ii) Non-PBS-related pretreatments and (iii)

PBS-related-pretreatments CVs

Non-PBS-related pretreatments (Fig 116(ii)) showed redox peaks clearly however the

252

two cathodic peaks merged into a broad peak at faster scan rate than 100 mVs-1

indicating quasi-reversible surface quinone redox reactions Only a broad merged

reduction peak was observed for all PBS-related pretreatments (Fig 116(iii)) at all scan

rates

Any PBS-related pretreatments resulted led to a broad cathodic peak (from + 01 V to

approximately ndash 03 V) made up of both Fesalphen reduction and reduction of surface

confined quinones arising from the electrochemical pretreatment PBS pretreatment

always greatly increases the surface area leading to micro fissures and consequently a

greater variety of microenvironments for electrochemically generated quinone

functionality

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR

Most were found to be only weakly catalytic (Fig 117) since either n or nα or

both of them were lt 1 Only double-pretreatment with NaOH and H2SO4 led to

sufficient ORR catalysis with n = 185 plusmn 017 nα = 079 plusmn 023 kdeg = 112 x 10-7 plusmn 771 x

10-9 ms-1 and Epc2(HDV) = - 057 V The double pretreatment showed a prewave at ndash 035

V and it disappeared at faster scan rate indicating a slow single-electron transfer to O2

Ipc was linearly proportional to radicυ hence the catalysis was diffusion limited

253

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS

Fig 117 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

727 Anthraquinone (AQ) composite electrodes

Anthraquinone-grafted graphite electrodes were pretreated with H2SO4 Both

untreated and H2SO4-pretreated electrodes showed couples of peaks (Fig 118(i)) one of

which Epc(AQ) was compatible with Em = - 049 reported by McCreery [44] These

peaks were therefore derived from anthraquinone chemically attached to the graphite

254

(i) N2

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

-1 -05 0 05 1

E V

J A

cm

-2

Untreated FRE01M H2SO4 FREUntreated Graphite

(ii) O2

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

-1 -05 0 05 1

E VJ

Ac

m-2

Untreated FRE01M H2SO4 FREUntreated Graphite

Fig 118 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl CVs of untreated graphite electrodes are also shown together

The other couple of peaks Epc(SQ) and Epa(SQ) was observed at - 003 plusmn 003 V and +

020 plusmn 003 V respectively They were due to quinone groups on the carbon surface

H2SO4-pretreatment did not alter the voltammogram pattern but only increased the

capacitance by x3 and enhanced the peak current Both electrodes showed Ip linearly

proportional to radicυ hence the redox reactions involve proton diffusion ORR by those

electrodes is shown in Fig 118(ii) together with the voltammogram of graphite

composite electrodes Both untreated and pretreated AQ-grafted composite electrodes

positively shifted Epc by 60 and 230 mV respectively relative to that of untreated

graphite composite electrodes However their voltammetric curves resembled those for

untreated graphite composite electrodes Besides this their Ip dropped by 20 relative

255

to untreated graphite composite electrodes and showed n asymp 1 and nα asymp 05 Therefore

either was not catalytic in this study This outcome does not correspond to the finding

by Manisankar who confirmed the presence of ORR catalysis at AQ-carbon paste

electrodes at pH 7 with a reduction prewave at ndash 04 and a peak at ndash 07 V [193] To the

contrary this result fits to the data presented by Schiffrin who observed ORR

suppression at AQGC electrodes at pH lt 7 He reported that voltammetry curves for

ORR at GC and GCAQ became similar at this pH hence implied the possibility of

disproportionation of semiquinone intermediates [194]

728 Lac and BOD enzyme membrane electrodes

In the absence of O2 neither Lac nor BOD showed any peaks in their cyclic

voltammograms However they showed ORR catalytic activity even in the absence of

mediator (Fig 119) DET is therefore possible as already confirmed by many [183

186-188] Carbon surface groups [195] also might mediate electron transfer [188]

256

-14E-04

-12E-04

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

-1 -09 -08 -07 -06 -05 -04 -03 -02 -01 0

E V

J Acm -2

LacBOD

Fig 119 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated

PBS at 005 Vs-1 between 0 and -09 V vs AgAgCl

Ipc was linearly proportional to radicυ therefore ORR was diffusion limited Epc were -054

plusmn 002 and ndash 067 plusmn 006 V at Lac and BOD respectively They reduced ORR

overpotential by 120 and 250 mV relative to that of GCEs however the Epc shifted with

υ hence they were quasi-reversible It is known that MCOs catalyse four-electron ORR

[175 184 236] however n = 005 and nα asymp 1 in this study This is probably due to the

difficulty in electrochemical measurement of enzyme catalysis It is however possible

to find nα because the rate-limiting step is the single electron transfer between the

electrode and the blue copper atom of the enzyme Assuming this kdeg was calculated for

four-electron ORR at the enzyme electrodes and the result is shown in Fig 120

257

0

1

2

nα

00E+00

40E-09

80E-09

12E-08

16E-08

20E-08

kordm

ms

-1

nα 077 081

kordm ms-1 134E-08 168E-09

Lac Bod

Fig 120 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Lac had kdeg = 134 x 10-8 plusmn 251 x 10-9 while BOD had kdeg = 168 x 10-9 plusmn 271 x 10-10

Lac electrodes showed better ORR efficiency despite of non-optimal pH condition Lac

is most active at pH 5 while BOD at pH 7 The amount of enzyme loaded on the

electrode surface also might have affected the catalytic activity It was however difficult

to control the loading because the enzyme was attached by dip coating Using mediators

could have also improved the ORR efficiency Many authors used mediators such as

ABTS [62 66] and Os-pyridine complexes linked to conductive polymer [97 237] to

assist the electron transfer between the electrode and enzyme

73 MOST EFFICIENT ORR CATALYSTS

The kinetic and thermodynamic parameters (kdeg nα Epc2(HDV)) were organised

258

by magnitude and are summarised in figures 25-27 and tables 1 amp 2 below For each

parameter the best five candidates were selected to present the effects of pretreatment

and their relation with each parameter The best three were chosen from them on the

basis of the parameter size and the number of top-ranked parameters they were

occupying kdeg was given the highest priority for selection process

731 Best five electrodes for nα

Pretreated GCEs were found to have prominent nα (Fig 121)

00

05

10

15

20

25

30

Electrode type

n α

nα 278 178 174 168 114

GC H2SO4-PBS GC NaOH-H2SO4-PBS

GC NaOH-PBS GC PBS GP NaOH-H2SO4

Fig 121 The best five electrodes for nα

The GCEs doubly pretreated with H2SO4 and PBS was found best with nα = 278 Its kdeg

was 279 x 10-7 ms-1 and also biggest among pretreated GCEs Correlation between nα

259

and kdeg has not been confirmed since it is possible for the intial electron transfer to be

accelerated whilst still remaining the RDS

732 Best five electrodes for kdeg

Top six kdegs were dominated by pretreated Co(salophen) composite electrodes

(Fig 122)

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

Electrode type

kdeg

ms

-1

kordm ms-1 119E-05 747E-06 684E-06 587E-06 296E-06

CoS NaOH CoS NaOH-H2SO4

CoS H2SO4-PBS CoS NaOH-H2SO4-PBS

CoS NaOH-PBS

Fig 122 The best five electrodes for kdeg

PBS pretreatment was found to suppress kdeg in Co(salophen) composite electrodes Clear

correlation between kdeg and nα has not yet confirmed however it was found that

favourable kdeg tends to lower overpotential

260

733 Best five electrodes for Epc2(HDV)

Although it is not an exact correspondence some correlation was observed

between lower overpotential and larger kdeg or lower overpotential and larger nα but not

between kdegand nα A range of pretreated Co(salophen) composite electrodes which

showed largest kdeg and a pretreated GCE which showed the largest nα were found to be

dominating low overpotential (Fig 123)

-040

-039

-038

-037

-036

-035

Electrode type

E pc

2(H

DV)

V

Epc2(HDV) -037 -037 -039 -039 -040

CoS H2SO4-PBS CoS NaOH CoS NaOH-H2SO4

GC H2SO4-PBS CoS NaOH-PBS

Fig 123 The best five electrodes for Epc2(HDV)

734 Best three candidates for ORR catalytic electrodes

Pretreated Co(salophen) composite electrodes were the best candidates for

ORR catalysis because of largest kdeg and lowest overpotential Top three catalytic

261

cathodes were listed in Table 17

kordm ms-1 Epc2(HDV) V nα

Co(salophen) NaOH 119E-05 -037 083

Co(salophen) NaOH-H2SO4 747E-06 -039 074

Co(salophen) H2SO4-PBS 684E-06 -037 091

Average 875E-06 -037 083

SD 278E-06 001 009

Table 17 Three candidates for catalytic cathodes for ORR

NaOH-pretreated Co(salophen) was found to be most efficient ORR catalytic cathode

because it has the largest kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 Epc2(HDV) = - 037 V and

was the second smallest of all kdeg of the three candidate Co(salophen) electrodes were

compared with those of catalytic metals and shown in Table 18

Electrode type kdeg ms-1 Experiment condition

Co(salophen) NaOH 119 x 10-5 In 001M PBS pH 74

Co(salophen) NaOH ndashH2SO4 747 x 10-6 In 001M PBS pH 74

Co(salophen) H2SO4-PBS 684 x 10-6 In 001M PBS pH 74

Au 4 x 10-3 In 005 M H2SO4 [173]

Au 3 x 10-5 In 01 M KOH

Ag 8 x 10-3 In 01 M KOH

Pt 4 x 10-3 In 01 M KOH

Table 18 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

262

NaOH-pretreated Co(salophen) composite electrode showed good catalysis at neutral

pH It could manage to achieve as efficient ORR as Au in acidic solution

74 CONCLUSION TO CATHODE STUDY

The large overpotential of ORR has been known to degrade the fuel cell

performance [3 4 62 165-167] Finding efficient ORR catalysts therefore has been a

subject of intensive research In this study various catalytic cathodes were fabricated

using inorganic organic and biological catalysts and they were electrochemically

pretreated in different media to improve the catalytic activity Studies were conducted

using various electrochemical methods (i) CV (ii) HDV and (iii) HDA The data

obtained was analysed both qualitatively and quantitatively The ORR catalytic

efficiency was calculated according to the three parameters (i) kdeg (ii) nα and (iii)

Epc2(HDV) and based on that most catalytic cathode was determined

None of the cathodes was catalytic enough without pretreatment or unless at high

overpotential In many cases electrochemical pretreatments were found to attach

various surface oxygen groups activated the electrodes and led to two-electron ORR

catalysis Their influence on electrochemistry varies by the electrode type and media

used for pretreatment It was found that PBS-related pretreatments were effective to

GCEs When the pretreatment is combined with H2SO4 this might lead to serial ORR

where H2O2 produced is further reduced to water NaOH-related pretreatments were

effective for graphite composite electrodes PtC composite electrodes were not active

due to the high resistance within the activated carbon The performance by RhC was

263

only marginally improved by H2SO4 pretreatment Any NaOH- or PBS- related

pretreatments dramatically increased kdeg of Co(salophen) composite electrodes and

reduced the overpotential by 100 mV Fe(salophen) composite electrodes were not

sufficiently catalytic without a double pretreatment with NaOH and H2SO4

Anthraquinone-grafted graphite composite electrodes were not catalytic at neutral pH

even after pretreatment Multi-copper oxidases were catalytic at DET however its RLS

was electron transfer between the electrode and the enzyme hence appropriate

mediators should be used to achieve higher catalysis

Many of pretreated Co(salophen) composite electrodes were strongly catalytic toward

ORR Among them NaOH-pretreated Co(slophen) electrodes were found to be most

catalytic at neutral pH with kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 nα = 083 and Epc2(HDV) =

- 037 V Its catalytic efficiency was close to that of Au in the acid solution

264

8 CONCLUSION AND FURTHER STUDIES

Miniaturised glucose-oxygen fuel cells are ideal to power small implantable

medical devices Typical specification include at least 1 microW power output and 1-year

operational life Therefore fuel cells must have efficient catalysis and electron transfer

good conductivity chemical and mechanical stability as well as biocompatibility The

electrodes must be feasible to build and scaleable In this study electrodes were built

using bulk modification method because it was simple to construct and scale-up and

they were easy to modify and mechanically stable The anodes were built with 10

GOx with 77 TTF-TCNQ composite and the cathodes were built with graphite

composite containing ORR catalysts using bulk modification method TTF-TCNQ was

used because it was conductive as well as mediating and necessary for electron transfer

of the GOx anodes Problems were enzyme stability and ORR catalytic efficiency

Enzyme mass loading is known to be effective to improve catalytic efficiency and

operational life however this also decreases conductivity of the composite electrodes

The key to optimising enzyme electrodes is in the balance and compromise among the

conductivity mediation enzyme activity mechanical and chemical stability and

difficulty in the fabrication process CNF composites showed reasonable conductivity at

30 content and this might allow higher enzyme loading with mediators without losing

conductivity CNT is also a candidate material because it has comparable or larger

conductivity than TTF-TCNQ and can operate without added mediator species Ideally

enzyme electrodes should be functional with minimum components simple to build

renew and store in dry so that they become easily portable Moreover when they are

further miniaturised this will allow the medical application under harsher conditions so

265

that powering disposable implantable sensors in economically-deprived regions might

become possible Many physiological enzymes however easily lose catalytic activity

outside the optimal condition Incorporation of enzyme stabilisers makes the enzyme

robust and long lasting as well as scavenging reactive species In the glucose-oxygen

fuel cells O2 has been known to reduce the catalytic output current of GOx anodes

PEI-DTT stabiliser mixtures were found to be good for this because they conferred

stability on GOx against oxidative stress prevented O2 electron deprivation and

improved the catalytic current output It was however a smaller effect than when

conductive hydrogel was used Conductive hydrogel can be a good candidate if it

becomes resistant against degradation ORR at the cathode has posed a long-term

problem in fuel cells because ORR is inefficient and consumes potential generated

within the fuel cell Pt has been used as an ORR catalyst but it is expensive and is

becoming scarce Use of the fuel cell in the physiological condition also imposes further

operational limitations Pretreated metal complexes such as Co salophen were found to

be good candidates for ORR-catalytic cathodes Surrounding complexes with

electron-rich environment such as conductive film might improve ORR further Once

candidate anode and cathode are identified and built a biofuel cell unit can be made To

increase the power each cell is connected together and made into a stack The prototype

was made as below in Fig 124 by connecting twenty cell units

266

Fig 124 Fuel cell stack made with various GOx anodes and Co(salophen) cathodes

Each cell contained various GOx anodes and pretreated Co(salophen) electrode in 10 ml

of oxygenated 01 M glucose 001 M PBS solution pH 74 The output voltage was

found to be 02 V and it did not change for one week The fuel cell must be studied

further with an adjustable resistor for voltage-current (V-I) and power-current (P-I)

curves so that OCV can be determined properly The passive type GOx fuel cell could

output 15 mWcm-2 at 03 V [18]

In vivo studies are also necessary for implantable purposes to examine the catalytic

efficiency electrode fouling and biocompatibility This can be studied systematically by

changing the type and concentration of interfering substances such as ascorbate

acetoaminophen and urate [18] Heller tested miniturised GOx-BOD fuel cells tested in

the buffer grape and calf serum It showed only 5 power decrease in buffer however

50 drop within 20 hours in grape and 50 drop within 2 hours in calf serum [19] To

avoid electrode fouling the electrode surface can be coated with a membrane such as

267

Nafion However this imposes another problem because it can interfere with the

reactant flux to the electrode surface In the real physiological system this might cause

a serious problem because glucose and O2 concentration is much lower than in vitro

Furthermore in the physiological conditions active transport of the reactant is not

available and this will cause local concentration polarisation It seems that there are

many obstacles lying in the medical application of miniaturised glucose-oxygen fuel

cells although they have widespread potentials applications

268

9 BIBLIOGRAPHY

1 Carrette L KA Friedrich and U Stimming Fuel cells Principles types fuels and applications

Chemphyschem 2000 1(4) p 162-193

2 Gregoire Padro CE and JO Keller Hydrogen Energy in Kirk-Othmer Encyclopedia of Chemical

Technology 2005 John Wiley amp Sons Inc p 837-866

3 Ohta K T Kazama and M Nishizawa All about fuel cells in Newton 2006 p 31-63

4 Tanabe S Mastering basics of the fuel cells 2009 Denkishoin

5 Tokyo-Gas Fuel cell academy 2009 2901 [cited Available from httpwwwtokyo-gascojppefc

6 Cropper MAJ S Geiger and DM Jollie Fuel cells a survey of current developments Journal of Power

Sources 2004 131(1-2) p 57-61

7 Kordesch K et al Intermittent use of a low-cost alkaline fuel cell-hybrid system for electric vehicles

Journal of Power Sources 1999 80(1-2) p 190-197

8 UTC-Power The fuel cell for Apollo 11

9 Connor S Warning Oil supplies are running out fast in The Independent 2009

10 Mori Y Mobile fuel cell providing new advantages for mobile electronic appliances Toshiba review 2007

62(6) p 44-47

11 Mori H Electronic components and materials Toshiba review 2009 64(3) p 62-63

12 Imperial-College-London Racing Green - Vehicles 2009 [cited Available from

httpwwwunionicacukrccracinggreenvehicleshtml

13 Cleave V Formula Zero kart race could drive fuel cell technology New Scientist 2008 [cited

Available from

httpwwwnewscientistcomarticledn14596-formula-zero-kart-race-could-drive-fuel-cell-technologyhtml

14 HyNor Hydrogen highway opens in Norway 2009 [cited Available from

httpwwwhynornohydrogen-highway-opens-in-norway

15 BMW Hydrogen 7 - The future is closer than you think 2009 [cited Available from

httpwwwbmwcomcomeninsightstechnologyefficient_dynamicsphase_2clean_energybmw_hydroge

n_7html

httpwwwbmwcojpjpjabrandtechnologycleanenergywallpaperhtml

16 Sony Side view - Sony developes Bio Battery generating electricity from sugar 2007 Sony

17 Burton N Sugar-powered electronics in RSC Chemical technology news 2008

18 Sakai H A high-power glucoseoxygen biofuel cell operating under quiescent conditions Energy amp

Environmental Science 2009 2(1) p 133-138

19 Heller A Miniature biofuel cells Physical Chemistry Chemical Physics 2004 6(2) p 209-216

20 Toshiba Toshiba announces worlds smallest direct methanol fuel cell with energy output of 100 milliwatts

269

2004 [cited Available from httpwwwtoshibacojpaboutpress2004_06pr2401htm

21 Osakai T K Kanoh and S Kuwabata Basic Electrochemistry 2007 Kagakudojin Ltd Kyoto Japan

22 Wilson K and J Walker Practical Biochemistry 1992 Cambridge UK Cambridge University Press

23 Murray RK et al Harpers Biochemistry 25 ed 2000 Stamford Connecticut Appleton amp Lange

24 Sawyer DT A Sobkowiak and JL Roberts Jr Electrochemistry for chemists Revised ed 1995

Wiley-Interscience

25 IUPAC IUPAC GOLD BOOK 2009 [cited Available from httpgoldbookiupacorgindexhtml

26 Kitamura T and K Fujisawa English-Japanese The students dictionary of physics 1995 Tokyo ALC

Inc

27 Gates BC Catalysis in Kirk-Othmer Encyclopedia of Chemical Technology 2002 John Wiley amp Sons p

200-254

28 Taguchi S and E Nakano Kinetic Study of Bromination of Malonic Acid Methyl Malonic Acid

and Ethyl Malonic Acid An Undergraduate Physical Chemistry Laboratory Experimentfor

Understanding of the Infuluence of Thermodynamic Parameter on Chemical Kinetics The Chemical

Education Jounal 1999 3(1) p 1-20

29 Pletcher D A First course in electrode processes 1991 The Electrochemical Consultancy (Romsey) Ltd

23 - 26 135 - 145

30 Bard AJ and LR Faulker Electrochemical Methods Fundamentals and Applications 2000 NY USA

John Wiley and Sons Inc

31 Daintith J A concise dictionary of chemistry 2 ed 1990 Oxford UK Oxford University Press

32 Tinoco IJ et al Physical Chemistry 2002 Prentice Hall p 157

33 Watanabe K and S Nakabayashi Introduction to electrochemistry The chemistry of electron transfer 13

ed 2005 Tokyo Japan The Chemical Society of Japan

34 Hamelers B et al Microbial fuel cells Methodology and technology Environmental science amp

technology 2006 40(17) p 5181-5192

35 Koike T Chapter 10 Free energy in Physical chemistry I amp II Lecture handouts for dept of pharmacy

2010 University of Hiroshima p 57-62

36 Lopes T E Gonzalez and E Antolini An overview of platinum-based catalysts as methanol-resistant

oxygen reduction materials for direct methanol fuel cells Journal of alloys and compounds 2008 461(1-2)

p 253-262

37 Vork FTA and E Barendrecht The reduction of dioxygen at polypyrrole-modified electrodes with

incorporated Pt particles Electrochimica Acta 1990 35(1) p 135-139

38 Marino W B Scharifker and A Leone ELECTRODEPOSITION AND

ELECTROCHEMICAL-BEHAVIOR OF PALLADIUM PARTICLES AT POLYANILINE AND

POLYPYRROLE FILMS Journal of the Electrochemical Society 1992 139(2) p 438-443

39 Tsakova V How to affect number size and location of metal particles deposited in conducting polymer

270

layers Journal of Solid State Electrochemistry 2008 12(11) p 1421-1434

40 Lemest Y et al Dioxygen Fixation by a Mixed-Valence Dicobalt Cofacial Porphyrin Journal of the

Chemical Society-Chemical Communications 1983(22) p 1286-1287

41 Kim E et al Superoxo micro-peroxo and micro-oxo complexes from hemeO2 and heme-CuO2 reactivity

Copper ligand influences in cytochrome c oxidase models 2003 p 3623-3628

42 Fukuzumi S et al Mechanism of Four-Electron Reduction of Dioxygen to Water by Ferrocene

Derivatives in the Presence of Perchloric Acid in Benzonitrile Catalyzed by Cofacial Dicobalt Porphyrins

Journal of the American Chemical Society 2004 126(33) p 10441-10449

43 Solomon EI et al O2 and N2O Activation by Bi- Tri- and Tetranuclear Cu Clusters in Biology

Accounts of Chemical Research 2007 40(7) p 581-591

44 Wildgoose GG et al Anthraquinone-derivatised carbon powder reagentless voltammetric pH electrodes

Talanta 2003 60(5) p 887-893

45 McCreery R Carbon electrodes Structural effects on electron transfer kinetics in Electroanalytical

Chemistry A series advances 1990 Marcel Dekker Inc p 221-374

46 Kneten K R McCreery and C McDermott ELECTRON-TRANSFER KINETICS OF AQUATED

FE+3+2 EU+3+2 AND V+3+2 AT CARBON ELECTRODES - INNER-SPHERE CATALYSIS BY

SURFACE OXIDES Journal of the Electrochemical Society 1993 140(9) p 2593-2599

47 Engstrom RC ELECTROCHEMICAL PRETREATMENT OF GLASSY-CARBON ELECTRODES

Analytical Chemistry 1982 54(13) p 2310-2314

48 Lawrence E Hendersons dictionary of biological terms 10 ed 1989 UK Longman group UK Ltd

49 Eijsink V Directed evolution of enzyme stability Biomolecular engineering 2005 22(1-3) p 21-30

50 Pantoliano M et al PROTEIN ENGINEERING OF SUBTILISIN BPN - ENHANCED STABILIZATION

THROUGH THE INTRODUCTION OF 2 CYSTEINES TO FORM A DISULFIDE BOND Biochemistry

1987 26(8) p 2077-2082

51 Bagotzky VS NV Osetrova and AM Skundin Fuel cells State-of-the-art and major scientific and

engineering problems Russian Journal of Electrochemistry 2003 39(9) p 919-934

52 Heller A Integrated medical feedback systems for drug delivery Aiche Journal 2005 51(4) p

1054-1066

53 Nakamura M et al High-performance ultrathin solid oxide fuel cells for low-temperature operation

Journal of the Electrochemical Society 2007 154(1) p B20-B24

54 Lapedes DN McGraw-Hill Dictionary of Physics and Mathematics in McGraw-Hill 1978 McGraw-Hill

Inc

55 Svoboda V et al Enzyme catalysed biofuel cells Energy amp Environmental Science 2008 1(3) p

320-337

56 Barton SC J Gallaway and P Atanassov Enzymatic biofuel cells for Implantable and microscale devices

Chemical Reviews 2004 104(10) p 4867-4886

271

57 Schroder U Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency

Physical chemistry chemical physics 2007 9(21) p 2619-2629

58 Chaudhuri SK and DR Lovley Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells Nature Biotechnology 2003 21(10) p 1229-1232

59 Vincent K F Armstrong and J Cracknell Enzymes as working or inspirational electrocatalysts for fuel

cells and electrolysis Chemical reviews 2008 108(7) p 2439-2461

60 Tsujimura S and K Kano Recent development of enzyme-based biofuel cells GS Yuasa technical report

2008 5(2) p 1-6

61 Cass AEG Biosensors A Practical Approach 1990 Oxford University Press Oxford UK p 49-53

62 Palmore GTR and HH Kim Electro-enzymatic reduction of dioxygen to water in the cathode

compartment of a biofuel cell Journal of Electroanalytical Chemistry 1999 464(1) p 110-117

63 Bullen RA et al Biofuel cells and their development Biosensors amp Bioelectronics 2006 21(11) p

2015-2045

64 Bertschy H et al A methanoldioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as

catalysts application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at

low overpotentials Journal of electroanalytical chemistry and interfacial electrochemistry 1998 443(1) p

155-161

65 Bartlett PN P Tebbutt and RG Whitaker Kinetic aspects of the use of modified electrodes and

mediators in bioelectrochemistry Progress in reaction kinetics 1991 16(2) p 55-155

66 Kano K T Ikeda and S Tsujimura GlucoseO-2 biofuel cell operating at physiological conditions Denki

kagaku oyobi kōgyō butsuri kagaku 2002 70(12) p 940-942

67 Heller A Electrochemical glucose sensors and their applications in diabetes management Chemical

Reviews 2008 108(7) p 2482-2505

68 Ilankumaran V et al Trends in cardiac pacemaker batteries Indian pacing and electrophysiology journal

2004 4(4) p 201-12

69 Schlindwein W and R Linford Medical applications of solid state lonics Solid State Ionics 2006

177(19-25) p 1559-1565

70 Latham R R Linford and W Schlindwein Biomedical applications of batteries Solid State Ionics 2004

172(1-4) p 7-11

71 Soykan O Power sources for implantable medical devices Business Briefing Medical Device

Manufacturing and Technology London 2002

72 Heller A Potentially implantable miniature batteries Analytical and Bioanalytical Chemistry 2006

385(3) p 469-473

73 Taniguchi M Achilles heel of the next-generation electric vehicles - Lithium World map of the resrouce

wars 2008 [cited 2009 Available from

httpbusinessnikkeibpcojparticlemanage20080407152450P=1

272

74 Yeatman EM Advances In Power Sources For Wireless Sensor Nodes International Workshop on Body

Sensor Networks 2004

75 Sato N et al Novel MEMS power generator with integrated thermoelectric and vibrational devices in

Solid-State Sensors Actuators and Microsystems 2005 Digest of Technical Papers TRANSDUCERS 05

The 13th International Conference on 2005

76 Hudak NS and GG Amatucci Small-scale energy harvesting through thermoelectric vibration and

radiofrequency power conversion Journal of Applied Physics 2008 103(10)

77 Seiko-Instruments SEIKO Worlds First 2009 [cited Available from

httpwwwseikowatchescomheritageworlds_firsthtml

78 Kishi M et al Micro thermoelectric modules and their application to wristwatches as an energy source in

Thermoelectrics 1999 Eighteenth International Conference on 1999

79 Lee J et al Ionic conduction in Zn-3(PO4)(2)()4H(2)O enables efficient discharge of the zinc anode in

serum Journal of the American Chemical Society 2005 127(42) p 14590-14591

80 Maxell Button battery table 2009 [cited Available from

httpwwwmaxellcojpproductsmaterialsbutton_batteryhtml

81 Wilson R and APF Turner Glucose-Oxidase - an Ideal Enzyme Biosensors amp Bioelectronics 1992 7(3)

p 165-185

82 Swoboda BEP The relationship between molecular conformation and the binding of flavin-adenine

dinucleotide in glucose oxidase Biochimica et Biophysica Acta (BBA) - Protein Structure 1969 175(2) p

365-379

83 Wohlfahrt G et al 18 and 19 angstrom resolution structures of the Penicillium amagasakiense and

Aspergillus niger glucose oxidases as a basis for modelling substrate complexes Acta Crystallographica

Section D-Biological Crystallography 1999 55 p 969-977

84 Patel RN Biocatalysis in the pharmaceutical and biotechnology industries 1st ed 2006 CRC Press

85 Gouda M Reversible denaturation behavior of immobilized glucose oxidase Applied Biochemistry and

Biotechnology 2002 102 p 471-480

86 Hecht HJ et al Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 3 Angstrom

Resolution Journal of Molecular Biology 1993 229(1) p 153-172

87 Zubrik A et al Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the

transition to the denatured state with residual structure The Journal of biological chemistry 2004 279(46)

p 47601-47609

88 Kojima S et al Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose

biosensor Bioscience biotechnology and biochemistry 2006 70(3) p 654-659

89 Duine J The PQQ story Journal of bioscience and bioengineering 1999 88(3) p 231-236

90 Kyoto-University KEGG - Kyoto Encyclopedia of Genes and Genomes 2009 Kyoto University

91 Delbem MF WJ Baader and SHP Serrano Mechanism of 34-dihydroxybenzaldehyde

273

electropolymerization at carbon paste electrodes catalytic detection of NADH 2002 scielo p 741-747

92 Zhao S et al Electrochemistry of Organic Conducting Salt Electrodes - a Unified Mechanistic

Description Journal of Physical Chemistry 1992 96(13) p 5641-5652

93 Lemuel BW Jr CC Liu and LN Niren Electrochemical measurements with glucose oxidase

immobilized in polyacrylamide gel Constant current voltametry 1971 p 629-639

94 Sato A et al Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Biochemical Journal

2001 357 p 893-898

95 Sigma-Aldrich Glucose Oxidase from Aspergillus niger Type II-S 15000-50000 unitsg solid (without

added oxygen) 2009 [cited

96 Cosnier S Biosensors based on electropolymerized films new trends Analytical and Bioanalytical

Chemistry 2003 377(3) p 507-520

97 Willner I et al Integrated Enzyme-Based Biofuel Cells-A Review Fuel Cells 2009 9(1) p 7-24

98 Heleg-Shabtai V et al Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers

assembled onto Au-electrodes Journal of the American Chemical Society 1996 118(42) p 10321-10322

99 Georganopoulou DG et al Development and comparison of biosensors for in-vivo applications Faraday

Discussions 2000 p 291-303

100 Fujii S et al Properties of glucose sensors prepared by the electropolymerization of pyrrole containing a

gluconyl group Analytical sciences 1999 15(12) p 1175-1176

101 Hoshi T Deposition of electron mediators by electrochemical polymerization Electrochemistry 2003

71(5) p 346-347

102 Arai G M Masuda and I Yasumori Direct Electrical Response between Glucose-Oxidase and

Poly(Mercapto-P-Benzoquinone) Films Bulletin of the Chemical Society of Japan 1994 67(11) p

2962-2966

103 Arai G K Shoji and I Yasumori Electrochemical characteristics of glucose oxidase immobilized in

poly(quinone) redox polymers Journal of Electroanalytical Chemistry 2006 591(1) p 1-6

104 Leonida MD et al Polymeric FAD used as enzyme-friendly mediator in lactate detection Analytical and

Bioanalytical Chemistry 2003 376(6) p 832-837

105 Kissinger PT and WR Heineman Laboratory techniques in electroanalytical chemistry 1996 Marcel

Dekker Inc NY USA p 294-332

106 OHare D and H Zhao Characterisation and modeling of conducting composite electrodes The journal of

physical chemistry C 2008 112(25) p 9351-9357

107 Wang J et al Metal-Dispersed Carbon Paste Electrodes Analytical Chemistry 1992 64(11) p

1285-1288

108 Khurana MK CP Winlove and D OHare Detection mechanism of metallized carbon epoxy oxidase

enzyme based sensors Electroanalysis 2003 15(12) p 1023-1030

109 Sasaki M et al Fabrication and Electrical Characterization of

274

Tetrathiafulvalene-tetracyanoquinodimethane Molecular Wires Japanese Journal of Applied Physics 2003

42 p 2488-2491

110 Iizuka M et al Highly oriented TTF-TCNQ crystal growth using an electric-field induced evaporation

technique Molecular crystals and liquid crystals science and technology Section A Molecular crystals and

liquid crystals 2000 349 p 367-370

111 Saito I and K Yoshida Development of new features on organic conductive material TCI mail 2005

127

112 Cass AEG et al FERROCENE-MEDIATED ENZYME ELECTRODE FOR AMPEROMETRIC

DETERMINATION OF GLUCOSE Analytical Chemistry 1984 56(4) p 667-671

113 Kulys J V Simkeviciene and IJ Higgins Concerning the Toxicity of 2 Compounds Used as Mediators in

Biosensor Devices - 7788-Tetracyanoquinodimethane (Tcnq) and Tetrathiafulvalene (Ttf) Biosensors amp

Bioelectronics 1992 7(7) p 495-501

114 Malinauskas A et al Conductive organic complex salt TTF-TCNQ as a mediator for biosensors An

overview Electroanalysis 2007 19(24) p 2491-2498

115 Llopis X et al Integration of a glucose biosensor based on an epoxy-graphite-TTF center dot

TCNQ-GOD biocomposite into a FIA system Sensors and Actuators B-Chemical 2005 107(2) p 742-748

116 Bourgeois J D Thomas and C Bourdillon COVALENT LINKAGE OF GLUCOSE-OXIDASE ON

MODIFIED GLASSY-CARBON ELECTRODES - KINETIC PHENOMENA Journal of the American

Chemical Society 1980 102(12) p 4231-4235

117 Yun Y et al Nanotube electrodes and biosensors Nano Today 2007 2(6) p 30-37

118 Kim BC et al Preparation of biocatalytic nanofibres with high activity and stability via enzyme

aggregate coating on polymer nanofibres Nanotechnology 2005 16(7) p S382-S388

119 Kim J and JW Grate Single-Enzyme Nanoparticles Armored by a Nanometer-Scale OrganicInorganic

Network 2003 p 1219-1222

120 OFagain C Enzyme stabilization - recent experimental progress Enzyme and Microbial Technology

2003 33(2-3) p 137-149

121 Sillery E et al Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a

platinum catalyst Chemical communications 2002(8) p 866-867

122 Gouda M et al Enhancement of stability of immobilized glucose oxidase by modification of free thiols

generated by reducing disulfide bonds and using additives Biosensors amp bioelectronics 2004 19(6) p

621-625

123 Manning M STABILITY OF PROTEIN PHARMACEUTICALS Pharmaceutical Research 1989 6(11) p

903-918

124 Cao L Immobilised enzymes science or art Current opinion in chemical biology 2005 9(2) p 217-226

125 Daniel R The role of dynamics in enzyme activity Annual review of biophysics and biomolecular

structure 2003 32 p 69-92

275

126 Andersson MM JD Breccia and R Hatti-Kaul Stabilizing effect of chemical additives against oxidation

of lactate dehydrogenase Biotechnology and Applied Biochemistry 2000 32 p 145-153

127 Souza JM and R Radi Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Archives of Biochemistry and Biophysics 1998 360(2) p 187-194

128 Andersson MA and R Hatti-Kaul Protein stabilising effect of polyethyleneimine Journal of

Biotechnology 1999 72(1-2) p 21-31

129 Sigma-Aldrich Poly(ethylenimine) 2009 [cited Available from

httpwwwsigmaaldrichcomstructureimages27mfcd00084427gif

130 Andersson MM R Hatti-Kaul and W Brown Dynamic and static light scattering and fluorescence

studies of the interactions between lactate dehydrogenase and poly(ethyleneimine) Journal of Physical

Chemistry B 2000 104(15) p 3660-3667

131 Wang J L Chen and J Liu Critical comparison of metallized and mediator-based carbon paste glucose

biosensors Electroanalysis 1997 9(4) p 298-301

132 Cleland WW Dithiothreitol a New Protective Reagent for SH Groups Biochemistry 1964 3(4) p

480-482

133 Atanasov P E Wilkins and Q Yang An integrated needle-type biosensor for intravascular glucose and

lactate monitoring Electroanalysis 1998 10(11) p 752-757

134 Sigma-Aldrich DL-Dithiothreitol 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=43815|SIGMAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

135 Ghisalba O and A Brunella Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate

reductase as biocatalyst in the preparative synthesis of 2 -deoxyribonucleoside-5 -triphosphates Journal

of molecular catalysis B Enzymatic 2000 10(1-3) p 215-222

136 Gollhofer D EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM

ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Enzyme and Microbial Technology 1995 17(3) p 235-240

137 Sigma-Aldrich Arsenite standard solution - 005 M in H2O 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=70039|FLUKAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

138 Arakawa T MECHANISM OF POLY(ETHYLENE GLYCOL) INTERACTION WITH PROTEINS

Biochemistry 1985 24(24) p 6756-6762

139 Lee C Improvement of protein stability in protein microarrays Biotechnology Letters 2002 24(10) p

839-844

140 Lee L THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE GLYCOLS)

Biochemistry 1987 26(24) p 7813-7819

141 Costa SA et al Studies of stabilization of native catalase using additives Enzyme and Microbial

276

Technology 2002 30(3) p 387-391

142 Lee J and L Lee THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE

GLYCOLS) Biochemistry 1987 26(24) p 7813-7819

143 Gekko K and SN Timasheff Mechanism of protein stabilization by glycerol preferential hydration in

glycerol-water mixtures Biochemistry 1981 20(16) p 4667-4676

144 Leandro P Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes

produced in a prokaryotic expression system Molecular genetics and metabolism 2001 73(2) p 173-178

145 Bartlett PN VQ Bradford and RG Whitaker Enzyme Electrode Studies of Glucose-Oxidase Modified

with a Redox Mediator Talanta 1991 38(1) p 57-63

146 Joseph S et al Drug metabolism biosensors electrochemical reactivities of cytochrome P450(cam)

immobilised in synthetic vesicular systems Journal of Pharmaceutical and Biomedical Analysis 1998

17(6-7) p 1101-1110

147 Cornish-Bowden A Fundamentals of enzyme kinetics 3rd ed 2004 London Portland Press Ltd

148 Wilkinson G STATISTICAL ESTIMATIONS IN ENZYME KINETICS The Biochemical journal 1961

80(2) p 324

149 Ikeda T and K Kano Fundamentals and practices of mediated bioelectrocatalysis Analytical sciences

2000 16(10) p 1013-1021

150 Duggleby RG and LP Daniel [3] Analysis of enzyme progress curves by nonlinear regression in

Methods in Enzymology 1995 Academic Press p 61-90

151 Eisenthal R and A Cornish-Bowden DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR

ESTIMATING ENZYME KINETIC-PARAMETERS The Biochemical journal 1974 139(3) p 715-720

152 Baici A Enzyme kinetics the velocity of reactions Biochem J 2006 p 1-3

153 Yamazaki A et al Improvement of precision in pharmacological studies using the non-linear regression

method

estimation of enzyme inhibition constants as an example Folia Pharmacologica Japonica 2008 131(3) p 195-203

154 Cornish-Bowden A Teaching Enzyme Kinetics and Mechanism in the 21st Century in 3rd Symposium

on ESCES 2008 Rudensheim Germany

155 Weisshaar DE and T Kuwana Considerations for Polishing Glassy-Carbon to a Scratch-Free Finish

Analytical Chemistry 1985 57(1) p 378-379

156 Stedman Stedmans medical dictionary 25th ed 2002 Lippincott Williams amp Wilkins

157 Tamura T Buffer preparation for biology Buffer preparation for biology ed 6 2008 Tokyo Yodosha

Ltd

158 Battino R and HL Clever The Solubility of Gases in Liquids 1966 p 395-463

159 Pandolfo A and A Hollenkamp Carbon properties and their role in supercapacitors Journal of power

sources 2006 157(1) p 11-27

160 Maynard AD Nanotechnology assessing the risks Nano Today 2006 1(2) p 22-33

277

161 Hindle A E Hall and N Martens AN ASSESSMENT OF MEDIATORS AS OXIDANTS FOR

GLUCOSE-OXIDASE IN THE PRESENCE OF OXYGEN Biosensors amp bioelectronics 1995 10(3-4) p

393-403

162 Mo J et al Comparison of oxygren-rich and mediator-based glucose-oxidase carbon-paste electrodes

Analytica Chimica Acta 2001 441(2) p 183-189

163 Turner A and G Palleschi AMPEROMETRIC TETRATHIAFULVALENE-MEDIATED LACTATE

ELECTRODE USING LACTATE OXIDASE ABSORBED ON CARBON FOIL Analytica Chimica Acta

1990 234(2) p 459-463

164 Mizutani F et al Glucose-Sensing Electrode Based on Carbon Paste Containing Ferrocene and

Polyethylene Glycol-Modified Enzyme Bulletin of the Chemical Society of Japan 1991 64(9) p

2849-2851

165 Zhang J PEM Fuel Cell Electrocatalysts and Catalyst Layers Fundamentals and Applications 2008

London Springer-Verlag

166 Kinoshita K Electrochemical oxygen technology 1992 John Willey amp Sons Inc

167 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of Power

Sources 2005 152 p 1-15

168 Sawyer DT Oxygen Chemistry International series of monographs on chemistry 1991 Oxford

University Press

169 Winter M Chemical Bonding Oxford Chemistry Primers 1997 New York Oxford University Press Inc

170 McLendon G and AE Martell Inorganic Oxygen Carriers as Models for Biological-Systems

Coordination Chemistry Reviews 1976 19(1) p 1-39

171 Toda T et al Enhancement of the electroreduction of oxygen on Pt alloys with Fe Ni and Co Journal of

the Electrochemical Society 1999 146(10) p 3750-3756

172 Norskov JK et al Origin of the overpotential for oxygen reduction at a fuel-cell cathode Journal of

Physical Chemistry B 2004 108(46) p 17886-17892

173 Fischer P and J Heitbaum MECHANISTIC ASPECTS OF CATHODIC OXYGEN REDUCTION Journal

of Electroanalytical Chemistry 1980 112(2) p 231-238

174 Okada T et al Oxygen reduction characteristics on pyrolytic graphite electrodes modified with

electro-polymerized salen compounds Chemistry Letters 1998(8) p 841-842

175 Sakurai T and K Kataoka Basic and applied features of multicopper oxidases CueO bilirubin oxidase

and laccase 2007 p 220-229

176 Sanders J Supramolecular catalysis in transition Chemistry a European journal 1998 4(8) p

1378-1383

177 Hart H LE Craine and DJ Hart Organic Chemistry A Short Course 10 ed 1998 Houghton Miffin

Company USA

178 Hayatsu H CELLULOSE BEARING COVALENTLY LINKED COPPER PHTHALOCYANINE

278

TRISULPHONATE AS AN ADSORBENT SELECTIVE FOR POLYCYCLIC COMPOUNDS AND ITS USE

IN STUDIES OF ENVIRONMENTAL MUTAGENS AND CARCINOGENS Journal of chromatography A

1992 597(1-2) p 37-56

179 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of power

sources 2005 152(1) p 1-15

180 Kingsborough RP and TM Swager Electrocatalytic Conducting Polymers Oxygen Reduction by a

Polythiophene-Cobalt Salen Hybrid Chemistry of Materials 2000 12(4) p 872-874

181 Ortiz B and SM Park Electrochemical and spectroelectrochemical studies of cobalt salen and salophen

as oxygen reduction catalysts Bulletin of the Korean Chemical Society 2000 21(4) p 405-411

182 Mano N et al An oxygen cathode operating in a physiological solution Journal of the American

Chemical Society 2002 124(22) p 6480-6486

183 Shleev S et al Direct electron transfer between copper-containing proteins and electrodes Biosensors

and Bioelectronics 2005 20(12) p 2517-2554

184 Solomon EI et al Oxygen binding activation and reduction to water by copper proteins Angewandte

Chemie-International Edition 2001 40(24) p 4570-4590

185 Heath R F Armstrong and C Blanford A stable electrode for high-potential electrocatalytic O-2

reduction based on rational attachment of a blue copper oxidase to a graphite surface Chemical

communications 2007(17) p 1710-1712

186 Miura Y et al Direct Electrochemistry of CueO and Its Mutants at Residues to and Near Type I Cu for

Oxygen-Reducing Biocathode Fuel cells 2009 9(1) p 70-78

187 Tarasevich MR VA Bogdanovskaya and LN Kuznetsova Bioelectrocatalytic reduction of oxygen in

the presence of laccase adsorbed on carbon electrodes Russian Journal of Electrochemistry 2001 37(8) p

833-837

188 Tsujimura S et al Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase

at carbon electrodes Electrochemistry 2004 72(6) p 437-439

189 Fernandez JL et al Optimization of wired enzyme O-2-electroreduction catalyst compositions by

scanning electrochemical microscopy Angewandte Chemie-International Edition 2004 43(46) p

6355-6357

190 Soukharev V N Mano and A Heller A four-electron O-2-electroreduction biocatalyst superior to

platinum and a biofuel cell operating at 088 V Journal of the American Chemical Society 2004 126(27)

p 8368-8369

191 Miura Y et al Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on

a highly oriented pyrolytic graphite electrode Chemistry Letters 2007 36(1) p 132-133

192 Seinberg JM et al Spontaneous modification of glassy carbon surface with anthraquinone from the

solutions of its diazonium derivative An oxygen reduction study Journal of Electroanalytical Chemistry

2008 624(1-2) p 151-160

279

193 Manisankar P and A Gomathi Electrocatalytic reduction of dioxygen at the surface of carbon paste

electrodes modified with 910-anthraquinone derivatives and dyes Electroanalysis 2005 17(12) p

1051-1057

194 Jurmann G DJ Schiffrin and K Tammeveski The pH-dependence of oxygen reduction on

quinone-modified glassy carbon electrodes Electrochimica Acta 2007 53(2) p 390-399

195 Abiman P R Compton and G Wildgoose Characterising chemical functionality on carbon surfaces

Journal of materials chemistry 2009 19(28) p 4875-4886

196 Thorogood CA et al Differentiating between ortho- and para-Quinone Surface Groups on Graphite

Glassy Carbon and Carbon Nanotubes Using Organic and Inorganic Voltammetric and X-ray

Photoelectron Spectroscopy Labels Chemistry of Materials 2007 19(20) p 4964-4974

197 Sigma-Aldrich Fast red AL salt 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildoN4=380660|SIALampN5=SEARCH_CONCAT_PN

O|BRAND_KEYampF=SPEC

198 Sljukic B C Banks and R Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

199 Granger M et al Polycrystalline diamond electrodes basic properties and applications as amperometric

detectors in flow injection analysis and liquid chromatography Analytica Chimica Acta 1999 397(1-3) p

145-161

200 Compton R J Foord and F Marken Electroanalysis at diamond-like and doped-diamond electrodes

Electroanalysis 2003 15(17) p 1349-1363

201 Arima S Do Science - Why diamond is so hard in Asahicom 2006

202 Dumitrescu I P Unwin and J Macpherson Electrochemistry at carbon nanotubes perspective and issues

Chemical communications 2009(45) p 6886-6901

203 Kinoshita K Carbon Electrochemical and Physicochemical Properties 1988 Wiley-Interscience

204 Kastening B et al Electronic properties and double layer of activated carbon Electrochimica Acta 1997

42(18) p 2789-2799

205 Swain G and R Ramesham THE ELECTROCHEMICAL ACTIVITY OF BORON-DOPED

POLYCRYSTALLINE DIAMOND THIN-FILM ELECTRODES Analytical chemistry 1993 65(4) p

345-351

206 Kaneto K et al Electrical conductivities of multi-wall carbon nano tubes Synthetic metals 1999

103(1-3) p 2543-2546

207 Meyer J et al Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes Nano

letters 2008 8(11) p 3582-3586

208 Harris P New perspectives on the structure of graphitic carbons Critical Reviews in Solid State and

Materials Sciences 2005 30(4) p 235-253

209 Akasaka T Chemical illustration - Carbon materials 2007 Hokkaido Japan

280

210 Iijima S M Yudasaka and F Nihey Carbon nanotube technology NEC Technical Journal 2007 2(1) p

52-56

211 Day T N Wilson and J Macpherson Electrochemical and conductivity measurements of single-wall

carbon nanotube network electrodes Journal of the American Chemical Society 2004 126(51) p

16724-16725

212 Fujita H A TEM image of the atomic structure of the diamond surface 2010 Britannica - The Online

Encyclopedia Osaka Japan

213 McCreery RL et al Control of reactivity at carbon electrode surfaces Colloids and Surfaces A

Physicochemical and Engineering Aspects 1994 93 p 211-219

214 Rice R C Allred and R McCreery FAST HETEROGENEOUS ELECTRON-TRANSFER RATES FOR

GLASSY-CARBON ELECTRODES WITHOUT POLISHING OR ACTIVATION PROCEDURES Journal of

electroanalytical chemistry and interfacial electrochemistry 1989 263(1) p 163-169

215 Swain Electrochemical formation of high surface area carbon fibers Analytical chemistry 1991 63(5) p

517-519

216 Nagaoka T and T Yoshino Surface-Properties of Electrochemically Pretreated Glassy-Carbon Analytical

Chemistry 1986 58(6) p 1037-1042

217 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 2002 58(9) p 1953-1955

218 Motta N and AR Guadalupe Activated Carbon-Paste Electrodes for Biosensors Analytical Chemistry

1994 66(4) p 566-571

219 Leung E et al A novel in vivo nitric oxide sensor Chemical Communications 1996(1) p 23-24

220 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 1986 58(9) p 1953-1955

221 Kissinger PT and WR Heineman Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy

Current Separations 1989 9 p 15-18

222 Wang J Analytical Electrochemistry 3 ed 2006 Wiley-VCH

223 BAS Measurement of electron transfer kinetics using rotating disk voltammetry Electrocehmistry

Principles methods and applications 1995 2(43)

224 Miomandre F et al Electrochemical behaviour of iron(III) salen and poly(iron-salen) Application to the

electrocatalytic reduction of hydrogen peroxide and oxygen Journal of electroanalytical chemistry and

interfacial electrochemistry 2001 516(1-2) p 66-72

225 Brighton-University Inorganic Chemistry Practical C170 Salen lab script 2009 Dept of Pharmacy and

Biomolecular Sciences Brighton University Brighton

226 Ranganathan S TC Kuo and RL McCreery Facile Preparation of Active Glassy Carbon Electrodes

with Activated Carbon and Organic Solvents 1999 p 3574-3580

227 Ohare D CP Winlove and KH Parker Electrochemical method for direct measurement of oxygen

281

concentration and diffusivity in the intervertebral disc electrochemical characterization and tissue-sensor

interactions J Biomed Eng 1991 13(4) p 304-312

228 Chen PH and RL McCreery Control of electron transfer kinetics at glassy carbon electrodes by specific

surface modification Analytical Chemistry 1996 68(22) p 3958-3965

229 Beilby AL TA Sasaki and HM Stern Electrochemical Pretreatment of Carbon Electrodes as a

Function of Potential Ph and Time Analytical Chemistry 1995 67(5) p 976-980

230 Dai H and K Shiu Voltammetric studies of electrochemical pretreatment of rotating-disc glassy carbon

electrodes in phosphate buffer Journal of electroanalytical chemistry and interfacial electrochemistry 1996

419(1) p 7-14

231 Musameh M N Lawrence and J Wang Electrochemical activation of carbon nanotubes

Electrochemistry communications 2005 7(1) p 14-18

232 Sljukic B CE Banks and RG Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

233 Tammeveski K et al Surface redox catalysis for O-2 reduction on quinone-modified glassy carbon

electrodes Journal of Electroanalytical Chemistry 2001 515(1-2) p 101-112

234 Yang HH and RL McCreery Elucidation of the mechanism of dioxygen reduction on metal-free carbon

electrodes Journal of the Electrochemical Society 2000 147(9) p 3420-3428

235 Regisser F et al Randomly oriented graphite electrode 1 Effect of electrochemical pretreatment on the

electrochemical behavior and chemical composition of the electrode Journal of electroanalytical chemistry

and interfacial electrochemistry 1996 415(1-2) p 47-54

236 Shleev S et al Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic

graphite electrode Electrochemistry Communications 2004 6(9) p 934-939

237 Mano N F Mao and A Heller A miniature biofuel cell operating in a physiological buffer Journal of the

American Chemical Society 2002 124(44) p 12962-12963

3

ABSTRACT

Miniaturized glucose-oxygen biofuel cells are useful to power implantable

medical devices such as biosensors They are small more biocompatible and run

continuously on glucose and oxygen providing cleaner energy at neutral environment

A typical glucose-oxygen biofuel cell consists of an anode with glucose oxidase (GOx)

and a cathode with various oxygen reducing catalysts This thesis describes

experimental investigations of the major issues of such systems viz complex electrode

fabrication enzyme instability and inefficient oxygen reduction Electrodes were built

using the simple and scaleable bulk modification method where all the material was

simply mixed and bound together into composites with epoxy resin For the anodes the

composite made of 10 GOx with 77 TTF-TCNQ was found optimal The GOx

electrodes were modified with various enzyme stabilisers (PEI DTT PEG GLC FAD

and mixture of PEIDTT and PEIFAD) and 2 of PEI-DTT (11 ww) was most

effective in the presence of O2 Its maximum output current density was 18 x 10-2 plusmn 99

x 10-3 Am-2 It also showed the resistant against O2 electron deprivation and enzyme

inhibition Its KMwas 5 mM For the cathodes various oxygen reducing catalysts

(metalised carbon anthroquinone modified carbon laccase and bilirubin oxidase) were

incorporated into graphite composite and the electrodes were pretreated in different

media in order to enhance their catalytic activity None showed four-electron O2

reduction NaOH-pretreated cobalt (II) salophen composite electrodes showed

two-electron O2 reduction and were most catalytic Its standard catalytic rate constant

was 12 x 10-5 plusmn 12 x 10-6 ms-1 Of the catalysts examined metal complex composites

gave the best results for oxygen-reducing cathodes and their pretreatment led to the

4

synergetic effect because it increased the concentration of catalytic surface oxygen

groups and enhanced oxygen reduction

5

ありがとう Thank you Terima kashi ขอบคณ คะ Merci 고마워 謝謝 Danke

やったね

copy鳥

山

明

集

英

社

copy Akira Toriyama Shueisha Inc

6

ACKNOWLEDGEMENTS

I would like to express deepest gratitude to my supervisor Dr Danny OrsquoHare

for his guidance on and patience for my research Frankly speaking the project on

glucose-oxygen fuel cells was very challenging and I came to realise the hard reality of

creating new clean energy despite of the world huge demand for such energy in a time

of global warming I could not help but feel that the research was turning into a

wild-goose chase however there was much to learn and gain from the experience here

For that and the people I met during this time I feel deeply grateful for the opportunity

given and I am convinced that this will enlighten my life

I would like to specially thank Dr Pei Ling Leow and Mr Parinya Seelanan for

their sincere support understanding and encouragement Without their help I would not

have been able to achieve my PhD I would like to thank to the current and past

members of Biosensor group Ms Nitin Bagha Dr Christopher Bell Dr Eleni Bitziou

Mr Jin-Young Kim Mr Wei-Chih Lin Dr Beinn Muir Mr Raphael Trouillon Dr

Martyn Boutelle Mr Yao-Min Chang Dr Delphine Feuerstein Ms Michelle Rogers

Dr Costas Anastassiou Dr Arun Arora Dr Martin Arundell Dr Catherine Dobson Dr

Melanie Fennan Dr Severin Harvey Dr Alex Lindsay Dr Bhavik Patel Dr Hong

Zhao Dr Emma Corcoles and Dr Parastoo Hashemi for their support during research

and sharing the wonderful time for many occasions I would like to thank Mr David

Roche for his sincere devotion to our work and being a great lab company I would like

to give my warm thanks to Dr Andrew Bond Dr Stephanie Cremers Ms Christina

Hood Dr Asimina Kazakidi Dr Peter Knox Dr Bettina Nier Ms Eileen Su Mr

Che-Fai Yeong Mr Krause Oliver and Dr Clifford Talbot for appropriate support fun

7

and encouragement I would also like to thank Dr Kate Hobson Mr Martin Holloway

Mr Ken Keating Mr Alan Nyant Mr Richard Oxenham Ms Paula Smith Ms Dima

Cvetkova Dr Anna Thomas-Betts and Mr Nick Watkins for their academic and

laboratory support Finally I would like to say thank you to all my family for their trust

and unconditional support my teacher Prof Fukuyama my friends Dr Pieta

Nasanen-Gilmore Jingzi Kofi Mariama Masa Maya Miki Pedro Sandra Sascha and

Wei Fong for being always close supportive and encouraging even when we were apart

8

CONTENTS

ABSTRACT 3

ACKNOWLEDGEMENTS 6

CONTENTS 8

LIST OF ABBREVIATIONS 15

LIST OF FIGURES 20

LIST OF TABLES 26

LIST OF EQUATIONS 27

1 INTRODUCTION 31

11 INTRODUCTION TO FUEL CELLS 31

111 History of the fuel cells 31 112 Principles of the fuel cells 33 113 Electric power from the fuel cells 35

12 CATALYSTS 37

121 Faradayrsquos second law ndash Current and reaction rate37 122 Transition state theory Eyring equation and reaction rate 38 123 Overcoming the activation free energy 40 124 Variations in catalysts42

13 ELECTRODE POTENTIAL AND REACTION RATE 44

131 Fermi level electrode potential and electron transfer 44 132 Electrode potential equilibrium potential and Nernst equation47

9

133 Electrode potential and activation free energy 50 134 Potential shift and standard rate constant 52 135 Butler-Volmer equation overpotential and current 53 136 Butler-Volmer equation vs Tafel equation 55

14 OVERPOTENTIAL AND FUEL CELLS 57

141 Four overpotentials that decrease the EMF57 142 Internal current 58 143 Activation overpotential58 144 Diffusion overpotential 59 145 Resistance overpotential60 146 Total overpotential and cell voltage 62

15 BIOFUEL CELLS 63

151 Microbial and enzymatic fuel cells 63 152 Enzyme fuel cells and mediators64

16 MINIATURISED MEDICAL POWER SOURCES 66

161 Realisation of miniaturised fuel cells and medical power supply 66 162 Miniaturised potential medical power sources67

17 OUTLINE OF THE WORK 69

171 Introduction to miniaturised glucose-oxygen fuel cells 69 172 Outline71

2 ANODE INTRODUCTION 73

21 GLUCOSE OXIDATION ENZYMES 73

22 DESIGNING ENZYME ELECTRODES 77

221 Physical adsorption 78 222 Reconstituting apo-enzyme78 223 Tethered Osmium-complexes linked to conductive hydrogel 80 224 Entrapment in the conductive polymer 81 225 Bulk modification83

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties 84 226 Enzyme covalent attachment and mass loading 88

23 PROTEIN INACTIVATION AND STABILISATION 90

10

24 PROTEIN DESTABILISATION 92

241 Deamidation 92 242 Oxidation93 243 Proteolysis and racemisation94 244 Physical instabilities95

25 ENZYME STABILISERS 95

251 Polyehyleneimine (PEI) 96 252 Dithiothreitol (DTT)97 253 Polyethylene glycol (PEG)98 254 Polyols99 255 FAD100

26 ENZYME KINETICS AND ELECTROCHEMISTRY 101

261 Michaelis-Menten kinetics 101 262 Electrochemistry and Michaelis-Menten kinetics 105 263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics108

2631 Lineweaver-Burk linear regression 108 2632 Eadie-Hofstee linear regression 109 2633 Electrochemical Hanes-Woolf linear regression 110

264 Cornish-Bowden-Wharton method 112 265 Nonlinear regression methods113

3 ANODE MATERIAL AND METHOD115

31 ELECTRODE FABRICATION 115

311 TTF-TCNQ synthesis115 312 Electrode template fabrication 115 313 Electrode composite fabrication117

3131 Composite for conductance study 117 3132 Bulk modified GOx enzyme electrode composite 118 3133 GOx enzyme electrode composite with enzyme stabilisers 119

314 Electrode wiring and insulation120

32 CONDUCTIVITY EXPERIMENT 121

33 POLISHING COMPOSITE ELECTRODES 122

34 GLUCOSE AMPEROMETRY 123

341 Glucose solution123

11

342 Electrochemical system123 343 Glucose amperometry with GOx electrodes 124

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA 125

4 ANODE RESULTS AND DISCUSSION 127

41 AIMS OF ANODE STUDIES 127

42 CONDUCTANCE OF THE COMPOSITE 128

421 Conductance of various conductive materials128 422 Influence of enzyme concentration on conductivity 130

43 CATALYTIC ACTIVITY OF GOX COMPOSITE ELECTRODES 132

431 Influence of GOx concentration on the catalytic activity of GOx electrodes132 432 Influence of O2 on the catalytic activity of GOx electrodes134

44 THE EFFICACY OF GOX STABILISERS 139

441 Introduction139 442 PEI140 443 DTT142 444 PEG 143 445 GLC144 446 FAD146 447 PEI and DTT mixture147 448 PEI and FAD mixture149 449 Most effective stabiliser content for GOx anodes 150

45 CONCLUSION TO ANODE STUDY 152

5 CATHODE INTRODUCTION 155

51 OXYGEN REDUCTION REACTION (ORR) 155

52 REACTIVITY OF OXYGEN 157

53 ORR CATALYSTS 159

531 Noble metal ORR catalysts 160 532 Macrocycle and Schiff-base complex ORR catalysts 165

12

533 Multicopper enzymes as ORR catalyst 168 5331 BOD 171 5332 Lac 173 5333 CueO 173

534 Anthraquinone ORR catalyst 174

54 CARBON PROPERTIES 177

541 Graphite180 542 Glassy carbon (GC)181 543 Activated carbon (AC) 182 544 Carbon fibre (CF)182 545 Carbon nanotubes (CNTs)183 546 Boron-doped diamond (BDD)184

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON 185

551 Electrochemical properties of carbon185 552 Electrochemical pretreatments (ECPs) of the carbon185

56 ELECTROCHEMICAL TECHNIQUES FOR ORR 187

561 Cyclic voltammetry (CV)188 562 Non-faradic current and electric double layer capacitance 189 563 Diagnosis of redox reactions in cyclic voltammetry 190 564 Coupled reactions in cyclic voltammetry194 565 Voltammograms for the surface-confined redox groups 195 566 Hydrodynamic voltammetry (HDV) and Levich equation197 567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the charge

transfer rate constant 200 568 Finding the standard rate constant (kdeg)205 569 Summary of the electrochemical techniques206

57 SUMMARY OF THE CHAPTER 207

6 CATHODE MATERIAL AND METHOD 208

61 ELECTRODE FABRICATION 208

611 Graphite PtC and RhC electrodes208 612 Anthraquinone(AQ)-carbon composite electrodes208

6121 Derivatisation of graphite with AQ 208 6122 Fabrication of AQ-carbon composite electrode 209

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes209

13

6131 Information on synthesis of metallosalophen 209 6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes 210

62 COMPOSITE RDE CUTTING 210

63 ELECTRODE POLISHING 210

631 Composite RDE polishing210 632 Glassy carbon electrode polishing211

64 ENZYME RDES 212

641 Membrane preparation 212 642 Membrane enzyme electrodes212

65 ELECTROCHEMICAL EXPERIMENTS 213

651 Electrochemical system213 652 Electrode pretreatments214

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes 214 6522 Pretreatments of AQ-carbon PtC and RhC 215

653 Electrochemical experiments 215 654 Data analysis 216

7 CATHODE RESULTS AND DISCUSSION 218

71 AIMS OF THE CATHODE STUDIES 218

72 INFLUENCE AND EFFICACY OF ECPS 219

721 Glassy carbon electrodes (GCEs)219 7211 Untreated GCEs 219 7212 Influence of electrochemical pretreatments on GCE 220 7213 Effect of GCE pretreatment on ORR 227

722 Graphite composite electrodes 230 7221 Untreated graphite composite electrodes 230 7222 Influence of electrochemical pretreatments on graphite composite electrodes 232 7223 The effect of graphite composite electrode pretreatment on ORR 235

723 Platinum on carbon composite electrodes (PtC) 240 724 Rhodium on carbon composite electrodes (RhC)240

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR 241 725 Co(salophen) composite electrodes243

7251 Untreated Co(salophen) composite electrodes 243 7252 Influence of electrochemical pretreatments on Co(salophen) composite 244 7253 The effect of Co(salophen) composite electrode pretreatment on ORR 246

726 Fe(salophen) composite electrodes 248 7261 Untreated Fe(salophen) composite electrodes 248 7262 Influence of electrochemical pretreatments on Fe(salophen) composite 250

14

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR 252 727 Anthraquinone (AQ) composite electrodes253 728 Lac and BOD enzyme membrane electrodes 255

73 MOST EFFICIENT ORR CATALYSTS 257

731 Best five electrodes for nα 258 732 Best five electrodes for kdeg 259 733 Best five electrodes for Epc2(HDV) 260 734 Best three candidates for ORR catalytic electrodes 260

74 CONCLUSION TO CATHODE STUDY 262

8 CONCLUSION AND FURTHER STUDIES 264

9 BIBLIOGRAPHY 268

15

LIST OF ABBREVIATIONS

Α Transfer coefficient

A Electrode surface area

ABTS 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

AC Activated carbon

AFC Alkaline fuel cell

AQ Anthraquinone

Asn Asparagine

Asp Aspartic acid

BDD Boron-doped diamond

BOD Bilirubin oxidase

C Reactant concentration

C O (0t) Electrode surface oxidant concentration

C R (0t) Electrode surface reactant concentration

C(0t) Electrode surface concentration

C Bulk concentration

Cd Double layer capacitance

CF Carbon fibre

CNH Carbon nanohorn

CNT Carbon nanotubes

Co(salen) N-N-bis(salicylaldehyde)-ethylenediimino cobalt(II)

Co(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminocobalt (II)

CO Bulk oxidant concentration

CR Bulk reactant concentration

CueO Copper efflux oxidase

CV Cyclic voltammetry

Cys Cysteine

(DEAE)-dextran Diethylaminoethyl-dextran

DET Direct electron transfer

DHG Dehydrogenase

DMFC Direct methanol fuel cell

DTE Dithioerythriotol

DTT Dithiothreitol

[E0] Initial enzyme concentration

16

E Arbitrary electrode potential Enzyme

Edeg Standard electrode potential

Edeg Formal potential

Ea Anode potential

EC Electrochemical-chemical

Ec Cathode potential

Ecell Cell voltage

ECP Electrochemical pretreatment

Ee Electrode equilibrium potential

EF Fermi potential

EMF Electro motive force

Ep2 Half-peak potential

Epa Anodic peak potential

Epc Cathodic peak potential

Epc2(HDV) Half-wave reduction potential in HDV

ES Enzyme-substrate complex

Eλ Switching potential

∆E Potential difference Potential shift

∆Edeg Standard peak separation

δEp Peak width

εF Fermi energy

F Faraday constant

FAD Flavin adenine dinucleotide

Fe(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminoiron (II)

GC Glassy carbon

GCE Glassy carbon electrode

GHP 1-(6-D-gluconamidohexyl) pyrrole

GLC Glycerol

Gln Glutamine

GOx Glucose oxidase

GOx(O) Oxidised GOx

GOx(R) Reduced GOx

GSH Gluthatione

∆G Gibbs free energy shift

∆Gdeg Standard Gibbs free energy change

∆GdegDagger Standard activation free energy

17

∆GDagger Gibbs free energy of activation

∆GadegDagger Anode standard activation free energy

∆GaDagger Anode activation free energy

∆GodegDagger Oxidation standard activation free energy

∆GoDagger Oxidation activation free energy

∆GRdegDagger Reduction standard activation free energy

∆GRDagger Reduction activation free energy

h Planck constant

HDA Hydrodynamic amperometry

HDV Hydrodynamic voltammetry

His Histidine

HOMO Highest occupied molecular orbital

∆Hdeg Standard enthalpy change

I Electric current Net current

I0 Exchange current

Ia Anodic current

Ic Cathodic current

Id Disc limiting current

IK Kinetic current

IL Limiting current Diffusion-limited current

Im Maximum steady state limiting current

Ip Peak current

Ipa Anodic peak current

Ipc Cathodic peak current

I-V Current-Voltage

J Current density

Jm Maximum limiting current density

k Rate constant

K Equilibrium constant

k(E) Rate constant at the applied potential E

kdeg Standard heterogeneous rate constant

kdegb Backward reaction standard rate constant

kdegf Forward reaction standard rate constant

KDagger The equilibrium constant between the activated complexes and the reactants

k1 Rate constant for ES formation from E and S

k-1 Rate constant for ES dissociation into E + S

18

k2 Rate constant for P formation from ES

kB Boltzmann constant

kb Backward reaction rate constant

kf Forward reaction rate constant

kf(E) Rate constant for forward reactions at the applied potential E

KM Michaelis enzyme dissociation constant

Lac Laccase

LDH Lactate dehydrogenase

LMCT Ligand-to-metal charge transfer

LPG Liquefied petroleum gas

LUMO Lowest unoccupied molecular orbital

MCO Multicopper oxidase

MDH Methanol dehydrogenase

Me2SQ Dimethylmercaptobenzoquinone

MEMS Microelectromechanical systems

MeSQ Methylmercapttobenzoquinone

MET Mediated electron transfer

Met Methionine

mPQQ-GDH Membrane-bound PQQ dehydrogenase

MWNT Multi-walled nanotube

n Overall number of electrons transferred

na The number of electrons transferred up to the rate limiting step

NAD Nicotinamide adenine dinucleotide

NAD-GDH Nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

OCV Open circuit voltage

ORR Oxygen reduction reaction

P Electric power Product

PAFC Phosphoric acid fuel cell

PBS Phosphate buffer saline

PC Pyrolytic carbon

PDMS Polydimethylsiloxane

PEFC Polymer electrolyte fuel cell

PEG Polyethylene glycol

PEI Polyethyleneimine

P-I Power-Current

POx Pyranose oxidase

19

PQ Phenanthrenequinone

PQQ pyrrolo-quinoline quinone

PQQ-GDH Quinoprotein glucose dehydrogenase

Pro Proline

PtC Platinum on carbon

Q Charge

RDE Rotating disc electrode

RhC Rhodium on carbon

RLS Rate limiting step

rpm Rotaion per minute

S Substrate

SEN Single-enzyme nanoparticle

sPQQ-GDH Soluble PQQ dehydrogenase

SQ Mercaptobenzoquinone

SWNT Single-walled nanotube

∆Sdeg Standard entropy change

t Time

T1 Type I (blue Cu in Lac)

T2 Type II ( trinuclear Cu in Lac)

T3 Type III (trinuclear Cu in Lac)

TCNQ 7788-Tetracyanoquinodimethane

TEM Transmission electron microscopy

Trp Tryptophan

TTF Tetrathiafulvalene

TTF-TCNQ Tetrathiafulvalene 7788-tetracyanoquinodimethane

Tyr Tyrosine

V Voltage Maximum limiting catalytic velocity

VHT Vacuum heat treatment

η Overpotential

ν Vibrational frequency Kinematic viscosity

υ Reaction rate Scan rate Enzyme catalytic rate

ω Angular velocity

20

LIST OF FIGURES

Fig No Page Description

Fig 1 32 Historical fuel cells

Fig 2 33 Photographs of modern fuel cells

DMFC Cogeneration system Hydrogen car Glucose-Oxygen fuel cell

Fig 3 34 The schema of glucose-oxygen fuel cell

Fig 4 41 Transition state diagram with the reaction coordinate

Fig 5 45 Fermi level and electron transfer

Fig 6 46 Fermi level and electron transfer at an arbitrary electrode potential

Fig 7 48 Standard free energy change along the electrode potential at

(a) E = Edeg (b) E gt Edeg and (c) E lt Edeg

Fig 8 51 The shift in ∆GdegDagger accompanied by ∆E

Fig 9 57 Tafel plot

Fig 10 59 Activation overpotential vs current

Fig 11 60 Diffusion overpotential vs current

Fig 12 61 Resistance overpotential vs current

Fig 13 62 V-I curve for a typical low temperature fuel cell

Fig 14 65 Mediated electron transfer reaction by the enzyme electrode

Fig 15 70 The schema for a glucose-oxidising anode

Fig 16 75 The structure and redox reactions of FAD NAD and PQQ

Fig 17 79 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode

Fig 18 81 GOx wired to conductive hydrogel complexed with Os

Fig 19 83 Monomer structures of conductive polymers

1-(6-D-gluconamidohexyl) pyrrole and Mercapto-p-benzoquinone

Fig 20 85 TTF-TCNQ structure and its charge transfer

Fig 21 86 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 20 mVs-1

Fig 22 89 The procedure for the enzyme mass-loaded biocatalytic nanofibre

Fig 23 90 The fabrication procedure of single-enzyme nanoparticles

Fig 24 92 The chemical structures of the side groups of Gln and Asn

Fig 25 93 The chemical structures of the side groups of Cys Met Tyr Trp and His

Fig 26 95 The chemical structures of Asp and Pro

Fig 27 96 The structure of PEI

Fig 28 98 The structures of DTT from a linear to a cyclic disulfide form

Fig 29 98 The structures of PEG

21

Fig 30 100 The structure of glycerol

Fig 31 104 A typical Michaelis-Menten hyperbolic curve of the enzyme

Fig 32 107 Electrochemical Michaelis-Menten hyperbolic curve of the enzyme electrode

Fig 33 109 Electrochemical Lineweaver-Burk linear regression of the enzyme electrode

Fig 34 110 Electrochemical Eadie-Hofstee linear regression of the enzyme electrode

Fig 35 111 Electrochemical Hanes-Woolf linear regression of the enzyme electrode

Fig 36 113 Electrochemical Cornish-Bowden-Wharton plot of the enzyme electrode

Fig 37 116 The photographs of PDMS electrode templates

Fig 38 118 The photographs of empty and filled PDMS templates

Fig 39 119 The schema for bulk modification method

Fig 40 121 The photographs of insulated composite electrodes

Fig 41 122 The photographs of measuring electrode resistance

Fig 42 125 The photograph of glucose amperometry with a multichannel potentiostat

Fig 43 126 The analytical output by SigmaPlot Enzyme Kinetics

Fig 44 129 Conductance of various electrode composite

Fig 45 131 Conductance of GOx composite with varying GOx content

Fig 46 133 Difference in the glucose-response amperometric curve by the enzyme electrodes

containing various GOx content 5 10 and 15 in the absence of O2

Fig 47 134 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the absence of O2

Fig 48 135 (i) Difference in the glucose-response amperometric curve by 10 GOx composite

electrodes in the absence and presence of O2

(ii) The re-plotted amperometric curves in the presence of N2 air and O2

Fig 49 136 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the presence N2 O2 and air

Fig 50 140 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEI in the presence and absence of O2

Fig 51 142 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 DTT in the presence and absence of O2

Fig 52 145 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEG in the presence and absence of O2

Fig 53 146 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 GLC in the presence and absence of O2

Fig 54 147 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 FAD in the presence and absence of O2

22

Fig 55 143 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-DTT

(051 11 1505 and 22) in the presence and absence of O2

Fig 56 148 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051 11

1505 and 22)

Fig 57 149 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-FAD (21

22 and 24 ) in the presence and absence of O2

Fig 58 151 Jm KM in the absence and presence of O2 for various stabilisers

PEIDTT (11 0515 221505 ) and GLC 1

Fig 59 156 Oxygen reduction pathways 2 and 4 electron reduction pathways

Fig 60 157 Degradation of superoxide anion radical in aqueous solutions

Fig 61 158 An energy level diagram for ground state dioxygen molecule

Fig 62 159 Diagrams for the state of π orbitals in the ground state oxygen and peroxide anion

radical

Fig 63 162 Three models of oxygen adsorption Griffith Pauling and Bridge models

Fig 64 163 Models for oxygen adsorption on metals and ORR pathways

Fig 65 164 A volcano-shaped trend between ORR rate and oxygen binding energy of various

transition metals

Fig 66 165 Examples of transition metallomacrocycles Heme and copper phthalocyanine

Fig 67 166 Schiff base complexes

Co(II) salen Co(II) salophen and Co(III) salen conductive polymer

Fig 68 167 The molecular structure of dicobalt face-to-face diporphyrin [Co2(FTF4)]

Fig 69 168 ORR pathways by Co2(FTF4)

Fig 70 170 The structure of laccase from Trametes versicolor

Fig 71 171 Redox reaction cycle of MCOs with O2

Fig 72 172 Common mediators for BOD ABTS and Os complex

Fig 73 174 Two-electron ORR by anthraquinone

Fig 74 176 The structures of anthraquinone and its derivative Fast red AL salt

Fig 75 177 HDV of ORR at various quinone electrodes at pH 7

Unmodified GC AQGC and PQGC

Fig 76 178 The carbon structures graphite and diamond

Fig 77 179 The carbon sections basal and edge planes

Fig 78 181 A TEM image of graphene bilayer at 106 Aring resolution

Fig 79 182 A TEM image and a schema of GC

Fig 80 183 Computer graphic models of various CNTs Bundled SWNTs MWNT and CNHs

23

Fig 81 184 A TEM image of diamond

Fig 82 187 The CVs showing the influence of ECP on GCE at pH 7 in the presence and absence

of O2 The CVs for both pretreated and unpretreated GCEs are shown

Fig 83 188 The triangular waveform for CV and a typical voltammetric curves for BQ

Fig 84 189 The CV showing the double layer capacitance at graphite electrode surface at pH 7

Fig 85 194 A voltammogram examples of a quasi-reversible reaction at various scan rates

Fig 86 195 Voltammograms of two-electron transfer in the EE systems with different ∆Edegrsquo

Fig 87 196 A voltammogram of the reversible redox reaction by surface-confined groups

Fig 88 198 A diagram of hydrodynamic electrochemical method using a RDE

Fig 89 200 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH 74

Fig 90 201 Levich plot examples for reversible and quasi-reversible systems

Fig 91 203 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in oxygenated

001 M PBS at pH 74 at pretreated GCE

Fig 92 204 Koutecky-Levich plot for O2 reduction at a pretreated GCE in oxygenated 001 M PBS at

pH 74 with applied potential ndash 04 06 and 08 V

Fig 93 206 Log kf(E) plot for O2 reduction at various pretreated GCEs

Fig 94 207 The protocol for finding kinetic and thermodynamic parameters

Fig 95 211 The photographs of fabricated composite RDEs

Fig 96 213 The photograph of RDE experiment setting with a rotor and gas inlet

Fig 97 220 CVs of untreated GCEs at pH 74 in 001 M PBS in the absence and presence of O2

at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 98 221 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of O2

between + 09 and - 09 V vs AgAgCl at 005 Vs-1

(i) CVs for all the pretreated GCEs

(ii) CVs excludes PBS pretreatments

Fig 99 223 The bar graphs for the capacitance of GCEs in 015 M NaCl buffered with 001 M

phosphate to pH 74

Fig 100 225 Overlaid CVs during various pretreatments for GCEs involving

01 M H2SO4 alone 001 M PBS at pH 74 alone and both pretreatments

Fig 101 227 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M oxygenated

PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 102 229 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Fig 103 231 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M PBS in

the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

24

Fig 104 233 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M N2-sparged PBS

between + 09 and - 09 V vs AgAgCl

Fig 105 236 Overlaid CVs of ORR at various pretreated graphite composite electrodes at pH 74

in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 106 237 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at pH 74

in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various scan rates

50 100 200 and 500 mVs-1

Fig 107 238 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

Fig 108 239 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

Fig 109 241 CVs of untreated and H2SO4-pretreated RhC composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

Fig 110 242 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M PBS in

the absence and presence O2 at 005 Vs-1 between + 09 and - 09 V vs AgAgCl

Fig 111 243 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M PBS in the

absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 112 245 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 113 247 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 114 248 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite electrodes

Fig 115 249 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

Fig 116 251 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 117 253 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 118 254 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M PBS in

theabsence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 119 256 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated PBS at

005 Vs-1 between 0 and -09 V vs AgAgCl

Fig 120 257 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Fig 121 258 The bar graphs of the best five nα

25

Fig 122 259 The bar graphs of the best five kdeg

Fig 123 260 The bar graphs of the best five Epc2(HDV)

Fig 124 266 A photograph of the fuel cell stack made with GOx anodes and Co(salophen)

cathodes

26

LIST OF TABLES

Table No Page Description

Table 1 35 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Table 2 87 Electrochemical behaviour of TTF-TCNQ at pH 74

Table 3 104 Michaelis-Menten equation at various [S]

Table 4 117 Composite composition table

Table 5 119 Blended stabiliser composition

Table 6 139 Four possible causes of glucose amperometric current reduction

Table 7 152 Comparison of maximum output current and KM with some published data

Table 8 159 Bond orders bond lengths and bond energies of some dioxygen species

Table 9 180 The electric conductivity of various carbon forms and sections

Table 10 186 Various ECP examples

Table 11 192 CV diagnosis for reversible system at 25 ˚C

Table 12 193 CV diagnosis for irreversible system at 25 ˚C

Table 13 196 CV diagnosis for reversible system by surface-confined groups at 25 ˚C

Table 14 214 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Table 15 216 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC

Table 16 217 Parameter values for Koutecky-Levich equation

O2 diffusion coefficient O2 concentration and PBS kinematic viscosity

Table 17 261 Three candidate electrodes for high ORR catalysis

Table 18 261 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

27

LIST OF EQUATIONS

Eq No Page Description

Eq 1 35 Anode glucose oxidation reaction

Eq 2 35 Cathode oxygen reduction reaction

Eq 3 35 Overall glucose-oxygen fuel cell reaction

Eq 4 36 Electric power equation

Eq 5 36 Electric current equation in electrochemistry

Eq 6 38 Faradayrsquos second law of electrolysis Charge equation

Eq 7 38 Faradayrsquos second law of electrolysis Reaction equation

Eq 8 39 Transition state theory equation Rate constant equation

Eq 9 39 Transition state theory equation Equilibrium constant

Eq 10 39 Transition state theory equation Atomic vibration frequency equation

Eq 11 40 Transition state theory equation Erying equation

Eq 12 42 Gibbs free energy shift equation in thermodynamic terms

Eq 13 42 Gibbs free energy shift equation in electrochemical terms

Eq 14 49 A typical redox reaction

Eq 15 50 Nernst equation

Eq 16 50 Electrode potential shift

Eq 17 50 Free energy shift in electrochemical terms

Eq 18 52 Anodic oxidation activation free energy shift in terms of potential shift

Eq 19 52 Cathodic reduction activation free energy shift in terms of potential shift

Eq 20 53 Forward reduction rate constant equation in terms of kdeg and potential shift

Eq 21 53 Backward oxidation rate constant equation in terms of kdeg and potential shift

Eq 22 54 Butler-Volmer equation

Eq 23 54 Overpotential equation

Eq 24 54 Exchange current equation

Eq 25 55 Butler-Volmer equation in terms of electrode equilibrium constant

Eq 26 55 Butler-Volmer equation in terms of overpotential

Eq 27 56 Butler-Volmer equation for small overpotential

Eq 28 56 Tafel equation for large overpotential

Eq 29 60 Diffusion overpotential equation

Eq 30 61 Ohmrsquos law

Eq 31 63 Cell voltage equation

Eq 32 63 Gibbs free energy available from the fuel cell

28

Eq 33 73 Glucose oxidation reaction by GOx

Eq 34 73 Glucose oxidation reaction by NAD-DHG

Eq 35 87 TTF-TCNQ reaction At equilibrium state

Eq 36 87 TTF-TCNQ reaction TCNQ reduction at lt + 015 V vs AgAgCl

Eq 37 87 TTF-TCNQ reaction TCNQ lattice reduction at lt - 015 V vs AgAgCl

Eq 38 87 TTF-TCNQ reaction Anodic TCNQ salt formation at gt + 031 V vs AgAgCl

Eq 39 87 TTF-TCNQ reaction TTF lattice oxidation at gt + 035 V vs AgAgCl

Eq 40 87 TTF-TCNQ reaction TTF further oxidation at gt + 038 V vs AgAgCl

Eq 41 101 Enzyme kinetics A typical enzyme reaction

Eq 42 102 Enzyme kinetics Enzyme-substrate complex formation at steady state

Eq 43 102 Enzyme kinetics Rate of enzyme-substrate complex formation in terms of k-1 and k2

Eq 44 102 Enzyme kinetics

Enzyme-substrate complex equation in terms of [E0] k1 k-1 k2 and [S]

Eq 45 102 Enzyme kinetics Enzyme catalytic reaction

Eq 46 102 Enzyme kinetics Enzyme catalytic reaction equation

Eq 47 102 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of enzyme-substrate complex

Eq 48 103 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of [E0] k1 k-1 k2 and [S]

Eq 49 103 Enzyme kinetics Michaelis-Menten constant definition

Eq 50 103 Michaelis-Menten equation

Eq 51 104 Michaelis-Menten equation when [S] ge KM

Eq 52 104 Michaelis-Menten equation when [S] = KM

Eq 53 104 Michaelis-Menten equation when [S] le KM

Eq 54 106 Electrochemical Michaelis-Menten equation

Eq 55 106 Electrochemical Michaelis-Menten equation for current density

Eq 56 108 Electrochemical Lineweaver-Burk linear regression equation

Eq 57 109 Electrochemical Eadie-Hofstee linear regression equation

Eq 58 111 Electrochemical Hanes-Woolf linear regression equation

Eq 59 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for Jm

Eq 60 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for KM

Eq 61 128 Enzyme electrode specificity constant

Eq 62 139 The causes of anodic current reduction A typical GOx-O2 reaction

Eq 63 139 The causes of anodic current reduction TTF reduction by GOx

Eq 64 139 The causes of anodic current reduction GOx allosteric inhibition

Eq 65 139 The causes of anodic current reduction TTF oxidation by O2

29

Eq 66 139 The causes of anodic current reduction TTF oxidation by GOx

Eq 67 161 O2 Dissociative mechanism Simultaneous oxygen adsorption and molecular fission

Eq 68 161 O2 Dissociative mechanism Surface hydroxide formation

Eq 69 161 O2 Dissociative mechanism Oxygen reduction to water

Eq 70 161 O2 Associative mechanism Oxygen adsorption

Eq 71 161 O2 Associative mechanism Peroxide formation

Eq 72 161 O2 Associative mechanism Peroxide reduction

Eq 73 161 O2 Associative mechanism Surface oxygen reduction to hydroxide

Eq 74 161 O2 Associative mechanism Surface hydroxide reduction to water

Eq 75 175 Anthraquinone O2 reduction Semiquinone anion radical formation

Eq 76 175 Anthraquinone O2 reduction O2 reduction by a semiquinone anion radical

Eq 77 175 Anthraquinone O2 reduction Spontaneous superoxide reduction to O2

Eq 78 175 Anthraquinone O2 reduction

Spontaneous superoxide reduction to hydroperoxide

Eq 79 190 Electric double layer capacitance per unit area

Eq 80 192 CV diagnosis ndash Reversible system Ip vs υfrac12

Eq 81 192 CV diagnosis ndash Reversible system Ep

Eq 82 192 CV diagnosis ndash Reversible system ∆Ep

Eq 83 192 CV diagnosis ndash Reversible system δEp

Eq 84 192 CV diagnosis ndash Reversible system Reversal parameter

Eq 85 193 CV diagnosis ndash Irreversible system Ip vs υ

Eq 86 193 CV diagnosis ndash Irreversible system Ep a function of kdeg α and υ

Eq 87 193 CV diagnosis ndash Irreversible system ∆Ep

Eq 88 193 CV diagnosis ndash Irreversible system δEp

Eq 89 195 CV diagnosis ndash EE system Peak separation of EE system

Eq 90 196 CV diagnosis ndash Surface-confined groups Ip vs υ

Eq 91 196 CV diagnosis ndash Surface-confined groups Ep

Eq 92 196 CV diagnosis ndash Surface-confined groups ∆Ep2

Eq 93 198 Diffusion layer thickness in terms of ω

Eq 94 199 Levich equation in terms of ω

Eq 95 199 Levich equation in terms of diffusion layer thickness

Eq 96 202 Koutecky-Levich equation

Eq 97 204 The rate constant for arbitrary potential E

Eq 98 205 k(E) in terms of kdeg and η for a forward reaction

Eq 99 205 kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

Eq 100 238 Surface semiquinone radical formation

30

Eq 101 238 ORR by surface semiquinone radical

31

1 INTRODUCTION

11 INTRODUCTION TO FUEL CELLS

111 History of the fuel cells

Fuel cells are electrochemical devices which directly convert fuel into

electricity via chemical reactions The invention dates back to 19th century when a

British physicist Sir William Grove and a German-Swiss chemist Christian Schoumlnbein

discovered the reverse reaction of water electrolysis The current output by this system

was however very small Therefore the discovery stayed in the shadow of the steam and

internal combustion engines and it needed another century to gain its attention

In the 1930s a British engineer Francis Bacon started working towards

alkaline fuel cells (AFCs) and in the 1950s General Electric developed polymer

electrolyte fuel cells (PEFCs) Those fuel cells were loaded on the spaceships in the

1960s by NASA because they were compact provided clean power with large energy

density and generated drinkable water as a by-product [1-5] Since then fuel cells have

been continuously used for space and marine technologies [3] In the 1970s studies on

fuel cells were further expanded for consumer applications [1 3 5 6] The first

air-breathing fuel cell hybrid car was built by Kordesch [1 7] and phosphoric acid fuel

cells (PAFCs) were developed and introduced to offices and hospitals [2 3 5]

32

Fig 1 Some historical fuel cell applications (i) Alkaline fuel cell for Apollo 11 [8] (ii)

Kordeschrsquos Austin A40 city hybrid car [7]

Since the 1990s fuel cells have been drawing keen attention as flexible renewable

energy in conjunction with the depletion of fossil fuel global warming and the demand

in cleaner energy [6 9] Great effort has been made to miniaturise fuel cells for

individual users such as mobile devices vehicles and households [1 3 5 6] The recent

advance in such technologies has led to direct methanol fuel cells (DMFCs) for

mobile-phone handsets [10 11] cogeneration systems to supply household power [1 5]

hydrogen fuel cell cars [12-15] and bio-batteries running on glucose and air oxygen for

portable music players [16-18] Glucose-oxygen biofuel cells have been also further

miniaturised for medical implantable purposes [18 19]

33

Fig 2 Modern fuel cell technologies (i) Toshibarsquos world smallest DMFC [20] (ii)

TokyoGasrsquos cogeneration system Enefarm (iii) BMWrsquos Hydrogen 7 (iV)

Sonyrsquos Bio Battery generating electricity from sugar [16]

112 Principles of the fuel cells

A fuel cell unit simply consists of an anode a cathode electrolyte and a

conducting wire A conducting wire connects an anode and a cathode to form an

external circuit This allows coupling of electrochemical reactions taking place at each

electrode and provides a conductive path for electrons between them Electrolyte

allows charged species to move between two electrodes but blocks out the entry of

electrons This completes the electric circuit and permits electric conduction At the

anode fuel is oxidised by an oxidation reaction and this supplies electrons to the

cathode through the external circuit The number of electrons generated depends on the

34

type of fuels reactions and environment in which the reactions take place In the anode

reaction ions are also released to balance overall charge They move to the cathode

though the electrolyte At the cathode ions from the electrolyte and electrons from the

circuit meet and react together with an oxidiser supplied The completion of the coupled

reactions generate electricity and products [3 4 21] Fig 3 shows a glucose-oxygen

fuel cell as an example

Fig 3 A glucose-oxygen fuel cell

At the anode glucose is oxidised into gluconolactone This gives out two electrons and

two protons At the cathode oxygen gas receives them and is reduced to water Each

reaction has a unique redox potential called standard electrode potential Edeg and

electromotive force (EMF) is the potential difference between such electrode potentials

It is also called open circuit voltage (OCV) This is the theoretical size of potential

difference available from the fuel cell [3 4] The coupled reactions for glucose-oxygen

fuel cell is shown in Table 1 Each redox potential was measured against silver

35

silver-chloride reference electrode (AgAgCl) at pH 7 25degC therefore shown in formal

potential (Edegrsquo)

Table 1 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Anode Edegrsquo = - 042 V (Eq 1)

(via FADFADH2 redox [22 23])

Cathode [24] Edegrsquo = 062 V (Eq 2)

Overall ∆Edegrsquo = 104 V (Eq 3)

The reaction potential at each electrode is measured separately as half cell potential

against the universal reference electrode such as AgAgCl because it is impossible to

directly measure both inner potentials simultaneously

113 Electric power from the fuel cells

Electric power (P) is the amount of work can be handled by electric current per

unit time It is defined as in Eq 33 The size of power is determined by the size of

electric current (I) and voltage (V) since P is proportional to I and P

36

Electric power

VIP times= middotmiddotmiddotmiddotmiddot Eq 4

P = Electric power W

I = Electric current A

V = Voltage V

Electric current is the amount of charge (Q) flows per unit time Voltage is the electric

potential energy per unit charge and synonymous with potential difference (∆E) EMF

or OCV Increase in I andor V increases P V is however constrained by the type of

electrode reactions as discussed previously I depends on (i) reaction rate (ii) the

electrode surface and (iii) reactant concentration as shown in Eq 5

Electric current in electrochemistry

nFAkCnFAI == υ middotmiddotmiddotmiddotmiddot Eq 5

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

A = Electrode surface area m-2

υ = Reaction rate (1st or pseudo 1st order) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

It is indispensable to have large rate constant to increase output current This demands

lowering activation energy for a chemical transition during the reaction and can be

achieved by use of the catalysts

37

12 CATALYSTS

121 Faradayrsquos second law ndash Current and reaction rate

Catalysts accelerate specific chemical reactions without altering the change in

overall standard Gibbs free energy In other words they change the reaction kinetics but

not the thermodynamics They only provide an alternative lower-energy pathway to

shorten the reaction time without changing themselves before and after the reaction

[25-27] In fuel cells catalysts are used to increase the current output by accelerating the

electrode reaction The relationship between current and rate of reaction is defined by

Faradayrsquos second law of electrolysis (Eq 5 - Eq 7) This expresses the size of electric

charge generated in terms of the number of moles of reactant consumed or product

produced

38

Faradayrsquos second law of electrolysis

nFNQ = middotmiddotmiddotmiddotmiddot Eq 6

AtNCktimes

=times=υ middotmiddotmiddotmiddotmiddot Eq 7

Q = Charge C

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

N = The number of moles of reactant or product mol

υ = Reaction rate (1st or pseudo 1st ordedr) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

t = Time s

A = Electrode surface area m-2

Faradayrsquos second law of electrolysis states that an increase in reaction rate increases the

current output To increase the reaction rate intrinsically its Gibbs free energy of

activation (∆GDagger) must be lowered The relationship between k and ∆GDagger is stated in

Erying equation [28]

122 Transition state theory Eyring equation and reaction rate

When a reaction proceeds it must go through a critical state called transition

state of maximum potential energy also called the activated complex Activated

complexes are unstable molecules existing for only a few molecular vibrations before

decaying into the products Transition state theory assumes that the reactants and the

39

activated complexes are in quasi-equilibrium and the reaction rate is constrained by the

rate at which the activated complex decays into the products This implies that the

reaction rate depends on the equilibrium constant between the activated complexes and

the reactants (KDagger) and the atomic vibrational frequency (ν) as shown in Eq 9 ν is

defined by statistical mechanics in terms of Boltzmann constant (kB) Planck constant

(h) and the temperature (Eq 10) [28]

Transition state theory and reaction rate [28]

[ ]RTexpDagger Dagger

Dagger GC

CK ∆minus== middotmiddotmiddotmiddotmiddot Eq 8

Dagger Kk ν= middotmiddotmiddotmiddotmiddot Eq 9

hTkB=ν middotmiddotmiddotmiddotmiddot Eq 10

KDagger = Equilibrium constant between the reactants and the activated

complexes

s-1

CDagger = Activated complex concentration molm-3

C = Reactant concentration molm-3

∆GDagger = Gibbs free energy of activation Jmol-1

R = Gas constant = 8314 Jmol-1K-1

T = Thermodynamic temperature K

k = Rate constant (1st or pseudo 1st reaction) s-1

ν = Atomic vibrational frequency s-1

kB = Boltzmann constant = 13807 x 10-23 JK-1

h = Planck constant = 662621 x 10-34 Js

40

Based on this the Erying equation expresses the relationship between the reaction rate

and Gibbs activation free energy as shown in Eq 11 [25 28 29]

Erying equation TG

B

hTkk

RDagger ∆minus

= κ middotmiddotmiddotmiddotmiddot Eq 11

where κ = transmission coefficient

This tells that to increase the rate constant reaction temperature must be increased or

the activation free energy must be lowered

123 Overcoming the activation free energy

Activation free energy is a potential energy gap between the reactant and

activated complex At the transition state intra-molecular reorganisation takes place

through the bond destruction and formation This demands the energy and slows the

kinetics The reaction does not proceed unless this energy barrier is overcome Hence

energy input in someway or lowering activation free energy is required for the reaction

to proceed andor accelerate the reaction Heat energy can provide atomic and molecular

kinetic energy so that the system can overcome the energy barrier as indicated in Eq 11

[4 21 29] Applying electric potential also has the similar effect It provides the electric

energy to overcome the energy barrier The rate constant therefore can be altered

extrinsically by temperature andor electrode potential [21 29 30] Alternatively

catalysts can reduce ∆GDagger so that the reaction rate can be increased intrinsically They

enable or accelerate a reaction by providing alternative lower-energy reaction pathway

41

This does not change the reaction path or the standard enthalpy change (∆Hdeg) of the

system however lowers the activation free energy for this path so that the reactants can

be converted more quickly to the products as shown in Fig 4

Fig 4 Energy transition for catalytic reaction through the transition state

Fig 4 shows the energy state transition during a reaction with and without a catalyst

The reaction coordinate represents the reaction progress along the reaction pathway

∆Hdeg is a state function [31] therefore determined only by the initial and final state of

the system but independent of the reaction path ∆Hdeg is divided into standard Gibbs free

energy change (∆Gdeg) and the term of standard entropy change (∆Sdeg) as Eq 12 shows [4

21 23 26 32]

The key insight therefore from transition state theory is that for a catalyst to function it

42

must spontaneously bind the transition state

Gibbs free energy change

deg∆minusdeg∆=deg∆ STHG middotmiddotmiddotmiddotmiddot Eq 12

deg∆minus= EnF middotmiddotmiddotmiddotmiddot Eq 13

∆Gdeg is the standard free energy difference which became available for work through

the reaction and must be lt 0 for a spontaneous reaction ∆Gdeg can be related to the

standard electrical potential difference as Eq 13 shows [4 21 29 30 33 34] It is the

maximum EMF theoretically available from the system ∆Sdeg is the difference in the

degree of disorder in the system The entropy term (T∆Sdeg) is the energy not available for

work and temperature dependent This and Eq 13 imply that ∆Gdeg is also altered by

temperature as shown in Fig 4 [35]

124 Variations in catalysts

Given the general applicability of transition state theory it is not surprising that

catalysts have extensive variation and applications the reaction phase can be

homogeneous or heterogeneous catalysts can be inorganic organic or biological

Inorganic catalysts are metals such as transition or noble metals or complexes

on metal ions in various oxidation states Elemental metal catalysts typically catalyse

reactions through surface adsorption Metal particles have extensive variation in

physical properties such as particle size shape and surface area They often come in

dispersed on the porous support material for improved catalytic performance due to

43

increased surface area [36] and cost efficiency [1] Composition and structure of the

particles thickness of support material and condition of metal deposition all affect

reactions [27 37-39] A complex is a compound in which a ligand is loosely bonded to

a metal centre through coordination bonds An unsaturated complex has a dissociated

ligand and catalysis takes place when a substrate binds to that in the single orientation

[27 40] Complexes also have important roles in biological system [41-43] Organic

catalysts are non-metal non-biological catalysts They are often functionalised solid

surfaces for surface catalysis [27] In electrochemistry such catalysts can be chemically

bonded to conductive material [44] Simple electrochemical pretreatment can generate

or attach chemical groups onto the electrode surface and they mediate electron transfer

[45-47] Biological catalysts are generally called enzymes Enzymes are biological

polymers mostly made of protein and usually have high reaction and substrate

specificity They often have cofactors which are not proteinous but essential for their

catalytic activities Enzymes are involved in virtually all biological activities [23 25 27

48] and in a few industrial applications due to their substrate specificity [27]

Catalysts are by definition not supposed to be consumed in their reactions

however they could lose their catalytic power Impurities fouling and poisoning are

common deactivating factors for the metal catalysts Competing reactions also convert

them into inactive forms Particle migration and agglomeration reduce their catalytic

surface area hence their overall activity [27] Biological catalysts are very sensitive to

the environment Slight changes in pH and temperature can deactivate enzymes Their

stability is also affected by presence of metal ions surfactants oxidative stress and

organic solvent molecules [49] Metal catalysts can be possibly regenerated by

redispersion [27] however it is very difficult to recover biological catalytic functions

44

Therefore enzyme stabilisers are often added to extend their life Protein engineering

also can confer advantageous properties to the enzymes Amino acid sequences leading

to favourable protein folding functions or stability are determined in silico and they are

introduced to existing enzymes by genetic engineering such as site-directed mutagenesis

Protein engineering of serine protease subtilisin is a classic example [50]

13 ELECTRODE POTENTIAL AND REACTION RATE

131 Fermi level electrode potential and electron transfer

In the sect 123 the relationship between the electric potential and activation free

energy was briefly discussed As heat energy can provide the kinetic energy to atoms

and molecules for a reaction to proceed the electric energy also can provide kinetic

energy to electrons for electron transfer to proceed This leads to the redox reaction at

the electrode surface

Each substance has a set of discrete energy levels Each energy level can

accommodate up to two electrons Electrons fill these energy levels in ascending order

from the lower level This can divide these energy levels into two groups electron-filled

lower energy level and empty higher energy level without electrons In between the

critical energy level the called Fermi level lies It is designated as εF (Fermi energy) or

EF (Fermi potential) The Fermi level contains electrons of highest energy A substance

can transfer only these electrons to the external system as a donor while as an acceptor

it can accommodate external electrons only within the empty energy level which is

above its Fermi level (Fig 5)

45

Fig 5 Fermi level and electron transfer

The energy state of electrons however varies by substance therefore electrons at Fermi

level so-called highest occupied molecular orbital (HOMO) of a donor may need to

gain various kinetic energy to jump into the lowest empty energy level so-called lowest

unoccupied molecular orbital (LUMO) of an acceptor for electron transfer (Fig 6)

46

Fig 6 Electrode potential and electron transfer

As soon as the energy of electrons at Fermi level of a substance or an electrode meets

the energy level of LUMO of the other redox molecule electron transfer proceeds as

long as there is room to accommodate electrons in the acceptor This is the cathode

reaction The potential can be swept back by applying positive potential to the electrode

This lowers the energy of LUMO of the electrode so that an electron in HOMO of the

donor can be transferred back to the electrode This is the anode reaction If the electron

transfer is fast in both directions compared with other reaction steps it is a

thermodynamically reversible system and the standard electrode potential (Edeg) for this

redox reaction lies somewhere between and close to those two electrode potentials

which have enabled electron transfer At Edeg both anode and cathode reactions have the

same reaction rate hence they are at equilibrium [33] and there is no net current [21 30]

47

132 Electrode potential equilibrium potential and Nernst equation

Activation free energy of an electrode reaction can be altered by applying

potential The extent of its alteration and the direction of the reaction depend on the size

and direction of potential applied Fig 7 shows the difference in the standard activation

free energy (∆GdegDagger) for redox reactions at various electrode potentials [30]

48

Fig 7 Standard free energy and electrode potential [30]

(a) At E = Edeg (b) E gt Edeg and (c) E lt Edeg

More positive potential lowers the activation free energy for oxidation hence shifts the

reaction in favour of oxidation and vice versa This implies that applying extra potential

to the electrode perturbs the equilibrium and to restore it the electrode equilibrium

49

potential (Ee) must be shifted This phenomenon is clearly stated in Nernst equation (Eq

15 on p 50) as a relationship between Ee and the equilibrium constant (K) at the

electrode surface At equilibrium both oxidation and reduction reactions take place at

the same rate and there is no net current therefore the surface concentration (C(0t))

approximates bulk concentration (C) Nernst equation shows that Ee is always under

the influence of such concentration but can be established provided the reaction is fast

enough to restore the equilibrium [21 30] In the dilute solutions of the electroactive

species Edeg is substituted with the formal potential (Edegrsquo) which considers the influence

of inter-solute and solvent interactions [21 29]

Redox reaction

middotmiddotmiddotmiddotmiddot Eq 14

O = Oxidant

ne- = The number of electrons transferred

R = Reactant

kf = Forward reaction rate constant (1st or pseudo 1st reaction) s-1

kb = Backward reaction rate constant (1st or pseudo 1st reaction) s-1

50

Nernst equation

lowast

lowast

+deg=R

Oe

CC

nFRTEE ln middotmiddotmiddotmiddotmiddot Eq 15

Ee = Equilibrium potential V

Edegrsquo = Formal potential V

R = Gas constant = 8314 Jmol-1K-1

T = Absolute temperature K

n = The number of electron transferred

F = Faraday constant = 96487 Cmol-1

CO = Bulk oxidant concentration molm-3

CR = Bulk reductant concentration molm-3

133 Electrode potential and activation free energy

When E ne Edegrsquo the difference in these potentials is expressed as the electrode

potential shift (∆E) as in Eq 16 and its free energy shift (∆G) as in Eq 17

Electrode potential shift

degminus=∆ EEE middotmiddotmiddotmiddotmiddot Eq 16

Free energy shift

)( degminus=∆=∆ EEnFEnFG middotmiddotmiddotmiddotmiddot Eq 17

When ∆E is applied positively it lowers the anode oxidation activation free energy

(∆GaDagger) by fraction of the total energy change (∆G) as shown in Fig 8 on p 51 The

fraction for the oxidation reaction is defined as (1-α) The transfer coefficient (α) is a

51

kinetic parameter which states the degree of symmetry in the potential energy barrier

for redox reactions The symmetry in energy barrier is present when the slopes of two

potential energy curves are the same and it is defined as α = 05 α is dependent on

redox potential and could vary within the range between 0 and 1 In most cases α = 05

and appears to be constant when overpotential is small [21 30]

Fig 8 The shift of ∆E and ∆GdegDagger [21 30]

With α the shift in electrode activation free energy can be expressed in terms of

potential shift for both oxidation and reduction reactions as shown in Eq 18 and Eq 19

52

∆GDagger in terms of (E-Edegrsquo)

Anode Oxidation )()1(DaggerDagger degminusminusminus∆=∆ EEnFGG aa αo middotmiddotmiddotmiddotmiddot Eq 18

Cathode Reduction )(DaggerDagger degminus+∆=∆ EEnFGG cc αo middotmiddotmiddotmiddotmiddot Eq 19

∆GoDagger = Anode oxidation activation free energy Jmol-1

∆GodegDagger = Anode oxidation standard activation free energy Jmol-1

α = Transfer coefficient molm-3

n = The number of electrons transferred Jmol-1

F = Faraday constant = 96485 Cmol-1

E = Arbitrary electrode potential V

Edegrsquo = Formal potential V

∆GRDagger = Cathode reduction activation free energy Jmol-1

∆GRdegDagger = Cathode reduction standard activation free energy Jmol-1

134 Potential shift and standard rate constant

By substituting the activation free energy in Eyring equation (Eq 11) with the

shift in electrode activation free energy in Eq 18 and Eq 19 it is possible to express k

in terms of potential shift and standard rate constant (kdeg) (Eq 20 and Eq 21) kdeg is the

rate constant at Edegrsquo[21 30]

53

k in terms of (E-Edegrsquo) and kdeg

Forward reduction cathode rate constant (kf) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bf

Daggercexpκ

⎥⎦⎤

⎢⎣⎡ degminusminus

⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )(expexp

Dagger

EERT

nFRT

GhTk cB ακ

o

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= )(exp EERT

nFk fα middotmiddotmiddotmiddotmiddot Eq 20

Backward oxidation anode rate constant (kb) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bb

Daggeraexpκ

⎥⎦⎤

⎢⎣⎡ degminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )()1(expexp

Dagger

EERT

nFRT

GhTk aB ακ

o

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= )()1(exp EE

RTnFkb

α middotmiddotmiddotmiddotmiddot Eq 21

kf and kb = Forward and backward reaction rate constant s-1

kdegf and kdegb = Forward and backward reaction standard rate constant s-1

135 Butler-Volmer equation overpotential and current

At the equilibrium potential E = Ee kf = kb hence anodic current (Ia) =

cathodic current (Ic) and therefore the net current (I =Ia ndash Ic) is macroscopically zero (I =

0) However both forward and backward reactions still occur microscopically at the

electrode surface The absolute value of this electric current is called exchange current

(I0) and is not directly measurable When CO = CR at E = Ee C O (0t) = C R (0t) and kdegf

= kdegb = kdeg hence the net current any potential E can be defined as below by

Butler-Volmer equation (Eq 22)

54

Butler-Volmer equation

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡ degminus

minusminus⎥⎦

⎤⎢⎣⎡ degminusminus

deg= )()1(exp)(exp )0()0( EERT

nFCEERT

nFCnFAkI tRtOαα middotmiddotmiddotmiddotmiddot Eq 22

The Butler-Volmer equation states the relationship between the electric current

and the potential shift This can be further rephrased in terms of overpotential (η)

through defining I0 using Nernst equation (Eq 15) η is the potential required to drive a

reaction in a favourable direction from its equilibrium state and defined as n Eq 23

Overpotential (η)

eEE minus=η middotmiddotmiddotmiddotmiddot Eq 23

At E = Ee both anode and cathode exchange currents are the same therefore both

exchange currents can be defined together as I0 (see below) and I0 can be expressed in

terms within Nernst equation by substituting (Ee-Edegrsquo) as in Eq 24

Exchange current

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= lowast )(exp0 EERT

nFCnFAkI eO

α

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= lowast )()1(exp EE

RTnFCnFAk e

Rα

αα )()( 1RO CCnFAk minus= o middotmiddotmiddotmiddotmiddot Eq 24

Now the Butler-Volmer equation (Eq 22) can be expressed in terms of η (= E ndash Ee) (Eq

25) by expressing it with I0 and the with re-arranged Nernst equation

55

⎥⎦⎤

⎢⎣⎡ degminus=lowast

lowast

)(exp EERTnF

CC e

R

O The term

)0(

CC t in Eq 25 indicates the influence of mass

transport For purely kinetically controlled region the electrode reaction is independent

of mass transport effects therefore C(0t) asymp C and Bulter-Volmer equation approximates

to Eq 26

Butler-Volmer equation and η

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminusminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminus

minus= lowast

lowast

lowast

minusminus

lowast

lowast

lowast

αααα

R

O

O

tO

R

O

R

tR

CCEE

RTnF

CC

CCEE

RTnF

CC

II )(exp)()1(exp )0()1(

)0(0

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= lowastlowast ηαηαRT

nFC

CRT

nFC

CI

O

tO

R

tR exp)1(exp )0()0(0

middotmiddotmiddotmiddotmiddot Eq 25

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= ηαηαRT

nFRT

nFI exp)1(exp0 middotmiddotmiddotmiddotmiddot Eq 26

The Butler-Volmer equation shows the dependence of current and hence the reaction

rate on the overpotential This implies that small kdeg can be overcome by overpotential

The kinetically-limited region of the electrode reaction is however very small because

the rate constant sharply increases exponentially with overpotential and soon the

reaction became diffusion limited [21 30] At this state the surface equilibrium is

totally perturbed and the overpotential is used to drive the diffusion for the one-way

reaction

136 Butler-Volmer equation vs Tafel equation

At small η lt RTnF Butler-Volmer equation further approximates to a linear

equation as shown in Eq 27

56

Small η Butler-Volmer equation

η⎟⎠⎞

⎜⎝⎛=

RTFnII 0

middotmiddotmiddotmiddotmiddot Eq 27

At large |η| gt RTnF the rate of the backward reaction is negligible hence one

of the exponential terms in Butler-Volmer equation becomes negligible and the

equations are solved for η for both anode and cathode reactions as below

Large η Tafel equations

InFRTI

nFRT lnln 0 plusmn= mη in the form of ||ln Iba +=η middotmiddotmiddotmiddotmiddot Eq 28

Anode oxidation InF

RTInF

RT ln)1(

ln)1( 0 αα

ηminus

+minus

=

Cathode reduction InF

RTInF

RT lnln 0 ααη minus=

In the system which follows the Tafel equations I0 (and hence kdeg ) is very small and the

electrode reaction does not proceed unless large η is supplied In this potential range the

reaction is already totally irreversible [4 21 30] An irreversible slow reaction is

graphically presented in Tafel plot in which ln (I) is plotted against η as shown in Fig

9

57

Fig 9 Tafel plot [4 21 30]

I0 is deduced from the y-intercept of the extrapolated slope Once |I| has become too

large and reached to the mass transport limit at large |η| the slope deviates from the

linear trend [30]

14 OVERPOTENTIAL AND FUEL CELLS

141 Four overpotentials that decrease the EMF

As discussed so far overpotential is extra potential required to drive a reaction

In the fuel cells this can only be taken from the potential generated within the system

causing potential drop and reduces EMF The overpotential in the system is attributed to

the four causes [1 4] (i) internal current (ii) activation overpotential (iii) diffusion

overpotential and (iv) resistance overpotential

58

142 Internal current

Internal current is a reverse current caused by crossover and leakage current

Crossover happens when the fuel flows to the opposite electrode through the electrolyte

Leakage current occurs when the current flows through the electrolyte rather than the

external circuit Internal current therefore happens without a load and is observed as the

negative offset from the theoretical value of EMF This is noted especially in

low-temperature fuel cells such as direct methanol fuel cells (DMFCs) [4 51] In such

systems The inner current can be several 10 Am-2 and potential drop could be more

than 03 V [4] In order to prevent fuel mixture and crossover a separator is used to

partition the cell Alternatively it is also possible to confer high specificity to electrodes

as seen in glucose - oxygen fuel cells Such systems do not require

compartmentalisation and this is advantageous for miniaturisation [52]

143 Activation overpotential

Activation overpotential is a potential loss due to the slow reaction kinetics as

discussed in sect 135 This is unavoidable to some extent and is typically the biggest

problem in most fuel cells involving O2 cathode reduction because this reaction is

kinetically hindered Finding or inventing catalysts with high kdeg is the key solution for

this A sluggish reaction exhibits a sharp rise in activation overpotential for a slight

increase in low current After the steep increase the current increase becomes linear and

constant as shown in Fig 10 until the reaction reaches the limit of mass transport at the

higher overpotential [4 30 34]

59

Fig 10 Activation overpotential against current [4]

144 Diffusion overpotential

Diffusion overpotential is a potential loss due to concentration gradient This

tries to drive flux of electroactive species toward and from the electrode surface It

happens when the reaction kinetics is faster than mass transport [4 21 30] In fuel cells

diffusion overpotential is caused by fuel depletion due to the inefficient fuel convection

or supply [21 34] This problem is observed in reformers because the fuel refining and

conversion such as liquefied petroleum gas (LPG) to hydrogen gas take place outside

the system and it makes difficult to respond to the sudden fuel demand from the system

[4] It also happens when fuel diffusion is interfered with by bad electrode geometry

electrode fouling and adsorption [21 27] Diffusion overpotential is defined as Eq 29

60

Diffusion overpotential equation

⎟⎟⎠

⎞⎜⎜⎝

⎛minus=

Ldiffusion I

InFRT 1lnη middotmiddotmiddotmiddotmiddot Eq 29

ηdiffusion = Diffusion overpotential V

IL = Limiting current A

Limiting current (IL) is generated when a reaction takes place at the rate limited by mass

transport Diffusion overpotential sharply increases when reaction kinetics have reached

to the maximum and very large current are drawn as shown in Fig 11 [21 30 34 53]

Fig 11 Diffusion overpotential against current [4]

145 Resistance overpotential

Resistance overpotential arises within electrodes separators connectors circuit

or electrolyte [4 34] It is called Ohmic resistance or IR drop because it follows Ohmrsquos

61

law (Eq 30) where resistance overpotential is linearly proportional to the electric

current as shown in Fig 12 [4 30]

Ohmrsquos law

IRV = middotmiddotmiddotmiddotmiddot Eq 30

V = Voltage consumed due to the resistance V

I = Electric current drawn due to the resistance A

R = Resistance Ω

| |

Fig 12 Resistance overpotential against current [4]

Causes of internal resistance are scattered through the system therefore there are many

possible ways to resolve this One can possibly reduce it by using more conductive

material The difficulty lies in finding the conductive material which can be modified as

well as carry out the functions as one desires under the required conditions [53]

Resistance can be reduced by reducing the distance between two electrodes [54]

however this might cause short circuit and fuel leakage This becomes a serious issue in

62

use of solid electrolyte such as polymer electrolyte because both performance efficiency

and mechanical stability of electrolyte must be achieved [53] Excess in inert supporting

electrolyte minimises solution resistance and matrix effect It sustains a thin electric

double layer uniform ionic strength and buffering effect against pH change [21 30]

However electrolyte could become a problem when electrode material dissolves into

electrolytic solution and reacts there because it might change nature of electrolyte [1

21]

146 Total overpotential and cell voltage

Total overpotential is the sum of all four overpotentials Current ndash Voltage (IV)

polarisation curve visualises the tendency of each potential drop as shown in Fig 13 [1

4]

Fig 13 V-I curve for a typical low temperature fuel cell [4]

63

The cell voltage (Ecell) is determined by subtracting the cathode potential (Ec)

from the anode potential (Ea) (Eq 31) The actual cell voltage is found by subtracting

the total overpotential from the theoretical cell voltage The energy available from this

cell voltage can be calculated as in Eq 32

Cell voltage

cacell EEE minus= middotmiddotmiddotmiddotmiddot Eq 31

totalltheoreticacellactualcell EE ηminus= )()(

cellcell nFEG minus=∆ middotmiddotmiddotmiddotmiddot Eq 32

15 BIOFUEL CELLS

151 Microbial and enzymatic fuel cells

Biofuel cells are fuel cells which use biocatalysts for fuel reactions

Biocatalysts can be either the whole living cells or isolated enzymes The former is

called a microbial fuel cell and the latter is called an enzymatic fuel cell [55-57] They

often function under ambient conditions and allow wider choice of fuels Microbial fuel

cells use common carbohydrates such as glucose and lactate [55] but also have

potentials in extracting power from waste and sediments [34 55 57 58] Harnessing

such microbial life activities has some advantages They can complete fuel oxidation

within the cell It was reported that a microorganism Rhodoferax ferrireducen has over

80 glucose conversion rate [58] Microbial fuel cells also have long-term stability

[56-58] of up to five years [55] However their output current and power densities are

64

small due to high internal resistance [55-57] Enzymatic fuel cells in contrast have

higher current and power densities but short life incomplete reactions require

mediators to shuttle electron between the enzyme catalytic centre and the electrode and

become deactivated easily [55 56 58] Addition of mediators and their immobilisation

with enzyme complicates fuel cells design further However they can be more

advantageous due to their substrate specificity Both anode and cathode to reside in the

same compartment of mixed fuels without crossover This enables the miniaturisation of

fuel cells [52 59 60] and is particularly useful for medical implantation Stability and

lifetime of the enzyme fuel cells can be improved by appropriate enzyme

immobilisation fixing up the suitable conditions for their function [55] and adding

some enzyme stabilisers Addition of the right mediators and increased enzyme loading

will also increase output current and power density [55 59]

152 Enzyme fuel cells and mediators

Enzyme reactions are available for wide range of substrates yet each enzyme

has very high specificity for its substrates Many also function at neutral atmospheric

conditions therefore miniaturised enzyme fuel cells are of interest for in vivo

application Many redox reactions however take place at the catalytic centre which is

deeply buried in the protein body This proteinous content is an electronic insulator and

interrupts the electrical communication between the redox centre and the electrode It is

therefore difficult for many enzymes to establish efficient electron transfer without

mediators as shown in Fig 14 This is called mediated electron transfer (MET)

65

Fig 14 Mediated electron transfer reaction [55 61]

It is possible for some enzymes such as multicopper oxidases (MCOs) to perform direct

electron transfer (DET) without mediators however mediators ensure good electrical

communication and cell performance However the presence of mediators as well as the

enzyme specificity could complicate the fuel cell composition and its reaction path due

to the reaction cascade [55 59 62 63] Palmore built a methanol - oxygen enzyme fuel

cells and this involved four enzymes two mediators and six reactions to complete the

full reaction path [64] It is more advantageous to use enzymatic fuel cells for very

specific applications [59] such as medical implantation where there are environment

constraints but where applications demand only small power output through simple

reactions

Mediators are usually small redox active species which can be organic or

metal complexes In the enzymatic fuel cells they should be carefully selected because

improper mediators can cause unwanted overpotential or can destabilise the system

They must have (i) small overpotential (ii) fast reversible electron transfer [55 56 59

60 63 65] and (iii) stability [62 65] However there should be small but reasonable

66

potential gap between the enzyme and the mediator to drive electron transfer [60 66]

The gap of 50 mV is optimal [19 64] because of the small potential loss yet less

competing with enzyme reactions It is also possible to mediate electron transfer by

building a conductive nanowire between the electrode and the redox centre [60 63 67]

or a connection by plugging metal nanoparticles into the enzyme [60 67]

16 MINIATURISED MEDICAL POWER SOURCES

161 Realisation of miniaturised fuel cells and medical power supply

Medical application is one of the obvious targets for miniaturised biofuel cells

Glucose-oxygen biofuel cells which continuously run on glucose and oxygen were

thought to be ideal for implantable medical devices because both oxygen and glucose

are abundant in the body [63] They were initially intended to power cardiac assist

devices and pacemakers The studies on such systems have been made for more than 40

years however none has been made into practical use because their power density

stability operation life and biocompatibility were inadequate [19 67-69] Their output

power ranges between microW ndash mWcm-2 and they operate only for a few days to a few

weeks Most applications need a few W power for a longer duration [18 60 67] A

pacemaker requires 1microW [56] for more than 8 ndash 10 years with 996 reliability Such

power has been supplied by lithium (Li) iodine battery [19 56 70] which is

characterised by extended operational life [68] Li is fairly stable light-weight metal

and has high energy density of 386 kAhkg-1 Its standard potential is highly reducing ndash

323 V vs AgAgCl These properties enable Li batteries to power most devices

67

including pacemakers laptops and vehicles [19 21] However there are some problems

in Li batteries They are still bulky because they need to store their own fuel and it has

to be sealed with the metal case [19 68] They occupy half of the space in the

pacemaker volume [68 71] They use corrosive [72] and irritating materials There is

potential of explosion [68] leakage and the necessity of exchange Li also has potential

resource constraint due to the geopolitical issues as seen for oil and platinum More than

80 of its production is based in South America and China [73] With increased public

concern over using greener safer energy and saving resources as well as the global

political intervention in that it is natural to consider about using alternative

technologies to power implantable medical devices where it is possible

162 Miniaturised potential medical power sources

There are numerous implantable electrical medical devices with different

power requirements These include cardiac pacemakers defibrillators neurostimulators

for pain control drug delivery device hearing aids [70] and biosensors [74]

Implantable autonomous body sensors for physiological parameters such as glucose

concentration local pH flow and pressure [19] require power as low as 1 microW [56 70

74] and the operation period of 1 year at least [74] Such power can be supplied by

various implantable energy harvesting devices using body glucose [19] motion

vibration [75] and temperature differences [75] Heller developed the smallest

glucose-oxygen biofuel cells made of carbon fibres with the dimension of 17-microm

diameter and 2-cm length Anode glucose oxidase and cathode laccase were wired to

conductive hydrogel backbone with a flexible osmium (Os) mediator complex Its

68

output was 048 mWcm-2 with 06 V at 37 degC and it lasted for 1 week [19 67]

Kinetic energy of motion and vibration can be converted into electricity with

electrostatic transduction using microelectromechanical systems (MEMS) This

technology allows integration of various microelectronic devices on the same substrate

[76] NTT Corporation developed a system generating power from body motion and

temperature It consists of a thermoelectric and a comb-shaped Au-Si vibrational device

on a monolithic Si substrate The voltage produced by a thermoelectric device is

boosted by capacitance which was generated by the vibrational device then its charge

is stored in the capacitor [75] Another vibrational device was reported to have 20 x 20 x

2 mm volume and output 6 microW and 200 V at 10 Hz low frequency and 39 ms-2

acceleration The principal difficulty in using MEMS is to adjust and synchronise

flexibly the resonance frequency of vibrational device to the human body frequency

which is low and has constant fluctuation in order to increase energy conversion

efficiency The typical vibration has the frequency ranging from 1 to 100 Hz and

acceleration from 1 - 10 ms-2 [76]

Thermoelectric devices generate power from temperature difference Each

module consists of a number of thermocouples on Si substrate Thermocouples are

made of different material so that it can generate voltage from the heat flow following

temperature gradient This is called Seebeck effect and its size depends on the material

and number of thermocouples used and the spacing within the device [75 76] Typical

ambient temperature difference is less than 10K [74 76] and its power is 1microWcm-2 [74]

Seiko Instrument developed a wristwatch called Seiko Thermic which was powered by

human body temperature in 1998 [77 78] It consists of n-type Bismuth-Tellurium

(BiTe) and p-type Bismuth-Antimony-Tellurium on rods reinforced by Ni on oxidised

69

silicon wafer It generated microWcm-2 power at extremely low 0047 conversion

efficiency With a booster circuit it powered a wristwatch operating at 1microW and 15 V

It could however operate only when ambient temperatures were below 27 degC [78] and

so could not be used in tropical regions

Heller also developed an implantable miniature Nafion-coated zinc ndash silver

chloride (ZnAgCl) battery The anode was made of zinc fibre coated with Nafion It

was 76-microm diameter 2-cm length and 75 x 10-2 cm2 surface area The cathode was

made of AgCl wire covered with chloride-permeable membrane On discharge anode

zinc formed Zn3(PO4)2middot4H2O hopeite complex with Nafion growing non-porous

lamellae on the surface The lamellale had a unique property which was permeable to

zinc ions but impermeable to O2 therefore the battery became corrosion free It output

was 13 microAcm-2 at 1 V and operated for three weeks under physiological condition The

advantage of this battery was its size which was 100-fold smaller than the smallest

conventional batteries [79] The smallest silver oxide button buttery has the volume 30

mm3 [19 80] with energy density 04 mWh mm-3 at 15 V The smallest Li-ion buttery

has the volume 190 mm3 [80] with energy density 025 mWh mm-3 at 4 V [21]

17 OUTLINE OF THE WORK

171 Introduction to miniaturised glucose-oxygen fuel cells

A miniaturised glucose-oxygen biofuel cell runs on glucose and oxygen This

would be ideal to power implantable autonomous low-power body sensors since both

glucose and oxygen are abundantly present in the body The range in their physiological

70

concentration is between 4 ndash 8 mM for glucose and 01 ndash 02 mM for oxygen [56 67]

The aim of this study was to build miniaturised glucose-oxygen fuel cells with reliable

enzyme anodes and powerful oxygen-reducing cathodes using straightforward and

scaleable technology The general structure and mechanism of glucose-oxygen fuel cells

were described in sect 112

In this study both anode and cathode reactions took place at the room

temperature and physiological pH in the presence of catalysts Glucose oxidation was

catalysed by the enzyme glucose oxidase (GOx) from Aspergillus niger and its electron

transfer and conduction were facilitated by an conducting organic salt called

tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) (Fig 15)

Fig 15 The schema for a glucose-oxidising anode

GOx and TTF-TCNQ were mixed into a composite employing a feasible electrode

fabrication method called bulk modification method Various enzyme stabilisers were

added to the composite at different concentration to improve its performance The

influence of various enzyme stabilisers on anode performance was studied in the

71

presence of both N2 and O2 and the most ideal anode composition was determined

Cathodes were also fabricated in the similar manner using graphite as conductive

material and mixing with various oxygen reducing catalysts except for the enzyme

membrane cathodes The cathode composites were pretreated in various media to

enhance their activity For enzyme membrane electrodes glassy carbon electrodes were

used instead of graphite composite and enzyme solution was loaded onto the surface

and trapped with membrane Catalytic power and mechanism of each cathode were

studied with various electrochemical methods and the most catalytic cathode was

determined

172 Outline

Chapter 1 is a general introduction about fuel cells It explains the history and

basic concept of fuel cells and outlines the study

Chapter 2 explains about the enzyme anodes The introduction part refers to

glucose oxidising enzymes enzyme immobilisation TTF-TCNQ enzyme inactivation

enzyme stabilisers and enzyme kinetics The material and method section reports the

protocol for the enzyme electrode fabrication electrode conduction study and

amperometry study for enzyme electrodes Result and discussion section mentions

enzyme electrode conduction enzyme kinetic studies based on amperometry

mechanism of enzyme stabilisation in each stabiliser and ideal electrode composition

Chapter 3 discusses oxygen-reducing cathodes The introduction part reviews

the mechanism of the oxygen reduction reaction oxygen reactivity oxygen reduction

catalysts carbon properties electrochemical pretreatments electrochemical techniques

72

and how to determine the catalytic efficiency of cathodes The material and method

section describes the protocol for the oxygen-reducing electrode fabrication preparation

and electrochemical pretreatment electrochemical studies electrochemical studies for

qualitative analysis and to obtain the kinetic parameters and analysis to determine the

efficiency of catalytic cathodes The result and discussion section describes the

voltammetric behaviour of each pretreated cathode influence of pretreatment and

catalytic efficiency The most catalytic cathode was determined at the end

Chapter 4 refers to the overall conclusion and further studies

73

2 ANODE INTRODUCTION

21 GLUCOSE OXIDATION ENZYMES

Glucose-oxygen fuel cells extract power by oxidising glucose at the anode The

anode contains glucose-oxidising enzymes There are several enzymes available for

glucose oxidation (i) β-D glucose oxidase (GOx) (EC 1134) [56 81] (ii) pyranose

oxidase (POx) (EC 11310) [56 81] (iii) nicotinamide adenine dinucleotide-dependent

glucose dehydrogenase (NAD-GDH) (EC 11147) [18 56] and (iv) quinoprotein

glucose dehydrogenase (PQQ-GDH) (EC 1152) [56 81] These enzymes are

classified into the family of oxidoreductases which catalyse either transfer of (i)

hydride (H-) (ii) oxygen atoms or (ii) electrons from one molecule to another Oxidases

transfer electrons from substrates to O2 and form H2O or H2O2 as products

Dehydrogenases transfer electrons from substrates to coenzymes without involving O2

(Eq 33 and Eq 34)

Glucose oxidation by GOx and NAD-DHG

GOx middotmiddotmiddotmiddotmiddot Eq 33

NAD-DHG middotmiddotmiddotmiddotmiddot Eq 34

To function catalytically enzymes require non-peptide inorganic or organic cofactors or

prosthetic groups Non-peptide organic cofactors are also called coenzymes Enzymes

lacking such groups are called apoenzymes and hardly functional Cofactors are

generally loosely bound to the apoenzymes and freely diffusible but on catalysis they

74

bind to the enzymes as the second substrates Prosthetic groups are always tightly bound

to the apoenzymes through covalent or non-covalent bonding [22 23 48] GOx and

POx are fungal flavoprotein enzymes containing flavin adenine dinucleotide (FAD)

cofactors FAD in GOx is tightly bound to the apoenzyme but not covalently bonded

hence still dissociable [67 82-84] NAD-GDH contains dissociable NAD+ [65] while

PQQ-GDH contains pyrrolo-quinoline quinone (PQQ) redox cofactor [56 65] which is

moderately bound to the apoenzyme [65 67] The structure of each cofactor is shown in

Fig 16

75

N

N

NH

N O

O

R

N

N

NH

HN O

O

R

NH

N

NH

HN O

O

R

H+ + e- H+ + e-

2H+ +2e-

N+

NH2

O

R

H

N

NH2

OH

R

HH

N

NH

O

O

OHO

OH

O

OH

O

N

NH

O

HO

OHO

OH

O

OH

O

N

NH

OH

HO

OHO

OH

O

OH

O

H+ + e- H+ + e-

FAD FADH2

NAD NADH

PQQ PQQH2

Fig 16 The structure of FAD NAD and PQQ and their redox reactions

Moieties involved in the redox reactions are circled in gray [23 65]

Cofactors also stabilise the enzyme structure Irreversible thermal GOx denaturisation

involves the perturbation of FAD biding site causing FAD dissociation although 70 of

its secondary structure is retained Chemical denaturisation untangles the dimmer

structure of GOx [65 81 83 85] and dissociates monomers and FAD [86 87] Excess

FAD does not prevent or regain function after thermal inactivation [87] However FAD

can rapidly bind to apoenzymes at 25 degC pH 61 and stabilise the tertiary structure

through conformational change and give resistance to proteolysis [82]

76

The choice of enzyme is based on the reaction involved enzyme availability

stability enzymatic properties physical properties and operational environment In the

absence of O2 or when there is concern about system degradation due to the competition

of electrons between O2 and artificial electron acceptors or due to the by-product H2O2

GDHs are more preferred to GOx [23 60 65-67 88] However the use of GDHs poses

different disadvantages Membrane-bound PQQ dehydrogenase (mPQQ-GDH) has very

high glucose specificity however it is difficult to solubilise and purify hence not for

commercial use [88] Soluble PQQ dehydrogenase (sPQQ-GDH) has been used in

commercial glucose sensors [67 89] however it suffers from low thermostability and

very low glucose specificity [88] Moreover PQQ-GDHs add complication to the

system because metal ions such as calcium ions (Ca2+) and magnesium ions (Mg2+) is

required to activate them [89 90] In the case of NAD-GDH NAD must be provided

because it is a dissociable cofactor but must bind first to the apoenzyme to form a

catalytic complex for glucose oxidation In addition to this complication NAD was also

reported to be more prone to causing electrodes poisoning without a quinone acceptor

[65] NAD forms adduct with phosphate in the buffer its decomposition releases free

adenine and electrochemically oxidised adenine adsorbs onto the electrode surface

causing electrode poisoning [91]

The redox potential of enzyme is also important because it is the driving force

for electron transfer and the power although it is difficult to match both catalytic

efficiency and electrochemical efficiency It is also difficult to determine the redox

potential exactly because the redox potential of cofactors is influenced by

microenvironment such as where they are embedded in the enzyme as well as

77

macroenvironment such as pH and temperature For example the redox potential of

FAD itself is ndash 042 V vs AgAgCl at pH 7 25 degC [22 23] however that of

flavoproteins could range from ndash 069 to ndash 005 V [65] and FAD-containing GOx has

the redox potential approximately ndash 03 V [67 92] It was also reported glucose

gluconolactone redox potential was ndash 056 V [93] PQQ itself has - 013V in absence of

Ca2+ and ndash 012 V in presence of 1M CaCl2 [94] while sPQQ-GDH has the redox

potential approximately - 019 V in presence of Ca2+ [66 67] Furthermore the enzyme

redox potential leads to a complex issue when mediators which assist electron transfer

between the enzyme redox centre and the electrode are involved because the improper

combination of the enzyme and mediator causes inefficient electron transfer and hence

potential drop

For enzyme availability stability and simplicity in handling GOx has been

most popular glucose-oxidising enzyme It has optimal pH 55 and temperature 35 ndash

40 degC [93] but is stable between pH 4 ndash 7 [95] and up to 50 - 55 degC [82 87] GOx has

higher substrate specificity and is most active toward β-D-glucose oxidation [56 86 88]

and very stable therefore its use has prevailed among various sensor applications [60

67]

22 DESIGNING ENZYME ELECTRODES

Construction of enzyme electrodes is complex because it simultaneously

incorporates enzymes mediators conductive materials and stabilisers ensuring that all

of them work harmoniously under restricted performance conditions There are several

78

immobilisation techniques available These include physical adsorption cross-linking

covalent bonding and entrapment in gels or membranes [96]

221 Physical adsorption

Physical adsorption is a simplest immobilisation method Electrodes are dipped

in the solution containing enzymes and mediators or such solution can be applied to the

electrode surface In this method however the enzyme tends to leach due to the weak

interaction between the electrode and enzyme [55] In order to avoid the loss of

electrode material the electrode surface is often covered or coated with material One

classic method is that the solution on the electrode surface is trapped with a

semipermeable dialysis membrane [61 65 92] This allows enzymes free from the

possible denaturisation caused by immobilisation on the solid material Enzymes inside

the dialysis membrane also can be fixed onto the cellulose acetate membrane or the

enzyme layer can be cross-linked with bifunctional reagents such as glutaraldehyde

[61]

222 Reconstituting apo-enzyme

Willner constructed enzyme electrodes by reconstituting apo-enzyme on the

gold (Au) electrodes surface with quinone mediators The electrode consisted of three

layers which were made of PQQ synthetic FAD and apo-GOx [97] PQQ were

mediating groups and attached onto Au electrode by cystamine PQQ attached on Au

was then covalently linked to synthetic FAD called N6-(2-aminoethyl)-FAD Covalently

79

linked FAD self-assembles with apo-GOx so that holo-GOx assembly is formed on the

top of the PQQ mediating layer attached to the Au surface The reconstitution procedure

is shown in Fig 17

Fig 17 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode [97]

The output current density was 300 microAcm-2 at pH 7 35 degC and it dropped by 5 in the

presence of O2 although the decrease was negligible at 01 mM physiological O2

concentration [98] The reconstitution of apo-enzymes is a flexible system potentially

applicable to enzyme electrodes [97] The fabrication process is cumbersome however

the enzymes are neatly aligned and electrically connected in the correct orientation

therefore electrocommunication between the enzymes and electrodes can be

maximised

80

223 Tethered Osmium-complexes linked to conductive hydrogel

It has been reported that embedding enzymes in the conductive hydrogel

complexed with tethered osmium (Os) mediator led to high output current [19 60 66

67] The conductive hydrogel is made of cross-linked polyvinylpyridine or

polyvinylimidazole They are soluble as monomers however they gain their

water-swelling property in cross-linked polymer This is ideal for enzyme mobility and

electrical conductivity Electron transfer is realised by the Os complex which is tethered

to the conductive hydrogel back bone The tether is made of 10 ndash 15 atoms [19 67] in

order to allow flexile movement of mediators Tethered Os complexes sweep the large

volume made of multiple layers of hydrogel form electrostatic adducts with enzymes

[19] and facilitate collide-and-hop electron conduction [19 99] Polymer tether and Os

complexes can be flexibly tailored for different enzymes and different operational

environments [19] One of its great advantages is that the redox potential for electron

transfer can be adjusted by changing the ligand structure of Os complex [19 60 97]

Heller constructed such electrodes made of carbon fibers whose dimension was 7 microm

diameter 2 cm length 08 mm2 surface area and 26 x 10-3 mm3 volume GOx was

wired to poly(4-vinylpyridine) conductive hydrogel backbone by the tethered complex

of tris-dialkylated NNrsquo-biimidazole Os2+3+ (Fig 18)

81

Fig 18 GOx wired to conductive hydrogel complexed with Os [19 67]

The redox potential of its redox centre was ndash 02 V vs AgAgCl The glucose oxidation

took place at ndash 036V and its output current density was 11 mAcm-2 [19] Disadvantage

of the tethered enzyme is short life It was stable for one week in the physiological

buffer then 5 current drop per day was observed When it was implanted in grape sap

50 current drop occurred within 20 hours In calf serum 50 current dropped

occurred in 2 hours with electrode fouling [19]

224 Entrapment in the conductive polymer

Entrapping enzymes in conductive polymer such as polypyrrole polyaniline

and polythiophene [65] is a popular method for enzyme electrodes [96] This is made by

82

electropolymerisation in which potential is applied to the solution containing enzymes

and monomors for certain duration It is a simple method but can be complicated if the

conditions for polymer growth are not compatible with enzyme entrapment [99]

Enzyme leakage occurs when enzymes are not trapped securely but it can be reduced

when derivatised polymers are used [96 100] Their conductivity is affected by

monomer structure [100] acidity and electrolyte [101] It can be degraded by products

of enzymatic reactions [65] Fujii fabricated GOx electrodes which were stable for

more than four months though a gradual sensitivity drop was observed GOx was

entrapped in saccharide-containing polypyrrole made of 1-(6-D-gluconamidohexyl)

pyrrole (GHP) (Fig 19(i)) on Pt electrodes It was reported that entrapped GOx was

secured by hydrogen bonding present between sugar gluconyl groups and enzymes

Enzyme leaching was also reduced by improved polymerisation with extended

conductive hydrocarbon chain [100] The output current density was 75 microAcm-2 at 055

V pH 74 in the presence of 56 mM glucose and 175 microA cm-2 under glucose saturated

condition [100] Arai entrapped various enzymes in polymer of mercaptobenzoquinone

(SQ) (Fig 19(ii)) and its methylated derivatives methylmercapttobenzoquinone

(MeSQ) and dimethylmercaptobenzoquinone (Me2SQ) on Au electrode surface

83

OH

H

H

OH

OH

H

OH

H

CH2OHN(CH2)6NHCHOH

(i)O

O

SH

Fig 19 Monomer structures of conductive polymers

(i) 1-(6-D-gluconamidohexyl) pyrrole [100]

(ii) Mercapto-p-benzoquinone [101]

Quinones have been widely used as good mediators [21 60 61 65] Poly(SQ) is a

conductive polymer functionalised with quinone mediating groups and can be easily

attached to Au electrode due to the presence of thiol group (-SH) [100] It was revealed

that more methylated SQ polymer led to better performance The maximum current

density for SQ- MeSQ- and Me2SQ-GOx anodes was 006 522 and 544 microAcm-2 and

their electrode enzyme dissociation constant KM was 13 18 and 20 mM respectively

[102 103] FAD cofactors have been also electropolymerised to entrap lactate

dehydrogenase and lactate oxidase It was reported that polymerised FAD extended the

shelf and operational life of electrodes to more than one year and also reduced O2

influence [104]

225 Bulk modification

Another popular electrode fabrication involves bulk modification of solid

composite This is simply made by mixing electrode material and inert binding reagent

84

such as epoxy resin Typical composite electrodes consist of 70 conductive material

by weight [105] Bulk-modified electrodes are robust stable inexpensive and easy to

modify build and reuse [105-107] therefore often used for enzyme electrodes [108]

For GOx electrodes tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ)

has been used as both mediating and conducting material

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties

TTF-TCNQ is a black crystalline charge transfer complex having both

mediating and conducting properties Its handling is also easy and often used for GOx

electrodes [65] Its conductivity is 102 ndash 103 Scm-1 [109 110] and comparable to that of

graphite [65 105] TTF-TCNQ complex consists of TTF electron donor and TCNQ

electron acceptor and they are arranged in a segregated stack structure TTF-TCNQ has

metal-like electric conductivity and this derives from intra-stack and inter-stack

molecular interactions Within the stack π-orbitals of adjacent molecules overlap This

builds the major conductive path in the direction of stack [61 65 110 111] Between

the stacks there is partial charge transfer between different chemical groups and this

gives the metallic feature in which charge carriers are mobile among the stacks

[110-112] as shown in Fig 20

85

S

S S

S

N

C

C

N

C

C

N

N

S

S S

S

7 6

N

C

C

N

C

C

N

N

-

TTF - electron donor

TCNQ - electron acceptor

6 - localised 6 - delocalised

+ e-

+ e-

Fig 20 TTF-TCNQ structure and its charge transfer [61]

TTF-TCNQ is a good electrode material because it has low background current and is

efficient at electron transfer [65] is stable [65] insoluble in water and not toxic [113]

However it exhibits complex electrochemistry (Fig 21) and its behaviours have not yet

been fully elucidated

86

vs AgAgCl

TCNQTCNQ e⎯⎯rarr⎯minus minusbull+

+minus+ ⎯⎯rarr⎯minus 2e TTFTTF

minus+minusbull⎯⎯rarr⎯

minus 2e TCNQTCNQ

+minus⎯⎯rarr⎯minus

TTFTTF e

Fig 21 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 01M potassium

phosphate 01 M KCl at 20 mVs-1 [92] Modified

The range of its effective redox potential varies according to the authors [65 92 114

115] Following the Lennoxrsquos study its potential extremes should lie within the range

between - 015 V and + 031 V vs AgAgCl (Table 2) because beyond these potential

ranges irreversible lattice reduction or oxidation takes place Even within the range

crystal dissolution salt formation with buffer ions and its deposition are still present and

could change the electrochemical profile TTF-TCNQ stability is affected by many

factors such as potential applied buffer component and concentration ion solubility

mass transport and analyte [92]

87

Table 2 Electrochemical behaviour of TTF-TCNQ at pH 74

Equilibrium TCNQTTFTCNQ-TTF +⎯rarr⎯ middotmiddotmiddotmiddotmiddot Eq 35

TCNQ reduction + 015 V MTCNQTCNQTCNQ Me ⎯⎯rarr⎯⎯⎯rarr⎯+minus +minusbull+ middotmiddotmiddotmiddotmiddot Eq 36

Lattice reduction - 015 V minus+minusbull ⎯⎯rarr⎯minus 2e TCNQTCNQ middotmiddotmiddotmiddotmiddot Eq 37

Anode salt formation + 031 V MTCNQTCNQ M⎯⎯rarr⎯++minusbull middotmiddotmiddotmiddotmiddot Eq 38

Lattice oxidation + 035 V +minus⎯⎯rarr⎯minus

TTFTTF e middotmiddotmiddotmiddotmiddot Eq 39

Further oxidation + 038 V +minus+ ⎯⎯rarr⎯minus 2e TTFTTF middotmiddotmiddotmiddotmiddot Eq 40

In the presence of a redox couple it also exhibits different electron transfer mechanism

When Edegrsquo of the redox couple is Edegrsquo gt 0 V TTF-TCNQ shows metal-like behaviour

facilitating direct electron transfer (DET) while it presents mediated electron transfer

(MET) for the redox couple of Edegrsquo lt 0 V Lennox made a study on TTF-TCNQ GOx

electrodes and reported that large anodic current was observed at ndash 015 V He also

reported that stable current was obtained when constant potential + 02 V was applied

for 24 hours at pH 7 in the presence of glucose He concluded that hydrophobic protein

core solubilised TCNQ and it formed enzyme-mediator complex GOx oxidation could

be catalysed at low potential because it was still in the range of the stable redox

potential He also conducted experiments with GOx and methanol dehydrogenase

(MDH) modified TTF-TCNQ electrodes in the presence of O2 The performance was

interfered by O2 in both electrodes against his expectation because DHG is in general

resistant to O2 He gave a conclusion that reduced mediators were oxidised by O2 before

electrooxidation took place [92]

88

226 Enzyme covalent attachment and mass loading

Enzymes can be covalently attached to the electrode however it becomes

complicated when mediators and stabilisers are must be also attached for practical fuel

cell applications This is however an attractive technique once ideal means of

co-immobilisation are established because enzymes can be retained firmly on the

electrode surface Enzymes can be covalently linked to oxidised or aminated surface

through amide bonds [116 117] Covalent linkage often lowers enzyme activity because

it partially alters the enzyme structure and confines the enzyme conformation [55]

however Kim succeeded in fabricating stable biocatalytic nanofibre by loading enzyme

aggregate to the enzyme monolayer which was covalently attached to the nanofibres

(Fig 22) Nanofibres with ester groups were synthesised from polystyrene and

poly(styrene-co-maleic anhydride) using electrospinning Seed enzyme was covalently

attached to the chemically modified nanofibre to form an enzyme monolayer on the

nanofibre This enzyme-attached nanofibre was further modified with the mixture of

glutaraldehyde and enzymes so that enzyme mass load was achieved by loading enzyme

aggregate to the enzyme monolayer covalently attached to the fibre Minter reported

that enzyme immobilisation with cross-linking increased the enzyme stability but

limited the enzyme conformation and thus reduced the activity However the activity

reported was nine times higher than that with an enzyme monolayer only Moreover the

activity was retained more than one month under rigorous shaking [118] The activity of

each enzyme particle might have been reduced however mass loading increased the

reactant flux hence increased the overall activity

89

Fig 22 The procedure for the enzyme mass-loaded biocatalytic nanofibre [118]

He also fabricated single-enzyme nanoparticles (SENs) in which each enzyme particle

was encapsulated within a hybrid organic and inorganic network The surface amino

groups of the enzyme were converted into surface vinyl groups and then polymerised

The polymer was then cross-linked to form the shell around the enzyme particle (Fig

23)

90

Fig 23 Fabrication procedure of single-enzyme nanoparticles [55]

It was reported that high stability was achieved after polymer cross-linking The enzyme

activity was approximately half of the free enzyme however there was no mass

transport interference [55] due to their porous structure Km in SENs was nearly

equivalent that of the free enzyme but the storage stability went beyond five months

[119] Eight-fold increase on storage stability after GOx covalently linked to

poly(phosphorothioate) was also reported [120]

23 PROTEIN INACTIVATION AND STABILISATION

Enzyme inactivation is one of the biggest problems in enzyme electrodes

Enzymes are excellent catalysts in their native environment however they are only

designed to work optimally under specific conditions therefore very sensitive to the

slight change in their environment and easily undergo inactivation The issue is

persistent in the enzyme electrodes because their fabrication introduces the enzymes to

the unusual environment yet enzyme catalytic activity must be maintained to obtain

sufficient power output and operational life The problem can be alleviated by (i)

91

protein engineering [56 121] (ii) environmental engineering [56 60 63 67] and (iii)

addition of appropriate enzyme stabilisers [120 122] Protein engineering involves the

alteration of enzyme structure to gain desired functions stability or resistance However

it is very challenging to improve both stability and activity since protein stability is

conferred by its rigidity while its activity depends on the conformational flexibility

Alteration of protein properties is realised by genetic engineering such as site-directed

mutangenesis [123] or directed evolution [49] Environmental engineering is closely

linked to the enzyme immobilisation It aims to achieve the same goals as in protein

engineering but extrinsically The environment which rigidifies the protein structure

such as cross-linking or inclusion in electrode composite tends to increase the enzyme

stability and prolong its life In contrast the environment which more favours over

flexible protein conformation such as hydrogel and membrane trap exhibits higher

enzyme activity but shorter life However it was reported that enzyme cross-linking and

the presence of hydrophilic macromolecules improved both stability and activity while

electrostatic interaction improved selectivity [124] Addition of stabilisers is more

targeted smaller scale environmental engineering Intrinsic conformational stability of

the protein cannot be improved [120] however it can target and eliminate specific

inactivating factors increases the local specific protein stability and prevents or slows

its unfolding There are two factors to improve for enzymes to achieve ideal operation

(i) activity by hydration and (ii) increased lifetime by increased protein rigidity which

might involve the removal of bond-destabilising factors or amplifying the bond strength

within the enzymes In fact both must be probably achieved simultaneously for gaining

the optimal state because they interrelate to each other in terms of protein dynamics As

mentioned earlier protein hydration improves enzyme activity but also allows its

92

regeneration hence affects the protein internal dynamics Protein dehydration distorted

the protein structure [125]

24 PROTEIN DESTABILISATION

There are many protein destabilising factors apart from dehydration and these

can be classified into chemical and physical instability Chemical instability happens

due to chemical modification such as deamidation oxidation proteolysis and

racemisation

241 Deamidation

Deamidation is the hydrolysis of amide link in the amino acid side groups such

as glutamine (Gln) and asparagine (Asn) (Fig 24)

Glutamine (Gln) Asparagine (Asn)

Fig 24 Side groups of Gln and Asn

It is favoured by increase in pH temperature and ionic strength The buffer

concentration influences the rate of deamidation at pH 7 ndash 12 Urea also accelerates

deamidation It often implies the protein aging and then modifies the protein function

93

242 Oxidation

Oxidation takes place at the side chains of cystine (Cys) methionine (Met)

tyrosine (Ty) tryptophan (Trp) and histidine (His)

Cysteine (Cys) Methionine (Met)

Tyrosine (Tyr) Tryptophan (Trp) Histidine (His)

Fig 25 Side groups of Cys Met Tyr Trp and His

Atmospheric oxygen can oxidise Met residues H2O2 modifies indole thiol (SH) [123

126] disulfide (SS) imidazole phenol and thioether (RSR) groups at neutral or slightly

alkaline conditions At acidic condition it oxidises Met into Met sulfoxide (R-S=O-R)

Under extremely oxidising condition Met sulfoxide is further oxidised into Met sulfone

(R-S(=O)2-R) Met oxidation leads to loss of biological activity however it does not

abolish the enzyme activity Therefore if oxidised Met is reduced back by a reductant

such as thiols it will restore the full biological activity Met reactivity depends on its

position within the protein Cys plays an important role in protein folding because it

forms intermolecular disulfide bonds however very susceptible to oxidation It can

undergo autooxidation in the absence of oxidants It is oxidised in several stages in

94

which thiol R-SH group is oxidised to (i) sulfenic acid (R-S-OH) (ii) disulfide

(R-S-S-R) (iii) sulfinic acid (R-S=O-OH) and (iv) sulfonic acid (R-S(=O)2-OH) [123

127] Oxidised thiol groups can be reduced back however they can be also reduced

back in the wrong way This leads to protein misfolding and loss of activity There are

many factors that influence the rate of oxidation They are (i) temperature (ii) pH (iii)

buffer medium (iv) metal catalysts (v) O2 tension (vi) steric condition (vii) neighbouring

groups (viii) photooxidation The presence of metal ions such as Fe2+ and Cu2+

accelerates oxidation by air [123] and the presence of O2 also might accelerate thiol

oxidation with other oxide such as nitric oxide radical [127] Metal-catalysed oxidation

involves a site-specific metal binding to Trp His and Met This leads to severer form of

oxidation causing protein aggregation Peroxide-catalysed oxidation on the other hand

is milder and tends to affect Cys and Trp but does not form aggregates [126] Increase in

pH increases pKa hence accelerates dissociation of ndashSH All of Cys Met Typ Tyr and

His are susceptible to photooxidatoin however His photooxidation is fatal leading to

enzymatic inactivity [123]

243 Proteolysis and racemisation

Proteolysis and racemisation takes place at aspartic acid (Asp) residue The

bond between Asp and proline (Pro) is especially susceptible to hydrolysis Asp also

easily undergoes base-catalysed isomerisation to isoAsp through a ring formation [123]

95

Aspartic acid (Asp) Proline (Pro)

Fig 26 Side group of Asp and the amino acid structure of Pro

244 Physical instabilities

Physical instability includes denaturation adsorption aggregation and

precipitation and [123] This refers to loss of tertiary structure and does not involve

chemical modification It is defined as Gibbs free energy change between the native and

unfolded state of protein Physical instability is induced by change in temperature

extreme pH organic solvent denaturant [123] and solvation [120 125]

25 ENZYME STABILISERS

By clarifying the possible major causes of instability the choice of optimal

approach and stabilisers can be made Considering the case of glucose-oxygen fuel cells

the stabilisers should be selected on the basis of following points (i) conformational

stability for longer operation and shelf life (ii) hydrophilic environment for optimal

enzyme functions (iii) optimal interaction between enzyme and substrate and enzyme

and environment Conformational stability is to secure protein folding and avoid

denaturation This is achieved by thiol group protection reduction of oxidised thiol

96

groups removal of oxidising species an increase in internal hydrophobic interaction

and environmental hydrophilic and electrostatic interaction Optimal enzyme activities

can be achieved by an increase in hydrogen bonding and electrostatic interactions

251 Polyehyleneimine (PEI)

Polyethyleneimine (PEI) has been reported as an efficient antioxidant as well

as chelating agent It is also cationic (Fig 27) [120] therefore provides electrostatic

stabilisation with anionic protein [126] such as GOx [67 85] Anti-microbial activity

has been also reported [126] It is not effective against thermal inactivation [128]

H+

N+H3N+H3

N+H3H3N+

H2+ H+

H+

H+

H2

+

H2+

Fig 27 Structure of PEI [129]

PEI is available in different molecular weights [120] and smaller molecules are more

effective in binding and flexibility [130] However its optimal loading concentration

varies [120 128 131] PEI protects thiol groups efficiently [120] from both metal- and

peroxide- catalysed oxidation despite of their different oxidation mechanisms [126] PEI

also forms a complex with the enzyme and coats it with polycations This occludes the

97

oxidants from the protein surface [126 130] Complex formation might be involving

hydrogen bonding between carboxylic acid groups in the enzyme and nitrogen atoms in

PEI [130] This possibly confers stability in dried state [128] It was reported that PEI

increased the maximum limiting catalytic velocity (V) for GOx [131] and lactate

dehydrogenase (LDH) [128] However it also increased Km for LDH and shifted its

optimal pH slightly toward acidic [128]

252 Dithiothreitol (DTT)

Dithiothreitol (DTT) is another small water-soluble [132] thiol protecting

reagent [132 133] with the reducing potential at ndash 033 V at pH 7 It has only little

odour [132] and has low toxicity [134] Its epimer is dithioerythriotol (DTE) Both are

excellent reducing reagents with two thiol groups (Fig 28) [132] however DTT might

be marginally better in reducing power [135] It reduces back disulfide quantitatively

[127 132] at lower concentration than other thiol-containing reducing reagents such as

Cys and gluthatione (GSH) But it cannot restore some of the lost catalytic activity

[136] because it only reduces disulfides but not other thiol oxides Therefore its

efficacy depends on (i) the oxidation mechanism involved in the protein inactivation

(ii) the velocity at which the inactivation proceeds and (iii) how thiol-contaning amino

acids are present in the protein DTT is not effective against mild oxidation caused by

low concentration of H2O2 or isolated Cys residues [127] It is probably because that

DTT reduces thiol oxide by converting itself from a linear into a cyclic disulfide form

which is called trans-45-dihydroxy-o-dithiane with (Fig 28) [132]

98

Fig 28 Structures of DTT from a linear to a cyclic disulfide form [132]

On the other hand arsenite was effective in reducing only mildly oxidated thiol groups

but not disulfides [127] It is however toxic [137] and not suitable for practical use

Neither of DTT or arsenite was effective if thiol oxidation is outcompeting [127]

253 Polyethylene glycol (PEG)

Polyethylene glycol (PEG) (Fig 29) has been reported as an enzyme stabiliser

however also has mixed reception and its stabilisation mechanism has not yet been

clearly understood

Fig 29 Structures of PEG

OrsquoFagain reported that PEG increased storage and thermo stability [120] while

99

Andersson reported the increase in LDH activity with PEG 10000 Mw but was

followed by a decrease after 10-day storage and then eventually became worse than

those without stabilisers The stabilisation was also found to be concentration dependent

[128] As for protein stabilisation mechanism it was proposed that PEG preferentially

hydrates the protein and stabilises its native structure in the solution [138-140]

According to Arakawa this causes the exclusion of PEG from protein surface due to the

unfavourable interaction between them hence increases water density around the

protein This results in the water shell around the protein surface and increases the

hydrogen bonds [138 139] Better hydration favoured larger PEG Mw due to the larger

steric exclusion [138] on the other hand lower PEG Mw was also reported to be better

because of its better solubility and viscosity [139] Optimal concentration is unknown

however one proposal is that higher concentration is more advantageous to stabilise the

denatured protein because it can penetrate and stabilise the protein hydrophobic core

exposed during denaturation [138] In contrast Lee reported that this penetration affects

the hydrophobic core of active enzyme and decreased the activity and denatured the

protein [139] Both however concluded that a fine balance between preferential

hydration and hydrophobic interaction is important and PEG concentration must be up

to 30 [138 139] It was also reported that PEG stabilised the protein of low water

content better because it prevented the water evaporation while polyols tended to strip

water from the protein dehydrated microenvironment [139]

254 Polyols

Polyols such as glycerol (GLC) (Fig 30) were reported to increase shelf life

100

[141] thermostability [120] and also provide preferential solvation as seen in PEG

[141-143] Higher GLC concentration resulted in better protein activity [143 144] and

reduced protein aggregation due to the tighter protein packing [144] However observed

hydration was lower than expected for high GLC concentration therefore Gekko

proposed that the contribution of higher GLC concentration did not derive form the

increase in steric exclusion Instead GLC might penetrate the solvation layer of protein

and stabilise the structured water network around the protein [143] Andersson also

compared the influence on protein activity after the storage among several candidate

stabilisers diethylaminoethyl-dextran (DEAE)-dextran Mw 500000 sorbitol and PEI

DEAE-dextran increased the activity both before and after the storage while sorbitol

decreased it However PEI was only found to be superior to all [128]

OH

OHHO

Fig 30 Structure of glycerol

255 FAD

FAD (Fig 16 p 75) is also another possible stabiliser as well as mediator

because it can regenerate denatured aged enzymes which have lost its cofactors [82

108] However it has not been clarified well and Bartlett questioned its efficacy due to

the contradictory results he obtained [145] It was reported that addition of FAD

increased the electrode sensitivity by 70 especially in the concentration range below

101

3mM and activity by 8 times although it also increased Km by 2 times [108]

26 ENZYME KINETICS AND ELECTROCHEMISTRY

261 Michaelis-Menten kinetics

A simple enzyme reaction can be expressed in terms of equilibrium between

the free enzyme (E) substrate (S) enzyme-substrate complex (ES) and product release

(P) The enzyme is regenerated on completion of the catalytic cycle

General enzyme reaction mechanism

middotmiddotmiddotmiddotmiddot Eq 41

E = Enzyme concentration mol

S = Substrate concentration mol

ES = Enzyme-substrate complex concentration mol

P = Product concentration mol

k1 = Rate constant for ES formation from E and S mol-1s-1

k-1 = Rate constant for ES dissociation into E + S s-1

k2 = Rate constant for P formation from ES s-1

The rate of ES formation is defined as

102

][][]])[[]([][2101 ESkESkSESEk

dtESd

minusminusminus= minus middotmiddotmiddotmiddotmiddot Eq 42

Where [E0] is the initial enzyme concentration

When the reaction is at steady state 0][=

dtESd

])[(][][]])[[]([ 212101 ESkkESkESkSESEk +=+=minus minusminus middotmiddotmiddotmiddotmiddot Eq 43

Re-arranging the above equation for [ES] as below

][]][[

][121

01

SkkkSEk

ES++

=minus

middotmiddotmiddotmiddotmiddot Eq 44

When the rate of product formation is defined as follows

middotmiddotmiddotmiddotmiddot Eq 45

][2 ESktP=

∆∆

=υ middotmiddotmiddotmiddotmiddot Eq 46

Where υ is the catalytic rate

When all the enzyme active sites are occupied such a condition is called saturated and

defined as [E0] = [ES] The maximum catalytic rate (V) is therefore available as below

Maximum catalytic rate V

][][ 202 ESkEkV == middotmiddotmiddotmiddotmiddot Eq 47

103

Express υ in terms of [S] by inserting Eq 44 into Eq 46

][

]][[

1

21

02

Sk

kkSEk

++

=minus

υ middotmiddotmiddotmiddotmiddot Eq 48

Michaelis-Menten constant (KM) is a dissociation constant about ES and defined as

Michaelis-Menten constant

1

21

kkk

K M+

= minus middotmiddotmiddotmiddotmiddot Eq 49

Michaelis-Menten equation (Eq 50) is obtained when υ is expressed in terms of V and

KM by inserting Eq 47 and Eq 49 into Eq 48

Michaelis-Menten equation

][][SK

SV

M +=υ middotmiddotmiddotmiddotmiddot Eq 50

This is a hyperbola equation to lead the enzyme catalytic rate Most enzymes typically

show this hyperbolic curve (Fig 31) when they are free from allosteric controls or

enzyme inactivation

104

0E+00

1E-09

2E-09

3E-09

4E-09

5E-09

0E+00 2E-03 4E-03 6E-03 8E-03 1E-02 1E-02 1E-02 2E-02

[S] M

Fig 31 A typical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

As shown in Fig 31 Michaelis-Menten plot has different kinetic profiles at high and

low [S] Mathematical expression of its transition over [S] is shown in Table 3

Table 3 Michaelis-Menten equation at various [S]

[S] ge KM Vcongυ middotmiddotmiddotmiddotmiddot Eq 51

[S] = KM 2V

=υ middotmiddotmiddotmiddotmiddot Eq 52

[S] le KM MM K

SEkK

SV ]][[][ 02=congυ middotmiddotmiddotmiddotmiddot Eq 53

At high saturated substrate concentration the catalytic rate becomes zero-order

therefore Michaelis-Menten curve becomes plateau When [S] = KM the rate becomes

105

half of the maximum catalytic rate At low substrate concentration the catalytic rate is

linearly dependent on [S] therefore the kinetics becomes first-order about [S]

262 Electrochemistry and Michaelis-Menten kinetics

In electrochemistry enzyme activity is studied by amperometry This involves

measuring measuring the current generated by the enzyme electrode reaction at a fixed

potential over an arbitrary period It therefore shows the transition in current response

over time For enzyme electrodes substrate concentration is changed over time so that

the current response for a different substrate concentration can be measured over a long

period With intact enzyme and appropriate heterogeneous electron transfer present

most enzyme electrodes follow Michaelis-Menten kinetics (Fig 32) in the amperometry

in the absence of allosteric control or enzyme inactivation Therefore the amperometry

data can be analysed in the similar way to that of biochemical data and for this

Michaelis-Menten equation is expressed in electrochemical terms [146] In

electrochemical enzyme kinetics enzyme catalytic rate (υ) is obtained in terms of

electric current therefore V is substituted with maximum steady state limiting current

(Im) or its current density (Jm)

106

Electrochemical Michaelis-Menten equation

][][

SKSI

IM

m

+times

= middotmiddotmiddotmiddotmiddot Eq 54

][][

SKSJ

JAI

M

m

+times

== middotmiddotmiddotmiddotmiddot Eq 55

I = Current A

Im = Max steady-state limiting current A

[S] = Substrate concentration mol

KM = Michaelis-Menten constant mol

A = Electrode surface area m-2

J = Current density Am-2

Jm = Max steady-state limiting current density Am-2

There is often an offset at zero substrate concentration due to competing reactions and

trace contaminants and this must be subtracted before analysis

107

V

0E+00

2E-04

4E-04

6E-04

8E-04

1E-03

0 2 4 6 8 10 12 14 16

frac12 Jm

KM

Jm Am-2

[S] mM

Jm

Fig 32 Electrochemical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

KM and Jm (or V in biochemical enzyme kinetics) are important parameters to analyse

both enzyme catalytic power and stability Electrochemical Michaelis-Menten equation

and curves can be used for rough estimation of KM and Jm however it often leads to

inaccurate result because measuring initial rate for high substrate concentration is very

difficult in this method hence very prone to errors Therefore Michaelis-Menten

equation is linearised using one of the following four methods (i) Lineweaver-Burk (ii)

Eadie-Hofstee (iii) Hanes-Woolf and (iv) Cornish-Bowden-Wharton equations

Michaelis-Menten plot is however good for intuitive diagnosis of general enzyme

behaviours

108

263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics

2631 Lineweaver-Burk linear regression

Lineweaver-Burk linear regression is one of the classic regression methods

taking double reciprocal of Michaelis-Menten equation (Eq 56)

Electrochemical Lineweaver-Burk linear regression

mm

M

JSJK

J1

][11

+times= middotmiddotmiddotmiddotmiddot Eq 56

It plots 1J against 1[S] as shown in Fig 33 The value of each parameter is found by

extrapolating the line The reciprocal of Jm is found at y-intercept and KM can be derived

from either x-intercept or the slope once Jm is found

109

-50E+02

00E+00

50E+02

10E+03

15E+03

20E+03

-06 -05 -04 -03 -02 -01 00 01 02 03 04

1[S] mM-1

1J A-1m

2

Fig 33 Electrochemical Lineweaver-Burk linear regression

Based on the real data Refer to sect 343 p 124

Lineweaver-Burk linear regression is however largely influenced by the errors in j at

low substrate concentrations [22 147] Its reciprocal could vary by a factor of 80 over

the range of substrate concentration varying one-third to three times the KM [148]

Hence it is not an ideal method for the analysis

2632 Eadie-Hofstee linear regression

Eadie-Hofstee linear regression plots J against J[S] This plot is strongly

affected by the errors in υ because υ is present in both coordinates (Eq 57)

Electrochemical Eadie-Hofstee linear regression

mM JSJKJ +timesminus=

][ middotmiddotmiddotmiddotmiddot Eq 57

110

Again the plot must be extrapolated to find Jm at y-intercept KM can be derived from

the slope (Fig 34)

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

0E+00 5E-05 1E-04 2E-04 2E-04 3E-04

J [S] AmM-1

J Am

-2

Jm

-Km

Fig 34 Electrochemical Eadie-Hofstee linear regression Based on the real data

Refer to sect 343 p 124

Eadie-Hofstee linear regression will give very strict evaluation [148] and might be used

to detect the deviations from Michaelis-Menten behaviour only if the data is precise and

accurate [147]

2633 Electrochemical Hanes-Woolf linear regression

Hanes-Woolf linear regression plots [S]J against [S] KM is derived from the

x-intercept and Jm is found from the slope

111

Electrochemical Hanes-Woolf linear regression

m

M

m JKS

JJS

+times= ][1][ middotmiddotmiddotmiddotmiddot Eq 58

This is a better method compared to the previous two regression methods because all

the parameter values can be derived from the reasonable range of [S] and they donrsquot

heavily rely on the extrapolation [147] The data is normalised by [S] therefore does not

cause many errors

-20E+08

00E+00

20E+08

40E+08

60E+08

80E+08

10E+09

12E+09

14E+09

-40E+00 00E+00 40E+00 80E+00 12E+01 16E+01

[S] mM[S] J m

MA

-1m2

Fig 35 Electrochemical Hanes-Woolf linear regression Based on the real data

Refer to sect 343 p 124

However either of these three linear regressions might not be adequate for the enzyme

electrode analysis [65] because the data reflect not only the single-substrate

Michaelis-Menten kinetics but also include the influence from other factors such as

enzyme immobilisation mediator reactions and electrode composition as well as

112

general experimental errors Their contributions to the overall kinetics therefore must be

considered [65 149] although the enzyme electrode reactions can approximate the

Michaelis-Menten model when one substrate is a variable and other variables are fixed

over an experiment [147 149] Under such a complex system it is not ideal to use those

graphical linear regression methods because they assume that the results follow

Michaelis-Menten model and that all the experimental errors follow normal distribution

[150] without giving any measure in the precision [148] Hence it is more suitable to

use a non-parametric direct linear plot or nonlinear regression methods because they

respect the influence and errors from various sources without any bias

264 Cornish-Bowden-Wharton method

Cornish-Bowden-Wharton method is distribution-free non-parametric direct

linear plot In this method observations are plotted as lines in parameter space rather

than points in observation space [151] It then takes the median of Jm and KM from every

possible non-duplicate pair of observed [S] and υ (Eq 59 and Eq 60) as the best-fit

estimate

Electrochemical Cornish- Bowden-Wharton non-parametric method

])[][(])[]([

1221

122121 SJSJ

SSJJJm minusminus

= middotmiddotmiddotmiddotmiddot Eq 59

])[][()](][[

1221

122121 SJSJ

JJSSKM minusminus

= middotmiddotmiddotmiddotmiddot Eq 60

It is a simple method without complex calculation insensitive to outliers and no

113

weighting is required [147 152]

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

14E-03

16E-03

18E-03

20E-03

-5 -3 -1 1 3 5

KM mM

Jm Am-2Median Jm

Median KM

Fig 36 Electrochemical Cornish-Bowden-Wharton plot Based on the real data

Refer to sect 343 p 124

265 Nonlinear regression methods

Nonlinear regression methods manipulate the parameters to minimise the sum

of squared difference between the measured and expected values [150] They fit the

integrated equation to the enzyme progress curve and calculate the approximate location

of the minimum for the sum of squared difference at a particular point in the slope It

then recalculates the new minimum location to improve the prediction until there is no

further improvement This is called gradient method based on Gauss-Newton algorithm

or non-linear least square method [150 153] This is computationally intensive due to

114

the recursive operations however advanced computational technology has made it

possible The parameters obtained may not be perfectly correct [150] The method still

assumes errors normally distributed [153] and Cornish-Bowden has criticized about the

ambiguous statistical assumption of such computer programmes [154] However

non-linear regression methods are insensitive to outliers [150 153] have better estimate

accuracy compared to conventional linear regression methods and the estimations

mostly fit Michaelis-Menten model Graphical methods arose at a time when nonlinear

regression methods were computationally not available however it is now possible

thanks for the great technological advance and more recommended to use in analysis to

yield better precision and reliability [153]

115

3 ANODE MATERIAL AND METHOD

31 ELECTRODE FABRICATION

311 TTF-TCNQ synthesis

Tetrathiafulvalene ge980 (TTF) and 7788-Tetracyanoquinodimethane

ge980 (TCNQ) were purchased from Sigma Aldrich Spectroscopic grade acetonitrile

and diethyl ether were purchased from VWR International TTF and TCNQ were

weighted to 1 g each and dissolved separately in 50 ml of acetonitrile TTF-acetonitrile

solution had yellow colour while TCNQ-acetonitrile solution was green Both were

heated on the hot plate under the fume hood On the full dissolution two solutions were

quickly mixed to form black solid crystals The mixture was immediately taken off the

hot plate and cooled down at the room temperature for a few hours The crystals were

collected on Buchner funnel and washed under vacuum twice with acetonitrile twice

with diethyl ether and then twice again with acetonitrile The washed crystals were left

to dry for overnight with a cover The dried crystals were collected and kept in the

sealed container for future use [61]

312 Electrode template fabrication

Sylgard 184 Silicone Elastomer Kit were purchased from Dow Corning

Corporation for polydimethylsiloxane (PDMS) Base and hardener were mixed at 101

(vv) for use CY1301 and aradur hardener HY 1300 were purchased from Robnor

116

resins to make epoxy resin They were mixed at 31 (vv) for use

Silicone electrode templates (Fig 16(i)) were made to shape electrode discs

Each template had round holes in line Two types of templates template A and B were

made Template A had holes with the dimension 2-mm diameter x 1-mm thickness

while template B had holes with the dimension with 5-mm diameter x 2-mm thickness

In order to make PDMS templates for electrode discs different templates were made to

shape the holes in PDMS (Fig 16(ii)) Discs were made with epoxy resin and were

fixed onto the plastic Petri dish PDMS was poured into disk-mounted Petri dish

degassed for 20 minutes and then cured at 60 degC Cured PDMS electrode templates

were taken out from the epoxy resin-Petri dish templates and then cleaned with

analytical grade ethanol for use

Fig 37 PDMS electrode templates (i) PDMS template and (ii) their epoxy resin-Petri

dish template

117

313 Electrode composite fabrication

3131 Composite for conductance study

Graphite at microcrystal grade (2-15 micron 999995 purity) was purchased

from Alfa Aesar GmbH amp CoKG Glassy carbon (GC) powder was kindly donated from

Dr Zhao Carbon nanofibre (CNF) was also kindly donated from Loughborough

University Composite electrodes were built from the mixture of the carbon conductive

or TTF-TCNQ and epoxy resin Each composite mixture had different composition ratio

[108] as below in Table 4

Composite Ratio I Ratio II Ratio III Ratio IV Ratio V

Graphite Epoxy resin 73 64 55

GC Epoxy resin 64 73

CNF Epoxy resin 37 46 55 64 73

TTF-TCNQ Epoxy resin 64 73 82

Table 4 Composite composition

TTF-TCNQ was mixed with epoxy resin at 64 73 and 82 Graphite was mixed at 73

64 and 55 GC powder was mixed at 64 and 73 CNT was mixed at 37 46 55 64

and 73 (ww) Three to five copies were fabricated for each electrode type Composite

mixture was packed into the template A (Fig 38) and cured at room temperature for 72

hours [106]

118

Fig 38 PDMS template (i) packed with electrode material and (ii) empty one

3132 Bulk modified GOx enzyme electrode composite

Glucose oxidase (GOx) [Type II-S 15000-50000 unitsg] from Aspergillus

niger was purchased from Sigma Aldrich Conductive composite paste was first made

with TTF-TCNQ and epoxy resin at 73 as in sect 3131 GOx was then incorporated into

the conductive composite mixture at varying mixing ratio 5 10 and 15 (ww) by

bulk-modification method (Fig 39) 10 GOx composite electrodes were also made

with graphite Enzyme composite mixture was packed into the template B and cured at

4 degC for 96 hours

119

R

RR R

R

RR R

RRR

Conducting particles

Insulating binder

Chemical modification

Add biological receptor R

Cast extrude or print into suitable form

R

R

RR

R

R

R

R

R

R

RR

Fig 39 Schema for bulk modification method

3133 GOx enzyme electrode composite with enzyme stabilisers

Poly(ethyleneimine) (PEI) solution [50 (wv) in H2O] DL-dithiothreitol

(DTT) Poly(ethylene glycol) (PEG) [average mol wt 200] glycerol (GLC) [ge99] and

flavin adenine dinucleotide disodium (FAD) salt hydrate (ge95) were purchased from

Sigma Aldrich Each stabiliser was mixed with GOx at different fraction 0 1 2 and

4 PEI was also blended with DTT or FAD for synergetic effects as below in Table 5

Blend type Ratio I Ratio II Ratio III Ratio VI

PEIDTT 0515 1010 1505 2020

PEIFAD 21 22 24

Table 5 Blended stabiliser composition

120

PEI was mixed with DTT at the ratio of 0515 11 1505 and 22 PEI was also

mixed with FAD at 21 22 and 24 Each enzyme-stabiliser mixture was weighted to

10 of the whole composite It was then mixed with the conductive composite mixture

made earlier The conductive composite mixture was made of TTF-TCNQ and epoxy

resin at 73 The final composite mixture was packed into the template B and cured at

4 degC for 96 hours

314 Electrode wiring and insulation

Silver wire (temper annealed 025-mm diameter) was purchased from Advent

Portex polyethylene tubing (028 x 0165 mm) purchased from Scientific Laboratory

Supplies Silver epoxy was purchased from ITW chemotronics Silver epoxy came in

two parts A and B and they were mixed at 11 (vv) for use Silver wire was cut for

5-cm length and its tip was cleaned with analytical grade ethanol dried and coved with

insulating polyethylene tubes Cured electrode composite discs were popped out of the

PDMS template An insulated silver wire was fixed onto to each electrode disc with

conductive silver epoxy and it was left at room temperature for 24 hours to cure silver

epoxy After silver epoxy had been cured the wired electrodes were coated and

insulated with degassed epoxy resin and cured at room temperature for 3 days for

non-enzyme composite electrodes and 4degC for 4 days for enzyme composite electrodes

7-9 days were taken to build the complete composite electrodes (Fig 40) The enzyme

electrodes were stored at 4degC when not in use

121

Fig 40 Insulated composite electrodes (i) non-enzyme (ii) GOx electrodes

32 CONDUCTIVITY EXPERIMENT

Copper foil with electrically conductive acrylic adhesive was purchased from

RS Components Digital multimeter from Metra was used to measure resistance

Electrode surface was covered with adhesive copper foil evenly and both ends were

clamped with crocodile clips (Fig 41(i)) to measure electrode resistance of the

composite as in Fig 41(ii)

122

Fig 41 Measuring electrode resistance (i) connecting the electrodes to the

multimeter (ii) measuring the electrode resistance with the multimeter

33 POLISHING COMPOSITE ELECTRODES

MicroPolish II aluminium oxide abrasive powder (particle range 1 03 and

005 microm) silicone carbide (SiC) grinding paper for metallography wet or dry (grit 600

1200 and 2400) and MicroCloth Oslash73 mm for MiniMet for fine polishing were

purchased from Buehler Self adhesive cloth for intermediate polishing was purchased

from Kemet Electrodes were polished immediately before the experiment until

mirror-like flat surface was obtained This erases the electrode history and gives

assigned electrode surface area [105] Composite electrodes were hand polished

successively with SiC grinding paper in 400 1200 and 2400 grit During polishing

deionised water of 18 MΩ was added occasionally to the polishing surface This

prevents damaging electrode surface with frictional heat It was followed by successive

polishing with alumina oxide slurry with particle size 1 03 and 005 microm Slurry was

123

sonicated to dissolve agglomerates before use [155] Kemet polishing pad was used for

rougher polishing with 1 and 03 microm powder Felt-like MicroCloth was used for

polishing with 005microm Electrodes were sonicated in the deionised water briefly after

polishing at each grit or grade

34 GLUCOSE AMPEROMETRY

341 Glucose solution

Phosphate buffer saline (PBS) powder for 001 M at pH 74 containing 0138

M NaCl and 00027 M KCl [156] was purchased from Sigma Aldrich PBS was kept in

the fridge when not in use [157] 1M glucose solution was made by mixing

D-(+)-Glucose with 001 M PBS and left for 12 hours at 4degC for mutarotation It is

mandatory because GOx has stereochemical specificity and only active toward

β-D-glucose while glucose solution consists of two enantiomers α- and β-D glucose

and their concentration changes over time until they reach the equilibrium at 3862

Experiments should be carried out in the equilibrated solution for accuracy

342 Electrochemical system

A single-channel CHI 650A and multi-channel CHI 1030 potentiostats were

used for glucose amperometry with three-electrode system in which the

electrochemical cell contained (a) working electrode(s) an AgCl reference electrode

and a homemade Pt counter electrode The AgCl reference electrode was purchased

124

from IJ Cambria AgCl reference electrode was saturated with 3M KCl Pt counter

electrode was made of Pt wire and Pt mesh was fixed at its tip to give large surface area

Pt mesh at its tip

343 Glucose amperometry with GOx electrodes

Glucose amperometry was conducted by adding a known volume of 1M

glucose aliquot into 100 ml of 001 M PBS electrolyte solution containing GOx anodes

with convection applied Convection was however ceased on during current

measurement to avoid the noise The electrolytic solution was gassed with either N2 O2

or compressed air for 15 minutes before the experiment O2 concentration is 123 mM in

fully oxygenated PBS [158] and 026 mM in air [112] Gas was continuously supplied

through Dreschelrsquos gas washing bottle containing PBS during experiment The response

of GOx anodes to glucose was obtained as steady state current The amperometry was

carried on until no further current increase was observed against glucose added Each

experiment took more than 8 - 12 hours The sample size ranged from two to three in

most cases

125

Fig 42 Amperometry setting with CHI 1030 multichannel potentiostat

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA

Normality of enzyme function was confirmed by plotting Michaelis-Menten

curve based on the amperometry data obtained The capacitive current before glucose

addition was subtracted from the data Enzyme kinetic parameters the maximum

current density (Jm) and the enzyme dissociation Michaelis constant (KM) were

automatically estimated with standard error using an analytical software called

SigmaPlot Enzyme Kinetics version 13 The estimation is based on non-linear

regression analysis about Michaelis-Menten kinetics as shown in Fig 43

126

Glucose M

0 1e-2 2e-2 3e-2 4e-2 5e-2

J A

m-2

00

20e-3

40e-3

60e-3

80e-3

Vmax = 0008445 Km = 0005842

Fig 43 Analytical output by SigmaPlot Enzyme Kinetics Based on the real data

Influence of enzyme immobilisation electrode composition O2 and stabilisers on GOx

electrode performance was studied The most efficient GOx anodes was determined on

the basis of the parameters obtained

127

4 ANODE RESULTS AND DISCUSSION

41 AIMS OF ANODE STUDIES

This section describes the fundamental study on the enzyme composite

electrodes and the influence of enzyme stabilisers on the performance and stability of

GOx electrodes Firstly the difference in conductivity as a function of various

conductive materials and the component fraction were studied They did not contain

enzyme The most suitable composite was then selected and used to build the enzyme

electrodes Secondly GOx electrodes were built at various GOx fractions in the

composite The best component fraction was determined from the data obtained in

amperometry Amperometry was conducted to obtain two enzyme kinetic parameters

the maximum current density (Jm) and the enzyme dissociation Michaelis constant (KM)

Jm indicates the size of catalytic velocity KM indicates the extent of enzyme-substrate

dissociation Smaller KM values indicate better enzyme-substrate affinity and enzyme

stability With the component ratio selected enzyme electrodes were then constructed

using various types and concentrations of enzyme stabiliser Amperometry was again

conducted to study the influence of enzyme stabilisers on the GOx anode performance

and stability The catalytic efficiency was determined by the fraction M

mK

J This is

based on the concept of specificity constant M

catK

k as defined below

128

Enzyme electrode specificity constant

MMM

cat

KEV

Kk

Kk

sdot==

][2 middotmiddotmiddotmiddotmiddot Eq 61

where kd = k2 Rate constant for P formation from ES

The specificity constant is a common measure of the relative catalytic rate at low

substrate concentration [22] M

mK

J therefore can be used as a comprehensive

indicator for both catalytic velocity and enzyme stability under the fixed enzyme

concentration In this study GOx concentration was fixed to approximately 10 weight

of the total composite According to the M

mK

J obtained the five most efficient and

stable GOx electrodes were determined The possible enzyme stabilisation mechanisms

of each enzyme stabiliser were also discussed together in the light of published

hypothesises

42 CONDUCTANCE OF THE COMPOSITE

421 Conductance of various conductive materials

Various electrodes were built with different conductive materials graphite GC

CNF and TTF-TCNQ at different component fraction The electrode dimension was

fixed to 314 mm2 x 1 mm Their conductance was measured and presented in a box plot

as shown in Fig 44 The sample size ranged from 3 to 6 The median is indicated by a

short line within the box

129

00E+00

50E-02

10E-01

15E-01

20E-01

25E-01

30E-01

35E-01

40E-01

45E-01C

ondu

ctan

ce

S

Lower quartile S 103E-03 549E-03 219E-01 111E-01 142E-01 599E-04 390E-03 115E-02 972E-03

Max S 281E-03 338E-02 268E-01 312E-01 415E-01 810E-04 113E-02 467E-02 262E-02

Min S 739E-04 467E-03 200E-01 933E-02 855E-02 418E-04 296E-03 264E-03 587E-03

Upper quartile S 206E-03 282E-02 253E-01 220E-01 253E-01 795E-04 807E-03 232E-02 199E-02

Median S 131E-03 847E-03 238E-01 129E-01 216E-01 780E-04 484E-03 155E-02 136E-02

TTF-TCNQ64

TTF-TCNQ73

Graphite 55 Graphite 64 Graphite 73 CNF 37 CNF 46 CNF 55 GC 73

Fig 44 Conductance of various electrode composite n = 3 ndash 6

Graphite composites showed higher conductance than other composites It is explained

by graphite having the highest intrinsic conductivity ranging between 103 ndash 104 Scm-1

[105 159] although being compounded in a composite lowered the conductivity by 4 ndash

5 order of magnitude compared to that of raw material The conduction did not show a

linear relationship with the electrode composition ratio for graphite composite because

the graphite composite 55 had larger conductance of 024 S than that of 64 and 73 It

also showed the smallest deviation than the other graphite composites while the

deviation increased with increase in the graphite content One possible cause of this is

the presence of patchy conductive paths due to the uneven distribution of conductive

islands within the composite [105 106] The conduction of the composite is therefore

often locally biased unpredictable and also leads to low reproducibility TTF-TCNQ

composite at 73 showed the conductance of 85 mS smaller than the graphite

composited 55 approximately by 30 times Conductivity of TTF-TCNQ is 102 ndash 103

130

Scm-1 [109 110] and smaller than that of graphite by one order of magnitude so this is

consistent with the intrinsic conductivity of the conducting paticles

There were also different interactions observed among conductive materials

with insulating materials Mixing epoxy resin with graphite was easier than mixing with

other conductive materials Some even did not cure at the same mixing ratios The

carbon nanofibre (CNF) composite mixture did not cure when it exceeded more than

50 weight content With 40 CNF content however it showed the conductance of 48

mS It was not comparable to graphite composite despite its similar conductivity of 103

S [105] however it was similar to that of TTF-TCNQ 73 composite 50 CNF content

even showed larger conductance of 155 mS than 70 TTF-TCNQ CNFs have been

reported to have mediating function [117] therefore with this high conduction property

at low content CNF might be very useful to enzyme composite and even superior to

TTF-TCNQ

422 Influence of enzyme concentration on conductivity

Conductance of the GOx composite was measured The conductive composite

consisted of either TTF-TCNQ 73 or graphite composite 73 GOx content was varied

by 5 10 and 15 for TTF-TCNQ composite and 10 only for graphite composite

The electrode dimension was fixed to 196 mm2 x 2 mm The sample numbers ranged

from 2 ndash 4 The study revealed that the presence of enzyme interfered with conduction

in GOx concentration dependent manner for TTF-TCNQ composite as shown in Fig 45

131

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01C

ondu

ctan

ce

S

Low er quartile S 131E-02 924E-03 124E-05 165E-01

Max S 484E-02 143E-02 368E-05 593E-01

Min S 120E-02 754E-03 390E-06 122E-01

Upper quartile S 441E-02 126E-02 288E-05 400E-01

Median S 281E-02 109E-02 208E-05 207E-01

TTF-TCNQGOx 5 TTF-TCNQGOx 10 TTF-TCNQGOx 15 GraphiteGOx 10

Fig 45 Conductance of GOx composite with various GOx content n= 2 - 4

This was due to insulating property of the protein although higher enzyme loading is

preferred for better catalytic performance as discussed in sect 226 on p 88 The issue

may be circumvented by fixing the enzyme onto the electrode surface It is however

more technically tedious and not as feasible for mass manufacture as bulk modification

It is also difficult to modify conductive salt for covalent attachment of the enzyme This

is because the conductive property of conducting salt is very sensitive to its chemical

structure and slight modification can lead to insulating properties [61] A similar

phenomenon has been also observed in CNT functionalisation [117]

Graphite-GOx composite showed larger conductance than TTF-TCNQ-GOx

composite by 20 times at the same component fraction This was due to the better

conductive property of graphite However graphite does not have mediating property

and the use of graphite as conductive material for enzyme electrodes requires the

132

loading of mediator separately The key to designing enzyme electrodes is in the balance

and compromise among the conductivity mediation enzyme activity mechanical and

chemical stability and difficulty in fabrication process A good candidate material to

make up for the TTF-TCNQ lower conductivity is CNT It has been reported that CNF

has both good conductivity and mediating functions [117] and might be suitable for bulk

modification More safety study on use of CNF is however necessary [117 160]

43 CATALYTIC ACTIVITY OF GOx COMPOSITE ELECTRODES

431 Influence of GOx concentration on the catalytic activity of GOx electrodes

Amperometry was conducted for GOx composite electrodes in N2 O2 and air

for various GOx content The conductive composite consisted of either TTF-TCNQ 73

or graphite composite 73 GOx content was varied by 5 10 and 15 for

TTF-TCNQ composite and 10 only for graphite composite The sample size was 3 for

each electrode type Control experiments were also performed with enzyme-free

composite electrodes and there was no glucose response observed There was also no

stable current observed for 10 GOx-graphite electrodes despite of its high

conductivity reported in sect 422 while Michaelis-Menten curve was observed in any

TTF-TCNQ-GOx composite electrodes This confirmed the necessity of mediating

function for enzyme anodes and its presence in TTF-TCNQ

All the TTF-TCNQ-GOx electrodes showed the Michaelis-Menten current

response curves however differed in the current as shown in Fig 46

133

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

35E-02

00E+00 10E-02 20E-02 30E-02 40E-02 50E-02

Glucose M

J A

m-2

GOx 5GOx 10GOx 15

Fig 46 Difference in the glucose-response amperometric curve of enzyme

electrodes for various GOx content 5 10 and 15 in the presence N2

The 15 GOx composite showed the smallest glucose response current due to the

interfered conduction by high protein content while 5 GOx composite showed smaller

response than 10 composite due to the enzyme deficiency Their Jm only counted for

26 and 41 of that of 10 GOx composite (Fig 47(i)) 10 GOx electrode was

therefore most catalytic KM for 5 and 10 GOx were 71 and 73 mM and little

different (Fig 47(ii)) This implies that the size of catalytic activity can be flexibly

altered by only slight change in KM

134

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

Jm

Am

-2

Jm Am-2 729E-03 180E-02 466E-03

GOx 5 GOx 10 GOx 15

00E+00

20E-03

40E-03

60E-03

80E-03

10E-02

12E-02

14E-02

Km

M

KM M 710E-03 729E-03 187E-03

GOx 5 GOx 10 GOx 15

Fig 47 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 n = 1 - 3

The results also tell that an increase in GOx loading could enhance the catalysis hence

output current unless the increase is not excessive and gt 15 at least The loading limit

should lie somewhere between 10 and 15 for this composite Similar electrodes

were also built by Alegret with 75 epoxy resin 10 TTT-TCNQ 10 graphite and

5 GOx [115] Their Jm was around 7 mAm-2 and little different from that of 5 GOx

with 70 TTF-TCNQ composite It suggests that less TTF-TCNQ content might be

sufficient as long as the conductivity is sustained by more conductive material such as

graphite

432 Influence of O2 on the catalytic activity of GOx electrodes

O2 and air suppressed the current output by GOx composite electrodes and

delayed the response to the glucose as shown in Fig 48 A current increase was

observed immediately after glucose addition for N2 but not for O2 and air They showed

135

sigmoidal current curves with a glucose response lag

(i)

(ii)

-50E-03

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

00E+00 50E-03 10E-02 15E-02 20E-02 25E-02 30E-02 35E-02 40E-02 45E-02

Glucose M

J A

m-2

GOx 10 N2GOx 10 O2GOx 10 Air

Fig 48 Difference in the glucose-response amperometric curve of 10 GOx

composite electrodes in the presence (i) N2 and O2 and (ii) re-plotted

amperometric curves for N2 air and O2

136

As expected O2 and air decreased Jm and increased KM as shown in Fig 49

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

GOx 5 GOx 10 GOx 15

Jm

Am

-2

N2O2Air

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

70E-02

80E-02

GOx 5 GOx 10 GOx 15

Km

M

N2O2Air

Fig 49 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 O2 and air n = 1 - 3

Both O2 and air decreased the current of 15 GOx composite electrodes Jm dropped to

approximately 20 of that for N2 KM was increased by 10 and 2 times for O2 and air

respectively The glucose response lag was 8 mM for O2 but none for air Air showed

milder suppression toward current output than O2 and this was expected because O2

counts for only 20 of the air [31] and should cause less influence on GOx activity

Similar phenomena were observed for 10 GOx composite electrodes For O2 and air

Jm was 13 and 40 of that of N2 and decreased to 23 x 10-3 and 67 x 10-3 from 18 x

10-2 Am-2 respectively KM was 73 for N2 and 10 mM for both O2 and air although there

was a response lag of 8 and 1 mM respectively Little difference in KM was observed

among all cases and this implies that KM itself hence enzyme stability or substrate

affinity is little affected by O2 but slight change in KM has large influence on the

catalytic activity This implies that change in catalytic activity induced by O2 is

137

reversible 5 GOx composite electrodes showed slightly unexpected result KM was

increased by approximately 4 times in both O2 and air Jm also decreased however Jm

for air was smaller than O2 by 36 despite of less O2 content There was a response lag

by 10 mM for O2 and 2 mM for air

The overall conclusions from these results is that O2 reduces GOx catalytic rate

andor electron transfer rate and hence reduces the current output but the effect is

reversible because it hardly alters KM value implying that O2 does not affect the enzyme

stability or its affinity to the substrate This phenomenon is typical in non-competitive

inhibition for enzyme kinetics Non-competitive inhibition occurs when an inhibitor

binds to the enzyme-substrate complex and slows its catalysis It therefore changes

catalytic velocity but not KM In the glucose amperometry this implies two possibilities

as a cause of Jm reduction (i) allosteric binding of O2 to GOx-glucose complex or (ii)

interference in electron transfer after the catalysis In the former case O2 does not

inhibit glucose binding to GOx but allosterically interacts with its complex and hinders

its catalysis hence it reduces catalytic current output In the latter case either glucose

binding or GOx catalysis is not affected by O2 but competition for electrons produced

by GOx catalysis occurs between the natural and artificial mediators namely O2 and

TTF-TCNQ and this reduces the electron flow to the electrode therefore it is reflected

as the reduction of catalytic current In addition to this the presence of glucose response

lag also suggests the presence of electron competition [161-163] It was reported that O2

reacts with reduced GOx faster than any other mediators with the second order rate

reaction of 15 x l06 M-1s-l [112 161 163] It is therefore quite possible that O2 swiftly

deprives electrons from reduced GOx andor reduced mediators Reduced GOx reacts

with O2 much faster than with TTF-TCNQ because it was observed that O2 had

138

diminished glucose response current at low glucose concentration Most GOx hence

reacts with O2 first then oxidised TTF-TCNQ once most O2 has been converted into

H2O2 This is compatible with the hypothesis suggested by Hindle [161] Even after the

lag the reaction could go on with residual O2 because KM did not recover fully to the N2

level although a familiar Michaelis-Menten hyperbolic curve was regained This simply

indicates that O2 concentration became smaller than TTF-TCNQ to oxidise GOx The

glucose response lag also decreased with an increase in GOx loading and was smaller

for air than O2 This implies the presence of competition because the lag depends on the

concentration of reduced GOx and electron thieves The large KM increase in the 5

GOx composite electrodes was probably due to the deficiency in the electron transfer

rate to suppress O2 activity How O2 interacts with its environment is unknown however

O2 reduces the catalytic output current due to the following possible mechanisms (i)

electron competition between O2 and TTF+ (ii) non-competitive enzyme inhibition by

allosteric binding of O2 to GOx (iii) direct interaction between O2 and reduced TTF or

(iv) backward reaction between reduced TTF and oxidised GOx

139

Table 6 Four possible causes of glucose amperometric current reduction

(i) middotmiddotmiddotmiddotmiddot Eq 62

middotmiddotmiddotmiddotmiddot Eq 63

(ii) middotmiddotmiddotmiddotmiddot Eq 64

(iii) middotmiddotmiddotmiddotmiddot Eq 65

(iv) middotmiddotmiddotmiddotmiddot Eq 66

Diagnosis can be made by doing experiments with different concentration of O2 and

mediators

44 THE EFFICACY OF GOX STABILISERS

441 Introduction

Various GOx stabilisers PEI DTT PEG GLC and FAD were mixed with GOx

composite at the concentration varying among 1 2 and 4 PEI was also mixed with

DTT or FAD and their synergetic effects were studied All the GOx electrodes were

built on the basis of TTF-TCNQ 73 conductive composite with 10 weight

GOx-stabiliser mixture Control composite electrodes were built with 2 stabilisers but

without GOx Glucose amperometry was conducted as before in the both presence and

absence of O2 Two kinetic parameters Jm and KM were obtained through non-linear

regression analysis The standard kinetic parameter values were set to those obtained in

140

the absence of O2 for GOx electrodes containing no stabiliser They are Jm = 18 x 10-2 plusmn

73 x 10-3 Am-2 and KM = 73 plusmn 57 mM respectively The glucose response lag

observed for this electrode was 10 mM Comprehensive catalytic efficiency was

determined by the size of the fraction M

mK

J under the influence of high O2 and the

most catalytic GOx composite electrodes were chosen on the basis of M

mK

J The

sample size ranged between 2 and 3 None of control experiments showed any glucose

response

442 PEI

Kinetic parameters for various PEI concentrations are shown below in Fig 50

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser PEI 1 PEI 2 PEI 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEI 1 PEI 2 PEI 4

KM

M

N2 O2

Fig 50 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEI in the presence and absence of O2 n = 1 - 3

141

In the absence of O2 PEI lowered Jm without largely affecting KM compared to the

standard value This might imply the presence of allosteric non-competitive inhibition

of glucose catalysis It is certainly possible because PEI can lock in the enzyme

conformation with hydrogen and electrostatic bonding [120] or form a complex with

GOx [126 130] This interferes in its catalysis although it also might confer the

resistance against oxidative stress as reported [126] In the presence of O2 two out of

three electrodes of PEI 1 and 4 did not show Michaelis-Menten curves The data

presented here is therefore based on a single sample This may mean that the electrodes

at those PEI concentrations are in fact prone to the catalytic suppression by O2

Moreover 4 PEI data presented here looks unreliable due to the simultaneous increase

in Jm and KM by 3 ndash 4 times compared to those obtained in the presence of N2 It may

need more data to confirm the phenomenon and be premature to conclude it is however

probably more reasonable to think that PEI 4 is not suitable for GOx anodes At other

PEI concentrations electrodes showed more or less similar kinetic parameter values to

those without stabilisers since Jm decreased and KM increased in the presence of O2

There was no O2-scavenging function observed because their glucose response lag and

KM resembled those without stabilisers Among them 2 PEI containing electrodes

sustained 50 of catalytic activity obtained in N2 and showed marginally better Jm than

stabiliser-free electrodes in the presence of O2 It might therefore have some resistance

against O2 It can be however concluded that PEI is not suitable as an enzyme stabiliser

and not effective enough to enhance the catalytic current output because their Jm was

already smaller by 60 at minimum in N2 compared to the standard value and little

improvement in catalytic performance was observed in the presence of O2

142

443 DTT

Kinetic parameters for various DTT concentrations are shown below in Fig 51

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

Jm

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

KM

M

N2 O2

Fig 51 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 DTT in the presence and absence of O2 n = 3

All DTT containing electrodes showed smaller Jm and KM than the standard values in

the absence of O2 This might imply the possible presence of uncompetitive inhibition

by DTT although the size of inhibition was not in concentration dependent manner In

uncompetitive enzyme inhibition inhibitors bind both free enzyme and

enzyme-substrate complexes hence decreases both catalytic velocity and KM [22] In the

presence of O2 all Jm KM and glucose response lag were larger than those of

stabiliser-free electrodes The increase in glucose response lag was probably due to both

electron competition and uncompetitive inhibition while the inhibition by DTT itself

was less influential on catalytic velocity than non-competitive inhibition by O2 itself

143

and this resulted in larger Jm despite of their larger KM The truth of O2 enzyme

inhibition might be reversible Met oxidation as discussed in sect 242 on p 93 because

atmospheric O2 could oxidise Met residue [123] and DTT might be capable of reducing

it back although DTT has been reported to be effective only in reducing disulphide [127

132] The increase in Jm in the presence of O2 compared to N2 was probably due to the

reduced content of DTT working toward stabilising enzyme structure This freed up

some GOx portion from rigid conformation

444 PEG

Kinetic parameters for various PEG concentrations are shown below in Fig 52

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEG 1 PEG 2 PEG 4

J m

Am

-2

N2 O2

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

No Stabiliser PEG 1 PEG 2 PEG 4

KM

M

N2 O2

Fig 52 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEG in the presence and absence of O2 n = 2 - 3

PEG decreased Jm to 10 of the standard value at maximum in the absence of O2

144

although 4 PEG electrodes showed much larger Jm than the other PEG electrodes

unexpectedly 4 PEG however showed glucose response lag gt 10 mM even in the

absence of O2 The outcome of low Jm is compatible with the result reported by Lee

[139] while among PEI electrodes Jm however increased with PEG content and this

agreed with the results presented by Arakawa [138] The hypothesis by both authors

therefore might be correct but stabilisation mechanism might differ at different

concentrations Since KM was lowered at low PEG content compared to the standard

value PEG might have penetrated the protein core and stabilised the enzyme structure

[164] however it also lowered GOx activity because the protein conformation has been

locked in At 4 PEG concentration PEG still penetrated the protein core but residual

PEG also brought the preferential hydration and this allowed more flexible enzyme

conformation and increased the catalytic activity The residual PEG however might also

have an adverse effect on electron transfer rates at low glucose concentration In the

presence of O2 KM was increased compared to when in N2 and larger than that of

stabiliser-free electrodes while Jm only marginally better compared to that except for 4

PEG It is altogether not clear about the interaction between GOx and PEG however

only high PEG content effectively enhances the catalytic current output

445 GLC

Kinetic parameters for various GLC concentrations are shown below in Fig 53

145

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser GLC 1 GLC 2 GLC 4

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

20E-02

No Stabiliser GLC 1 GLC 2 GLC 4

KM

M

N2 O2

Fig 53 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 GLC in the presence and absence of O2 n = 3

In the absence of N2 GLC showed smaller Jm and KM compared to the standard values

This indicates the presence of uncompetitive inhibition and the decrease in Jm showed

the reciprocal dependency on GLC concentration It is also quite possible that

concentration-dependent inhibition was due to the tighter protein packing by GLC as

previously reported [144] This might contribute more to non-competitive inhibition

though GLC-containing electrodes showed more resistance against O2 because their Jm

was larger than that of stabiliser-free electrodes and again it showed reciprocal

dependence on GLC concentration In both N2 and O2 cases Jm decreased with increase

in GLC content KM also showed the dependence on GLC concentration in the presence

of O2 and increased with GLC concentration Glucose response lag was also decreased

in all cases Among all 1 GLC was most effective because it lowered KM for both N2

and O2 and increased Jm for O2 although it decreased Jm by 40 in N2 It also decreased

glucose response lag from 8 to 5 mM and this might indicate that glycerol reduced and

scavenged O2

146

446 FAD

Kinetic parameters for various FAD concentrations are shown below in Fig 54

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

No Stabiliser FAD 1 FAD 2 FAD 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser FAD 1 FAD 2 FAD 4

KM

M

N2 O2

Fig 54 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 FAD in the presence and absence of O2 n = 1 - 3

FAD may be reasonably effective as an enzyme stabiliser at concentration gt 1 because

its Jm was larger in both absence and presence of O2 compared to that of stabiliser-free

electrodes It also slightly decreased glucose response lag therefore it might be able to

react with oxidising species as previously reported [108] It is however only effective at

higher concentration because two out of three 1 FAD electrodes did not show

Michaelis-Menten curves in the absence of O2 and none worked in its presence although

4 FAD was not most effective either The relationship between increase and decrease

in Jm and KM in N2 is unknown Improvement in electron transfer was not particularly

observed because higher FAD content decreased the current output although FAD has a

147

function as a mediator Mechanism of GOx stabilisation by FAD is not clear as Bartlett

reported [145] however 2 FAD was shown to be most effective among all and the only

one which showed M

mK

J gt 1 for both N2 and O2

447 PEI and DTT mixture

Kinetic parameters for various PEI-DTT concentrations are shown below in

Fig 55

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

KM

M

N2 O2

Fig 55 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

PEI-DTT (051 11 1505 and 22) in the presence and absence of O2 n =

1 - 2

PEI-DTT stabiliser mix showed the most dramatic effect and promising as a stabiliser

formulation In the presence of O2 it had larger Jm and smaller or equivalent KM than

those found in stabiliser-free electrodes Glucose response lag was also decreased from

148

8 to 3 mM and this implies the presence of O2 scavengers which were new synergetic

effects achieved and not observed in use of either stabilisers as discussed in sect 442 and

443 In the absence of O2 most PEI-DTT showed smaller Jm and marginally smaller

KM than those of the standard values except for some as it can be seen Fig 55 This

probably implies the presence of non-competitive-inhibition like effect due to the

stabilisation of enzyme structure by PEI-DTT Since it was difficult to determine the

comprehensively most effective mixture and the relative relationship in the increase and

the decrease in the two kinetic parameter values were not obvious M

mK

J was found

and compared for each PEI-DTT mixture as shown below in Fig 56

00

05

10

15

20

25

30

35

40

45

50

1505 11 0515 22

PEIDTT ()

Jm

KM

(Am

-2M

-1)

N2O2

Fig 56 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051

11 1505 and 22)

It is clear that DTT must exceed 1 to achieve in the fraction to achieve efficient O2

resistance Since the equivalent glucose response lag was observed in all mixtures this

implies that at least 1 DTT is required for simultaneous reduction of Met amino acid

149

residue On the other hand gt 2 PEI seems required for high catalytic efficiency in N2

or equal weight PEIDTT mixing is preferable to increase catalytic efficiency in both N2

and O2 Since PEIDTT at 22 data presented here is based on a single sample the study

on more samples is required to confirm the results however PEIDTT mixture with

DTT gt 1 is effective to improve catalytic efficiency and output current

448 PEI and FAD mixture

Kinetic parameters for various PEI-FAD concentrations are shown below in

Fig 57

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

J m

Am

-2

N2 O2

00E+00

20E-02

40E-02

60E-02

80E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

KM

M

N2 O2

Fig 57 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with PEI-FAD (21 22 and 24 ) in the presence and absence of O2 n = 2

Firstly the result clearly showed total 6 stabiliser content hindered catalysis because it

showed extremely low Jm compared to other PEIFAD containing electrodes It only

counts for 15 and 9 of Jm obtained for other PEIDTT concentration in presence and

150

absence of O2 respectively Secondly there was no large difference in the performance

between PEIFAD at 21 and 22 hence PEIFAD 21 was most effective of all This

might involve non-competitive inhibition because Jm was smaller by 70 while KM was

smaller by only 20 compared to the standard values in the absence of O2 In the

presence of O2 it showed larger Jm by 7 times however also increased KM by 3 times

compared to those of stabiliser-free electrodes and did not decrease the glucose response

lag

449 Most effective stabiliser content for GOx anodes

To determine the size of comprehensive catalytic efficiency M

mK

J was

found for all the stabilisers and its value was compared for O2 for catalytic efficiency in

the presence of O2 Among all five most efficient catalysts were selected and presented

below in Fig 58

151

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

J m

Am

-2

N2 Am-2O2 Am-2

N2 Am-2 180E-02 125E-02 827E-03 292E-02 710E-03 104E-02O2 Am-2 168E-02 181E-02 151E-02 267E-02 122E-02 157E-02

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505 GLC 1

00E+00

50E-03

10E-02

15E-02

KM

M

N2 MO2 M

N2 M 729E-03 671E-03 576E-03 647E-03 938E-03 328E-03

O2 M 104E-02 471E-03 433E-03 801E-03 658E-03 886E-03

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505

GLC 1

Fig 58 Jm KM in the absence and presence of O2 for various stabilisers PEIDTT

(11 0515 221505 ) and GLC 1

PEIDTT mixture was found to be most effect as GOx stabiliser as discussed in sect 447

For Jm PEIDTT 22 showed the largest in both absence and presence of O2 and this

was followed by PEIDTT 11 For KM PEIDTT 0515 showed the smallest

comprehensively for both N2 and O2 and this followed by PEIDTT 11 PEIDTT

22 and GLC 2 showed relatively large KM in the presence of O2 It can be therefore

concluded that PEIDTT 11 is the most effective in stabilising GOx has largest

resistance against O2 and increases the catalytic output current It showed Jm = 13 x 10-2

plusmn 19 x 10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2

with KM = 47 plusmn 42 mM for O2 The current output obtained is much lower than some

reported results by Willner Heller and Sasaki [18 19 97] by 2 ndash 3 order of magnitudes

their electrodes however involved more complex fabrication methods or system as

discussed sect 22 on p 77 On the other hand it was slightly better than the result

reported by Merkoci who composed a similar composite with 5 GOx and was similar

to the results reported by Arai who built GOx electrodes by more complex methods KM

152

obtained is also smaller by 2 ndash 4 times than some of results by Arai [103]

Max current output Am-2 KM mM Method involved Author

125 x 10-3 ndash 181 x 10-2 47 ndash 67 10 GOx composite Data botained

55 x 10-3 Not available 5 GOx composite Merkoci [115]

30 Not available Apo-GOx reconstituted Willner [97]

11 x 10 Not available Os-complex hydrogel Heller [19]

18 Not available Saccharide-modified polypyrrole Fujii [100]

54 x 10-2 Not available QS conductive polymer Arai [103]

41 x 10 13 - 20 Poly(acrylic) acid hydrogel modified

carbon fibre GDH electrodes

Sakai [18]

Table 7 Comparison of maximum output current and KM with some published data

45 CONCLUSION TO ANODE STUDY

Bulk modified enzyme electrodes are good because they are mechanically very

stable and their fabrication is straightforward and scaleable requiring only mixing

electrode components conductive material mediators enzymes and stabiliser with

binding material TTF-TCNQ is useful material for GOx electrodes because it is

conductive as well as mediating The electrode component ratio is important for its

function and conductivity High binder and protein content hinder the conduction It was

found that 73 weight ratio of TTF-TCNQepoxy resin with 10 GOx loading optimal

The balance among the conductivity mediation enzyme activity mechanical and

153

chemical stability and difficulty in fabrication process is key to the successful

fabrication

O2 is known to be the natural mediator of GOx In the glucose-oxygen fuel

cells GOx anode must be resistant to O2 because O2 is an electron acceptor at the

cathode O2 lowers the current output by competing for electron transfer between GOx

and artificial mediators as well as non-competitive inhibition Various GOx stabilisers

were examined to alleviate such O2 influence

PEI DTT PEG GLC FAD PEIDTT and PEIFAD mixtures were studied as

candidate stabilisers to enhance catalytic activity and the current output PEI was not

effective It stabilised the structure but made conformation more rigid and did not

increase the output current in both the absence and presence of O2 DTT was reasonably

effective It showed uncompetitive inhibition in N2 but stabilised the enzyme structure

It therefore increased Jm and KM in the presence of O2 but increase in KM was probably

due to alleviating inhibition by O2 and loosening up the enzyme conformation PEG was

not effective It probably stabilised the enzyme structure but not show much

improvement for O2 competition It might also deprive electrons from reduced GOx

GLC was effective The effect was concentration dependent Lower concentration was

more effective It also scavenged O2 FAD was reasonably effective but only at higher

concentration PEI-FAD was also reasonably effective It decreased Jm but Km was

marginally equivalent to that of stabiliser-free electrodes for N2 But it increased Jm for

O2 Among all PEIDTT at 11 mixture was found to be most effective because it

stabilised the GOx and scavenged O2 Such powerful influence was not observed in the

use of either stabilisers hence it is a synergetic effect It showed Jm = 13 x 10-2 plusmn 19 x

154

10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2 with KM

= 47 plusmn 42 mM for O2 Although it lowered Jm by 30 in the absence of O2 compared

to the standard value of 18 x 10-2 plusmn 73 x 10-3 Am-2 it also decreased KM in both

absence and presence of O2 and increased Jm by 8 times compared to that of

stabiliser-free electrodes PEIDTT 11 is therefore most effective in stabilising GOx

has largest resistance against O2 and increases the catalytic output current

155

5 CATHODE INTRODUCTION

51 OXYGEN REDUCTION REACTION (ORR)

The oxygen reduction reaction (ORR) takes place at the cathode as oxygen

accepts electrons from the anode Oxygen is an ideal oxidiser for the cathode because it

is readily available and its reaction product water is harmless [62] However ORR has

been a long-time bottleneck especially for low-temperature fuel cells because it causes

large potential loss [3 4 62 165-167] due to a strong double bond in the dioxygen

molecule Its difficult bond dissociation causes slow irreversible kinetics [24 165 166]

so a catalyst is required for this reaction to proceed at useful rates [62 165] ORR is a

multi-electron reaction which involves various reduction pathways and different

oxygen intermediates (Fig 16) In aqueous solution either the direct four- or

two-electron ORR pathway may proceed [165 166] The direct four-electron transfer

pathway reduces O2 into H2O in acid and neutral mediaor OH- in alkaline media This

occurs via several reduction steps [24 165 166] which may involve adsorbed peroxide

intermediates These peroxide intermediates never diffuse away in the solution in this

pathway [166] The two-electron transfer pathway reduces O2 into peroxide species as

final products These peroxide species either diffuse away are reduced further or

decomposed into H2O or OH- [24 165 166] This is not favourable because it leads to

lower energy conversion efficiency and its product hydrogen peroxide may degrade the

catalysts and the fuel cells [165 167] Four- and two-electron transfers also may occur

in series or in parallel [165]

156

Fig 59 Oxygen reduction pathways [165 166]

Reduction kinetics and mechanisms depend on the type of catalysts electrode material

electrode surface condition solution pH temperature contaminant and reactant

concentration [24 165 166] Rate-limiting step also varies by type of the catalysts

[166] However the first electron transfer to dioxygen leading to the formation of

superoxide anion radical (O2־) is the rate limiting step [168] O2־ is very rapidly

protonated in water and converted to hydroperoxyl radical (HO2)

157

Fig 60 Degradation of superoxide anion radical in aqueous solutions [24]

In aqueous solution O2־ is unstable and rapidly converted to O2 and H2O2 via a

proton-driven disproportionation process (Fig 60) [24]

52 REACTIVITY OF OXYGEN

A dioxygen molecule is a homodiatomic molecule consisting of two oxygen

atoms double bonded It has two parallel unpaired electrons in degenerate anti-bonding

π designated as π orbitals therefore it is diradical and paramagnetic (Fig 61) [169]

158

Fig 61 An energy level diagram for ground state dioxygen molecule [169]

When one electron is transferred to a dioxygen molecule the electron is placed in π

orbital and it turns into a reactive O2[24] ־ reducing the bond order from 2 to 15

Oxygen reduction changes the molecular symmetry or stretches the double bond

causing bond disruption [165 166] Electron paring in one of the π orbitals also

changes their orbital energy state and lifts the degeneracy as in Fig 62 [170]

Hereogeneous catalysis involves chemisorption of the oxygen and therefore some

transfer of electron density from the oxygen to the catalyst This facilitates the rate

determining initial electron transfer Oxygen reduction catalysts are reviewed in the

next section

159

(O2) 3Σg- O2־

Fig 62 Diagrams for the state of π orbitals in the ground state oxygen (3Σg-) (left)

and peroxide anion radical (right) [24]

O2 species Bond Order Bond length pm Bond energy kJmol-1

O2 2 1207 493

O2+ 2frac12 1116 643

O2- 1frac12 135 395

O22- 1 149 -

Table 8 Bond orders bond lengths and bond energies of some dioxygen species [169]

53 ORR CATALYSTS

Catalysts are required to reduce the overpotential and improve the efficiency of

160

ORR [3 4 62 165-167] It was reported that 80 of the whole potential loss can be

attributed to cathode ORR overpotential [171] The choice of ORR catalysts varies by

applications [36 171] and direct four-electron transfer pathway is most preferred for

ORR at the lowest possible overpotential in every fuel cell application however its

kinetics and reduction pathway are limited by the type of catalysts and experimental

conditions [24 165 166] Miniaturised glucose-oxygen fuel cells operate in the neutral

aqueous solution at ambient or body temperature with relatively low electrolyte and

oxygen concentrations therefore they require powerful ORR catalysts Platinum (Pt)

has been most ideal however its availability and cost are major limiting factors Many

studies have been made on other noble metals [172 173] transition metal macrocyclic

compounds [174] multicopper oxidases [62 175] and quinones [165] as alternatives

ORR catalysts to Pt These ORR catalysts are reviewed below

531 Noble metal ORR catalysts

Noble and transition metals are well-known ORR catalysts There are two

possible ORR mechanisms for metal surface dissociative and associative

161

Dissociative mechanism

middotmiddotmiddotmiddotmiddot Eq 67

middotmiddotmiddotmiddotmiddot Eq 68

middotmiddotmiddotmiddotmiddot Eq 69

( indicates the catalyst surface)

Associative mechanism

middotmiddotmiddotmiddotmiddot Eq 70

middotmiddotmiddotmiddotmiddot Eq 71

middotmiddotmiddotmiddotmiddot Eq 72

middotmiddotmiddotmiddotmiddot Eq 73

middotmiddotmiddotmiddotmiddot Eq 74

In dissociative mechanism the O2 double bond is ruptured rapidly on its adsorption on

the metal surface [165 166 172] This involves a strong interaction between oxygen

and metal atoms [166] and does not generate H2O2 [165] Electron and proton transfer

take place simultaneously and this is the rate-limiting step [166 171] In the associative

mechanism O2 does not dissociate before proton transfer Adsorbed O2 stays on the

metal surface [165 172] and two-electron and proton transfer [166 173] take place via

the formation of superoxide or peroxide intermediate [165 166] This reaction is

thermodynamically easier than the dissociative pathway and its final product is H2O2 or

162

H2O if both pathways occur in series [165] O2 adsorption on the metal surface is an

inner sphere interaction which involves complex formation with the metal surface [30]

There are three ways in which O2 adsorbs (i) Griffiths (ii) Pauling and (iii) bridge

model (Fig 63)

Griffiths model Side-on Pauling model End-on Bridge model

OO

M

Fig 63 Three models of oxygen adsorption [165 166 173]

The Griffiths model is called lsquoside-onrsquo type O2 functions as a bidentate ligand against a

single metal atom in this complex and its π orbitals laterally interact with empty metal

dz2 orbitals causing ligand-to-metal charge transfer (LMCT) πrarrdz

2 At the same time

electrons partially filling metal dxz or dyz orbitals are back donated to oxygen π orbitals

[166 171] Therefore a strong metal-oxygen interaction is present in this model This

effectively disrupts the O=O bond [166] and facilitates the electron transfer from the

metal ion by changing its oxidation number The Pauling model is called the lsquoend-onrsquo

type [165 166 173] O2 is a monodentate ligand in this model and only one of the

oxygen atoms in the dioxygen molecule can be bonded to the metal surface An oxygen

π orbital interacts with metal dz2 orbitals and this allows either the direct or peroxide

ORR pathway after partial charge transfer [166] The bridge model resembles Griffiths

model in the way the metal atom and oxygen interact however it requires two adjacent

163

adsorption sites so that O2 can ligate two different metal atoms The bridge-model

interaction is easily interfered with by the surface contamination and the oxide layer

because the surface species block the adsorption sites for ORR catalysis [173]

Fig 64 Models for oxygen adsorption and ORR pathways [166]

Both the Griffiths and bridge models follow dissociative mechanism (pathway I and III

respectively Fig 64) due to the lateral interaction leading to the dissociation of O2

double bond The Pauling model on the other hand follows the associative mechanism

(pathway II Fig 64) This is further divided into two other pathways IIa and IIb

according to its end product H2O2 or H2O (Fig 64) respectively [166] O2 adsorption

and the ORR mechanism therefore depend on (i) the extent of oxygen-metal

interaction and (ii) the availability of oxygen adsorption sites A volcano-shaped trend

between ORR rate and oxygen-metal interaction has been plotted by Norskov as in Fig

65 [172]

164

Fig 65 A volcano-shaped trend between ORR rate and energy required for oxygen

binding on various transition metal surface [172]

Many have reported platinum (Pt) as the most ideal metal catalyst due to its high

catalytic ORR activity [165 166 171 172] Fig 65 shows that Pt and Pd (palladium)

have largest ORR rate yet medium oxygen adsorption energy and iridium (Ir) and

rhodium (Rd) follow them On the other hand metals with either higher or lower

oxygen adsorption energy result in lower ORR rate This is because metals with high

oxygen adsorption energy such as gold (Au) imposes too large energy barrier for the

formation of a stable oxygen-metal bond while too small adsorption energy as in nickel

(Ni) leads to excessively stable oxygen-metal interaction and slows the proton transfer

Therefore associative pathway is dominant at Au surface while oxygen dissociation is

deactivated at Ni surface but instead this increases the surface oxide coverage [172

173] Pt performs best because it has moderate oxygen-metal surface interaction [165]

Pt alloys with other transition metals such as Ni Co and Fe have been also reported to

increase the catalytic activity [36 167 171 172] because alloying dilutes the Pt-O

165

interaction and alters the overall oxygen-metal surface interaction [171 172] This is a

useful property to add because Pt is known to form a surface oxide layer leading to both

associative and dissociative catalytic pathways [165 171] This surface state is

potential-dependent and stays persistenly at less than 08 V vs AgAgCl [172]

532 Macrocycle and Schiff-base complex ORR catalysts

Macrocyclic transition metal complexes have been widely investigated as

potential ORR catalyst both for fuel cells and as catalysts to activate dioxygen for

synthetic purposes They have been widely reviewed by [165 176] Macrocyclic

complexes consist of (i) transition metals such as Fe Co Ni or Cu (ii) chelating atoms

N4 N2O2 N2S2 O4 and S4 and conjugate π systems such as porphyrin and

phthalocyanine [165] Examples of transition metal macrocycles are shown in Fig 66

Fig 66 Examples of transition metallomacrocycles Heme (left) [177] and copper

phthalocyanine (right) [178]

166

Schiff base complexes such as metallosalens and metallosalophens are other

well-known chelating ORR catalysts [174 179] Transition metal macrocycles and

Schiff bases can be also polymerised into conductive films [180] (Fig 67)

Fig 67 Schiff base complexes

Co(II) salen Co(II) salophen and

Co(II) salen modified poly(34-ethylenedioxythiophene) [174 180]

Interaction between the oxygen molecule and the metal complex is similar to that with

the noble metals and involves partial charge transfer between the filled metal dz orbitals

and the π orbital of O2 as described in sect 531 The catalytic activity of the metal

complexes depends on the type of central metal atom ligand and the size of the

π-electon systems The reducing ability of the ligand is important because the central

metal atom must transfer considerable electron density to oxygen atoms during the

167

catalysis [170] The thermodynamically most stable metal-oxygen complexes are

bionuclear complexes such as dicobalt face-to-face 4-atom-bridged diporphyrin

(Co2(FTF4)) (Fig 68) [165-167]

Fig 68 Molecular structure of dicobalt face-to-face diporphyrin [166]

Mononuclear Co complexes take the associative pathway [165] forming superoxo

cobaltic complexs (Co-O-O־) [181 170] while their dimers or binuclear complexes can

take either direct four-electron or the parallel pathways (Fig 69) [165 167] because

they can lock the oxygen moiety in rigid orientation [170] Co2(FTF4) favours

four-electron transfer and its ORR pathway depends on the distance between two

cofacial metal atoms [165-167] Spacing distance can be altered by inserting a spacer

molecule [165 166]

168

Fig 69 ORR pathway by Co2(FTF4) [166]

However Co2(FTF4) is not stable enough for practical use [165 166] Practical ORR

catalysts for fuel cell application must be stable inexpensive and feasible to synthesise

533 Multicopper enzymes as ORR catalyst

Multicopper oxidases (MCOs) are copper-containing enzymes such as bilirubin

oxidase (BOD) laccase (Lac) and copper efflux oxidase (CueO) Oxygen is widely used

as an electron acceptor in nature because it is ubiquitous and oxidation is a good mean

to obtain energy as in combustion Millions of years of evolution have therefore

provided enzymes with good catalytic activity for oxygen reduction Although oxygen

169

reduction in biological system is complex due to involving various substrates and

catalytic steps as well as it tends to be incomplete and leads to hydrogen peroxide or

superoxide formation as the product the enzymes catalyse the rate-determining initial

electron transfer For application in biofuel cells it is essential that the enzymes are

widely available and possess good stability activity in immobilised form and ideally

four-electron ORR catalysis MCOs have been prevalent in biofuel cell applications

because they are active at neutral pH catalyse four-electron ORR and bear

immobilisation Fungal laccase and bilirubin oxidase have been most widely studied

MCOs [56 59 60]

Most MCOs have four Cu atoms which are designated as type I (T1) type II (T2) and

a pair of type III (T3) T2 and T3 together form a trinuclear Cu centre while T1-Cu

called blue copper [175 182 183] is located away from the trinuclear Cu centre but

adjacent to the hydrophobic substrate-binding site Fig 70 shows the location of the

trinuclear Cu centre (copper coloured) and the blue copper (in blue) Oxygen binding

and ORR take place at the trinuclear Cu centre [175] while T1-Cu receives electrons

from the substrate and mediates electron transfer between substrate and the trinuclear

Cu centre [175 184]

170

Fig 70 Laccase from Trametes versicolor [185]

Enzyme catalytic efficiency depends on the redox potential [175] of T1-Cu It has been

reported that fungal laccase has the highest redox potential between 053 ndash 058 V vs

AgAgCl [62 183] T1-Cu also allows DET due to being located close to the surface

[183 186 187]

MCO reaction cycle starts from substrate oxidation to form a fully reduced state MCO

is initially at native form or so-called intermediate II Native form is characterised by

doubly OH--bridged structure (Fig 71(ii)) In the absence of substrate the native form

decays to the resting form which is at fully oxidised state (Fig 71(i)) After

four-electron reduction of enzyme the fully reduced MCO (Fig 71(iii)) reacts with O2

The first reduction step involves two-electron transfer from each of T3-Cu+ to O2 This

forms peroxide intermediate or intermediate I (Fig 71(iv)) and lowers the energy

barrier to disrupt O=O It is followed by swift protonation of adsorbed peroxide at

T3-Cu site This protonation reaction is induced by the charge transfer from T1- and

171

T2-Cu+ This leads to the complete reduction of oxygen to water and the enzyme native

form is retrieved

(i)

(ii)

(iii)

(iv)

Fig 71 Redox reaction cycle of MCOs with O2 [175]

All MCOs have this common ORR mechanisms despite the different origin structure

biological functions substrate reducing power and optimal environment

5331 BOD

BOD is an anionic monomer [182] most active at neutral pH [183 188] and

172

capable of both DET and MET The mediators must have their redox potential close or

slightly lower than that of BOD 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

(ABTS) (Fig 72(i)) [62 97] Os-pyridine complexes linked to conductive polymer (Fig

72(ii)) [66 189] and cyanometals [18 56] have been used as its mediators [56 185]

ABTS is a divalent anion which works as co-substrate of MCOs and its redox potential

is 067 V vs AgAgCl [56] Os-pyridine complex with conductive polymer follows the

similar idea for the wired GOx anodes discussed in sect 223 on p 80

4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine) has been used

especially for the cathode and its redox potential is 055 V vs AgAgCl

Fig 72 Common mediators for BOD (i) ABTS [56 62]

(ii) Os[4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine)] [190]

173

Tsujimura showed that ORR catalytic constant was 6-times greater in the presence of

Os-pyridine complex than in DET [66 188] Reduction starts around 05 V in most

cases [66 97 183 188] The size of current density varies roughly between 05 - 1

mAcm-2 [56 97 188] and 95 mAcm-2 was achieved by Heller [189]

5332 Lac

Fungal laccase is another anionic monomer [183] It is less expensive than

BOD and has broad substrate specificity including xenobiotics [175] although it has

optimum pH around 5 and activity hindered by chloride [19 182] Its T1-Cu redox

potential ranges between 053 ndash 058 V vs AgAgCl [62 183] Highest redox potential

066 V and current density 057 mAcm-2 were achieved by the use of anthracene

plugging molecule which provides strong binding between Lac and pyrolytic graphite

edge electrode as well as long-term activity 57 of the initial current was retained after

eight weeks [185]

5333 CueO

CueO is a less common enzyme in biofuel cells however it is active for a wide

range of pH between 2 - 8 [186] and also it has better catalytic activity than other

MCOs because it has five Cu atoms This extra Cu atom is believed to have mediating

function and 4 mAcm-2 has been achieved at pH 5 [191] However it has lower redox

potential around 03 V [186 191] Protein engineering has been introduced to increase

T1-Cu redox potential [186]

174

534 Anthraquinone ORR catalyst

Anthraquinone (AQ) is known to catalyse two-electron transfer in ORR (Fig

73) [44 165 192-194] and it is used in industries to produce hydrogen peroxide [165

192]

Fig 73 Two-electron ORR by anthraquinone [193]

ORR by anthraquinone is an electrochemical-chemical (EC) process [165 192] in

which quinone must be first electrochemically reduced to an active species

semiquinone anion radical (Q־) The active semiquinone radical can then react with

dioxygen so that it converts the dioxygen into O2־ via forming of the intermediate

superoxo-C-O(O2) species This is the rate-limiting step O2־ produced by a

semiquinone radical either disproportionates (Eq 77) or it is further reduced into HO2-

(Eq 78) [192]

175

Oxygen reduction reaction by anthraquinone

ltlt Rate limiting step gtgt

middotmiddotmiddotmiddotmiddot Eq 75

middotmiddotmiddotmiddotmiddot Eq 76

ltlt Following spontaneous reactions gtgt

middotmiddotmiddotmiddotmiddot Eq 77

middotmiddotmiddotmiddotmiddot Eq 78

(Q indicates quinone on the surface)

Derivatised anthraquinone such as Fast red (Fig 74) can be attached to the carbon either

physically or covalently Grafted quinones are very stable [192 194 195] and resistant

against continuous potential cycling [194] metal ions and surfactant despite of some

fouling present [44]

176

O

O

N N Cl

frac12 ZnCl2

1

2

4

6

5

3

10

9 7

8

12

14

13

11

O

O

(i) (ii)

Fig 74 The structures of (i) anthraquinone [196] and (ii) Fast red AL salt [44 197]

However its ORR is heavily affected by pH [194] because its catalytic efficiency

depends on the concentration of surface Q־ species [192 194] The concentration

varies by different pH and the catalytic efficiency drops at both high and low pH due to

the depletion of Q־ When pH falls below its dissociation constant Q־ is not

reproducible anymore and ORR is inhibited This phenomenon appears at as high as pH

7 and the ORR vanishes at pH lt 7 [194] AQ-grafted GC (AQGC) shows only sluggish

kinetics at pH lt 10 Its voltammogram also becomes complex due to the presence of

native quinone groups originated from the carbon surface [192] ORR mediated by the

native quinone groups is observed as a prewave and this varies by different quinone

derivatives such as phenanthrenequinone (PQ) (Fig 75) electrode composition and pH

[194]

177

Fig 75 Hydrodynamic voltammogram for ORR at various quinone electrodes with various

rotation speeds [194] (i) unmodified GC (ii) AQGC and (iii) PQGC at pH 7

54 CARBON PROPERTIES

Carbon is widely used for electrochemical applications because it is

inexpensive mechanically stable [105] and sufficiently chemically inert yet reactive

due to the presence of surface defects It has wide potential window between 1 and -13

V [21 30] Carbon is available in various solid forms such as graphite glassy carbon

(GC) pyrolytic carbon (PC) activated carbon (AC) carbon fibres (CFs) carbon

nanotubes (CNT) and diamond It is easy to renew the surface and allows facile

chemical modification [195] However it is also prone to adsorption and surface

oxidation simply by air and water exposure and its electron transfer is under the

constant influence of the electrode surface condition and history [198] This leads to the

deviation and the low reproducibility in carbon electrochemical experiments [105]

178

Physical properties of carbon vary by its form and section The carbon consists of either

(i) layers of the conductive planar graphene sheets or (ii) the insulating

tetrahedral-puckered forms as seen in the diamond (Fig 76) Conductivity in diamond

is gained by doping with boron [199 200]

Fig 76 Carbon structures (i) graphite (ii) diamond [201]

The electrical conductivity is anisotropic and closely correlates with the carbon

structure and section The carbon section is classified into (i) basal and (ii) edge planes

The basal plane extends along the graphene plane while the edge plane is perpendicular

to this

179

Fig 77 Carbon section (i) basal plane (ii) edge plane [105]

The basal plane has larger conductivity due to being parallel to the vast graphene

network while the edge plane is superior in electrochemical reactivity due to being

richer in reactive graphene sheet termini and carbon defects The ratio between the basal

and edge planes therefore affects the conductivity and electrochemical properties of

the carbon and this varies by the carbon form and its treatment [45 105 195 202 203]

The electrical conductivities for various carbon forms and sections is shown in Table 9

180

The type of carbon Electrical conductivity Scm-1

Graphite [105 159] 1 x 103 - 104

Highly ordered PG basal plane 250 x 104

Highly ordered PG edge plane 588

PG basal plane 400 x 103

PG edge plane 333

Polycrystalline graphite 100 x 103

GC [105 159 203] 01 - 250 x 102

Activated carbon [204] 1 x 10

Carbon fibre 133 x 103

Boron-doped diamond [199 205] 01 - 1 x 102

Multi-wall carbon nanotube [206] 1 ndash 2 x 103

Pt 909 x 104

Cu 588 x 105

Table 9 The electric conductivity of various carbon forms and sections Data from

[105] unless defined specifically

541 Graphite

Graphite is black-coloured material with pores and high gas permeability It is

available in many physical forms as powders moulded structures and thin sheets

therefore it is used in many commercial applications [203] Powdered graphite is

convenient to make carbon paste and composite electrodes [105] Graphite consists of

181

layers of graphene sheets each of which is a huge π-conjugate system π-conjugate

system is made of hexagonal rings of sp2 hybridised carbon atoms [105 195] (Fig 76(i)

and Fig 78) and allows free electron movement within graphene This gives carbon

metallic properties such as electric and thermal conductivity

Fig 78 A direct image of a graphene bilayer by transmission electron microscopy

(TEM) at resolution of 106 Aring and with a 2-Aring scale bar [207]

Each graphene sheet is structurally stable in horizontal direction but fragile in vertical

direction because each layer is only held by van der Waalsrsquo force Therefore the

graphene sheets easily slide and collapse [31]

542 Glassy carbon (GC)

Glassy carbon (GC) is a non-graphitised carbon made of randomly interwoven

sp2-carbon fibres which are tangled and heavily cross-linked (Fig 79) It contains many

closed voids [203] and fullerene-like elements [208] It is impermeable to gases [203

182

208] chemically more inert [208] and mechanically stronger but electrically more

resistive than other carbons [203]

Fig 79 GC (i) High resolution TEM image (ii) A schema of GC [208]

543 Activated carbon (AC)

Activated carbon (AC) is thermally and chemically activated carbon It is

highly porous and has large internal surface area This is an excellent adsorbent for

solvent recovery and gas refining [203 208]

544 Carbon fibre (CF)

Carbon fibres (CFs) have small diameter ranging between 5 to 30 microm They are

lightweight have lower fabrication cost and are superior in mechanical properties

therefore they have been used for sporting automotive and military equipments They

are available in many forms such as tow mat felt cloth and thread They consist of a

random arrangement of flat or crumpled graphite sheets but their structure and

183

molecular composition depend on the type of precursors [203 208]

545 Carbon nanotubes (CNTs)

Carbon nanotubes (CNTs) are made rolled-up sheets of sp2-graphene in nano to

micro-meter range They are available in various formats single-walled nanotube

(SWNT) multi-walled nanotube (MWNT) carbon nanohorn (CNH) (Fig 80) and

various forms powder arrays and posts [117]

Fig 80 Various CNTs (i) Bundled SWNTs (ii) MWNT (iii) CNHs [209]

CNTs have been drawing keen attention due to their wide surface area conductive or

semiconductive properties light weight mechanical stability and facile

functionalisation [117 195 202 210] They have been used as support for the dispersed

metal catalysts [3] and electrode materials [117 202] The drawbacks of CNTs are the

high presence of impurities alteration of their properties on acid cleaning [202 211]

and unknown toxicity [117 160 202]

184

546 Boron-doped diamond (BDD)

Diamond is different from other carbons because it is transparent and not

electrically conductive It has a cubic lattice structure made of closely-packed repetitive

units of sp3-hybridised carbon atoms as seen in Fig 81

Fig 81 A TEM image of diamond [212]

Each unit consists of four σ-bonded sp3-carbon atoms in tetrahedral coordination as in

Fig 76(ii) Each bond is localised and this gives structural stability but does not conduct

electricity By doping it with boron diamond can become semiconductive Such

diamond is called boron-doped diamond (BDD) and boron functions as an electron

acceptor BDD is mechanical more stable chemically more inert sensitive to analyte

and has wide potential window lower back ground current and stable electrochemical

responses [195 199 205]

185

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON

551 Electrochemical properties of carbon

The edge plane of carbon which contains graphene termini and carbon defects

is chemically reactive and accounts for carbon electrochemistry while the vast network

of graphene sheets is responsible for its electrical conductivity The termini and defects

include some sp3 hydrocarbons and they are oxidised into various oxygen functional

groups such as carboxyl quinoyl hydroxyl lactone groups ethers cyclic esters and

lactone-like groups [195] These electrode functional groups facilitate electron transfer

provide bonding between the electrode and the catalysts or enzymes or simply cause

electrode capacitance or resistance Various carbon pretreatments such as

electrochemical pretreatment (ECP) vacuum heat treatment (VHT) carbon fracturing

and polishing laser irradiation [195 213-215] are used to attach active surface

functional groups and increase the rate of electron transfer in various electrochemical

reactions [198 216]

552 Electrochemical pretreatments (ECPs) of the carbon

Electrochemical pretreatments (ECPs) provide feasible electrode surface

modification Various ECPs have been used to introduce carbon-oxygen surface

functional groups Most common method is potential cycling in the acid or basic media

or the combination of anodisation and cathodisation as shown in Table 10

186

Table 10 ECPs examples

Potential cycling at 02 Vs-1 between 0 and 02 V in 01 M H2SO4 [105]

Anodisation at 18 V in pH 7 phosphate buffer for 5 minutes followed by 1-minute cathodisation at

ndash 15 V [216 217]

Potential cycling between 06 and 2 V at 5 Vs-1 for 15 to 30 minutes in 05 M NaOH [218] or their

combination [219]

ECPs introduce the electrodes various new properties They (i) attach electrochemically

active functional groups and increase the oxygencarbon ratio [105] (ii) introduce

carbon defects and active sites [105 218] (iii) increase the surface area [105 198 215

216 218] (iv) form a surface film and (v) increase the background current [105] and

double layer capacitance [105 215 216] Most notable effect of EPCs is the increase in

electron transfer rate in many electrochemical reactions This is observed as a decreases

in peak separation and an increase in peak current in voltammetry as seen in Fig 82

[105 216 218 220] As for ORR however it was reported that surface quinone groups

attached by EPC or originated from the carbon itself reduce ORR overpotential but do

not mediate ORR as in quinone groups physically or covalently attached [216 220]

187

Fig 82 The influence of ECP on GCE and its voltammogram at 01 Vs-1 in pH 7

phosphate buffer in the presence of (i) N2 and (ii) O2 The voltammograms

of both pretreated and unpretreated GCEs are shown [216] The

presentation has been altered for convenience

56 ELECTROCHEMICAL TECHNIQUES FOR ORR

ORR has been studied by two electrochemistry techniques cyclic voltammetry

(CV) and hydrodynamic voltammetry (HDV) using the rotating disc electrode (RDE)

These techniques provide overlapping but complementary information CV is a dynamic

technique and allows calculation of the number of electrons up to and including the rate

determining step (nα) With numerical simulation and data fitting CV can be used to

estimate the standard heterogeneous rate constant (kdeg) RDE is a steady state technique

and can give the overall number of electrons transferred (n) The well-established

Koutecky-Levich plot (see sect 567 on p 200) provides a robust method for determining

kdeg provided the standard electrode potential (Edeg) for the reaction is known

188

561 Cyclic voltammetry (CV)

Cyclic voltammetry (CV) is widely used for initial exploration and

characterisation of a redox active system It is of limited electroanalytical value

however kinetic parameters can be obtained from the current-voltage (I-V) plot In

cyclic voltammetry potential is swept linearly with a triangular waveform (Fig 83(i))

and applied cyclically The resulting time-dependent current is typically plotted versus

applied potential (Fig 83(ii)) [30 221] The potential range and scan rate are decided in

advance according to the type of analytes reactions and materials of interest

Fig 83 Schemas for (i) a Triangular waveform (ii) Typical cyclic voltammogram for

benzoquinone (BQ) CV was taken at 005 Vs-1 between + 025 and ndash 05 V vs

AgAgCl for 1 mM BQ in 1M KCl at graphite composite electrode Switching

potential (Eλ) is ndash 05 V and time taken to reach Eλ (t) is 15 seconds Based on

the real data

189

562 Non-faradic current and electric double layer capacitance

Transient techniques such as CV always contain two types of electric current

non-faradaic and faradaic current The non-faradaic charging current is due to the

electric double layer which is formed at the electrode-solution interface This occurs

because such an interface behaves like a capacitor as counter ions accumulate at the

interface in order to balance the potential at the electrode This does not account for the

charge transfer reactions involved however provides the information about the

electrode surface [30] and also can affect the rate of electrode process and distort the

voltammogram Cd can be estimated by electrochemical impedance spectroscopy or

deduced by averaging Cd over the potential range as in Fig 84 and Eq 33 [21 30]

Fig 84 The double layer capacitance (Cd) observed in CV for deoxygenated pH 74 at

the acid-pretreated graphite electrode See Eq 33 for the calculation and

symbols Based on the real data

190

Electric double layer capacitance per unit area

AII

AEQareaunitperC ccca

d sdotminus

=sdot

=υ2

)()( middotmiddotmiddotmiddotmiddot Eq 79

Cd = Double layer capacitance per unit area microFcm-2

Q = Charge C

E = Potential V

A = Electrode surface area cm-2

Ica = Anodic non-faradic current A

Icc = Cathodic non-faradic current A

υ = Scan rate Vs-1

Cd needs to be either negligible or subtracted before interpretation of faradaic current

which accounts for the charge transfer phenomena in redox reactions In order to

minimise the influence of Cd on the voltammogram the scan must be taken at slow scan

rate and the electrolyte concentration must be always kept reasonably high [30]

563 Diagnosis of redox reactions in cyclic voltammetry

A typical voltammogram contains current peaks as seen in Fig 83(ii) and these

are due to the redox reactions in the system Diagnosis of the redox system using CV is

based on (i) peak separation (ii) peak current height (iii) peak shape or peak width (iv)

the number of peaks and (v) the dependency of peak shape on the scan rate The

analysis of such peaks involves the following four parameters anodic peak current (Ipa)

cathode peak current (Ipc) anodic peak potential (Epa) and cathode peak potential (Epc)

This reveals the type of electrochemical reactions involved which are either (i)

191

reversible (ii) quasi reversible or (iii) irreversible When the peak current is proportional

to υfrac12 the redox reaction is diffusion-limited in the solution while a linear dependence

shows that the redox active species is confined to the electrode surface

In the reversible system charge transfer between the redox species and the electrode is

rapid [221] therefore it has fast kinetics and the reaction is diffusion-limited The

electrode potential follows the Nernst equation (Eq 15 on p 50) because the electrode

potential is dependent on the concentration equilibrium at the electrode surface and the

peak potential is independent of the scan rate The features on the reversible system are

listed in Table 11 The peak current (Ip) is linearly proportional to υ12 as the redox

reaction is diffusion-limited The peak separation (∆Ep) is the difference between Epa

and Epc and it is 59 mV at 25 ˚C for the single-charge transfer [29 30] The peak width

(δEp) is the difference between Ep and half-peak potential (Ep2) Ep2 is the potential

where the peak current is half It is 565 mV for a single charge transfer The ratio

between Ipc and Ipa is called a reversal parameter [105] It is unity at any scan rate in the

reversible system

192

Table 11 Reversible system at 25 ˚C [30]

2121235 )10692( υlowasttimes= CADnI p middotmiddotmiddotmiddotmiddot Eq 80

mVn

E p528

= independent of υ middotmiddotmiddotmiddotmiddot Eq 81

mVn

EEE pcpap59)( =minus=∆ middotmiddotmiddotmiddotmiddot Eq 82

mVn

EEE ppp556

2 =minus=δ middotmiddotmiddotmiddotmiddot Eq 83

1=pa

pc

II middotmiddotmiddotmiddotmiddot Eq 84

Ip = Peak current A

n = The number of charges transferred

A = Electrode surface area m-2

D = Analyte diffusion coefficient m-2s-1

ν = Solution kinematic viscosity m-2s-1

Ep = Peak potential V

∆Ep = Peak separation V

Epa = Anodic peak potential V

Epc = Cathodic peak potential V

δEp = Peak width V

Ep2 = Half-wave potential V

Ipa = Anodic peak current A

Ipc = Cathodic peak current A

In a totally irreversible system charge transfer between the redox species and the

electrode takes place only slowly [221] The electrode must be activated by applying

193

large potential to drive a charge transfer reaction at a reasonable rate Ep becomes

dependent on scan rate and this increases ∆Ep and skews the voltammogram [30]

However Ip is still proportional to υfrac12 because at high overpotential the redox reaction

is diffusion-limited [29]

Table 12 Irreversible system at 25 ˚C [222]

2121215 )()10992( υα αlowasttimes= CADnnI p middotmiddotmiddotmiddotmiddot Eq 85

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛

deg+minusdeg=

2121

lnln780RT

Fnk

DFn

RTEE pυα

αα

α

middotmiddotmiddotmiddotmiddot Eq 86

mVn

E pαα

59gt∆ middotmiddotmiddotmiddotmiddot Eq 87

mVn

E pαα

δ 747= middotmiddotmiddotmiddotmiddot Eq 88

α = Transfer coefficient

nα = The number of charge transferred up to the limiting step

( Please refer to Table 11 for other terms and symbols)

A quasi-reversible system is controlled by both kinetics and diffusion Its

electrochemical reversibility is relative to the scan rate [21 30] The peak potential

depends on scan rate for rapid scans and the peak shape and peak parameters become

dependent on kinetic parameters such as α and Λ where 21

1

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛

deg=Λ

minus υαα

RTFDD

k

RO

[30]

Since Λ is a function of k˚υ-frac12 where kdeg is the standard rate constant the voltammogram

hence kdeg is affected by the scan rate as seen in Fig 85

194

Fig 85 Voltammograms of a quasi-reversible reaction at various scan rates [21]

564 Coupled reactions in cyclic voltammetry

Redox reactions are sometimes coupled with different electrochemical or

chemical reactions Voltammogram with altering scan rates can indicate the presence of

such reactions A redox reaction coupled with another redox reaction is called EE

mechanism while with homogeneous chemical reaction it is called EC mechanism If

the coupled reaction is catalytic then it is called ECrsquo mechanism [21]

Many ORR catalysts show coupled reactions with ECrsquo mechanism ORR is a

quasi-reversible reaction because the reaction is limited by oxygen diffusion yet O2

adsorption and reduction heavily relies on overpotential or catalytic power of catalysts

Catalysed reactions show distinctive voltammetric features There is only a single-sided

large current but with no reverse current because they catalyse a reaction only in a

single direction The reversal parameter therefore becomes non-unity [30]

195

ORR catalysts sometimes contain multiple reducible centres or an atom with various

oxidation states as in seen transition metal They might exhibit a multi-peak

voltammogram under non-catalytic condition The shape of voltammogram depends on

the difference in the standard potential (∆E˚rsquo) between two redox centres or states (Eq

89) as seen in Fig 86 [21 30]

21 ooo EEE minus=∆ middotmiddotmiddotmiddotmiddot Eq 89

Fig 86 Voltammograms of two-electron transfer EE systems of different ∆Edegrsquo [21]

565 Voltammograms for the surface-confined redox groups

Catalysts encapsulated in or attached to the electrode provide catalytic groups

or moieties on the electrode surface Such surface-confined redox species show different

voltammograms from solution redox species because their redox reactions do not

196

involve diffusional redox species They exhibit a characteristic voltammogram of

perfect symmetry with Epa = Epc as seen in Fig 87 and their peak current is linearly

proportional to υ rather than υfrac12 as in Eq 90 [21 30] The features of surface-confined

groups are listed in Table 13

Fig 87 A voltammogram of the reversible redox reaction by surface-confined groups [21]

Table 13 Reversible system by surface-confined groups at 25 ˚C [30]

RTAFn

I p 4

22 Γ=

υ middotmiddotmiddotmiddotmiddot Eq 90

oadsp EE = middotmiddotmiddotmiddotmiddot Eq 91

mVn

E p690

2 =∆ middotmiddotmiddotmiddotmiddot Eq 92

Where Γ isiInitial concentration of the absorbed species

However such an ideal voltammogram is seldom observed because the density of redox

species is so high in the surface-confined environment compared to in the solution and a

197

voltammogram is also influenced by the interaction among those packed redox species

A multi-electron system of the surface-confined groups is also not this case [21]

566 Hydrodynamic voltammetry (HDV) and Levich equation

Hydrodynamic methods purposely introduce convective flow to the system

using RDEs This allows the study of precise kinetics because the diffusion is under

precise control Unknown influence by mass transfer and double layer capacitance on

the electrode reaction is eliminated and steady state is quickly achieved [29 30] In

RDE method laminar flow is applied perpendicular to the electrode surface by spinning

a disc electrode at arbitrary rotation velocity (Fig 88) The rotation rate is expressed in

either rpm (rotation per minute) or angular velocity (ω) ω is defined as ω = 2πf where f

is frequency With disc rotation diffusion becomes a function of electrode rotation rate

as in Eq 93 However the limit of disc rotation rate must be set in order to avoid the

thick hydrodynamic boundary layer and turbulent flows to maintain steady state

Allowable range is between 100 rpm lt f lt 10000 rpm or 10 s-1 lt ω lt 1000 s-1 [30]

198

ω

Fig 88 RDE is rotated at ω and laminar flows applied to the electrode surface

Diffusion layer thickness and disc rotation rate [30]

612131611 νωδ minus= D middotmiddotmiddotmiddotmiddot Eq 93

δ = Diffusion layer thickness cm

D = Analyte diffusion coefficient m2s-1

ω = RDE angular velocity s-1

ν = Solution kinematic viscosity m2s-1

Under such a condition disc limiting current (Id) becomes a function of disc rotation

rate as described in Levich equation (Fig 85) [30] This allows finding the total number

of charge transferred (n)

199

Levich equation [30]

612132620 CnFADI Lminus= νω middotmiddotmiddotmiddotmiddot Eq 94

δ

nFAC= middotmiddotmiddotmiddotmiddot Eq 95

IL = Levich current A

n = Total number of charge transferred

F = Faraday constant Cmol-1

A = Electrode surface area m2

C = Bulk analyte concentration molm-3

( Please refer to Eq 93 for other terms and symbols)

Due to the steady state condition the current response in HDV becomes sigmoidal [223]

as seen in Fig 89

200

Fig 89 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH

74 Based on the real data

567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the

charge transfer rate constant

Levich equation (Eq 94) shows that Id is proportional to ωfrac12 therefore also a

function of diffusion layer thickness (Eq 95) This is true for reversible kinetics

because the electrode reaction is limited by diffusion which is a function of ωfrac12 and

therefore Id also becomes dependent on ωfrac12 at any potential However in

quasi-reversible system Id depends on diffusion but also affected by its kinetics [223]

Therefore when Id reaches the kinetic limitation for charge transfer the slope gradually

deviates from its Levich current as seen in Fig 90 [30]

201

Fig 90 Levich plots for reversible (blue) and quasi-reversible (red) systems [30]

In addition its charge transfer kinetics is altered by the potential applied As Tafel plot

(Fig 9 on p 57) presents the current is exponentially dependent on the overpotential

The electrode reaction is kinetically limited at low overpotential then gradually

becomes diffusion-limited as overpotential increases [223] This implies that Id in

quasi-reversible system is always under the influence of potential applied because the

potential alters its kinetics

When Id becomes completely free from the mass transport influence such current is

called kinetic current (IK) because Id now purely depends on only its kinetics Arbitrary

Id then defined as the sum of diffusion-limited IL and kinetically-limited IK The

double-reciprocal equation of this summation is defined as Koutecky-Levich equation

as in Eq 96

202

Koutecky-Levich equation [30]

LKd III111

+=

216132

11620

11ων

times⎟⎟⎠

⎞⎜⎜⎝

⎛+= minus nCFADI K

middotmiddotmiddotmiddotmiddot Eq 96

Id = Disc limiting current A

IK = Kinetic current A

IL = Levich current A

( Please refer to Eq 93 and Eq 94 for other terms and symbols)

By extrapolating the Koutecky-Levich plot IK can be easily estimated [30] from the

reciprocal of its y-intersect In order to plot Koutecky-Levich equation and find IK a

sequence of arbitrary Id is recorded with altering disc rotation velocity at arbitrary

potential This technique is called hydrodynamic amperometry (HDA) and it is shown

in Fig 91

203

Fig 91 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in

oxygenated 001 M PBS at pH 74 at pretreated GCE Based on the real data

Based on the data obtained by HDA Koutecky-Levich plot is plotted as in Fig 92

204

0E+00

5E-02

1E-01

2E-01

2E-01

3E-01

-002 000 002 004 006 008 010 012 014 016 018

ωˉfrac12 (sˉ

frac12)

1 IK (A-1)

- 04 V

- 06 V- 08 V

05 x 10-1

10 x 10-1

15 x 10-1

0

20 x 10-1

25 x 10-1

-1

I d(A

-1)

Fig 92 Koutecky-Levich plot for O2 reduction at pretreated GCE in oxygenated 001 M

PBS at pH 74 with applied potential ndash 04 06 and 08 V Based on the real data

With Ik from Koutecky-Levich plot and nα from CV the rate constant for arbitrary

potential (k(E)) can be calculated using Eq 97

The rate constant for arbitrary potential E [30]

lowast=nFAC

Ik K

E )( middotmiddotmiddotmiddotmiddot Eq 97

k(E) = Rate constant for the applied potential E ms-1

( Please refer to Eq 94 Eq 95 and Eq 96 for other terms and symbols)

205

568 Finding the standard rate constant (kdeg)

Finding the standard rate constant (kdeg) for ORR of each catalyst is the ultimate

aim of this long tedious cathode analysis because kdeg tells the pure catalytic power

independent of reactant diffusion and overpotential (η) k(E) can be expressed in terms of

kdeg and η as in Eq 98 therefore it is possible to estimate kdeg with known η and k(E)

k(E) in terms of kdeg and η for a forward reaction [30 181]

⎥⎦⎤

⎢⎣⎡ minusminus

deg= )(exp 0)( EE

RTnFkk Ef

α

⎥⎦⎤

⎢⎣⎡ minus

deg= ηαRT

nFk )1(exp middotmiddotmiddotmiddotmiddot Eq 98

kf(E) = Rate constant for a reduction reaction at the applied potential E ms-1

kdeg = Standard rate constant ms-1

η = Overpotential V

( Please refer to Table 11 and Table 12 for other terms and symbols)

η can be found by subtracting Edegrsquo of the reaction involved from the applied potential in

HDA n obtained by HDV is used to specify the reaction involved hence its Edegrsquo In

order to estimate kdeg Eq 98 is first converted into logarithmic expression as in Eq 99

kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

ηαRT

nFkk Efminus

minusdeg= loglog )( middotmiddotmiddotmiddotmiddot Eq 99

206

Log(k(E)) is plotted against a range of η and by extrapolating the plot log(kdeg) is obtained

from its y-intersect where η = 0 hence kdeg can be calculated (Fig 93) [181]

-12

-10

-8

-6

-4

-2

0

-10 -08 -06 -04 -02 00 02 04

η V

log kf(E)

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS NaOH PBS NaOH H2SO4 PBS

NaOH H2SO4

NaOH H2SO4 PBSH2SO4 PBS

H2SO4

NaOH PBS

log kdeg

η V

Fig 93 Log kf(E) plot for O2 reduction at various pretreated GCEs Based on the real data

569 Summary of the electrochemical techniques

CV and hydrodynamic methods are complementary techniques for qualitative

examination of redox reactions and extraction of thermodynamic and kinetic parameters

The protocol for calculating kdeg from hydrodynamic methods and nα from CV is shown

in Fig 94

207

Fig 94 The protocol for finding kinetic and thermodynamic parameters

57 SUMMARY OF THE CHAPTER

The chemistry and electrochemistry of O2 has been reviewed The mechanisms of most

widely investigated catalysts noble metals macrocyclic complexes and enzymes have

been reviewed and their suitability for application in biofuel cells has been discussed

The key physico-chemical parameters which characterise these reactions have been

identified and methods for their determination have been described

208

6 CATHODE MATERIAL AND METHOD

61 ELECTRODE FABRICATION

611 Graphite PtC and RhC electrodes

Microcrystalline graphite of grade (2-15 micron 999995 purity) was

purchased from Alfa Aesar GmbH amp CoKG 5 wt-loaded rhodium on activated

charcoal (RhC) and platinum on activated carbon (PtC) were purchased from Sigma

Aldrich CY1301 and Aradur hardener HY 1300 were purchased from Robnor resins

and they were mixed at 31 (vv) for use RDE templates were specially designed and

made on site They were made of Teflon with the dimension 4-mm ID 20-mm OD

and 15-mm height The depth of composite-holding cavity for each RDE was 25 mm

The composite was made by mixing graphite RhC or PtC with epoxy resin at 73 by

hand Composite mixture was packed into five RDE templates and cured at room

temperature for 72 hours [106]

612 Anthraquinone(AQ)-carbon composite electrodes

6121 Derivatisation of graphite with AQ

Fast red AL salt with 25 dye content and hypophosphorous acid at 50 wt

in H2O were purchased from Sigma Aldrich HPLC grade acetonitrile was purchased

from VWR international 5 mM of Fast Red AL salt solution was prepared in 50 mM

209

hypophosphorous acid 10 ml of the Fast Red solution was mixed with 2 g of graphite

The mixture was left at 5 degC for 30 minutes with regular stirring at every 10 minutes It

was finally washed thoroughly with deionised water followed by vacuum suction in

acetonitrile flow The washed derivatised AQ-carbon was left to dry overnight It was

kept in the sealed container for future use [44]

6122 Fabrication of AQ-carbon composite electrode

AQ-graphite was mixed with epoxy resin at 64 by hand The composite was

packed into five RDE templates and cured at room temperature for 72 hours

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes

6131 Information on synthesis of metallosalophen

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminocobalt (II)] and

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminoiron (II)] denoted as Co(salophen)

and Fe(salophen) respectively were kindly donated by Dr Peter Cragg (School of

Pharmacy Brighton University) They were synthesised by dissolving metal acetate in

the ligand solution which was made by condensation of 21 molar ratio of

salicylaldehyde and diethylenediamine in ethanol or methanol [224 225]

210

6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes

Analytical grade dichloromethane was purchased from VWR international In

the presence of small quantity of dichloromethane 5 wt of Co(salophen) or

Fe(salophen) was mixed and homogenised with graphite [181] After the

dichloromethane evaporated the metallosalophen-graphite mixture was further mixed

with epoxy resin at 64 [219] by hand The composite was packed into five RDE

templates and cured at room temperature for 72 hours

62 COMPOSITE RDE CUTTING

The electrode discs of all the RDE composite electrodes were cut into 1

mm-thick sections using a Buehler IsoMet Low diamond saw with a 04 mm-thick

diamond blade

63 ELECTRODE POLISHING

631 Composite RDE polishing

Composite RDEs were polished successively with SiC grinding paper of 400

1200 and 2400 grit followed by with alumina oxide slurry of particle size 1 03 and

005 microm as in sect 33 on p 122 Electrode polishing was done just before the experiment

in order to avoid air oxidation [195]

211

Fig 95 Fabricated composite RDEs

632 Glassy carbon electrode polishing

Glassy carbon electrodes (GCE) (5-mm disc diameter) were purchased from

Pine Instruments Activated carbon (AC) was purchased from Chemviron Carbon GC

cleaning solvent mixture was made by mixing AC and HPLC grade acetonitrile in a

ratio of 13 (vv) The mixture was covered and left at least for 30 minutes then filtered

before use [226] GCEs were successively polished with alumina oxide powder slurry of

1- 03- and 005-microm particles on MicroCloth pad Polished GCEs were sonicated in

filtered ACacetonitrile mixture for 10 ndash 15 minutes to remove alumina powder debris

and contaminants [226] The GCEs were then sonicated in deoxygenated deionised

water Air contact of the electrode surface was strictly avoided [195]

212

64 ENZYME RDEs

641 Membrane preparation

Cellulose acetate dialysis tubing visking 3632 was purchased from Medicell

International Ltd Sodium carbonate (Na2CO3) was obtained from Sigma Aldrich

Dialysis membrane was cut into 10x10-mm2 pieces which were boiled in 1 wv

aqueous Na2CO3 solution for 10 minutes The membrane pieces were rinsed thoroughly

with deionised water and stored [61] in PBS in a sealed container at 4degC until its use

642 Membrane enzyme electrodes

Laccase (Lac) from Trametes versicolor gt20 unitsmg-1 and lyophilised

bilirubin oxidase (BOD) from Myrothecium verrucaria 15 ndash 65 units mg-1 were

purchased from Sigma Aldrich 4 mgml-1 of Lac and BOD solution was made with PBS

respectively BOD immediately dissolved in PBS while a vortex mixer was needed to

dissolve Lac In order to attach enzyme on the GCE surface the tip of polished cleaned

GCEs was dipped and left in the enzyme solution for two hours with gentle stirring The

electrodes were then lightly washed with deionised water Pretreated dialysis membrane

was carefully placed onto the surface of the enzyme-modified GCE and fixed with

rubber O-ring

213

65 ELECTROCHEMICAL EXPERIMENTS

651 Electrochemical system

CHI 650A with single-channel and CHI 1030A with multi-channel

potentiostats were used for electrode pretreatment and electrochemical experiments CV

HDV and HAD An adjustable rotor from Pine Instrument was used to rotate a RDE

Three-electrode system (sect 342 on p 123) was employed with an AgCl reference and a

Pt counter electrodes

Fig 96 RDE experiment setting with a rotor and gas inlet

214

652 Electrode pretreatments

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes

Graphite GC Co(salophen) and Fe(salophen) electrodes were pretreated in

seven different ways in Table 14 Untreated electrodes were also prepared as controls

All the pretreatments were made in series

Treatment type 1st pretreatment 2nd pretreatment 3rd pretreatment

Type I Untreated -

Type II Acidic 01 M H2SO4

Type III Basic 01 M NaOH

Type IV Neutral 001 M PBS

Type V Base followed by Acid 01 M NaOH 01 M H2SO4

Type VI Acid followed by Neutral 01 M H2SO4 001 M PBS

Type VII Base followed by Neutral 01 M NaOH 001 M PBS

Type VIII Base Acid then Neutral 01 M H2SO4 01 M NaOH 001 M PBS

Table 14 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Concentrated sulphuric acid at 90 - 98 and sodium hydroxide tablets were purchased

from VWR international 001 M PBS was purchased from Sigma Aldrich All the

pretreatments were based on potential cycling in 100-ml electrochemical cell except

untreated ones Acidic pretreatment was performed at 5 Vs-1 between 15 V and ndash 05 V

215

in 01 M H2SO4 [105 219] for 30 minutes Basic pretreatment was performed at 5 Vs-1

between 06 V and 2 V in 01 M NaOH [218 219] for 30 minutes Neutral pretreatment

was done at 5 Vs-1 between 15 V and ndash 05 V in deoxygenated 001 M PBS pH 74

[219 220] Constant N2 flow was applied to PBS through the pretreatment In addition

pretreatments were performed in various combinations as in Table 14 Type V Base

followed by Acid Type VI Acid followed by Neutral Type VII Base followed by

Neutral and Type VIII Base followed by Acid then Neutral [219] The influence of

pretreatments on electrode surface and ORR was studied by CV HDV and HDA

6522 Pretreatments of AQ-carbon PtC and RhC

AQ-carbon PtC and RhC composite electrodes were pretreated only in 01 M

H2SO4 at 5 Vs-1 between 15 V and ndash 05 V in for 30 minutes Untreated electrodes were

also prepared as controls

653 Electrochemical experiments

All experiments were carried out under both N2 and O2 saturation 100 ml of

001 M PBS electrolyte solution was sparged with either N2 or O2 for 15 minutes before

the experiment Gas was continuously supplied through a Dreschel gas washing bottle

containing PBS throughout the experiment

CV was performed at 002 005 01 02 and 05 Vs-1 between + 09 V and ndash

09 V HDV was performed at fixed scan rate of 005 Vs-1 sweeping the potential from

+ 09 V to ndash 09 V with disc rotation velocity altered through 900 1600 and 2116 rpm

216

HDA was performed at a range of fixed potentials - 02 - 04 - 06 and ndash 08 V at disc

rotation velocity of 400 625 900 1156 1225 1600 2116 and 2500 rpm All

experiments were repeated twice or three times The sample size ranges from one to

three The median values were taken for the analysis Obtaining a set of data for each

electrode sample took from three weeks to more than one month

654 Data analysis

CV data was used to calculate nα and reaction reversibility HDV data was used

to calculate n using Levich equation (Eq 30 on p 61) Edegrsquo for ORR involved at pH 7

25 degC was specified by n value as in Table 15

n Redox type Redox potential vs AgAgCl

1 Edegrsquo(O2H2O2) 0082 V

2 Edegrsquo(O2H2O) 0616 V

Table 15 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC [24]

η was calculated by subtracting Edegrsquo (ORR) from the potential applied in HDA HDA

data was used to estimate kf(E) at each applied potential using Koutecky-Levich plot (Eq

30 on p 61 and Fig 92 on p 204) N2 data was subtracted from O2 data as background

Other parameters required for Koutecky-Levich plot were set as in Table 16 All

parameters assume the condition of 001 M PBS at 24 degC

217

Parameter Value Reference

O2 diffusion coefficient 23 x 10-9 m2s-1 [227]

O2 concentration 123 molm-3 [158]

Kinematic viscosity (ν) 884 x 10-7 m2s-1 [30]

Table 16 Parameter values for Koutecky-Levich equation

kdeg was estimated by plotting log(kf(E)) vs η (Eq 99 and Fig 93 on p 206) kdeg for

various and pretreated cathodes were compared The best catalyst shows the largest kdeg

The procedure for finding kdeg is summarised in (Fig 94 on p 207)

218

7 CATHODE RESULTS AND DISCUSSION

71 AIMS OF THE CATHODE STUDIES

This section describes the electrochemical characterisation of various modified

electrodes which were considered as candidate cathodes for the biofuel cells Various

carbons were used as electrode materials in all cases The electrochemical behaviour of

carbon depends strongly on history and also can be radically altered by pre-treatment

The effects of electrochemical pretreatments (ECPs) on the kinetic parameters will be

described for both glassy carbon and graphite composites The cathode modifiers

considered in this section include noble metals (Pt Rh) inorganic Schiff bases

(Co(salophen) Fe(salophen)) an organic modifier (anthroquinone) and the enzymes

bilirubin oxidase (BOD) and laccase (Lac) In each case of non-biological catalysts the

effects of ECPs were also quantified using cyclic voltammetry (CV) and hydrodynamic

methods with rotating disc electrodes (RDEs)

CV allowed the investigation of

Capacitance from the double layer region

Qualitative description of the mechanism such as number of discrete reaction

steps reversibility and surface confinement

nα the number of electrons transferred up to and including the rate limiting step

(RLS)

219

while hydrodynamic methods were used to quantify

n the total number of electrons transferred

kdeg the heterogeneous standard rate constant

Epc2(HDV) the half-wave reduction potential in HDV

The data was taken under both N2 and O2 to allow characterisation of the effects of

electrochemical pretreatment and its effects on the kinetics of O2 reduction The best

candidate catalytic cathode was determined according to kdeg nα and Epc2(HDV)

72 INFLUENCE AND EFFICACY OF ECPS

721 Glassy carbon electrodes (GCEs)

7211 Untreated GCEs

Voltammetry curves of untreated GCEs at pH 74 in the presence of N2 and O2

are shown in Fig 97

220

Fig 97 CVs of untreated GCEs at pH 74 in 001 M PBS in the presence of (i) N2 (ii)

O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Untreated GCEs did not show any peak in the absence of O2 (Fig 97(i)) indicating that

there was no electrochemically active redox species on the electrode surface and in the

solution CV was in the presence of O2 (Fig 97(ii)) showed a peak at ndash 08 V but

without a reverse anodic current This indicates the presence of catalytic activity

however it occurred at only high overpotential and is therefore not efficient for ORR

This result is consistent with previous work [215 216]

7212 Influence of electrochemical pretreatments on GCE

GCEs were pretreated in various media in order to improve ORR efficiency It

has been reported that ECPs activate the electrode surface by introducing

electrochemically active functional groups and that this improves electrochemical

(i) N2

-3E-05

-2E-05

-1E-05

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1E-05

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

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1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

221

reversibility and [105 218] and the electron transfer rate of inner-sphere catalysis [228]

Fig 82 shows the voltammetry curves in the absence of O2 for various pretreated GCEs

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-60E-05

-50E-05

-40E-05

-30E-05

-20E-05

-10E-05

00E+00

10E-05

20E-05

30E-05

40E-05

50E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH

Fig 98 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of

O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1 Arrows indicate the

peak position (i) includes CVs for all the pretreated GCEs and (ii) excludes

PBS pretreatments for convenience

222

As the figure shows any pretreatment resulted in an increase in capacitance and

introduced characteristic voltammetric peaks which were not observed in the untreated

GCEs In all cases the peak current was linearly proportional to υ indicating that the

pretreatments led to surface-confined redox active functional groups most likely to be

quinone groups That ECPs give rise to surface quinones is well established [105 218

220 229 230] The Epc shifted with υ and was separated from Epa hence redox

reactions by these surface quinone groups are quasi-reversible The effect of

pretreatments however varied by the type of medium used in pretreatments

Above all PBS-related pretreatments gave qualitatively different results which

were characterised by a great increase in average capacitance calculated from CVs in

001 M PBS at pH 74 at 50 100 and 200 mVs-1 in the double layer region of the CV at

04 V Summary results are shown in Fig 82(i) and Fig 99

223

GCE Capacitance vs Pretreatment

0100200300400500600700800900

1000

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS

NaOH PBS

NaOH H2SO4

PBS

Pretreatment

Capa

cita

nce

microF

cm-2

Fig 99 The capacitance of GCEs in 015 M NaCl buffered with 001 M phosphate to

pH 74 Error bars represent the standard deviation (n = 3)

The average capacitance of the untreated GCEs was 76 microFcm-1 and is within the typical

ranges reported hitherto [221] while PBS pretreatment increased the capacitance to 700

- 800 microFcm-1 A similar phenomenon was reported by Nagaoka and by Swain [215

216] There are two possible reasons for this (i) PBS treatment caused the

microfractures of GC and increased the electrode area and therefore double layer [105

215 216 218] andor (ii) attached the electrochemically inactive charged surface group

such as phenol and carboxylate [216 229]

In the pretreatment involving NaOH the carbon dissolution was observed This

is harsher pretreatment so erodes the electrode and makes the electrode surface more

porous [216 218] NaOH also forms unstable oxide film and eventually removes

graphite oxide [229] and surface impurities [218] However the resultant capacitance

224

was unexpectedly smaller than that of sole PBS pretreatment Therefore NaOH

pretreatment increased the capacitance by increasing the exposed electrode area [218

231] while PBS pretreatment increased capacitance by attaching redox inactive charged

surface groups The largest capacitance after the double pretreatment involving NaOH

and PBS was therefore derived from the layer of charged groups attached on the

electrode surface area made larger by NaOH pretreatment

H2SO4 pretreatment on the other hand resulted in a smallest capacitance

increase The electrode behaviour was also slightly altered from that of other

PBS-related pretreatments when the pretreatments involved H2SO4 together The

capacitance became smaller as shown in Fig 99 and the peak positions were shifted to

more positive potential side (Fig 82(i)) To investigate the causes of this phenomena

the voltammograms during such pretreatments were studied as Fig 100 below

225

Fig 100 Overlaid CVs during various pretreatments for GCEs involving 01 M H2SO4

alone (red) 001 M PBS at pH 74 alone (green) and both pretreatments (pink)

It is clearly shown that either pretreatment resulted in distinctive peaks for the

attachment of active surface quinone groups However the peak potentials were

different when in sole H2SO4 or PBS-related pretreatments This is because as proposed

by Nagaoka those quinone groups were present in the different chemical environment

leading to different behaviours [216] H2SO4 pretreatment attached fewer inactive

surface oxygen groups while PBS pretreatment attached more Therefore H2SO4

pretreatment led to the smaller capacitance change than PBS pretreatment This implies

the presence of different electrode surface conditions hence quinones present in such

different environment behave differently showing different peak positions The

behaviours of surface quinone groups depends on the conditions of the pretreatment

involved [229 230 232] This fact can also explain the phenomena caused by the

226

double pretreatment with H2SO4 and PBS as shown in Fig 82(i) More quinone groups

had been attached by H2SO4 pretreatment in advance therefore only smaller area than

usual became available for PBS pretreatment to stably attach their charged oxygen

surface groups This is the most likely causes of the reduced capacitance and different

peak positions in such a double pretreatment compared to other PBS-related

pretreatments Furthermore quinone peak positions should be close to those found for

the H2SO4 pretreatment alone because quinone groups attached by preceding H2SO4

pretreatment dominate the electrode surface and should be have more influence on the

electrochemistry However they also resulted in larger capacitance because the

subsequent PBS pretreatment attached inactive oxygen surface group which led to an

increase in fixed charge density This is a typical phenomenon of synergetic effects as

already seen in the double pretreatment with NaOH and PBS

On the other hand voltammetric curves for the double pretreatment of NaOH

and H2SO4 did resemble to those by sole NaOH pretreatment Similar phenomena were

observed between the double pretreatment with NaOH and PBS and the triple

pretreatment with NaOH H2SO4 and PBS It seems that preceding NaOH pretreatment

overwhelmed the subsequent H2SO4 pretreatment although some influence by H2SO4

can be observed in terms of capacitance as shown in Fig 99 This implies that the

preceding NaOH pretreatment made most of the electrode area not available for

subsequent H2SO4 pretreatment However if NaOH pretreatment creates micropores in

the GC protons from subsequent H2SO4 pretreatment can still penetrate such

micropores These micropores are accessible for protons but not most ions [216]

227

7213 Effect of GCE pretreatment on ORR

Voltammograms for ORR at various pretreated GCEs at pH 74 are shown in

Fig 101 below

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO4

01M NaOH01M NaOH + 01M H2SO4

001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS

01M NaOH + 01 M H2SO4 + 001M PBS

Fig 101 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

In the figure any pretreated GCE shows larger cathode peaks between ndash 03 and ndash 05 V

without reverse anodic current They were due to O2 reduction by surface quinone

groups whose peaks were observed at ndash 01 V in Fig 82(i) in the absence of O2 This

indicates that ORR at any pretreated GCEs is regulated by ECrsquo mechanism [30] Ipc was

linearly proportional to radicυ in all cases indicating the diffusion-limited charge transfer

228

However untreated GCE was catalytic only at high overpotential since Epc2(HDV) = ndash 08

V This is compatible to the observation made by Tammeveski [233] It was seldom

catalytic since n = 102 plusmn 011 and nα = 059 plusmn 004 This single-electron ORR implies

the presence of O2־ disproportionation from which little power can be extracted [233]

kdeg was could not be calculated because the standard Koutecky-Levich plot could not be

obtained since it did not show a normal linear slope In contrrast all the other

pretreatments improved ORR as seen in Fig 101 They showed large cathodic peaks at

smaller overpotentials PBS-related pretreatments in particular increased the catalytic

current by gt 15 times and positively shifted Epc2(HDV) by gt 300 mV relative to that of

untreated GCEs Similar phenomena were reported elsewhere [220 234] All the GCE

pretreatments resulted in an increase in both nα and n The increase in kdeg accompanied

by an increase in nα was also observed as a general tendency as shown in Fig 102

229

0

2

4

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

kordm

ms

-1

nα 059 107 094 106 168 278 174 178

kordm ms-1 000E+00 715E-09 529E-09 631E-08 131E-07 279E-07 654E-08 113E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

- nα

kdeg ms-1

Untreated H2SO4 NaOHNaOHH2SO4

H2SO4PBS

NaOHPBS

NaOHH2SO4PBS

Fig 102 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Above all the H2SO4-PBS double pretreatment was most effective giving kdeg = 279 x

10-7 plusmn 507 x 10-9 ms-1 and Epc2(HDV) = - 039 plusmn 002 V However its nα was calculated

to be 278 while n = 185 This is an unusual result It might imply the presence of serial

ORR pathway in which H2O2 is further reduced or it is simply impossible to obtain

correct parameters by using Eq 88 on p 193 for ORR at pretreated GCEs due to their

excessively narrow peak width Pretreatment of GCE was proved to increase kdeg of ORR

but how this improves its catalysis has been uncertain for a long time between two

hypotheses whether they mediate electron transfer or they alter the adsorption of O2

and O2[233 232 220] ־ The latter or both is more likely because as Fig 102 shows

PBS-related pretreatments increased the capacitance which was derived from redox

inactive surface oxygen groups yet made significant contribution to an increase in kdeg It

230

is therefore natural to deduce that the largest kdeg and nα were the consequence of

synergetic effect derived from different surface oxygen groups attached by H2SO4 and

PBS

722 Graphite composite electrodes

7221 Untreated graphite composite electrodes

Graphite composite electrodes are different from GCEs They are made of

graphite particles which are more porous and permeable to the solution and gas This

practically results in larger electrode surface area therefore becomes more subject to

facile adsorption and surface modification This leads to the different surface conditions

and electrode behaviours from GCEs as proposed by Sljukic [232] CVs of untreated

graphite composite electrodes at pH 74 in the presence of N2 and O2 are shown in Fig

103 together with those of untreated GCEs

231

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

Fig 103 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl Small peaks on graphite electrodes are indicated by arrows

Different electrode behaviour is noticeable especially in nitrogenated solution because

untreated graphite composite electrodes showed small peaks at + 100 mV and - 100 mV

as indicated by arrows No peak was present fo untreated GCE This indicates the

presence of some reactive surface quinone groups on graphite composite without

pretreatment This is because graphite composite electrodes have more reactive edge

plane graphite exposed and the porous electrode structure allows larger electrode area to

be exposed There was however little difference in averaged capacitance between

graphite composite and GCEs This indicates that an increase in capacitance does not

necessarily correlate to an increase in the surface area as proposed by Regisser [235]

Voltsmmograms for ORR at untreated GC and graphite composite electrodes resembles

each other though for the latter the reduction peak is around 50 mV more positive It can

232

be seen that graphite composites are slightly more catalytic toward ORR presumably

because of the intrinsic quinone groups derived from the graphite electrode surface

7222 Influence of electrochemical pretreatments on graphite composite electrodes

Cvs for the graphite composite electrodes differed from those of GCEs Peaks

were observed in all graphite cases including untreated ones as shown in Fig 104

233

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

(ii)

01M H2SO401M NaOH01M NaOH + 01M H2SO4Untreated

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBSPBS

(iii)01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS001M PBS

Fig 104 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M

nitrogen-sparged PBS between + 09 and - 09 V vs AgAgCl (i) All

pretreatments at 005 Vs-1 (ii) Non-PBS-related pretreatment and (iii)

PBS-related-pretreatment CVs at 002 Vs-1

The capacitance became larger after pretreatments Any multi-pretreatment involving

NaOH and H2SO4 resulted in the largest capacitance which was 20 times larger than

234

that of untreated graphite composite electrodes The capacitance increase was also

larger than that of any pretreated GCEs except for PBS-pretreated ones PBS

pretreatment led to the smallest capacitance increase in graphite composite electrodes in

contrast to the GCEs Instead H2SO4 pretreatment led to the largest capacitance

increase Moreover this outcome was found to correspond to a specific proportional

relation between Ip and υ Ip of electrodes involving any pretreatment in PBS NaOH or

their combinstions were linearly proportional to radicυ This can only arise from on or more

diffusion limited step or steps in the redox reactions of the surface groups most likely

proton diffusion though a porus film or to microscopic porous environments Surface

oxygen groups have previously been reported to involve involve proton uptake and this

leads to a broad peak [216 230] H2SO4-pretreated electrodes showed Ip proportional to

υ and the redox reaction is independent of proton diffusion

These phenomena show that different pretreatments provide different chemical

environment for active quinone groups leading to different quinone behaviours and

voltammetry peaks [229 230 232] In the multi-pretreatment with NaOH and H2SO4

synergetic effects became obvious Its redox reaction was diffusion-limited and showed

voltammograms with two partially resolved sets of peaks yet its peak still partially

overlaps with that of diffusion-independent H2SO4-pretreated electrodes as seen in Fig

104(i) When this double-pretreatment was further followed by PBS pretreatment the

peak derived from quinones attached by H2SO4 disappeared This implies that

subsequent PBS pretreatment suppressed the activity of proton-independent quinones

and the effect by H2SO4 The similar phenomenon was observed when electrodes were

pretreated doubly by H2SO4 and PBS (Fig 104(ii)) Every case presented

235

quasi-reversible redox systems because peak potentials shifted with scan rates Small

multiple peaks were present in slower scan rates merged into one broad peak at faster

scan rate as the contribution of capacitance charging and increased iR drop obscured

voltammetic detail

7223 The effect of graphite composite electrode pretreatment on ORR

ORR at pretreated graphite composite electrodes was found to be less effective

than for pretreated GCEs Only NaOH-related pretreatments were effective (Fig 105)

enough to achieve two-electron ORR Untreated graphite composite electrodes were

catalytic only at high overpotential despite the presence of surface quinone groups PBS

pretreatment was hardly effective in contrast to the results obtained for GCEs

Electrochemical pre=treatment in H2SO4 and its double pretreatment with PBS reduced

Epc2(HDV) by 40 ndash 90 mV however the electrodes showed poor catalytic performance nα

lt 05 and n lt 2 consistent with the initial electron transfer still being rate determining

236

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 105 Overlaid CVs of ORR at various pretreated graphite composite electrodes

at pH 74 in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005

Vs-1

Pretreatment of the graphite composites with just H2SO4 showed a single merged peak

at slower scan rate while they showed an addition prewave as indicated by arrows in

Fig 106 at faster scan rate

237

-16E-03

-14E-03

-12E-03

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

-1 -08 -06 -04 -02 0 02

E V

J Acm-2

50 mV100 mV200 mV500 mV

Fig 106 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at

pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various

scan rates 50 100 200 and 500 mVs-1

Coupled with the fact of n asymp 1 this prewave is due to the rate-limiting slow formation of

semiquinone radicals [233] A semiquinone radical is formed when one electron is

transferred to a quinone group and its formation is necessary to mediate ORR as shown

in Eq 100

238

middotmiddotmiddotmiddotmiddot Eq 100

[233] middotmiddotmiddotmiddotmiddot Eq 101

Q = Surface quinone group

O2־ = Semiquinone radical

Any NaOH-related pretreatment on the other hand showed a prewave around ndash 035 V

at slower scan rate as shown in Fig 107 but this merged into a broad peak at faster scan

rate

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

01M NaOH01M NaOH + 01M H2SO4 01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 107 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

239

Ipc was linearly proportional to radicυ except for the triple pretreatment which was difficult

to define due to the ambiguous peak positions over different scan rates The prewave

was due to the slow reduction of O2 into O2־ (Eq 101) rather than semiquinone radical

formation (Eq 100) as seen in H2SO4-pretreated electrodes They had n asymp 2 and nα asymp 1

hence they catalysed two-electron ORR Those pretreatments positively shifted

Epc2(HDV) by 173 plusmn 26 mV on average from ndash 730 mV at the untreated graphite

composite electrodes The largest catalytic activity was achieved by doubly pretreated

electrodes with NaOH and H2SO4 with kdeg = 382 x 10-7 plusmn 32 x 10-8 ms-1 (Fig 108)

0

2

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

35E-07

40E-07

45E-07

kordm

ms

-1

nα 042 035 080 114 045 040 101 072

kordm ms-1 000E+00 000E+00 233E-07 382E-07 000E+00 000E+00 132E-07 172E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBSUntreated H2SO4 NaOH

NaOHH2SO4

H2SO4

PBS NaOHPBS

NaOHH2SO4

PBS

- - - - nα

kdeg ms-1

Fig 108 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

240

723 Platinum on carbon composite electrodes (PtC)

Noble metals are popular ORR catalysts [172 173] with Pt showing the highest

catalytic power [165 166 171 172] It was reported that Pt has a 98 O2-to-H2O

conversion rate [174] However for PtC composite electrodes the voltammetry was

dominated by showed solution resistance both in the presence and absence of O2 and

therefore were not either catalytic or conductive H2SO4 pretreatment was not at all

effective This is probably due to the large electronic resistance of activated carbon itself

[204] as well as intrinsic resistance of composite conductors

724 Rhodium on carbon composite electrodes (RhC)

Rhodium is a platinum group metal and has been reported as a good ORR

catalysis after Pt and Ir [172] Voltammograms for untreated RhC composite electrodes

only showed the capacitance but no redox peaks in nitrogenated solution The

capacitance decreased by 80 for every ten-fold increase in the scan rate Non-faradic

current is normally linearly proportional to υ however it exponentially decreased with υ

in the untreated RhC composite electrodes It did not show any peak in the presence of

O2 and was not catalytic toward ORR (Fig 110(i)) H2SO4-pretreated RhC composite

also did not show any voltammetric peaks Its capacitance was roughly x30 larger than

that of untreated electrodes and again exponentially decreased with an increase in υ

ORR catalysis was however present (Fig 110(ii)) therefore it is reasonable to deduce

that H2SO4 attached the surface quinone groups but the large capacitance charging

prevented voltammetic observation of quinones

241

(i) Untreated

-16E-03

-12E-03

-80E-04

-40E-04

00E+00

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

N2O2

(ii) H2SO4

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

10E-03

-1 -08 -06 -04 -02 0 02 04 06 08 1

E VJ

Ac

m-2

N2O2

Fig 109 CVs of (i) untreated and (ii) H2SO4-pretreated RhC composite electrodes at

pH 74 in 001 M PBS in the presence of N2 and O2 at 005 Vs-1 between + 09

and -09 V vs AgAgCl

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR

Untreated RhC composite electrodes did not show ORR catalysis however a

steep decrease in cathodic current was observed without any peak as the potential

decreases This steep current decrease was probably caused by O2 adsorption on the Rh

particle surface H2SO4-treated RhC on the other hand was found to be catalytic with n

= 177 and nα = 068 plusmn 011 A reduction prewave was observed at around ndash 05 V and

this merged into a broad peak at faster scan rates This prewave was due to ORR by

surface quinone groups attached by H2SO4 because it overlaps with the peak observed

in H2SO4-treated graphite composite electrodes as shown in (Fig 110) This prewave

242

therefore indicates the slow first electron transfer to form O2־

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

-1 -05 0 05 1

E V

J Acm-2

RhC H2SO4

Graphite H2SO4

Fig 110 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

O2־ was probably further reduced to H2O2 by Rh particles because n = 177 plusmn 011 The

catalytic activity was much larger compared with H2SO4-pretreated graphite composite

electrodes as shown in Fig 110 with kdeg =257 x 10-7 plusmn 230 x 10-8 ms-1

243

725 Co(salophen) composite electrodes

7251 Untreated Co(salophen) composite electrodes

Untreated Co(salophen) electrode showed couples of distinctive peaks Epc(1) =

053 Epc(2) = -024 Epa(3) = -016 and Epa(4) = 016 V as shown in Fig 111(i) These

peaks correspond to stepwise cobalt redox reactions Co3+2+ and Co2+1+ respectively

The peak potentials hardly changed over different scan rates consistent with previously

published results [181] Similar peaks have been observed in deoxygenated organic

solvents [174 181] as well as for Co(salen) [181]

(i) N2

-2E-05

-1E-05

0E+00

1E-05

2E-05

3E-05

4E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

2E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fig 111 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

244

In the presence of O2 it showed a reduction peak at ndash 04 V as shown in Fig 111(ii)

ORR potential was shifted by approximately 300 mV relative to that of untreated

graphite composite This result reasonably compatible to the result reported by Ortiz

[181] At this potential O2 forms an adduct with Co2+(salophen) and then is reduced to

O2־ while Co2+ undergoes one-electron oxidation to Co3+ [170] To be catalytic Co3+

formed here must be reduced back to Co2+ to reduce O2־ further into H2O2 [181] or a

binuclear complex must be formed with another Co2+ [170] Untreated Co(salophen)

composite electrodes were however not strongly catalytic due to n = 138 plusmn 009 and nα

= 055 plusmn 012 Ip was linearly proportional to radicυ and the peak potentials Epc(2) and Epa(3)

varied only by plusmn 8 over different five scan rates This indicates nearly reversible

multi-electron transfer redox system of Co(salophen) composite electrodes

7252 Influence of electrochemical pretreatments on Co(salophen) composite

There was a particular redox pattern found in voltammetry waves according to

the type of pretreatments (Fig 112)

245

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 112 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in

001 M nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

NaOH-pretreatment showed similar voltammetric faradaic features as those in untreated

electrodes (Fig 112) but increased the capacitance by six times relative to that of

untreated electrodes The size of capacitance was 382 microFcm-1 and approximately equal

to that of NaOH-pretreated graphite composite electrodes This implies that NaOH

attached electrochemically inactive surface oxygen groups and the attachment of active

surface quinone groups were not clearly confirmed

PBS-pretreatment on the other hand attached quinone groups because Epa asymp 04 V at

which similar peaks were observed in PBS-pretreated graphite composite (Fig 104(iii)

p 233) Their cathodic peaks were however broader because they merged into peaks

246

for Co-redox processes The effect of PBS pretreatment was much smaller compared to

that of H2SO4-related pretreatment in the deoxygenated solution Single NaOH

treatment and any PBS-related pretreatment showed Ip linearly proportionally to radicυ

while any H2SO4-related pretreatment but without involving PBS-pretreatment showed

a proportional relation with υ This indicates that H2SO4 and PBS or NaOH

pretreatments lead to different surface redox mechanism Peaks due to H2SO4-related

pretreatments were prominent and are indicated by a red circle in Fig 112

7253 The effect of Co(salophen) composite electrode pretreatment on ORR

Untreated electrodes were not catalytic Parameters for H2SO4-pretreated

Co(salophen) composite found to have n asymp 1 and nα le 1 although they look catalytic in

Fig 113 reducing its ORR overpotential by 400 ndash 500 mV compared to GCEs This

inconsistent result arose because it was impossible to find the correct parameter using

the conventional calculation using Eq 88 on p 193 and need more sophisticated

method such as a simulation probramme The other pretreatments were on the other

hand found strikingly effective toward ORR as shown in Fig 113

247

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

40E-04

60E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 113 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Such pretreated Co(salophen) electrodes showed Epc asymp - 03 V having reduced the

overpotential against unpretreated Co(salophen) by 100 mV and 300 mV against

untreated graphite composite electrodes ORR was synergistically catalysed by Co2+ and

surface semiquinone radicals [181 233] They showed n asymp 2 and nα gt 07 hence they

catalysed two-electron ORR Ipc was linearly proportional to radicυ hence the redox

reactions were diffusion limited except for the double pretreatment with NaOH and

H2SO4 Large catalytic activity was achieved by sole NaOH and by the double

pretreatment with NaOH and H2SO4 or the combination of H2SO4 and PBS Among

them sole NaOH pretreatment was revealed to be most effective as shown in Fig 114

and showed kdeg = 119 x 10-5 plusmn 118 x 10-6 nα = 083 plusmn 014 and Epc2(HDV) = 037 V

248

0

1

2

nα

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

kordm

ms

-1

nα 055 028 083 074 068 091 071 078

kordm ms-1 000E+00 000E+00 119E-05 747E-06 271E-06 684E-06 296E-06 587E-06

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

Fig 114 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite

electrodes

One possible reason for this largest catalytic activity by NaOH pretreatment is that

NaOH might attach more reductive surface OH groups [218] which became involved in

reducing back Co3+ into Co2+ during ORR [181]

726 Fe(salophen) composite electrodes

7261 Untreated Fe(salophen) composite electrodes

Untreated Fe(salophen) electrodes showed a couple of distinctive peaks Epa = -

024 plusmn 001 V and Epc = - 036 plusmn 001 V These peaks are due to Fe3+2+ redox reaction as

249

shown in Fig 115(i) The voltammetry curve and redox peaks are reasonably similar to

those of Fe(salen) reported [224]

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

(ii) O2

-6E-04

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

Fig 115 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74

in 001 M PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and

- 09 V vs AgAgCl

However the redox system is not perfectly reversible here because ∆Ep slightly

increases with an increase in the scan rate and its Ip was linearly proportional to radicυ It is

therefore a quasi-reversible system Untreated Fe(salophen) electrodes was not as

catalytic as Co(salophen) electrodes Epc was ndash 079 plusmn 007 V and little different from

that of untreated graphite composite electrode as shown in Fig 115(ii) although Ipc was

slightly bigger The low ORR catalytic activity of Fe(salophen) was due to the low ORR

rate of Fe itself (Fig 65 on p 164) [172]

250

7262 Influence of electrochemical pretreatments on Fe(salophen) composite

The effect of pretreatments to Fe(salophen) was striking They resulted in

additional voltammetric features due to the Fe redox peaks at Epc (Fe) = - 03 V and Epa

(Fe) = - 02 V and by attaching the surface quinone groups at Epc (Q) = 015 V and Epa (Q) =

02 V as shown in Fig 116(i)

251

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

60E-05

80E-05

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

Untreated01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS001M PBS

-1E-04

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

1E-04

1E-04

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

Fig 116 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1 (i) All

pretreatments (ii) Non-PBS-related pretreatments and (iii)

PBS-related-pretreatments CVs

Non-PBS-related pretreatments (Fig 116(ii)) showed redox peaks clearly however the

252

two cathodic peaks merged into a broad peak at faster scan rate than 100 mVs-1

indicating quasi-reversible surface quinone redox reactions Only a broad merged

reduction peak was observed for all PBS-related pretreatments (Fig 116(iii)) at all scan

rates

Any PBS-related pretreatments resulted led to a broad cathodic peak (from + 01 V to

approximately ndash 03 V) made up of both Fesalphen reduction and reduction of surface

confined quinones arising from the electrochemical pretreatment PBS pretreatment

always greatly increases the surface area leading to micro fissures and consequently a

greater variety of microenvironments for electrochemically generated quinone

functionality

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR

Most were found to be only weakly catalytic (Fig 117) since either n or nα or

both of them were lt 1 Only double-pretreatment with NaOH and H2SO4 led to

sufficient ORR catalysis with n = 185 plusmn 017 nα = 079 plusmn 023 kdeg = 112 x 10-7 plusmn 771 x

10-9 ms-1 and Epc2(HDV) = - 057 V The double pretreatment showed a prewave at ndash 035

V and it disappeared at faster scan rate indicating a slow single-electron transfer to O2

Ipc was linearly proportional to radicυ hence the catalysis was diffusion limited

253

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS

Fig 117 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

727 Anthraquinone (AQ) composite electrodes

Anthraquinone-grafted graphite electrodes were pretreated with H2SO4 Both

untreated and H2SO4-pretreated electrodes showed couples of peaks (Fig 118(i)) one of

which Epc(AQ) was compatible with Em = - 049 reported by McCreery [44] These

peaks were therefore derived from anthraquinone chemically attached to the graphite

254

(i) N2

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

-1 -05 0 05 1

E V

J A

cm

-2

Untreated FRE01M H2SO4 FREUntreated Graphite

(ii) O2

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

-1 -05 0 05 1

E VJ

Ac

m-2

Untreated FRE01M H2SO4 FREUntreated Graphite

Fig 118 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl CVs of untreated graphite electrodes are also shown together

The other couple of peaks Epc(SQ) and Epa(SQ) was observed at - 003 plusmn 003 V and +

020 plusmn 003 V respectively They were due to quinone groups on the carbon surface

H2SO4-pretreatment did not alter the voltammogram pattern but only increased the

capacitance by x3 and enhanced the peak current Both electrodes showed Ip linearly

proportional to radicυ hence the redox reactions involve proton diffusion ORR by those

electrodes is shown in Fig 118(ii) together with the voltammogram of graphite

composite electrodes Both untreated and pretreated AQ-grafted composite electrodes

positively shifted Epc by 60 and 230 mV respectively relative to that of untreated

graphite composite electrodes However their voltammetric curves resembled those for

untreated graphite composite electrodes Besides this their Ip dropped by 20 relative

255

to untreated graphite composite electrodes and showed n asymp 1 and nα asymp 05 Therefore

either was not catalytic in this study This outcome does not correspond to the finding

by Manisankar who confirmed the presence of ORR catalysis at AQ-carbon paste

electrodes at pH 7 with a reduction prewave at ndash 04 and a peak at ndash 07 V [193] To the

contrary this result fits to the data presented by Schiffrin who observed ORR

suppression at AQGC electrodes at pH lt 7 He reported that voltammetry curves for

ORR at GC and GCAQ became similar at this pH hence implied the possibility of

disproportionation of semiquinone intermediates [194]

728 Lac and BOD enzyme membrane electrodes

In the absence of O2 neither Lac nor BOD showed any peaks in their cyclic

voltammograms However they showed ORR catalytic activity even in the absence of

mediator (Fig 119) DET is therefore possible as already confirmed by many [183

186-188] Carbon surface groups [195] also might mediate electron transfer [188]

256

-14E-04

-12E-04

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

-1 -09 -08 -07 -06 -05 -04 -03 -02 -01 0

E V

J Acm -2

LacBOD

Fig 119 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated

PBS at 005 Vs-1 between 0 and -09 V vs AgAgCl

Ipc was linearly proportional to radicυ therefore ORR was diffusion limited Epc were -054

plusmn 002 and ndash 067 plusmn 006 V at Lac and BOD respectively They reduced ORR

overpotential by 120 and 250 mV relative to that of GCEs however the Epc shifted with

υ hence they were quasi-reversible It is known that MCOs catalyse four-electron ORR

[175 184 236] however n = 005 and nα asymp 1 in this study This is probably due to the

difficulty in electrochemical measurement of enzyme catalysis It is however possible

to find nα because the rate-limiting step is the single electron transfer between the

electrode and the blue copper atom of the enzyme Assuming this kdeg was calculated for

four-electron ORR at the enzyme electrodes and the result is shown in Fig 120

257

0

1

2

nα

00E+00

40E-09

80E-09

12E-08

16E-08

20E-08

kordm

ms

-1

nα 077 081

kordm ms-1 134E-08 168E-09

Lac Bod

Fig 120 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Lac had kdeg = 134 x 10-8 plusmn 251 x 10-9 while BOD had kdeg = 168 x 10-9 plusmn 271 x 10-10

Lac electrodes showed better ORR efficiency despite of non-optimal pH condition Lac

is most active at pH 5 while BOD at pH 7 The amount of enzyme loaded on the

electrode surface also might have affected the catalytic activity It was however difficult

to control the loading because the enzyme was attached by dip coating Using mediators

could have also improved the ORR efficiency Many authors used mediators such as

ABTS [62 66] and Os-pyridine complexes linked to conductive polymer [97 237] to

assist the electron transfer between the electrode and enzyme

73 MOST EFFICIENT ORR CATALYSTS

The kinetic and thermodynamic parameters (kdeg nα Epc2(HDV)) were organised

258

by magnitude and are summarised in figures 25-27 and tables 1 amp 2 below For each

parameter the best five candidates were selected to present the effects of pretreatment

and their relation with each parameter The best three were chosen from them on the

basis of the parameter size and the number of top-ranked parameters they were

occupying kdeg was given the highest priority for selection process

731 Best five electrodes for nα

Pretreated GCEs were found to have prominent nα (Fig 121)

00

05

10

15

20

25

30

Electrode type

n α

nα 278 178 174 168 114

GC H2SO4-PBS GC NaOH-H2SO4-PBS

GC NaOH-PBS GC PBS GP NaOH-H2SO4

Fig 121 The best five electrodes for nα

The GCEs doubly pretreated with H2SO4 and PBS was found best with nα = 278 Its kdeg

was 279 x 10-7 ms-1 and also biggest among pretreated GCEs Correlation between nα

259

and kdeg has not been confirmed since it is possible for the intial electron transfer to be

accelerated whilst still remaining the RDS

732 Best five electrodes for kdeg

Top six kdegs were dominated by pretreated Co(salophen) composite electrodes

(Fig 122)

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

Electrode type

kdeg

ms

-1

kordm ms-1 119E-05 747E-06 684E-06 587E-06 296E-06

CoS NaOH CoS NaOH-H2SO4

CoS H2SO4-PBS CoS NaOH-H2SO4-PBS

CoS NaOH-PBS

Fig 122 The best five electrodes for kdeg

PBS pretreatment was found to suppress kdeg in Co(salophen) composite electrodes Clear

correlation between kdeg and nα has not yet confirmed however it was found that

favourable kdeg tends to lower overpotential

260

733 Best five electrodes for Epc2(HDV)

Although it is not an exact correspondence some correlation was observed

between lower overpotential and larger kdeg or lower overpotential and larger nα but not

between kdegand nα A range of pretreated Co(salophen) composite electrodes which

showed largest kdeg and a pretreated GCE which showed the largest nα were found to be

dominating low overpotential (Fig 123)

-040

-039

-038

-037

-036

-035

Electrode type

E pc

2(H

DV)

V

Epc2(HDV) -037 -037 -039 -039 -040

CoS H2SO4-PBS CoS NaOH CoS NaOH-H2SO4

GC H2SO4-PBS CoS NaOH-PBS

Fig 123 The best five electrodes for Epc2(HDV)

734 Best three candidates for ORR catalytic electrodes

Pretreated Co(salophen) composite electrodes were the best candidates for

ORR catalysis because of largest kdeg and lowest overpotential Top three catalytic

261

cathodes were listed in Table 17

kordm ms-1 Epc2(HDV) V nα

Co(salophen) NaOH 119E-05 -037 083

Co(salophen) NaOH-H2SO4 747E-06 -039 074

Co(salophen) H2SO4-PBS 684E-06 -037 091

Average 875E-06 -037 083

SD 278E-06 001 009

Table 17 Three candidates for catalytic cathodes for ORR

NaOH-pretreated Co(salophen) was found to be most efficient ORR catalytic cathode

because it has the largest kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 Epc2(HDV) = - 037 V and

was the second smallest of all kdeg of the three candidate Co(salophen) electrodes were

compared with those of catalytic metals and shown in Table 18

Electrode type kdeg ms-1 Experiment condition

Co(salophen) NaOH 119 x 10-5 In 001M PBS pH 74

Co(salophen) NaOH ndashH2SO4 747 x 10-6 In 001M PBS pH 74

Co(salophen) H2SO4-PBS 684 x 10-6 In 001M PBS pH 74

Au 4 x 10-3 In 005 M H2SO4 [173]

Au 3 x 10-5 In 01 M KOH

Ag 8 x 10-3 In 01 M KOH

Pt 4 x 10-3 In 01 M KOH

Table 18 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

262

NaOH-pretreated Co(salophen) composite electrode showed good catalysis at neutral

pH It could manage to achieve as efficient ORR as Au in acidic solution

74 CONCLUSION TO CATHODE STUDY

The large overpotential of ORR has been known to degrade the fuel cell

performance [3 4 62 165-167] Finding efficient ORR catalysts therefore has been a

subject of intensive research In this study various catalytic cathodes were fabricated

using inorganic organic and biological catalysts and they were electrochemically

pretreated in different media to improve the catalytic activity Studies were conducted

using various electrochemical methods (i) CV (ii) HDV and (iii) HDA The data

obtained was analysed both qualitatively and quantitatively The ORR catalytic

efficiency was calculated according to the three parameters (i) kdeg (ii) nα and (iii)

Epc2(HDV) and based on that most catalytic cathode was determined

None of the cathodes was catalytic enough without pretreatment or unless at high

overpotential In many cases electrochemical pretreatments were found to attach

various surface oxygen groups activated the electrodes and led to two-electron ORR

catalysis Their influence on electrochemistry varies by the electrode type and media

used for pretreatment It was found that PBS-related pretreatments were effective to

GCEs When the pretreatment is combined with H2SO4 this might lead to serial ORR

where H2O2 produced is further reduced to water NaOH-related pretreatments were

effective for graphite composite electrodes PtC composite electrodes were not active

due to the high resistance within the activated carbon The performance by RhC was

263

only marginally improved by H2SO4 pretreatment Any NaOH- or PBS- related

pretreatments dramatically increased kdeg of Co(salophen) composite electrodes and

reduced the overpotential by 100 mV Fe(salophen) composite electrodes were not

sufficiently catalytic without a double pretreatment with NaOH and H2SO4

Anthraquinone-grafted graphite composite electrodes were not catalytic at neutral pH

even after pretreatment Multi-copper oxidases were catalytic at DET however its RLS

was electron transfer between the electrode and the enzyme hence appropriate

mediators should be used to achieve higher catalysis

Many of pretreated Co(salophen) composite electrodes were strongly catalytic toward

ORR Among them NaOH-pretreated Co(slophen) electrodes were found to be most

catalytic at neutral pH with kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 nα = 083 and Epc2(HDV) =

- 037 V Its catalytic efficiency was close to that of Au in the acid solution

264

8 CONCLUSION AND FURTHER STUDIES

Miniaturised glucose-oxygen fuel cells are ideal to power small implantable

medical devices Typical specification include at least 1 microW power output and 1-year

operational life Therefore fuel cells must have efficient catalysis and electron transfer

good conductivity chemical and mechanical stability as well as biocompatibility The

electrodes must be feasible to build and scaleable In this study electrodes were built

using bulk modification method because it was simple to construct and scale-up and

they were easy to modify and mechanically stable The anodes were built with 10

GOx with 77 TTF-TCNQ composite and the cathodes were built with graphite

composite containing ORR catalysts using bulk modification method TTF-TCNQ was

used because it was conductive as well as mediating and necessary for electron transfer

of the GOx anodes Problems were enzyme stability and ORR catalytic efficiency

Enzyme mass loading is known to be effective to improve catalytic efficiency and

operational life however this also decreases conductivity of the composite electrodes

The key to optimising enzyme electrodes is in the balance and compromise among the

conductivity mediation enzyme activity mechanical and chemical stability and

difficulty in the fabrication process CNF composites showed reasonable conductivity at

30 content and this might allow higher enzyme loading with mediators without losing

conductivity CNT is also a candidate material because it has comparable or larger

conductivity than TTF-TCNQ and can operate without added mediator species Ideally

enzyme electrodes should be functional with minimum components simple to build

renew and store in dry so that they become easily portable Moreover when they are

further miniaturised this will allow the medical application under harsher conditions so

265

that powering disposable implantable sensors in economically-deprived regions might

become possible Many physiological enzymes however easily lose catalytic activity

outside the optimal condition Incorporation of enzyme stabilisers makes the enzyme

robust and long lasting as well as scavenging reactive species In the glucose-oxygen

fuel cells O2 has been known to reduce the catalytic output current of GOx anodes

PEI-DTT stabiliser mixtures were found to be good for this because they conferred

stability on GOx against oxidative stress prevented O2 electron deprivation and

improved the catalytic current output It was however a smaller effect than when

conductive hydrogel was used Conductive hydrogel can be a good candidate if it

becomes resistant against degradation ORR at the cathode has posed a long-term

problem in fuel cells because ORR is inefficient and consumes potential generated

within the fuel cell Pt has been used as an ORR catalyst but it is expensive and is

becoming scarce Use of the fuel cell in the physiological condition also imposes further

operational limitations Pretreated metal complexes such as Co salophen were found to

be good candidates for ORR-catalytic cathodes Surrounding complexes with

electron-rich environment such as conductive film might improve ORR further Once

candidate anode and cathode are identified and built a biofuel cell unit can be made To

increase the power each cell is connected together and made into a stack The prototype

was made as below in Fig 124 by connecting twenty cell units

266

Fig 124 Fuel cell stack made with various GOx anodes and Co(salophen) cathodes

Each cell contained various GOx anodes and pretreated Co(salophen) electrode in 10 ml

of oxygenated 01 M glucose 001 M PBS solution pH 74 The output voltage was

found to be 02 V and it did not change for one week The fuel cell must be studied

further with an adjustable resistor for voltage-current (V-I) and power-current (P-I)

curves so that OCV can be determined properly The passive type GOx fuel cell could

output 15 mWcm-2 at 03 V [18]

In vivo studies are also necessary for implantable purposes to examine the catalytic

efficiency electrode fouling and biocompatibility This can be studied systematically by

changing the type and concentration of interfering substances such as ascorbate

acetoaminophen and urate [18] Heller tested miniturised GOx-BOD fuel cells tested in

the buffer grape and calf serum It showed only 5 power decrease in buffer however

50 drop within 20 hours in grape and 50 drop within 2 hours in calf serum [19] To

avoid electrode fouling the electrode surface can be coated with a membrane such as

267

Nafion However this imposes another problem because it can interfere with the

reactant flux to the electrode surface In the real physiological system this might cause

a serious problem because glucose and O2 concentration is much lower than in vitro

Furthermore in the physiological conditions active transport of the reactant is not

available and this will cause local concentration polarisation It seems that there are

many obstacles lying in the medical application of miniaturised glucose-oxygen fuel

cells although they have widespread potentials applications

268

9 BIBLIOGRAPHY

1 Carrette L KA Friedrich and U Stimming Fuel cells Principles types fuels and applications

Chemphyschem 2000 1(4) p 162-193

2 Gregoire Padro CE and JO Keller Hydrogen Energy in Kirk-Othmer Encyclopedia of Chemical

Technology 2005 John Wiley amp Sons Inc p 837-866

3 Ohta K T Kazama and M Nishizawa All about fuel cells in Newton 2006 p 31-63

4 Tanabe S Mastering basics of the fuel cells 2009 Denkishoin

5 Tokyo-Gas Fuel cell academy 2009 2901 [cited Available from httpwwwtokyo-gascojppefc

6 Cropper MAJ S Geiger and DM Jollie Fuel cells a survey of current developments Journal of Power

Sources 2004 131(1-2) p 57-61

7 Kordesch K et al Intermittent use of a low-cost alkaline fuel cell-hybrid system for electric vehicles

Journal of Power Sources 1999 80(1-2) p 190-197

8 UTC-Power The fuel cell for Apollo 11

9 Connor S Warning Oil supplies are running out fast in The Independent 2009

10 Mori Y Mobile fuel cell providing new advantages for mobile electronic appliances Toshiba review 2007

62(6) p 44-47

11 Mori H Electronic components and materials Toshiba review 2009 64(3) p 62-63

12 Imperial-College-London Racing Green - Vehicles 2009 [cited Available from

httpwwwunionicacukrccracinggreenvehicleshtml

13 Cleave V Formula Zero kart race could drive fuel cell technology New Scientist 2008 [cited

Available from

httpwwwnewscientistcomarticledn14596-formula-zero-kart-race-could-drive-fuel-cell-technologyhtml

14 HyNor Hydrogen highway opens in Norway 2009 [cited Available from

httpwwwhynornohydrogen-highway-opens-in-norway

15 BMW Hydrogen 7 - The future is closer than you think 2009 [cited Available from

httpwwwbmwcomcomeninsightstechnologyefficient_dynamicsphase_2clean_energybmw_hydroge

n_7html

httpwwwbmwcojpjpjabrandtechnologycleanenergywallpaperhtml

16 Sony Side view - Sony developes Bio Battery generating electricity from sugar 2007 Sony

17 Burton N Sugar-powered electronics in RSC Chemical technology news 2008

18 Sakai H A high-power glucoseoxygen biofuel cell operating under quiescent conditions Energy amp

Environmental Science 2009 2(1) p 133-138

19 Heller A Miniature biofuel cells Physical Chemistry Chemical Physics 2004 6(2) p 209-216

20 Toshiba Toshiba announces worlds smallest direct methanol fuel cell with energy output of 100 milliwatts

269

2004 [cited Available from httpwwwtoshibacojpaboutpress2004_06pr2401htm

21 Osakai T K Kanoh and S Kuwabata Basic Electrochemistry 2007 Kagakudojin Ltd Kyoto Japan

22 Wilson K and J Walker Practical Biochemistry 1992 Cambridge UK Cambridge University Press

23 Murray RK et al Harpers Biochemistry 25 ed 2000 Stamford Connecticut Appleton amp Lange

24 Sawyer DT A Sobkowiak and JL Roberts Jr Electrochemistry for chemists Revised ed 1995

Wiley-Interscience

25 IUPAC IUPAC GOLD BOOK 2009 [cited Available from httpgoldbookiupacorgindexhtml

26 Kitamura T and K Fujisawa English-Japanese The students dictionary of physics 1995 Tokyo ALC

Inc

27 Gates BC Catalysis in Kirk-Othmer Encyclopedia of Chemical Technology 2002 John Wiley amp Sons p

200-254

28 Taguchi S and E Nakano Kinetic Study of Bromination of Malonic Acid Methyl Malonic Acid

and Ethyl Malonic Acid An Undergraduate Physical Chemistry Laboratory Experimentfor

Understanding of the Infuluence of Thermodynamic Parameter on Chemical Kinetics The Chemical

Education Jounal 1999 3(1) p 1-20

29 Pletcher D A First course in electrode processes 1991 The Electrochemical Consultancy (Romsey) Ltd

23 - 26 135 - 145

30 Bard AJ and LR Faulker Electrochemical Methods Fundamentals and Applications 2000 NY USA

John Wiley and Sons Inc

31 Daintith J A concise dictionary of chemistry 2 ed 1990 Oxford UK Oxford University Press

32 Tinoco IJ et al Physical Chemistry 2002 Prentice Hall p 157

33 Watanabe K and S Nakabayashi Introduction to electrochemistry The chemistry of electron transfer 13

ed 2005 Tokyo Japan The Chemical Society of Japan

34 Hamelers B et al Microbial fuel cells Methodology and technology Environmental science amp

technology 2006 40(17) p 5181-5192

35 Koike T Chapter 10 Free energy in Physical chemistry I amp II Lecture handouts for dept of pharmacy

2010 University of Hiroshima p 57-62

36 Lopes T E Gonzalez and E Antolini An overview of platinum-based catalysts as methanol-resistant

oxygen reduction materials for direct methanol fuel cells Journal of alloys and compounds 2008 461(1-2)

p 253-262

37 Vork FTA and E Barendrecht The reduction of dioxygen at polypyrrole-modified electrodes with

incorporated Pt particles Electrochimica Acta 1990 35(1) p 135-139

38 Marino W B Scharifker and A Leone ELECTRODEPOSITION AND

ELECTROCHEMICAL-BEHAVIOR OF PALLADIUM PARTICLES AT POLYANILINE AND

POLYPYRROLE FILMS Journal of the Electrochemical Society 1992 139(2) p 438-443

39 Tsakova V How to affect number size and location of metal particles deposited in conducting polymer

270

layers Journal of Solid State Electrochemistry 2008 12(11) p 1421-1434

40 Lemest Y et al Dioxygen Fixation by a Mixed-Valence Dicobalt Cofacial Porphyrin Journal of the

Chemical Society-Chemical Communications 1983(22) p 1286-1287

41 Kim E et al Superoxo micro-peroxo and micro-oxo complexes from hemeO2 and heme-CuO2 reactivity

Copper ligand influences in cytochrome c oxidase models 2003 p 3623-3628

42 Fukuzumi S et al Mechanism of Four-Electron Reduction of Dioxygen to Water by Ferrocene

Derivatives in the Presence of Perchloric Acid in Benzonitrile Catalyzed by Cofacial Dicobalt Porphyrins

Journal of the American Chemical Society 2004 126(33) p 10441-10449

43 Solomon EI et al O2 and N2O Activation by Bi- Tri- and Tetranuclear Cu Clusters in Biology

Accounts of Chemical Research 2007 40(7) p 581-591

44 Wildgoose GG et al Anthraquinone-derivatised carbon powder reagentless voltammetric pH electrodes

Talanta 2003 60(5) p 887-893

45 McCreery R Carbon electrodes Structural effects on electron transfer kinetics in Electroanalytical

Chemistry A series advances 1990 Marcel Dekker Inc p 221-374

46 Kneten K R McCreery and C McDermott ELECTRON-TRANSFER KINETICS OF AQUATED

FE+3+2 EU+3+2 AND V+3+2 AT CARBON ELECTRODES - INNER-SPHERE CATALYSIS BY

SURFACE OXIDES Journal of the Electrochemical Society 1993 140(9) p 2593-2599

47 Engstrom RC ELECTROCHEMICAL PRETREATMENT OF GLASSY-CARBON ELECTRODES

Analytical Chemistry 1982 54(13) p 2310-2314

48 Lawrence E Hendersons dictionary of biological terms 10 ed 1989 UK Longman group UK Ltd

49 Eijsink V Directed evolution of enzyme stability Biomolecular engineering 2005 22(1-3) p 21-30

50 Pantoliano M et al PROTEIN ENGINEERING OF SUBTILISIN BPN - ENHANCED STABILIZATION

THROUGH THE INTRODUCTION OF 2 CYSTEINES TO FORM A DISULFIDE BOND Biochemistry

1987 26(8) p 2077-2082

51 Bagotzky VS NV Osetrova and AM Skundin Fuel cells State-of-the-art and major scientific and

engineering problems Russian Journal of Electrochemistry 2003 39(9) p 919-934

52 Heller A Integrated medical feedback systems for drug delivery Aiche Journal 2005 51(4) p

1054-1066

53 Nakamura M et al High-performance ultrathin solid oxide fuel cells for low-temperature operation

Journal of the Electrochemical Society 2007 154(1) p B20-B24

54 Lapedes DN McGraw-Hill Dictionary of Physics and Mathematics in McGraw-Hill 1978 McGraw-Hill

Inc

55 Svoboda V et al Enzyme catalysed biofuel cells Energy amp Environmental Science 2008 1(3) p

320-337

56 Barton SC J Gallaway and P Atanassov Enzymatic biofuel cells for Implantable and microscale devices

Chemical Reviews 2004 104(10) p 4867-4886

271

57 Schroder U Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency

Physical chemistry chemical physics 2007 9(21) p 2619-2629

58 Chaudhuri SK and DR Lovley Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells Nature Biotechnology 2003 21(10) p 1229-1232

59 Vincent K F Armstrong and J Cracknell Enzymes as working or inspirational electrocatalysts for fuel

cells and electrolysis Chemical reviews 2008 108(7) p 2439-2461

60 Tsujimura S and K Kano Recent development of enzyme-based biofuel cells GS Yuasa technical report

2008 5(2) p 1-6

61 Cass AEG Biosensors A Practical Approach 1990 Oxford University Press Oxford UK p 49-53

62 Palmore GTR and HH Kim Electro-enzymatic reduction of dioxygen to water in the cathode

compartment of a biofuel cell Journal of Electroanalytical Chemistry 1999 464(1) p 110-117

63 Bullen RA et al Biofuel cells and their development Biosensors amp Bioelectronics 2006 21(11) p

2015-2045

64 Bertschy H et al A methanoldioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as

catalysts application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at

low overpotentials Journal of electroanalytical chemistry and interfacial electrochemistry 1998 443(1) p

155-161

65 Bartlett PN P Tebbutt and RG Whitaker Kinetic aspects of the use of modified electrodes and

mediators in bioelectrochemistry Progress in reaction kinetics 1991 16(2) p 55-155

66 Kano K T Ikeda and S Tsujimura GlucoseO-2 biofuel cell operating at physiological conditions Denki

kagaku oyobi kōgyō butsuri kagaku 2002 70(12) p 940-942

67 Heller A Electrochemical glucose sensors and their applications in diabetes management Chemical

Reviews 2008 108(7) p 2482-2505

68 Ilankumaran V et al Trends in cardiac pacemaker batteries Indian pacing and electrophysiology journal

2004 4(4) p 201-12

69 Schlindwein W and R Linford Medical applications of solid state lonics Solid State Ionics 2006

177(19-25) p 1559-1565

70 Latham R R Linford and W Schlindwein Biomedical applications of batteries Solid State Ionics 2004

172(1-4) p 7-11

71 Soykan O Power sources for implantable medical devices Business Briefing Medical Device

Manufacturing and Technology London 2002

72 Heller A Potentially implantable miniature batteries Analytical and Bioanalytical Chemistry 2006

385(3) p 469-473

73 Taniguchi M Achilles heel of the next-generation electric vehicles - Lithium World map of the resrouce

wars 2008 [cited 2009 Available from

httpbusinessnikkeibpcojparticlemanage20080407152450P=1

272

74 Yeatman EM Advances In Power Sources For Wireless Sensor Nodes International Workshop on Body

Sensor Networks 2004

75 Sato N et al Novel MEMS power generator with integrated thermoelectric and vibrational devices in

Solid-State Sensors Actuators and Microsystems 2005 Digest of Technical Papers TRANSDUCERS 05

The 13th International Conference on 2005

76 Hudak NS and GG Amatucci Small-scale energy harvesting through thermoelectric vibration and

radiofrequency power conversion Journal of Applied Physics 2008 103(10)

77 Seiko-Instruments SEIKO Worlds First 2009 [cited Available from

httpwwwseikowatchescomheritageworlds_firsthtml

78 Kishi M et al Micro thermoelectric modules and their application to wristwatches as an energy source in

Thermoelectrics 1999 Eighteenth International Conference on 1999

79 Lee J et al Ionic conduction in Zn-3(PO4)(2)()4H(2)O enables efficient discharge of the zinc anode in

serum Journal of the American Chemical Society 2005 127(42) p 14590-14591

80 Maxell Button battery table 2009 [cited Available from

httpwwwmaxellcojpproductsmaterialsbutton_batteryhtml

81 Wilson R and APF Turner Glucose-Oxidase - an Ideal Enzyme Biosensors amp Bioelectronics 1992 7(3)

p 165-185

82 Swoboda BEP The relationship between molecular conformation and the binding of flavin-adenine

dinucleotide in glucose oxidase Biochimica et Biophysica Acta (BBA) - Protein Structure 1969 175(2) p

365-379

83 Wohlfahrt G et al 18 and 19 angstrom resolution structures of the Penicillium amagasakiense and

Aspergillus niger glucose oxidases as a basis for modelling substrate complexes Acta Crystallographica

Section D-Biological Crystallography 1999 55 p 969-977

84 Patel RN Biocatalysis in the pharmaceutical and biotechnology industries 1st ed 2006 CRC Press

85 Gouda M Reversible denaturation behavior of immobilized glucose oxidase Applied Biochemistry and

Biotechnology 2002 102 p 471-480

86 Hecht HJ et al Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 3 Angstrom

Resolution Journal of Molecular Biology 1993 229(1) p 153-172

87 Zubrik A et al Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the

transition to the denatured state with residual structure The Journal of biological chemistry 2004 279(46)

p 47601-47609

88 Kojima S et al Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose

biosensor Bioscience biotechnology and biochemistry 2006 70(3) p 654-659

89 Duine J The PQQ story Journal of bioscience and bioengineering 1999 88(3) p 231-236

90 Kyoto-University KEGG - Kyoto Encyclopedia of Genes and Genomes 2009 Kyoto University

91 Delbem MF WJ Baader and SHP Serrano Mechanism of 34-dihydroxybenzaldehyde

273

electropolymerization at carbon paste electrodes catalytic detection of NADH 2002 scielo p 741-747

92 Zhao S et al Electrochemistry of Organic Conducting Salt Electrodes - a Unified Mechanistic

Description Journal of Physical Chemistry 1992 96(13) p 5641-5652

93 Lemuel BW Jr CC Liu and LN Niren Electrochemical measurements with glucose oxidase

immobilized in polyacrylamide gel Constant current voltametry 1971 p 629-639

94 Sato A et al Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Biochemical Journal

2001 357 p 893-898

95 Sigma-Aldrich Glucose Oxidase from Aspergillus niger Type II-S 15000-50000 unitsg solid (without

added oxygen) 2009 [cited

96 Cosnier S Biosensors based on electropolymerized films new trends Analytical and Bioanalytical

Chemistry 2003 377(3) p 507-520

97 Willner I et al Integrated Enzyme-Based Biofuel Cells-A Review Fuel Cells 2009 9(1) p 7-24

98 Heleg-Shabtai V et al Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers

assembled onto Au-electrodes Journal of the American Chemical Society 1996 118(42) p 10321-10322

99 Georganopoulou DG et al Development and comparison of biosensors for in-vivo applications Faraday

Discussions 2000 p 291-303

100 Fujii S et al Properties of glucose sensors prepared by the electropolymerization of pyrrole containing a

gluconyl group Analytical sciences 1999 15(12) p 1175-1176

101 Hoshi T Deposition of electron mediators by electrochemical polymerization Electrochemistry 2003

71(5) p 346-347

102 Arai G M Masuda and I Yasumori Direct Electrical Response between Glucose-Oxidase and

Poly(Mercapto-P-Benzoquinone) Films Bulletin of the Chemical Society of Japan 1994 67(11) p

2962-2966

103 Arai G K Shoji and I Yasumori Electrochemical characteristics of glucose oxidase immobilized in

poly(quinone) redox polymers Journal of Electroanalytical Chemistry 2006 591(1) p 1-6

104 Leonida MD et al Polymeric FAD used as enzyme-friendly mediator in lactate detection Analytical and

Bioanalytical Chemistry 2003 376(6) p 832-837

105 Kissinger PT and WR Heineman Laboratory techniques in electroanalytical chemistry 1996 Marcel

Dekker Inc NY USA p 294-332

106 OHare D and H Zhao Characterisation and modeling of conducting composite electrodes The journal of

physical chemistry C 2008 112(25) p 9351-9357

107 Wang J et al Metal-Dispersed Carbon Paste Electrodes Analytical Chemistry 1992 64(11) p

1285-1288

108 Khurana MK CP Winlove and D OHare Detection mechanism of metallized carbon epoxy oxidase

enzyme based sensors Electroanalysis 2003 15(12) p 1023-1030

109 Sasaki M et al Fabrication and Electrical Characterization of

274

Tetrathiafulvalene-tetracyanoquinodimethane Molecular Wires Japanese Journal of Applied Physics 2003

42 p 2488-2491

110 Iizuka M et al Highly oriented TTF-TCNQ crystal growth using an electric-field induced evaporation

technique Molecular crystals and liquid crystals science and technology Section A Molecular crystals and

liquid crystals 2000 349 p 367-370

111 Saito I and K Yoshida Development of new features on organic conductive material TCI mail 2005

127

112 Cass AEG et al FERROCENE-MEDIATED ENZYME ELECTRODE FOR AMPEROMETRIC

DETERMINATION OF GLUCOSE Analytical Chemistry 1984 56(4) p 667-671

113 Kulys J V Simkeviciene and IJ Higgins Concerning the Toxicity of 2 Compounds Used as Mediators in

Biosensor Devices - 7788-Tetracyanoquinodimethane (Tcnq) and Tetrathiafulvalene (Ttf) Biosensors amp

Bioelectronics 1992 7(7) p 495-501

114 Malinauskas A et al Conductive organic complex salt TTF-TCNQ as a mediator for biosensors An

overview Electroanalysis 2007 19(24) p 2491-2498

115 Llopis X et al Integration of a glucose biosensor based on an epoxy-graphite-TTF center dot

TCNQ-GOD biocomposite into a FIA system Sensors and Actuators B-Chemical 2005 107(2) p 742-748

116 Bourgeois J D Thomas and C Bourdillon COVALENT LINKAGE OF GLUCOSE-OXIDASE ON

MODIFIED GLASSY-CARBON ELECTRODES - KINETIC PHENOMENA Journal of the American

Chemical Society 1980 102(12) p 4231-4235

117 Yun Y et al Nanotube electrodes and biosensors Nano Today 2007 2(6) p 30-37

118 Kim BC et al Preparation of biocatalytic nanofibres with high activity and stability via enzyme

aggregate coating on polymer nanofibres Nanotechnology 2005 16(7) p S382-S388

119 Kim J and JW Grate Single-Enzyme Nanoparticles Armored by a Nanometer-Scale OrganicInorganic

Network 2003 p 1219-1222

120 OFagain C Enzyme stabilization - recent experimental progress Enzyme and Microbial Technology

2003 33(2-3) p 137-149

121 Sillery E et al Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a

platinum catalyst Chemical communications 2002(8) p 866-867

122 Gouda M et al Enhancement of stability of immobilized glucose oxidase by modification of free thiols

generated by reducing disulfide bonds and using additives Biosensors amp bioelectronics 2004 19(6) p

621-625

123 Manning M STABILITY OF PROTEIN PHARMACEUTICALS Pharmaceutical Research 1989 6(11) p

903-918

124 Cao L Immobilised enzymes science or art Current opinion in chemical biology 2005 9(2) p 217-226

125 Daniel R The role of dynamics in enzyme activity Annual review of biophysics and biomolecular

structure 2003 32 p 69-92

275

126 Andersson MM JD Breccia and R Hatti-Kaul Stabilizing effect of chemical additives against oxidation

of lactate dehydrogenase Biotechnology and Applied Biochemistry 2000 32 p 145-153

127 Souza JM and R Radi Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Archives of Biochemistry and Biophysics 1998 360(2) p 187-194

128 Andersson MA and R Hatti-Kaul Protein stabilising effect of polyethyleneimine Journal of

Biotechnology 1999 72(1-2) p 21-31

129 Sigma-Aldrich Poly(ethylenimine) 2009 [cited Available from

httpwwwsigmaaldrichcomstructureimages27mfcd00084427gif

130 Andersson MM R Hatti-Kaul and W Brown Dynamic and static light scattering and fluorescence

studies of the interactions between lactate dehydrogenase and poly(ethyleneimine) Journal of Physical

Chemistry B 2000 104(15) p 3660-3667

131 Wang J L Chen and J Liu Critical comparison of metallized and mediator-based carbon paste glucose

biosensors Electroanalysis 1997 9(4) p 298-301

132 Cleland WW Dithiothreitol a New Protective Reagent for SH Groups Biochemistry 1964 3(4) p

480-482

133 Atanasov P E Wilkins and Q Yang An integrated needle-type biosensor for intravascular glucose and

lactate monitoring Electroanalysis 1998 10(11) p 752-757

134 Sigma-Aldrich DL-Dithiothreitol 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=43815|SIGMAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

135 Ghisalba O and A Brunella Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate

reductase as biocatalyst in the preparative synthesis of 2 -deoxyribonucleoside-5 -triphosphates Journal

of molecular catalysis B Enzymatic 2000 10(1-3) p 215-222

136 Gollhofer D EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM

ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Enzyme and Microbial Technology 1995 17(3) p 235-240

137 Sigma-Aldrich Arsenite standard solution - 005 M in H2O 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=70039|FLUKAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

138 Arakawa T MECHANISM OF POLY(ETHYLENE GLYCOL) INTERACTION WITH PROTEINS

Biochemistry 1985 24(24) p 6756-6762

139 Lee C Improvement of protein stability in protein microarrays Biotechnology Letters 2002 24(10) p

839-844

140 Lee L THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE GLYCOLS)

Biochemistry 1987 26(24) p 7813-7819

141 Costa SA et al Studies of stabilization of native catalase using additives Enzyme and Microbial

276

Technology 2002 30(3) p 387-391

142 Lee J and L Lee THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE

GLYCOLS) Biochemistry 1987 26(24) p 7813-7819

143 Gekko K and SN Timasheff Mechanism of protein stabilization by glycerol preferential hydration in

glycerol-water mixtures Biochemistry 1981 20(16) p 4667-4676

144 Leandro P Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes

produced in a prokaryotic expression system Molecular genetics and metabolism 2001 73(2) p 173-178

145 Bartlett PN VQ Bradford and RG Whitaker Enzyme Electrode Studies of Glucose-Oxidase Modified

with a Redox Mediator Talanta 1991 38(1) p 57-63

146 Joseph S et al Drug metabolism biosensors electrochemical reactivities of cytochrome P450(cam)

immobilised in synthetic vesicular systems Journal of Pharmaceutical and Biomedical Analysis 1998

17(6-7) p 1101-1110

147 Cornish-Bowden A Fundamentals of enzyme kinetics 3rd ed 2004 London Portland Press Ltd

148 Wilkinson G STATISTICAL ESTIMATIONS IN ENZYME KINETICS The Biochemical journal 1961

80(2) p 324

149 Ikeda T and K Kano Fundamentals and practices of mediated bioelectrocatalysis Analytical sciences

2000 16(10) p 1013-1021

150 Duggleby RG and LP Daniel [3] Analysis of enzyme progress curves by nonlinear regression in

Methods in Enzymology 1995 Academic Press p 61-90

151 Eisenthal R and A Cornish-Bowden DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR

ESTIMATING ENZYME KINETIC-PARAMETERS The Biochemical journal 1974 139(3) p 715-720

152 Baici A Enzyme kinetics the velocity of reactions Biochem J 2006 p 1-3

153 Yamazaki A et al Improvement of precision in pharmacological studies using the non-linear regression

method

estimation of enzyme inhibition constants as an example Folia Pharmacologica Japonica 2008 131(3) p 195-203

154 Cornish-Bowden A Teaching Enzyme Kinetics and Mechanism in the 21st Century in 3rd Symposium

on ESCES 2008 Rudensheim Germany

155 Weisshaar DE and T Kuwana Considerations for Polishing Glassy-Carbon to a Scratch-Free Finish

Analytical Chemistry 1985 57(1) p 378-379

156 Stedman Stedmans medical dictionary 25th ed 2002 Lippincott Williams amp Wilkins

157 Tamura T Buffer preparation for biology Buffer preparation for biology ed 6 2008 Tokyo Yodosha

Ltd

158 Battino R and HL Clever The Solubility of Gases in Liquids 1966 p 395-463

159 Pandolfo A and A Hollenkamp Carbon properties and their role in supercapacitors Journal of power

sources 2006 157(1) p 11-27

160 Maynard AD Nanotechnology assessing the risks Nano Today 2006 1(2) p 22-33

277

161 Hindle A E Hall and N Martens AN ASSESSMENT OF MEDIATORS AS OXIDANTS FOR

GLUCOSE-OXIDASE IN THE PRESENCE OF OXYGEN Biosensors amp bioelectronics 1995 10(3-4) p

393-403

162 Mo J et al Comparison of oxygren-rich and mediator-based glucose-oxidase carbon-paste electrodes

Analytica Chimica Acta 2001 441(2) p 183-189

163 Turner A and G Palleschi AMPEROMETRIC TETRATHIAFULVALENE-MEDIATED LACTATE

ELECTRODE USING LACTATE OXIDASE ABSORBED ON CARBON FOIL Analytica Chimica Acta

1990 234(2) p 459-463

164 Mizutani F et al Glucose-Sensing Electrode Based on Carbon Paste Containing Ferrocene and

Polyethylene Glycol-Modified Enzyme Bulletin of the Chemical Society of Japan 1991 64(9) p

2849-2851

165 Zhang J PEM Fuel Cell Electrocatalysts and Catalyst Layers Fundamentals and Applications 2008

London Springer-Verlag

166 Kinoshita K Electrochemical oxygen technology 1992 John Willey amp Sons Inc

167 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of Power

Sources 2005 152 p 1-15

168 Sawyer DT Oxygen Chemistry International series of monographs on chemistry 1991 Oxford

University Press

169 Winter M Chemical Bonding Oxford Chemistry Primers 1997 New York Oxford University Press Inc

170 McLendon G and AE Martell Inorganic Oxygen Carriers as Models for Biological-Systems

Coordination Chemistry Reviews 1976 19(1) p 1-39

171 Toda T et al Enhancement of the electroreduction of oxygen on Pt alloys with Fe Ni and Co Journal of

the Electrochemical Society 1999 146(10) p 3750-3756

172 Norskov JK et al Origin of the overpotential for oxygen reduction at a fuel-cell cathode Journal of

Physical Chemistry B 2004 108(46) p 17886-17892

173 Fischer P and J Heitbaum MECHANISTIC ASPECTS OF CATHODIC OXYGEN REDUCTION Journal

of Electroanalytical Chemistry 1980 112(2) p 231-238

174 Okada T et al Oxygen reduction characteristics on pyrolytic graphite electrodes modified with

electro-polymerized salen compounds Chemistry Letters 1998(8) p 841-842

175 Sakurai T and K Kataoka Basic and applied features of multicopper oxidases CueO bilirubin oxidase

and laccase 2007 p 220-229

176 Sanders J Supramolecular catalysis in transition Chemistry a European journal 1998 4(8) p

1378-1383

177 Hart H LE Craine and DJ Hart Organic Chemistry A Short Course 10 ed 1998 Houghton Miffin

Company USA

178 Hayatsu H CELLULOSE BEARING COVALENTLY LINKED COPPER PHTHALOCYANINE

278

TRISULPHONATE AS AN ADSORBENT SELECTIVE FOR POLYCYCLIC COMPOUNDS AND ITS USE

IN STUDIES OF ENVIRONMENTAL MUTAGENS AND CARCINOGENS Journal of chromatography A

1992 597(1-2) p 37-56

179 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of power

sources 2005 152(1) p 1-15

180 Kingsborough RP and TM Swager Electrocatalytic Conducting Polymers Oxygen Reduction by a

Polythiophene-Cobalt Salen Hybrid Chemistry of Materials 2000 12(4) p 872-874

181 Ortiz B and SM Park Electrochemical and spectroelectrochemical studies of cobalt salen and salophen

as oxygen reduction catalysts Bulletin of the Korean Chemical Society 2000 21(4) p 405-411

182 Mano N et al An oxygen cathode operating in a physiological solution Journal of the American

Chemical Society 2002 124(22) p 6480-6486

183 Shleev S et al Direct electron transfer between copper-containing proteins and electrodes Biosensors

and Bioelectronics 2005 20(12) p 2517-2554

184 Solomon EI et al Oxygen binding activation and reduction to water by copper proteins Angewandte

Chemie-International Edition 2001 40(24) p 4570-4590

185 Heath R F Armstrong and C Blanford A stable electrode for high-potential electrocatalytic O-2

reduction based on rational attachment of a blue copper oxidase to a graphite surface Chemical

communications 2007(17) p 1710-1712

186 Miura Y et al Direct Electrochemistry of CueO and Its Mutants at Residues to and Near Type I Cu for

Oxygen-Reducing Biocathode Fuel cells 2009 9(1) p 70-78

187 Tarasevich MR VA Bogdanovskaya and LN Kuznetsova Bioelectrocatalytic reduction of oxygen in

the presence of laccase adsorbed on carbon electrodes Russian Journal of Electrochemistry 2001 37(8) p

833-837

188 Tsujimura S et al Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase

at carbon electrodes Electrochemistry 2004 72(6) p 437-439

189 Fernandez JL et al Optimization of wired enzyme O-2-electroreduction catalyst compositions by

scanning electrochemical microscopy Angewandte Chemie-International Edition 2004 43(46) p

6355-6357

190 Soukharev V N Mano and A Heller A four-electron O-2-electroreduction biocatalyst superior to

platinum and a biofuel cell operating at 088 V Journal of the American Chemical Society 2004 126(27)

p 8368-8369

191 Miura Y et al Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on

a highly oriented pyrolytic graphite electrode Chemistry Letters 2007 36(1) p 132-133

192 Seinberg JM et al Spontaneous modification of glassy carbon surface with anthraquinone from the

solutions of its diazonium derivative An oxygen reduction study Journal of Electroanalytical Chemistry

2008 624(1-2) p 151-160

279

193 Manisankar P and A Gomathi Electrocatalytic reduction of dioxygen at the surface of carbon paste

electrodes modified with 910-anthraquinone derivatives and dyes Electroanalysis 2005 17(12) p

1051-1057

194 Jurmann G DJ Schiffrin and K Tammeveski The pH-dependence of oxygen reduction on

quinone-modified glassy carbon electrodes Electrochimica Acta 2007 53(2) p 390-399

195 Abiman P R Compton and G Wildgoose Characterising chemical functionality on carbon surfaces

Journal of materials chemistry 2009 19(28) p 4875-4886

196 Thorogood CA et al Differentiating between ortho- and para-Quinone Surface Groups on Graphite

Glassy Carbon and Carbon Nanotubes Using Organic and Inorganic Voltammetric and X-ray

Photoelectron Spectroscopy Labels Chemistry of Materials 2007 19(20) p 4964-4974

197 Sigma-Aldrich Fast red AL salt 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildoN4=380660|SIALampN5=SEARCH_CONCAT_PN

O|BRAND_KEYampF=SPEC

198 Sljukic B C Banks and R Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

199 Granger M et al Polycrystalline diamond electrodes basic properties and applications as amperometric

detectors in flow injection analysis and liquid chromatography Analytica Chimica Acta 1999 397(1-3) p

145-161

200 Compton R J Foord and F Marken Electroanalysis at diamond-like and doped-diamond electrodes

Electroanalysis 2003 15(17) p 1349-1363

201 Arima S Do Science - Why diamond is so hard in Asahicom 2006

202 Dumitrescu I P Unwin and J Macpherson Electrochemistry at carbon nanotubes perspective and issues

Chemical communications 2009(45) p 6886-6901

203 Kinoshita K Carbon Electrochemical and Physicochemical Properties 1988 Wiley-Interscience

204 Kastening B et al Electronic properties and double layer of activated carbon Electrochimica Acta 1997

42(18) p 2789-2799

205 Swain G and R Ramesham THE ELECTROCHEMICAL ACTIVITY OF BORON-DOPED

POLYCRYSTALLINE DIAMOND THIN-FILM ELECTRODES Analytical chemistry 1993 65(4) p

345-351

206 Kaneto K et al Electrical conductivities of multi-wall carbon nano tubes Synthetic metals 1999

103(1-3) p 2543-2546

207 Meyer J et al Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes Nano

letters 2008 8(11) p 3582-3586

208 Harris P New perspectives on the structure of graphitic carbons Critical Reviews in Solid State and

Materials Sciences 2005 30(4) p 235-253

209 Akasaka T Chemical illustration - Carbon materials 2007 Hokkaido Japan

280

210 Iijima S M Yudasaka and F Nihey Carbon nanotube technology NEC Technical Journal 2007 2(1) p

52-56

211 Day T N Wilson and J Macpherson Electrochemical and conductivity measurements of single-wall

carbon nanotube network electrodes Journal of the American Chemical Society 2004 126(51) p

16724-16725

212 Fujita H A TEM image of the atomic structure of the diamond surface 2010 Britannica - The Online

Encyclopedia Osaka Japan

213 McCreery RL et al Control of reactivity at carbon electrode surfaces Colloids and Surfaces A

Physicochemical and Engineering Aspects 1994 93 p 211-219

214 Rice R C Allred and R McCreery FAST HETEROGENEOUS ELECTRON-TRANSFER RATES FOR

GLASSY-CARBON ELECTRODES WITHOUT POLISHING OR ACTIVATION PROCEDURES Journal of

electroanalytical chemistry and interfacial electrochemistry 1989 263(1) p 163-169

215 Swain Electrochemical formation of high surface area carbon fibers Analytical chemistry 1991 63(5) p

517-519

216 Nagaoka T and T Yoshino Surface-Properties of Electrochemically Pretreated Glassy-Carbon Analytical

Chemistry 1986 58(6) p 1037-1042

217 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 2002 58(9) p 1953-1955

218 Motta N and AR Guadalupe Activated Carbon-Paste Electrodes for Biosensors Analytical Chemistry

1994 66(4) p 566-571

219 Leung E et al A novel in vivo nitric oxide sensor Chemical Communications 1996(1) p 23-24

220 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 1986 58(9) p 1953-1955

221 Kissinger PT and WR Heineman Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy

Current Separations 1989 9 p 15-18

222 Wang J Analytical Electrochemistry 3 ed 2006 Wiley-VCH

223 BAS Measurement of electron transfer kinetics using rotating disk voltammetry Electrocehmistry

Principles methods and applications 1995 2(43)

224 Miomandre F et al Electrochemical behaviour of iron(III) salen and poly(iron-salen) Application to the

electrocatalytic reduction of hydrogen peroxide and oxygen Journal of electroanalytical chemistry and

interfacial electrochemistry 2001 516(1-2) p 66-72

225 Brighton-University Inorganic Chemistry Practical C170 Salen lab script 2009 Dept of Pharmacy and

Biomolecular Sciences Brighton University Brighton

226 Ranganathan S TC Kuo and RL McCreery Facile Preparation of Active Glassy Carbon Electrodes

with Activated Carbon and Organic Solvents 1999 p 3574-3580

227 Ohare D CP Winlove and KH Parker Electrochemical method for direct measurement of oxygen

281

concentration and diffusivity in the intervertebral disc electrochemical characterization and tissue-sensor

interactions J Biomed Eng 1991 13(4) p 304-312

228 Chen PH and RL McCreery Control of electron transfer kinetics at glassy carbon electrodes by specific

surface modification Analytical Chemistry 1996 68(22) p 3958-3965

229 Beilby AL TA Sasaki and HM Stern Electrochemical Pretreatment of Carbon Electrodes as a

Function of Potential Ph and Time Analytical Chemistry 1995 67(5) p 976-980

230 Dai H and K Shiu Voltammetric studies of electrochemical pretreatment of rotating-disc glassy carbon

electrodes in phosphate buffer Journal of electroanalytical chemistry and interfacial electrochemistry 1996

419(1) p 7-14

231 Musameh M N Lawrence and J Wang Electrochemical activation of carbon nanotubes

Electrochemistry communications 2005 7(1) p 14-18

232 Sljukic B CE Banks and RG Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

233 Tammeveski K et al Surface redox catalysis for O-2 reduction on quinone-modified glassy carbon

electrodes Journal of Electroanalytical Chemistry 2001 515(1-2) p 101-112

234 Yang HH and RL McCreery Elucidation of the mechanism of dioxygen reduction on metal-free carbon

electrodes Journal of the Electrochemical Society 2000 147(9) p 3420-3428

235 Regisser F et al Randomly oriented graphite electrode 1 Effect of electrochemical pretreatment on the

electrochemical behavior and chemical composition of the electrode Journal of electroanalytical chemistry

and interfacial electrochemistry 1996 415(1-2) p 47-54

236 Shleev S et al Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic

graphite electrode Electrochemistry Communications 2004 6(9) p 934-939

237 Mano N F Mao and A Heller A miniature biofuel cell operating in a physiological buffer Journal of the

American Chemical Society 2002 124(44) p 12962-12963

4

synergetic effect because it increased the concentration of catalytic surface oxygen

groups and enhanced oxygen reduction

5

ありがとう Thank you Terima kashi ขอบคณ คะ Merci 고마워 謝謝 Danke

やったね

copy鳥

山

明

集

英

社

copy Akira Toriyama Shueisha Inc

6

ACKNOWLEDGEMENTS

I would like to express deepest gratitude to my supervisor Dr Danny OrsquoHare

for his guidance on and patience for my research Frankly speaking the project on

glucose-oxygen fuel cells was very challenging and I came to realise the hard reality of

creating new clean energy despite of the world huge demand for such energy in a time

of global warming I could not help but feel that the research was turning into a

wild-goose chase however there was much to learn and gain from the experience here

For that and the people I met during this time I feel deeply grateful for the opportunity

given and I am convinced that this will enlighten my life

I would like to specially thank Dr Pei Ling Leow and Mr Parinya Seelanan for

their sincere support understanding and encouragement Without their help I would not

have been able to achieve my PhD I would like to thank to the current and past

members of Biosensor group Ms Nitin Bagha Dr Christopher Bell Dr Eleni Bitziou

Mr Jin-Young Kim Mr Wei-Chih Lin Dr Beinn Muir Mr Raphael Trouillon Dr

Martyn Boutelle Mr Yao-Min Chang Dr Delphine Feuerstein Ms Michelle Rogers

Dr Costas Anastassiou Dr Arun Arora Dr Martin Arundell Dr Catherine Dobson Dr

Melanie Fennan Dr Severin Harvey Dr Alex Lindsay Dr Bhavik Patel Dr Hong

Zhao Dr Emma Corcoles and Dr Parastoo Hashemi for their support during research

and sharing the wonderful time for many occasions I would like to thank Mr David

Roche for his sincere devotion to our work and being a great lab company I would like

to give my warm thanks to Dr Andrew Bond Dr Stephanie Cremers Ms Christina

Hood Dr Asimina Kazakidi Dr Peter Knox Dr Bettina Nier Ms Eileen Su Mr

Che-Fai Yeong Mr Krause Oliver and Dr Clifford Talbot for appropriate support fun

7

and encouragement I would also like to thank Dr Kate Hobson Mr Martin Holloway

Mr Ken Keating Mr Alan Nyant Mr Richard Oxenham Ms Paula Smith Ms Dima

Cvetkova Dr Anna Thomas-Betts and Mr Nick Watkins for their academic and

laboratory support Finally I would like to say thank you to all my family for their trust

and unconditional support my teacher Prof Fukuyama my friends Dr Pieta

Nasanen-Gilmore Jingzi Kofi Mariama Masa Maya Miki Pedro Sandra Sascha and

Wei Fong for being always close supportive and encouraging even when we were apart

8

CONTENTS

ABSTRACT 3

ACKNOWLEDGEMENTS 6

CONTENTS 8

LIST OF ABBREVIATIONS 15

LIST OF FIGURES 20

LIST OF TABLES 26

LIST OF EQUATIONS 27

1 INTRODUCTION 31

11 INTRODUCTION TO FUEL CELLS 31

111 History of the fuel cells 31 112 Principles of the fuel cells 33 113 Electric power from the fuel cells 35

12 CATALYSTS 37

121 Faradayrsquos second law ndash Current and reaction rate37 122 Transition state theory Eyring equation and reaction rate 38 123 Overcoming the activation free energy 40 124 Variations in catalysts42

13 ELECTRODE POTENTIAL AND REACTION RATE 44

131 Fermi level electrode potential and electron transfer 44 132 Electrode potential equilibrium potential and Nernst equation47

9

133 Electrode potential and activation free energy 50 134 Potential shift and standard rate constant 52 135 Butler-Volmer equation overpotential and current 53 136 Butler-Volmer equation vs Tafel equation 55

14 OVERPOTENTIAL AND FUEL CELLS 57

141 Four overpotentials that decrease the EMF57 142 Internal current 58 143 Activation overpotential58 144 Diffusion overpotential 59 145 Resistance overpotential60 146 Total overpotential and cell voltage 62

15 BIOFUEL CELLS 63

151 Microbial and enzymatic fuel cells 63 152 Enzyme fuel cells and mediators64

16 MINIATURISED MEDICAL POWER SOURCES 66

161 Realisation of miniaturised fuel cells and medical power supply 66 162 Miniaturised potential medical power sources67

17 OUTLINE OF THE WORK 69

171 Introduction to miniaturised glucose-oxygen fuel cells 69 172 Outline71

2 ANODE INTRODUCTION 73

21 GLUCOSE OXIDATION ENZYMES 73

22 DESIGNING ENZYME ELECTRODES 77

221 Physical adsorption 78 222 Reconstituting apo-enzyme78 223 Tethered Osmium-complexes linked to conductive hydrogel 80 224 Entrapment in the conductive polymer 81 225 Bulk modification83

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties 84 226 Enzyme covalent attachment and mass loading 88

23 PROTEIN INACTIVATION AND STABILISATION 90

10

24 PROTEIN DESTABILISATION 92

241 Deamidation 92 242 Oxidation93 243 Proteolysis and racemisation94 244 Physical instabilities95

25 ENZYME STABILISERS 95

251 Polyehyleneimine (PEI) 96 252 Dithiothreitol (DTT)97 253 Polyethylene glycol (PEG)98 254 Polyols99 255 FAD100

26 ENZYME KINETICS AND ELECTROCHEMISTRY 101

261 Michaelis-Menten kinetics 101 262 Electrochemistry and Michaelis-Menten kinetics 105 263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics108

2631 Lineweaver-Burk linear regression 108 2632 Eadie-Hofstee linear regression 109 2633 Electrochemical Hanes-Woolf linear regression 110

264 Cornish-Bowden-Wharton method 112 265 Nonlinear regression methods113

3 ANODE MATERIAL AND METHOD115

31 ELECTRODE FABRICATION 115

311 TTF-TCNQ synthesis115 312 Electrode template fabrication 115 313 Electrode composite fabrication117

3131 Composite for conductance study 117 3132 Bulk modified GOx enzyme electrode composite 118 3133 GOx enzyme electrode composite with enzyme stabilisers 119

314 Electrode wiring and insulation120

32 CONDUCTIVITY EXPERIMENT 121

33 POLISHING COMPOSITE ELECTRODES 122

34 GLUCOSE AMPEROMETRY 123

341 Glucose solution123

11

342 Electrochemical system123 343 Glucose amperometry with GOx electrodes 124

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA 125

4 ANODE RESULTS AND DISCUSSION 127

41 AIMS OF ANODE STUDIES 127

42 CONDUCTANCE OF THE COMPOSITE 128

421 Conductance of various conductive materials128 422 Influence of enzyme concentration on conductivity 130

43 CATALYTIC ACTIVITY OF GOX COMPOSITE ELECTRODES 132

431 Influence of GOx concentration on the catalytic activity of GOx electrodes132 432 Influence of O2 on the catalytic activity of GOx electrodes134

44 THE EFFICACY OF GOX STABILISERS 139

441 Introduction139 442 PEI140 443 DTT142 444 PEG 143 445 GLC144 446 FAD146 447 PEI and DTT mixture147 448 PEI and FAD mixture149 449 Most effective stabiliser content for GOx anodes 150

45 CONCLUSION TO ANODE STUDY 152

5 CATHODE INTRODUCTION 155

51 OXYGEN REDUCTION REACTION (ORR) 155

52 REACTIVITY OF OXYGEN 157

53 ORR CATALYSTS 159

531 Noble metal ORR catalysts 160 532 Macrocycle and Schiff-base complex ORR catalysts 165

12

533 Multicopper enzymes as ORR catalyst 168 5331 BOD 171 5332 Lac 173 5333 CueO 173

534 Anthraquinone ORR catalyst 174

54 CARBON PROPERTIES 177

541 Graphite180 542 Glassy carbon (GC)181 543 Activated carbon (AC) 182 544 Carbon fibre (CF)182 545 Carbon nanotubes (CNTs)183 546 Boron-doped diamond (BDD)184

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON 185

551 Electrochemical properties of carbon185 552 Electrochemical pretreatments (ECPs) of the carbon185

56 ELECTROCHEMICAL TECHNIQUES FOR ORR 187

561 Cyclic voltammetry (CV)188 562 Non-faradic current and electric double layer capacitance 189 563 Diagnosis of redox reactions in cyclic voltammetry 190 564 Coupled reactions in cyclic voltammetry194 565 Voltammograms for the surface-confined redox groups 195 566 Hydrodynamic voltammetry (HDV) and Levich equation197 567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the charge

transfer rate constant 200 568 Finding the standard rate constant (kdeg)205 569 Summary of the electrochemical techniques206

57 SUMMARY OF THE CHAPTER 207

6 CATHODE MATERIAL AND METHOD 208

61 ELECTRODE FABRICATION 208

611 Graphite PtC and RhC electrodes208 612 Anthraquinone(AQ)-carbon composite electrodes208

6121 Derivatisation of graphite with AQ 208 6122 Fabrication of AQ-carbon composite electrode 209

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes209

13

6131 Information on synthesis of metallosalophen 209 6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes 210

62 COMPOSITE RDE CUTTING 210

63 ELECTRODE POLISHING 210

631 Composite RDE polishing210 632 Glassy carbon electrode polishing211

64 ENZYME RDES 212

641 Membrane preparation 212 642 Membrane enzyme electrodes212

65 ELECTROCHEMICAL EXPERIMENTS 213

651 Electrochemical system213 652 Electrode pretreatments214

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes 214 6522 Pretreatments of AQ-carbon PtC and RhC 215

653 Electrochemical experiments 215 654 Data analysis 216

7 CATHODE RESULTS AND DISCUSSION 218

71 AIMS OF THE CATHODE STUDIES 218

72 INFLUENCE AND EFFICACY OF ECPS 219

721 Glassy carbon electrodes (GCEs)219 7211 Untreated GCEs 219 7212 Influence of electrochemical pretreatments on GCE 220 7213 Effect of GCE pretreatment on ORR 227

722 Graphite composite electrodes 230 7221 Untreated graphite composite electrodes 230 7222 Influence of electrochemical pretreatments on graphite composite electrodes 232 7223 The effect of graphite composite electrode pretreatment on ORR 235

723 Platinum on carbon composite electrodes (PtC) 240 724 Rhodium on carbon composite electrodes (RhC)240

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR 241 725 Co(salophen) composite electrodes243

7251 Untreated Co(salophen) composite electrodes 243 7252 Influence of electrochemical pretreatments on Co(salophen) composite 244 7253 The effect of Co(salophen) composite electrode pretreatment on ORR 246

726 Fe(salophen) composite electrodes 248 7261 Untreated Fe(salophen) composite electrodes 248 7262 Influence of electrochemical pretreatments on Fe(salophen) composite 250

14

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR 252 727 Anthraquinone (AQ) composite electrodes253 728 Lac and BOD enzyme membrane electrodes 255

73 MOST EFFICIENT ORR CATALYSTS 257

731 Best five electrodes for nα 258 732 Best five electrodes for kdeg 259 733 Best five electrodes for Epc2(HDV) 260 734 Best three candidates for ORR catalytic electrodes 260

74 CONCLUSION TO CATHODE STUDY 262

8 CONCLUSION AND FURTHER STUDIES 264

9 BIBLIOGRAPHY 268

15

LIST OF ABBREVIATIONS

Α Transfer coefficient

A Electrode surface area

ABTS 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

AC Activated carbon

AFC Alkaline fuel cell

AQ Anthraquinone

Asn Asparagine

Asp Aspartic acid

BDD Boron-doped diamond

BOD Bilirubin oxidase

C Reactant concentration

C O (0t) Electrode surface oxidant concentration

C R (0t) Electrode surface reactant concentration

C(0t) Electrode surface concentration

C Bulk concentration

Cd Double layer capacitance

CF Carbon fibre

CNH Carbon nanohorn

CNT Carbon nanotubes

Co(salen) N-N-bis(salicylaldehyde)-ethylenediimino cobalt(II)

Co(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminocobalt (II)

CO Bulk oxidant concentration

CR Bulk reactant concentration

CueO Copper efflux oxidase

CV Cyclic voltammetry

Cys Cysteine

(DEAE)-dextran Diethylaminoethyl-dextran

DET Direct electron transfer

DHG Dehydrogenase

DMFC Direct methanol fuel cell

DTE Dithioerythriotol

DTT Dithiothreitol

[E0] Initial enzyme concentration

16

E Arbitrary electrode potential Enzyme

Edeg Standard electrode potential

Edeg Formal potential

Ea Anode potential

EC Electrochemical-chemical

Ec Cathode potential

Ecell Cell voltage

ECP Electrochemical pretreatment

Ee Electrode equilibrium potential

EF Fermi potential

EMF Electro motive force

Ep2 Half-peak potential

Epa Anodic peak potential

Epc Cathodic peak potential

Epc2(HDV) Half-wave reduction potential in HDV

ES Enzyme-substrate complex

Eλ Switching potential

∆E Potential difference Potential shift

∆Edeg Standard peak separation

δEp Peak width

εF Fermi energy

F Faraday constant

FAD Flavin adenine dinucleotide

Fe(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminoiron (II)

GC Glassy carbon

GCE Glassy carbon electrode

GHP 1-(6-D-gluconamidohexyl) pyrrole

GLC Glycerol

Gln Glutamine

GOx Glucose oxidase

GOx(O) Oxidised GOx

GOx(R) Reduced GOx

GSH Gluthatione

∆G Gibbs free energy shift

∆Gdeg Standard Gibbs free energy change

∆GdegDagger Standard activation free energy

17

∆GDagger Gibbs free energy of activation

∆GadegDagger Anode standard activation free energy

∆GaDagger Anode activation free energy

∆GodegDagger Oxidation standard activation free energy

∆GoDagger Oxidation activation free energy

∆GRdegDagger Reduction standard activation free energy

∆GRDagger Reduction activation free energy

h Planck constant

HDA Hydrodynamic amperometry

HDV Hydrodynamic voltammetry

His Histidine

HOMO Highest occupied molecular orbital

∆Hdeg Standard enthalpy change

I Electric current Net current

I0 Exchange current

Ia Anodic current

Ic Cathodic current

Id Disc limiting current

IK Kinetic current

IL Limiting current Diffusion-limited current

Im Maximum steady state limiting current

Ip Peak current

Ipa Anodic peak current

Ipc Cathodic peak current

I-V Current-Voltage

J Current density

Jm Maximum limiting current density

k Rate constant

K Equilibrium constant

k(E) Rate constant at the applied potential E

kdeg Standard heterogeneous rate constant

kdegb Backward reaction standard rate constant

kdegf Forward reaction standard rate constant

KDagger The equilibrium constant between the activated complexes and the reactants

k1 Rate constant for ES formation from E and S

k-1 Rate constant for ES dissociation into E + S

18

k2 Rate constant for P formation from ES

kB Boltzmann constant

kb Backward reaction rate constant

kf Forward reaction rate constant

kf(E) Rate constant for forward reactions at the applied potential E

KM Michaelis enzyme dissociation constant

Lac Laccase

LDH Lactate dehydrogenase

LMCT Ligand-to-metal charge transfer

LPG Liquefied petroleum gas

LUMO Lowest unoccupied molecular orbital

MCO Multicopper oxidase

MDH Methanol dehydrogenase

Me2SQ Dimethylmercaptobenzoquinone

MEMS Microelectromechanical systems

MeSQ Methylmercapttobenzoquinone

MET Mediated electron transfer

Met Methionine

mPQQ-GDH Membrane-bound PQQ dehydrogenase

MWNT Multi-walled nanotube

n Overall number of electrons transferred

na The number of electrons transferred up to the rate limiting step

NAD Nicotinamide adenine dinucleotide

NAD-GDH Nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

OCV Open circuit voltage

ORR Oxygen reduction reaction

P Electric power Product

PAFC Phosphoric acid fuel cell

PBS Phosphate buffer saline

PC Pyrolytic carbon

PDMS Polydimethylsiloxane

PEFC Polymer electrolyte fuel cell

PEG Polyethylene glycol

PEI Polyethyleneimine

P-I Power-Current

POx Pyranose oxidase

19

PQ Phenanthrenequinone

PQQ pyrrolo-quinoline quinone

PQQ-GDH Quinoprotein glucose dehydrogenase

Pro Proline

PtC Platinum on carbon

Q Charge

RDE Rotating disc electrode

RhC Rhodium on carbon

RLS Rate limiting step

rpm Rotaion per minute

S Substrate

SEN Single-enzyme nanoparticle

sPQQ-GDH Soluble PQQ dehydrogenase

SQ Mercaptobenzoquinone

SWNT Single-walled nanotube

∆Sdeg Standard entropy change

t Time

T1 Type I (blue Cu in Lac)

T2 Type II ( trinuclear Cu in Lac)

T3 Type III (trinuclear Cu in Lac)

TCNQ 7788-Tetracyanoquinodimethane

TEM Transmission electron microscopy

Trp Tryptophan

TTF Tetrathiafulvalene

TTF-TCNQ Tetrathiafulvalene 7788-tetracyanoquinodimethane

Tyr Tyrosine

V Voltage Maximum limiting catalytic velocity

VHT Vacuum heat treatment

η Overpotential

ν Vibrational frequency Kinematic viscosity

υ Reaction rate Scan rate Enzyme catalytic rate

ω Angular velocity

20

LIST OF FIGURES

Fig No Page Description

Fig 1 32 Historical fuel cells

Fig 2 33 Photographs of modern fuel cells

DMFC Cogeneration system Hydrogen car Glucose-Oxygen fuel cell

Fig 3 34 The schema of glucose-oxygen fuel cell

Fig 4 41 Transition state diagram with the reaction coordinate

Fig 5 45 Fermi level and electron transfer

Fig 6 46 Fermi level and electron transfer at an arbitrary electrode potential

Fig 7 48 Standard free energy change along the electrode potential at

(a) E = Edeg (b) E gt Edeg and (c) E lt Edeg

Fig 8 51 The shift in ∆GdegDagger accompanied by ∆E

Fig 9 57 Tafel plot

Fig 10 59 Activation overpotential vs current

Fig 11 60 Diffusion overpotential vs current

Fig 12 61 Resistance overpotential vs current

Fig 13 62 V-I curve for a typical low temperature fuel cell

Fig 14 65 Mediated electron transfer reaction by the enzyme electrode

Fig 15 70 The schema for a glucose-oxidising anode

Fig 16 75 The structure and redox reactions of FAD NAD and PQQ

Fig 17 79 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode

Fig 18 81 GOx wired to conductive hydrogel complexed with Os

Fig 19 83 Monomer structures of conductive polymers

1-(6-D-gluconamidohexyl) pyrrole and Mercapto-p-benzoquinone

Fig 20 85 TTF-TCNQ structure and its charge transfer

Fig 21 86 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 20 mVs-1

Fig 22 89 The procedure for the enzyme mass-loaded biocatalytic nanofibre

Fig 23 90 The fabrication procedure of single-enzyme nanoparticles

Fig 24 92 The chemical structures of the side groups of Gln and Asn

Fig 25 93 The chemical structures of the side groups of Cys Met Tyr Trp and His

Fig 26 95 The chemical structures of Asp and Pro

Fig 27 96 The structure of PEI

Fig 28 98 The structures of DTT from a linear to a cyclic disulfide form

Fig 29 98 The structures of PEG

21

Fig 30 100 The structure of glycerol

Fig 31 104 A typical Michaelis-Menten hyperbolic curve of the enzyme

Fig 32 107 Electrochemical Michaelis-Menten hyperbolic curve of the enzyme electrode

Fig 33 109 Electrochemical Lineweaver-Burk linear regression of the enzyme electrode

Fig 34 110 Electrochemical Eadie-Hofstee linear regression of the enzyme electrode

Fig 35 111 Electrochemical Hanes-Woolf linear regression of the enzyme electrode

Fig 36 113 Electrochemical Cornish-Bowden-Wharton plot of the enzyme electrode

Fig 37 116 The photographs of PDMS electrode templates

Fig 38 118 The photographs of empty and filled PDMS templates

Fig 39 119 The schema for bulk modification method

Fig 40 121 The photographs of insulated composite electrodes

Fig 41 122 The photographs of measuring electrode resistance

Fig 42 125 The photograph of glucose amperometry with a multichannel potentiostat

Fig 43 126 The analytical output by SigmaPlot Enzyme Kinetics

Fig 44 129 Conductance of various electrode composite

Fig 45 131 Conductance of GOx composite with varying GOx content

Fig 46 133 Difference in the glucose-response amperometric curve by the enzyme electrodes

containing various GOx content 5 10 and 15 in the absence of O2

Fig 47 134 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the absence of O2

Fig 48 135 (i) Difference in the glucose-response amperometric curve by 10 GOx composite

electrodes in the absence and presence of O2

(ii) The re-plotted amperometric curves in the presence of N2 air and O2

Fig 49 136 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the presence N2 O2 and air

Fig 50 140 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEI in the presence and absence of O2

Fig 51 142 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 DTT in the presence and absence of O2

Fig 52 145 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEG in the presence and absence of O2

Fig 53 146 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 GLC in the presence and absence of O2

Fig 54 147 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 FAD in the presence and absence of O2

22

Fig 55 143 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-DTT

(051 11 1505 and 22) in the presence and absence of O2

Fig 56 148 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051 11

1505 and 22)

Fig 57 149 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-FAD (21

22 and 24 ) in the presence and absence of O2

Fig 58 151 Jm KM in the absence and presence of O2 for various stabilisers

PEIDTT (11 0515 221505 ) and GLC 1

Fig 59 156 Oxygen reduction pathways 2 and 4 electron reduction pathways

Fig 60 157 Degradation of superoxide anion radical in aqueous solutions

Fig 61 158 An energy level diagram for ground state dioxygen molecule

Fig 62 159 Diagrams for the state of π orbitals in the ground state oxygen and peroxide anion

radical

Fig 63 162 Three models of oxygen adsorption Griffith Pauling and Bridge models

Fig 64 163 Models for oxygen adsorption on metals and ORR pathways

Fig 65 164 A volcano-shaped trend between ORR rate and oxygen binding energy of various

transition metals

Fig 66 165 Examples of transition metallomacrocycles Heme and copper phthalocyanine

Fig 67 166 Schiff base complexes

Co(II) salen Co(II) salophen and Co(III) salen conductive polymer

Fig 68 167 The molecular structure of dicobalt face-to-face diporphyrin [Co2(FTF4)]

Fig 69 168 ORR pathways by Co2(FTF4)

Fig 70 170 The structure of laccase from Trametes versicolor

Fig 71 171 Redox reaction cycle of MCOs with O2

Fig 72 172 Common mediators for BOD ABTS and Os complex

Fig 73 174 Two-electron ORR by anthraquinone

Fig 74 176 The structures of anthraquinone and its derivative Fast red AL salt

Fig 75 177 HDV of ORR at various quinone electrodes at pH 7

Unmodified GC AQGC and PQGC

Fig 76 178 The carbon structures graphite and diamond

Fig 77 179 The carbon sections basal and edge planes

Fig 78 181 A TEM image of graphene bilayer at 106 Aring resolution

Fig 79 182 A TEM image and a schema of GC

Fig 80 183 Computer graphic models of various CNTs Bundled SWNTs MWNT and CNHs

23

Fig 81 184 A TEM image of diamond

Fig 82 187 The CVs showing the influence of ECP on GCE at pH 7 in the presence and absence

of O2 The CVs for both pretreated and unpretreated GCEs are shown

Fig 83 188 The triangular waveform for CV and a typical voltammetric curves for BQ

Fig 84 189 The CV showing the double layer capacitance at graphite electrode surface at pH 7

Fig 85 194 A voltammogram examples of a quasi-reversible reaction at various scan rates

Fig 86 195 Voltammograms of two-electron transfer in the EE systems with different ∆Edegrsquo

Fig 87 196 A voltammogram of the reversible redox reaction by surface-confined groups

Fig 88 198 A diagram of hydrodynamic electrochemical method using a RDE

Fig 89 200 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH 74

Fig 90 201 Levich plot examples for reversible and quasi-reversible systems

Fig 91 203 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in oxygenated

001 M PBS at pH 74 at pretreated GCE

Fig 92 204 Koutecky-Levich plot for O2 reduction at a pretreated GCE in oxygenated 001 M PBS at

pH 74 with applied potential ndash 04 06 and 08 V

Fig 93 206 Log kf(E) plot for O2 reduction at various pretreated GCEs

Fig 94 207 The protocol for finding kinetic and thermodynamic parameters

Fig 95 211 The photographs of fabricated composite RDEs

Fig 96 213 The photograph of RDE experiment setting with a rotor and gas inlet

Fig 97 220 CVs of untreated GCEs at pH 74 in 001 M PBS in the absence and presence of O2

at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 98 221 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of O2

between + 09 and - 09 V vs AgAgCl at 005 Vs-1

(i) CVs for all the pretreated GCEs

(ii) CVs excludes PBS pretreatments

Fig 99 223 The bar graphs for the capacitance of GCEs in 015 M NaCl buffered with 001 M

phosphate to pH 74

Fig 100 225 Overlaid CVs during various pretreatments for GCEs involving

01 M H2SO4 alone 001 M PBS at pH 74 alone and both pretreatments

Fig 101 227 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M oxygenated

PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 102 229 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Fig 103 231 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M PBS in

the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

24

Fig 104 233 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M N2-sparged PBS

between + 09 and - 09 V vs AgAgCl

Fig 105 236 Overlaid CVs of ORR at various pretreated graphite composite electrodes at pH 74

in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 106 237 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at pH 74

in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various scan rates

50 100 200 and 500 mVs-1

Fig 107 238 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

Fig 108 239 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

Fig 109 241 CVs of untreated and H2SO4-pretreated RhC composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

Fig 110 242 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M PBS in

the absence and presence O2 at 005 Vs-1 between + 09 and - 09 V vs AgAgCl

Fig 111 243 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M PBS in the

absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 112 245 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 113 247 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 114 248 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite electrodes

Fig 115 249 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

Fig 116 251 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 117 253 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 118 254 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M PBS in

theabsence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 119 256 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated PBS at

005 Vs-1 between 0 and -09 V vs AgAgCl

Fig 120 257 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Fig 121 258 The bar graphs of the best five nα

25

Fig 122 259 The bar graphs of the best five kdeg

Fig 123 260 The bar graphs of the best five Epc2(HDV)

Fig 124 266 A photograph of the fuel cell stack made with GOx anodes and Co(salophen)

cathodes

26

LIST OF TABLES

Table No Page Description

Table 1 35 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Table 2 87 Electrochemical behaviour of TTF-TCNQ at pH 74

Table 3 104 Michaelis-Menten equation at various [S]

Table 4 117 Composite composition table

Table 5 119 Blended stabiliser composition

Table 6 139 Four possible causes of glucose amperometric current reduction

Table 7 152 Comparison of maximum output current and KM with some published data

Table 8 159 Bond orders bond lengths and bond energies of some dioxygen species

Table 9 180 The electric conductivity of various carbon forms and sections

Table 10 186 Various ECP examples

Table 11 192 CV diagnosis for reversible system at 25 ˚C

Table 12 193 CV diagnosis for irreversible system at 25 ˚C

Table 13 196 CV diagnosis for reversible system by surface-confined groups at 25 ˚C

Table 14 214 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Table 15 216 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC

Table 16 217 Parameter values for Koutecky-Levich equation

O2 diffusion coefficient O2 concentration and PBS kinematic viscosity

Table 17 261 Three candidate electrodes for high ORR catalysis

Table 18 261 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

27

LIST OF EQUATIONS

Eq No Page Description

Eq 1 35 Anode glucose oxidation reaction

Eq 2 35 Cathode oxygen reduction reaction

Eq 3 35 Overall glucose-oxygen fuel cell reaction

Eq 4 36 Electric power equation

Eq 5 36 Electric current equation in electrochemistry

Eq 6 38 Faradayrsquos second law of electrolysis Charge equation

Eq 7 38 Faradayrsquos second law of electrolysis Reaction equation

Eq 8 39 Transition state theory equation Rate constant equation

Eq 9 39 Transition state theory equation Equilibrium constant

Eq 10 39 Transition state theory equation Atomic vibration frequency equation

Eq 11 40 Transition state theory equation Erying equation

Eq 12 42 Gibbs free energy shift equation in thermodynamic terms

Eq 13 42 Gibbs free energy shift equation in electrochemical terms

Eq 14 49 A typical redox reaction

Eq 15 50 Nernst equation

Eq 16 50 Electrode potential shift

Eq 17 50 Free energy shift in electrochemical terms

Eq 18 52 Anodic oxidation activation free energy shift in terms of potential shift

Eq 19 52 Cathodic reduction activation free energy shift in terms of potential shift

Eq 20 53 Forward reduction rate constant equation in terms of kdeg and potential shift

Eq 21 53 Backward oxidation rate constant equation in terms of kdeg and potential shift

Eq 22 54 Butler-Volmer equation

Eq 23 54 Overpotential equation

Eq 24 54 Exchange current equation

Eq 25 55 Butler-Volmer equation in terms of electrode equilibrium constant

Eq 26 55 Butler-Volmer equation in terms of overpotential

Eq 27 56 Butler-Volmer equation for small overpotential

Eq 28 56 Tafel equation for large overpotential

Eq 29 60 Diffusion overpotential equation

Eq 30 61 Ohmrsquos law

Eq 31 63 Cell voltage equation

Eq 32 63 Gibbs free energy available from the fuel cell

28

Eq 33 73 Glucose oxidation reaction by GOx

Eq 34 73 Glucose oxidation reaction by NAD-DHG

Eq 35 87 TTF-TCNQ reaction At equilibrium state

Eq 36 87 TTF-TCNQ reaction TCNQ reduction at lt + 015 V vs AgAgCl

Eq 37 87 TTF-TCNQ reaction TCNQ lattice reduction at lt - 015 V vs AgAgCl

Eq 38 87 TTF-TCNQ reaction Anodic TCNQ salt formation at gt + 031 V vs AgAgCl

Eq 39 87 TTF-TCNQ reaction TTF lattice oxidation at gt + 035 V vs AgAgCl

Eq 40 87 TTF-TCNQ reaction TTF further oxidation at gt + 038 V vs AgAgCl

Eq 41 101 Enzyme kinetics A typical enzyme reaction

Eq 42 102 Enzyme kinetics Enzyme-substrate complex formation at steady state

Eq 43 102 Enzyme kinetics Rate of enzyme-substrate complex formation in terms of k-1 and k2

Eq 44 102 Enzyme kinetics

Enzyme-substrate complex equation in terms of [E0] k1 k-1 k2 and [S]

Eq 45 102 Enzyme kinetics Enzyme catalytic reaction

Eq 46 102 Enzyme kinetics Enzyme catalytic reaction equation

Eq 47 102 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of enzyme-substrate complex

Eq 48 103 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of [E0] k1 k-1 k2 and [S]

Eq 49 103 Enzyme kinetics Michaelis-Menten constant definition

Eq 50 103 Michaelis-Menten equation

Eq 51 104 Michaelis-Menten equation when [S] ge KM

Eq 52 104 Michaelis-Menten equation when [S] = KM

Eq 53 104 Michaelis-Menten equation when [S] le KM

Eq 54 106 Electrochemical Michaelis-Menten equation

Eq 55 106 Electrochemical Michaelis-Menten equation for current density

Eq 56 108 Electrochemical Lineweaver-Burk linear regression equation

Eq 57 109 Electrochemical Eadie-Hofstee linear regression equation

Eq 58 111 Electrochemical Hanes-Woolf linear regression equation

Eq 59 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for Jm

Eq 60 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for KM

Eq 61 128 Enzyme electrode specificity constant

Eq 62 139 The causes of anodic current reduction A typical GOx-O2 reaction

Eq 63 139 The causes of anodic current reduction TTF reduction by GOx

Eq 64 139 The causes of anodic current reduction GOx allosteric inhibition

Eq 65 139 The causes of anodic current reduction TTF oxidation by O2

29

Eq 66 139 The causes of anodic current reduction TTF oxidation by GOx

Eq 67 161 O2 Dissociative mechanism Simultaneous oxygen adsorption and molecular fission

Eq 68 161 O2 Dissociative mechanism Surface hydroxide formation

Eq 69 161 O2 Dissociative mechanism Oxygen reduction to water

Eq 70 161 O2 Associative mechanism Oxygen adsorption

Eq 71 161 O2 Associative mechanism Peroxide formation

Eq 72 161 O2 Associative mechanism Peroxide reduction

Eq 73 161 O2 Associative mechanism Surface oxygen reduction to hydroxide

Eq 74 161 O2 Associative mechanism Surface hydroxide reduction to water

Eq 75 175 Anthraquinone O2 reduction Semiquinone anion radical formation

Eq 76 175 Anthraquinone O2 reduction O2 reduction by a semiquinone anion radical

Eq 77 175 Anthraquinone O2 reduction Spontaneous superoxide reduction to O2

Eq 78 175 Anthraquinone O2 reduction

Spontaneous superoxide reduction to hydroperoxide

Eq 79 190 Electric double layer capacitance per unit area

Eq 80 192 CV diagnosis ndash Reversible system Ip vs υfrac12

Eq 81 192 CV diagnosis ndash Reversible system Ep

Eq 82 192 CV diagnosis ndash Reversible system ∆Ep

Eq 83 192 CV diagnosis ndash Reversible system δEp

Eq 84 192 CV diagnosis ndash Reversible system Reversal parameter

Eq 85 193 CV diagnosis ndash Irreversible system Ip vs υ

Eq 86 193 CV diagnosis ndash Irreversible system Ep a function of kdeg α and υ

Eq 87 193 CV diagnosis ndash Irreversible system ∆Ep

Eq 88 193 CV diagnosis ndash Irreversible system δEp

Eq 89 195 CV diagnosis ndash EE system Peak separation of EE system

Eq 90 196 CV diagnosis ndash Surface-confined groups Ip vs υ

Eq 91 196 CV diagnosis ndash Surface-confined groups Ep

Eq 92 196 CV diagnosis ndash Surface-confined groups ∆Ep2

Eq 93 198 Diffusion layer thickness in terms of ω

Eq 94 199 Levich equation in terms of ω

Eq 95 199 Levich equation in terms of diffusion layer thickness

Eq 96 202 Koutecky-Levich equation

Eq 97 204 The rate constant for arbitrary potential E

Eq 98 205 k(E) in terms of kdeg and η for a forward reaction

Eq 99 205 kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

Eq 100 238 Surface semiquinone radical formation

30

Eq 101 238 ORR by surface semiquinone radical

31

1 INTRODUCTION

11 INTRODUCTION TO FUEL CELLS

111 History of the fuel cells

Fuel cells are electrochemical devices which directly convert fuel into

electricity via chemical reactions The invention dates back to 19th century when a

British physicist Sir William Grove and a German-Swiss chemist Christian Schoumlnbein

discovered the reverse reaction of water electrolysis The current output by this system

was however very small Therefore the discovery stayed in the shadow of the steam and

internal combustion engines and it needed another century to gain its attention

In the 1930s a British engineer Francis Bacon started working towards

alkaline fuel cells (AFCs) and in the 1950s General Electric developed polymer

electrolyte fuel cells (PEFCs) Those fuel cells were loaded on the spaceships in the

1960s by NASA because they were compact provided clean power with large energy

density and generated drinkable water as a by-product [1-5] Since then fuel cells have

been continuously used for space and marine technologies [3] In the 1970s studies on

fuel cells were further expanded for consumer applications [1 3 5 6] The first

air-breathing fuel cell hybrid car was built by Kordesch [1 7] and phosphoric acid fuel

cells (PAFCs) were developed and introduced to offices and hospitals [2 3 5]

32

Fig 1 Some historical fuel cell applications (i) Alkaline fuel cell for Apollo 11 [8] (ii)

Kordeschrsquos Austin A40 city hybrid car [7]

Since the 1990s fuel cells have been drawing keen attention as flexible renewable

energy in conjunction with the depletion of fossil fuel global warming and the demand

in cleaner energy [6 9] Great effort has been made to miniaturise fuel cells for

individual users such as mobile devices vehicles and households [1 3 5 6] The recent

advance in such technologies has led to direct methanol fuel cells (DMFCs) for

mobile-phone handsets [10 11] cogeneration systems to supply household power [1 5]

hydrogen fuel cell cars [12-15] and bio-batteries running on glucose and air oxygen for

portable music players [16-18] Glucose-oxygen biofuel cells have been also further

miniaturised for medical implantable purposes [18 19]

33

Fig 2 Modern fuel cell technologies (i) Toshibarsquos world smallest DMFC [20] (ii)

TokyoGasrsquos cogeneration system Enefarm (iii) BMWrsquos Hydrogen 7 (iV)

Sonyrsquos Bio Battery generating electricity from sugar [16]

112 Principles of the fuel cells

A fuel cell unit simply consists of an anode a cathode electrolyte and a

conducting wire A conducting wire connects an anode and a cathode to form an

external circuit This allows coupling of electrochemical reactions taking place at each

electrode and provides a conductive path for electrons between them Electrolyte

allows charged species to move between two electrodes but blocks out the entry of

electrons This completes the electric circuit and permits electric conduction At the

anode fuel is oxidised by an oxidation reaction and this supplies electrons to the

cathode through the external circuit The number of electrons generated depends on the

34

type of fuels reactions and environment in which the reactions take place In the anode

reaction ions are also released to balance overall charge They move to the cathode

though the electrolyte At the cathode ions from the electrolyte and electrons from the

circuit meet and react together with an oxidiser supplied The completion of the coupled

reactions generate electricity and products [3 4 21] Fig 3 shows a glucose-oxygen

fuel cell as an example

Fig 3 A glucose-oxygen fuel cell

At the anode glucose is oxidised into gluconolactone This gives out two electrons and

two protons At the cathode oxygen gas receives them and is reduced to water Each

reaction has a unique redox potential called standard electrode potential Edeg and

electromotive force (EMF) is the potential difference between such electrode potentials

It is also called open circuit voltage (OCV) This is the theoretical size of potential

difference available from the fuel cell [3 4] The coupled reactions for glucose-oxygen

fuel cell is shown in Table 1 Each redox potential was measured against silver

35

silver-chloride reference electrode (AgAgCl) at pH 7 25degC therefore shown in formal

potential (Edegrsquo)

Table 1 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Anode Edegrsquo = - 042 V (Eq 1)

(via FADFADH2 redox [22 23])

Cathode [24] Edegrsquo = 062 V (Eq 2)

Overall ∆Edegrsquo = 104 V (Eq 3)

The reaction potential at each electrode is measured separately as half cell potential

against the universal reference electrode such as AgAgCl because it is impossible to

directly measure both inner potentials simultaneously

113 Electric power from the fuel cells

Electric power (P) is the amount of work can be handled by electric current per

unit time It is defined as in Eq 33 The size of power is determined by the size of

electric current (I) and voltage (V) since P is proportional to I and P

36

Electric power

VIP times= middotmiddotmiddotmiddotmiddot Eq 4

P = Electric power W

I = Electric current A

V = Voltage V

Electric current is the amount of charge (Q) flows per unit time Voltage is the electric

potential energy per unit charge and synonymous with potential difference (∆E) EMF

or OCV Increase in I andor V increases P V is however constrained by the type of

electrode reactions as discussed previously I depends on (i) reaction rate (ii) the

electrode surface and (iii) reactant concentration as shown in Eq 5

Electric current in electrochemistry

nFAkCnFAI == υ middotmiddotmiddotmiddotmiddot Eq 5

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

A = Electrode surface area m-2

υ = Reaction rate (1st or pseudo 1st order) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

It is indispensable to have large rate constant to increase output current This demands

lowering activation energy for a chemical transition during the reaction and can be

achieved by use of the catalysts

37

12 CATALYSTS

121 Faradayrsquos second law ndash Current and reaction rate

Catalysts accelerate specific chemical reactions without altering the change in

overall standard Gibbs free energy In other words they change the reaction kinetics but

not the thermodynamics They only provide an alternative lower-energy pathway to

shorten the reaction time without changing themselves before and after the reaction

[25-27] In fuel cells catalysts are used to increase the current output by accelerating the

electrode reaction The relationship between current and rate of reaction is defined by

Faradayrsquos second law of electrolysis (Eq 5 - Eq 7) This expresses the size of electric

charge generated in terms of the number of moles of reactant consumed or product

produced

38

Faradayrsquos second law of electrolysis

nFNQ = middotmiddotmiddotmiddotmiddot Eq 6

AtNCktimes

=times=υ middotmiddotmiddotmiddotmiddot Eq 7

Q = Charge C

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

N = The number of moles of reactant or product mol

υ = Reaction rate (1st or pseudo 1st ordedr) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

t = Time s

A = Electrode surface area m-2

Faradayrsquos second law of electrolysis states that an increase in reaction rate increases the

current output To increase the reaction rate intrinsically its Gibbs free energy of

activation (∆GDagger) must be lowered The relationship between k and ∆GDagger is stated in

Erying equation [28]

122 Transition state theory Eyring equation and reaction rate

When a reaction proceeds it must go through a critical state called transition

state of maximum potential energy also called the activated complex Activated

complexes are unstable molecules existing for only a few molecular vibrations before

decaying into the products Transition state theory assumes that the reactants and the

39

activated complexes are in quasi-equilibrium and the reaction rate is constrained by the

rate at which the activated complex decays into the products This implies that the

reaction rate depends on the equilibrium constant between the activated complexes and

the reactants (KDagger) and the atomic vibrational frequency (ν) as shown in Eq 9 ν is

defined by statistical mechanics in terms of Boltzmann constant (kB) Planck constant

(h) and the temperature (Eq 10) [28]

Transition state theory and reaction rate [28]

[ ]RTexpDagger Dagger

Dagger GC

CK ∆minus== middotmiddotmiddotmiddotmiddot Eq 8

Dagger Kk ν= middotmiddotmiddotmiddotmiddot Eq 9

hTkB=ν middotmiddotmiddotmiddotmiddot Eq 10

KDagger = Equilibrium constant between the reactants and the activated

complexes

s-1

CDagger = Activated complex concentration molm-3

C = Reactant concentration molm-3

∆GDagger = Gibbs free energy of activation Jmol-1

R = Gas constant = 8314 Jmol-1K-1

T = Thermodynamic temperature K

k = Rate constant (1st or pseudo 1st reaction) s-1

ν = Atomic vibrational frequency s-1

kB = Boltzmann constant = 13807 x 10-23 JK-1

h = Planck constant = 662621 x 10-34 Js

40

Based on this the Erying equation expresses the relationship between the reaction rate

and Gibbs activation free energy as shown in Eq 11 [25 28 29]

Erying equation TG

B

hTkk

RDagger ∆minus

= κ middotmiddotmiddotmiddotmiddot Eq 11

where κ = transmission coefficient

This tells that to increase the rate constant reaction temperature must be increased or

the activation free energy must be lowered

123 Overcoming the activation free energy

Activation free energy is a potential energy gap between the reactant and

activated complex At the transition state intra-molecular reorganisation takes place

through the bond destruction and formation This demands the energy and slows the

kinetics The reaction does not proceed unless this energy barrier is overcome Hence

energy input in someway or lowering activation free energy is required for the reaction

to proceed andor accelerate the reaction Heat energy can provide atomic and molecular

kinetic energy so that the system can overcome the energy barrier as indicated in Eq 11

[4 21 29] Applying electric potential also has the similar effect It provides the electric

energy to overcome the energy barrier The rate constant therefore can be altered

extrinsically by temperature andor electrode potential [21 29 30] Alternatively

catalysts can reduce ∆GDagger so that the reaction rate can be increased intrinsically They

enable or accelerate a reaction by providing alternative lower-energy reaction pathway

41

This does not change the reaction path or the standard enthalpy change (∆Hdeg) of the

system however lowers the activation free energy for this path so that the reactants can

be converted more quickly to the products as shown in Fig 4

Fig 4 Energy transition for catalytic reaction through the transition state

Fig 4 shows the energy state transition during a reaction with and without a catalyst

The reaction coordinate represents the reaction progress along the reaction pathway

∆Hdeg is a state function [31] therefore determined only by the initial and final state of

the system but independent of the reaction path ∆Hdeg is divided into standard Gibbs free

energy change (∆Gdeg) and the term of standard entropy change (∆Sdeg) as Eq 12 shows [4

21 23 26 32]

The key insight therefore from transition state theory is that for a catalyst to function it

42

must spontaneously bind the transition state

Gibbs free energy change

deg∆minusdeg∆=deg∆ STHG middotmiddotmiddotmiddotmiddot Eq 12

deg∆minus= EnF middotmiddotmiddotmiddotmiddot Eq 13

∆Gdeg is the standard free energy difference which became available for work through

the reaction and must be lt 0 for a spontaneous reaction ∆Gdeg can be related to the

standard electrical potential difference as Eq 13 shows [4 21 29 30 33 34] It is the

maximum EMF theoretically available from the system ∆Sdeg is the difference in the

degree of disorder in the system The entropy term (T∆Sdeg) is the energy not available for

work and temperature dependent This and Eq 13 imply that ∆Gdeg is also altered by

temperature as shown in Fig 4 [35]

124 Variations in catalysts

Given the general applicability of transition state theory it is not surprising that

catalysts have extensive variation and applications the reaction phase can be

homogeneous or heterogeneous catalysts can be inorganic organic or biological

Inorganic catalysts are metals such as transition or noble metals or complexes

on metal ions in various oxidation states Elemental metal catalysts typically catalyse

reactions through surface adsorption Metal particles have extensive variation in

physical properties such as particle size shape and surface area They often come in

dispersed on the porous support material for improved catalytic performance due to

43

increased surface area [36] and cost efficiency [1] Composition and structure of the

particles thickness of support material and condition of metal deposition all affect

reactions [27 37-39] A complex is a compound in which a ligand is loosely bonded to

a metal centre through coordination bonds An unsaturated complex has a dissociated

ligand and catalysis takes place when a substrate binds to that in the single orientation

[27 40] Complexes also have important roles in biological system [41-43] Organic

catalysts are non-metal non-biological catalysts They are often functionalised solid

surfaces for surface catalysis [27] In electrochemistry such catalysts can be chemically

bonded to conductive material [44] Simple electrochemical pretreatment can generate

or attach chemical groups onto the electrode surface and they mediate electron transfer

[45-47] Biological catalysts are generally called enzymes Enzymes are biological

polymers mostly made of protein and usually have high reaction and substrate

specificity They often have cofactors which are not proteinous but essential for their

catalytic activities Enzymes are involved in virtually all biological activities [23 25 27

48] and in a few industrial applications due to their substrate specificity [27]

Catalysts are by definition not supposed to be consumed in their reactions

however they could lose their catalytic power Impurities fouling and poisoning are

common deactivating factors for the metal catalysts Competing reactions also convert

them into inactive forms Particle migration and agglomeration reduce their catalytic

surface area hence their overall activity [27] Biological catalysts are very sensitive to

the environment Slight changes in pH and temperature can deactivate enzymes Their

stability is also affected by presence of metal ions surfactants oxidative stress and

organic solvent molecules [49] Metal catalysts can be possibly regenerated by

redispersion [27] however it is very difficult to recover biological catalytic functions

44

Therefore enzyme stabilisers are often added to extend their life Protein engineering

also can confer advantageous properties to the enzymes Amino acid sequences leading

to favourable protein folding functions or stability are determined in silico and they are

introduced to existing enzymes by genetic engineering such as site-directed mutagenesis

Protein engineering of serine protease subtilisin is a classic example [50]

13 ELECTRODE POTENTIAL AND REACTION RATE

131 Fermi level electrode potential and electron transfer

In the sect 123 the relationship between the electric potential and activation free

energy was briefly discussed As heat energy can provide the kinetic energy to atoms

and molecules for a reaction to proceed the electric energy also can provide kinetic

energy to electrons for electron transfer to proceed This leads to the redox reaction at

the electrode surface

Each substance has a set of discrete energy levels Each energy level can

accommodate up to two electrons Electrons fill these energy levels in ascending order

from the lower level This can divide these energy levels into two groups electron-filled

lower energy level and empty higher energy level without electrons In between the

critical energy level the called Fermi level lies It is designated as εF (Fermi energy) or

EF (Fermi potential) The Fermi level contains electrons of highest energy A substance

can transfer only these electrons to the external system as a donor while as an acceptor

it can accommodate external electrons only within the empty energy level which is

above its Fermi level (Fig 5)

45

Fig 5 Fermi level and electron transfer

The energy state of electrons however varies by substance therefore electrons at Fermi

level so-called highest occupied molecular orbital (HOMO) of a donor may need to

gain various kinetic energy to jump into the lowest empty energy level so-called lowest

unoccupied molecular orbital (LUMO) of an acceptor for electron transfer (Fig 6)

46

Fig 6 Electrode potential and electron transfer

As soon as the energy of electrons at Fermi level of a substance or an electrode meets

the energy level of LUMO of the other redox molecule electron transfer proceeds as

long as there is room to accommodate electrons in the acceptor This is the cathode

reaction The potential can be swept back by applying positive potential to the electrode

This lowers the energy of LUMO of the electrode so that an electron in HOMO of the

donor can be transferred back to the electrode This is the anode reaction If the electron

transfer is fast in both directions compared with other reaction steps it is a

thermodynamically reversible system and the standard electrode potential (Edeg) for this

redox reaction lies somewhere between and close to those two electrode potentials

which have enabled electron transfer At Edeg both anode and cathode reactions have the

same reaction rate hence they are at equilibrium [33] and there is no net current [21 30]

47

132 Electrode potential equilibrium potential and Nernst equation

Activation free energy of an electrode reaction can be altered by applying

potential The extent of its alteration and the direction of the reaction depend on the size

and direction of potential applied Fig 7 shows the difference in the standard activation

free energy (∆GdegDagger) for redox reactions at various electrode potentials [30]

48

Fig 7 Standard free energy and electrode potential [30]

(a) At E = Edeg (b) E gt Edeg and (c) E lt Edeg

More positive potential lowers the activation free energy for oxidation hence shifts the

reaction in favour of oxidation and vice versa This implies that applying extra potential

to the electrode perturbs the equilibrium and to restore it the electrode equilibrium

49

potential (Ee) must be shifted This phenomenon is clearly stated in Nernst equation (Eq

15 on p 50) as a relationship between Ee and the equilibrium constant (K) at the

electrode surface At equilibrium both oxidation and reduction reactions take place at

the same rate and there is no net current therefore the surface concentration (C(0t))

approximates bulk concentration (C) Nernst equation shows that Ee is always under

the influence of such concentration but can be established provided the reaction is fast

enough to restore the equilibrium [21 30] In the dilute solutions of the electroactive

species Edeg is substituted with the formal potential (Edegrsquo) which considers the influence

of inter-solute and solvent interactions [21 29]

Redox reaction

middotmiddotmiddotmiddotmiddot Eq 14

O = Oxidant

ne- = The number of electrons transferred

R = Reactant

kf = Forward reaction rate constant (1st or pseudo 1st reaction) s-1

kb = Backward reaction rate constant (1st or pseudo 1st reaction) s-1

50

Nernst equation

lowast

lowast

+deg=R

Oe

CC

nFRTEE ln middotmiddotmiddotmiddotmiddot Eq 15

Ee = Equilibrium potential V

Edegrsquo = Formal potential V

R = Gas constant = 8314 Jmol-1K-1

T = Absolute temperature K

n = The number of electron transferred

F = Faraday constant = 96487 Cmol-1

CO = Bulk oxidant concentration molm-3

CR = Bulk reductant concentration molm-3

133 Electrode potential and activation free energy

When E ne Edegrsquo the difference in these potentials is expressed as the electrode

potential shift (∆E) as in Eq 16 and its free energy shift (∆G) as in Eq 17

Electrode potential shift

degminus=∆ EEE middotmiddotmiddotmiddotmiddot Eq 16

Free energy shift

)( degminus=∆=∆ EEnFEnFG middotmiddotmiddotmiddotmiddot Eq 17

When ∆E is applied positively it lowers the anode oxidation activation free energy

(∆GaDagger) by fraction of the total energy change (∆G) as shown in Fig 8 on p 51 The

fraction for the oxidation reaction is defined as (1-α) The transfer coefficient (α) is a

51

kinetic parameter which states the degree of symmetry in the potential energy barrier

for redox reactions The symmetry in energy barrier is present when the slopes of two

potential energy curves are the same and it is defined as α = 05 α is dependent on

redox potential and could vary within the range between 0 and 1 In most cases α = 05

and appears to be constant when overpotential is small [21 30]

Fig 8 The shift of ∆E and ∆GdegDagger [21 30]

With α the shift in electrode activation free energy can be expressed in terms of

potential shift for both oxidation and reduction reactions as shown in Eq 18 and Eq 19

52

∆GDagger in terms of (E-Edegrsquo)

Anode Oxidation )()1(DaggerDagger degminusminusminus∆=∆ EEnFGG aa αo middotmiddotmiddotmiddotmiddot Eq 18

Cathode Reduction )(DaggerDagger degminus+∆=∆ EEnFGG cc αo middotmiddotmiddotmiddotmiddot Eq 19

∆GoDagger = Anode oxidation activation free energy Jmol-1

∆GodegDagger = Anode oxidation standard activation free energy Jmol-1

α = Transfer coefficient molm-3

n = The number of electrons transferred Jmol-1

F = Faraday constant = 96485 Cmol-1

E = Arbitrary electrode potential V

Edegrsquo = Formal potential V

∆GRDagger = Cathode reduction activation free energy Jmol-1

∆GRdegDagger = Cathode reduction standard activation free energy Jmol-1

134 Potential shift and standard rate constant

By substituting the activation free energy in Eyring equation (Eq 11) with the

shift in electrode activation free energy in Eq 18 and Eq 19 it is possible to express k

in terms of potential shift and standard rate constant (kdeg) (Eq 20 and Eq 21) kdeg is the

rate constant at Edegrsquo[21 30]

53

k in terms of (E-Edegrsquo) and kdeg

Forward reduction cathode rate constant (kf) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bf

Daggercexpκ

⎥⎦⎤

⎢⎣⎡ degminusminus

⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )(expexp

Dagger

EERT

nFRT

GhTk cB ακ

o

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= )(exp EERT

nFk fα middotmiddotmiddotmiddotmiddot Eq 20

Backward oxidation anode rate constant (kb) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bb

Daggeraexpκ

⎥⎦⎤

⎢⎣⎡ degminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )()1(expexp

Dagger

EERT

nFRT

GhTk aB ακ

o

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= )()1(exp EE

RTnFkb

α middotmiddotmiddotmiddotmiddot Eq 21

kf and kb = Forward and backward reaction rate constant s-1

kdegf and kdegb = Forward and backward reaction standard rate constant s-1

135 Butler-Volmer equation overpotential and current

At the equilibrium potential E = Ee kf = kb hence anodic current (Ia) =

cathodic current (Ic) and therefore the net current (I =Ia ndash Ic) is macroscopically zero (I =

0) However both forward and backward reactions still occur microscopically at the

electrode surface The absolute value of this electric current is called exchange current

(I0) and is not directly measurable When CO = CR at E = Ee C O (0t) = C R (0t) and kdegf

= kdegb = kdeg hence the net current any potential E can be defined as below by

Butler-Volmer equation (Eq 22)

54

Butler-Volmer equation

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡ degminus

minusminus⎥⎦

⎤⎢⎣⎡ degminusminus

deg= )()1(exp)(exp )0()0( EERT

nFCEERT

nFCnFAkI tRtOαα middotmiddotmiddotmiddotmiddot Eq 22

The Butler-Volmer equation states the relationship between the electric current

and the potential shift This can be further rephrased in terms of overpotential (η)

through defining I0 using Nernst equation (Eq 15) η is the potential required to drive a

reaction in a favourable direction from its equilibrium state and defined as n Eq 23

Overpotential (η)

eEE minus=η middotmiddotmiddotmiddotmiddot Eq 23

At E = Ee both anode and cathode exchange currents are the same therefore both

exchange currents can be defined together as I0 (see below) and I0 can be expressed in

terms within Nernst equation by substituting (Ee-Edegrsquo) as in Eq 24

Exchange current

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= lowast )(exp0 EERT

nFCnFAkI eO

α

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= lowast )()1(exp EE

RTnFCnFAk e

Rα

αα )()( 1RO CCnFAk minus= o middotmiddotmiddotmiddotmiddot Eq 24

Now the Butler-Volmer equation (Eq 22) can be expressed in terms of η (= E ndash Ee) (Eq

25) by expressing it with I0 and the with re-arranged Nernst equation

55

⎥⎦⎤

⎢⎣⎡ degminus=lowast

lowast

)(exp EERTnF

CC e

R

O The term

)0(

CC t in Eq 25 indicates the influence of mass

transport For purely kinetically controlled region the electrode reaction is independent

of mass transport effects therefore C(0t) asymp C and Bulter-Volmer equation approximates

to Eq 26

Butler-Volmer equation and η

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminusminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminus

minus= lowast

lowast

lowast

minusminus

lowast

lowast

lowast

αααα

R

O

O

tO

R

O

R

tR

CCEE

RTnF

CC

CCEE

RTnF

CC

II )(exp)()1(exp )0()1(

)0(0

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= lowastlowast ηαηαRT

nFC

CRT

nFC

CI

O

tO

R

tR exp)1(exp )0()0(0

middotmiddotmiddotmiddotmiddot Eq 25

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= ηαηαRT

nFRT

nFI exp)1(exp0 middotmiddotmiddotmiddotmiddot Eq 26

The Butler-Volmer equation shows the dependence of current and hence the reaction

rate on the overpotential This implies that small kdeg can be overcome by overpotential

The kinetically-limited region of the electrode reaction is however very small because

the rate constant sharply increases exponentially with overpotential and soon the

reaction became diffusion limited [21 30] At this state the surface equilibrium is

totally perturbed and the overpotential is used to drive the diffusion for the one-way

reaction

136 Butler-Volmer equation vs Tafel equation

At small η lt RTnF Butler-Volmer equation further approximates to a linear

equation as shown in Eq 27

56

Small η Butler-Volmer equation

η⎟⎠⎞

⎜⎝⎛=

RTFnII 0

middotmiddotmiddotmiddotmiddot Eq 27

At large |η| gt RTnF the rate of the backward reaction is negligible hence one

of the exponential terms in Butler-Volmer equation becomes negligible and the

equations are solved for η for both anode and cathode reactions as below

Large η Tafel equations

InFRTI

nFRT lnln 0 plusmn= mη in the form of ||ln Iba +=η middotmiddotmiddotmiddotmiddot Eq 28

Anode oxidation InF

RTInF

RT ln)1(

ln)1( 0 αα

ηminus

+minus

=

Cathode reduction InF

RTInF

RT lnln 0 ααη minus=

In the system which follows the Tafel equations I0 (and hence kdeg ) is very small and the

electrode reaction does not proceed unless large η is supplied In this potential range the

reaction is already totally irreversible [4 21 30] An irreversible slow reaction is

graphically presented in Tafel plot in which ln (I) is plotted against η as shown in Fig

9

57

Fig 9 Tafel plot [4 21 30]

I0 is deduced from the y-intercept of the extrapolated slope Once |I| has become too

large and reached to the mass transport limit at large |η| the slope deviates from the

linear trend [30]

14 OVERPOTENTIAL AND FUEL CELLS

141 Four overpotentials that decrease the EMF

As discussed so far overpotential is extra potential required to drive a reaction

In the fuel cells this can only be taken from the potential generated within the system

causing potential drop and reduces EMF The overpotential in the system is attributed to

the four causes [1 4] (i) internal current (ii) activation overpotential (iii) diffusion

overpotential and (iv) resistance overpotential

58

142 Internal current

Internal current is a reverse current caused by crossover and leakage current

Crossover happens when the fuel flows to the opposite electrode through the electrolyte

Leakage current occurs when the current flows through the electrolyte rather than the

external circuit Internal current therefore happens without a load and is observed as the

negative offset from the theoretical value of EMF This is noted especially in

low-temperature fuel cells such as direct methanol fuel cells (DMFCs) [4 51] In such

systems The inner current can be several 10 Am-2 and potential drop could be more

than 03 V [4] In order to prevent fuel mixture and crossover a separator is used to

partition the cell Alternatively it is also possible to confer high specificity to electrodes

as seen in glucose - oxygen fuel cells Such systems do not require

compartmentalisation and this is advantageous for miniaturisation [52]

143 Activation overpotential

Activation overpotential is a potential loss due to the slow reaction kinetics as

discussed in sect 135 This is unavoidable to some extent and is typically the biggest

problem in most fuel cells involving O2 cathode reduction because this reaction is

kinetically hindered Finding or inventing catalysts with high kdeg is the key solution for

this A sluggish reaction exhibits a sharp rise in activation overpotential for a slight

increase in low current After the steep increase the current increase becomes linear and

constant as shown in Fig 10 until the reaction reaches the limit of mass transport at the

higher overpotential [4 30 34]

59

Fig 10 Activation overpotential against current [4]

144 Diffusion overpotential

Diffusion overpotential is a potential loss due to concentration gradient This

tries to drive flux of electroactive species toward and from the electrode surface It

happens when the reaction kinetics is faster than mass transport [4 21 30] In fuel cells

diffusion overpotential is caused by fuel depletion due to the inefficient fuel convection

or supply [21 34] This problem is observed in reformers because the fuel refining and

conversion such as liquefied petroleum gas (LPG) to hydrogen gas take place outside

the system and it makes difficult to respond to the sudden fuel demand from the system

[4] It also happens when fuel diffusion is interfered with by bad electrode geometry

electrode fouling and adsorption [21 27] Diffusion overpotential is defined as Eq 29

60

Diffusion overpotential equation

⎟⎟⎠

⎞⎜⎜⎝

⎛minus=

Ldiffusion I

InFRT 1lnη middotmiddotmiddotmiddotmiddot Eq 29

ηdiffusion = Diffusion overpotential V

IL = Limiting current A

Limiting current (IL) is generated when a reaction takes place at the rate limited by mass

transport Diffusion overpotential sharply increases when reaction kinetics have reached

to the maximum and very large current are drawn as shown in Fig 11 [21 30 34 53]

Fig 11 Diffusion overpotential against current [4]

145 Resistance overpotential

Resistance overpotential arises within electrodes separators connectors circuit

or electrolyte [4 34] It is called Ohmic resistance or IR drop because it follows Ohmrsquos

61

law (Eq 30) where resistance overpotential is linearly proportional to the electric

current as shown in Fig 12 [4 30]

Ohmrsquos law

IRV = middotmiddotmiddotmiddotmiddot Eq 30

V = Voltage consumed due to the resistance V

I = Electric current drawn due to the resistance A

R = Resistance Ω

| |

Fig 12 Resistance overpotential against current [4]

Causes of internal resistance are scattered through the system therefore there are many

possible ways to resolve this One can possibly reduce it by using more conductive

material The difficulty lies in finding the conductive material which can be modified as

well as carry out the functions as one desires under the required conditions [53]

Resistance can be reduced by reducing the distance between two electrodes [54]

however this might cause short circuit and fuel leakage This becomes a serious issue in

62

use of solid electrolyte such as polymer electrolyte because both performance efficiency

and mechanical stability of electrolyte must be achieved [53] Excess in inert supporting

electrolyte minimises solution resistance and matrix effect It sustains a thin electric

double layer uniform ionic strength and buffering effect against pH change [21 30]

However electrolyte could become a problem when electrode material dissolves into

electrolytic solution and reacts there because it might change nature of electrolyte [1

21]

146 Total overpotential and cell voltage

Total overpotential is the sum of all four overpotentials Current ndash Voltage (IV)

polarisation curve visualises the tendency of each potential drop as shown in Fig 13 [1

4]

Fig 13 V-I curve for a typical low temperature fuel cell [4]

63

The cell voltage (Ecell) is determined by subtracting the cathode potential (Ec)

from the anode potential (Ea) (Eq 31) The actual cell voltage is found by subtracting

the total overpotential from the theoretical cell voltage The energy available from this

cell voltage can be calculated as in Eq 32

Cell voltage

cacell EEE minus= middotmiddotmiddotmiddotmiddot Eq 31

totalltheoreticacellactualcell EE ηminus= )()(

cellcell nFEG minus=∆ middotmiddotmiddotmiddotmiddot Eq 32

15 BIOFUEL CELLS

151 Microbial and enzymatic fuel cells

Biofuel cells are fuel cells which use biocatalysts for fuel reactions

Biocatalysts can be either the whole living cells or isolated enzymes The former is

called a microbial fuel cell and the latter is called an enzymatic fuel cell [55-57] They

often function under ambient conditions and allow wider choice of fuels Microbial fuel

cells use common carbohydrates such as glucose and lactate [55] but also have

potentials in extracting power from waste and sediments [34 55 57 58] Harnessing

such microbial life activities has some advantages They can complete fuel oxidation

within the cell It was reported that a microorganism Rhodoferax ferrireducen has over

80 glucose conversion rate [58] Microbial fuel cells also have long-term stability

[56-58] of up to five years [55] However their output current and power densities are

64

small due to high internal resistance [55-57] Enzymatic fuel cells in contrast have

higher current and power densities but short life incomplete reactions require

mediators to shuttle electron between the enzyme catalytic centre and the electrode and

become deactivated easily [55 56 58] Addition of mediators and their immobilisation

with enzyme complicates fuel cells design further However they can be more

advantageous due to their substrate specificity Both anode and cathode to reside in the

same compartment of mixed fuels without crossover This enables the miniaturisation of

fuel cells [52 59 60] and is particularly useful for medical implantation Stability and

lifetime of the enzyme fuel cells can be improved by appropriate enzyme

immobilisation fixing up the suitable conditions for their function [55] and adding

some enzyme stabilisers Addition of the right mediators and increased enzyme loading

will also increase output current and power density [55 59]

152 Enzyme fuel cells and mediators

Enzyme reactions are available for wide range of substrates yet each enzyme

has very high specificity for its substrates Many also function at neutral atmospheric

conditions therefore miniaturised enzyme fuel cells are of interest for in vivo

application Many redox reactions however take place at the catalytic centre which is

deeply buried in the protein body This proteinous content is an electronic insulator and

interrupts the electrical communication between the redox centre and the electrode It is

therefore difficult for many enzymes to establish efficient electron transfer without

mediators as shown in Fig 14 This is called mediated electron transfer (MET)

65

Fig 14 Mediated electron transfer reaction [55 61]

It is possible for some enzymes such as multicopper oxidases (MCOs) to perform direct

electron transfer (DET) without mediators however mediators ensure good electrical

communication and cell performance However the presence of mediators as well as the

enzyme specificity could complicate the fuel cell composition and its reaction path due

to the reaction cascade [55 59 62 63] Palmore built a methanol - oxygen enzyme fuel

cells and this involved four enzymes two mediators and six reactions to complete the

full reaction path [64] It is more advantageous to use enzymatic fuel cells for very

specific applications [59] such as medical implantation where there are environment

constraints but where applications demand only small power output through simple

reactions

Mediators are usually small redox active species which can be organic or

metal complexes In the enzymatic fuel cells they should be carefully selected because

improper mediators can cause unwanted overpotential or can destabilise the system

They must have (i) small overpotential (ii) fast reversible electron transfer [55 56 59

60 63 65] and (iii) stability [62 65] However there should be small but reasonable

66

potential gap between the enzyme and the mediator to drive electron transfer [60 66]

The gap of 50 mV is optimal [19 64] because of the small potential loss yet less

competing with enzyme reactions It is also possible to mediate electron transfer by

building a conductive nanowire between the electrode and the redox centre [60 63 67]

or a connection by plugging metal nanoparticles into the enzyme [60 67]

16 MINIATURISED MEDICAL POWER SOURCES

161 Realisation of miniaturised fuel cells and medical power supply

Medical application is one of the obvious targets for miniaturised biofuel cells

Glucose-oxygen biofuel cells which continuously run on glucose and oxygen were

thought to be ideal for implantable medical devices because both oxygen and glucose

are abundant in the body [63] They were initially intended to power cardiac assist

devices and pacemakers The studies on such systems have been made for more than 40

years however none has been made into practical use because their power density

stability operation life and biocompatibility were inadequate [19 67-69] Their output

power ranges between microW ndash mWcm-2 and they operate only for a few days to a few

weeks Most applications need a few W power for a longer duration [18 60 67] A

pacemaker requires 1microW [56] for more than 8 ndash 10 years with 996 reliability Such

power has been supplied by lithium (Li) iodine battery [19 56 70] which is

characterised by extended operational life [68] Li is fairly stable light-weight metal

and has high energy density of 386 kAhkg-1 Its standard potential is highly reducing ndash

323 V vs AgAgCl These properties enable Li batteries to power most devices

67

including pacemakers laptops and vehicles [19 21] However there are some problems

in Li batteries They are still bulky because they need to store their own fuel and it has

to be sealed with the metal case [19 68] They occupy half of the space in the

pacemaker volume [68 71] They use corrosive [72] and irritating materials There is

potential of explosion [68] leakage and the necessity of exchange Li also has potential

resource constraint due to the geopolitical issues as seen for oil and platinum More than

80 of its production is based in South America and China [73] With increased public

concern over using greener safer energy and saving resources as well as the global

political intervention in that it is natural to consider about using alternative

technologies to power implantable medical devices where it is possible

162 Miniaturised potential medical power sources

There are numerous implantable electrical medical devices with different

power requirements These include cardiac pacemakers defibrillators neurostimulators

for pain control drug delivery device hearing aids [70] and biosensors [74]

Implantable autonomous body sensors for physiological parameters such as glucose

concentration local pH flow and pressure [19] require power as low as 1 microW [56 70

74] and the operation period of 1 year at least [74] Such power can be supplied by

various implantable energy harvesting devices using body glucose [19] motion

vibration [75] and temperature differences [75] Heller developed the smallest

glucose-oxygen biofuel cells made of carbon fibres with the dimension of 17-microm

diameter and 2-cm length Anode glucose oxidase and cathode laccase were wired to

conductive hydrogel backbone with a flexible osmium (Os) mediator complex Its

68

output was 048 mWcm-2 with 06 V at 37 degC and it lasted for 1 week [19 67]

Kinetic energy of motion and vibration can be converted into electricity with

electrostatic transduction using microelectromechanical systems (MEMS) This

technology allows integration of various microelectronic devices on the same substrate

[76] NTT Corporation developed a system generating power from body motion and

temperature It consists of a thermoelectric and a comb-shaped Au-Si vibrational device

on a monolithic Si substrate The voltage produced by a thermoelectric device is

boosted by capacitance which was generated by the vibrational device then its charge

is stored in the capacitor [75] Another vibrational device was reported to have 20 x 20 x

2 mm volume and output 6 microW and 200 V at 10 Hz low frequency and 39 ms-2

acceleration The principal difficulty in using MEMS is to adjust and synchronise

flexibly the resonance frequency of vibrational device to the human body frequency

which is low and has constant fluctuation in order to increase energy conversion

efficiency The typical vibration has the frequency ranging from 1 to 100 Hz and

acceleration from 1 - 10 ms-2 [76]

Thermoelectric devices generate power from temperature difference Each

module consists of a number of thermocouples on Si substrate Thermocouples are

made of different material so that it can generate voltage from the heat flow following

temperature gradient This is called Seebeck effect and its size depends on the material

and number of thermocouples used and the spacing within the device [75 76] Typical

ambient temperature difference is less than 10K [74 76] and its power is 1microWcm-2 [74]

Seiko Instrument developed a wristwatch called Seiko Thermic which was powered by

human body temperature in 1998 [77 78] It consists of n-type Bismuth-Tellurium

(BiTe) and p-type Bismuth-Antimony-Tellurium on rods reinforced by Ni on oxidised

69

silicon wafer It generated microWcm-2 power at extremely low 0047 conversion

efficiency With a booster circuit it powered a wristwatch operating at 1microW and 15 V

It could however operate only when ambient temperatures were below 27 degC [78] and

so could not be used in tropical regions

Heller also developed an implantable miniature Nafion-coated zinc ndash silver

chloride (ZnAgCl) battery The anode was made of zinc fibre coated with Nafion It

was 76-microm diameter 2-cm length and 75 x 10-2 cm2 surface area The cathode was

made of AgCl wire covered with chloride-permeable membrane On discharge anode

zinc formed Zn3(PO4)2middot4H2O hopeite complex with Nafion growing non-porous

lamellae on the surface The lamellale had a unique property which was permeable to

zinc ions but impermeable to O2 therefore the battery became corrosion free It output

was 13 microAcm-2 at 1 V and operated for three weeks under physiological condition The

advantage of this battery was its size which was 100-fold smaller than the smallest

conventional batteries [79] The smallest silver oxide button buttery has the volume 30

mm3 [19 80] with energy density 04 mWh mm-3 at 15 V The smallest Li-ion buttery

has the volume 190 mm3 [80] with energy density 025 mWh mm-3 at 4 V [21]

17 OUTLINE OF THE WORK

171 Introduction to miniaturised glucose-oxygen fuel cells

A miniaturised glucose-oxygen biofuel cell runs on glucose and oxygen This

would be ideal to power implantable autonomous low-power body sensors since both

glucose and oxygen are abundantly present in the body The range in their physiological

70

concentration is between 4 ndash 8 mM for glucose and 01 ndash 02 mM for oxygen [56 67]

The aim of this study was to build miniaturised glucose-oxygen fuel cells with reliable

enzyme anodes and powerful oxygen-reducing cathodes using straightforward and

scaleable technology The general structure and mechanism of glucose-oxygen fuel cells

were described in sect 112

In this study both anode and cathode reactions took place at the room

temperature and physiological pH in the presence of catalysts Glucose oxidation was

catalysed by the enzyme glucose oxidase (GOx) from Aspergillus niger and its electron

transfer and conduction were facilitated by an conducting organic salt called

tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) (Fig 15)

Fig 15 The schema for a glucose-oxidising anode

GOx and TTF-TCNQ were mixed into a composite employing a feasible electrode

fabrication method called bulk modification method Various enzyme stabilisers were

added to the composite at different concentration to improve its performance The

influence of various enzyme stabilisers on anode performance was studied in the

71

presence of both N2 and O2 and the most ideal anode composition was determined

Cathodes were also fabricated in the similar manner using graphite as conductive

material and mixing with various oxygen reducing catalysts except for the enzyme

membrane cathodes The cathode composites were pretreated in various media to

enhance their activity For enzyme membrane electrodes glassy carbon electrodes were

used instead of graphite composite and enzyme solution was loaded onto the surface

and trapped with membrane Catalytic power and mechanism of each cathode were

studied with various electrochemical methods and the most catalytic cathode was

determined

172 Outline

Chapter 1 is a general introduction about fuel cells It explains the history and

basic concept of fuel cells and outlines the study

Chapter 2 explains about the enzyme anodes The introduction part refers to

glucose oxidising enzymes enzyme immobilisation TTF-TCNQ enzyme inactivation

enzyme stabilisers and enzyme kinetics The material and method section reports the

protocol for the enzyme electrode fabrication electrode conduction study and

amperometry study for enzyme electrodes Result and discussion section mentions

enzyme electrode conduction enzyme kinetic studies based on amperometry

mechanism of enzyme stabilisation in each stabiliser and ideal electrode composition

Chapter 3 discusses oxygen-reducing cathodes The introduction part reviews

the mechanism of the oxygen reduction reaction oxygen reactivity oxygen reduction

catalysts carbon properties electrochemical pretreatments electrochemical techniques

72

and how to determine the catalytic efficiency of cathodes The material and method

section describes the protocol for the oxygen-reducing electrode fabrication preparation

and electrochemical pretreatment electrochemical studies electrochemical studies for

qualitative analysis and to obtain the kinetic parameters and analysis to determine the

efficiency of catalytic cathodes The result and discussion section describes the

voltammetric behaviour of each pretreated cathode influence of pretreatment and

catalytic efficiency The most catalytic cathode was determined at the end

Chapter 4 refers to the overall conclusion and further studies

73

2 ANODE INTRODUCTION

21 GLUCOSE OXIDATION ENZYMES

Glucose-oxygen fuel cells extract power by oxidising glucose at the anode The

anode contains glucose-oxidising enzymes There are several enzymes available for

glucose oxidation (i) β-D glucose oxidase (GOx) (EC 1134) [56 81] (ii) pyranose

oxidase (POx) (EC 11310) [56 81] (iii) nicotinamide adenine dinucleotide-dependent

glucose dehydrogenase (NAD-GDH) (EC 11147) [18 56] and (iv) quinoprotein

glucose dehydrogenase (PQQ-GDH) (EC 1152) [56 81] These enzymes are

classified into the family of oxidoreductases which catalyse either transfer of (i)

hydride (H-) (ii) oxygen atoms or (ii) electrons from one molecule to another Oxidases

transfer electrons from substrates to O2 and form H2O or H2O2 as products

Dehydrogenases transfer electrons from substrates to coenzymes without involving O2

(Eq 33 and Eq 34)

Glucose oxidation by GOx and NAD-DHG

GOx middotmiddotmiddotmiddotmiddot Eq 33

NAD-DHG middotmiddotmiddotmiddotmiddot Eq 34

To function catalytically enzymes require non-peptide inorganic or organic cofactors or

prosthetic groups Non-peptide organic cofactors are also called coenzymes Enzymes

lacking such groups are called apoenzymes and hardly functional Cofactors are

generally loosely bound to the apoenzymes and freely diffusible but on catalysis they

74

bind to the enzymes as the second substrates Prosthetic groups are always tightly bound

to the apoenzymes through covalent or non-covalent bonding [22 23 48] GOx and

POx are fungal flavoprotein enzymes containing flavin adenine dinucleotide (FAD)

cofactors FAD in GOx is tightly bound to the apoenzyme but not covalently bonded

hence still dissociable [67 82-84] NAD-GDH contains dissociable NAD+ [65] while

PQQ-GDH contains pyrrolo-quinoline quinone (PQQ) redox cofactor [56 65] which is

moderately bound to the apoenzyme [65 67] The structure of each cofactor is shown in

Fig 16

75

N

N

NH

N O

O

R

N

N

NH

HN O

O

R

NH

N

NH

HN O

O

R

H+ + e- H+ + e-

2H+ +2e-

N+

NH2

O

R

H

N

NH2

OH

R

HH

N

NH

O

O

OHO

OH

O

OH

O

N

NH

O

HO

OHO

OH

O

OH

O

N

NH

OH

HO

OHO

OH

O

OH

O

H+ + e- H+ + e-

FAD FADH2

NAD NADH

PQQ PQQH2

Fig 16 The structure of FAD NAD and PQQ and their redox reactions

Moieties involved in the redox reactions are circled in gray [23 65]

Cofactors also stabilise the enzyme structure Irreversible thermal GOx denaturisation

involves the perturbation of FAD biding site causing FAD dissociation although 70 of

its secondary structure is retained Chemical denaturisation untangles the dimmer

structure of GOx [65 81 83 85] and dissociates monomers and FAD [86 87] Excess

FAD does not prevent or regain function after thermal inactivation [87] However FAD

can rapidly bind to apoenzymes at 25 degC pH 61 and stabilise the tertiary structure

through conformational change and give resistance to proteolysis [82]

76

The choice of enzyme is based on the reaction involved enzyme availability

stability enzymatic properties physical properties and operational environment In the

absence of O2 or when there is concern about system degradation due to the competition

of electrons between O2 and artificial electron acceptors or due to the by-product H2O2

GDHs are more preferred to GOx [23 60 65-67 88] However the use of GDHs poses

different disadvantages Membrane-bound PQQ dehydrogenase (mPQQ-GDH) has very

high glucose specificity however it is difficult to solubilise and purify hence not for

commercial use [88] Soluble PQQ dehydrogenase (sPQQ-GDH) has been used in

commercial glucose sensors [67 89] however it suffers from low thermostability and

very low glucose specificity [88] Moreover PQQ-GDHs add complication to the

system because metal ions such as calcium ions (Ca2+) and magnesium ions (Mg2+) is

required to activate them [89 90] In the case of NAD-GDH NAD must be provided

because it is a dissociable cofactor but must bind first to the apoenzyme to form a

catalytic complex for glucose oxidation In addition to this complication NAD was also

reported to be more prone to causing electrodes poisoning without a quinone acceptor

[65] NAD forms adduct with phosphate in the buffer its decomposition releases free

adenine and electrochemically oxidised adenine adsorbs onto the electrode surface

causing electrode poisoning [91]

The redox potential of enzyme is also important because it is the driving force

for electron transfer and the power although it is difficult to match both catalytic

efficiency and electrochemical efficiency It is also difficult to determine the redox

potential exactly because the redox potential of cofactors is influenced by

microenvironment such as where they are embedded in the enzyme as well as

77

macroenvironment such as pH and temperature For example the redox potential of

FAD itself is ndash 042 V vs AgAgCl at pH 7 25 degC [22 23] however that of

flavoproteins could range from ndash 069 to ndash 005 V [65] and FAD-containing GOx has

the redox potential approximately ndash 03 V [67 92] It was also reported glucose

gluconolactone redox potential was ndash 056 V [93] PQQ itself has - 013V in absence of

Ca2+ and ndash 012 V in presence of 1M CaCl2 [94] while sPQQ-GDH has the redox

potential approximately - 019 V in presence of Ca2+ [66 67] Furthermore the enzyme

redox potential leads to a complex issue when mediators which assist electron transfer

between the enzyme redox centre and the electrode are involved because the improper

combination of the enzyme and mediator causes inefficient electron transfer and hence

potential drop

For enzyme availability stability and simplicity in handling GOx has been

most popular glucose-oxidising enzyme It has optimal pH 55 and temperature 35 ndash

40 degC [93] but is stable between pH 4 ndash 7 [95] and up to 50 - 55 degC [82 87] GOx has

higher substrate specificity and is most active toward β-D-glucose oxidation [56 86 88]

and very stable therefore its use has prevailed among various sensor applications [60

67]

22 DESIGNING ENZYME ELECTRODES

Construction of enzyme electrodes is complex because it simultaneously

incorporates enzymes mediators conductive materials and stabilisers ensuring that all

of them work harmoniously under restricted performance conditions There are several

78

immobilisation techniques available These include physical adsorption cross-linking

covalent bonding and entrapment in gels or membranes [96]

221 Physical adsorption

Physical adsorption is a simplest immobilisation method Electrodes are dipped

in the solution containing enzymes and mediators or such solution can be applied to the

electrode surface In this method however the enzyme tends to leach due to the weak

interaction between the electrode and enzyme [55] In order to avoid the loss of

electrode material the electrode surface is often covered or coated with material One

classic method is that the solution on the electrode surface is trapped with a

semipermeable dialysis membrane [61 65 92] This allows enzymes free from the

possible denaturisation caused by immobilisation on the solid material Enzymes inside

the dialysis membrane also can be fixed onto the cellulose acetate membrane or the

enzyme layer can be cross-linked with bifunctional reagents such as glutaraldehyde

[61]

222 Reconstituting apo-enzyme

Willner constructed enzyme electrodes by reconstituting apo-enzyme on the

gold (Au) electrodes surface with quinone mediators The electrode consisted of three

layers which were made of PQQ synthetic FAD and apo-GOx [97] PQQ were

mediating groups and attached onto Au electrode by cystamine PQQ attached on Au

was then covalently linked to synthetic FAD called N6-(2-aminoethyl)-FAD Covalently

79

linked FAD self-assembles with apo-GOx so that holo-GOx assembly is formed on the

top of the PQQ mediating layer attached to the Au surface The reconstitution procedure

is shown in Fig 17

Fig 17 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode [97]

The output current density was 300 microAcm-2 at pH 7 35 degC and it dropped by 5 in the

presence of O2 although the decrease was negligible at 01 mM physiological O2

concentration [98] The reconstitution of apo-enzymes is a flexible system potentially

applicable to enzyme electrodes [97] The fabrication process is cumbersome however

the enzymes are neatly aligned and electrically connected in the correct orientation

therefore electrocommunication between the enzymes and electrodes can be

maximised

80

223 Tethered Osmium-complexes linked to conductive hydrogel

It has been reported that embedding enzymes in the conductive hydrogel

complexed with tethered osmium (Os) mediator led to high output current [19 60 66

67] The conductive hydrogel is made of cross-linked polyvinylpyridine or

polyvinylimidazole They are soluble as monomers however they gain their

water-swelling property in cross-linked polymer This is ideal for enzyme mobility and

electrical conductivity Electron transfer is realised by the Os complex which is tethered

to the conductive hydrogel back bone The tether is made of 10 ndash 15 atoms [19 67] in

order to allow flexile movement of mediators Tethered Os complexes sweep the large

volume made of multiple layers of hydrogel form electrostatic adducts with enzymes

[19] and facilitate collide-and-hop electron conduction [19 99] Polymer tether and Os

complexes can be flexibly tailored for different enzymes and different operational

environments [19] One of its great advantages is that the redox potential for electron

transfer can be adjusted by changing the ligand structure of Os complex [19 60 97]

Heller constructed such electrodes made of carbon fibers whose dimension was 7 microm

diameter 2 cm length 08 mm2 surface area and 26 x 10-3 mm3 volume GOx was

wired to poly(4-vinylpyridine) conductive hydrogel backbone by the tethered complex

of tris-dialkylated NNrsquo-biimidazole Os2+3+ (Fig 18)

81

Fig 18 GOx wired to conductive hydrogel complexed with Os [19 67]

The redox potential of its redox centre was ndash 02 V vs AgAgCl The glucose oxidation

took place at ndash 036V and its output current density was 11 mAcm-2 [19] Disadvantage

of the tethered enzyme is short life It was stable for one week in the physiological

buffer then 5 current drop per day was observed When it was implanted in grape sap

50 current drop occurred within 20 hours In calf serum 50 current dropped

occurred in 2 hours with electrode fouling [19]

224 Entrapment in the conductive polymer

Entrapping enzymes in conductive polymer such as polypyrrole polyaniline

and polythiophene [65] is a popular method for enzyme electrodes [96] This is made by

82

electropolymerisation in which potential is applied to the solution containing enzymes

and monomors for certain duration It is a simple method but can be complicated if the

conditions for polymer growth are not compatible with enzyme entrapment [99]

Enzyme leakage occurs when enzymes are not trapped securely but it can be reduced

when derivatised polymers are used [96 100] Their conductivity is affected by

monomer structure [100] acidity and electrolyte [101] It can be degraded by products

of enzymatic reactions [65] Fujii fabricated GOx electrodes which were stable for

more than four months though a gradual sensitivity drop was observed GOx was

entrapped in saccharide-containing polypyrrole made of 1-(6-D-gluconamidohexyl)

pyrrole (GHP) (Fig 19(i)) on Pt electrodes It was reported that entrapped GOx was

secured by hydrogen bonding present between sugar gluconyl groups and enzymes

Enzyme leaching was also reduced by improved polymerisation with extended

conductive hydrocarbon chain [100] The output current density was 75 microAcm-2 at 055

V pH 74 in the presence of 56 mM glucose and 175 microA cm-2 under glucose saturated

condition [100] Arai entrapped various enzymes in polymer of mercaptobenzoquinone

(SQ) (Fig 19(ii)) and its methylated derivatives methylmercapttobenzoquinone

(MeSQ) and dimethylmercaptobenzoquinone (Me2SQ) on Au electrode surface

83

OH

H

H

OH

OH

H

OH

H

CH2OHN(CH2)6NHCHOH

(i)O

O

SH

Fig 19 Monomer structures of conductive polymers

(i) 1-(6-D-gluconamidohexyl) pyrrole [100]

(ii) Mercapto-p-benzoquinone [101]

Quinones have been widely used as good mediators [21 60 61 65] Poly(SQ) is a

conductive polymer functionalised with quinone mediating groups and can be easily

attached to Au electrode due to the presence of thiol group (-SH) [100] It was revealed

that more methylated SQ polymer led to better performance The maximum current

density for SQ- MeSQ- and Me2SQ-GOx anodes was 006 522 and 544 microAcm-2 and

their electrode enzyme dissociation constant KM was 13 18 and 20 mM respectively

[102 103] FAD cofactors have been also electropolymerised to entrap lactate

dehydrogenase and lactate oxidase It was reported that polymerised FAD extended the

shelf and operational life of electrodes to more than one year and also reduced O2

influence [104]

225 Bulk modification

Another popular electrode fabrication involves bulk modification of solid

composite This is simply made by mixing electrode material and inert binding reagent

84

such as epoxy resin Typical composite electrodes consist of 70 conductive material

by weight [105] Bulk-modified electrodes are robust stable inexpensive and easy to

modify build and reuse [105-107] therefore often used for enzyme electrodes [108]

For GOx electrodes tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ)

has been used as both mediating and conducting material

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties

TTF-TCNQ is a black crystalline charge transfer complex having both

mediating and conducting properties Its handling is also easy and often used for GOx

electrodes [65] Its conductivity is 102 ndash 103 Scm-1 [109 110] and comparable to that of

graphite [65 105] TTF-TCNQ complex consists of TTF electron donor and TCNQ

electron acceptor and they are arranged in a segregated stack structure TTF-TCNQ has

metal-like electric conductivity and this derives from intra-stack and inter-stack

molecular interactions Within the stack π-orbitals of adjacent molecules overlap This

builds the major conductive path in the direction of stack [61 65 110 111] Between

the stacks there is partial charge transfer between different chemical groups and this

gives the metallic feature in which charge carriers are mobile among the stacks

[110-112] as shown in Fig 20

85

S

S S

S

N

C

C

N

C

C

N

N

S

S S

S

7 6

N

C

C

N

C

C

N

N

-

TTF - electron donor

TCNQ - electron acceptor

6 - localised 6 - delocalised

+ e-

+ e-

Fig 20 TTF-TCNQ structure and its charge transfer [61]

TTF-TCNQ is a good electrode material because it has low background current and is

efficient at electron transfer [65] is stable [65] insoluble in water and not toxic [113]

However it exhibits complex electrochemistry (Fig 21) and its behaviours have not yet

been fully elucidated

86

vs AgAgCl

TCNQTCNQ e⎯⎯rarr⎯minus minusbull+

+minus+ ⎯⎯rarr⎯minus 2e TTFTTF

minus+minusbull⎯⎯rarr⎯

minus 2e TCNQTCNQ

+minus⎯⎯rarr⎯minus

TTFTTF e

Fig 21 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 01M potassium

phosphate 01 M KCl at 20 mVs-1 [92] Modified

The range of its effective redox potential varies according to the authors [65 92 114

115] Following the Lennoxrsquos study its potential extremes should lie within the range

between - 015 V and + 031 V vs AgAgCl (Table 2) because beyond these potential

ranges irreversible lattice reduction or oxidation takes place Even within the range

crystal dissolution salt formation with buffer ions and its deposition are still present and

could change the electrochemical profile TTF-TCNQ stability is affected by many

factors such as potential applied buffer component and concentration ion solubility

mass transport and analyte [92]

87

Table 2 Electrochemical behaviour of TTF-TCNQ at pH 74

Equilibrium TCNQTTFTCNQ-TTF +⎯rarr⎯ middotmiddotmiddotmiddotmiddot Eq 35

TCNQ reduction + 015 V MTCNQTCNQTCNQ Me ⎯⎯rarr⎯⎯⎯rarr⎯+minus +minusbull+ middotmiddotmiddotmiddotmiddot Eq 36

Lattice reduction - 015 V minus+minusbull ⎯⎯rarr⎯minus 2e TCNQTCNQ middotmiddotmiddotmiddotmiddot Eq 37

Anode salt formation + 031 V MTCNQTCNQ M⎯⎯rarr⎯++minusbull middotmiddotmiddotmiddotmiddot Eq 38

Lattice oxidation + 035 V +minus⎯⎯rarr⎯minus

TTFTTF e middotmiddotmiddotmiddotmiddot Eq 39

Further oxidation + 038 V +minus+ ⎯⎯rarr⎯minus 2e TTFTTF middotmiddotmiddotmiddotmiddot Eq 40

In the presence of a redox couple it also exhibits different electron transfer mechanism

When Edegrsquo of the redox couple is Edegrsquo gt 0 V TTF-TCNQ shows metal-like behaviour

facilitating direct electron transfer (DET) while it presents mediated electron transfer

(MET) for the redox couple of Edegrsquo lt 0 V Lennox made a study on TTF-TCNQ GOx

electrodes and reported that large anodic current was observed at ndash 015 V He also

reported that stable current was obtained when constant potential + 02 V was applied

for 24 hours at pH 7 in the presence of glucose He concluded that hydrophobic protein

core solubilised TCNQ and it formed enzyme-mediator complex GOx oxidation could

be catalysed at low potential because it was still in the range of the stable redox

potential He also conducted experiments with GOx and methanol dehydrogenase

(MDH) modified TTF-TCNQ electrodes in the presence of O2 The performance was

interfered by O2 in both electrodes against his expectation because DHG is in general

resistant to O2 He gave a conclusion that reduced mediators were oxidised by O2 before

electrooxidation took place [92]

88

226 Enzyme covalent attachment and mass loading

Enzymes can be covalently attached to the electrode however it becomes

complicated when mediators and stabilisers are must be also attached for practical fuel

cell applications This is however an attractive technique once ideal means of

co-immobilisation are established because enzymes can be retained firmly on the

electrode surface Enzymes can be covalently linked to oxidised or aminated surface

through amide bonds [116 117] Covalent linkage often lowers enzyme activity because

it partially alters the enzyme structure and confines the enzyme conformation [55]

however Kim succeeded in fabricating stable biocatalytic nanofibre by loading enzyme

aggregate to the enzyme monolayer which was covalently attached to the nanofibres

(Fig 22) Nanofibres with ester groups were synthesised from polystyrene and

poly(styrene-co-maleic anhydride) using electrospinning Seed enzyme was covalently

attached to the chemically modified nanofibre to form an enzyme monolayer on the

nanofibre This enzyme-attached nanofibre was further modified with the mixture of

glutaraldehyde and enzymes so that enzyme mass load was achieved by loading enzyme

aggregate to the enzyme monolayer covalently attached to the fibre Minter reported

that enzyme immobilisation with cross-linking increased the enzyme stability but

limited the enzyme conformation and thus reduced the activity However the activity

reported was nine times higher than that with an enzyme monolayer only Moreover the

activity was retained more than one month under rigorous shaking [118] The activity of

each enzyme particle might have been reduced however mass loading increased the

reactant flux hence increased the overall activity

89

Fig 22 The procedure for the enzyme mass-loaded biocatalytic nanofibre [118]

He also fabricated single-enzyme nanoparticles (SENs) in which each enzyme particle

was encapsulated within a hybrid organic and inorganic network The surface amino

groups of the enzyme were converted into surface vinyl groups and then polymerised

The polymer was then cross-linked to form the shell around the enzyme particle (Fig

23)

90

Fig 23 Fabrication procedure of single-enzyme nanoparticles [55]

It was reported that high stability was achieved after polymer cross-linking The enzyme

activity was approximately half of the free enzyme however there was no mass

transport interference [55] due to their porous structure Km in SENs was nearly

equivalent that of the free enzyme but the storage stability went beyond five months

[119] Eight-fold increase on storage stability after GOx covalently linked to

poly(phosphorothioate) was also reported [120]

23 PROTEIN INACTIVATION AND STABILISATION

Enzyme inactivation is one of the biggest problems in enzyme electrodes

Enzymes are excellent catalysts in their native environment however they are only

designed to work optimally under specific conditions therefore very sensitive to the

slight change in their environment and easily undergo inactivation The issue is

persistent in the enzyme electrodes because their fabrication introduces the enzymes to

the unusual environment yet enzyme catalytic activity must be maintained to obtain

sufficient power output and operational life The problem can be alleviated by (i)

91

protein engineering [56 121] (ii) environmental engineering [56 60 63 67] and (iii)

addition of appropriate enzyme stabilisers [120 122] Protein engineering involves the

alteration of enzyme structure to gain desired functions stability or resistance However

it is very challenging to improve both stability and activity since protein stability is

conferred by its rigidity while its activity depends on the conformational flexibility

Alteration of protein properties is realised by genetic engineering such as site-directed

mutangenesis [123] or directed evolution [49] Environmental engineering is closely

linked to the enzyme immobilisation It aims to achieve the same goals as in protein

engineering but extrinsically The environment which rigidifies the protein structure

such as cross-linking or inclusion in electrode composite tends to increase the enzyme

stability and prolong its life In contrast the environment which more favours over

flexible protein conformation such as hydrogel and membrane trap exhibits higher

enzyme activity but shorter life However it was reported that enzyme cross-linking and

the presence of hydrophilic macromolecules improved both stability and activity while

electrostatic interaction improved selectivity [124] Addition of stabilisers is more

targeted smaller scale environmental engineering Intrinsic conformational stability of

the protein cannot be improved [120] however it can target and eliminate specific

inactivating factors increases the local specific protein stability and prevents or slows

its unfolding There are two factors to improve for enzymes to achieve ideal operation

(i) activity by hydration and (ii) increased lifetime by increased protein rigidity which

might involve the removal of bond-destabilising factors or amplifying the bond strength

within the enzymes In fact both must be probably achieved simultaneously for gaining

the optimal state because they interrelate to each other in terms of protein dynamics As

mentioned earlier protein hydration improves enzyme activity but also allows its

92

regeneration hence affects the protein internal dynamics Protein dehydration distorted

the protein structure [125]

24 PROTEIN DESTABILISATION

There are many protein destabilising factors apart from dehydration and these

can be classified into chemical and physical instability Chemical instability happens

due to chemical modification such as deamidation oxidation proteolysis and

racemisation

241 Deamidation

Deamidation is the hydrolysis of amide link in the amino acid side groups such

as glutamine (Gln) and asparagine (Asn) (Fig 24)

Glutamine (Gln) Asparagine (Asn)

Fig 24 Side groups of Gln and Asn

It is favoured by increase in pH temperature and ionic strength The buffer

concentration influences the rate of deamidation at pH 7 ndash 12 Urea also accelerates

deamidation It often implies the protein aging and then modifies the protein function

93

242 Oxidation

Oxidation takes place at the side chains of cystine (Cys) methionine (Met)

tyrosine (Ty) tryptophan (Trp) and histidine (His)

Cysteine (Cys) Methionine (Met)

Tyrosine (Tyr) Tryptophan (Trp) Histidine (His)

Fig 25 Side groups of Cys Met Tyr Trp and His

Atmospheric oxygen can oxidise Met residues H2O2 modifies indole thiol (SH) [123

126] disulfide (SS) imidazole phenol and thioether (RSR) groups at neutral or slightly

alkaline conditions At acidic condition it oxidises Met into Met sulfoxide (R-S=O-R)

Under extremely oxidising condition Met sulfoxide is further oxidised into Met sulfone

(R-S(=O)2-R) Met oxidation leads to loss of biological activity however it does not

abolish the enzyme activity Therefore if oxidised Met is reduced back by a reductant

such as thiols it will restore the full biological activity Met reactivity depends on its

position within the protein Cys plays an important role in protein folding because it

forms intermolecular disulfide bonds however very susceptible to oxidation It can

undergo autooxidation in the absence of oxidants It is oxidised in several stages in

94

which thiol R-SH group is oxidised to (i) sulfenic acid (R-S-OH) (ii) disulfide

(R-S-S-R) (iii) sulfinic acid (R-S=O-OH) and (iv) sulfonic acid (R-S(=O)2-OH) [123

127] Oxidised thiol groups can be reduced back however they can be also reduced

back in the wrong way This leads to protein misfolding and loss of activity There are

many factors that influence the rate of oxidation They are (i) temperature (ii) pH (iii)

buffer medium (iv) metal catalysts (v) O2 tension (vi) steric condition (vii) neighbouring

groups (viii) photooxidation The presence of metal ions such as Fe2+ and Cu2+

accelerates oxidation by air [123] and the presence of O2 also might accelerate thiol

oxidation with other oxide such as nitric oxide radical [127] Metal-catalysed oxidation

involves a site-specific metal binding to Trp His and Met This leads to severer form of

oxidation causing protein aggregation Peroxide-catalysed oxidation on the other hand

is milder and tends to affect Cys and Trp but does not form aggregates [126] Increase in

pH increases pKa hence accelerates dissociation of ndashSH All of Cys Met Typ Tyr and

His are susceptible to photooxidatoin however His photooxidation is fatal leading to

enzymatic inactivity [123]

243 Proteolysis and racemisation

Proteolysis and racemisation takes place at aspartic acid (Asp) residue The

bond between Asp and proline (Pro) is especially susceptible to hydrolysis Asp also

easily undergoes base-catalysed isomerisation to isoAsp through a ring formation [123]

95

Aspartic acid (Asp) Proline (Pro)

Fig 26 Side group of Asp and the amino acid structure of Pro

244 Physical instabilities

Physical instability includes denaturation adsorption aggregation and

precipitation and [123] This refers to loss of tertiary structure and does not involve

chemical modification It is defined as Gibbs free energy change between the native and

unfolded state of protein Physical instability is induced by change in temperature

extreme pH organic solvent denaturant [123] and solvation [120 125]

25 ENZYME STABILISERS

By clarifying the possible major causes of instability the choice of optimal

approach and stabilisers can be made Considering the case of glucose-oxygen fuel cells

the stabilisers should be selected on the basis of following points (i) conformational

stability for longer operation and shelf life (ii) hydrophilic environment for optimal

enzyme functions (iii) optimal interaction between enzyme and substrate and enzyme

and environment Conformational stability is to secure protein folding and avoid

denaturation This is achieved by thiol group protection reduction of oxidised thiol

96

groups removal of oxidising species an increase in internal hydrophobic interaction

and environmental hydrophilic and electrostatic interaction Optimal enzyme activities

can be achieved by an increase in hydrogen bonding and electrostatic interactions

251 Polyehyleneimine (PEI)

Polyethyleneimine (PEI) has been reported as an efficient antioxidant as well

as chelating agent It is also cationic (Fig 27) [120] therefore provides electrostatic

stabilisation with anionic protein [126] such as GOx [67 85] Anti-microbial activity

has been also reported [126] It is not effective against thermal inactivation [128]

H+

N+H3N+H3

N+H3H3N+

H2+ H+

H+

H+

H2

+

H2+

Fig 27 Structure of PEI [129]

PEI is available in different molecular weights [120] and smaller molecules are more

effective in binding and flexibility [130] However its optimal loading concentration

varies [120 128 131] PEI protects thiol groups efficiently [120] from both metal- and

peroxide- catalysed oxidation despite of their different oxidation mechanisms [126] PEI

also forms a complex with the enzyme and coats it with polycations This occludes the

97

oxidants from the protein surface [126 130] Complex formation might be involving

hydrogen bonding between carboxylic acid groups in the enzyme and nitrogen atoms in

PEI [130] This possibly confers stability in dried state [128] It was reported that PEI

increased the maximum limiting catalytic velocity (V) for GOx [131] and lactate

dehydrogenase (LDH) [128] However it also increased Km for LDH and shifted its

optimal pH slightly toward acidic [128]

252 Dithiothreitol (DTT)

Dithiothreitol (DTT) is another small water-soluble [132] thiol protecting

reagent [132 133] with the reducing potential at ndash 033 V at pH 7 It has only little

odour [132] and has low toxicity [134] Its epimer is dithioerythriotol (DTE) Both are

excellent reducing reagents with two thiol groups (Fig 28) [132] however DTT might

be marginally better in reducing power [135] It reduces back disulfide quantitatively

[127 132] at lower concentration than other thiol-containing reducing reagents such as

Cys and gluthatione (GSH) But it cannot restore some of the lost catalytic activity

[136] because it only reduces disulfides but not other thiol oxides Therefore its

efficacy depends on (i) the oxidation mechanism involved in the protein inactivation

(ii) the velocity at which the inactivation proceeds and (iii) how thiol-contaning amino

acids are present in the protein DTT is not effective against mild oxidation caused by

low concentration of H2O2 or isolated Cys residues [127] It is probably because that

DTT reduces thiol oxide by converting itself from a linear into a cyclic disulfide form

which is called trans-45-dihydroxy-o-dithiane with (Fig 28) [132]

98

Fig 28 Structures of DTT from a linear to a cyclic disulfide form [132]

On the other hand arsenite was effective in reducing only mildly oxidated thiol groups

but not disulfides [127] It is however toxic [137] and not suitable for practical use

Neither of DTT or arsenite was effective if thiol oxidation is outcompeting [127]

253 Polyethylene glycol (PEG)

Polyethylene glycol (PEG) (Fig 29) has been reported as an enzyme stabiliser

however also has mixed reception and its stabilisation mechanism has not yet been

clearly understood

Fig 29 Structures of PEG

OrsquoFagain reported that PEG increased storage and thermo stability [120] while

99

Andersson reported the increase in LDH activity with PEG 10000 Mw but was

followed by a decrease after 10-day storage and then eventually became worse than

those without stabilisers The stabilisation was also found to be concentration dependent

[128] As for protein stabilisation mechanism it was proposed that PEG preferentially

hydrates the protein and stabilises its native structure in the solution [138-140]

According to Arakawa this causes the exclusion of PEG from protein surface due to the

unfavourable interaction between them hence increases water density around the

protein This results in the water shell around the protein surface and increases the

hydrogen bonds [138 139] Better hydration favoured larger PEG Mw due to the larger

steric exclusion [138] on the other hand lower PEG Mw was also reported to be better

because of its better solubility and viscosity [139] Optimal concentration is unknown

however one proposal is that higher concentration is more advantageous to stabilise the

denatured protein because it can penetrate and stabilise the protein hydrophobic core

exposed during denaturation [138] In contrast Lee reported that this penetration affects

the hydrophobic core of active enzyme and decreased the activity and denatured the

protein [139] Both however concluded that a fine balance between preferential

hydration and hydrophobic interaction is important and PEG concentration must be up

to 30 [138 139] It was also reported that PEG stabilised the protein of low water

content better because it prevented the water evaporation while polyols tended to strip

water from the protein dehydrated microenvironment [139]

254 Polyols

Polyols such as glycerol (GLC) (Fig 30) were reported to increase shelf life

100

[141] thermostability [120] and also provide preferential solvation as seen in PEG

[141-143] Higher GLC concentration resulted in better protein activity [143 144] and

reduced protein aggregation due to the tighter protein packing [144] However observed

hydration was lower than expected for high GLC concentration therefore Gekko

proposed that the contribution of higher GLC concentration did not derive form the

increase in steric exclusion Instead GLC might penetrate the solvation layer of protein

and stabilise the structured water network around the protein [143] Andersson also

compared the influence on protein activity after the storage among several candidate

stabilisers diethylaminoethyl-dextran (DEAE)-dextran Mw 500000 sorbitol and PEI

DEAE-dextran increased the activity both before and after the storage while sorbitol

decreased it However PEI was only found to be superior to all [128]

OH

OHHO

Fig 30 Structure of glycerol

255 FAD

FAD (Fig 16 p 75) is also another possible stabiliser as well as mediator

because it can regenerate denatured aged enzymes which have lost its cofactors [82

108] However it has not been clarified well and Bartlett questioned its efficacy due to

the contradictory results he obtained [145] It was reported that addition of FAD

increased the electrode sensitivity by 70 especially in the concentration range below

101

3mM and activity by 8 times although it also increased Km by 2 times [108]

26 ENZYME KINETICS AND ELECTROCHEMISTRY

261 Michaelis-Menten kinetics

A simple enzyme reaction can be expressed in terms of equilibrium between

the free enzyme (E) substrate (S) enzyme-substrate complex (ES) and product release

(P) The enzyme is regenerated on completion of the catalytic cycle

General enzyme reaction mechanism

middotmiddotmiddotmiddotmiddot Eq 41

E = Enzyme concentration mol

S = Substrate concentration mol

ES = Enzyme-substrate complex concentration mol

P = Product concentration mol

k1 = Rate constant for ES formation from E and S mol-1s-1

k-1 = Rate constant for ES dissociation into E + S s-1

k2 = Rate constant for P formation from ES s-1

The rate of ES formation is defined as

102

][][]])[[]([][2101 ESkESkSESEk

dtESd

minusminusminus= minus middotmiddotmiddotmiddotmiddot Eq 42

Where [E0] is the initial enzyme concentration

When the reaction is at steady state 0][=

dtESd

])[(][][]])[[]([ 212101 ESkkESkESkSESEk +=+=minus minusminus middotmiddotmiddotmiddotmiddot Eq 43

Re-arranging the above equation for [ES] as below

][]][[

][121

01

SkkkSEk

ES++

=minus

middotmiddotmiddotmiddotmiddot Eq 44

When the rate of product formation is defined as follows

middotmiddotmiddotmiddotmiddot Eq 45

][2 ESktP=

∆∆

=υ middotmiddotmiddotmiddotmiddot Eq 46

Where υ is the catalytic rate

When all the enzyme active sites are occupied such a condition is called saturated and

defined as [E0] = [ES] The maximum catalytic rate (V) is therefore available as below

Maximum catalytic rate V

][][ 202 ESkEkV == middotmiddotmiddotmiddotmiddot Eq 47

103

Express υ in terms of [S] by inserting Eq 44 into Eq 46

][

]][[

1

21

02

Sk

kkSEk

++

=minus

υ middotmiddotmiddotmiddotmiddot Eq 48

Michaelis-Menten constant (KM) is a dissociation constant about ES and defined as

Michaelis-Menten constant

1

21

kkk

K M+

= minus middotmiddotmiddotmiddotmiddot Eq 49

Michaelis-Menten equation (Eq 50) is obtained when υ is expressed in terms of V and

KM by inserting Eq 47 and Eq 49 into Eq 48

Michaelis-Menten equation

][][SK

SV

M +=υ middotmiddotmiddotmiddotmiddot Eq 50

This is a hyperbola equation to lead the enzyme catalytic rate Most enzymes typically

show this hyperbolic curve (Fig 31) when they are free from allosteric controls or

enzyme inactivation

104

0E+00

1E-09

2E-09

3E-09

4E-09

5E-09

0E+00 2E-03 4E-03 6E-03 8E-03 1E-02 1E-02 1E-02 2E-02

[S] M

Fig 31 A typical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

As shown in Fig 31 Michaelis-Menten plot has different kinetic profiles at high and

low [S] Mathematical expression of its transition over [S] is shown in Table 3

Table 3 Michaelis-Menten equation at various [S]

[S] ge KM Vcongυ middotmiddotmiddotmiddotmiddot Eq 51

[S] = KM 2V

=υ middotmiddotmiddotmiddotmiddot Eq 52

[S] le KM MM K

SEkK

SV ]][[][ 02=congυ middotmiddotmiddotmiddotmiddot Eq 53

At high saturated substrate concentration the catalytic rate becomes zero-order

therefore Michaelis-Menten curve becomes plateau When [S] = KM the rate becomes

105

half of the maximum catalytic rate At low substrate concentration the catalytic rate is

linearly dependent on [S] therefore the kinetics becomes first-order about [S]

262 Electrochemistry and Michaelis-Menten kinetics

In electrochemistry enzyme activity is studied by amperometry This involves

measuring measuring the current generated by the enzyme electrode reaction at a fixed

potential over an arbitrary period It therefore shows the transition in current response

over time For enzyme electrodes substrate concentration is changed over time so that

the current response for a different substrate concentration can be measured over a long

period With intact enzyme and appropriate heterogeneous electron transfer present

most enzyme electrodes follow Michaelis-Menten kinetics (Fig 32) in the amperometry

in the absence of allosteric control or enzyme inactivation Therefore the amperometry

data can be analysed in the similar way to that of biochemical data and for this

Michaelis-Menten equation is expressed in electrochemical terms [146] In

electrochemical enzyme kinetics enzyme catalytic rate (υ) is obtained in terms of

electric current therefore V is substituted with maximum steady state limiting current

(Im) or its current density (Jm)

106

Electrochemical Michaelis-Menten equation

][][

SKSI

IM

m

+times

= middotmiddotmiddotmiddotmiddot Eq 54

][][

SKSJ

JAI

M

m

+times

== middotmiddotmiddotmiddotmiddot Eq 55

I = Current A

Im = Max steady-state limiting current A

[S] = Substrate concentration mol

KM = Michaelis-Menten constant mol

A = Electrode surface area m-2

J = Current density Am-2

Jm = Max steady-state limiting current density Am-2

There is often an offset at zero substrate concentration due to competing reactions and

trace contaminants and this must be subtracted before analysis

107

V

0E+00

2E-04

4E-04

6E-04

8E-04

1E-03

0 2 4 6 8 10 12 14 16

frac12 Jm

KM

Jm Am-2

[S] mM

Jm

Fig 32 Electrochemical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

KM and Jm (or V in biochemical enzyme kinetics) are important parameters to analyse

both enzyme catalytic power and stability Electrochemical Michaelis-Menten equation

and curves can be used for rough estimation of KM and Jm however it often leads to

inaccurate result because measuring initial rate for high substrate concentration is very

difficult in this method hence very prone to errors Therefore Michaelis-Menten

equation is linearised using one of the following four methods (i) Lineweaver-Burk (ii)

Eadie-Hofstee (iii) Hanes-Woolf and (iv) Cornish-Bowden-Wharton equations

Michaelis-Menten plot is however good for intuitive diagnosis of general enzyme

behaviours

108

263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics

2631 Lineweaver-Burk linear regression

Lineweaver-Burk linear regression is one of the classic regression methods

taking double reciprocal of Michaelis-Menten equation (Eq 56)

Electrochemical Lineweaver-Burk linear regression

mm

M

JSJK

J1

][11

+times= middotmiddotmiddotmiddotmiddot Eq 56

It plots 1J against 1[S] as shown in Fig 33 The value of each parameter is found by

extrapolating the line The reciprocal of Jm is found at y-intercept and KM can be derived

from either x-intercept or the slope once Jm is found

109

-50E+02

00E+00

50E+02

10E+03

15E+03

20E+03

-06 -05 -04 -03 -02 -01 00 01 02 03 04

1[S] mM-1

1J A-1m

2

Fig 33 Electrochemical Lineweaver-Burk linear regression

Based on the real data Refer to sect 343 p 124

Lineweaver-Burk linear regression is however largely influenced by the errors in j at

low substrate concentrations [22 147] Its reciprocal could vary by a factor of 80 over

the range of substrate concentration varying one-third to three times the KM [148]

Hence it is not an ideal method for the analysis

2632 Eadie-Hofstee linear regression

Eadie-Hofstee linear regression plots J against J[S] This plot is strongly

affected by the errors in υ because υ is present in both coordinates (Eq 57)

Electrochemical Eadie-Hofstee linear regression

mM JSJKJ +timesminus=

][ middotmiddotmiddotmiddotmiddot Eq 57

110

Again the plot must be extrapolated to find Jm at y-intercept KM can be derived from

the slope (Fig 34)

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

0E+00 5E-05 1E-04 2E-04 2E-04 3E-04

J [S] AmM-1

J Am

-2

Jm

-Km

Fig 34 Electrochemical Eadie-Hofstee linear regression Based on the real data

Refer to sect 343 p 124

Eadie-Hofstee linear regression will give very strict evaluation [148] and might be used

to detect the deviations from Michaelis-Menten behaviour only if the data is precise and

accurate [147]

2633 Electrochemical Hanes-Woolf linear regression

Hanes-Woolf linear regression plots [S]J against [S] KM is derived from the

x-intercept and Jm is found from the slope

111

Electrochemical Hanes-Woolf linear regression

m

M

m JKS

JJS

+times= ][1][ middotmiddotmiddotmiddotmiddot Eq 58

This is a better method compared to the previous two regression methods because all

the parameter values can be derived from the reasonable range of [S] and they donrsquot

heavily rely on the extrapolation [147] The data is normalised by [S] therefore does not

cause many errors

-20E+08

00E+00

20E+08

40E+08

60E+08

80E+08

10E+09

12E+09

14E+09

-40E+00 00E+00 40E+00 80E+00 12E+01 16E+01

[S] mM[S] J m

MA

-1m2

Fig 35 Electrochemical Hanes-Woolf linear regression Based on the real data

Refer to sect 343 p 124

However either of these three linear regressions might not be adequate for the enzyme

electrode analysis [65] because the data reflect not only the single-substrate

Michaelis-Menten kinetics but also include the influence from other factors such as

enzyme immobilisation mediator reactions and electrode composition as well as

112

general experimental errors Their contributions to the overall kinetics therefore must be

considered [65 149] although the enzyme electrode reactions can approximate the

Michaelis-Menten model when one substrate is a variable and other variables are fixed

over an experiment [147 149] Under such a complex system it is not ideal to use those

graphical linear regression methods because they assume that the results follow

Michaelis-Menten model and that all the experimental errors follow normal distribution

[150] without giving any measure in the precision [148] Hence it is more suitable to

use a non-parametric direct linear plot or nonlinear regression methods because they

respect the influence and errors from various sources without any bias

264 Cornish-Bowden-Wharton method

Cornish-Bowden-Wharton method is distribution-free non-parametric direct

linear plot In this method observations are plotted as lines in parameter space rather

than points in observation space [151] It then takes the median of Jm and KM from every

possible non-duplicate pair of observed [S] and υ (Eq 59 and Eq 60) as the best-fit

estimate

Electrochemical Cornish- Bowden-Wharton non-parametric method

])[][(])[]([

1221

122121 SJSJ

SSJJJm minusminus

= middotmiddotmiddotmiddotmiddot Eq 59

])[][()](][[

1221

122121 SJSJ

JJSSKM minusminus

= middotmiddotmiddotmiddotmiddot Eq 60

It is a simple method without complex calculation insensitive to outliers and no

113

weighting is required [147 152]

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

14E-03

16E-03

18E-03

20E-03

-5 -3 -1 1 3 5

KM mM

Jm Am-2Median Jm

Median KM

Fig 36 Electrochemical Cornish-Bowden-Wharton plot Based on the real data

Refer to sect 343 p 124

265 Nonlinear regression methods

Nonlinear regression methods manipulate the parameters to minimise the sum

of squared difference between the measured and expected values [150] They fit the

integrated equation to the enzyme progress curve and calculate the approximate location

of the minimum for the sum of squared difference at a particular point in the slope It

then recalculates the new minimum location to improve the prediction until there is no

further improvement This is called gradient method based on Gauss-Newton algorithm

or non-linear least square method [150 153] This is computationally intensive due to

114

the recursive operations however advanced computational technology has made it

possible The parameters obtained may not be perfectly correct [150] The method still

assumes errors normally distributed [153] and Cornish-Bowden has criticized about the

ambiguous statistical assumption of such computer programmes [154] However

non-linear regression methods are insensitive to outliers [150 153] have better estimate

accuracy compared to conventional linear regression methods and the estimations

mostly fit Michaelis-Menten model Graphical methods arose at a time when nonlinear

regression methods were computationally not available however it is now possible

thanks for the great technological advance and more recommended to use in analysis to

yield better precision and reliability [153]

115

3 ANODE MATERIAL AND METHOD

31 ELECTRODE FABRICATION

311 TTF-TCNQ synthesis

Tetrathiafulvalene ge980 (TTF) and 7788-Tetracyanoquinodimethane

ge980 (TCNQ) were purchased from Sigma Aldrich Spectroscopic grade acetonitrile

and diethyl ether were purchased from VWR International TTF and TCNQ were

weighted to 1 g each and dissolved separately in 50 ml of acetonitrile TTF-acetonitrile

solution had yellow colour while TCNQ-acetonitrile solution was green Both were

heated on the hot plate under the fume hood On the full dissolution two solutions were

quickly mixed to form black solid crystals The mixture was immediately taken off the

hot plate and cooled down at the room temperature for a few hours The crystals were

collected on Buchner funnel and washed under vacuum twice with acetonitrile twice

with diethyl ether and then twice again with acetonitrile The washed crystals were left

to dry for overnight with a cover The dried crystals were collected and kept in the

sealed container for future use [61]

312 Electrode template fabrication

Sylgard 184 Silicone Elastomer Kit were purchased from Dow Corning

Corporation for polydimethylsiloxane (PDMS) Base and hardener were mixed at 101

(vv) for use CY1301 and aradur hardener HY 1300 were purchased from Robnor

116

resins to make epoxy resin They were mixed at 31 (vv) for use

Silicone electrode templates (Fig 16(i)) were made to shape electrode discs

Each template had round holes in line Two types of templates template A and B were

made Template A had holes with the dimension 2-mm diameter x 1-mm thickness

while template B had holes with the dimension with 5-mm diameter x 2-mm thickness

In order to make PDMS templates for electrode discs different templates were made to

shape the holes in PDMS (Fig 16(ii)) Discs were made with epoxy resin and were

fixed onto the plastic Petri dish PDMS was poured into disk-mounted Petri dish

degassed for 20 minutes and then cured at 60 degC Cured PDMS electrode templates

were taken out from the epoxy resin-Petri dish templates and then cleaned with

analytical grade ethanol for use

Fig 37 PDMS electrode templates (i) PDMS template and (ii) their epoxy resin-Petri

dish template

117

313 Electrode composite fabrication

3131 Composite for conductance study

Graphite at microcrystal grade (2-15 micron 999995 purity) was purchased

from Alfa Aesar GmbH amp CoKG Glassy carbon (GC) powder was kindly donated from

Dr Zhao Carbon nanofibre (CNF) was also kindly donated from Loughborough

University Composite electrodes were built from the mixture of the carbon conductive

or TTF-TCNQ and epoxy resin Each composite mixture had different composition ratio

[108] as below in Table 4

Composite Ratio I Ratio II Ratio III Ratio IV Ratio V

Graphite Epoxy resin 73 64 55

GC Epoxy resin 64 73

CNF Epoxy resin 37 46 55 64 73

TTF-TCNQ Epoxy resin 64 73 82

Table 4 Composite composition

TTF-TCNQ was mixed with epoxy resin at 64 73 and 82 Graphite was mixed at 73

64 and 55 GC powder was mixed at 64 and 73 CNT was mixed at 37 46 55 64

and 73 (ww) Three to five copies were fabricated for each electrode type Composite

mixture was packed into the template A (Fig 38) and cured at room temperature for 72

hours [106]

118

Fig 38 PDMS template (i) packed with electrode material and (ii) empty one

3132 Bulk modified GOx enzyme electrode composite

Glucose oxidase (GOx) [Type II-S 15000-50000 unitsg] from Aspergillus

niger was purchased from Sigma Aldrich Conductive composite paste was first made

with TTF-TCNQ and epoxy resin at 73 as in sect 3131 GOx was then incorporated into

the conductive composite mixture at varying mixing ratio 5 10 and 15 (ww) by

bulk-modification method (Fig 39) 10 GOx composite electrodes were also made

with graphite Enzyme composite mixture was packed into the template B and cured at

4 degC for 96 hours

119

R

RR R

R

RR R

RRR

Conducting particles

Insulating binder

Chemical modification

Add biological receptor R

Cast extrude or print into suitable form

R

R

RR

R

R

R

R

R

R

RR

Fig 39 Schema for bulk modification method

3133 GOx enzyme electrode composite with enzyme stabilisers

Poly(ethyleneimine) (PEI) solution [50 (wv) in H2O] DL-dithiothreitol

(DTT) Poly(ethylene glycol) (PEG) [average mol wt 200] glycerol (GLC) [ge99] and

flavin adenine dinucleotide disodium (FAD) salt hydrate (ge95) were purchased from

Sigma Aldrich Each stabiliser was mixed with GOx at different fraction 0 1 2 and

4 PEI was also blended with DTT or FAD for synergetic effects as below in Table 5

Blend type Ratio I Ratio II Ratio III Ratio VI

PEIDTT 0515 1010 1505 2020

PEIFAD 21 22 24

Table 5 Blended stabiliser composition

120

PEI was mixed with DTT at the ratio of 0515 11 1505 and 22 PEI was also

mixed with FAD at 21 22 and 24 Each enzyme-stabiliser mixture was weighted to

10 of the whole composite It was then mixed with the conductive composite mixture

made earlier The conductive composite mixture was made of TTF-TCNQ and epoxy

resin at 73 The final composite mixture was packed into the template B and cured at

4 degC for 96 hours

314 Electrode wiring and insulation

Silver wire (temper annealed 025-mm diameter) was purchased from Advent

Portex polyethylene tubing (028 x 0165 mm) purchased from Scientific Laboratory

Supplies Silver epoxy was purchased from ITW chemotronics Silver epoxy came in

two parts A and B and they were mixed at 11 (vv) for use Silver wire was cut for

5-cm length and its tip was cleaned with analytical grade ethanol dried and coved with

insulating polyethylene tubes Cured electrode composite discs were popped out of the

PDMS template An insulated silver wire was fixed onto to each electrode disc with

conductive silver epoxy and it was left at room temperature for 24 hours to cure silver

epoxy After silver epoxy had been cured the wired electrodes were coated and

insulated with degassed epoxy resin and cured at room temperature for 3 days for

non-enzyme composite electrodes and 4degC for 4 days for enzyme composite electrodes

7-9 days were taken to build the complete composite electrodes (Fig 40) The enzyme

electrodes were stored at 4degC when not in use

121

Fig 40 Insulated composite electrodes (i) non-enzyme (ii) GOx electrodes

32 CONDUCTIVITY EXPERIMENT

Copper foil with electrically conductive acrylic adhesive was purchased from

RS Components Digital multimeter from Metra was used to measure resistance

Electrode surface was covered with adhesive copper foil evenly and both ends were

clamped with crocodile clips (Fig 41(i)) to measure electrode resistance of the

composite as in Fig 41(ii)

122

Fig 41 Measuring electrode resistance (i) connecting the electrodes to the

multimeter (ii) measuring the electrode resistance with the multimeter

33 POLISHING COMPOSITE ELECTRODES

MicroPolish II aluminium oxide abrasive powder (particle range 1 03 and

005 microm) silicone carbide (SiC) grinding paper for metallography wet or dry (grit 600

1200 and 2400) and MicroCloth Oslash73 mm for MiniMet for fine polishing were

purchased from Buehler Self adhesive cloth for intermediate polishing was purchased

from Kemet Electrodes were polished immediately before the experiment until

mirror-like flat surface was obtained This erases the electrode history and gives

assigned electrode surface area [105] Composite electrodes were hand polished

successively with SiC grinding paper in 400 1200 and 2400 grit During polishing

deionised water of 18 MΩ was added occasionally to the polishing surface This

prevents damaging electrode surface with frictional heat It was followed by successive

polishing with alumina oxide slurry with particle size 1 03 and 005 microm Slurry was

123

sonicated to dissolve agglomerates before use [155] Kemet polishing pad was used for

rougher polishing with 1 and 03 microm powder Felt-like MicroCloth was used for

polishing with 005microm Electrodes were sonicated in the deionised water briefly after

polishing at each grit or grade

34 GLUCOSE AMPEROMETRY

341 Glucose solution

Phosphate buffer saline (PBS) powder for 001 M at pH 74 containing 0138

M NaCl and 00027 M KCl [156] was purchased from Sigma Aldrich PBS was kept in

the fridge when not in use [157] 1M glucose solution was made by mixing

D-(+)-Glucose with 001 M PBS and left for 12 hours at 4degC for mutarotation It is

mandatory because GOx has stereochemical specificity and only active toward

β-D-glucose while glucose solution consists of two enantiomers α- and β-D glucose

and their concentration changes over time until they reach the equilibrium at 3862

Experiments should be carried out in the equilibrated solution for accuracy

342 Electrochemical system

A single-channel CHI 650A and multi-channel CHI 1030 potentiostats were

used for glucose amperometry with three-electrode system in which the

electrochemical cell contained (a) working electrode(s) an AgCl reference electrode

and a homemade Pt counter electrode The AgCl reference electrode was purchased

124

from IJ Cambria AgCl reference electrode was saturated with 3M KCl Pt counter

electrode was made of Pt wire and Pt mesh was fixed at its tip to give large surface area

Pt mesh at its tip

343 Glucose amperometry with GOx electrodes

Glucose amperometry was conducted by adding a known volume of 1M

glucose aliquot into 100 ml of 001 M PBS electrolyte solution containing GOx anodes

with convection applied Convection was however ceased on during current

measurement to avoid the noise The electrolytic solution was gassed with either N2 O2

or compressed air for 15 minutes before the experiment O2 concentration is 123 mM in

fully oxygenated PBS [158] and 026 mM in air [112] Gas was continuously supplied

through Dreschelrsquos gas washing bottle containing PBS during experiment The response

of GOx anodes to glucose was obtained as steady state current The amperometry was

carried on until no further current increase was observed against glucose added Each

experiment took more than 8 - 12 hours The sample size ranged from two to three in

most cases

125

Fig 42 Amperometry setting with CHI 1030 multichannel potentiostat

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA

Normality of enzyme function was confirmed by plotting Michaelis-Menten

curve based on the amperometry data obtained The capacitive current before glucose

addition was subtracted from the data Enzyme kinetic parameters the maximum

current density (Jm) and the enzyme dissociation Michaelis constant (KM) were

automatically estimated with standard error using an analytical software called

SigmaPlot Enzyme Kinetics version 13 The estimation is based on non-linear

regression analysis about Michaelis-Menten kinetics as shown in Fig 43

126

Glucose M

0 1e-2 2e-2 3e-2 4e-2 5e-2

J A

m-2

00

20e-3

40e-3

60e-3

80e-3

Vmax = 0008445 Km = 0005842

Fig 43 Analytical output by SigmaPlot Enzyme Kinetics Based on the real data

Influence of enzyme immobilisation electrode composition O2 and stabilisers on GOx

electrode performance was studied The most efficient GOx anodes was determined on

the basis of the parameters obtained

127

4 ANODE RESULTS AND DISCUSSION

41 AIMS OF ANODE STUDIES

This section describes the fundamental study on the enzyme composite

electrodes and the influence of enzyme stabilisers on the performance and stability of

GOx electrodes Firstly the difference in conductivity as a function of various

conductive materials and the component fraction were studied They did not contain

enzyme The most suitable composite was then selected and used to build the enzyme

electrodes Secondly GOx electrodes were built at various GOx fractions in the

composite The best component fraction was determined from the data obtained in

amperometry Amperometry was conducted to obtain two enzyme kinetic parameters

the maximum current density (Jm) and the enzyme dissociation Michaelis constant (KM)

Jm indicates the size of catalytic velocity KM indicates the extent of enzyme-substrate

dissociation Smaller KM values indicate better enzyme-substrate affinity and enzyme

stability With the component ratio selected enzyme electrodes were then constructed

using various types and concentrations of enzyme stabiliser Amperometry was again

conducted to study the influence of enzyme stabilisers on the GOx anode performance

and stability The catalytic efficiency was determined by the fraction M

mK

J This is

based on the concept of specificity constant M

catK

k as defined below

128

Enzyme electrode specificity constant

MMM

cat

KEV

Kk

Kk

sdot==

][2 middotmiddotmiddotmiddotmiddot Eq 61

where kd = k2 Rate constant for P formation from ES

The specificity constant is a common measure of the relative catalytic rate at low

substrate concentration [22] M

mK

J therefore can be used as a comprehensive

indicator for both catalytic velocity and enzyme stability under the fixed enzyme

concentration In this study GOx concentration was fixed to approximately 10 weight

of the total composite According to the M

mK

J obtained the five most efficient and

stable GOx electrodes were determined The possible enzyme stabilisation mechanisms

of each enzyme stabiliser were also discussed together in the light of published

hypothesises

42 CONDUCTANCE OF THE COMPOSITE

421 Conductance of various conductive materials

Various electrodes were built with different conductive materials graphite GC

CNF and TTF-TCNQ at different component fraction The electrode dimension was

fixed to 314 mm2 x 1 mm Their conductance was measured and presented in a box plot

as shown in Fig 44 The sample size ranged from 3 to 6 The median is indicated by a

short line within the box

129

00E+00

50E-02

10E-01

15E-01

20E-01

25E-01

30E-01

35E-01

40E-01

45E-01C

ondu

ctan

ce

S

Lower quartile S 103E-03 549E-03 219E-01 111E-01 142E-01 599E-04 390E-03 115E-02 972E-03

Max S 281E-03 338E-02 268E-01 312E-01 415E-01 810E-04 113E-02 467E-02 262E-02

Min S 739E-04 467E-03 200E-01 933E-02 855E-02 418E-04 296E-03 264E-03 587E-03

Upper quartile S 206E-03 282E-02 253E-01 220E-01 253E-01 795E-04 807E-03 232E-02 199E-02

Median S 131E-03 847E-03 238E-01 129E-01 216E-01 780E-04 484E-03 155E-02 136E-02

TTF-TCNQ64

TTF-TCNQ73

Graphite 55 Graphite 64 Graphite 73 CNF 37 CNF 46 CNF 55 GC 73

Fig 44 Conductance of various electrode composite n = 3 ndash 6

Graphite composites showed higher conductance than other composites It is explained

by graphite having the highest intrinsic conductivity ranging between 103 ndash 104 Scm-1

[105 159] although being compounded in a composite lowered the conductivity by 4 ndash

5 order of magnitude compared to that of raw material The conduction did not show a

linear relationship with the electrode composition ratio for graphite composite because

the graphite composite 55 had larger conductance of 024 S than that of 64 and 73 It

also showed the smallest deviation than the other graphite composites while the

deviation increased with increase in the graphite content One possible cause of this is

the presence of patchy conductive paths due to the uneven distribution of conductive

islands within the composite [105 106] The conduction of the composite is therefore

often locally biased unpredictable and also leads to low reproducibility TTF-TCNQ

composite at 73 showed the conductance of 85 mS smaller than the graphite

composited 55 approximately by 30 times Conductivity of TTF-TCNQ is 102 ndash 103

130

Scm-1 [109 110] and smaller than that of graphite by one order of magnitude so this is

consistent with the intrinsic conductivity of the conducting paticles

There were also different interactions observed among conductive materials

with insulating materials Mixing epoxy resin with graphite was easier than mixing with

other conductive materials Some even did not cure at the same mixing ratios The

carbon nanofibre (CNF) composite mixture did not cure when it exceeded more than

50 weight content With 40 CNF content however it showed the conductance of 48

mS It was not comparable to graphite composite despite its similar conductivity of 103

S [105] however it was similar to that of TTF-TCNQ 73 composite 50 CNF content

even showed larger conductance of 155 mS than 70 TTF-TCNQ CNFs have been

reported to have mediating function [117] therefore with this high conduction property

at low content CNF might be very useful to enzyme composite and even superior to

TTF-TCNQ

422 Influence of enzyme concentration on conductivity

Conductance of the GOx composite was measured The conductive composite

consisted of either TTF-TCNQ 73 or graphite composite 73 GOx content was varied

by 5 10 and 15 for TTF-TCNQ composite and 10 only for graphite composite

The electrode dimension was fixed to 196 mm2 x 2 mm The sample numbers ranged

from 2 ndash 4 The study revealed that the presence of enzyme interfered with conduction

in GOx concentration dependent manner for TTF-TCNQ composite as shown in Fig 45

131

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01C

ondu

ctan

ce

S

Low er quartile S 131E-02 924E-03 124E-05 165E-01

Max S 484E-02 143E-02 368E-05 593E-01

Min S 120E-02 754E-03 390E-06 122E-01

Upper quartile S 441E-02 126E-02 288E-05 400E-01

Median S 281E-02 109E-02 208E-05 207E-01

TTF-TCNQGOx 5 TTF-TCNQGOx 10 TTF-TCNQGOx 15 GraphiteGOx 10

Fig 45 Conductance of GOx composite with various GOx content n= 2 - 4

This was due to insulating property of the protein although higher enzyme loading is

preferred for better catalytic performance as discussed in sect 226 on p 88 The issue

may be circumvented by fixing the enzyme onto the electrode surface It is however

more technically tedious and not as feasible for mass manufacture as bulk modification

It is also difficult to modify conductive salt for covalent attachment of the enzyme This

is because the conductive property of conducting salt is very sensitive to its chemical

structure and slight modification can lead to insulating properties [61] A similar

phenomenon has been also observed in CNT functionalisation [117]

Graphite-GOx composite showed larger conductance than TTF-TCNQ-GOx

composite by 20 times at the same component fraction This was due to the better

conductive property of graphite However graphite does not have mediating property

and the use of graphite as conductive material for enzyme electrodes requires the

132

loading of mediator separately The key to designing enzyme electrodes is in the balance

and compromise among the conductivity mediation enzyme activity mechanical and

chemical stability and difficulty in fabrication process A good candidate material to

make up for the TTF-TCNQ lower conductivity is CNT It has been reported that CNF

has both good conductivity and mediating functions [117] and might be suitable for bulk

modification More safety study on use of CNF is however necessary [117 160]

43 CATALYTIC ACTIVITY OF GOx COMPOSITE ELECTRODES

431 Influence of GOx concentration on the catalytic activity of GOx electrodes

Amperometry was conducted for GOx composite electrodes in N2 O2 and air

for various GOx content The conductive composite consisted of either TTF-TCNQ 73

or graphite composite 73 GOx content was varied by 5 10 and 15 for

TTF-TCNQ composite and 10 only for graphite composite The sample size was 3 for

each electrode type Control experiments were also performed with enzyme-free

composite electrodes and there was no glucose response observed There was also no

stable current observed for 10 GOx-graphite electrodes despite of its high

conductivity reported in sect 422 while Michaelis-Menten curve was observed in any

TTF-TCNQ-GOx composite electrodes This confirmed the necessity of mediating

function for enzyme anodes and its presence in TTF-TCNQ

All the TTF-TCNQ-GOx electrodes showed the Michaelis-Menten current

response curves however differed in the current as shown in Fig 46

133

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

35E-02

00E+00 10E-02 20E-02 30E-02 40E-02 50E-02

Glucose M

J A

m-2

GOx 5GOx 10GOx 15

Fig 46 Difference in the glucose-response amperometric curve of enzyme

electrodes for various GOx content 5 10 and 15 in the presence N2

The 15 GOx composite showed the smallest glucose response current due to the

interfered conduction by high protein content while 5 GOx composite showed smaller

response than 10 composite due to the enzyme deficiency Their Jm only counted for

26 and 41 of that of 10 GOx composite (Fig 47(i)) 10 GOx electrode was

therefore most catalytic KM for 5 and 10 GOx were 71 and 73 mM and little

different (Fig 47(ii)) This implies that the size of catalytic activity can be flexibly

altered by only slight change in KM

134

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

Jm

Am

-2

Jm Am-2 729E-03 180E-02 466E-03

GOx 5 GOx 10 GOx 15

00E+00

20E-03

40E-03

60E-03

80E-03

10E-02

12E-02

14E-02

Km

M

KM M 710E-03 729E-03 187E-03

GOx 5 GOx 10 GOx 15

Fig 47 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 n = 1 - 3

The results also tell that an increase in GOx loading could enhance the catalysis hence

output current unless the increase is not excessive and gt 15 at least The loading limit

should lie somewhere between 10 and 15 for this composite Similar electrodes

were also built by Alegret with 75 epoxy resin 10 TTT-TCNQ 10 graphite and

5 GOx [115] Their Jm was around 7 mAm-2 and little different from that of 5 GOx

with 70 TTF-TCNQ composite It suggests that less TTF-TCNQ content might be

sufficient as long as the conductivity is sustained by more conductive material such as

graphite

432 Influence of O2 on the catalytic activity of GOx electrodes

O2 and air suppressed the current output by GOx composite electrodes and

delayed the response to the glucose as shown in Fig 48 A current increase was

observed immediately after glucose addition for N2 but not for O2 and air They showed

135

sigmoidal current curves with a glucose response lag

(i)

(ii)

-50E-03

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

00E+00 50E-03 10E-02 15E-02 20E-02 25E-02 30E-02 35E-02 40E-02 45E-02

Glucose M

J A

m-2

GOx 10 N2GOx 10 O2GOx 10 Air

Fig 48 Difference in the glucose-response amperometric curve of 10 GOx

composite electrodes in the presence (i) N2 and O2 and (ii) re-plotted

amperometric curves for N2 air and O2

136

As expected O2 and air decreased Jm and increased KM as shown in Fig 49

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

GOx 5 GOx 10 GOx 15

Jm

Am

-2

N2O2Air

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

70E-02

80E-02

GOx 5 GOx 10 GOx 15

Km

M

N2O2Air

Fig 49 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 O2 and air n = 1 - 3

Both O2 and air decreased the current of 15 GOx composite electrodes Jm dropped to

approximately 20 of that for N2 KM was increased by 10 and 2 times for O2 and air

respectively The glucose response lag was 8 mM for O2 but none for air Air showed

milder suppression toward current output than O2 and this was expected because O2

counts for only 20 of the air [31] and should cause less influence on GOx activity

Similar phenomena were observed for 10 GOx composite electrodes For O2 and air

Jm was 13 and 40 of that of N2 and decreased to 23 x 10-3 and 67 x 10-3 from 18 x

10-2 Am-2 respectively KM was 73 for N2 and 10 mM for both O2 and air although there

was a response lag of 8 and 1 mM respectively Little difference in KM was observed

among all cases and this implies that KM itself hence enzyme stability or substrate

affinity is little affected by O2 but slight change in KM has large influence on the

catalytic activity This implies that change in catalytic activity induced by O2 is

137

reversible 5 GOx composite electrodes showed slightly unexpected result KM was

increased by approximately 4 times in both O2 and air Jm also decreased however Jm

for air was smaller than O2 by 36 despite of less O2 content There was a response lag

by 10 mM for O2 and 2 mM for air

The overall conclusions from these results is that O2 reduces GOx catalytic rate

andor electron transfer rate and hence reduces the current output but the effect is

reversible because it hardly alters KM value implying that O2 does not affect the enzyme

stability or its affinity to the substrate This phenomenon is typical in non-competitive

inhibition for enzyme kinetics Non-competitive inhibition occurs when an inhibitor

binds to the enzyme-substrate complex and slows its catalysis It therefore changes

catalytic velocity but not KM In the glucose amperometry this implies two possibilities

as a cause of Jm reduction (i) allosteric binding of O2 to GOx-glucose complex or (ii)

interference in electron transfer after the catalysis In the former case O2 does not

inhibit glucose binding to GOx but allosterically interacts with its complex and hinders

its catalysis hence it reduces catalytic current output In the latter case either glucose

binding or GOx catalysis is not affected by O2 but competition for electrons produced

by GOx catalysis occurs between the natural and artificial mediators namely O2 and

TTF-TCNQ and this reduces the electron flow to the electrode therefore it is reflected

as the reduction of catalytic current In addition to this the presence of glucose response

lag also suggests the presence of electron competition [161-163] It was reported that O2

reacts with reduced GOx faster than any other mediators with the second order rate

reaction of 15 x l06 M-1s-l [112 161 163] It is therefore quite possible that O2 swiftly

deprives electrons from reduced GOx andor reduced mediators Reduced GOx reacts

with O2 much faster than with TTF-TCNQ because it was observed that O2 had

138

diminished glucose response current at low glucose concentration Most GOx hence

reacts with O2 first then oxidised TTF-TCNQ once most O2 has been converted into

H2O2 This is compatible with the hypothesis suggested by Hindle [161] Even after the

lag the reaction could go on with residual O2 because KM did not recover fully to the N2

level although a familiar Michaelis-Menten hyperbolic curve was regained This simply

indicates that O2 concentration became smaller than TTF-TCNQ to oxidise GOx The

glucose response lag also decreased with an increase in GOx loading and was smaller

for air than O2 This implies the presence of competition because the lag depends on the

concentration of reduced GOx and electron thieves The large KM increase in the 5

GOx composite electrodes was probably due to the deficiency in the electron transfer

rate to suppress O2 activity How O2 interacts with its environment is unknown however

O2 reduces the catalytic output current due to the following possible mechanisms (i)

electron competition between O2 and TTF+ (ii) non-competitive enzyme inhibition by

allosteric binding of O2 to GOx (iii) direct interaction between O2 and reduced TTF or

(iv) backward reaction between reduced TTF and oxidised GOx

139

Table 6 Four possible causes of glucose amperometric current reduction

(i) middotmiddotmiddotmiddotmiddot Eq 62

middotmiddotmiddotmiddotmiddot Eq 63

(ii) middotmiddotmiddotmiddotmiddot Eq 64

(iii) middotmiddotmiddotmiddotmiddot Eq 65

(iv) middotmiddotmiddotmiddotmiddot Eq 66

Diagnosis can be made by doing experiments with different concentration of O2 and

mediators

44 THE EFFICACY OF GOX STABILISERS

441 Introduction

Various GOx stabilisers PEI DTT PEG GLC and FAD were mixed with GOx

composite at the concentration varying among 1 2 and 4 PEI was also mixed with

DTT or FAD and their synergetic effects were studied All the GOx electrodes were

built on the basis of TTF-TCNQ 73 conductive composite with 10 weight

GOx-stabiliser mixture Control composite electrodes were built with 2 stabilisers but

without GOx Glucose amperometry was conducted as before in the both presence and

absence of O2 Two kinetic parameters Jm and KM were obtained through non-linear

regression analysis The standard kinetic parameter values were set to those obtained in

140

the absence of O2 for GOx electrodes containing no stabiliser They are Jm = 18 x 10-2 plusmn

73 x 10-3 Am-2 and KM = 73 plusmn 57 mM respectively The glucose response lag

observed for this electrode was 10 mM Comprehensive catalytic efficiency was

determined by the size of the fraction M

mK

J under the influence of high O2 and the

most catalytic GOx composite electrodes were chosen on the basis of M

mK

J The

sample size ranged between 2 and 3 None of control experiments showed any glucose

response

442 PEI

Kinetic parameters for various PEI concentrations are shown below in Fig 50

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser PEI 1 PEI 2 PEI 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEI 1 PEI 2 PEI 4

KM

M

N2 O2

Fig 50 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEI in the presence and absence of O2 n = 1 - 3

141

In the absence of O2 PEI lowered Jm without largely affecting KM compared to the

standard value This might imply the presence of allosteric non-competitive inhibition

of glucose catalysis It is certainly possible because PEI can lock in the enzyme

conformation with hydrogen and electrostatic bonding [120] or form a complex with

GOx [126 130] This interferes in its catalysis although it also might confer the

resistance against oxidative stress as reported [126] In the presence of O2 two out of

three electrodes of PEI 1 and 4 did not show Michaelis-Menten curves The data

presented here is therefore based on a single sample This may mean that the electrodes

at those PEI concentrations are in fact prone to the catalytic suppression by O2

Moreover 4 PEI data presented here looks unreliable due to the simultaneous increase

in Jm and KM by 3 ndash 4 times compared to those obtained in the presence of N2 It may

need more data to confirm the phenomenon and be premature to conclude it is however

probably more reasonable to think that PEI 4 is not suitable for GOx anodes At other

PEI concentrations electrodes showed more or less similar kinetic parameter values to

those without stabilisers since Jm decreased and KM increased in the presence of O2

There was no O2-scavenging function observed because their glucose response lag and

KM resembled those without stabilisers Among them 2 PEI containing electrodes

sustained 50 of catalytic activity obtained in N2 and showed marginally better Jm than

stabiliser-free electrodes in the presence of O2 It might therefore have some resistance

against O2 It can be however concluded that PEI is not suitable as an enzyme stabiliser

and not effective enough to enhance the catalytic current output because their Jm was

already smaller by 60 at minimum in N2 compared to the standard value and little

improvement in catalytic performance was observed in the presence of O2

142

443 DTT

Kinetic parameters for various DTT concentrations are shown below in Fig 51

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

Jm

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

KM

M

N2 O2

Fig 51 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 DTT in the presence and absence of O2 n = 3

All DTT containing electrodes showed smaller Jm and KM than the standard values in

the absence of O2 This might imply the possible presence of uncompetitive inhibition

by DTT although the size of inhibition was not in concentration dependent manner In

uncompetitive enzyme inhibition inhibitors bind both free enzyme and

enzyme-substrate complexes hence decreases both catalytic velocity and KM [22] In the

presence of O2 all Jm KM and glucose response lag were larger than those of

stabiliser-free electrodes The increase in glucose response lag was probably due to both

electron competition and uncompetitive inhibition while the inhibition by DTT itself

was less influential on catalytic velocity than non-competitive inhibition by O2 itself

143

and this resulted in larger Jm despite of their larger KM The truth of O2 enzyme

inhibition might be reversible Met oxidation as discussed in sect 242 on p 93 because

atmospheric O2 could oxidise Met residue [123] and DTT might be capable of reducing

it back although DTT has been reported to be effective only in reducing disulphide [127

132] The increase in Jm in the presence of O2 compared to N2 was probably due to the

reduced content of DTT working toward stabilising enzyme structure This freed up

some GOx portion from rigid conformation

444 PEG

Kinetic parameters for various PEG concentrations are shown below in Fig 52

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEG 1 PEG 2 PEG 4

J m

Am

-2

N2 O2

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

No Stabiliser PEG 1 PEG 2 PEG 4

KM

M

N2 O2

Fig 52 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEG in the presence and absence of O2 n = 2 - 3

PEG decreased Jm to 10 of the standard value at maximum in the absence of O2

144

although 4 PEG electrodes showed much larger Jm than the other PEG electrodes

unexpectedly 4 PEG however showed glucose response lag gt 10 mM even in the

absence of O2 The outcome of low Jm is compatible with the result reported by Lee

[139] while among PEI electrodes Jm however increased with PEG content and this

agreed with the results presented by Arakawa [138] The hypothesis by both authors

therefore might be correct but stabilisation mechanism might differ at different

concentrations Since KM was lowered at low PEG content compared to the standard

value PEG might have penetrated the protein core and stabilised the enzyme structure

[164] however it also lowered GOx activity because the protein conformation has been

locked in At 4 PEG concentration PEG still penetrated the protein core but residual

PEG also brought the preferential hydration and this allowed more flexible enzyme

conformation and increased the catalytic activity The residual PEG however might also

have an adverse effect on electron transfer rates at low glucose concentration In the

presence of O2 KM was increased compared to when in N2 and larger than that of

stabiliser-free electrodes while Jm only marginally better compared to that except for 4

PEG It is altogether not clear about the interaction between GOx and PEG however

only high PEG content effectively enhances the catalytic current output

445 GLC

Kinetic parameters for various GLC concentrations are shown below in Fig 53

145

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser GLC 1 GLC 2 GLC 4

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

20E-02

No Stabiliser GLC 1 GLC 2 GLC 4

KM

M

N2 O2

Fig 53 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 GLC in the presence and absence of O2 n = 3

In the absence of N2 GLC showed smaller Jm and KM compared to the standard values

This indicates the presence of uncompetitive inhibition and the decrease in Jm showed

the reciprocal dependency on GLC concentration It is also quite possible that

concentration-dependent inhibition was due to the tighter protein packing by GLC as

previously reported [144] This might contribute more to non-competitive inhibition

though GLC-containing electrodes showed more resistance against O2 because their Jm

was larger than that of stabiliser-free electrodes and again it showed reciprocal

dependence on GLC concentration In both N2 and O2 cases Jm decreased with increase

in GLC content KM also showed the dependence on GLC concentration in the presence

of O2 and increased with GLC concentration Glucose response lag was also decreased

in all cases Among all 1 GLC was most effective because it lowered KM for both N2

and O2 and increased Jm for O2 although it decreased Jm by 40 in N2 It also decreased

glucose response lag from 8 to 5 mM and this might indicate that glycerol reduced and

scavenged O2

146

446 FAD

Kinetic parameters for various FAD concentrations are shown below in Fig 54

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

No Stabiliser FAD 1 FAD 2 FAD 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser FAD 1 FAD 2 FAD 4

KM

M

N2 O2

Fig 54 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 FAD in the presence and absence of O2 n = 1 - 3

FAD may be reasonably effective as an enzyme stabiliser at concentration gt 1 because

its Jm was larger in both absence and presence of O2 compared to that of stabiliser-free

electrodes It also slightly decreased glucose response lag therefore it might be able to

react with oxidising species as previously reported [108] It is however only effective at

higher concentration because two out of three 1 FAD electrodes did not show

Michaelis-Menten curves in the absence of O2 and none worked in its presence although

4 FAD was not most effective either The relationship between increase and decrease

in Jm and KM in N2 is unknown Improvement in electron transfer was not particularly

observed because higher FAD content decreased the current output although FAD has a

147

function as a mediator Mechanism of GOx stabilisation by FAD is not clear as Bartlett

reported [145] however 2 FAD was shown to be most effective among all and the only

one which showed M

mK

J gt 1 for both N2 and O2

447 PEI and DTT mixture

Kinetic parameters for various PEI-DTT concentrations are shown below in

Fig 55

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

KM

M

N2 O2

Fig 55 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

PEI-DTT (051 11 1505 and 22) in the presence and absence of O2 n =

1 - 2

PEI-DTT stabiliser mix showed the most dramatic effect and promising as a stabiliser

formulation In the presence of O2 it had larger Jm and smaller or equivalent KM than

those found in stabiliser-free electrodes Glucose response lag was also decreased from

148

8 to 3 mM and this implies the presence of O2 scavengers which were new synergetic

effects achieved and not observed in use of either stabilisers as discussed in sect 442 and

443 In the absence of O2 most PEI-DTT showed smaller Jm and marginally smaller

KM than those of the standard values except for some as it can be seen Fig 55 This

probably implies the presence of non-competitive-inhibition like effect due to the

stabilisation of enzyme structure by PEI-DTT Since it was difficult to determine the

comprehensively most effective mixture and the relative relationship in the increase and

the decrease in the two kinetic parameter values were not obvious M

mK

J was found

and compared for each PEI-DTT mixture as shown below in Fig 56

00

05

10

15

20

25

30

35

40

45

50

1505 11 0515 22

PEIDTT ()

Jm

KM

(Am

-2M

-1)

N2O2

Fig 56 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051

11 1505 and 22)

It is clear that DTT must exceed 1 to achieve in the fraction to achieve efficient O2

resistance Since the equivalent glucose response lag was observed in all mixtures this

implies that at least 1 DTT is required for simultaneous reduction of Met amino acid

149

residue On the other hand gt 2 PEI seems required for high catalytic efficiency in N2

or equal weight PEIDTT mixing is preferable to increase catalytic efficiency in both N2

and O2 Since PEIDTT at 22 data presented here is based on a single sample the study

on more samples is required to confirm the results however PEIDTT mixture with

DTT gt 1 is effective to improve catalytic efficiency and output current

448 PEI and FAD mixture

Kinetic parameters for various PEI-FAD concentrations are shown below in

Fig 57

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

J m

Am

-2

N2 O2

00E+00

20E-02

40E-02

60E-02

80E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

KM

M

N2 O2

Fig 57 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with PEI-FAD (21 22 and 24 ) in the presence and absence of O2 n = 2

Firstly the result clearly showed total 6 stabiliser content hindered catalysis because it

showed extremely low Jm compared to other PEIFAD containing electrodes It only

counts for 15 and 9 of Jm obtained for other PEIDTT concentration in presence and

150

absence of O2 respectively Secondly there was no large difference in the performance

between PEIFAD at 21 and 22 hence PEIFAD 21 was most effective of all This

might involve non-competitive inhibition because Jm was smaller by 70 while KM was

smaller by only 20 compared to the standard values in the absence of O2 In the

presence of O2 it showed larger Jm by 7 times however also increased KM by 3 times

compared to those of stabiliser-free electrodes and did not decrease the glucose response

lag

449 Most effective stabiliser content for GOx anodes

To determine the size of comprehensive catalytic efficiency M

mK

J was

found for all the stabilisers and its value was compared for O2 for catalytic efficiency in

the presence of O2 Among all five most efficient catalysts were selected and presented

below in Fig 58

151

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

J m

Am

-2

N2 Am-2O2 Am-2

N2 Am-2 180E-02 125E-02 827E-03 292E-02 710E-03 104E-02O2 Am-2 168E-02 181E-02 151E-02 267E-02 122E-02 157E-02

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505 GLC 1

00E+00

50E-03

10E-02

15E-02

KM

M

N2 MO2 M

N2 M 729E-03 671E-03 576E-03 647E-03 938E-03 328E-03

O2 M 104E-02 471E-03 433E-03 801E-03 658E-03 886E-03

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505

GLC 1

Fig 58 Jm KM in the absence and presence of O2 for various stabilisers PEIDTT

(11 0515 221505 ) and GLC 1

PEIDTT mixture was found to be most effect as GOx stabiliser as discussed in sect 447

For Jm PEIDTT 22 showed the largest in both absence and presence of O2 and this

was followed by PEIDTT 11 For KM PEIDTT 0515 showed the smallest

comprehensively for both N2 and O2 and this followed by PEIDTT 11 PEIDTT

22 and GLC 2 showed relatively large KM in the presence of O2 It can be therefore

concluded that PEIDTT 11 is the most effective in stabilising GOx has largest

resistance against O2 and increases the catalytic output current It showed Jm = 13 x 10-2

plusmn 19 x 10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2

with KM = 47 plusmn 42 mM for O2 The current output obtained is much lower than some

reported results by Willner Heller and Sasaki [18 19 97] by 2 ndash 3 order of magnitudes

their electrodes however involved more complex fabrication methods or system as

discussed sect 22 on p 77 On the other hand it was slightly better than the result

reported by Merkoci who composed a similar composite with 5 GOx and was similar

to the results reported by Arai who built GOx electrodes by more complex methods KM

152

obtained is also smaller by 2 ndash 4 times than some of results by Arai [103]

Max current output Am-2 KM mM Method involved Author

125 x 10-3 ndash 181 x 10-2 47 ndash 67 10 GOx composite Data botained

55 x 10-3 Not available 5 GOx composite Merkoci [115]

30 Not available Apo-GOx reconstituted Willner [97]

11 x 10 Not available Os-complex hydrogel Heller [19]

18 Not available Saccharide-modified polypyrrole Fujii [100]

54 x 10-2 Not available QS conductive polymer Arai [103]

41 x 10 13 - 20 Poly(acrylic) acid hydrogel modified

carbon fibre GDH electrodes

Sakai [18]

Table 7 Comparison of maximum output current and KM with some published data

45 CONCLUSION TO ANODE STUDY

Bulk modified enzyme electrodes are good because they are mechanically very

stable and their fabrication is straightforward and scaleable requiring only mixing

electrode components conductive material mediators enzymes and stabiliser with

binding material TTF-TCNQ is useful material for GOx electrodes because it is

conductive as well as mediating The electrode component ratio is important for its

function and conductivity High binder and protein content hinder the conduction It was

found that 73 weight ratio of TTF-TCNQepoxy resin with 10 GOx loading optimal

The balance among the conductivity mediation enzyme activity mechanical and

153

chemical stability and difficulty in fabrication process is key to the successful

fabrication

O2 is known to be the natural mediator of GOx In the glucose-oxygen fuel

cells GOx anode must be resistant to O2 because O2 is an electron acceptor at the

cathode O2 lowers the current output by competing for electron transfer between GOx

and artificial mediators as well as non-competitive inhibition Various GOx stabilisers

were examined to alleviate such O2 influence

PEI DTT PEG GLC FAD PEIDTT and PEIFAD mixtures were studied as

candidate stabilisers to enhance catalytic activity and the current output PEI was not

effective It stabilised the structure but made conformation more rigid and did not

increase the output current in both the absence and presence of O2 DTT was reasonably

effective It showed uncompetitive inhibition in N2 but stabilised the enzyme structure

It therefore increased Jm and KM in the presence of O2 but increase in KM was probably

due to alleviating inhibition by O2 and loosening up the enzyme conformation PEG was

not effective It probably stabilised the enzyme structure but not show much

improvement for O2 competition It might also deprive electrons from reduced GOx

GLC was effective The effect was concentration dependent Lower concentration was

more effective It also scavenged O2 FAD was reasonably effective but only at higher

concentration PEI-FAD was also reasonably effective It decreased Jm but Km was

marginally equivalent to that of stabiliser-free electrodes for N2 But it increased Jm for

O2 Among all PEIDTT at 11 mixture was found to be most effective because it

stabilised the GOx and scavenged O2 Such powerful influence was not observed in the

use of either stabilisers hence it is a synergetic effect It showed Jm = 13 x 10-2 plusmn 19 x

154

10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2 with KM

= 47 plusmn 42 mM for O2 Although it lowered Jm by 30 in the absence of O2 compared

to the standard value of 18 x 10-2 plusmn 73 x 10-3 Am-2 it also decreased KM in both

absence and presence of O2 and increased Jm by 8 times compared to that of

stabiliser-free electrodes PEIDTT 11 is therefore most effective in stabilising GOx

has largest resistance against O2 and increases the catalytic output current

155

5 CATHODE INTRODUCTION

51 OXYGEN REDUCTION REACTION (ORR)

The oxygen reduction reaction (ORR) takes place at the cathode as oxygen

accepts electrons from the anode Oxygen is an ideal oxidiser for the cathode because it

is readily available and its reaction product water is harmless [62] However ORR has

been a long-time bottleneck especially for low-temperature fuel cells because it causes

large potential loss [3 4 62 165-167] due to a strong double bond in the dioxygen

molecule Its difficult bond dissociation causes slow irreversible kinetics [24 165 166]

so a catalyst is required for this reaction to proceed at useful rates [62 165] ORR is a

multi-electron reaction which involves various reduction pathways and different

oxygen intermediates (Fig 16) In aqueous solution either the direct four- or

two-electron ORR pathway may proceed [165 166] The direct four-electron transfer

pathway reduces O2 into H2O in acid and neutral mediaor OH- in alkaline media This

occurs via several reduction steps [24 165 166] which may involve adsorbed peroxide

intermediates These peroxide intermediates never diffuse away in the solution in this

pathway [166] The two-electron transfer pathway reduces O2 into peroxide species as

final products These peroxide species either diffuse away are reduced further or

decomposed into H2O or OH- [24 165 166] This is not favourable because it leads to

lower energy conversion efficiency and its product hydrogen peroxide may degrade the

catalysts and the fuel cells [165 167] Four- and two-electron transfers also may occur

in series or in parallel [165]

156

Fig 59 Oxygen reduction pathways [165 166]

Reduction kinetics and mechanisms depend on the type of catalysts electrode material

electrode surface condition solution pH temperature contaminant and reactant

concentration [24 165 166] Rate-limiting step also varies by type of the catalysts

[166] However the first electron transfer to dioxygen leading to the formation of

superoxide anion radical (O2־) is the rate limiting step [168] O2־ is very rapidly

protonated in water and converted to hydroperoxyl radical (HO2)

157

Fig 60 Degradation of superoxide anion radical in aqueous solutions [24]

In aqueous solution O2־ is unstable and rapidly converted to O2 and H2O2 via a

proton-driven disproportionation process (Fig 60) [24]

52 REACTIVITY OF OXYGEN

A dioxygen molecule is a homodiatomic molecule consisting of two oxygen

atoms double bonded It has two parallel unpaired electrons in degenerate anti-bonding

π designated as π orbitals therefore it is diradical and paramagnetic (Fig 61) [169]

158

Fig 61 An energy level diagram for ground state dioxygen molecule [169]

When one electron is transferred to a dioxygen molecule the electron is placed in π

orbital and it turns into a reactive O2[24] ־ reducing the bond order from 2 to 15

Oxygen reduction changes the molecular symmetry or stretches the double bond

causing bond disruption [165 166] Electron paring in one of the π orbitals also

changes their orbital energy state and lifts the degeneracy as in Fig 62 [170]

Hereogeneous catalysis involves chemisorption of the oxygen and therefore some

transfer of electron density from the oxygen to the catalyst This facilitates the rate

determining initial electron transfer Oxygen reduction catalysts are reviewed in the

next section

159

(O2) 3Σg- O2־

Fig 62 Diagrams for the state of π orbitals in the ground state oxygen (3Σg-) (left)

and peroxide anion radical (right) [24]

O2 species Bond Order Bond length pm Bond energy kJmol-1

O2 2 1207 493

O2+ 2frac12 1116 643

O2- 1frac12 135 395

O22- 1 149 -

Table 8 Bond orders bond lengths and bond energies of some dioxygen species [169]

53 ORR CATALYSTS

Catalysts are required to reduce the overpotential and improve the efficiency of

160

ORR [3 4 62 165-167] It was reported that 80 of the whole potential loss can be

attributed to cathode ORR overpotential [171] The choice of ORR catalysts varies by

applications [36 171] and direct four-electron transfer pathway is most preferred for

ORR at the lowest possible overpotential in every fuel cell application however its

kinetics and reduction pathway are limited by the type of catalysts and experimental

conditions [24 165 166] Miniaturised glucose-oxygen fuel cells operate in the neutral

aqueous solution at ambient or body temperature with relatively low electrolyte and

oxygen concentrations therefore they require powerful ORR catalysts Platinum (Pt)

has been most ideal however its availability and cost are major limiting factors Many

studies have been made on other noble metals [172 173] transition metal macrocyclic

compounds [174] multicopper oxidases [62 175] and quinones [165] as alternatives

ORR catalysts to Pt These ORR catalysts are reviewed below

531 Noble metal ORR catalysts

Noble and transition metals are well-known ORR catalysts There are two

possible ORR mechanisms for metal surface dissociative and associative

161

Dissociative mechanism

middotmiddotmiddotmiddotmiddot Eq 67

middotmiddotmiddotmiddotmiddot Eq 68

middotmiddotmiddotmiddotmiddot Eq 69

( indicates the catalyst surface)

Associative mechanism

middotmiddotmiddotmiddotmiddot Eq 70

middotmiddotmiddotmiddotmiddot Eq 71

middotmiddotmiddotmiddotmiddot Eq 72

middotmiddotmiddotmiddotmiddot Eq 73

middotmiddotmiddotmiddotmiddot Eq 74

In dissociative mechanism the O2 double bond is ruptured rapidly on its adsorption on

the metal surface [165 166 172] This involves a strong interaction between oxygen

and metal atoms [166] and does not generate H2O2 [165] Electron and proton transfer

take place simultaneously and this is the rate-limiting step [166 171] In the associative

mechanism O2 does not dissociate before proton transfer Adsorbed O2 stays on the

metal surface [165 172] and two-electron and proton transfer [166 173] take place via

the formation of superoxide or peroxide intermediate [165 166] This reaction is

thermodynamically easier than the dissociative pathway and its final product is H2O2 or

162

H2O if both pathways occur in series [165] O2 adsorption on the metal surface is an

inner sphere interaction which involves complex formation with the metal surface [30]

There are three ways in which O2 adsorbs (i) Griffiths (ii) Pauling and (iii) bridge

model (Fig 63)

Griffiths model Side-on Pauling model End-on Bridge model

OO

M

Fig 63 Three models of oxygen adsorption [165 166 173]

The Griffiths model is called lsquoside-onrsquo type O2 functions as a bidentate ligand against a

single metal atom in this complex and its π orbitals laterally interact with empty metal

dz2 orbitals causing ligand-to-metal charge transfer (LMCT) πrarrdz

2 At the same time

electrons partially filling metal dxz or dyz orbitals are back donated to oxygen π orbitals

[166 171] Therefore a strong metal-oxygen interaction is present in this model This

effectively disrupts the O=O bond [166] and facilitates the electron transfer from the

metal ion by changing its oxidation number The Pauling model is called the lsquoend-onrsquo

type [165 166 173] O2 is a monodentate ligand in this model and only one of the

oxygen atoms in the dioxygen molecule can be bonded to the metal surface An oxygen

π orbital interacts with metal dz2 orbitals and this allows either the direct or peroxide

ORR pathway after partial charge transfer [166] The bridge model resembles Griffiths

model in the way the metal atom and oxygen interact however it requires two adjacent

163

adsorption sites so that O2 can ligate two different metal atoms The bridge-model

interaction is easily interfered with by the surface contamination and the oxide layer

because the surface species block the adsorption sites for ORR catalysis [173]

Fig 64 Models for oxygen adsorption and ORR pathways [166]

Both the Griffiths and bridge models follow dissociative mechanism (pathway I and III

respectively Fig 64) due to the lateral interaction leading to the dissociation of O2

double bond The Pauling model on the other hand follows the associative mechanism

(pathway II Fig 64) This is further divided into two other pathways IIa and IIb

according to its end product H2O2 or H2O (Fig 64) respectively [166] O2 adsorption

and the ORR mechanism therefore depend on (i) the extent of oxygen-metal

interaction and (ii) the availability of oxygen adsorption sites A volcano-shaped trend

between ORR rate and oxygen-metal interaction has been plotted by Norskov as in Fig

65 [172]

164

Fig 65 A volcano-shaped trend between ORR rate and energy required for oxygen

binding on various transition metal surface [172]

Many have reported platinum (Pt) as the most ideal metal catalyst due to its high

catalytic ORR activity [165 166 171 172] Fig 65 shows that Pt and Pd (palladium)

have largest ORR rate yet medium oxygen adsorption energy and iridium (Ir) and

rhodium (Rd) follow them On the other hand metals with either higher or lower

oxygen adsorption energy result in lower ORR rate This is because metals with high

oxygen adsorption energy such as gold (Au) imposes too large energy barrier for the

formation of a stable oxygen-metal bond while too small adsorption energy as in nickel

(Ni) leads to excessively stable oxygen-metal interaction and slows the proton transfer

Therefore associative pathway is dominant at Au surface while oxygen dissociation is

deactivated at Ni surface but instead this increases the surface oxide coverage [172

173] Pt performs best because it has moderate oxygen-metal surface interaction [165]

Pt alloys with other transition metals such as Ni Co and Fe have been also reported to

increase the catalytic activity [36 167 171 172] because alloying dilutes the Pt-O

165

interaction and alters the overall oxygen-metal surface interaction [171 172] This is a

useful property to add because Pt is known to form a surface oxide layer leading to both

associative and dissociative catalytic pathways [165 171] This surface state is

potential-dependent and stays persistenly at less than 08 V vs AgAgCl [172]

532 Macrocycle and Schiff-base complex ORR catalysts

Macrocyclic transition metal complexes have been widely investigated as

potential ORR catalyst both for fuel cells and as catalysts to activate dioxygen for

synthetic purposes They have been widely reviewed by [165 176] Macrocyclic

complexes consist of (i) transition metals such as Fe Co Ni or Cu (ii) chelating atoms

N4 N2O2 N2S2 O4 and S4 and conjugate π systems such as porphyrin and

phthalocyanine [165] Examples of transition metal macrocycles are shown in Fig 66

Fig 66 Examples of transition metallomacrocycles Heme (left) [177] and copper

phthalocyanine (right) [178]

166

Schiff base complexes such as metallosalens and metallosalophens are other

well-known chelating ORR catalysts [174 179] Transition metal macrocycles and

Schiff bases can be also polymerised into conductive films [180] (Fig 67)

Fig 67 Schiff base complexes

Co(II) salen Co(II) salophen and

Co(II) salen modified poly(34-ethylenedioxythiophene) [174 180]

Interaction between the oxygen molecule and the metal complex is similar to that with

the noble metals and involves partial charge transfer between the filled metal dz orbitals

and the π orbital of O2 as described in sect 531 The catalytic activity of the metal

complexes depends on the type of central metal atom ligand and the size of the

π-electon systems The reducing ability of the ligand is important because the central

metal atom must transfer considerable electron density to oxygen atoms during the

167

catalysis [170] The thermodynamically most stable metal-oxygen complexes are

bionuclear complexes such as dicobalt face-to-face 4-atom-bridged diporphyrin

(Co2(FTF4)) (Fig 68) [165-167]

Fig 68 Molecular structure of dicobalt face-to-face diporphyrin [166]

Mononuclear Co complexes take the associative pathway [165] forming superoxo

cobaltic complexs (Co-O-O־) [181 170] while their dimers or binuclear complexes can

take either direct four-electron or the parallel pathways (Fig 69) [165 167] because

they can lock the oxygen moiety in rigid orientation [170] Co2(FTF4) favours

four-electron transfer and its ORR pathway depends on the distance between two

cofacial metal atoms [165-167] Spacing distance can be altered by inserting a spacer

molecule [165 166]

168

Fig 69 ORR pathway by Co2(FTF4) [166]

However Co2(FTF4) is not stable enough for practical use [165 166] Practical ORR

catalysts for fuel cell application must be stable inexpensive and feasible to synthesise

533 Multicopper enzymes as ORR catalyst

Multicopper oxidases (MCOs) are copper-containing enzymes such as bilirubin

oxidase (BOD) laccase (Lac) and copper efflux oxidase (CueO) Oxygen is widely used

as an electron acceptor in nature because it is ubiquitous and oxidation is a good mean

to obtain energy as in combustion Millions of years of evolution have therefore

provided enzymes with good catalytic activity for oxygen reduction Although oxygen

169

reduction in biological system is complex due to involving various substrates and

catalytic steps as well as it tends to be incomplete and leads to hydrogen peroxide or

superoxide formation as the product the enzymes catalyse the rate-determining initial

electron transfer For application in biofuel cells it is essential that the enzymes are

widely available and possess good stability activity in immobilised form and ideally

four-electron ORR catalysis MCOs have been prevalent in biofuel cell applications

because they are active at neutral pH catalyse four-electron ORR and bear

immobilisation Fungal laccase and bilirubin oxidase have been most widely studied

MCOs [56 59 60]

Most MCOs have four Cu atoms which are designated as type I (T1) type II (T2) and

a pair of type III (T3) T2 and T3 together form a trinuclear Cu centre while T1-Cu

called blue copper [175 182 183] is located away from the trinuclear Cu centre but

adjacent to the hydrophobic substrate-binding site Fig 70 shows the location of the

trinuclear Cu centre (copper coloured) and the blue copper (in blue) Oxygen binding

and ORR take place at the trinuclear Cu centre [175] while T1-Cu receives electrons

from the substrate and mediates electron transfer between substrate and the trinuclear

Cu centre [175 184]

170

Fig 70 Laccase from Trametes versicolor [185]

Enzyme catalytic efficiency depends on the redox potential [175] of T1-Cu It has been

reported that fungal laccase has the highest redox potential between 053 ndash 058 V vs

AgAgCl [62 183] T1-Cu also allows DET due to being located close to the surface

[183 186 187]

MCO reaction cycle starts from substrate oxidation to form a fully reduced state MCO

is initially at native form or so-called intermediate II Native form is characterised by

doubly OH--bridged structure (Fig 71(ii)) In the absence of substrate the native form

decays to the resting form which is at fully oxidised state (Fig 71(i)) After

four-electron reduction of enzyme the fully reduced MCO (Fig 71(iii)) reacts with O2

The first reduction step involves two-electron transfer from each of T3-Cu+ to O2 This

forms peroxide intermediate or intermediate I (Fig 71(iv)) and lowers the energy

barrier to disrupt O=O It is followed by swift protonation of adsorbed peroxide at

T3-Cu site This protonation reaction is induced by the charge transfer from T1- and

171

T2-Cu+ This leads to the complete reduction of oxygen to water and the enzyme native

form is retrieved

(i)

(ii)

(iii)

(iv)

Fig 71 Redox reaction cycle of MCOs with O2 [175]

All MCOs have this common ORR mechanisms despite the different origin structure

biological functions substrate reducing power and optimal environment

5331 BOD

BOD is an anionic monomer [182] most active at neutral pH [183 188] and

172

capable of both DET and MET The mediators must have their redox potential close or

slightly lower than that of BOD 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

(ABTS) (Fig 72(i)) [62 97] Os-pyridine complexes linked to conductive polymer (Fig

72(ii)) [66 189] and cyanometals [18 56] have been used as its mediators [56 185]

ABTS is a divalent anion which works as co-substrate of MCOs and its redox potential

is 067 V vs AgAgCl [56] Os-pyridine complex with conductive polymer follows the

similar idea for the wired GOx anodes discussed in sect 223 on p 80

4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine) has been used

especially for the cathode and its redox potential is 055 V vs AgAgCl

Fig 72 Common mediators for BOD (i) ABTS [56 62]

(ii) Os[4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine)] [190]

173

Tsujimura showed that ORR catalytic constant was 6-times greater in the presence of

Os-pyridine complex than in DET [66 188] Reduction starts around 05 V in most

cases [66 97 183 188] The size of current density varies roughly between 05 - 1

mAcm-2 [56 97 188] and 95 mAcm-2 was achieved by Heller [189]

5332 Lac

Fungal laccase is another anionic monomer [183] It is less expensive than

BOD and has broad substrate specificity including xenobiotics [175] although it has

optimum pH around 5 and activity hindered by chloride [19 182] Its T1-Cu redox

potential ranges between 053 ndash 058 V vs AgAgCl [62 183] Highest redox potential

066 V and current density 057 mAcm-2 were achieved by the use of anthracene

plugging molecule which provides strong binding between Lac and pyrolytic graphite

edge electrode as well as long-term activity 57 of the initial current was retained after

eight weeks [185]

5333 CueO

CueO is a less common enzyme in biofuel cells however it is active for a wide

range of pH between 2 - 8 [186] and also it has better catalytic activity than other

MCOs because it has five Cu atoms This extra Cu atom is believed to have mediating

function and 4 mAcm-2 has been achieved at pH 5 [191] However it has lower redox

potential around 03 V [186 191] Protein engineering has been introduced to increase

T1-Cu redox potential [186]

174

534 Anthraquinone ORR catalyst

Anthraquinone (AQ) is known to catalyse two-electron transfer in ORR (Fig

73) [44 165 192-194] and it is used in industries to produce hydrogen peroxide [165

192]

Fig 73 Two-electron ORR by anthraquinone [193]

ORR by anthraquinone is an electrochemical-chemical (EC) process [165 192] in

which quinone must be first electrochemically reduced to an active species

semiquinone anion radical (Q־) The active semiquinone radical can then react with

dioxygen so that it converts the dioxygen into O2־ via forming of the intermediate

superoxo-C-O(O2) species This is the rate-limiting step O2־ produced by a

semiquinone radical either disproportionates (Eq 77) or it is further reduced into HO2-

(Eq 78) [192]

175

Oxygen reduction reaction by anthraquinone

ltlt Rate limiting step gtgt

middotmiddotmiddotmiddotmiddot Eq 75

middotmiddotmiddotmiddotmiddot Eq 76

ltlt Following spontaneous reactions gtgt

middotmiddotmiddotmiddotmiddot Eq 77

middotmiddotmiddotmiddotmiddot Eq 78

(Q indicates quinone on the surface)

Derivatised anthraquinone such as Fast red (Fig 74) can be attached to the carbon either

physically or covalently Grafted quinones are very stable [192 194 195] and resistant

against continuous potential cycling [194] metal ions and surfactant despite of some

fouling present [44]

176

O

O

N N Cl

frac12 ZnCl2

1

2

4

6

5

3

10

9 7

8

12

14

13

11

O

O

(i) (ii)

Fig 74 The structures of (i) anthraquinone [196] and (ii) Fast red AL salt [44 197]

However its ORR is heavily affected by pH [194] because its catalytic efficiency

depends on the concentration of surface Q־ species [192 194] The concentration

varies by different pH and the catalytic efficiency drops at both high and low pH due to

the depletion of Q־ When pH falls below its dissociation constant Q־ is not

reproducible anymore and ORR is inhibited This phenomenon appears at as high as pH

7 and the ORR vanishes at pH lt 7 [194] AQ-grafted GC (AQGC) shows only sluggish

kinetics at pH lt 10 Its voltammogram also becomes complex due to the presence of

native quinone groups originated from the carbon surface [192] ORR mediated by the

native quinone groups is observed as a prewave and this varies by different quinone

derivatives such as phenanthrenequinone (PQ) (Fig 75) electrode composition and pH

[194]

177

Fig 75 Hydrodynamic voltammogram for ORR at various quinone electrodes with various

rotation speeds [194] (i) unmodified GC (ii) AQGC and (iii) PQGC at pH 7

54 CARBON PROPERTIES

Carbon is widely used for electrochemical applications because it is

inexpensive mechanically stable [105] and sufficiently chemically inert yet reactive

due to the presence of surface defects It has wide potential window between 1 and -13

V [21 30] Carbon is available in various solid forms such as graphite glassy carbon

(GC) pyrolytic carbon (PC) activated carbon (AC) carbon fibres (CFs) carbon

nanotubes (CNT) and diamond It is easy to renew the surface and allows facile

chemical modification [195] However it is also prone to adsorption and surface

oxidation simply by air and water exposure and its electron transfer is under the

constant influence of the electrode surface condition and history [198] This leads to the

deviation and the low reproducibility in carbon electrochemical experiments [105]

178

Physical properties of carbon vary by its form and section The carbon consists of either

(i) layers of the conductive planar graphene sheets or (ii) the insulating

tetrahedral-puckered forms as seen in the diamond (Fig 76) Conductivity in diamond

is gained by doping with boron [199 200]

Fig 76 Carbon structures (i) graphite (ii) diamond [201]

The electrical conductivity is anisotropic and closely correlates with the carbon

structure and section The carbon section is classified into (i) basal and (ii) edge planes

The basal plane extends along the graphene plane while the edge plane is perpendicular

to this

179

Fig 77 Carbon section (i) basal plane (ii) edge plane [105]

The basal plane has larger conductivity due to being parallel to the vast graphene

network while the edge plane is superior in electrochemical reactivity due to being

richer in reactive graphene sheet termini and carbon defects The ratio between the basal

and edge planes therefore affects the conductivity and electrochemical properties of

the carbon and this varies by the carbon form and its treatment [45 105 195 202 203]

The electrical conductivities for various carbon forms and sections is shown in Table 9

180

The type of carbon Electrical conductivity Scm-1

Graphite [105 159] 1 x 103 - 104

Highly ordered PG basal plane 250 x 104

Highly ordered PG edge plane 588

PG basal plane 400 x 103

PG edge plane 333

Polycrystalline graphite 100 x 103

GC [105 159 203] 01 - 250 x 102

Activated carbon [204] 1 x 10

Carbon fibre 133 x 103

Boron-doped diamond [199 205] 01 - 1 x 102

Multi-wall carbon nanotube [206] 1 ndash 2 x 103

Pt 909 x 104

Cu 588 x 105

Table 9 The electric conductivity of various carbon forms and sections Data from

[105] unless defined specifically

541 Graphite

Graphite is black-coloured material with pores and high gas permeability It is

available in many physical forms as powders moulded structures and thin sheets

therefore it is used in many commercial applications [203] Powdered graphite is

convenient to make carbon paste and composite electrodes [105] Graphite consists of

181

layers of graphene sheets each of which is a huge π-conjugate system π-conjugate

system is made of hexagonal rings of sp2 hybridised carbon atoms [105 195] (Fig 76(i)

and Fig 78) and allows free electron movement within graphene This gives carbon

metallic properties such as electric and thermal conductivity

Fig 78 A direct image of a graphene bilayer by transmission electron microscopy

(TEM) at resolution of 106 Aring and with a 2-Aring scale bar [207]

Each graphene sheet is structurally stable in horizontal direction but fragile in vertical

direction because each layer is only held by van der Waalsrsquo force Therefore the

graphene sheets easily slide and collapse [31]

542 Glassy carbon (GC)

Glassy carbon (GC) is a non-graphitised carbon made of randomly interwoven

sp2-carbon fibres which are tangled and heavily cross-linked (Fig 79) It contains many

closed voids [203] and fullerene-like elements [208] It is impermeable to gases [203

182

208] chemically more inert [208] and mechanically stronger but electrically more

resistive than other carbons [203]

Fig 79 GC (i) High resolution TEM image (ii) A schema of GC [208]

543 Activated carbon (AC)

Activated carbon (AC) is thermally and chemically activated carbon It is

highly porous and has large internal surface area This is an excellent adsorbent for

solvent recovery and gas refining [203 208]

544 Carbon fibre (CF)

Carbon fibres (CFs) have small diameter ranging between 5 to 30 microm They are

lightweight have lower fabrication cost and are superior in mechanical properties

therefore they have been used for sporting automotive and military equipments They

are available in many forms such as tow mat felt cloth and thread They consist of a

random arrangement of flat or crumpled graphite sheets but their structure and

183

molecular composition depend on the type of precursors [203 208]

545 Carbon nanotubes (CNTs)

Carbon nanotubes (CNTs) are made rolled-up sheets of sp2-graphene in nano to

micro-meter range They are available in various formats single-walled nanotube

(SWNT) multi-walled nanotube (MWNT) carbon nanohorn (CNH) (Fig 80) and

various forms powder arrays and posts [117]

Fig 80 Various CNTs (i) Bundled SWNTs (ii) MWNT (iii) CNHs [209]

CNTs have been drawing keen attention due to their wide surface area conductive or

semiconductive properties light weight mechanical stability and facile

functionalisation [117 195 202 210] They have been used as support for the dispersed

metal catalysts [3] and electrode materials [117 202] The drawbacks of CNTs are the

high presence of impurities alteration of their properties on acid cleaning [202 211]

and unknown toxicity [117 160 202]

184

546 Boron-doped diamond (BDD)

Diamond is different from other carbons because it is transparent and not

electrically conductive It has a cubic lattice structure made of closely-packed repetitive

units of sp3-hybridised carbon atoms as seen in Fig 81

Fig 81 A TEM image of diamond [212]

Each unit consists of four σ-bonded sp3-carbon atoms in tetrahedral coordination as in

Fig 76(ii) Each bond is localised and this gives structural stability but does not conduct

electricity By doping it with boron diamond can become semiconductive Such

diamond is called boron-doped diamond (BDD) and boron functions as an electron

acceptor BDD is mechanical more stable chemically more inert sensitive to analyte

and has wide potential window lower back ground current and stable electrochemical

responses [195 199 205]

185

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON

551 Electrochemical properties of carbon

The edge plane of carbon which contains graphene termini and carbon defects

is chemically reactive and accounts for carbon electrochemistry while the vast network

of graphene sheets is responsible for its electrical conductivity The termini and defects

include some sp3 hydrocarbons and they are oxidised into various oxygen functional

groups such as carboxyl quinoyl hydroxyl lactone groups ethers cyclic esters and

lactone-like groups [195] These electrode functional groups facilitate electron transfer

provide bonding between the electrode and the catalysts or enzymes or simply cause

electrode capacitance or resistance Various carbon pretreatments such as

electrochemical pretreatment (ECP) vacuum heat treatment (VHT) carbon fracturing

and polishing laser irradiation [195 213-215] are used to attach active surface

functional groups and increase the rate of electron transfer in various electrochemical

reactions [198 216]

552 Electrochemical pretreatments (ECPs) of the carbon

Electrochemical pretreatments (ECPs) provide feasible electrode surface

modification Various ECPs have been used to introduce carbon-oxygen surface

functional groups Most common method is potential cycling in the acid or basic media

or the combination of anodisation and cathodisation as shown in Table 10

186

Table 10 ECPs examples

Potential cycling at 02 Vs-1 between 0 and 02 V in 01 M H2SO4 [105]

Anodisation at 18 V in pH 7 phosphate buffer for 5 minutes followed by 1-minute cathodisation at

ndash 15 V [216 217]

Potential cycling between 06 and 2 V at 5 Vs-1 for 15 to 30 minutes in 05 M NaOH [218] or their

combination [219]

ECPs introduce the electrodes various new properties They (i) attach electrochemically

active functional groups and increase the oxygencarbon ratio [105] (ii) introduce

carbon defects and active sites [105 218] (iii) increase the surface area [105 198 215

216 218] (iv) form a surface film and (v) increase the background current [105] and

double layer capacitance [105 215 216] Most notable effect of EPCs is the increase in

electron transfer rate in many electrochemical reactions This is observed as a decreases

in peak separation and an increase in peak current in voltammetry as seen in Fig 82

[105 216 218 220] As for ORR however it was reported that surface quinone groups

attached by EPC or originated from the carbon itself reduce ORR overpotential but do

not mediate ORR as in quinone groups physically or covalently attached [216 220]

187

Fig 82 The influence of ECP on GCE and its voltammogram at 01 Vs-1 in pH 7

phosphate buffer in the presence of (i) N2 and (ii) O2 The voltammograms

of both pretreated and unpretreated GCEs are shown [216] The

presentation has been altered for convenience

56 ELECTROCHEMICAL TECHNIQUES FOR ORR

ORR has been studied by two electrochemistry techniques cyclic voltammetry

(CV) and hydrodynamic voltammetry (HDV) using the rotating disc electrode (RDE)

These techniques provide overlapping but complementary information CV is a dynamic

technique and allows calculation of the number of electrons up to and including the rate

determining step (nα) With numerical simulation and data fitting CV can be used to

estimate the standard heterogeneous rate constant (kdeg) RDE is a steady state technique

and can give the overall number of electrons transferred (n) The well-established

Koutecky-Levich plot (see sect 567 on p 200) provides a robust method for determining

kdeg provided the standard electrode potential (Edeg) for the reaction is known

188

561 Cyclic voltammetry (CV)

Cyclic voltammetry (CV) is widely used for initial exploration and

characterisation of a redox active system It is of limited electroanalytical value

however kinetic parameters can be obtained from the current-voltage (I-V) plot In

cyclic voltammetry potential is swept linearly with a triangular waveform (Fig 83(i))

and applied cyclically The resulting time-dependent current is typically plotted versus

applied potential (Fig 83(ii)) [30 221] The potential range and scan rate are decided in

advance according to the type of analytes reactions and materials of interest

Fig 83 Schemas for (i) a Triangular waveform (ii) Typical cyclic voltammogram for

benzoquinone (BQ) CV was taken at 005 Vs-1 between + 025 and ndash 05 V vs

AgAgCl for 1 mM BQ in 1M KCl at graphite composite electrode Switching

potential (Eλ) is ndash 05 V and time taken to reach Eλ (t) is 15 seconds Based on

the real data

189

562 Non-faradic current and electric double layer capacitance

Transient techniques such as CV always contain two types of electric current

non-faradaic and faradaic current The non-faradaic charging current is due to the

electric double layer which is formed at the electrode-solution interface This occurs

because such an interface behaves like a capacitor as counter ions accumulate at the

interface in order to balance the potential at the electrode This does not account for the

charge transfer reactions involved however provides the information about the

electrode surface [30] and also can affect the rate of electrode process and distort the

voltammogram Cd can be estimated by electrochemical impedance spectroscopy or

deduced by averaging Cd over the potential range as in Fig 84 and Eq 33 [21 30]

Fig 84 The double layer capacitance (Cd) observed in CV for deoxygenated pH 74 at

the acid-pretreated graphite electrode See Eq 33 for the calculation and

symbols Based on the real data

190

Electric double layer capacitance per unit area

AII

AEQareaunitperC ccca

d sdotminus

=sdot

=υ2

)()( middotmiddotmiddotmiddotmiddot Eq 79

Cd = Double layer capacitance per unit area microFcm-2

Q = Charge C

E = Potential V

A = Electrode surface area cm-2

Ica = Anodic non-faradic current A

Icc = Cathodic non-faradic current A

υ = Scan rate Vs-1

Cd needs to be either negligible or subtracted before interpretation of faradaic current

which accounts for the charge transfer phenomena in redox reactions In order to

minimise the influence of Cd on the voltammogram the scan must be taken at slow scan

rate and the electrolyte concentration must be always kept reasonably high [30]

563 Diagnosis of redox reactions in cyclic voltammetry

A typical voltammogram contains current peaks as seen in Fig 83(ii) and these

are due to the redox reactions in the system Diagnosis of the redox system using CV is

based on (i) peak separation (ii) peak current height (iii) peak shape or peak width (iv)

the number of peaks and (v) the dependency of peak shape on the scan rate The

analysis of such peaks involves the following four parameters anodic peak current (Ipa)

cathode peak current (Ipc) anodic peak potential (Epa) and cathode peak potential (Epc)

This reveals the type of electrochemical reactions involved which are either (i)

191

reversible (ii) quasi reversible or (iii) irreversible When the peak current is proportional

to υfrac12 the redox reaction is diffusion-limited in the solution while a linear dependence

shows that the redox active species is confined to the electrode surface

In the reversible system charge transfer between the redox species and the electrode is

rapid [221] therefore it has fast kinetics and the reaction is diffusion-limited The

electrode potential follows the Nernst equation (Eq 15 on p 50) because the electrode

potential is dependent on the concentration equilibrium at the electrode surface and the

peak potential is independent of the scan rate The features on the reversible system are

listed in Table 11 The peak current (Ip) is linearly proportional to υ12 as the redox

reaction is diffusion-limited The peak separation (∆Ep) is the difference between Epa

and Epc and it is 59 mV at 25 ˚C for the single-charge transfer [29 30] The peak width

(δEp) is the difference between Ep and half-peak potential (Ep2) Ep2 is the potential

where the peak current is half It is 565 mV for a single charge transfer The ratio

between Ipc and Ipa is called a reversal parameter [105] It is unity at any scan rate in the

reversible system

192

Table 11 Reversible system at 25 ˚C [30]

2121235 )10692( υlowasttimes= CADnI p middotmiddotmiddotmiddotmiddot Eq 80

mVn

E p528

= independent of υ middotmiddotmiddotmiddotmiddot Eq 81

mVn

EEE pcpap59)( =minus=∆ middotmiddotmiddotmiddotmiddot Eq 82

mVn

EEE ppp556

2 =minus=δ middotmiddotmiddotmiddotmiddot Eq 83

1=pa

pc

II middotmiddotmiddotmiddotmiddot Eq 84

Ip = Peak current A

n = The number of charges transferred

A = Electrode surface area m-2

D = Analyte diffusion coefficient m-2s-1

ν = Solution kinematic viscosity m-2s-1

Ep = Peak potential V

∆Ep = Peak separation V

Epa = Anodic peak potential V

Epc = Cathodic peak potential V

δEp = Peak width V

Ep2 = Half-wave potential V

Ipa = Anodic peak current A

Ipc = Cathodic peak current A

In a totally irreversible system charge transfer between the redox species and the

electrode takes place only slowly [221] The electrode must be activated by applying

193

large potential to drive a charge transfer reaction at a reasonable rate Ep becomes

dependent on scan rate and this increases ∆Ep and skews the voltammogram [30]

However Ip is still proportional to υfrac12 because at high overpotential the redox reaction

is diffusion-limited [29]

Table 12 Irreversible system at 25 ˚C [222]

2121215 )()10992( υα αlowasttimes= CADnnI p middotmiddotmiddotmiddotmiddot Eq 85

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛

deg+minusdeg=

2121

lnln780RT

Fnk

DFn

RTEE pυα

αα

α

middotmiddotmiddotmiddotmiddot Eq 86

mVn

E pαα

59gt∆ middotmiddotmiddotmiddotmiddot Eq 87

mVn

E pαα

δ 747= middotmiddotmiddotmiddotmiddot Eq 88

α = Transfer coefficient

nα = The number of charge transferred up to the limiting step

( Please refer to Table 11 for other terms and symbols)

A quasi-reversible system is controlled by both kinetics and diffusion Its

electrochemical reversibility is relative to the scan rate [21 30] The peak potential

depends on scan rate for rapid scans and the peak shape and peak parameters become

dependent on kinetic parameters such as α and Λ where 21

1

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛

deg=Λ

minus υαα

RTFDD

k

RO

[30]

Since Λ is a function of k˚υ-frac12 where kdeg is the standard rate constant the voltammogram

hence kdeg is affected by the scan rate as seen in Fig 85

194

Fig 85 Voltammograms of a quasi-reversible reaction at various scan rates [21]

564 Coupled reactions in cyclic voltammetry

Redox reactions are sometimes coupled with different electrochemical or

chemical reactions Voltammogram with altering scan rates can indicate the presence of

such reactions A redox reaction coupled with another redox reaction is called EE

mechanism while with homogeneous chemical reaction it is called EC mechanism If

the coupled reaction is catalytic then it is called ECrsquo mechanism [21]

Many ORR catalysts show coupled reactions with ECrsquo mechanism ORR is a

quasi-reversible reaction because the reaction is limited by oxygen diffusion yet O2

adsorption and reduction heavily relies on overpotential or catalytic power of catalysts

Catalysed reactions show distinctive voltammetric features There is only a single-sided

large current but with no reverse current because they catalyse a reaction only in a

single direction The reversal parameter therefore becomes non-unity [30]

195

ORR catalysts sometimes contain multiple reducible centres or an atom with various

oxidation states as in seen transition metal They might exhibit a multi-peak

voltammogram under non-catalytic condition The shape of voltammogram depends on

the difference in the standard potential (∆E˚rsquo) between two redox centres or states (Eq

89) as seen in Fig 86 [21 30]

21 ooo EEE minus=∆ middotmiddotmiddotmiddotmiddot Eq 89

Fig 86 Voltammograms of two-electron transfer EE systems of different ∆Edegrsquo [21]

565 Voltammograms for the surface-confined redox groups

Catalysts encapsulated in or attached to the electrode provide catalytic groups

or moieties on the electrode surface Such surface-confined redox species show different

voltammograms from solution redox species because their redox reactions do not

196

involve diffusional redox species They exhibit a characteristic voltammogram of

perfect symmetry with Epa = Epc as seen in Fig 87 and their peak current is linearly

proportional to υ rather than υfrac12 as in Eq 90 [21 30] The features of surface-confined

groups are listed in Table 13

Fig 87 A voltammogram of the reversible redox reaction by surface-confined groups [21]

Table 13 Reversible system by surface-confined groups at 25 ˚C [30]

RTAFn

I p 4

22 Γ=

υ middotmiddotmiddotmiddotmiddot Eq 90

oadsp EE = middotmiddotmiddotmiddotmiddot Eq 91

mVn

E p690

2 =∆ middotmiddotmiddotmiddotmiddot Eq 92

Where Γ isiInitial concentration of the absorbed species

However such an ideal voltammogram is seldom observed because the density of redox

species is so high in the surface-confined environment compared to in the solution and a

197

voltammogram is also influenced by the interaction among those packed redox species

A multi-electron system of the surface-confined groups is also not this case [21]

566 Hydrodynamic voltammetry (HDV) and Levich equation

Hydrodynamic methods purposely introduce convective flow to the system

using RDEs This allows the study of precise kinetics because the diffusion is under

precise control Unknown influence by mass transfer and double layer capacitance on

the electrode reaction is eliminated and steady state is quickly achieved [29 30] In

RDE method laminar flow is applied perpendicular to the electrode surface by spinning

a disc electrode at arbitrary rotation velocity (Fig 88) The rotation rate is expressed in

either rpm (rotation per minute) or angular velocity (ω) ω is defined as ω = 2πf where f

is frequency With disc rotation diffusion becomes a function of electrode rotation rate

as in Eq 93 However the limit of disc rotation rate must be set in order to avoid the

thick hydrodynamic boundary layer and turbulent flows to maintain steady state

Allowable range is between 100 rpm lt f lt 10000 rpm or 10 s-1 lt ω lt 1000 s-1 [30]

198

ω

Fig 88 RDE is rotated at ω and laminar flows applied to the electrode surface

Diffusion layer thickness and disc rotation rate [30]

612131611 νωδ minus= D middotmiddotmiddotmiddotmiddot Eq 93

δ = Diffusion layer thickness cm

D = Analyte diffusion coefficient m2s-1

ω = RDE angular velocity s-1

ν = Solution kinematic viscosity m2s-1

Under such a condition disc limiting current (Id) becomes a function of disc rotation

rate as described in Levich equation (Fig 85) [30] This allows finding the total number

of charge transferred (n)

199

Levich equation [30]

612132620 CnFADI Lminus= νω middotmiddotmiddotmiddotmiddot Eq 94

δ

nFAC= middotmiddotmiddotmiddotmiddot Eq 95

IL = Levich current A

n = Total number of charge transferred

F = Faraday constant Cmol-1

A = Electrode surface area m2

C = Bulk analyte concentration molm-3

( Please refer to Eq 93 for other terms and symbols)

Due to the steady state condition the current response in HDV becomes sigmoidal [223]

as seen in Fig 89

200

Fig 89 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH

74 Based on the real data

567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the

charge transfer rate constant

Levich equation (Eq 94) shows that Id is proportional to ωfrac12 therefore also a

function of diffusion layer thickness (Eq 95) This is true for reversible kinetics

because the electrode reaction is limited by diffusion which is a function of ωfrac12 and

therefore Id also becomes dependent on ωfrac12 at any potential However in

quasi-reversible system Id depends on diffusion but also affected by its kinetics [223]

Therefore when Id reaches the kinetic limitation for charge transfer the slope gradually

deviates from its Levich current as seen in Fig 90 [30]

201

Fig 90 Levich plots for reversible (blue) and quasi-reversible (red) systems [30]

In addition its charge transfer kinetics is altered by the potential applied As Tafel plot

(Fig 9 on p 57) presents the current is exponentially dependent on the overpotential

The electrode reaction is kinetically limited at low overpotential then gradually

becomes diffusion-limited as overpotential increases [223] This implies that Id in

quasi-reversible system is always under the influence of potential applied because the

potential alters its kinetics

When Id becomes completely free from the mass transport influence such current is

called kinetic current (IK) because Id now purely depends on only its kinetics Arbitrary

Id then defined as the sum of diffusion-limited IL and kinetically-limited IK The

double-reciprocal equation of this summation is defined as Koutecky-Levich equation

as in Eq 96

202

Koutecky-Levich equation [30]

LKd III111

+=

216132

11620

11ων

times⎟⎟⎠

⎞⎜⎜⎝

⎛+= minus nCFADI K

middotmiddotmiddotmiddotmiddot Eq 96

Id = Disc limiting current A

IK = Kinetic current A

IL = Levich current A

( Please refer to Eq 93 and Eq 94 for other terms and symbols)

By extrapolating the Koutecky-Levich plot IK can be easily estimated [30] from the

reciprocal of its y-intersect In order to plot Koutecky-Levich equation and find IK a

sequence of arbitrary Id is recorded with altering disc rotation velocity at arbitrary

potential This technique is called hydrodynamic amperometry (HDA) and it is shown

in Fig 91

203

Fig 91 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in

oxygenated 001 M PBS at pH 74 at pretreated GCE Based on the real data

Based on the data obtained by HDA Koutecky-Levich plot is plotted as in Fig 92

204

0E+00

5E-02

1E-01

2E-01

2E-01

3E-01

-002 000 002 004 006 008 010 012 014 016 018

ωˉfrac12 (sˉ

frac12)

1 IK (A-1)

- 04 V

- 06 V- 08 V

05 x 10-1

10 x 10-1

15 x 10-1

0

20 x 10-1

25 x 10-1

-1

I d(A

-1)

Fig 92 Koutecky-Levich plot for O2 reduction at pretreated GCE in oxygenated 001 M

PBS at pH 74 with applied potential ndash 04 06 and 08 V Based on the real data

With Ik from Koutecky-Levich plot and nα from CV the rate constant for arbitrary

potential (k(E)) can be calculated using Eq 97

The rate constant for arbitrary potential E [30]

lowast=nFAC

Ik K

E )( middotmiddotmiddotmiddotmiddot Eq 97

k(E) = Rate constant for the applied potential E ms-1

( Please refer to Eq 94 Eq 95 and Eq 96 for other terms and symbols)

205

568 Finding the standard rate constant (kdeg)

Finding the standard rate constant (kdeg) for ORR of each catalyst is the ultimate

aim of this long tedious cathode analysis because kdeg tells the pure catalytic power

independent of reactant diffusion and overpotential (η) k(E) can be expressed in terms of

kdeg and η as in Eq 98 therefore it is possible to estimate kdeg with known η and k(E)

k(E) in terms of kdeg and η for a forward reaction [30 181]

⎥⎦⎤

⎢⎣⎡ minusminus

deg= )(exp 0)( EE

RTnFkk Ef

α

⎥⎦⎤

⎢⎣⎡ minus

deg= ηαRT

nFk )1(exp middotmiddotmiddotmiddotmiddot Eq 98

kf(E) = Rate constant for a reduction reaction at the applied potential E ms-1

kdeg = Standard rate constant ms-1

η = Overpotential V

( Please refer to Table 11 and Table 12 for other terms and symbols)

η can be found by subtracting Edegrsquo of the reaction involved from the applied potential in

HDA n obtained by HDV is used to specify the reaction involved hence its Edegrsquo In

order to estimate kdeg Eq 98 is first converted into logarithmic expression as in Eq 99

kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

ηαRT

nFkk Efminus

minusdeg= loglog )( middotmiddotmiddotmiddotmiddot Eq 99

206

Log(k(E)) is plotted against a range of η and by extrapolating the plot log(kdeg) is obtained

from its y-intersect where η = 0 hence kdeg can be calculated (Fig 93) [181]

-12

-10

-8

-6

-4

-2

0

-10 -08 -06 -04 -02 00 02 04

η V

log kf(E)

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS NaOH PBS NaOH H2SO4 PBS

NaOH H2SO4

NaOH H2SO4 PBSH2SO4 PBS

H2SO4

NaOH PBS

log kdeg

η V

Fig 93 Log kf(E) plot for O2 reduction at various pretreated GCEs Based on the real data

569 Summary of the electrochemical techniques

CV and hydrodynamic methods are complementary techniques for qualitative

examination of redox reactions and extraction of thermodynamic and kinetic parameters

The protocol for calculating kdeg from hydrodynamic methods and nα from CV is shown

in Fig 94

207

Fig 94 The protocol for finding kinetic and thermodynamic parameters

57 SUMMARY OF THE CHAPTER

The chemistry and electrochemistry of O2 has been reviewed The mechanisms of most

widely investigated catalysts noble metals macrocyclic complexes and enzymes have

been reviewed and their suitability for application in biofuel cells has been discussed

The key physico-chemical parameters which characterise these reactions have been

identified and methods for their determination have been described

208

6 CATHODE MATERIAL AND METHOD

61 ELECTRODE FABRICATION

611 Graphite PtC and RhC electrodes

Microcrystalline graphite of grade (2-15 micron 999995 purity) was

purchased from Alfa Aesar GmbH amp CoKG 5 wt-loaded rhodium on activated

charcoal (RhC) and platinum on activated carbon (PtC) were purchased from Sigma

Aldrich CY1301 and Aradur hardener HY 1300 were purchased from Robnor resins

and they were mixed at 31 (vv) for use RDE templates were specially designed and

made on site They were made of Teflon with the dimension 4-mm ID 20-mm OD

and 15-mm height The depth of composite-holding cavity for each RDE was 25 mm

The composite was made by mixing graphite RhC or PtC with epoxy resin at 73 by

hand Composite mixture was packed into five RDE templates and cured at room

temperature for 72 hours [106]

612 Anthraquinone(AQ)-carbon composite electrodes

6121 Derivatisation of graphite with AQ

Fast red AL salt with 25 dye content and hypophosphorous acid at 50 wt

in H2O were purchased from Sigma Aldrich HPLC grade acetonitrile was purchased

from VWR international 5 mM of Fast Red AL salt solution was prepared in 50 mM

209

hypophosphorous acid 10 ml of the Fast Red solution was mixed with 2 g of graphite

The mixture was left at 5 degC for 30 minutes with regular stirring at every 10 minutes It

was finally washed thoroughly with deionised water followed by vacuum suction in

acetonitrile flow The washed derivatised AQ-carbon was left to dry overnight It was

kept in the sealed container for future use [44]

6122 Fabrication of AQ-carbon composite electrode

AQ-graphite was mixed with epoxy resin at 64 by hand The composite was

packed into five RDE templates and cured at room temperature for 72 hours

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes

6131 Information on synthesis of metallosalophen

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminocobalt (II)] and

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminoiron (II)] denoted as Co(salophen)

and Fe(salophen) respectively were kindly donated by Dr Peter Cragg (School of

Pharmacy Brighton University) They were synthesised by dissolving metal acetate in

the ligand solution which was made by condensation of 21 molar ratio of

salicylaldehyde and diethylenediamine in ethanol or methanol [224 225]

210

6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes

Analytical grade dichloromethane was purchased from VWR international In

the presence of small quantity of dichloromethane 5 wt of Co(salophen) or

Fe(salophen) was mixed and homogenised with graphite [181] After the

dichloromethane evaporated the metallosalophen-graphite mixture was further mixed

with epoxy resin at 64 [219] by hand The composite was packed into five RDE

templates and cured at room temperature for 72 hours

62 COMPOSITE RDE CUTTING

The electrode discs of all the RDE composite electrodes were cut into 1

mm-thick sections using a Buehler IsoMet Low diamond saw with a 04 mm-thick

diamond blade

63 ELECTRODE POLISHING

631 Composite RDE polishing

Composite RDEs were polished successively with SiC grinding paper of 400

1200 and 2400 grit followed by with alumina oxide slurry of particle size 1 03 and

005 microm as in sect 33 on p 122 Electrode polishing was done just before the experiment

in order to avoid air oxidation [195]

211

Fig 95 Fabricated composite RDEs

632 Glassy carbon electrode polishing

Glassy carbon electrodes (GCE) (5-mm disc diameter) were purchased from

Pine Instruments Activated carbon (AC) was purchased from Chemviron Carbon GC

cleaning solvent mixture was made by mixing AC and HPLC grade acetonitrile in a

ratio of 13 (vv) The mixture was covered and left at least for 30 minutes then filtered

before use [226] GCEs were successively polished with alumina oxide powder slurry of

1- 03- and 005-microm particles on MicroCloth pad Polished GCEs were sonicated in

filtered ACacetonitrile mixture for 10 ndash 15 minutes to remove alumina powder debris

and contaminants [226] The GCEs were then sonicated in deoxygenated deionised

water Air contact of the electrode surface was strictly avoided [195]

212

64 ENZYME RDEs

641 Membrane preparation

Cellulose acetate dialysis tubing visking 3632 was purchased from Medicell

International Ltd Sodium carbonate (Na2CO3) was obtained from Sigma Aldrich

Dialysis membrane was cut into 10x10-mm2 pieces which were boiled in 1 wv

aqueous Na2CO3 solution for 10 minutes The membrane pieces were rinsed thoroughly

with deionised water and stored [61] in PBS in a sealed container at 4degC until its use

642 Membrane enzyme electrodes

Laccase (Lac) from Trametes versicolor gt20 unitsmg-1 and lyophilised

bilirubin oxidase (BOD) from Myrothecium verrucaria 15 ndash 65 units mg-1 were

purchased from Sigma Aldrich 4 mgml-1 of Lac and BOD solution was made with PBS

respectively BOD immediately dissolved in PBS while a vortex mixer was needed to

dissolve Lac In order to attach enzyme on the GCE surface the tip of polished cleaned

GCEs was dipped and left in the enzyme solution for two hours with gentle stirring The

electrodes were then lightly washed with deionised water Pretreated dialysis membrane

was carefully placed onto the surface of the enzyme-modified GCE and fixed with

rubber O-ring

213

65 ELECTROCHEMICAL EXPERIMENTS

651 Electrochemical system

CHI 650A with single-channel and CHI 1030A with multi-channel

potentiostats were used for electrode pretreatment and electrochemical experiments CV

HDV and HAD An adjustable rotor from Pine Instrument was used to rotate a RDE

Three-electrode system (sect 342 on p 123) was employed with an AgCl reference and a

Pt counter electrodes

Fig 96 RDE experiment setting with a rotor and gas inlet

214

652 Electrode pretreatments

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes

Graphite GC Co(salophen) and Fe(salophen) electrodes were pretreated in

seven different ways in Table 14 Untreated electrodes were also prepared as controls

All the pretreatments were made in series

Treatment type 1st pretreatment 2nd pretreatment 3rd pretreatment

Type I Untreated -

Type II Acidic 01 M H2SO4

Type III Basic 01 M NaOH

Type IV Neutral 001 M PBS

Type V Base followed by Acid 01 M NaOH 01 M H2SO4

Type VI Acid followed by Neutral 01 M H2SO4 001 M PBS

Type VII Base followed by Neutral 01 M NaOH 001 M PBS

Type VIII Base Acid then Neutral 01 M H2SO4 01 M NaOH 001 M PBS

Table 14 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Concentrated sulphuric acid at 90 - 98 and sodium hydroxide tablets were purchased

from VWR international 001 M PBS was purchased from Sigma Aldrich All the

pretreatments were based on potential cycling in 100-ml electrochemical cell except

untreated ones Acidic pretreatment was performed at 5 Vs-1 between 15 V and ndash 05 V

215

in 01 M H2SO4 [105 219] for 30 minutes Basic pretreatment was performed at 5 Vs-1

between 06 V and 2 V in 01 M NaOH [218 219] for 30 minutes Neutral pretreatment

was done at 5 Vs-1 between 15 V and ndash 05 V in deoxygenated 001 M PBS pH 74

[219 220] Constant N2 flow was applied to PBS through the pretreatment In addition

pretreatments were performed in various combinations as in Table 14 Type V Base

followed by Acid Type VI Acid followed by Neutral Type VII Base followed by

Neutral and Type VIII Base followed by Acid then Neutral [219] The influence of

pretreatments on electrode surface and ORR was studied by CV HDV and HDA

6522 Pretreatments of AQ-carbon PtC and RhC

AQ-carbon PtC and RhC composite electrodes were pretreated only in 01 M

H2SO4 at 5 Vs-1 between 15 V and ndash 05 V in for 30 minutes Untreated electrodes were

also prepared as controls

653 Electrochemical experiments

All experiments were carried out under both N2 and O2 saturation 100 ml of

001 M PBS electrolyte solution was sparged with either N2 or O2 for 15 minutes before

the experiment Gas was continuously supplied through a Dreschel gas washing bottle

containing PBS throughout the experiment

CV was performed at 002 005 01 02 and 05 Vs-1 between + 09 V and ndash

09 V HDV was performed at fixed scan rate of 005 Vs-1 sweeping the potential from

+ 09 V to ndash 09 V with disc rotation velocity altered through 900 1600 and 2116 rpm

216

HDA was performed at a range of fixed potentials - 02 - 04 - 06 and ndash 08 V at disc

rotation velocity of 400 625 900 1156 1225 1600 2116 and 2500 rpm All

experiments were repeated twice or three times The sample size ranges from one to

three The median values were taken for the analysis Obtaining a set of data for each

electrode sample took from three weeks to more than one month

654 Data analysis

CV data was used to calculate nα and reaction reversibility HDV data was used

to calculate n using Levich equation (Eq 30 on p 61) Edegrsquo for ORR involved at pH 7

25 degC was specified by n value as in Table 15

n Redox type Redox potential vs AgAgCl

1 Edegrsquo(O2H2O2) 0082 V

2 Edegrsquo(O2H2O) 0616 V

Table 15 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC [24]

η was calculated by subtracting Edegrsquo (ORR) from the potential applied in HDA HDA

data was used to estimate kf(E) at each applied potential using Koutecky-Levich plot (Eq

30 on p 61 and Fig 92 on p 204) N2 data was subtracted from O2 data as background

Other parameters required for Koutecky-Levich plot were set as in Table 16 All

parameters assume the condition of 001 M PBS at 24 degC

217

Parameter Value Reference

O2 diffusion coefficient 23 x 10-9 m2s-1 [227]

O2 concentration 123 molm-3 [158]

Kinematic viscosity (ν) 884 x 10-7 m2s-1 [30]

Table 16 Parameter values for Koutecky-Levich equation

kdeg was estimated by plotting log(kf(E)) vs η (Eq 99 and Fig 93 on p 206) kdeg for

various and pretreated cathodes were compared The best catalyst shows the largest kdeg

The procedure for finding kdeg is summarised in (Fig 94 on p 207)

218

7 CATHODE RESULTS AND DISCUSSION

71 AIMS OF THE CATHODE STUDIES

This section describes the electrochemical characterisation of various modified

electrodes which were considered as candidate cathodes for the biofuel cells Various

carbons were used as electrode materials in all cases The electrochemical behaviour of

carbon depends strongly on history and also can be radically altered by pre-treatment

The effects of electrochemical pretreatments (ECPs) on the kinetic parameters will be

described for both glassy carbon and graphite composites The cathode modifiers

considered in this section include noble metals (Pt Rh) inorganic Schiff bases

(Co(salophen) Fe(salophen)) an organic modifier (anthroquinone) and the enzymes

bilirubin oxidase (BOD) and laccase (Lac) In each case of non-biological catalysts the

effects of ECPs were also quantified using cyclic voltammetry (CV) and hydrodynamic

methods with rotating disc electrodes (RDEs)

CV allowed the investigation of

Capacitance from the double layer region

Qualitative description of the mechanism such as number of discrete reaction

steps reversibility and surface confinement

nα the number of electrons transferred up to and including the rate limiting step

(RLS)

219

while hydrodynamic methods were used to quantify

n the total number of electrons transferred

kdeg the heterogeneous standard rate constant

Epc2(HDV) the half-wave reduction potential in HDV

The data was taken under both N2 and O2 to allow characterisation of the effects of

electrochemical pretreatment and its effects on the kinetics of O2 reduction The best

candidate catalytic cathode was determined according to kdeg nα and Epc2(HDV)

72 INFLUENCE AND EFFICACY OF ECPS

721 Glassy carbon electrodes (GCEs)

7211 Untreated GCEs

Voltammetry curves of untreated GCEs at pH 74 in the presence of N2 and O2

are shown in Fig 97

220

Fig 97 CVs of untreated GCEs at pH 74 in 001 M PBS in the presence of (i) N2 (ii)

O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Untreated GCEs did not show any peak in the absence of O2 (Fig 97(i)) indicating that

there was no electrochemically active redox species on the electrode surface and in the

solution CV was in the presence of O2 (Fig 97(ii)) showed a peak at ndash 08 V but

without a reverse anodic current This indicates the presence of catalytic activity

however it occurred at only high overpotential and is therefore not efficient for ORR

This result is consistent with previous work [215 216]

7212 Influence of electrochemical pretreatments on GCE

GCEs were pretreated in various media in order to improve ORR efficiency It

has been reported that ECPs activate the electrode surface by introducing

electrochemically active functional groups and that this improves electrochemical

(i) N2

-3E-05

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E V

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(ii) O2

-5E-04

-4E-04

-3E-04

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1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

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cm

-2

221

reversibility and [105 218] and the electron transfer rate of inner-sphere catalysis [228]

Fig 82 shows the voltammetry curves in the absence of O2 for various pretreated GCEs

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-60E-05

-50E-05

-40E-05

-30E-05

-20E-05

-10E-05

00E+00

10E-05

20E-05

30E-05

40E-05

50E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH

Fig 98 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of

O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1 Arrows indicate the

peak position (i) includes CVs for all the pretreated GCEs and (ii) excludes

PBS pretreatments for convenience

222

As the figure shows any pretreatment resulted in an increase in capacitance and

introduced characteristic voltammetric peaks which were not observed in the untreated

GCEs In all cases the peak current was linearly proportional to υ indicating that the

pretreatments led to surface-confined redox active functional groups most likely to be

quinone groups That ECPs give rise to surface quinones is well established [105 218

220 229 230] The Epc shifted with υ and was separated from Epa hence redox

reactions by these surface quinone groups are quasi-reversible The effect of

pretreatments however varied by the type of medium used in pretreatments

Above all PBS-related pretreatments gave qualitatively different results which

were characterised by a great increase in average capacitance calculated from CVs in

001 M PBS at pH 74 at 50 100 and 200 mVs-1 in the double layer region of the CV at

04 V Summary results are shown in Fig 82(i) and Fig 99

223

GCE Capacitance vs Pretreatment

0100200300400500600700800900

1000

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS

NaOH PBS

NaOH H2SO4

PBS

Pretreatment

Capa

cita

nce

microF

cm-2

Fig 99 The capacitance of GCEs in 015 M NaCl buffered with 001 M phosphate to

pH 74 Error bars represent the standard deviation (n = 3)

The average capacitance of the untreated GCEs was 76 microFcm-1 and is within the typical

ranges reported hitherto [221] while PBS pretreatment increased the capacitance to 700

- 800 microFcm-1 A similar phenomenon was reported by Nagaoka and by Swain [215

216] There are two possible reasons for this (i) PBS treatment caused the

microfractures of GC and increased the electrode area and therefore double layer [105

215 216 218] andor (ii) attached the electrochemically inactive charged surface group

such as phenol and carboxylate [216 229]

In the pretreatment involving NaOH the carbon dissolution was observed This

is harsher pretreatment so erodes the electrode and makes the electrode surface more

porous [216 218] NaOH also forms unstable oxide film and eventually removes

graphite oxide [229] and surface impurities [218] However the resultant capacitance

224

was unexpectedly smaller than that of sole PBS pretreatment Therefore NaOH

pretreatment increased the capacitance by increasing the exposed electrode area [218

231] while PBS pretreatment increased capacitance by attaching redox inactive charged

surface groups The largest capacitance after the double pretreatment involving NaOH

and PBS was therefore derived from the layer of charged groups attached on the

electrode surface area made larger by NaOH pretreatment

H2SO4 pretreatment on the other hand resulted in a smallest capacitance

increase The electrode behaviour was also slightly altered from that of other

PBS-related pretreatments when the pretreatments involved H2SO4 together The

capacitance became smaller as shown in Fig 99 and the peak positions were shifted to

more positive potential side (Fig 82(i)) To investigate the causes of this phenomena

the voltammograms during such pretreatments were studied as Fig 100 below

225

Fig 100 Overlaid CVs during various pretreatments for GCEs involving 01 M H2SO4

alone (red) 001 M PBS at pH 74 alone (green) and both pretreatments (pink)

It is clearly shown that either pretreatment resulted in distinctive peaks for the

attachment of active surface quinone groups However the peak potentials were

different when in sole H2SO4 or PBS-related pretreatments This is because as proposed

by Nagaoka those quinone groups were present in the different chemical environment

leading to different behaviours [216] H2SO4 pretreatment attached fewer inactive

surface oxygen groups while PBS pretreatment attached more Therefore H2SO4

pretreatment led to the smaller capacitance change than PBS pretreatment This implies

the presence of different electrode surface conditions hence quinones present in such

different environment behave differently showing different peak positions The

behaviours of surface quinone groups depends on the conditions of the pretreatment

involved [229 230 232] This fact can also explain the phenomena caused by the

226

double pretreatment with H2SO4 and PBS as shown in Fig 82(i) More quinone groups

had been attached by H2SO4 pretreatment in advance therefore only smaller area than

usual became available for PBS pretreatment to stably attach their charged oxygen

surface groups This is the most likely causes of the reduced capacitance and different

peak positions in such a double pretreatment compared to other PBS-related

pretreatments Furthermore quinone peak positions should be close to those found for

the H2SO4 pretreatment alone because quinone groups attached by preceding H2SO4

pretreatment dominate the electrode surface and should be have more influence on the

electrochemistry However they also resulted in larger capacitance because the

subsequent PBS pretreatment attached inactive oxygen surface group which led to an

increase in fixed charge density This is a typical phenomenon of synergetic effects as

already seen in the double pretreatment with NaOH and PBS

On the other hand voltammetric curves for the double pretreatment of NaOH

and H2SO4 did resemble to those by sole NaOH pretreatment Similar phenomena were

observed between the double pretreatment with NaOH and PBS and the triple

pretreatment with NaOH H2SO4 and PBS It seems that preceding NaOH pretreatment

overwhelmed the subsequent H2SO4 pretreatment although some influence by H2SO4

can be observed in terms of capacitance as shown in Fig 99 This implies that the

preceding NaOH pretreatment made most of the electrode area not available for

subsequent H2SO4 pretreatment However if NaOH pretreatment creates micropores in

the GC protons from subsequent H2SO4 pretreatment can still penetrate such

micropores These micropores are accessible for protons but not most ions [216]

227

7213 Effect of GCE pretreatment on ORR

Voltammograms for ORR at various pretreated GCEs at pH 74 are shown in

Fig 101 below

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO4

01M NaOH01M NaOH + 01M H2SO4

001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS

01M NaOH + 01 M H2SO4 + 001M PBS

Fig 101 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

In the figure any pretreated GCE shows larger cathode peaks between ndash 03 and ndash 05 V

without reverse anodic current They were due to O2 reduction by surface quinone

groups whose peaks were observed at ndash 01 V in Fig 82(i) in the absence of O2 This

indicates that ORR at any pretreated GCEs is regulated by ECrsquo mechanism [30] Ipc was

linearly proportional to radicυ in all cases indicating the diffusion-limited charge transfer

228

However untreated GCE was catalytic only at high overpotential since Epc2(HDV) = ndash 08

V This is compatible to the observation made by Tammeveski [233] It was seldom

catalytic since n = 102 plusmn 011 and nα = 059 plusmn 004 This single-electron ORR implies

the presence of O2־ disproportionation from which little power can be extracted [233]

kdeg was could not be calculated because the standard Koutecky-Levich plot could not be

obtained since it did not show a normal linear slope In contrrast all the other

pretreatments improved ORR as seen in Fig 101 They showed large cathodic peaks at

smaller overpotentials PBS-related pretreatments in particular increased the catalytic

current by gt 15 times and positively shifted Epc2(HDV) by gt 300 mV relative to that of

untreated GCEs Similar phenomena were reported elsewhere [220 234] All the GCE

pretreatments resulted in an increase in both nα and n The increase in kdeg accompanied

by an increase in nα was also observed as a general tendency as shown in Fig 102

229

0

2

4

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

kordm

ms

-1

nα 059 107 094 106 168 278 174 178

kordm ms-1 000E+00 715E-09 529E-09 631E-08 131E-07 279E-07 654E-08 113E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

- nα

kdeg ms-1

Untreated H2SO4 NaOHNaOHH2SO4

H2SO4PBS

NaOHPBS

NaOHH2SO4PBS

Fig 102 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Above all the H2SO4-PBS double pretreatment was most effective giving kdeg = 279 x

10-7 plusmn 507 x 10-9 ms-1 and Epc2(HDV) = - 039 plusmn 002 V However its nα was calculated

to be 278 while n = 185 This is an unusual result It might imply the presence of serial

ORR pathway in which H2O2 is further reduced or it is simply impossible to obtain

correct parameters by using Eq 88 on p 193 for ORR at pretreated GCEs due to their

excessively narrow peak width Pretreatment of GCE was proved to increase kdeg of ORR

but how this improves its catalysis has been uncertain for a long time between two

hypotheses whether they mediate electron transfer or they alter the adsorption of O2

and O2[233 232 220] ־ The latter or both is more likely because as Fig 102 shows

PBS-related pretreatments increased the capacitance which was derived from redox

inactive surface oxygen groups yet made significant contribution to an increase in kdeg It

230

is therefore natural to deduce that the largest kdeg and nα were the consequence of

synergetic effect derived from different surface oxygen groups attached by H2SO4 and

PBS

722 Graphite composite electrodes

7221 Untreated graphite composite electrodes

Graphite composite electrodes are different from GCEs They are made of

graphite particles which are more porous and permeable to the solution and gas This

practically results in larger electrode surface area therefore becomes more subject to

facile adsorption and surface modification This leads to the different surface conditions

and electrode behaviours from GCEs as proposed by Sljukic [232] CVs of untreated

graphite composite electrodes at pH 74 in the presence of N2 and O2 are shown in Fig

103 together with those of untreated GCEs

231

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

Fig 103 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl Small peaks on graphite electrodes are indicated by arrows

Different electrode behaviour is noticeable especially in nitrogenated solution because

untreated graphite composite electrodes showed small peaks at + 100 mV and - 100 mV

as indicated by arrows No peak was present fo untreated GCE This indicates the

presence of some reactive surface quinone groups on graphite composite without

pretreatment This is because graphite composite electrodes have more reactive edge

plane graphite exposed and the porous electrode structure allows larger electrode area to

be exposed There was however little difference in averaged capacitance between

graphite composite and GCEs This indicates that an increase in capacitance does not

necessarily correlate to an increase in the surface area as proposed by Regisser [235]

Voltsmmograms for ORR at untreated GC and graphite composite electrodes resembles

each other though for the latter the reduction peak is around 50 mV more positive It can

232

be seen that graphite composites are slightly more catalytic toward ORR presumably

because of the intrinsic quinone groups derived from the graphite electrode surface

7222 Influence of electrochemical pretreatments on graphite composite electrodes

Cvs for the graphite composite electrodes differed from those of GCEs Peaks

were observed in all graphite cases including untreated ones as shown in Fig 104

233

-20E-04

-15E-04

-10E-04

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-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

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Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

(ii)

01M H2SO401M NaOH01M NaOH + 01M H2SO4Untreated

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBSPBS

(iii)01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS001M PBS

Fig 104 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M

nitrogen-sparged PBS between + 09 and - 09 V vs AgAgCl (i) All

pretreatments at 005 Vs-1 (ii) Non-PBS-related pretreatment and (iii)

PBS-related-pretreatment CVs at 002 Vs-1

The capacitance became larger after pretreatments Any multi-pretreatment involving

NaOH and H2SO4 resulted in the largest capacitance which was 20 times larger than

234

that of untreated graphite composite electrodes The capacitance increase was also

larger than that of any pretreated GCEs except for PBS-pretreated ones PBS

pretreatment led to the smallest capacitance increase in graphite composite electrodes in

contrast to the GCEs Instead H2SO4 pretreatment led to the largest capacitance

increase Moreover this outcome was found to correspond to a specific proportional

relation between Ip and υ Ip of electrodes involving any pretreatment in PBS NaOH or

their combinstions were linearly proportional to radicυ This can only arise from on or more

diffusion limited step or steps in the redox reactions of the surface groups most likely

proton diffusion though a porus film or to microscopic porous environments Surface

oxygen groups have previously been reported to involve involve proton uptake and this

leads to a broad peak [216 230] H2SO4-pretreated electrodes showed Ip proportional to

υ and the redox reaction is independent of proton diffusion

These phenomena show that different pretreatments provide different chemical

environment for active quinone groups leading to different quinone behaviours and

voltammetry peaks [229 230 232] In the multi-pretreatment with NaOH and H2SO4

synergetic effects became obvious Its redox reaction was diffusion-limited and showed

voltammograms with two partially resolved sets of peaks yet its peak still partially

overlaps with that of diffusion-independent H2SO4-pretreated electrodes as seen in Fig

104(i) When this double-pretreatment was further followed by PBS pretreatment the

peak derived from quinones attached by H2SO4 disappeared This implies that

subsequent PBS pretreatment suppressed the activity of proton-independent quinones

and the effect by H2SO4 The similar phenomenon was observed when electrodes were

pretreated doubly by H2SO4 and PBS (Fig 104(ii)) Every case presented

235

quasi-reversible redox systems because peak potentials shifted with scan rates Small

multiple peaks were present in slower scan rates merged into one broad peak at faster

scan rate as the contribution of capacitance charging and increased iR drop obscured

voltammetic detail

7223 The effect of graphite composite electrode pretreatment on ORR

ORR at pretreated graphite composite electrodes was found to be less effective

than for pretreated GCEs Only NaOH-related pretreatments were effective (Fig 105)

enough to achieve two-electron ORR Untreated graphite composite electrodes were

catalytic only at high overpotential despite the presence of surface quinone groups PBS

pretreatment was hardly effective in contrast to the results obtained for GCEs

Electrochemical pre=treatment in H2SO4 and its double pretreatment with PBS reduced

Epc2(HDV) by 40 ndash 90 mV however the electrodes showed poor catalytic performance nα

lt 05 and n lt 2 consistent with the initial electron transfer still being rate determining

236

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 105 Overlaid CVs of ORR at various pretreated graphite composite electrodes

at pH 74 in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005

Vs-1

Pretreatment of the graphite composites with just H2SO4 showed a single merged peak

at slower scan rate while they showed an addition prewave as indicated by arrows in

Fig 106 at faster scan rate

237

-16E-03

-14E-03

-12E-03

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

-1 -08 -06 -04 -02 0 02

E V

J Acm-2

50 mV100 mV200 mV500 mV

Fig 106 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at

pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various

scan rates 50 100 200 and 500 mVs-1

Coupled with the fact of n asymp 1 this prewave is due to the rate-limiting slow formation of

semiquinone radicals [233] A semiquinone radical is formed when one electron is

transferred to a quinone group and its formation is necessary to mediate ORR as shown

in Eq 100

238

middotmiddotmiddotmiddotmiddot Eq 100

[233] middotmiddotmiddotmiddotmiddot Eq 101

Q = Surface quinone group

O2־ = Semiquinone radical

Any NaOH-related pretreatment on the other hand showed a prewave around ndash 035 V

at slower scan rate as shown in Fig 107 but this merged into a broad peak at faster scan

rate

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

01M NaOH01M NaOH + 01M H2SO4 01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 107 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

239

Ipc was linearly proportional to radicυ except for the triple pretreatment which was difficult

to define due to the ambiguous peak positions over different scan rates The prewave

was due to the slow reduction of O2 into O2־ (Eq 101) rather than semiquinone radical

formation (Eq 100) as seen in H2SO4-pretreated electrodes They had n asymp 2 and nα asymp 1

hence they catalysed two-electron ORR Those pretreatments positively shifted

Epc2(HDV) by 173 plusmn 26 mV on average from ndash 730 mV at the untreated graphite

composite electrodes The largest catalytic activity was achieved by doubly pretreated

electrodes with NaOH and H2SO4 with kdeg = 382 x 10-7 plusmn 32 x 10-8 ms-1 (Fig 108)

0

2

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

35E-07

40E-07

45E-07

kordm

ms

-1

nα 042 035 080 114 045 040 101 072

kordm ms-1 000E+00 000E+00 233E-07 382E-07 000E+00 000E+00 132E-07 172E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBSUntreated H2SO4 NaOH

NaOHH2SO4

H2SO4

PBS NaOHPBS

NaOHH2SO4

PBS

- - - - nα

kdeg ms-1

Fig 108 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

240

723 Platinum on carbon composite electrodes (PtC)

Noble metals are popular ORR catalysts [172 173] with Pt showing the highest

catalytic power [165 166 171 172] It was reported that Pt has a 98 O2-to-H2O

conversion rate [174] However for PtC composite electrodes the voltammetry was

dominated by showed solution resistance both in the presence and absence of O2 and

therefore were not either catalytic or conductive H2SO4 pretreatment was not at all

effective This is probably due to the large electronic resistance of activated carbon itself

[204] as well as intrinsic resistance of composite conductors

724 Rhodium on carbon composite electrodes (RhC)

Rhodium is a platinum group metal and has been reported as a good ORR

catalysis after Pt and Ir [172] Voltammograms for untreated RhC composite electrodes

only showed the capacitance but no redox peaks in nitrogenated solution The

capacitance decreased by 80 for every ten-fold increase in the scan rate Non-faradic

current is normally linearly proportional to υ however it exponentially decreased with υ

in the untreated RhC composite electrodes It did not show any peak in the presence of

O2 and was not catalytic toward ORR (Fig 110(i)) H2SO4-pretreated RhC composite

also did not show any voltammetric peaks Its capacitance was roughly x30 larger than

that of untreated electrodes and again exponentially decreased with an increase in υ

ORR catalysis was however present (Fig 110(ii)) therefore it is reasonable to deduce

that H2SO4 attached the surface quinone groups but the large capacitance charging

prevented voltammetic observation of quinones

241

(i) Untreated

-16E-03

-12E-03

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-40E-04

00E+00

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

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cm

-2

N2O2

(ii) H2SO4

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

10E-03

-1 -08 -06 -04 -02 0 02 04 06 08 1

E VJ

Ac

m-2

N2O2

Fig 109 CVs of (i) untreated and (ii) H2SO4-pretreated RhC composite electrodes at

pH 74 in 001 M PBS in the presence of N2 and O2 at 005 Vs-1 between + 09

and -09 V vs AgAgCl

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR

Untreated RhC composite electrodes did not show ORR catalysis however a

steep decrease in cathodic current was observed without any peak as the potential

decreases This steep current decrease was probably caused by O2 adsorption on the Rh

particle surface H2SO4-treated RhC on the other hand was found to be catalytic with n

= 177 and nα = 068 plusmn 011 A reduction prewave was observed at around ndash 05 V and

this merged into a broad peak at faster scan rates This prewave was due to ORR by

surface quinone groups attached by H2SO4 because it overlaps with the peak observed

in H2SO4-treated graphite composite electrodes as shown in (Fig 110) This prewave

242

therefore indicates the slow first electron transfer to form O2־

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

-1 -05 0 05 1

E V

J Acm-2

RhC H2SO4

Graphite H2SO4

Fig 110 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

O2־ was probably further reduced to H2O2 by Rh particles because n = 177 plusmn 011 The

catalytic activity was much larger compared with H2SO4-pretreated graphite composite

electrodes as shown in Fig 110 with kdeg =257 x 10-7 plusmn 230 x 10-8 ms-1

243

725 Co(salophen) composite electrodes

7251 Untreated Co(salophen) composite electrodes

Untreated Co(salophen) electrode showed couples of distinctive peaks Epc(1) =

053 Epc(2) = -024 Epa(3) = -016 and Epa(4) = 016 V as shown in Fig 111(i) These

peaks correspond to stepwise cobalt redox reactions Co3+2+ and Co2+1+ respectively

The peak potentials hardly changed over different scan rates consistent with previously

published results [181] Similar peaks have been observed in deoxygenated organic

solvents [174 181] as well as for Co(salen) [181]

(i) N2

-2E-05

-1E-05

0E+00

1E-05

2E-05

3E-05

4E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

2E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fig 111 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

244

In the presence of O2 it showed a reduction peak at ndash 04 V as shown in Fig 111(ii)

ORR potential was shifted by approximately 300 mV relative to that of untreated

graphite composite This result reasonably compatible to the result reported by Ortiz

[181] At this potential O2 forms an adduct with Co2+(salophen) and then is reduced to

O2־ while Co2+ undergoes one-electron oxidation to Co3+ [170] To be catalytic Co3+

formed here must be reduced back to Co2+ to reduce O2־ further into H2O2 [181] or a

binuclear complex must be formed with another Co2+ [170] Untreated Co(salophen)

composite electrodes were however not strongly catalytic due to n = 138 plusmn 009 and nα

= 055 plusmn 012 Ip was linearly proportional to radicυ and the peak potentials Epc(2) and Epa(3)

varied only by plusmn 8 over different five scan rates This indicates nearly reversible

multi-electron transfer redox system of Co(salophen) composite electrodes

7252 Influence of electrochemical pretreatments on Co(salophen) composite

There was a particular redox pattern found in voltammetry waves according to

the type of pretreatments (Fig 112)

245

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 112 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in

001 M nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

NaOH-pretreatment showed similar voltammetric faradaic features as those in untreated

electrodes (Fig 112) but increased the capacitance by six times relative to that of

untreated electrodes The size of capacitance was 382 microFcm-1 and approximately equal

to that of NaOH-pretreated graphite composite electrodes This implies that NaOH

attached electrochemically inactive surface oxygen groups and the attachment of active

surface quinone groups were not clearly confirmed

PBS-pretreatment on the other hand attached quinone groups because Epa asymp 04 V at

which similar peaks were observed in PBS-pretreated graphite composite (Fig 104(iii)

p 233) Their cathodic peaks were however broader because they merged into peaks

246

for Co-redox processes The effect of PBS pretreatment was much smaller compared to

that of H2SO4-related pretreatment in the deoxygenated solution Single NaOH

treatment and any PBS-related pretreatment showed Ip linearly proportionally to radicυ

while any H2SO4-related pretreatment but without involving PBS-pretreatment showed

a proportional relation with υ This indicates that H2SO4 and PBS or NaOH

pretreatments lead to different surface redox mechanism Peaks due to H2SO4-related

pretreatments were prominent and are indicated by a red circle in Fig 112

7253 The effect of Co(salophen) composite electrode pretreatment on ORR

Untreated electrodes were not catalytic Parameters for H2SO4-pretreated

Co(salophen) composite found to have n asymp 1 and nα le 1 although they look catalytic in

Fig 113 reducing its ORR overpotential by 400 ndash 500 mV compared to GCEs This

inconsistent result arose because it was impossible to find the correct parameter using

the conventional calculation using Eq 88 on p 193 and need more sophisticated

method such as a simulation probramme The other pretreatments were on the other

hand found strikingly effective toward ORR as shown in Fig 113

247

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

40E-04

60E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 113 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Such pretreated Co(salophen) electrodes showed Epc asymp - 03 V having reduced the

overpotential against unpretreated Co(salophen) by 100 mV and 300 mV against

untreated graphite composite electrodes ORR was synergistically catalysed by Co2+ and

surface semiquinone radicals [181 233] They showed n asymp 2 and nα gt 07 hence they

catalysed two-electron ORR Ipc was linearly proportional to radicυ hence the redox

reactions were diffusion limited except for the double pretreatment with NaOH and

H2SO4 Large catalytic activity was achieved by sole NaOH and by the double

pretreatment with NaOH and H2SO4 or the combination of H2SO4 and PBS Among

them sole NaOH pretreatment was revealed to be most effective as shown in Fig 114

and showed kdeg = 119 x 10-5 plusmn 118 x 10-6 nα = 083 plusmn 014 and Epc2(HDV) = 037 V

248

0

1

2

nα

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

kordm

ms

-1

nα 055 028 083 074 068 091 071 078

kordm ms-1 000E+00 000E+00 119E-05 747E-06 271E-06 684E-06 296E-06 587E-06

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

Fig 114 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite

electrodes

One possible reason for this largest catalytic activity by NaOH pretreatment is that

NaOH might attach more reductive surface OH groups [218] which became involved in

reducing back Co3+ into Co2+ during ORR [181]

726 Fe(salophen) composite electrodes

7261 Untreated Fe(salophen) composite electrodes

Untreated Fe(salophen) electrodes showed a couple of distinctive peaks Epa = -

024 plusmn 001 V and Epc = - 036 plusmn 001 V These peaks are due to Fe3+2+ redox reaction as

249

shown in Fig 115(i) The voltammetry curve and redox peaks are reasonably similar to

those of Fe(salen) reported [224]

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

(ii) O2

-6E-04

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

Fig 115 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74

in 001 M PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and

- 09 V vs AgAgCl

However the redox system is not perfectly reversible here because ∆Ep slightly

increases with an increase in the scan rate and its Ip was linearly proportional to radicυ It is

therefore a quasi-reversible system Untreated Fe(salophen) electrodes was not as

catalytic as Co(salophen) electrodes Epc was ndash 079 plusmn 007 V and little different from

that of untreated graphite composite electrode as shown in Fig 115(ii) although Ipc was

slightly bigger The low ORR catalytic activity of Fe(salophen) was due to the low ORR

rate of Fe itself (Fig 65 on p 164) [172]

250

7262 Influence of electrochemical pretreatments on Fe(salophen) composite

The effect of pretreatments to Fe(salophen) was striking They resulted in

additional voltammetric features due to the Fe redox peaks at Epc (Fe) = - 03 V and Epa

(Fe) = - 02 V and by attaching the surface quinone groups at Epc (Q) = 015 V and Epa (Q) =

02 V as shown in Fig 116(i)

251

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

60E-05

80E-05

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

Untreated01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS001M PBS

-1E-04

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

1E-04

1E-04

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

Fig 116 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1 (i) All

pretreatments (ii) Non-PBS-related pretreatments and (iii)

PBS-related-pretreatments CVs

Non-PBS-related pretreatments (Fig 116(ii)) showed redox peaks clearly however the

252

two cathodic peaks merged into a broad peak at faster scan rate than 100 mVs-1

indicating quasi-reversible surface quinone redox reactions Only a broad merged

reduction peak was observed for all PBS-related pretreatments (Fig 116(iii)) at all scan

rates

Any PBS-related pretreatments resulted led to a broad cathodic peak (from + 01 V to

approximately ndash 03 V) made up of both Fesalphen reduction and reduction of surface

confined quinones arising from the electrochemical pretreatment PBS pretreatment

always greatly increases the surface area leading to micro fissures and consequently a

greater variety of microenvironments for electrochemically generated quinone

functionality

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR

Most were found to be only weakly catalytic (Fig 117) since either n or nα or

both of them were lt 1 Only double-pretreatment with NaOH and H2SO4 led to

sufficient ORR catalysis with n = 185 plusmn 017 nα = 079 plusmn 023 kdeg = 112 x 10-7 plusmn 771 x

10-9 ms-1 and Epc2(HDV) = - 057 V The double pretreatment showed a prewave at ndash 035

V and it disappeared at faster scan rate indicating a slow single-electron transfer to O2

Ipc was linearly proportional to radicυ hence the catalysis was diffusion limited

253

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS

Fig 117 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

727 Anthraquinone (AQ) composite electrodes

Anthraquinone-grafted graphite electrodes were pretreated with H2SO4 Both

untreated and H2SO4-pretreated electrodes showed couples of peaks (Fig 118(i)) one of

which Epc(AQ) was compatible with Em = - 049 reported by McCreery [44] These

peaks were therefore derived from anthraquinone chemically attached to the graphite

254

(i) N2

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

-1 -05 0 05 1

E V

J A

cm

-2

Untreated FRE01M H2SO4 FREUntreated Graphite

(ii) O2

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

-1 -05 0 05 1

E VJ

Ac

m-2

Untreated FRE01M H2SO4 FREUntreated Graphite

Fig 118 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl CVs of untreated graphite electrodes are also shown together

The other couple of peaks Epc(SQ) and Epa(SQ) was observed at - 003 plusmn 003 V and +

020 plusmn 003 V respectively They were due to quinone groups on the carbon surface

H2SO4-pretreatment did not alter the voltammogram pattern but only increased the

capacitance by x3 and enhanced the peak current Both electrodes showed Ip linearly

proportional to radicυ hence the redox reactions involve proton diffusion ORR by those

electrodes is shown in Fig 118(ii) together with the voltammogram of graphite

composite electrodes Both untreated and pretreated AQ-grafted composite electrodes

positively shifted Epc by 60 and 230 mV respectively relative to that of untreated

graphite composite electrodes However their voltammetric curves resembled those for

untreated graphite composite electrodes Besides this their Ip dropped by 20 relative

255

to untreated graphite composite electrodes and showed n asymp 1 and nα asymp 05 Therefore

either was not catalytic in this study This outcome does not correspond to the finding

by Manisankar who confirmed the presence of ORR catalysis at AQ-carbon paste

electrodes at pH 7 with a reduction prewave at ndash 04 and a peak at ndash 07 V [193] To the

contrary this result fits to the data presented by Schiffrin who observed ORR

suppression at AQGC electrodes at pH lt 7 He reported that voltammetry curves for

ORR at GC and GCAQ became similar at this pH hence implied the possibility of

disproportionation of semiquinone intermediates [194]

728 Lac and BOD enzyme membrane electrodes

In the absence of O2 neither Lac nor BOD showed any peaks in their cyclic

voltammograms However they showed ORR catalytic activity even in the absence of

mediator (Fig 119) DET is therefore possible as already confirmed by many [183

186-188] Carbon surface groups [195] also might mediate electron transfer [188]

256

-14E-04

-12E-04

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

-1 -09 -08 -07 -06 -05 -04 -03 -02 -01 0

E V

J Acm -2

LacBOD

Fig 119 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated

PBS at 005 Vs-1 between 0 and -09 V vs AgAgCl

Ipc was linearly proportional to radicυ therefore ORR was diffusion limited Epc were -054

plusmn 002 and ndash 067 plusmn 006 V at Lac and BOD respectively They reduced ORR

overpotential by 120 and 250 mV relative to that of GCEs however the Epc shifted with

υ hence they were quasi-reversible It is known that MCOs catalyse four-electron ORR

[175 184 236] however n = 005 and nα asymp 1 in this study This is probably due to the

difficulty in electrochemical measurement of enzyme catalysis It is however possible

to find nα because the rate-limiting step is the single electron transfer between the

electrode and the blue copper atom of the enzyme Assuming this kdeg was calculated for

four-electron ORR at the enzyme electrodes and the result is shown in Fig 120

257

0

1

2

nα

00E+00

40E-09

80E-09

12E-08

16E-08

20E-08

kordm

ms

-1

nα 077 081

kordm ms-1 134E-08 168E-09

Lac Bod

Fig 120 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Lac had kdeg = 134 x 10-8 plusmn 251 x 10-9 while BOD had kdeg = 168 x 10-9 plusmn 271 x 10-10

Lac electrodes showed better ORR efficiency despite of non-optimal pH condition Lac

is most active at pH 5 while BOD at pH 7 The amount of enzyme loaded on the

electrode surface also might have affected the catalytic activity It was however difficult

to control the loading because the enzyme was attached by dip coating Using mediators

could have also improved the ORR efficiency Many authors used mediators such as

ABTS [62 66] and Os-pyridine complexes linked to conductive polymer [97 237] to

assist the electron transfer between the electrode and enzyme

73 MOST EFFICIENT ORR CATALYSTS

The kinetic and thermodynamic parameters (kdeg nα Epc2(HDV)) were organised

258

by magnitude and are summarised in figures 25-27 and tables 1 amp 2 below For each

parameter the best five candidates were selected to present the effects of pretreatment

and their relation with each parameter The best three were chosen from them on the

basis of the parameter size and the number of top-ranked parameters they were

occupying kdeg was given the highest priority for selection process

731 Best five electrodes for nα

Pretreated GCEs were found to have prominent nα (Fig 121)

00

05

10

15

20

25

30

Electrode type

n α

nα 278 178 174 168 114

GC H2SO4-PBS GC NaOH-H2SO4-PBS

GC NaOH-PBS GC PBS GP NaOH-H2SO4

Fig 121 The best five electrodes for nα

The GCEs doubly pretreated with H2SO4 and PBS was found best with nα = 278 Its kdeg

was 279 x 10-7 ms-1 and also biggest among pretreated GCEs Correlation between nα

259

and kdeg has not been confirmed since it is possible for the intial electron transfer to be

accelerated whilst still remaining the RDS

732 Best five electrodes for kdeg

Top six kdegs were dominated by pretreated Co(salophen) composite electrodes

(Fig 122)

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

Electrode type

kdeg

ms

-1

kordm ms-1 119E-05 747E-06 684E-06 587E-06 296E-06

CoS NaOH CoS NaOH-H2SO4

CoS H2SO4-PBS CoS NaOH-H2SO4-PBS

CoS NaOH-PBS

Fig 122 The best five electrodes for kdeg

PBS pretreatment was found to suppress kdeg in Co(salophen) composite electrodes Clear

correlation between kdeg and nα has not yet confirmed however it was found that

favourable kdeg tends to lower overpotential

260

733 Best five electrodes for Epc2(HDV)

Although it is not an exact correspondence some correlation was observed

between lower overpotential and larger kdeg or lower overpotential and larger nα but not

between kdegand nα A range of pretreated Co(salophen) composite electrodes which

showed largest kdeg and a pretreated GCE which showed the largest nα were found to be

dominating low overpotential (Fig 123)

-040

-039

-038

-037

-036

-035

Electrode type

E pc

2(H

DV)

V

Epc2(HDV) -037 -037 -039 -039 -040

CoS H2SO4-PBS CoS NaOH CoS NaOH-H2SO4

GC H2SO4-PBS CoS NaOH-PBS

Fig 123 The best five electrodes for Epc2(HDV)

734 Best three candidates for ORR catalytic electrodes

Pretreated Co(salophen) composite electrodes were the best candidates for

ORR catalysis because of largest kdeg and lowest overpotential Top three catalytic

261

cathodes were listed in Table 17

kordm ms-1 Epc2(HDV) V nα

Co(salophen) NaOH 119E-05 -037 083

Co(salophen) NaOH-H2SO4 747E-06 -039 074

Co(salophen) H2SO4-PBS 684E-06 -037 091

Average 875E-06 -037 083

SD 278E-06 001 009

Table 17 Three candidates for catalytic cathodes for ORR

NaOH-pretreated Co(salophen) was found to be most efficient ORR catalytic cathode

because it has the largest kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 Epc2(HDV) = - 037 V and

was the second smallest of all kdeg of the three candidate Co(salophen) electrodes were

compared with those of catalytic metals and shown in Table 18

Electrode type kdeg ms-1 Experiment condition

Co(salophen) NaOH 119 x 10-5 In 001M PBS pH 74

Co(salophen) NaOH ndashH2SO4 747 x 10-6 In 001M PBS pH 74

Co(salophen) H2SO4-PBS 684 x 10-6 In 001M PBS pH 74

Au 4 x 10-3 In 005 M H2SO4 [173]

Au 3 x 10-5 In 01 M KOH

Ag 8 x 10-3 In 01 M KOH

Pt 4 x 10-3 In 01 M KOH

Table 18 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

262

NaOH-pretreated Co(salophen) composite electrode showed good catalysis at neutral

pH It could manage to achieve as efficient ORR as Au in acidic solution

74 CONCLUSION TO CATHODE STUDY

The large overpotential of ORR has been known to degrade the fuel cell

performance [3 4 62 165-167] Finding efficient ORR catalysts therefore has been a

subject of intensive research In this study various catalytic cathodes were fabricated

using inorganic organic and biological catalysts and they were electrochemically

pretreated in different media to improve the catalytic activity Studies were conducted

using various electrochemical methods (i) CV (ii) HDV and (iii) HDA The data

obtained was analysed both qualitatively and quantitatively The ORR catalytic

efficiency was calculated according to the three parameters (i) kdeg (ii) nα and (iii)

Epc2(HDV) and based on that most catalytic cathode was determined

None of the cathodes was catalytic enough without pretreatment or unless at high

overpotential In many cases electrochemical pretreatments were found to attach

various surface oxygen groups activated the electrodes and led to two-electron ORR

catalysis Their influence on electrochemistry varies by the electrode type and media

used for pretreatment It was found that PBS-related pretreatments were effective to

GCEs When the pretreatment is combined with H2SO4 this might lead to serial ORR

where H2O2 produced is further reduced to water NaOH-related pretreatments were

effective for graphite composite electrodes PtC composite electrodes were not active

due to the high resistance within the activated carbon The performance by RhC was

263

only marginally improved by H2SO4 pretreatment Any NaOH- or PBS- related

pretreatments dramatically increased kdeg of Co(salophen) composite electrodes and

reduced the overpotential by 100 mV Fe(salophen) composite electrodes were not

sufficiently catalytic without a double pretreatment with NaOH and H2SO4

Anthraquinone-grafted graphite composite electrodes were not catalytic at neutral pH

even after pretreatment Multi-copper oxidases were catalytic at DET however its RLS

was electron transfer between the electrode and the enzyme hence appropriate

mediators should be used to achieve higher catalysis

Many of pretreated Co(salophen) composite electrodes were strongly catalytic toward

ORR Among them NaOH-pretreated Co(slophen) electrodes were found to be most

catalytic at neutral pH with kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 nα = 083 and Epc2(HDV) =

- 037 V Its catalytic efficiency was close to that of Au in the acid solution

264

8 CONCLUSION AND FURTHER STUDIES

Miniaturised glucose-oxygen fuel cells are ideal to power small implantable

medical devices Typical specification include at least 1 microW power output and 1-year

operational life Therefore fuel cells must have efficient catalysis and electron transfer

good conductivity chemical and mechanical stability as well as biocompatibility The

electrodes must be feasible to build and scaleable In this study electrodes were built

using bulk modification method because it was simple to construct and scale-up and

they were easy to modify and mechanically stable The anodes were built with 10

GOx with 77 TTF-TCNQ composite and the cathodes were built with graphite

composite containing ORR catalysts using bulk modification method TTF-TCNQ was

used because it was conductive as well as mediating and necessary for electron transfer

of the GOx anodes Problems were enzyme stability and ORR catalytic efficiency

Enzyme mass loading is known to be effective to improve catalytic efficiency and

operational life however this also decreases conductivity of the composite electrodes

The key to optimising enzyme electrodes is in the balance and compromise among the

conductivity mediation enzyme activity mechanical and chemical stability and

difficulty in the fabrication process CNF composites showed reasonable conductivity at

30 content and this might allow higher enzyme loading with mediators without losing

conductivity CNT is also a candidate material because it has comparable or larger

conductivity than TTF-TCNQ and can operate without added mediator species Ideally

enzyme electrodes should be functional with minimum components simple to build

renew and store in dry so that they become easily portable Moreover when they are

further miniaturised this will allow the medical application under harsher conditions so

265

that powering disposable implantable sensors in economically-deprived regions might

become possible Many physiological enzymes however easily lose catalytic activity

outside the optimal condition Incorporation of enzyme stabilisers makes the enzyme

robust and long lasting as well as scavenging reactive species In the glucose-oxygen

fuel cells O2 has been known to reduce the catalytic output current of GOx anodes

PEI-DTT stabiliser mixtures were found to be good for this because they conferred

stability on GOx against oxidative stress prevented O2 electron deprivation and

improved the catalytic current output It was however a smaller effect than when

conductive hydrogel was used Conductive hydrogel can be a good candidate if it

becomes resistant against degradation ORR at the cathode has posed a long-term

problem in fuel cells because ORR is inefficient and consumes potential generated

within the fuel cell Pt has been used as an ORR catalyst but it is expensive and is

becoming scarce Use of the fuel cell in the physiological condition also imposes further

operational limitations Pretreated metal complexes such as Co salophen were found to

be good candidates for ORR-catalytic cathodes Surrounding complexes with

electron-rich environment such as conductive film might improve ORR further Once

candidate anode and cathode are identified and built a biofuel cell unit can be made To

increase the power each cell is connected together and made into a stack The prototype

was made as below in Fig 124 by connecting twenty cell units

266

Fig 124 Fuel cell stack made with various GOx anodes and Co(salophen) cathodes

Each cell contained various GOx anodes and pretreated Co(salophen) electrode in 10 ml

of oxygenated 01 M glucose 001 M PBS solution pH 74 The output voltage was

found to be 02 V and it did not change for one week The fuel cell must be studied

further with an adjustable resistor for voltage-current (V-I) and power-current (P-I)

curves so that OCV can be determined properly The passive type GOx fuel cell could

output 15 mWcm-2 at 03 V [18]

In vivo studies are also necessary for implantable purposes to examine the catalytic

efficiency electrode fouling and biocompatibility This can be studied systematically by

changing the type and concentration of interfering substances such as ascorbate

acetoaminophen and urate [18] Heller tested miniturised GOx-BOD fuel cells tested in

the buffer grape and calf serum It showed only 5 power decrease in buffer however

50 drop within 20 hours in grape and 50 drop within 2 hours in calf serum [19] To

avoid electrode fouling the electrode surface can be coated with a membrane such as

267

Nafion However this imposes another problem because it can interfere with the

reactant flux to the electrode surface In the real physiological system this might cause

a serious problem because glucose and O2 concentration is much lower than in vitro

Furthermore in the physiological conditions active transport of the reactant is not

available and this will cause local concentration polarisation It seems that there are

many obstacles lying in the medical application of miniaturised glucose-oxygen fuel

cells although they have widespread potentials applications

268

9 BIBLIOGRAPHY

1 Carrette L KA Friedrich and U Stimming Fuel cells Principles types fuels and applications

Chemphyschem 2000 1(4) p 162-193

2 Gregoire Padro CE and JO Keller Hydrogen Energy in Kirk-Othmer Encyclopedia of Chemical

Technology 2005 John Wiley amp Sons Inc p 837-866

3 Ohta K T Kazama and M Nishizawa All about fuel cells in Newton 2006 p 31-63

4 Tanabe S Mastering basics of the fuel cells 2009 Denkishoin

5 Tokyo-Gas Fuel cell academy 2009 2901 [cited Available from httpwwwtokyo-gascojppefc

6 Cropper MAJ S Geiger and DM Jollie Fuel cells a survey of current developments Journal of Power

Sources 2004 131(1-2) p 57-61

7 Kordesch K et al Intermittent use of a low-cost alkaline fuel cell-hybrid system for electric vehicles

Journal of Power Sources 1999 80(1-2) p 190-197

8 UTC-Power The fuel cell for Apollo 11

9 Connor S Warning Oil supplies are running out fast in The Independent 2009

10 Mori Y Mobile fuel cell providing new advantages for mobile electronic appliances Toshiba review 2007

62(6) p 44-47

11 Mori H Electronic components and materials Toshiba review 2009 64(3) p 62-63

12 Imperial-College-London Racing Green - Vehicles 2009 [cited Available from

httpwwwunionicacukrccracinggreenvehicleshtml

13 Cleave V Formula Zero kart race could drive fuel cell technology New Scientist 2008 [cited

Available from

httpwwwnewscientistcomarticledn14596-formula-zero-kart-race-could-drive-fuel-cell-technologyhtml

14 HyNor Hydrogen highway opens in Norway 2009 [cited Available from

httpwwwhynornohydrogen-highway-opens-in-norway

15 BMW Hydrogen 7 - The future is closer than you think 2009 [cited Available from

httpwwwbmwcomcomeninsightstechnologyefficient_dynamicsphase_2clean_energybmw_hydroge

n_7html

httpwwwbmwcojpjpjabrandtechnologycleanenergywallpaperhtml

16 Sony Side view - Sony developes Bio Battery generating electricity from sugar 2007 Sony

17 Burton N Sugar-powered electronics in RSC Chemical technology news 2008

18 Sakai H A high-power glucoseoxygen biofuel cell operating under quiescent conditions Energy amp

Environmental Science 2009 2(1) p 133-138

19 Heller A Miniature biofuel cells Physical Chemistry Chemical Physics 2004 6(2) p 209-216

20 Toshiba Toshiba announces worlds smallest direct methanol fuel cell with energy output of 100 milliwatts

269

2004 [cited Available from httpwwwtoshibacojpaboutpress2004_06pr2401htm

21 Osakai T K Kanoh and S Kuwabata Basic Electrochemistry 2007 Kagakudojin Ltd Kyoto Japan

22 Wilson K and J Walker Practical Biochemistry 1992 Cambridge UK Cambridge University Press

23 Murray RK et al Harpers Biochemistry 25 ed 2000 Stamford Connecticut Appleton amp Lange

24 Sawyer DT A Sobkowiak and JL Roberts Jr Electrochemistry for chemists Revised ed 1995

Wiley-Interscience

25 IUPAC IUPAC GOLD BOOK 2009 [cited Available from httpgoldbookiupacorgindexhtml

26 Kitamura T and K Fujisawa English-Japanese The students dictionary of physics 1995 Tokyo ALC

Inc

27 Gates BC Catalysis in Kirk-Othmer Encyclopedia of Chemical Technology 2002 John Wiley amp Sons p

200-254

28 Taguchi S and E Nakano Kinetic Study of Bromination of Malonic Acid Methyl Malonic Acid

and Ethyl Malonic Acid An Undergraduate Physical Chemistry Laboratory Experimentfor

Understanding of the Infuluence of Thermodynamic Parameter on Chemical Kinetics The Chemical

Education Jounal 1999 3(1) p 1-20

29 Pletcher D A First course in electrode processes 1991 The Electrochemical Consultancy (Romsey) Ltd

23 - 26 135 - 145

30 Bard AJ and LR Faulker Electrochemical Methods Fundamentals and Applications 2000 NY USA

John Wiley and Sons Inc

31 Daintith J A concise dictionary of chemistry 2 ed 1990 Oxford UK Oxford University Press

32 Tinoco IJ et al Physical Chemistry 2002 Prentice Hall p 157

33 Watanabe K and S Nakabayashi Introduction to electrochemistry The chemistry of electron transfer 13

ed 2005 Tokyo Japan The Chemical Society of Japan

34 Hamelers B et al Microbial fuel cells Methodology and technology Environmental science amp

technology 2006 40(17) p 5181-5192

35 Koike T Chapter 10 Free energy in Physical chemistry I amp II Lecture handouts for dept of pharmacy

2010 University of Hiroshima p 57-62

36 Lopes T E Gonzalez and E Antolini An overview of platinum-based catalysts as methanol-resistant

oxygen reduction materials for direct methanol fuel cells Journal of alloys and compounds 2008 461(1-2)

p 253-262

37 Vork FTA and E Barendrecht The reduction of dioxygen at polypyrrole-modified electrodes with

incorporated Pt particles Electrochimica Acta 1990 35(1) p 135-139

38 Marino W B Scharifker and A Leone ELECTRODEPOSITION AND

ELECTROCHEMICAL-BEHAVIOR OF PALLADIUM PARTICLES AT POLYANILINE AND

POLYPYRROLE FILMS Journal of the Electrochemical Society 1992 139(2) p 438-443

39 Tsakova V How to affect number size and location of metal particles deposited in conducting polymer

270

layers Journal of Solid State Electrochemistry 2008 12(11) p 1421-1434

40 Lemest Y et al Dioxygen Fixation by a Mixed-Valence Dicobalt Cofacial Porphyrin Journal of the

Chemical Society-Chemical Communications 1983(22) p 1286-1287

41 Kim E et al Superoxo micro-peroxo and micro-oxo complexes from hemeO2 and heme-CuO2 reactivity

Copper ligand influences in cytochrome c oxidase models 2003 p 3623-3628

42 Fukuzumi S et al Mechanism of Four-Electron Reduction of Dioxygen to Water by Ferrocene

Derivatives in the Presence of Perchloric Acid in Benzonitrile Catalyzed by Cofacial Dicobalt Porphyrins

Journal of the American Chemical Society 2004 126(33) p 10441-10449

43 Solomon EI et al O2 and N2O Activation by Bi- Tri- and Tetranuclear Cu Clusters in Biology

Accounts of Chemical Research 2007 40(7) p 581-591

44 Wildgoose GG et al Anthraquinone-derivatised carbon powder reagentless voltammetric pH electrodes

Talanta 2003 60(5) p 887-893

45 McCreery R Carbon electrodes Structural effects on electron transfer kinetics in Electroanalytical

Chemistry A series advances 1990 Marcel Dekker Inc p 221-374

46 Kneten K R McCreery and C McDermott ELECTRON-TRANSFER KINETICS OF AQUATED

FE+3+2 EU+3+2 AND V+3+2 AT CARBON ELECTRODES - INNER-SPHERE CATALYSIS BY

SURFACE OXIDES Journal of the Electrochemical Society 1993 140(9) p 2593-2599

47 Engstrom RC ELECTROCHEMICAL PRETREATMENT OF GLASSY-CARBON ELECTRODES

Analytical Chemistry 1982 54(13) p 2310-2314

48 Lawrence E Hendersons dictionary of biological terms 10 ed 1989 UK Longman group UK Ltd

49 Eijsink V Directed evolution of enzyme stability Biomolecular engineering 2005 22(1-3) p 21-30

50 Pantoliano M et al PROTEIN ENGINEERING OF SUBTILISIN BPN - ENHANCED STABILIZATION

THROUGH THE INTRODUCTION OF 2 CYSTEINES TO FORM A DISULFIDE BOND Biochemistry

1987 26(8) p 2077-2082

51 Bagotzky VS NV Osetrova and AM Skundin Fuel cells State-of-the-art and major scientific and

engineering problems Russian Journal of Electrochemistry 2003 39(9) p 919-934

52 Heller A Integrated medical feedback systems for drug delivery Aiche Journal 2005 51(4) p

1054-1066

53 Nakamura M et al High-performance ultrathin solid oxide fuel cells for low-temperature operation

Journal of the Electrochemical Society 2007 154(1) p B20-B24

54 Lapedes DN McGraw-Hill Dictionary of Physics and Mathematics in McGraw-Hill 1978 McGraw-Hill

Inc

55 Svoboda V et al Enzyme catalysed biofuel cells Energy amp Environmental Science 2008 1(3) p

320-337

56 Barton SC J Gallaway and P Atanassov Enzymatic biofuel cells for Implantable and microscale devices

Chemical Reviews 2004 104(10) p 4867-4886

271

57 Schroder U Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency

Physical chemistry chemical physics 2007 9(21) p 2619-2629

58 Chaudhuri SK and DR Lovley Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells Nature Biotechnology 2003 21(10) p 1229-1232

59 Vincent K F Armstrong and J Cracknell Enzymes as working or inspirational electrocatalysts for fuel

cells and electrolysis Chemical reviews 2008 108(7) p 2439-2461

60 Tsujimura S and K Kano Recent development of enzyme-based biofuel cells GS Yuasa technical report

2008 5(2) p 1-6

61 Cass AEG Biosensors A Practical Approach 1990 Oxford University Press Oxford UK p 49-53

62 Palmore GTR and HH Kim Electro-enzymatic reduction of dioxygen to water in the cathode

compartment of a biofuel cell Journal of Electroanalytical Chemistry 1999 464(1) p 110-117

63 Bullen RA et al Biofuel cells and their development Biosensors amp Bioelectronics 2006 21(11) p

2015-2045

64 Bertschy H et al A methanoldioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as

catalysts application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at

low overpotentials Journal of electroanalytical chemistry and interfacial electrochemistry 1998 443(1) p

155-161

65 Bartlett PN P Tebbutt and RG Whitaker Kinetic aspects of the use of modified electrodes and

mediators in bioelectrochemistry Progress in reaction kinetics 1991 16(2) p 55-155

66 Kano K T Ikeda and S Tsujimura GlucoseO-2 biofuel cell operating at physiological conditions Denki

kagaku oyobi kōgyō butsuri kagaku 2002 70(12) p 940-942

67 Heller A Electrochemical glucose sensors and their applications in diabetes management Chemical

Reviews 2008 108(7) p 2482-2505

68 Ilankumaran V et al Trends in cardiac pacemaker batteries Indian pacing and electrophysiology journal

2004 4(4) p 201-12

69 Schlindwein W and R Linford Medical applications of solid state lonics Solid State Ionics 2006

177(19-25) p 1559-1565

70 Latham R R Linford and W Schlindwein Biomedical applications of batteries Solid State Ionics 2004

172(1-4) p 7-11

71 Soykan O Power sources for implantable medical devices Business Briefing Medical Device

Manufacturing and Technology London 2002

72 Heller A Potentially implantable miniature batteries Analytical and Bioanalytical Chemistry 2006

385(3) p 469-473

73 Taniguchi M Achilles heel of the next-generation electric vehicles - Lithium World map of the resrouce

wars 2008 [cited 2009 Available from

httpbusinessnikkeibpcojparticlemanage20080407152450P=1

272

74 Yeatman EM Advances In Power Sources For Wireless Sensor Nodes International Workshop on Body

Sensor Networks 2004

75 Sato N et al Novel MEMS power generator with integrated thermoelectric and vibrational devices in

Solid-State Sensors Actuators and Microsystems 2005 Digest of Technical Papers TRANSDUCERS 05

The 13th International Conference on 2005

76 Hudak NS and GG Amatucci Small-scale energy harvesting through thermoelectric vibration and

radiofrequency power conversion Journal of Applied Physics 2008 103(10)

77 Seiko-Instruments SEIKO Worlds First 2009 [cited Available from

httpwwwseikowatchescomheritageworlds_firsthtml

78 Kishi M et al Micro thermoelectric modules and their application to wristwatches as an energy source in

Thermoelectrics 1999 Eighteenth International Conference on 1999

79 Lee J et al Ionic conduction in Zn-3(PO4)(2)()4H(2)O enables efficient discharge of the zinc anode in

serum Journal of the American Chemical Society 2005 127(42) p 14590-14591

80 Maxell Button battery table 2009 [cited Available from

httpwwwmaxellcojpproductsmaterialsbutton_batteryhtml

81 Wilson R and APF Turner Glucose-Oxidase - an Ideal Enzyme Biosensors amp Bioelectronics 1992 7(3)

p 165-185

82 Swoboda BEP The relationship between molecular conformation and the binding of flavin-adenine

dinucleotide in glucose oxidase Biochimica et Biophysica Acta (BBA) - Protein Structure 1969 175(2) p

365-379

83 Wohlfahrt G et al 18 and 19 angstrom resolution structures of the Penicillium amagasakiense and

Aspergillus niger glucose oxidases as a basis for modelling substrate complexes Acta Crystallographica

Section D-Biological Crystallography 1999 55 p 969-977

84 Patel RN Biocatalysis in the pharmaceutical and biotechnology industries 1st ed 2006 CRC Press

85 Gouda M Reversible denaturation behavior of immobilized glucose oxidase Applied Biochemistry and

Biotechnology 2002 102 p 471-480

86 Hecht HJ et al Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 3 Angstrom

Resolution Journal of Molecular Biology 1993 229(1) p 153-172

87 Zubrik A et al Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the

transition to the denatured state with residual structure The Journal of biological chemistry 2004 279(46)

p 47601-47609

88 Kojima S et al Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose

biosensor Bioscience biotechnology and biochemistry 2006 70(3) p 654-659

89 Duine J The PQQ story Journal of bioscience and bioengineering 1999 88(3) p 231-236

90 Kyoto-University KEGG - Kyoto Encyclopedia of Genes and Genomes 2009 Kyoto University

91 Delbem MF WJ Baader and SHP Serrano Mechanism of 34-dihydroxybenzaldehyde

273

electropolymerization at carbon paste electrodes catalytic detection of NADH 2002 scielo p 741-747

92 Zhao S et al Electrochemistry of Organic Conducting Salt Electrodes - a Unified Mechanistic

Description Journal of Physical Chemistry 1992 96(13) p 5641-5652

93 Lemuel BW Jr CC Liu and LN Niren Electrochemical measurements with glucose oxidase

immobilized in polyacrylamide gel Constant current voltametry 1971 p 629-639

94 Sato A et al Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Biochemical Journal

2001 357 p 893-898

95 Sigma-Aldrich Glucose Oxidase from Aspergillus niger Type II-S 15000-50000 unitsg solid (without

added oxygen) 2009 [cited

96 Cosnier S Biosensors based on electropolymerized films new trends Analytical and Bioanalytical

Chemistry 2003 377(3) p 507-520

97 Willner I et al Integrated Enzyme-Based Biofuel Cells-A Review Fuel Cells 2009 9(1) p 7-24

98 Heleg-Shabtai V et al Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers

assembled onto Au-electrodes Journal of the American Chemical Society 1996 118(42) p 10321-10322

99 Georganopoulou DG et al Development and comparison of biosensors for in-vivo applications Faraday

Discussions 2000 p 291-303

100 Fujii S et al Properties of glucose sensors prepared by the electropolymerization of pyrrole containing a

gluconyl group Analytical sciences 1999 15(12) p 1175-1176

101 Hoshi T Deposition of electron mediators by electrochemical polymerization Electrochemistry 2003

71(5) p 346-347

102 Arai G M Masuda and I Yasumori Direct Electrical Response between Glucose-Oxidase and

Poly(Mercapto-P-Benzoquinone) Films Bulletin of the Chemical Society of Japan 1994 67(11) p

2962-2966

103 Arai G K Shoji and I Yasumori Electrochemical characteristics of glucose oxidase immobilized in

poly(quinone) redox polymers Journal of Electroanalytical Chemistry 2006 591(1) p 1-6

104 Leonida MD et al Polymeric FAD used as enzyme-friendly mediator in lactate detection Analytical and

Bioanalytical Chemistry 2003 376(6) p 832-837

105 Kissinger PT and WR Heineman Laboratory techniques in electroanalytical chemistry 1996 Marcel

Dekker Inc NY USA p 294-332

106 OHare D and H Zhao Characterisation and modeling of conducting composite electrodes The journal of

physical chemistry C 2008 112(25) p 9351-9357

107 Wang J et al Metal-Dispersed Carbon Paste Electrodes Analytical Chemistry 1992 64(11) p

1285-1288

108 Khurana MK CP Winlove and D OHare Detection mechanism of metallized carbon epoxy oxidase

enzyme based sensors Electroanalysis 2003 15(12) p 1023-1030

109 Sasaki M et al Fabrication and Electrical Characterization of

274

Tetrathiafulvalene-tetracyanoquinodimethane Molecular Wires Japanese Journal of Applied Physics 2003

42 p 2488-2491

110 Iizuka M et al Highly oriented TTF-TCNQ crystal growth using an electric-field induced evaporation

technique Molecular crystals and liquid crystals science and technology Section A Molecular crystals and

liquid crystals 2000 349 p 367-370

111 Saito I and K Yoshida Development of new features on organic conductive material TCI mail 2005

127

112 Cass AEG et al FERROCENE-MEDIATED ENZYME ELECTRODE FOR AMPEROMETRIC

DETERMINATION OF GLUCOSE Analytical Chemistry 1984 56(4) p 667-671

113 Kulys J V Simkeviciene and IJ Higgins Concerning the Toxicity of 2 Compounds Used as Mediators in

Biosensor Devices - 7788-Tetracyanoquinodimethane (Tcnq) and Tetrathiafulvalene (Ttf) Biosensors amp

Bioelectronics 1992 7(7) p 495-501

114 Malinauskas A et al Conductive organic complex salt TTF-TCNQ as a mediator for biosensors An

overview Electroanalysis 2007 19(24) p 2491-2498

115 Llopis X et al Integration of a glucose biosensor based on an epoxy-graphite-TTF center dot

TCNQ-GOD biocomposite into a FIA system Sensors and Actuators B-Chemical 2005 107(2) p 742-748

116 Bourgeois J D Thomas and C Bourdillon COVALENT LINKAGE OF GLUCOSE-OXIDASE ON

MODIFIED GLASSY-CARBON ELECTRODES - KINETIC PHENOMENA Journal of the American

Chemical Society 1980 102(12) p 4231-4235

117 Yun Y et al Nanotube electrodes and biosensors Nano Today 2007 2(6) p 30-37

118 Kim BC et al Preparation of biocatalytic nanofibres with high activity and stability via enzyme

aggregate coating on polymer nanofibres Nanotechnology 2005 16(7) p S382-S388

119 Kim J and JW Grate Single-Enzyme Nanoparticles Armored by a Nanometer-Scale OrganicInorganic

Network 2003 p 1219-1222

120 OFagain C Enzyme stabilization - recent experimental progress Enzyme and Microbial Technology

2003 33(2-3) p 137-149

121 Sillery E et al Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a

platinum catalyst Chemical communications 2002(8) p 866-867

122 Gouda M et al Enhancement of stability of immobilized glucose oxidase by modification of free thiols

generated by reducing disulfide bonds and using additives Biosensors amp bioelectronics 2004 19(6) p

621-625

123 Manning M STABILITY OF PROTEIN PHARMACEUTICALS Pharmaceutical Research 1989 6(11) p

903-918

124 Cao L Immobilised enzymes science or art Current opinion in chemical biology 2005 9(2) p 217-226

125 Daniel R The role of dynamics in enzyme activity Annual review of biophysics and biomolecular

structure 2003 32 p 69-92

275

126 Andersson MM JD Breccia and R Hatti-Kaul Stabilizing effect of chemical additives against oxidation

of lactate dehydrogenase Biotechnology and Applied Biochemistry 2000 32 p 145-153

127 Souza JM and R Radi Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Archives of Biochemistry and Biophysics 1998 360(2) p 187-194

128 Andersson MA and R Hatti-Kaul Protein stabilising effect of polyethyleneimine Journal of

Biotechnology 1999 72(1-2) p 21-31

129 Sigma-Aldrich Poly(ethylenimine) 2009 [cited Available from

httpwwwsigmaaldrichcomstructureimages27mfcd00084427gif

130 Andersson MM R Hatti-Kaul and W Brown Dynamic and static light scattering and fluorescence

studies of the interactions between lactate dehydrogenase and poly(ethyleneimine) Journal of Physical

Chemistry B 2000 104(15) p 3660-3667

131 Wang J L Chen and J Liu Critical comparison of metallized and mediator-based carbon paste glucose

biosensors Electroanalysis 1997 9(4) p 298-301

132 Cleland WW Dithiothreitol a New Protective Reagent for SH Groups Biochemistry 1964 3(4) p

480-482

133 Atanasov P E Wilkins and Q Yang An integrated needle-type biosensor for intravascular glucose and

lactate monitoring Electroanalysis 1998 10(11) p 752-757

134 Sigma-Aldrich DL-Dithiothreitol 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=43815|SIGMAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

135 Ghisalba O and A Brunella Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate

reductase as biocatalyst in the preparative synthesis of 2 -deoxyribonucleoside-5 -triphosphates Journal

of molecular catalysis B Enzymatic 2000 10(1-3) p 215-222

136 Gollhofer D EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM

ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Enzyme and Microbial Technology 1995 17(3) p 235-240

137 Sigma-Aldrich Arsenite standard solution - 005 M in H2O 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=70039|FLUKAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

138 Arakawa T MECHANISM OF POLY(ETHYLENE GLYCOL) INTERACTION WITH PROTEINS

Biochemistry 1985 24(24) p 6756-6762

139 Lee C Improvement of protein stability in protein microarrays Biotechnology Letters 2002 24(10) p

839-844

140 Lee L THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE GLYCOLS)

Biochemistry 1987 26(24) p 7813-7819

141 Costa SA et al Studies of stabilization of native catalase using additives Enzyme and Microbial

276

Technology 2002 30(3) p 387-391

142 Lee J and L Lee THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE

GLYCOLS) Biochemistry 1987 26(24) p 7813-7819

143 Gekko K and SN Timasheff Mechanism of protein stabilization by glycerol preferential hydration in

glycerol-water mixtures Biochemistry 1981 20(16) p 4667-4676

144 Leandro P Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes

produced in a prokaryotic expression system Molecular genetics and metabolism 2001 73(2) p 173-178

145 Bartlett PN VQ Bradford and RG Whitaker Enzyme Electrode Studies of Glucose-Oxidase Modified

with a Redox Mediator Talanta 1991 38(1) p 57-63

146 Joseph S et al Drug metabolism biosensors electrochemical reactivities of cytochrome P450(cam)

immobilised in synthetic vesicular systems Journal of Pharmaceutical and Biomedical Analysis 1998

17(6-7) p 1101-1110

147 Cornish-Bowden A Fundamentals of enzyme kinetics 3rd ed 2004 London Portland Press Ltd

148 Wilkinson G STATISTICAL ESTIMATIONS IN ENZYME KINETICS The Biochemical journal 1961

80(2) p 324

149 Ikeda T and K Kano Fundamentals and practices of mediated bioelectrocatalysis Analytical sciences

2000 16(10) p 1013-1021

150 Duggleby RG and LP Daniel [3] Analysis of enzyme progress curves by nonlinear regression in

Methods in Enzymology 1995 Academic Press p 61-90

151 Eisenthal R and A Cornish-Bowden DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR

ESTIMATING ENZYME KINETIC-PARAMETERS The Biochemical journal 1974 139(3) p 715-720

152 Baici A Enzyme kinetics the velocity of reactions Biochem J 2006 p 1-3

153 Yamazaki A et al Improvement of precision in pharmacological studies using the non-linear regression

method

estimation of enzyme inhibition constants as an example Folia Pharmacologica Japonica 2008 131(3) p 195-203

154 Cornish-Bowden A Teaching Enzyme Kinetics and Mechanism in the 21st Century in 3rd Symposium

on ESCES 2008 Rudensheim Germany

155 Weisshaar DE and T Kuwana Considerations for Polishing Glassy-Carbon to a Scratch-Free Finish

Analytical Chemistry 1985 57(1) p 378-379

156 Stedman Stedmans medical dictionary 25th ed 2002 Lippincott Williams amp Wilkins

157 Tamura T Buffer preparation for biology Buffer preparation for biology ed 6 2008 Tokyo Yodosha

Ltd

158 Battino R and HL Clever The Solubility of Gases in Liquids 1966 p 395-463

159 Pandolfo A and A Hollenkamp Carbon properties and their role in supercapacitors Journal of power

sources 2006 157(1) p 11-27

160 Maynard AD Nanotechnology assessing the risks Nano Today 2006 1(2) p 22-33

277

161 Hindle A E Hall and N Martens AN ASSESSMENT OF MEDIATORS AS OXIDANTS FOR

GLUCOSE-OXIDASE IN THE PRESENCE OF OXYGEN Biosensors amp bioelectronics 1995 10(3-4) p

393-403

162 Mo J et al Comparison of oxygren-rich and mediator-based glucose-oxidase carbon-paste electrodes

Analytica Chimica Acta 2001 441(2) p 183-189

163 Turner A and G Palleschi AMPEROMETRIC TETRATHIAFULVALENE-MEDIATED LACTATE

ELECTRODE USING LACTATE OXIDASE ABSORBED ON CARBON FOIL Analytica Chimica Acta

1990 234(2) p 459-463

164 Mizutani F et al Glucose-Sensing Electrode Based on Carbon Paste Containing Ferrocene and

Polyethylene Glycol-Modified Enzyme Bulletin of the Chemical Society of Japan 1991 64(9) p

2849-2851

165 Zhang J PEM Fuel Cell Electrocatalysts and Catalyst Layers Fundamentals and Applications 2008

London Springer-Verlag

166 Kinoshita K Electrochemical oxygen technology 1992 John Willey amp Sons Inc

167 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of Power

Sources 2005 152 p 1-15

168 Sawyer DT Oxygen Chemistry International series of monographs on chemistry 1991 Oxford

University Press

169 Winter M Chemical Bonding Oxford Chemistry Primers 1997 New York Oxford University Press Inc

170 McLendon G and AE Martell Inorganic Oxygen Carriers as Models for Biological-Systems

Coordination Chemistry Reviews 1976 19(1) p 1-39

171 Toda T et al Enhancement of the electroreduction of oxygen on Pt alloys with Fe Ni and Co Journal of

the Electrochemical Society 1999 146(10) p 3750-3756

172 Norskov JK et al Origin of the overpotential for oxygen reduction at a fuel-cell cathode Journal of

Physical Chemistry B 2004 108(46) p 17886-17892

173 Fischer P and J Heitbaum MECHANISTIC ASPECTS OF CATHODIC OXYGEN REDUCTION Journal

of Electroanalytical Chemistry 1980 112(2) p 231-238

174 Okada T et al Oxygen reduction characteristics on pyrolytic graphite electrodes modified with

electro-polymerized salen compounds Chemistry Letters 1998(8) p 841-842

175 Sakurai T and K Kataoka Basic and applied features of multicopper oxidases CueO bilirubin oxidase

and laccase 2007 p 220-229

176 Sanders J Supramolecular catalysis in transition Chemistry a European journal 1998 4(8) p

1378-1383

177 Hart H LE Craine and DJ Hart Organic Chemistry A Short Course 10 ed 1998 Houghton Miffin

Company USA

178 Hayatsu H CELLULOSE BEARING COVALENTLY LINKED COPPER PHTHALOCYANINE

278

TRISULPHONATE AS AN ADSORBENT SELECTIVE FOR POLYCYCLIC COMPOUNDS AND ITS USE

IN STUDIES OF ENVIRONMENTAL MUTAGENS AND CARCINOGENS Journal of chromatography A

1992 597(1-2) p 37-56

179 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of power

sources 2005 152(1) p 1-15

180 Kingsborough RP and TM Swager Electrocatalytic Conducting Polymers Oxygen Reduction by a

Polythiophene-Cobalt Salen Hybrid Chemistry of Materials 2000 12(4) p 872-874

181 Ortiz B and SM Park Electrochemical and spectroelectrochemical studies of cobalt salen and salophen

as oxygen reduction catalysts Bulletin of the Korean Chemical Society 2000 21(4) p 405-411

182 Mano N et al An oxygen cathode operating in a physiological solution Journal of the American

Chemical Society 2002 124(22) p 6480-6486

183 Shleev S et al Direct electron transfer between copper-containing proteins and electrodes Biosensors

and Bioelectronics 2005 20(12) p 2517-2554

184 Solomon EI et al Oxygen binding activation and reduction to water by copper proteins Angewandte

Chemie-International Edition 2001 40(24) p 4570-4590

185 Heath R F Armstrong and C Blanford A stable electrode for high-potential electrocatalytic O-2

reduction based on rational attachment of a blue copper oxidase to a graphite surface Chemical

communications 2007(17) p 1710-1712

186 Miura Y et al Direct Electrochemistry of CueO and Its Mutants at Residues to and Near Type I Cu for

Oxygen-Reducing Biocathode Fuel cells 2009 9(1) p 70-78

187 Tarasevich MR VA Bogdanovskaya and LN Kuznetsova Bioelectrocatalytic reduction of oxygen in

the presence of laccase adsorbed on carbon electrodes Russian Journal of Electrochemistry 2001 37(8) p

833-837

188 Tsujimura S et al Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase

at carbon electrodes Electrochemistry 2004 72(6) p 437-439

189 Fernandez JL et al Optimization of wired enzyme O-2-electroreduction catalyst compositions by

scanning electrochemical microscopy Angewandte Chemie-International Edition 2004 43(46) p

6355-6357

190 Soukharev V N Mano and A Heller A four-electron O-2-electroreduction biocatalyst superior to

platinum and a biofuel cell operating at 088 V Journal of the American Chemical Society 2004 126(27)

p 8368-8369

191 Miura Y et al Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on

a highly oriented pyrolytic graphite electrode Chemistry Letters 2007 36(1) p 132-133

192 Seinberg JM et al Spontaneous modification of glassy carbon surface with anthraquinone from the

solutions of its diazonium derivative An oxygen reduction study Journal of Electroanalytical Chemistry

2008 624(1-2) p 151-160

279

193 Manisankar P and A Gomathi Electrocatalytic reduction of dioxygen at the surface of carbon paste

electrodes modified with 910-anthraquinone derivatives and dyes Electroanalysis 2005 17(12) p

1051-1057

194 Jurmann G DJ Schiffrin and K Tammeveski The pH-dependence of oxygen reduction on

quinone-modified glassy carbon electrodes Electrochimica Acta 2007 53(2) p 390-399

195 Abiman P R Compton and G Wildgoose Characterising chemical functionality on carbon surfaces

Journal of materials chemistry 2009 19(28) p 4875-4886

196 Thorogood CA et al Differentiating between ortho- and para-Quinone Surface Groups on Graphite

Glassy Carbon and Carbon Nanotubes Using Organic and Inorganic Voltammetric and X-ray

Photoelectron Spectroscopy Labels Chemistry of Materials 2007 19(20) p 4964-4974

197 Sigma-Aldrich Fast red AL salt 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildoN4=380660|SIALampN5=SEARCH_CONCAT_PN

O|BRAND_KEYampF=SPEC

198 Sljukic B C Banks and R Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

199 Granger M et al Polycrystalline diamond electrodes basic properties and applications as amperometric

detectors in flow injection analysis and liquid chromatography Analytica Chimica Acta 1999 397(1-3) p

145-161

200 Compton R J Foord and F Marken Electroanalysis at diamond-like and doped-diamond electrodes

Electroanalysis 2003 15(17) p 1349-1363

201 Arima S Do Science - Why diamond is so hard in Asahicom 2006

202 Dumitrescu I P Unwin and J Macpherson Electrochemistry at carbon nanotubes perspective and issues

Chemical communications 2009(45) p 6886-6901

203 Kinoshita K Carbon Electrochemical and Physicochemical Properties 1988 Wiley-Interscience

204 Kastening B et al Electronic properties and double layer of activated carbon Electrochimica Acta 1997

42(18) p 2789-2799

205 Swain G and R Ramesham THE ELECTROCHEMICAL ACTIVITY OF BORON-DOPED

POLYCRYSTALLINE DIAMOND THIN-FILM ELECTRODES Analytical chemistry 1993 65(4) p

345-351

206 Kaneto K et al Electrical conductivities of multi-wall carbon nano tubes Synthetic metals 1999

103(1-3) p 2543-2546

207 Meyer J et al Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes Nano

letters 2008 8(11) p 3582-3586

208 Harris P New perspectives on the structure of graphitic carbons Critical Reviews in Solid State and

Materials Sciences 2005 30(4) p 235-253

209 Akasaka T Chemical illustration - Carbon materials 2007 Hokkaido Japan

280

210 Iijima S M Yudasaka and F Nihey Carbon nanotube technology NEC Technical Journal 2007 2(1) p

52-56

211 Day T N Wilson and J Macpherson Electrochemical and conductivity measurements of single-wall

carbon nanotube network electrodes Journal of the American Chemical Society 2004 126(51) p

16724-16725

212 Fujita H A TEM image of the atomic structure of the diamond surface 2010 Britannica - The Online

Encyclopedia Osaka Japan

213 McCreery RL et al Control of reactivity at carbon electrode surfaces Colloids and Surfaces A

Physicochemical and Engineering Aspects 1994 93 p 211-219

214 Rice R C Allred and R McCreery FAST HETEROGENEOUS ELECTRON-TRANSFER RATES FOR

GLASSY-CARBON ELECTRODES WITHOUT POLISHING OR ACTIVATION PROCEDURES Journal of

electroanalytical chemistry and interfacial electrochemistry 1989 263(1) p 163-169

215 Swain Electrochemical formation of high surface area carbon fibers Analytical chemistry 1991 63(5) p

517-519

216 Nagaoka T and T Yoshino Surface-Properties of Electrochemically Pretreated Glassy-Carbon Analytical

Chemistry 1986 58(6) p 1037-1042

217 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 2002 58(9) p 1953-1955

218 Motta N and AR Guadalupe Activated Carbon-Paste Electrodes for Biosensors Analytical Chemistry

1994 66(4) p 566-571

219 Leung E et al A novel in vivo nitric oxide sensor Chemical Communications 1996(1) p 23-24

220 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 1986 58(9) p 1953-1955

221 Kissinger PT and WR Heineman Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy

Current Separations 1989 9 p 15-18

222 Wang J Analytical Electrochemistry 3 ed 2006 Wiley-VCH

223 BAS Measurement of electron transfer kinetics using rotating disk voltammetry Electrocehmistry

Principles methods and applications 1995 2(43)

224 Miomandre F et al Electrochemical behaviour of iron(III) salen and poly(iron-salen) Application to the

electrocatalytic reduction of hydrogen peroxide and oxygen Journal of electroanalytical chemistry and

interfacial electrochemistry 2001 516(1-2) p 66-72

225 Brighton-University Inorganic Chemistry Practical C170 Salen lab script 2009 Dept of Pharmacy and

Biomolecular Sciences Brighton University Brighton

226 Ranganathan S TC Kuo and RL McCreery Facile Preparation of Active Glassy Carbon Electrodes

with Activated Carbon and Organic Solvents 1999 p 3574-3580

227 Ohare D CP Winlove and KH Parker Electrochemical method for direct measurement of oxygen

281

concentration and diffusivity in the intervertebral disc electrochemical characterization and tissue-sensor

interactions J Biomed Eng 1991 13(4) p 304-312

228 Chen PH and RL McCreery Control of electron transfer kinetics at glassy carbon electrodes by specific

surface modification Analytical Chemistry 1996 68(22) p 3958-3965

229 Beilby AL TA Sasaki and HM Stern Electrochemical Pretreatment of Carbon Electrodes as a

Function of Potential Ph and Time Analytical Chemistry 1995 67(5) p 976-980

230 Dai H and K Shiu Voltammetric studies of electrochemical pretreatment of rotating-disc glassy carbon

electrodes in phosphate buffer Journal of electroanalytical chemistry and interfacial electrochemistry 1996

419(1) p 7-14

231 Musameh M N Lawrence and J Wang Electrochemical activation of carbon nanotubes

Electrochemistry communications 2005 7(1) p 14-18

232 Sljukic B CE Banks and RG Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

233 Tammeveski K et al Surface redox catalysis for O-2 reduction on quinone-modified glassy carbon

electrodes Journal of Electroanalytical Chemistry 2001 515(1-2) p 101-112

234 Yang HH and RL McCreery Elucidation of the mechanism of dioxygen reduction on metal-free carbon

electrodes Journal of the Electrochemical Society 2000 147(9) p 3420-3428

235 Regisser F et al Randomly oriented graphite electrode 1 Effect of electrochemical pretreatment on the

electrochemical behavior and chemical composition of the electrode Journal of electroanalytical chemistry

and interfacial electrochemistry 1996 415(1-2) p 47-54

236 Shleev S et al Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic

graphite electrode Electrochemistry Communications 2004 6(9) p 934-939

237 Mano N F Mao and A Heller A miniature biofuel cell operating in a physiological buffer Journal of the

American Chemical Society 2002 124(44) p 12962-12963

5

ありがとう Thank you Terima kashi ขอบคณ คะ Merci 고마워 謝謝 Danke

やったね

copy鳥

山

明

集

英

社

copy Akira Toriyama Shueisha Inc

6

ACKNOWLEDGEMENTS

I would like to express deepest gratitude to my supervisor Dr Danny OrsquoHare

for his guidance on and patience for my research Frankly speaking the project on

glucose-oxygen fuel cells was very challenging and I came to realise the hard reality of

creating new clean energy despite of the world huge demand for such energy in a time

of global warming I could not help but feel that the research was turning into a

wild-goose chase however there was much to learn and gain from the experience here

For that and the people I met during this time I feel deeply grateful for the opportunity

given and I am convinced that this will enlighten my life

I would like to specially thank Dr Pei Ling Leow and Mr Parinya Seelanan for

their sincere support understanding and encouragement Without their help I would not

have been able to achieve my PhD I would like to thank to the current and past

members of Biosensor group Ms Nitin Bagha Dr Christopher Bell Dr Eleni Bitziou

Mr Jin-Young Kim Mr Wei-Chih Lin Dr Beinn Muir Mr Raphael Trouillon Dr

Martyn Boutelle Mr Yao-Min Chang Dr Delphine Feuerstein Ms Michelle Rogers

Dr Costas Anastassiou Dr Arun Arora Dr Martin Arundell Dr Catherine Dobson Dr

Melanie Fennan Dr Severin Harvey Dr Alex Lindsay Dr Bhavik Patel Dr Hong

Zhao Dr Emma Corcoles and Dr Parastoo Hashemi for their support during research

and sharing the wonderful time for many occasions I would like to thank Mr David

Roche for his sincere devotion to our work and being a great lab company I would like

to give my warm thanks to Dr Andrew Bond Dr Stephanie Cremers Ms Christina

Hood Dr Asimina Kazakidi Dr Peter Knox Dr Bettina Nier Ms Eileen Su Mr

Che-Fai Yeong Mr Krause Oliver and Dr Clifford Talbot for appropriate support fun

7

and encouragement I would also like to thank Dr Kate Hobson Mr Martin Holloway

Mr Ken Keating Mr Alan Nyant Mr Richard Oxenham Ms Paula Smith Ms Dima

Cvetkova Dr Anna Thomas-Betts and Mr Nick Watkins for their academic and

laboratory support Finally I would like to say thank you to all my family for their trust

and unconditional support my teacher Prof Fukuyama my friends Dr Pieta

Nasanen-Gilmore Jingzi Kofi Mariama Masa Maya Miki Pedro Sandra Sascha and

Wei Fong for being always close supportive and encouraging even when we were apart

8

CONTENTS

ABSTRACT 3

ACKNOWLEDGEMENTS 6

CONTENTS 8

LIST OF ABBREVIATIONS 15

LIST OF FIGURES 20

LIST OF TABLES 26

LIST OF EQUATIONS 27

1 INTRODUCTION 31

11 INTRODUCTION TO FUEL CELLS 31

111 History of the fuel cells 31 112 Principles of the fuel cells 33 113 Electric power from the fuel cells 35

12 CATALYSTS 37

121 Faradayrsquos second law ndash Current and reaction rate37 122 Transition state theory Eyring equation and reaction rate 38 123 Overcoming the activation free energy 40 124 Variations in catalysts42

13 ELECTRODE POTENTIAL AND REACTION RATE 44

131 Fermi level electrode potential and electron transfer 44 132 Electrode potential equilibrium potential and Nernst equation47

9

133 Electrode potential and activation free energy 50 134 Potential shift and standard rate constant 52 135 Butler-Volmer equation overpotential and current 53 136 Butler-Volmer equation vs Tafel equation 55

14 OVERPOTENTIAL AND FUEL CELLS 57

141 Four overpotentials that decrease the EMF57 142 Internal current 58 143 Activation overpotential58 144 Diffusion overpotential 59 145 Resistance overpotential60 146 Total overpotential and cell voltage 62

15 BIOFUEL CELLS 63

151 Microbial and enzymatic fuel cells 63 152 Enzyme fuel cells and mediators64

16 MINIATURISED MEDICAL POWER SOURCES 66

161 Realisation of miniaturised fuel cells and medical power supply 66 162 Miniaturised potential medical power sources67

17 OUTLINE OF THE WORK 69

171 Introduction to miniaturised glucose-oxygen fuel cells 69 172 Outline71

2 ANODE INTRODUCTION 73

21 GLUCOSE OXIDATION ENZYMES 73

22 DESIGNING ENZYME ELECTRODES 77

221 Physical adsorption 78 222 Reconstituting apo-enzyme78 223 Tethered Osmium-complexes linked to conductive hydrogel 80 224 Entrapment in the conductive polymer 81 225 Bulk modification83

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties 84 226 Enzyme covalent attachment and mass loading 88

23 PROTEIN INACTIVATION AND STABILISATION 90

10

24 PROTEIN DESTABILISATION 92

241 Deamidation 92 242 Oxidation93 243 Proteolysis and racemisation94 244 Physical instabilities95

25 ENZYME STABILISERS 95

251 Polyehyleneimine (PEI) 96 252 Dithiothreitol (DTT)97 253 Polyethylene glycol (PEG)98 254 Polyols99 255 FAD100

26 ENZYME KINETICS AND ELECTROCHEMISTRY 101

261 Michaelis-Menten kinetics 101 262 Electrochemistry and Michaelis-Menten kinetics 105 263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics108

2631 Lineweaver-Burk linear regression 108 2632 Eadie-Hofstee linear regression 109 2633 Electrochemical Hanes-Woolf linear regression 110

264 Cornish-Bowden-Wharton method 112 265 Nonlinear regression methods113

3 ANODE MATERIAL AND METHOD115

31 ELECTRODE FABRICATION 115

311 TTF-TCNQ synthesis115 312 Electrode template fabrication 115 313 Electrode composite fabrication117

3131 Composite for conductance study 117 3132 Bulk modified GOx enzyme electrode composite 118 3133 GOx enzyme electrode composite with enzyme stabilisers 119

314 Electrode wiring and insulation120

32 CONDUCTIVITY EXPERIMENT 121

33 POLISHING COMPOSITE ELECTRODES 122

34 GLUCOSE AMPEROMETRY 123

341 Glucose solution123

11

342 Electrochemical system123 343 Glucose amperometry with GOx electrodes 124

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA 125

4 ANODE RESULTS AND DISCUSSION 127

41 AIMS OF ANODE STUDIES 127

42 CONDUCTANCE OF THE COMPOSITE 128

421 Conductance of various conductive materials128 422 Influence of enzyme concentration on conductivity 130

43 CATALYTIC ACTIVITY OF GOX COMPOSITE ELECTRODES 132

431 Influence of GOx concentration on the catalytic activity of GOx electrodes132 432 Influence of O2 on the catalytic activity of GOx electrodes134

44 THE EFFICACY OF GOX STABILISERS 139

441 Introduction139 442 PEI140 443 DTT142 444 PEG 143 445 GLC144 446 FAD146 447 PEI and DTT mixture147 448 PEI and FAD mixture149 449 Most effective stabiliser content for GOx anodes 150

45 CONCLUSION TO ANODE STUDY 152

5 CATHODE INTRODUCTION 155

51 OXYGEN REDUCTION REACTION (ORR) 155

52 REACTIVITY OF OXYGEN 157

53 ORR CATALYSTS 159

531 Noble metal ORR catalysts 160 532 Macrocycle and Schiff-base complex ORR catalysts 165

12

533 Multicopper enzymes as ORR catalyst 168 5331 BOD 171 5332 Lac 173 5333 CueO 173

534 Anthraquinone ORR catalyst 174

54 CARBON PROPERTIES 177

541 Graphite180 542 Glassy carbon (GC)181 543 Activated carbon (AC) 182 544 Carbon fibre (CF)182 545 Carbon nanotubes (CNTs)183 546 Boron-doped diamond (BDD)184

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON 185

551 Electrochemical properties of carbon185 552 Electrochemical pretreatments (ECPs) of the carbon185

56 ELECTROCHEMICAL TECHNIQUES FOR ORR 187

561 Cyclic voltammetry (CV)188 562 Non-faradic current and electric double layer capacitance 189 563 Diagnosis of redox reactions in cyclic voltammetry 190 564 Coupled reactions in cyclic voltammetry194 565 Voltammograms for the surface-confined redox groups 195 566 Hydrodynamic voltammetry (HDV) and Levich equation197 567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the charge

transfer rate constant 200 568 Finding the standard rate constant (kdeg)205 569 Summary of the electrochemical techniques206

57 SUMMARY OF THE CHAPTER 207

6 CATHODE MATERIAL AND METHOD 208

61 ELECTRODE FABRICATION 208

611 Graphite PtC and RhC electrodes208 612 Anthraquinone(AQ)-carbon composite electrodes208

6121 Derivatisation of graphite with AQ 208 6122 Fabrication of AQ-carbon composite electrode 209

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes209

13

6131 Information on synthesis of metallosalophen 209 6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes 210

62 COMPOSITE RDE CUTTING 210

63 ELECTRODE POLISHING 210

631 Composite RDE polishing210 632 Glassy carbon electrode polishing211

64 ENZYME RDES 212

641 Membrane preparation 212 642 Membrane enzyme electrodes212

65 ELECTROCHEMICAL EXPERIMENTS 213

651 Electrochemical system213 652 Electrode pretreatments214

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes 214 6522 Pretreatments of AQ-carbon PtC and RhC 215

653 Electrochemical experiments 215 654 Data analysis 216

7 CATHODE RESULTS AND DISCUSSION 218

71 AIMS OF THE CATHODE STUDIES 218

72 INFLUENCE AND EFFICACY OF ECPS 219

721 Glassy carbon electrodes (GCEs)219 7211 Untreated GCEs 219 7212 Influence of electrochemical pretreatments on GCE 220 7213 Effect of GCE pretreatment on ORR 227

722 Graphite composite electrodes 230 7221 Untreated graphite composite electrodes 230 7222 Influence of electrochemical pretreatments on graphite composite electrodes 232 7223 The effect of graphite composite electrode pretreatment on ORR 235

723 Platinum on carbon composite electrodes (PtC) 240 724 Rhodium on carbon composite electrodes (RhC)240

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR 241 725 Co(salophen) composite electrodes243

7251 Untreated Co(salophen) composite electrodes 243 7252 Influence of electrochemical pretreatments on Co(salophen) composite 244 7253 The effect of Co(salophen) composite electrode pretreatment on ORR 246

726 Fe(salophen) composite electrodes 248 7261 Untreated Fe(salophen) composite electrodes 248 7262 Influence of electrochemical pretreatments on Fe(salophen) composite 250

14

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR 252 727 Anthraquinone (AQ) composite electrodes253 728 Lac and BOD enzyme membrane electrodes 255

73 MOST EFFICIENT ORR CATALYSTS 257

731 Best five electrodes for nα 258 732 Best five electrodes for kdeg 259 733 Best five electrodes for Epc2(HDV) 260 734 Best three candidates for ORR catalytic electrodes 260

74 CONCLUSION TO CATHODE STUDY 262

8 CONCLUSION AND FURTHER STUDIES 264

9 BIBLIOGRAPHY 268

15

LIST OF ABBREVIATIONS

Α Transfer coefficient

A Electrode surface area

ABTS 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

AC Activated carbon

AFC Alkaline fuel cell

AQ Anthraquinone

Asn Asparagine

Asp Aspartic acid

BDD Boron-doped diamond

BOD Bilirubin oxidase

C Reactant concentration

C O (0t) Electrode surface oxidant concentration

C R (0t) Electrode surface reactant concentration

C(0t) Electrode surface concentration

C Bulk concentration

Cd Double layer capacitance

CF Carbon fibre

CNH Carbon nanohorn

CNT Carbon nanotubes

Co(salen) N-N-bis(salicylaldehyde)-ethylenediimino cobalt(II)

Co(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminocobalt (II)

CO Bulk oxidant concentration

CR Bulk reactant concentration

CueO Copper efflux oxidase

CV Cyclic voltammetry

Cys Cysteine

(DEAE)-dextran Diethylaminoethyl-dextran

DET Direct electron transfer

DHG Dehydrogenase

DMFC Direct methanol fuel cell

DTE Dithioerythriotol

DTT Dithiothreitol

[E0] Initial enzyme concentration

16

E Arbitrary electrode potential Enzyme

Edeg Standard electrode potential

Edeg Formal potential

Ea Anode potential

EC Electrochemical-chemical

Ec Cathode potential

Ecell Cell voltage

ECP Electrochemical pretreatment

Ee Electrode equilibrium potential

EF Fermi potential

EMF Electro motive force

Ep2 Half-peak potential

Epa Anodic peak potential

Epc Cathodic peak potential

Epc2(HDV) Half-wave reduction potential in HDV

ES Enzyme-substrate complex

Eλ Switching potential

∆E Potential difference Potential shift

∆Edeg Standard peak separation

δEp Peak width

εF Fermi energy

F Faraday constant

FAD Flavin adenine dinucleotide

Fe(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminoiron (II)

GC Glassy carbon

GCE Glassy carbon electrode

GHP 1-(6-D-gluconamidohexyl) pyrrole

GLC Glycerol

Gln Glutamine

GOx Glucose oxidase

GOx(O) Oxidised GOx

GOx(R) Reduced GOx

GSH Gluthatione

∆G Gibbs free energy shift

∆Gdeg Standard Gibbs free energy change

∆GdegDagger Standard activation free energy

17

∆GDagger Gibbs free energy of activation

∆GadegDagger Anode standard activation free energy

∆GaDagger Anode activation free energy

∆GodegDagger Oxidation standard activation free energy

∆GoDagger Oxidation activation free energy

∆GRdegDagger Reduction standard activation free energy

∆GRDagger Reduction activation free energy

h Planck constant

HDA Hydrodynamic amperometry

HDV Hydrodynamic voltammetry

His Histidine

HOMO Highest occupied molecular orbital

∆Hdeg Standard enthalpy change

I Electric current Net current

I0 Exchange current

Ia Anodic current

Ic Cathodic current

Id Disc limiting current

IK Kinetic current

IL Limiting current Diffusion-limited current

Im Maximum steady state limiting current

Ip Peak current

Ipa Anodic peak current

Ipc Cathodic peak current

I-V Current-Voltage

J Current density

Jm Maximum limiting current density

k Rate constant

K Equilibrium constant

k(E) Rate constant at the applied potential E

kdeg Standard heterogeneous rate constant

kdegb Backward reaction standard rate constant

kdegf Forward reaction standard rate constant

KDagger The equilibrium constant between the activated complexes and the reactants

k1 Rate constant for ES formation from E and S

k-1 Rate constant for ES dissociation into E + S

18

k2 Rate constant for P formation from ES

kB Boltzmann constant

kb Backward reaction rate constant

kf Forward reaction rate constant

kf(E) Rate constant for forward reactions at the applied potential E

KM Michaelis enzyme dissociation constant

Lac Laccase

LDH Lactate dehydrogenase

LMCT Ligand-to-metal charge transfer

LPG Liquefied petroleum gas

LUMO Lowest unoccupied molecular orbital

MCO Multicopper oxidase

MDH Methanol dehydrogenase

Me2SQ Dimethylmercaptobenzoquinone

MEMS Microelectromechanical systems

MeSQ Methylmercapttobenzoquinone

MET Mediated electron transfer

Met Methionine

mPQQ-GDH Membrane-bound PQQ dehydrogenase

MWNT Multi-walled nanotube

n Overall number of electrons transferred

na The number of electrons transferred up to the rate limiting step

NAD Nicotinamide adenine dinucleotide

NAD-GDH Nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

OCV Open circuit voltage

ORR Oxygen reduction reaction

P Electric power Product

PAFC Phosphoric acid fuel cell

PBS Phosphate buffer saline

PC Pyrolytic carbon

PDMS Polydimethylsiloxane

PEFC Polymer electrolyte fuel cell

PEG Polyethylene glycol

PEI Polyethyleneimine

P-I Power-Current

POx Pyranose oxidase

19

PQ Phenanthrenequinone

PQQ pyrrolo-quinoline quinone

PQQ-GDH Quinoprotein glucose dehydrogenase

Pro Proline

PtC Platinum on carbon

Q Charge

RDE Rotating disc electrode

RhC Rhodium on carbon

RLS Rate limiting step

rpm Rotaion per minute

S Substrate

SEN Single-enzyme nanoparticle

sPQQ-GDH Soluble PQQ dehydrogenase

SQ Mercaptobenzoquinone

SWNT Single-walled nanotube

∆Sdeg Standard entropy change

t Time

T1 Type I (blue Cu in Lac)

T2 Type II ( trinuclear Cu in Lac)

T3 Type III (trinuclear Cu in Lac)

TCNQ 7788-Tetracyanoquinodimethane

TEM Transmission electron microscopy

Trp Tryptophan

TTF Tetrathiafulvalene

TTF-TCNQ Tetrathiafulvalene 7788-tetracyanoquinodimethane

Tyr Tyrosine

V Voltage Maximum limiting catalytic velocity

VHT Vacuum heat treatment

η Overpotential

ν Vibrational frequency Kinematic viscosity

υ Reaction rate Scan rate Enzyme catalytic rate

ω Angular velocity

20

LIST OF FIGURES

Fig No Page Description

Fig 1 32 Historical fuel cells

Fig 2 33 Photographs of modern fuel cells

DMFC Cogeneration system Hydrogen car Glucose-Oxygen fuel cell

Fig 3 34 The schema of glucose-oxygen fuel cell

Fig 4 41 Transition state diagram with the reaction coordinate

Fig 5 45 Fermi level and electron transfer

Fig 6 46 Fermi level and electron transfer at an arbitrary electrode potential

Fig 7 48 Standard free energy change along the electrode potential at

(a) E = Edeg (b) E gt Edeg and (c) E lt Edeg

Fig 8 51 The shift in ∆GdegDagger accompanied by ∆E

Fig 9 57 Tafel plot

Fig 10 59 Activation overpotential vs current

Fig 11 60 Diffusion overpotential vs current

Fig 12 61 Resistance overpotential vs current

Fig 13 62 V-I curve for a typical low temperature fuel cell

Fig 14 65 Mediated electron transfer reaction by the enzyme electrode

Fig 15 70 The schema for a glucose-oxidising anode

Fig 16 75 The structure and redox reactions of FAD NAD and PQQ

Fig 17 79 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode

Fig 18 81 GOx wired to conductive hydrogel complexed with Os

Fig 19 83 Monomer structures of conductive polymers

1-(6-D-gluconamidohexyl) pyrrole and Mercapto-p-benzoquinone

Fig 20 85 TTF-TCNQ structure and its charge transfer

Fig 21 86 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 20 mVs-1

Fig 22 89 The procedure for the enzyme mass-loaded biocatalytic nanofibre

Fig 23 90 The fabrication procedure of single-enzyme nanoparticles

Fig 24 92 The chemical structures of the side groups of Gln and Asn

Fig 25 93 The chemical structures of the side groups of Cys Met Tyr Trp and His

Fig 26 95 The chemical structures of Asp and Pro

Fig 27 96 The structure of PEI

Fig 28 98 The structures of DTT from a linear to a cyclic disulfide form

Fig 29 98 The structures of PEG

21

Fig 30 100 The structure of glycerol

Fig 31 104 A typical Michaelis-Menten hyperbolic curve of the enzyme

Fig 32 107 Electrochemical Michaelis-Menten hyperbolic curve of the enzyme electrode

Fig 33 109 Electrochemical Lineweaver-Burk linear regression of the enzyme electrode

Fig 34 110 Electrochemical Eadie-Hofstee linear regression of the enzyme electrode

Fig 35 111 Electrochemical Hanes-Woolf linear regression of the enzyme electrode

Fig 36 113 Electrochemical Cornish-Bowden-Wharton plot of the enzyme electrode

Fig 37 116 The photographs of PDMS electrode templates

Fig 38 118 The photographs of empty and filled PDMS templates

Fig 39 119 The schema for bulk modification method

Fig 40 121 The photographs of insulated composite electrodes

Fig 41 122 The photographs of measuring electrode resistance

Fig 42 125 The photograph of glucose amperometry with a multichannel potentiostat

Fig 43 126 The analytical output by SigmaPlot Enzyme Kinetics

Fig 44 129 Conductance of various electrode composite

Fig 45 131 Conductance of GOx composite with varying GOx content

Fig 46 133 Difference in the glucose-response amperometric curve by the enzyme electrodes

containing various GOx content 5 10 and 15 in the absence of O2

Fig 47 134 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the absence of O2

Fig 48 135 (i) Difference in the glucose-response amperometric curve by 10 GOx composite

electrodes in the absence and presence of O2

(ii) The re-plotted amperometric curves in the presence of N2 air and O2

Fig 49 136 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the presence N2 O2 and air

Fig 50 140 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEI in the presence and absence of O2

Fig 51 142 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 DTT in the presence and absence of O2

Fig 52 145 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEG in the presence and absence of O2

Fig 53 146 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 GLC in the presence and absence of O2

Fig 54 147 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 FAD in the presence and absence of O2

22

Fig 55 143 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-DTT

(051 11 1505 and 22) in the presence and absence of O2

Fig 56 148 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051 11

1505 and 22)

Fig 57 149 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-FAD (21

22 and 24 ) in the presence and absence of O2

Fig 58 151 Jm KM in the absence and presence of O2 for various stabilisers

PEIDTT (11 0515 221505 ) and GLC 1

Fig 59 156 Oxygen reduction pathways 2 and 4 electron reduction pathways

Fig 60 157 Degradation of superoxide anion radical in aqueous solutions

Fig 61 158 An energy level diagram for ground state dioxygen molecule

Fig 62 159 Diagrams for the state of π orbitals in the ground state oxygen and peroxide anion

radical

Fig 63 162 Three models of oxygen adsorption Griffith Pauling and Bridge models

Fig 64 163 Models for oxygen adsorption on metals and ORR pathways

Fig 65 164 A volcano-shaped trend between ORR rate and oxygen binding energy of various

transition metals

Fig 66 165 Examples of transition metallomacrocycles Heme and copper phthalocyanine

Fig 67 166 Schiff base complexes

Co(II) salen Co(II) salophen and Co(III) salen conductive polymer

Fig 68 167 The molecular structure of dicobalt face-to-face diporphyrin [Co2(FTF4)]

Fig 69 168 ORR pathways by Co2(FTF4)

Fig 70 170 The structure of laccase from Trametes versicolor

Fig 71 171 Redox reaction cycle of MCOs with O2

Fig 72 172 Common mediators for BOD ABTS and Os complex

Fig 73 174 Two-electron ORR by anthraquinone

Fig 74 176 The structures of anthraquinone and its derivative Fast red AL salt

Fig 75 177 HDV of ORR at various quinone electrodes at pH 7

Unmodified GC AQGC and PQGC

Fig 76 178 The carbon structures graphite and diamond

Fig 77 179 The carbon sections basal and edge planes

Fig 78 181 A TEM image of graphene bilayer at 106 Aring resolution

Fig 79 182 A TEM image and a schema of GC

Fig 80 183 Computer graphic models of various CNTs Bundled SWNTs MWNT and CNHs

23

Fig 81 184 A TEM image of diamond

Fig 82 187 The CVs showing the influence of ECP on GCE at pH 7 in the presence and absence

of O2 The CVs for both pretreated and unpretreated GCEs are shown

Fig 83 188 The triangular waveform for CV and a typical voltammetric curves for BQ

Fig 84 189 The CV showing the double layer capacitance at graphite electrode surface at pH 7

Fig 85 194 A voltammogram examples of a quasi-reversible reaction at various scan rates

Fig 86 195 Voltammograms of two-electron transfer in the EE systems with different ∆Edegrsquo

Fig 87 196 A voltammogram of the reversible redox reaction by surface-confined groups

Fig 88 198 A diagram of hydrodynamic electrochemical method using a RDE

Fig 89 200 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH 74

Fig 90 201 Levich plot examples for reversible and quasi-reversible systems

Fig 91 203 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in oxygenated

001 M PBS at pH 74 at pretreated GCE

Fig 92 204 Koutecky-Levich plot for O2 reduction at a pretreated GCE in oxygenated 001 M PBS at

pH 74 with applied potential ndash 04 06 and 08 V

Fig 93 206 Log kf(E) plot for O2 reduction at various pretreated GCEs

Fig 94 207 The protocol for finding kinetic and thermodynamic parameters

Fig 95 211 The photographs of fabricated composite RDEs

Fig 96 213 The photograph of RDE experiment setting with a rotor and gas inlet

Fig 97 220 CVs of untreated GCEs at pH 74 in 001 M PBS in the absence and presence of O2

at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 98 221 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of O2

between + 09 and - 09 V vs AgAgCl at 005 Vs-1

(i) CVs for all the pretreated GCEs

(ii) CVs excludes PBS pretreatments

Fig 99 223 The bar graphs for the capacitance of GCEs in 015 M NaCl buffered with 001 M

phosphate to pH 74

Fig 100 225 Overlaid CVs during various pretreatments for GCEs involving

01 M H2SO4 alone 001 M PBS at pH 74 alone and both pretreatments

Fig 101 227 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M oxygenated

PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 102 229 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Fig 103 231 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M PBS in

the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

24

Fig 104 233 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M N2-sparged PBS

between + 09 and - 09 V vs AgAgCl

Fig 105 236 Overlaid CVs of ORR at various pretreated graphite composite electrodes at pH 74

in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 106 237 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at pH 74

in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various scan rates

50 100 200 and 500 mVs-1

Fig 107 238 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

Fig 108 239 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

Fig 109 241 CVs of untreated and H2SO4-pretreated RhC composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

Fig 110 242 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M PBS in

the absence and presence O2 at 005 Vs-1 between + 09 and - 09 V vs AgAgCl

Fig 111 243 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M PBS in the

absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 112 245 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 113 247 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 114 248 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite electrodes

Fig 115 249 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

Fig 116 251 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 117 253 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 118 254 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M PBS in

theabsence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 119 256 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated PBS at

005 Vs-1 between 0 and -09 V vs AgAgCl

Fig 120 257 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Fig 121 258 The bar graphs of the best five nα

25

Fig 122 259 The bar graphs of the best five kdeg

Fig 123 260 The bar graphs of the best five Epc2(HDV)

Fig 124 266 A photograph of the fuel cell stack made with GOx anodes and Co(salophen)

cathodes

26

LIST OF TABLES

Table No Page Description

Table 1 35 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Table 2 87 Electrochemical behaviour of TTF-TCNQ at pH 74

Table 3 104 Michaelis-Menten equation at various [S]

Table 4 117 Composite composition table

Table 5 119 Blended stabiliser composition

Table 6 139 Four possible causes of glucose amperometric current reduction

Table 7 152 Comparison of maximum output current and KM with some published data

Table 8 159 Bond orders bond lengths and bond energies of some dioxygen species

Table 9 180 The electric conductivity of various carbon forms and sections

Table 10 186 Various ECP examples

Table 11 192 CV diagnosis for reversible system at 25 ˚C

Table 12 193 CV diagnosis for irreversible system at 25 ˚C

Table 13 196 CV diagnosis for reversible system by surface-confined groups at 25 ˚C

Table 14 214 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Table 15 216 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC

Table 16 217 Parameter values for Koutecky-Levich equation

O2 diffusion coefficient O2 concentration and PBS kinematic viscosity

Table 17 261 Three candidate electrodes for high ORR catalysis

Table 18 261 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

27

LIST OF EQUATIONS

Eq No Page Description

Eq 1 35 Anode glucose oxidation reaction

Eq 2 35 Cathode oxygen reduction reaction

Eq 3 35 Overall glucose-oxygen fuel cell reaction

Eq 4 36 Electric power equation

Eq 5 36 Electric current equation in electrochemistry

Eq 6 38 Faradayrsquos second law of electrolysis Charge equation

Eq 7 38 Faradayrsquos second law of electrolysis Reaction equation

Eq 8 39 Transition state theory equation Rate constant equation

Eq 9 39 Transition state theory equation Equilibrium constant

Eq 10 39 Transition state theory equation Atomic vibration frequency equation

Eq 11 40 Transition state theory equation Erying equation

Eq 12 42 Gibbs free energy shift equation in thermodynamic terms

Eq 13 42 Gibbs free energy shift equation in electrochemical terms

Eq 14 49 A typical redox reaction

Eq 15 50 Nernst equation

Eq 16 50 Electrode potential shift

Eq 17 50 Free energy shift in electrochemical terms

Eq 18 52 Anodic oxidation activation free energy shift in terms of potential shift

Eq 19 52 Cathodic reduction activation free energy shift in terms of potential shift

Eq 20 53 Forward reduction rate constant equation in terms of kdeg and potential shift

Eq 21 53 Backward oxidation rate constant equation in terms of kdeg and potential shift

Eq 22 54 Butler-Volmer equation

Eq 23 54 Overpotential equation

Eq 24 54 Exchange current equation

Eq 25 55 Butler-Volmer equation in terms of electrode equilibrium constant

Eq 26 55 Butler-Volmer equation in terms of overpotential

Eq 27 56 Butler-Volmer equation for small overpotential

Eq 28 56 Tafel equation for large overpotential

Eq 29 60 Diffusion overpotential equation

Eq 30 61 Ohmrsquos law

Eq 31 63 Cell voltage equation

Eq 32 63 Gibbs free energy available from the fuel cell

28

Eq 33 73 Glucose oxidation reaction by GOx

Eq 34 73 Glucose oxidation reaction by NAD-DHG

Eq 35 87 TTF-TCNQ reaction At equilibrium state

Eq 36 87 TTF-TCNQ reaction TCNQ reduction at lt + 015 V vs AgAgCl

Eq 37 87 TTF-TCNQ reaction TCNQ lattice reduction at lt - 015 V vs AgAgCl

Eq 38 87 TTF-TCNQ reaction Anodic TCNQ salt formation at gt + 031 V vs AgAgCl

Eq 39 87 TTF-TCNQ reaction TTF lattice oxidation at gt + 035 V vs AgAgCl

Eq 40 87 TTF-TCNQ reaction TTF further oxidation at gt + 038 V vs AgAgCl

Eq 41 101 Enzyme kinetics A typical enzyme reaction

Eq 42 102 Enzyme kinetics Enzyme-substrate complex formation at steady state

Eq 43 102 Enzyme kinetics Rate of enzyme-substrate complex formation in terms of k-1 and k2

Eq 44 102 Enzyme kinetics

Enzyme-substrate complex equation in terms of [E0] k1 k-1 k2 and [S]

Eq 45 102 Enzyme kinetics Enzyme catalytic reaction

Eq 46 102 Enzyme kinetics Enzyme catalytic reaction equation

Eq 47 102 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of enzyme-substrate complex

Eq 48 103 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of [E0] k1 k-1 k2 and [S]

Eq 49 103 Enzyme kinetics Michaelis-Menten constant definition

Eq 50 103 Michaelis-Menten equation

Eq 51 104 Michaelis-Menten equation when [S] ge KM

Eq 52 104 Michaelis-Menten equation when [S] = KM

Eq 53 104 Michaelis-Menten equation when [S] le KM

Eq 54 106 Electrochemical Michaelis-Menten equation

Eq 55 106 Electrochemical Michaelis-Menten equation for current density

Eq 56 108 Electrochemical Lineweaver-Burk linear regression equation

Eq 57 109 Electrochemical Eadie-Hofstee linear regression equation

Eq 58 111 Electrochemical Hanes-Woolf linear regression equation

Eq 59 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for Jm

Eq 60 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for KM

Eq 61 128 Enzyme electrode specificity constant

Eq 62 139 The causes of anodic current reduction A typical GOx-O2 reaction

Eq 63 139 The causes of anodic current reduction TTF reduction by GOx

Eq 64 139 The causes of anodic current reduction GOx allosteric inhibition

Eq 65 139 The causes of anodic current reduction TTF oxidation by O2

29

Eq 66 139 The causes of anodic current reduction TTF oxidation by GOx

Eq 67 161 O2 Dissociative mechanism Simultaneous oxygen adsorption and molecular fission

Eq 68 161 O2 Dissociative mechanism Surface hydroxide formation

Eq 69 161 O2 Dissociative mechanism Oxygen reduction to water

Eq 70 161 O2 Associative mechanism Oxygen adsorption

Eq 71 161 O2 Associative mechanism Peroxide formation

Eq 72 161 O2 Associative mechanism Peroxide reduction

Eq 73 161 O2 Associative mechanism Surface oxygen reduction to hydroxide

Eq 74 161 O2 Associative mechanism Surface hydroxide reduction to water

Eq 75 175 Anthraquinone O2 reduction Semiquinone anion radical formation

Eq 76 175 Anthraquinone O2 reduction O2 reduction by a semiquinone anion radical

Eq 77 175 Anthraquinone O2 reduction Spontaneous superoxide reduction to O2

Eq 78 175 Anthraquinone O2 reduction

Spontaneous superoxide reduction to hydroperoxide

Eq 79 190 Electric double layer capacitance per unit area

Eq 80 192 CV diagnosis ndash Reversible system Ip vs υfrac12

Eq 81 192 CV diagnosis ndash Reversible system Ep

Eq 82 192 CV diagnosis ndash Reversible system ∆Ep

Eq 83 192 CV diagnosis ndash Reversible system δEp

Eq 84 192 CV diagnosis ndash Reversible system Reversal parameter

Eq 85 193 CV diagnosis ndash Irreversible system Ip vs υ

Eq 86 193 CV diagnosis ndash Irreversible system Ep a function of kdeg α and υ

Eq 87 193 CV diagnosis ndash Irreversible system ∆Ep

Eq 88 193 CV diagnosis ndash Irreversible system δEp

Eq 89 195 CV diagnosis ndash EE system Peak separation of EE system

Eq 90 196 CV diagnosis ndash Surface-confined groups Ip vs υ

Eq 91 196 CV diagnosis ndash Surface-confined groups Ep

Eq 92 196 CV diagnosis ndash Surface-confined groups ∆Ep2

Eq 93 198 Diffusion layer thickness in terms of ω

Eq 94 199 Levich equation in terms of ω

Eq 95 199 Levich equation in terms of diffusion layer thickness

Eq 96 202 Koutecky-Levich equation

Eq 97 204 The rate constant for arbitrary potential E

Eq 98 205 k(E) in terms of kdeg and η for a forward reaction

Eq 99 205 kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

Eq 100 238 Surface semiquinone radical formation

30

Eq 101 238 ORR by surface semiquinone radical

31

1 INTRODUCTION

11 INTRODUCTION TO FUEL CELLS

111 History of the fuel cells

Fuel cells are electrochemical devices which directly convert fuel into

electricity via chemical reactions The invention dates back to 19th century when a

British physicist Sir William Grove and a German-Swiss chemist Christian Schoumlnbein

discovered the reverse reaction of water electrolysis The current output by this system

was however very small Therefore the discovery stayed in the shadow of the steam and

internal combustion engines and it needed another century to gain its attention

In the 1930s a British engineer Francis Bacon started working towards

alkaline fuel cells (AFCs) and in the 1950s General Electric developed polymer

electrolyte fuel cells (PEFCs) Those fuel cells were loaded on the spaceships in the

1960s by NASA because they were compact provided clean power with large energy

density and generated drinkable water as a by-product [1-5] Since then fuel cells have

been continuously used for space and marine technologies [3] In the 1970s studies on

fuel cells were further expanded for consumer applications [1 3 5 6] The first

air-breathing fuel cell hybrid car was built by Kordesch [1 7] and phosphoric acid fuel

cells (PAFCs) were developed and introduced to offices and hospitals [2 3 5]

32

Fig 1 Some historical fuel cell applications (i) Alkaline fuel cell for Apollo 11 [8] (ii)

Kordeschrsquos Austin A40 city hybrid car [7]

Since the 1990s fuel cells have been drawing keen attention as flexible renewable

energy in conjunction with the depletion of fossil fuel global warming and the demand

in cleaner energy [6 9] Great effort has been made to miniaturise fuel cells for

individual users such as mobile devices vehicles and households [1 3 5 6] The recent

advance in such technologies has led to direct methanol fuel cells (DMFCs) for

mobile-phone handsets [10 11] cogeneration systems to supply household power [1 5]

hydrogen fuel cell cars [12-15] and bio-batteries running on glucose and air oxygen for

portable music players [16-18] Glucose-oxygen biofuel cells have been also further

miniaturised for medical implantable purposes [18 19]

33

Fig 2 Modern fuel cell technologies (i) Toshibarsquos world smallest DMFC [20] (ii)

TokyoGasrsquos cogeneration system Enefarm (iii) BMWrsquos Hydrogen 7 (iV)

Sonyrsquos Bio Battery generating electricity from sugar [16]

112 Principles of the fuel cells

A fuel cell unit simply consists of an anode a cathode electrolyte and a

conducting wire A conducting wire connects an anode and a cathode to form an

external circuit This allows coupling of electrochemical reactions taking place at each

electrode and provides a conductive path for electrons between them Electrolyte

allows charged species to move between two electrodes but blocks out the entry of

electrons This completes the electric circuit and permits electric conduction At the

anode fuel is oxidised by an oxidation reaction and this supplies electrons to the

cathode through the external circuit The number of electrons generated depends on the

34

type of fuels reactions and environment in which the reactions take place In the anode

reaction ions are also released to balance overall charge They move to the cathode

though the electrolyte At the cathode ions from the electrolyte and electrons from the

circuit meet and react together with an oxidiser supplied The completion of the coupled

reactions generate electricity and products [3 4 21] Fig 3 shows a glucose-oxygen

fuel cell as an example

Fig 3 A glucose-oxygen fuel cell

At the anode glucose is oxidised into gluconolactone This gives out two electrons and

two protons At the cathode oxygen gas receives them and is reduced to water Each

reaction has a unique redox potential called standard electrode potential Edeg and

electromotive force (EMF) is the potential difference between such electrode potentials

It is also called open circuit voltage (OCV) This is the theoretical size of potential

difference available from the fuel cell [3 4] The coupled reactions for glucose-oxygen

fuel cell is shown in Table 1 Each redox potential was measured against silver

35

silver-chloride reference electrode (AgAgCl) at pH 7 25degC therefore shown in formal

potential (Edegrsquo)

Table 1 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Anode Edegrsquo = - 042 V (Eq 1)

(via FADFADH2 redox [22 23])

Cathode [24] Edegrsquo = 062 V (Eq 2)

Overall ∆Edegrsquo = 104 V (Eq 3)

The reaction potential at each electrode is measured separately as half cell potential

against the universal reference electrode such as AgAgCl because it is impossible to

directly measure both inner potentials simultaneously

113 Electric power from the fuel cells

Electric power (P) is the amount of work can be handled by electric current per

unit time It is defined as in Eq 33 The size of power is determined by the size of

electric current (I) and voltage (V) since P is proportional to I and P

36

Electric power

VIP times= middotmiddotmiddotmiddotmiddot Eq 4

P = Electric power W

I = Electric current A

V = Voltage V

Electric current is the amount of charge (Q) flows per unit time Voltage is the electric

potential energy per unit charge and synonymous with potential difference (∆E) EMF

or OCV Increase in I andor V increases P V is however constrained by the type of

electrode reactions as discussed previously I depends on (i) reaction rate (ii) the

electrode surface and (iii) reactant concentration as shown in Eq 5

Electric current in electrochemistry

nFAkCnFAI == υ middotmiddotmiddotmiddotmiddot Eq 5

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

A = Electrode surface area m-2

υ = Reaction rate (1st or pseudo 1st order) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

It is indispensable to have large rate constant to increase output current This demands

lowering activation energy for a chemical transition during the reaction and can be

achieved by use of the catalysts

37

12 CATALYSTS

121 Faradayrsquos second law ndash Current and reaction rate

Catalysts accelerate specific chemical reactions without altering the change in

overall standard Gibbs free energy In other words they change the reaction kinetics but

not the thermodynamics They only provide an alternative lower-energy pathway to

shorten the reaction time without changing themselves before and after the reaction

[25-27] In fuel cells catalysts are used to increase the current output by accelerating the

electrode reaction The relationship between current and rate of reaction is defined by

Faradayrsquos second law of electrolysis (Eq 5 - Eq 7) This expresses the size of electric

charge generated in terms of the number of moles of reactant consumed or product

produced

38

Faradayrsquos second law of electrolysis

nFNQ = middotmiddotmiddotmiddotmiddot Eq 6

AtNCktimes

=times=υ middotmiddotmiddotmiddotmiddot Eq 7

Q = Charge C

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

N = The number of moles of reactant or product mol

υ = Reaction rate (1st or pseudo 1st ordedr) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

t = Time s

A = Electrode surface area m-2

Faradayrsquos second law of electrolysis states that an increase in reaction rate increases the

current output To increase the reaction rate intrinsically its Gibbs free energy of

activation (∆GDagger) must be lowered The relationship between k and ∆GDagger is stated in

Erying equation [28]

122 Transition state theory Eyring equation and reaction rate

When a reaction proceeds it must go through a critical state called transition

state of maximum potential energy also called the activated complex Activated

complexes are unstable molecules existing for only a few molecular vibrations before

decaying into the products Transition state theory assumes that the reactants and the

39

activated complexes are in quasi-equilibrium and the reaction rate is constrained by the

rate at which the activated complex decays into the products This implies that the

reaction rate depends on the equilibrium constant between the activated complexes and

the reactants (KDagger) and the atomic vibrational frequency (ν) as shown in Eq 9 ν is

defined by statistical mechanics in terms of Boltzmann constant (kB) Planck constant

(h) and the temperature (Eq 10) [28]

Transition state theory and reaction rate [28]

[ ]RTexpDagger Dagger

Dagger GC

CK ∆minus== middotmiddotmiddotmiddotmiddot Eq 8

Dagger Kk ν= middotmiddotmiddotmiddotmiddot Eq 9

hTkB=ν middotmiddotmiddotmiddotmiddot Eq 10

KDagger = Equilibrium constant between the reactants and the activated

complexes

s-1

CDagger = Activated complex concentration molm-3

C = Reactant concentration molm-3

∆GDagger = Gibbs free energy of activation Jmol-1

R = Gas constant = 8314 Jmol-1K-1

T = Thermodynamic temperature K

k = Rate constant (1st or pseudo 1st reaction) s-1

ν = Atomic vibrational frequency s-1

kB = Boltzmann constant = 13807 x 10-23 JK-1

h = Planck constant = 662621 x 10-34 Js

40

Based on this the Erying equation expresses the relationship between the reaction rate

and Gibbs activation free energy as shown in Eq 11 [25 28 29]

Erying equation TG

B

hTkk

RDagger ∆minus

= κ middotmiddotmiddotmiddotmiddot Eq 11

where κ = transmission coefficient

This tells that to increase the rate constant reaction temperature must be increased or

the activation free energy must be lowered

123 Overcoming the activation free energy

Activation free energy is a potential energy gap between the reactant and

activated complex At the transition state intra-molecular reorganisation takes place

through the bond destruction and formation This demands the energy and slows the

kinetics The reaction does not proceed unless this energy barrier is overcome Hence

energy input in someway or lowering activation free energy is required for the reaction

to proceed andor accelerate the reaction Heat energy can provide atomic and molecular

kinetic energy so that the system can overcome the energy barrier as indicated in Eq 11

[4 21 29] Applying electric potential also has the similar effect It provides the electric

energy to overcome the energy barrier The rate constant therefore can be altered

extrinsically by temperature andor electrode potential [21 29 30] Alternatively

catalysts can reduce ∆GDagger so that the reaction rate can be increased intrinsically They

enable or accelerate a reaction by providing alternative lower-energy reaction pathway

41

This does not change the reaction path or the standard enthalpy change (∆Hdeg) of the

system however lowers the activation free energy for this path so that the reactants can

be converted more quickly to the products as shown in Fig 4

Fig 4 Energy transition for catalytic reaction through the transition state

Fig 4 shows the energy state transition during a reaction with and without a catalyst

The reaction coordinate represents the reaction progress along the reaction pathway

∆Hdeg is a state function [31] therefore determined only by the initial and final state of

the system but independent of the reaction path ∆Hdeg is divided into standard Gibbs free

energy change (∆Gdeg) and the term of standard entropy change (∆Sdeg) as Eq 12 shows [4

21 23 26 32]

The key insight therefore from transition state theory is that for a catalyst to function it

42

must spontaneously bind the transition state

Gibbs free energy change

deg∆minusdeg∆=deg∆ STHG middotmiddotmiddotmiddotmiddot Eq 12

deg∆minus= EnF middotmiddotmiddotmiddotmiddot Eq 13

∆Gdeg is the standard free energy difference which became available for work through

the reaction and must be lt 0 for a spontaneous reaction ∆Gdeg can be related to the

standard electrical potential difference as Eq 13 shows [4 21 29 30 33 34] It is the

maximum EMF theoretically available from the system ∆Sdeg is the difference in the

degree of disorder in the system The entropy term (T∆Sdeg) is the energy not available for

work and temperature dependent This and Eq 13 imply that ∆Gdeg is also altered by

temperature as shown in Fig 4 [35]

124 Variations in catalysts

Given the general applicability of transition state theory it is not surprising that

catalysts have extensive variation and applications the reaction phase can be

homogeneous or heterogeneous catalysts can be inorganic organic or biological

Inorganic catalysts are metals such as transition or noble metals or complexes

on metal ions in various oxidation states Elemental metal catalysts typically catalyse

reactions through surface adsorption Metal particles have extensive variation in

physical properties such as particle size shape and surface area They often come in

dispersed on the porous support material for improved catalytic performance due to

43

increased surface area [36] and cost efficiency [1] Composition and structure of the

particles thickness of support material and condition of metal deposition all affect

reactions [27 37-39] A complex is a compound in which a ligand is loosely bonded to

a metal centre through coordination bonds An unsaturated complex has a dissociated

ligand and catalysis takes place when a substrate binds to that in the single orientation

[27 40] Complexes also have important roles in biological system [41-43] Organic

catalysts are non-metal non-biological catalysts They are often functionalised solid

surfaces for surface catalysis [27] In electrochemistry such catalysts can be chemically

bonded to conductive material [44] Simple electrochemical pretreatment can generate

or attach chemical groups onto the electrode surface and they mediate electron transfer

[45-47] Biological catalysts are generally called enzymes Enzymes are biological

polymers mostly made of protein and usually have high reaction and substrate

specificity They often have cofactors which are not proteinous but essential for their

catalytic activities Enzymes are involved in virtually all biological activities [23 25 27

48] and in a few industrial applications due to their substrate specificity [27]

Catalysts are by definition not supposed to be consumed in their reactions

however they could lose their catalytic power Impurities fouling and poisoning are

common deactivating factors for the metal catalysts Competing reactions also convert

them into inactive forms Particle migration and agglomeration reduce their catalytic

surface area hence their overall activity [27] Biological catalysts are very sensitive to

the environment Slight changes in pH and temperature can deactivate enzymes Their

stability is also affected by presence of metal ions surfactants oxidative stress and

organic solvent molecules [49] Metal catalysts can be possibly regenerated by

redispersion [27] however it is very difficult to recover biological catalytic functions

44

Therefore enzyme stabilisers are often added to extend their life Protein engineering

also can confer advantageous properties to the enzymes Amino acid sequences leading

to favourable protein folding functions or stability are determined in silico and they are

introduced to existing enzymes by genetic engineering such as site-directed mutagenesis

Protein engineering of serine protease subtilisin is a classic example [50]

13 ELECTRODE POTENTIAL AND REACTION RATE

131 Fermi level electrode potential and electron transfer

In the sect 123 the relationship between the electric potential and activation free

energy was briefly discussed As heat energy can provide the kinetic energy to atoms

and molecules for a reaction to proceed the electric energy also can provide kinetic

energy to electrons for electron transfer to proceed This leads to the redox reaction at

the electrode surface

Each substance has a set of discrete energy levels Each energy level can

accommodate up to two electrons Electrons fill these energy levels in ascending order

from the lower level This can divide these energy levels into two groups electron-filled

lower energy level and empty higher energy level without electrons In between the

critical energy level the called Fermi level lies It is designated as εF (Fermi energy) or

EF (Fermi potential) The Fermi level contains electrons of highest energy A substance

can transfer only these electrons to the external system as a donor while as an acceptor

it can accommodate external electrons only within the empty energy level which is

above its Fermi level (Fig 5)

45

Fig 5 Fermi level and electron transfer

The energy state of electrons however varies by substance therefore electrons at Fermi

level so-called highest occupied molecular orbital (HOMO) of a donor may need to

gain various kinetic energy to jump into the lowest empty energy level so-called lowest

unoccupied molecular orbital (LUMO) of an acceptor for electron transfer (Fig 6)

46

Fig 6 Electrode potential and electron transfer

As soon as the energy of electrons at Fermi level of a substance or an electrode meets

the energy level of LUMO of the other redox molecule electron transfer proceeds as

long as there is room to accommodate electrons in the acceptor This is the cathode

reaction The potential can be swept back by applying positive potential to the electrode

This lowers the energy of LUMO of the electrode so that an electron in HOMO of the

donor can be transferred back to the electrode This is the anode reaction If the electron

transfer is fast in both directions compared with other reaction steps it is a

thermodynamically reversible system and the standard electrode potential (Edeg) for this

redox reaction lies somewhere between and close to those two electrode potentials

which have enabled electron transfer At Edeg both anode and cathode reactions have the

same reaction rate hence they are at equilibrium [33] and there is no net current [21 30]

47

132 Electrode potential equilibrium potential and Nernst equation

Activation free energy of an electrode reaction can be altered by applying

potential The extent of its alteration and the direction of the reaction depend on the size

and direction of potential applied Fig 7 shows the difference in the standard activation

free energy (∆GdegDagger) for redox reactions at various electrode potentials [30]

48

Fig 7 Standard free energy and electrode potential [30]

(a) At E = Edeg (b) E gt Edeg and (c) E lt Edeg

More positive potential lowers the activation free energy for oxidation hence shifts the

reaction in favour of oxidation and vice versa This implies that applying extra potential

to the electrode perturbs the equilibrium and to restore it the electrode equilibrium

49

potential (Ee) must be shifted This phenomenon is clearly stated in Nernst equation (Eq

15 on p 50) as a relationship between Ee and the equilibrium constant (K) at the

electrode surface At equilibrium both oxidation and reduction reactions take place at

the same rate and there is no net current therefore the surface concentration (C(0t))

approximates bulk concentration (C) Nernst equation shows that Ee is always under

the influence of such concentration but can be established provided the reaction is fast

enough to restore the equilibrium [21 30] In the dilute solutions of the electroactive

species Edeg is substituted with the formal potential (Edegrsquo) which considers the influence

of inter-solute and solvent interactions [21 29]

Redox reaction

middotmiddotmiddotmiddotmiddot Eq 14

O = Oxidant

ne- = The number of electrons transferred

R = Reactant

kf = Forward reaction rate constant (1st or pseudo 1st reaction) s-1

kb = Backward reaction rate constant (1st or pseudo 1st reaction) s-1

50

Nernst equation

lowast

lowast

+deg=R

Oe

CC

nFRTEE ln middotmiddotmiddotmiddotmiddot Eq 15

Ee = Equilibrium potential V

Edegrsquo = Formal potential V

R = Gas constant = 8314 Jmol-1K-1

T = Absolute temperature K

n = The number of electron transferred

F = Faraday constant = 96487 Cmol-1

CO = Bulk oxidant concentration molm-3

CR = Bulk reductant concentration molm-3

133 Electrode potential and activation free energy

When E ne Edegrsquo the difference in these potentials is expressed as the electrode

potential shift (∆E) as in Eq 16 and its free energy shift (∆G) as in Eq 17

Electrode potential shift

degminus=∆ EEE middotmiddotmiddotmiddotmiddot Eq 16

Free energy shift

)( degminus=∆=∆ EEnFEnFG middotmiddotmiddotmiddotmiddot Eq 17

When ∆E is applied positively it lowers the anode oxidation activation free energy

(∆GaDagger) by fraction of the total energy change (∆G) as shown in Fig 8 on p 51 The

fraction for the oxidation reaction is defined as (1-α) The transfer coefficient (α) is a

51

kinetic parameter which states the degree of symmetry in the potential energy barrier

for redox reactions The symmetry in energy barrier is present when the slopes of two

potential energy curves are the same and it is defined as α = 05 α is dependent on

redox potential and could vary within the range between 0 and 1 In most cases α = 05

and appears to be constant when overpotential is small [21 30]

Fig 8 The shift of ∆E and ∆GdegDagger [21 30]

With α the shift in electrode activation free energy can be expressed in terms of

potential shift for both oxidation and reduction reactions as shown in Eq 18 and Eq 19

52

∆GDagger in terms of (E-Edegrsquo)

Anode Oxidation )()1(DaggerDagger degminusminusminus∆=∆ EEnFGG aa αo middotmiddotmiddotmiddotmiddot Eq 18

Cathode Reduction )(DaggerDagger degminus+∆=∆ EEnFGG cc αo middotmiddotmiddotmiddotmiddot Eq 19

∆GoDagger = Anode oxidation activation free energy Jmol-1

∆GodegDagger = Anode oxidation standard activation free energy Jmol-1

α = Transfer coefficient molm-3

n = The number of electrons transferred Jmol-1

F = Faraday constant = 96485 Cmol-1

E = Arbitrary electrode potential V

Edegrsquo = Formal potential V

∆GRDagger = Cathode reduction activation free energy Jmol-1

∆GRdegDagger = Cathode reduction standard activation free energy Jmol-1

134 Potential shift and standard rate constant

By substituting the activation free energy in Eyring equation (Eq 11) with the

shift in electrode activation free energy in Eq 18 and Eq 19 it is possible to express k

in terms of potential shift and standard rate constant (kdeg) (Eq 20 and Eq 21) kdeg is the

rate constant at Edegrsquo[21 30]

53

k in terms of (E-Edegrsquo) and kdeg

Forward reduction cathode rate constant (kf) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bf

Daggercexpκ

⎥⎦⎤

⎢⎣⎡ degminusminus

⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )(expexp

Dagger

EERT

nFRT

GhTk cB ακ

o

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= )(exp EERT

nFk fα middotmiddotmiddotmiddotmiddot Eq 20

Backward oxidation anode rate constant (kb) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bb

Daggeraexpκ

⎥⎦⎤

⎢⎣⎡ degminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )()1(expexp

Dagger

EERT

nFRT

GhTk aB ακ

o

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= )()1(exp EE

RTnFkb

α middotmiddotmiddotmiddotmiddot Eq 21

kf and kb = Forward and backward reaction rate constant s-1

kdegf and kdegb = Forward and backward reaction standard rate constant s-1

135 Butler-Volmer equation overpotential and current

At the equilibrium potential E = Ee kf = kb hence anodic current (Ia) =

cathodic current (Ic) and therefore the net current (I =Ia ndash Ic) is macroscopically zero (I =

0) However both forward and backward reactions still occur microscopically at the

electrode surface The absolute value of this electric current is called exchange current

(I0) and is not directly measurable When CO = CR at E = Ee C O (0t) = C R (0t) and kdegf

= kdegb = kdeg hence the net current any potential E can be defined as below by

Butler-Volmer equation (Eq 22)

54

Butler-Volmer equation

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡ degminus

minusminus⎥⎦

⎤⎢⎣⎡ degminusminus

deg= )()1(exp)(exp )0()0( EERT

nFCEERT

nFCnFAkI tRtOαα middotmiddotmiddotmiddotmiddot Eq 22

The Butler-Volmer equation states the relationship between the electric current

and the potential shift This can be further rephrased in terms of overpotential (η)

through defining I0 using Nernst equation (Eq 15) η is the potential required to drive a

reaction in a favourable direction from its equilibrium state and defined as n Eq 23

Overpotential (η)

eEE minus=η middotmiddotmiddotmiddotmiddot Eq 23

At E = Ee both anode and cathode exchange currents are the same therefore both

exchange currents can be defined together as I0 (see below) and I0 can be expressed in

terms within Nernst equation by substituting (Ee-Edegrsquo) as in Eq 24

Exchange current

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= lowast )(exp0 EERT

nFCnFAkI eO

α

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= lowast )()1(exp EE

RTnFCnFAk e

Rα

αα )()( 1RO CCnFAk minus= o middotmiddotmiddotmiddotmiddot Eq 24

Now the Butler-Volmer equation (Eq 22) can be expressed in terms of η (= E ndash Ee) (Eq

25) by expressing it with I0 and the with re-arranged Nernst equation

55

⎥⎦⎤

⎢⎣⎡ degminus=lowast

lowast

)(exp EERTnF

CC e

R

O The term

)0(

CC t in Eq 25 indicates the influence of mass

transport For purely kinetically controlled region the electrode reaction is independent

of mass transport effects therefore C(0t) asymp C and Bulter-Volmer equation approximates

to Eq 26

Butler-Volmer equation and η

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminusminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminus

minus= lowast

lowast

lowast

minusminus

lowast

lowast

lowast

αααα

R

O

O

tO

R

O

R

tR

CCEE

RTnF

CC

CCEE

RTnF

CC

II )(exp)()1(exp )0()1(

)0(0

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= lowastlowast ηαηαRT

nFC

CRT

nFC

CI

O

tO

R

tR exp)1(exp )0()0(0

middotmiddotmiddotmiddotmiddot Eq 25

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= ηαηαRT

nFRT

nFI exp)1(exp0 middotmiddotmiddotmiddotmiddot Eq 26

The Butler-Volmer equation shows the dependence of current and hence the reaction

rate on the overpotential This implies that small kdeg can be overcome by overpotential

The kinetically-limited region of the electrode reaction is however very small because

the rate constant sharply increases exponentially with overpotential and soon the

reaction became diffusion limited [21 30] At this state the surface equilibrium is

totally perturbed and the overpotential is used to drive the diffusion for the one-way

reaction

136 Butler-Volmer equation vs Tafel equation

At small η lt RTnF Butler-Volmer equation further approximates to a linear

equation as shown in Eq 27

56

Small η Butler-Volmer equation

η⎟⎠⎞

⎜⎝⎛=

RTFnII 0

middotmiddotmiddotmiddotmiddot Eq 27

At large |η| gt RTnF the rate of the backward reaction is negligible hence one

of the exponential terms in Butler-Volmer equation becomes negligible and the

equations are solved for η for both anode and cathode reactions as below

Large η Tafel equations

InFRTI

nFRT lnln 0 plusmn= mη in the form of ||ln Iba +=η middotmiddotmiddotmiddotmiddot Eq 28

Anode oxidation InF

RTInF

RT ln)1(

ln)1( 0 αα

ηminus

+minus

=

Cathode reduction InF

RTInF

RT lnln 0 ααη minus=

In the system which follows the Tafel equations I0 (and hence kdeg ) is very small and the

electrode reaction does not proceed unless large η is supplied In this potential range the

reaction is already totally irreversible [4 21 30] An irreversible slow reaction is

graphically presented in Tafel plot in which ln (I) is plotted against η as shown in Fig

9

57

Fig 9 Tafel plot [4 21 30]

I0 is deduced from the y-intercept of the extrapolated slope Once |I| has become too

large and reached to the mass transport limit at large |η| the slope deviates from the

linear trend [30]

14 OVERPOTENTIAL AND FUEL CELLS

141 Four overpotentials that decrease the EMF

As discussed so far overpotential is extra potential required to drive a reaction

In the fuel cells this can only be taken from the potential generated within the system

causing potential drop and reduces EMF The overpotential in the system is attributed to

the four causes [1 4] (i) internal current (ii) activation overpotential (iii) diffusion

overpotential and (iv) resistance overpotential

58

142 Internal current

Internal current is a reverse current caused by crossover and leakage current

Crossover happens when the fuel flows to the opposite electrode through the electrolyte

Leakage current occurs when the current flows through the electrolyte rather than the

external circuit Internal current therefore happens without a load and is observed as the

negative offset from the theoretical value of EMF This is noted especially in

low-temperature fuel cells such as direct methanol fuel cells (DMFCs) [4 51] In such

systems The inner current can be several 10 Am-2 and potential drop could be more

than 03 V [4] In order to prevent fuel mixture and crossover a separator is used to

partition the cell Alternatively it is also possible to confer high specificity to electrodes

as seen in glucose - oxygen fuel cells Such systems do not require

compartmentalisation and this is advantageous for miniaturisation [52]

143 Activation overpotential

Activation overpotential is a potential loss due to the slow reaction kinetics as

discussed in sect 135 This is unavoidable to some extent and is typically the biggest

problem in most fuel cells involving O2 cathode reduction because this reaction is

kinetically hindered Finding or inventing catalysts with high kdeg is the key solution for

this A sluggish reaction exhibits a sharp rise in activation overpotential for a slight

increase in low current After the steep increase the current increase becomes linear and

constant as shown in Fig 10 until the reaction reaches the limit of mass transport at the

higher overpotential [4 30 34]

59

Fig 10 Activation overpotential against current [4]

144 Diffusion overpotential

Diffusion overpotential is a potential loss due to concentration gradient This

tries to drive flux of electroactive species toward and from the electrode surface It

happens when the reaction kinetics is faster than mass transport [4 21 30] In fuel cells

diffusion overpotential is caused by fuel depletion due to the inefficient fuel convection

or supply [21 34] This problem is observed in reformers because the fuel refining and

conversion such as liquefied petroleum gas (LPG) to hydrogen gas take place outside

the system and it makes difficult to respond to the sudden fuel demand from the system

[4] It also happens when fuel diffusion is interfered with by bad electrode geometry

electrode fouling and adsorption [21 27] Diffusion overpotential is defined as Eq 29

60

Diffusion overpotential equation

⎟⎟⎠

⎞⎜⎜⎝

⎛minus=

Ldiffusion I

InFRT 1lnη middotmiddotmiddotmiddotmiddot Eq 29

ηdiffusion = Diffusion overpotential V

IL = Limiting current A

Limiting current (IL) is generated when a reaction takes place at the rate limited by mass

transport Diffusion overpotential sharply increases when reaction kinetics have reached

to the maximum and very large current are drawn as shown in Fig 11 [21 30 34 53]

Fig 11 Diffusion overpotential against current [4]

145 Resistance overpotential

Resistance overpotential arises within electrodes separators connectors circuit

or electrolyte [4 34] It is called Ohmic resistance or IR drop because it follows Ohmrsquos

61

law (Eq 30) where resistance overpotential is linearly proportional to the electric

current as shown in Fig 12 [4 30]

Ohmrsquos law

IRV = middotmiddotmiddotmiddotmiddot Eq 30

V = Voltage consumed due to the resistance V

I = Electric current drawn due to the resistance A

R = Resistance Ω

| |

Fig 12 Resistance overpotential against current [4]

Causes of internal resistance are scattered through the system therefore there are many

possible ways to resolve this One can possibly reduce it by using more conductive

material The difficulty lies in finding the conductive material which can be modified as

well as carry out the functions as one desires under the required conditions [53]

Resistance can be reduced by reducing the distance between two electrodes [54]

however this might cause short circuit and fuel leakage This becomes a serious issue in

62

use of solid electrolyte such as polymer electrolyte because both performance efficiency

and mechanical stability of electrolyte must be achieved [53] Excess in inert supporting

electrolyte minimises solution resistance and matrix effect It sustains a thin electric

double layer uniform ionic strength and buffering effect against pH change [21 30]

However electrolyte could become a problem when electrode material dissolves into

electrolytic solution and reacts there because it might change nature of electrolyte [1

21]

146 Total overpotential and cell voltage

Total overpotential is the sum of all four overpotentials Current ndash Voltage (IV)

polarisation curve visualises the tendency of each potential drop as shown in Fig 13 [1

4]

Fig 13 V-I curve for a typical low temperature fuel cell [4]

63

The cell voltage (Ecell) is determined by subtracting the cathode potential (Ec)

from the anode potential (Ea) (Eq 31) The actual cell voltage is found by subtracting

the total overpotential from the theoretical cell voltage The energy available from this

cell voltage can be calculated as in Eq 32

Cell voltage

cacell EEE minus= middotmiddotmiddotmiddotmiddot Eq 31

totalltheoreticacellactualcell EE ηminus= )()(

cellcell nFEG minus=∆ middotmiddotmiddotmiddotmiddot Eq 32

15 BIOFUEL CELLS

151 Microbial and enzymatic fuel cells

Biofuel cells are fuel cells which use biocatalysts for fuel reactions

Biocatalysts can be either the whole living cells or isolated enzymes The former is

called a microbial fuel cell and the latter is called an enzymatic fuel cell [55-57] They

often function under ambient conditions and allow wider choice of fuels Microbial fuel

cells use common carbohydrates such as glucose and lactate [55] but also have

potentials in extracting power from waste and sediments [34 55 57 58] Harnessing

such microbial life activities has some advantages They can complete fuel oxidation

within the cell It was reported that a microorganism Rhodoferax ferrireducen has over

80 glucose conversion rate [58] Microbial fuel cells also have long-term stability

[56-58] of up to five years [55] However their output current and power densities are

64

small due to high internal resistance [55-57] Enzymatic fuel cells in contrast have

higher current and power densities but short life incomplete reactions require

mediators to shuttle electron between the enzyme catalytic centre and the electrode and

become deactivated easily [55 56 58] Addition of mediators and their immobilisation

with enzyme complicates fuel cells design further However they can be more

advantageous due to their substrate specificity Both anode and cathode to reside in the

same compartment of mixed fuels without crossover This enables the miniaturisation of

fuel cells [52 59 60] and is particularly useful for medical implantation Stability and

lifetime of the enzyme fuel cells can be improved by appropriate enzyme

immobilisation fixing up the suitable conditions for their function [55] and adding

some enzyme stabilisers Addition of the right mediators and increased enzyme loading

will also increase output current and power density [55 59]

152 Enzyme fuel cells and mediators

Enzyme reactions are available for wide range of substrates yet each enzyme

has very high specificity for its substrates Many also function at neutral atmospheric

conditions therefore miniaturised enzyme fuel cells are of interest for in vivo

application Many redox reactions however take place at the catalytic centre which is

deeply buried in the protein body This proteinous content is an electronic insulator and

interrupts the electrical communication between the redox centre and the electrode It is

therefore difficult for many enzymes to establish efficient electron transfer without

mediators as shown in Fig 14 This is called mediated electron transfer (MET)

65

Fig 14 Mediated electron transfer reaction [55 61]

It is possible for some enzymes such as multicopper oxidases (MCOs) to perform direct

electron transfer (DET) without mediators however mediators ensure good electrical

communication and cell performance However the presence of mediators as well as the

enzyme specificity could complicate the fuel cell composition and its reaction path due

to the reaction cascade [55 59 62 63] Palmore built a methanol - oxygen enzyme fuel

cells and this involved four enzymes two mediators and six reactions to complete the

full reaction path [64] It is more advantageous to use enzymatic fuel cells for very

specific applications [59] such as medical implantation where there are environment

constraints but where applications demand only small power output through simple

reactions

Mediators are usually small redox active species which can be organic or

metal complexes In the enzymatic fuel cells they should be carefully selected because

improper mediators can cause unwanted overpotential or can destabilise the system

They must have (i) small overpotential (ii) fast reversible electron transfer [55 56 59

60 63 65] and (iii) stability [62 65] However there should be small but reasonable

66

potential gap between the enzyme and the mediator to drive electron transfer [60 66]

The gap of 50 mV is optimal [19 64] because of the small potential loss yet less

competing with enzyme reactions It is also possible to mediate electron transfer by

building a conductive nanowire between the electrode and the redox centre [60 63 67]

or a connection by plugging metal nanoparticles into the enzyme [60 67]

16 MINIATURISED MEDICAL POWER SOURCES

161 Realisation of miniaturised fuel cells and medical power supply

Medical application is one of the obvious targets for miniaturised biofuel cells

Glucose-oxygen biofuel cells which continuously run on glucose and oxygen were

thought to be ideal for implantable medical devices because both oxygen and glucose

are abundant in the body [63] They were initially intended to power cardiac assist

devices and pacemakers The studies on such systems have been made for more than 40

years however none has been made into practical use because their power density

stability operation life and biocompatibility were inadequate [19 67-69] Their output

power ranges between microW ndash mWcm-2 and they operate only for a few days to a few

weeks Most applications need a few W power for a longer duration [18 60 67] A

pacemaker requires 1microW [56] for more than 8 ndash 10 years with 996 reliability Such

power has been supplied by lithium (Li) iodine battery [19 56 70] which is

characterised by extended operational life [68] Li is fairly stable light-weight metal

and has high energy density of 386 kAhkg-1 Its standard potential is highly reducing ndash

323 V vs AgAgCl These properties enable Li batteries to power most devices

67

including pacemakers laptops and vehicles [19 21] However there are some problems

in Li batteries They are still bulky because they need to store their own fuel and it has

to be sealed with the metal case [19 68] They occupy half of the space in the

pacemaker volume [68 71] They use corrosive [72] and irritating materials There is

potential of explosion [68] leakage and the necessity of exchange Li also has potential

resource constraint due to the geopolitical issues as seen for oil and platinum More than

80 of its production is based in South America and China [73] With increased public

concern over using greener safer energy and saving resources as well as the global

political intervention in that it is natural to consider about using alternative

technologies to power implantable medical devices where it is possible

162 Miniaturised potential medical power sources

There are numerous implantable electrical medical devices with different

power requirements These include cardiac pacemakers defibrillators neurostimulators

for pain control drug delivery device hearing aids [70] and biosensors [74]

Implantable autonomous body sensors for physiological parameters such as glucose

concentration local pH flow and pressure [19] require power as low as 1 microW [56 70

74] and the operation period of 1 year at least [74] Such power can be supplied by

various implantable energy harvesting devices using body glucose [19] motion

vibration [75] and temperature differences [75] Heller developed the smallest

glucose-oxygen biofuel cells made of carbon fibres with the dimension of 17-microm

diameter and 2-cm length Anode glucose oxidase and cathode laccase were wired to

conductive hydrogel backbone with a flexible osmium (Os) mediator complex Its

68

output was 048 mWcm-2 with 06 V at 37 degC and it lasted for 1 week [19 67]

Kinetic energy of motion and vibration can be converted into electricity with

electrostatic transduction using microelectromechanical systems (MEMS) This

technology allows integration of various microelectronic devices on the same substrate

[76] NTT Corporation developed a system generating power from body motion and

temperature It consists of a thermoelectric and a comb-shaped Au-Si vibrational device

on a monolithic Si substrate The voltage produced by a thermoelectric device is

boosted by capacitance which was generated by the vibrational device then its charge

is stored in the capacitor [75] Another vibrational device was reported to have 20 x 20 x

2 mm volume and output 6 microW and 200 V at 10 Hz low frequency and 39 ms-2

acceleration The principal difficulty in using MEMS is to adjust and synchronise

flexibly the resonance frequency of vibrational device to the human body frequency

which is low and has constant fluctuation in order to increase energy conversion

efficiency The typical vibration has the frequency ranging from 1 to 100 Hz and

acceleration from 1 - 10 ms-2 [76]

Thermoelectric devices generate power from temperature difference Each

module consists of a number of thermocouples on Si substrate Thermocouples are

made of different material so that it can generate voltage from the heat flow following

temperature gradient This is called Seebeck effect and its size depends on the material

and number of thermocouples used and the spacing within the device [75 76] Typical

ambient temperature difference is less than 10K [74 76] and its power is 1microWcm-2 [74]

Seiko Instrument developed a wristwatch called Seiko Thermic which was powered by

human body temperature in 1998 [77 78] It consists of n-type Bismuth-Tellurium

(BiTe) and p-type Bismuth-Antimony-Tellurium on rods reinforced by Ni on oxidised

69

silicon wafer It generated microWcm-2 power at extremely low 0047 conversion

efficiency With a booster circuit it powered a wristwatch operating at 1microW and 15 V

It could however operate only when ambient temperatures were below 27 degC [78] and

so could not be used in tropical regions

Heller also developed an implantable miniature Nafion-coated zinc ndash silver

chloride (ZnAgCl) battery The anode was made of zinc fibre coated with Nafion It

was 76-microm diameter 2-cm length and 75 x 10-2 cm2 surface area The cathode was

made of AgCl wire covered with chloride-permeable membrane On discharge anode

zinc formed Zn3(PO4)2middot4H2O hopeite complex with Nafion growing non-porous

lamellae on the surface The lamellale had a unique property which was permeable to

zinc ions but impermeable to O2 therefore the battery became corrosion free It output

was 13 microAcm-2 at 1 V and operated for three weeks under physiological condition The

advantage of this battery was its size which was 100-fold smaller than the smallest

conventional batteries [79] The smallest silver oxide button buttery has the volume 30

mm3 [19 80] with energy density 04 mWh mm-3 at 15 V The smallest Li-ion buttery

has the volume 190 mm3 [80] with energy density 025 mWh mm-3 at 4 V [21]

17 OUTLINE OF THE WORK

171 Introduction to miniaturised glucose-oxygen fuel cells

A miniaturised glucose-oxygen biofuel cell runs on glucose and oxygen This

would be ideal to power implantable autonomous low-power body sensors since both

glucose and oxygen are abundantly present in the body The range in their physiological

70

concentration is between 4 ndash 8 mM for glucose and 01 ndash 02 mM for oxygen [56 67]

The aim of this study was to build miniaturised glucose-oxygen fuel cells with reliable

enzyme anodes and powerful oxygen-reducing cathodes using straightforward and

scaleable technology The general structure and mechanism of glucose-oxygen fuel cells

were described in sect 112

In this study both anode and cathode reactions took place at the room

temperature and physiological pH in the presence of catalysts Glucose oxidation was

catalysed by the enzyme glucose oxidase (GOx) from Aspergillus niger and its electron

transfer and conduction were facilitated by an conducting organic salt called

tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) (Fig 15)

Fig 15 The schema for a glucose-oxidising anode

GOx and TTF-TCNQ were mixed into a composite employing a feasible electrode

fabrication method called bulk modification method Various enzyme stabilisers were

added to the composite at different concentration to improve its performance The

influence of various enzyme stabilisers on anode performance was studied in the

71

presence of both N2 and O2 and the most ideal anode composition was determined

Cathodes were also fabricated in the similar manner using graphite as conductive

material and mixing with various oxygen reducing catalysts except for the enzyme

membrane cathodes The cathode composites were pretreated in various media to

enhance their activity For enzyme membrane electrodes glassy carbon electrodes were

used instead of graphite composite and enzyme solution was loaded onto the surface

and trapped with membrane Catalytic power and mechanism of each cathode were

studied with various electrochemical methods and the most catalytic cathode was

determined

172 Outline

Chapter 1 is a general introduction about fuel cells It explains the history and

basic concept of fuel cells and outlines the study

Chapter 2 explains about the enzyme anodes The introduction part refers to

glucose oxidising enzymes enzyme immobilisation TTF-TCNQ enzyme inactivation

enzyme stabilisers and enzyme kinetics The material and method section reports the

protocol for the enzyme electrode fabrication electrode conduction study and

amperometry study for enzyme electrodes Result and discussion section mentions

enzyme electrode conduction enzyme kinetic studies based on amperometry

mechanism of enzyme stabilisation in each stabiliser and ideal electrode composition

Chapter 3 discusses oxygen-reducing cathodes The introduction part reviews

the mechanism of the oxygen reduction reaction oxygen reactivity oxygen reduction

catalysts carbon properties electrochemical pretreatments electrochemical techniques

72

and how to determine the catalytic efficiency of cathodes The material and method

section describes the protocol for the oxygen-reducing electrode fabrication preparation

and electrochemical pretreatment electrochemical studies electrochemical studies for

qualitative analysis and to obtain the kinetic parameters and analysis to determine the

efficiency of catalytic cathodes The result and discussion section describes the

voltammetric behaviour of each pretreated cathode influence of pretreatment and

catalytic efficiency The most catalytic cathode was determined at the end

Chapter 4 refers to the overall conclusion and further studies

73

2 ANODE INTRODUCTION

21 GLUCOSE OXIDATION ENZYMES

Glucose-oxygen fuel cells extract power by oxidising glucose at the anode The

anode contains glucose-oxidising enzymes There are several enzymes available for

glucose oxidation (i) β-D glucose oxidase (GOx) (EC 1134) [56 81] (ii) pyranose

oxidase (POx) (EC 11310) [56 81] (iii) nicotinamide adenine dinucleotide-dependent

glucose dehydrogenase (NAD-GDH) (EC 11147) [18 56] and (iv) quinoprotein

glucose dehydrogenase (PQQ-GDH) (EC 1152) [56 81] These enzymes are

classified into the family of oxidoreductases which catalyse either transfer of (i)

hydride (H-) (ii) oxygen atoms or (ii) electrons from one molecule to another Oxidases

transfer electrons from substrates to O2 and form H2O or H2O2 as products

Dehydrogenases transfer electrons from substrates to coenzymes without involving O2

(Eq 33 and Eq 34)

Glucose oxidation by GOx and NAD-DHG

GOx middotmiddotmiddotmiddotmiddot Eq 33

NAD-DHG middotmiddotmiddotmiddotmiddot Eq 34

To function catalytically enzymes require non-peptide inorganic or organic cofactors or

prosthetic groups Non-peptide organic cofactors are also called coenzymes Enzymes

lacking such groups are called apoenzymes and hardly functional Cofactors are

generally loosely bound to the apoenzymes and freely diffusible but on catalysis they

74

bind to the enzymes as the second substrates Prosthetic groups are always tightly bound

to the apoenzymes through covalent or non-covalent bonding [22 23 48] GOx and

POx are fungal flavoprotein enzymes containing flavin adenine dinucleotide (FAD)

cofactors FAD in GOx is tightly bound to the apoenzyme but not covalently bonded

hence still dissociable [67 82-84] NAD-GDH contains dissociable NAD+ [65] while

PQQ-GDH contains pyrrolo-quinoline quinone (PQQ) redox cofactor [56 65] which is

moderately bound to the apoenzyme [65 67] The structure of each cofactor is shown in

Fig 16

75

N

N

NH

N O

O

R

N

N

NH

HN O

O

R

NH

N

NH

HN O

O

R

H+ + e- H+ + e-

2H+ +2e-

N+

NH2

O

R

H

N

NH2

OH

R

HH

N

NH

O

O

OHO

OH

O

OH

O

N

NH

O

HO

OHO

OH

O

OH

O

N

NH

OH

HO

OHO

OH

O

OH

O

H+ + e- H+ + e-

FAD FADH2

NAD NADH

PQQ PQQH2

Fig 16 The structure of FAD NAD and PQQ and their redox reactions

Moieties involved in the redox reactions are circled in gray [23 65]

Cofactors also stabilise the enzyme structure Irreversible thermal GOx denaturisation

involves the perturbation of FAD biding site causing FAD dissociation although 70 of

its secondary structure is retained Chemical denaturisation untangles the dimmer

structure of GOx [65 81 83 85] and dissociates monomers and FAD [86 87] Excess

FAD does not prevent or regain function after thermal inactivation [87] However FAD

can rapidly bind to apoenzymes at 25 degC pH 61 and stabilise the tertiary structure

through conformational change and give resistance to proteolysis [82]

76

The choice of enzyme is based on the reaction involved enzyme availability

stability enzymatic properties physical properties and operational environment In the

absence of O2 or when there is concern about system degradation due to the competition

of electrons between O2 and artificial electron acceptors or due to the by-product H2O2

GDHs are more preferred to GOx [23 60 65-67 88] However the use of GDHs poses

different disadvantages Membrane-bound PQQ dehydrogenase (mPQQ-GDH) has very

high glucose specificity however it is difficult to solubilise and purify hence not for

commercial use [88] Soluble PQQ dehydrogenase (sPQQ-GDH) has been used in

commercial glucose sensors [67 89] however it suffers from low thermostability and

very low glucose specificity [88] Moreover PQQ-GDHs add complication to the

system because metal ions such as calcium ions (Ca2+) and magnesium ions (Mg2+) is

required to activate them [89 90] In the case of NAD-GDH NAD must be provided

because it is a dissociable cofactor but must bind first to the apoenzyme to form a

catalytic complex for glucose oxidation In addition to this complication NAD was also

reported to be more prone to causing electrodes poisoning without a quinone acceptor

[65] NAD forms adduct with phosphate in the buffer its decomposition releases free

adenine and electrochemically oxidised adenine adsorbs onto the electrode surface

causing electrode poisoning [91]

The redox potential of enzyme is also important because it is the driving force

for electron transfer and the power although it is difficult to match both catalytic

efficiency and electrochemical efficiency It is also difficult to determine the redox

potential exactly because the redox potential of cofactors is influenced by

microenvironment such as where they are embedded in the enzyme as well as

77

macroenvironment such as pH and temperature For example the redox potential of

FAD itself is ndash 042 V vs AgAgCl at pH 7 25 degC [22 23] however that of

flavoproteins could range from ndash 069 to ndash 005 V [65] and FAD-containing GOx has

the redox potential approximately ndash 03 V [67 92] It was also reported glucose

gluconolactone redox potential was ndash 056 V [93] PQQ itself has - 013V in absence of

Ca2+ and ndash 012 V in presence of 1M CaCl2 [94] while sPQQ-GDH has the redox

potential approximately - 019 V in presence of Ca2+ [66 67] Furthermore the enzyme

redox potential leads to a complex issue when mediators which assist electron transfer

between the enzyme redox centre and the electrode are involved because the improper

combination of the enzyme and mediator causes inefficient electron transfer and hence

potential drop

For enzyme availability stability and simplicity in handling GOx has been

most popular glucose-oxidising enzyme It has optimal pH 55 and temperature 35 ndash

40 degC [93] but is stable between pH 4 ndash 7 [95] and up to 50 - 55 degC [82 87] GOx has

higher substrate specificity and is most active toward β-D-glucose oxidation [56 86 88]

and very stable therefore its use has prevailed among various sensor applications [60

67]

22 DESIGNING ENZYME ELECTRODES

Construction of enzyme electrodes is complex because it simultaneously

incorporates enzymes mediators conductive materials and stabilisers ensuring that all

of them work harmoniously under restricted performance conditions There are several

78

immobilisation techniques available These include physical adsorption cross-linking

covalent bonding and entrapment in gels or membranes [96]

221 Physical adsorption

Physical adsorption is a simplest immobilisation method Electrodes are dipped

in the solution containing enzymes and mediators or such solution can be applied to the

electrode surface In this method however the enzyme tends to leach due to the weak

interaction between the electrode and enzyme [55] In order to avoid the loss of

electrode material the electrode surface is often covered or coated with material One

classic method is that the solution on the electrode surface is trapped with a

semipermeable dialysis membrane [61 65 92] This allows enzymes free from the

possible denaturisation caused by immobilisation on the solid material Enzymes inside

the dialysis membrane also can be fixed onto the cellulose acetate membrane or the

enzyme layer can be cross-linked with bifunctional reagents such as glutaraldehyde

[61]

222 Reconstituting apo-enzyme

Willner constructed enzyme electrodes by reconstituting apo-enzyme on the

gold (Au) electrodes surface with quinone mediators The electrode consisted of three

layers which were made of PQQ synthetic FAD and apo-GOx [97] PQQ were

mediating groups and attached onto Au electrode by cystamine PQQ attached on Au

was then covalently linked to synthetic FAD called N6-(2-aminoethyl)-FAD Covalently

79

linked FAD self-assembles with apo-GOx so that holo-GOx assembly is formed on the

top of the PQQ mediating layer attached to the Au surface The reconstitution procedure

is shown in Fig 17

Fig 17 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode [97]

The output current density was 300 microAcm-2 at pH 7 35 degC and it dropped by 5 in the

presence of O2 although the decrease was negligible at 01 mM physiological O2

concentration [98] The reconstitution of apo-enzymes is a flexible system potentially

applicable to enzyme electrodes [97] The fabrication process is cumbersome however

the enzymes are neatly aligned and electrically connected in the correct orientation

therefore electrocommunication between the enzymes and electrodes can be

maximised

80

223 Tethered Osmium-complexes linked to conductive hydrogel

It has been reported that embedding enzymes in the conductive hydrogel

complexed with tethered osmium (Os) mediator led to high output current [19 60 66

67] The conductive hydrogel is made of cross-linked polyvinylpyridine or

polyvinylimidazole They are soluble as monomers however they gain their

water-swelling property in cross-linked polymer This is ideal for enzyme mobility and

electrical conductivity Electron transfer is realised by the Os complex which is tethered

to the conductive hydrogel back bone The tether is made of 10 ndash 15 atoms [19 67] in

order to allow flexile movement of mediators Tethered Os complexes sweep the large

volume made of multiple layers of hydrogel form electrostatic adducts with enzymes

[19] and facilitate collide-and-hop electron conduction [19 99] Polymer tether and Os

complexes can be flexibly tailored for different enzymes and different operational

environments [19] One of its great advantages is that the redox potential for electron

transfer can be adjusted by changing the ligand structure of Os complex [19 60 97]

Heller constructed such electrodes made of carbon fibers whose dimension was 7 microm

diameter 2 cm length 08 mm2 surface area and 26 x 10-3 mm3 volume GOx was

wired to poly(4-vinylpyridine) conductive hydrogel backbone by the tethered complex

of tris-dialkylated NNrsquo-biimidazole Os2+3+ (Fig 18)

81

Fig 18 GOx wired to conductive hydrogel complexed with Os [19 67]

The redox potential of its redox centre was ndash 02 V vs AgAgCl The glucose oxidation

took place at ndash 036V and its output current density was 11 mAcm-2 [19] Disadvantage

of the tethered enzyme is short life It was stable for one week in the physiological

buffer then 5 current drop per day was observed When it was implanted in grape sap

50 current drop occurred within 20 hours In calf serum 50 current dropped

occurred in 2 hours with electrode fouling [19]

224 Entrapment in the conductive polymer

Entrapping enzymes in conductive polymer such as polypyrrole polyaniline

and polythiophene [65] is a popular method for enzyme electrodes [96] This is made by

82

electropolymerisation in which potential is applied to the solution containing enzymes

and monomors for certain duration It is a simple method but can be complicated if the

conditions for polymer growth are not compatible with enzyme entrapment [99]

Enzyme leakage occurs when enzymes are not trapped securely but it can be reduced

when derivatised polymers are used [96 100] Their conductivity is affected by

monomer structure [100] acidity and electrolyte [101] It can be degraded by products

of enzymatic reactions [65] Fujii fabricated GOx electrodes which were stable for

more than four months though a gradual sensitivity drop was observed GOx was

entrapped in saccharide-containing polypyrrole made of 1-(6-D-gluconamidohexyl)

pyrrole (GHP) (Fig 19(i)) on Pt electrodes It was reported that entrapped GOx was

secured by hydrogen bonding present between sugar gluconyl groups and enzymes

Enzyme leaching was also reduced by improved polymerisation with extended

conductive hydrocarbon chain [100] The output current density was 75 microAcm-2 at 055

V pH 74 in the presence of 56 mM glucose and 175 microA cm-2 under glucose saturated

condition [100] Arai entrapped various enzymes in polymer of mercaptobenzoquinone

(SQ) (Fig 19(ii)) and its methylated derivatives methylmercapttobenzoquinone

(MeSQ) and dimethylmercaptobenzoquinone (Me2SQ) on Au electrode surface

83

OH

H

H

OH

OH

H

OH

H

CH2OHN(CH2)6NHCHOH

(i)O

O

SH

Fig 19 Monomer structures of conductive polymers

(i) 1-(6-D-gluconamidohexyl) pyrrole [100]

(ii) Mercapto-p-benzoquinone [101]

Quinones have been widely used as good mediators [21 60 61 65] Poly(SQ) is a

conductive polymer functionalised with quinone mediating groups and can be easily

attached to Au electrode due to the presence of thiol group (-SH) [100] It was revealed

that more methylated SQ polymer led to better performance The maximum current

density for SQ- MeSQ- and Me2SQ-GOx anodes was 006 522 and 544 microAcm-2 and

their electrode enzyme dissociation constant KM was 13 18 and 20 mM respectively

[102 103] FAD cofactors have been also electropolymerised to entrap lactate

dehydrogenase and lactate oxidase It was reported that polymerised FAD extended the

shelf and operational life of electrodes to more than one year and also reduced O2

influence [104]

225 Bulk modification

Another popular electrode fabrication involves bulk modification of solid

composite This is simply made by mixing electrode material and inert binding reagent

84

such as epoxy resin Typical composite electrodes consist of 70 conductive material

by weight [105] Bulk-modified electrodes are robust stable inexpensive and easy to

modify build and reuse [105-107] therefore often used for enzyme electrodes [108]

For GOx electrodes tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ)

has been used as both mediating and conducting material

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties

TTF-TCNQ is a black crystalline charge transfer complex having both

mediating and conducting properties Its handling is also easy and often used for GOx

electrodes [65] Its conductivity is 102 ndash 103 Scm-1 [109 110] and comparable to that of

graphite [65 105] TTF-TCNQ complex consists of TTF electron donor and TCNQ

electron acceptor and they are arranged in a segregated stack structure TTF-TCNQ has

metal-like electric conductivity and this derives from intra-stack and inter-stack

molecular interactions Within the stack π-orbitals of adjacent molecules overlap This

builds the major conductive path in the direction of stack [61 65 110 111] Between

the stacks there is partial charge transfer between different chemical groups and this

gives the metallic feature in which charge carriers are mobile among the stacks

[110-112] as shown in Fig 20

85

S

S S

S

N

C

C

N

C

C

N

N

S

S S

S

7 6

N

C

C

N

C

C

N

N

-

TTF - electron donor

TCNQ - electron acceptor

6 - localised 6 - delocalised

+ e-

+ e-

Fig 20 TTF-TCNQ structure and its charge transfer [61]

TTF-TCNQ is a good electrode material because it has low background current and is

efficient at electron transfer [65] is stable [65] insoluble in water and not toxic [113]

However it exhibits complex electrochemistry (Fig 21) and its behaviours have not yet

been fully elucidated

86

vs AgAgCl

TCNQTCNQ e⎯⎯rarr⎯minus minusbull+

+minus+ ⎯⎯rarr⎯minus 2e TTFTTF

minus+minusbull⎯⎯rarr⎯

minus 2e TCNQTCNQ

+minus⎯⎯rarr⎯minus

TTFTTF e

Fig 21 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 01M potassium

phosphate 01 M KCl at 20 mVs-1 [92] Modified

The range of its effective redox potential varies according to the authors [65 92 114

115] Following the Lennoxrsquos study its potential extremes should lie within the range

between - 015 V and + 031 V vs AgAgCl (Table 2) because beyond these potential

ranges irreversible lattice reduction or oxidation takes place Even within the range

crystal dissolution salt formation with buffer ions and its deposition are still present and

could change the electrochemical profile TTF-TCNQ stability is affected by many

factors such as potential applied buffer component and concentration ion solubility

mass transport and analyte [92]

87

Table 2 Electrochemical behaviour of TTF-TCNQ at pH 74

Equilibrium TCNQTTFTCNQ-TTF +⎯rarr⎯ middotmiddotmiddotmiddotmiddot Eq 35

TCNQ reduction + 015 V MTCNQTCNQTCNQ Me ⎯⎯rarr⎯⎯⎯rarr⎯+minus +minusbull+ middotmiddotmiddotmiddotmiddot Eq 36

Lattice reduction - 015 V minus+minusbull ⎯⎯rarr⎯minus 2e TCNQTCNQ middotmiddotmiddotmiddotmiddot Eq 37

Anode salt formation + 031 V MTCNQTCNQ M⎯⎯rarr⎯++minusbull middotmiddotmiddotmiddotmiddot Eq 38

Lattice oxidation + 035 V +minus⎯⎯rarr⎯minus

TTFTTF e middotmiddotmiddotmiddotmiddot Eq 39

Further oxidation + 038 V +minus+ ⎯⎯rarr⎯minus 2e TTFTTF middotmiddotmiddotmiddotmiddot Eq 40

In the presence of a redox couple it also exhibits different electron transfer mechanism

When Edegrsquo of the redox couple is Edegrsquo gt 0 V TTF-TCNQ shows metal-like behaviour

facilitating direct electron transfer (DET) while it presents mediated electron transfer

(MET) for the redox couple of Edegrsquo lt 0 V Lennox made a study on TTF-TCNQ GOx

electrodes and reported that large anodic current was observed at ndash 015 V He also

reported that stable current was obtained when constant potential + 02 V was applied

for 24 hours at pH 7 in the presence of glucose He concluded that hydrophobic protein

core solubilised TCNQ and it formed enzyme-mediator complex GOx oxidation could

be catalysed at low potential because it was still in the range of the stable redox

potential He also conducted experiments with GOx and methanol dehydrogenase

(MDH) modified TTF-TCNQ electrodes in the presence of O2 The performance was

interfered by O2 in both electrodes against his expectation because DHG is in general

resistant to O2 He gave a conclusion that reduced mediators were oxidised by O2 before

electrooxidation took place [92]

88

226 Enzyme covalent attachment and mass loading

Enzymes can be covalently attached to the electrode however it becomes

complicated when mediators and stabilisers are must be also attached for practical fuel

cell applications This is however an attractive technique once ideal means of

co-immobilisation are established because enzymes can be retained firmly on the

electrode surface Enzymes can be covalently linked to oxidised or aminated surface

through amide bonds [116 117] Covalent linkage often lowers enzyme activity because

it partially alters the enzyme structure and confines the enzyme conformation [55]

however Kim succeeded in fabricating stable biocatalytic nanofibre by loading enzyme

aggregate to the enzyme monolayer which was covalently attached to the nanofibres

(Fig 22) Nanofibres with ester groups were synthesised from polystyrene and

poly(styrene-co-maleic anhydride) using electrospinning Seed enzyme was covalently

attached to the chemically modified nanofibre to form an enzyme monolayer on the

nanofibre This enzyme-attached nanofibre was further modified with the mixture of

glutaraldehyde and enzymes so that enzyme mass load was achieved by loading enzyme

aggregate to the enzyme monolayer covalently attached to the fibre Minter reported

that enzyme immobilisation with cross-linking increased the enzyme stability but

limited the enzyme conformation and thus reduced the activity However the activity

reported was nine times higher than that with an enzyme monolayer only Moreover the

activity was retained more than one month under rigorous shaking [118] The activity of

each enzyme particle might have been reduced however mass loading increased the

reactant flux hence increased the overall activity

89

Fig 22 The procedure for the enzyme mass-loaded biocatalytic nanofibre [118]

He also fabricated single-enzyme nanoparticles (SENs) in which each enzyme particle

was encapsulated within a hybrid organic and inorganic network The surface amino

groups of the enzyme were converted into surface vinyl groups and then polymerised

The polymer was then cross-linked to form the shell around the enzyme particle (Fig

23)

90

Fig 23 Fabrication procedure of single-enzyme nanoparticles [55]

It was reported that high stability was achieved after polymer cross-linking The enzyme

activity was approximately half of the free enzyme however there was no mass

transport interference [55] due to their porous structure Km in SENs was nearly

equivalent that of the free enzyme but the storage stability went beyond five months

[119] Eight-fold increase on storage stability after GOx covalently linked to

poly(phosphorothioate) was also reported [120]

23 PROTEIN INACTIVATION AND STABILISATION

Enzyme inactivation is one of the biggest problems in enzyme electrodes

Enzymes are excellent catalysts in their native environment however they are only

designed to work optimally under specific conditions therefore very sensitive to the

slight change in their environment and easily undergo inactivation The issue is

persistent in the enzyme electrodes because their fabrication introduces the enzymes to

the unusual environment yet enzyme catalytic activity must be maintained to obtain

sufficient power output and operational life The problem can be alleviated by (i)

91

protein engineering [56 121] (ii) environmental engineering [56 60 63 67] and (iii)

addition of appropriate enzyme stabilisers [120 122] Protein engineering involves the

alteration of enzyme structure to gain desired functions stability or resistance However

it is very challenging to improve both stability and activity since protein stability is

conferred by its rigidity while its activity depends on the conformational flexibility

Alteration of protein properties is realised by genetic engineering such as site-directed

mutangenesis [123] or directed evolution [49] Environmental engineering is closely

linked to the enzyme immobilisation It aims to achieve the same goals as in protein

engineering but extrinsically The environment which rigidifies the protein structure

such as cross-linking or inclusion in electrode composite tends to increase the enzyme

stability and prolong its life In contrast the environment which more favours over

flexible protein conformation such as hydrogel and membrane trap exhibits higher

enzyme activity but shorter life However it was reported that enzyme cross-linking and

the presence of hydrophilic macromolecules improved both stability and activity while

electrostatic interaction improved selectivity [124] Addition of stabilisers is more

targeted smaller scale environmental engineering Intrinsic conformational stability of

the protein cannot be improved [120] however it can target and eliminate specific

inactivating factors increases the local specific protein stability and prevents or slows

its unfolding There are two factors to improve for enzymes to achieve ideal operation

(i) activity by hydration and (ii) increased lifetime by increased protein rigidity which

might involve the removal of bond-destabilising factors or amplifying the bond strength

within the enzymes In fact both must be probably achieved simultaneously for gaining

the optimal state because they interrelate to each other in terms of protein dynamics As

mentioned earlier protein hydration improves enzyme activity but also allows its

92

regeneration hence affects the protein internal dynamics Protein dehydration distorted

the protein structure [125]

24 PROTEIN DESTABILISATION

There are many protein destabilising factors apart from dehydration and these

can be classified into chemical and physical instability Chemical instability happens

due to chemical modification such as deamidation oxidation proteolysis and

racemisation

241 Deamidation

Deamidation is the hydrolysis of amide link in the amino acid side groups such

as glutamine (Gln) and asparagine (Asn) (Fig 24)

Glutamine (Gln) Asparagine (Asn)

Fig 24 Side groups of Gln and Asn

It is favoured by increase in pH temperature and ionic strength The buffer

concentration influences the rate of deamidation at pH 7 ndash 12 Urea also accelerates

deamidation It often implies the protein aging and then modifies the protein function

93

242 Oxidation

Oxidation takes place at the side chains of cystine (Cys) methionine (Met)

tyrosine (Ty) tryptophan (Trp) and histidine (His)

Cysteine (Cys) Methionine (Met)

Tyrosine (Tyr) Tryptophan (Trp) Histidine (His)

Fig 25 Side groups of Cys Met Tyr Trp and His

Atmospheric oxygen can oxidise Met residues H2O2 modifies indole thiol (SH) [123

126] disulfide (SS) imidazole phenol and thioether (RSR) groups at neutral or slightly

alkaline conditions At acidic condition it oxidises Met into Met sulfoxide (R-S=O-R)

Under extremely oxidising condition Met sulfoxide is further oxidised into Met sulfone

(R-S(=O)2-R) Met oxidation leads to loss of biological activity however it does not

abolish the enzyme activity Therefore if oxidised Met is reduced back by a reductant

such as thiols it will restore the full biological activity Met reactivity depends on its

position within the protein Cys plays an important role in protein folding because it

forms intermolecular disulfide bonds however very susceptible to oxidation It can

undergo autooxidation in the absence of oxidants It is oxidised in several stages in

94

which thiol R-SH group is oxidised to (i) sulfenic acid (R-S-OH) (ii) disulfide

(R-S-S-R) (iii) sulfinic acid (R-S=O-OH) and (iv) sulfonic acid (R-S(=O)2-OH) [123

127] Oxidised thiol groups can be reduced back however they can be also reduced

back in the wrong way This leads to protein misfolding and loss of activity There are

many factors that influence the rate of oxidation They are (i) temperature (ii) pH (iii)

buffer medium (iv) metal catalysts (v) O2 tension (vi) steric condition (vii) neighbouring

groups (viii) photooxidation The presence of metal ions such as Fe2+ and Cu2+

accelerates oxidation by air [123] and the presence of O2 also might accelerate thiol

oxidation with other oxide such as nitric oxide radical [127] Metal-catalysed oxidation

involves a site-specific metal binding to Trp His and Met This leads to severer form of

oxidation causing protein aggregation Peroxide-catalysed oxidation on the other hand

is milder and tends to affect Cys and Trp but does not form aggregates [126] Increase in

pH increases pKa hence accelerates dissociation of ndashSH All of Cys Met Typ Tyr and

His are susceptible to photooxidatoin however His photooxidation is fatal leading to

enzymatic inactivity [123]

243 Proteolysis and racemisation

Proteolysis and racemisation takes place at aspartic acid (Asp) residue The

bond between Asp and proline (Pro) is especially susceptible to hydrolysis Asp also

easily undergoes base-catalysed isomerisation to isoAsp through a ring formation [123]

95

Aspartic acid (Asp) Proline (Pro)

Fig 26 Side group of Asp and the amino acid structure of Pro

244 Physical instabilities

Physical instability includes denaturation adsorption aggregation and

precipitation and [123] This refers to loss of tertiary structure and does not involve

chemical modification It is defined as Gibbs free energy change between the native and

unfolded state of protein Physical instability is induced by change in temperature

extreme pH organic solvent denaturant [123] and solvation [120 125]

25 ENZYME STABILISERS

By clarifying the possible major causes of instability the choice of optimal

approach and stabilisers can be made Considering the case of glucose-oxygen fuel cells

the stabilisers should be selected on the basis of following points (i) conformational

stability for longer operation and shelf life (ii) hydrophilic environment for optimal

enzyme functions (iii) optimal interaction between enzyme and substrate and enzyme

and environment Conformational stability is to secure protein folding and avoid

denaturation This is achieved by thiol group protection reduction of oxidised thiol

96

groups removal of oxidising species an increase in internal hydrophobic interaction

and environmental hydrophilic and electrostatic interaction Optimal enzyme activities

can be achieved by an increase in hydrogen bonding and electrostatic interactions

251 Polyehyleneimine (PEI)

Polyethyleneimine (PEI) has been reported as an efficient antioxidant as well

as chelating agent It is also cationic (Fig 27) [120] therefore provides electrostatic

stabilisation with anionic protein [126] such as GOx [67 85] Anti-microbial activity

has been also reported [126] It is not effective against thermal inactivation [128]

H+

N+H3N+H3

N+H3H3N+

H2+ H+

H+

H+

H2

+

H2+

Fig 27 Structure of PEI [129]

PEI is available in different molecular weights [120] and smaller molecules are more

effective in binding and flexibility [130] However its optimal loading concentration

varies [120 128 131] PEI protects thiol groups efficiently [120] from both metal- and

peroxide- catalysed oxidation despite of their different oxidation mechanisms [126] PEI

also forms a complex with the enzyme and coats it with polycations This occludes the

97

oxidants from the protein surface [126 130] Complex formation might be involving

hydrogen bonding between carboxylic acid groups in the enzyme and nitrogen atoms in

PEI [130] This possibly confers stability in dried state [128] It was reported that PEI

increased the maximum limiting catalytic velocity (V) for GOx [131] and lactate

dehydrogenase (LDH) [128] However it also increased Km for LDH and shifted its

optimal pH slightly toward acidic [128]

252 Dithiothreitol (DTT)

Dithiothreitol (DTT) is another small water-soluble [132] thiol protecting

reagent [132 133] with the reducing potential at ndash 033 V at pH 7 It has only little

odour [132] and has low toxicity [134] Its epimer is dithioerythriotol (DTE) Both are

excellent reducing reagents with two thiol groups (Fig 28) [132] however DTT might

be marginally better in reducing power [135] It reduces back disulfide quantitatively

[127 132] at lower concentration than other thiol-containing reducing reagents such as

Cys and gluthatione (GSH) But it cannot restore some of the lost catalytic activity

[136] because it only reduces disulfides but not other thiol oxides Therefore its

efficacy depends on (i) the oxidation mechanism involved in the protein inactivation

(ii) the velocity at which the inactivation proceeds and (iii) how thiol-contaning amino

acids are present in the protein DTT is not effective against mild oxidation caused by

low concentration of H2O2 or isolated Cys residues [127] It is probably because that

DTT reduces thiol oxide by converting itself from a linear into a cyclic disulfide form

which is called trans-45-dihydroxy-o-dithiane with (Fig 28) [132]

98

Fig 28 Structures of DTT from a linear to a cyclic disulfide form [132]

On the other hand arsenite was effective in reducing only mildly oxidated thiol groups

but not disulfides [127] It is however toxic [137] and not suitable for practical use

Neither of DTT or arsenite was effective if thiol oxidation is outcompeting [127]

253 Polyethylene glycol (PEG)

Polyethylene glycol (PEG) (Fig 29) has been reported as an enzyme stabiliser

however also has mixed reception and its stabilisation mechanism has not yet been

clearly understood

Fig 29 Structures of PEG

OrsquoFagain reported that PEG increased storage and thermo stability [120] while

99

Andersson reported the increase in LDH activity with PEG 10000 Mw but was

followed by a decrease after 10-day storage and then eventually became worse than

those without stabilisers The stabilisation was also found to be concentration dependent

[128] As for protein stabilisation mechanism it was proposed that PEG preferentially

hydrates the protein and stabilises its native structure in the solution [138-140]

According to Arakawa this causes the exclusion of PEG from protein surface due to the

unfavourable interaction between them hence increases water density around the

protein This results in the water shell around the protein surface and increases the

hydrogen bonds [138 139] Better hydration favoured larger PEG Mw due to the larger

steric exclusion [138] on the other hand lower PEG Mw was also reported to be better

because of its better solubility and viscosity [139] Optimal concentration is unknown

however one proposal is that higher concentration is more advantageous to stabilise the

denatured protein because it can penetrate and stabilise the protein hydrophobic core

exposed during denaturation [138] In contrast Lee reported that this penetration affects

the hydrophobic core of active enzyme and decreased the activity and denatured the

protein [139] Both however concluded that a fine balance between preferential

hydration and hydrophobic interaction is important and PEG concentration must be up

to 30 [138 139] It was also reported that PEG stabilised the protein of low water

content better because it prevented the water evaporation while polyols tended to strip

water from the protein dehydrated microenvironment [139]

254 Polyols

Polyols such as glycerol (GLC) (Fig 30) were reported to increase shelf life

100

[141] thermostability [120] and also provide preferential solvation as seen in PEG

[141-143] Higher GLC concentration resulted in better protein activity [143 144] and

reduced protein aggregation due to the tighter protein packing [144] However observed

hydration was lower than expected for high GLC concentration therefore Gekko

proposed that the contribution of higher GLC concentration did not derive form the

increase in steric exclusion Instead GLC might penetrate the solvation layer of protein

and stabilise the structured water network around the protein [143] Andersson also

compared the influence on protein activity after the storage among several candidate

stabilisers diethylaminoethyl-dextran (DEAE)-dextran Mw 500000 sorbitol and PEI

DEAE-dextran increased the activity both before and after the storage while sorbitol

decreased it However PEI was only found to be superior to all [128]

OH

OHHO

Fig 30 Structure of glycerol

255 FAD

FAD (Fig 16 p 75) is also another possible stabiliser as well as mediator

because it can regenerate denatured aged enzymes which have lost its cofactors [82

108] However it has not been clarified well and Bartlett questioned its efficacy due to

the contradictory results he obtained [145] It was reported that addition of FAD

increased the electrode sensitivity by 70 especially in the concentration range below

101

3mM and activity by 8 times although it also increased Km by 2 times [108]

26 ENZYME KINETICS AND ELECTROCHEMISTRY

261 Michaelis-Menten kinetics

A simple enzyme reaction can be expressed in terms of equilibrium between

the free enzyme (E) substrate (S) enzyme-substrate complex (ES) and product release

(P) The enzyme is regenerated on completion of the catalytic cycle

General enzyme reaction mechanism

middotmiddotmiddotmiddotmiddot Eq 41

E = Enzyme concentration mol

S = Substrate concentration mol

ES = Enzyme-substrate complex concentration mol

P = Product concentration mol

k1 = Rate constant for ES formation from E and S mol-1s-1

k-1 = Rate constant for ES dissociation into E + S s-1

k2 = Rate constant for P formation from ES s-1

The rate of ES formation is defined as

102

][][]])[[]([][2101 ESkESkSESEk

dtESd

minusminusminus= minus middotmiddotmiddotmiddotmiddot Eq 42

Where [E0] is the initial enzyme concentration

When the reaction is at steady state 0][=

dtESd

])[(][][]])[[]([ 212101 ESkkESkESkSESEk +=+=minus minusminus middotmiddotmiddotmiddotmiddot Eq 43

Re-arranging the above equation for [ES] as below

][]][[

][121

01

SkkkSEk

ES++

=minus

middotmiddotmiddotmiddotmiddot Eq 44

When the rate of product formation is defined as follows

middotmiddotmiddotmiddotmiddot Eq 45

][2 ESktP=

∆∆

=υ middotmiddotmiddotmiddotmiddot Eq 46

Where υ is the catalytic rate

When all the enzyme active sites are occupied such a condition is called saturated and

defined as [E0] = [ES] The maximum catalytic rate (V) is therefore available as below

Maximum catalytic rate V

][][ 202 ESkEkV == middotmiddotmiddotmiddotmiddot Eq 47

103

Express υ in terms of [S] by inserting Eq 44 into Eq 46

][

]][[

1

21

02

Sk

kkSEk

++

=minus

υ middotmiddotmiddotmiddotmiddot Eq 48

Michaelis-Menten constant (KM) is a dissociation constant about ES and defined as

Michaelis-Menten constant

1

21

kkk

K M+

= minus middotmiddotmiddotmiddotmiddot Eq 49

Michaelis-Menten equation (Eq 50) is obtained when υ is expressed in terms of V and

KM by inserting Eq 47 and Eq 49 into Eq 48

Michaelis-Menten equation

][][SK

SV

M +=υ middotmiddotmiddotmiddotmiddot Eq 50

This is a hyperbola equation to lead the enzyme catalytic rate Most enzymes typically

show this hyperbolic curve (Fig 31) when they are free from allosteric controls or

enzyme inactivation

104

0E+00

1E-09

2E-09

3E-09

4E-09

5E-09

0E+00 2E-03 4E-03 6E-03 8E-03 1E-02 1E-02 1E-02 2E-02

[S] M

Fig 31 A typical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

As shown in Fig 31 Michaelis-Menten plot has different kinetic profiles at high and

low [S] Mathematical expression of its transition over [S] is shown in Table 3

Table 3 Michaelis-Menten equation at various [S]

[S] ge KM Vcongυ middotmiddotmiddotmiddotmiddot Eq 51

[S] = KM 2V

=υ middotmiddotmiddotmiddotmiddot Eq 52

[S] le KM MM K

SEkK

SV ]][[][ 02=congυ middotmiddotmiddotmiddotmiddot Eq 53

At high saturated substrate concentration the catalytic rate becomes zero-order

therefore Michaelis-Menten curve becomes plateau When [S] = KM the rate becomes

105

half of the maximum catalytic rate At low substrate concentration the catalytic rate is

linearly dependent on [S] therefore the kinetics becomes first-order about [S]

262 Electrochemistry and Michaelis-Menten kinetics

In electrochemistry enzyme activity is studied by amperometry This involves

measuring measuring the current generated by the enzyme electrode reaction at a fixed

potential over an arbitrary period It therefore shows the transition in current response

over time For enzyme electrodes substrate concentration is changed over time so that

the current response for a different substrate concentration can be measured over a long

period With intact enzyme and appropriate heterogeneous electron transfer present

most enzyme electrodes follow Michaelis-Menten kinetics (Fig 32) in the amperometry

in the absence of allosteric control or enzyme inactivation Therefore the amperometry

data can be analysed in the similar way to that of biochemical data and for this

Michaelis-Menten equation is expressed in electrochemical terms [146] In

electrochemical enzyme kinetics enzyme catalytic rate (υ) is obtained in terms of

electric current therefore V is substituted with maximum steady state limiting current

(Im) or its current density (Jm)

106

Electrochemical Michaelis-Menten equation

][][

SKSI

IM

m

+times

= middotmiddotmiddotmiddotmiddot Eq 54

][][

SKSJ

JAI

M

m

+times

== middotmiddotmiddotmiddotmiddot Eq 55

I = Current A

Im = Max steady-state limiting current A

[S] = Substrate concentration mol

KM = Michaelis-Menten constant mol

A = Electrode surface area m-2

J = Current density Am-2

Jm = Max steady-state limiting current density Am-2

There is often an offset at zero substrate concentration due to competing reactions and

trace contaminants and this must be subtracted before analysis

107

V

0E+00

2E-04

4E-04

6E-04

8E-04

1E-03

0 2 4 6 8 10 12 14 16

frac12 Jm

KM

Jm Am-2

[S] mM

Jm

Fig 32 Electrochemical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

KM and Jm (or V in biochemical enzyme kinetics) are important parameters to analyse

both enzyme catalytic power and stability Electrochemical Michaelis-Menten equation

and curves can be used for rough estimation of KM and Jm however it often leads to

inaccurate result because measuring initial rate for high substrate concentration is very

difficult in this method hence very prone to errors Therefore Michaelis-Menten

equation is linearised using one of the following four methods (i) Lineweaver-Burk (ii)

Eadie-Hofstee (iii) Hanes-Woolf and (iv) Cornish-Bowden-Wharton equations

Michaelis-Menten plot is however good for intuitive diagnosis of general enzyme

behaviours

108

263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics

2631 Lineweaver-Burk linear regression

Lineweaver-Burk linear regression is one of the classic regression methods

taking double reciprocal of Michaelis-Menten equation (Eq 56)

Electrochemical Lineweaver-Burk linear regression

mm

M

JSJK

J1

][11

+times= middotmiddotmiddotmiddotmiddot Eq 56

It plots 1J against 1[S] as shown in Fig 33 The value of each parameter is found by

extrapolating the line The reciprocal of Jm is found at y-intercept and KM can be derived

from either x-intercept or the slope once Jm is found

109

-50E+02

00E+00

50E+02

10E+03

15E+03

20E+03

-06 -05 -04 -03 -02 -01 00 01 02 03 04

1[S] mM-1

1J A-1m

2

Fig 33 Electrochemical Lineweaver-Burk linear regression

Based on the real data Refer to sect 343 p 124

Lineweaver-Burk linear regression is however largely influenced by the errors in j at

low substrate concentrations [22 147] Its reciprocal could vary by a factor of 80 over

the range of substrate concentration varying one-third to three times the KM [148]

Hence it is not an ideal method for the analysis

2632 Eadie-Hofstee linear regression

Eadie-Hofstee linear regression plots J against J[S] This plot is strongly

affected by the errors in υ because υ is present in both coordinates (Eq 57)

Electrochemical Eadie-Hofstee linear regression

mM JSJKJ +timesminus=

][ middotmiddotmiddotmiddotmiddot Eq 57

110

Again the plot must be extrapolated to find Jm at y-intercept KM can be derived from

the slope (Fig 34)

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

0E+00 5E-05 1E-04 2E-04 2E-04 3E-04

J [S] AmM-1

J Am

-2

Jm

-Km

Fig 34 Electrochemical Eadie-Hofstee linear regression Based on the real data

Refer to sect 343 p 124

Eadie-Hofstee linear regression will give very strict evaluation [148] and might be used

to detect the deviations from Michaelis-Menten behaviour only if the data is precise and

accurate [147]

2633 Electrochemical Hanes-Woolf linear regression

Hanes-Woolf linear regression plots [S]J against [S] KM is derived from the

x-intercept and Jm is found from the slope

111

Electrochemical Hanes-Woolf linear regression

m

M

m JKS

JJS

+times= ][1][ middotmiddotmiddotmiddotmiddot Eq 58

This is a better method compared to the previous two regression methods because all

the parameter values can be derived from the reasonable range of [S] and they donrsquot

heavily rely on the extrapolation [147] The data is normalised by [S] therefore does not

cause many errors

-20E+08

00E+00

20E+08

40E+08

60E+08

80E+08

10E+09

12E+09

14E+09

-40E+00 00E+00 40E+00 80E+00 12E+01 16E+01

[S] mM[S] J m

MA

-1m2

Fig 35 Electrochemical Hanes-Woolf linear regression Based on the real data

Refer to sect 343 p 124

However either of these three linear regressions might not be adequate for the enzyme

electrode analysis [65] because the data reflect not only the single-substrate

Michaelis-Menten kinetics but also include the influence from other factors such as

enzyme immobilisation mediator reactions and electrode composition as well as

112

general experimental errors Their contributions to the overall kinetics therefore must be

considered [65 149] although the enzyme electrode reactions can approximate the

Michaelis-Menten model when one substrate is a variable and other variables are fixed

over an experiment [147 149] Under such a complex system it is not ideal to use those

graphical linear regression methods because they assume that the results follow

Michaelis-Menten model and that all the experimental errors follow normal distribution

[150] without giving any measure in the precision [148] Hence it is more suitable to

use a non-parametric direct linear plot or nonlinear regression methods because they

respect the influence and errors from various sources without any bias

264 Cornish-Bowden-Wharton method

Cornish-Bowden-Wharton method is distribution-free non-parametric direct

linear plot In this method observations are plotted as lines in parameter space rather

than points in observation space [151] It then takes the median of Jm and KM from every

possible non-duplicate pair of observed [S] and υ (Eq 59 and Eq 60) as the best-fit

estimate

Electrochemical Cornish- Bowden-Wharton non-parametric method

])[][(])[]([

1221

122121 SJSJ

SSJJJm minusminus

= middotmiddotmiddotmiddotmiddot Eq 59

])[][()](][[

1221

122121 SJSJ

JJSSKM minusminus

= middotmiddotmiddotmiddotmiddot Eq 60

It is a simple method without complex calculation insensitive to outliers and no

113

weighting is required [147 152]

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

14E-03

16E-03

18E-03

20E-03

-5 -3 -1 1 3 5

KM mM

Jm Am-2Median Jm

Median KM

Fig 36 Electrochemical Cornish-Bowden-Wharton plot Based on the real data

Refer to sect 343 p 124

265 Nonlinear regression methods

Nonlinear regression methods manipulate the parameters to minimise the sum

of squared difference between the measured and expected values [150] They fit the

integrated equation to the enzyme progress curve and calculate the approximate location

of the minimum for the sum of squared difference at a particular point in the slope It

then recalculates the new minimum location to improve the prediction until there is no

further improvement This is called gradient method based on Gauss-Newton algorithm

or non-linear least square method [150 153] This is computationally intensive due to

114

the recursive operations however advanced computational technology has made it

possible The parameters obtained may not be perfectly correct [150] The method still

assumes errors normally distributed [153] and Cornish-Bowden has criticized about the

ambiguous statistical assumption of such computer programmes [154] However

non-linear regression methods are insensitive to outliers [150 153] have better estimate

accuracy compared to conventional linear regression methods and the estimations

mostly fit Michaelis-Menten model Graphical methods arose at a time when nonlinear

regression methods were computationally not available however it is now possible

thanks for the great technological advance and more recommended to use in analysis to

yield better precision and reliability [153]

115

3 ANODE MATERIAL AND METHOD

31 ELECTRODE FABRICATION

311 TTF-TCNQ synthesis

Tetrathiafulvalene ge980 (TTF) and 7788-Tetracyanoquinodimethane

ge980 (TCNQ) were purchased from Sigma Aldrich Spectroscopic grade acetonitrile

and diethyl ether were purchased from VWR International TTF and TCNQ were

weighted to 1 g each and dissolved separately in 50 ml of acetonitrile TTF-acetonitrile

solution had yellow colour while TCNQ-acetonitrile solution was green Both were

heated on the hot plate under the fume hood On the full dissolution two solutions were

quickly mixed to form black solid crystals The mixture was immediately taken off the

hot plate and cooled down at the room temperature for a few hours The crystals were

collected on Buchner funnel and washed under vacuum twice with acetonitrile twice

with diethyl ether and then twice again with acetonitrile The washed crystals were left

to dry for overnight with a cover The dried crystals were collected and kept in the

sealed container for future use [61]

312 Electrode template fabrication

Sylgard 184 Silicone Elastomer Kit were purchased from Dow Corning

Corporation for polydimethylsiloxane (PDMS) Base and hardener were mixed at 101

(vv) for use CY1301 and aradur hardener HY 1300 were purchased from Robnor

116

resins to make epoxy resin They were mixed at 31 (vv) for use

Silicone electrode templates (Fig 16(i)) were made to shape electrode discs

Each template had round holes in line Two types of templates template A and B were

made Template A had holes with the dimension 2-mm diameter x 1-mm thickness

while template B had holes with the dimension with 5-mm diameter x 2-mm thickness

In order to make PDMS templates for electrode discs different templates were made to

shape the holes in PDMS (Fig 16(ii)) Discs were made with epoxy resin and were

fixed onto the plastic Petri dish PDMS was poured into disk-mounted Petri dish

degassed for 20 minutes and then cured at 60 degC Cured PDMS electrode templates

were taken out from the epoxy resin-Petri dish templates and then cleaned with

analytical grade ethanol for use

Fig 37 PDMS electrode templates (i) PDMS template and (ii) their epoxy resin-Petri

dish template

117

313 Electrode composite fabrication

3131 Composite for conductance study

Graphite at microcrystal grade (2-15 micron 999995 purity) was purchased

from Alfa Aesar GmbH amp CoKG Glassy carbon (GC) powder was kindly donated from

Dr Zhao Carbon nanofibre (CNF) was also kindly donated from Loughborough

University Composite electrodes were built from the mixture of the carbon conductive

or TTF-TCNQ and epoxy resin Each composite mixture had different composition ratio

[108] as below in Table 4

Composite Ratio I Ratio II Ratio III Ratio IV Ratio V

Graphite Epoxy resin 73 64 55

GC Epoxy resin 64 73

CNF Epoxy resin 37 46 55 64 73

TTF-TCNQ Epoxy resin 64 73 82

Table 4 Composite composition

TTF-TCNQ was mixed with epoxy resin at 64 73 and 82 Graphite was mixed at 73

64 and 55 GC powder was mixed at 64 and 73 CNT was mixed at 37 46 55 64

and 73 (ww) Three to five copies were fabricated for each electrode type Composite

mixture was packed into the template A (Fig 38) and cured at room temperature for 72

hours [106]

118

Fig 38 PDMS template (i) packed with electrode material and (ii) empty one

3132 Bulk modified GOx enzyme electrode composite

Glucose oxidase (GOx) [Type II-S 15000-50000 unitsg] from Aspergillus

niger was purchased from Sigma Aldrich Conductive composite paste was first made

with TTF-TCNQ and epoxy resin at 73 as in sect 3131 GOx was then incorporated into

the conductive composite mixture at varying mixing ratio 5 10 and 15 (ww) by

bulk-modification method (Fig 39) 10 GOx composite electrodes were also made

with graphite Enzyme composite mixture was packed into the template B and cured at

4 degC for 96 hours

119

R

RR R

R

RR R

RRR

Conducting particles

Insulating binder

Chemical modification

Add biological receptor R

Cast extrude or print into suitable form

R

R

RR

R

R

R

R

R

R

RR

Fig 39 Schema for bulk modification method

3133 GOx enzyme electrode composite with enzyme stabilisers

Poly(ethyleneimine) (PEI) solution [50 (wv) in H2O] DL-dithiothreitol

(DTT) Poly(ethylene glycol) (PEG) [average mol wt 200] glycerol (GLC) [ge99] and

flavin adenine dinucleotide disodium (FAD) salt hydrate (ge95) were purchased from

Sigma Aldrich Each stabiliser was mixed with GOx at different fraction 0 1 2 and

4 PEI was also blended with DTT or FAD for synergetic effects as below in Table 5

Blend type Ratio I Ratio II Ratio III Ratio VI

PEIDTT 0515 1010 1505 2020

PEIFAD 21 22 24

Table 5 Blended stabiliser composition

120

PEI was mixed with DTT at the ratio of 0515 11 1505 and 22 PEI was also

mixed with FAD at 21 22 and 24 Each enzyme-stabiliser mixture was weighted to

10 of the whole composite It was then mixed with the conductive composite mixture

made earlier The conductive composite mixture was made of TTF-TCNQ and epoxy

resin at 73 The final composite mixture was packed into the template B and cured at

4 degC for 96 hours

314 Electrode wiring and insulation

Silver wire (temper annealed 025-mm diameter) was purchased from Advent

Portex polyethylene tubing (028 x 0165 mm) purchased from Scientific Laboratory

Supplies Silver epoxy was purchased from ITW chemotronics Silver epoxy came in

two parts A and B and they were mixed at 11 (vv) for use Silver wire was cut for

5-cm length and its tip was cleaned with analytical grade ethanol dried and coved with

insulating polyethylene tubes Cured electrode composite discs were popped out of the

PDMS template An insulated silver wire was fixed onto to each electrode disc with

conductive silver epoxy and it was left at room temperature for 24 hours to cure silver

epoxy After silver epoxy had been cured the wired electrodes were coated and

insulated with degassed epoxy resin and cured at room temperature for 3 days for

non-enzyme composite electrodes and 4degC for 4 days for enzyme composite electrodes

7-9 days were taken to build the complete composite electrodes (Fig 40) The enzyme

electrodes were stored at 4degC when not in use

121

Fig 40 Insulated composite electrodes (i) non-enzyme (ii) GOx electrodes

32 CONDUCTIVITY EXPERIMENT

Copper foil with electrically conductive acrylic adhesive was purchased from

RS Components Digital multimeter from Metra was used to measure resistance

Electrode surface was covered with adhesive copper foil evenly and both ends were

clamped with crocodile clips (Fig 41(i)) to measure electrode resistance of the

composite as in Fig 41(ii)

122

Fig 41 Measuring electrode resistance (i) connecting the electrodes to the

multimeter (ii) measuring the electrode resistance with the multimeter

33 POLISHING COMPOSITE ELECTRODES

MicroPolish II aluminium oxide abrasive powder (particle range 1 03 and

005 microm) silicone carbide (SiC) grinding paper for metallography wet or dry (grit 600

1200 and 2400) and MicroCloth Oslash73 mm for MiniMet for fine polishing were

purchased from Buehler Self adhesive cloth for intermediate polishing was purchased

from Kemet Electrodes were polished immediately before the experiment until

mirror-like flat surface was obtained This erases the electrode history and gives

assigned electrode surface area [105] Composite electrodes were hand polished

successively with SiC grinding paper in 400 1200 and 2400 grit During polishing

deionised water of 18 MΩ was added occasionally to the polishing surface This

prevents damaging electrode surface with frictional heat It was followed by successive

polishing with alumina oxide slurry with particle size 1 03 and 005 microm Slurry was

123

sonicated to dissolve agglomerates before use [155] Kemet polishing pad was used for

rougher polishing with 1 and 03 microm powder Felt-like MicroCloth was used for

polishing with 005microm Electrodes were sonicated in the deionised water briefly after

polishing at each grit or grade

34 GLUCOSE AMPEROMETRY

341 Glucose solution

Phosphate buffer saline (PBS) powder for 001 M at pH 74 containing 0138

M NaCl and 00027 M KCl [156] was purchased from Sigma Aldrich PBS was kept in

the fridge when not in use [157] 1M glucose solution was made by mixing

D-(+)-Glucose with 001 M PBS and left for 12 hours at 4degC for mutarotation It is

mandatory because GOx has stereochemical specificity and only active toward

β-D-glucose while glucose solution consists of two enantiomers α- and β-D glucose

and their concentration changes over time until they reach the equilibrium at 3862

Experiments should be carried out in the equilibrated solution for accuracy

342 Electrochemical system

A single-channel CHI 650A and multi-channel CHI 1030 potentiostats were

used for glucose amperometry with three-electrode system in which the

electrochemical cell contained (a) working electrode(s) an AgCl reference electrode

and a homemade Pt counter electrode The AgCl reference electrode was purchased

124

from IJ Cambria AgCl reference electrode was saturated with 3M KCl Pt counter

electrode was made of Pt wire and Pt mesh was fixed at its tip to give large surface area

Pt mesh at its tip

343 Glucose amperometry with GOx electrodes

Glucose amperometry was conducted by adding a known volume of 1M

glucose aliquot into 100 ml of 001 M PBS electrolyte solution containing GOx anodes

with convection applied Convection was however ceased on during current

measurement to avoid the noise The electrolytic solution was gassed with either N2 O2

or compressed air for 15 minutes before the experiment O2 concentration is 123 mM in

fully oxygenated PBS [158] and 026 mM in air [112] Gas was continuously supplied

through Dreschelrsquos gas washing bottle containing PBS during experiment The response

of GOx anodes to glucose was obtained as steady state current The amperometry was

carried on until no further current increase was observed against glucose added Each

experiment took more than 8 - 12 hours The sample size ranged from two to three in

most cases

125

Fig 42 Amperometry setting with CHI 1030 multichannel potentiostat

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA

Normality of enzyme function was confirmed by plotting Michaelis-Menten

curve based on the amperometry data obtained The capacitive current before glucose

addition was subtracted from the data Enzyme kinetic parameters the maximum

current density (Jm) and the enzyme dissociation Michaelis constant (KM) were

automatically estimated with standard error using an analytical software called

SigmaPlot Enzyme Kinetics version 13 The estimation is based on non-linear

regression analysis about Michaelis-Menten kinetics as shown in Fig 43

126

Glucose M

0 1e-2 2e-2 3e-2 4e-2 5e-2

J A

m-2

00

20e-3

40e-3

60e-3

80e-3

Vmax = 0008445 Km = 0005842

Fig 43 Analytical output by SigmaPlot Enzyme Kinetics Based on the real data

Influence of enzyme immobilisation electrode composition O2 and stabilisers on GOx

electrode performance was studied The most efficient GOx anodes was determined on

the basis of the parameters obtained

127

4 ANODE RESULTS AND DISCUSSION

41 AIMS OF ANODE STUDIES

This section describes the fundamental study on the enzyme composite

electrodes and the influence of enzyme stabilisers on the performance and stability of

GOx electrodes Firstly the difference in conductivity as a function of various

conductive materials and the component fraction were studied They did not contain

enzyme The most suitable composite was then selected and used to build the enzyme

electrodes Secondly GOx electrodes were built at various GOx fractions in the

composite The best component fraction was determined from the data obtained in

amperometry Amperometry was conducted to obtain two enzyme kinetic parameters

the maximum current density (Jm) and the enzyme dissociation Michaelis constant (KM)

Jm indicates the size of catalytic velocity KM indicates the extent of enzyme-substrate

dissociation Smaller KM values indicate better enzyme-substrate affinity and enzyme

stability With the component ratio selected enzyme electrodes were then constructed

using various types and concentrations of enzyme stabiliser Amperometry was again

conducted to study the influence of enzyme stabilisers on the GOx anode performance

and stability The catalytic efficiency was determined by the fraction M

mK

J This is

based on the concept of specificity constant M

catK

k as defined below

128

Enzyme electrode specificity constant

MMM

cat

KEV

Kk

Kk

sdot==

][2 middotmiddotmiddotmiddotmiddot Eq 61

where kd = k2 Rate constant for P formation from ES

The specificity constant is a common measure of the relative catalytic rate at low

substrate concentration [22] M

mK

J therefore can be used as a comprehensive

indicator for both catalytic velocity and enzyme stability under the fixed enzyme

concentration In this study GOx concentration was fixed to approximately 10 weight

of the total composite According to the M

mK

J obtained the five most efficient and

stable GOx electrodes were determined The possible enzyme stabilisation mechanisms

of each enzyme stabiliser were also discussed together in the light of published

hypothesises

42 CONDUCTANCE OF THE COMPOSITE

421 Conductance of various conductive materials

Various electrodes were built with different conductive materials graphite GC

CNF and TTF-TCNQ at different component fraction The electrode dimension was

fixed to 314 mm2 x 1 mm Their conductance was measured and presented in a box plot

as shown in Fig 44 The sample size ranged from 3 to 6 The median is indicated by a

short line within the box

129

00E+00

50E-02

10E-01

15E-01

20E-01

25E-01

30E-01

35E-01

40E-01

45E-01C

ondu

ctan

ce

S

Lower quartile S 103E-03 549E-03 219E-01 111E-01 142E-01 599E-04 390E-03 115E-02 972E-03

Max S 281E-03 338E-02 268E-01 312E-01 415E-01 810E-04 113E-02 467E-02 262E-02

Min S 739E-04 467E-03 200E-01 933E-02 855E-02 418E-04 296E-03 264E-03 587E-03

Upper quartile S 206E-03 282E-02 253E-01 220E-01 253E-01 795E-04 807E-03 232E-02 199E-02

Median S 131E-03 847E-03 238E-01 129E-01 216E-01 780E-04 484E-03 155E-02 136E-02

TTF-TCNQ64

TTF-TCNQ73

Graphite 55 Graphite 64 Graphite 73 CNF 37 CNF 46 CNF 55 GC 73

Fig 44 Conductance of various electrode composite n = 3 ndash 6

Graphite composites showed higher conductance than other composites It is explained

by graphite having the highest intrinsic conductivity ranging between 103 ndash 104 Scm-1

[105 159] although being compounded in a composite lowered the conductivity by 4 ndash

5 order of magnitude compared to that of raw material The conduction did not show a

linear relationship with the electrode composition ratio for graphite composite because

the graphite composite 55 had larger conductance of 024 S than that of 64 and 73 It

also showed the smallest deviation than the other graphite composites while the

deviation increased with increase in the graphite content One possible cause of this is

the presence of patchy conductive paths due to the uneven distribution of conductive

islands within the composite [105 106] The conduction of the composite is therefore

often locally biased unpredictable and also leads to low reproducibility TTF-TCNQ

composite at 73 showed the conductance of 85 mS smaller than the graphite

composited 55 approximately by 30 times Conductivity of TTF-TCNQ is 102 ndash 103

130

Scm-1 [109 110] and smaller than that of graphite by one order of magnitude so this is

consistent with the intrinsic conductivity of the conducting paticles

There were also different interactions observed among conductive materials

with insulating materials Mixing epoxy resin with graphite was easier than mixing with

other conductive materials Some even did not cure at the same mixing ratios The

carbon nanofibre (CNF) composite mixture did not cure when it exceeded more than

50 weight content With 40 CNF content however it showed the conductance of 48

mS It was not comparable to graphite composite despite its similar conductivity of 103

S [105] however it was similar to that of TTF-TCNQ 73 composite 50 CNF content

even showed larger conductance of 155 mS than 70 TTF-TCNQ CNFs have been

reported to have mediating function [117] therefore with this high conduction property

at low content CNF might be very useful to enzyme composite and even superior to

TTF-TCNQ

422 Influence of enzyme concentration on conductivity

Conductance of the GOx composite was measured The conductive composite

consisted of either TTF-TCNQ 73 or graphite composite 73 GOx content was varied

by 5 10 and 15 for TTF-TCNQ composite and 10 only for graphite composite

The electrode dimension was fixed to 196 mm2 x 2 mm The sample numbers ranged

from 2 ndash 4 The study revealed that the presence of enzyme interfered with conduction

in GOx concentration dependent manner for TTF-TCNQ composite as shown in Fig 45

131

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01C

ondu

ctan

ce

S

Low er quartile S 131E-02 924E-03 124E-05 165E-01

Max S 484E-02 143E-02 368E-05 593E-01

Min S 120E-02 754E-03 390E-06 122E-01

Upper quartile S 441E-02 126E-02 288E-05 400E-01

Median S 281E-02 109E-02 208E-05 207E-01

TTF-TCNQGOx 5 TTF-TCNQGOx 10 TTF-TCNQGOx 15 GraphiteGOx 10

Fig 45 Conductance of GOx composite with various GOx content n= 2 - 4

This was due to insulating property of the protein although higher enzyme loading is

preferred for better catalytic performance as discussed in sect 226 on p 88 The issue

may be circumvented by fixing the enzyme onto the electrode surface It is however

more technically tedious and not as feasible for mass manufacture as bulk modification

It is also difficult to modify conductive salt for covalent attachment of the enzyme This

is because the conductive property of conducting salt is very sensitive to its chemical

structure and slight modification can lead to insulating properties [61] A similar

phenomenon has been also observed in CNT functionalisation [117]

Graphite-GOx composite showed larger conductance than TTF-TCNQ-GOx

composite by 20 times at the same component fraction This was due to the better

conductive property of graphite However graphite does not have mediating property

and the use of graphite as conductive material for enzyme electrodes requires the

132

loading of mediator separately The key to designing enzyme electrodes is in the balance

and compromise among the conductivity mediation enzyme activity mechanical and

chemical stability and difficulty in fabrication process A good candidate material to

make up for the TTF-TCNQ lower conductivity is CNT It has been reported that CNF

has both good conductivity and mediating functions [117] and might be suitable for bulk

modification More safety study on use of CNF is however necessary [117 160]

43 CATALYTIC ACTIVITY OF GOx COMPOSITE ELECTRODES

431 Influence of GOx concentration on the catalytic activity of GOx electrodes

Amperometry was conducted for GOx composite electrodes in N2 O2 and air

for various GOx content The conductive composite consisted of either TTF-TCNQ 73

or graphite composite 73 GOx content was varied by 5 10 and 15 for

TTF-TCNQ composite and 10 only for graphite composite The sample size was 3 for

each electrode type Control experiments were also performed with enzyme-free

composite electrodes and there was no glucose response observed There was also no

stable current observed for 10 GOx-graphite electrodes despite of its high

conductivity reported in sect 422 while Michaelis-Menten curve was observed in any

TTF-TCNQ-GOx composite electrodes This confirmed the necessity of mediating

function for enzyme anodes and its presence in TTF-TCNQ

All the TTF-TCNQ-GOx electrodes showed the Michaelis-Menten current

response curves however differed in the current as shown in Fig 46

133

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

35E-02

00E+00 10E-02 20E-02 30E-02 40E-02 50E-02

Glucose M

J A

m-2

GOx 5GOx 10GOx 15

Fig 46 Difference in the glucose-response amperometric curve of enzyme

electrodes for various GOx content 5 10 and 15 in the presence N2

The 15 GOx composite showed the smallest glucose response current due to the

interfered conduction by high protein content while 5 GOx composite showed smaller

response than 10 composite due to the enzyme deficiency Their Jm only counted for

26 and 41 of that of 10 GOx composite (Fig 47(i)) 10 GOx electrode was

therefore most catalytic KM for 5 and 10 GOx were 71 and 73 mM and little

different (Fig 47(ii)) This implies that the size of catalytic activity can be flexibly

altered by only slight change in KM

134

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

Jm

Am

-2

Jm Am-2 729E-03 180E-02 466E-03

GOx 5 GOx 10 GOx 15

00E+00

20E-03

40E-03

60E-03

80E-03

10E-02

12E-02

14E-02

Km

M

KM M 710E-03 729E-03 187E-03

GOx 5 GOx 10 GOx 15

Fig 47 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 n = 1 - 3

The results also tell that an increase in GOx loading could enhance the catalysis hence

output current unless the increase is not excessive and gt 15 at least The loading limit

should lie somewhere between 10 and 15 for this composite Similar electrodes

were also built by Alegret with 75 epoxy resin 10 TTT-TCNQ 10 graphite and

5 GOx [115] Their Jm was around 7 mAm-2 and little different from that of 5 GOx

with 70 TTF-TCNQ composite It suggests that less TTF-TCNQ content might be

sufficient as long as the conductivity is sustained by more conductive material such as

graphite

432 Influence of O2 on the catalytic activity of GOx electrodes

O2 and air suppressed the current output by GOx composite electrodes and

delayed the response to the glucose as shown in Fig 48 A current increase was

observed immediately after glucose addition for N2 but not for O2 and air They showed

135

sigmoidal current curves with a glucose response lag

(i)

(ii)

-50E-03

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

00E+00 50E-03 10E-02 15E-02 20E-02 25E-02 30E-02 35E-02 40E-02 45E-02

Glucose M

J A

m-2

GOx 10 N2GOx 10 O2GOx 10 Air

Fig 48 Difference in the glucose-response amperometric curve of 10 GOx

composite electrodes in the presence (i) N2 and O2 and (ii) re-plotted

amperometric curves for N2 air and O2

136

As expected O2 and air decreased Jm and increased KM as shown in Fig 49

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

GOx 5 GOx 10 GOx 15

Jm

Am

-2

N2O2Air

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

70E-02

80E-02

GOx 5 GOx 10 GOx 15

Km

M

N2O2Air

Fig 49 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 O2 and air n = 1 - 3

Both O2 and air decreased the current of 15 GOx composite electrodes Jm dropped to

approximately 20 of that for N2 KM was increased by 10 and 2 times for O2 and air

respectively The glucose response lag was 8 mM for O2 but none for air Air showed

milder suppression toward current output than O2 and this was expected because O2

counts for only 20 of the air [31] and should cause less influence on GOx activity

Similar phenomena were observed for 10 GOx composite electrodes For O2 and air

Jm was 13 and 40 of that of N2 and decreased to 23 x 10-3 and 67 x 10-3 from 18 x

10-2 Am-2 respectively KM was 73 for N2 and 10 mM for both O2 and air although there

was a response lag of 8 and 1 mM respectively Little difference in KM was observed

among all cases and this implies that KM itself hence enzyme stability or substrate

affinity is little affected by O2 but slight change in KM has large influence on the

catalytic activity This implies that change in catalytic activity induced by O2 is

137

reversible 5 GOx composite electrodes showed slightly unexpected result KM was

increased by approximately 4 times in both O2 and air Jm also decreased however Jm

for air was smaller than O2 by 36 despite of less O2 content There was a response lag

by 10 mM for O2 and 2 mM for air

The overall conclusions from these results is that O2 reduces GOx catalytic rate

andor electron transfer rate and hence reduces the current output but the effect is

reversible because it hardly alters KM value implying that O2 does not affect the enzyme

stability or its affinity to the substrate This phenomenon is typical in non-competitive

inhibition for enzyme kinetics Non-competitive inhibition occurs when an inhibitor

binds to the enzyme-substrate complex and slows its catalysis It therefore changes

catalytic velocity but not KM In the glucose amperometry this implies two possibilities

as a cause of Jm reduction (i) allosteric binding of O2 to GOx-glucose complex or (ii)

interference in electron transfer after the catalysis In the former case O2 does not

inhibit glucose binding to GOx but allosterically interacts with its complex and hinders

its catalysis hence it reduces catalytic current output In the latter case either glucose

binding or GOx catalysis is not affected by O2 but competition for electrons produced

by GOx catalysis occurs between the natural and artificial mediators namely O2 and

TTF-TCNQ and this reduces the electron flow to the electrode therefore it is reflected

as the reduction of catalytic current In addition to this the presence of glucose response

lag also suggests the presence of electron competition [161-163] It was reported that O2

reacts with reduced GOx faster than any other mediators with the second order rate

reaction of 15 x l06 M-1s-l [112 161 163] It is therefore quite possible that O2 swiftly

deprives electrons from reduced GOx andor reduced mediators Reduced GOx reacts

with O2 much faster than with TTF-TCNQ because it was observed that O2 had

138

diminished glucose response current at low glucose concentration Most GOx hence

reacts with O2 first then oxidised TTF-TCNQ once most O2 has been converted into

H2O2 This is compatible with the hypothesis suggested by Hindle [161] Even after the

lag the reaction could go on with residual O2 because KM did not recover fully to the N2

level although a familiar Michaelis-Menten hyperbolic curve was regained This simply

indicates that O2 concentration became smaller than TTF-TCNQ to oxidise GOx The

glucose response lag also decreased with an increase in GOx loading and was smaller

for air than O2 This implies the presence of competition because the lag depends on the

concentration of reduced GOx and electron thieves The large KM increase in the 5

GOx composite electrodes was probably due to the deficiency in the electron transfer

rate to suppress O2 activity How O2 interacts with its environment is unknown however

O2 reduces the catalytic output current due to the following possible mechanisms (i)

electron competition between O2 and TTF+ (ii) non-competitive enzyme inhibition by

allosteric binding of O2 to GOx (iii) direct interaction between O2 and reduced TTF or

(iv) backward reaction between reduced TTF and oxidised GOx

139

Table 6 Four possible causes of glucose amperometric current reduction

(i) middotmiddotmiddotmiddotmiddot Eq 62

middotmiddotmiddotmiddotmiddot Eq 63

(ii) middotmiddotmiddotmiddotmiddot Eq 64

(iii) middotmiddotmiddotmiddotmiddot Eq 65

(iv) middotmiddotmiddotmiddotmiddot Eq 66

Diagnosis can be made by doing experiments with different concentration of O2 and

mediators

44 THE EFFICACY OF GOX STABILISERS

441 Introduction

Various GOx stabilisers PEI DTT PEG GLC and FAD were mixed with GOx

composite at the concentration varying among 1 2 and 4 PEI was also mixed with

DTT or FAD and their synergetic effects were studied All the GOx electrodes were

built on the basis of TTF-TCNQ 73 conductive composite with 10 weight

GOx-stabiliser mixture Control composite electrodes were built with 2 stabilisers but

without GOx Glucose amperometry was conducted as before in the both presence and

absence of O2 Two kinetic parameters Jm and KM were obtained through non-linear

regression analysis The standard kinetic parameter values were set to those obtained in

140

the absence of O2 for GOx electrodes containing no stabiliser They are Jm = 18 x 10-2 plusmn

73 x 10-3 Am-2 and KM = 73 plusmn 57 mM respectively The glucose response lag

observed for this electrode was 10 mM Comprehensive catalytic efficiency was

determined by the size of the fraction M

mK

J under the influence of high O2 and the

most catalytic GOx composite electrodes were chosen on the basis of M

mK

J The

sample size ranged between 2 and 3 None of control experiments showed any glucose

response

442 PEI

Kinetic parameters for various PEI concentrations are shown below in Fig 50

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser PEI 1 PEI 2 PEI 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEI 1 PEI 2 PEI 4

KM

M

N2 O2

Fig 50 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEI in the presence and absence of O2 n = 1 - 3

141

In the absence of O2 PEI lowered Jm without largely affecting KM compared to the

standard value This might imply the presence of allosteric non-competitive inhibition

of glucose catalysis It is certainly possible because PEI can lock in the enzyme

conformation with hydrogen and electrostatic bonding [120] or form a complex with

GOx [126 130] This interferes in its catalysis although it also might confer the

resistance against oxidative stress as reported [126] In the presence of O2 two out of

three electrodes of PEI 1 and 4 did not show Michaelis-Menten curves The data

presented here is therefore based on a single sample This may mean that the electrodes

at those PEI concentrations are in fact prone to the catalytic suppression by O2

Moreover 4 PEI data presented here looks unreliable due to the simultaneous increase

in Jm and KM by 3 ndash 4 times compared to those obtained in the presence of N2 It may

need more data to confirm the phenomenon and be premature to conclude it is however

probably more reasonable to think that PEI 4 is not suitable for GOx anodes At other

PEI concentrations electrodes showed more or less similar kinetic parameter values to

those without stabilisers since Jm decreased and KM increased in the presence of O2

There was no O2-scavenging function observed because their glucose response lag and

KM resembled those without stabilisers Among them 2 PEI containing electrodes

sustained 50 of catalytic activity obtained in N2 and showed marginally better Jm than

stabiliser-free electrodes in the presence of O2 It might therefore have some resistance

against O2 It can be however concluded that PEI is not suitable as an enzyme stabiliser

and not effective enough to enhance the catalytic current output because their Jm was

already smaller by 60 at minimum in N2 compared to the standard value and little

improvement in catalytic performance was observed in the presence of O2

142

443 DTT

Kinetic parameters for various DTT concentrations are shown below in Fig 51

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

Jm

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

KM

M

N2 O2

Fig 51 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 DTT in the presence and absence of O2 n = 3

All DTT containing electrodes showed smaller Jm and KM than the standard values in

the absence of O2 This might imply the possible presence of uncompetitive inhibition

by DTT although the size of inhibition was not in concentration dependent manner In

uncompetitive enzyme inhibition inhibitors bind both free enzyme and

enzyme-substrate complexes hence decreases both catalytic velocity and KM [22] In the

presence of O2 all Jm KM and glucose response lag were larger than those of

stabiliser-free electrodes The increase in glucose response lag was probably due to both

electron competition and uncompetitive inhibition while the inhibition by DTT itself

was less influential on catalytic velocity than non-competitive inhibition by O2 itself

143

and this resulted in larger Jm despite of their larger KM The truth of O2 enzyme

inhibition might be reversible Met oxidation as discussed in sect 242 on p 93 because

atmospheric O2 could oxidise Met residue [123] and DTT might be capable of reducing

it back although DTT has been reported to be effective only in reducing disulphide [127

132] The increase in Jm in the presence of O2 compared to N2 was probably due to the

reduced content of DTT working toward stabilising enzyme structure This freed up

some GOx portion from rigid conformation

444 PEG

Kinetic parameters for various PEG concentrations are shown below in Fig 52

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEG 1 PEG 2 PEG 4

J m

Am

-2

N2 O2

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

No Stabiliser PEG 1 PEG 2 PEG 4

KM

M

N2 O2

Fig 52 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEG in the presence and absence of O2 n = 2 - 3

PEG decreased Jm to 10 of the standard value at maximum in the absence of O2

144

although 4 PEG electrodes showed much larger Jm than the other PEG electrodes

unexpectedly 4 PEG however showed glucose response lag gt 10 mM even in the

absence of O2 The outcome of low Jm is compatible with the result reported by Lee

[139] while among PEI electrodes Jm however increased with PEG content and this

agreed with the results presented by Arakawa [138] The hypothesis by both authors

therefore might be correct but stabilisation mechanism might differ at different

concentrations Since KM was lowered at low PEG content compared to the standard

value PEG might have penetrated the protein core and stabilised the enzyme structure

[164] however it also lowered GOx activity because the protein conformation has been

locked in At 4 PEG concentration PEG still penetrated the protein core but residual

PEG also brought the preferential hydration and this allowed more flexible enzyme

conformation and increased the catalytic activity The residual PEG however might also

have an adverse effect on electron transfer rates at low glucose concentration In the

presence of O2 KM was increased compared to when in N2 and larger than that of

stabiliser-free electrodes while Jm only marginally better compared to that except for 4

PEG It is altogether not clear about the interaction between GOx and PEG however

only high PEG content effectively enhances the catalytic current output

445 GLC

Kinetic parameters for various GLC concentrations are shown below in Fig 53

145

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser GLC 1 GLC 2 GLC 4

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

20E-02

No Stabiliser GLC 1 GLC 2 GLC 4

KM

M

N2 O2

Fig 53 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 GLC in the presence and absence of O2 n = 3

In the absence of N2 GLC showed smaller Jm and KM compared to the standard values

This indicates the presence of uncompetitive inhibition and the decrease in Jm showed

the reciprocal dependency on GLC concentration It is also quite possible that

concentration-dependent inhibition was due to the tighter protein packing by GLC as

previously reported [144] This might contribute more to non-competitive inhibition

though GLC-containing electrodes showed more resistance against O2 because their Jm

was larger than that of stabiliser-free electrodes and again it showed reciprocal

dependence on GLC concentration In both N2 and O2 cases Jm decreased with increase

in GLC content KM also showed the dependence on GLC concentration in the presence

of O2 and increased with GLC concentration Glucose response lag was also decreased

in all cases Among all 1 GLC was most effective because it lowered KM for both N2

and O2 and increased Jm for O2 although it decreased Jm by 40 in N2 It also decreased

glucose response lag from 8 to 5 mM and this might indicate that glycerol reduced and

scavenged O2

146

446 FAD

Kinetic parameters for various FAD concentrations are shown below in Fig 54

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

No Stabiliser FAD 1 FAD 2 FAD 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser FAD 1 FAD 2 FAD 4

KM

M

N2 O2

Fig 54 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 FAD in the presence and absence of O2 n = 1 - 3

FAD may be reasonably effective as an enzyme stabiliser at concentration gt 1 because

its Jm was larger in both absence and presence of O2 compared to that of stabiliser-free

electrodes It also slightly decreased glucose response lag therefore it might be able to

react with oxidising species as previously reported [108] It is however only effective at

higher concentration because two out of three 1 FAD electrodes did not show

Michaelis-Menten curves in the absence of O2 and none worked in its presence although

4 FAD was not most effective either The relationship between increase and decrease

in Jm and KM in N2 is unknown Improvement in electron transfer was not particularly

observed because higher FAD content decreased the current output although FAD has a

147

function as a mediator Mechanism of GOx stabilisation by FAD is not clear as Bartlett

reported [145] however 2 FAD was shown to be most effective among all and the only

one which showed M

mK

J gt 1 for both N2 and O2

447 PEI and DTT mixture

Kinetic parameters for various PEI-DTT concentrations are shown below in

Fig 55

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

KM

M

N2 O2

Fig 55 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

PEI-DTT (051 11 1505 and 22) in the presence and absence of O2 n =

1 - 2

PEI-DTT stabiliser mix showed the most dramatic effect and promising as a stabiliser

formulation In the presence of O2 it had larger Jm and smaller or equivalent KM than

those found in stabiliser-free electrodes Glucose response lag was also decreased from

148

8 to 3 mM and this implies the presence of O2 scavengers which were new synergetic

effects achieved and not observed in use of either stabilisers as discussed in sect 442 and

443 In the absence of O2 most PEI-DTT showed smaller Jm and marginally smaller

KM than those of the standard values except for some as it can be seen Fig 55 This

probably implies the presence of non-competitive-inhibition like effect due to the

stabilisation of enzyme structure by PEI-DTT Since it was difficult to determine the

comprehensively most effective mixture and the relative relationship in the increase and

the decrease in the two kinetic parameter values were not obvious M

mK

J was found

and compared for each PEI-DTT mixture as shown below in Fig 56

00

05

10

15

20

25

30

35

40

45

50

1505 11 0515 22

PEIDTT ()

Jm

KM

(Am

-2M

-1)

N2O2

Fig 56 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051

11 1505 and 22)

It is clear that DTT must exceed 1 to achieve in the fraction to achieve efficient O2

resistance Since the equivalent glucose response lag was observed in all mixtures this

implies that at least 1 DTT is required for simultaneous reduction of Met amino acid

149

residue On the other hand gt 2 PEI seems required for high catalytic efficiency in N2

or equal weight PEIDTT mixing is preferable to increase catalytic efficiency in both N2

and O2 Since PEIDTT at 22 data presented here is based on a single sample the study

on more samples is required to confirm the results however PEIDTT mixture with

DTT gt 1 is effective to improve catalytic efficiency and output current

448 PEI and FAD mixture

Kinetic parameters for various PEI-FAD concentrations are shown below in

Fig 57

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

J m

Am

-2

N2 O2

00E+00

20E-02

40E-02

60E-02

80E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

KM

M

N2 O2

Fig 57 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with PEI-FAD (21 22 and 24 ) in the presence and absence of O2 n = 2

Firstly the result clearly showed total 6 stabiliser content hindered catalysis because it

showed extremely low Jm compared to other PEIFAD containing electrodes It only

counts for 15 and 9 of Jm obtained for other PEIDTT concentration in presence and

150

absence of O2 respectively Secondly there was no large difference in the performance

between PEIFAD at 21 and 22 hence PEIFAD 21 was most effective of all This

might involve non-competitive inhibition because Jm was smaller by 70 while KM was

smaller by only 20 compared to the standard values in the absence of O2 In the

presence of O2 it showed larger Jm by 7 times however also increased KM by 3 times

compared to those of stabiliser-free electrodes and did not decrease the glucose response

lag

449 Most effective stabiliser content for GOx anodes

To determine the size of comprehensive catalytic efficiency M

mK

J was

found for all the stabilisers and its value was compared for O2 for catalytic efficiency in

the presence of O2 Among all five most efficient catalysts were selected and presented

below in Fig 58

151

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

J m

Am

-2

N2 Am-2O2 Am-2

N2 Am-2 180E-02 125E-02 827E-03 292E-02 710E-03 104E-02O2 Am-2 168E-02 181E-02 151E-02 267E-02 122E-02 157E-02

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505 GLC 1

00E+00

50E-03

10E-02

15E-02

KM

M

N2 MO2 M

N2 M 729E-03 671E-03 576E-03 647E-03 938E-03 328E-03

O2 M 104E-02 471E-03 433E-03 801E-03 658E-03 886E-03

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505

GLC 1

Fig 58 Jm KM in the absence and presence of O2 for various stabilisers PEIDTT

(11 0515 221505 ) and GLC 1

PEIDTT mixture was found to be most effect as GOx stabiliser as discussed in sect 447

For Jm PEIDTT 22 showed the largest in both absence and presence of O2 and this

was followed by PEIDTT 11 For KM PEIDTT 0515 showed the smallest

comprehensively for both N2 and O2 and this followed by PEIDTT 11 PEIDTT

22 and GLC 2 showed relatively large KM in the presence of O2 It can be therefore

concluded that PEIDTT 11 is the most effective in stabilising GOx has largest

resistance against O2 and increases the catalytic output current It showed Jm = 13 x 10-2

plusmn 19 x 10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2

with KM = 47 plusmn 42 mM for O2 The current output obtained is much lower than some

reported results by Willner Heller and Sasaki [18 19 97] by 2 ndash 3 order of magnitudes

their electrodes however involved more complex fabrication methods or system as

discussed sect 22 on p 77 On the other hand it was slightly better than the result

reported by Merkoci who composed a similar composite with 5 GOx and was similar

to the results reported by Arai who built GOx electrodes by more complex methods KM

152

obtained is also smaller by 2 ndash 4 times than some of results by Arai [103]

Max current output Am-2 KM mM Method involved Author

125 x 10-3 ndash 181 x 10-2 47 ndash 67 10 GOx composite Data botained

55 x 10-3 Not available 5 GOx composite Merkoci [115]

30 Not available Apo-GOx reconstituted Willner [97]

11 x 10 Not available Os-complex hydrogel Heller [19]

18 Not available Saccharide-modified polypyrrole Fujii [100]

54 x 10-2 Not available QS conductive polymer Arai [103]

41 x 10 13 - 20 Poly(acrylic) acid hydrogel modified

carbon fibre GDH electrodes

Sakai [18]

Table 7 Comparison of maximum output current and KM with some published data

45 CONCLUSION TO ANODE STUDY

Bulk modified enzyme electrodes are good because they are mechanically very

stable and their fabrication is straightforward and scaleable requiring only mixing

electrode components conductive material mediators enzymes and stabiliser with

binding material TTF-TCNQ is useful material for GOx electrodes because it is

conductive as well as mediating The electrode component ratio is important for its

function and conductivity High binder and protein content hinder the conduction It was

found that 73 weight ratio of TTF-TCNQepoxy resin with 10 GOx loading optimal

The balance among the conductivity mediation enzyme activity mechanical and

153

chemical stability and difficulty in fabrication process is key to the successful

fabrication

O2 is known to be the natural mediator of GOx In the glucose-oxygen fuel

cells GOx anode must be resistant to O2 because O2 is an electron acceptor at the

cathode O2 lowers the current output by competing for electron transfer between GOx

and artificial mediators as well as non-competitive inhibition Various GOx stabilisers

were examined to alleviate such O2 influence

PEI DTT PEG GLC FAD PEIDTT and PEIFAD mixtures were studied as

candidate stabilisers to enhance catalytic activity and the current output PEI was not

effective It stabilised the structure but made conformation more rigid and did not

increase the output current in both the absence and presence of O2 DTT was reasonably

effective It showed uncompetitive inhibition in N2 but stabilised the enzyme structure

It therefore increased Jm and KM in the presence of O2 but increase in KM was probably

due to alleviating inhibition by O2 and loosening up the enzyme conformation PEG was

not effective It probably stabilised the enzyme structure but not show much

improvement for O2 competition It might also deprive electrons from reduced GOx

GLC was effective The effect was concentration dependent Lower concentration was

more effective It also scavenged O2 FAD was reasonably effective but only at higher

concentration PEI-FAD was also reasonably effective It decreased Jm but Km was

marginally equivalent to that of stabiliser-free electrodes for N2 But it increased Jm for

O2 Among all PEIDTT at 11 mixture was found to be most effective because it

stabilised the GOx and scavenged O2 Such powerful influence was not observed in the

use of either stabilisers hence it is a synergetic effect It showed Jm = 13 x 10-2 plusmn 19 x

154

10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2 with KM

= 47 plusmn 42 mM for O2 Although it lowered Jm by 30 in the absence of O2 compared

to the standard value of 18 x 10-2 plusmn 73 x 10-3 Am-2 it also decreased KM in both

absence and presence of O2 and increased Jm by 8 times compared to that of

stabiliser-free electrodes PEIDTT 11 is therefore most effective in stabilising GOx

has largest resistance against O2 and increases the catalytic output current

155

5 CATHODE INTRODUCTION

51 OXYGEN REDUCTION REACTION (ORR)

The oxygen reduction reaction (ORR) takes place at the cathode as oxygen

accepts electrons from the anode Oxygen is an ideal oxidiser for the cathode because it

is readily available and its reaction product water is harmless [62] However ORR has

been a long-time bottleneck especially for low-temperature fuel cells because it causes

large potential loss [3 4 62 165-167] due to a strong double bond in the dioxygen

molecule Its difficult bond dissociation causes slow irreversible kinetics [24 165 166]

so a catalyst is required for this reaction to proceed at useful rates [62 165] ORR is a

multi-electron reaction which involves various reduction pathways and different

oxygen intermediates (Fig 16) In aqueous solution either the direct four- or

two-electron ORR pathway may proceed [165 166] The direct four-electron transfer

pathway reduces O2 into H2O in acid and neutral mediaor OH- in alkaline media This

occurs via several reduction steps [24 165 166] which may involve adsorbed peroxide

intermediates These peroxide intermediates never diffuse away in the solution in this

pathway [166] The two-electron transfer pathway reduces O2 into peroxide species as

final products These peroxide species either diffuse away are reduced further or

decomposed into H2O or OH- [24 165 166] This is not favourable because it leads to

lower energy conversion efficiency and its product hydrogen peroxide may degrade the

catalysts and the fuel cells [165 167] Four- and two-electron transfers also may occur

in series or in parallel [165]

156

Fig 59 Oxygen reduction pathways [165 166]

Reduction kinetics and mechanisms depend on the type of catalysts electrode material

electrode surface condition solution pH temperature contaminant and reactant

concentration [24 165 166] Rate-limiting step also varies by type of the catalysts

[166] However the first electron transfer to dioxygen leading to the formation of

superoxide anion radical (O2־) is the rate limiting step [168] O2־ is very rapidly

protonated in water and converted to hydroperoxyl radical (HO2)

157

Fig 60 Degradation of superoxide anion radical in aqueous solutions [24]

In aqueous solution O2־ is unstable and rapidly converted to O2 and H2O2 via a

proton-driven disproportionation process (Fig 60) [24]

52 REACTIVITY OF OXYGEN

A dioxygen molecule is a homodiatomic molecule consisting of two oxygen

atoms double bonded It has two parallel unpaired electrons in degenerate anti-bonding

π designated as π orbitals therefore it is diradical and paramagnetic (Fig 61) [169]

158

Fig 61 An energy level diagram for ground state dioxygen molecule [169]

When one electron is transferred to a dioxygen molecule the electron is placed in π

orbital and it turns into a reactive O2[24] ־ reducing the bond order from 2 to 15

Oxygen reduction changes the molecular symmetry or stretches the double bond

causing bond disruption [165 166] Electron paring in one of the π orbitals also

changes their orbital energy state and lifts the degeneracy as in Fig 62 [170]

Hereogeneous catalysis involves chemisorption of the oxygen and therefore some

transfer of electron density from the oxygen to the catalyst This facilitates the rate

determining initial electron transfer Oxygen reduction catalysts are reviewed in the

next section

159

(O2) 3Σg- O2־

Fig 62 Diagrams for the state of π orbitals in the ground state oxygen (3Σg-) (left)

and peroxide anion radical (right) [24]

O2 species Bond Order Bond length pm Bond energy kJmol-1

O2 2 1207 493

O2+ 2frac12 1116 643

O2- 1frac12 135 395

O22- 1 149 -

Table 8 Bond orders bond lengths and bond energies of some dioxygen species [169]

53 ORR CATALYSTS

Catalysts are required to reduce the overpotential and improve the efficiency of

160

ORR [3 4 62 165-167] It was reported that 80 of the whole potential loss can be

attributed to cathode ORR overpotential [171] The choice of ORR catalysts varies by

applications [36 171] and direct four-electron transfer pathway is most preferred for

ORR at the lowest possible overpotential in every fuel cell application however its

kinetics and reduction pathway are limited by the type of catalysts and experimental

conditions [24 165 166] Miniaturised glucose-oxygen fuel cells operate in the neutral

aqueous solution at ambient or body temperature with relatively low electrolyte and

oxygen concentrations therefore they require powerful ORR catalysts Platinum (Pt)

has been most ideal however its availability and cost are major limiting factors Many

studies have been made on other noble metals [172 173] transition metal macrocyclic

compounds [174] multicopper oxidases [62 175] and quinones [165] as alternatives

ORR catalysts to Pt These ORR catalysts are reviewed below

531 Noble metal ORR catalysts

Noble and transition metals are well-known ORR catalysts There are two

possible ORR mechanisms for metal surface dissociative and associative

161

Dissociative mechanism

middotmiddotmiddotmiddotmiddot Eq 67

middotmiddotmiddotmiddotmiddot Eq 68

middotmiddotmiddotmiddotmiddot Eq 69

( indicates the catalyst surface)

Associative mechanism

middotmiddotmiddotmiddotmiddot Eq 70

middotmiddotmiddotmiddotmiddot Eq 71

middotmiddotmiddotmiddotmiddot Eq 72

middotmiddotmiddotmiddotmiddot Eq 73

middotmiddotmiddotmiddotmiddot Eq 74

In dissociative mechanism the O2 double bond is ruptured rapidly on its adsorption on

the metal surface [165 166 172] This involves a strong interaction between oxygen

and metal atoms [166] and does not generate H2O2 [165] Electron and proton transfer

take place simultaneously and this is the rate-limiting step [166 171] In the associative

mechanism O2 does not dissociate before proton transfer Adsorbed O2 stays on the

metal surface [165 172] and two-electron and proton transfer [166 173] take place via

the formation of superoxide or peroxide intermediate [165 166] This reaction is

thermodynamically easier than the dissociative pathway and its final product is H2O2 or

162

H2O if both pathways occur in series [165] O2 adsorption on the metal surface is an

inner sphere interaction which involves complex formation with the metal surface [30]

There are three ways in which O2 adsorbs (i) Griffiths (ii) Pauling and (iii) bridge

model (Fig 63)

Griffiths model Side-on Pauling model End-on Bridge model

OO

M

Fig 63 Three models of oxygen adsorption [165 166 173]

The Griffiths model is called lsquoside-onrsquo type O2 functions as a bidentate ligand against a

single metal atom in this complex and its π orbitals laterally interact with empty metal

dz2 orbitals causing ligand-to-metal charge transfer (LMCT) πrarrdz

2 At the same time

electrons partially filling metal dxz or dyz orbitals are back donated to oxygen π orbitals

[166 171] Therefore a strong metal-oxygen interaction is present in this model This

effectively disrupts the O=O bond [166] and facilitates the electron transfer from the

metal ion by changing its oxidation number The Pauling model is called the lsquoend-onrsquo

type [165 166 173] O2 is a monodentate ligand in this model and only one of the

oxygen atoms in the dioxygen molecule can be bonded to the metal surface An oxygen

π orbital interacts with metal dz2 orbitals and this allows either the direct or peroxide

ORR pathway after partial charge transfer [166] The bridge model resembles Griffiths

model in the way the metal atom and oxygen interact however it requires two adjacent

163

adsorption sites so that O2 can ligate two different metal atoms The bridge-model

interaction is easily interfered with by the surface contamination and the oxide layer

because the surface species block the adsorption sites for ORR catalysis [173]

Fig 64 Models for oxygen adsorption and ORR pathways [166]

Both the Griffiths and bridge models follow dissociative mechanism (pathway I and III

respectively Fig 64) due to the lateral interaction leading to the dissociation of O2

double bond The Pauling model on the other hand follows the associative mechanism

(pathway II Fig 64) This is further divided into two other pathways IIa and IIb

according to its end product H2O2 or H2O (Fig 64) respectively [166] O2 adsorption

and the ORR mechanism therefore depend on (i) the extent of oxygen-metal

interaction and (ii) the availability of oxygen adsorption sites A volcano-shaped trend

between ORR rate and oxygen-metal interaction has been plotted by Norskov as in Fig

65 [172]

164

Fig 65 A volcano-shaped trend between ORR rate and energy required for oxygen

binding on various transition metal surface [172]

Many have reported platinum (Pt) as the most ideal metal catalyst due to its high

catalytic ORR activity [165 166 171 172] Fig 65 shows that Pt and Pd (palladium)

have largest ORR rate yet medium oxygen adsorption energy and iridium (Ir) and

rhodium (Rd) follow them On the other hand metals with either higher or lower

oxygen adsorption energy result in lower ORR rate This is because metals with high

oxygen adsorption energy such as gold (Au) imposes too large energy barrier for the

formation of a stable oxygen-metal bond while too small adsorption energy as in nickel

(Ni) leads to excessively stable oxygen-metal interaction and slows the proton transfer

Therefore associative pathway is dominant at Au surface while oxygen dissociation is

deactivated at Ni surface but instead this increases the surface oxide coverage [172

173] Pt performs best because it has moderate oxygen-metal surface interaction [165]

Pt alloys with other transition metals such as Ni Co and Fe have been also reported to

increase the catalytic activity [36 167 171 172] because alloying dilutes the Pt-O

165

interaction and alters the overall oxygen-metal surface interaction [171 172] This is a

useful property to add because Pt is known to form a surface oxide layer leading to both

associative and dissociative catalytic pathways [165 171] This surface state is

potential-dependent and stays persistenly at less than 08 V vs AgAgCl [172]

532 Macrocycle and Schiff-base complex ORR catalysts

Macrocyclic transition metal complexes have been widely investigated as

potential ORR catalyst both for fuel cells and as catalysts to activate dioxygen for

synthetic purposes They have been widely reviewed by [165 176] Macrocyclic

complexes consist of (i) transition metals such as Fe Co Ni or Cu (ii) chelating atoms

N4 N2O2 N2S2 O4 and S4 and conjugate π systems such as porphyrin and

phthalocyanine [165] Examples of transition metal macrocycles are shown in Fig 66

Fig 66 Examples of transition metallomacrocycles Heme (left) [177] and copper

phthalocyanine (right) [178]

166

Schiff base complexes such as metallosalens and metallosalophens are other

well-known chelating ORR catalysts [174 179] Transition metal macrocycles and

Schiff bases can be also polymerised into conductive films [180] (Fig 67)

Fig 67 Schiff base complexes

Co(II) salen Co(II) salophen and

Co(II) salen modified poly(34-ethylenedioxythiophene) [174 180]

Interaction between the oxygen molecule and the metal complex is similar to that with

the noble metals and involves partial charge transfer between the filled metal dz orbitals

and the π orbital of O2 as described in sect 531 The catalytic activity of the metal

complexes depends on the type of central metal atom ligand and the size of the

π-electon systems The reducing ability of the ligand is important because the central

metal atom must transfer considerable electron density to oxygen atoms during the

167

catalysis [170] The thermodynamically most stable metal-oxygen complexes are

bionuclear complexes such as dicobalt face-to-face 4-atom-bridged diporphyrin

(Co2(FTF4)) (Fig 68) [165-167]

Fig 68 Molecular structure of dicobalt face-to-face diporphyrin [166]

Mononuclear Co complexes take the associative pathway [165] forming superoxo

cobaltic complexs (Co-O-O־) [181 170] while their dimers or binuclear complexes can

take either direct four-electron or the parallel pathways (Fig 69) [165 167] because

they can lock the oxygen moiety in rigid orientation [170] Co2(FTF4) favours

four-electron transfer and its ORR pathway depends on the distance between two

cofacial metal atoms [165-167] Spacing distance can be altered by inserting a spacer

molecule [165 166]

168

Fig 69 ORR pathway by Co2(FTF4) [166]

However Co2(FTF4) is not stable enough for practical use [165 166] Practical ORR

catalysts for fuel cell application must be stable inexpensive and feasible to synthesise

533 Multicopper enzymes as ORR catalyst

Multicopper oxidases (MCOs) are copper-containing enzymes such as bilirubin

oxidase (BOD) laccase (Lac) and copper efflux oxidase (CueO) Oxygen is widely used

as an electron acceptor in nature because it is ubiquitous and oxidation is a good mean

to obtain energy as in combustion Millions of years of evolution have therefore

provided enzymes with good catalytic activity for oxygen reduction Although oxygen

169

reduction in biological system is complex due to involving various substrates and

catalytic steps as well as it tends to be incomplete and leads to hydrogen peroxide or

superoxide formation as the product the enzymes catalyse the rate-determining initial

electron transfer For application in biofuel cells it is essential that the enzymes are

widely available and possess good stability activity in immobilised form and ideally

four-electron ORR catalysis MCOs have been prevalent in biofuel cell applications

because they are active at neutral pH catalyse four-electron ORR and bear

immobilisation Fungal laccase and bilirubin oxidase have been most widely studied

MCOs [56 59 60]

Most MCOs have four Cu atoms which are designated as type I (T1) type II (T2) and

a pair of type III (T3) T2 and T3 together form a trinuclear Cu centre while T1-Cu

called blue copper [175 182 183] is located away from the trinuclear Cu centre but

adjacent to the hydrophobic substrate-binding site Fig 70 shows the location of the

trinuclear Cu centre (copper coloured) and the blue copper (in blue) Oxygen binding

and ORR take place at the trinuclear Cu centre [175] while T1-Cu receives electrons

from the substrate and mediates electron transfer between substrate and the trinuclear

Cu centre [175 184]

170

Fig 70 Laccase from Trametes versicolor [185]

Enzyme catalytic efficiency depends on the redox potential [175] of T1-Cu It has been

reported that fungal laccase has the highest redox potential between 053 ndash 058 V vs

AgAgCl [62 183] T1-Cu also allows DET due to being located close to the surface

[183 186 187]

MCO reaction cycle starts from substrate oxidation to form a fully reduced state MCO

is initially at native form or so-called intermediate II Native form is characterised by

doubly OH--bridged structure (Fig 71(ii)) In the absence of substrate the native form

decays to the resting form which is at fully oxidised state (Fig 71(i)) After

four-electron reduction of enzyme the fully reduced MCO (Fig 71(iii)) reacts with O2

The first reduction step involves two-electron transfer from each of T3-Cu+ to O2 This

forms peroxide intermediate or intermediate I (Fig 71(iv)) and lowers the energy

barrier to disrupt O=O It is followed by swift protonation of adsorbed peroxide at

T3-Cu site This protonation reaction is induced by the charge transfer from T1- and

171

T2-Cu+ This leads to the complete reduction of oxygen to water and the enzyme native

form is retrieved

(i)

(ii)

(iii)

(iv)

Fig 71 Redox reaction cycle of MCOs with O2 [175]

All MCOs have this common ORR mechanisms despite the different origin structure

biological functions substrate reducing power and optimal environment

5331 BOD

BOD is an anionic monomer [182] most active at neutral pH [183 188] and

172

capable of both DET and MET The mediators must have their redox potential close or

slightly lower than that of BOD 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

(ABTS) (Fig 72(i)) [62 97] Os-pyridine complexes linked to conductive polymer (Fig

72(ii)) [66 189] and cyanometals [18 56] have been used as its mediators [56 185]

ABTS is a divalent anion which works as co-substrate of MCOs and its redox potential

is 067 V vs AgAgCl [56] Os-pyridine complex with conductive polymer follows the

similar idea for the wired GOx anodes discussed in sect 223 on p 80

4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine) has been used

especially for the cathode and its redox potential is 055 V vs AgAgCl

Fig 72 Common mediators for BOD (i) ABTS [56 62]

(ii) Os[4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine)] [190]

173

Tsujimura showed that ORR catalytic constant was 6-times greater in the presence of

Os-pyridine complex than in DET [66 188] Reduction starts around 05 V in most

cases [66 97 183 188] The size of current density varies roughly between 05 - 1

mAcm-2 [56 97 188] and 95 mAcm-2 was achieved by Heller [189]

5332 Lac

Fungal laccase is another anionic monomer [183] It is less expensive than

BOD and has broad substrate specificity including xenobiotics [175] although it has

optimum pH around 5 and activity hindered by chloride [19 182] Its T1-Cu redox

potential ranges between 053 ndash 058 V vs AgAgCl [62 183] Highest redox potential

066 V and current density 057 mAcm-2 were achieved by the use of anthracene

plugging molecule which provides strong binding between Lac and pyrolytic graphite

edge electrode as well as long-term activity 57 of the initial current was retained after

eight weeks [185]

5333 CueO

CueO is a less common enzyme in biofuel cells however it is active for a wide

range of pH between 2 - 8 [186] and also it has better catalytic activity than other

MCOs because it has five Cu atoms This extra Cu atom is believed to have mediating

function and 4 mAcm-2 has been achieved at pH 5 [191] However it has lower redox

potential around 03 V [186 191] Protein engineering has been introduced to increase

T1-Cu redox potential [186]

174

534 Anthraquinone ORR catalyst

Anthraquinone (AQ) is known to catalyse two-electron transfer in ORR (Fig

73) [44 165 192-194] and it is used in industries to produce hydrogen peroxide [165

192]

Fig 73 Two-electron ORR by anthraquinone [193]

ORR by anthraquinone is an electrochemical-chemical (EC) process [165 192] in

which quinone must be first electrochemically reduced to an active species

semiquinone anion radical (Q־) The active semiquinone radical can then react with

dioxygen so that it converts the dioxygen into O2־ via forming of the intermediate

superoxo-C-O(O2) species This is the rate-limiting step O2־ produced by a

semiquinone radical either disproportionates (Eq 77) or it is further reduced into HO2-

(Eq 78) [192]

175

Oxygen reduction reaction by anthraquinone

ltlt Rate limiting step gtgt

middotmiddotmiddotmiddotmiddot Eq 75

middotmiddotmiddotmiddotmiddot Eq 76

ltlt Following spontaneous reactions gtgt

middotmiddotmiddotmiddotmiddot Eq 77

middotmiddotmiddotmiddotmiddot Eq 78

(Q indicates quinone on the surface)

Derivatised anthraquinone such as Fast red (Fig 74) can be attached to the carbon either

physically or covalently Grafted quinones are very stable [192 194 195] and resistant

against continuous potential cycling [194] metal ions and surfactant despite of some

fouling present [44]

176

O

O

N N Cl

frac12 ZnCl2

1

2

4

6

5

3

10

9 7

8

12

14

13

11

O

O

(i) (ii)

Fig 74 The structures of (i) anthraquinone [196] and (ii) Fast red AL salt [44 197]

However its ORR is heavily affected by pH [194] because its catalytic efficiency

depends on the concentration of surface Q־ species [192 194] The concentration

varies by different pH and the catalytic efficiency drops at both high and low pH due to

the depletion of Q־ When pH falls below its dissociation constant Q־ is not

reproducible anymore and ORR is inhibited This phenomenon appears at as high as pH

7 and the ORR vanishes at pH lt 7 [194] AQ-grafted GC (AQGC) shows only sluggish

kinetics at pH lt 10 Its voltammogram also becomes complex due to the presence of

native quinone groups originated from the carbon surface [192] ORR mediated by the

native quinone groups is observed as a prewave and this varies by different quinone

derivatives such as phenanthrenequinone (PQ) (Fig 75) electrode composition and pH

[194]

177

Fig 75 Hydrodynamic voltammogram for ORR at various quinone electrodes with various

rotation speeds [194] (i) unmodified GC (ii) AQGC and (iii) PQGC at pH 7

54 CARBON PROPERTIES

Carbon is widely used for electrochemical applications because it is

inexpensive mechanically stable [105] and sufficiently chemically inert yet reactive

due to the presence of surface defects It has wide potential window between 1 and -13

V [21 30] Carbon is available in various solid forms such as graphite glassy carbon

(GC) pyrolytic carbon (PC) activated carbon (AC) carbon fibres (CFs) carbon

nanotubes (CNT) and diamond It is easy to renew the surface and allows facile

chemical modification [195] However it is also prone to adsorption and surface

oxidation simply by air and water exposure and its electron transfer is under the

constant influence of the electrode surface condition and history [198] This leads to the

deviation and the low reproducibility in carbon electrochemical experiments [105]

178

Physical properties of carbon vary by its form and section The carbon consists of either

(i) layers of the conductive planar graphene sheets or (ii) the insulating

tetrahedral-puckered forms as seen in the diamond (Fig 76) Conductivity in diamond

is gained by doping with boron [199 200]

Fig 76 Carbon structures (i) graphite (ii) diamond [201]

The electrical conductivity is anisotropic and closely correlates with the carbon

structure and section The carbon section is classified into (i) basal and (ii) edge planes

The basal plane extends along the graphene plane while the edge plane is perpendicular

to this

179

Fig 77 Carbon section (i) basal plane (ii) edge plane [105]

The basal plane has larger conductivity due to being parallel to the vast graphene

network while the edge plane is superior in electrochemical reactivity due to being

richer in reactive graphene sheet termini and carbon defects The ratio between the basal

and edge planes therefore affects the conductivity and electrochemical properties of

the carbon and this varies by the carbon form and its treatment [45 105 195 202 203]

The electrical conductivities for various carbon forms and sections is shown in Table 9

180

The type of carbon Electrical conductivity Scm-1

Graphite [105 159] 1 x 103 - 104

Highly ordered PG basal plane 250 x 104

Highly ordered PG edge plane 588

PG basal plane 400 x 103

PG edge plane 333

Polycrystalline graphite 100 x 103

GC [105 159 203] 01 - 250 x 102

Activated carbon [204] 1 x 10

Carbon fibre 133 x 103

Boron-doped diamond [199 205] 01 - 1 x 102

Multi-wall carbon nanotube [206] 1 ndash 2 x 103

Pt 909 x 104

Cu 588 x 105

Table 9 The electric conductivity of various carbon forms and sections Data from

[105] unless defined specifically

541 Graphite

Graphite is black-coloured material with pores and high gas permeability It is

available in many physical forms as powders moulded structures and thin sheets

therefore it is used in many commercial applications [203] Powdered graphite is

convenient to make carbon paste and composite electrodes [105] Graphite consists of

181

layers of graphene sheets each of which is a huge π-conjugate system π-conjugate

system is made of hexagonal rings of sp2 hybridised carbon atoms [105 195] (Fig 76(i)

and Fig 78) and allows free electron movement within graphene This gives carbon

metallic properties such as electric and thermal conductivity

Fig 78 A direct image of a graphene bilayer by transmission electron microscopy

(TEM) at resolution of 106 Aring and with a 2-Aring scale bar [207]

Each graphene sheet is structurally stable in horizontal direction but fragile in vertical

direction because each layer is only held by van der Waalsrsquo force Therefore the

graphene sheets easily slide and collapse [31]

542 Glassy carbon (GC)

Glassy carbon (GC) is a non-graphitised carbon made of randomly interwoven

sp2-carbon fibres which are tangled and heavily cross-linked (Fig 79) It contains many

closed voids [203] and fullerene-like elements [208] It is impermeable to gases [203

182

208] chemically more inert [208] and mechanically stronger but electrically more

resistive than other carbons [203]

Fig 79 GC (i) High resolution TEM image (ii) A schema of GC [208]

543 Activated carbon (AC)

Activated carbon (AC) is thermally and chemically activated carbon It is

highly porous and has large internal surface area This is an excellent adsorbent for

solvent recovery and gas refining [203 208]

544 Carbon fibre (CF)

Carbon fibres (CFs) have small diameter ranging between 5 to 30 microm They are

lightweight have lower fabrication cost and are superior in mechanical properties

therefore they have been used for sporting automotive and military equipments They

are available in many forms such as tow mat felt cloth and thread They consist of a

random arrangement of flat or crumpled graphite sheets but their structure and

183

molecular composition depend on the type of precursors [203 208]

545 Carbon nanotubes (CNTs)

Carbon nanotubes (CNTs) are made rolled-up sheets of sp2-graphene in nano to

micro-meter range They are available in various formats single-walled nanotube

(SWNT) multi-walled nanotube (MWNT) carbon nanohorn (CNH) (Fig 80) and

various forms powder arrays and posts [117]

Fig 80 Various CNTs (i) Bundled SWNTs (ii) MWNT (iii) CNHs [209]

CNTs have been drawing keen attention due to their wide surface area conductive or

semiconductive properties light weight mechanical stability and facile

functionalisation [117 195 202 210] They have been used as support for the dispersed

metal catalysts [3] and electrode materials [117 202] The drawbacks of CNTs are the

high presence of impurities alteration of their properties on acid cleaning [202 211]

and unknown toxicity [117 160 202]

184

546 Boron-doped diamond (BDD)

Diamond is different from other carbons because it is transparent and not

electrically conductive It has a cubic lattice structure made of closely-packed repetitive

units of sp3-hybridised carbon atoms as seen in Fig 81

Fig 81 A TEM image of diamond [212]

Each unit consists of four σ-bonded sp3-carbon atoms in tetrahedral coordination as in

Fig 76(ii) Each bond is localised and this gives structural stability but does not conduct

electricity By doping it with boron diamond can become semiconductive Such

diamond is called boron-doped diamond (BDD) and boron functions as an electron

acceptor BDD is mechanical more stable chemically more inert sensitive to analyte

and has wide potential window lower back ground current and stable electrochemical

responses [195 199 205]

185

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON

551 Electrochemical properties of carbon

The edge plane of carbon which contains graphene termini and carbon defects

is chemically reactive and accounts for carbon electrochemistry while the vast network

of graphene sheets is responsible for its electrical conductivity The termini and defects

include some sp3 hydrocarbons and they are oxidised into various oxygen functional

groups such as carboxyl quinoyl hydroxyl lactone groups ethers cyclic esters and

lactone-like groups [195] These electrode functional groups facilitate electron transfer

provide bonding between the electrode and the catalysts or enzymes or simply cause

electrode capacitance or resistance Various carbon pretreatments such as

electrochemical pretreatment (ECP) vacuum heat treatment (VHT) carbon fracturing

and polishing laser irradiation [195 213-215] are used to attach active surface

functional groups and increase the rate of electron transfer in various electrochemical

reactions [198 216]

552 Electrochemical pretreatments (ECPs) of the carbon

Electrochemical pretreatments (ECPs) provide feasible electrode surface

modification Various ECPs have been used to introduce carbon-oxygen surface

functional groups Most common method is potential cycling in the acid or basic media

or the combination of anodisation and cathodisation as shown in Table 10

186

Table 10 ECPs examples

Potential cycling at 02 Vs-1 between 0 and 02 V in 01 M H2SO4 [105]

Anodisation at 18 V in pH 7 phosphate buffer for 5 minutes followed by 1-minute cathodisation at

ndash 15 V [216 217]

Potential cycling between 06 and 2 V at 5 Vs-1 for 15 to 30 minutes in 05 M NaOH [218] or their

combination [219]

ECPs introduce the electrodes various new properties They (i) attach electrochemically

active functional groups and increase the oxygencarbon ratio [105] (ii) introduce

carbon defects and active sites [105 218] (iii) increase the surface area [105 198 215

216 218] (iv) form a surface film and (v) increase the background current [105] and

double layer capacitance [105 215 216] Most notable effect of EPCs is the increase in

electron transfer rate in many electrochemical reactions This is observed as a decreases

in peak separation and an increase in peak current in voltammetry as seen in Fig 82

[105 216 218 220] As for ORR however it was reported that surface quinone groups

attached by EPC or originated from the carbon itself reduce ORR overpotential but do

not mediate ORR as in quinone groups physically or covalently attached [216 220]

187

Fig 82 The influence of ECP on GCE and its voltammogram at 01 Vs-1 in pH 7

phosphate buffer in the presence of (i) N2 and (ii) O2 The voltammograms

of both pretreated and unpretreated GCEs are shown [216] The

presentation has been altered for convenience

56 ELECTROCHEMICAL TECHNIQUES FOR ORR

ORR has been studied by two electrochemistry techniques cyclic voltammetry

(CV) and hydrodynamic voltammetry (HDV) using the rotating disc electrode (RDE)

These techniques provide overlapping but complementary information CV is a dynamic

technique and allows calculation of the number of electrons up to and including the rate

determining step (nα) With numerical simulation and data fitting CV can be used to

estimate the standard heterogeneous rate constant (kdeg) RDE is a steady state technique

and can give the overall number of electrons transferred (n) The well-established

Koutecky-Levich plot (see sect 567 on p 200) provides a robust method for determining

kdeg provided the standard electrode potential (Edeg) for the reaction is known

188

561 Cyclic voltammetry (CV)

Cyclic voltammetry (CV) is widely used for initial exploration and

characterisation of a redox active system It is of limited electroanalytical value

however kinetic parameters can be obtained from the current-voltage (I-V) plot In

cyclic voltammetry potential is swept linearly with a triangular waveform (Fig 83(i))

and applied cyclically The resulting time-dependent current is typically plotted versus

applied potential (Fig 83(ii)) [30 221] The potential range and scan rate are decided in

advance according to the type of analytes reactions and materials of interest

Fig 83 Schemas for (i) a Triangular waveform (ii) Typical cyclic voltammogram for

benzoquinone (BQ) CV was taken at 005 Vs-1 between + 025 and ndash 05 V vs

AgAgCl for 1 mM BQ in 1M KCl at graphite composite electrode Switching

potential (Eλ) is ndash 05 V and time taken to reach Eλ (t) is 15 seconds Based on

the real data

189

562 Non-faradic current and electric double layer capacitance

Transient techniques such as CV always contain two types of electric current

non-faradaic and faradaic current The non-faradaic charging current is due to the

electric double layer which is formed at the electrode-solution interface This occurs

because such an interface behaves like a capacitor as counter ions accumulate at the

interface in order to balance the potential at the electrode This does not account for the

charge transfer reactions involved however provides the information about the

electrode surface [30] and also can affect the rate of electrode process and distort the

voltammogram Cd can be estimated by electrochemical impedance spectroscopy or

deduced by averaging Cd over the potential range as in Fig 84 and Eq 33 [21 30]

Fig 84 The double layer capacitance (Cd) observed in CV for deoxygenated pH 74 at

the acid-pretreated graphite electrode See Eq 33 for the calculation and

symbols Based on the real data

190

Electric double layer capacitance per unit area

AII

AEQareaunitperC ccca

d sdotminus

=sdot

=υ2

)()( middotmiddotmiddotmiddotmiddot Eq 79

Cd = Double layer capacitance per unit area microFcm-2

Q = Charge C

E = Potential V

A = Electrode surface area cm-2

Ica = Anodic non-faradic current A

Icc = Cathodic non-faradic current A

υ = Scan rate Vs-1

Cd needs to be either negligible or subtracted before interpretation of faradaic current

which accounts for the charge transfer phenomena in redox reactions In order to

minimise the influence of Cd on the voltammogram the scan must be taken at slow scan

rate and the electrolyte concentration must be always kept reasonably high [30]

563 Diagnosis of redox reactions in cyclic voltammetry

A typical voltammogram contains current peaks as seen in Fig 83(ii) and these

are due to the redox reactions in the system Diagnosis of the redox system using CV is

based on (i) peak separation (ii) peak current height (iii) peak shape or peak width (iv)

the number of peaks and (v) the dependency of peak shape on the scan rate The

analysis of such peaks involves the following four parameters anodic peak current (Ipa)

cathode peak current (Ipc) anodic peak potential (Epa) and cathode peak potential (Epc)

This reveals the type of electrochemical reactions involved which are either (i)

191

reversible (ii) quasi reversible or (iii) irreversible When the peak current is proportional

to υfrac12 the redox reaction is diffusion-limited in the solution while a linear dependence

shows that the redox active species is confined to the electrode surface

In the reversible system charge transfer between the redox species and the electrode is

rapid [221] therefore it has fast kinetics and the reaction is diffusion-limited The

electrode potential follows the Nernst equation (Eq 15 on p 50) because the electrode

potential is dependent on the concentration equilibrium at the electrode surface and the

peak potential is independent of the scan rate The features on the reversible system are

listed in Table 11 The peak current (Ip) is linearly proportional to υ12 as the redox

reaction is diffusion-limited The peak separation (∆Ep) is the difference between Epa

and Epc and it is 59 mV at 25 ˚C for the single-charge transfer [29 30] The peak width

(δEp) is the difference between Ep and half-peak potential (Ep2) Ep2 is the potential

where the peak current is half It is 565 mV for a single charge transfer The ratio

between Ipc and Ipa is called a reversal parameter [105] It is unity at any scan rate in the

reversible system

192

Table 11 Reversible system at 25 ˚C [30]

2121235 )10692( υlowasttimes= CADnI p middotmiddotmiddotmiddotmiddot Eq 80

mVn

E p528

= independent of υ middotmiddotmiddotmiddotmiddot Eq 81

mVn

EEE pcpap59)( =minus=∆ middotmiddotmiddotmiddotmiddot Eq 82

mVn

EEE ppp556

2 =minus=δ middotmiddotmiddotmiddotmiddot Eq 83

1=pa

pc

II middotmiddotmiddotmiddotmiddot Eq 84

Ip = Peak current A

n = The number of charges transferred

A = Electrode surface area m-2

D = Analyte diffusion coefficient m-2s-1

ν = Solution kinematic viscosity m-2s-1

Ep = Peak potential V

∆Ep = Peak separation V

Epa = Anodic peak potential V

Epc = Cathodic peak potential V

δEp = Peak width V

Ep2 = Half-wave potential V

Ipa = Anodic peak current A

Ipc = Cathodic peak current A

In a totally irreversible system charge transfer between the redox species and the

electrode takes place only slowly [221] The electrode must be activated by applying

193

large potential to drive a charge transfer reaction at a reasonable rate Ep becomes

dependent on scan rate and this increases ∆Ep and skews the voltammogram [30]

However Ip is still proportional to υfrac12 because at high overpotential the redox reaction

is diffusion-limited [29]

Table 12 Irreversible system at 25 ˚C [222]

2121215 )()10992( υα αlowasttimes= CADnnI p middotmiddotmiddotmiddotmiddot Eq 85

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛

deg+minusdeg=

2121

lnln780RT

Fnk

DFn

RTEE pυα

αα

α

middotmiddotmiddotmiddotmiddot Eq 86

mVn

E pαα

59gt∆ middotmiddotmiddotmiddotmiddot Eq 87

mVn

E pαα

δ 747= middotmiddotmiddotmiddotmiddot Eq 88

α = Transfer coefficient

nα = The number of charge transferred up to the limiting step

( Please refer to Table 11 for other terms and symbols)

A quasi-reversible system is controlled by both kinetics and diffusion Its

electrochemical reversibility is relative to the scan rate [21 30] The peak potential

depends on scan rate for rapid scans and the peak shape and peak parameters become

dependent on kinetic parameters such as α and Λ where 21

1

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛

deg=Λ

minus υαα

RTFDD

k

RO

[30]

Since Λ is a function of k˚υ-frac12 where kdeg is the standard rate constant the voltammogram

hence kdeg is affected by the scan rate as seen in Fig 85

194

Fig 85 Voltammograms of a quasi-reversible reaction at various scan rates [21]

564 Coupled reactions in cyclic voltammetry

Redox reactions are sometimes coupled with different electrochemical or

chemical reactions Voltammogram with altering scan rates can indicate the presence of

such reactions A redox reaction coupled with another redox reaction is called EE

mechanism while with homogeneous chemical reaction it is called EC mechanism If

the coupled reaction is catalytic then it is called ECrsquo mechanism [21]

Many ORR catalysts show coupled reactions with ECrsquo mechanism ORR is a

quasi-reversible reaction because the reaction is limited by oxygen diffusion yet O2

adsorption and reduction heavily relies on overpotential or catalytic power of catalysts

Catalysed reactions show distinctive voltammetric features There is only a single-sided

large current but with no reverse current because they catalyse a reaction only in a

single direction The reversal parameter therefore becomes non-unity [30]

195

ORR catalysts sometimes contain multiple reducible centres or an atom with various

oxidation states as in seen transition metal They might exhibit a multi-peak

voltammogram under non-catalytic condition The shape of voltammogram depends on

the difference in the standard potential (∆E˚rsquo) between two redox centres or states (Eq

89) as seen in Fig 86 [21 30]

21 ooo EEE minus=∆ middotmiddotmiddotmiddotmiddot Eq 89

Fig 86 Voltammograms of two-electron transfer EE systems of different ∆Edegrsquo [21]

565 Voltammograms for the surface-confined redox groups

Catalysts encapsulated in or attached to the electrode provide catalytic groups

or moieties on the electrode surface Such surface-confined redox species show different

voltammograms from solution redox species because their redox reactions do not

196

involve diffusional redox species They exhibit a characteristic voltammogram of

perfect symmetry with Epa = Epc as seen in Fig 87 and their peak current is linearly

proportional to υ rather than υfrac12 as in Eq 90 [21 30] The features of surface-confined

groups are listed in Table 13

Fig 87 A voltammogram of the reversible redox reaction by surface-confined groups [21]

Table 13 Reversible system by surface-confined groups at 25 ˚C [30]

RTAFn

I p 4

22 Γ=

υ middotmiddotmiddotmiddotmiddot Eq 90

oadsp EE = middotmiddotmiddotmiddotmiddot Eq 91

mVn

E p690

2 =∆ middotmiddotmiddotmiddotmiddot Eq 92

Where Γ isiInitial concentration of the absorbed species

However such an ideal voltammogram is seldom observed because the density of redox

species is so high in the surface-confined environment compared to in the solution and a

197

voltammogram is also influenced by the interaction among those packed redox species

A multi-electron system of the surface-confined groups is also not this case [21]

566 Hydrodynamic voltammetry (HDV) and Levich equation

Hydrodynamic methods purposely introduce convective flow to the system

using RDEs This allows the study of precise kinetics because the diffusion is under

precise control Unknown influence by mass transfer and double layer capacitance on

the electrode reaction is eliminated and steady state is quickly achieved [29 30] In

RDE method laminar flow is applied perpendicular to the electrode surface by spinning

a disc electrode at arbitrary rotation velocity (Fig 88) The rotation rate is expressed in

either rpm (rotation per minute) or angular velocity (ω) ω is defined as ω = 2πf where f

is frequency With disc rotation diffusion becomes a function of electrode rotation rate

as in Eq 93 However the limit of disc rotation rate must be set in order to avoid the

thick hydrodynamic boundary layer and turbulent flows to maintain steady state

Allowable range is between 100 rpm lt f lt 10000 rpm or 10 s-1 lt ω lt 1000 s-1 [30]

198

ω

Fig 88 RDE is rotated at ω and laminar flows applied to the electrode surface

Diffusion layer thickness and disc rotation rate [30]

612131611 νωδ minus= D middotmiddotmiddotmiddotmiddot Eq 93

δ = Diffusion layer thickness cm

D = Analyte diffusion coefficient m2s-1

ω = RDE angular velocity s-1

ν = Solution kinematic viscosity m2s-1

Under such a condition disc limiting current (Id) becomes a function of disc rotation

rate as described in Levich equation (Fig 85) [30] This allows finding the total number

of charge transferred (n)

199

Levich equation [30]

612132620 CnFADI Lminus= νω middotmiddotmiddotmiddotmiddot Eq 94

δ

nFAC= middotmiddotmiddotmiddotmiddot Eq 95

IL = Levich current A

n = Total number of charge transferred

F = Faraday constant Cmol-1

A = Electrode surface area m2

C = Bulk analyte concentration molm-3

( Please refer to Eq 93 for other terms and symbols)

Due to the steady state condition the current response in HDV becomes sigmoidal [223]

as seen in Fig 89

200

Fig 89 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH

74 Based on the real data

567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the

charge transfer rate constant

Levich equation (Eq 94) shows that Id is proportional to ωfrac12 therefore also a

function of diffusion layer thickness (Eq 95) This is true for reversible kinetics

because the electrode reaction is limited by diffusion which is a function of ωfrac12 and

therefore Id also becomes dependent on ωfrac12 at any potential However in

quasi-reversible system Id depends on diffusion but also affected by its kinetics [223]

Therefore when Id reaches the kinetic limitation for charge transfer the slope gradually

deviates from its Levich current as seen in Fig 90 [30]

201

Fig 90 Levich plots for reversible (blue) and quasi-reversible (red) systems [30]

In addition its charge transfer kinetics is altered by the potential applied As Tafel plot

(Fig 9 on p 57) presents the current is exponentially dependent on the overpotential

The electrode reaction is kinetically limited at low overpotential then gradually

becomes diffusion-limited as overpotential increases [223] This implies that Id in

quasi-reversible system is always under the influence of potential applied because the

potential alters its kinetics

When Id becomes completely free from the mass transport influence such current is

called kinetic current (IK) because Id now purely depends on only its kinetics Arbitrary

Id then defined as the sum of diffusion-limited IL and kinetically-limited IK The

double-reciprocal equation of this summation is defined as Koutecky-Levich equation

as in Eq 96

202

Koutecky-Levich equation [30]

LKd III111

+=

216132

11620

11ων

times⎟⎟⎠

⎞⎜⎜⎝

⎛+= minus nCFADI K

middotmiddotmiddotmiddotmiddot Eq 96

Id = Disc limiting current A

IK = Kinetic current A

IL = Levich current A

( Please refer to Eq 93 and Eq 94 for other terms and symbols)

By extrapolating the Koutecky-Levich plot IK can be easily estimated [30] from the

reciprocal of its y-intersect In order to plot Koutecky-Levich equation and find IK a

sequence of arbitrary Id is recorded with altering disc rotation velocity at arbitrary

potential This technique is called hydrodynamic amperometry (HDA) and it is shown

in Fig 91

203

Fig 91 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in

oxygenated 001 M PBS at pH 74 at pretreated GCE Based on the real data

Based on the data obtained by HDA Koutecky-Levich plot is plotted as in Fig 92

204

0E+00

5E-02

1E-01

2E-01

2E-01

3E-01

-002 000 002 004 006 008 010 012 014 016 018

ωˉfrac12 (sˉ

frac12)

1 IK (A-1)

- 04 V

- 06 V- 08 V

05 x 10-1

10 x 10-1

15 x 10-1

0

20 x 10-1

25 x 10-1

-1

I d(A

-1)

Fig 92 Koutecky-Levich plot for O2 reduction at pretreated GCE in oxygenated 001 M

PBS at pH 74 with applied potential ndash 04 06 and 08 V Based on the real data

With Ik from Koutecky-Levich plot and nα from CV the rate constant for arbitrary

potential (k(E)) can be calculated using Eq 97

The rate constant for arbitrary potential E [30]

lowast=nFAC

Ik K

E )( middotmiddotmiddotmiddotmiddot Eq 97

k(E) = Rate constant for the applied potential E ms-1

( Please refer to Eq 94 Eq 95 and Eq 96 for other terms and symbols)

205

568 Finding the standard rate constant (kdeg)

Finding the standard rate constant (kdeg) for ORR of each catalyst is the ultimate

aim of this long tedious cathode analysis because kdeg tells the pure catalytic power

independent of reactant diffusion and overpotential (η) k(E) can be expressed in terms of

kdeg and η as in Eq 98 therefore it is possible to estimate kdeg with known η and k(E)

k(E) in terms of kdeg and η for a forward reaction [30 181]

⎥⎦⎤

⎢⎣⎡ minusminus

deg= )(exp 0)( EE

RTnFkk Ef

α

⎥⎦⎤

⎢⎣⎡ minus

deg= ηαRT

nFk )1(exp middotmiddotmiddotmiddotmiddot Eq 98

kf(E) = Rate constant for a reduction reaction at the applied potential E ms-1

kdeg = Standard rate constant ms-1

η = Overpotential V

( Please refer to Table 11 and Table 12 for other terms and symbols)

η can be found by subtracting Edegrsquo of the reaction involved from the applied potential in

HDA n obtained by HDV is used to specify the reaction involved hence its Edegrsquo In

order to estimate kdeg Eq 98 is first converted into logarithmic expression as in Eq 99

kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

ηαRT

nFkk Efminus

minusdeg= loglog )( middotmiddotmiddotmiddotmiddot Eq 99

206

Log(k(E)) is plotted against a range of η and by extrapolating the plot log(kdeg) is obtained

from its y-intersect where η = 0 hence kdeg can be calculated (Fig 93) [181]

-12

-10

-8

-6

-4

-2

0

-10 -08 -06 -04 -02 00 02 04

η V

log kf(E)

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS NaOH PBS NaOH H2SO4 PBS

NaOH H2SO4

NaOH H2SO4 PBSH2SO4 PBS

H2SO4

NaOH PBS

log kdeg

η V

Fig 93 Log kf(E) plot for O2 reduction at various pretreated GCEs Based on the real data

569 Summary of the electrochemical techniques

CV and hydrodynamic methods are complementary techniques for qualitative

examination of redox reactions and extraction of thermodynamic and kinetic parameters

The protocol for calculating kdeg from hydrodynamic methods and nα from CV is shown

in Fig 94

207

Fig 94 The protocol for finding kinetic and thermodynamic parameters

57 SUMMARY OF THE CHAPTER

The chemistry and electrochemistry of O2 has been reviewed The mechanisms of most

widely investigated catalysts noble metals macrocyclic complexes and enzymes have

been reviewed and their suitability for application in biofuel cells has been discussed

The key physico-chemical parameters which characterise these reactions have been

identified and methods for their determination have been described

208

6 CATHODE MATERIAL AND METHOD

61 ELECTRODE FABRICATION

611 Graphite PtC and RhC electrodes

Microcrystalline graphite of grade (2-15 micron 999995 purity) was

purchased from Alfa Aesar GmbH amp CoKG 5 wt-loaded rhodium on activated

charcoal (RhC) and platinum on activated carbon (PtC) were purchased from Sigma

Aldrich CY1301 and Aradur hardener HY 1300 were purchased from Robnor resins

and they were mixed at 31 (vv) for use RDE templates were specially designed and

made on site They were made of Teflon with the dimension 4-mm ID 20-mm OD

and 15-mm height The depth of composite-holding cavity for each RDE was 25 mm

The composite was made by mixing graphite RhC or PtC with epoxy resin at 73 by

hand Composite mixture was packed into five RDE templates and cured at room

temperature for 72 hours [106]

612 Anthraquinone(AQ)-carbon composite electrodes

6121 Derivatisation of graphite with AQ

Fast red AL salt with 25 dye content and hypophosphorous acid at 50 wt

in H2O were purchased from Sigma Aldrich HPLC grade acetonitrile was purchased

from VWR international 5 mM of Fast Red AL salt solution was prepared in 50 mM

209

hypophosphorous acid 10 ml of the Fast Red solution was mixed with 2 g of graphite

The mixture was left at 5 degC for 30 minutes with regular stirring at every 10 minutes It

was finally washed thoroughly with deionised water followed by vacuum suction in

acetonitrile flow The washed derivatised AQ-carbon was left to dry overnight It was

kept in the sealed container for future use [44]

6122 Fabrication of AQ-carbon composite electrode

AQ-graphite was mixed with epoxy resin at 64 by hand The composite was

packed into five RDE templates and cured at room temperature for 72 hours

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes

6131 Information on synthesis of metallosalophen

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminocobalt (II)] and

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminoiron (II)] denoted as Co(salophen)

and Fe(salophen) respectively were kindly donated by Dr Peter Cragg (School of

Pharmacy Brighton University) They were synthesised by dissolving metal acetate in

the ligand solution which was made by condensation of 21 molar ratio of

salicylaldehyde and diethylenediamine in ethanol or methanol [224 225]

210

6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes

Analytical grade dichloromethane was purchased from VWR international In

the presence of small quantity of dichloromethane 5 wt of Co(salophen) or

Fe(salophen) was mixed and homogenised with graphite [181] After the

dichloromethane evaporated the metallosalophen-graphite mixture was further mixed

with epoxy resin at 64 [219] by hand The composite was packed into five RDE

templates and cured at room temperature for 72 hours

62 COMPOSITE RDE CUTTING

The electrode discs of all the RDE composite electrodes were cut into 1

mm-thick sections using a Buehler IsoMet Low diamond saw with a 04 mm-thick

diamond blade

63 ELECTRODE POLISHING

631 Composite RDE polishing

Composite RDEs were polished successively with SiC grinding paper of 400

1200 and 2400 grit followed by with alumina oxide slurry of particle size 1 03 and

005 microm as in sect 33 on p 122 Electrode polishing was done just before the experiment

in order to avoid air oxidation [195]

211

Fig 95 Fabricated composite RDEs

632 Glassy carbon electrode polishing

Glassy carbon electrodes (GCE) (5-mm disc diameter) were purchased from

Pine Instruments Activated carbon (AC) was purchased from Chemviron Carbon GC

cleaning solvent mixture was made by mixing AC and HPLC grade acetonitrile in a

ratio of 13 (vv) The mixture was covered and left at least for 30 minutes then filtered

before use [226] GCEs were successively polished with alumina oxide powder slurry of

1- 03- and 005-microm particles on MicroCloth pad Polished GCEs were sonicated in

filtered ACacetonitrile mixture for 10 ndash 15 minutes to remove alumina powder debris

and contaminants [226] The GCEs were then sonicated in deoxygenated deionised

water Air contact of the electrode surface was strictly avoided [195]

212

64 ENZYME RDEs

641 Membrane preparation

Cellulose acetate dialysis tubing visking 3632 was purchased from Medicell

International Ltd Sodium carbonate (Na2CO3) was obtained from Sigma Aldrich

Dialysis membrane was cut into 10x10-mm2 pieces which were boiled in 1 wv

aqueous Na2CO3 solution for 10 minutes The membrane pieces were rinsed thoroughly

with deionised water and stored [61] in PBS in a sealed container at 4degC until its use

642 Membrane enzyme electrodes

Laccase (Lac) from Trametes versicolor gt20 unitsmg-1 and lyophilised

bilirubin oxidase (BOD) from Myrothecium verrucaria 15 ndash 65 units mg-1 were

purchased from Sigma Aldrich 4 mgml-1 of Lac and BOD solution was made with PBS

respectively BOD immediately dissolved in PBS while a vortex mixer was needed to

dissolve Lac In order to attach enzyme on the GCE surface the tip of polished cleaned

GCEs was dipped and left in the enzyme solution for two hours with gentle stirring The

electrodes were then lightly washed with deionised water Pretreated dialysis membrane

was carefully placed onto the surface of the enzyme-modified GCE and fixed with

rubber O-ring

213

65 ELECTROCHEMICAL EXPERIMENTS

651 Electrochemical system

CHI 650A with single-channel and CHI 1030A with multi-channel

potentiostats were used for electrode pretreatment and electrochemical experiments CV

HDV and HAD An adjustable rotor from Pine Instrument was used to rotate a RDE

Three-electrode system (sect 342 on p 123) was employed with an AgCl reference and a

Pt counter electrodes

Fig 96 RDE experiment setting with a rotor and gas inlet

214

652 Electrode pretreatments

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes

Graphite GC Co(salophen) and Fe(salophen) electrodes were pretreated in

seven different ways in Table 14 Untreated electrodes were also prepared as controls

All the pretreatments were made in series

Treatment type 1st pretreatment 2nd pretreatment 3rd pretreatment

Type I Untreated -

Type II Acidic 01 M H2SO4

Type III Basic 01 M NaOH

Type IV Neutral 001 M PBS

Type V Base followed by Acid 01 M NaOH 01 M H2SO4

Type VI Acid followed by Neutral 01 M H2SO4 001 M PBS

Type VII Base followed by Neutral 01 M NaOH 001 M PBS

Type VIII Base Acid then Neutral 01 M H2SO4 01 M NaOH 001 M PBS

Table 14 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Concentrated sulphuric acid at 90 - 98 and sodium hydroxide tablets were purchased

from VWR international 001 M PBS was purchased from Sigma Aldrich All the

pretreatments were based on potential cycling in 100-ml electrochemical cell except

untreated ones Acidic pretreatment was performed at 5 Vs-1 between 15 V and ndash 05 V

215

in 01 M H2SO4 [105 219] for 30 minutes Basic pretreatment was performed at 5 Vs-1

between 06 V and 2 V in 01 M NaOH [218 219] for 30 minutes Neutral pretreatment

was done at 5 Vs-1 between 15 V and ndash 05 V in deoxygenated 001 M PBS pH 74

[219 220] Constant N2 flow was applied to PBS through the pretreatment In addition

pretreatments were performed in various combinations as in Table 14 Type V Base

followed by Acid Type VI Acid followed by Neutral Type VII Base followed by

Neutral and Type VIII Base followed by Acid then Neutral [219] The influence of

pretreatments on electrode surface and ORR was studied by CV HDV and HDA

6522 Pretreatments of AQ-carbon PtC and RhC

AQ-carbon PtC and RhC composite electrodes were pretreated only in 01 M

H2SO4 at 5 Vs-1 between 15 V and ndash 05 V in for 30 minutes Untreated electrodes were

also prepared as controls

653 Electrochemical experiments

All experiments were carried out under both N2 and O2 saturation 100 ml of

001 M PBS electrolyte solution was sparged with either N2 or O2 for 15 minutes before

the experiment Gas was continuously supplied through a Dreschel gas washing bottle

containing PBS throughout the experiment

CV was performed at 002 005 01 02 and 05 Vs-1 between + 09 V and ndash

09 V HDV was performed at fixed scan rate of 005 Vs-1 sweeping the potential from

+ 09 V to ndash 09 V with disc rotation velocity altered through 900 1600 and 2116 rpm

216

HDA was performed at a range of fixed potentials - 02 - 04 - 06 and ndash 08 V at disc

rotation velocity of 400 625 900 1156 1225 1600 2116 and 2500 rpm All

experiments were repeated twice or three times The sample size ranges from one to

three The median values were taken for the analysis Obtaining a set of data for each

electrode sample took from three weeks to more than one month

654 Data analysis

CV data was used to calculate nα and reaction reversibility HDV data was used

to calculate n using Levich equation (Eq 30 on p 61) Edegrsquo for ORR involved at pH 7

25 degC was specified by n value as in Table 15

n Redox type Redox potential vs AgAgCl

1 Edegrsquo(O2H2O2) 0082 V

2 Edegrsquo(O2H2O) 0616 V

Table 15 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC [24]

η was calculated by subtracting Edegrsquo (ORR) from the potential applied in HDA HDA

data was used to estimate kf(E) at each applied potential using Koutecky-Levich plot (Eq

30 on p 61 and Fig 92 on p 204) N2 data was subtracted from O2 data as background

Other parameters required for Koutecky-Levich plot were set as in Table 16 All

parameters assume the condition of 001 M PBS at 24 degC

217

Parameter Value Reference

O2 diffusion coefficient 23 x 10-9 m2s-1 [227]

O2 concentration 123 molm-3 [158]

Kinematic viscosity (ν) 884 x 10-7 m2s-1 [30]

Table 16 Parameter values for Koutecky-Levich equation

kdeg was estimated by plotting log(kf(E)) vs η (Eq 99 and Fig 93 on p 206) kdeg for

various and pretreated cathodes were compared The best catalyst shows the largest kdeg

The procedure for finding kdeg is summarised in (Fig 94 on p 207)

218

7 CATHODE RESULTS AND DISCUSSION

71 AIMS OF THE CATHODE STUDIES

This section describes the electrochemical characterisation of various modified

electrodes which were considered as candidate cathodes for the biofuel cells Various

carbons were used as electrode materials in all cases The electrochemical behaviour of

carbon depends strongly on history and also can be radically altered by pre-treatment

The effects of electrochemical pretreatments (ECPs) on the kinetic parameters will be

described for both glassy carbon and graphite composites The cathode modifiers

considered in this section include noble metals (Pt Rh) inorganic Schiff bases

(Co(salophen) Fe(salophen)) an organic modifier (anthroquinone) and the enzymes

bilirubin oxidase (BOD) and laccase (Lac) In each case of non-biological catalysts the

effects of ECPs were also quantified using cyclic voltammetry (CV) and hydrodynamic

methods with rotating disc electrodes (RDEs)

CV allowed the investigation of

Capacitance from the double layer region

Qualitative description of the mechanism such as number of discrete reaction

steps reversibility and surface confinement

nα the number of electrons transferred up to and including the rate limiting step

(RLS)

219

while hydrodynamic methods were used to quantify

n the total number of electrons transferred

kdeg the heterogeneous standard rate constant

Epc2(HDV) the half-wave reduction potential in HDV

The data was taken under both N2 and O2 to allow characterisation of the effects of

electrochemical pretreatment and its effects on the kinetics of O2 reduction The best

candidate catalytic cathode was determined according to kdeg nα and Epc2(HDV)

72 INFLUENCE AND EFFICACY OF ECPS

721 Glassy carbon electrodes (GCEs)

7211 Untreated GCEs

Voltammetry curves of untreated GCEs at pH 74 in the presence of N2 and O2

are shown in Fig 97

220

Fig 97 CVs of untreated GCEs at pH 74 in 001 M PBS in the presence of (i) N2 (ii)

O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Untreated GCEs did not show any peak in the absence of O2 (Fig 97(i)) indicating that

there was no electrochemically active redox species on the electrode surface and in the

solution CV was in the presence of O2 (Fig 97(ii)) showed a peak at ndash 08 V but

without a reverse anodic current This indicates the presence of catalytic activity

however it occurred at only high overpotential and is therefore not efficient for ORR

This result is consistent with previous work [215 216]

7212 Influence of electrochemical pretreatments on GCE

GCEs were pretreated in various media in order to improve ORR efficiency It

has been reported that ECPs activate the electrode surface by introducing

electrochemically active functional groups and that this improves electrochemical

(i) N2

-3E-05

-2E-05

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1E-05

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

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cm

-2

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

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1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

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cm

-2

221

reversibility and [105 218] and the electron transfer rate of inner-sphere catalysis [228]

Fig 82 shows the voltammetry curves in the absence of O2 for various pretreated GCEs

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-60E-05

-50E-05

-40E-05

-30E-05

-20E-05

-10E-05

00E+00

10E-05

20E-05

30E-05

40E-05

50E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH

Fig 98 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of

O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1 Arrows indicate the

peak position (i) includes CVs for all the pretreated GCEs and (ii) excludes

PBS pretreatments for convenience

222

As the figure shows any pretreatment resulted in an increase in capacitance and

introduced characteristic voltammetric peaks which were not observed in the untreated

GCEs In all cases the peak current was linearly proportional to υ indicating that the

pretreatments led to surface-confined redox active functional groups most likely to be

quinone groups That ECPs give rise to surface quinones is well established [105 218

220 229 230] The Epc shifted with υ and was separated from Epa hence redox

reactions by these surface quinone groups are quasi-reversible The effect of

pretreatments however varied by the type of medium used in pretreatments

Above all PBS-related pretreatments gave qualitatively different results which

were characterised by a great increase in average capacitance calculated from CVs in

001 M PBS at pH 74 at 50 100 and 200 mVs-1 in the double layer region of the CV at

04 V Summary results are shown in Fig 82(i) and Fig 99

223

GCE Capacitance vs Pretreatment

0100200300400500600700800900

1000

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS

NaOH PBS

NaOH H2SO4

PBS

Pretreatment

Capa

cita

nce

microF

cm-2

Fig 99 The capacitance of GCEs in 015 M NaCl buffered with 001 M phosphate to

pH 74 Error bars represent the standard deviation (n = 3)

The average capacitance of the untreated GCEs was 76 microFcm-1 and is within the typical

ranges reported hitherto [221] while PBS pretreatment increased the capacitance to 700

- 800 microFcm-1 A similar phenomenon was reported by Nagaoka and by Swain [215

216] There are two possible reasons for this (i) PBS treatment caused the

microfractures of GC and increased the electrode area and therefore double layer [105

215 216 218] andor (ii) attached the electrochemically inactive charged surface group

such as phenol and carboxylate [216 229]

In the pretreatment involving NaOH the carbon dissolution was observed This

is harsher pretreatment so erodes the electrode and makes the electrode surface more

porous [216 218] NaOH also forms unstable oxide film and eventually removes

graphite oxide [229] and surface impurities [218] However the resultant capacitance

224

was unexpectedly smaller than that of sole PBS pretreatment Therefore NaOH

pretreatment increased the capacitance by increasing the exposed electrode area [218

231] while PBS pretreatment increased capacitance by attaching redox inactive charged

surface groups The largest capacitance after the double pretreatment involving NaOH

and PBS was therefore derived from the layer of charged groups attached on the

electrode surface area made larger by NaOH pretreatment

H2SO4 pretreatment on the other hand resulted in a smallest capacitance

increase The electrode behaviour was also slightly altered from that of other

PBS-related pretreatments when the pretreatments involved H2SO4 together The

capacitance became smaller as shown in Fig 99 and the peak positions were shifted to

more positive potential side (Fig 82(i)) To investigate the causes of this phenomena

the voltammograms during such pretreatments were studied as Fig 100 below

225

Fig 100 Overlaid CVs during various pretreatments for GCEs involving 01 M H2SO4

alone (red) 001 M PBS at pH 74 alone (green) and both pretreatments (pink)

It is clearly shown that either pretreatment resulted in distinctive peaks for the

attachment of active surface quinone groups However the peak potentials were

different when in sole H2SO4 or PBS-related pretreatments This is because as proposed

by Nagaoka those quinone groups were present in the different chemical environment

leading to different behaviours [216] H2SO4 pretreatment attached fewer inactive

surface oxygen groups while PBS pretreatment attached more Therefore H2SO4

pretreatment led to the smaller capacitance change than PBS pretreatment This implies

the presence of different electrode surface conditions hence quinones present in such

different environment behave differently showing different peak positions The

behaviours of surface quinone groups depends on the conditions of the pretreatment

involved [229 230 232] This fact can also explain the phenomena caused by the

226

double pretreatment with H2SO4 and PBS as shown in Fig 82(i) More quinone groups

had been attached by H2SO4 pretreatment in advance therefore only smaller area than

usual became available for PBS pretreatment to stably attach their charged oxygen

surface groups This is the most likely causes of the reduced capacitance and different

peak positions in such a double pretreatment compared to other PBS-related

pretreatments Furthermore quinone peak positions should be close to those found for

the H2SO4 pretreatment alone because quinone groups attached by preceding H2SO4

pretreatment dominate the electrode surface and should be have more influence on the

electrochemistry However they also resulted in larger capacitance because the

subsequent PBS pretreatment attached inactive oxygen surface group which led to an

increase in fixed charge density This is a typical phenomenon of synergetic effects as

already seen in the double pretreatment with NaOH and PBS

On the other hand voltammetric curves for the double pretreatment of NaOH

and H2SO4 did resemble to those by sole NaOH pretreatment Similar phenomena were

observed between the double pretreatment with NaOH and PBS and the triple

pretreatment with NaOH H2SO4 and PBS It seems that preceding NaOH pretreatment

overwhelmed the subsequent H2SO4 pretreatment although some influence by H2SO4

can be observed in terms of capacitance as shown in Fig 99 This implies that the

preceding NaOH pretreatment made most of the electrode area not available for

subsequent H2SO4 pretreatment However if NaOH pretreatment creates micropores in

the GC protons from subsequent H2SO4 pretreatment can still penetrate such

micropores These micropores are accessible for protons but not most ions [216]

227

7213 Effect of GCE pretreatment on ORR

Voltammograms for ORR at various pretreated GCEs at pH 74 are shown in

Fig 101 below

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO4

01M NaOH01M NaOH + 01M H2SO4

001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS

01M NaOH + 01 M H2SO4 + 001M PBS

Fig 101 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

In the figure any pretreated GCE shows larger cathode peaks between ndash 03 and ndash 05 V

without reverse anodic current They were due to O2 reduction by surface quinone

groups whose peaks were observed at ndash 01 V in Fig 82(i) in the absence of O2 This

indicates that ORR at any pretreated GCEs is regulated by ECrsquo mechanism [30] Ipc was

linearly proportional to radicυ in all cases indicating the diffusion-limited charge transfer

228

However untreated GCE was catalytic only at high overpotential since Epc2(HDV) = ndash 08

V This is compatible to the observation made by Tammeveski [233] It was seldom

catalytic since n = 102 plusmn 011 and nα = 059 plusmn 004 This single-electron ORR implies

the presence of O2־ disproportionation from which little power can be extracted [233]

kdeg was could not be calculated because the standard Koutecky-Levich plot could not be

obtained since it did not show a normal linear slope In contrrast all the other

pretreatments improved ORR as seen in Fig 101 They showed large cathodic peaks at

smaller overpotentials PBS-related pretreatments in particular increased the catalytic

current by gt 15 times and positively shifted Epc2(HDV) by gt 300 mV relative to that of

untreated GCEs Similar phenomena were reported elsewhere [220 234] All the GCE

pretreatments resulted in an increase in both nα and n The increase in kdeg accompanied

by an increase in nα was also observed as a general tendency as shown in Fig 102

229

0

2

4

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

kordm

ms

-1

nα 059 107 094 106 168 278 174 178

kordm ms-1 000E+00 715E-09 529E-09 631E-08 131E-07 279E-07 654E-08 113E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

- nα

kdeg ms-1

Untreated H2SO4 NaOHNaOHH2SO4

H2SO4PBS

NaOHPBS

NaOHH2SO4PBS

Fig 102 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Above all the H2SO4-PBS double pretreatment was most effective giving kdeg = 279 x

10-7 plusmn 507 x 10-9 ms-1 and Epc2(HDV) = - 039 plusmn 002 V However its nα was calculated

to be 278 while n = 185 This is an unusual result It might imply the presence of serial

ORR pathway in which H2O2 is further reduced or it is simply impossible to obtain

correct parameters by using Eq 88 on p 193 for ORR at pretreated GCEs due to their

excessively narrow peak width Pretreatment of GCE was proved to increase kdeg of ORR

but how this improves its catalysis has been uncertain for a long time between two

hypotheses whether they mediate electron transfer or they alter the adsorption of O2

and O2[233 232 220] ־ The latter or both is more likely because as Fig 102 shows

PBS-related pretreatments increased the capacitance which was derived from redox

inactive surface oxygen groups yet made significant contribution to an increase in kdeg It

230

is therefore natural to deduce that the largest kdeg and nα were the consequence of

synergetic effect derived from different surface oxygen groups attached by H2SO4 and

PBS

722 Graphite composite electrodes

7221 Untreated graphite composite electrodes

Graphite composite electrodes are different from GCEs They are made of

graphite particles which are more porous and permeable to the solution and gas This

practically results in larger electrode surface area therefore becomes more subject to

facile adsorption and surface modification This leads to the different surface conditions

and electrode behaviours from GCEs as proposed by Sljukic [232] CVs of untreated

graphite composite electrodes at pH 74 in the presence of N2 and O2 are shown in Fig

103 together with those of untreated GCEs

231

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

Fig 103 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl Small peaks on graphite electrodes are indicated by arrows

Different electrode behaviour is noticeable especially in nitrogenated solution because

untreated graphite composite electrodes showed small peaks at + 100 mV and - 100 mV

as indicated by arrows No peak was present fo untreated GCE This indicates the

presence of some reactive surface quinone groups on graphite composite without

pretreatment This is because graphite composite electrodes have more reactive edge

plane graphite exposed and the porous electrode structure allows larger electrode area to

be exposed There was however little difference in averaged capacitance between

graphite composite and GCEs This indicates that an increase in capacitance does not

necessarily correlate to an increase in the surface area as proposed by Regisser [235]

Voltsmmograms for ORR at untreated GC and graphite composite electrodes resembles

each other though for the latter the reduction peak is around 50 mV more positive It can

232

be seen that graphite composites are slightly more catalytic toward ORR presumably

because of the intrinsic quinone groups derived from the graphite electrode surface

7222 Influence of electrochemical pretreatments on graphite composite electrodes

Cvs for the graphite composite electrodes differed from those of GCEs Peaks

were observed in all graphite cases including untreated ones as shown in Fig 104

233

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

(ii)

01M H2SO401M NaOH01M NaOH + 01M H2SO4Untreated

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBSPBS

(iii)01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS001M PBS

Fig 104 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M

nitrogen-sparged PBS between + 09 and - 09 V vs AgAgCl (i) All

pretreatments at 005 Vs-1 (ii) Non-PBS-related pretreatment and (iii)

PBS-related-pretreatment CVs at 002 Vs-1

The capacitance became larger after pretreatments Any multi-pretreatment involving

NaOH and H2SO4 resulted in the largest capacitance which was 20 times larger than

234

that of untreated graphite composite electrodes The capacitance increase was also

larger than that of any pretreated GCEs except for PBS-pretreated ones PBS

pretreatment led to the smallest capacitance increase in graphite composite electrodes in

contrast to the GCEs Instead H2SO4 pretreatment led to the largest capacitance

increase Moreover this outcome was found to correspond to a specific proportional

relation between Ip and υ Ip of electrodes involving any pretreatment in PBS NaOH or

their combinstions were linearly proportional to radicυ This can only arise from on or more

diffusion limited step or steps in the redox reactions of the surface groups most likely

proton diffusion though a porus film or to microscopic porous environments Surface

oxygen groups have previously been reported to involve involve proton uptake and this

leads to a broad peak [216 230] H2SO4-pretreated electrodes showed Ip proportional to

υ and the redox reaction is independent of proton diffusion

These phenomena show that different pretreatments provide different chemical

environment for active quinone groups leading to different quinone behaviours and

voltammetry peaks [229 230 232] In the multi-pretreatment with NaOH and H2SO4

synergetic effects became obvious Its redox reaction was diffusion-limited and showed

voltammograms with two partially resolved sets of peaks yet its peak still partially

overlaps with that of diffusion-independent H2SO4-pretreated electrodes as seen in Fig

104(i) When this double-pretreatment was further followed by PBS pretreatment the

peak derived from quinones attached by H2SO4 disappeared This implies that

subsequent PBS pretreatment suppressed the activity of proton-independent quinones

and the effect by H2SO4 The similar phenomenon was observed when electrodes were

pretreated doubly by H2SO4 and PBS (Fig 104(ii)) Every case presented

235

quasi-reversible redox systems because peak potentials shifted with scan rates Small

multiple peaks were present in slower scan rates merged into one broad peak at faster

scan rate as the contribution of capacitance charging and increased iR drop obscured

voltammetic detail

7223 The effect of graphite composite electrode pretreatment on ORR

ORR at pretreated graphite composite electrodes was found to be less effective

than for pretreated GCEs Only NaOH-related pretreatments were effective (Fig 105)

enough to achieve two-electron ORR Untreated graphite composite electrodes were

catalytic only at high overpotential despite the presence of surface quinone groups PBS

pretreatment was hardly effective in contrast to the results obtained for GCEs

Electrochemical pre=treatment in H2SO4 and its double pretreatment with PBS reduced

Epc2(HDV) by 40 ndash 90 mV however the electrodes showed poor catalytic performance nα

lt 05 and n lt 2 consistent with the initial electron transfer still being rate determining

236

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 105 Overlaid CVs of ORR at various pretreated graphite composite electrodes

at pH 74 in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005

Vs-1

Pretreatment of the graphite composites with just H2SO4 showed a single merged peak

at slower scan rate while they showed an addition prewave as indicated by arrows in

Fig 106 at faster scan rate

237

-16E-03

-14E-03

-12E-03

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

-1 -08 -06 -04 -02 0 02

E V

J Acm-2

50 mV100 mV200 mV500 mV

Fig 106 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at

pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various

scan rates 50 100 200 and 500 mVs-1

Coupled with the fact of n asymp 1 this prewave is due to the rate-limiting slow formation of

semiquinone radicals [233] A semiquinone radical is formed when one electron is

transferred to a quinone group and its formation is necessary to mediate ORR as shown

in Eq 100

238

middotmiddotmiddotmiddotmiddot Eq 100

[233] middotmiddotmiddotmiddotmiddot Eq 101

Q = Surface quinone group

O2־ = Semiquinone radical

Any NaOH-related pretreatment on the other hand showed a prewave around ndash 035 V

at slower scan rate as shown in Fig 107 but this merged into a broad peak at faster scan

rate

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

01M NaOH01M NaOH + 01M H2SO4 01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 107 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

239

Ipc was linearly proportional to radicυ except for the triple pretreatment which was difficult

to define due to the ambiguous peak positions over different scan rates The prewave

was due to the slow reduction of O2 into O2־ (Eq 101) rather than semiquinone radical

formation (Eq 100) as seen in H2SO4-pretreated electrodes They had n asymp 2 and nα asymp 1

hence they catalysed two-electron ORR Those pretreatments positively shifted

Epc2(HDV) by 173 plusmn 26 mV on average from ndash 730 mV at the untreated graphite

composite electrodes The largest catalytic activity was achieved by doubly pretreated

electrodes with NaOH and H2SO4 with kdeg = 382 x 10-7 plusmn 32 x 10-8 ms-1 (Fig 108)

0

2

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

35E-07

40E-07

45E-07

kordm

ms

-1

nα 042 035 080 114 045 040 101 072

kordm ms-1 000E+00 000E+00 233E-07 382E-07 000E+00 000E+00 132E-07 172E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBSUntreated H2SO4 NaOH

NaOHH2SO4

H2SO4

PBS NaOHPBS

NaOHH2SO4

PBS

- - - - nα

kdeg ms-1

Fig 108 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

240

723 Platinum on carbon composite electrodes (PtC)

Noble metals are popular ORR catalysts [172 173] with Pt showing the highest

catalytic power [165 166 171 172] It was reported that Pt has a 98 O2-to-H2O

conversion rate [174] However for PtC composite electrodes the voltammetry was

dominated by showed solution resistance both in the presence and absence of O2 and

therefore were not either catalytic or conductive H2SO4 pretreatment was not at all

effective This is probably due to the large electronic resistance of activated carbon itself

[204] as well as intrinsic resistance of composite conductors

724 Rhodium on carbon composite electrodes (RhC)

Rhodium is a platinum group metal and has been reported as a good ORR

catalysis after Pt and Ir [172] Voltammograms for untreated RhC composite electrodes

only showed the capacitance but no redox peaks in nitrogenated solution The

capacitance decreased by 80 for every ten-fold increase in the scan rate Non-faradic

current is normally linearly proportional to υ however it exponentially decreased with υ

in the untreated RhC composite electrodes It did not show any peak in the presence of

O2 and was not catalytic toward ORR (Fig 110(i)) H2SO4-pretreated RhC composite

also did not show any voltammetric peaks Its capacitance was roughly x30 larger than

that of untreated electrodes and again exponentially decreased with an increase in υ

ORR catalysis was however present (Fig 110(ii)) therefore it is reasonable to deduce

that H2SO4 attached the surface quinone groups but the large capacitance charging

prevented voltammetic observation of quinones

241

(i) Untreated

-16E-03

-12E-03

-80E-04

-40E-04

00E+00

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

N2O2

(ii) H2SO4

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

10E-03

-1 -08 -06 -04 -02 0 02 04 06 08 1

E VJ

Ac

m-2

N2O2

Fig 109 CVs of (i) untreated and (ii) H2SO4-pretreated RhC composite electrodes at

pH 74 in 001 M PBS in the presence of N2 and O2 at 005 Vs-1 between + 09

and -09 V vs AgAgCl

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR

Untreated RhC composite electrodes did not show ORR catalysis however a

steep decrease in cathodic current was observed without any peak as the potential

decreases This steep current decrease was probably caused by O2 adsorption on the Rh

particle surface H2SO4-treated RhC on the other hand was found to be catalytic with n

= 177 and nα = 068 plusmn 011 A reduction prewave was observed at around ndash 05 V and

this merged into a broad peak at faster scan rates This prewave was due to ORR by

surface quinone groups attached by H2SO4 because it overlaps with the peak observed

in H2SO4-treated graphite composite electrodes as shown in (Fig 110) This prewave

242

therefore indicates the slow first electron transfer to form O2־

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

-1 -05 0 05 1

E V

J Acm-2

RhC H2SO4

Graphite H2SO4

Fig 110 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

O2־ was probably further reduced to H2O2 by Rh particles because n = 177 plusmn 011 The

catalytic activity was much larger compared with H2SO4-pretreated graphite composite

electrodes as shown in Fig 110 with kdeg =257 x 10-7 plusmn 230 x 10-8 ms-1

243

725 Co(salophen) composite electrodes

7251 Untreated Co(salophen) composite electrodes

Untreated Co(salophen) electrode showed couples of distinctive peaks Epc(1) =

053 Epc(2) = -024 Epa(3) = -016 and Epa(4) = 016 V as shown in Fig 111(i) These

peaks correspond to stepwise cobalt redox reactions Co3+2+ and Co2+1+ respectively

The peak potentials hardly changed over different scan rates consistent with previously

published results [181] Similar peaks have been observed in deoxygenated organic

solvents [174 181] as well as for Co(salen) [181]

(i) N2

-2E-05

-1E-05

0E+00

1E-05

2E-05

3E-05

4E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

2E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fig 111 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

244

In the presence of O2 it showed a reduction peak at ndash 04 V as shown in Fig 111(ii)

ORR potential was shifted by approximately 300 mV relative to that of untreated

graphite composite This result reasonably compatible to the result reported by Ortiz

[181] At this potential O2 forms an adduct with Co2+(salophen) and then is reduced to

O2־ while Co2+ undergoes one-electron oxidation to Co3+ [170] To be catalytic Co3+

formed here must be reduced back to Co2+ to reduce O2־ further into H2O2 [181] or a

binuclear complex must be formed with another Co2+ [170] Untreated Co(salophen)

composite electrodes were however not strongly catalytic due to n = 138 plusmn 009 and nα

= 055 plusmn 012 Ip was linearly proportional to radicυ and the peak potentials Epc(2) and Epa(3)

varied only by plusmn 8 over different five scan rates This indicates nearly reversible

multi-electron transfer redox system of Co(salophen) composite electrodes

7252 Influence of electrochemical pretreatments on Co(salophen) composite

There was a particular redox pattern found in voltammetry waves according to

the type of pretreatments (Fig 112)

245

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 112 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in

001 M nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

NaOH-pretreatment showed similar voltammetric faradaic features as those in untreated

electrodes (Fig 112) but increased the capacitance by six times relative to that of

untreated electrodes The size of capacitance was 382 microFcm-1 and approximately equal

to that of NaOH-pretreated graphite composite electrodes This implies that NaOH

attached electrochemically inactive surface oxygen groups and the attachment of active

surface quinone groups were not clearly confirmed

PBS-pretreatment on the other hand attached quinone groups because Epa asymp 04 V at

which similar peaks were observed in PBS-pretreated graphite composite (Fig 104(iii)

p 233) Their cathodic peaks were however broader because they merged into peaks

246

for Co-redox processes The effect of PBS pretreatment was much smaller compared to

that of H2SO4-related pretreatment in the deoxygenated solution Single NaOH

treatment and any PBS-related pretreatment showed Ip linearly proportionally to radicυ

while any H2SO4-related pretreatment but without involving PBS-pretreatment showed

a proportional relation with υ This indicates that H2SO4 and PBS or NaOH

pretreatments lead to different surface redox mechanism Peaks due to H2SO4-related

pretreatments were prominent and are indicated by a red circle in Fig 112

7253 The effect of Co(salophen) composite electrode pretreatment on ORR

Untreated electrodes were not catalytic Parameters for H2SO4-pretreated

Co(salophen) composite found to have n asymp 1 and nα le 1 although they look catalytic in

Fig 113 reducing its ORR overpotential by 400 ndash 500 mV compared to GCEs This

inconsistent result arose because it was impossible to find the correct parameter using

the conventional calculation using Eq 88 on p 193 and need more sophisticated

method such as a simulation probramme The other pretreatments were on the other

hand found strikingly effective toward ORR as shown in Fig 113

247

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

40E-04

60E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 113 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Such pretreated Co(salophen) electrodes showed Epc asymp - 03 V having reduced the

overpotential against unpretreated Co(salophen) by 100 mV and 300 mV against

untreated graphite composite electrodes ORR was synergistically catalysed by Co2+ and

surface semiquinone radicals [181 233] They showed n asymp 2 and nα gt 07 hence they

catalysed two-electron ORR Ipc was linearly proportional to radicυ hence the redox

reactions were diffusion limited except for the double pretreatment with NaOH and

H2SO4 Large catalytic activity was achieved by sole NaOH and by the double

pretreatment with NaOH and H2SO4 or the combination of H2SO4 and PBS Among

them sole NaOH pretreatment was revealed to be most effective as shown in Fig 114

and showed kdeg = 119 x 10-5 plusmn 118 x 10-6 nα = 083 plusmn 014 and Epc2(HDV) = 037 V

248

0

1

2

nα

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

kordm

ms

-1

nα 055 028 083 074 068 091 071 078

kordm ms-1 000E+00 000E+00 119E-05 747E-06 271E-06 684E-06 296E-06 587E-06

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

Fig 114 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite

electrodes

One possible reason for this largest catalytic activity by NaOH pretreatment is that

NaOH might attach more reductive surface OH groups [218] which became involved in

reducing back Co3+ into Co2+ during ORR [181]

726 Fe(salophen) composite electrodes

7261 Untreated Fe(salophen) composite electrodes

Untreated Fe(salophen) electrodes showed a couple of distinctive peaks Epa = -

024 plusmn 001 V and Epc = - 036 plusmn 001 V These peaks are due to Fe3+2+ redox reaction as

249

shown in Fig 115(i) The voltammetry curve and redox peaks are reasonably similar to

those of Fe(salen) reported [224]

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

(ii) O2

-6E-04

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

Fig 115 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74

in 001 M PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and

- 09 V vs AgAgCl

However the redox system is not perfectly reversible here because ∆Ep slightly

increases with an increase in the scan rate and its Ip was linearly proportional to radicυ It is

therefore a quasi-reversible system Untreated Fe(salophen) electrodes was not as

catalytic as Co(salophen) electrodes Epc was ndash 079 plusmn 007 V and little different from

that of untreated graphite composite electrode as shown in Fig 115(ii) although Ipc was

slightly bigger The low ORR catalytic activity of Fe(salophen) was due to the low ORR

rate of Fe itself (Fig 65 on p 164) [172]

250

7262 Influence of electrochemical pretreatments on Fe(salophen) composite

The effect of pretreatments to Fe(salophen) was striking They resulted in

additional voltammetric features due to the Fe redox peaks at Epc (Fe) = - 03 V and Epa

(Fe) = - 02 V and by attaching the surface quinone groups at Epc (Q) = 015 V and Epa (Q) =

02 V as shown in Fig 116(i)

251

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

60E-05

80E-05

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

Untreated01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS001M PBS

-1E-04

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

1E-04

1E-04

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

Fig 116 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1 (i) All

pretreatments (ii) Non-PBS-related pretreatments and (iii)

PBS-related-pretreatments CVs

Non-PBS-related pretreatments (Fig 116(ii)) showed redox peaks clearly however the

252

two cathodic peaks merged into a broad peak at faster scan rate than 100 mVs-1

indicating quasi-reversible surface quinone redox reactions Only a broad merged

reduction peak was observed for all PBS-related pretreatments (Fig 116(iii)) at all scan

rates

Any PBS-related pretreatments resulted led to a broad cathodic peak (from + 01 V to

approximately ndash 03 V) made up of both Fesalphen reduction and reduction of surface

confined quinones arising from the electrochemical pretreatment PBS pretreatment

always greatly increases the surface area leading to micro fissures and consequently a

greater variety of microenvironments for electrochemically generated quinone

functionality

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR

Most were found to be only weakly catalytic (Fig 117) since either n or nα or

both of them were lt 1 Only double-pretreatment with NaOH and H2SO4 led to

sufficient ORR catalysis with n = 185 plusmn 017 nα = 079 plusmn 023 kdeg = 112 x 10-7 plusmn 771 x

10-9 ms-1 and Epc2(HDV) = - 057 V The double pretreatment showed a prewave at ndash 035

V and it disappeared at faster scan rate indicating a slow single-electron transfer to O2

Ipc was linearly proportional to radicυ hence the catalysis was diffusion limited

253

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS

Fig 117 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

727 Anthraquinone (AQ) composite electrodes

Anthraquinone-grafted graphite electrodes were pretreated with H2SO4 Both

untreated and H2SO4-pretreated electrodes showed couples of peaks (Fig 118(i)) one of

which Epc(AQ) was compatible with Em = - 049 reported by McCreery [44] These

peaks were therefore derived from anthraquinone chemically attached to the graphite

254

(i) N2

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

-1 -05 0 05 1

E V

J A

cm

-2

Untreated FRE01M H2SO4 FREUntreated Graphite

(ii) O2

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

-1 -05 0 05 1

E VJ

Ac

m-2

Untreated FRE01M H2SO4 FREUntreated Graphite

Fig 118 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl CVs of untreated graphite electrodes are also shown together

The other couple of peaks Epc(SQ) and Epa(SQ) was observed at - 003 plusmn 003 V and +

020 plusmn 003 V respectively They were due to quinone groups on the carbon surface

H2SO4-pretreatment did not alter the voltammogram pattern but only increased the

capacitance by x3 and enhanced the peak current Both electrodes showed Ip linearly

proportional to radicυ hence the redox reactions involve proton diffusion ORR by those

electrodes is shown in Fig 118(ii) together with the voltammogram of graphite

composite electrodes Both untreated and pretreated AQ-grafted composite electrodes

positively shifted Epc by 60 and 230 mV respectively relative to that of untreated

graphite composite electrodes However their voltammetric curves resembled those for

untreated graphite composite electrodes Besides this their Ip dropped by 20 relative

255

to untreated graphite composite electrodes and showed n asymp 1 and nα asymp 05 Therefore

either was not catalytic in this study This outcome does not correspond to the finding

by Manisankar who confirmed the presence of ORR catalysis at AQ-carbon paste

electrodes at pH 7 with a reduction prewave at ndash 04 and a peak at ndash 07 V [193] To the

contrary this result fits to the data presented by Schiffrin who observed ORR

suppression at AQGC electrodes at pH lt 7 He reported that voltammetry curves for

ORR at GC and GCAQ became similar at this pH hence implied the possibility of

disproportionation of semiquinone intermediates [194]

728 Lac and BOD enzyme membrane electrodes

In the absence of O2 neither Lac nor BOD showed any peaks in their cyclic

voltammograms However they showed ORR catalytic activity even in the absence of

mediator (Fig 119) DET is therefore possible as already confirmed by many [183

186-188] Carbon surface groups [195] also might mediate electron transfer [188]

256

-14E-04

-12E-04

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

-1 -09 -08 -07 -06 -05 -04 -03 -02 -01 0

E V

J Acm -2

LacBOD

Fig 119 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated

PBS at 005 Vs-1 between 0 and -09 V vs AgAgCl

Ipc was linearly proportional to radicυ therefore ORR was diffusion limited Epc were -054

plusmn 002 and ndash 067 plusmn 006 V at Lac and BOD respectively They reduced ORR

overpotential by 120 and 250 mV relative to that of GCEs however the Epc shifted with

υ hence they were quasi-reversible It is known that MCOs catalyse four-electron ORR

[175 184 236] however n = 005 and nα asymp 1 in this study This is probably due to the

difficulty in electrochemical measurement of enzyme catalysis It is however possible

to find nα because the rate-limiting step is the single electron transfer between the

electrode and the blue copper atom of the enzyme Assuming this kdeg was calculated for

four-electron ORR at the enzyme electrodes and the result is shown in Fig 120

257

0

1

2

nα

00E+00

40E-09

80E-09

12E-08

16E-08

20E-08

kordm

ms

-1

nα 077 081

kordm ms-1 134E-08 168E-09

Lac Bod

Fig 120 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Lac had kdeg = 134 x 10-8 plusmn 251 x 10-9 while BOD had kdeg = 168 x 10-9 plusmn 271 x 10-10

Lac electrodes showed better ORR efficiency despite of non-optimal pH condition Lac

is most active at pH 5 while BOD at pH 7 The amount of enzyme loaded on the

electrode surface also might have affected the catalytic activity It was however difficult

to control the loading because the enzyme was attached by dip coating Using mediators

could have also improved the ORR efficiency Many authors used mediators such as

ABTS [62 66] and Os-pyridine complexes linked to conductive polymer [97 237] to

assist the electron transfer between the electrode and enzyme

73 MOST EFFICIENT ORR CATALYSTS

The kinetic and thermodynamic parameters (kdeg nα Epc2(HDV)) were organised

258

by magnitude and are summarised in figures 25-27 and tables 1 amp 2 below For each

parameter the best five candidates were selected to present the effects of pretreatment

and their relation with each parameter The best three were chosen from them on the

basis of the parameter size and the number of top-ranked parameters they were

occupying kdeg was given the highest priority for selection process

731 Best five electrodes for nα

Pretreated GCEs were found to have prominent nα (Fig 121)

00

05

10

15

20

25

30

Electrode type

n α

nα 278 178 174 168 114

GC H2SO4-PBS GC NaOH-H2SO4-PBS

GC NaOH-PBS GC PBS GP NaOH-H2SO4

Fig 121 The best five electrodes for nα

The GCEs doubly pretreated with H2SO4 and PBS was found best with nα = 278 Its kdeg

was 279 x 10-7 ms-1 and also biggest among pretreated GCEs Correlation between nα

259

and kdeg has not been confirmed since it is possible for the intial electron transfer to be

accelerated whilst still remaining the RDS

732 Best five electrodes for kdeg

Top six kdegs were dominated by pretreated Co(salophen) composite electrodes

(Fig 122)

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

Electrode type

kdeg

ms

-1

kordm ms-1 119E-05 747E-06 684E-06 587E-06 296E-06

CoS NaOH CoS NaOH-H2SO4

CoS H2SO4-PBS CoS NaOH-H2SO4-PBS

CoS NaOH-PBS

Fig 122 The best five electrodes for kdeg

PBS pretreatment was found to suppress kdeg in Co(salophen) composite electrodes Clear

correlation between kdeg and nα has not yet confirmed however it was found that

favourable kdeg tends to lower overpotential

260

733 Best five electrodes for Epc2(HDV)

Although it is not an exact correspondence some correlation was observed

between lower overpotential and larger kdeg or lower overpotential and larger nα but not

between kdegand nα A range of pretreated Co(salophen) composite electrodes which

showed largest kdeg and a pretreated GCE which showed the largest nα were found to be

dominating low overpotential (Fig 123)

-040

-039

-038

-037

-036

-035

Electrode type

E pc

2(H

DV)

V

Epc2(HDV) -037 -037 -039 -039 -040

CoS H2SO4-PBS CoS NaOH CoS NaOH-H2SO4

GC H2SO4-PBS CoS NaOH-PBS

Fig 123 The best five electrodes for Epc2(HDV)

734 Best three candidates for ORR catalytic electrodes

Pretreated Co(salophen) composite electrodes were the best candidates for

ORR catalysis because of largest kdeg and lowest overpotential Top three catalytic

261

cathodes were listed in Table 17

kordm ms-1 Epc2(HDV) V nα

Co(salophen) NaOH 119E-05 -037 083

Co(salophen) NaOH-H2SO4 747E-06 -039 074

Co(salophen) H2SO4-PBS 684E-06 -037 091

Average 875E-06 -037 083

SD 278E-06 001 009

Table 17 Three candidates for catalytic cathodes for ORR

NaOH-pretreated Co(salophen) was found to be most efficient ORR catalytic cathode

because it has the largest kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 Epc2(HDV) = - 037 V and

was the second smallest of all kdeg of the three candidate Co(salophen) electrodes were

compared with those of catalytic metals and shown in Table 18

Electrode type kdeg ms-1 Experiment condition

Co(salophen) NaOH 119 x 10-5 In 001M PBS pH 74

Co(salophen) NaOH ndashH2SO4 747 x 10-6 In 001M PBS pH 74

Co(salophen) H2SO4-PBS 684 x 10-6 In 001M PBS pH 74

Au 4 x 10-3 In 005 M H2SO4 [173]

Au 3 x 10-5 In 01 M KOH

Ag 8 x 10-3 In 01 M KOH

Pt 4 x 10-3 In 01 M KOH

Table 18 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

262

NaOH-pretreated Co(salophen) composite electrode showed good catalysis at neutral

pH It could manage to achieve as efficient ORR as Au in acidic solution

74 CONCLUSION TO CATHODE STUDY

The large overpotential of ORR has been known to degrade the fuel cell

performance [3 4 62 165-167] Finding efficient ORR catalysts therefore has been a

subject of intensive research In this study various catalytic cathodes were fabricated

using inorganic organic and biological catalysts and they were electrochemically

pretreated in different media to improve the catalytic activity Studies were conducted

using various electrochemical methods (i) CV (ii) HDV and (iii) HDA The data

obtained was analysed both qualitatively and quantitatively The ORR catalytic

efficiency was calculated according to the three parameters (i) kdeg (ii) nα and (iii)

Epc2(HDV) and based on that most catalytic cathode was determined

None of the cathodes was catalytic enough without pretreatment or unless at high

overpotential In many cases electrochemical pretreatments were found to attach

various surface oxygen groups activated the electrodes and led to two-electron ORR

catalysis Their influence on electrochemistry varies by the electrode type and media

used for pretreatment It was found that PBS-related pretreatments were effective to

GCEs When the pretreatment is combined with H2SO4 this might lead to serial ORR

where H2O2 produced is further reduced to water NaOH-related pretreatments were

effective for graphite composite electrodes PtC composite electrodes were not active

due to the high resistance within the activated carbon The performance by RhC was

263

only marginally improved by H2SO4 pretreatment Any NaOH- or PBS- related

pretreatments dramatically increased kdeg of Co(salophen) composite electrodes and

reduced the overpotential by 100 mV Fe(salophen) composite electrodes were not

sufficiently catalytic without a double pretreatment with NaOH and H2SO4

Anthraquinone-grafted graphite composite electrodes were not catalytic at neutral pH

even after pretreatment Multi-copper oxidases were catalytic at DET however its RLS

was electron transfer between the electrode and the enzyme hence appropriate

mediators should be used to achieve higher catalysis

Many of pretreated Co(salophen) composite electrodes were strongly catalytic toward

ORR Among them NaOH-pretreated Co(slophen) electrodes were found to be most

catalytic at neutral pH with kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 nα = 083 and Epc2(HDV) =

- 037 V Its catalytic efficiency was close to that of Au in the acid solution

264

8 CONCLUSION AND FURTHER STUDIES

Miniaturised glucose-oxygen fuel cells are ideal to power small implantable

medical devices Typical specification include at least 1 microW power output and 1-year

operational life Therefore fuel cells must have efficient catalysis and electron transfer

good conductivity chemical and mechanical stability as well as biocompatibility The

electrodes must be feasible to build and scaleable In this study electrodes were built

using bulk modification method because it was simple to construct and scale-up and

they were easy to modify and mechanically stable The anodes were built with 10

GOx with 77 TTF-TCNQ composite and the cathodes were built with graphite

composite containing ORR catalysts using bulk modification method TTF-TCNQ was

used because it was conductive as well as mediating and necessary for electron transfer

of the GOx anodes Problems were enzyme stability and ORR catalytic efficiency

Enzyme mass loading is known to be effective to improve catalytic efficiency and

operational life however this also decreases conductivity of the composite electrodes

The key to optimising enzyme electrodes is in the balance and compromise among the

conductivity mediation enzyme activity mechanical and chemical stability and

difficulty in the fabrication process CNF composites showed reasonable conductivity at

30 content and this might allow higher enzyme loading with mediators without losing

conductivity CNT is also a candidate material because it has comparable or larger

conductivity than TTF-TCNQ and can operate without added mediator species Ideally

enzyme electrodes should be functional with minimum components simple to build

renew and store in dry so that they become easily portable Moreover when they are

further miniaturised this will allow the medical application under harsher conditions so

265

that powering disposable implantable sensors in economically-deprived regions might

become possible Many physiological enzymes however easily lose catalytic activity

outside the optimal condition Incorporation of enzyme stabilisers makes the enzyme

robust and long lasting as well as scavenging reactive species In the glucose-oxygen

fuel cells O2 has been known to reduce the catalytic output current of GOx anodes

PEI-DTT stabiliser mixtures were found to be good for this because they conferred

stability on GOx against oxidative stress prevented O2 electron deprivation and

improved the catalytic current output It was however a smaller effect than when

conductive hydrogel was used Conductive hydrogel can be a good candidate if it

becomes resistant against degradation ORR at the cathode has posed a long-term

problem in fuel cells because ORR is inefficient and consumes potential generated

within the fuel cell Pt has been used as an ORR catalyst but it is expensive and is

becoming scarce Use of the fuel cell in the physiological condition also imposes further

operational limitations Pretreated metal complexes such as Co salophen were found to

be good candidates for ORR-catalytic cathodes Surrounding complexes with

electron-rich environment such as conductive film might improve ORR further Once

candidate anode and cathode are identified and built a biofuel cell unit can be made To

increase the power each cell is connected together and made into a stack The prototype

was made as below in Fig 124 by connecting twenty cell units

266

Fig 124 Fuel cell stack made with various GOx anodes and Co(salophen) cathodes

Each cell contained various GOx anodes and pretreated Co(salophen) electrode in 10 ml

of oxygenated 01 M glucose 001 M PBS solution pH 74 The output voltage was

found to be 02 V and it did not change for one week The fuel cell must be studied

further with an adjustable resistor for voltage-current (V-I) and power-current (P-I)

curves so that OCV can be determined properly The passive type GOx fuel cell could

output 15 mWcm-2 at 03 V [18]

In vivo studies are also necessary for implantable purposes to examine the catalytic

efficiency electrode fouling and biocompatibility This can be studied systematically by

changing the type and concentration of interfering substances such as ascorbate

acetoaminophen and urate [18] Heller tested miniturised GOx-BOD fuel cells tested in

the buffer grape and calf serum It showed only 5 power decrease in buffer however

50 drop within 20 hours in grape and 50 drop within 2 hours in calf serum [19] To

avoid electrode fouling the electrode surface can be coated with a membrane such as

267

Nafion However this imposes another problem because it can interfere with the

reactant flux to the electrode surface In the real physiological system this might cause

a serious problem because glucose and O2 concentration is much lower than in vitro

Furthermore in the physiological conditions active transport of the reactant is not

available and this will cause local concentration polarisation It seems that there are

many obstacles lying in the medical application of miniaturised glucose-oxygen fuel

cells although they have widespread potentials applications

268

9 BIBLIOGRAPHY

1 Carrette L KA Friedrich and U Stimming Fuel cells Principles types fuels and applications

Chemphyschem 2000 1(4) p 162-193

2 Gregoire Padro CE and JO Keller Hydrogen Energy in Kirk-Othmer Encyclopedia of Chemical

Technology 2005 John Wiley amp Sons Inc p 837-866

3 Ohta K T Kazama and M Nishizawa All about fuel cells in Newton 2006 p 31-63

4 Tanabe S Mastering basics of the fuel cells 2009 Denkishoin

5 Tokyo-Gas Fuel cell academy 2009 2901 [cited Available from httpwwwtokyo-gascojppefc

6 Cropper MAJ S Geiger and DM Jollie Fuel cells a survey of current developments Journal of Power

Sources 2004 131(1-2) p 57-61

7 Kordesch K et al Intermittent use of a low-cost alkaline fuel cell-hybrid system for electric vehicles

Journal of Power Sources 1999 80(1-2) p 190-197

8 UTC-Power The fuel cell for Apollo 11

9 Connor S Warning Oil supplies are running out fast in The Independent 2009

10 Mori Y Mobile fuel cell providing new advantages for mobile electronic appliances Toshiba review 2007

62(6) p 44-47

11 Mori H Electronic components and materials Toshiba review 2009 64(3) p 62-63

12 Imperial-College-London Racing Green - Vehicles 2009 [cited Available from

httpwwwunionicacukrccracinggreenvehicleshtml

13 Cleave V Formula Zero kart race could drive fuel cell technology New Scientist 2008 [cited

Available from

httpwwwnewscientistcomarticledn14596-formula-zero-kart-race-could-drive-fuel-cell-technologyhtml

14 HyNor Hydrogen highway opens in Norway 2009 [cited Available from

httpwwwhynornohydrogen-highway-opens-in-norway

15 BMW Hydrogen 7 - The future is closer than you think 2009 [cited Available from

httpwwwbmwcomcomeninsightstechnologyefficient_dynamicsphase_2clean_energybmw_hydroge

n_7html

httpwwwbmwcojpjpjabrandtechnologycleanenergywallpaperhtml

16 Sony Side view - Sony developes Bio Battery generating electricity from sugar 2007 Sony

17 Burton N Sugar-powered electronics in RSC Chemical technology news 2008

18 Sakai H A high-power glucoseoxygen biofuel cell operating under quiescent conditions Energy amp

Environmental Science 2009 2(1) p 133-138

19 Heller A Miniature biofuel cells Physical Chemistry Chemical Physics 2004 6(2) p 209-216

20 Toshiba Toshiba announces worlds smallest direct methanol fuel cell with energy output of 100 milliwatts

269

2004 [cited Available from httpwwwtoshibacojpaboutpress2004_06pr2401htm

21 Osakai T K Kanoh and S Kuwabata Basic Electrochemistry 2007 Kagakudojin Ltd Kyoto Japan

22 Wilson K and J Walker Practical Biochemistry 1992 Cambridge UK Cambridge University Press

23 Murray RK et al Harpers Biochemistry 25 ed 2000 Stamford Connecticut Appleton amp Lange

24 Sawyer DT A Sobkowiak and JL Roberts Jr Electrochemistry for chemists Revised ed 1995

Wiley-Interscience

25 IUPAC IUPAC GOLD BOOK 2009 [cited Available from httpgoldbookiupacorgindexhtml

26 Kitamura T and K Fujisawa English-Japanese The students dictionary of physics 1995 Tokyo ALC

Inc

27 Gates BC Catalysis in Kirk-Othmer Encyclopedia of Chemical Technology 2002 John Wiley amp Sons p

200-254

28 Taguchi S and E Nakano Kinetic Study of Bromination of Malonic Acid Methyl Malonic Acid

and Ethyl Malonic Acid An Undergraduate Physical Chemistry Laboratory Experimentfor

Understanding of the Infuluence of Thermodynamic Parameter on Chemical Kinetics The Chemical

Education Jounal 1999 3(1) p 1-20

29 Pletcher D A First course in electrode processes 1991 The Electrochemical Consultancy (Romsey) Ltd

23 - 26 135 - 145

30 Bard AJ and LR Faulker Electrochemical Methods Fundamentals and Applications 2000 NY USA

John Wiley and Sons Inc

31 Daintith J A concise dictionary of chemistry 2 ed 1990 Oxford UK Oxford University Press

32 Tinoco IJ et al Physical Chemistry 2002 Prentice Hall p 157

33 Watanabe K and S Nakabayashi Introduction to electrochemistry The chemistry of electron transfer 13

ed 2005 Tokyo Japan The Chemical Society of Japan

34 Hamelers B et al Microbial fuel cells Methodology and technology Environmental science amp

technology 2006 40(17) p 5181-5192

35 Koike T Chapter 10 Free energy in Physical chemistry I amp II Lecture handouts for dept of pharmacy

2010 University of Hiroshima p 57-62

36 Lopes T E Gonzalez and E Antolini An overview of platinum-based catalysts as methanol-resistant

oxygen reduction materials for direct methanol fuel cells Journal of alloys and compounds 2008 461(1-2)

p 253-262

37 Vork FTA and E Barendrecht The reduction of dioxygen at polypyrrole-modified electrodes with

incorporated Pt particles Electrochimica Acta 1990 35(1) p 135-139

38 Marino W B Scharifker and A Leone ELECTRODEPOSITION AND

ELECTROCHEMICAL-BEHAVIOR OF PALLADIUM PARTICLES AT POLYANILINE AND

POLYPYRROLE FILMS Journal of the Electrochemical Society 1992 139(2) p 438-443

39 Tsakova V How to affect number size and location of metal particles deposited in conducting polymer

270

layers Journal of Solid State Electrochemistry 2008 12(11) p 1421-1434

40 Lemest Y et al Dioxygen Fixation by a Mixed-Valence Dicobalt Cofacial Porphyrin Journal of the

Chemical Society-Chemical Communications 1983(22) p 1286-1287

41 Kim E et al Superoxo micro-peroxo and micro-oxo complexes from hemeO2 and heme-CuO2 reactivity

Copper ligand influences in cytochrome c oxidase models 2003 p 3623-3628

42 Fukuzumi S et al Mechanism of Four-Electron Reduction of Dioxygen to Water by Ferrocene

Derivatives in the Presence of Perchloric Acid in Benzonitrile Catalyzed by Cofacial Dicobalt Porphyrins

Journal of the American Chemical Society 2004 126(33) p 10441-10449

43 Solomon EI et al O2 and N2O Activation by Bi- Tri- and Tetranuclear Cu Clusters in Biology

Accounts of Chemical Research 2007 40(7) p 581-591

44 Wildgoose GG et al Anthraquinone-derivatised carbon powder reagentless voltammetric pH electrodes

Talanta 2003 60(5) p 887-893

45 McCreery R Carbon electrodes Structural effects on electron transfer kinetics in Electroanalytical

Chemistry A series advances 1990 Marcel Dekker Inc p 221-374

46 Kneten K R McCreery and C McDermott ELECTRON-TRANSFER KINETICS OF AQUATED

FE+3+2 EU+3+2 AND V+3+2 AT CARBON ELECTRODES - INNER-SPHERE CATALYSIS BY

SURFACE OXIDES Journal of the Electrochemical Society 1993 140(9) p 2593-2599

47 Engstrom RC ELECTROCHEMICAL PRETREATMENT OF GLASSY-CARBON ELECTRODES

Analytical Chemistry 1982 54(13) p 2310-2314

48 Lawrence E Hendersons dictionary of biological terms 10 ed 1989 UK Longman group UK Ltd

49 Eijsink V Directed evolution of enzyme stability Biomolecular engineering 2005 22(1-3) p 21-30

50 Pantoliano M et al PROTEIN ENGINEERING OF SUBTILISIN BPN - ENHANCED STABILIZATION

THROUGH THE INTRODUCTION OF 2 CYSTEINES TO FORM A DISULFIDE BOND Biochemistry

1987 26(8) p 2077-2082

51 Bagotzky VS NV Osetrova and AM Skundin Fuel cells State-of-the-art and major scientific and

engineering problems Russian Journal of Electrochemistry 2003 39(9) p 919-934

52 Heller A Integrated medical feedback systems for drug delivery Aiche Journal 2005 51(4) p

1054-1066

53 Nakamura M et al High-performance ultrathin solid oxide fuel cells for low-temperature operation

Journal of the Electrochemical Society 2007 154(1) p B20-B24

54 Lapedes DN McGraw-Hill Dictionary of Physics and Mathematics in McGraw-Hill 1978 McGraw-Hill

Inc

55 Svoboda V et al Enzyme catalysed biofuel cells Energy amp Environmental Science 2008 1(3) p

320-337

56 Barton SC J Gallaway and P Atanassov Enzymatic biofuel cells for Implantable and microscale devices

Chemical Reviews 2004 104(10) p 4867-4886

271

57 Schroder U Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency

Physical chemistry chemical physics 2007 9(21) p 2619-2629

58 Chaudhuri SK and DR Lovley Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells Nature Biotechnology 2003 21(10) p 1229-1232

59 Vincent K F Armstrong and J Cracknell Enzymes as working or inspirational electrocatalysts for fuel

cells and electrolysis Chemical reviews 2008 108(7) p 2439-2461

60 Tsujimura S and K Kano Recent development of enzyme-based biofuel cells GS Yuasa technical report

2008 5(2) p 1-6

61 Cass AEG Biosensors A Practical Approach 1990 Oxford University Press Oxford UK p 49-53

62 Palmore GTR and HH Kim Electro-enzymatic reduction of dioxygen to water in the cathode

compartment of a biofuel cell Journal of Electroanalytical Chemistry 1999 464(1) p 110-117

63 Bullen RA et al Biofuel cells and their development Biosensors amp Bioelectronics 2006 21(11) p

2015-2045

64 Bertschy H et al A methanoldioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as

catalysts application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at

low overpotentials Journal of electroanalytical chemistry and interfacial electrochemistry 1998 443(1) p

155-161

65 Bartlett PN P Tebbutt and RG Whitaker Kinetic aspects of the use of modified electrodes and

mediators in bioelectrochemistry Progress in reaction kinetics 1991 16(2) p 55-155

66 Kano K T Ikeda and S Tsujimura GlucoseO-2 biofuel cell operating at physiological conditions Denki

kagaku oyobi kōgyō butsuri kagaku 2002 70(12) p 940-942

67 Heller A Electrochemical glucose sensors and their applications in diabetes management Chemical

Reviews 2008 108(7) p 2482-2505

68 Ilankumaran V et al Trends in cardiac pacemaker batteries Indian pacing and electrophysiology journal

2004 4(4) p 201-12

69 Schlindwein W and R Linford Medical applications of solid state lonics Solid State Ionics 2006

177(19-25) p 1559-1565

70 Latham R R Linford and W Schlindwein Biomedical applications of batteries Solid State Ionics 2004

172(1-4) p 7-11

71 Soykan O Power sources for implantable medical devices Business Briefing Medical Device

Manufacturing and Technology London 2002

72 Heller A Potentially implantable miniature batteries Analytical and Bioanalytical Chemistry 2006

385(3) p 469-473

73 Taniguchi M Achilles heel of the next-generation electric vehicles - Lithium World map of the resrouce

wars 2008 [cited 2009 Available from

httpbusinessnikkeibpcojparticlemanage20080407152450P=1

272

74 Yeatman EM Advances In Power Sources For Wireless Sensor Nodes International Workshop on Body

Sensor Networks 2004

75 Sato N et al Novel MEMS power generator with integrated thermoelectric and vibrational devices in

Solid-State Sensors Actuators and Microsystems 2005 Digest of Technical Papers TRANSDUCERS 05

The 13th International Conference on 2005

76 Hudak NS and GG Amatucci Small-scale energy harvesting through thermoelectric vibration and

radiofrequency power conversion Journal of Applied Physics 2008 103(10)

77 Seiko-Instruments SEIKO Worlds First 2009 [cited Available from

httpwwwseikowatchescomheritageworlds_firsthtml

78 Kishi M et al Micro thermoelectric modules and their application to wristwatches as an energy source in

Thermoelectrics 1999 Eighteenth International Conference on 1999

79 Lee J et al Ionic conduction in Zn-3(PO4)(2)()4H(2)O enables efficient discharge of the zinc anode in

serum Journal of the American Chemical Society 2005 127(42) p 14590-14591

80 Maxell Button battery table 2009 [cited Available from

httpwwwmaxellcojpproductsmaterialsbutton_batteryhtml

81 Wilson R and APF Turner Glucose-Oxidase - an Ideal Enzyme Biosensors amp Bioelectronics 1992 7(3)

p 165-185

82 Swoboda BEP The relationship between molecular conformation and the binding of flavin-adenine

dinucleotide in glucose oxidase Biochimica et Biophysica Acta (BBA) - Protein Structure 1969 175(2) p

365-379

83 Wohlfahrt G et al 18 and 19 angstrom resolution structures of the Penicillium amagasakiense and

Aspergillus niger glucose oxidases as a basis for modelling substrate complexes Acta Crystallographica

Section D-Biological Crystallography 1999 55 p 969-977

84 Patel RN Biocatalysis in the pharmaceutical and biotechnology industries 1st ed 2006 CRC Press

85 Gouda M Reversible denaturation behavior of immobilized glucose oxidase Applied Biochemistry and

Biotechnology 2002 102 p 471-480

86 Hecht HJ et al Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 3 Angstrom

Resolution Journal of Molecular Biology 1993 229(1) p 153-172

87 Zubrik A et al Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the

transition to the denatured state with residual structure The Journal of biological chemistry 2004 279(46)

p 47601-47609

88 Kojima S et al Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose

biosensor Bioscience biotechnology and biochemistry 2006 70(3) p 654-659

89 Duine J The PQQ story Journal of bioscience and bioengineering 1999 88(3) p 231-236

90 Kyoto-University KEGG - Kyoto Encyclopedia of Genes and Genomes 2009 Kyoto University

91 Delbem MF WJ Baader and SHP Serrano Mechanism of 34-dihydroxybenzaldehyde

273

electropolymerization at carbon paste electrodes catalytic detection of NADH 2002 scielo p 741-747

92 Zhao S et al Electrochemistry of Organic Conducting Salt Electrodes - a Unified Mechanistic

Description Journal of Physical Chemistry 1992 96(13) p 5641-5652

93 Lemuel BW Jr CC Liu and LN Niren Electrochemical measurements with glucose oxidase

immobilized in polyacrylamide gel Constant current voltametry 1971 p 629-639

94 Sato A et al Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Biochemical Journal

2001 357 p 893-898

95 Sigma-Aldrich Glucose Oxidase from Aspergillus niger Type II-S 15000-50000 unitsg solid (without

added oxygen) 2009 [cited

96 Cosnier S Biosensors based on electropolymerized films new trends Analytical and Bioanalytical

Chemistry 2003 377(3) p 507-520

97 Willner I et al Integrated Enzyme-Based Biofuel Cells-A Review Fuel Cells 2009 9(1) p 7-24

98 Heleg-Shabtai V et al Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers

assembled onto Au-electrodes Journal of the American Chemical Society 1996 118(42) p 10321-10322

99 Georganopoulou DG et al Development and comparison of biosensors for in-vivo applications Faraday

Discussions 2000 p 291-303

100 Fujii S et al Properties of glucose sensors prepared by the electropolymerization of pyrrole containing a

gluconyl group Analytical sciences 1999 15(12) p 1175-1176

101 Hoshi T Deposition of electron mediators by electrochemical polymerization Electrochemistry 2003

71(5) p 346-347

102 Arai G M Masuda and I Yasumori Direct Electrical Response between Glucose-Oxidase and

Poly(Mercapto-P-Benzoquinone) Films Bulletin of the Chemical Society of Japan 1994 67(11) p

2962-2966

103 Arai G K Shoji and I Yasumori Electrochemical characteristics of glucose oxidase immobilized in

poly(quinone) redox polymers Journal of Electroanalytical Chemistry 2006 591(1) p 1-6

104 Leonida MD et al Polymeric FAD used as enzyme-friendly mediator in lactate detection Analytical and

Bioanalytical Chemistry 2003 376(6) p 832-837

105 Kissinger PT and WR Heineman Laboratory techniques in electroanalytical chemistry 1996 Marcel

Dekker Inc NY USA p 294-332

106 OHare D and H Zhao Characterisation and modeling of conducting composite electrodes The journal of

physical chemistry C 2008 112(25) p 9351-9357

107 Wang J et al Metal-Dispersed Carbon Paste Electrodes Analytical Chemistry 1992 64(11) p

1285-1288

108 Khurana MK CP Winlove and D OHare Detection mechanism of metallized carbon epoxy oxidase

enzyme based sensors Electroanalysis 2003 15(12) p 1023-1030

109 Sasaki M et al Fabrication and Electrical Characterization of

274

Tetrathiafulvalene-tetracyanoquinodimethane Molecular Wires Japanese Journal of Applied Physics 2003

42 p 2488-2491

110 Iizuka M et al Highly oriented TTF-TCNQ crystal growth using an electric-field induced evaporation

technique Molecular crystals and liquid crystals science and technology Section A Molecular crystals and

liquid crystals 2000 349 p 367-370

111 Saito I and K Yoshida Development of new features on organic conductive material TCI mail 2005

127

112 Cass AEG et al FERROCENE-MEDIATED ENZYME ELECTRODE FOR AMPEROMETRIC

DETERMINATION OF GLUCOSE Analytical Chemistry 1984 56(4) p 667-671

113 Kulys J V Simkeviciene and IJ Higgins Concerning the Toxicity of 2 Compounds Used as Mediators in

Biosensor Devices - 7788-Tetracyanoquinodimethane (Tcnq) and Tetrathiafulvalene (Ttf) Biosensors amp

Bioelectronics 1992 7(7) p 495-501

114 Malinauskas A et al Conductive organic complex salt TTF-TCNQ as a mediator for biosensors An

overview Electroanalysis 2007 19(24) p 2491-2498

115 Llopis X et al Integration of a glucose biosensor based on an epoxy-graphite-TTF center dot

TCNQ-GOD biocomposite into a FIA system Sensors and Actuators B-Chemical 2005 107(2) p 742-748

116 Bourgeois J D Thomas and C Bourdillon COVALENT LINKAGE OF GLUCOSE-OXIDASE ON

MODIFIED GLASSY-CARBON ELECTRODES - KINETIC PHENOMENA Journal of the American

Chemical Society 1980 102(12) p 4231-4235

117 Yun Y et al Nanotube electrodes and biosensors Nano Today 2007 2(6) p 30-37

118 Kim BC et al Preparation of biocatalytic nanofibres with high activity and stability via enzyme

aggregate coating on polymer nanofibres Nanotechnology 2005 16(7) p S382-S388

119 Kim J and JW Grate Single-Enzyme Nanoparticles Armored by a Nanometer-Scale OrganicInorganic

Network 2003 p 1219-1222

120 OFagain C Enzyme stabilization - recent experimental progress Enzyme and Microbial Technology

2003 33(2-3) p 137-149

121 Sillery E et al Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a

platinum catalyst Chemical communications 2002(8) p 866-867

122 Gouda M et al Enhancement of stability of immobilized glucose oxidase by modification of free thiols

generated by reducing disulfide bonds and using additives Biosensors amp bioelectronics 2004 19(6) p

621-625

123 Manning M STABILITY OF PROTEIN PHARMACEUTICALS Pharmaceutical Research 1989 6(11) p

903-918

124 Cao L Immobilised enzymes science or art Current opinion in chemical biology 2005 9(2) p 217-226

125 Daniel R The role of dynamics in enzyme activity Annual review of biophysics and biomolecular

structure 2003 32 p 69-92

275

126 Andersson MM JD Breccia and R Hatti-Kaul Stabilizing effect of chemical additives against oxidation

of lactate dehydrogenase Biotechnology and Applied Biochemistry 2000 32 p 145-153

127 Souza JM and R Radi Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Archives of Biochemistry and Biophysics 1998 360(2) p 187-194

128 Andersson MA and R Hatti-Kaul Protein stabilising effect of polyethyleneimine Journal of

Biotechnology 1999 72(1-2) p 21-31

129 Sigma-Aldrich Poly(ethylenimine) 2009 [cited Available from

httpwwwsigmaaldrichcomstructureimages27mfcd00084427gif

130 Andersson MM R Hatti-Kaul and W Brown Dynamic and static light scattering and fluorescence

studies of the interactions between lactate dehydrogenase and poly(ethyleneimine) Journal of Physical

Chemistry B 2000 104(15) p 3660-3667

131 Wang J L Chen and J Liu Critical comparison of metallized and mediator-based carbon paste glucose

biosensors Electroanalysis 1997 9(4) p 298-301

132 Cleland WW Dithiothreitol a New Protective Reagent for SH Groups Biochemistry 1964 3(4) p

480-482

133 Atanasov P E Wilkins and Q Yang An integrated needle-type biosensor for intravascular glucose and

lactate monitoring Electroanalysis 1998 10(11) p 752-757

134 Sigma-Aldrich DL-Dithiothreitol 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=43815|SIGMAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

135 Ghisalba O and A Brunella Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate

reductase as biocatalyst in the preparative synthesis of 2 -deoxyribonucleoside-5 -triphosphates Journal

of molecular catalysis B Enzymatic 2000 10(1-3) p 215-222

136 Gollhofer D EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM

ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Enzyme and Microbial Technology 1995 17(3) p 235-240

137 Sigma-Aldrich Arsenite standard solution - 005 M in H2O 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=70039|FLUKAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

138 Arakawa T MECHANISM OF POLY(ETHYLENE GLYCOL) INTERACTION WITH PROTEINS

Biochemistry 1985 24(24) p 6756-6762

139 Lee C Improvement of protein stability in protein microarrays Biotechnology Letters 2002 24(10) p

839-844

140 Lee L THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE GLYCOLS)

Biochemistry 1987 26(24) p 7813-7819

141 Costa SA et al Studies of stabilization of native catalase using additives Enzyme and Microbial

276

Technology 2002 30(3) p 387-391

142 Lee J and L Lee THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE

GLYCOLS) Biochemistry 1987 26(24) p 7813-7819

143 Gekko K and SN Timasheff Mechanism of protein stabilization by glycerol preferential hydration in

glycerol-water mixtures Biochemistry 1981 20(16) p 4667-4676

144 Leandro P Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes

produced in a prokaryotic expression system Molecular genetics and metabolism 2001 73(2) p 173-178

145 Bartlett PN VQ Bradford and RG Whitaker Enzyme Electrode Studies of Glucose-Oxidase Modified

with a Redox Mediator Talanta 1991 38(1) p 57-63

146 Joseph S et al Drug metabolism biosensors electrochemical reactivities of cytochrome P450(cam)

immobilised in synthetic vesicular systems Journal of Pharmaceutical and Biomedical Analysis 1998

17(6-7) p 1101-1110

147 Cornish-Bowden A Fundamentals of enzyme kinetics 3rd ed 2004 London Portland Press Ltd

148 Wilkinson G STATISTICAL ESTIMATIONS IN ENZYME KINETICS The Biochemical journal 1961

80(2) p 324

149 Ikeda T and K Kano Fundamentals and practices of mediated bioelectrocatalysis Analytical sciences

2000 16(10) p 1013-1021

150 Duggleby RG and LP Daniel [3] Analysis of enzyme progress curves by nonlinear regression in

Methods in Enzymology 1995 Academic Press p 61-90

151 Eisenthal R and A Cornish-Bowden DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR

ESTIMATING ENZYME KINETIC-PARAMETERS The Biochemical journal 1974 139(3) p 715-720

152 Baici A Enzyme kinetics the velocity of reactions Biochem J 2006 p 1-3

153 Yamazaki A et al Improvement of precision in pharmacological studies using the non-linear regression

method

estimation of enzyme inhibition constants as an example Folia Pharmacologica Japonica 2008 131(3) p 195-203

154 Cornish-Bowden A Teaching Enzyme Kinetics and Mechanism in the 21st Century in 3rd Symposium

on ESCES 2008 Rudensheim Germany

155 Weisshaar DE and T Kuwana Considerations for Polishing Glassy-Carbon to a Scratch-Free Finish

Analytical Chemistry 1985 57(1) p 378-379

156 Stedman Stedmans medical dictionary 25th ed 2002 Lippincott Williams amp Wilkins

157 Tamura T Buffer preparation for biology Buffer preparation for biology ed 6 2008 Tokyo Yodosha

Ltd

158 Battino R and HL Clever The Solubility of Gases in Liquids 1966 p 395-463

159 Pandolfo A and A Hollenkamp Carbon properties and their role in supercapacitors Journal of power

sources 2006 157(1) p 11-27

160 Maynard AD Nanotechnology assessing the risks Nano Today 2006 1(2) p 22-33

277

161 Hindle A E Hall and N Martens AN ASSESSMENT OF MEDIATORS AS OXIDANTS FOR

GLUCOSE-OXIDASE IN THE PRESENCE OF OXYGEN Biosensors amp bioelectronics 1995 10(3-4) p

393-403

162 Mo J et al Comparison of oxygren-rich and mediator-based glucose-oxidase carbon-paste electrodes

Analytica Chimica Acta 2001 441(2) p 183-189

163 Turner A and G Palleschi AMPEROMETRIC TETRATHIAFULVALENE-MEDIATED LACTATE

ELECTRODE USING LACTATE OXIDASE ABSORBED ON CARBON FOIL Analytica Chimica Acta

1990 234(2) p 459-463

164 Mizutani F et al Glucose-Sensing Electrode Based on Carbon Paste Containing Ferrocene and

Polyethylene Glycol-Modified Enzyme Bulletin of the Chemical Society of Japan 1991 64(9) p

2849-2851

165 Zhang J PEM Fuel Cell Electrocatalysts and Catalyst Layers Fundamentals and Applications 2008

London Springer-Verlag

166 Kinoshita K Electrochemical oxygen technology 1992 John Willey amp Sons Inc

167 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of Power

Sources 2005 152 p 1-15

168 Sawyer DT Oxygen Chemistry International series of monographs on chemistry 1991 Oxford

University Press

169 Winter M Chemical Bonding Oxford Chemistry Primers 1997 New York Oxford University Press Inc

170 McLendon G and AE Martell Inorganic Oxygen Carriers as Models for Biological-Systems

Coordination Chemistry Reviews 1976 19(1) p 1-39

171 Toda T et al Enhancement of the electroreduction of oxygen on Pt alloys with Fe Ni and Co Journal of

the Electrochemical Society 1999 146(10) p 3750-3756

172 Norskov JK et al Origin of the overpotential for oxygen reduction at a fuel-cell cathode Journal of

Physical Chemistry B 2004 108(46) p 17886-17892

173 Fischer P and J Heitbaum MECHANISTIC ASPECTS OF CATHODIC OXYGEN REDUCTION Journal

of Electroanalytical Chemistry 1980 112(2) p 231-238

174 Okada T et al Oxygen reduction characteristics on pyrolytic graphite electrodes modified with

electro-polymerized salen compounds Chemistry Letters 1998(8) p 841-842

175 Sakurai T and K Kataoka Basic and applied features of multicopper oxidases CueO bilirubin oxidase

and laccase 2007 p 220-229

176 Sanders J Supramolecular catalysis in transition Chemistry a European journal 1998 4(8) p

1378-1383

177 Hart H LE Craine and DJ Hart Organic Chemistry A Short Course 10 ed 1998 Houghton Miffin

Company USA

178 Hayatsu H CELLULOSE BEARING COVALENTLY LINKED COPPER PHTHALOCYANINE

278

TRISULPHONATE AS AN ADSORBENT SELECTIVE FOR POLYCYCLIC COMPOUNDS AND ITS USE

IN STUDIES OF ENVIRONMENTAL MUTAGENS AND CARCINOGENS Journal of chromatography A

1992 597(1-2) p 37-56

179 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of power

sources 2005 152(1) p 1-15

180 Kingsborough RP and TM Swager Electrocatalytic Conducting Polymers Oxygen Reduction by a

Polythiophene-Cobalt Salen Hybrid Chemistry of Materials 2000 12(4) p 872-874

181 Ortiz B and SM Park Electrochemical and spectroelectrochemical studies of cobalt salen and salophen

as oxygen reduction catalysts Bulletin of the Korean Chemical Society 2000 21(4) p 405-411

182 Mano N et al An oxygen cathode operating in a physiological solution Journal of the American

Chemical Society 2002 124(22) p 6480-6486

183 Shleev S et al Direct electron transfer between copper-containing proteins and electrodes Biosensors

and Bioelectronics 2005 20(12) p 2517-2554

184 Solomon EI et al Oxygen binding activation and reduction to water by copper proteins Angewandte

Chemie-International Edition 2001 40(24) p 4570-4590

185 Heath R F Armstrong and C Blanford A stable electrode for high-potential electrocatalytic O-2

reduction based on rational attachment of a blue copper oxidase to a graphite surface Chemical

communications 2007(17) p 1710-1712

186 Miura Y et al Direct Electrochemistry of CueO and Its Mutants at Residues to and Near Type I Cu for

Oxygen-Reducing Biocathode Fuel cells 2009 9(1) p 70-78

187 Tarasevich MR VA Bogdanovskaya and LN Kuznetsova Bioelectrocatalytic reduction of oxygen in

the presence of laccase adsorbed on carbon electrodes Russian Journal of Electrochemistry 2001 37(8) p

833-837

188 Tsujimura S et al Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase

at carbon electrodes Electrochemistry 2004 72(6) p 437-439

189 Fernandez JL et al Optimization of wired enzyme O-2-electroreduction catalyst compositions by

scanning electrochemical microscopy Angewandte Chemie-International Edition 2004 43(46) p

6355-6357

190 Soukharev V N Mano and A Heller A four-electron O-2-electroreduction biocatalyst superior to

platinum and a biofuel cell operating at 088 V Journal of the American Chemical Society 2004 126(27)

p 8368-8369

191 Miura Y et al Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on

a highly oriented pyrolytic graphite electrode Chemistry Letters 2007 36(1) p 132-133

192 Seinberg JM et al Spontaneous modification of glassy carbon surface with anthraquinone from the

solutions of its diazonium derivative An oxygen reduction study Journal of Electroanalytical Chemistry

2008 624(1-2) p 151-160

279

193 Manisankar P and A Gomathi Electrocatalytic reduction of dioxygen at the surface of carbon paste

electrodes modified with 910-anthraquinone derivatives and dyes Electroanalysis 2005 17(12) p

1051-1057

194 Jurmann G DJ Schiffrin and K Tammeveski The pH-dependence of oxygen reduction on

quinone-modified glassy carbon electrodes Electrochimica Acta 2007 53(2) p 390-399

195 Abiman P R Compton and G Wildgoose Characterising chemical functionality on carbon surfaces

Journal of materials chemistry 2009 19(28) p 4875-4886

196 Thorogood CA et al Differentiating between ortho- and para-Quinone Surface Groups on Graphite

Glassy Carbon and Carbon Nanotubes Using Organic and Inorganic Voltammetric and X-ray

Photoelectron Spectroscopy Labels Chemistry of Materials 2007 19(20) p 4964-4974

197 Sigma-Aldrich Fast red AL salt 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildoN4=380660|SIALampN5=SEARCH_CONCAT_PN

O|BRAND_KEYampF=SPEC

198 Sljukic B C Banks and R Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

199 Granger M et al Polycrystalline diamond electrodes basic properties and applications as amperometric

detectors in flow injection analysis and liquid chromatography Analytica Chimica Acta 1999 397(1-3) p

145-161

200 Compton R J Foord and F Marken Electroanalysis at diamond-like and doped-diamond electrodes

Electroanalysis 2003 15(17) p 1349-1363

201 Arima S Do Science - Why diamond is so hard in Asahicom 2006

202 Dumitrescu I P Unwin and J Macpherson Electrochemistry at carbon nanotubes perspective and issues

Chemical communications 2009(45) p 6886-6901

203 Kinoshita K Carbon Electrochemical and Physicochemical Properties 1988 Wiley-Interscience

204 Kastening B et al Electronic properties and double layer of activated carbon Electrochimica Acta 1997

42(18) p 2789-2799

205 Swain G and R Ramesham THE ELECTROCHEMICAL ACTIVITY OF BORON-DOPED

POLYCRYSTALLINE DIAMOND THIN-FILM ELECTRODES Analytical chemistry 1993 65(4) p

345-351

206 Kaneto K et al Electrical conductivities of multi-wall carbon nano tubes Synthetic metals 1999

103(1-3) p 2543-2546

207 Meyer J et al Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes Nano

letters 2008 8(11) p 3582-3586

208 Harris P New perspectives on the structure of graphitic carbons Critical Reviews in Solid State and

Materials Sciences 2005 30(4) p 235-253

209 Akasaka T Chemical illustration - Carbon materials 2007 Hokkaido Japan

280

210 Iijima S M Yudasaka and F Nihey Carbon nanotube technology NEC Technical Journal 2007 2(1) p

52-56

211 Day T N Wilson and J Macpherson Electrochemical and conductivity measurements of single-wall

carbon nanotube network electrodes Journal of the American Chemical Society 2004 126(51) p

16724-16725

212 Fujita H A TEM image of the atomic structure of the diamond surface 2010 Britannica - The Online

Encyclopedia Osaka Japan

213 McCreery RL et al Control of reactivity at carbon electrode surfaces Colloids and Surfaces A

Physicochemical and Engineering Aspects 1994 93 p 211-219

214 Rice R C Allred and R McCreery FAST HETEROGENEOUS ELECTRON-TRANSFER RATES FOR

GLASSY-CARBON ELECTRODES WITHOUT POLISHING OR ACTIVATION PROCEDURES Journal of

electroanalytical chemistry and interfacial electrochemistry 1989 263(1) p 163-169

215 Swain Electrochemical formation of high surface area carbon fibers Analytical chemistry 1991 63(5) p

517-519

216 Nagaoka T and T Yoshino Surface-Properties of Electrochemically Pretreated Glassy-Carbon Analytical

Chemistry 1986 58(6) p 1037-1042

217 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 2002 58(9) p 1953-1955

218 Motta N and AR Guadalupe Activated Carbon-Paste Electrodes for Biosensors Analytical Chemistry

1994 66(4) p 566-571

219 Leung E et al A novel in vivo nitric oxide sensor Chemical Communications 1996(1) p 23-24

220 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 1986 58(9) p 1953-1955

221 Kissinger PT and WR Heineman Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy

Current Separations 1989 9 p 15-18

222 Wang J Analytical Electrochemistry 3 ed 2006 Wiley-VCH

223 BAS Measurement of electron transfer kinetics using rotating disk voltammetry Electrocehmistry

Principles methods and applications 1995 2(43)

224 Miomandre F et al Electrochemical behaviour of iron(III) salen and poly(iron-salen) Application to the

electrocatalytic reduction of hydrogen peroxide and oxygen Journal of electroanalytical chemistry and

interfacial electrochemistry 2001 516(1-2) p 66-72

225 Brighton-University Inorganic Chemistry Practical C170 Salen lab script 2009 Dept of Pharmacy and

Biomolecular Sciences Brighton University Brighton

226 Ranganathan S TC Kuo and RL McCreery Facile Preparation of Active Glassy Carbon Electrodes

with Activated Carbon and Organic Solvents 1999 p 3574-3580

227 Ohare D CP Winlove and KH Parker Electrochemical method for direct measurement of oxygen

281

concentration and diffusivity in the intervertebral disc electrochemical characterization and tissue-sensor

interactions J Biomed Eng 1991 13(4) p 304-312

228 Chen PH and RL McCreery Control of electron transfer kinetics at glassy carbon electrodes by specific

surface modification Analytical Chemistry 1996 68(22) p 3958-3965

229 Beilby AL TA Sasaki and HM Stern Electrochemical Pretreatment of Carbon Electrodes as a

Function of Potential Ph and Time Analytical Chemistry 1995 67(5) p 976-980

230 Dai H and K Shiu Voltammetric studies of electrochemical pretreatment of rotating-disc glassy carbon

electrodes in phosphate buffer Journal of electroanalytical chemistry and interfacial electrochemistry 1996

419(1) p 7-14

231 Musameh M N Lawrence and J Wang Electrochemical activation of carbon nanotubes

Electrochemistry communications 2005 7(1) p 14-18

232 Sljukic B CE Banks and RG Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

233 Tammeveski K et al Surface redox catalysis for O-2 reduction on quinone-modified glassy carbon

electrodes Journal of Electroanalytical Chemistry 2001 515(1-2) p 101-112

234 Yang HH and RL McCreery Elucidation of the mechanism of dioxygen reduction on metal-free carbon

electrodes Journal of the Electrochemical Society 2000 147(9) p 3420-3428

235 Regisser F et al Randomly oriented graphite electrode 1 Effect of electrochemical pretreatment on the

electrochemical behavior and chemical composition of the electrode Journal of electroanalytical chemistry

and interfacial electrochemistry 1996 415(1-2) p 47-54

236 Shleev S et al Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic

graphite electrode Electrochemistry Communications 2004 6(9) p 934-939

237 Mano N F Mao and A Heller A miniature biofuel cell operating in a physiological buffer Journal of the

American Chemical Society 2002 124(44) p 12962-12963

6

ACKNOWLEDGEMENTS

I would like to express deepest gratitude to my supervisor Dr Danny OrsquoHare

for his guidance on and patience for my research Frankly speaking the project on

glucose-oxygen fuel cells was very challenging and I came to realise the hard reality of

creating new clean energy despite of the world huge demand for such energy in a time

of global warming I could not help but feel that the research was turning into a

wild-goose chase however there was much to learn and gain from the experience here

For that and the people I met during this time I feel deeply grateful for the opportunity

given and I am convinced that this will enlighten my life

I would like to specially thank Dr Pei Ling Leow and Mr Parinya Seelanan for

their sincere support understanding and encouragement Without their help I would not

have been able to achieve my PhD I would like to thank to the current and past

members of Biosensor group Ms Nitin Bagha Dr Christopher Bell Dr Eleni Bitziou

Mr Jin-Young Kim Mr Wei-Chih Lin Dr Beinn Muir Mr Raphael Trouillon Dr

Martyn Boutelle Mr Yao-Min Chang Dr Delphine Feuerstein Ms Michelle Rogers

Dr Costas Anastassiou Dr Arun Arora Dr Martin Arundell Dr Catherine Dobson Dr

Melanie Fennan Dr Severin Harvey Dr Alex Lindsay Dr Bhavik Patel Dr Hong

Zhao Dr Emma Corcoles and Dr Parastoo Hashemi for their support during research

and sharing the wonderful time for many occasions I would like to thank Mr David

Roche for his sincere devotion to our work and being a great lab company I would like

to give my warm thanks to Dr Andrew Bond Dr Stephanie Cremers Ms Christina

Hood Dr Asimina Kazakidi Dr Peter Knox Dr Bettina Nier Ms Eileen Su Mr

Che-Fai Yeong Mr Krause Oliver and Dr Clifford Talbot for appropriate support fun

7

and encouragement I would also like to thank Dr Kate Hobson Mr Martin Holloway

Mr Ken Keating Mr Alan Nyant Mr Richard Oxenham Ms Paula Smith Ms Dima

Cvetkova Dr Anna Thomas-Betts and Mr Nick Watkins for their academic and

laboratory support Finally I would like to say thank you to all my family for their trust

and unconditional support my teacher Prof Fukuyama my friends Dr Pieta

Nasanen-Gilmore Jingzi Kofi Mariama Masa Maya Miki Pedro Sandra Sascha and

Wei Fong for being always close supportive and encouraging even when we were apart

8

CONTENTS

ABSTRACT 3

ACKNOWLEDGEMENTS 6

CONTENTS 8

LIST OF ABBREVIATIONS 15

LIST OF FIGURES 20

LIST OF TABLES 26

LIST OF EQUATIONS 27

1 INTRODUCTION 31

11 INTRODUCTION TO FUEL CELLS 31

111 History of the fuel cells 31 112 Principles of the fuel cells 33 113 Electric power from the fuel cells 35

12 CATALYSTS 37

121 Faradayrsquos second law ndash Current and reaction rate37 122 Transition state theory Eyring equation and reaction rate 38 123 Overcoming the activation free energy 40 124 Variations in catalysts42

13 ELECTRODE POTENTIAL AND REACTION RATE 44

131 Fermi level electrode potential and electron transfer 44 132 Electrode potential equilibrium potential and Nernst equation47

9

133 Electrode potential and activation free energy 50 134 Potential shift and standard rate constant 52 135 Butler-Volmer equation overpotential and current 53 136 Butler-Volmer equation vs Tafel equation 55

14 OVERPOTENTIAL AND FUEL CELLS 57

141 Four overpotentials that decrease the EMF57 142 Internal current 58 143 Activation overpotential58 144 Diffusion overpotential 59 145 Resistance overpotential60 146 Total overpotential and cell voltage 62

15 BIOFUEL CELLS 63

151 Microbial and enzymatic fuel cells 63 152 Enzyme fuel cells and mediators64

16 MINIATURISED MEDICAL POWER SOURCES 66

161 Realisation of miniaturised fuel cells and medical power supply 66 162 Miniaturised potential medical power sources67

17 OUTLINE OF THE WORK 69

171 Introduction to miniaturised glucose-oxygen fuel cells 69 172 Outline71

2 ANODE INTRODUCTION 73

21 GLUCOSE OXIDATION ENZYMES 73

22 DESIGNING ENZYME ELECTRODES 77

221 Physical adsorption 78 222 Reconstituting apo-enzyme78 223 Tethered Osmium-complexes linked to conductive hydrogel 80 224 Entrapment in the conductive polymer 81 225 Bulk modification83

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties 84 226 Enzyme covalent attachment and mass loading 88

23 PROTEIN INACTIVATION AND STABILISATION 90

10

24 PROTEIN DESTABILISATION 92

241 Deamidation 92 242 Oxidation93 243 Proteolysis and racemisation94 244 Physical instabilities95

25 ENZYME STABILISERS 95

251 Polyehyleneimine (PEI) 96 252 Dithiothreitol (DTT)97 253 Polyethylene glycol (PEG)98 254 Polyols99 255 FAD100

26 ENZYME KINETICS AND ELECTROCHEMISTRY 101

261 Michaelis-Menten kinetics 101 262 Electrochemistry and Michaelis-Menten kinetics 105 263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics108

2631 Lineweaver-Burk linear regression 108 2632 Eadie-Hofstee linear regression 109 2633 Electrochemical Hanes-Woolf linear regression 110

264 Cornish-Bowden-Wharton method 112 265 Nonlinear regression methods113

3 ANODE MATERIAL AND METHOD115

31 ELECTRODE FABRICATION 115

311 TTF-TCNQ synthesis115 312 Electrode template fabrication 115 313 Electrode composite fabrication117

3131 Composite for conductance study 117 3132 Bulk modified GOx enzyme electrode composite 118 3133 GOx enzyme electrode composite with enzyme stabilisers 119

314 Electrode wiring and insulation120

32 CONDUCTIVITY EXPERIMENT 121

33 POLISHING COMPOSITE ELECTRODES 122

34 GLUCOSE AMPEROMETRY 123

341 Glucose solution123

11

342 Electrochemical system123 343 Glucose amperometry with GOx electrodes 124

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA 125

4 ANODE RESULTS AND DISCUSSION 127

41 AIMS OF ANODE STUDIES 127

42 CONDUCTANCE OF THE COMPOSITE 128

421 Conductance of various conductive materials128 422 Influence of enzyme concentration on conductivity 130

43 CATALYTIC ACTIVITY OF GOX COMPOSITE ELECTRODES 132

431 Influence of GOx concentration on the catalytic activity of GOx electrodes132 432 Influence of O2 on the catalytic activity of GOx electrodes134

44 THE EFFICACY OF GOX STABILISERS 139

441 Introduction139 442 PEI140 443 DTT142 444 PEG 143 445 GLC144 446 FAD146 447 PEI and DTT mixture147 448 PEI and FAD mixture149 449 Most effective stabiliser content for GOx anodes 150

45 CONCLUSION TO ANODE STUDY 152

5 CATHODE INTRODUCTION 155

51 OXYGEN REDUCTION REACTION (ORR) 155

52 REACTIVITY OF OXYGEN 157

53 ORR CATALYSTS 159

531 Noble metal ORR catalysts 160 532 Macrocycle and Schiff-base complex ORR catalysts 165

12

533 Multicopper enzymes as ORR catalyst 168 5331 BOD 171 5332 Lac 173 5333 CueO 173

534 Anthraquinone ORR catalyst 174

54 CARBON PROPERTIES 177

541 Graphite180 542 Glassy carbon (GC)181 543 Activated carbon (AC) 182 544 Carbon fibre (CF)182 545 Carbon nanotubes (CNTs)183 546 Boron-doped diamond (BDD)184

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON 185

551 Electrochemical properties of carbon185 552 Electrochemical pretreatments (ECPs) of the carbon185

56 ELECTROCHEMICAL TECHNIQUES FOR ORR 187

561 Cyclic voltammetry (CV)188 562 Non-faradic current and electric double layer capacitance 189 563 Diagnosis of redox reactions in cyclic voltammetry 190 564 Coupled reactions in cyclic voltammetry194 565 Voltammograms for the surface-confined redox groups 195 566 Hydrodynamic voltammetry (HDV) and Levich equation197 567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the charge

transfer rate constant 200 568 Finding the standard rate constant (kdeg)205 569 Summary of the electrochemical techniques206

57 SUMMARY OF THE CHAPTER 207

6 CATHODE MATERIAL AND METHOD 208

61 ELECTRODE FABRICATION 208

611 Graphite PtC and RhC electrodes208 612 Anthraquinone(AQ)-carbon composite electrodes208

6121 Derivatisation of graphite with AQ 208 6122 Fabrication of AQ-carbon composite electrode 209

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes209

13

6131 Information on synthesis of metallosalophen 209 6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes 210

62 COMPOSITE RDE CUTTING 210

63 ELECTRODE POLISHING 210

631 Composite RDE polishing210 632 Glassy carbon electrode polishing211

64 ENZYME RDES 212

641 Membrane preparation 212 642 Membrane enzyme electrodes212

65 ELECTROCHEMICAL EXPERIMENTS 213

651 Electrochemical system213 652 Electrode pretreatments214

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes 214 6522 Pretreatments of AQ-carbon PtC and RhC 215

653 Electrochemical experiments 215 654 Data analysis 216

7 CATHODE RESULTS AND DISCUSSION 218

71 AIMS OF THE CATHODE STUDIES 218

72 INFLUENCE AND EFFICACY OF ECPS 219

721 Glassy carbon electrodes (GCEs)219 7211 Untreated GCEs 219 7212 Influence of electrochemical pretreatments on GCE 220 7213 Effect of GCE pretreatment on ORR 227

722 Graphite composite electrodes 230 7221 Untreated graphite composite electrodes 230 7222 Influence of electrochemical pretreatments on graphite composite electrodes 232 7223 The effect of graphite composite electrode pretreatment on ORR 235

723 Platinum on carbon composite electrodes (PtC) 240 724 Rhodium on carbon composite electrodes (RhC)240

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR 241 725 Co(salophen) composite electrodes243

7251 Untreated Co(salophen) composite electrodes 243 7252 Influence of electrochemical pretreatments on Co(salophen) composite 244 7253 The effect of Co(salophen) composite electrode pretreatment on ORR 246

726 Fe(salophen) composite electrodes 248 7261 Untreated Fe(salophen) composite electrodes 248 7262 Influence of electrochemical pretreatments on Fe(salophen) composite 250

14

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR 252 727 Anthraquinone (AQ) composite electrodes253 728 Lac and BOD enzyme membrane electrodes 255

73 MOST EFFICIENT ORR CATALYSTS 257

731 Best five electrodes for nα 258 732 Best five electrodes for kdeg 259 733 Best five electrodes for Epc2(HDV) 260 734 Best three candidates for ORR catalytic electrodes 260

74 CONCLUSION TO CATHODE STUDY 262

8 CONCLUSION AND FURTHER STUDIES 264

9 BIBLIOGRAPHY 268

15

LIST OF ABBREVIATIONS

Α Transfer coefficient

A Electrode surface area

ABTS 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

AC Activated carbon

AFC Alkaline fuel cell

AQ Anthraquinone

Asn Asparagine

Asp Aspartic acid

BDD Boron-doped diamond

BOD Bilirubin oxidase

C Reactant concentration

C O (0t) Electrode surface oxidant concentration

C R (0t) Electrode surface reactant concentration

C(0t) Electrode surface concentration

C Bulk concentration

Cd Double layer capacitance

CF Carbon fibre

CNH Carbon nanohorn

CNT Carbon nanotubes

Co(salen) N-N-bis(salicylaldehyde)-ethylenediimino cobalt(II)

Co(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminocobalt (II)

CO Bulk oxidant concentration

CR Bulk reactant concentration

CueO Copper efflux oxidase

CV Cyclic voltammetry

Cys Cysteine

(DEAE)-dextran Diethylaminoethyl-dextran

DET Direct electron transfer

DHG Dehydrogenase

DMFC Direct methanol fuel cell

DTE Dithioerythriotol

DTT Dithiothreitol

[E0] Initial enzyme concentration

16

E Arbitrary electrode potential Enzyme

Edeg Standard electrode potential

Edeg Formal potential

Ea Anode potential

EC Electrochemical-chemical

Ec Cathode potential

Ecell Cell voltage

ECP Electrochemical pretreatment

Ee Electrode equilibrium potential

EF Fermi potential

EMF Electro motive force

Ep2 Half-peak potential

Epa Anodic peak potential

Epc Cathodic peak potential

Epc2(HDV) Half-wave reduction potential in HDV

ES Enzyme-substrate complex

Eλ Switching potential

∆E Potential difference Potential shift

∆Edeg Standard peak separation

δEp Peak width

εF Fermi energy

F Faraday constant

FAD Flavin adenine dinucleotide

Fe(salophen) N-N-bis(salicylaldehyde)-12-phenylenediiminoiron (II)

GC Glassy carbon

GCE Glassy carbon electrode

GHP 1-(6-D-gluconamidohexyl) pyrrole

GLC Glycerol

Gln Glutamine

GOx Glucose oxidase

GOx(O) Oxidised GOx

GOx(R) Reduced GOx

GSH Gluthatione

∆G Gibbs free energy shift

∆Gdeg Standard Gibbs free energy change

∆GdegDagger Standard activation free energy

17

∆GDagger Gibbs free energy of activation

∆GadegDagger Anode standard activation free energy

∆GaDagger Anode activation free energy

∆GodegDagger Oxidation standard activation free energy

∆GoDagger Oxidation activation free energy

∆GRdegDagger Reduction standard activation free energy

∆GRDagger Reduction activation free energy

h Planck constant

HDA Hydrodynamic amperometry

HDV Hydrodynamic voltammetry

His Histidine

HOMO Highest occupied molecular orbital

∆Hdeg Standard enthalpy change

I Electric current Net current

I0 Exchange current

Ia Anodic current

Ic Cathodic current

Id Disc limiting current

IK Kinetic current

IL Limiting current Diffusion-limited current

Im Maximum steady state limiting current

Ip Peak current

Ipa Anodic peak current

Ipc Cathodic peak current

I-V Current-Voltage

J Current density

Jm Maximum limiting current density

k Rate constant

K Equilibrium constant

k(E) Rate constant at the applied potential E

kdeg Standard heterogeneous rate constant

kdegb Backward reaction standard rate constant

kdegf Forward reaction standard rate constant

KDagger The equilibrium constant between the activated complexes and the reactants

k1 Rate constant for ES formation from E and S

k-1 Rate constant for ES dissociation into E + S

18

k2 Rate constant for P formation from ES

kB Boltzmann constant

kb Backward reaction rate constant

kf Forward reaction rate constant

kf(E) Rate constant for forward reactions at the applied potential E

KM Michaelis enzyme dissociation constant

Lac Laccase

LDH Lactate dehydrogenase

LMCT Ligand-to-metal charge transfer

LPG Liquefied petroleum gas

LUMO Lowest unoccupied molecular orbital

MCO Multicopper oxidase

MDH Methanol dehydrogenase

Me2SQ Dimethylmercaptobenzoquinone

MEMS Microelectromechanical systems

MeSQ Methylmercapttobenzoquinone

MET Mediated electron transfer

Met Methionine

mPQQ-GDH Membrane-bound PQQ dehydrogenase

MWNT Multi-walled nanotube

n Overall number of electrons transferred

na The number of electrons transferred up to the rate limiting step

NAD Nicotinamide adenine dinucleotide

NAD-GDH Nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

OCV Open circuit voltage

ORR Oxygen reduction reaction

P Electric power Product

PAFC Phosphoric acid fuel cell

PBS Phosphate buffer saline

PC Pyrolytic carbon

PDMS Polydimethylsiloxane

PEFC Polymer electrolyte fuel cell

PEG Polyethylene glycol

PEI Polyethyleneimine

P-I Power-Current

POx Pyranose oxidase

19

PQ Phenanthrenequinone

PQQ pyrrolo-quinoline quinone

PQQ-GDH Quinoprotein glucose dehydrogenase

Pro Proline

PtC Platinum on carbon

Q Charge

RDE Rotating disc electrode

RhC Rhodium on carbon

RLS Rate limiting step

rpm Rotaion per minute

S Substrate

SEN Single-enzyme nanoparticle

sPQQ-GDH Soluble PQQ dehydrogenase

SQ Mercaptobenzoquinone

SWNT Single-walled nanotube

∆Sdeg Standard entropy change

t Time

T1 Type I (blue Cu in Lac)

T2 Type II ( trinuclear Cu in Lac)

T3 Type III (trinuclear Cu in Lac)

TCNQ 7788-Tetracyanoquinodimethane

TEM Transmission electron microscopy

Trp Tryptophan

TTF Tetrathiafulvalene

TTF-TCNQ Tetrathiafulvalene 7788-tetracyanoquinodimethane

Tyr Tyrosine

V Voltage Maximum limiting catalytic velocity

VHT Vacuum heat treatment

η Overpotential

ν Vibrational frequency Kinematic viscosity

υ Reaction rate Scan rate Enzyme catalytic rate

ω Angular velocity

20

LIST OF FIGURES

Fig No Page Description

Fig 1 32 Historical fuel cells

Fig 2 33 Photographs of modern fuel cells

DMFC Cogeneration system Hydrogen car Glucose-Oxygen fuel cell

Fig 3 34 The schema of glucose-oxygen fuel cell

Fig 4 41 Transition state diagram with the reaction coordinate

Fig 5 45 Fermi level and electron transfer

Fig 6 46 Fermi level and electron transfer at an arbitrary electrode potential

Fig 7 48 Standard free energy change along the electrode potential at

(a) E = Edeg (b) E gt Edeg and (c) E lt Edeg

Fig 8 51 The shift in ∆GdegDagger accompanied by ∆E

Fig 9 57 Tafel plot

Fig 10 59 Activation overpotential vs current

Fig 11 60 Diffusion overpotential vs current

Fig 12 61 Resistance overpotential vs current

Fig 13 62 V-I curve for a typical low temperature fuel cell

Fig 14 65 Mediated electron transfer reaction by the enzyme electrode

Fig 15 70 The schema for a glucose-oxidising anode

Fig 16 75 The structure and redox reactions of FAD NAD and PQQ

Fig 17 79 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode

Fig 18 81 GOx wired to conductive hydrogel complexed with Os

Fig 19 83 Monomer structures of conductive polymers

1-(6-D-gluconamidohexyl) pyrrole and Mercapto-p-benzoquinone

Fig 20 85 TTF-TCNQ structure and its charge transfer

Fig 21 86 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 20 mVs-1

Fig 22 89 The procedure for the enzyme mass-loaded biocatalytic nanofibre

Fig 23 90 The fabrication procedure of single-enzyme nanoparticles

Fig 24 92 The chemical structures of the side groups of Gln and Asn

Fig 25 93 The chemical structures of the side groups of Cys Met Tyr Trp and His

Fig 26 95 The chemical structures of Asp and Pro

Fig 27 96 The structure of PEI

Fig 28 98 The structures of DTT from a linear to a cyclic disulfide form

Fig 29 98 The structures of PEG

21

Fig 30 100 The structure of glycerol

Fig 31 104 A typical Michaelis-Menten hyperbolic curve of the enzyme

Fig 32 107 Electrochemical Michaelis-Menten hyperbolic curve of the enzyme electrode

Fig 33 109 Electrochemical Lineweaver-Burk linear regression of the enzyme electrode

Fig 34 110 Electrochemical Eadie-Hofstee linear regression of the enzyme electrode

Fig 35 111 Electrochemical Hanes-Woolf linear regression of the enzyme electrode

Fig 36 113 Electrochemical Cornish-Bowden-Wharton plot of the enzyme electrode

Fig 37 116 The photographs of PDMS electrode templates

Fig 38 118 The photographs of empty and filled PDMS templates

Fig 39 119 The schema for bulk modification method

Fig 40 121 The photographs of insulated composite electrodes

Fig 41 122 The photographs of measuring electrode resistance

Fig 42 125 The photograph of glucose amperometry with a multichannel potentiostat

Fig 43 126 The analytical output by SigmaPlot Enzyme Kinetics

Fig 44 129 Conductance of various electrode composite

Fig 45 131 Conductance of GOx composite with varying GOx content

Fig 46 133 Difference in the glucose-response amperometric curve by the enzyme electrodes

containing various GOx content 5 10 and 15 in the absence of O2

Fig 47 134 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the absence of O2

Fig 48 135 (i) Difference in the glucose-response amperometric curve by 10 GOx composite

electrodes in the absence and presence of O2

(ii) The re-plotted amperometric curves in the presence of N2 air and O2

Fig 49 136 Kinetic parameters Jm and KM for enzyme electrodes with various GOx content

5 10 and 15 in the presence N2 O2 and air

Fig 50 140 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEI in the presence and absence of O2

Fig 51 142 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 DTT in the presence and absence of O2

Fig 52 145 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 PEG in the presence and absence of O2

Fig 53 146 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 GLC in the presence and absence of O2

Fig 54 147 Kinetic parameters Jm and KM for GOx electrodes

without and with 1 2 and 4 FAD in the presence and absence of O2

22

Fig 55 143 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-DTT

(051 11 1505 and 22) in the presence and absence of O2

Fig 56 148 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051 11

1505 and 22)

Fig 57 149 Kinetic parameters Jm and KM for GOx electrodes without and with PEI-FAD (21

22 and 24 ) in the presence and absence of O2

Fig 58 151 Jm KM in the absence and presence of O2 for various stabilisers

PEIDTT (11 0515 221505 ) and GLC 1

Fig 59 156 Oxygen reduction pathways 2 and 4 electron reduction pathways

Fig 60 157 Degradation of superoxide anion radical in aqueous solutions

Fig 61 158 An energy level diagram for ground state dioxygen molecule

Fig 62 159 Diagrams for the state of π orbitals in the ground state oxygen and peroxide anion

radical

Fig 63 162 Three models of oxygen adsorption Griffith Pauling and Bridge models

Fig 64 163 Models for oxygen adsorption on metals and ORR pathways

Fig 65 164 A volcano-shaped trend between ORR rate and oxygen binding energy of various

transition metals

Fig 66 165 Examples of transition metallomacrocycles Heme and copper phthalocyanine

Fig 67 166 Schiff base complexes

Co(II) salen Co(II) salophen and Co(III) salen conductive polymer

Fig 68 167 The molecular structure of dicobalt face-to-face diporphyrin [Co2(FTF4)]

Fig 69 168 ORR pathways by Co2(FTF4)

Fig 70 170 The structure of laccase from Trametes versicolor

Fig 71 171 Redox reaction cycle of MCOs with O2

Fig 72 172 Common mediators for BOD ABTS and Os complex

Fig 73 174 Two-electron ORR by anthraquinone

Fig 74 176 The structures of anthraquinone and its derivative Fast red AL salt

Fig 75 177 HDV of ORR at various quinone electrodes at pH 7

Unmodified GC AQGC and PQGC

Fig 76 178 The carbon structures graphite and diamond

Fig 77 179 The carbon sections basal and edge planes

Fig 78 181 A TEM image of graphene bilayer at 106 Aring resolution

Fig 79 182 A TEM image and a schema of GC

Fig 80 183 Computer graphic models of various CNTs Bundled SWNTs MWNT and CNHs

23

Fig 81 184 A TEM image of diamond

Fig 82 187 The CVs showing the influence of ECP on GCE at pH 7 in the presence and absence

of O2 The CVs for both pretreated and unpretreated GCEs are shown

Fig 83 188 The triangular waveform for CV and a typical voltammetric curves for BQ

Fig 84 189 The CV showing the double layer capacitance at graphite electrode surface at pH 7

Fig 85 194 A voltammogram examples of a quasi-reversible reaction at various scan rates

Fig 86 195 Voltammograms of two-electron transfer in the EE systems with different ∆Edegrsquo

Fig 87 196 A voltammogram of the reversible redox reaction by surface-confined groups

Fig 88 198 A diagram of hydrodynamic electrochemical method using a RDE

Fig 89 200 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH 74

Fig 90 201 Levich plot examples for reversible and quasi-reversible systems

Fig 91 203 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in oxygenated

001 M PBS at pH 74 at pretreated GCE

Fig 92 204 Koutecky-Levich plot for O2 reduction at a pretreated GCE in oxygenated 001 M PBS at

pH 74 with applied potential ndash 04 06 and 08 V

Fig 93 206 Log kf(E) plot for O2 reduction at various pretreated GCEs

Fig 94 207 The protocol for finding kinetic and thermodynamic parameters

Fig 95 211 The photographs of fabricated composite RDEs

Fig 96 213 The photograph of RDE experiment setting with a rotor and gas inlet

Fig 97 220 CVs of untreated GCEs at pH 74 in 001 M PBS in the absence and presence of O2

at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 98 221 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of O2

between + 09 and - 09 V vs AgAgCl at 005 Vs-1

(i) CVs for all the pretreated GCEs

(ii) CVs excludes PBS pretreatments

Fig 99 223 The bar graphs for the capacitance of GCEs in 015 M NaCl buffered with 001 M

phosphate to pH 74

Fig 100 225 Overlaid CVs during various pretreatments for GCEs involving

01 M H2SO4 alone 001 M PBS at pH 74 alone and both pretreatments

Fig 101 227 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M oxygenated

PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 102 229 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Fig 103 231 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M PBS in

the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

24

Fig 104 233 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M N2-sparged PBS

between + 09 and - 09 V vs AgAgCl

Fig 105 236 Overlaid CVs of ORR at various pretreated graphite composite electrodes at pH 74

in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 106 237 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at pH 74

in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various scan rates

50 100 200 and 500 mVs-1

Fig 107 238 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

Fig 108 239 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

Fig 109 241 CVs of untreated and H2SO4-pretreated RhC composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

Fig 110 242 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M PBS in

the absence and presence O2 at 005 Vs-1 between + 09 and - 09 V vs AgAgCl

Fig 111 243 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M PBS in the

absence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 112 245 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 113 247 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 114 248 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite electrodes

Fig 115 249 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74 in 001

M PBS in the absence and presence of O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

Fig 116 251 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 117 253 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Fig 118 254 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M PBS in

theabsence and presence of O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Fig 119 256 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated PBS at

005 Vs-1 between 0 and -09 V vs AgAgCl

Fig 120 257 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Fig 121 258 The bar graphs of the best five nα

25

Fig 122 259 The bar graphs of the best five kdeg

Fig 123 260 The bar graphs of the best five Epc2(HDV)

Fig 124 266 A photograph of the fuel cell stack made with GOx anodes and Co(salophen)

cathodes

26

LIST OF TABLES

Table No Page Description

Table 1 35 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Table 2 87 Electrochemical behaviour of TTF-TCNQ at pH 74

Table 3 104 Michaelis-Menten equation at various [S]

Table 4 117 Composite composition table

Table 5 119 Blended stabiliser composition

Table 6 139 Four possible causes of glucose amperometric current reduction

Table 7 152 Comparison of maximum output current and KM with some published data

Table 8 159 Bond orders bond lengths and bond energies of some dioxygen species

Table 9 180 The electric conductivity of various carbon forms and sections

Table 10 186 Various ECP examples

Table 11 192 CV diagnosis for reversible system at 25 ˚C

Table 12 193 CV diagnosis for irreversible system at 25 ˚C

Table 13 196 CV diagnosis for reversible system by surface-confined groups at 25 ˚C

Table 14 214 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Table 15 216 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC

Table 16 217 Parameter values for Koutecky-Levich equation

O2 diffusion coefficient O2 concentration and PBS kinematic viscosity

Table 17 261 Three candidate electrodes for high ORR catalysis

Table 18 261 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

27

LIST OF EQUATIONS

Eq No Page Description

Eq 1 35 Anode glucose oxidation reaction

Eq 2 35 Cathode oxygen reduction reaction

Eq 3 35 Overall glucose-oxygen fuel cell reaction

Eq 4 36 Electric power equation

Eq 5 36 Electric current equation in electrochemistry

Eq 6 38 Faradayrsquos second law of electrolysis Charge equation

Eq 7 38 Faradayrsquos second law of electrolysis Reaction equation

Eq 8 39 Transition state theory equation Rate constant equation

Eq 9 39 Transition state theory equation Equilibrium constant

Eq 10 39 Transition state theory equation Atomic vibration frequency equation

Eq 11 40 Transition state theory equation Erying equation

Eq 12 42 Gibbs free energy shift equation in thermodynamic terms

Eq 13 42 Gibbs free energy shift equation in electrochemical terms

Eq 14 49 A typical redox reaction

Eq 15 50 Nernst equation

Eq 16 50 Electrode potential shift

Eq 17 50 Free energy shift in electrochemical terms

Eq 18 52 Anodic oxidation activation free energy shift in terms of potential shift

Eq 19 52 Cathodic reduction activation free energy shift in terms of potential shift

Eq 20 53 Forward reduction rate constant equation in terms of kdeg and potential shift

Eq 21 53 Backward oxidation rate constant equation in terms of kdeg and potential shift

Eq 22 54 Butler-Volmer equation

Eq 23 54 Overpotential equation

Eq 24 54 Exchange current equation

Eq 25 55 Butler-Volmer equation in terms of electrode equilibrium constant

Eq 26 55 Butler-Volmer equation in terms of overpotential

Eq 27 56 Butler-Volmer equation for small overpotential

Eq 28 56 Tafel equation for large overpotential

Eq 29 60 Diffusion overpotential equation

Eq 30 61 Ohmrsquos law

Eq 31 63 Cell voltage equation

Eq 32 63 Gibbs free energy available from the fuel cell

28

Eq 33 73 Glucose oxidation reaction by GOx

Eq 34 73 Glucose oxidation reaction by NAD-DHG

Eq 35 87 TTF-TCNQ reaction At equilibrium state

Eq 36 87 TTF-TCNQ reaction TCNQ reduction at lt + 015 V vs AgAgCl

Eq 37 87 TTF-TCNQ reaction TCNQ lattice reduction at lt - 015 V vs AgAgCl

Eq 38 87 TTF-TCNQ reaction Anodic TCNQ salt formation at gt + 031 V vs AgAgCl

Eq 39 87 TTF-TCNQ reaction TTF lattice oxidation at gt + 035 V vs AgAgCl

Eq 40 87 TTF-TCNQ reaction TTF further oxidation at gt + 038 V vs AgAgCl

Eq 41 101 Enzyme kinetics A typical enzyme reaction

Eq 42 102 Enzyme kinetics Enzyme-substrate complex formation at steady state

Eq 43 102 Enzyme kinetics Rate of enzyme-substrate complex formation in terms of k-1 and k2

Eq 44 102 Enzyme kinetics

Enzyme-substrate complex equation in terms of [E0] k1 k-1 k2 and [S]

Eq 45 102 Enzyme kinetics Enzyme catalytic reaction

Eq 46 102 Enzyme kinetics Enzyme catalytic reaction equation

Eq 47 102 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of enzyme-substrate complex

Eq 48 103 Enzyme kinetics

Enzyme maximum catalytic rate V in terms of [E0] k1 k-1 k2 and [S]

Eq 49 103 Enzyme kinetics Michaelis-Menten constant definition

Eq 50 103 Michaelis-Menten equation

Eq 51 104 Michaelis-Menten equation when [S] ge KM

Eq 52 104 Michaelis-Menten equation when [S] = KM

Eq 53 104 Michaelis-Menten equation when [S] le KM

Eq 54 106 Electrochemical Michaelis-Menten equation

Eq 55 106 Electrochemical Michaelis-Menten equation for current density

Eq 56 108 Electrochemical Lineweaver-Burk linear regression equation

Eq 57 109 Electrochemical Eadie-Hofstee linear regression equation

Eq 58 111 Electrochemical Hanes-Woolf linear regression equation

Eq 59 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for Jm

Eq 60 112 Electrochemical Cornish- Bowden-Wharton non-parametric method for KM

Eq 61 128 Enzyme electrode specificity constant

Eq 62 139 The causes of anodic current reduction A typical GOx-O2 reaction

Eq 63 139 The causes of anodic current reduction TTF reduction by GOx

Eq 64 139 The causes of anodic current reduction GOx allosteric inhibition

Eq 65 139 The causes of anodic current reduction TTF oxidation by O2

29

Eq 66 139 The causes of anodic current reduction TTF oxidation by GOx

Eq 67 161 O2 Dissociative mechanism Simultaneous oxygen adsorption and molecular fission

Eq 68 161 O2 Dissociative mechanism Surface hydroxide formation

Eq 69 161 O2 Dissociative mechanism Oxygen reduction to water

Eq 70 161 O2 Associative mechanism Oxygen adsorption

Eq 71 161 O2 Associative mechanism Peroxide formation

Eq 72 161 O2 Associative mechanism Peroxide reduction

Eq 73 161 O2 Associative mechanism Surface oxygen reduction to hydroxide

Eq 74 161 O2 Associative mechanism Surface hydroxide reduction to water

Eq 75 175 Anthraquinone O2 reduction Semiquinone anion radical formation

Eq 76 175 Anthraquinone O2 reduction O2 reduction by a semiquinone anion radical

Eq 77 175 Anthraquinone O2 reduction Spontaneous superoxide reduction to O2

Eq 78 175 Anthraquinone O2 reduction

Spontaneous superoxide reduction to hydroperoxide

Eq 79 190 Electric double layer capacitance per unit area

Eq 80 192 CV diagnosis ndash Reversible system Ip vs υfrac12

Eq 81 192 CV diagnosis ndash Reversible system Ep

Eq 82 192 CV diagnosis ndash Reversible system ∆Ep

Eq 83 192 CV diagnosis ndash Reversible system δEp

Eq 84 192 CV diagnosis ndash Reversible system Reversal parameter

Eq 85 193 CV diagnosis ndash Irreversible system Ip vs υ

Eq 86 193 CV diagnosis ndash Irreversible system Ep a function of kdeg α and υ

Eq 87 193 CV diagnosis ndash Irreversible system ∆Ep

Eq 88 193 CV diagnosis ndash Irreversible system δEp

Eq 89 195 CV diagnosis ndash EE system Peak separation of EE system

Eq 90 196 CV diagnosis ndash Surface-confined groups Ip vs υ

Eq 91 196 CV diagnosis ndash Surface-confined groups Ep

Eq 92 196 CV diagnosis ndash Surface-confined groups ∆Ep2

Eq 93 198 Diffusion layer thickness in terms of ω

Eq 94 199 Levich equation in terms of ω

Eq 95 199 Levich equation in terms of diffusion layer thickness

Eq 96 202 Koutecky-Levich equation

Eq 97 204 The rate constant for arbitrary potential E

Eq 98 205 k(E) in terms of kdeg and η for a forward reaction

Eq 99 205 kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

Eq 100 238 Surface semiquinone radical formation

30

Eq 101 238 ORR by surface semiquinone radical

31

1 INTRODUCTION

11 INTRODUCTION TO FUEL CELLS

111 History of the fuel cells

Fuel cells are electrochemical devices which directly convert fuel into

electricity via chemical reactions The invention dates back to 19th century when a

British physicist Sir William Grove and a German-Swiss chemist Christian Schoumlnbein

discovered the reverse reaction of water electrolysis The current output by this system

was however very small Therefore the discovery stayed in the shadow of the steam and

internal combustion engines and it needed another century to gain its attention

In the 1930s a British engineer Francis Bacon started working towards

alkaline fuel cells (AFCs) and in the 1950s General Electric developed polymer

electrolyte fuel cells (PEFCs) Those fuel cells were loaded on the spaceships in the

1960s by NASA because they were compact provided clean power with large energy

density and generated drinkable water as a by-product [1-5] Since then fuel cells have

been continuously used for space and marine technologies [3] In the 1970s studies on

fuel cells were further expanded for consumer applications [1 3 5 6] The first

air-breathing fuel cell hybrid car was built by Kordesch [1 7] and phosphoric acid fuel

cells (PAFCs) were developed and introduced to offices and hospitals [2 3 5]

32

Fig 1 Some historical fuel cell applications (i) Alkaline fuel cell for Apollo 11 [8] (ii)

Kordeschrsquos Austin A40 city hybrid car [7]

Since the 1990s fuel cells have been drawing keen attention as flexible renewable

energy in conjunction with the depletion of fossil fuel global warming and the demand

in cleaner energy [6 9] Great effort has been made to miniaturise fuel cells for

individual users such as mobile devices vehicles and households [1 3 5 6] The recent

advance in such technologies has led to direct methanol fuel cells (DMFCs) for

mobile-phone handsets [10 11] cogeneration systems to supply household power [1 5]

hydrogen fuel cell cars [12-15] and bio-batteries running on glucose and air oxygen for

portable music players [16-18] Glucose-oxygen biofuel cells have been also further

miniaturised for medical implantable purposes [18 19]

33

Fig 2 Modern fuel cell technologies (i) Toshibarsquos world smallest DMFC [20] (ii)

TokyoGasrsquos cogeneration system Enefarm (iii) BMWrsquos Hydrogen 7 (iV)

Sonyrsquos Bio Battery generating electricity from sugar [16]

112 Principles of the fuel cells

A fuel cell unit simply consists of an anode a cathode electrolyte and a

conducting wire A conducting wire connects an anode and a cathode to form an

external circuit This allows coupling of electrochemical reactions taking place at each

electrode and provides a conductive path for electrons between them Electrolyte

allows charged species to move between two electrodes but blocks out the entry of

electrons This completes the electric circuit and permits electric conduction At the

anode fuel is oxidised by an oxidation reaction and this supplies electrons to the

cathode through the external circuit The number of electrons generated depends on the

34

type of fuels reactions and environment in which the reactions take place In the anode

reaction ions are also released to balance overall charge They move to the cathode

though the electrolyte At the cathode ions from the electrolyte and electrons from the

circuit meet and react together with an oxidiser supplied The completion of the coupled

reactions generate electricity and products [3 4 21] Fig 3 shows a glucose-oxygen

fuel cell as an example

Fig 3 A glucose-oxygen fuel cell

At the anode glucose is oxidised into gluconolactone This gives out two electrons and

two protons At the cathode oxygen gas receives them and is reduced to water Each

reaction has a unique redox potential called standard electrode potential Edeg and

electromotive force (EMF) is the potential difference between such electrode potentials

It is also called open circuit voltage (OCV) This is the theoretical size of potential

difference available from the fuel cell [3 4] The coupled reactions for glucose-oxygen

fuel cell is shown in Table 1 Each redox potential was measured against silver

35

silver-chloride reference electrode (AgAgCl) at pH 7 25degC therefore shown in formal

potential (Edegrsquo)

Table 1 Glucose-oxygen coupled reactions at pH 7 25 degC vs AgAgCl

Anode Edegrsquo = - 042 V (Eq 1)

(via FADFADH2 redox [22 23])

Cathode [24] Edegrsquo = 062 V (Eq 2)

Overall ∆Edegrsquo = 104 V (Eq 3)

The reaction potential at each electrode is measured separately as half cell potential

against the universal reference electrode such as AgAgCl because it is impossible to

directly measure both inner potentials simultaneously

113 Electric power from the fuel cells

Electric power (P) is the amount of work can be handled by electric current per

unit time It is defined as in Eq 33 The size of power is determined by the size of

electric current (I) and voltage (V) since P is proportional to I and P

36

Electric power

VIP times= middotmiddotmiddotmiddotmiddot Eq 4

P = Electric power W

I = Electric current A

V = Voltage V

Electric current is the amount of charge (Q) flows per unit time Voltage is the electric

potential energy per unit charge and synonymous with potential difference (∆E) EMF

or OCV Increase in I andor V increases P V is however constrained by the type of

electrode reactions as discussed previously I depends on (i) reaction rate (ii) the

electrode surface and (iii) reactant concentration as shown in Eq 5

Electric current in electrochemistry

nFAkCnFAI == υ middotmiddotmiddotmiddotmiddot Eq 5

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

A = Electrode surface area m-2

υ = Reaction rate (1st or pseudo 1st order) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

It is indispensable to have large rate constant to increase output current This demands

lowering activation energy for a chemical transition during the reaction and can be

achieved by use of the catalysts

37

12 CATALYSTS

121 Faradayrsquos second law ndash Current and reaction rate

Catalysts accelerate specific chemical reactions without altering the change in

overall standard Gibbs free energy In other words they change the reaction kinetics but

not the thermodynamics They only provide an alternative lower-energy pathway to

shorten the reaction time without changing themselves before and after the reaction

[25-27] In fuel cells catalysts are used to increase the current output by accelerating the

electrode reaction The relationship between current and rate of reaction is defined by

Faradayrsquos second law of electrolysis (Eq 5 - Eq 7) This expresses the size of electric

charge generated in terms of the number of moles of reactant consumed or product

produced

38

Faradayrsquos second law of electrolysis

nFNQ = middotmiddotmiddotmiddotmiddot Eq 6

AtNCktimes

=times=υ middotmiddotmiddotmiddotmiddot Eq 7

Q = Charge C

n = The number of electrons transferred

F = Faraday constant 96485 Cmol-1

N = The number of moles of reactant or product mol

υ = Reaction rate (1st or pseudo 1st ordedr) molm-3s-1

k = Rate constant (1st or pseudo 1st order) s-1

C = Reactant concentration molm-3

t = Time s

A = Electrode surface area m-2

Faradayrsquos second law of electrolysis states that an increase in reaction rate increases the

current output To increase the reaction rate intrinsically its Gibbs free energy of

activation (∆GDagger) must be lowered The relationship between k and ∆GDagger is stated in

Erying equation [28]

122 Transition state theory Eyring equation and reaction rate

When a reaction proceeds it must go through a critical state called transition

state of maximum potential energy also called the activated complex Activated

complexes are unstable molecules existing for only a few molecular vibrations before

decaying into the products Transition state theory assumes that the reactants and the

39

activated complexes are in quasi-equilibrium and the reaction rate is constrained by the

rate at which the activated complex decays into the products This implies that the

reaction rate depends on the equilibrium constant between the activated complexes and

the reactants (KDagger) and the atomic vibrational frequency (ν) as shown in Eq 9 ν is

defined by statistical mechanics in terms of Boltzmann constant (kB) Planck constant

(h) and the temperature (Eq 10) [28]

Transition state theory and reaction rate [28]

[ ]RTexpDagger Dagger

Dagger GC

CK ∆minus== middotmiddotmiddotmiddotmiddot Eq 8

Dagger Kk ν= middotmiddotmiddotmiddotmiddot Eq 9

hTkB=ν middotmiddotmiddotmiddotmiddot Eq 10

KDagger = Equilibrium constant between the reactants and the activated

complexes

s-1

CDagger = Activated complex concentration molm-3

C = Reactant concentration molm-3

∆GDagger = Gibbs free energy of activation Jmol-1

R = Gas constant = 8314 Jmol-1K-1

T = Thermodynamic temperature K

k = Rate constant (1st or pseudo 1st reaction) s-1

ν = Atomic vibrational frequency s-1

kB = Boltzmann constant = 13807 x 10-23 JK-1

h = Planck constant = 662621 x 10-34 Js

40

Based on this the Erying equation expresses the relationship between the reaction rate

and Gibbs activation free energy as shown in Eq 11 [25 28 29]

Erying equation TG

B

hTkk

RDagger ∆minus

= κ middotmiddotmiddotmiddotmiddot Eq 11

where κ = transmission coefficient

This tells that to increase the rate constant reaction temperature must be increased or

the activation free energy must be lowered

123 Overcoming the activation free energy

Activation free energy is a potential energy gap between the reactant and

activated complex At the transition state intra-molecular reorganisation takes place

through the bond destruction and formation This demands the energy and slows the

kinetics The reaction does not proceed unless this energy barrier is overcome Hence

energy input in someway or lowering activation free energy is required for the reaction

to proceed andor accelerate the reaction Heat energy can provide atomic and molecular

kinetic energy so that the system can overcome the energy barrier as indicated in Eq 11

[4 21 29] Applying electric potential also has the similar effect It provides the electric

energy to overcome the energy barrier The rate constant therefore can be altered

extrinsically by temperature andor electrode potential [21 29 30] Alternatively

catalysts can reduce ∆GDagger so that the reaction rate can be increased intrinsically They

enable or accelerate a reaction by providing alternative lower-energy reaction pathway

41

This does not change the reaction path or the standard enthalpy change (∆Hdeg) of the

system however lowers the activation free energy for this path so that the reactants can

be converted more quickly to the products as shown in Fig 4

Fig 4 Energy transition for catalytic reaction through the transition state

Fig 4 shows the energy state transition during a reaction with and without a catalyst

The reaction coordinate represents the reaction progress along the reaction pathway

∆Hdeg is a state function [31] therefore determined only by the initial and final state of

the system but independent of the reaction path ∆Hdeg is divided into standard Gibbs free

energy change (∆Gdeg) and the term of standard entropy change (∆Sdeg) as Eq 12 shows [4

21 23 26 32]

The key insight therefore from transition state theory is that for a catalyst to function it

42

must spontaneously bind the transition state

Gibbs free energy change

deg∆minusdeg∆=deg∆ STHG middotmiddotmiddotmiddotmiddot Eq 12

deg∆minus= EnF middotmiddotmiddotmiddotmiddot Eq 13

∆Gdeg is the standard free energy difference which became available for work through

the reaction and must be lt 0 for a spontaneous reaction ∆Gdeg can be related to the

standard electrical potential difference as Eq 13 shows [4 21 29 30 33 34] It is the

maximum EMF theoretically available from the system ∆Sdeg is the difference in the

degree of disorder in the system The entropy term (T∆Sdeg) is the energy not available for

work and temperature dependent This and Eq 13 imply that ∆Gdeg is also altered by

temperature as shown in Fig 4 [35]

124 Variations in catalysts

Given the general applicability of transition state theory it is not surprising that

catalysts have extensive variation and applications the reaction phase can be

homogeneous or heterogeneous catalysts can be inorganic organic or biological

Inorganic catalysts are metals such as transition or noble metals or complexes

on metal ions in various oxidation states Elemental metal catalysts typically catalyse

reactions through surface adsorption Metal particles have extensive variation in

physical properties such as particle size shape and surface area They often come in

dispersed on the porous support material for improved catalytic performance due to

43

increased surface area [36] and cost efficiency [1] Composition and structure of the

particles thickness of support material and condition of metal deposition all affect

reactions [27 37-39] A complex is a compound in which a ligand is loosely bonded to

a metal centre through coordination bonds An unsaturated complex has a dissociated

ligand and catalysis takes place when a substrate binds to that in the single orientation

[27 40] Complexes also have important roles in biological system [41-43] Organic

catalysts are non-metal non-biological catalysts They are often functionalised solid

surfaces for surface catalysis [27] In electrochemistry such catalysts can be chemically

bonded to conductive material [44] Simple electrochemical pretreatment can generate

or attach chemical groups onto the electrode surface and they mediate electron transfer

[45-47] Biological catalysts are generally called enzymes Enzymes are biological

polymers mostly made of protein and usually have high reaction and substrate

specificity They often have cofactors which are not proteinous but essential for their

catalytic activities Enzymes are involved in virtually all biological activities [23 25 27

48] and in a few industrial applications due to their substrate specificity [27]

Catalysts are by definition not supposed to be consumed in their reactions

however they could lose their catalytic power Impurities fouling and poisoning are

common deactivating factors for the metal catalysts Competing reactions also convert

them into inactive forms Particle migration and agglomeration reduce their catalytic

surface area hence their overall activity [27] Biological catalysts are very sensitive to

the environment Slight changes in pH and temperature can deactivate enzymes Their

stability is also affected by presence of metal ions surfactants oxidative stress and

organic solvent molecules [49] Metal catalysts can be possibly regenerated by

redispersion [27] however it is very difficult to recover biological catalytic functions

44

Therefore enzyme stabilisers are often added to extend their life Protein engineering

also can confer advantageous properties to the enzymes Amino acid sequences leading

to favourable protein folding functions or stability are determined in silico and they are

introduced to existing enzymes by genetic engineering such as site-directed mutagenesis

Protein engineering of serine protease subtilisin is a classic example [50]

13 ELECTRODE POTENTIAL AND REACTION RATE

131 Fermi level electrode potential and electron transfer

In the sect 123 the relationship between the electric potential and activation free

energy was briefly discussed As heat energy can provide the kinetic energy to atoms

and molecules for a reaction to proceed the electric energy also can provide kinetic

energy to electrons for electron transfer to proceed This leads to the redox reaction at

the electrode surface

Each substance has a set of discrete energy levels Each energy level can

accommodate up to two electrons Electrons fill these energy levels in ascending order

from the lower level This can divide these energy levels into two groups electron-filled

lower energy level and empty higher energy level without electrons In between the

critical energy level the called Fermi level lies It is designated as εF (Fermi energy) or

EF (Fermi potential) The Fermi level contains electrons of highest energy A substance

can transfer only these electrons to the external system as a donor while as an acceptor

it can accommodate external electrons only within the empty energy level which is

above its Fermi level (Fig 5)

45

Fig 5 Fermi level and electron transfer

The energy state of electrons however varies by substance therefore electrons at Fermi

level so-called highest occupied molecular orbital (HOMO) of a donor may need to

gain various kinetic energy to jump into the lowest empty energy level so-called lowest

unoccupied molecular orbital (LUMO) of an acceptor for electron transfer (Fig 6)

46

Fig 6 Electrode potential and electron transfer

As soon as the energy of electrons at Fermi level of a substance or an electrode meets

the energy level of LUMO of the other redox molecule electron transfer proceeds as

long as there is room to accommodate electrons in the acceptor This is the cathode

reaction The potential can be swept back by applying positive potential to the electrode

This lowers the energy of LUMO of the electrode so that an electron in HOMO of the

donor can be transferred back to the electrode This is the anode reaction If the electron

transfer is fast in both directions compared with other reaction steps it is a

thermodynamically reversible system and the standard electrode potential (Edeg) for this

redox reaction lies somewhere between and close to those two electrode potentials

which have enabled electron transfer At Edeg both anode and cathode reactions have the

same reaction rate hence they are at equilibrium [33] and there is no net current [21 30]

47

132 Electrode potential equilibrium potential and Nernst equation

Activation free energy of an electrode reaction can be altered by applying

potential The extent of its alteration and the direction of the reaction depend on the size

and direction of potential applied Fig 7 shows the difference in the standard activation

free energy (∆GdegDagger) for redox reactions at various electrode potentials [30]

48

Fig 7 Standard free energy and electrode potential [30]

(a) At E = Edeg (b) E gt Edeg and (c) E lt Edeg

More positive potential lowers the activation free energy for oxidation hence shifts the

reaction in favour of oxidation and vice versa This implies that applying extra potential

to the electrode perturbs the equilibrium and to restore it the electrode equilibrium

49

potential (Ee) must be shifted This phenomenon is clearly stated in Nernst equation (Eq

15 on p 50) as a relationship between Ee and the equilibrium constant (K) at the

electrode surface At equilibrium both oxidation and reduction reactions take place at

the same rate and there is no net current therefore the surface concentration (C(0t))

approximates bulk concentration (C) Nernst equation shows that Ee is always under

the influence of such concentration but can be established provided the reaction is fast

enough to restore the equilibrium [21 30] In the dilute solutions of the electroactive

species Edeg is substituted with the formal potential (Edegrsquo) which considers the influence

of inter-solute and solvent interactions [21 29]

Redox reaction

middotmiddotmiddotmiddotmiddot Eq 14

O = Oxidant

ne- = The number of electrons transferred

R = Reactant

kf = Forward reaction rate constant (1st or pseudo 1st reaction) s-1

kb = Backward reaction rate constant (1st or pseudo 1st reaction) s-1

50

Nernst equation

lowast

lowast

+deg=R

Oe

CC

nFRTEE ln middotmiddotmiddotmiddotmiddot Eq 15

Ee = Equilibrium potential V

Edegrsquo = Formal potential V

R = Gas constant = 8314 Jmol-1K-1

T = Absolute temperature K

n = The number of electron transferred

F = Faraday constant = 96487 Cmol-1

CO = Bulk oxidant concentration molm-3

CR = Bulk reductant concentration molm-3

133 Electrode potential and activation free energy

When E ne Edegrsquo the difference in these potentials is expressed as the electrode

potential shift (∆E) as in Eq 16 and its free energy shift (∆G) as in Eq 17

Electrode potential shift

degminus=∆ EEE middotmiddotmiddotmiddotmiddot Eq 16

Free energy shift

)( degminus=∆=∆ EEnFEnFG middotmiddotmiddotmiddotmiddot Eq 17

When ∆E is applied positively it lowers the anode oxidation activation free energy

(∆GaDagger) by fraction of the total energy change (∆G) as shown in Fig 8 on p 51 The

fraction for the oxidation reaction is defined as (1-α) The transfer coefficient (α) is a

51

kinetic parameter which states the degree of symmetry in the potential energy barrier

for redox reactions The symmetry in energy barrier is present when the slopes of two

potential energy curves are the same and it is defined as α = 05 α is dependent on

redox potential and could vary within the range between 0 and 1 In most cases α = 05

and appears to be constant when overpotential is small [21 30]

Fig 8 The shift of ∆E and ∆GdegDagger [21 30]

With α the shift in electrode activation free energy can be expressed in terms of

potential shift for both oxidation and reduction reactions as shown in Eq 18 and Eq 19

52

∆GDagger in terms of (E-Edegrsquo)

Anode Oxidation )()1(DaggerDagger degminusminusminus∆=∆ EEnFGG aa αo middotmiddotmiddotmiddotmiddot Eq 18

Cathode Reduction )(DaggerDagger degminus+∆=∆ EEnFGG cc αo middotmiddotmiddotmiddotmiddot Eq 19

∆GoDagger = Anode oxidation activation free energy Jmol-1

∆GodegDagger = Anode oxidation standard activation free energy Jmol-1

α = Transfer coefficient molm-3

n = The number of electrons transferred Jmol-1

F = Faraday constant = 96485 Cmol-1

E = Arbitrary electrode potential V

Edegrsquo = Formal potential V

∆GRDagger = Cathode reduction activation free energy Jmol-1

∆GRdegDagger = Cathode reduction standard activation free energy Jmol-1

134 Potential shift and standard rate constant

By substituting the activation free energy in Eyring equation (Eq 11) with the

shift in electrode activation free energy in Eq 18 and Eq 19 it is possible to express k

in terms of potential shift and standard rate constant (kdeg) (Eq 20 and Eq 21) kdeg is the

rate constant at Edegrsquo[21 30]

53

k in terms of (E-Edegrsquo) and kdeg

Forward reduction cathode rate constant (kf) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bf

Daggercexpκ

⎥⎦⎤

⎢⎣⎡ degminusminus

⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )(expexp

Dagger

EERT

nFRT

GhTk cB ακ

o

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= )(exp EERT

nFk fα middotmiddotmiddotmiddotmiddot Eq 20

Backward oxidation anode rate constant (kb) at E

⎟⎠⎞

⎜⎝⎛ ∆minus= RT

GhTk

k Bb

Daggeraexpκ

⎥⎦⎤

⎢⎣⎡ degminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛ ∆minus= )()1(expexp

Dagger

EERT

nFRT

GhTk aB ακ

o

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= )()1(exp EE

RTnFkb

α middotmiddotmiddotmiddotmiddot Eq 21

kf and kb = Forward and backward reaction rate constant s-1

kdegf and kdegb = Forward and backward reaction standard rate constant s-1

135 Butler-Volmer equation overpotential and current

At the equilibrium potential E = Ee kf = kb hence anodic current (Ia) =

cathodic current (Ic) and therefore the net current (I =Ia ndash Ic) is macroscopically zero (I =

0) However both forward and backward reactions still occur microscopically at the

electrode surface The absolute value of this electric current is called exchange current

(I0) and is not directly measurable When CO = CR at E = Ee C O (0t) = C R (0t) and kdegf

= kdegb = kdeg hence the net current any potential E can be defined as below by

Butler-Volmer equation (Eq 22)

54

Butler-Volmer equation

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡ degminus

minusminus⎥⎦

⎤⎢⎣⎡ degminusminus

deg= )()1(exp)(exp )0()0( EERT

nFCEERT

nFCnFAkI tRtOαα middotmiddotmiddotmiddotmiddot Eq 22

The Butler-Volmer equation states the relationship between the electric current

and the potential shift This can be further rephrased in terms of overpotential (η)

through defining I0 using Nernst equation (Eq 15) η is the potential required to drive a

reaction in a favourable direction from its equilibrium state and defined as n Eq 23

Overpotential (η)

eEE minus=η middotmiddotmiddotmiddotmiddot Eq 23

At E = Ee both anode and cathode exchange currents are the same therefore both

exchange currents can be defined together as I0 (see below) and I0 can be expressed in

terms within Nernst equation by substituting (Ee-Edegrsquo) as in Eq 24

Exchange current

⎥⎦⎤

⎢⎣⎡ degminusminus

deg= lowast )(exp0 EERT

nFCnFAkI eO

α

⎥⎦⎤

⎢⎣⎡ degminus

minusdeg= lowast )()1(exp EE

RTnFCnFAk e

Rα

αα )()( 1RO CCnFAk minus= o middotmiddotmiddotmiddotmiddot Eq 24

Now the Butler-Volmer equation (Eq 22) can be expressed in terms of η (= E ndash Ee) (Eq

25) by expressing it with I0 and the with re-arranged Nernst equation

55

⎥⎦⎤

⎢⎣⎡ degminus=lowast

lowast

)(exp EERTnF

CC e

R

O The term

)0(

CC t in Eq 25 indicates the influence of mass

transport For purely kinetically controlled region the electrode reaction is independent

of mass transport effects therefore C(0t) asymp C and Bulter-Volmer equation approximates

to Eq 26

Butler-Volmer equation and η

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminusminus

minus⎟⎟⎠

⎞⎜⎜⎝

⎛times⎥⎦

⎤⎢⎣⎡ degminus

minus= lowast

lowast

lowast

minusminus

lowast

lowast

lowast

αααα

R

O

O

tO

R

O

R

tR

CCEE

RTnF

CC

CCEE

RTnF

CC

II )(exp)()1(exp )0()1(

)0(0

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= lowastlowast ηαηαRT

nFC

CRT

nFC

CI

O

tO

R

tR exp)1(exp )0()0(0

middotmiddotmiddotmiddotmiddot Eq 25

⎭⎬⎫

⎩⎨⎧

⎥⎦⎤

⎢⎣⎡minusminus⎥⎦

⎤⎢⎣⎡ minus

= ηαηαRT

nFRT

nFI exp)1(exp0 middotmiddotmiddotmiddotmiddot Eq 26

The Butler-Volmer equation shows the dependence of current and hence the reaction

rate on the overpotential This implies that small kdeg can be overcome by overpotential

The kinetically-limited region of the electrode reaction is however very small because

the rate constant sharply increases exponentially with overpotential and soon the

reaction became diffusion limited [21 30] At this state the surface equilibrium is

totally perturbed and the overpotential is used to drive the diffusion for the one-way

reaction

136 Butler-Volmer equation vs Tafel equation

At small η lt RTnF Butler-Volmer equation further approximates to a linear

equation as shown in Eq 27

56

Small η Butler-Volmer equation

η⎟⎠⎞

⎜⎝⎛=

RTFnII 0

middotmiddotmiddotmiddotmiddot Eq 27

At large |η| gt RTnF the rate of the backward reaction is negligible hence one

of the exponential terms in Butler-Volmer equation becomes negligible and the

equations are solved for η for both anode and cathode reactions as below

Large η Tafel equations

InFRTI

nFRT lnln 0 plusmn= mη in the form of ||ln Iba +=η middotmiddotmiddotmiddotmiddot Eq 28

Anode oxidation InF

RTInF

RT ln)1(

ln)1( 0 αα

ηminus

+minus

=

Cathode reduction InF

RTInF

RT lnln 0 ααη minus=

In the system which follows the Tafel equations I0 (and hence kdeg ) is very small and the

electrode reaction does not proceed unless large η is supplied In this potential range the

reaction is already totally irreversible [4 21 30] An irreversible slow reaction is

graphically presented in Tafel plot in which ln (I) is plotted against η as shown in Fig

9

57

Fig 9 Tafel plot [4 21 30]

I0 is deduced from the y-intercept of the extrapolated slope Once |I| has become too

large and reached to the mass transport limit at large |η| the slope deviates from the

linear trend [30]

14 OVERPOTENTIAL AND FUEL CELLS

141 Four overpotentials that decrease the EMF

As discussed so far overpotential is extra potential required to drive a reaction

In the fuel cells this can only be taken from the potential generated within the system

causing potential drop and reduces EMF The overpotential in the system is attributed to

the four causes [1 4] (i) internal current (ii) activation overpotential (iii) diffusion

overpotential and (iv) resistance overpotential

58

142 Internal current

Internal current is a reverse current caused by crossover and leakage current

Crossover happens when the fuel flows to the opposite electrode through the electrolyte

Leakage current occurs when the current flows through the electrolyte rather than the

external circuit Internal current therefore happens without a load and is observed as the

negative offset from the theoretical value of EMF This is noted especially in

low-temperature fuel cells such as direct methanol fuel cells (DMFCs) [4 51] In such

systems The inner current can be several 10 Am-2 and potential drop could be more

than 03 V [4] In order to prevent fuel mixture and crossover a separator is used to

partition the cell Alternatively it is also possible to confer high specificity to electrodes

as seen in glucose - oxygen fuel cells Such systems do not require

compartmentalisation and this is advantageous for miniaturisation [52]

143 Activation overpotential

Activation overpotential is a potential loss due to the slow reaction kinetics as

discussed in sect 135 This is unavoidable to some extent and is typically the biggest

problem in most fuel cells involving O2 cathode reduction because this reaction is

kinetically hindered Finding or inventing catalysts with high kdeg is the key solution for

this A sluggish reaction exhibits a sharp rise in activation overpotential for a slight

increase in low current After the steep increase the current increase becomes linear and

constant as shown in Fig 10 until the reaction reaches the limit of mass transport at the

higher overpotential [4 30 34]

59

Fig 10 Activation overpotential against current [4]

144 Diffusion overpotential

Diffusion overpotential is a potential loss due to concentration gradient This

tries to drive flux of electroactive species toward and from the electrode surface It

happens when the reaction kinetics is faster than mass transport [4 21 30] In fuel cells

diffusion overpotential is caused by fuel depletion due to the inefficient fuel convection

or supply [21 34] This problem is observed in reformers because the fuel refining and

conversion such as liquefied petroleum gas (LPG) to hydrogen gas take place outside

the system and it makes difficult to respond to the sudden fuel demand from the system

[4] It also happens when fuel diffusion is interfered with by bad electrode geometry

electrode fouling and adsorption [21 27] Diffusion overpotential is defined as Eq 29

60

Diffusion overpotential equation

⎟⎟⎠

⎞⎜⎜⎝

⎛minus=

Ldiffusion I

InFRT 1lnη middotmiddotmiddotmiddotmiddot Eq 29

ηdiffusion = Diffusion overpotential V

IL = Limiting current A

Limiting current (IL) is generated when a reaction takes place at the rate limited by mass

transport Diffusion overpotential sharply increases when reaction kinetics have reached

to the maximum and very large current are drawn as shown in Fig 11 [21 30 34 53]

Fig 11 Diffusion overpotential against current [4]

145 Resistance overpotential

Resistance overpotential arises within electrodes separators connectors circuit

or electrolyte [4 34] It is called Ohmic resistance or IR drop because it follows Ohmrsquos

61

law (Eq 30) where resistance overpotential is linearly proportional to the electric

current as shown in Fig 12 [4 30]

Ohmrsquos law

IRV = middotmiddotmiddotmiddotmiddot Eq 30

V = Voltage consumed due to the resistance V

I = Electric current drawn due to the resistance A

R = Resistance Ω

| |

Fig 12 Resistance overpotential against current [4]

Causes of internal resistance are scattered through the system therefore there are many

possible ways to resolve this One can possibly reduce it by using more conductive

material The difficulty lies in finding the conductive material which can be modified as

well as carry out the functions as one desires under the required conditions [53]

Resistance can be reduced by reducing the distance between two electrodes [54]

however this might cause short circuit and fuel leakage This becomes a serious issue in

62

use of solid electrolyte such as polymer electrolyte because both performance efficiency

and mechanical stability of electrolyte must be achieved [53] Excess in inert supporting

electrolyte minimises solution resistance and matrix effect It sustains a thin electric

double layer uniform ionic strength and buffering effect against pH change [21 30]

However electrolyte could become a problem when electrode material dissolves into

electrolytic solution and reacts there because it might change nature of electrolyte [1

21]

146 Total overpotential and cell voltage

Total overpotential is the sum of all four overpotentials Current ndash Voltage (IV)

polarisation curve visualises the tendency of each potential drop as shown in Fig 13 [1

4]

Fig 13 V-I curve for a typical low temperature fuel cell [4]

63

The cell voltage (Ecell) is determined by subtracting the cathode potential (Ec)

from the anode potential (Ea) (Eq 31) The actual cell voltage is found by subtracting

the total overpotential from the theoretical cell voltage The energy available from this

cell voltage can be calculated as in Eq 32

Cell voltage

cacell EEE minus= middotmiddotmiddotmiddotmiddot Eq 31

totalltheoreticacellactualcell EE ηminus= )()(

cellcell nFEG minus=∆ middotmiddotmiddotmiddotmiddot Eq 32

15 BIOFUEL CELLS

151 Microbial and enzymatic fuel cells

Biofuel cells are fuel cells which use biocatalysts for fuel reactions

Biocatalysts can be either the whole living cells or isolated enzymes The former is

called a microbial fuel cell and the latter is called an enzymatic fuel cell [55-57] They

often function under ambient conditions and allow wider choice of fuels Microbial fuel

cells use common carbohydrates such as glucose and lactate [55] but also have

potentials in extracting power from waste and sediments [34 55 57 58] Harnessing

such microbial life activities has some advantages They can complete fuel oxidation

within the cell It was reported that a microorganism Rhodoferax ferrireducen has over

80 glucose conversion rate [58] Microbial fuel cells also have long-term stability

[56-58] of up to five years [55] However their output current and power densities are

64

small due to high internal resistance [55-57] Enzymatic fuel cells in contrast have

higher current and power densities but short life incomplete reactions require

mediators to shuttle electron between the enzyme catalytic centre and the electrode and

become deactivated easily [55 56 58] Addition of mediators and their immobilisation

with enzyme complicates fuel cells design further However they can be more

advantageous due to their substrate specificity Both anode and cathode to reside in the

same compartment of mixed fuels without crossover This enables the miniaturisation of

fuel cells [52 59 60] and is particularly useful for medical implantation Stability and

lifetime of the enzyme fuel cells can be improved by appropriate enzyme

immobilisation fixing up the suitable conditions for their function [55] and adding

some enzyme stabilisers Addition of the right mediators and increased enzyme loading

will also increase output current and power density [55 59]

152 Enzyme fuel cells and mediators

Enzyme reactions are available for wide range of substrates yet each enzyme

has very high specificity for its substrates Many also function at neutral atmospheric

conditions therefore miniaturised enzyme fuel cells are of interest for in vivo

application Many redox reactions however take place at the catalytic centre which is

deeply buried in the protein body This proteinous content is an electronic insulator and

interrupts the electrical communication between the redox centre and the electrode It is

therefore difficult for many enzymes to establish efficient electron transfer without

mediators as shown in Fig 14 This is called mediated electron transfer (MET)

65

Fig 14 Mediated electron transfer reaction [55 61]

It is possible for some enzymes such as multicopper oxidases (MCOs) to perform direct

electron transfer (DET) without mediators however mediators ensure good electrical

communication and cell performance However the presence of mediators as well as the

enzyme specificity could complicate the fuel cell composition and its reaction path due

to the reaction cascade [55 59 62 63] Palmore built a methanol - oxygen enzyme fuel

cells and this involved four enzymes two mediators and six reactions to complete the

full reaction path [64] It is more advantageous to use enzymatic fuel cells for very

specific applications [59] such as medical implantation where there are environment

constraints but where applications demand only small power output through simple

reactions

Mediators are usually small redox active species which can be organic or

metal complexes In the enzymatic fuel cells they should be carefully selected because

improper mediators can cause unwanted overpotential or can destabilise the system

They must have (i) small overpotential (ii) fast reversible electron transfer [55 56 59

60 63 65] and (iii) stability [62 65] However there should be small but reasonable

66

potential gap between the enzyme and the mediator to drive electron transfer [60 66]

The gap of 50 mV is optimal [19 64] because of the small potential loss yet less

competing with enzyme reactions It is also possible to mediate electron transfer by

building a conductive nanowire between the electrode and the redox centre [60 63 67]

or a connection by plugging metal nanoparticles into the enzyme [60 67]

16 MINIATURISED MEDICAL POWER SOURCES

161 Realisation of miniaturised fuel cells and medical power supply

Medical application is one of the obvious targets for miniaturised biofuel cells

Glucose-oxygen biofuel cells which continuously run on glucose and oxygen were

thought to be ideal for implantable medical devices because both oxygen and glucose

are abundant in the body [63] They were initially intended to power cardiac assist

devices and pacemakers The studies on such systems have been made for more than 40

years however none has been made into practical use because their power density

stability operation life and biocompatibility were inadequate [19 67-69] Their output

power ranges between microW ndash mWcm-2 and they operate only for a few days to a few

weeks Most applications need a few W power for a longer duration [18 60 67] A

pacemaker requires 1microW [56] for more than 8 ndash 10 years with 996 reliability Such

power has been supplied by lithium (Li) iodine battery [19 56 70] which is

characterised by extended operational life [68] Li is fairly stable light-weight metal

and has high energy density of 386 kAhkg-1 Its standard potential is highly reducing ndash

323 V vs AgAgCl These properties enable Li batteries to power most devices

67

including pacemakers laptops and vehicles [19 21] However there are some problems

in Li batteries They are still bulky because they need to store their own fuel and it has

to be sealed with the metal case [19 68] They occupy half of the space in the

pacemaker volume [68 71] They use corrosive [72] and irritating materials There is

potential of explosion [68] leakage and the necessity of exchange Li also has potential

resource constraint due to the geopolitical issues as seen for oil and platinum More than

80 of its production is based in South America and China [73] With increased public

concern over using greener safer energy and saving resources as well as the global

political intervention in that it is natural to consider about using alternative

technologies to power implantable medical devices where it is possible

162 Miniaturised potential medical power sources

There are numerous implantable electrical medical devices with different

power requirements These include cardiac pacemakers defibrillators neurostimulators

for pain control drug delivery device hearing aids [70] and biosensors [74]

Implantable autonomous body sensors for physiological parameters such as glucose

concentration local pH flow and pressure [19] require power as low as 1 microW [56 70

74] and the operation period of 1 year at least [74] Such power can be supplied by

various implantable energy harvesting devices using body glucose [19] motion

vibration [75] and temperature differences [75] Heller developed the smallest

glucose-oxygen biofuel cells made of carbon fibres with the dimension of 17-microm

diameter and 2-cm length Anode glucose oxidase and cathode laccase were wired to

conductive hydrogel backbone with a flexible osmium (Os) mediator complex Its

68

output was 048 mWcm-2 with 06 V at 37 degC and it lasted for 1 week [19 67]

Kinetic energy of motion and vibration can be converted into electricity with

electrostatic transduction using microelectromechanical systems (MEMS) This

technology allows integration of various microelectronic devices on the same substrate

[76] NTT Corporation developed a system generating power from body motion and

temperature It consists of a thermoelectric and a comb-shaped Au-Si vibrational device

on a monolithic Si substrate The voltage produced by a thermoelectric device is

boosted by capacitance which was generated by the vibrational device then its charge

is stored in the capacitor [75] Another vibrational device was reported to have 20 x 20 x

2 mm volume and output 6 microW and 200 V at 10 Hz low frequency and 39 ms-2

acceleration The principal difficulty in using MEMS is to adjust and synchronise

flexibly the resonance frequency of vibrational device to the human body frequency

which is low and has constant fluctuation in order to increase energy conversion

efficiency The typical vibration has the frequency ranging from 1 to 100 Hz and

acceleration from 1 - 10 ms-2 [76]

Thermoelectric devices generate power from temperature difference Each

module consists of a number of thermocouples on Si substrate Thermocouples are

made of different material so that it can generate voltage from the heat flow following

temperature gradient This is called Seebeck effect and its size depends on the material

and number of thermocouples used and the spacing within the device [75 76] Typical

ambient temperature difference is less than 10K [74 76] and its power is 1microWcm-2 [74]

Seiko Instrument developed a wristwatch called Seiko Thermic which was powered by

human body temperature in 1998 [77 78] It consists of n-type Bismuth-Tellurium

(BiTe) and p-type Bismuth-Antimony-Tellurium on rods reinforced by Ni on oxidised

69

silicon wafer It generated microWcm-2 power at extremely low 0047 conversion

efficiency With a booster circuit it powered a wristwatch operating at 1microW and 15 V

It could however operate only when ambient temperatures were below 27 degC [78] and

so could not be used in tropical regions

Heller also developed an implantable miniature Nafion-coated zinc ndash silver

chloride (ZnAgCl) battery The anode was made of zinc fibre coated with Nafion It

was 76-microm diameter 2-cm length and 75 x 10-2 cm2 surface area The cathode was

made of AgCl wire covered with chloride-permeable membrane On discharge anode

zinc formed Zn3(PO4)2middot4H2O hopeite complex with Nafion growing non-porous

lamellae on the surface The lamellale had a unique property which was permeable to

zinc ions but impermeable to O2 therefore the battery became corrosion free It output

was 13 microAcm-2 at 1 V and operated for three weeks under physiological condition The

advantage of this battery was its size which was 100-fold smaller than the smallest

conventional batteries [79] The smallest silver oxide button buttery has the volume 30

mm3 [19 80] with energy density 04 mWh mm-3 at 15 V The smallest Li-ion buttery

has the volume 190 mm3 [80] with energy density 025 mWh mm-3 at 4 V [21]

17 OUTLINE OF THE WORK

171 Introduction to miniaturised glucose-oxygen fuel cells

A miniaturised glucose-oxygen biofuel cell runs on glucose and oxygen This

would be ideal to power implantable autonomous low-power body sensors since both

glucose and oxygen are abundantly present in the body The range in their physiological

70

concentration is between 4 ndash 8 mM for glucose and 01 ndash 02 mM for oxygen [56 67]

The aim of this study was to build miniaturised glucose-oxygen fuel cells with reliable

enzyme anodes and powerful oxygen-reducing cathodes using straightforward and

scaleable technology The general structure and mechanism of glucose-oxygen fuel cells

were described in sect 112

In this study both anode and cathode reactions took place at the room

temperature and physiological pH in the presence of catalysts Glucose oxidation was

catalysed by the enzyme glucose oxidase (GOx) from Aspergillus niger and its electron

transfer and conduction were facilitated by an conducting organic salt called

tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) (Fig 15)

Fig 15 The schema for a glucose-oxidising anode

GOx and TTF-TCNQ were mixed into a composite employing a feasible electrode

fabrication method called bulk modification method Various enzyme stabilisers were

added to the composite at different concentration to improve its performance The

influence of various enzyme stabilisers on anode performance was studied in the

71

presence of both N2 and O2 and the most ideal anode composition was determined

Cathodes were also fabricated in the similar manner using graphite as conductive

material and mixing with various oxygen reducing catalysts except for the enzyme

membrane cathodes The cathode composites were pretreated in various media to

enhance their activity For enzyme membrane electrodes glassy carbon electrodes were

used instead of graphite composite and enzyme solution was loaded onto the surface

and trapped with membrane Catalytic power and mechanism of each cathode were

studied with various electrochemical methods and the most catalytic cathode was

determined

172 Outline

Chapter 1 is a general introduction about fuel cells It explains the history and

basic concept of fuel cells and outlines the study

Chapter 2 explains about the enzyme anodes The introduction part refers to

glucose oxidising enzymes enzyme immobilisation TTF-TCNQ enzyme inactivation

enzyme stabilisers and enzyme kinetics The material and method section reports the

protocol for the enzyme electrode fabrication electrode conduction study and

amperometry study for enzyme electrodes Result and discussion section mentions

enzyme electrode conduction enzyme kinetic studies based on amperometry

mechanism of enzyme stabilisation in each stabiliser and ideal electrode composition

Chapter 3 discusses oxygen-reducing cathodes The introduction part reviews

the mechanism of the oxygen reduction reaction oxygen reactivity oxygen reduction

catalysts carbon properties electrochemical pretreatments electrochemical techniques

72

and how to determine the catalytic efficiency of cathodes The material and method

section describes the protocol for the oxygen-reducing electrode fabrication preparation

and electrochemical pretreatment electrochemical studies electrochemical studies for

qualitative analysis and to obtain the kinetic parameters and analysis to determine the

efficiency of catalytic cathodes The result and discussion section describes the

voltammetric behaviour of each pretreated cathode influence of pretreatment and

catalytic efficiency The most catalytic cathode was determined at the end

Chapter 4 refers to the overall conclusion and further studies

73

2 ANODE INTRODUCTION

21 GLUCOSE OXIDATION ENZYMES

Glucose-oxygen fuel cells extract power by oxidising glucose at the anode The

anode contains glucose-oxidising enzymes There are several enzymes available for

glucose oxidation (i) β-D glucose oxidase (GOx) (EC 1134) [56 81] (ii) pyranose

oxidase (POx) (EC 11310) [56 81] (iii) nicotinamide adenine dinucleotide-dependent

glucose dehydrogenase (NAD-GDH) (EC 11147) [18 56] and (iv) quinoprotein

glucose dehydrogenase (PQQ-GDH) (EC 1152) [56 81] These enzymes are

classified into the family of oxidoreductases which catalyse either transfer of (i)

hydride (H-) (ii) oxygen atoms or (ii) electrons from one molecule to another Oxidases

transfer electrons from substrates to O2 and form H2O or H2O2 as products

Dehydrogenases transfer electrons from substrates to coenzymes without involving O2

(Eq 33 and Eq 34)

Glucose oxidation by GOx and NAD-DHG

GOx middotmiddotmiddotmiddotmiddot Eq 33

NAD-DHG middotmiddotmiddotmiddotmiddot Eq 34

To function catalytically enzymes require non-peptide inorganic or organic cofactors or

prosthetic groups Non-peptide organic cofactors are also called coenzymes Enzymes

lacking such groups are called apoenzymes and hardly functional Cofactors are

generally loosely bound to the apoenzymes and freely diffusible but on catalysis they

74

bind to the enzymes as the second substrates Prosthetic groups are always tightly bound

to the apoenzymes through covalent or non-covalent bonding [22 23 48] GOx and

POx are fungal flavoprotein enzymes containing flavin adenine dinucleotide (FAD)

cofactors FAD in GOx is tightly bound to the apoenzyme but not covalently bonded

hence still dissociable [67 82-84] NAD-GDH contains dissociable NAD+ [65] while

PQQ-GDH contains pyrrolo-quinoline quinone (PQQ) redox cofactor [56 65] which is

moderately bound to the apoenzyme [65 67] The structure of each cofactor is shown in

Fig 16

75

N

N

NH

N O

O

R

N

N

NH

HN O

O

R

NH

N

NH

HN O

O

R

H+ + e- H+ + e-

2H+ +2e-

N+

NH2

O

R

H

N

NH2

OH

R

HH

N

NH

O

O

OHO

OH

O

OH

O

N

NH

O

HO

OHO

OH

O

OH

O

N

NH

OH

HO

OHO

OH

O

OH

O

H+ + e- H+ + e-

FAD FADH2

NAD NADH

PQQ PQQH2

Fig 16 The structure of FAD NAD and PQQ and their redox reactions

Moieties involved in the redox reactions are circled in gray [23 65]

Cofactors also stabilise the enzyme structure Irreversible thermal GOx denaturisation

involves the perturbation of FAD biding site causing FAD dissociation although 70 of

its secondary structure is retained Chemical denaturisation untangles the dimmer

structure of GOx [65 81 83 85] and dissociates monomers and FAD [86 87] Excess

FAD does not prevent or regain function after thermal inactivation [87] However FAD

can rapidly bind to apoenzymes at 25 degC pH 61 and stabilise the tertiary structure

through conformational change and give resistance to proteolysis [82]

76

The choice of enzyme is based on the reaction involved enzyme availability

stability enzymatic properties physical properties and operational environment In the

absence of O2 or when there is concern about system degradation due to the competition

of electrons between O2 and artificial electron acceptors or due to the by-product H2O2

GDHs are more preferred to GOx [23 60 65-67 88] However the use of GDHs poses

different disadvantages Membrane-bound PQQ dehydrogenase (mPQQ-GDH) has very

high glucose specificity however it is difficult to solubilise and purify hence not for

commercial use [88] Soluble PQQ dehydrogenase (sPQQ-GDH) has been used in

commercial glucose sensors [67 89] however it suffers from low thermostability and

very low glucose specificity [88] Moreover PQQ-GDHs add complication to the

system because metal ions such as calcium ions (Ca2+) and magnesium ions (Mg2+) is

required to activate them [89 90] In the case of NAD-GDH NAD must be provided

because it is a dissociable cofactor but must bind first to the apoenzyme to form a

catalytic complex for glucose oxidation In addition to this complication NAD was also

reported to be more prone to causing electrodes poisoning without a quinone acceptor

[65] NAD forms adduct with phosphate in the buffer its decomposition releases free

adenine and electrochemically oxidised adenine adsorbs onto the electrode surface

causing electrode poisoning [91]

The redox potential of enzyme is also important because it is the driving force

for electron transfer and the power although it is difficult to match both catalytic

efficiency and electrochemical efficiency It is also difficult to determine the redox

potential exactly because the redox potential of cofactors is influenced by

microenvironment such as where they are embedded in the enzyme as well as

77

macroenvironment such as pH and temperature For example the redox potential of

FAD itself is ndash 042 V vs AgAgCl at pH 7 25 degC [22 23] however that of

flavoproteins could range from ndash 069 to ndash 005 V [65] and FAD-containing GOx has

the redox potential approximately ndash 03 V [67 92] It was also reported glucose

gluconolactone redox potential was ndash 056 V [93] PQQ itself has - 013V in absence of

Ca2+ and ndash 012 V in presence of 1M CaCl2 [94] while sPQQ-GDH has the redox

potential approximately - 019 V in presence of Ca2+ [66 67] Furthermore the enzyme

redox potential leads to a complex issue when mediators which assist electron transfer

between the enzyme redox centre and the electrode are involved because the improper

combination of the enzyme and mediator causes inefficient electron transfer and hence

potential drop

For enzyme availability stability and simplicity in handling GOx has been

most popular glucose-oxidising enzyme It has optimal pH 55 and temperature 35 ndash

40 degC [93] but is stable between pH 4 ndash 7 [95] and up to 50 - 55 degC [82 87] GOx has

higher substrate specificity and is most active toward β-D-glucose oxidation [56 86 88]

and very stable therefore its use has prevailed among various sensor applications [60

67]

22 DESIGNING ENZYME ELECTRODES

Construction of enzyme electrodes is complex because it simultaneously

incorporates enzymes mediators conductive materials and stabilisers ensuring that all

of them work harmoniously under restricted performance conditions There are several

78

immobilisation techniques available These include physical adsorption cross-linking

covalent bonding and entrapment in gels or membranes [96]

221 Physical adsorption

Physical adsorption is a simplest immobilisation method Electrodes are dipped

in the solution containing enzymes and mediators or such solution can be applied to the

electrode surface In this method however the enzyme tends to leach due to the weak

interaction between the electrode and enzyme [55] In order to avoid the loss of

electrode material the electrode surface is often covered or coated with material One

classic method is that the solution on the electrode surface is trapped with a

semipermeable dialysis membrane [61 65 92] This allows enzymes free from the

possible denaturisation caused by immobilisation on the solid material Enzymes inside

the dialysis membrane also can be fixed onto the cellulose acetate membrane or the

enzyme layer can be cross-linked with bifunctional reagents such as glutaraldehyde

[61]

222 Reconstituting apo-enzyme

Willner constructed enzyme electrodes by reconstituting apo-enzyme on the

gold (Au) electrodes surface with quinone mediators The electrode consisted of three

layers which were made of PQQ synthetic FAD and apo-GOx [97] PQQ were

mediating groups and attached onto Au electrode by cystamine PQQ attached on Au

was then covalently linked to synthetic FAD called N6-(2-aminoethyl)-FAD Covalently

79

linked FAD self-assembles with apo-GOx so that holo-GOx assembly is formed on the

top of the PQQ mediating layer attached to the Au surface The reconstitution procedure

is shown in Fig 17

Fig 17 Apo-GOx reconstruction with PQQ electron transfer relay on Au electrode [97]

The output current density was 300 microAcm-2 at pH 7 35 degC and it dropped by 5 in the

presence of O2 although the decrease was negligible at 01 mM physiological O2

concentration [98] The reconstitution of apo-enzymes is a flexible system potentially

applicable to enzyme electrodes [97] The fabrication process is cumbersome however

the enzymes are neatly aligned and electrically connected in the correct orientation

therefore electrocommunication between the enzymes and electrodes can be

maximised

80

223 Tethered Osmium-complexes linked to conductive hydrogel

It has been reported that embedding enzymes in the conductive hydrogel

complexed with tethered osmium (Os) mediator led to high output current [19 60 66

67] The conductive hydrogel is made of cross-linked polyvinylpyridine or

polyvinylimidazole They are soluble as monomers however they gain their

water-swelling property in cross-linked polymer This is ideal for enzyme mobility and

electrical conductivity Electron transfer is realised by the Os complex which is tethered

to the conductive hydrogel back bone The tether is made of 10 ndash 15 atoms [19 67] in

order to allow flexile movement of mediators Tethered Os complexes sweep the large

volume made of multiple layers of hydrogel form electrostatic adducts with enzymes

[19] and facilitate collide-and-hop electron conduction [19 99] Polymer tether and Os

complexes can be flexibly tailored for different enzymes and different operational

environments [19] One of its great advantages is that the redox potential for electron

transfer can be adjusted by changing the ligand structure of Os complex [19 60 97]

Heller constructed such electrodes made of carbon fibers whose dimension was 7 microm

diameter 2 cm length 08 mm2 surface area and 26 x 10-3 mm3 volume GOx was

wired to poly(4-vinylpyridine) conductive hydrogel backbone by the tethered complex

of tris-dialkylated NNrsquo-biimidazole Os2+3+ (Fig 18)

81

Fig 18 GOx wired to conductive hydrogel complexed with Os [19 67]

The redox potential of its redox centre was ndash 02 V vs AgAgCl The glucose oxidation

took place at ndash 036V and its output current density was 11 mAcm-2 [19] Disadvantage

of the tethered enzyme is short life It was stable for one week in the physiological

buffer then 5 current drop per day was observed When it was implanted in grape sap

50 current drop occurred within 20 hours In calf serum 50 current dropped

occurred in 2 hours with electrode fouling [19]

224 Entrapment in the conductive polymer

Entrapping enzymes in conductive polymer such as polypyrrole polyaniline

and polythiophene [65] is a popular method for enzyme electrodes [96] This is made by

82

electropolymerisation in which potential is applied to the solution containing enzymes

and monomors for certain duration It is a simple method but can be complicated if the

conditions for polymer growth are not compatible with enzyme entrapment [99]

Enzyme leakage occurs when enzymes are not trapped securely but it can be reduced

when derivatised polymers are used [96 100] Their conductivity is affected by

monomer structure [100] acidity and electrolyte [101] It can be degraded by products

of enzymatic reactions [65] Fujii fabricated GOx electrodes which were stable for

more than four months though a gradual sensitivity drop was observed GOx was

entrapped in saccharide-containing polypyrrole made of 1-(6-D-gluconamidohexyl)

pyrrole (GHP) (Fig 19(i)) on Pt electrodes It was reported that entrapped GOx was

secured by hydrogen bonding present between sugar gluconyl groups and enzymes

Enzyme leaching was also reduced by improved polymerisation with extended

conductive hydrocarbon chain [100] The output current density was 75 microAcm-2 at 055

V pH 74 in the presence of 56 mM glucose and 175 microA cm-2 under glucose saturated

condition [100] Arai entrapped various enzymes in polymer of mercaptobenzoquinone

(SQ) (Fig 19(ii)) and its methylated derivatives methylmercapttobenzoquinone

(MeSQ) and dimethylmercaptobenzoquinone (Me2SQ) on Au electrode surface

83

OH

H

H

OH

OH

H

OH

H

CH2OHN(CH2)6NHCHOH

(i)O

O

SH

Fig 19 Monomer structures of conductive polymers

(i) 1-(6-D-gluconamidohexyl) pyrrole [100]

(ii) Mercapto-p-benzoquinone [101]

Quinones have been widely used as good mediators [21 60 61 65] Poly(SQ) is a

conductive polymer functionalised with quinone mediating groups and can be easily

attached to Au electrode due to the presence of thiol group (-SH) [100] It was revealed

that more methylated SQ polymer led to better performance The maximum current

density for SQ- MeSQ- and Me2SQ-GOx anodes was 006 522 and 544 microAcm-2 and

their electrode enzyme dissociation constant KM was 13 18 and 20 mM respectively

[102 103] FAD cofactors have been also electropolymerised to entrap lactate

dehydrogenase and lactate oxidase It was reported that polymerised FAD extended the

shelf and operational life of electrodes to more than one year and also reduced O2

influence [104]

225 Bulk modification

Another popular electrode fabrication involves bulk modification of solid

composite This is simply made by mixing electrode material and inert binding reagent

84

such as epoxy resin Typical composite electrodes consist of 70 conductive material

by weight [105] Bulk-modified electrodes are robust stable inexpensive and easy to

modify build and reuse [105-107] therefore often used for enzyme electrodes [108]

For GOx electrodes tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ)

has been used as both mediating and conducting material

2251 Tetrathiafulvalene 7788-tetracyanoquinodimethane (TTF-TCNQ) properties

TTF-TCNQ is a black crystalline charge transfer complex having both

mediating and conducting properties Its handling is also easy and often used for GOx

electrodes [65] Its conductivity is 102 ndash 103 Scm-1 [109 110] and comparable to that of

graphite [65 105] TTF-TCNQ complex consists of TTF electron donor and TCNQ

electron acceptor and they are arranged in a segregated stack structure TTF-TCNQ has

metal-like electric conductivity and this derives from intra-stack and inter-stack

molecular interactions Within the stack π-orbitals of adjacent molecules overlap This

builds the major conductive path in the direction of stack [61 65 110 111] Between

the stacks there is partial charge transfer between different chemical groups and this

gives the metallic feature in which charge carriers are mobile among the stacks

[110-112] as shown in Fig 20

85

S

S S

S

N

C

C

N

C

C

N

N

S

S S

S

7 6

N

C

C

N

C

C

N

N

-

TTF - electron donor

TCNQ - electron acceptor

6 - localised 6 - delocalised

+ e-

+ e-

Fig 20 TTF-TCNQ structure and its charge transfer [61]

TTF-TCNQ is a good electrode material because it has low background current and is

efficient at electron transfer [65] is stable [65] insoluble in water and not toxic [113]

However it exhibits complex electrochemistry (Fig 21) and its behaviours have not yet

been fully elucidated

86

vs AgAgCl

TCNQTCNQ e⎯⎯rarr⎯minus minusbull+

+minus+ ⎯⎯rarr⎯minus 2e TTFTTF

minus+minusbull⎯⎯rarr⎯

minus 2e TCNQTCNQ

+minus⎯⎯rarr⎯minus

TTFTTF e

Fig 21 CV of TTF-TCNQ Silicon oil paste electrode at pH 74 01M potassium

phosphate 01 M KCl at 20 mVs-1 [92] Modified

The range of its effective redox potential varies according to the authors [65 92 114

115] Following the Lennoxrsquos study its potential extremes should lie within the range

between - 015 V and + 031 V vs AgAgCl (Table 2) because beyond these potential

ranges irreversible lattice reduction or oxidation takes place Even within the range

crystal dissolution salt formation with buffer ions and its deposition are still present and

could change the electrochemical profile TTF-TCNQ stability is affected by many

factors such as potential applied buffer component and concentration ion solubility

mass transport and analyte [92]

87

Table 2 Electrochemical behaviour of TTF-TCNQ at pH 74

Equilibrium TCNQTTFTCNQ-TTF +⎯rarr⎯ middotmiddotmiddotmiddotmiddot Eq 35

TCNQ reduction + 015 V MTCNQTCNQTCNQ Me ⎯⎯rarr⎯⎯⎯rarr⎯+minus +minusbull+ middotmiddotmiddotmiddotmiddot Eq 36

Lattice reduction - 015 V minus+minusbull ⎯⎯rarr⎯minus 2e TCNQTCNQ middotmiddotmiddotmiddotmiddot Eq 37

Anode salt formation + 031 V MTCNQTCNQ M⎯⎯rarr⎯++minusbull middotmiddotmiddotmiddotmiddot Eq 38

Lattice oxidation + 035 V +minus⎯⎯rarr⎯minus

TTFTTF e middotmiddotmiddotmiddotmiddot Eq 39

Further oxidation + 038 V +minus+ ⎯⎯rarr⎯minus 2e TTFTTF middotmiddotmiddotmiddotmiddot Eq 40

In the presence of a redox couple it also exhibits different electron transfer mechanism

When Edegrsquo of the redox couple is Edegrsquo gt 0 V TTF-TCNQ shows metal-like behaviour

facilitating direct electron transfer (DET) while it presents mediated electron transfer

(MET) for the redox couple of Edegrsquo lt 0 V Lennox made a study on TTF-TCNQ GOx

electrodes and reported that large anodic current was observed at ndash 015 V He also

reported that stable current was obtained when constant potential + 02 V was applied

for 24 hours at pH 7 in the presence of glucose He concluded that hydrophobic protein

core solubilised TCNQ and it formed enzyme-mediator complex GOx oxidation could

be catalysed at low potential because it was still in the range of the stable redox

potential He also conducted experiments with GOx and methanol dehydrogenase

(MDH) modified TTF-TCNQ electrodes in the presence of O2 The performance was

interfered by O2 in both electrodes against his expectation because DHG is in general

resistant to O2 He gave a conclusion that reduced mediators were oxidised by O2 before

electrooxidation took place [92]

88

226 Enzyme covalent attachment and mass loading

Enzymes can be covalently attached to the electrode however it becomes

complicated when mediators and stabilisers are must be also attached for practical fuel

cell applications This is however an attractive technique once ideal means of

co-immobilisation are established because enzymes can be retained firmly on the

electrode surface Enzymes can be covalently linked to oxidised or aminated surface

through amide bonds [116 117] Covalent linkage often lowers enzyme activity because

it partially alters the enzyme structure and confines the enzyme conformation [55]

however Kim succeeded in fabricating stable biocatalytic nanofibre by loading enzyme

aggregate to the enzyme monolayer which was covalently attached to the nanofibres

(Fig 22) Nanofibres with ester groups were synthesised from polystyrene and

poly(styrene-co-maleic anhydride) using electrospinning Seed enzyme was covalently

attached to the chemically modified nanofibre to form an enzyme monolayer on the

nanofibre This enzyme-attached nanofibre was further modified with the mixture of

glutaraldehyde and enzymes so that enzyme mass load was achieved by loading enzyme

aggregate to the enzyme monolayer covalently attached to the fibre Minter reported

that enzyme immobilisation with cross-linking increased the enzyme stability but

limited the enzyme conformation and thus reduced the activity However the activity

reported was nine times higher than that with an enzyme monolayer only Moreover the

activity was retained more than one month under rigorous shaking [118] The activity of

each enzyme particle might have been reduced however mass loading increased the

reactant flux hence increased the overall activity

89

Fig 22 The procedure for the enzyme mass-loaded biocatalytic nanofibre [118]

He also fabricated single-enzyme nanoparticles (SENs) in which each enzyme particle

was encapsulated within a hybrid organic and inorganic network The surface amino

groups of the enzyme were converted into surface vinyl groups and then polymerised

The polymer was then cross-linked to form the shell around the enzyme particle (Fig

23)

90

Fig 23 Fabrication procedure of single-enzyme nanoparticles [55]

It was reported that high stability was achieved after polymer cross-linking The enzyme

activity was approximately half of the free enzyme however there was no mass

transport interference [55] due to their porous structure Km in SENs was nearly

equivalent that of the free enzyme but the storage stability went beyond five months

[119] Eight-fold increase on storage stability after GOx covalently linked to

poly(phosphorothioate) was also reported [120]

23 PROTEIN INACTIVATION AND STABILISATION

Enzyme inactivation is one of the biggest problems in enzyme electrodes

Enzymes are excellent catalysts in their native environment however they are only

designed to work optimally under specific conditions therefore very sensitive to the

slight change in their environment and easily undergo inactivation The issue is

persistent in the enzyme electrodes because their fabrication introduces the enzymes to

the unusual environment yet enzyme catalytic activity must be maintained to obtain

sufficient power output and operational life The problem can be alleviated by (i)

91

protein engineering [56 121] (ii) environmental engineering [56 60 63 67] and (iii)

addition of appropriate enzyme stabilisers [120 122] Protein engineering involves the

alteration of enzyme structure to gain desired functions stability or resistance However

it is very challenging to improve both stability and activity since protein stability is

conferred by its rigidity while its activity depends on the conformational flexibility

Alteration of protein properties is realised by genetic engineering such as site-directed

mutangenesis [123] or directed evolution [49] Environmental engineering is closely

linked to the enzyme immobilisation It aims to achieve the same goals as in protein

engineering but extrinsically The environment which rigidifies the protein structure

such as cross-linking or inclusion in electrode composite tends to increase the enzyme

stability and prolong its life In contrast the environment which more favours over

flexible protein conformation such as hydrogel and membrane trap exhibits higher

enzyme activity but shorter life However it was reported that enzyme cross-linking and

the presence of hydrophilic macromolecules improved both stability and activity while

electrostatic interaction improved selectivity [124] Addition of stabilisers is more

targeted smaller scale environmental engineering Intrinsic conformational stability of

the protein cannot be improved [120] however it can target and eliminate specific

inactivating factors increases the local specific protein stability and prevents or slows

its unfolding There are two factors to improve for enzymes to achieve ideal operation

(i) activity by hydration and (ii) increased lifetime by increased protein rigidity which

might involve the removal of bond-destabilising factors or amplifying the bond strength

within the enzymes In fact both must be probably achieved simultaneously for gaining

the optimal state because they interrelate to each other in terms of protein dynamics As

mentioned earlier protein hydration improves enzyme activity but also allows its

92

regeneration hence affects the protein internal dynamics Protein dehydration distorted

the protein structure [125]

24 PROTEIN DESTABILISATION

There are many protein destabilising factors apart from dehydration and these

can be classified into chemical and physical instability Chemical instability happens

due to chemical modification such as deamidation oxidation proteolysis and

racemisation

241 Deamidation

Deamidation is the hydrolysis of amide link in the amino acid side groups such

as glutamine (Gln) and asparagine (Asn) (Fig 24)

Glutamine (Gln) Asparagine (Asn)

Fig 24 Side groups of Gln and Asn

It is favoured by increase in pH temperature and ionic strength The buffer

concentration influences the rate of deamidation at pH 7 ndash 12 Urea also accelerates

deamidation It often implies the protein aging and then modifies the protein function

93

242 Oxidation

Oxidation takes place at the side chains of cystine (Cys) methionine (Met)

tyrosine (Ty) tryptophan (Trp) and histidine (His)

Cysteine (Cys) Methionine (Met)

Tyrosine (Tyr) Tryptophan (Trp) Histidine (His)

Fig 25 Side groups of Cys Met Tyr Trp and His

Atmospheric oxygen can oxidise Met residues H2O2 modifies indole thiol (SH) [123

126] disulfide (SS) imidazole phenol and thioether (RSR) groups at neutral or slightly

alkaline conditions At acidic condition it oxidises Met into Met sulfoxide (R-S=O-R)

Under extremely oxidising condition Met sulfoxide is further oxidised into Met sulfone

(R-S(=O)2-R) Met oxidation leads to loss of biological activity however it does not

abolish the enzyme activity Therefore if oxidised Met is reduced back by a reductant

such as thiols it will restore the full biological activity Met reactivity depends on its

position within the protein Cys plays an important role in protein folding because it

forms intermolecular disulfide bonds however very susceptible to oxidation It can

undergo autooxidation in the absence of oxidants It is oxidised in several stages in

94

which thiol R-SH group is oxidised to (i) sulfenic acid (R-S-OH) (ii) disulfide

(R-S-S-R) (iii) sulfinic acid (R-S=O-OH) and (iv) sulfonic acid (R-S(=O)2-OH) [123

127] Oxidised thiol groups can be reduced back however they can be also reduced

back in the wrong way This leads to protein misfolding and loss of activity There are

many factors that influence the rate of oxidation They are (i) temperature (ii) pH (iii)

buffer medium (iv) metal catalysts (v) O2 tension (vi) steric condition (vii) neighbouring

groups (viii) photooxidation The presence of metal ions such as Fe2+ and Cu2+

accelerates oxidation by air [123] and the presence of O2 also might accelerate thiol

oxidation with other oxide such as nitric oxide radical [127] Metal-catalysed oxidation

involves a site-specific metal binding to Trp His and Met This leads to severer form of

oxidation causing protein aggregation Peroxide-catalysed oxidation on the other hand

is milder and tends to affect Cys and Trp but does not form aggregates [126] Increase in

pH increases pKa hence accelerates dissociation of ndashSH All of Cys Met Typ Tyr and

His are susceptible to photooxidatoin however His photooxidation is fatal leading to

enzymatic inactivity [123]

243 Proteolysis and racemisation

Proteolysis and racemisation takes place at aspartic acid (Asp) residue The

bond between Asp and proline (Pro) is especially susceptible to hydrolysis Asp also

easily undergoes base-catalysed isomerisation to isoAsp through a ring formation [123]

95

Aspartic acid (Asp) Proline (Pro)

Fig 26 Side group of Asp and the amino acid structure of Pro

244 Physical instabilities

Physical instability includes denaturation adsorption aggregation and

precipitation and [123] This refers to loss of tertiary structure and does not involve

chemical modification It is defined as Gibbs free energy change between the native and

unfolded state of protein Physical instability is induced by change in temperature

extreme pH organic solvent denaturant [123] and solvation [120 125]

25 ENZYME STABILISERS

By clarifying the possible major causes of instability the choice of optimal

approach and stabilisers can be made Considering the case of glucose-oxygen fuel cells

the stabilisers should be selected on the basis of following points (i) conformational

stability for longer operation and shelf life (ii) hydrophilic environment for optimal

enzyme functions (iii) optimal interaction between enzyme and substrate and enzyme

and environment Conformational stability is to secure protein folding and avoid

denaturation This is achieved by thiol group protection reduction of oxidised thiol

96

groups removal of oxidising species an increase in internal hydrophobic interaction

and environmental hydrophilic and electrostatic interaction Optimal enzyme activities

can be achieved by an increase in hydrogen bonding and electrostatic interactions

251 Polyehyleneimine (PEI)

Polyethyleneimine (PEI) has been reported as an efficient antioxidant as well

as chelating agent It is also cationic (Fig 27) [120] therefore provides electrostatic

stabilisation with anionic protein [126] such as GOx [67 85] Anti-microbial activity

has been also reported [126] It is not effective against thermal inactivation [128]

H+

N+H3N+H3

N+H3H3N+

H2+ H+

H+

H+

H2

+

H2+

Fig 27 Structure of PEI [129]

PEI is available in different molecular weights [120] and smaller molecules are more

effective in binding and flexibility [130] However its optimal loading concentration

varies [120 128 131] PEI protects thiol groups efficiently [120] from both metal- and

peroxide- catalysed oxidation despite of their different oxidation mechanisms [126] PEI

also forms a complex with the enzyme and coats it with polycations This occludes the

97

oxidants from the protein surface [126 130] Complex formation might be involving

hydrogen bonding between carboxylic acid groups in the enzyme and nitrogen atoms in

PEI [130] This possibly confers stability in dried state [128] It was reported that PEI

increased the maximum limiting catalytic velocity (V) for GOx [131] and lactate

dehydrogenase (LDH) [128] However it also increased Km for LDH and shifted its

optimal pH slightly toward acidic [128]

252 Dithiothreitol (DTT)

Dithiothreitol (DTT) is another small water-soluble [132] thiol protecting

reagent [132 133] with the reducing potential at ndash 033 V at pH 7 It has only little

odour [132] and has low toxicity [134] Its epimer is dithioerythriotol (DTE) Both are

excellent reducing reagents with two thiol groups (Fig 28) [132] however DTT might

be marginally better in reducing power [135] It reduces back disulfide quantitatively

[127 132] at lower concentration than other thiol-containing reducing reagents such as

Cys and gluthatione (GSH) But it cannot restore some of the lost catalytic activity

[136] because it only reduces disulfides but not other thiol oxides Therefore its

efficacy depends on (i) the oxidation mechanism involved in the protein inactivation

(ii) the velocity at which the inactivation proceeds and (iii) how thiol-contaning amino

acids are present in the protein DTT is not effective against mild oxidation caused by

low concentration of H2O2 or isolated Cys residues [127] It is probably because that

DTT reduces thiol oxide by converting itself from a linear into a cyclic disulfide form

which is called trans-45-dihydroxy-o-dithiane with (Fig 28) [132]

98

Fig 28 Structures of DTT from a linear to a cyclic disulfide form [132]

On the other hand arsenite was effective in reducing only mildly oxidated thiol groups

but not disulfides [127] It is however toxic [137] and not suitable for practical use

Neither of DTT or arsenite was effective if thiol oxidation is outcompeting [127]

253 Polyethylene glycol (PEG)

Polyethylene glycol (PEG) (Fig 29) has been reported as an enzyme stabiliser

however also has mixed reception and its stabilisation mechanism has not yet been

clearly understood

Fig 29 Structures of PEG

OrsquoFagain reported that PEG increased storage and thermo stability [120] while

99

Andersson reported the increase in LDH activity with PEG 10000 Mw but was

followed by a decrease after 10-day storage and then eventually became worse than

those without stabilisers The stabilisation was also found to be concentration dependent

[128] As for protein stabilisation mechanism it was proposed that PEG preferentially

hydrates the protein and stabilises its native structure in the solution [138-140]

According to Arakawa this causes the exclusion of PEG from protein surface due to the

unfavourable interaction between them hence increases water density around the

protein This results in the water shell around the protein surface and increases the

hydrogen bonds [138 139] Better hydration favoured larger PEG Mw due to the larger

steric exclusion [138] on the other hand lower PEG Mw was also reported to be better

because of its better solubility and viscosity [139] Optimal concentration is unknown

however one proposal is that higher concentration is more advantageous to stabilise the

denatured protein because it can penetrate and stabilise the protein hydrophobic core

exposed during denaturation [138] In contrast Lee reported that this penetration affects

the hydrophobic core of active enzyme and decreased the activity and denatured the

protein [139] Both however concluded that a fine balance between preferential

hydration and hydrophobic interaction is important and PEG concentration must be up

to 30 [138 139] It was also reported that PEG stabilised the protein of low water

content better because it prevented the water evaporation while polyols tended to strip

water from the protein dehydrated microenvironment [139]

254 Polyols

Polyols such as glycerol (GLC) (Fig 30) were reported to increase shelf life

100

[141] thermostability [120] and also provide preferential solvation as seen in PEG

[141-143] Higher GLC concentration resulted in better protein activity [143 144] and

reduced protein aggregation due to the tighter protein packing [144] However observed

hydration was lower than expected for high GLC concentration therefore Gekko

proposed that the contribution of higher GLC concentration did not derive form the

increase in steric exclusion Instead GLC might penetrate the solvation layer of protein

and stabilise the structured water network around the protein [143] Andersson also

compared the influence on protein activity after the storage among several candidate

stabilisers diethylaminoethyl-dextran (DEAE)-dextran Mw 500000 sorbitol and PEI

DEAE-dextran increased the activity both before and after the storage while sorbitol

decreased it However PEI was only found to be superior to all [128]

OH

OHHO

Fig 30 Structure of glycerol

255 FAD

FAD (Fig 16 p 75) is also another possible stabiliser as well as mediator

because it can regenerate denatured aged enzymes which have lost its cofactors [82

108] However it has not been clarified well and Bartlett questioned its efficacy due to

the contradictory results he obtained [145] It was reported that addition of FAD

increased the electrode sensitivity by 70 especially in the concentration range below

101

3mM and activity by 8 times although it also increased Km by 2 times [108]

26 ENZYME KINETICS AND ELECTROCHEMISTRY

261 Michaelis-Menten kinetics

A simple enzyme reaction can be expressed in terms of equilibrium between

the free enzyme (E) substrate (S) enzyme-substrate complex (ES) and product release

(P) The enzyme is regenerated on completion of the catalytic cycle

General enzyme reaction mechanism

middotmiddotmiddotmiddotmiddot Eq 41

E = Enzyme concentration mol

S = Substrate concentration mol

ES = Enzyme-substrate complex concentration mol

P = Product concentration mol

k1 = Rate constant for ES formation from E and S mol-1s-1

k-1 = Rate constant for ES dissociation into E + S s-1

k2 = Rate constant for P formation from ES s-1

The rate of ES formation is defined as

102

][][]])[[]([][2101 ESkESkSESEk

dtESd

minusminusminus= minus middotmiddotmiddotmiddotmiddot Eq 42

Where [E0] is the initial enzyme concentration

When the reaction is at steady state 0][=

dtESd

])[(][][]])[[]([ 212101 ESkkESkESkSESEk +=+=minus minusminus middotmiddotmiddotmiddotmiddot Eq 43

Re-arranging the above equation for [ES] as below

][]][[

][121

01

SkkkSEk

ES++

=minus

middotmiddotmiddotmiddotmiddot Eq 44

When the rate of product formation is defined as follows

middotmiddotmiddotmiddotmiddot Eq 45

][2 ESktP=

∆∆

=υ middotmiddotmiddotmiddotmiddot Eq 46

Where υ is the catalytic rate

When all the enzyme active sites are occupied such a condition is called saturated and

defined as [E0] = [ES] The maximum catalytic rate (V) is therefore available as below

Maximum catalytic rate V

][][ 202 ESkEkV == middotmiddotmiddotmiddotmiddot Eq 47

103

Express υ in terms of [S] by inserting Eq 44 into Eq 46

][

]][[

1

21

02

Sk

kkSEk

++

=minus

υ middotmiddotmiddotmiddotmiddot Eq 48

Michaelis-Menten constant (KM) is a dissociation constant about ES and defined as

Michaelis-Menten constant

1

21

kkk

K M+

= minus middotmiddotmiddotmiddotmiddot Eq 49

Michaelis-Menten equation (Eq 50) is obtained when υ is expressed in terms of V and

KM by inserting Eq 47 and Eq 49 into Eq 48

Michaelis-Menten equation

][][SK

SV

M +=υ middotmiddotmiddotmiddotmiddot Eq 50

This is a hyperbola equation to lead the enzyme catalytic rate Most enzymes typically

show this hyperbolic curve (Fig 31) when they are free from allosteric controls or

enzyme inactivation

104

0E+00

1E-09

2E-09

3E-09

4E-09

5E-09

0E+00 2E-03 4E-03 6E-03 8E-03 1E-02 1E-02 1E-02 2E-02

[S] M

Fig 31 A typical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

As shown in Fig 31 Michaelis-Menten plot has different kinetic profiles at high and

low [S] Mathematical expression of its transition over [S] is shown in Table 3

Table 3 Michaelis-Menten equation at various [S]

[S] ge KM Vcongυ middotmiddotmiddotmiddotmiddot Eq 51

[S] = KM 2V

=υ middotmiddotmiddotmiddotmiddot Eq 52

[S] le KM MM K

SEkK

SV ]][[][ 02=congυ middotmiddotmiddotmiddotmiddot Eq 53

At high saturated substrate concentration the catalytic rate becomes zero-order

therefore Michaelis-Menten curve becomes plateau When [S] = KM the rate becomes

105

half of the maximum catalytic rate At low substrate concentration the catalytic rate is

linearly dependent on [S] therefore the kinetics becomes first-order about [S]

262 Electrochemistry and Michaelis-Menten kinetics

In electrochemistry enzyme activity is studied by amperometry This involves

measuring measuring the current generated by the enzyme electrode reaction at a fixed

potential over an arbitrary period It therefore shows the transition in current response

over time For enzyme electrodes substrate concentration is changed over time so that

the current response for a different substrate concentration can be measured over a long

period With intact enzyme and appropriate heterogeneous electron transfer present

most enzyme electrodes follow Michaelis-Menten kinetics (Fig 32) in the amperometry

in the absence of allosteric control or enzyme inactivation Therefore the amperometry

data can be analysed in the similar way to that of biochemical data and for this

Michaelis-Menten equation is expressed in electrochemical terms [146] In

electrochemical enzyme kinetics enzyme catalytic rate (υ) is obtained in terms of

electric current therefore V is substituted with maximum steady state limiting current

(Im) or its current density (Jm)

106

Electrochemical Michaelis-Menten equation

][][

SKSI

IM

m

+times

= middotmiddotmiddotmiddotmiddot Eq 54

][][

SKSJ

JAI

M

m

+times

== middotmiddotmiddotmiddotmiddot Eq 55

I = Current A

Im = Max steady-state limiting current A

[S] = Substrate concentration mol

KM = Michaelis-Menten constant mol

A = Electrode surface area m-2

J = Current density Am-2

Jm = Max steady-state limiting current density Am-2

There is often an offset at zero substrate concentration due to competing reactions and

trace contaminants and this must be subtracted before analysis

107

V

0E+00

2E-04

4E-04

6E-04

8E-04

1E-03

0 2 4 6 8 10 12 14 16

frac12 Jm

KM

Jm Am-2

[S] mM

Jm

Fig 32 Electrochemical Michaelis-Menten hyperbolic curve of enzyme kinetics

Based on the real data Refer to sect 343 p 124

KM and Jm (or V in biochemical enzyme kinetics) are important parameters to analyse

both enzyme catalytic power and stability Electrochemical Michaelis-Menten equation

and curves can be used for rough estimation of KM and Jm however it often leads to

inaccurate result because measuring initial rate for high substrate concentration is very

difficult in this method hence very prone to errors Therefore Michaelis-Menten

equation is linearised using one of the following four methods (i) Lineweaver-Burk (ii)

Eadie-Hofstee (iii) Hanes-Woolf and (iv) Cornish-Bowden-Wharton equations

Michaelis-Menten plot is however good for intuitive diagnosis of general enzyme

behaviours

108

263 Linear regression methods for electrochemical Michaelis-Menten enzyme kinetics

2631 Lineweaver-Burk linear regression

Lineweaver-Burk linear regression is one of the classic regression methods

taking double reciprocal of Michaelis-Menten equation (Eq 56)

Electrochemical Lineweaver-Burk linear regression

mm

M

JSJK

J1

][11

+times= middotmiddotmiddotmiddotmiddot Eq 56

It plots 1J against 1[S] as shown in Fig 33 The value of each parameter is found by

extrapolating the line The reciprocal of Jm is found at y-intercept and KM can be derived

from either x-intercept or the slope once Jm is found

109

-50E+02

00E+00

50E+02

10E+03

15E+03

20E+03

-06 -05 -04 -03 -02 -01 00 01 02 03 04

1[S] mM-1

1J A-1m

2

Fig 33 Electrochemical Lineweaver-Burk linear regression

Based on the real data Refer to sect 343 p 124

Lineweaver-Burk linear regression is however largely influenced by the errors in j at

low substrate concentrations [22 147] Its reciprocal could vary by a factor of 80 over

the range of substrate concentration varying one-third to three times the KM [148]

Hence it is not an ideal method for the analysis

2632 Eadie-Hofstee linear regression

Eadie-Hofstee linear regression plots J against J[S] This plot is strongly

affected by the errors in υ because υ is present in both coordinates (Eq 57)

Electrochemical Eadie-Hofstee linear regression

mM JSJKJ +timesminus=

][ middotmiddotmiddotmiddotmiddot Eq 57

110

Again the plot must be extrapolated to find Jm at y-intercept KM can be derived from

the slope (Fig 34)

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

0E+00 5E-05 1E-04 2E-04 2E-04 3E-04

J [S] AmM-1

J Am

-2

Jm

-Km

Fig 34 Electrochemical Eadie-Hofstee linear regression Based on the real data

Refer to sect 343 p 124

Eadie-Hofstee linear regression will give very strict evaluation [148] and might be used

to detect the deviations from Michaelis-Menten behaviour only if the data is precise and

accurate [147]

2633 Electrochemical Hanes-Woolf linear regression

Hanes-Woolf linear regression plots [S]J against [S] KM is derived from the

x-intercept and Jm is found from the slope

111

Electrochemical Hanes-Woolf linear regression

m

M

m JKS

JJS

+times= ][1][ middotmiddotmiddotmiddotmiddot Eq 58

This is a better method compared to the previous two regression methods because all

the parameter values can be derived from the reasonable range of [S] and they donrsquot

heavily rely on the extrapolation [147] The data is normalised by [S] therefore does not

cause many errors

-20E+08

00E+00

20E+08

40E+08

60E+08

80E+08

10E+09

12E+09

14E+09

-40E+00 00E+00 40E+00 80E+00 12E+01 16E+01

[S] mM[S] J m

MA

-1m2

Fig 35 Electrochemical Hanes-Woolf linear regression Based on the real data

Refer to sect 343 p 124

However either of these three linear regressions might not be adequate for the enzyme

electrode analysis [65] because the data reflect not only the single-substrate

Michaelis-Menten kinetics but also include the influence from other factors such as

enzyme immobilisation mediator reactions and electrode composition as well as

112

general experimental errors Their contributions to the overall kinetics therefore must be

considered [65 149] although the enzyme electrode reactions can approximate the

Michaelis-Menten model when one substrate is a variable and other variables are fixed

over an experiment [147 149] Under such a complex system it is not ideal to use those

graphical linear regression methods because they assume that the results follow

Michaelis-Menten model and that all the experimental errors follow normal distribution

[150] without giving any measure in the precision [148] Hence it is more suitable to

use a non-parametric direct linear plot or nonlinear regression methods because they

respect the influence and errors from various sources without any bias

264 Cornish-Bowden-Wharton method

Cornish-Bowden-Wharton method is distribution-free non-parametric direct

linear plot In this method observations are plotted as lines in parameter space rather

than points in observation space [151] It then takes the median of Jm and KM from every

possible non-duplicate pair of observed [S] and υ (Eq 59 and Eq 60) as the best-fit

estimate

Electrochemical Cornish- Bowden-Wharton non-parametric method

])[][(])[]([

1221

122121 SJSJ

SSJJJm minusminus

= middotmiddotmiddotmiddotmiddot Eq 59

])[][()](][[

1221

122121 SJSJ

JJSSKM minusminus

= middotmiddotmiddotmiddotmiddot Eq 60

It is a simple method without complex calculation insensitive to outliers and no

113

weighting is required [147 152]

00E+00

20E-04

40E-04

60E-04

80E-04

10E-03

12E-03

14E-03

16E-03

18E-03

20E-03

-5 -3 -1 1 3 5

KM mM

Jm Am-2Median Jm

Median KM

Fig 36 Electrochemical Cornish-Bowden-Wharton plot Based on the real data

Refer to sect 343 p 124

265 Nonlinear regression methods

Nonlinear regression methods manipulate the parameters to minimise the sum

of squared difference between the measured and expected values [150] They fit the

integrated equation to the enzyme progress curve and calculate the approximate location

of the minimum for the sum of squared difference at a particular point in the slope It

then recalculates the new minimum location to improve the prediction until there is no

further improvement This is called gradient method based on Gauss-Newton algorithm

or non-linear least square method [150 153] This is computationally intensive due to

114

the recursive operations however advanced computational technology has made it

possible The parameters obtained may not be perfectly correct [150] The method still

assumes errors normally distributed [153] and Cornish-Bowden has criticized about the

ambiguous statistical assumption of such computer programmes [154] However

non-linear regression methods are insensitive to outliers [150 153] have better estimate

accuracy compared to conventional linear regression methods and the estimations

mostly fit Michaelis-Menten model Graphical methods arose at a time when nonlinear

regression methods were computationally not available however it is now possible

thanks for the great technological advance and more recommended to use in analysis to

yield better precision and reliability [153]

115

3 ANODE MATERIAL AND METHOD

31 ELECTRODE FABRICATION

311 TTF-TCNQ synthesis

Tetrathiafulvalene ge980 (TTF) and 7788-Tetracyanoquinodimethane

ge980 (TCNQ) were purchased from Sigma Aldrich Spectroscopic grade acetonitrile

and diethyl ether were purchased from VWR International TTF and TCNQ were

weighted to 1 g each and dissolved separately in 50 ml of acetonitrile TTF-acetonitrile

solution had yellow colour while TCNQ-acetonitrile solution was green Both were

heated on the hot plate under the fume hood On the full dissolution two solutions were

quickly mixed to form black solid crystals The mixture was immediately taken off the

hot plate and cooled down at the room temperature for a few hours The crystals were

collected on Buchner funnel and washed under vacuum twice with acetonitrile twice

with diethyl ether and then twice again with acetonitrile The washed crystals were left

to dry for overnight with a cover The dried crystals were collected and kept in the

sealed container for future use [61]

312 Electrode template fabrication

Sylgard 184 Silicone Elastomer Kit were purchased from Dow Corning

Corporation for polydimethylsiloxane (PDMS) Base and hardener were mixed at 101

(vv) for use CY1301 and aradur hardener HY 1300 were purchased from Robnor

116

resins to make epoxy resin They were mixed at 31 (vv) for use

Silicone electrode templates (Fig 16(i)) were made to shape electrode discs

Each template had round holes in line Two types of templates template A and B were

made Template A had holes with the dimension 2-mm diameter x 1-mm thickness

while template B had holes with the dimension with 5-mm diameter x 2-mm thickness

In order to make PDMS templates for electrode discs different templates were made to

shape the holes in PDMS (Fig 16(ii)) Discs were made with epoxy resin and were

fixed onto the plastic Petri dish PDMS was poured into disk-mounted Petri dish

degassed for 20 minutes and then cured at 60 degC Cured PDMS electrode templates

were taken out from the epoxy resin-Petri dish templates and then cleaned with

analytical grade ethanol for use

Fig 37 PDMS electrode templates (i) PDMS template and (ii) their epoxy resin-Petri

dish template

117

313 Electrode composite fabrication

3131 Composite for conductance study

Graphite at microcrystal grade (2-15 micron 999995 purity) was purchased

from Alfa Aesar GmbH amp CoKG Glassy carbon (GC) powder was kindly donated from

Dr Zhao Carbon nanofibre (CNF) was also kindly donated from Loughborough

University Composite electrodes were built from the mixture of the carbon conductive

or TTF-TCNQ and epoxy resin Each composite mixture had different composition ratio

[108] as below in Table 4

Composite Ratio I Ratio II Ratio III Ratio IV Ratio V

Graphite Epoxy resin 73 64 55

GC Epoxy resin 64 73

CNF Epoxy resin 37 46 55 64 73

TTF-TCNQ Epoxy resin 64 73 82

Table 4 Composite composition

TTF-TCNQ was mixed with epoxy resin at 64 73 and 82 Graphite was mixed at 73

64 and 55 GC powder was mixed at 64 and 73 CNT was mixed at 37 46 55 64

and 73 (ww) Three to five copies were fabricated for each electrode type Composite

mixture was packed into the template A (Fig 38) and cured at room temperature for 72

hours [106]

118

Fig 38 PDMS template (i) packed with electrode material and (ii) empty one

3132 Bulk modified GOx enzyme electrode composite

Glucose oxidase (GOx) [Type II-S 15000-50000 unitsg] from Aspergillus

niger was purchased from Sigma Aldrich Conductive composite paste was first made

with TTF-TCNQ and epoxy resin at 73 as in sect 3131 GOx was then incorporated into

the conductive composite mixture at varying mixing ratio 5 10 and 15 (ww) by

bulk-modification method (Fig 39) 10 GOx composite electrodes were also made

with graphite Enzyme composite mixture was packed into the template B and cured at

4 degC for 96 hours

119

R

RR R

R

RR R

RRR

Conducting particles

Insulating binder

Chemical modification

Add biological receptor R

Cast extrude or print into suitable form

R

R

RR

R

R

R

R

R

R

RR

Fig 39 Schema for bulk modification method

3133 GOx enzyme electrode composite with enzyme stabilisers

Poly(ethyleneimine) (PEI) solution [50 (wv) in H2O] DL-dithiothreitol

(DTT) Poly(ethylene glycol) (PEG) [average mol wt 200] glycerol (GLC) [ge99] and

flavin adenine dinucleotide disodium (FAD) salt hydrate (ge95) were purchased from

Sigma Aldrich Each stabiliser was mixed with GOx at different fraction 0 1 2 and

4 PEI was also blended with DTT or FAD for synergetic effects as below in Table 5

Blend type Ratio I Ratio II Ratio III Ratio VI

PEIDTT 0515 1010 1505 2020

PEIFAD 21 22 24

Table 5 Blended stabiliser composition

120

PEI was mixed with DTT at the ratio of 0515 11 1505 and 22 PEI was also

mixed with FAD at 21 22 and 24 Each enzyme-stabiliser mixture was weighted to

10 of the whole composite It was then mixed with the conductive composite mixture

made earlier The conductive composite mixture was made of TTF-TCNQ and epoxy

resin at 73 The final composite mixture was packed into the template B and cured at

4 degC for 96 hours

314 Electrode wiring and insulation

Silver wire (temper annealed 025-mm diameter) was purchased from Advent

Portex polyethylene tubing (028 x 0165 mm) purchased from Scientific Laboratory

Supplies Silver epoxy was purchased from ITW chemotronics Silver epoxy came in

two parts A and B and they were mixed at 11 (vv) for use Silver wire was cut for

5-cm length and its tip was cleaned with analytical grade ethanol dried and coved with

insulating polyethylene tubes Cured electrode composite discs were popped out of the

PDMS template An insulated silver wire was fixed onto to each electrode disc with

conductive silver epoxy and it was left at room temperature for 24 hours to cure silver

epoxy After silver epoxy had been cured the wired electrodes were coated and

insulated with degassed epoxy resin and cured at room temperature for 3 days for

non-enzyme composite electrodes and 4degC for 4 days for enzyme composite electrodes

7-9 days were taken to build the complete composite electrodes (Fig 40) The enzyme

electrodes were stored at 4degC when not in use

121

Fig 40 Insulated composite electrodes (i) non-enzyme (ii) GOx electrodes

32 CONDUCTIVITY EXPERIMENT

Copper foil with electrically conductive acrylic adhesive was purchased from

RS Components Digital multimeter from Metra was used to measure resistance

Electrode surface was covered with adhesive copper foil evenly and both ends were

clamped with crocodile clips (Fig 41(i)) to measure electrode resistance of the

composite as in Fig 41(ii)

122

Fig 41 Measuring electrode resistance (i) connecting the electrodes to the

multimeter (ii) measuring the electrode resistance with the multimeter

33 POLISHING COMPOSITE ELECTRODES

MicroPolish II aluminium oxide abrasive powder (particle range 1 03 and

005 microm) silicone carbide (SiC) grinding paper for metallography wet or dry (grit 600

1200 and 2400) and MicroCloth Oslash73 mm for MiniMet for fine polishing were

purchased from Buehler Self adhesive cloth for intermediate polishing was purchased

from Kemet Electrodes were polished immediately before the experiment until

mirror-like flat surface was obtained This erases the electrode history and gives

assigned electrode surface area [105] Composite electrodes were hand polished

successively with SiC grinding paper in 400 1200 and 2400 grit During polishing

deionised water of 18 MΩ was added occasionally to the polishing surface This

prevents damaging electrode surface with frictional heat It was followed by successive

polishing with alumina oxide slurry with particle size 1 03 and 005 microm Slurry was

123

sonicated to dissolve agglomerates before use [155] Kemet polishing pad was used for

rougher polishing with 1 and 03 microm powder Felt-like MicroCloth was used for

polishing with 005microm Electrodes were sonicated in the deionised water briefly after

polishing at each grit or grade

34 GLUCOSE AMPEROMETRY

341 Glucose solution

Phosphate buffer saline (PBS) powder for 001 M at pH 74 containing 0138

M NaCl and 00027 M KCl [156] was purchased from Sigma Aldrich PBS was kept in

the fridge when not in use [157] 1M glucose solution was made by mixing

D-(+)-Glucose with 001 M PBS and left for 12 hours at 4degC for mutarotation It is

mandatory because GOx has stereochemical specificity and only active toward

β-D-glucose while glucose solution consists of two enantiomers α- and β-D glucose

and their concentration changes over time until they reach the equilibrium at 3862

Experiments should be carried out in the equilibrated solution for accuracy

342 Electrochemical system

A single-channel CHI 650A and multi-channel CHI 1030 potentiostats were

used for glucose amperometry with three-electrode system in which the

electrochemical cell contained (a) working electrode(s) an AgCl reference electrode

and a homemade Pt counter electrode The AgCl reference electrode was purchased

124

from IJ Cambria AgCl reference electrode was saturated with 3M KCl Pt counter

electrode was made of Pt wire and Pt mesh was fixed at its tip to give large surface area

Pt mesh at its tip

343 Glucose amperometry with GOx electrodes

Glucose amperometry was conducted by adding a known volume of 1M

glucose aliquot into 100 ml of 001 M PBS electrolyte solution containing GOx anodes

with convection applied Convection was however ceased on during current

measurement to avoid the noise The electrolytic solution was gassed with either N2 O2

or compressed air for 15 minutes before the experiment O2 concentration is 123 mM in

fully oxygenated PBS [158] and 026 mM in air [112] Gas was continuously supplied

through Dreschelrsquos gas washing bottle containing PBS during experiment The response

of GOx anodes to glucose was obtained as steady state current The amperometry was

carried on until no further current increase was observed against glucose added Each

experiment took more than 8 - 12 hours The sample size ranged from two to three in

most cases

125

Fig 42 Amperometry setting with CHI 1030 multichannel potentiostat

35 ANALYSIS OF GLUCOSE AMPEROMETRY DATA

Normality of enzyme function was confirmed by plotting Michaelis-Menten

curve based on the amperometry data obtained The capacitive current before glucose

addition was subtracted from the data Enzyme kinetic parameters the maximum

current density (Jm) and the enzyme dissociation Michaelis constant (KM) were

automatically estimated with standard error using an analytical software called

SigmaPlot Enzyme Kinetics version 13 The estimation is based on non-linear

regression analysis about Michaelis-Menten kinetics as shown in Fig 43

126

Glucose M

0 1e-2 2e-2 3e-2 4e-2 5e-2

J A

m-2

00

20e-3

40e-3

60e-3

80e-3

Vmax = 0008445 Km = 0005842

Fig 43 Analytical output by SigmaPlot Enzyme Kinetics Based on the real data

Influence of enzyme immobilisation electrode composition O2 and stabilisers on GOx

electrode performance was studied The most efficient GOx anodes was determined on

the basis of the parameters obtained

127

4 ANODE RESULTS AND DISCUSSION

41 AIMS OF ANODE STUDIES

This section describes the fundamental study on the enzyme composite

electrodes and the influence of enzyme stabilisers on the performance and stability of

GOx electrodes Firstly the difference in conductivity as a function of various

conductive materials and the component fraction were studied They did not contain

enzyme The most suitable composite was then selected and used to build the enzyme

electrodes Secondly GOx electrodes were built at various GOx fractions in the

composite The best component fraction was determined from the data obtained in

amperometry Amperometry was conducted to obtain two enzyme kinetic parameters

the maximum current density (Jm) and the enzyme dissociation Michaelis constant (KM)

Jm indicates the size of catalytic velocity KM indicates the extent of enzyme-substrate

dissociation Smaller KM values indicate better enzyme-substrate affinity and enzyme

stability With the component ratio selected enzyme electrodes were then constructed

using various types and concentrations of enzyme stabiliser Amperometry was again

conducted to study the influence of enzyme stabilisers on the GOx anode performance

and stability The catalytic efficiency was determined by the fraction M

mK

J This is

based on the concept of specificity constant M

catK

k as defined below

128

Enzyme electrode specificity constant

MMM

cat

KEV

Kk

Kk

sdot==

][2 middotmiddotmiddotmiddotmiddot Eq 61

where kd = k2 Rate constant for P formation from ES

The specificity constant is a common measure of the relative catalytic rate at low

substrate concentration [22] M

mK

J therefore can be used as a comprehensive

indicator for both catalytic velocity and enzyme stability under the fixed enzyme

concentration In this study GOx concentration was fixed to approximately 10 weight

of the total composite According to the M

mK

J obtained the five most efficient and

stable GOx electrodes were determined The possible enzyme stabilisation mechanisms

of each enzyme stabiliser were also discussed together in the light of published

hypothesises

42 CONDUCTANCE OF THE COMPOSITE

421 Conductance of various conductive materials

Various electrodes were built with different conductive materials graphite GC

CNF and TTF-TCNQ at different component fraction The electrode dimension was

fixed to 314 mm2 x 1 mm Their conductance was measured and presented in a box plot

as shown in Fig 44 The sample size ranged from 3 to 6 The median is indicated by a

short line within the box

129

00E+00

50E-02

10E-01

15E-01

20E-01

25E-01

30E-01

35E-01

40E-01

45E-01C

ondu

ctan

ce

S

Lower quartile S 103E-03 549E-03 219E-01 111E-01 142E-01 599E-04 390E-03 115E-02 972E-03

Max S 281E-03 338E-02 268E-01 312E-01 415E-01 810E-04 113E-02 467E-02 262E-02

Min S 739E-04 467E-03 200E-01 933E-02 855E-02 418E-04 296E-03 264E-03 587E-03

Upper quartile S 206E-03 282E-02 253E-01 220E-01 253E-01 795E-04 807E-03 232E-02 199E-02

Median S 131E-03 847E-03 238E-01 129E-01 216E-01 780E-04 484E-03 155E-02 136E-02

TTF-TCNQ64

TTF-TCNQ73

Graphite 55 Graphite 64 Graphite 73 CNF 37 CNF 46 CNF 55 GC 73

Fig 44 Conductance of various electrode composite n = 3 ndash 6

Graphite composites showed higher conductance than other composites It is explained

by graphite having the highest intrinsic conductivity ranging between 103 ndash 104 Scm-1

[105 159] although being compounded in a composite lowered the conductivity by 4 ndash

5 order of magnitude compared to that of raw material The conduction did not show a

linear relationship with the electrode composition ratio for graphite composite because

the graphite composite 55 had larger conductance of 024 S than that of 64 and 73 It

also showed the smallest deviation than the other graphite composites while the

deviation increased with increase in the graphite content One possible cause of this is

the presence of patchy conductive paths due to the uneven distribution of conductive

islands within the composite [105 106] The conduction of the composite is therefore

often locally biased unpredictable and also leads to low reproducibility TTF-TCNQ

composite at 73 showed the conductance of 85 mS smaller than the graphite

composited 55 approximately by 30 times Conductivity of TTF-TCNQ is 102 ndash 103

130

Scm-1 [109 110] and smaller than that of graphite by one order of magnitude so this is

consistent with the intrinsic conductivity of the conducting paticles

There were also different interactions observed among conductive materials

with insulating materials Mixing epoxy resin with graphite was easier than mixing with

other conductive materials Some even did not cure at the same mixing ratios The

carbon nanofibre (CNF) composite mixture did not cure when it exceeded more than

50 weight content With 40 CNF content however it showed the conductance of 48

mS It was not comparable to graphite composite despite its similar conductivity of 103

S [105] however it was similar to that of TTF-TCNQ 73 composite 50 CNF content

even showed larger conductance of 155 mS than 70 TTF-TCNQ CNFs have been

reported to have mediating function [117] therefore with this high conduction property

at low content CNF might be very useful to enzyme composite and even superior to

TTF-TCNQ

422 Influence of enzyme concentration on conductivity

Conductance of the GOx composite was measured The conductive composite

consisted of either TTF-TCNQ 73 or graphite composite 73 GOx content was varied

by 5 10 and 15 for TTF-TCNQ composite and 10 only for graphite composite

The electrode dimension was fixed to 196 mm2 x 2 mm The sample numbers ranged

from 2 ndash 4 The study revealed that the presence of enzyme interfered with conduction

in GOx concentration dependent manner for TTF-TCNQ composite as shown in Fig 45

131

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01C

ondu

ctan

ce

S

Low er quartile S 131E-02 924E-03 124E-05 165E-01

Max S 484E-02 143E-02 368E-05 593E-01

Min S 120E-02 754E-03 390E-06 122E-01

Upper quartile S 441E-02 126E-02 288E-05 400E-01

Median S 281E-02 109E-02 208E-05 207E-01

TTF-TCNQGOx 5 TTF-TCNQGOx 10 TTF-TCNQGOx 15 GraphiteGOx 10

Fig 45 Conductance of GOx composite with various GOx content n= 2 - 4

This was due to insulating property of the protein although higher enzyme loading is

preferred for better catalytic performance as discussed in sect 226 on p 88 The issue

may be circumvented by fixing the enzyme onto the electrode surface It is however

more technically tedious and not as feasible for mass manufacture as bulk modification

It is also difficult to modify conductive salt for covalent attachment of the enzyme This

is because the conductive property of conducting salt is very sensitive to its chemical

structure and slight modification can lead to insulating properties [61] A similar

phenomenon has been also observed in CNT functionalisation [117]

Graphite-GOx composite showed larger conductance than TTF-TCNQ-GOx

composite by 20 times at the same component fraction This was due to the better

conductive property of graphite However graphite does not have mediating property

and the use of graphite as conductive material for enzyme electrodes requires the

132

loading of mediator separately The key to designing enzyme electrodes is in the balance

and compromise among the conductivity mediation enzyme activity mechanical and

chemical stability and difficulty in fabrication process A good candidate material to

make up for the TTF-TCNQ lower conductivity is CNT It has been reported that CNF

has both good conductivity and mediating functions [117] and might be suitable for bulk

modification More safety study on use of CNF is however necessary [117 160]

43 CATALYTIC ACTIVITY OF GOx COMPOSITE ELECTRODES

431 Influence of GOx concentration on the catalytic activity of GOx electrodes

Amperometry was conducted for GOx composite electrodes in N2 O2 and air

for various GOx content The conductive composite consisted of either TTF-TCNQ 73

or graphite composite 73 GOx content was varied by 5 10 and 15 for

TTF-TCNQ composite and 10 only for graphite composite The sample size was 3 for

each electrode type Control experiments were also performed with enzyme-free

composite electrodes and there was no glucose response observed There was also no

stable current observed for 10 GOx-graphite electrodes despite of its high

conductivity reported in sect 422 while Michaelis-Menten curve was observed in any

TTF-TCNQ-GOx composite electrodes This confirmed the necessity of mediating

function for enzyme anodes and its presence in TTF-TCNQ

All the TTF-TCNQ-GOx electrodes showed the Michaelis-Menten current

response curves however differed in the current as shown in Fig 46

133

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

35E-02

00E+00 10E-02 20E-02 30E-02 40E-02 50E-02

Glucose M

J A

m-2

GOx 5GOx 10GOx 15

Fig 46 Difference in the glucose-response amperometric curve of enzyme

electrodes for various GOx content 5 10 and 15 in the presence N2

The 15 GOx composite showed the smallest glucose response current due to the

interfered conduction by high protein content while 5 GOx composite showed smaller

response than 10 composite due to the enzyme deficiency Their Jm only counted for

26 and 41 of that of 10 GOx composite (Fig 47(i)) 10 GOx electrode was

therefore most catalytic KM for 5 and 10 GOx were 71 and 73 mM and little

different (Fig 47(ii)) This implies that the size of catalytic activity can be flexibly

altered by only slight change in KM

134

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

Jm

Am

-2

Jm Am-2 729E-03 180E-02 466E-03

GOx 5 GOx 10 GOx 15

00E+00

20E-03

40E-03

60E-03

80E-03

10E-02

12E-02

14E-02

Km

M

KM M 710E-03 729E-03 187E-03

GOx 5 GOx 10 GOx 15

Fig 47 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 n = 1 - 3

The results also tell that an increase in GOx loading could enhance the catalysis hence

output current unless the increase is not excessive and gt 15 at least The loading limit

should lie somewhere between 10 and 15 for this composite Similar electrodes

were also built by Alegret with 75 epoxy resin 10 TTT-TCNQ 10 graphite and

5 GOx [115] Their Jm was around 7 mAm-2 and little different from that of 5 GOx

with 70 TTF-TCNQ composite It suggests that less TTF-TCNQ content might be

sufficient as long as the conductivity is sustained by more conductive material such as

graphite

432 Influence of O2 on the catalytic activity of GOx electrodes

O2 and air suppressed the current output by GOx composite electrodes and

delayed the response to the glucose as shown in Fig 48 A current increase was

observed immediately after glucose addition for N2 but not for O2 and air They showed

135

sigmoidal current curves with a glucose response lag

(i)

(ii)

-50E-03

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

00E+00 50E-03 10E-02 15E-02 20E-02 25E-02 30E-02 35E-02 40E-02 45E-02

Glucose M

J A

m-2

GOx 10 N2GOx 10 O2GOx 10 Air

Fig 48 Difference in the glucose-response amperometric curve of 10 GOx

composite electrodes in the presence (i) N2 and O2 and (ii) re-plotted

amperometric curves for N2 air and O2

136

As expected O2 and air decreased Jm and increased KM as shown in Fig 49

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

GOx 5 GOx 10 GOx 15

Jm

Am

-2

N2O2Air

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

70E-02

80E-02

GOx 5 GOx 10 GOx 15

Km

M

N2O2Air

Fig 49 Kinetic parameters Jm and KM for enzyme electrodes with various GOx

content 5 10 and 15 in the presence N2 O2 and air n = 1 - 3

Both O2 and air decreased the current of 15 GOx composite electrodes Jm dropped to

approximately 20 of that for N2 KM was increased by 10 and 2 times for O2 and air

respectively The glucose response lag was 8 mM for O2 but none for air Air showed

milder suppression toward current output than O2 and this was expected because O2

counts for only 20 of the air [31] and should cause less influence on GOx activity

Similar phenomena were observed for 10 GOx composite electrodes For O2 and air

Jm was 13 and 40 of that of N2 and decreased to 23 x 10-3 and 67 x 10-3 from 18 x

10-2 Am-2 respectively KM was 73 for N2 and 10 mM for both O2 and air although there

was a response lag of 8 and 1 mM respectively Little difference in KM was observed

among all cases and this implies that KM itself hence enzyme stability or substrate

affinity is little affected by O2 but slight change in KM has large influence on the

catalytic activity This implies that change in catalytic activity induced by O2 is

137

reversible 5 GOx composite electrodes showed slightly unexpected result KM was

increased by approximately 4 times in both O2 and air Jm also decreased however Jm

for air was smaller than O2 by 36 despite of less O2 content There was a response lag

by 10 mM for O2 and 2 mM for air

The overall conclusions from these results is that O2 reduces GOx catalytic rate

andor electron transfer rate and hence reduces the current output but the effect is

reversible because it hardly alters KM value implying that O2 does not affect the enzyme

stability or its affinity to the substrate This phenomenon is typical in non-competitive

inhibition for enzyme kinetics Non-competitive inhibition occurs when an inhibitor

binds to the enzyme-substrate complex and slows its catalysis It therefore changes

catalytic velocity but not KM In the glucose amperometry this implies two possibilities

as a cause of Jm reduction (i) allosteric binding of O2 to GOx-glucose complex or (ii)

interference in electron transfer after the catalysis In the former case O2 does not

inhibit glucose binding to GOx but allosterically interacts with its complex and hinders

its catalysis hence it reduces catalytic current output In the latter case either glucose

binding or GOx catalysis is not affected by O2 but competition for electrons produced

by GOx catalysis occurs between the natural and artificial mediators namely O2 and

TTF-TCNQ and this reduces the electron flow to the electrode therefore it is reflected

as the reduction of catalytic current In addition to this the presence of glucose response

lag also suggests the presence of electron competition [161-163] It was reported that O2

reacts with reduced GOx faster than any other mediators with the second order rate

reaction of 15 x l06 M-1s-l [112 161 163] It is therefore quite possible that O2 swiftly

deprives electrons from reduced GOx andor reduced mediators Reduced GOx reacts

with O2 much faster than with TTF-TCNQ because it was observed that O2 had

138

diminished glucose response current at low glucose concentration Most GOx hence

reacts with O2 first then oxidised TTF-TCNQ once most O2 has been converted into

H2O2 This is compatible with the hypothesis suggested by Hindle [161] Even after the

lag the reaction could go on with residual O2 because KM did not recover fully to the N2

level although a familiar Michaelis-Menten hyperbolic curve was regained This simply

indicates that O2 concentration became smaller than TTF-TCNQ to oxidise GOx The

glucose response lag also decreased with an increase in GOx loading and was smaller

for air than O2 This implies the presence of competition because the lag depends on the

concentration of reduced GOx and electron thieves The large KM increase in the 5

GOx composite electrodes was probably due to the deficiency in the electron transfer

rate to suppress O2 activity How O2 interacts with its environment is unknown however

O2 reduces the catalytic output current due to the following possible mechanisms (i)

electron competition between O2 and TTF+ (ii) non-competitive enzyme inhibition by

allosteric binding of O2 to GOx (iii) direct interaction between O2 and reduced TTF or

(iv) backward reaction between reduced TTF and oxidised GOx

139

Table 6 Four possible causes of glucose amperometric current reduction

(i) middotmiddotmiddotmiddotmiddot Eq 62

middotmiddotmiddotmiddotmiddot Eq 63

(ii) middotmiddotmiddotmiddotmiddot Eq 64

(iii) middotmiddotmiddotmiddotmiddot Eq 65

(iv) middotmiddotmiddotmiddotmiddot Eq 66

Diagnosis can be made by doing experiments with different concentration of O2 and

mediators

44 THE EFFICACY OF GOX STABILISERS

441 Introduction

Various GOx stabilisers PEI DTT PEG GLC and FAD were mixed with GOx

composite at the concentration varying among 1 2 and 4 PEI was also mixed with

DTT or FAD and their synergetic effects were studied All the GOx electrodes were

built on the basis of TTF-TCNQ 73 conductive composite with 10 weight

GOx-stabiliser mixture Control composite electrodes were built with 2 stabilisers but

without GOx Glucose amperometry was conducted as before in the both presence and

absence of O2 Two kinetic parameters Jm and KM were obtained through non-linear

regression analysis The standard kinetic parameter values were set to those obtained in

140

the absence of O2 for GOx electrodes containing no stabiliser They are Jm = 18 x 10-2 plusmn

73 x 10-3 Am-2 and KM = 73 plusmn 57 mM respectively The glucose response lag

observed for this electrode was 10 mM Comprehensive catalytic efficiency was

determined by the size of the fraction M

mK

J under the influence of high O2 and the

most catalytic GOx composite electrodes were chosen on the basis of M

mK

J The

sample size ranged between 2 and 3 None of control experiments showed any glucose

response

442 PEI

Kinetic parameters for various PEI concentrations are shown below in Fig 50

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser PEI 1 PEI 2 PEI 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEI 1 PEI 2 PEI 4

KM

M

N2 O2

Fig 50 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEI in the presence and absence of O2 n = 1 - 3

141

In the absence of O2 PEI lowered Jm without largely affecting KM compared to the

standard value This might imply the presence of allosteric non-competitive inhibition

of glucose catalysis It is certainly possible because PEI can lock in the enzyme

conformation with hydrogen and electrostatic bonding [120] or form a complex with

GOx [126 130] This interferes in its catalysis although it also might confer the

resistance against oxidative stress as reported [126] In the presence of O2 two out of

three electrodes of PEI 1 and 4 did not show Michaelis-Menten curves The data

presented here is therefore based on a single sample This may mean that the electrodes

at those PEI concentrations are in fact prone to the catalytic suppression by O2

Moreover 4 PEI data presented here looks unreliable due to the simultaneous increase

in Jm and KM by 3 ndash 4 times compared to those obtained in the presence of N2 It may

need more data to confirm the phenomenon and be premature to conclude it is however

probably more reasonable to think that PEI 4 is not suitable for GOx anodes At other

PEI concentrations electrodes showed more or less similar kinetic parameter values to

those without stabilisers since Jm decreased and KM increased in the presence of O2

There was no O2-scavenging function observed because their glucose response lag and

KM resembled those without stabilisers Among them 2 PEI containing electrodes

sustained 50 of catalytic activity obtained in N2 and showed marginally better Jm than

stabiliser-free electrodes in the presence of O2 It might therefore have some resistance

against O2 It can be however concluded that PEI is not suitable as an enzyme stabiliser

and not effective enough to enhance the catalytic current output because their Jm was

already smaller by 60 at minimum in N2 compared to the standard value and little

improvement in catalytic performance was observed in the presence of O2

142

443 DTT

Kinetic parameters for various DTT concentrations are shown below in Fig 51

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

Jm

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser DTT 1 DTT 2 DTT 4

KM

M

N2 O2

Fig 51 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 DTT in the presence and absence of O2 n = 3

All DTT containing electrodes showed smaller Jm and KM than the standard values in

the absence of O2 This might imply the possible presence of uncompetitive inhibition

by DTT although the size of inhibition was not in concentration dependent manner In

uncompetitive enzyme inhibition inhibitors bind both free enzyme and

enzyme-substrate complexes hence decreases both catalytic velocity and KM [22] In the

presence of O2 all Jm KM and glucose response lag were larger than those of

stabiliser-free electrodes The increase in glucose response lag was probably due to both

electron competition and uncompetitive inhibition while the inhibition by DTT itself

was less influential on catalytic velocity than non-competitive inhibition by O2 itself

143

and this resulted in larger Jm despite of their larger KM The truth of O2 enzyme

inhibition might be reversible Met oxidation as discussed in sect 242 on p 93 because

atmospheric O2 could oxidise Met residue [123] and DTT might be capable of reducing

it back although DTT has been reported to be effective only in reducing disulphide [127

132] The increase in Jm in the presence of O2 compared to N2 was probably due to the

reduced content of DTT working toward stabilising enzyme structure This freed up

some GOx portion from rigid conformation

444 PEG

Kinetic parameters for various PEG concentrations are shown below in Fig 52

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEG 1 PEG 2 PEG 4

J m

Am

-2

N2 O2

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

60E-02

No Stabiliser PEG 1 PEG 2 PEG 4

KM

M

N2 O2

Fig 52 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

1 2 and 4 PEG in the presence and absence of O2 n = 2 - 3

PEG decreased Jm to 10 of the standard value at maximum in the absence of O2

144

although 4 PEG electrodes showed much larger Jm than the other PEG electrodes

unexpectedly 4 PEG however showed glucose response lag gt 10 mM even in the

absence of O2 The outcome of low Jm is compatible with the result reported by Lee

[139] while among PEI electrodes Jm however increased with PEG content and this

agreed with the results presented by Arakawa [138] The hypothesis by both authors

therefore might be correct but stabilisation mechanism might differ at different

concentrations Since KM was lowered at low PEG content compared to the standard

value PEG might have penetrated the protein core and stabilised the enzyme structure

[164] however it also lowered GOx activity because the protein conformation has been

locked in At 4 PEG concentration PEG still penetrated the protein core but residual

PEG also brought the preferential hydration and this allowed more flexible enzyme

conformation and increased the catalytic activity The residual PEG however might also

have an adverse effect on electron transfer rates at low glucose concentration In the

presence of O2 KM was increased compared to when in N2 and larger than that of

stabiliser-free electrodes while Jm only marginally better compared to that except for 4

PEG It is altogether not clear about the interaction between GOx and PEG however

only high PEG content effectively enhances the catalytic current output

445 GLC

Kinetic parameters for various GLC concentrations are shown below in Fig 53

145

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser GLC 1 GLC 2 GLC 4

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

20E-02

No Stabiliser GLC 1 GLC 2 GLC 4

KM

M

N2 O2

Fig 53 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 GLC in the presence and absence of O2 n = 3

In the absence of N2 GLC showed smaller Jm and KM compared to the standard values

This indicates the presence of uncompetitive inhibition and the decrease in Jm showed

the reciprocal dependency on GLC concentration It is also quite possible that

concentration-dependent inhibition was due to the tighter protein packing by GLC as

previously reported [144] This might contribute more to non-competitive inhibition

though GLC-containing electrodes showed more resistance against O2 because their Jm

was larger than that of stabiliser-free electrodes and again it showed reciprocal

dependence on GLC concentration In both N2 and O2 cases Jm decreased with increase

in GLC content KM also showed the dependence on GLC concentration in the presence

of O2 and increased with GLC concentration Glucose response lag was also decreased

in all cases Among all 1 GLC was most effective because it lowered KM for both N2

and O2 and increased Jm for O2 although it decreased Jm by 40 in N2 It also decreased

glucose response lag from 8 to 5 mM and this might indicate that glycerol reduced and

scavenged O2

146

446 FAD

Kinetic parameters for various FAD concentrations are shown below in Fig 54

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

No Stabiliser FAD 1 FAD 2 FAD 4

J m

Am

-2

N2 O2

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

No Stabiliser FAD 1 FAD 2 FAD 4

KM

M

N2 O2

Fig 54 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with 1 2 and 4 FAD in the presence and absence of O2 n = 1 - 3

FAD may be reasonably effective as an enzyme stabiliser at concentration gt 1 because

its Jm was larger in both absence and presence of O2 compared to that of stabiliser-free

electrodes It also slightly decreased glucose response lag therefore it might be able to

react with oxidising species as previously reported [108] It is however only effective at

higher concentration because two out of three 1 FAD electrodes did not show

Michaelis-Menten curves in the absence of O2 and none worked in its presence although

4 FAD was not most effective either The relationship between increase and decrease

in Jm and KM in N2 is unknown Improvement in electron transfer was not particularly

observed because higher FAD content decreased the current output although FAD has a

147

function as a mediator Mechanism of GOx stabilisation by FAD is not clear as Bartlett

reported [145] however 2 FAD was shown to be most effective among all and the only

one which showed M

mK

J gt 1 for both N2 and O2

447 PEI and DTT mixture

Kinetic parameters for various PEI-DTT concentrations are shown below in

Fig 55

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

50E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

J m

Am

-2

N2 O2

00E+00

40E-03

80E-03

12E-02

16E-02

NoStabiliser

PEIDTT1505

PEIDTT11

PEIDTT0515

PEIDTT22

KM

M

N2 O2

Fig 55 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and with

PEI-DTT (051 11 1505 and 22) in the presence and absence of O2 n =

1 - 2

PEI-DTT stabiliser mix showed the most dramatic effect and promising as a stabiliser

formulation In the presence of O2 it had larger Jm and smaller or equivalent KM than

those found in stabiliser-free electrodes Glucose response lag was also decreased from

148

8 to 3 mM and this implies the presence of O2 scavengers which were new synergetic

effects achieved and not observed in use of either stabilisers as discussed in sect 442 and

443 In the absence of O2 most PEI-DTT showed smaller Jm and marginally smaller

KM than those of the standard values except for some as it can be seen Fig 55 This

probably implies the presence of non-competitive-inhibition like effect due to the

stabilisation of enzyme structure by PEI-DTT Since it was difficult to determine the

comprehensively most effective mixture and the relative relationship in the increase and

the decrease in the two kinetic parameter values were not obvious M

mK

J was found

and compared for each PEI-DTT mixture as shown below in Fig 56

00

05

10

15

20

25

30

35

40

45

50

1505 11 0515 22

PEIDTT ()

Jm

KM

(Am

-2M

-1)

N2O2

Fig 56 Jm KM in the absence and presence of O2 for various PEIDTT ratios (051

11 1505 and 22)

It is clear that DTT must exceed 1 to achieve in the fraction to achieve efficient O2

resistance Since the equivalent glucose response lag was observed in all mixtures this

implies that at least 1 DTT is required for simultaneous reduction of Met amino acid

149

residue On the other hand gt 2 PEI seems required for high catalytic efficiency in N2

or equal weight PEIDTT mixing is preferable to increase catalytic efficiency in both N2

and O2 Since PEIDTT at 22 data presented here is based on a single sample the study

on more samples is required to confirm the results however PEIDTT mixture with

DTT gt 1 is effective to improve catalytic efficiency and output current

448 PEI and FAD mixture

Kinetic parameters for various PEI-FAD concentrations are shown below in

Fig 57

(i) Jm (ii) KM

00E+00

50E-03

10E-02

15E-02

20E-02

25E-02

30E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

J m

Am

-2

N2 O2

00E+00

20E-02

40E-02

60E-02

80E-02

No Stabiliser PEIFAD21

PEIFAD22

PEIFAD24

KM

M

N2 O2

Fig 57 Kinetic parameters Jm and KM for GOx electrodes without stabiliser and

with PEI-FAD (21 22 and 24 ) in the presence and absence of O2 n = 2

Firstly the result clearly showed total 6 stabiliser content hindered catalysis because it

showed extremely low Jm compared to other PEIFAD containing electrodes It only

counts for 15 and 9 of Jm obtained for other PEIDTT concentration in presence and

150

absence of O2 respectively Secondly there was no large difference in the performance

between PEIFAD at 21 and 22 hence PEIFAD 21 was most effective of all This

might involve non-competitive inhibition because Jm was smaller by 70 while KM was

smaller by only 20 compared to the standard values in the absence of O2 In the

presence of O2 it showed larger Jm by 7 times however also increased KM by 3 times

compared to those of stabiliser-free electrodes and did not decrease the glucose response

lag

449 Most effective stabiliser content for GOx anodes

To determine the size of comprehensive catalytic efficiency M

mK

J was

found for all the stabilisers and its value was compared for O2 for catalytic efficiency in

the presence of O2 Among all five most efficient catalysts were selected and presented

below in Fig 58

151

(i) Jm (ii) KM

00E+00

10E-02

20E-02

30E-02

40E-02

J m

Am

-2

N2 Am-2O2 Am-2

N2 Am-2 180E-02 125E-02 827E-03 292E-02 710E-03 104E-02O2 Am-2 168E-02 181E-02 151E-02 267E-02 122E-02 157E-02

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505 GLC 1

00E+00

50E-03

10E-02

15E-02

KM

M

N2 MO2 M

N2 M 729E-03 671E-03 576E-03 647E-03 938E-03 328E-03

O2 M 104E-02 471E-03 433E-03 801E-03 658E-03 886E-03

NoStabliser

PEIDTT11

PEIDTT0515

PEIDTT22

PEIDTT1505

GLC 1

Fig 58 Jm KM in the absence and presence of O2 for various stabilisers PEIDTT

(11 0515 221505 ) and GLC 1

PEIDTT mixture was found to be most effect as GOx stabiliser as discussed in sect 447

For Jm PEIDTT 22 showed the largest in both absence and presence of O2 and this

was followed by PEIDTT 11 For KM PEIDTT 0515 showed the smallest

comprehensively for both N2 and O2 and this followed by PEIDTT 11 PEIDTT

22 and GLC 2 showed relatively large KM in the presence of O2 It can be therefore

concluded that PEIDTT 11 is the most effective in stabilising GOx has largest

resistance against O2 and increases the catalytic output current It showed Jm = 13 x 10-2

plusmn 19 x 10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2

with KM = 47 plusmn 42 mM for O2 The current output obtained is much lower than some

reported results by Willner Heller and Sasaki [18 19 97] by 2 ndash 3 order of magnitudes

their electrodes however involved more complex fabrication methods or system as

discussed sect 22 on p 77 On the other hand it was slightly better than the result

reported by Merkoci who composed a similar composite with 5 GOx and was similar

to the results reported by Arai who built GOx electrodes by more complex methods KM

152

obtained is also smaller by 2 ndash 4 times than some of results by Arai [103]

Max current output Am-2 KM mM Method involved Author

125 x 10-3 ndash 181 x 10-2 47 ndash 67 10 GOx composite Data botained

55 x 10-3 Not available 5 GOx composite Merkoci [115]

30 Not available Apo-GOx reconstituted Willner [97]

11 x 10 Not available Os-complex hydrogel Heller [19]

18 Not available Saccharide-modified polypyrrole Fujii [100]

54 x 10-2 Not available QS conductive polymer Arai [103]

41 x 10 13 - 20 Poly(acrylic) acid hydrogel modified

carbon fibre GDH electrodes

Sakai [18]

Table 7 Comparison of maximum output current and KM with some published data

45 CONCLUSION TO ANODE STUDY

Bulk modified enzyme electrodes are good because they are mechanically very

stable and their fabrication is straightforward and scaleable requiring only mixing

electrode components conductive material mediators enzymes and stabiliser with

binding material TTF-TCNQ is useful material for GOx electrodes because it is

conductive as well as mediating The electrode component ratio is important for its

function and conductivity High binder and protein content hinder the conduction It was

found that 73 weight ratio of TTF-TCNQepoxy resin with 10 GOx loading optimal

The balance among the conductivity mediation enzyme activity mechanical and

153

chemical stability and difficulty in fabrication process is key to the successful

fabrication

O2 is known to be the natural mediator of GOx In the glucose-oxygen fuel

cells GOx anode must be resistant to O2 because O2 is an electron acceptor at the

cathode O2 lowers the current output by competing for electron transfer between GOx

and artificial mediators as well as non-competitive inhibition Various GOx stabilisers

were examined to alleviate such O2 influence

PEI DTT PEG GLC FAD PEIDTT and PEIFAD mixtures were studied as

candidate stabilisers to enhance catalytic activity and the current output PEI was not

effective It stabilised the structure but made conformation more rigid and did not

increase the output current in both the absence and presence of O2 DTT was reasonably

effective It showed uncompetitive inhibition in N2 but stabilised the enzyme structure

It therefore increased Jm and KM in the presence of O2 but increase in KM was probably

due to alleviating inhibition by O2 and loosening up the enzyme conformation PEG was

not effective It probably stabilised the enzyme structure but not show much

improvement for O2 competition It might also deprive electrons from reduced GOx

GLC was effective The effect was concentration dependent Lower concentration was

more effective It also scavenged O2 FAD was reasonably effective but only at higher

concentration PEI-FAD was also reasonably effective It decreased Jm but Km was

marginally equivalent to that of stabiliser-free electrodes for N2 But it increased Jm for

O2 Among all PEIDTT at 11 mixture was found to be most effective because it

stabilised the GOx and scavenged O2 Such powerful influence was not observed in the

use of either stabilisers hence it is a synergetic effect It showed Jm = 13 x 10-2 plusmn 19 x

154

10-3 Am-2 with KM = 67 plusmn 42 mM for N2 and Jm = 18 x 10-2 plusmn 99 x 10-3 Am-2 with KM

= 47 plusmn 42 mM for O2 Although it lowered Jm by 30 in the absence of O2 compared

to the standard value of 18 x 10-2 plusmn 73 x 10-3 Am-2 it also decreased KM in both

absence and presence of O2 and increased Jm by 8 times compared to that of

stabiliser-free electrodes PEIDTT 11 is therefore most effective in stabilising GOx

has largest resistance against O2 and increases the catalytic output current

155

5 CATHODE INTRODUCTION

51 OXYGEN REDUCTION REACTION (ORR)

The oxygen reduction reaction (ORR) takes place at the cathode as oxygen

accepts electrons from the anode Oxygen is an ideal oxidiser for the cathode because it

is readily available and its reaction product water is harmless [62] However ORR has

been a long-time bottleneck especially for low-temperature fuel cells because it causes

large potential loss [3 4 62 165-167] due to a strong double bond in the dioxygen

molecule Its difficult bond dissociation causes slow irreversible kinetics [24 165 166]

so a catalyst is required for this reaction to proceed at useful rates [62 165] ORR is a

multi-electron reaction which involves various reduction pathways and different

oxygen intermediates (Fig 16) In aqueous solution either the direct four- or

two-electron ORR pathway may proceed [165 166] The direct four-electron transfer

pathway reduces O2 into H2O in acid and neutral mediaor OH- in alkaline media This

occurs via several reduction steps [24 165 166] which may involve adsorbed peroxide

intermediates These peroxide intermediates never diffuse away in the solution in this

pathway [166] The two-electron transfer pathway reduces O2 into peroxide species as

final products These peroxide species either diffuse away are reduced further or

decomposed into H2O or OH- [24 165 166] This is not favourable because it leads to

lower energy conversion efficiency and its product hydrogen peroxide may degrade the

catalysts and the fuel cells [165 167] Four- and two-electron transfers also may occur

in series or in parallel [165]

156

Fig 59 Oxygen reduction pathways [165 166]

Reduction kinetics and mechanisms depend on the type of catalysts electrode material

electrode surface condition solution pH temperature contaminant and reactant

concentration [24 165 166] Rate-limiting step also varies by type of the catalysts

[166] However the first electron transfer to dioxygen leading to the formation of

superoxide anion radical (O2־) is the rate limiting step [168] O2־ is very rapidly

protonated in water and converted to hydroperoxyl radical (HO2)

157

Fig 60 Degradation of superoxide anion radical in aqueous solutions [24]

In aqueous solution O2־ is unstable and rapidly converted to O2 and H2O2 via a

proton-driven disproportionation process (Fig 60) [24]

52 REACTIVITY OF OXYGEN

A dioxygen molecule is a homodiatomic molecule consisting of two oxygen

atoms double bonded It has two parallel unpaired electrons in degenerate anti-bonding

π designated as π orbitals therefore it is diradical and paramagnetic (Fig 61) [169]

158

Fig 61 An energy level diagram for ground state dioxygen molecule [169]

When one electron is transferred to a dioxygen molecule the electron is placed in π

orbital and it turns into a reactive O2[24] ־ reducing the bond order from 2 to 15

Oxygen reduction changes the molecular symmetry or stretches the double bond

causing bond disruption [165 166] Electron paring in one of the π orbitals also

changes their orbital energy state and lifts the degeneracy as in Fig 62 [170]

Hereogeneous catalysis involves chemisorption of the oxygen and therefore some

transfer of electron density from the oxygen to the catalyst This facilitates the rate

determining initial electron transfer Oxygen reduction catalysts are reviewed in the

next section

159

(O2) 3Σg- O2־

Fig 62 Diagrams for the state of π orbitals in the ground state oxygen (3Σg-) (left)

and peroxide anion radical (right) [24]

O2 species Bond Order Bond length pm Bond energy kJmol-1

O2 2 1207 493

O2+ 2frac12 1116 643

O2- 1frac12 135 395

O22- 1 149 -

Table 8 Bond orders bond lengths and bond energies of some dioxygen species [169]

53 ORR CATALYSTS

Catalysts are required to reduce the overpotential and improve the efficiency of

160

ORR [3 4 62 165-167] It was reported that 80 of the whole potential loss can be

attributed to cathode ORR overpotential [171] The choice of ORR catalysts varies by

applications [36 171] and direct four-electron transfer pathway is most preferred for

ORR at the lowest possible overpotential in every fuel cell application however its

kinetics and reduction pathway are limited by the type of catalysts and experimental

conditions [24 165 166] Miniaturised glucose-oxygen fuel cells operate in the neutral

aqueous solution at ambient or body temperature with relatively low electrolyte and

oxygen concentrations therefore they require powerful ORR catalysts Platinum (Pt)

has been most ideal however its availability and cost are major limiting factors Many

studies have been made on other noble metals [172 173] transition metal macrocyclic

compounds [174] multicopper oxidases [62 175] and quinones [165] as alternatives

ORR catalysts to Pt These ORR catalysts are reviewed below

531 Noble metal ORR catalysts

Noble and transition metals are well-known ORR catalysts There are two

possible ORR mechanisms for metal surface dissociative and associative

161

Dissociative mechanism

middotmiddotmiddotmiddotmiddot Eq 67

middotmiddotmiddotmiddotmiddot Eq 68

middotmiddotmiddotmiddotmiddot Eq 69

( indicates the catalyst surface)

Associative mechanism

middotmiddotmiddotmiddotmiddot Eq 70

middotmiddotmiddotmiddotmiddot Eq 71

middotmiddotmiddotmiddotmiddot Eq 72

middotmiddotmiddotmiddotmiddot Eq 73

middotmiddotmiddotmiddotmiddot Eq 74

In dissociative mechanism the O2 double bond is ruptured rapidly on its adsorption on

the metal surface [165 166 172] This involves a strong interaction between oxygen

and metal atoms [166] and does not generate H2O2 [165] Electron and proton transfer

take place simultaneously and this is the rate-limiting step [166 171] In the associative

mechanism O2 does not dissociate before proton transfer Adsorbed O2 stays on the

metal surface [165 172] and two-electron and proton transfer [166 173] take place via

the formation of superoxide or peroxide intermediate [165 166] This reaction is

thermodynamically easier than the dissociative pathway and its final product is H2O2 or

162

H2O if both pathways occur in series [165] O2 adsorption on the metal surface is an

inner sphere interaction which involves complex formation with the metal surface [30]

There are three ways in which O2 adsorbs (i) Griffiths (ii) Pauling and (iii) bridge

model (Fig 63)

Griffiths model Side-on Pauling model End-on Bridge model

OO

M

Fig 63 Three models of oxygen adsorption [165 166 173]

The Griffiths model is called lsquoside-onrsquo type O2 functions as a bidentate ligand against a

single metal atom in this complex and its π orbitals laterally interact with empty metal

dz2 orbitals causing ligand-to-metal charge transfer (LMCT) πrarrdz

2 At the same time

electrons partially filling metal dxz or dyz orbitals are back donated to oxygen π orbitals

[166 171] Therefore a strong metal-oxygen interaction is present in this model This

effectively disrupts the O=O bond [166] and facilitates the electron transfer from the

metal ion by changing its oxidation number The Pauling model is called the lsquoend-onrsquo

type [165 166 173] O2 is a monodentate ligand in this model and only one of the

oxygen atoms in the dioxygen molecule can be bonded to the metal surface An oxygen

π orbital interacts with metal dz2 orbitals and this allows either the direct or peroxide

ORR pathway after partial charge transfer [166] The bridge model resembles Griffiths

model in the way the metal atom and oxygen interact however it requires two adjacent

163

adsorption sites so that O2 can ligate two different metal atoms The bridge-model

interaction is easily interfered with by the surface contamination and the oxide layer

because the surface species block the adsorption sites for ORR catalysis [173]

Fig 64 Models for oxygen adsorption and ORR pathways [166]

Both the Griffiths and bridge models follow dissociative mechanism (pathway I and III

respectively Fig 64) due to the lateral interaction leading to the dissociation of O2

double bond The Pauling model on the other hand follows the associative mechanism

(pathway II Fig 64) This is further divided into two other pathways IIa and IIb

according to its end product H2O2 or H2O (Fig 64) respectively [166] O2 adsorption

and the ORR mechanism therefore depend on (i) the extent of oxygen-metal

interaction and (ii) the availability of oxygen adsorption sites A volcano-shaped trend

between ORR rate and oxygen-metal interaction has been plotted by Norskov as in Fig

65 [172]

164

Fig 65 A volcano-shaped trend between ORR rate and energy required for oxygen

binding on various transition metal surface [172]

Many have reported platinum (Pt) as the most ideal metal catalyst due to its high

catalytic ORR activity [165 166 171 172] Fig 65 shows that Pt and Pd (palladium)

have largest ORR rate yet medium oxygen adsorption energy and iridium (Ir) and

rhodium (Rd) follow them On the other hand metals with either higher or lower

oxygen adsorption energy result in lower ORR rate This is because metals with high

oxygen adsorption energy such as gold (Au) imposes too large energy barrier for the

formation of a stable oxygen-metal bond while too small adsorption energy as in nickel

(Ni) leads to excessively stable oxygen-metal interaction and slows the proton transfer

Therefore associative pathway is dominant at Au surface while oxygen dissociation is

deactivated at Ni surface but instead this increases the surface oxide coverage [172

173] Pt performs best because it has moderate oxygen-metal surface interaction [165]

Pt alloys with other transition metals such as Ni Co and Fe have been also reported to

increase the catalytic activity [36 167 171 172] because alloying dilutes the Pt-O

165

interaction and alters the overall oxygen-metal surface interaction [171 172] This is a

useful property to add because Pt is known to form a surface oxide layer leading to both

associative and dissociative catalytic pathways [165 171] This surface state is

potential-dependent and stays persistenly at less than 08 V vs AgAgCl [172]

532 Macrocycle and Schiff-base complex ORR catalysts

Macrocyclic transition metal complexes have been widely investigated as

potential ORR catalyst both for fuel cells and as catalysts to activate dioxygen for

synthetic purposes They have been widely reviewed by [165 176] Macrocyclic

complexes consist of (i) transition metals such as Fe Co Ni or Cu (ii) chelating atoms

N4 N2O2 N2S2 O4 and S4 and conjugate π systems such as porphyrin and

phthalocyanine [165] Examples of transition metal macrocycles are shown in Fig 66

Fig 66 Examples of transition metallomacrocycles Heme (left) [177] and copper

phthalocyanine (right) [178]

166

Schiff base complexes such as metallosalens and metallosalophens are other

well-known chelating ORR catalysts [174 179] Transition metal macrocycles and

Schiff bases can be also polymerised into conductive films [180] (Fig 67)

Fig 67 Schiff base complexes

Co(II) salen Co(II) salophen and

Co(II) salen modified poly(34-ethylenedioxythiophene) [174 180]

Interaction between the oxygen molecule and the metal complex is similar to that with

the noble metals and involves partial charge transfer between the filled metal dz orbitals

and the π orbital of O2 as described in sect 531 The catalytic activity of the metal

complexes depends on the type of central metal atom ligand and the size of the

π-electon systems The reducing ability of the ligand is important because the central

metal atom must transfer considerable electron density to oxygen atoms during the

167

catalysis [170] The thermodynamically most stable metal-oxygen complexes are

bionuclear complexes such as dicobalt face-to-face 4-atom-bridged diporphyrin

(Co2(FTF4)) (Fig 68) [165-167]

Fig 68 Molecular structure of dicobalt face-to-face diporphyrin [166]

Mononuclear Co complexes take the associative pathway [165] forming superoxo

cobaltic complexs (Co-O-O־) [181 170] while their dimers or binuclear complexes can

take either direct four-electron or the parallel pathways (Fig 69) [165 167] because

they can lock the oxygen moiety in rigid orientation [170] Co2(FTF4) favours

four-electron transfer and its ORR pathway depends on the distance between two

cofacial metal atoms [165-167] Spacing distance can be altered by inserting a spacer

molecule [165 166]

168

Fig 69 ORR pathway by Co2(FTF4) [166]

However Co2(FTF4) is not stable enough for practical use [165 166] Practical ORR

catalysts for fuel cell application must be stable inexpensive and feasible to synthesise

533 Multicopper enzymes as ORR catalyst

Multicopper oxidases (MCOs) are copper-containing enzymes such as bilirubin

oxidase (BOD) laccase (Lac) and copper efflux oxidase (CueO) Oxygen is widely used

as an electron acceptor in nature because it is ubiquitous and oxidation is a good mean

to obtain energy as in combustion Millions of years of evolution have therefore

provided enzymes with good catalytic activity for oxygen reduction Although oxygen

169

reduction in biological system is complex due to involving various substrates and

catalytic steps as well as it tends to be incomplete and leads to hydrogen peroxide or

superoxide formation as the product the enzymes catalyse the rate-determining initial

electron transfer For application in biofuel cells it is essential that the enzymes are

widely available and possess good stability activity in immobilised form and ideally

four-electron ORR catalysis MCOs have been prevalent in biofuel cell applications

because they are active at neutral pH catalyse four-electron ORR and bear

immobilisation Fungal laccase and bilirubin oxidase have been most widely studied

MCOs [56 59 60]

Most MCOs have four Cu atoms which are designated as type I (T1) type II (T2) and

a pair of type III (T3) T2 and T3 together form a trinuclear Cu centre while T1-Cu

called blue copper [175 182 183] is located away from the trinuclear Cu centre but

adjacent to the hydrophobic substrate-binding site Fig 70 shows the location of the

trinuclear Cu centre (copper coloured) and the blue copper (in blue) Oxygen binding

and ORR take place at the trinuclear Cu centre [175] while T1-Cu receives electrons

from the substrate and mediates electron transfer between substrate and the trinuclear

Cu centre [175 184]

170

Fig 70 Laccase from Trametes versicolor [185]

Enzyme catalytic efficiency depends on the redox potential [175] of T1-Cu It has been

reported that fungal laccase has the highest redox potential between 053 ndash 058 V vs

AgAgCl [62 183] T1-Cu also allows DET due to being located close to the surface

[183 186 187]

MCO reaction cycle starts from substrate oxidation to form a fully reduced state MCO

is initially at native form or so-called intermediate II Native form is characterised by

doubly OH--bridged structure (Fig 71(ii)) In the absence of substrate the native form

decays to the resting form which is at fully oxidised state (Fig 71(i)) After

four-electron reduction of enzyme the fully reduced MCO (Fig 71(iii)) reacts with O2

The first reduction step involves two-electron transfer from each of T3-Cu+ to O2 This

forms peroxide intermediate or intermediate I (Fig 71(iv)) and lowers the energy

barrier to disrupt O=O It is followed by swift protonation of adsorbed peroxide at

T3-Cu site This protonation reaction is induced by the charge transfer from T1- and

171

T2-Cu+ This leads to the complete reduction of oxygen to water and the enzyme native

form is retrieved

(i)

(ii)

(iii)

(iv)

Fig 71 Redox reaction cycle of MCOs with O2 [175]

All MCOs have this common ORR mechanisms despite the different origin structure

biological functions substrate reducing power and optimal environment

5331 BOD

BOD is an anionic monomer [182] most active at neutral pH [183 188] and

172

capable of both DET and MET The mediators must have their redox potential close or

slightly lower than that of BOD 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid

(ABTS) (Fig 72(i)) [62 97] Os-pyridine complexes linked to conductive polymer (Fig

72(ii)) [66 189] and cyanometals [18 56] have been used as its mediators [56 185]

ABTS is a divalent anion which works as co-substrate of MCOs and its redox potential

is 067 V vs AgAgCl [56] Os-pyridine complex with conductive polymer follows the

similar idea for the wired GOx anodes discussed in sect 223 on p 80

4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine) has been used

especially for the cathode and its redox potential is 055 V vs AgAgCl

Fig 72 Common mediators for BOD (i) ABTS [56 62]

(ii) Os[4-aminomethyl-4rsquo-methyl-22rsquo-bipyridine with poly(4-vinylpyridine)] [190]

173

Tsujimura showed that ORR catalytic constant was 6-times greater in the presence of

Os-pyridine complex than in DET [66 188] Reduction starts around 05 V in most

cases [66 97 183 188] The size of current density varies roughly between 05 - 1

mAcm-2 [56 97 188] and 95 mAcm-2 was achieved by Heller [189]

5332 Lac

Fungal laccase is another anionic monomer [183] It is less expensive than

BOD and has broad substrate specificity including xenobiotics [175] although it has

optimum pH around 5 and activity hindered by chloride [19 182] Its T1-Cu redox

potential ranges between 053 ndash 058 V vs AgAgCl [62 183] Highest redox potential

066 V and current density 057 mAcm-2 were achieved by the use of anthracene

plugging molecule which provides strong binding between Lac and pyrolytic graphite

edge electrode as well as long-term activity 57 of the initial current was retained after

eight weeks [185]

5333 CueO

CueO is a less common enzyme in biofuel cells however it is active for a wide

range of pH between 2 - 8 [186] and also it has better catalytic activity than other

MCOs because it has five Cu atoms This extra Cu atom is believed to have mediating

function and 4 mAcm-2 has been achieved at pH 5 [191] However it has lower redox

potential around 03 V [186 191] Protein engineering has been introduced to increase

T1-Cu redox potential [186]

174

534 Anthraquinone ORR catalyst

Anthraquinone (AQ) is known to catalyse two-electron transfer in ORR (Fig

73) [44 165 192-194] and it is used in industries to produce hydrogen peroxide [165

192]

Fig 73 Two-electron ORR by anthraquinone [193]

ORR by anthraquinone is an electrochemical-chemical (EC) process [165 192] in

which quinone must be first electrochemically reduced to an active species

semiquinone anion radical (Q־) The active semiquinone radical can then react with

dioxygen so that it converts the dioxygen into O2־ via forming of the intermediate

superoxo-C-O(O2) species This is the rate-limiting step O2־ produced by a

semiquinone radical either disproportionates (Eq 77) or it is further reduced into HO2-

(Eq 78) [192]

175

Oxygen reduction reaction by anthraquinone

ltlt Rate limiting step gtgt

middotmiddotmiddotmiddotmiddot Eq 75

middotmiddotmiddotmiddotmiddot Eq 76

ltlt Following spontaneous reactions gtgt

middotmiddotmiddotmiddotmiddot Eq 77

middotmiddotmiddotmiddotmiddot Eq 78

(Q indicates quinone on the surface)

Derivatised anthraquinone such as Fast red (Fig 74) can be attached to the carbon either

physically or covalently Grafted quinones are very stable [192 194 195] and resistant

against continuous potential cycling [194] metal ions and surfactant despite of some

fouling present [44]

176

O

O

N N Cl

frac12 ZnCl2

1

2

4

6

5

3

10

9 7

8

12

14

13

11

O

O

(i) (ii)

Fig 74 The structures of (i) anthraquinone [196] and (ii) Fast red AL salt [44 197]

However its ORR is heavily affected by pH [194] because its catalytic efficiency

depends on the concentration of surface Q־ species [192 194] The concentration

varies by different pH and the catalytic efficiency drops at both high and low pH due to

the depletion of Q־ When pH falls below its dissociation constant Q־ is not

reproducible anymore and ORR is inhibited This phenomenon appears at as high as pH

7 and the ORR vanishes at pH lt 7 [194] AQ-grafted GC (AQGC) shows only sluggish

kinetics at pH lt 10 Its voltammogram also becomes complex due to the presence of

native quinone groups originated from the carbon surface [192] ORR mediated by the

native quinone groups is observed as a prewave and this varies by different quinone

derivatives such as phenanthrenequinone (PQ) (Fig 75) electrode composition and pH

[194]

177

Fig 75 Hydrodynamic voltammogram for ORR at various quinone electrodes with various

rotation speeds [194] (i) unmodified GC (ii) AQGC and (iii) PQGC at pH 7

54 CARBON PROPERTIES

Carbon is widely used for electrochemical applications because it is

inexpensive mechanically stable [105] and sufficiently chemically inert yet reactive

due to the presence of surface defects It has wide potential window between 1 and -13

V [21 30] Carbon is available in various solid forms such as graphite glassy carbon

(GC) pyrolytic carbon (PC) activated carbon (AC) carbon fibres (CFs) carbon

nanotubes (CNT) and diamond It is easy to renew the surface and allows facile

chemical modification [195] However it is also prone to adsorption and surface

oxidation simply by air and water exposure and its electron transfer is under the

constant influence of the electrode surface condition and history [198] This leads to the

deviation and the low reproducibility in carbon electrochemical experiments [105]

178

Physical properties of carbon vary by its form and section The carbon consists of either

(i) layers of the conductive planar graphene sheets or (ii) the insulating

tetrahedral-puckered forms as seen in the diamond (Fig 76) Conductivity in diamond

is gained by doping with boron [199 200]

Fig 76 Carbon structures (i) graphite (ii) diamond [201]

The electrical conductivity is anisotropic and closely correlates with the carbon

structure and section The carbon section is classified into (i) basal and (ii) edge planes

The basal plane extends along the graphene plane while the edge plane is perpendicular

to this

179

Fig 77 Carbon section (i) basal plane (ii) edge plane [105]

The basal plane has larger conductivity due to being parallel to the vast graphene

network while the edge plane is superior in electrochemical reactivity due to being

richer in reactive graphene sheet termini and carbon defects The ratio between the basal

and edge planes therefore affects the conductivity and electrochemical properties of

the carbon and this varies by the carbon form and its treatment [45 105 195 202 203]

The electrical conductivities for various carbon forms and sections is shown in Table 9

180

The type of carbon Electrical conductivity Scm-1

Graphite [105 159] 1 x 103 - 104

Highly ordered PG basal plane 250 x 104

Highly ordered PG edge plane 588

PG basal plane 400 x 103

PG edge plane 333

Polycrystalline graphite 100 x 103

GC [105 159 203] 01 - 250 x 102

Activated carbon [204] 1 x 10

Carbon fibre 133 x 103

Boron-doped diamond [199 205] 01 - 1 x 102

Multi-wall carbon nanotube [206] 1 ndash 2 x 103

Pt 909 x 104

Cu 588 x 105

Table 9 The electric conductivity of various carbon forms and sections Data from

[105] unless defined specifically

541 Graphite

Graphite is black-coloured material with pores and high gas permeability It is

available in many physical forms as powders moulded structures and thin sheets

therefore it is used in many commercial applications [203] Powdered graphite is

convenient to make carbon paste and composite electrodes [105] Graphite consists of

181

layers of graphene sheets each of which is a huge π-conjugate system π-conjugate

system is made of hexagonal rings of sp2 hybridised carbon atoms [105 195] (Fig 76(i)

and Fig 78) and allows free electron movement within graphene This gives carbon

metallic properties such as electric and thermal conductivity

Fig 78 A direct image of a graphene bilayer by transmission electron microscopy

(TEM) at resolution of 106 Aring and with a 2-Aring scale bar [207]

Each graphene sheet is structurally stable in horizontal direction but fragile in vertical

direction because each layer is only held by van der Waalsrsquo force Therefore the

graphene sheets easily slide and collapse [31]

542 Glassy carbon (GC)

Glassy carbon (GC) is a non-graphitised carbon made of randomly interwoven

sp2-carbon fibres which are tangled and heavily cross-linked (Fig 79) It contains many

closed voids [203] and fullerene-like elements [208] It is impermeable to gases [203

182

208] chemically more inert [208] and mechanically stronger but electrically more

resistive than other carbons [203]

Fig 79 GC (i) High resolution TEM image (ii) A schema of GC [208]

543 Activated carbon (AC)

Activated carbon (AC) is thermally and chemically activated carbon It is

highly porous and has large internal surface area This is an excellent adsorbent for

solvent recovery and gas refining [203 208]

544 Carbon fibre (CF)

Carbon fibres (CFs) have small diameter ranging between 5 to 30 microm They are

lightweight have lower fabrication cost and are superior in mechanical properties

therefore they have been used for sporting automotive and military equipments They

are available in many forms such as tow mat felt cloth and thread They consist of a

random arrangement of flat or crumpled graphite sheets but their structure and

183

molecular composition depend on the type of precursors [203 208]

545 Carbon nanotubes (CNTs)

Carbon nanotubes (CNTs) are made rolled-up sheets of sp2-graphene in nano to

micro-meter range They are available in various formats single-walled nanotube

(SWNT) multi-walled nanotube (MWNT) carbon nanohorn (CNH) (Fig 80) and

various forms powder arrays and posts [117]

Fig 80 Various CNTs (i) Bundled SWNTs (ii) MWNT (iii) CNHs [209]

CNTs have been drawing keen attention due to their wide surface area conductive or

semiconductive properties light weight mechanical stability and facile

functionalisation [117 195 202 210] They have been used as support for the dispersed

metal catalysts [3] and electrode materials [117 202] The drawbacks of CNTs are the

high presence of impurities alteration of their properties on acid cleaning [202 211]

and unknown toxicity [117 160 202]

184

546 Boron-doped diamond (BDD)

Diamond is different from other carbons because it is transparent and not

electrically conductive It has a cubic lattice structure made of closely-packed repetitive

units of sp3-hybridised carbon atoms as seen in Fig 81

Fig 81 A TEM image of diamond [212]

Each unit consists of four σ-bonded sp3-carbon atoms in tetrahedral coordination as in

Fig 76(ii) Each bond is localised and this gives structural stability but does not conduct

electricity By doping it with boron diamond can become semiconductive Such

diamond is called boron-doped diamond (BDD) and boron functions as an electron

acceptor BDD is mechanical more stable chemically more inert sensitive to analyte

and has wide potential window lower back ground current and stable electrochemical

responses [195 199 205]

185

55 ELECTROCHEMISTRY AND ELECTROCHEMICAL PRETREATMENTS OF CARBON

551 Electrochemical properties of carbon

The edge plane of carbon which contains graphene termini and carbon defects

is chemically reactive and accounts for carbon electrochemistry while the vast network

of graphene sheets is responsible for its electrical conductivity The termini and defects

include some sp3 hydrocarbons and they are oxidised into various oxygen functional

groups such as carboxyl quinoyl hydroxyl lactone groups ethers cyclic esters and

lactone-like groups [195] These electrode functional groups facilitate electron transfer

provide bonding between the electrode and the catalysts or enzymes or simply cause

electrode capacitance or resistance Various carbon pretreatments such as

electrochemical pretreatment (ECP) vacuum heat treatment (VHT) carbon fracturing

and polishing laser irradiation [195 213-215] are used to attach active surface

functional groups and increase the rate of electron transfer in various electrochemical

reactions [198 216]

552 Electrochemical pretreatments (ECPs) of the carbon

Electrochemical pretreatments (ECPs) provide feasible electrode surface

modification Various ECPs have been used to introduce carbon-oxygen surface

functional groups Most common method is potential cycling in the acid or basic media

or the combination of anodisation and cathodisation as shown in Table 10

186

Table 10 ECPs examples

Potential cycling at 02 Vs-1 between 0 and 02 V in 01 M H2SO4 [105]

Anodisation at 18 V in pH 7 phosphate buffer for 5 minutes followed by 1-minute cathodisation at

ndash 15 V [216 217]

Potential cycling between 06 and 2 V at 5 Vs-1 for 15 to 30 minutes in 05 M NaOH [218] or their

combination [219]

ECPs introduce the electrodes various new properties They (i) attach electrochemically

active functional groups and increase the oxygencarbon ratio [105] (ii) introduce

carbon defects and active sites [105 218] (iii) increase the surface area [105 198 215

216 218] (iv) form a surface film and (v) increase the background current [105] and

double layer capacitance [105 215 216] Most notable effect of EPCs is the increase in

electron transfer rate in many electrochemical reactions This is observed as a decreases

in peak separation and an increase in peak current in voltammetry as seen in Fig 82

[105 216 218 220] As for ORR however it was reported that surface quinone groups

attached by EPC or originated from the carbon itself reduce ORR overpotential but do

not mediate ORR as in quinone groups physically or covalently attached [216 220]

187

Fig 82 The influence of ECP on GCE and its voltammogram at 01 Vs-1 in pH 7

phosphate buffer in the presence of (i) N2 and (ii) O2 The voltammograms

of both pretreated and unpretreated GCEs are shown [216] The

presentation has been altered for convenience

56 ELECTROCHEMICAL TECHNIQUES FOR ORR

ORR has been studied by two electrochemistry techniques cyclic voltammetry

(CV) and hydrodynamic voltammetry (HDV) using the rotating disc electrode (RDE)

These techniques provide overlapping but complementary information CV is a dynamic

technique and allows calculation of the number of electrons up to and including the rate

determining step (nα) With numerical simulation and data fitting CV can be used to

estimate the standard heterogeneous rate constant (kdeg) RDE is a steady state technique

and can give the overall number of electrons transferred (n) The well-established

Koutecky-Levich plot (see sect 567 on p 200) provides a robust method for determining

kdeg provided the standard electrode potential (Edeg) for the reaction is known

188

561 Cyclic voltammetry (CV)

Cyclic voltammetry (CV) is widely used for initial exploration and

characterisation of a redox active system It is of limited electroanalytical value

however kinetic parameters can be obtained from the current-voltage (I-V) plot In

cyclic voltammetry potential is swept linearly with a triangular waveform (Fig 83(i))

and applied cyclically The resulting time-dependent current is typically plotted versus

applied potential (Fig 83(ii)) [30 221] The potential range and scan rate are decided in

advance according to the type of analytes reactions and materials of interest

Fig 83 Schemas for (i) a Triangular waveform (ii) Typical cyclic voltammogram for

benzoquinone (BQ) CV was taken at 005 Vs-1 between + 025 and ndash 05 V vs

AgAgCl for 1 mM BQ in 1M KCl at graphite composite electrode Switching

potential (Eλ) is ndash 05 V and time taken to reach Eλ (t) is 15 seconds Based on

the real data

189

562 Non-faradic current and electric double layer capacitance

Transient techniques such as CV always contain two types of electric current

non-faradaic and faradaic current The non-faradaic charging current is due to the

electric double layer which is formed at the electrode-solution interface This occurs

because such an interface behaves like a capacitor as counter ions accumulate at the

interface in order to balance the potential at the electrode This does not account for the

charge transfer reactions involved however provides the information about the

electrode surface [30] and also can affect the rate of electrode process and distort the

voltammogram Cd can be estimated by electrochemical impedance spectroscopy or

deduced by averaging Cd over the potential range as in Fig 84 and Eq 33 [21 30]

Fig 84 The double layer capacitance (Cd) observed in CV for deoxygenated pH 74 at

the acid-pretreated graphite electrode See Eq 33 for the calculation and

symbols Based on the real data

190

Electric double layer capacitance per unit area

AII

AEQareaunitperC ccca

d sdotminus

=sdot

=υ2

)()( middotmiddotmiddotmiddotmiddot Eq 79

Cd = Double layer capacitance per unit area microFcm-2

Q = Charge C

E = Potential V

A = Electrode surface area cm-2

Ica = Anodic non-faradic current A

Icc = Cathodic non-faradic current A

υ = Scan rate Vs-1

Cd needs to be either negligible or subtracted before interpretation of faradaic current

which accounts for the charge transfer phenomena in redox reactions In order to

minimise the influence of Cd on the voltammogram the scan must be taken at slow scan

rate and the electrolyte concentration must be always kept reasonably high [30]

563 Diagnosis of redox reactions in cyclic voltammetry

A typical voltammogram contains current peaks as seen in Fig 83(ii) and these

are due to the redox reactions in the system Diagnosis of the redox system using CV is

based on (i) peak separation (ii) peak current height (iii) peak shape or peak width (iv)

the number of peaks and (v) the dependency of peak shape on the scan rate The

analysis of such peaks involves the following four parameters anodic peak current (Ipa)

cathode peak current (Ipc) anodic peak potential (Epa) and cathode peak potential (Epc)

This reveals the type of electrochemical reactions involved which are either (i)

191

reversible (ii) quasi reversible or (iii) irreversible When the peak current is proportional

to υfrac12 the redox reaction is diffusion-limited in the solution while a linear dependence

shows that the redox active species is confined to the electrode surface

In the reversible system charge transfer between the redox species and the electrode is

rapid [221] therefore it has fast kinetics and the reaction is diffusion-limited The

electrode potential follows the Nernst equation (Eq 15 on p 50) because the electrode

potential is dependent on the concentration equilibrium at the electrode surface and the

peak potential is independent of the scan rate The features on the reversible system are

listed in Table 11 The peak current (Ip) is linearly proportional to υ12 as the redox

reaction is diffusion-limited The peak separation (∆Ep) is the difference between Epa

and Epc and it is 59 mV at 25 ˚C for the single-charge transfer [29 30] The peak width

(δEp) is the difference between Ep and half-peak potential (Ep2) Ep2 is the potential

where the peak current is half It is 565 mV for a single charge transfer The ratio

between Ipc and Ipa is called a reversal parameter [105] It is unity at any scan rate in the

reversible system

192

Table 11 Reversible system at 25 ˚C [30]

2121235 )10692( υlowasttimes= CADnI p middotmiddotmiddotmiddotmiddot Eq 80

mVn

E p528

= independent of υ middotmiddotmiddotmiddotmiddot Eq 81

mVn

EEE pcpap59)( =minus=∆ middotmiddotmiddotmiddotmiddot Eq 82

mVn

EEE ppp556

2 =minus=δ middotmiddotmiddotmiddotmiddot Eq 83

1=pa

pc

II middotmiddotmiddotmiddotmiddot Eq 84

Ip = Peak current A

n = The number of charges transferred

A = Electrode surface area m-2

D = Analyte diffusion coefficient m-2s-1

ν = Solution kinematic viscosity m-2s-1

Ep = Peak potential V

∆Ep = Peak separation V

Epa = Anodic peak potential V

Epc = Cathodic peak potential V

δEp = Peak width V

Ep2 = Half-wave potential V

Ipa = Anodic peak current A

Ipc = Cathodic peak current A

In a totally irreversible system charge transfer between the redox species and the

electrode takes place only slowly [221] The electrode must be activated by applying

193

large potential to drive a charge transfer reaction at a reasonable rate Ep becomes

dependent on scan rate and this increases ∆Ep and skews the voltammogram [30]

However Ip is still proportional to υfrac12 because at high overpotential the redox reaction

is diffusion-limited [29]

Table 12 Irreversible system at 25 ˚C [222]

2121215 )()10992( υα αlowasttimes= CADnnI p middotmiddotmiddotmiddotmiddot Eq 85

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛

deg+minusdeg=

2121

lnln780RT

Fnk

DFn

RTEE pυα

αα

α

middotmiddotmiddotmiddotmiddot Eq 86

mVn

E pαα

59gt∆ middotmiddotmiddotmiddotmiddot Eq 87

mVn

E pαα

δ 747= middotmiddotmiddotmiddotmiddot Eq 88

α = Transfer coefficient

nα = The number of charge transferred up to the limiting step

( Please refer to Table 11 for other terms and symbols)

A quasi-reversible system is controlled by both kinetics and diffusion Its

electrochemical reversibility is relative to the scan rate [21 30] The peak potential

depends on scan rate for rapid scans and the peak shape and peak parameters become

dependent on kinetic parameters such as α and Λ where 21

1

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛

deg=Λ

minus υαα

RTFDD

k

RO

[30]

Since Λ is a function of k˚υ-frac12 where kdeg is the standard rate constant the voltammogram

hence kdeg is affected by the scan rate as seen in Fig 85

194

Fig 85 Voltammograms of a quasi-reversible reaction at various scan rates [21]

564 Coupled reactions in cyclic voltammetry

Redox reactions are sometimes coupled with different electrochemical or

chemical reactions Voltammogram with altering scan rates can indicate the presence of

such reactions A redox reaction coupled with another redox reaction is called EE

mechanism while with homogeneous chemical reaction it is called EC mechanism If

the coupled reaction is catalytic then it is called ECrsquo mechanism [21]

Many ORR catalysts show coupled reactions with ECrsquo mechanism ORR is a

quasi-reversible reaction because the reaction is limited by oxygen diffusion yet O2

adsorption and reduction heavily relies on overpotential or catalytic power of catalysts

Catalysed reactions show distinctive voltammetric features There is only a single-sided

large current but with no reverse current because they catalyse a reaction only in a

single direction The reversal parameter therefore becomes non-unity [30]

195

ORR catalysts sometimes contain multiple reducible centres or an atom with various

oxidation states as in seen transition metal They might exhibit a multi-peak

voltammogram under non-catalytic condition The shape of voltammogram depends on

the difference in the standard potential (∆E˚rsquo) between two redox centres or states (Eq

89) as seen in Fig 86 [21 30]

21 ooo EEE minus=∆ middotmiddotmiddotmiddotmiddot Eq 89

Fig 86 Voltammograms of two-electron transfer EE systems of different ∆Edegrsquo [21]

565 Voltammograms for the surface-confined redox groups

Catalysts encapsulated in or attached to the electrode provide catalytic groups

or moieties on the electrode surface Such surface-confined redox species show different

voltammograms from solution redox species because their redox reactions do not

196

involve diffusional redox species They exhibit a characteristic voltammogram of

perfect symmetry with Epa = Epc as seen in Fig 87 and their peak current is linearly

proportional to υ rather than υfrac12 as in Eq 90 [21 30] The features of surface-confined

groups are listed in Table 13

Fig 87 A voltammogram of the reversible redox reaction by surface-confined groups [21]

Table 13 Reversible system by surface-confined groups at 25 ˚C [30]

RTAFn

I p 4

22 Γ=

υ middotmiddotmiddotmiddotmiddot Eq 90

oadsp EE = middotmiddotmiddotmiddotmiddot Eq 91

mVn

E p690

2 =∆ middotmiddotmiddotmiddotmiddot Eq 92

Where Γ isiInitial concentration of the absorbed species

However such an ideal voltammogram is seldom observed because the density of redox

species is so high in the surface-confined environment compared to in the solution and a

197

voltammogram is also influenced by the interaction among those packed redox species

A multi-electron system of the surface-confined groups is also not this case [21]

566 Hydrodynamic voltammetry (HDV) and Levich equation

Hydrodynamic methods purposely introduce convective flow to the system

using RDEs This allows the study of precise kinetics because the diffusion is under

precise control Unknown influence by mass transfer and double layer capacitance on

the electrode reaction is eliminated and steady state is quickly achieved [29 30] In

RDE method laminar flow is applied perpendicular to the electrode surface by spinning

a disc electrode at arbitrary rotation velocity (Fig 88) The rotation rate is expressed in

either rpm (rotation per minute) or angular velocity (ω) ω is defined as ω = 2πf where f

is frequency With disc rotation diffusion becomes a function of electrode rotation rate

as in Eq 93 However the limit of disc rotation rate must be set in order to avoid the

thick hydrodynamic boundary layer and turbulent flows to maintain steady state

Allowable range is between 100 rpm lt f lt 10000 rpm or 10 s-1 lt ω lt 1000 s-1 [30]

198

ω

Fig 88 RDE is rotated at ω and laminar flows applied to the electrode surface

Diffusion layer thickness and disc rotation rate [30]

612131611 νωδ minus= D middotmiddotmiddotmiddotmiddot Eq 93

δ = Diffusion layer thickness cm

D = Analyte diffusion coefficient m2s-1

ω = RDE angular velocity s-1

ν = Solution kinematic viscosity m2s-1

Under such a condition disc limiting current (Id) becomes a function of disc rotation

rate as described in Levich equation (Fig 85) [30] This allows finding the total number

of charge transferred (n)

199

Levich equation [30]

612132620 CnFADI Lminus= νω middotmiddotmiddotmiddotmiddot Eq 94

δ

nFAC= middotmiddotmiddotmiddotmiddot Eq 95

IL = Levich current A

n = Total number of charge transferred

F = Faraday constant Cmol-1

A = Electrode surface area m2

C = Bulk analyte concentration molm-3

( Please refer to Eq 93 for other terms and symbols)

Due to the steady state condition the current response in HDV becomes sigmoidal [223]

as seen in Fig 89

200

Fig 89 HDV at 005 Vs-1 sweeping from 09 V to ndash 09 V vs AgAgCl at 900 1600 and

2116 rpm for O2 reduction at pretreated GCE in oxygenated 001 M PBS at pH

74 Based on the real data

567 Hydrodynamic amperometry (HDA) Koutecky-Levich equation and the

charge transfer rate constant

Levich equation (Eq 94) shows that Id is proportional to ωfrac12 therefore also a

function of diffusion layer thickness (Eq 95) This is true for reversible kinetics

because the electrode reaction is limited by diffusion which is a function of ωfrac12 and

therefore Id also becomes dependent on ωfrac12 at any potential However in

quasi-reversible system Id depends on diffusion but also affected by its kinetics [223]

Therefore when Id reaches the kinetic limitation for charge transfer the slope gradually

deviates from its Levich current as seen in Fig 90 [30]

201

Fig 90 Levich plots for reversible (blue) and quasi-reversible (red) systems [30]

In addition its charge transfer kinetics is altered by the potential applied As Tafel plot

(Fig 9 on p 57) presents the current is exponentially dependent on the overpotential

The electrode reaction is kinetically limited at low overpotential then gradually

becomes diffusion-limited as overpotential increases [223] This implies that Id in

quasi-reversible system is always under the influence of potential applied because the

potential alters its kinetics

When Id becomes completely free from the mass transport influence such current is

called kinetic current (IK) because Id now purely depends on only its kinetics Arbitrary

Id then defined as the sum of diffusion-limited IL and kinetically-limited IK The

double-reciprocal equation of this summation is defined as Koutecky-Levich equation

as in Eq 96

202

Koutecky-Levich equation [30]

LKd III111

+=

216132

11620

11ων

times⎟⎟⎠

⎞⎜⎜⎝

⎛+= minus nCFADI K

middotmiddotmiddotmiddotmiddot Eq 96

Id = Disc limiting current A

IK = Kinetic current A

IL = Levich current A

( Please refer to Eq 93 and Eq 94 for other terms and symbols)

By extrapolating the Koutecky-Levich plot IK can be easily estimated [30] from the

reciprocal of its y-intersect In order to plot Koutecky-Levich equation and find IK a

sequence of arbitrary Id is recorded with altering disc rotation velocity at arbitrary

potential This technique is called hydrodynamic amperometry (HDA) and it is shown

in Fig 91

203

Fig 91 HDA with various disc rotation velocities at ndash 08 V for O2 reduction in

oxygenated 001 M PBS at pH 74 at pretreated GCE Based on the real data

Based on the data obtained by HDA Koutecky-Levich plot is plotted as in Fig 92

204

0E+00

5E-02

1E-01

2E-01

2E-01

3E-01

-002 000 002 004 006 008 010 012 014 016 018

ωˉfrac12 (sˉ

frac12)

1 IK (A-1)

- 04 V

- 06 V- 08 V

05 x 10-1

10 x 10-1

15 x 10-1

0

20 x 10-1

25 x 10-1

-1

I d(A

-1)

Fig 92 Koutecky-Levich plot for O2 reduction at pretreated GCE in oxygenated 001 M

PBS at pH 74 with applied potential ndash 04 06 and 08 V Based on the real data

With Ik from Koutecky-Levich plot and nα from CV the rate constant for arbitrary

potential (k(E)) can be calculated using Eq 97

The rate constant for arbitrary potential E [30]

lowast=nFAC

Ik K

E )( middotmiddotmiddotmiddotmiddot Eq 97

k(E) = Rate constant for the applied potential E ms-1

( Please refer to Eq 94 Eq 95 and Eq 96 for other terms and symbols)

205

568 Finding the standard rate constant (kdeg)

Finding the standard rate constant (kdeg) for ORR of each catalyst is the ultimate

aim of this long tedious cathode analysis because kdeg tells the pure catalytic power

independent of reactant diffusion and overpotential (η) k(E) can be expressed in terms of

kdeg and η as in Eq 98 therefore it is possible to estimate kdeg with known η and k(E)

k(E) in terms of kdeg and η for a forward reaction [30 181]

⎥⎦⎤

⎢⎣⎡ minusminus

deg= )(exp 0)( EE

RTnFkk Ef

α

⎥⎦⎤

⎢⎣⎡ minus

deg= ηαRT

nFk )1(exp middotmiddotmiddotmiddotmiddot Eq 98

kf(E) = Rate constant for a reduction reaction at the applied potential E ms-1

kdeg = Standard rate constant ms-1

η = Overpotential V

( Please refer to Table 11 and Table 12 for other terms and symbols)

η can be found by subtracting Edegrsquo of the reaction involved from the applied potential in

HDA n obtained by HDV is used to specify the reaction involved hence its Edegrsquo In

order to estimate kdeg Eq 98 is first converted into logarithmic expression as in Eq 99

kf(E) in terms of kdeg and η for a heterogeneous reduction reaction

ηαRT

nFkk Efminus

minusdeg= loglog )( middotmiddotmiddotmiddotmiddot Eq 99

206

Log(k(E)) is plotted against a range of η and by extrapolating the plot log(kdeg) is obtained

from its y-intersect where η = 0 hence kdeg can be calculated (Fig 93) [181]

-12

-10

-8

-6

-4

-2

0

-10 -08 -06 -04 -02 00 02 04

η V

log kf(E)

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS NaOH PBS NaOH H2SO4 PBS

NaOH H2SO4

NaOH H2SO4 PBSH2SO4 PBS

H2SO4

NaOH PBS

log kdeg

η V

Fig 93 Log kf(E) plot for O2 reduction at various pretreated GCEs Based on the real data

569 Summary of the electrochemical techniques

CV and hydrodynamic methods are complementary techniques for qualitative

examination of redox reactions and extraction of thermodynamic and kinetic parameters

The protocol for calculating kdeg from hydrodynamic methods and nα from CV is shown

in Fig 94

207

Fig 94 The protocol for finding kinetic and thermodynamic parameters

57 SUMMARY OF THE CHAPTER

The chemistry and electrochemistry of O2 has been reviewed The mechanisms of most

widely investigated catalysts noble metals macrocyclic complexes and enzymes have

been reviewed and their suitability for application in biofuel cells has been discussed

The key physico-chemical parameters which characterise these reactions have been

identified and methods for their determination have been described

208

6 CATHODE MATERIAL AND METHOD

61 ELECTRODE FABRICATION

611 Graphite PtC and RhC electrodes

Microcrystalline graphite of grade (2-15 micron 999995 purity) was

purchased from Alfa Aesar GmbH amp CoKG 5 wt-loaded rhodium on activated

charcoal (RhC) and platinum on activated carbon (PtC) were purchased from Sigma

Aldrich CY1301 and Aradur hardener HY 1300 were purchased from Robnor resins

and they were mixed at 31 (vv) for use RDE templates were specially designed and

made on site They were made of Teflon with the dimension 4-mm ID 20-mm OD

and 15-mm height The depth of composite-holding cavity for each RDE was 25 mm

The composite was made by mixing graphite RhC or PtC with epoxy resin at 73 by

hand Composite mixture was packed into five RDE templates and cured at room

temperature for 72 hours [106]

612 Anthraquinone(AQ)-carbon composite electrodes

6121 Derivatisation of graphite with AQ

Fast red AL salt with 25 dye content and hypophosphorous acid at 50 wt

in H2O were purchased from Sigma Aldrich HPLC grade acetonitrile was purchased

from VWR international 5 mM of Fast Red AL salt solution was prepared in 50 mM

209

hypophosphorous acid 10 ml of the Fast Red solution was mixed with 2 g of graphite

The mixture was left at 5 degC for 30 minutes with regular stirring at every 10 minutes It

was finally washed thoroughly with deionised water followed by vacuum suction in

acetonitrile flow The washed derivatised AQ-carbon was left to dry overnight It was

kept in the sealed container for future use [44]

6122 Fabrication of AQ-carbon composite electrode

AQ-graphite was mixed with epoxy resin at 64 by hand The composite was

packed into five RDE templates and cured at room temperature for 72 hours

613 Co(salophen)- and Fe(salophen)-graphite composite electrodes

6131 Information on synthesis of metallosalophen

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminocobalt (II)] and

[N-Nrsquo-bis(salicyldaldehyde)-12-phenylenediiminoiron (II)] denoted as Co(salophen)

and Fe(salophen) respectively were kindly donated by Dr Peter Cragg (School of

Pharmacy Brighton University) They were synthesised by dissolving metal acetate in

the ligand solution which was made by condensation of 21 molar ratio of

salicylaldehyde and diethylenediamine in ethanol or methanol [224 225]

210

6132 Fabrication of Co(salophen)- and Fe(salophen)-graphite composite electrodes

Analytical grade dichloromethane was purchased from VWR international In

the presence of small quantity of dichloromethane 5 wt of Co(salophen) or

Fe(salophen) was mixed and homogenised with graphite [181] After the

dichloromethane evaporated the metallosalophen-graphite mixture was further mixed

with epoxy resin at 64 [219] by hand The composite was packed into five RDE

templates and cured at room temperature for 72 hours

62 COMPOSITE RDE CUTTING

The electrode discs of all the RDE composite electrodes were cut into 1

mm-thick sections using a Buehler IsoMet Low diamond saw with a 04 mm-thick

diamond blade

63 ELECTRODE POLISHING

631 Composite RDE polishing

Composite RDEs were polished successively with SiC grinding paper of 400

1200 and 2400 grit followed by with alumina oxide slurry of particle size 1 03 and

005 microm as in sect 33 on p 122 Electrode polishing was done just before the experiment

in order to avoid air oxidation [195]

211

Fig 95 Fabricated composite RDEs

632 Glassy carbon electrode polishing

Glassy carbon electrodes (GCE) (5-mm disc diameter) were purchased from

Pine Instruments Activated carbon (AC) was purchased from Chemviron Carbon GC

cleaning solvent mixture was made by mixing AC and HPLC grade acetonitrile in a

ratio of 13 (vv) The mixture was covered and left at least for 30 minutes then filtered

before use [226] GCEs were successively polished with alumina oxide powder slurry of

1- 03- and 005-microm particles on MicroCloth pad Polished GCEs were sonicated in

filtered ACacetonitrile mixture for 10 ndash 15 minutes to remove alumina powder debris

and contaminants [226] The GCEs were then sonicated in deoxygenated deionised

water Air contact of the electrode surface was strictly avoided [195]

212

64 ENZYME RDEs

641 Membrane preparation

Cellulose acetate dialysis tubing visking 3632 was purchased from Medicell

International Ltd Sodium carbonate (Na2CO3) was obtained from Sigma Aldrich

Dialysis membrane was cut into 10x10-mm2 pieces which were boiled in 1 wv

aqueous Na2CO3 solution for 10 minutes The membrane pieces were rinsed thoroughly

with deionised water and stored [61] in PBS in a sealed container at 4degC until its use

642 Membrane enzyme electrodes

Laccase (Lac) from Trametes versicolor gt20 unitsmg-1 and lyophilised

bilirubin oxidase (BOD) from Myrothecium verrucaria 15 ndash 65 units mg-1 were

purchased from Sigma Aldrich 4 mgml-1 of Lac and BOD solution was made with PBS

respectively BOD immediately dissolved in PBS while a vortex mixer was needed to

dissolve Lac In order to attach enzyme on the GCE surface the tip of polished cleaned

GCEs was dipped and left in the enzyme solution for two hours with gentle stirring The

electrodes were then lightly washed with deionised water Pretreated dialysis membrane

was carefully placed onto the surface of the enzyme-modified GCE and fixed with

rubber O-ring

213

65 ELECTROCHEMICAL EXPERIMENTS

651 Electrochemical system

CHI 650A with single-channel and CHI 1030A with multi-channel

potentiostats were used for electrode pretreatment and electrochemical experiments CV

HDV and HAD An adjustable rotor from Pine Instrument was used to rotate a RDE

Three-electrode system (sect 342 on p 123) was employed with an AgCl reference and a

Pt counter electrodes

Fig 96 RDE experiment setting with a rotor and gas inlet

214

652 Electrode pretreatments

6521 Pretreatments of Graphite GC Co(salophen) and Fe(salophen) electrodes

Graphite GC Co(salophen) and Fe(salophen) electrodes were pretreated in

seven different ways in Table 14 Untreated electrodes were also prepared as controls

All the pretreatments were made in series

Treatment type 1st pretreatment 2nd pretreatment 3rd pretreatment

Type I Untreated -

Type II Acidic 01 M H2SO4

Type III Basic 01 M NaOH

Type IV Neutral 001 M PBS

Type V Base followed by Acid 01 M NaOH 01 M H2SO4

Type VI Acid followed by Neutral 01 M H2SO4 001 M PBS

Type VII Base followed by Neutral 01 M NaOH 001 M PBS

Type VIII Base Acid then Neutral 01 M H2SO4 01 M NaOH 001 M PBS

Table 14 Pretreatment types for graphite GC Co(salophen) and Fe(salophen) electrodes

Concentrated sulphuric acid at 90 - 98 and sodium hydroxide tablets were purchased

from VWR international 001 M PBS was purchased from Sigma Aldrich All the

pretreatments were based on potential cycling in 100-ml electrochemical cell except

untreated ones Acidic pretreatment was performed at 5 Vs-1 between 15 V and ndash 05 V

215

in 01 M H2SO4 [105 219] for 30 minutes Basic pretreatment was performed at 5 Vs-1

between 06 V and 2 V in 01 M NaOH [218 219] for 30 minutes Neutral pretreatment

was done at 5 Vs-1 between 15 V and ndash 05 V in deoxygenated 001 M PBS pH 74

[219 220] Constant N2 flow was applied to PBS through the pretreatment In addition

pretreatments were performed in various combinations as in Table 14 Type V Base

followed by Acid Type VI Acid followed by Neutral Type VII Base followed by

Neutral and Type VIII Base followed by Acid then Neutral [219] The influence of

pretreatments on electrode surface and ORR was studied by CV HDV and HDA

6522 Pretreatments of AQ-carbon PtC and RhC

AQ-carbon PtC and RhC composite electrodes were pretreated only in 01 M

H2SO4 at 5 Vs-1 between 15 V and ndash 05 V in for 30 minutes Untreated electrodes were

also prepared as controls

653 Electrochemical experiments

All experiments were carried out under both N2 and O2 saturation 100 ml of

001 M PBS electrolyte solution was sparged with either N2 or O2 for 15 minutes before

the experiment Gas was continuously supplied through a Dreschel gas washing bottle

containing PBS throughout the experiment

CV was performed at 002 005 01 02 and 05 Vs-1 between + 09 V and ndash

09 V HDV was performed at fixed scan rate of 005 Vs-1 sweeping the potential from

+ 09 V to ndash 09 V with disc rotation velocity altered through 900 1600 and 2116 rpm

216

HDA was performed at a range of fixed potentials - 02 - 04 - 06 and ndash 08 V at disc

rotation velocity of 400 625 900 1156 1225 1600 2116 and 2500 rpm All

experiments were repeated twice or three times The sample size ranges from one to

three The median values were taken for the analysis Obtaining a set of data for each

electrode sample took from three weeks to more than one month

654 Data analysis

CV data was used to calculate nα and reaction reversibility HDV data was used

to calculate n using Levich equation (Eq 30 on p 61) Edegrsquo for ORR involved at pH 7

25 degC was specified by n value as in Table 15

n Redox type Redox potential vs AgAgCl

1 Edegrsquo(O2H2O2) 0082 V

2 Edegrsquo(O2H2O) 0616 V

Table 15 Redox potentials for O2H2O2and O2H2O at pH 7 25 degC [24]

η was calculated by subtracting Edegrsquo (ORR) from the potential applied in HDA HDA

data was used to estimate kf(E) at each applied potential using Koutecky-Levich plot (Eq

30 on p 61 and Fig 92 on p 204) N2 data was subtracted from O2 data as background

Other parameters required for Koutecky-Levich plot were set as in Table 16 All

parameters assume the condition of 001 M PBS at 24 degC

217

Parameter Value Reference

O2 diffusion coefficient 23 x 10-9 m2s-1 [227]

O2 concentration 123 molm-3 [158]

Kinematic viscosity (ν) 884 x 10-7 m2s-1 [30]

Table 16 Parameter values for Koutecky-Levich equation

kdeg was estimated by plotting log(kf(E)) vs η (Eq 99 and Fig 93 on p 206) kdeg for

various and pretreated cathodes were compared The best catalyst shows the largest kdeg

The procedure for finding kdeg is summarised in (Fig 94 on p 207)

218

7 CATHODE RESULTS AND DISCUSSION

71 AIMS OF THE CATHODE STUDIES

This section describes the electrochemical characterisation of various modified

electrodes which were considered as candidate cathodes for the biofuel cells Various

carbons were used as electrode materials in all cases The electrochemical behaviour of

carbon depends strongly on history and also can be radically altered by pre-treatment

The effects of electrochemical pretreatments (ECPs) on the kinetic parameters will be

described for both glassy carbon and graphite composites The cathode modifiers

considered in this section include noble metals (Pt Rh) inorganic Schiff bases

(Co(salophen) Fe(salophen)) an organic modifier (anthroquinone) and the enzymes

bilirubin oxidase (BOD) and laccase (Lac) In each case of non-biological catalysts the

effects of ECPs were also quantified using cyclic voltammetry (CV) and hydrodynamic

methods with rotating disc electrodes (RDEs)

CV allowed the investigation of

Capacitance from the double layer region

Qualitative description of the mechanism such as number of discrete reaction

steps reversibility and surface confinement

nα the number of electrons transferred up to and including the rate limiting step

(RLS)

219

while hydrodynamic methods were used to quantify

n the total number of electrons transferred

kdeg the heterogeneous standard rate constant

Epc2(HDV) the half-wave reduction potential in HDV

The data was taken under both N2 and O2 to allow characterisation of the effects of

electrochemical pretreatment and its effects on the kinetics of O2 reduction The best

candidate catalytic cathode was determined according to kdeg nα and Epc2(HDV)

72 INFLUENCE AND EFFICACY OF ECPS

721 Glassy carbon electrodes (GCEs)

7211 Untreated GCEs

Voltammetry curves of untreated GCEs at pH 74 in the presence of N2 and O2

are shown in Fig 97

220

Fig 97 CVs of untreated GCEs at pH 74 in 001 M PBS in the presence of (i) N2 (ii)

O2 at 005 Vs-1 between + 09 and -09 V vs AgAgCl

Untreated GCEs did not show any peak in the absence of O2 (Fig 97(i)) indicating that

there was no electrochemically active redox species on the electrode surface and in the

solution CV was in the presence of O2 (Fig 97(ii)) showed a peak at ndash 08 V but

without a reverse anodic current This indicates the presence of catalytic activity

however it occurred at only high overpotential and is therefore not efficient for ORR

This result is consistent with previous work [215 216]

7212 Influence of electrochemical pretreatments on GCE

GCEs were pretreated in various media in order to improve ORR efficiency It

has been reported that ECPs activate the electrode surface by introducing

electrochemically active functional groups and that this improves electrochemical

(i) N2

-3E-05

-2E-05

-1E-05

0E+00

1E-05

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

221

reversibility and [105 218] and the electron transfer rate of inner-sphere catalysis [228]

Fig 82 shows the voltammetry curves in the absence of O2 for various pretreated GCEs

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-60E-05

-50E-05

-40E-05

-30E-05

-20E-05

-10E-05

00E+00

10E-05

20E-05

30E-05

40E-05

50E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH

Fig 98 Overlaid CVs of pretreated GCEs at pH 74 in 001 M PBS in the absence of

O2 between + 09 and - 09 V vs AgAgCl at 005 Vs-1 Arrows indicate the

peak position (i) includes CVs for all the pretreated GCEs and (ii) excludes

PBS pretreatments for convenience

222

As the figure shows any pretreatment resulted in an increase in capacitance and

introduced characteristic voltammetric peaks which were not observed in the untreated

GCEs In all cases the peak current was linearly proportional to υ indicating that the

pretreatments led to surface-confined redox active functional groups most likely to be

quinone groups That ECPs give rise to surface quinones is well established [105 218

220 229 230] The Epc shifted with υ and was separated from Epa hence redox

reactions by these surface quinone groups are quasi-reversible The effect of

pretreatments however varied by the type of medium used in pretreatments

Above all PBS-related pretreatments gave qualitatively different results which

were characterised by a great increase in average capacitance calculated from CVs in

001 M PBS at pH 74 at 50 100 and 200 mVs-1 in the double layer region of the CV at

04 V Summary results are shown in Fig 82(i) and Fig 99

223

GCE Capacitance vs Pretreatment

0100200300400500600700800900

1000

Untreated H2SO4 NaOH NaOH H2SO4

PBS H2SO4 PBS

NaOH PBS

NaOH H2SO4

PBS

Pretreatment

Capa

cita

nce

microF

cm-2

Fig 99 The capacitance of GCEs in 015 M NaCl buffered with 001 M phosphate to

pH 74 Error bars represent the standard deviation (n = 3)

The average capacitance of the untreated GCEs was 76 microFcm-1 and is within the typical

ranges reported hitherto [221] while PBS pretreatment increased the capacitance to 700

- 800 microFcm-1 A similar phenomenon was reported by Nagaoka and by Swain [215

216] There are two possible reasons for this (i) PBS treatment caused the

microfractures of GC and increased the electrode area and therefore double layer [105

215 216 218] andor (ii) attached the electrochemically inactive charged surface group

such as phenol and carboxylate [216 229]

In the pretreatment involving NaOH the carbon dissolution was observed This

is harsher pretreatment so erodes the electrode and makes the electrode surface more

porous [216 218] NaOH also forms unstable oxide film and eventually removes

graphite oxide [229] and surface impurities [218] However the resultant capacitance

224

was unexpectedly smaller than that of sole PBS pretreatment Therefore NaOH

pretreatment increased the capacitance by increasing the exposed electrode area [218

231] while PBS pretreatment increased capacitance by attaching redox inactive charged

surface groups The largest capacitance after the double pretreatment involving NaOH

and PBS was therefore derived from the layer of charged groups attached on the

electrode surface area made larger by NaOH pretreatment

H2SO4 pretreatment on the other hand resulted in a smallest capacitance

increase The electrode behaviour was also slightly altered from that of other

PBS-related pretreatments when the pretreatments involved H2SO4 together The

capacitance became smaller as shown in Fig 99 and the peak positions were shifted to

more positive potential side (Fig 82(i)) To investigate the causes of this phenomena

the voltammograms during such pretreatments were studied as Fig 100 below

225

Fig 100 Overlaid CVs during various pretreatments for GCEs involving 01 M H2SO4

alone (red) 001 M PBS at pH 74 alone (green) and both pretreatments (pink)

It is clearly shown that either pretreatment resulted in distinctive peaks for the

attachment of active surface quinone groups However the peak potentials were

different when in sole H2SO4 or PBS-related pretreatments This is because as proposed

by Nagaoka those quinone groups were present in the different chemical environment

leading to different behaviours [216] H2SO4 pretreatment attached fewer inactive

surface oxygen groups while PBS pretreatment attached more Therefore H2SO4

pretreatment led to the smaller capacitance change than PBS pretreatment This implies

the presence of different electrode surface conditions hence quinones present in such

different environment behave differently showing different peak positions The

behaviours of surface quinone groups depends on the conditions of the pretreatment

involved [229 230 232] This fact can also explain the phenomena caused by the

226

double pretreatment with H2SO4 and PBS as shown in Fig 82(i) More quinone groups

had been attached by H2SO4 pretreatment in advance therefore only smaller area than

usual became available for PBS pretreatment to stably attach their charged oxygen

surface groups This is the most likely causes of the reduced capacitance and different

peak positions in such a double pretreatment compared to other PBS-related

pretreatments Furthermore quinone peak positions should be close to those found for

the H2SO4 pretreatment alone because quinone groups attached by preceding H2SO4

pretreatment dominate the electrode surface and should be have more influence on the

electrochemistry However they also resulted in larger capacitance because the

subsequent PBS pretreatment attached inactive oxygen surface group which led to an

increase in fixed charge density This is a typical phenomenon of synergetic effects as

already seen in the double pretreatment with NaOH and PBS

On the other hand voltammetric curves for the double pretreatment of NaOH

and H2SO4 did resemble to those by sole NaOH pretreatment Similar phenomena were

observed between the double pretreatment with NaOH and PBS and the triple

pretreatment with NaOH H2SO4 and PBS It seems that preceding NaOH pretreatment

overwhelmed the subsequent H2SO4 pretreatment although some influence by H2SO4

can be observed in terms of capacitance as shown in Fig 99 This implies that the

preceding NaOH pretreatment made most of the electrode area not available for

subsequent H2SO4 pretreatment However if NaOH pretreatment creates micropores in

the GC protons from subsequent H2SO4 pretreatment can still penetrate such

micropores These micropores are accessible for protons but not most ions [216]

227

7213 Effect of GCE pretreatment on ORR

Voltammograms for ORR at various pretreated GCEs at pH 74 are shown in

Fig 101 below

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO4

01M NaOH01M NaOH + 01M H2SO4

001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS

01M NaOH + 01 M H2SO4 + 001M PBS

Fig 101 Overlaid CVs of ORR at various pretreated GCEs at pH 74 in 001 M

oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

In the figure any pretreated GCE shows larger cathode peaks between ndash 03 and ndash 05 V

without reverse anodic current They were due to O2 reduction by surface quinone

groups whose peaks were observed at ndash 01 V in Fig 82(i) in the absence of O2 This

indicates that ORR at any pretreated GCEs is regulated by ECrsquo mechanism [30] Ipc was

linearly proportional to radicυ in all cases indicating the diffusion-limited charge transfer

228

However untreated GCE was catalytic only at high overpotential since Epc2(HDV) = ndash 08

V This is compatible to the observation made by Tammeveski [233] It was seldom

catalytic since n = 102 plusmn 011 and nα = 059 plusmn 004 This single-electron ORR implies

the presence of O2־ disproportionation from which little power can be extracted [233]

kdeg was could not be calculated because the standard Koutecky-Levich plot could not be

obtained since it did not show a normal linear slope In contrrast all the other

pretreatments improved ORR as seen in Fig 101 They showed large cathodic peaks at

smaller overpotentials PBS-related pretreatments in particular increased the catalytic

current by gt 15 times and positively shifted Epc2(HDV) by gt 300 mV relative to that of

untreated GCEs Similar phenomena were reported elsewhere [220 234] All the GCE

pretreatments resulted in an increase in both nα and n The increase in kdeg accompanied

by an increase in nα was also observed as a general tendency as shown in Fig 102

229

0

2

4

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

kordm

ms

-1

nα 059 107 094 106 168 278 174 178

kordm ms-1 000E+00 715E-09 529E-09 631E-08 131E-07 279E-07 654E-08 113E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

- nα

kdeg ms-1

Untreated H2SO4 NaOHNaOHH2SO4

H2SO4PBS

NaOHPBS

NaOHH2SO4PBS

Fig 102 Catalytic efficiency (kdeg nα) of various pretreated GCEs

Above all the H2SO4-PBS double pretreatment was most effective giving kdeg = 279 x

10-7 plusmn 507 x 10-9 ms-1 and Epc2(HDV) = - 039 plusmn 002 V However its nα was calculated

to be 278 while n = 185 This is an unusual result It might imply the presence of serial

ORR pathway in which H2O2 is further reduced or it is simply impossible to obtain

correct parameters by using Eq 88 on p 193 for ORR at pretreated GCEs due to their

excessively narrow peak width Pretreatment of GCE was proved to increase kdeg of ORR

but how this improves its catalysis has been uncertain for a long time between two

hypotheses whether they mediate electron transfer or they alter the adsorption of O2

and O2[233 232 220] ־ The latter or both is more likely because as Fig 102 shows

PBS-related pretreatments increased the capacitance which was derived from redox

inactive surface oxygen groups yet made significant contribution to an increase in kdeg It

230

is therefore natural to deduce that the largest kdeg and nα were the consequence of

synergetic effect derived from different surface oxygen groups attached by H2SO4 and

PBS

722 Graphite composite electrodes

7221 Untreated graphite composite electrodes

Graphite composite electrodes are different from GCEs They are made of

graphite particles which are more porous and permeable to the solution and gas This

practically results in larger electrode surface area therefore becomes more subject to

facile adsorption and surface modification This leads to the different surface conditions

and electrode behaviours from GCEs as proposed by Sljukic [232] CVs of untreated

graphite composite electrodes at pH 74 in the presence of N2 and O2 are shown in Fig

103 together with those of untreated GCEs

231

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

(ii) O2

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Graphite

GC

Fig 103 CVs of untreated graphite composite and GC electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl Small peaks on graphite electrodes are indicated by arrows

Different electrode behaviour is noticeable especially in nitrogenated solution because

untreated graphite composite electrodes showed small peaks at + 100 mV and - 100 mV

as indicated by arrows No peak was present fo untreated GCE This indicates the

presence of some reactive surface quinone groups on graphite composite without

pretreatment This is because graphite composite electrodes have more reactive edge

plane graphite exposed and the porous electrode structure allows larger electrode area to

be exposed There was however little difference in averaged capacitance between

graphite composite and GCEs This indicates that an increase in capacitance does not

necessarily correlate to an increase in the surface area as proposed by Regisser [235]

Voltsmmograms for ORR at untreated GC and graphite composite electrodes resembles

each other though for the latter the reduction peak is around 50 mV more positive It can

232

be seen that graphite composites are slightly more catalytic toward ORR presumably

because of the intrinsic quinone groups derived from the graphite electrode surface

7222 Influence of electrochemical pretreatments on graphite composite electrodes

Cvs for the graphite composite electrodes differed from those of GCEs Peaks

were observed in all graphite cases including untreated ones as shown in Fig 104

233

-20E-04

-15E-04

-10E-04

-50E-05

00E+00

50E-05

10E-04

15E-04

20E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

(ii)

01M H2SO401M NaOH01M NaOH + 01M H2SO4Untreated

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBSPBS

(iii)01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS001M PBS

Fig 104 Overlaid CVs of graphite composite electrodes at pH 74 in 001 M

nitrogen-sparged PBS between + 09 and - 09 V vs AgAgCl (i) All

pretreatments at 005 Vs-1 (ii) Non-PBS-related pretreatment and (iii)

PBS-related-pretreatment CVs at 002 Vs-1

The capacitance became larger after pretreatments Any multi-pretreatment involving

NaOH and H2SO4 resulted in the largest capacitance which was 20 times larger than

234

that of untreated graphite composite electrodes The capacitance increase was also

larger than that of any pretreated GCEs except for PBS-pretreated ones PBS

pretreatment led to the smallest capacitance increase in graphite composite electrodes in

contrast to the GCEs Instead H2SO4 pretreatment led to the largest capacitance

increase Moreover this outcome was found to correspond to a specific proportional

relation between Ip and υ Ip of electrodes involving any pretreatment in PBS NaOH or

their combinstions were linearly proportional to radicυ This can only arise from on or more

diffusion limited step or steps in the redox reactions of the surface groups most likely

proton diffusion though a porus film or to microscopic porous environments Surface

oxygen groups have previously been reported to involve involve proton uptake and this

leads to a broad peak [216 230] H2SO4-pretreated electrodes showed Ip proportional to

υ and the redox reaction is independent of proton diffusion

These phenomena show that different pretreatments provide different chemical

environment for active quinone groups leading to different quinone behaviours and

voltammetry peaks [229 230 232] In the multi-pretreatment with NaOH and H2SO4

synergetic effects became obvious Its redox reaction was diffusion-limited and showed

voltammograms with two partially resolved sets of peaks yet its peak still partially

overlaps with that of diffusion-independent H2SO4-pretreated electrodes as seen in Fig

104(i) When this double-pretreatment was further followed by PBS pretreatment the

peak derived from quinones attached by H2SO4 disappeared This implies that

subsequent PBS pretreatment suppressed the activity of proton-independent quinones

and the effect by H2SO4 The similar phenomenon was observed when electrodes were

pretreated doubly by H2SO4 and PBS (Fig 104(ii)) Every case presented

235

quasi-reversible redox systems because peak potentials shifted with scan rates Small

multiple peaks were present in slower scan rates merged into one broad peak at faster

scan rate as the contribution of capacitance charging and increased iR drop obscured

voltammetic detail

7223 The effect of graphite composite electrode pretreatment on ORR

ORR at pretreated graphite composite electrodes was found to be less effective

than for pretreated GCEs Only NaOH-related pretreatments were effective (Fig 105)

enough to achieve two-electron ORR Untreated graphite composite electrodes were

catalytic only at high overpotential despite the presence of surface quinone groups PBS

pretreatment was hardly effective in contrast to the results obtained for GCEs

Electrochemical pre=treatment in H2SO4 and its double pretreatment with PBS reduced

Epc2(HDV) by 40 ndash 90 mV however the electrodes showed poor catalytic performance nα

lt 05 and n lt 2 consistent with the initial electron transfer still being rate determining

236

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 105 Overlaid CVs of ORR at various pretreated graphite composite electrodes

at pH 74 in 001 M PBS with O2 between + 09 and - 09 V vs AgAgCl at 005

Vs-1

Pretreatment of the graphite composites with just H2SO4 showed a single merged peak

at slower scan rate while they showed an addition prewave as indicated by arrows in

Fig 106 at faster scan rate

237

-16E-03

-14E-03

-12E-03

-10E-03

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

-1 -08 -06 -04 -02 0 02

E V

J Acm-2

50 mV100 mV200 mV500 mV

Fig 106 Overlaid CVs of ORR at H2SO4-pretreated graphite composite electrodes at

pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs AgAgCl at various

scan rates 50 100 200 and 500 mVs-1

Coupled with the fact of n asymp 1 this prewave is due to the rate-limiting slow formation of

semiquinone radicals [233] A semiquinone radical is formed when one electron is

transferred to a quinone group and its formation is necessary to mediate ORR as shown

in Eq 100

238

middotmiddotmiddotmiddotmiddot Eq 100

[233] middotmiddotmiddotmiddotmiddot Eq 101

Q = Surface quinone group

O2־ = Semiquinone radical

Any NaOH-related pretreatment on the other hand showed a prewave around ndash 035 V

at slower scan rate as shown in Fig 107 but this merged into a broad peak at faster scan

rate

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

01M NaOH01M NaOH + 01M H2SO4 01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 107 Overlaid CVs of ORR at graphite composite electrodes of NaOH-related

pretreatments at pH 74 in 001 M oxygenated PBS from + 09 to - 09 V vs

AgAgCl at 20 mVs-1

239

Ipc was linearly proportional to radicυ except for the triple pretreatment which was difficult

to define due to the ambiguous peak positions over different scan rates The prewave

was due to the slow reduction of O2 into O2־ (Eq 101) rather than semiquinone radical

formation (Eq 100) as seen in H2SO4-pretreated electrodes They had n asymp 2 and nα asymp 1

hence they catalysed two-electron ORR Those pretreatments positively shifted

Epc2(HDV) by 173 plusmn 26 mV on average from ndash 730 mV at the untreated graphite

composite electrodes The largest catalytic activity was achieved by doubly pretreated

electrodes with NaOH and H2SO4 with kdeg = 382 x 10-7 plusmn 32 x 10-8 ms-1 (Fig 108)

0

2

nα

00E+00

50E-08

10E-07

15E-07

20E-07

25E-07

30E-07

35E-07

40E-07

45E-07

kordm

ms

-1

nα 042 035 080 114 045 040 101 072

kordm ms-1 000E+00 000E+00 233E-07 382E-07 000E+00 000E+00 132E-07 172E-07

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBSUntreated H2SO4 NaOH

NaOHH2SO4

H2SO4

PBS NaOHPBS

NaOHH2SO4

PBS

- - - - nα

kdeg ms-1

Fig 108 Catalytic efficiency (kdeg nα) of various pretreated graphite composite electrodes

240

723 Platinum on carbon composite electrodes (PtC)

Noble metals are popular ORR catalysts [172 173] with Pt showing the highest

catalytic power [165 166 171 172] It was reported that Pt has a 98 O2-to-H2O

conversion rate [174] However for PtC composite electrodes the voltammetry was

dominated by showed solution resistance both in the presence and absence of O2 and

therefore were not either catalytic or conductive H2SO4 pretreatment was not at all

effective This is probably due to the large electronic resistance of activated carbon itself

[204] as well as intrinsic resistance of composite conductors

724 Rhodium on carbon composite electrodes (RhC)

Rhodium is a platinum group metal and has been reported as a good ORR

catalysis after Pt and Ir [172] Voltammograms for untreated RhC composite electrodes

only showed the capacitance but no redox peaks in nitrogenated solution The

capacitance decreased by 80 for every ten-fold increase in the scan rate Non-faradic

current is normally linearly proportional to υ however it exponentially decreased with υ

in the untreated RhC composite electrodes It did not show any peak in the presence of

O2 and was not catalytic toward ORR (Fig 110(i)) H2SO4-pretreated RhC composite

also did not show any voltammetric peaks Its capacitance was roughly x30 larger than

that of untreated electrodes and again exponentially decreased with an increase in υ

ORR catalysis was however present (Fig 110(ii)) therefore it is reasonable to deduce

that H2SO4 attached the surface quinone groups but the large capacitance charging

prevented voltammetic observation of quinones

241

(i) Untreated

-16E-03

-12E-03

-80E-04

-40E-04

00E+00

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

N2O2

(ii) H2SO4

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

10E-03

-1 -08 -06 -04 -02 0 02 04 06 08 1

E VJ

Ac

m-2

N2O2

Fig 109 CVs of (i) untreated and (ii) H2SO4-pretreated RhC composite electrodes at

pH 74 in 001 M PBS in the presence of N2 and O2 at 005 Vs-1 between + 09

and -09 V vs AgAgCl

7241 The effect of H2SO4 pretreatment of RhC composite electrodes on ORR

Untreated RhC composite electrodes did not show ORR catalysis however a

steep decrease in cathodic current was observed without any peak as the potential

decreases This steep current decrease was probably caused by O2 adsorption on the Rh

particle surface H2SO4-treated RhC on the other hand was found to be catalytic with n

= 177 and nα = 068 plusmn 011 A reduction prewave was observed at around ndash 05 V and

this merged into a broad peak at faster scan rates This prewave was due to ORR by

surface quinone groups attached by H2SO4 because it overlaps with the peak observed

in H2SO4-treated graphite composite electrodes as shown in (Fig 110) This prewave

242

therefore indicates the slow first electron transfer to form O2־

-20E-03

-15E-03

-10E-03

-50E-04

00E+00

50E-04

-1 -05 0 05 1

E V

J Acm-2

RhC H2SO4

Graphite H2SO4

Fig 110 ORR of H2SO4-pretreated RhC and graphite composite electrodes in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and - 09 V vs

AgAgCl

O2־ was probably further reduced to H2O2 by Rh particles because n = 177 plusmn 011 The

catalytic activity was much larger compared with H2SO4-pretreated graphite composite

electrodes as shown in Fig 110 with kdeg =257 x 10-7 plusmn 230 x 10-8 ms-1

243

725 Co(salophen) composite electrodes

7251 Untreated Co(salophen) composite electrodes

Untreated Co(salophen) electrode showed couples of distinctive peaks Epc(1) =

053 Epc(2) = -024 Epa(3) = -016 and Epa(4) = 016 V as shown in Fig 111(i) These

peaks correspond to stepwise cobalt redox reactions Co3+2+ and Co2+1+ respectively

The peak potentials hardly changed over different scan rates consistent with previously

published results [181] Similar peaks have been observed in deoxygenated organic

solvents [174 181] as well as for Co(salen) [181]

(i) N2

-2E-05

-1E-05

0E+00

1E-05

2E-05

3E-05

4E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

(ii) O2

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

2E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fig 111 CVs of untreated Co(salophen) composite electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl

244

In the presence of O2 it showed a reduction peak at ndash 04 V as shown in Fig 111(ii)

ORR potential was shifted by approximately 300 mV relative to that of untreated

graphite composite This result reasonably compatible to the result reported by Ortiz

[181] At this potential O2 forms an adduct with Co2+(salophen) and then is reduced to

O2־ while Co2+ undergoes one-electron oxidation to Co3+ [170] To be catalytic Co3+

formed here must be reduced back to Co2+ to reduce O2־ further into H2O2 [181] or a

binuclear complex must be formed with another Co2+ [170] Untreated Co(salophen)

composite electrodes were however not strongly catalytic due to n = 138 plusmn 009 and nα

= 055 plusmn 012 Ip was linearly proportional to radicυ and the peak potentials Epc(2) and Epa(3)

varied only by plusmn 8 over different five scan rates This indicates nearly reversible

multi-electron transfer redox system of Co(salophen) composite electrodes

7252 Influence of electrochemical pretreatments on Co(salophen) composite

There was a particular redox pattern found in voltammetry waves according to

the type of pretreatments (Fig 112)

245

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

40E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 112 Overlaid CVs of pretreated Co(salophen) composite electrodes at pH 74 in

001 M nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

NaOH-pretreatment showed similar voltammetric faradaic features as those in untreated

electrodes (Fig 112) but increased the capacitance by six times relative to that of

untreated electrodes The size of capacitance was 382 microFcm-1 and approximately equal

to that of NaOH-pretreated graphite composite electrodes This implies that NaOH

attached electrochemically inactive surface oxygen groups and the attachment of active

surface quinone groups were not clearly confirmed

PBS-pretreatment on the other hand attached quinone groups because Epa asymp 04 V at

which similar peaks were observed in PBS-pretreated graphite composite (Fig 104(iii)

p 233) Their cathodic peaks were however broader because they merged into peaks

246

for Co-redox processes The effect of PBS pretreatment was much smaller compared to

that of H2SO4-related pretreatment in the deoxygenated solution Single NaOH

treatment and any PBS-related pretreatment showed Ip linearly proportionally to radicυ

while any H2SO4-related pretreatment but without involving PBS-pretreatment showed

a proportional relation with υ This indicates that H2SO4 and PBS or NaOH

pretreatments lead to different surface redox mechanism Peaks due to H2SO4-related

pretreatments were prominent and are indicated by a red circle in Fig 112

7253 The effect of Co(salophen) composite electrode pretreatment on ORR

Untreated electrodes were not catalytic Parameters for H2SO4-pretreated

Co(salophen) composite found to have n asymp 1 and nα le 1 although they look catalytic in

Fig 113 reducing its ORR overpotential by 400 ndash 500 mV compared to GCEs This

inconsistent result arose because it was impossible to find the correct parameter using

the conventional calculation using Eq 88 on p 193 and need more sophisticated

method such as a simulation probramme The other pretreatments were on the other

hand found strikingly effective toward ORR as shown in Fig 113

247

-80E-04

-60E-04

-40E-04

-20E-04

00E+00

20E-04

40E-04

60E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Fig 113 Overlaid CVs of pretreated Co(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

Such pretreated Co(salophen) electrodes showed Epc asymp - 03 V having reduced the

overpotential against unpretreated Co(salophen) by 100 mV and 300 mV against

untreated graphite composite electrodes ORR was synergistically catalysed by Co2+ and

surface semiquinone radicals [181 233] They showed n asymp 2 and nα gt 07 hence they

catalysed two-electron ORR Ipc was linearly proportional to radicυ hence the redox

reactions were diffusion limited except for the double pretreatment with NaOH and

H2SO4 Large catalytic activity was achieved by sole NaOH and by the double

pretreatment with NaOH and H2SO4 or the combination of H2SO4 and PBS Among

them sole NaOH pretreatment was revealed to be most effective as shown in Fig 114

and showed kdeg = 119 x 10-5 plusmn 118 x 10-6 nα = 083 plusmn 014 and Epc2(HDV) = 037 V

248

0

1

2

nα

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

kordm

ms

-1

nα 055 028 083 074 068 091 071 078

kordm ms-1 000E+00 000E+00 119E-05 747E-06 271E-06 684E-06 296E-06 587E-06

Untreated H2SO4 NaOHNaOH-H2SO4 PBS

H2SO4-PBS NaOH-PBS

NaOH-H2SO4-

PBS

Fig 114 Catalytic efficiency (kdeg nα) of various pretreated Co(salophen) composite

electrodes

One possible reason for this largest catalytic activity by NaOH pretreatment is that

NaOH might attach more reductive surface OH groups [218] which became involved in

reducing back Co3+ into Co2+ during ORR [181]

726 Fe(salophen) composite electrodes

7261 Untreated Fe(salophen) composite electrodes

Untreated Fe(salophen) electrodes showed a couple of distinctive peaks Epa = -

024 plusmn 001 V and Epc = - 036 plusmn 001 V These peaks are due to Fe3+2+ redox reaction as

249

shown in Fig 115(i) The voltammetry curve and redox peaks are reasonably similar to

those of Fe(salen) reported [224]

(i) N2

-3E-05

-2E-05

-5E-06

5E-06

2E-05

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

(ii) O2

-6E-04

-5E-04

-4E-04

-3E-04

-2E-04

-1E-04

0E+00

1E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J A

cm

-2

Fe(salophen)

Graphite

Fig 115 CVs of untreated Fe(salophen) and graphite composite electrodes at pH 74

in 001 M PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and

- 09 V vs AgAgCl

However the redox system is not perfectly reversible here because ∆Ep slightly

increases with an increase in the scan rate and its Ip was linearly proportional to radicυ It is

therefore a quasi-reversible system Untreated Fe(salophen) electrodes was not as

catalytic as Co(salophen) electrodes Epc was ndash 079 plusmn 007 V and little different from

that of untreated graphite composite electrode as shown in Fig 115(ii) although Ipc was

slightly bigger The low ORR catalytic activity of Fe(salophen) was due to the low ORR

rate of Fe itself (Fig 65 on p 164) [172]

250

7262 Influence of electrochemical pretreatments on Fe(salophen) composite

The effect of pretreatments to Fe(salophen) was striking They resulted in

additional voltammetric features due to the Fe redox peaks at Epc (Fe) = - 03 V and Epa

(Fe) = - 02 V and by attaching the surface quinone groups at Epc (Q) = 015 V and Epa (Q) =

02 V as shown in Fig 116(i)

251

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

60E-05

80E-05

10E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M H2SO4 + 01M NaOH001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

-1 -05 0 05 1

E V

J Acm-2

Untreated01 M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS001M PBS

-1E-04

-8E-05

-6E-05

-4E-05

-2E-05

0E+00

2E-05

4E-05

6E-05

8E-05

1E-04

1E-04

-1 -05 0 05 1

E V

J Acm-2

01M H2SO401M NaOH01M H2SO4 + 01M NaOHUntreated

Fig 116 Overlaid CVs of Fe(salophen) composite electrodes at pH 74 in 001 M

nitrogenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1 (i) All

pretreatments (ii) Non-PBS-related pretreatments and (iii)

PBS-related-pretreatments CVs

Non-PBS-related pretreatments (Fig 116(ii)) showed redox peaks clearly however the

252

two cathodic peaks merged into a broad peak at faster scan rate than 100 mVs-1

indicating quasi-reversible surface quinone redox reactions Only a broad merged

reduction peak was observed for all PBS-related pretreatments (Fig 116(iii)) at all scan

rates

Any PBS-related pretreatments resulted led to a broad cathodic peak (from + 01 V to

approximately ndash 03 V) made up of both Fesalphen reduction and reduction of surface

confined quinones arising from the electrochemical pretreatment PBS pretreatment

always greatly increases the surface area leading to micro fissures and consequently a

greater variety of microenvironments for electrochemically generated quinone

functionality

7263 The effect of Fe(salophen) composite electrode pretreatment on ORR

Most were found to be only weakly catalytic (Fig 117) since either n or nα or

both of them were lt 1 Only double-pretreatment with NaOH and H2SO4 led to

sufficient ORR catalysis with n = 185 plusmn 017 nα = 079 plusmn 023 kdeg = 112 x 10-7 plusmn 771 x

10-9 ms-1 and Epc2(HDV) = - 057 V The double pretreatment showed a prewave at ndash 035

V and it disappeared at faster scan rate indicating a slow single-electron transfer to O2

Ipc was linearly proportional to radicυ hence the catalysis was diffusion limited

253

-70E-04

-60E-04

-50E-04

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

10E-04

20E-04

30E-04

-1 -08 -06 -04 -02 0 02 04 06 08 1

E V

J Acm-2

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4 001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01 M H2SO4 + 001M PBS

Untreated01M H2SO401M NaOH01M NaOH + 01M H2SO4001M PBS01M H2SO4 + 001M PBS01M NaOH + 001M PBS01M NaOH + 01M H2SO4 + 001M PBS

Fig 117 Overlaid CVs of pretreated Fe(salophen)composite electrodes at pH 74 in

001 M oxygenated PBS between + 09 and - 09 V vs AgAgCl at 005 Vs-1

727 Anthraquinone (AQ) composite electrodes

Anthraquinone-grafted graphite electrodes were pretreated with H2SO4 Both

untreated and H2SO4-pretreated electrodes showed couples of peaks (Fig 118(i)) one of

which Epc(AQ) was compatible with Em = - 049 reported by McCreery [44] These

peaks were therefore derived from anthraquinone chemically attached to the graphite

254

(i) N2

-60E-05

-40E-05

-20E-05

00E+00

20E-05

40E-05

-1 -05 0 05 1

E V

J A

cm

-2

Untreated FRE01M H2SO4 FREUntreated Graphite

(ii) O2

-40E-04

-30E-04

-20E-04

-10E-04

00E+00

-1 -05 0 05 1

E VJ

Ac

m-2

Untreated FRE01M H2SO4 FREUntreated Graphite

Fig 118 CVs of untreated and H2SO4-pretreated AQ electrodes at pH 74 in 001 M

PBS in the presence of (i) N2 (ii) O2 at 005 Vs-1 between + 09 and -09 V vs

AgAgCl CVs of untreated graphite electrodes are also shown together

The other couple of peaks Epc(SQ) and Epa(SQ) was observed at - 003 plusmn 003 V and +

020 plusmn 003 V respectively They were due to quinone groups on the carbon surface

H2SO4-pretreatment did not alter the voltammogram pattern but only increased the

capacitance by x3 and enhanced the peak current Both electrodes showed Ip linearly

proportional to radicυ hence the redox reactions involve proton diffusion ORR by those

electrodes is shown in Fig 118(ii) together with the voltammogram of graphite

composite electrodes Both untreated and pretreated AQ-grafted composite electrodes

positively shifted Epc by 60 and 230 mV respectively relative to that of untreated

graphite composite electrodes However their voltammetric curves resembled those for

untreated graphite composite electrodes Besides this their Ip dropped by 20 relative

255

to untreated graphite composite electrodes and showed n asymp 1 and nα asymp 05 Therefore

either was not catalytic in this study This outcome does not correspond to the finding

by Manisankar who confirmed the presence of ORR catalysis at AQ-carbon paste

electrodes at pH 7 with a reduction prewave at ndash 04 and a peak at ndash 07 V [193] To the

contrary this result fits to the data presented by Schiffrin who observed ORR

suppression at AQGC electrodes at pH lt 7 He reported that voltammetry curves for

ORR at GC and GCAQ became similar at this pH hence implied the possibility of

disproportionation of semiquinone intermediates [194]

728 Lac and BOD enzyme membrane electrodes

In the absence of O2 neither Lac nor BOD showed any peaks in their cyclic

voltammograms However they showed ORR catalytic activity even in the absence of

mediator (Fig 119) DET is therefore possible as already confirmed by many [183

186-188] Carbon surface groups [195] also might mediate electron transfer [188]

256

-14E-04

-12E-04

-10E-04

-80E-05

-60E-05

-40E-05

-20E-05

00E+00

20E-05

-1 -09 -08 -07 -06 -05 -04 -03 -02 -01 0

E V

J Acm -2

LacBOD

Fig 119 CVs of Lac and BOD enzyme GC electrodes at pH 74 in 001 M oxygenated

PBS at 005 Vs-1 between 0 and -09 V vs AgAgCl

Ipc was linearly proportional to radicυ therefore ORR was diffusion limited Epc were -054

plusmn 002 and ndash 067 plusmn 006 V at Lac and BOD respectively They reduced ORR

overpotential by 120 and 250 mV relative to that of GCEs however the Epc shifted with

υ hence they were quasi-reversible It is known that MCOs catalyse four-electron ORR

[175 184 236] however n = 005 and nα asymp 1 in this study This is probably due to the

difficulty in electrochemical measurement of enzyme catalysis It is however possible

to find nα because the rate-limiting step is the single electron transfer between the

electrode and the blue copper atom of the enzyme Assuming this kdeg was calculated for

four-electron ORR at the enzyme electrodes and the result is shown in Fig 120

257

0

1

2

nα

00E+00

40E-09

80E-09

12E-08

16E-08

20E-08

kordm

ms

-1

nα 077 081

kordm ms-1 134E-08 168E-09

Lac Bod

Fig 120 Catalytic efficiency (kdeg nα) of Lac and BOD enzyme GC electrodes

Lac had kdeg = 134 x 10-8 plusmn 251 x 10-9 while BOD had kdeg = 168 x 10-9 plusmn 271 x 10-10

Lac electrodes showed better ORR efficiency despite of non-optimal pH condition Lac

is most active at pH 5 while BOD at pH 7 The amount of enzyme loaded on the

electrode surface also might have affected the catalytic activity It was however difficult

to control the loading because the enzyme was attached by dip coating Using mediators

could have also improved the ORR efficiency Many authors used mediators such as

ABTS [62 66] and Os-pyridine complexes linked to conductive polymer [97 237] to

assist the electron transfer between the electrode and enzyme

73 MOST EFFICIENT ORR CATALYSTS

The kinetic and thermodynamic parameters (kdeg nα Epc2(HDV)) were organised

258

by magnitude and are summarised in figures 25-27 and tables 1 amp 2 below For each

parameter the best five candidates were selected to present the effects of pretreatment

and their relation with each parameter The best three were chosen from them on the

basis of the parameter size and the number of top-ranked parameters they were

occupying kdeg was given the highest priority for selection process

731 Best five electrodes for nα

Pretreated GCEs were found to have prominent nα (Fig 121)

00

05

10

15

20

25

30

Electrode type

n α

nα 278 178 174 168 114

GC H2SO4-PBS GC NaOH-H2SO4-PBS

GC NaOH-PBS GC PBS GP NaOH-H2SO4

Fig 121 The best five electrodes for nα

The GCEs doubly pretreated with H2SO4 and PBS was found best with nα = 278 Its kdeg

was 279 x 10-7 ms-1 and also biggest among pretreated GCEs Correlation between nα

259

and kdeg has not been confirmed since it is possible for the intial electron transfer to be

accelerated whilst still remaining the RDS

732 Best five electrodes for kdeg

Top six kdegs were dominated by pretreated Co(salophen) composite electrodes

(Fig 122)

00E+00

20E-06

40E-06

60E-06

80E-06

10E-05

12E-05

14E-05

Electrode type

kdeg

ms

-1

kordm ms-1 119E-05 747E-06 684E-06 587E-06 296E-06

CoS NaOH CoS NaOH-H2SO4

CoS H2SO4-PBS CoS NaOH-H2SO4-PBS

CoS NaOH-PBS

Fig 122 The best five electrodes for kdeg

PBS pretreatment was found to suppress kdeg in Co(salophen) composite electrodes Clear

correlation between kdeg and nα has not yet confirmed however it was found that

favourable kdeg tends to lower overpotential

260

733 Best five electrodes for Epc2(HDV)

Although it is not an exact correspondence some correlation was observed

between lower overpotential and larger kdeg or lower overpotential and larger nα but not

between kdegand nα A range of pretreated Co(salophen) composite electrodes which

showed largest kdeg and a pretreated GCE which showed the largest nα were found to be

dominating low overpotential (Fig 123)

-040

-039

-038

-037

-036

-035

Electrode type

E pc

2(H

DV)

V

Epc2(HDV) -037 -037 -039 -039 -040

CoS H2SO4-PBS CoS NaOH CoS NaOH-H2SO4

GC H2SO4-PBS CoS NaOH-PBS

Fig 123 The best five electrodes for Epc2(HDV)

734 Best three candidates for ORR catalytic electrodes

Pretreated Co(salophen) composite electrodes were the best candidates for

ORR catalysis because of largest kdeg and lowest overpotential Top three catalytic

261

cathodes were listed in Table 17

kordm ms-1 Epc2(HDV) V nα

Co(salophen) NaOH 119E-05 -037 083

Co(salophen) NaOH-H2SO4 747E-06 -039 074

Co(salophen) H2SO4-PBS 684E-06 -037 091

Average 875E-06 -037 083

SD 278E-06 001 009

Table 17 Three candidates for catalytic cathodes for ORR

NaOH-pretreated Co(salophen) was found to be most efficient ORR catalytic cathode

because it has the largest kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 Epc2(HDV) = - 037 V and

was the second smallest of all kdeg of the three candidate Co(salophen) electrodes were

compared with those of catalytic metals and shown in Table 18

Electrode type kdeg ms-1 Experiment condition

Co(salophen) NaOH 119 x 10-5 In 001M PBS pH 74

Co(salophen) NaOH ndashH2SO4 747 x 10-6 In 001M PBS pH 74

Co(salophen) H2SO4-PBS 684 x 10-6 In 001M PBS pH 74

Au 4 x 10-3 In 005 M H2SO4 [173]

Au 3 x 10-5 In 01 M KOH

Ag 8 x 10-3 In 01 M KOH

Pt 4 x 10-3 In 01 M KOH

Table 18 kdeg of various ORR metal catalysts and pretreated Co(salophen) electrodes

262

NaOH-pretreated Co(salophen) composite electrode showed good catalysis at neutral

pH It could manage to achieve as efficient ORR as Au in acidic solution

74 CONCLUSION TO CATHODE STUDY

The large overpotential of ORR has been known to degrade the fuel cell

performance [3 4 62 165-167] Finding efficient ORR catalysts therefore has been a

subject of intensive research In this study various catalytic cathodes were fabricated

using inorganic organic and biological catalysts and they were electrochemically

pretreated in different media to improve the catalytic activity Studies were conducted

using various electrochemical methods (i) CV (ii) HDV and (iii) HDA The data

obtained was analysed both qualitatively and quantitatively The ORR catalytic

efficiency was calculated according to the three parameters (i) kdeg (ii) nα and (iii)

Epc2(HDV) and based on that most catalytic cathode was determined

None of the cathodes was catalytic enough without pretreatment or unless at high

overpotential In many cases electrochemical pretreatments were found to attach

various surface oxygen groups activated the electrodes and led to two-electron ORR

catalysis Their influence on electrochemistry varies by the electrode type and media

used for pretreatment It was found that PBS-related pretreatments were effective to

GCEs When the pretreatment is combined with H2SO4 this might lead to serial ORR

where H2O2 produced is further reduced to water NaOH-related pretreatments were

effective for graphite composite electrodes PtC composite electrodes were not active

due to the high resistance within the activated carbon The performance by RhC was

263

only marginally improved by H2SO4 pretreatment Any NaOH- or PBS- related

pretreatments dramatically increased kdeg of Co(salophen) composite electrodes and

reduced the overpotential by 100 mV Fe(salophen) composite electrodes were not

sufficiently catalytic without a double pretreatment with NaOH and H2SO4

Anthraquinone-grafted graphite composite electrodes were not catalytic at neutral pH

even after pretreatment Multi-copper oxidases were catalytic at DET however its RLS

was electron transfer between the electrode and the enzyme hence appropriate

mediators should be used to achieve higher catalysis

Many of pretreated Co(salophen) composite electrodes were strongly catalytic toward

ORR Among them NaOH-pretreated Co(slophen) electrodes were found to be most

catalytic at neutral pH with kdeg = 119 x 10-5 plusmn 118 x 10-6 ms-1 nα = 083 and Epc2(HDV) =

- 037 V Its catalytic efficiency was close to that of Au in the acid solution

264

8 CONCLUSION AND FURTHER STUDIES

Miniaturised glucose-oxygen fuel cells are ideal to power small implantable

medical devices Typical specification include at least 1 microW power output and 1-year

operational life Therefore fuel cells must have efficient catalysis and electron transfer

good conductivity chemical and mechanical stability as well as biocompatibility The

electrodes must be feasible to build and scaleable In this study electrodes were built

using bulk modification method because it was simple to construct and scale-up and

they were easy to modify and mechanically stable The anodes were built with 10

GOx with 77 TTF-TCNQ composite and the cathodes were built with graphite

composite containing ORR catalysts using bulk modification method TTF-TCNQ was

used because it was conductive as well as mediating and necessary for electron transfer

of the GOx anodes Problems were enzyme stability and ORR catalytic efficiency

Enzyme mass loading is known to be effective to improve catalytic efficiency and

operational life however this also decreases conductivity of the composite electrodes

The key to optimising enzyme electrodes is in the balance and compromise among the

conductivity mediation enzyme activity mechanical and chemical stability and

difficulty in the fabrication process CNF composites showed reasonable conductivity at

30 content and this might allow higher enzyme loading with mediators without losing

conductivity CNT is also a candidate material because it has comparable or larger

conductivity than TTF-TCNQ and can operate without added mediator species Ideally

enzyme electrodes should be functional with minimum components simple to build

renew and store in dry so that they become easily portable Moreover when they are

further miniaturised this will allow the medical application under harsher conditions so

265

that powering disposable implantable sensors in economically-deprived regions might

become possible Many physiological enzymes however easily lose catalytic activity

outside the optimal condition Incorporation of enzyme stabilisers makes the enzyme

robust and long lasting as well as scavenging reactive species In the glucose-oxygen

fuel cells O2 has been known to reduce the catalytic output current of GOx anodes

PEI-DTT stabiliser mixtures were found to be good for this because they conferred

stability on GOx against oxidative stress prevented O2 electron deprivation and

improved the catalytic current output It was however a smaller effect than when

conductive hydrogel was used Conductive hydrogel can be a good candidate if it

becomes resistant against degradation ORR at the cathode has posed a long-term

problem in fuel cells because ORR is inefficient and consumes potential generated

within the fuel cell Pt has been used as an ORR catalyst but it is expensive and is

becoming scarce Use of the fuel cell in the physiological condition also imposes further

operational limitations Pretreated metal complexes such as Co salophen were found to

be good candidates for ORR-catalytic cathodes Surrounding complexes with

electron-rich environment such as conductive film might improve ORR further Once

candidate anode and cathode are identified and built a biofuel cell unit can be made To

increase the power each cell is connected together and made into a stack The prototype

was made as below in Fig 124 by connecting twenty cell units

266

Fig 124 Fuel cell stack made with various GOx anodes and Co(salophen) cathodes

Each cell contained various GOx anodes and pretreated Co(salophen) electrode in 10 ml

of oxygenated 01 M glucose 001 M PBS solution pH 74 The output voltage was

found to be 02 V and it did not change for one week The fuel cell must be studied

further with an adjustable resistor for voltage-current (V-I) and power-current (P-I)

curves so that OCV can be determined properly The passive type GOx fuel cell could

output 15 mWcm-2 at 03 V [18]

In vivo studies are also necessary for implantable purposes to examine the catalytic

efficiency electrode fouling and biocompatibility This can be studied systematically by

changing the type and concentration of interfering substances such as ascorbate

acetoaminophen and urate [18] Heller tested miniturised GOx-BOD fuel cells tested in

the buffer grape and calf serum It showed only 5 power decrease in buffer however

50 drop within 20 hours in grape and 50 drop within 2 hours in calf serum [19] To

avoid electrode fouling the electrode surface can be coated with a membrane such as

267

Nafion However this imposes another problem because it can interfere with the

reactant flux to the electrode surface In the real physiological system this might cause

a serious problem because glucose and O2 concentration is much lower than in vitro

Furthermore in the physiological conditions active transport of the reactant is not

available and this will cause local concentration polarisation It seems that there are

many obstacles lying in the medical application of miniaturised glucose-oxygen fuel

cells although they have widespread potentials applications

268

9 BIBLIOGRAPHY

1 Carrette L KA Friedrich and U Stimming Fuel cells Principles types fuels and applications

Chemphyschem 2000 1(4) p 162-193

2 Gregoire Padro CE and JO Keller Hydrogen Energy in Kirk-Othmer Encyclopedia of Chemical

Technology 2005 John Wiley amp Sons Inc p 837-866

3 Ohta K T Kazama and M Nishizawa All about fuel cells in Newton 2006 p 31-63

4 Tanabe S Mastering basics of the fuel cells 2009 Denkishoin

5 Tokyo-Gas Fuel cell academy 2009 2901 [cited Available from httpwwwtokyo-gascojppefc

6 Cropper MAJ S Geiger and DM Jollie Fuel cells a survey of current developments Journal of Power

Sources 2004 131(1-2) p 57-61

7 Kordesch K et al Intermittent use of a low-cost alkaline fuel cell-hybrid system for electric vehicles

Journal of Power Sources 1999 80(1-2) p 190-197

8 UTC-Power The fuel cell for Apollo 11

9 Connor S Warning Oil supplies are running out fast in The Independent 2009

10 Mori Y Mobile fuel cell providing new advantages for mobile electronic appliances Toshiba review 2007

62(6) p 44-47

11 Mori H Electronic components and materials Toshiba review 2009 64(3) p 62-63

12 Imperial-College-London Racing Green - Vehicles 2009 [cited Available from

httpwwwunionicacukrccracinggreenvehicleshtml

13 Cleave V Formula Zero kart race could drive fuel cell technology New Scientist 2008 [cited

Available from

httpwwwnewscientistcomarticledn14596-formula-zero-kart-race-could-drive-fuel-cell-technologyhtml

14 HyNor Hydrogen highway opens in Norway 2009 [cited Available from

httpwwwhynornohydrogen-highway-opens-in-norway

15 BMW Hydrogen 7 - The future is closer than you think 2009 [cited Available from

httpwwwbmwcomcomeninsightstechnologyefficient_dynamicsphase_2clean_energybmw_hydroge

n_7html

httpwwwbmwcojpjpjabrandtechnologycleanenergywallpaperhtml

16 Sony Side view - Sony developes Bio Battery generating electricity from sugar 2007 Sony

17 Burton N Sugar-powered electronics in RSC Chemical technology news 2008

18 Sakai H A high-power glucoseoxygen biofuel cell operating under quiescent conditions Energy amp

Environmental Science 2009 2(1) p 133-138

19 Heller A Miniature biofuel cells Physical Chemistry Chemical Physics 2004 6(2) p 209-216

20 Toshiba Toshiba announces worlds smallest direct methanol fuel cell with energy output of 100 milliwatts

269

2004 [cited Available from httpwwwtoshibacojpaboutpress2004_06pr2401htm

21 Osakai T K Kanoh and S Kuwabata Basic Electrochemistry 2007 Kagakudojin Ltd Kyoto Japan

22 Wilson K and J Walker Practical Biochemistry 1992 Cambridge UK Cambridge University Press

23 Murray RK et al Harpers Biochemistry 25 ed 2000 Stamford Connecticut Appleton amp Lange

24 Sawyer DT A Sobkowiak and JL Roberts Jr Electrochemistry for chemists Revised ed 1995

Wiley-Interscience

25 IUPAC IUPAC GOLD BOOK 2009 [cited Available from httpgoldbookiupacorgindexhtml

26 Kitamura T and K Fujisawa English-Japanese The students dictionary of physics 1995 Tokyo ALC

Inc

27 Gates BC Catalysis in Kirk-Othmer Encyclopedia of Chemical Technology 2002 John Wiley amp Sons p

200-254

28 Taguchi S and E Nakano Kinetic Study of Bromination of Malonic Acid Methyl Malonic Acid

and Ethyl Malonic Acid An Undergraduate Physical Chemistry Laboratory Experimentfor

Understanding of the Infuluence of Thermodynamic Parameter on Chemical Kinetics The Chemical

Education Jounal 1999 3(1) p 1-20

29 Pletcher D A First course in electrode processes 1991 The Electrochemical Consultancy (Romsey) Ltd

23 - 26 135 - 145

30 Bard AJ and LR Faulker Electrochemical Methods Fundamentals and Applications 2000 NY USA

John Wiley and Sons Inc

31 Daintith J A concise dictionary of chemistry 2 ed 1990 Oxford UK Oxford University Press

32 Tinoco IJ et al Physical Chemistry 2002 Prentice Hall p 157

33 Watanabe K and S Nakabayashi Introduction to electrochemistry The chemistry of electron transfer 13

ed 2005 Tokyo Japan The Chemical Society of Japan

34 Hamelers B et al Microbial fuel cells Methodology and technology Environmental science amp

technology 2006 40(17) p 5181-5192

35 Koike T Chapter 10 Free energy in Physical chemistry I amp II Lecture handouts for dept of pharmacy

2010 University of Hiroshima p 57-62

36 Lopes T E Gonzalez and E Antolini An overview of platinum-based catalysts as methanol-resistant

oxygen reduction materials for direct methanol fuel cells Journal of alloys and compounds 2008 461(1-2)

p 253-262

37 Vork FTA and E Barendrecht The reduction of dioxygen at polypyrrole-modified electrodes with

incorporated Pt particles Electrochimica Acta 1990 35(1) p 135-139

38 Marino W B Scharifker and A Leone ELECTRODEPOSITION AND

ELECTROCHEMICAL-BEHAVIOR OF PALLADIUM PARTICLES AT POLYANILINE AND

POLYPYRROLE FILMS Journal of the Electrochemical Society 1992 139(2) p 438-443

39 Tsakova V How to affect number size and location of metal particles deposited in conducting polymer

270

layers Journal of Solid State Electrochemistry 2008 12(11) p 1421-1434

40 Lemest Y et al Dioxygen Fixation by a Mixed-Valence Dicobalt Cofacial Porphyrin Journal of the

Chemical Society-Chemical Communications 1983(22) p 1286-1287

41 Kim E et al Superoxo micro-peroxo and micro-oxo complexes from hemeO2 and heme-CuO2 reactivity

Copper ligand influences in cytochrome c oxidase models 2003 p 3623-3628

42 Fukuzumi S et al Mechanism of Four-Electron Reduction of Dioxygen to Water by Ferrocene

Derivatives in the Presence of Perchloric Acid in Benzonitrile Catalyzed by Cofacial Dicobalt Porphyrins

Journal of the American Chemical Society 2004 126(33) p 10441-10449

43 Solomon EI et al O2 and N2O Activation by Bi- Tri- and Tetranuclear Cu Clusters in Biology

Accounts of Chemical Research 2007 40(7) p 581-591

44 Wildgoose GG et al Anthraquinone-derivatised carbon powder reagentless voltammetric pH electrodes

Talanta 2003 60(5) p 887-893

45 McCreery R Carbon electrodes Structural effects on electron transfer kinetics in Electroanalytical

Chemistry A series advances 1990 Marcel Dekker Inc p 221-374

46 Kneten K R McCreery and C McDermott ELECTRON-TRANSFER KINETICS OF AQUATED

FE+3+2 EU+3+2 AND V+3+2 AT CARBON ELECTRODES - INNER-SPHERE CATALYSIS BY

SURFACE OXIDES Journal of the Electrochemical Society 1993 140(9) p 2593-2599

47 Engstrom RC ELECTROCHEMICAL PRETREATMENT OF GLASSY-CARBON ELECTRODES

Analytical Chemistry 1982 54(13) p 2310-2314

48 Lawrence E Hendersons dictionary of biological terms 10 ed 1989 UK Longman group UK Ltd

49 Eijsink V Directed evolution of enzyme stability Biomolecular engineering 2005 22(1-3) p 21-30

50 Pantoliano M et al PROTEIN ENGINEERING OF SUBTILISIN BPN - ENHANCED STABILIZATION

THROUGH THE INTRODUCTION OF 2 CYSTEINES TO FORM A DISULFIDE BOND Biochemistry

1987 26(8) p 2077-2082

51 Bagotzky VS NV Osetrova and AM Skundin Fuel cells State-of-the-art and major scientific and

engineering problems Russian Journal of Electrochemistry 2003 39(9) p 919-934

52 Heller A Integrated medical feedback systems for drug delivery Aiche Journal 2005 51(4) p

1054-1066

53 Nakamura M et al High-performance ultrathin solid oxide fuel cells for low-temperature operation

Journal of the Electrochemical Society 2007 154(1) p B20-B24

54 Lapedes DN McGraw-Hill Dictionary of Physics and Mathematics in McGraw-Hill 1978 McGraw-Hill

Inc

55 Svoboda V et al Enzyme catalysed biofuel cells Energy amp Environmental Science 2008 1(3) p

320-337

56 Barton SC J Gallaway and P Atanassov Enzymatic biofuel cells for Implantable and microscale devices

Chemical Reviews 2004 104(10) p 4867-4886

271

57 Schroder U Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency

Physical chemistry chemical physics 2007 9(21) p 2619-2629

58 Chaudhuri SK and DR Lovley Electricity generation by direct oxidation of glucose in mediatorless

microbial fuel cells Nature Biotechnology 2003 21(10) p 1229-1232

59 Vincent K F Armstrong and J Cracknell Enzymes as working or inspirational electrocatalysts for fuel

cells and electrolysis Chemical reviews 2008 108(7) p 2439-2461

60 Tsujimura S and K Kano Recent development of enzyme-based biofuel cells GS Yuasa technical report

2008 5(2) p 1-6

61 Cass AEG Biosensors A Practical Approach 1990 Oxford University Press Oxford UK p 49-53

62 Palmore GTR and HH Kim Electro-enzymatic reduction of dioxygen to water in the cathode

compartment of a biofuel cell Journal of Electroanalytical Chemistry 1999 464(1) p 110-117

63 Bullen RA et al Biofuel cells and their development Biosensors amp Bioelectronics 2006 21(11) p

2015-2045

64 Bertschy H et al A methanoldioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as

catalysts application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at

low overpotentials Journal of electroanalytical chemistry and interfacial electrochemistry 1998 443(1) p

155-161

65 Bartlett PN P Tebbutt and RG Whitaker Kinetic aspects of the use of modified electrodes and

mediators in bioelectrochemistry Progress in reaction kinetics 1991 16(2) p 55-155

66 Kano K T Ikeda and S Tsujimura GlucoseO-2 biofuel cell operating at physiological conditions Denki

kagaku oyobi kōgyō butsuri kagaku 2002 70(12) p 940-942

67 Heller A Electrochemical glucose sensors and their applications in diabetes management Chemical

Reviews 2008 108(7) p 2482-2505

68 Ilankumaran V et al Trends in cardiac pacemaker batteries Indian pacing and electrophysiology journal

2004 4(4) p 201-12

69 Schlindwein W and R Linford Medical applications of solid state lonics Solid State Ionics 2006

177(19-25) p 1559-1565

70 Latham R R Linford and W Schlindwein Biomedical applications of batteries Solid State Ionics 2004

172(1-4) p 7-11

71 Soykan O Power sources for implantable medical devices Business Briefing Medical Device

Manufacturing and Technology London 2002

72 Heller A Potentially implantable miniature batteries Analytical and Bioanalytical Chemistry 2006

385(3) p 469-473

73 Taniguchi M Achilles heel of the next-generation electric vehicles - Lithium World map of the resrouce

wars 2008 [cited 2009 Available from

httpbusinessnikkeibpcojparticlemanage20080407152450P=1

272

74 Yeatman EM Advances In Power Sources For Wireless Sensor Nodes International Workshop on Body

Sensor Networks 2004

75 Sato N et al Novel MEMS power generator with integrated thermoelectric and vibrational devices in

Solid-State Sensors Actuators and Microsystems 2005 Digest of Technical Papers TRANSDUCERS 05

The 13th International Conference on 2005

76 Hudak NS and GG Amatucci Small-scale energy harvesting through thermoelectric vibration and

radiofrequency power conversion Journal of Applied Physics 2008 103(10)

77 Seiko-Instruments SEIKO Worlds First 2009 [cited Available from

httpwwwseikowatchescomheritageworlds_firsthtml

78 Kishi M et al Micro thermoelectric modules and their application to wristwatches as an energy source in

Thermoelectrics 1999 Eighteenth International Conference on 1999

79 Lee J et al Ionic conduction in Zn-3(PO4)(2)()4H(2)O enables efficient discharge of the zinc anode in

serum Journal of the American Chemical Society 2005 127(42) p 14590-14591

80 Maxell Button battery table 2009 [cited Available from

httpwwwmaxellcojpproductsmaterialsbutton_batteryhtml

81 Wilson R and APF Turner Glucose-Oxidase - an Ideal Enzyme Biosensors amp Bioelectronics 1992 7(3)

p 165-185

82 Swoboda BEP The relationship between molecular conformation and the binding of flavin-adenine

dinucleotide in glucose oxidase Biochimica et Biophysica Acta (BBA) - Protein Structure 1969 175(2) p

365-379

83 Wohlfahrt G et al 18 and 19 angstrom resolution structures of the Penicillium amagasakiense and

Aspergillus niger glucose oxidases as a basis for modelling substrate complexes Acta Crystallographica

Section D-Biological Crystallography 1999 55 p 969-977

84 Patel RN Biocatalysis in the pharmaceutical and biotechnology industries 1st ed 2006 CRC Press

85 Gouda M Reversible denaturation behavior of immobilized glucose oxidase Applied Biochemistry and

Biotechnology 2002 102 p 471-480

86 Hecht HJ et al Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 3 Angstrom

Resolution Journal of Molecular Biology 1993 229(1) p 153-172

87 Zubrik A et al Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the

transition to the denatured state with residual structure The Journal of biological chemistry 2004 279(46)

p 47601-47609

88 Kojima S et al Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose

biosensor Bioscience biotechnology and biochemistry 2006 70(3) p 654-659

89 Duine J The PQQ story Journal of bioscience and bioengineering 1999 88(3) p 231-236

90 Kyoto-University KEGG - Kyoto Encyclopedia of Genes and Genomes 2009 Kyoto University

91 Delbem MF WJ Baader and SHP Serrano Mechanism of 34-dihydroxybenzaldehyde

273

electropolymerization at carbon paste electrodes catalytic detection of NADH 2002 scielo p 741-747

92 Zhao S et al Electrochemistry of Organic Conducting Salt Electrodes - a Unified Mechanistic

Description Journal of Physical Chemistry 1992 96(13) p 5641-5652

93 Lemuel BW Jr CC Liu and LN Niren Electrochemical measurements with glucose oxidase

immobilized in polyacrylamide gel Constant current voltametry 1971 p 629-639

94 Sato A et al Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Biochemical Journal

2001 357 p 893-898

95 Sigma-Aldrich Glucose Oxidase from Aspergillus niger Type II-S 15000-50000 unitsg solid (without

added oxygen) 2009 [cited

96 Cosnier S Biosensors based on electropolymerized films new trends Analytical and Bioanalytical

Chemistry 2003 377(3) p 507-520

97 Willner I et al Integrated Enzyme-Based Biofuel Cells-A Review Fuel Cells 2009 9(1) p 7-24

98 Heleg-Shabtai V et al Electrical wiring of glucose oxidase by reconstitution of FAD-modified monolayers

assembled onto Au-electrodes Journal of the American Chemical Society 1996 118(42) p 10321-10322

99 Georganopoulou DG et al Development and comparison of biosensors for in-vivo applications Faraday

Discussions 2000 p 291-303

100 Fujii S et al Properties of glucose sensors prepared by the electropolymerization of pyrrole containing a

gluconyl group Analytical sciences 1999 15(12) p 1175-1176

101 Hoshi T Deposition of electron mediators by electrochemical polymerization Electrochemistry 2003

71(5) p 346-347

102 Arai G M Masuda and I Yasumori Direct Electrical Response between Glucose-Oxidase and

Poly(Mercapto-P-Benzoquinone) Films Bulletin of the Chemical Society of Japan 1994 67(11) p

2962-2966

103 Arai G K Shoji and I Yasumori Electrochemical characteristics of glucose oxidase immobilized in

poly(quinone) redox polymers Journal of Electroanalytical Chemistry 2006 591(1) p 1-6

104 Leonida MD et al Polymeric FAD used as enzyme-friendly mediator in lactate detection Analytical and

Bioanalytical Chemistry 2003 376(6) p 832-837

105 Kissinger PT and WR Heineman Laboratory techniques in electroanalytical chemistry 1996 Marcel

Dekker Inc NY USA p 294-332

106 OHare D and H Zhao Characterisation and modeling of conducting composite electrodes The journal of

physical chemistry C 2008 112(25) p 9351-9357

107 Wang J et al Metal-Dispersed Carbon Paste Electrodes Analytical Chemistry 1992 64(11) p

1285-1288

108 Khurana MK CP Winlove and D OHare Detection mechanism of metallized carbon epoxy oxidase

enzyme based sensors Electroanalysis 2003 15(12) p 1023-1030

109 Sasaki M et al Fabrication and Electrical Characterization of

274

Tetrathiafulvalene-tetracyanoquinodimethane Molecular Wires Japanese Journal of Applied Physics 2003

42 p 2488-2491

110 Iizuka M et al Highly oriented TTF-TCNQ crystal growth using an electric-field induced evaporation

technique Molecular crystals and liquid crystals science and technology Section A Molecular crystals and

liquid crystals 2000 349 p 367-370

111 Saito I and K Yoshida Development of new features on organic conductive material TCI mail 2005

127

112 Cass AEG et al FERROCENE-MEDIATED ENZYME ELECTRODE FOR AMPEROMETRIC

DETERMINATION OF GLUCOSE Analytical Chemistry 1984 56(4) p 667-671

113 Kulys J V Simkeviciene and IJ Higgins Concerning the Toxicity of 2 Compounds Used as Mediators in

Biosensor Devices - 7788-Tetracyanoquinodimethane (Tcnq) and Tetrathiafulvalene (Ttf) Biosensors amp

Bioelectronics 1992 7(7) p 495-501

114 Malinauskas A et al Conductive organic complex salt TTF-TCNQ as a mediator for biosensors An

overview Electroanalysis 2007 19(24) p 2491-2498

115 Llopis X et al Integration of a glucose biosensor based on an epoxy-graphite-TTF center dot

TCNQ-GOD biocomposite into a FIA system Sensors and Actuators B-Chemical 2005 107(2) p 742-748

116 Bourgeois J D Thomas and C Bourdillon COVALENT LINKAGE OF GLUCOSE-OXIDASE ON

MODIFIED GLASSY-CARBON ELECTRODES - KINETIC PHENOMENA Journal of the American

Chemical Society 1980 102(12) p 4231-4235

117 Yun Y et al Nanotube electrodes and biosensors Nano Today 2007 2(6) p 30-37

118 Kim BC et al Preparation of biocatalytic nanofibres with high activity and stability via enzyme

aggregate coating on polymer nanofibres Nanotechnology 2005 16(7) p S382-S388

119 Kim J and JW Grate Single-Enzyme Nanoparticles Armored by a Nanometer-Scale OrganicInorganic

Network 2003 p 1219-1222

120 OFagain C Enzyme stabilization - recent experimental progress Enzyme and Microbial Technology

2003 33(2-3) p 137-149

121 Sillery E et al Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a

platinum catalyst Chemical communications 2002(8) p 866-867

122 Gouda M et al Enhancement of stability of immobilized glucose oxidase by modification of free thiols

generated by reducing disulfide bonds and using additives Biosensors amp bioelectronics 2004 19(6) p

621-625

123 Manning M STABILITY OF PROTEIN PHARMACEUTICALS Pharmaceutical Research 1989 6(11) p

903-918

124 Cao L Immobilised enzymes science or art Current opinion in chemical biology 2005 9(2) p 217-226

125 Daniel R The role of dynamics in enzyme activity Annual review of biophysics and biomolecular

structure 2003 32 p 69-92

275

126 Andersson MM JD Breccia and R Hatti-Kaul Stabilizing effect of chemical additives against oxidation

of lactate dehydrogenase Biotechnology and Applied Biochemistry 2000 32 p 145-153

127 Souza JM and R Radi Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Archives of Biochemistry and Biophysics 1998 360(2) p 187-194

128 Andersson MA and R Hatti-Kaul Protein stabilising effect of polyethyleneimine Journal of

Biotechnology 1999 72(1-2) p 21-31

129 Sigma-Aldrich Poly(ethylenimine) 2009 [cited Available from

httpwwwsigmaaldrichcomstructureimages27mfcd00084427gif

130 Andersson MM R Hatti-Kaul and W Brown Dynamic and static light scattering and fluorescence

studies of the interactions between lactate dehydrogenase and poly(ethyleneimine) Journal of Physical

Chemistry B 2000 104(15) p 3660-3667

131 Wang J L Chen and J Liu Critical comparison of metallized and mediator-based carbon paste glucose

biosensors Electroanalysis 1997 9(4) p 298-301

132 Cleland WW Dithiothreitol a New Protective Reagent for SH Groups Biochemistry 1964 3(4) p

480-482

133 Atanasov P E Wilkins and Q Yang An integrated needle-type biosensor for intravascular glucose and

lactate monitoring Electroanalysis 1998 10(11) p 752-757

134 Sigma-Aldrich DL-Dithiothreitol 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=43815|SIGMAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

135 Ghisalba O and A Brunella Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate

reductase as biocatalyst in the preparative synthesis of 2 -deoxyribonucleoside-5 -triphosphates Journal

of molecular catalysis B Enzymatic 2000 10(1-3) p 215-222

136 Gollhofer D EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM

ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Enzyme and Microbial Technology 1995 17(3) p 235-240

137 Sigma-Aldrich Arsenite standard solution - 005 M in H2O 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=70039|FLUKAampN5=SEARCH_CO

NCAT_PNO|BRAND_KEYampF=SPEC

138 Arakawa T MECHANISM OF POLY(ETHYLENE GLYCOL) INTERACTION WITH PROTEINS

Biochemistry 1985 24(24) p 6756-6762

139 Lee C Improvement of protein stability in protein microarrays Biotechnology Letters 2002 24(10) p

839-844

140 Lee L THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE GLYCOLS)

Biochemistry 1987 26(24) p 7813-7819

141 Costa SA et al Studies of stabilization of native catalase using additives Enzyme and Microbial

276

Technology 2002 30(3) p 387-391

142 Lee J and L Lee THERMAL-STABILITY OF PROTEINS IN THE PRESENCE OF POLY(ETHYLENE

GLYCOLS) Biochemistry 1987 26(24) p 7813-7819

143 Gekko K and SN Timasheff Mechanism of protein stabilization by glycerol preferential hydration in

glycerol-water mixtures Biochemistry 1981 20(16) p 4667-4676

144 Leandro P Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes

produced in a prokaryotic expression system Molecular genetics and metabolism 2001 73(2) p 173-178

145 Bartlett PN VQ Bradford and RG Whitaker Enzyme Electrode Studies of Glucose-Oxidase Modified

with a Redox Mediator Talanta 1991 38(1) p 57-63

146 Joseph S et al Drug metabolism biosensors electrochemical reactivities of cytochrome P450(cam)

immobilised in synthetic vesicular systems Journal of Pharmaceutical and Biomedical Analysis 1998

17(6-7) p 1101-1110

147 Cornish-Bowden A Fundamentals of enzyme kinetics 3rd ed 2004 London Portland Press Ltd

148 Wilkinson G STATISTICAL ESTIMATIONS IN ENZYME KINETICS The Biochemical journal 1961

80(2) p 324

149 Ikeda T and K Kano Fundamentals and practices of mediated bioelectrocatalysis Analytical sciences

2000 16(10) p 1013-1021

150 Duggleby RG and LP Daniel [3] Analysis of enzyme progress curves by nonlinear regression in

Methods in Enzymology 1995 Academic Press p 61-90

151 Eisenthal R and A Cornish-Bowden DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR

ESTIMATING ENZYME KINETIC-PARAMETERS The Biochemical journal 1974 139(3) p 715-720

152 Baici A Enzyme kinetics the velocity of reactions Biochem J 2006 p 1-3

153 Yamazaki A et al Improvement of precision in pharmacological studies using the non-linear regression

method

estimation of enzyme inhibition constants as an example Folia Pharmacologica Japonica 2008 131(3) p 195-203

154 Cornish-Bowden A Teaching Enzyme Kinetics and Mechanism in the 21st Century in 3rd Symposium

on ESCES 2008 Rudensheim Germany

155 Weisshaar DE and T Kuwana Considerations for Polishing Glassy-Carbon to a Scratch-Free Finish

Analytical Chemistry 1985 57(1) p 378-379

156 Stedman Stedmans medical dictionary 25th ed 2002 Lippincott Williams amp Wilkins

157 Tamura T Buffer preparation for biology Buffer preparation for biology ed 6 2008 Tokyo Yodosha

Ltd

158 Battino R and HL Clever The Solubility of Gases in Liquids 1966 p 395-463

159 Pandolfo A and A Hollenkamp Carbon properties and their role in supercapacitors Journal of power

sources 2006 157(1) p 11-27

160 Maynard AD Nanotechnology assessing the risks Nano Today 2006 1(2) p 22-33

277

161 Hindle A E Hall and N Martens AN ASSESSMENT OF MEDIATORS AS OXIDANTS FOR

GLUCOSE-OXIDASE IN THE PRESENCE OF OXYGEN Biosensors amp bioelectronics 1995 10(3-4) p

393-403

162 Mo J et al Comparison of oxygren-rich and mediator-based glucose-oxidase carbon-paste electrodes

Analytica Chimica Acta 2001 441(2) p 183-189

163 Turner A and G Palleschi AMPEROMETRIC TETRATHIAFULVALENE-MEDIATED LACTATE

ELECTRODE USING LACTATE OXIDASE ABSORBED ON CARBON FOIL Analytica Chimica Acta

1990 234(2) p 459-463

164 Mizutani F et al Glucose-Sensing Electrode Based on Carbon Paste Containing Ferrocene and

Polyethylene Glycol-Modified Enzyme Bulletin of the Chemical Society of Japan 1991 64(9) p

2849-2851

165 Zhang J PEM Fuel Cell Electrocatalysts and Catalyst Layers Fundamentals and Applications 2008

London Springer-Verlag

166 Kinoshita K Electrochemical oxygen technology 1992 John Willey amp Sons Inc

167 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of Power

Sources 2005 152 p 1-15

168 Sawyer DT Oxygen Chemistry International series of monographs on chemistry 1991 Oxford

University Press

169 Winter M Chemical Bonding Oxford Chemistry Primers 1997 New York Oxford University Press Inc

170 McLendon G and AE Martell Inorganic Oxygen Carriers as Models for Biological-Systems

Coordination Chemistry Reviews 1976 19(1) p 1-39

171 Toda T et al Enhancement of the electroreduction of oxygen on Pt alloys with Fe Ni and Co Journal of

the Electrochemical Society 1999 146(10) p 3750-3756

172 Norskov JK et al Origin of the overpotential for oxygen reduction at a fuel-cell cathode Journal of

Physical Chemistry B 2004 108(46) p 17886-17892

173 Fischer P and J Heitbaum MECHANISTIC ASPECTS OF CATHODIC OXYGEN REDUCTION Journal

of Electroanalytical Chemistry 1980 112(2) p 231-238

174 Okada T et al Oxygen reduction characteristics on pyrolytic graphite electrodes modified with

electro-polymerized salen compounds Chemistry Letters 1998(8) p 841-842

175 Sakurai T and K Kataoka Basic and applied features of multicopper oxidases CueO bilirubin oxidase

and laccase 2007 p 220-229

176 Sanders J Supramolecular catalysis in transition Chemistry a European journal 1998 4(8) p

1378-1383

177 Hart H LE Craine and DJ Hart Organic Chemistry A Short Course 10 ed 1998 Houghton Miffin

Company USA

178 Hayatsu H CELLULOSE BEARING COVALENTLY LINKED COPPER PHTHALOCYANINE

278

TRISULPHONATE AS AN ADSORBENT SELECTIVE FOR POLYCYCLIC COMPOUNDS AND ITS USE

IN STUDIES OF ENVIRONMENTAL MUTAGENS AND CARCINOGENS Journal of chromatography A

1992 597(1-2) p 37-56

179 Wang B Recent development of non-platinum catalysts for oxygen reduction reaction Journal of power

sources 2005 152(1) p 1-15

180 Kingsborough RP and TM Swager Electrocatalytic Conducting Polymers Oxygen Reduction by a

Polythiophene-Cobalt Salen Hybrid Chemistry of Materials 2000 12(4) p 872-874

181 Ortiz B and SM Park Electrochemical and spectroelectrochemical studies of cobalt salen and salophen

as oxygen reduction catalysts Bulletin of the Korean Chemical Society 2000 21(4) p 405-411

182 Mano N et al An oxygen cathode operating in a physiological solution Journal of the American

Chemical Society 2002 124(22) p 6480-6486

183 Shleev S et al Direct electron transfer between copper-containing proteins and electrodes Biosensors

and Bioelectronics 2005 20(12) p 2517-2554

184 Solomon EI et al Oxygen binding activation and reduction to water by copper proteins Angewandte

Chemie-International Edition 2001 40(24) p 4570-4590

185 Heath R F Armstrong and C Blanford A stable electrode for high-potential electrocatalytic O-2

reduction based on rational attachment of a blue copper oxidase to a graphite surface Chemical

communications 2007(17) p 1710-1712

186 Miura Y et al Direct Electrochemistry of CueO and Its Mutants at Residues to and Near Type I Cu for

Oxygen-Reducing Biocathode Fuel cells 2009 9(1) p 70-78

187 Tarasevich MR VA Bogdanovskaya and LN Kuznetsova Bioelectrocatalytic reduction of oxygen in

the presence of laccase adsorbed on carbon electrodes Russian Journal of Electrochemistry 2001 37(8) p

833-837

188 Tsujimura S et al Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase

at carbon electrodes Electrochemistry 2004 72(6) p 437-439

189 Fernandez JL et al Optimization of wired enzyme O-2-electroreduction catalyst compositions by

scanning electrochemical microscopy Angewandte Chemie-International Edition 2004 43(46) p

6355-6357

190 Soukharev V N Mano and A Heller A four-electron O-2-electroreduction biocatalyst superior to

platinum and a biofuel cell operating at 088 V Journal of the American Chemical Society 2004 126(27)

p 8368-8369

191 Miura Y et al Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on

a highly oriented pyrolytic graphite electrode Chemistry Letters 2007 36(1) p 132-133

192 Seinberg JM et al Spontaneous modification of glassy carbon surface with anthraquinone from the

solutions of its diazonium derivative An oxygen reduction study Journal of Electroanalytical Chemistry

2008 624(1-2) p 151-160

279

193 Manisankar P and A Gomathi Electrocatalytic reduction of dioxygen at the surface of carbon paste

electrodes modified with 910-anthraquinone derivatives and dyes Electroanalysis 2005 17(12) p

1051-1057

194 Jurmann G DJ Schiffrin and K Tammeveski The pH-dependence of oxygen reduction on

quinone-modified glassy carbon electrodes Electrochimica Acta 2007 53(2) p 390-399

195 Abiman P R Compton and G Wildgoose Characterising chemical functionality on carbon surfaces

Journal of materials chemistry 2009 19(28) p 4875-4886

196 Thorogood CA et al Differentiating between ortho- and para-Quinone Surface Groups on Graphite

Glassy Carbon and Carbon Nanotubes Using Organic and Inorganic Voltammetric and X-ray

Photoelectron Spectroscopy Labels Chemistry of Materials 2007 19(20) p 4964-4974

197 Sigma-Aldrich Fast red AL salt 2009 [cited Available from

httpwwwsigmaaldrichcomcatalogProductDetaildoN4=380660|SIALampN5=SEARCH_CONCAT_PN

O|BRAND_KEYampF=SPEC

198 Sljukic B C Banks and R Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

199 Granger M et al Polycrystalline diamond electrodes basic properties and applications as amperometric

detectors in flow injection analysis and liquid chromatography Analytica Chimica Acta 1999 397(1-3) p

145-161

200 Compton R J Foord and F Marken Electroanalysis at diamond-like and doped-diamond electrodes

Electroanalysis 2003 15(17) p 1349-1363

201 Arima S Do Science - Why diamond is so hard in Asahicom 2006

202 Dumitrescu I P Unwin and J Macpherson Electrochemistry at carbon nanotubes perspective and issues

Chemical communications 2009(45) p 6886-6901

203 Kinoshita K Carbon Electrochemical and Physicochemical Properties 1988 Wiley-Interscience

204 Kastening B et al Electronic properties and double layer of activated carbon Electrochimica Acta 1997

42(18) p 2789-2799

205 Swain G and R Ramesham THE ELECTROCHEMICAL ACTIVITY OF BORON-DOPED

POLYCRYSTALLINE DIAMOND THIN-FILM ELECTRODES Analytical chemistry 1993 65(4) p

345-351

206 Kaneto K et al Electrical conductivities of multi-wall carbon nano tubes Synthetic metals 1999

103(1-3) p 2543-2546

207 Meyer J et al Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes Nano

letters 2008 8(11) p 3582-3586

208 Harris P New perspectives on the structure of graphitic carbons Critical Reviews in Solid State and

Materials Sciences 2005 30(4) p 235-253

209 Akasaka T Chemical illustration - Carbon materials 2007 Hokkaido Japan

280

210 Iijima S M Yudasaka and F Nihey Carbon nanotube technology NEC Technical Journal 2007 2(1) p

52-56

211 Day T N Wilson and J Macpherson Electrochemical and conductivity measurements of single-wall

carbon nanotube network electrodes Journal of the American Chemical Society 2004 126(51) p

16724-16725

212 Fujita H A TEM image of the atomic structure of the diamond surface 2010 Britannica - The Online

Encyclopedia Osaka Japan

213 McCreery RL et al Control of reactivity at carbon electrode surfaces Colloids and Surfaces A

Physicochemical and Engineering Aspects 1994 93 p 211-219

214 Rice R C Allred and R McCreery FAST HETEROGENEOUS ELECTRON-TRANSFER RATES FOR

GLASSY-CARBON ELECTRODES WITHOUT POLISHING OR ACTIVATION PROCEDURES Journal of

electroanalytical chemistry and interfacial electrochemistry 1989 263(1) p 163-169

215 Swain Electrochemical formation of high surface area carbon fibers Analytical chemistry 1991 63(5) p

517-519

216 Nagaoka T and T Yoshino Surface-Properties of Electrochemically Pretreated Glassy-Carbon Analytical

Chemistry 1986 58(6) p 1037-1042

217 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 2002 58(9) p 1953-1955

218 Motta N and AR Guadalupe Activated Carbon-Paste Electrodes for Biosensors Analytical Chemistry

1994 66(4) p 566-571

219 Leung E et al A novel in vivo nitric oxide sensor Chemical Communications 1996(1) p 23-24

220 Nagaoka T et al Oxygen reduction at electrochemically treated glassy carbon electrodes Analytical

Chemistry 1986 58(9) p 1953-1955

221 Kissinger PT and WR Heineman Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy

Current Separations 1989 9 p 15-18

222 Wang J Analytical Electrochemistry 3 ed 2006 Wiley-VCH

223 BAS Measurement of electron transfer kinetics using rotating disk voltammetry Electrocehmistry

Principles methods and applications 1995 2(43)

224 Miomandre F et al Electrochemical behaviour of iron(III) salen and poly(iron-salen) Application to the

electrocatalytic reduction of hydrogen peroxide and oxygen Journal of electroanalytical chemistry and

interfacial electrochemistry 2001 516(1-2) p 66-72

225 Brighton-University Inorganic Chemistry Practical C170 Salen lab script 2009 Dept of Pharmacy and

Biomolecular Sciences Brighton University Brighton

226 Ranganathan S TC Kuo and RL McCreery Facile Preparation of Active Glassy Carbon Electrodes

with Activated Carbon and Organic Solvents 1999 p 3574-3580

227 Ohare D CP Winlove and KH Parker Electrochemical method for direct measurement of oxygen

281

concentration and diffusivity in the intervertebral disc electrochemical characterization and tissue-sensor

interactions J Biomed Eng 1991 13(4) p 304-312

228 Chen PH and RL McCreery Control of electron transfer kinetics at glassy carbon electrodes by specific

surface modification Analytical Chemistry 1996 68(22) p 3958-3965

229 Beilby AL TA Sasaki and HM Stern Electrochemical Pretreatment of Carbon Electrodes as a

Function of Potential Ph and Time Analytical Chemistry 1995 67(5) p 976-980

230 Dai H and K Shiu Voltammetric studies of electrochemical pretreatment of rotating-disc glassy carbon

electrodes in phosphate buffer Journal of electroanalytical chemistry and interfacial electrochemistry 1996

419(1) p 7-14

231 Musameh M N Lawrence and J Wang Electrochemical activation of carbon nanotubes

Electrochemistry communications 2005 7(1) p 14-18

232 Sljukic B CE Banks and RG Compton An overview of the electrochemical reduction of oxygen at

carbon-based modified electrodes Journal of the Iranian Chemical Society 2005 2(1) p 1-25

233 Tammeveski K et al Surface redox catalysis for O-2 reduction on quinone-modified glassy carbon

electrodes Journal of Electroanalytical Chemistry 2001 515(1-2) p 101-112

234 Yang HH and RL McCreery Elucidation of the mechanism of dioxygen reduction on metal-free carbon

electrodes Journal of the Electrochemical Society 2000 147(9) p 3420-3428

235 Regisser F et al Randomly oriented graphite electrode 1 Effect of electrochemical pretreatment on the

electrochemical behavior and chemical composition of the electrode Journal of electroanalytical chemistry

and interfacial electrochemistry 1996 415(1-2) p 47-54

236 Shleev S et al Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic

graphite electrode Electrochemistry Communications 2004 6(9) p 934-939

237 Mano N F Mao and A Heller A miniature biofuel cell operating in a physiological buffer Journal of the

American Chemical Society 2002 124(44) p 12962-12963