Development of Amperometric Biosensors with Carbon Nanotube Composite Materials

YAO Yanli

A thesis submitted in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Principal Supervisor: Dr. SHIU Kwok-Keung

Hong Kong Baptist University

May 2008

Abstract

Carbon nanotubes (CNTs) have attracted much attention since their discovery. CNTs

offer unique mechanical, chemical, physical and electronic properties. Many investigations

have demonstrated that CNTs possess good electrocatalytic activities. This thesis work

mainly focuses on the development of amperometric glucose biosensors utilizing carbon

nanotubes and CNT composite materials. The electrochemical behaviors of the CNT

composite materials have been investigated and the biosensor performance was explored.

CNT-modified electrodes with different types of CNT materials have been prepared.

Electrochemical reactions involving ferricyanide and hydrogen peroxide were examined at

the CNT modified electrodes. Glucose biosensors have been prepared with different types of

CNT materials and the amperometric behaviors for glucose determination have been

examined. Single walled carbon nanotube biosensor usually exhibited a wider dynamic range

and higher sensitivity for glucose determination.

Carbon nanotubes have been combined with different materials, including Prussian

blue inorganic nanoparticles, copper nanoparticles and gold colloid, to prepare different

CNT-based electrode substrates. Different polymeric materials were utilized as enzyme

entrapment agents. Chitosan, PDDA and poly(toluidine blue O) films have been employed.

Amperometric glucose biosensors can be prepared by using different CNT composite

materials and the biosensor performance was investigated.

A highly sensitive glucose biosensor has been fabricated by electrochemical

deposition of chitosan (CHIT) and glucose oxidase (GOD) on single-walled carbon nanotube

(SWCNT) modified glassy carbon electrode with copper nanoparticles. The presence of

SWCNT and copper nanoparticles offered improvement in electron transfer at the electrode

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surface. Electrodeposited chitosan film provided biocompatible matrix for the

immobilization of enzymes. The biosensor displayed linear response to glucose from 0.02 to

5 mM with a sensitivity of 22.4 mA M–1 cm–2 at an applied potential of +0.65 V (vs.

Ag|AgCl). On the other hand, Au colloid was prepared and assembled on the CNT surface by

a positively charged polymer PDDA. The direct electrochemistry of GOD on the

GC/CNT+Au/PDDA modified electrode was observed.

A mediator glucose biosensor has been constructed by immobilizing glucose oxidase

at electropolymerized poly(toluidine blue O) film on carbon nanotube modified glass carbon

electrode. Toluidine blue O moieties served as mediators for direct oxidation of glucose

oxidase and as polymeric network to maintain the biosensor activity. The detection potential

could be decreased to –0.1 V (vs. Ag|AgCl), where common interferences such as ascorbic

acid, uric acid and Acetaminophen were not oxidized to cause interferences. The

amperometric glucose biosensor offered a sensitivity of 5.09 mAM–1cm–2 and a wider linear

range of 1-18 mM.

Utilizing the unique properties of multi-walled carbon nanotubes, an amperometric

biosensor based on horseradish peroxidase (HRP) was fabricated. Experimental results

indicated that there was direct electron transfer between HRP enzyme and CNT-modified

glassy carbon electrode. Electrocatalytic reduction of hydrogen peroxide at the

GC/CNT/HRP/Nafion electrode was investigated. Additionally, a mediator-free bienzyme

glucose biosensor based on horseradish peroxidase and glucose oxidase was constructed. A

zero applied potential offered an improvement in the selectivity of the biosensor. Under the

optimized conditions, the bienzyme biosensor exhibited a high sensitivity for glucose

detection.

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Table of contents

Declaration…………………………………..……………………………………………….i

Abstract……………………………………………………………………………................ii

Acknowledgements..………………………………………………………………...............vi

Table of Contents………………………………………………………………….................v

List of Tables………………………………………………………………………………..xii

List of Figures……………………………………………………………………………....xiii

List of Symbols and Abbreviations…………………………………………………………xxi

Chapter 1 Introduction……………………………………………………………...................1

1.1 Carbon nanotubes…………………………………………………………..................1

1.1.1 Synthesis and purification of CNTs…………………………………………3

1.1.2 Characterization of CNTs……………………………………........................5

1.1.3 Properties of CNTs………………………………………………..................6

1.1.3.1 Electronic properties………………………………………………….....6

1.1.3.2 Thermal stability and mechanical properties……………………………6

1.1.3.3 Electrochemical properties…………………………………...................7

1.1.4 Defects of CNTs…………………………………………………..................9

1.1.5 Functionalization of carbon nanotubes……………………………………....9

1.1.6 Applications of CNT materials…………………………………..................10

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1.1.6.1 Energy storage…………………………………………………………10

1.1.6.2 Nanoprobes………………………………………………………........11

1.1.6.3 Biosensors and chemical sensors………………………………………12

1.1.6.4 Metal supports and templates…………………………….....................12

1.2 Biosensors……………… ………………………………………………..................13

1.2.1 Immobilization of enzymes on electrode…………………………………...14

1.2.2 Biosensor development…………………………………………..................16

1.2.2.1 First-generation biosensors……………………………….....................16

1.2.2.2 Second-generation biosensors................................................................17

1.2.2.3 Third-generation biosensors…………………………………………...18

1.2.3 Carbon nanotube based biosensors……………………………....................18

1.3 Objectives………………………………………………………………...................20

References ………………………………………………………………….. …………23

Chapter 2 Electron transfer properties of different carbon nanotube materials and their

applications in glucose biosensors………………………………………………...35

2.1 Introduction………………………………………………………………………....35

2.2 Experimental……………………………………………………………..................37

2.2.1 Reagents and instruments…………………………………………..................37

2.2.2 Preparation of CNT-modified electrodes and biosensors…………..................38

2.2.3 Amperometric determination of glucose……………………………………...38

2.3 Results and discussion……………………………………………………………....39

2.3.1 Electrochemical behaviors of CNT-modified electrodes…………..................39

2.3.2 Electron transfer properties of CNT-modified electrodes…………………….41

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2.3.3 Oxidation/Reduction of hydrogen peroxide at CNT-modified

electrode………………………………………………………………………44

2.3.4 Preparation of CNT-modified glucose biosensors…………………………....48

2.3.5 Electrocatalytic oxidation of glucose at CNT-modified biosensors.................49

2.4 Conclusions……………………………………………………………….................52

References…………………………………………………………………….................53

Chapter 3 Spontaneous deposition of prussian blue on multi-walled carbon nanotubes and

their application in amperometric biosensor…………………………………….55

3.1 Introduction………………………………………………………………………….55

3.2 Experimental…………………………………………………………………………57

3.2.1 Chemicals and instruments………………………………………………........57

3.2.2 Preparation of CNT-PB modified electrode……………………………….....58

3.2.3 Spectroscopic characterization of electrode substrates……………................59

3.2.4 Preparation of CNT-PB modified glucose biosensor………………………...59

3.2.5 Amperometric determination………………………………………………....60

3.3 Results and discussion……………………………………………………………....60

3.3.1 Deposition of PB on different electrode substrates…………………………..60

3.3.2 Electrochemical characterization of PB……………………………………...65

3.3.3 Ultraviolet-visible absorption characteristics of CNT/PB modified electrode.66

3.3.4 Reduction of hydrogen peroxide at different electrodes………………………67

3.3.5 Preparation of amperometric glucose biosensor based on the CNT/PB/GOD

modified electrode……………………………………………………………69

3.3.6 Effects of pH on the electro-oxidation of glucose………………………….....71

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3.3.7 Effects of detection potential…………………………………………….......72

3.3.8 Detection of glucose and the stability of biosensor……………….................74

3.4 Conclusions………………………………………………………………………....76

References………………………………………………………………………………77

Chapter 4 Preparation of SWCNT-nanocopper-chitosan composites and its application in

the development of amperometric biosensors…………………………………...79

4.1 Introduction……………………………………………………………....................79

4.2 Experimental……………………………………………………………...................81

4.2.1 Reagents and instruments…………………………………………...................81

4.2.2 Preparation of GC/SWCNT/Cu/CHIT-GOD biosensor……………………….83

4.3 Results and discussion…………………………………………………....................84

4.3.1 Electrodeposition of copper and chitosan-GOD composites on SWCNT-

modified electrodes…………………………………………………………….84

4.3.2 Electrocatalytic oxidation of hydrogen peroxide and glucose at the modified

electrodes…………………………………………………………….................87

4.3.3 Effects of copper deposition time on glucose oxidation………….....................94

4.3.4 Effects of the detection potential on the biosensor performance……………...95

4.3.5 Effects of interfering species………………………………………..................97

4.3.6 Amperometric detection of glucose ………………………………....................99

4.4 Conclusions……………………………………………………………....................101

References……………………………………………………………………………....102

Chapter 5 Direct electrochemistry of glucose oxidase at carbon nanotube-gold colloid

modified electrode with poly(diallyldimethylammonium chloride) coating…105

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5.1 Introduction……………………………………………………………...................105

5.2 Experimental section……………………………………………………………….108

5.2.1 Reagents and instruments………………………………………......................108

5.2.2 Synthesis of gold colloid nanoparticles………………………….....................109

5.2.3 Fabrication of glucose biosensors…………………………………..................109

5.3 Results and discussion……………………………………………………………...110

5.3.1 TEM and SEM characterization of gold nanoparticle composites…………....110

5.3.2 UV-visible spectral characteristics………………………………....................112

5.3.3 Electrochemical behaviors of the biosensors………………………………....113

5.3.4 Electrochemical detection of glucose at the GC/CNT/Au/PDDA-GOD modified

electrode………………………………………………………….....................119

5.3.5 Detection of glucose employing an electron mediator……………..................125

5.3.6 Effects of interferences……………………………………………..................128

5.3.7 Stability and reproducibility of the biosensor……………………………….....130

5.4 Conclusions……………………………………………………………....................131

References…………………………………………………………………………….....133

Chapter 6 Low potential detection of glucose at carbon nanotube modified glassy carbon

electrode with electropolymerized poly(toluidine blue O) film…………………137

6.1 Introduction……………………………………………………………...................137

6.2 Experimental……………………………………………………………………….139

6.2.1 Reagents and Instruments…………………………………………………….139

6.2.2 Preparation of the glucose biosensor………………………………………....140

6.2.3 Amperometric measurement of glucose………………………………………141

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6.3 Results and Discussion…………………………………………………………....141

6.3.1 Electropolymerization of toluidine blue O. ………………………………....141

6.3.2 AFM characterization of the modification procedure…………………….....144

6.3.3 Redox response of hydrogen peroxide at CNT-modified electrode with PTBO

coating………………………………………………………………………146

6.3.4 Electrochemical characterization of the glucose biosensor………..................147

6.3.5 Effects of PTBO film thickness on the biosensor performance……………. .152

6.3.6 Effects of detection potential on the biosensor performance………………...153

6.3.7 Amperometric determination of glucose…………………………………......156

6.4 Conclusions…………………………………………………………….................158

References…………………………………………………………………………......159

Chapter 7 A mediator free bienzyme amperometric biosensor based on horseradish

peroxidase and glucose oxidase immobilized on carbon nanotube modified

electrode………………………………………………………………….......162

7.1 Introduction…………………………………………………………………….....162

7.2 Experimental……………………………………………………………………...163

7.2.1 Materials and reagents………………………………………………….........163

7.2.2 Fabrication of biosensor…………………………………………………......164

7.2.3 Electrochemical measurements……………………………………………...164

7.3 Results and discussion.…………………………………………………...............165

7.3.1 Electrochemical behavior of HRP…………………………………………...165

7.3.2 Electrochemical behavior of GC/CNT/Nafion-HRP for hydrogen

peroxide…………………………………………………………………….168

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7.3.3 Characteristics of GC/CNT/Nafion-HRP, GOD bienzyme biosensor……......170

7.3.4 Effects of enzyme composition…………………………………….................173

7.3.5 The effect of the detection potential………………………………..................174

7.3.6 Amperometric detection of glucose……………………………………….......175

7.3.7 Influence of interferences……………………………………………………..178

7.4 Conclusions…………………………………………………………………………179

References………………………………………………………………………………180

Chapter 8 Summary and future work……………………………………………………....183

8.1 Summary…………………………………………………………………………...183

8.2 Future work…………………………………………………………….................187

8.2.1 Fabrication of miniature biodevices………………………………………….187

8.2.2 Preparation of Au colloid…………………………………………………….187

References………………………………………………………………………...........189

CURRICULUM VITAE…………………………………………………………………...190

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