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High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis. Hanzi Li Comprehensive oral presentation Advisor: Dr. Scott Calabrese Barton Department of Chemical Engineering and Materials Science Michigan state University. Nov , 2011. Introduction and background. - PowerPoint PPT Presentation
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1
High-rate cofactor regeneration at nanostructured interfaces for
bioelectrocatalysisHanzi Li
Comprehensive oral presentation
Advisor: Dr. Scott Calabrese Barton
Department of Chemical Engineering and Materials Science Michigan state University
Nov , 2011
2
Introduction and background
Cathode
Anode
Power supply
e-
Dual Chamber Catalysis
Why electrode: Cofactor electrochemical regeneration
3
Dehydrogenase-based electrochemical conversion
• Dihydroxyaceton(DHA): Sunless tanning cream; Precursor to pharmaceuticals• Mannitol: Natural sugar alcohol sweetener; Additive to food and pharmaceuticals
GlycerolGlyDH
FructoseMtDH
Mannitol DHA
NAD+ NADH
NADH NAD+
4
• Thermodynamically, NADH oxidation should be observed at low potential.
Cofactor electroregeneration
ProductEnzyme
NADHNAD+
Substrate
-0.49 V/Ag|AgCl at pH 6
2electrodeNADH NAD e H
CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.
NAD+
5
Cofactor electroregeneration
• Cyclic voltammograms in 0.5 mM NADH at glassy carbon electrode, 50 mV/s, 0.1 M PBS, pH 6
60
50
40
30
20
10
0
-10
Curre
nt de
nsity
(µA/
cm2 )
1.21.00.80.60.40.20.0-0.2
Potential (V) vs. Ag/AgClGlassy carbon
Electrode
NADH NAD+
E0’ = -0.49 V/Ag|AgCl at pH 6
• Direct NADH oxidation requires high overpotential; Reaction rate is low.
Typical planar electrode:Glassy carbon electrode ( 3 mm diameter)
CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.
6
High-performance cofactor regeneration
NADH NAD+
Electrode
• Achieve high-rate kinetics for NADH oxidation by electrode modification
• Analyze the conversions in NADH oxidation using modified electrode as working electrode
7
Bioelectrocatalysis involving cofactor regeneration
Substrat
e
NAD+
NADHAnode
catalyst red
catalyst ox
Product
Enzym
e• Evaluate bioelectrocatalysis
based on NADH electrocatalysis
• Model glycerol oxidation and fructose reduction coupled with cofactor regeneration
8
Electropolymeried azine electrodes modified with carbon nanotubes for NADH oxidation
May 13th, 2011
Part 1
9
Electrode modification
High-surface area material to
increase active site density
NADH NAD+
Glassy carbon
Electrode
NADH NAD+
Glassy carbon
Electrode
High surface area material
NADH NAD+
Glassy carbon
Electrode
Catalystox Catalystred
Electrocatalyst
to decrease activation energy
NADH NAD+
Glassy carbon
Electrode
Catalyst ox Catalystred
High-surface area material
1. Gorton, L.; Dominguez, E. J Biotechnol 2002, 82, 371.2. Zhao, X.; Lu, X.; Tze, W. T. Y.; Wang, P. Biosensors and Bioelectronics 2010, 25, 2343. 3. Villarrubia, C. W. N.; Rincon, R. A.; Atanassov, P.; Radhakrishnan, V.; Davis, V. ECS Meeting Abstracts 2010, 1001, 443.
10
• CNT-GC: CNT were coated on glassy carbon electrode surface (3 mm diameter RDE) by drop-casting 5 µl CNT ink on the surface of GC electrode and drying in vacuum. Glassy carbon
Electrode
Drop Casting CNT ink
Carboxylated CNT (Nanocyl)
http://www.nanocyl.com/
Modify electrode with CNT
SEM image of CNT on electrode surface
1. Li, H.; Wen, H.; Calabrese Barton, S. In Electroanalysis, 2011.2. Wen, H.; Nallathambi, V.; Chakraborty, D.; Calabrese Barton, S. Microchim. Acta, 1.
111. Barton, S. C.; Sun, Y.; Chandra, B.; White, S.; Hone, J. Electrochemical and Solid-State Letters 2007, 10, B96.
2. Kinoshita, K.; Carbon: Electrochemical and Physicochemical Properties; 1st ed.; Wiley-Interscience, 1988.
CNT-GC: High-surface area material
Capacitance (mF/cm2) in 1 M sulfuric acid
Active surface area / Geometric surface area(Assuming 25 µF/cm2)
1600
1400
1200
1000
800
600
400
200
00.80.60.40.20.0
CNT loading (mg/cm2)
40
30
20
10
01.00.80.60.40.20.0
CNT Loading (mg/cm2)
12
Toluidine Blue O
Methylene Green
Glassy carbon
Electrode
Poly(azine) ox
Poly(azine) red
CNT
Cyclic voltammograms of PTBO (Right: Top) and PMG (Right: Bottom) electropolymerization on 0.85 mg cm2- CNT-coated GC, 20 cycles, 50 mV/s, 0.4 mM TBO, 0.01 M borate buffer pH 9.1, 0.1M NaNO3, 30 ºC
Coat electrocatalyst: Electropolymerization
1. Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.
2. Zeng, J.; Wei, W.; Wu, L.; Liu, X.; Liu, K.; Li, Y. Journal of Electroanalytical Chemistry 2006, 595, 152.
13
NADH NAD+
Glassy carbon
Electrode
Poly(azine) ox
Poly(azine) red
CNT
, oxNADH ads Pi k
,NADH adsPox
ox redNADH P NAD P H
max
exp ( ) /1 exp ( ) /
NADH
S NADH
V U bCi iK C V U b
1. Kar, P.; Barton, S. C. ECS Meeting Abstracts 2010, 1001, 405.2. Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.
NADH electrocatalysis
14
NADH electrocatalysis
NADH concentration study of PTBO-CNT-GC (a) and PMG-CNT-GC (b) at 50 mV/Ag|AgCl; Polarization curves of PTBO-CNT-GC (c) and PMG-CNT-GC (d) in 0.5 mM NADH. 0.1 M phosphate buffer pH 6.0, 900 rpm, 30 ºC. Markers: Experimental data; Solid line: Fitting using mass-transport corrected model; Dash line: Simulation for mass-transport corrected curves.
a&c: PTBO ; b&d: PMG 1: Bare GC; 2: 0.21 mg/cm2 CNT-GC; 3: 0.85 mg/cm2 CNT-GC
max
exp ( ) /1 exp ( ) /
NADH
S NADH
V U bCi iK C V U b
Electrodes imax (mA/cm2)
PTBO-0.21 mg/cm2 CNT-
GC 4.2 ± 0.8
PTBO-0.85 mg/cm2 CNT-
GC 8.4 ± 1.9
PMG-0.21 mg/cm2 CNT-GC 15 ± 3.2
PMG-0.85 mg/cm2 CNT-GC 26 ± 4.1
NADH electrocatalysis
15
16
Part 2
Analysis of the bulk rate of cofactor electroregeneration
17
CNT modified carbon paper (Toray)
Active surface area / Geometric surface area(Assuming 25 µF/cm2)
Capacitance was obtained in 0.01 M borate buffer pH 9.1, 0.1 M NaNO3, 30 ºC
18
NADH Oxidation Using PMG-CNT-Toray
NADH oxidation was performed with initial NADH concentration 0.94 mM in 20 ml pH 6 phosphate buffer, constant applied potential 0.5 V/ Ag|AgCl, 1200 rpm magnetically stirred, 30 ºC.
Batch reactor to study the conversion
NADH
NAD
+
Carbon Paper CNT-
PMG
PMG-CNT acts as electrocatalyst for NADH oxidation
• CNT-Toray: CNT were coated on carbon paper surface (2.5×2.5 cm2) by air-brushing 2 mg ml-1 CNT ink on the surface and drying in vacuum.
• 1.2×0.8 cm2 (Exposed surface area 1.0×0.8 cm2 , CNT loading 0.9 ± 0.1 mg/cm2) CNT-Toray was used for further modification and working electrode.
Electrocatalysis:
Decay
ox redNADH Catalyst NAD Catalyst H
max exp ( ) /1 exp ( ) /
NADHelectro
S NADH
V U bj A Cj ArnFV nFV K C V U b
decayNADH NADHr kC
k=(1.0± 0.1 ) ×10-3 min-1
Conversions in NADH bulk oxidation
19
NADH consumption:
NADH electro decayr r r
NADH concentration profile can be simulated.
20
NADH concentration was measured using UV-Vis spectra during NADH bulk oxidation
1.0
0.8
0.6
0.4
0.2
0.0
NA
DH
con
cent
ratio
n / m
M
140120100806040200
Reaction time / min
1.0
0.8
0.6
0.4
0.2
0.0
NA
D+ concentration / m
M
NADH concentration Expected NAD+
a
0 , ,_ , t measured decayedNADH NADH t NADH tExpected NAD tC C C C
Conversions in NADH bulk oxidation
21
www.bioassaysys.com
Initially: LDH, Lactate, Diaphorase, MTTox
Lactate
NAD+
NADH
Pyruvate Diaphorase
MTTox
MTTred
LDH
Very fast Relatively slow
565, ([ ] [ ])tA NADH NAD in the solution
Enzyme cycling assay for detecting bioactive NAD+
• During electraocatalysis and after electrocatalysis, enzyme assay was employed for bulk solution
0 , ,_ , t measured decayedNADH NADH t NADH tExpected NAD tC C C C
,_ , ( _ ), measuredNADH tActive NAD t Active NAD NADH tC C C
22
Applied potential Yield of NAD+ at end (%)500 mV 88 ± 2.3150 mV 82 ± 3.6
1.0
0.8
0.6
0.4
0.2
0.0
Enz
ymat
ical
ly a
ctiv
e N
AD
+ / m
M
1.00.80.60.40.20.0
Expected NAD+ / mM
500 mV / Ag|AgCl 150 mV / Ag|AgCl
Bioactive NAD+
23
Part 3
Immobilization of enzymes and cofactors on poly(azine)-CNT modified electrodes to achieve high-performance bioelectrocatalysis
24
Aryl amine
N6 –linked-NAD+/NADH by Vieille Lab
Lindberg, M.; Larsson, P.-O.; Mosbach, K. European Journal of Biochemistry 1973, 40, 187
NAD+
25
Typical RDE Set-up 40 µl - Electrolyte Set-up
900 rpm, 30 °C, At least 10 ml solution, Purged Ar
5 µmoles NADH is needed for 0.5 mM solution
40 µl, Room temperature0.02 µmoles NADH is needed for 0.5 mM
solution
Electrochemical activity of N6-linked NADH
electrode
ω
electrolyte
electrode
electrolyte
26
Polarization curvesSteady-state data from chronoamperometry , pH 6 PBS, Standard NADH solution: 0.5 mM
• The lower activity may due too Limited mass transporto O2 present
Electrochemical activity of N6-linked NADH
• Can be fixed byo Compare RDE data in 0
rpm and in air o (Experiment in N2 or Ar)
8
6
4
2
0
Cur
rent
den
sity
/ µA
cm
-2
0.50.40.30.20.10.0-0.1
E / V | Ag/AgCl
RDE New set-up
27
Biosensor based on electronic interfaceReference electrode
Malate
NAD+
NADH
Anode
catalyst red
catalyst ox
Oxaloacetate
MDH
Kinetics:
electrodeNADH NAD
MDHMalate NAD Oxaloacetate NADH
Step 1 relectro
Step 2 renzyme
• Evaluate the whole process by monitoring the responding current
28
Biosensor towards malate concentration
PMG-CNT-GC, chronoamperometry , E=0.4 V vs. Ag|AgCl, 900 rpm, pH 6 PBS, 30 oC, MDH 0.83 µM, initially NAD+ 10 mM
100
80
60
40
20
0
Cur
rent
den
sity
/ µA
cm
-2
300250200150100500
Malate Concentration / mM
#1 Electrode #2 Electrode
• Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH (immobilization method by Worden Lab)
29
Back-up plan for cofactor/enzyme immobilization
Zhou, H.; Zhang, Z.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. Langmuir 2010, 26, 6028.
• Cofactor is non-covalently attached to CNT via π-π stacking interaction
CVs obtained at the MWCNT-modified Pt electrodes in 0.1 PBS buffet before (blue curve) and after (black curve) at the electrodes were first immersed into the aqueous solution of 10 mM NAD+ for 1 h and then thoroughly rinsed with distilled water. Scant rate: 50 mV/s. Inset: structure of NAD+ cofactor .
30
Model glycerol oxidation and fructose reduction coupled with cofactor regeneration
Part 4
31
Linear model
Mass balance involving kinetics and diffusion within film :
2
2Nh enzyme
NAD ND r
t x
2
2Gly enzyme
Glycerol GlyD r
t x
Steady-state within film :
2
2Nh enzyme
d ND r
dx
2
2Gly enzyme
d GlyD r
dx
Boundary conditions:
0
d Glydx
0d NAD
dx
max 0
0
[ ] exp(( ) / ) 1[ ] 1 exp(( ) / )
x
Nh x
d NAD j Nh V U bdx K Nh V U b nF
0x
x l [ ] [ ]Glycerol Glycerol
Glycero
l
NAD+
NADHAnode
catalyst red
catalyst ox
Dihydroxyacetone
(DHA)
GlyDH
X=0 X=l
32
Non-dimensionalization
Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.
2'
2 NADa enzyme
d N D rdw
2'
2 Glycerola enzymed G D rdw
0dGdw
0dNdw
0x
x w
1G
'
NADaa electro
dN D rdw
2[ ][ ]Glycerol
catfa
Glycerol
k E LD
D Glycerol
max
0
exp(( ) / ) 1[ ] 1 exp(( ) / )NAD
aaNAD
j L V U bDD NADH V U b nF
0
2[ ][ ]NAD
catfa
NAD
k E LD
D NADH
Damkohler numbers
33
Porous model
Boundary conditions:
Mass balance: 2
' '2 NAD NAD
a enzyme aa elelctrod N D r D rdw
2'
2 Glycerola enzymed G D rdw
0dGdw
0dNdw
0x
x l
1G
0dNdw
34
Parameters
Parametera Value SourceNADH concentration, [NADH]0 10 mM SetGlycerol bulk concentration, [Glycerol]∞ 1 M SetReactor volume, Vol 10 cm3 SetElectrode geometric surface area, cm2 1 cm2 SetEnzyme concentration, [Enzyme] 1 mM SetEquilibrium constant for enzyme reaction, Keq 4×10-4 Vieille labTurnover number of glycerol oxidation, kf 9.1 s-1 Vieille labTurnover number of DHA reduction, kr 9.1 s-1 Vieille labMichaelis-menten constant for NAD+, KmA 12 µM Vieille labMichaelis-menten constant for glycerol, KmB 440 mM Vieille labMichaelis-menten constant for NADH, KmQ 14 µM Vieille labMichaelis-menten constant for DHA, KmP 13 mM Vieille labDissociation constant of NAD+, Kia 1.09 mM 2
Dissociation constant of glycerol, Kib 1.5×104 mM 2,3
Dissociation constant for NADH, Kiq 25 µM 2
Dissociation constant for DHA, Kip 11 mM 3
Film thickness, L 10 µm SetDiffusion coefficient for NADH/NAD+, DNh/DN 3.3×10-8 cm2 s-1 1
Diffusion coefficient for glycerol/DHAb, DGly 4.0×10-6 cm2 s-1 1
a: parameter values regarding NADH electrocatalytic reaction have been shown in Project 1b: assumed to be the same as methanol
1. Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.2. Nishise, H.; Nagao, A.; Tani, Y.; Yamada, H. Agricultural and Biological Chemistry 1984, 48,
1603.3. Gartner, G.; Kopperschlager, G. J. Gen. Microbiol. 1984, 130, 3225.
35
enzymeR A r dl
Simulation results
Linear model: Porous model:
'enzymeR A r dw 4porous
linear
RR
DaNAD+ = 16
Daglycerol = 0.0013;DaaNAD
+ = 406;
36
• Fabricated poly(azine)-CNT-GC demonstrates high-rate for NADH electrocatalysis.
• NADH bulk oxidation shows 80% conversion of 1 mM NADH in 1 hr. Bioactive NAD+ was verified.
• Calibration curve for immobilized cofactor evaluation and dehydrogenase-based biosensor are proposed
• Nondimensional Damkohler numbers can provide useful approach to simulate, predict and evaluate performance of bioreactor.
Summary
37
Thank you.
38
Supplemental information
39
Biosensor towards malate concentration
80
60
40
20Cur
rent
den
sity
/ µA
cm
-2
2000150010005000
Time / s
PMG-CNT-GC, chronoamperometry , E=0.4 V vs. Ag|AgCl, 900 rpm, pH 6 PBS, 30 oC, MDH 0.83 µM, initially NAD+ 10 mM
100
80
60
40
20
0
Cur
rent
den
sity
/ µA
cm
-2
300250200150100500
Malate Concentration / mM
#1 Electrode #2 Electrode
• Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH (immobilization method by Worden Lab)
40
Cystein
41
• The decay of NADH in 0.1 M phosphate buffer pH 6.0, magnetic stirred speed 1200 rpm, 30 ºC. a. At varies NADH initial concentrations, NADH decay was monitored using UV-Vis spectra at 340 nm; b. The slopes in a. varying with NADH initial concentration.
42
• Collaborators
• Dr. Mark Worden
• Dr. Claire Vieille
• Justin Beauchamp
Acknowledgements
• The National Science Foundation
(Award CBET-0756703)
43
www.bioassaysys.com
Lactate
NAD+
NADH
Pyruvate Diaphorase
MTTox
MTTred
LDH
Initially: LDH, Lactate, Diaphorase, MTTox
Very fast
Principle of LDH-MTT Assay
1. When NAD+ presents in the sample, it is converted to NADH in LDH and lactate.
2. MTTox uses NADH to oxidize into MTTred. The NADH is thus converted back to NAD+.
3. The enzyme cycle starts over.
Relatively slow
Once the cycle starts, NADH concentration in the assay is not changing = [NAD]+[NADH] in the sample
44
www.bioassaysys.com
Lactate
NAD+
NADH
Pyruvate Diaphorase
MTTox
MTTred
LDH
Initially: LDH, Lactate, Diaphorase, MTTox
565,0 15min 565, 15min 565, 0min 15min 0( ) ([ ] [ ] )t t red t red tA A A MTT MTT
Kinetics assay using LDH-MTT Assay Kit
565, 0 15min [ ]tA NADH
565 [ ]redA MTT
15min 0([ ] [ ] ) 15min [ ] 15minred t red tMTT MTT R k NADH • Linear kinetics within 15 mins
565, 0 15min ([ ] [ ])tA NADH NAD in the sample
0.8
0.6
0.4
0.2
0.0
A56
5
1086420
Pyridine necleotide/ µM
BioAssay using NAD only NADH only NAD NADH:NAD=1:1
45
High-surface area electrodes for NADH electrocatalysis
Modified electrodes
Data source Approach Applied potential E
vs. RHE (mV)
imax
(µA/cm2)
imax
(µA)
(Villarrubia, Rincon et al.
2010)
PMG -“Bucky
paper”
913 ------------ 600
(Yang and Liu 2009) PBCB-SWCNT-GC 685 ------------ 1.2
(Doaga, McCormac et al.
2009)
p-DAB-MB-SWCNT-
GC
663 8.49 0.6
(Zhu, Zhai et al. 2007) Meldola blue-CNT-
GC
505 1.6 0.4
(Huang, Jiang et al.
2007)
Thionine-CNT-
Nafion/GC
537 28.3 2
(Zeng, Wei et al. 2006) TBO-MWNT-GC 655 ------------ 45
Why Mannitol?
• Mannitol is a natural sugar alcohol sweetener.• Mannitol is especially useful as an additive to food and
pharmaceuticals– It has low caloric and cariogenic properties– It is not metabolized by the body– It has a cool sweet taste
• Currently mannitol is produced by hydrogenating a 1:1 fructose/glucose syrup– Very high temperatures, pressure and a Raney nickel catalyst– Needs highly purified substrates– Energy intensive– Costly purification– Low yield (15%)
• Enzymatic catalysis reducing fructose to mannitol– Potential applications to other dehydrogenases
Overall Objective
• Glucose fructose using a thermostable glucose isomerase – Triple mutant of Thermotoga neapolitana xylose isomerase (TNXI 1F1)
• Optimized for high activity at 60°C, and high activity at pH 6.0 while maintaining glucose activity
• Fructose mannitol• NADH regeneration from cathodic current pulls reaction towards
mannitol production
48
Adenine
Nicotinamide
Dinucleotide
49
Literature review about NADH electrocatalytic oxidation: The reported steady-state current densities for NADH oxidation were far less than 1 mA cm-2 under low overpotentialData source Approach Applied
potential E vs. RHE (mV)
Vmax(uA/cm2)
Vmax(uA)
(Palmore, Bertschy et al. 1998)
Free diffusing DI+ BV-GC ---------- --------- -----------
(Dilgin, Gorton et al. 2007) PTBO-GC: photoelectrocatalytic 755 25.4 5(Radoi, Compagnone et al.
2007)Bulk screen-printed electrodes
modified with Prussian blue (PB)445 3.54 0.25
(Zhang, Smith et al. 2004) MWCNT-Chitosan-GC 1037 85 6(Liu, Zhang et al. 2010) Magnetic chitosan microspheres -
Polythionine-GC705 141 10
(Zhao, Lu et al. 2010) Single-carbon fiber microelectrode with CNT
1331 ------------- 1.7
(Villarrubia, Rincon et al. 2010)
PMG SWCNTs-based “Bucky paper”
913 (pH 7 solution)
------------ 600
(Yang and Liu 2009) PBCB-SWCNT-GC 685 ------------ 1.2(Zeng, Wei et al. 2006) TBO/MWNTs adduct-GC 655 ------------ 45
(Doaga, McCormac et al. 2009)
p-DAB-MB/SWCNTs/GC 663 8.49 0.6
(Zhu, Zhai et al. 2007) Meldola’s blue adsorbed-CNT-GC 505 1.6 0.4(Huang, Jiang et al. 2007) Thionie incorporated by
DMF/CNTs-Nafion/GC537 28.3 2
(Kim, Kim et al. 2010) Iron oxide/carbon black-GC 643 16 4
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Curre
nt de
nsity
(mA/
cm2 )
0.40.30.20.10.0-0.1-0.2
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
PTBO-0.85 mg/cm2 CNT-GC PTBO-Bare GC
Proposed reason: Impact of Mass-transport
50
For the reduction of U in polarization curves
Take one PTBO-0.85 mg/cm2 CNT-GC and PTBO-GC as an example:
Polarization curve: 0.5 mM NADH , 900 rpm, pH 6 PBS, 30 oC
Controlled by electron-transfer rate (controlled by applied potential)
Controlled by mass-transport(not controlled by applied potential)
Mixed Control (By both applied potential and mass-
transport)
51
• High-surface area of CNT-GC: Good utilization of CNT
115 m2/g (capacitive surface area) vs. 80-140 m2/g (BET)• Carboxylated multiwall carbon nanotubes (CNT) instead of untreated CNT were used:
hydrophilic property of COOH-CNT make it possible to utilize the good properties of CNT for electrochemical experiments
• Dimethylformamid (DMF) is used as solvent to form CNT-ink:• Organic solvent, disperse CNT well; Can evaporate; Miscible in water
CNT-GC
Wen, H.; Nallathambi, V.; Chakraborty, D.; Barton, S. C. ECS Meeting Abstracts 2010, 1002, 366.
52
Characterization of PTBO and PMG films
• CV in pH 6 0.1 M PBS, 50 mV/s 30 oC
-4
-2
0
2
4
Curre
nt de
nsity
(mA/
cm2 )
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6E (V) vs. Ag/AgCl
PTBO/bare GC PTBO/ 0.21 mg/cm2 CNT-GC PTBO/ 0.85 mg/cm2 CNT-GC
-4
-2
0
2
4
Curre
nt de
nsity
(mA/
cm2 )
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6E (V) vs. Ag/AgCl
PMG/bare GC PMG/ 0.21 mg/cm2 CNT-GC PMG/ 0.85 mg/cm2 CNT-GC
Glassy carbon
Electrode
Poly(azine) ox
Poly(azine) red
CNT
53
• DMF: Dimethylformamid (CH3)2NC(O)H
54
Process of catalytic reaction of NADH
Or 2NADH NAD H e
1. Qi-Jin, C. and D. Shao-Jun, Journal of Molecular Catalysis A: Chemical, 1996. 105(3): p. 193-201.2. Cooney, M.J., et al., Energy & Environmental
Science, 2008. 1(3): p. 320-337.
The catalysis efficiency varies with polymers. Even though the mechanisms are not well developed, it is reported that the differences of azine chemical structures affect the electrocatalytic activity toward NADH oxidation. For instance, the additional electron acceptor groups in the aromatic ring always lead to higher electrocatalytic activity, while the additional proton donor groups cause lower electrocatalytic activity. [13, 36] Methylene green has an additional –NO2 group and toluidine blue has an additional -CH3 group. Thus PMG-modified electrodes tend to show higher activity especially at high positive potential region and high NADH concentration.
55
CNT-modified GC electrode: Capacitance measurement
• Carbon nanotube is coated on glassy carbon electrode surface (3 mm diameter RDE) by drop-
casting:
Each CNT layer: 5ug 1mg/ml CNT-DMF suspension Glassy
carbon
Electrod
e
Drop Casting CNT
Capacitance data were obtained by cyclic voltammetry in the 0.3 to 0.4 V vs. Ag/AgCl at 0.01 M borate buffer pH 9.1, 0.1 M NaNO3, 30 oC
Example of capacitance measurement: 0.50 mg/cm2 CNT loaded on GC
1.0
0.5
0.0
-0.5
-1.0
Curre
nt (m
A/cm
2 )
0.400.380.360.340.320.30E (V) vs. Ag/AgCl
30 mV/s 50 mV/s 60 mV/s 80 mV/s 100 mV/s
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Char
ging C
urre
nt (m
A/cm
2 )
120100806040200Scan rate (mV/s)
Charging current Linear fit: slope is 12.5 mF/cm2
56
Proposed structure of Poly (MB)
S
N
N NCH3
CH3
CH3
H3C
NH3C
S
N
NCH3
CH3
H N
S NCH3
CH3N
H3C
Karyakin, A.A., et al., 1999. 11(8): p. 553-557.
S
N
N NCH3
CH3
CH3
H3C
NO2
Methylene Green
57
Imax after MT correction
25
20
15
10
5
0
i max (
mA/cm
2 )
1.00.80.60.40.20.0Loading of CNT (mg/cm2)
PMG-CNT-GC PTBO-CNT-GC
58Solid lines : kinetic controlDotted lines : partially MT
limited
Effect of Mass transport
Mass balance:( ) S
dS S
k Ck C Cs
K C
0 ( )x dCi nFD nFk C Csx
Electrochemical
experiments:
Obtain i’max and K’s for pure kinetic control
MT Correction
59
Bioreactor based on electronic interface
Glycerol
NAD+
NADHAnode
catalyst red
catalyst ox
Dihydroxyaceto
ne(DHA)
GlyDH
Power supply
Reference
electrode
electrodeNADH NAD GlyDHGlycerol NAD DHA NADH H Step 1
r1
Step 2 r2
Kinetics:
1[ ][ ]electro
Nh
k SS NADHr inF K NADH nFV
[ ][ ][ ][ ] [ ][ ] [ ]
catenzyme
GlycerolNAD
k Enzyme Glycerol NADrK Glycerol Glycerol NAD K NAD
100
80
60
40
20
0
Glyc
erol
conc
entra
tion (
mM)
1086420Time (hr)
60
Concentration profile for substrate conversion
Initial values: t=0, [NADH] = [NADH]0 ; [NAD+]=0; [Glycerol] =
[Glycerol]0
[ ] [ ]electro enzyme
d NAD d NADH r rdt dt
[ ]enzyme
d Glycerol rdt
[NADH]0=20 mM; [Glycerol]0 =100
mM;V = 20 cm3;S = 1 cm2;E = 10 µM;
KNADH =7.0 mM;kcat = 9.1 s-1;
Kglycerol = 11 mMKNAD
+ = 25 µM;
3.02 hrs
• Key parameters:1. Sk1/nFV ( µM/s ) 2. [Enzyme] (µM or mM)
For the whole batch reactor:
61
Fabrication of PMG-CNT-Toray
1. CNT-Toray: Spray-coat (air-brushing) CNT ink on Toray paper surface and dry in vacuum.
Toray paper: 3.5 cm × 3.5 cm; 100 µm thicknessCNT ink: 20 mg CNT dispersed in 10 ml DMF
Exposed surface area of Toray to CNT ink: 2.5 cm × 2.5 cm
Resulted loading: 1.1 mg ± 0.11 CNT/ cm2
Bare Toray: 0.16 m2/cm3
For 0.9 cm2 bare Toray, active surface area: 14.4 cm2 Capacitance: 608 uF/cm2
62
63
How Sk1/nFV or/and [Enzyme] impact Time constant?
Sk1/nFV ( µM/s )
Sk1/V ( A/cm3 ) E (µM)
10 - 1000 0.002 – 0.2 1-100
Zoom in5
4
3
2
1
0
Time (
hr)
6 7 8 9100
2 3 4 5 6 7 8 91000
Sk1/nFV (uM/s)
[E] = 0.1 uM [E] = 1 uM [E] = 10 uM [E] = 100 uM
30
25
20
15
10
5
0
Time (
hr)
102 3 4 5 6 7 8 9
1002 3 4 5 6 7 8 9
1000
Sk1/nFV (uM/s)
[E] = 0.1 uM [E] = 1 uM [E] = 10 uM [E] = 100 uM
64
Nondimensionalization
1 2N F Nh
dx dy x yfrho K rho Kd d x y f yf
2F Nh
df yfM rho Md y f yf
DEQs
Boundary conditions:
t=0, x=1, y=0, f=1
Important parameters
:
Time constants for step 1,
step 2
and their ratio
0
[ ][ ]NADxNAD
0
[ ][ ]NADHyNAD
0
[ ][ ]FructosefFructose
t
Parameters:
Variables:
1
[ ]cat
Enzyme VK k nFSk
01
2 0
[ ][ ]
NADM KFructose
01
1
[ ]V NAD nFS k
02
[ ][ ]cat
Fructosek Enzyme
Equilibriu
m constant, representing key operation condition
s
0[ ]NAD
N
KNAD
0[ ]NADH
NhKNAD
0[ ]Fructose
FK
Fructose
65
Current work: Enzyme kinetics of MtDH
p
p
k
kE NADH F E M NAD
• Ordered bi bi
kineticsNADH Fructose Mannitol NAD
k1 k-1 k2 k-2 k3 k-3 k4 k-4
[ ][ ]([ ][ ] )
[ ] [ ][ ] [ ] [ ][ ]
[ ][ ] [ ][ ] [ ][ ][ ][ ][ ] [ ][ ][ ]
f req
f mQ f mPr ia mB r mB r mA r
eq eq
f mQ f fr mA r
eq ia eq iq ip ib eq
P QV V A BK
v V K P V K QV K K V K A V K B V A B
K K
V K A P V P Q V B P QV K B Q V A B PK K K K K K K
A: NAD
HB:
FructoseP:
MannitolQ:
NAD
1. Segel, Irwin H. (1993). New York: Wiley
66
Enzyme kinetics of MtDH
• Definition of parameters1
1ia
kKk
1. Segel, Irwin H. (1993). New York: Wiley 2. Seung Hoon, S., N. Ahluwalia, et al. (2008). "Applied Microbiology and Biotechnology: 81 (3) 485-495 81(3): 485-495.
A: NAD
HB:
FructoseP:
MannitolQ:
NAD
4
4iq
kKk
• The values of 10 parameters I extracted based on experimental data
KmA KmB KmP KmQ Kia Kiq Vf Vr Keq
[P]/Kip=0Kib
0.0371 39.89 8.06 0.0181 0.033 0.222 19.38 0.445 59.2 4.7e4Kip1. Vf and Vr: U/mg; All Km’s and Ki’s: mM; Keq: dimensionless 2. For 60 oC,
pH 6.1
1 2
4 1 2 1 1 2( )P
mQP P P
k k kKk k k k k k k k k
1 2 3 2 3
3 1 2 1 1 2
( )( )
P PmP
P P P
k k k k k k kKk k k k k k k k k
4 3
1 4 3 4 4 3( )P
mAP P P
k k kKk k k k k k k k k
4 2 3 3 2
2 4 3 4 4 3
( )( )
P PmB
P P P
k k k k k k kKk k k k k k k k k
2f iq mP f ip mQ
eqr ia mB r ib mA
V K K V K KK
V K K V K K