PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES

Creating Stable and Versatile Monolayer Systems on Carbon Substrates for Sensors and other

Applications

by

Guozhen Liu

B.Sc., M.Sc.

A thesis presented in fulfilment

of the requirements for the degree of

Doctor of Philosophy

School of Chemistry

The University of New South Wales

Sydney 2052, Australia

August 2006

i

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Liu

First name: Guozhen Other names:

Abbreviation for degree as given in the University Calendar: PhD

School: Chemistry Faculty: Science

Title: Creating Stable and Versatile Monolayer Systems on Carbon Substrates for Sensors and other Applications

Abstract 350 words maximum: (PLEASE TYPE)

The aim of this project is to develop strategies for fabrication of carbon electrode surfaces with a view to creating stable and versatile monolayer

systems for sensing and other applications. Glassy carbon (GC) electrodes have been successfully modified with versatile monolayers via the

electrochemical reduction of aryl diazonium salts. The surfaces modified with diazonium salt monolayers were properly characterised by

electrochemistry, AFM and XPS. The rates of heterogeneous electron transfer through organic monolayers on GC, Pyrolysed Photoresist Films

(PPF) and gold surfaces have been studied using ferrocene as the redox probe.

The diazonium salt monolayers created on GC surfaces demonstrated very stable ability and can serve as a good alternative to alkanethiol self-

assembled monolayers on gold electrodes for sensing purposes. Tripeptide Gly-Gly-His modified GC electrodes have been successfully used as

the electrochemical copper sensors and were found to be extremely stable. PPF has proved to be a good alternative to the GC electrode for the

commercialisation of the fabricated electrochemical sensors.

The most important and difficult task of this project is to fabricate glucose biosensors and immunosensors on carbon electrodes. The rigid and

conjugated molecular wires (MW) as the efficient conduit for electron transfer, and a molecule with poly(ethylene glycol) chains (PEG) as an

insulator for reducing the non-specific protein adsorption were successfully synthesised and introduced in the sensing systems. MW modified on

GC electrodes can be used to explore the deeply buried active site of glucose oxidase to achieve direct electron transfer of GOx from the active

centre FAD through the MW to the underlying GC electrode, and to fabricate third generation biosensors.

The interface comprising mixed monolayers of MW and PEG has the ability to facilitate efficient electron transfer. A label-free immunosensor

system has been successfully developed for electrochemical detection of biomolecular pairs such as biotin/antibiotin with low detection limitation

based on mixed monolayers of MW and PEG modified GC electrode surfaces. In addition, a displacement assay has shown that the free biotin

can compete with the attached biotin for binding antibiotin. SWNTs can be used as an alternative to MW to fabricate another label-free

immunosensor system due to the high efficiency of electron transfer that SWNTs have demonstrated.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future work (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral these only).

Signature: Witness: Date: .

The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing to the Registrar. Requests for a longer period of restriction may be considered in exceptional circumstances if accompanied by a letter of support from the Supervisor or Head of School. Such requests must be submitted with the thesis/dissertation.

FOR OFFICE USE ONLY Date of Completion of Requirements for the Award:

Registrar and Deputy Principal

THIS SHEET IS TO BE GLUED TO THE SIDE FRONT COVER OF THE THESIS

Certificate of Originality

‘I hereby declare that this submission is my own work and that, to the best of my

knowledge, it contains no material preciously published or written by another person, or

substantial proportions of material which have been accepted for the award of any other

degree or diploma at the University of New South Wales or any other educational

institution, except where due acknowledgement is made in the thesis. Any contribution

made to the research by others, with whom I have worked at the University of New

South Wales or elsewhere, is explicitly acknowledged in the thesis. I also declare that

the intellectual content of this thesis is the product of my own work, except to the extent

that assistance from others in the project’s design and conception or in style,

presentation and linguistic expression is acknowledged.’

Signed:………………………………….

Guozhen Liu

August 2006

ii

Copyright and DAI Statement

‘I hereby grant the University of New South Wales or its agents the right to archive and

to make available my thesis or dissertation in whole or part in the University libraries in

all forms of media, now or here after known, subject to the provisions of the Copyright

Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to

use in future works (such as articles or books) all or part of this thesis or dissertation.

I authorise University Microfilms to use the 350 words abstract of my thesis in

Dissertation Abstracts International.

I have either used no substantial portions of copyright material in my thesis or I have

obtained permission to use copy material; where permission has not been granted I have

applied/will apply for a partial restriction of the digital copy of my thesis or

dissertation.’

Signed:………………………………….

Guozhen Liu

August 2006

iii

Acknowledgements

It would have been definitely impossible to complete my PhD project and thesis work

without the help, support and encouragement from other people including friends and

families.

First of all, I sincerely thank my supervisor Assoc/Prof. J. Justin Gooding for his

consistent guidance, patience, and support throughout the past three years. I was always

impressed by his down-to-earth personality and earnest attitude towards scientific

research, and more importantly his enthusiastic approach and erudite knowledge have

enlightened me in many aspects in my research. He has also made an excellent model of

being a good person in the society for me. I feel very grateful to have known him and

worked with him, and I believe what I have learn from him will greatly benefit to my

future career and even my whole life.

Thanks to Prof. D. Brynn Hibbert for lots of inspiring advices during this project.

Thanks to Dr. Alison Downard at University of Canterbury, New Zealand for help with

making Pyrolysed Photoresist Films (PPF) and her kind hospitality during my visiting

her lab.

Thanks to Dr. Jingquan Liu for help with the organic synthesis and invaluable

comments and support throughout this project.

Thanks also to Assoc/Prof. Barbara Messerle, Dr. Jason Harper, Dr. Jim Hook, Dr.

Michael Jones, and Paul Eggers for discuss with organic synthesis.

Thanks to Dr. Till Böcking for help with the XPS data and all the suggestions.

I’d like to thank Dr. Edith Chow, Dr. Jingquan Liu, and Dr. Till Böcking for their

careful proof reading of this thesis.

iv

Thanks all the group members Alison Chou, Callie Reynolds Massey-Reed, Eillen Peh,

Kate Odenthal, Kris Kilian, Paul Eggers, Rongmei Liu, Dr. Till Böcking, Dr. Wenrong

Yang and former group members Dr. Edith Chow, Dr. Freya Mearns, Dr. Florian

Bender, Dr. Elicia Wong, Dr. Jingquan Liu, Dr. Jianfeng Li and Dr. Min Zhao for their

help and kindness.

Thanks to all of the friends I have met in both countries, Australia and China. I will

refrain from mentioning any names, for fear of leaving someone out. Nevertheless, you

are all greatly cherished and I am indebted to each one of you.

Thanks to the Endeavour International Postgraduate Research Scholarship (EIPRS) and

Australia Research Council (ARC) for financial support to undertake this project.

Finally I would like to thank my dear husband Kaiji Wang for his deep love, constant

support and patience. Without his accompany with me in Sydney, I could not have

carried out my PhD study. And I would like to thank my parents and all my families for

their constant love, support and encouragement in China.

v

Abstract

The aim of this project is to develop strategies for fabrication of carbon electrode

surfaces with a view to creating stable and versatile monolayer systems for sensing and

other applications. Glassy carbon (GC) electrodes have been successfully modified with

versatile monolayers via the electrochemical reduction of aryl diazonium salts. The

surfaces modified with diazonium salt monolayers were properly characterised by

electrochemistry, AFM and XPS. The rates of heterogeneous electron transfer through

organic monolayers on GC, Pyrolysed Photoresist Films (PPF) and gold surfaces have

been studied using ferrocene as the redox probe.

The diazonium salt monolayers created on GC surfaces demonstrated very stable ability

and can serve as a good alternative to alkanethiol self-assembled monolayers on gold

electrodes for sensing purposes. Tripeptide Gly-Gly-His modified GC electrodes have

been successfully used as the electrochemical copper sensors and were found to be

extremely stable. PPF has proved to be a good alternative to the GC electrode for the

commercialisation of the fabricated electrochemical sensors.

The most important and difficult task of this project is to fabricate glucose biosensors

and immunosensors on carbon electrodes. The rigid and conjugated molecular wires

(MW) as the efficient conduit for electron transfer, and a molecule with poly(ethylene

glycol) chains (PEG) as an insulator for reducing the non-specific protein adsorption

were successfully synthesised and introduced in the sensing systems. MW modified on

GC electrodes can be used to explore the deeply buried active site of glucose oxidase to

vi

achieve direct electron transfer of GOx from the active centre FAD through the MW to

the underlying GC electrode, and to fabricate third generation biosensors.

The interface comprising mixed monolayers of MW and PEG has the ability to facilitate

efficient electron transfer. A label-free immunosensor system has been successfully

developed for electrochemical detection of biomolecular pairs such as biotin and

antibiotin with low detection limitation based on mixed monolayers of MW and PEG

modified GC electrode surfaces. In addition, a displacement assay has shown that the

free biotin can compete with the attached biotin for binding antibiotin. SWNTs can be

used as an alternative to MW to fabricate another label-free immunosensor system due

to the high efficiency of electron transfer that SWNTs have demonstrated.

vii

Publications (during PhD)

1. The Modification of Glassy Carbon and Gold Electrodes with Aryl Diazonium Salt:

The Impact of the Electrode Materials on the Rate of Heterogeneous Electron

Transfer, Guozhen Liu, Jingquan Liu, Till Böcking, Paul. K. Eggers and J. Justin

Gooding, Chemical Physics, 2005, 319, 136-146.

2. Study of Factors Affecting the Performance of Voltammetric Copper Sensors based

on Gly-Gly-His Modified Glassy Carbon and Gold Electrodes, Guozhen Liu,

Quynh Thu Nguyen, Edith Chow, Till Böcking, D. Brynn Hibbert and J. Justin

Gooding, Electroanalysis, 2006, 18(12), 1141-1151.

3. An Electrochemical Interface Comprising Molecular Wires and Poly(ethylene

glycol) Spacer Units Self-Assembled on Carbon Electrodes for Studies of Protein

Electrochemistry, Guozhen Liu and J. Justin Gooding, Langmuir, 2006, 22(17),

7421-7430.

4. Diazonium salts: Stable Monolayers on Gold Electrodes for Sensing Applications,

Guozhen Liu, Till Böcking, and J. Justin Gooding, accepted by Journal of

Electroanalytical Chemistry.

5. Exploration of Deeply Buried Active Sites of Glucose Oxidase Using Molecular

Wires Immobilized on Carbon Electrodes, Guozhen Liu and J. Justin Gooding, to

be submitted to Chemical Communications.

6. Electrochemical Transduction of Biomolecular Recognition on Molecular Wire

Modified Carbon Surfaces, Guozhen Liu and J. Justin Gooding, in preparation.

7. A Label-Free Immunosensor on GC Electrodes, Guozhen Liu and J. Justin

Gooding, submitted for the patent.

viii

Table of Contents

Title Page……………………………………………………………………….……...(i)

Certificate of Originality..…………………………………………………….…….....(ii)

Copyright and DAI Statement………………………………………………….…..…(iii)

Acknowledgements……………………………………………….…..……………….(iv)

Abstract..……………………………………………………………………….…..…(vi)

Publications………………………………………………………………….…...….(viii)

Table of Contents………………………………………………………………...……(ix)

List of Abbreviations..……………………………………………………….…..…..(xxi)

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

1.1 Introduction.........................................................................................................2

1.2 Biosensors ............................................................................................................4

1.2.1 The General Principle of Biosensors…………………………………….4

1.2.2 Transducers………………………………………………………………5

1.2.3 Immobilisation…………………………………………………………...6

1.3 Classification of Biosensors ................................................................................7

1.3.1 Catalytic Biosensors……………………………………………………..7

1.3.1.1 The Principle of Catalytic Biosensors…………………………...7

1.3.1.2 Efforts towards Improving Electrical Communication between

the Enzyme and the Electrode………………………………………………….9

1.3.1.3 Issues with the Current Catalytic Biosensors…………………..10

1.3.2 Affinity Biosensors……………………………………………………..11

ix

1.3.2.1 Immunosensors…………………………………………………12

1.3.2.2 Antibody Structures…………………………………………….12

1.3.2.3 The Principle of Immuno-Interaction…………………………..13

1.3.2.4 Issues with Current Immunosensors……………………………14

1.4 Solutions for Existing Problems with Current Biosensors............................16

1.5 Creating More Stable Self-Assembled Monolayers on Electrode Surfaces

for the Construction of Sensors ...................................................................................16

1.5.1 Self-Assembled Monolayers on Gold Electrodes………………………17

1.5.2 Self-Assembled Monolayers on Glassy Carbon Surfaces……………...19

1.5.2.1 Glassy Carbon Surfaces………………………………………...19

1.5.2.2 Modification of Glassy Carbon Electrodes with Stable Self-

Assembled Monolayers……………………………………………………….20

1.6 Creating the Sensing Interfaces with the Ability to Resist Non-Specific

Adsorption .....................................................................................................................24

1.7 Using Molecular Wires to Establish Efficient Electron Transfer on Sensing

Interfaces........................................................................................................................26

1.7.1 Using Oligo(phenyl ethynylene) Bridges to Facilitate Electron Transfer

between Biomolecules and Sensing Interfaces……………………………………..27

1.7.2 Using Single-Walled Carbon Nanotubes to Facilitate Electron Transfer

between Biomolecules and Sensing Interfaces……………………………………..28

1.8 Aims of the Thesis .............................................................................................30

1.9 Overview of Chapters .......................................................................................31

1.10 References ..........................................................................................................33

x

Chapter Two Experimental Procedures and Instrumentation.……….….47

2.1 Chemicals, Reagents and Solutions .................................................................48

2.2 Synthesis.............................................................................................................53

2.2.1 Synthesis of 4-Carboxyphenyl Diazonium Tetrafluoroborate and 4-

Nitrophenyl Diazonium Tetrafluoroborate…………………………………………53

2.2.2 Synthesis of Benzenediazonium Tetrafluoroborate…………………….54

2.2.3 Synthesis of Ferrocenemethylamine……………………………………54

2.2.4 Synthesis of 1,1`-Ferrocenedimethylamine…………………………….55

2.2.5 Synthesis of Molecular Wires………………………………………….56

2.2.5.1 Synthesis of 1-Bromo-3-nitro-4-(4-aminophenylethynyl)benzene

(1)……………………………………………………………………………...57

2.2.5.2 Synthesis of Methyl 4-(trimethylsilylethynyl)benzoate (2a)…...58

2.2.5.3 Synthesis of Methyl 4-ethynylbenzoate (2)…………………….58

2.2.5.4 Synthesis of Methyl 2`-nitro-4, 4`-diphenylethynyl-4``-

aminobenzoate (3)…………………………………………………………….59

2.2.5.5 Synthesis of Methyl 2`-nitro-4, 4`-diphenylethynyl-4``-amino

Benzoic Acid (4)………………………………………………………………59

2.2.5.6 Synthesis of Methyl 2`-nitro-4, 4`-diphenylethynyl-4``-benzoic

acid Benzenediazonium Tetrafluoroborate (5)………………………………..60

2.2.6 Synthesis of 4-(2-(2-(2-Methoxyethoxy)ethyl)benzenediazonium

Tetrafluoroborate…………………………………………………………………...61

2.2.6.1 Synthesis of 2-(2-(2-Methoxyethoxy)ethoxy)ethyl p-

Toluenesulfonate (a).………………………………………………………….62

2.2.6.2 Synthesis of 4-(2-(2-(2-

Methoxyethoxy)ethoxy)ethyl)nitrobenzene (b)……………………………….62

xi

2.2.6.3 Synthesis of 4-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)aniline

(c)……………………………………………………………………………...63

2.2.6.4 Synthesis of 4-(2-(2-(2-

Methoxyethoxy)ethoxy)ethyl)benzenediazonium Tetrafluoroborate (d)……..63

2.3 Instrumentation.................................................................................................64

2.3.1 Electrochemical System………………………………………………..64

2.3.2 Nuclear Magnetic Resonance (NMR) Spectrometer…………………...66

2.3.3 X-ray Photoelectron Spectroscopy (XPS)……………………………...66

2.3.4 Atomic Force Microscopy (AFM)……………………………………...67

2.3.5 Fourier Transfer Ion Cyclotron Mass Spectrometry (FT-ICR MS)……67

2.3.6 LEO-Scanning Electron Microscope (LEO-SEM)……………………..67

2.4 Procedures .........................................................................................................67

2.4.1 Preparation of Glassy Carbon Electrodes and Calculation of the

Electrochemical Surface Area……………………………………………………...67

2.4.2 Derivatisation of Glassy Carbon Electrodes with Diazonium Salts……70

2.4.3 Preparation of Pyrolysed Photoresist Films…………………………….71

2.4.4 Preparation of Gold Electrodes and Calculation of the Electrochemical

Surface Area………………………………………………………………………..72

2.4.5 Preparation of Homogeneous Pure and Mixed Alkanethiol Self-

Assembled Monolayers on Gold Surfaces………………………………………….73

2.4.6 Covalent Attachment of Ferrocene Redox Probes onto Glassy Carbon

Electrodes Modified with Diazonium Salt Monolayers……………………………75

2.4.7 Determination of the Surface Coverage of Redox Species…………….76

2.4.8 Calculation of the Rate Constant of Heterogeneous Electron Transfer

using Laviron Theory...…………………………………………………………….76

xii

2.5 References ..........................................................................................................79

Chapter Three Covalent Modification of Electrode Surfaces by

Electrochemical Reduction of Aryl Diazonium Salts…………….………82

3.1 Introduction.......................................................................................................83

3.2 Experimental Section........................................................................................84

3.3 Results and Discussion......................................................................................84

3.3.1 Influence of Scan Rates on the Capacitance of Bare Glassy Carbon

Electrodes in Aqueous and Nonaqueous Electrolytes……………………………...84

3.3.2 Modification of Glassy Carbon Electrodes with Aryl Diazonium Salts by

Electrochemical Reductive Adsorption…………………………………………….87

3.3.3 Electrochemistry of a Glassy Carbon Electrode Modified with 4-

Carboxyphenyl Diazonium Tetrafluoroborate……………………………………...90

3.3.4 Effect of Solution pH on the Blocking Property of the 4-Carboxyphenyl

Groups Modified on Glassy Carbon Electrodes……………………………………94

3.3.5 Characterisation of 4-Carboxyphenyl Modified Glassy Carbon Surfaces

Using X-ray Photoelectron Spectroscopy………………………………………….95

3.3.6 Stability of Carboxyphenyl Monolayers Modified on Glassy Carbon

Electrodes…………………………………………………………………………..96

3.3.7 Electrochemistry of a Glassy Carbon Electrode Modified with Phenyl

Diazonium Tetrafluoroborate………………………………………………………99

3.3.8 Modification of Gold Electrodes with Aryl Diazonium Salts………...102

3.3.8.1 Electrochemistry of 4-Carboxyphenyl Modified Gold

Electrodes……………………………………………………………………102

xiii

3.3.8.2 Characterisation of 4-Carboxyphenyl Modified Gold Surfaces

Using XPS…………………………………………………………………...104

3.3.8.3 Robustness of Monolayers Modified on Gold Surfaces………105

3.3.8.4 Reductive Desorption of Diazonium Salt Monolayers………..109

3.4 Conclusions ......................................................................................................112

3.5 References ........................................................................................................112

Chapter Four Heterogeneous Electron Transfer Through Organic

Monolayers on Carbon and Gold Electrodes……….…………………...116

4.1 Introduction.....................................................................................................117

4.2 Experimental Section......................................................................................118

4.3 Results and Discussion....................................................................................119

4.3.1 Heterogeneous Electron Transfer Through Diazonium Salt Monolayers

Modified on Glassy Carbon Electrodes Using Ferrocene as the Redox Probe…...119


Modified on Gold Electrodes Using Ferrocene as the Redox Probe……………...124


Modified on Pyrolysed Photoresist Films………………………………………...126

4.3.3.1 Pyrolysed Photoresist Films (PPF)……………………………126

4.3.3.2 Heterogeneous Electron Transfer on PPF Surfaces…………...127

4.3.4 Heterogeneous Electron Transfer Through Alkanethiol Monolayers

Modified on Gold Electrodes……………………………………………………..131

4.3.5 Kinetics of Heterogeneous Electron Transfer Through Organic

Monolayers on Carbon and Gold Electrodes……………………………………...132

4.4 Conclusions ......................................................................................................140

xiv

4.5 References ........................................................................................................141

Chapter Five Fabrication of Electrochemical Copper Sensors Based on

Gly-Gly-His Modified Carbon Electrodes………………………………145

5.1 Introduction.....................................................................................................146



5.3.1 Electrochemistry of Cu2+ Complexed Gly-Gly-His on Glassy Carbon

Electrodes…………………………………………………………………………150

5.3.2 Characterisation of Gly-Gly-His Modified Glassy Carbon Surfaces Using

XPS………………………………………………………………………………..156

5.3.3 Calibration Curve of Gly-Gly-His Modified Glassy Carbon Electrodes

for the Detection of Cu2+………………………………………………………….162

5.3.4 Attachment of Gly-Gly-His onto Mixed Aryl Diazonium Salts Modified

Glassy Carbon Electrodes for the Detection of Cu2+……………………………...164

5.3.5 Electrochemistry of Cu2+ Complexed with Gly-Gly-His Modified on

Pyrolysed Photoresist Films………………………………………………………168

5.3.6 Stability of Gly-Gly-His Modified Glassy Carbon Electrodes for the

Detection of Cu2+……………………………………………………………….…172

5.4 Conclusions ......................................................................................................174

5.5 References ........................................................................................................175

xv

Chapter Six An Interface Comprising Molecular Wires and Poly(ethylene

glycol) Spacer Units Self-Assembled on Carbon Electrodes for Studies of

Protein Electrochemistry...………………………………………………180

6.1 Introduction.....................................................................................................181


6.2.1 Chemicals and Procedures…………………………………………….184

6.2.2 Bovine Serum Albumin Labelled Au Nanoparticles………………….185

6.3 Result and Discussion .....................................................................................185

6.3.1 Electrochemistry of PEG Modified Glassy Carbon Electrodes……….185

6.3.2 Non-Specific Protein Adsorption on PEG Modified Glassy Carbon

Surfaces…………………………………………………………………………...187

6.3.3 Electrochemistry of Glassy Carbon Electrodes Modified with Mixed

Monolayers of MW and PEG at the Molar Ratio of 1:20 ………………………...191

6.3.4 Electron Transfer Through Monolayers of MW on Glassy Carbon

Surfaces…………………………………………………………………………...194

6.3.5 Direct Electron Transfer between HRP and MW on Glassy Carbon

Surfaces…………………………………………………………………………...199

6.4 Conclusions ......................................................................................................204

6.5 References ........................................................................................................204

Chapter Seven Exploration of Deeply Buried Active Sites of Glucose

Oxidase Using Molecular Wires Self-Assembled on Carbon Electrodes.212

7.1 Introduction.....................................................................................................213


xvi


7.2.2 Direct Attachment of GOx to Glassy Carbon Electrodes Modified with

Mixed Monolayers of 4-Carboxyphenyl and MW………………………………..220


7.3.1 Electrochemistry of MW Modified Glassy Carbon Surfaces…………221

7.3.2 Exploration of Active Centres of GOx Using MW Modified on Glassy

Carbon Electrodes…………………………………………………………………222

7.3.2.1 Electrochemistry of GOx Coupled on Glassy Carbon

Electrodes……………………………………………………………………222

7.3.2.2 Effect of Mixed Monolayers on the Redox Response of GOx..226

7.3.2.3 Surface Coverage of GOx on MW Modified Glassy Carbon

Electrodes……………………………………………………………………229

7.3.2.4 The Rate Constant of Electron Transfer between FAD and Glassy

Carbon Electrodes……………………………………………………………230

7.3.2.5 Measurement of Biocatalytical Activity of GOx……………...231

7.4 Conclusions ......................................................................................................237

7.5 References ........................................................................................................237

Chapter Eight Development of a Label-Free Immunosensor on Molecular

Wire Modified Glassy Carbon Surfaces…………………….…………...241

8.1 Introduction.....................................................................................................242



8.2.2 Covalent Coupling of Ferrocenedimethylamine to Mixed Monolayers of

MW and PEG Modified Glassy Carbon Electrodes………………………………248

xvii

8.2.3 Immobilisation of Biotin and Anti-biotin on Ferrocenedimethylamine

Modified Glassy Carbon Electrode Surfaces……………………………………...248


8.3.1 Electrochemistry of Glassy Carbon Electrodes Modified with Mixed

Monolayers of MW and PEG at a Molar Ratio of 1:20…………………………...248

8.3.2 Electrochemistry of Ferrocenedimethylamine on Glassy Carbon

Electrodes Modified with Mixed Monolayers of MW and PEG at a Molar Ratio of

1:20………………………………………………………………………………..249

8.3.3 Heterogeneous Electron Transfer Through Mixed Monolayers of MW

and PEG Modified Glassy Carbon Electrodes Using Ferrocene as the Redox

Probe………………………………………………………………………………253

8.3.4 Electrochemistry of Ferrocene Modified Glassy Carbon Electrode

Surfaces after Immobilisation of Biotin and Antibiotin…………………………..254

8.3.5 Calibration Curve for the Detection of Antibiotin…………………….260

8.3.6 Displacement Immunoassay…………………………………………..262

8.3.7 Electrochemical Stimulation of Antibiotin Dissociation from the

Immunosensor Interface…………………………………………………………..266

8.4 Conclusions ......................................................................................................269

8.5 References ........................................................................................................270

Chapter Nine Towards the Fabrication of Immunosensors Using SWNTs

as the Conduit for Electron Transfer.…………….……………………...275

9.1 Introduction.....................................................................................................276



xviii

9.2.2 Preparation of the Cut SWNTs……..…………………………………279

9.2.3 Fabrication of the Cut SWNTs on the 4-Aminophenyl Modified Glassy

Carbon Electrodes…………………………………………………………………280


9.3.1 Modification of the Glassy Carbon Electrodes with 4-Nitrophenyl

Diazonium Tetrafluoroborate……………………………………………………..280

9.3.2 Conversion of the 4-Nitrophenyl Groups on Glassy Carbon Electrode

Surfaces into 4-Aminophenyl Groups…………………………………………….284

9.3.3 Attachment of Ferrocenecarboxylic Acids on 4-Aminophenyl Modified

Glassy Carbon Electrodes…………………………………………………………286

9.3.4 Characterisation of the SWNT Modified Glassy Carbon Surfaces by

AFM……………………………………………………………………………….289

9.3.5 Covalent Attachment of Ferrocenemethylamine to SWNT Modified

Glassy Carbon Electrodes…………………………………………………………290

9.3.6 Fabrication of a Label-Free Immunosensor on SWNTs Modified Glassy

Carbon Substrates…………………………………………………………………294

9.4 Conclusions ......................................................................................................298

9.5 References ........................................................................................................299

Chapter Ten Conclusions and Future Directions……………………...304

10.1 Introduction.....................................................................................................305

10.2 Brief Summary ................................................................................................306

10.3 Future Directions ............................................................................................308

10.3.1 Further Investigation of Heterogeneous Electron Transfer within and

between the Enzymes……………………………………………………………..308

xix

10.3.2 Optimisation of the Fabricated Immunosensors………………………308

10.3.3 Development of the Immunosensor Arrays…………………………...309

10.3.4 Diagnosis and Treatment of Pathogenic Cells Using the Nanoparticle-

Antibody Conjugate……………………………………………………………….313

10.4 References ........................................................................................................316

xx

List of Abbreviations

AFM Atomic force microscope

BSA Bovine Serum Albumin

CV Cyclic voltammetry

DCC 1,3-dicyclohexylcarbodiimide

DMF Dimethyl formamide

DMSO Dimethyl sulphoxide

E Electrode potential

Eo` Formal potential

EDC 1-ethyl-3-(3-dimethylamino)propyl) carbodiimide

ET Electron transfer

EtOAc Ethyl acetate

F Farady constant

FCA Ferrocenecarboxylic acid

GCE Glassy carbon electrode

Gly-Gly-His Glycine-Glycine-Histidine

h Hour

HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid)

kET Electron transfer rate constant

L Litre

LP Light petroleum

m Minute

MES 2-(N-morpholino)ethanesulfonic acid

xxi

mol Mole

MP-11 Horseradish peroxidase

NHS N-hydroxysuccinimide

NMR Nuclear magnetic resonance

OSWV Osteryoung Square Wave Voltammetry

PPF Pyrolysed Photoresist Film

Q Charge per unit area

R Gas constant

R Radius

R Resistance

s Second

SAMs Self-assembled monolayers

SEM Scanning electron microscopy

STM Scanning tunnelling microscopy

SWNT Single-walled carbon nanotube

T Absolute temperature

TEM Transmission electron microscopy

THF Tetrahydrofuran

UV Ultraviolet

xxii

Chapter 1-General Introduction

Chapter One

General Introduction

1


1.1 Introduction

A biosensor is a compact analytical device that is capable of detecting a target analyte

using a biological recognition element (biochemical receptor) immobilised directly onto

a physical or physicochemical transducer.1 Examples of biochemical receptors are

enzymes, antibodies or fragments of antibodies, membrane receptors, whole cells,

nucleic acids and DNA fragments, which are used to recognise and interact specifically

with the analyte in question. The response is sensed by the transducer, which has the

ability to translate a biological interaction into a usable electrical signal. The most

frequently used transducers are based on electrochemistry, fluorescence, interferometry,

resonance and reflectometry.2 Biosensors are usually classified into various basic

groups according either to the method of signal transduction or to the biorecognition

principle. Accordingly, biosensors can be categorised as electrochemical, optical,

piezoelectric and thermal sensors on the basis of the transducing element, and as

immuno-chemical, enzymatic, non-enzymatic receptor, whole-cell and DNA biosensors

on the basis of the biorecognition principle.

Biosensors have many potential advantages in comparison to many conventional

analytical approaches in terms of simplicity, low limits of detection and sensitivity. A

biosensor should be clearly distinguished from a bioanalytical system, which requires

additional steps, such as reagent addition. Biosensor technology offers the possibility of

identifying and quantifying specific compounds directly in liquid media or in very dirty

environments, although in some cases previous sample preparation is also needed.

Biosensors are widely used for quality assurance in agriculture, food and

pharmaceutical industries, monitoring environmental pollutants and biological agents,

2


medical diagnostics and biological assays. There are many biosensors under

development and also extensive literature on this area.1-8 Commercialisation is the best

indicator of the success of a biosensor.8-10 However, only a small number of all

biosensors developed are commercially available. Additionally, most commercial

biosensors are focused on medical applications, such as glucose-detecting biosensors.

Food, agriculture, military, veterinary and the environment are potential markets which

still need to be established. An essential requirement for the commercialisation of these

devices is a clear application need. Also, the technology must offer advantages over

established technologies. Issues such as market size, development costs, and ease of

manufacture should be addressed before the sensor reaches the development stage.

The biosensors market is growing significantly, from $113.5 million in 1997 to $197.6

million by 2004, as predicted for sales in Europe.11 Taking advantages of the steady new

developments in biochemistry, biotechnology, genetics, electronics, physics, and

microfabrication, research in biosensors has produced a huge amount of new ideas, and

devices in recent decades. As a result of the potential development, biosensors have

been obtaining wide applications for clinical diagnostics, analytical chemistry,

pharmaceutical development, the food industry and environmental monitoring.

This chapter will introduce the concept of biosensors, the problems existing with the

current biosensors, how to form more stable monolayer systems for the fabrication of

biosensors, and how to achieve direct electron transfer between biomolecules and the

electrodes via the efficient electron transfer linker to develop third generation

biosensors and label-free immunosensors.

3


1.2 Biosensors

1.2.1 The General Principle of Biosensors

Generally, a biosensor is a self-contained integrated analytical device incorporating a

biological or biologically derived sensing element associated with a physicochemical

transducer or transducing microsystem. The general design of a typical biosensor is

illustrated in Figure 1.1.

TRANSDUCER

Biorecognition(Proteins, enzymes,antibodies, DNA, RNA,…)

Sample Electrical signal(Electrochemical, acoustic,mechanical, photometric,…)

Reading

DISPLAY

Figure 1.1 The general scheme of a biosensor.

The biorecognition elements of biosensors are not only responsible for the selective

recognition of the analyte, but also the generation of the physicochemical signal

monitored on the transducer and the sensitivity of the final device.7 There are four main

groups of biorecognition elements: i) enzymes (biocatalyst), ii) antibodies (bioaffinity),

iii) nucleic acids, iv) receptors. A transducer is used to convert the biochemical

recognition reaction into a quantifiable output signal, which is critical for biosensors.

The two crucial components of the biosensor are usually integrated by the

4


immobilisation of the biorecognition components onto the surface of the transducer. The

following two sections will introduce the transducer and the immobilisation method in

more detail.

1.2.2 Transducers

A key part of the biosensor is the transducer, which makes use of a physicochemical

change accompanying the interaction between biological molecules. The

physicochemical change may be (i) the heat outputted (or absorbed) by the reaction, (ii)

movement of electrons produced in a redox reaction, (iii) light output during the

reaction, a light absorbance difference between the reactants and products or a change in

other optical properties, or (iv) effects due to the mass of the reactants or products.

Based on the physicochemical change accompanying the biorecognition reaction,

transducers are thus mainly divided into four categories: calorimetric, electrochemical,

optical and piezoelectric devices. All types of transducers have been used with all

biorecognition components.8, 12 All these transducers suffer from certain drawbacks, for

example, the optical transducer, though very sensitive, however, cannot be used in

turbid media. Calorimetric transducers cannot be utilised with systems with very little

heat change. Moreover, they are not easy to handle. Electrochemical transducers have

been found to overcome most disadvantages, which inhibit the use of other types of

transducers.

Electrochemical transducers have emerged as the most commonly used type of

transducers. High sensitivity and selectivity, ability to operate in turbid solutions, rapid

analysis and being more amenable to miniaturisation are the advantages of

electrochemical transducers. Furthermore, the continuous response of an electrode

5


system allows for on-line control, and the equipment required for electrochemical

transducers is simple and cheap compared to most other analytical devices. Thus this

thesis will exclusively investigate the electrochemical transducers.

1.2.3 Immobilisation

Immobilisation of biological elements as well as their orientation (i.e., accessibility of

the ligand binding site to the analyte) are important considerations in the design and

construction of biosensors. This is primarily due to the stoichiometric relationship

between biological elements and the finite surface area of the signal transducer. The

immobilisation method is determined by the nature of the biocomponent to be

immobilised. The type of transducing element used and the physical properties of the

analyte are also important factors that assist in selecting a method.13 In general, the

immobilisation procedure must maintain the biorecognition molecule close to the

transducer surface, while retaining its biological activity, in a reproducible manner.

Furthermore, it is desirable that the immobilisation layer gives the biological molecule

enhanced stability, is robust, is applicable to many different biomolecules, is chemically

resistant to the reactants and products of the biochemical reaction and gives control over

the distribution and orientation of the immobilised species.14

A wide variety of immobilisation methods, which have the dominant effect on the

performance of the biosensors, have been developed on various substrates.15-17 General

strategies for immobilising biological elements on the transducer surface include

adsorption,18-20 microencapsulation, entrapment,21, 22 cross linking, covalent bonding,23-

25 and the use of biological binding proteins such as protein A or protein G26, 27 or use of

the avidin/biotin system. Although all these immobilisation approaches are highly

6


versatile, the common drawback with these methods is the poor control over the

location and density of biorecognition molecules. Modification of surfaces with a self-

assembled monolayer can be achieved with molecule level control over the interface,

and hence the position of the recognition molecules in space can also be controlled with

molecular level precision.28 Thus, the self-assembly provides a potential strategy for

integrating biorecognition components with transducers. This thesis will use self-

assembly chemistry to immobilise the sensing interface in fabrication of biosensors.

1.3 Classification of Biosensors

Based on the biorecognition components used, biosensors can be classified as two

categories: catalytic biosensors and affinity biosensors.

1.3.1 Catalytic Biosensors

Catalytic biosensors are based on the recognition and binding of an analyte followed by

a catalysed chemical conversion of the analyte from a nondetectable form to a

detectable form, which are detected and recorded by a transducer. In the catalytic

biosensors, biocatalysts, such as enzymes and microbiological cells, are used to

recognise, bind, and chemically convert a molecule. To extend the range of detectable

analytes a second or third enzyme can be used that converts the nondetectable primary

product to a secondary detectable one.29

1.3.1.1 The Principle of Catalytic Biosensors

Enzyme electrodes have received the most attention in the overall progression of

catalytic biosensors, which are based on the activity of an enzyme catalysing a redox

chemical reaction, thus producing or consuming electrons. Figure 1.2 shows a scheme

7


of the principle of an enzyme biosensor. In an enzyme biosensor the substrate is the

analyte of interest. The analyte reacts with the enzyme and produces a product. The role

of the mediating species is to complete the catalytic cycle. In this reaction, a molecule

must either be produced or consumed that can be detected at the transducer. The most

widely investigated enzyme biosensors are glucose biosensors owing to their potential

interest especially in the field of human health care and bioprocess control. The glucose

biosensor uses enzyme glucose oxidase (GOx) to oxidase glucose in the presence of a

mediator to produce gluconolactone and a reduced form of the mediator. In nature the

mediator is oxygen with hydrogen peroxide being produced while many of the

commercial glucose biosensors use redox species ferrocene or ferricyanide.1 As there

are charges in redox state in the recognition reaction it is common for the transduction

of enzyme reactions to be electrochemical. In a conventional enzyme biosensor the

reduced form of the mediator is detected amperometrically at the electrode with the

current being proportional to the amount of substrate in the sample.

-ne-

Substrate

Product

Mediatorred

Mediatorox

ELECTRODE

-ne-

Substrate

Product

Substrate

Product

Mediatorred

Mediatorox

ELECTRODE

ELECTRODE

Figure 1.2 The scheme of an enzyme biosensor.

8


1.3.1.2 Efforts towards Improving Electrical Communication between the Enzyme

and the Electrode

As the enzyme reaction involves a change in oxidation state, the ultimate goal of

enzyme electrode research is to obviate the need for a mediator by oxidising and

reducing the enzyme directly at the electrode. Achieving direct electron transfer

between the active centre of the enzyme and an electrode is crucial for development of

novel enzyme biosensors or bioelectronics.30 Direct electron transfer was observed on

small redox proteins such as cytochrome c,31-36 microperoxidase37, 38 and azurin35 with

redox active sites being located close to the enzyme surface. With most oxidoreductase

enzymes however, the redox active centres are located at a sufficient distance from the

surface of the glycoprotein to prevent direct electron transfer. For example, in the case

of GOx, the closest approach of the redox active centre, flavin adenine dinucleotide

(FAD), to the enzyme surface is 13 Å.39 Therefore, it is far more difficult to incorporate

these proteins into a biosensor system due to the weak electrical communication to their

surrounding environment.

Various strategies have been employed, such as promoters,40 redox mediators,41 direct

covalent linkage of the protein or enzyme to the electrode,42 and protein adsorption,43 to

achieve direct and reversible electron transfer between the active centre of GOx and the

underlying electrodes by bringing the flavoenzyme close to the electrode surfaces.

Degani and Heller44, 45 have successfully established direct electrical communication

between the redox centres of the enzymes and electrodes through bonding electron

transfer relays. Later, based on the idea of entrapping GOx in a redox polymer which

shuttled the electrons by Heller and coworkers.46-48 Willner and coworkers49, 50

9


incorporated an electron transfer relay (PPQ) between the active centre of the enzyme

and the electrode and illustrated some important development. Firstly, the

communication between enzyme and electrodes was improved by coupling mediating

molecules to the redox active centre. Secondly, the active GOx could be reconstituted

around a surface immobilised FAD to give an immobilised enzyme with a defined

orientation on the surface. Willner’s final glucose biosensors had demonstrated

excellent performance, however the electron transfer still relied on the presence of a

mediating relay. Thus, the elegant wired enzyme electrode of Willner and coworkers

still represents a second generation biosensor.

1.3.1.3 Issues with the Current Catalytic Biosensors

By introducing the mediator to turn over the enzyme and carry electrons between

enzyme and the transducer, second generation biosensors have overcome the drawback

of first generation biosensors where the normal product of the reaction diffuses to the

transducer and causes the electrical response.51-54 However, new problems are faced

when the mediator is introduced into a biosensor system. Firstly, oxygen competition

with the mediator exists because oxygen is a very active native mediator. Secondly, the

concentration of the mediator is changing all the time, hence the measured response on

the transducer is variable. Thirdly, the working efficiency is limited by the diffusion

process of the mediator. For these reasons it is desirable to develop third generation

biosensors based on direct electron transfer.

With third generation biosensors, the absence of mediators is the main advantage,

providing them with superior selectivity and the lack of another reagent in the reaction

sequence. Another attractive feature of the system based on direct electron transfer is

10


the possibility of modulating the desired properties of an analytical device using protein

modification with genetic or chemical engineering techniques on one hand, and novel

interfacial technologies on the other hand. A few groups have been trying to fabricate

third generation biosensors based on direct electron transfer.42, 55-57 Gooding and

coworkers have extended the principle to achieve electron tunnelling directly to the

electrode from the FAD by introducing a norbornylogous bridge as the electron transfer

linker.58 When the apo-GOx was refolded on the FAD modified electrode incorporated

with the norbornylogous bridge, the enzyme was found to be biocatalytically active.

However, no biocatalytic event was observed under anaerobic conditions, indicating

that the direct electron transfer to the enzyme was very insignificant due to poor

electrical coupling between the redox active centre FAD and the electrode. Thus, how to

achieve significant direct electron transfer to GOx for fabrication third generation

biosensors still remains the challenge to researchers.

1.3.2 Affinity Biosensors

Another class of biosensors is the affinity bisensors, which are devices in which

receptor molecules bind analyte molecules, causing a physicochemical change that is

detected by a transducer. In the affinity biosensors, receptor molecules such as

antibodies, nucleic acids, receptor proteins, biomimetic materials and DNA are used to

bind molecules non-catalytically. The main advantages of these kinds of biosensors are

the wide range of affinities available, thus expanding the number of analytes that can be

selectively detected. Bioaffinity biosensors primarily depend on the use of antibodies

due to the availability of monoclonal and polyclonal antibodies toward a wide range of

analytes as well as their relative affinity and selectivity of these proteins for a specific

11


compound or a closely related group of compounds.7 So the following section will

concentrate on affinity biosensors based on immunoreactions on the sensing interface.

1.3.2.1 Immunosensors

Immunosensors are immunoreaction-based affinity biosensors, which use immuno-

compounds as biological receptors and are usually the result of the integration in one

device of an immunoassay and a directly associated transducer. Generally, immobilised

antibodies or antigens on a transducer form the biorecognition elements of an

immunosensor. So understanding the antibody structure is very important for the

development of an immunosensor.

1.3.2.2 Antibody Structures

Antibodies (Ab) are immunosystem related proteins synthesised by plasma cells, i.e.,

mature B-lymphocyte, in animals in response to the presence of a foreign substance,

called an antigen (Ag) with a molecular weight higher than 1.5 kDa. Antibodies are

divided into five major classes, IgG, IgA, IgM, IgD, and IgE, based on their constant

region structure and immune function. IgG, also known as –globulin, has a mass of

150 kDa and is the principal antibody in serum.

Antibodies are structurally very similar. The structure of an antibody can be generally

represented by the structure of an IgG molecule (Figure 1.3). An IgG consists of two

heavy and two light chains, which are interconnected by disulfide bonds to form a “Y”

shaped molecule. Each chain is composed of a variable and a constant region. Each

variable region includes three hypervariable segments that vary from one antibody to

another, conferring on antibodies a large range of antigen specificity. This antigen

12


specificity of an antibody is conferred by the variable regions of both the light and

heavy chains which contain the complementarity-determining regions that determine

much of the specificity of the molecule. Thus, an IgG molecule has two identical

binding locations for the antigen.59

Figure 1.3 Basic structure of an IgG molecule.

1.3.2.3 The Principle of Immuno-Interaction

The interaction of an antibody with an antigen forms the basis of all immuno-chemical

techniques. The molecular forces responsible for the Ab-Ag binding are based on non-

covalent interactions including: non-polar hydrophobic interactions, Coulomb

interaction, Van der Waals interaction, London dispersion attractive forces and steric

repulsion forces. The interaction is characterised with an association and a dissociation

reaction rate constant, ka and kd respectively.

Ab Ab-AgAg+ka

kd

13


The affinity constant Ka, which varies in strength from 104 to 1015 M-1 (typically of the

order of 108 to 1012 M-1)60 depending on the nature of antigens and the binding affinity

of the corresponding antibodies, can be described by:

]][[][

AgAbAgAb

kk

d

aAK

Where [Ab], [Ag] and [Ab-Ag] are the molar concentrations of the antibody, antigen

and antibody-antigen complex in solution, respectively.61 The antibody-antigen

interaction defines both the specificity and the detection limit of an immonosensor. The

ultimate detection limit of an immunoassay is determined by the antibody-antigen

binding constant. The greater the binding constant of the antibody, the lower detection

limit can be achieved.62

1.3.2.4 Issues with Current Immunosensors

With an immunosensor, once the antibody-antigen binding reaction has occurred, there

is still the need to transduce the biorecognition event. Unlike catalytic biosensors where

the biorecognition event produces a molecule which can be detected, in affinity sensors

the analyte simply binds. To transduce such biorecognition events either requires labels,

so familiar in the myriad of immunoassay formats, or a transduction method which can

detect the change that occurs at the sensing interface. Most immunosensor devices

reported to date perform indirect measurements by using competitive immunoassay

configurations and/or labels such as enzymes (e.g., alkaline phosphate,63 horseradish

peroxidase64), and chemiluminescent probes,65, 66 that convert an enzyme substrate into

a measurable product.67-69 In these configurations, the analyte and the enzyme-labelled

analyte compete for a limited number of binding sites on the immobilised antibodies.

Figure 1.4 shows the scheme of the electrochemical immunoelectrode competitive

14


configuration. Electrochemical immunoelectrodes are based on the activity of an

enzyme label catalysing a redox chemical reaction, thus producing or consuming

electrodes. The signal generation of labelled immunosensors is significantly facilitated.

However, this type of sensor is expensive, time-consuming, requires trained personnel

and is difficult to carry out measurements in real time. So it remains a challenge for

researchers to investigate the direct and label-free immunosensors.

Substrate

Product

ELECTRODE

Enzyme-labelled antigen

Antigen (analyte)

Immobilised antibody

e-

Substrate

Product

ELECTRODE

Enzyme-labelled antigen

Antigen (analyte)

Immobilised antibody

e-

Figure 1.4 Scheme of the electrochemical immunoelectrode competitive configuration.

Immunosensors for direct, label-free, measurements of various analytes are attractive

for many reasons and have been the subject of several research efforts.70-72 The major

attraction of the label-free immunosensors is that they are able to determine the analyte

directly in a sample with no or very little sample preparation. A key problem, though

associated with non-labelled affinity biosensors is non-specific binding, as there is no

discrimination between the measured signal from specific and non-specific interactions.

If such a distinction is to be realised the transducer has to be sensitive to conformational

changes of the antigen binding site or changes in charge distribution around this site

when the antigen binds. It is therefore a challenge to design the sensing surface in such

15


a way that ensures higher specific rather than non-specific binding in the fabrication of

label-free immunosensors.

1.4 Solutions for Existing Problems with Current Biosensors

The concept of biosensors (i.e, catalytic biosensors and affinity biosensors) and the

existing problems with current biosensor have been introduced in above sections. It is

necessary to find solutions for the existing problem with current biosensors. Based on

the principle of a biosensor, the integration of biorecognition components with

transducers through immobilisation is the interfacial reaction. Thus it is possible to

solve the existing problems with current catalytic biosensors and immunosensors if an

optimised sensing interface was constructed, which is critical for development of both

catalytic biosensors and affinity biosensors. For biosensing applications, the optimised

sensing interface should meet three requirements: i) the sensing interface should be very

stable, ii) the sensing interface has the ability to bind the specific analyte of interest but

resist the non-specific binding which affects the selectivity and sensitivity of a

biosensor, iii) efficient electrical communications can be established between the

biorecognition component and the transducer. The following sections will introduce

how to design this optimised sensing interface with these unique characteristics to solve

the existing problems with current biosensors.

1.5 Creating More Stable Self-Assembled Monolayers on Electrode Surfaces for the

Construction of Sensors

As introduced in section 1.2.3, self-assembling provides a potential strategy for

integrating biorecognition components with transducers to form the sensing interface.

Formation of monolayers by self-assembly makes it possible to control the sensing

16


interface with desirable properties. Self-assembled monolayers (SAMs) are ordered

monomolecular films which are spontaneously formed upon immersing a solid substrate

into a solution containing amphifunctional molecules. The amphifunctional molecule

has a head group, which usually has a high affinity for the solid surface, a tail, typically

an alkyl chain, and a terminal group which can be used to control the surface properties

of the resultant monolayer. So SAMs have two key features of self-assembly in

biological systems, namely that molecules have high affinity for each other and

predictable structures are formed when the molecular units associate. These unique

characteristics make SAMs an excellent model system for sensing construction.

1.5.1 Self-Assembled Monolayers on Gold Electrodes

The most studied and the best characterised self-assembled monolayers (SAMs) for

sensing applications are those formed by alkanethiols chemisorbed from solution onto

gold surfaces.73-75 The exact nature of the bond that forms between the gold and the

sulfur is still not clear but in the case of alkanethiols it can be considered as a oxidative

addition of the S-H bond to the gold surface followed by a reductive elimination of

hydrogen.76

200

21 HAuAuSRAuHSR nn

Evidence for hydrogen elimination has been hard to come by but the presence of a

thiolate has been confirmed by many groups.77-79 The formed Au-S bond strength is

about 40 kcal mol-1 78 and the free energy change for the adsorption of alkanethiols on

gold is approximately –5.5 kcal mol-1.80 The equivalent Au-S bond is also formed with a

disulphide.76 The alkanethiol chains typically tilt between 20-30 degrees from normal to

the Au(111) surface as shown in Figure 1.5 a. The tilt angle is dictated by the spacing

17


sites on the metal surface and is a consequence of the chains establishing van der Waals

contact.74, 76 Gold surfaces can be modified with pure SAMs (Figure 1.5 a) by dipping

the substrate into the solution of one alkanethiol. It is also applicable to modify the gold

surfaces with mixed SAMs (Figure 1.5 b) by dipping the substrate into mixed

alkanethiol solutions. A variety of advantages can be achieved by using mixed

monolayers, such as reducing the steric hindrance,37 reducing the interaction between

the molecules,81 or reducing the concentration of the redox probes on the monolayers.82

The choice of pure or mixed SAMs is dictated by their applications.

a)30o

b)

Au AuAu

Figure 1.5 The illustration of a (a) pure and (b) mixed alkanethiol self-assembled

monolayer.

SAMs of alkanethiols offer numerous advantages such as simple preparation, well-

defined organisation, densely-packed structures and the possibility of introducing a vast

variety of functional groups at the monolayer surface.76, 83-85 Unfortunately, dynamic

studies of alkanethiols on gold surfaces have revealed several serious disadvantages of

the thiol SAMs particularly of the concern of their thermal instability,86, 87 influence of

UV photoxidation88 and evidence of the changing structures over time.86, 88 Other lesser

concerns include the defect mobility behaviour, SAM pattern stability, stability in

solution, gold etching and adsorbate-solution interchange.84, 85, 89 It was confirmed that

alkanethiolates in SAMs on gold can be oxidised extensively in air and in the dark to

18


form sulfates and sulfonates.85, 88 All these critical issues have greatly limited the

possible commercial applications involving SAMs on gold surfaces. It would therefore

be desirable to develop an alternative substrate which is more stable and more

compatible with self-assembled monolayers, and then hopefully some attractive

applications of gold–thiol chemistry can be transferred to this substrate.

1.5.2 Self-Assembled Monolayers on Glassy Carbon Surfaces

Many researchers have proved that modification of glassy carbon (GC) surface is an

important objective in material science and electrochemistry due to the key advantages

with carbon materials,90 such as low cost, rich surface chemistry, wide potential range,

and compatibility with a variety of electrolytes. Thus modification of GC electrodes

with self-assembled monolayers of diazonium salts by electrochemically reductive

adsorption might be an alternative system to SAMs of alkanethiols on gold surfaces for

sensing construction. Before investigating this possibility, it is important to understand

the constitution of the GC surfaces.

1.5.2.1 Glassy Carbon Surfaces

Glassy or vitreous carbon is an attractive addition to the growing list of solid carbon

electrodes that are now available to electrochemists.91 Glassy carbon firstly named by

Yamada and Sato in 1962,92 is typically a hard, solid carbon material and is produced by

thermal degradation of selected organic polymers, such as resins of furfuryl alcohol,

phenol formaldehyde, acetone-furfural, or furfural alcohol-phenol copolymer in an inert

atmosphere.93 The formation of final structure of glassy carbon has been extensively

studied by Jenkins and Kawamura by means of X-ray diffraction, infrared spectroscopy,

and the determination of the hardness.94 They concluded from their studies that glassy

19


carbon is made up from aromatic ribbon molecules which are oriented randomly and are

tangled in a complicated manner as presented in Figure 1.6.95 According to Figure 1.6,

a substantial porosity exists in glassy carbon, and in this representation the structure

consists of long and randomly oriented microfibrils that twist, bend, and interlock to

form strong interfibrillar bonds. So its structure contains a significant volume of closed

voids, which accounts for its low density and low gas permeability. Glassy carbon has

many desirable properties for electrodes because it is impermeable to gases, highly

resistant to chemical attack, electrically conductive and available in relatively high

purity.96 In addition, glassy carbon electrodes have the widest potential range of the

many carbon electrodes or other solid electrodes.97 A wide potential window is very

important in electrochemistry,90 which gives that GC potential applications in

chemically modified electrodes.

Figure 1.6 Schematic representation of the structural model for glassy carbon.95

1.5.2.2 Modification of Glassy Carbon Electrodes with Stable Self-Assembled

Monolayers

Traditional pathways for modifying carbon surfaces involve coating the surface with a

polymer film98 or carbon surface oxidation,99 thus leading to the generation of

superficial carboxylic, quinonic, ketonic, or hydroxylic groups that can further react

20


with the substance to be attached.100, 101 The exact nature and number of oxygenated

functional groups thus formed are difficult to ascertain and control, and corrosion of the

carbon surface is often observed leading to undesirable large background currents in

electrochemical applications. Fortunately, electrochemically assisted covalent

modification of GC surfaces has been introduced to bind moieties directly to the carbon

lattice in past decade with many advantadges.102 Covalent modification of carbon

electrodes via electrochemically reductive adsorption of aryl diazonioum salts has been

explored by a few groups,102-120 which has proved to be the more simple, flexible and

promising surface modification strategy. The acceptance and application of this method

by a number of researchers is primarily due to the ease with which diazonium salts

bearing a wide range of functional groups can be synthesised, as well as the structure

and stability of the resulting layer.107 The attractiveness of aryl diazonium salts are

enhanced further by recent studies showing they can also be grafted to a variety of

metal121-128 and semiconductor129-133 surfaces as well as organic materials.134, 135 This

feature raises the exciting possibility of one monolayer forming system being suitable

for a large range of electrode materials for a diverse range of applications.

The mechanism of the electrografting of diazonium salts has been extensively reported

in the literature.125, 136 The binding of aryl groups to carbon electrodes is believed to be a

two-step process as shown in Scheme 1.1 which involves i) the electrochemical

reduction of the diazonium function with the formation of a phenyl radical, and ii) the

chemical grafting of the radical at the surface of the electrode with the formation of a

covalent carbon-carbon bond between a surface of the substrate and the phenyl group. It

is worth mentioning that the terminal functional R group can be versatile (i.e., a large

21


number of diazonium salts with different R functional groups can be synthesised),

which greatly broaden applications of this modification method in surface engineering.

+N2 R N2 RGC.e-

GCGC R+

Scheme 1.1 Schematic of covalently attached alkyl monolayers onto GC surfaces

The design of different head groups of monolayers by a large number of electroactive or

non-electroinactive functional groups on GC surfaces makes this functionalisation

strategy especially useful for biosensor applications.137 Functionalised phenyl films

have also been utilised in a number of fundamental studies at carbon electrodes

including investigations of electrochemical reactions of surface bound layers,104 long-

range electron tunnelling studies,138 the linking to biomolecules,139, 140 the bonding of

gold nanoparticles,141 and the limitation of protein adsorption.142 Considering the

number of studies exploiting this attachment scheme thus far, it is clear that the use of

this method to control the chemistry of carbon surfaces will become more widespread.

Thus, a complete understanding of the deposition and structure of these films is required

for their successful applications in sensing.

In order to understand the nature of the organic layers obtained with diverse diazonium

salts on carbon surfaces, different characterisation methods have been used, such as

cyclic voltammetry, X-ray photoelectron spectroscopy (XPS),104, 107, 108, 112 atomic force

microscopy (AFM),114 vibrational spectroscopy (polarisation modulation infrared

reflection adsorption spectroscopy (PMIRRAS),107 Raman spectroscopy,110, 143

rutherford backscattering (RBS),107 energy dispersion spectroscopy (EDS),120 and time-

22


of-flight secondary ion mass spectroscopy (ToF-SIMS).119 The results of these different

techniques leave little doubt about the presence on solid–state substrates of the aryl-R

groups bearing the R-substituents of the starting phenyldiazonium salt. In addition,

clarifying the character of the bond between the organic layer and substrate has attracted

much attention. The first indication is the strength of this bond on carbon which resists

ultrasonic cleaning in a variety of solvents and is stable for a month in ambient

conditions.107 The XPS observation,107 ToF-SIMS data119 and electrochemistry results

obtained by McCreery’s group144-147 clearly support the existence of a covalent bond

excluding a mere physisorption between carbon and the organic group.

Although the presence of an organic layer on the carbon surface and the existence of the

strong covalent bonding produced by electrical reduction of aryl diazonium salts have

been thoroughly characterised, there still exist divergent results on the modification

layer thickness. Some groups estimated the surface coverages of modified organic

layers by integration of cyclic voltammograms, Raman and RBS signals,110, 112 and

reported monolayers had been achieved. Monolayers also have been observed using

AFM by McCreery on pyrolyzed photoresist at certain conditions.115 However

multilayers have been observed using scanning probe microscopy in the reduction of

diethylaminophenyldiazonium ion by Kariuki and McDermott.103, 114 Downard obtained

four layers of 4-nitrophenyl groups on pyrolysed photoresist films by AFM.148 The

theoretical surface coverage of nitrophenyl groups was calculated to be 12 × 10-10 mol

cm-2 for ideal monolayer close-packing.110 Thus, it is difficult to make precise

comparisons between the above results since the conditions and carbon substrates vary

from one research to another. However, an agreement that it is possible to control the

thickness of the layers by controlling the charge passed during the electrochemical

23


modification has been reached.126 Thus it is clear that derivatisation conditions, such as

electrolysis time, grafting potential, the type of carbon substrate, and the nature of the

diazonium salt and its concentration, are critical to producing a monolayer without

progressing to multilayer films. The low concentrations ( 1 mM) and relatively short

electrolysis times are generally suitable for the monolayer formation on GC.115 In

general, it is applicable to form self-assembled monolayers of diazonium salts on carbon

substrates.

Based on above introduction, it can be realised that modification of GC surfaces with

SAMs of diazonium salts can serve as an excellent alternative to SAMs of alkanethiols

on gold substrates. Thus covalent modification of GC surfaces by electrically reductive

adsorption of aryl diazonium salts to form stable SAMs provides the desirable strategy

for construction of sensing interface. This strategy will be used throughout this thesis

for fabrication of stable sensing interface.

1.6 Creating the Sensing Interfaces with the Ability to Resist Non-Specific

Adsorption

Non-specific binding is a general problem though associated with biosensors as there is

no discrimination between the measured signal from specific and non-specific

interactions. Non-specific adsorption is an interfacial process and appears to correlate

with the hydrophobicity of the surface. SAMs are therefore important in understanding

and controlling proteins adsorption because they are model systems where the surface

properties are well defined and can be easily altered in a known way. For biosensing

applications, the chemical properties of the SAM surface must be tailored such that the

24


surface presents functional groups that will specifically bind to proteins of interest while

rejecting all other proteins.

Considerable effort has been expended in efforts to create surfaces that minimise the

non-specific adsorption of proteins by masking the surfaces with blocking agents such

as bovine serum albumin149 or by tailoring the end groups on the sensing interface.150

The most widely used and successful strategy is to form SAMs tailed with hydrophilic

end groups such as poly(ethylene glycol).151 Poly(ethylene glycol) (PEG) as shown in

Figure 1.7, is an important molecule with a number of unique properties, such as

biocompatibility, simple chemical nature, low toxicity, non-immunogenicity, and high

water solubility, and has attracted considerable attention for a lot of applications.152, 153

N

N-

O

O

O

O

17.5 Å

N

N-

O

O

O

O

17.5 Å

Figure 1.7 The structure of PEG molecules synthesised for the studies in this thesis.

The PEG head group has been shown to resist non-specific adsorption of a number of

proteins with a range of molecular weights and isoelectric points under a wide range of

solution conditions.150, 154-157 Although several theories have been proposed to explain

the resistance of these PEG-coated surfaces,158-160 the underlying mechanism for this

25


anti-nonspecific binding problem remains unclear. With the widely accepted agreement

that PEG has the ability to resist protein adsorption, it is hence applicable to introduce

PEG molecules to sensing interface for solving the non-specific binding problem with

biosensors.

1.7 Using Molecular Wires to Establish Efficient Electron Transfer on Sensing

Interfaces

For sensing applications, it is critical to establish the efficient electrical communication

between biorecognition components and the surrounding environments through a linker,

which should be concluded on the self-assembled monolayers, the platform for sensing.

Most importantly, the linker should have high efficiency for electron transfer in order to

provide efficient response time in sensing. Basically, this linker should be rigid allowing

it to stand free in space above a surface, and be sufficiently long to prevent close

proximity between the electrode surface and redox-active centre, thereby minimising

undesirable interactions between the two sites. This linker, however, should not be too

long because long chain molecules provide substantial barrier properties to electron

transfer and are strongly resistant to ion penetration.161 Thus in order to establish the

efficient electron transfer between biomolecules and the surrounding environment, it is

critical to design a rigid linker molecule with easily tailored lengths and high efficient

electron transfer.

Molecule wires consisting of a molecular chain with extended electronic conjugation

that would promote strong coupling between the two groups (molecules, electrodes, or

other entities) attached to the chain ends, such as oligo(phenyl enevinylene) bridges,162

norbornylogous163, 164 bridges and oligo(phenyl ethynylene)137, 165-167 bridges have the

26


important advantages of being structurally rigid, giving well-defined molecular

architectures and efficient electron transfer. Thus molecular wires are essential for the

need to form the efficient electrical communications between the biological molecules

and the electrode.

1.7.1 Using Oligo(phenyl ethynylene) Bridges to Facilitate Electron Transfer between

Biomolecules and Sensing Interfaces

Molecular wires (MW) based on oligo(phenyl ethynylene) derivatives have been

extensively studied by several research groups.165, 168-172 These kind of molecular wires

have demonstrated great potential in the development of molecular electronics due to its

high electron transfer efficiency.173-177 Tour and coworkers178, 179 have contributed

significantly to the synthesis of conjugated molecule wires with precise length and

constitution. With its completely rigid property and the easily tailored lengths,

functional groups, steric configuration, and the high efficiency for electron transfer,

Thus, MW of oligo(phenyl ethynylene) bridge as shown in Figure 1.8 is a potential

linker to achieve direct electron transfer between biomolecules and electrodes.

When the MW with terminal carboxylic acid groups in Figure 1.8 was self-assembled

on electrodes to form a monolayer, a variety of redox active species possessing an

amine functionality, such as ferrocenemethylamine, glucose oxidase, flavin adenine

dinucleotide, could be covalently attached on the monolayer surface through the amine

coupling. Then, many important properties of biosensors, such as charge transfer and

the electron transfer on the sensing interface, and the working mechanism of enzyme,

can be easily investigated. In this thesis, this MW was used as the efficient electron

transfer linker for the fabrication of biosensors and immunosensors. Electrons were

27


transferred by tunnelling via multiple off-resonance state within the MW, which

effectively enhances tunnelling between the electrodes by overlap between

neighbouring states, but still results in an exponential distance dependence. Due

qualitatively to the extended nature of the electronic orbitals in conjugated structures,

the electron-transfer rates for conjugated bridges were a shallow distance dependence as

compared to aliphatic structures in which the molecular orbitals are for the most part

localised to smaller number of atoms.165 So the MW can facilitate the tunnelling of

electrons down the molecule to the underlying electrode substrates. The synthesised

molecular wires do, however, suffer the disadvantage of high cost to produce in large

quantities. Thus, it is crucial to find an alternative to molecular wires.

N

N-

NO2

COOH

20 Å

N

N-

NO2

COOH

20 Å

Figure 1.8 The structure of molecular wires synthesised for the studies in this thesis.

1.7.2 Using Single-Walled Carbon Nanotubes to Facilitate Electron Transfer

between Biomolecules and Sensing Interfaces

Single-walled carbon nanotubes (SWNTs), as shown in Figure 1.9, have attracted

increasing attention since their discovery by Iijima180, 181 due to their unique

28


structural,182 mechanical183 and electric184 properties. Carbon nanotubes being small,

rigid and simple to produce in large quantities, and having functional groups at both

ends of the tube after being cut, can be metallic185-187 or semi-conducting. Their small

size and conductivity means they can be regarded as the smallest possible electrodes

with diameters less than one nanometer.188 Basic electrochemistry of SWNTs has been

studied by a few groups.189-192 Electrodes modified with carbon nanotubes have been

shown to have outstanding electrochemical properties193-195 and show fast electron

transfer properties.196 So SWNTs have demonstrated all properties owned by molecular

wires such as being rigid, easily tailored length by cutting, having terminal functional

groups and high efficiency for electron transfer, and can be recognised as another kind

of molecular wire. To be produced in large quantities is an important advantage of

SWNTs over MW. Thus carbon nanotubes are good materials for the development of

biosensors and can serve as a good alternative to the synthesised molecular wires.

a) b)

Figure 1.9 The general structure of single-walled carbon nanotubes in (a) side view, (b)

top view.

SWNTs are known to have a number of carboxylic acid groups at each end of the tubes

after being cut in a 3:1 mixture of concentrated H2SO4/HNO3, which are responsible for

29


the good electrochemical properties.197-199 A longer cutting time produces shorter

tubes198 and more oxygenated species formed at the ends of tubes.200 The presence of

terminal carboxylic acid groups at each end of the nanotubes makes it compatible with

SAMs. If a substrate is modified with a monolayer with amine terminated groups, the

cut SWNTs with terminal carboxylate groups can be covalently attached by the amide

bond coupling. After the attachment of SWNTs, a lot of other molecules with functional

groups can be further introduced to SAMs. Thus, it is applicable to replace MWs with

SWNTs for sensing construction.

1.8 Aims of the Thesis

The purpose of this thesis is to fabricate GC electrodes, as an alternative to gold

substrates, by covalent attachment of a variety of aryl diazonium salts with different

functional groups for creating stable and versatile monolayer systems for the

development of biosensors. Another very interesting aspect in this thesis is to introduce

the PEG molecules as an insulator and molecular wires as the electron transfer linker to

the sensing interface for sensing construction. The PEG insulator has the ability to

reduce the non-specific protein adsorption and the MW is a good conduit with high

efficiency of electron transfer. This thesis will attempt to achieve direct electron transfer

to GOx by using molecular wires to build the connection to the redox centre via the

substrate channel such that a close approach can be achieved between the isoalloxazin

ring of FAD and the conduit for electron transfer. This thesis will also attempt to

construct a sensing interface consisting molecular wire and PEG for fabrication a label-

free immunosensor which can overcome the problem of non-specific adsorption with

current immunosensors. Inspired by the unique properties possessed by SWNTs,

30


another similar label-free immunosensor will also be investigated by replacing MWs

with SWNTs.

1.9 Overview of Chapters

The general organisation of the thesis is as follows:

Chapter 2–Experimental Procedures and Instrumentations

This chapter gives a general description of the experimental techniques,

instrumentations, chemicals, reagents and synthesis of a few compounds.

Chapter 3–Covalent Modification of Electrode Surfaces by Electrochemical

Reduction of Aryl Diazonium Salts

The strategy of covalent modification of electrode surfaces by electrochemical reduction

of aryl diazonium salts with different functional groups to form stable monolayers has

been studied. The step-by-step modifications were characterised by electrochemistry

and XPS.

Chapter 4–Heterogeneous Electron Transfer Through Organic Monolayers on

Carbon and Gold Electrodes

The monolayer modified electrode surfaces with the terminal carboxylic acid groups

can be covalently attached with the redox probe ferrocenemethylamine by the amine

binding. The rates of the heterogeneous electron transfer for ferrocene through organic

monolayers on glassy carbon electrodes, pyrolised photoresist films and gold surfaces

were studied and discussed.

31


Chapter 5–Fabrication of Electrochemical Copper Sensors Based on Gly-Gly-His

Modified Carbon Electrodes

The stable monolayers formed by reductive adsorption of aryl diazonium salts on GC

electrodes were further tested by covalent modification of tripeptide Gly-Gly-His to 4-

carboxyphenyl modified carbon surfaces for the sensitive detection of a metal ion, Cu2+.

The detection limits of copper for the fabricated systems are presented and discussed in

this chapter. The fabricated Gly-Gly-His copper sensor was found to be very stable.

Chapter 6–An Interface Comprising Molecular Wires and Poly(ethylene glycol)

Spacer Units Self-Assembled on Carbon Electrodes for Studies of Protein

Electrochemistry

In this chapter, a generic interface composed of a mixed monolayer of

oligophenylethynylene MW and PEG is deposited on GC electrodes by reductive

adsorption of the respective aryl diazonium salts. PEG with the ability to reduce the

non-specific protein adsorption was demonstrated. The ability of the MW to facilitate

efficient electron transfer through the PEG layer to the underlying electrode was also

investigated by covalently attaching ferrocene methylamine to the end of the MW.

Direct electron transfer between HRP and underlying GC electrodes through MW has

been studied.

Chapter 7–Exploration of Deeply Buried Active Sites of Glucose Oxidase Using

Molecular Wire Self-Assembled on Carbon Electrodes

The efficient electron transfer of MW is further demonstrated by fabrication of a third

generation glucose biosensor using the synthesised MW as the electron transfer linker.

The electron transfer between the active centre FAD of GOx and the underlying GC

32


electrode through MW has been discussed. The biocatalytical activity for the fabricated

biosensors was also investigated.

Chapter 8–Development of a Label-Free Immunosensors on Molecular Wire

Modified Glassy Carbon Surfaces

A novel innunosensor system is developed on the MW and PEG modified GC interface.

The so-formed system can be used to detect anti-biotin based on the biotin-antibody

biorecognition to a low detection limit. In addition, the displacement assay has shown

the free biotin can compete with the attached biotin for binding antibiotin. Thus the so-

fabricated novel immunosensor system can be used for detection of biological analytes.

Chapter 9–Towards the Fabrication of Immunosensors Using SWNTs as the

Conductor

In this chapter the chemical modification of SWNTs is described together with their

attachment onto a GC electrode as an alternative to MW acting as the electron transfer

linker. The rate of electron transfer through the attached SWNTs was discussed. An

immunosensor incorporated with SWNTs has also been fabricated.

Chapter 10– Conclusions and Future Directions

In this chapter general conclusions are made and future directions are also discussed.

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46

Chapter 2-Experimental Procedures and Instrumentations

Chapter Two

Experimental Procedures and Instrumentation

47


This chapter describes the reagents, chemicals and solutions, the instrumentation, and

the general experimental procedures involved in this project. The synthesis of some

molecules which are commercially unavailable are also presented in this chapter.

2.1 Chemicals, Reagents and Solutions

The majority of the chemicals used in the electroanalytical experiments were of

analytical reagent grade and they were used without further purification except where

specified. All the aqueous solutions were prepared in Milli-Q water (18 M cm,

Millipore, Sydney, Australia). All the chemicals used in this PhD project are listed in

the Table 2.1.

Table 2.1 The chemicals used in this PhD project.

Chemical Formula Grade Source

Bovine Serum Albumin (BSA) ---- 98% Sigma

Microperoxidase (MP-11) ---- 90% Sigma

Horseradish peroxidase (HRP) ---- ---- Sigma

Glucose oxidase (GOx) ---- ---- Sigma

G-Glucose C6H6O6

A.C.S

reagentAldrich

Sulfo-NHS-Biotin ---- ---- Pierce

Anti-biotin from goat ---- ---- Sigma

Anti-pig IgG ---- ---- Sigma

Free biotin (Vitamin H) ---- ---- Sigma

l-Tryptophan ---- ---- Sigma

Argon Ar ---- Linde

Gold wire (1.0 mm diameter) Au >99.99% Aldrich

48



Silicon wafer Si ----Silicon Quest

International(USA)

N-(2-Hydroxyethyl)piperazine-

N’-(2-ethanesulfonic acid)

(HEPES)

C8H18N2O4S >99.5% Sigma

1,3-Dicyclohexylcarbodiimide

(DCC)C6H11N=C=NC6H11 99% Aldrich

Tri(ethylene glycol) monomethyl

etherC7H16O4 95% Aldrich

Aniline C6H5NH2 99% APS

1-Ethyl-3-(3-dimethyl

aminopropyl) carbodiimide

hydrochloride (EDC)

C8H17N3 98% Sigma

Methyl 4-iodobenzoate C8H7NO2 98% ACROS Organics

4-Nitrophenol C6H5NO3 99% Aldrich

N,N-diisopropylethylamine C8H19N 99.5% Aldrich

Tetrabutylammonium

tetrafluoroborateC16H36N 99% Aldrich

4-Aminophenylacetic acid C8H9NO2 98% Aldrich

4-Ethynylaniline C8H7N 97% Aldrich

2,5-Dibromonitrobenzene C6H3Br2N 99% Aldrich

1-Hexadecanethiol (HDT) C16H34S >95% Aldrich

3-Mercaptopropionic acid (MPA) C3H6O2S 99% Aldrich

4-Mercaptobenzoic acid C7H5O2S 97% Aldrich

Tetramethylene sulfone C4H8SO2 99% Aldrich

49



Chloroform-d CDCl3 99.9% D Aldrich

Cysteamine C2H7NS ---- Sigma

Deuterium oxide D2O 99.9 % D Ajax Finechem

Acetonitrile-d3 CD3CN 99.8% D CIL

Dichloromethane CH2Cl2 99.8% Sigma

Dimethyl sulphoxide-d6 (DMSO) (CD3)2SO 99.9% CIL

Dimethylacetylenedicarboxylate C6H6O4 98% ACROS

Ethanol CH3CH2OH 70% (W/W) Ajax Finechem

Methanol CH3OH A. R. APS

Diethyl Ether (CH3CH2)2O 99% Ajax Finechem

Ethylacetate CH3CH2OOCCH3 99.5% APS

Ethyleneimine CH2CH2NH 98.8% Aldrich

Ferrocenecarboxylic acid C11H10FeO2 98% Fluka

Ferrocene carboxaldehyde C11H10FeO 98% Aldrich

Ferrocene dicarboxaldehyde C12H10FeO2 98% Aldrich

Flavin adenine dinucleotide

(FAD)C27H31N9O15P2Na2 >94% Sigma

N-hydroxysuccinimide (NHS) C4H5NO3 98% Sigma

Tetrahydrofuran (THF) C4H8OAnalytical

reagentAPS Finechem

N,N-Dimethylformamide (DMF) HCON(CH3)2 99.9% Aldrich

(Trimethylsilyl)acetylene C5H10Si 98% Aldrich

50



Acetonitrile CH3CN 99.9% Ajax Finechem

Ammonium acetate C2H7NO2 97% Ajax Finechem

Glycyl-Glycyl-Histidine

(Gly-Gly-His)C10H15N5O4 ---- Sigma

2-(N-morpholino) ethanesulfonic

acid (MES) C6H13NO4S 99.5% Sigma

Single walled carbon nanotube

(SWNT)C ----

Carbon

Nanotechnologies

Inc USA

Pyridine CDCl3 99.9% D Aldrich

Copper(I) iodide CuI 98% Aldrich

Copper(II) sulfate hydrate CuSO4 >99% APS

Hydrochloric acid HCl 32% APS

Sulphuric acid H2SO4 95% APS

Nitric acid HNO3 70% APS

Perchloric acid HClO4 70% Aldrich

Hydrogen peroxide H2O2 30% Aldrich

Dipotassium hydrogen

phosphateK2HPO4 99.9% Ajax Finechem

Potassium dihydrogen

phosphateKH2PO4 >99% Aldrich

Potassium ferricyanide K3Fe(CN)6 99% M&B

51



Hexaamineruthenium ( )

chlorideRu(NH3)6Cl3 98% Aldrich

Potassium hydroxide KOHAnalytical

reagentAPS

Potassium chloride KCl >99% Aldrich

Lithium hydroxide LiOH >98% Aldrich

Lithium aluminium hydride LiAlH4 >99% Aldrich

Sodium nitrite NaNO2 97% APS

Sodium chloride NaClAnalytical

reagentAPS

Sodium tetrafluoroborate NaBF4 98% Aldrich

Tetrabutylammonium

tetrafluoroborateNBu4BF4 99% Aldrich

Nitrosonium tetrafluoroborate NOBF4 ---- Aldrich

Sodium hydroxide NaOHAnalytical

reagentAPS

Sodium sulphate NaSO4

Analytical

reagentAPS

Sodium cyanoborohydride NaBH3(CN) 95% Aldrich

Dichlorobis(triphenylphosphine)-

palladium(II)---- 99.99% Aldrich

Palladium on activated carbon Pd ---- Aldrich

52


2.2 Synthesis

Some molecules used in this project were synthesised because they are not

commercially available.

2.2.1 Synthesis of 4-Carboxyphenyl Diazonium Tetrafluoroborate and 4-Nitrophenyl

Diazonium Tetrafluoroborate

The synthesis of 4-nitrophenyl diazonium tetrafluoroborate and 4-carboxyphenyl

diazonium tetrafluoroborate were carried out following the method developed by

Saby et al.1 The corresponding aniline (0.01 mol) was dissolved by warming into 3 mL

of concentrated hydrochloric acid (10 M) and 12 mL of water. A precipitate was

obtained by cooling down to 0 oC in an ice/acetone bath. This precipitate disappeared

after slow addition of a solution containing 0.752 g of sodium nitrite (0.011 mol) in

2 mL of water with vigorous stirring. The solution was filtered, and 1.48 g (0.013 mol)

of sodium tetrafluoroborate was added with stirring. The thick slurry was cooled below

0 oC in an ice/acetone bath, filtered by suction, and washed with a cooled 5% NaBF4

solution to remove traces of acid and then washed with cold ether. The powder was

dried in vacuum. Recrystallisation was carried out with a mixture of acetonitrile and

cold ether to give the expected product. The diazonium salt was kept in a desiccator, at

4 oC over phosphorus pentaoxide. 1H NMR (300 MHz, CD3CN) for 4-carboxyphenyl

diazonium tetrafluoroborate: 8.42 (d, J 9.1 Hz, 2H), 8.58 (d, J 9.1 Hz, 2H). 1H NMR

(300 MHz, CD3CN) for (4-nitrophenyl) diazonium tetrafluoroborate: 8.62 (d, J 4.7 Hz,

2H), 8.75(d, J 9.4 Hz, 2H). The diazonium function was detected by IR spectroscopy at

about 2300 cm-1.

53


2.2.2 Synthesis of Benzenediazonium Tetrafluoroborate

The benzenediazonium tetrafluoroborate was synthesised by following the procedures

described by Downard et al.2 Aniline (0.49 mL, 5 mmol) was added to 2.5 mL of 35%

fluoroboric acid prediluted with 1.5 mL of water, and the solution was then cooled in

the 0 oC ice/acetone bath. A solution of 0.346 g (5 mmol) of sodium nitrite in 0.7 mL

water was added slowly to the stirred solution maintaining the temperature at around

10 oC. The mixture was then cooled to below 0 oC in an ice/acetone bath. The cream

precipitate was collected on a sintered glass filter which had been cooled with ice water

and washed with cold 5% fluoroboric acid (1 mL) followed by cold ether. The product

was dried in vacuo and stored in a fridge in the dark in a vacuumed desiccator. 1H NMR

(300 MHz, CD3CN): 7.94 (d, J 1.1 Hz, 2H), 8.26 (s, 1H), 8.46 (d, J 1.1 Hz, 2H).

2.2.3 Synthesis of Ferrocenemethylamine

Synthesis of ferrocenemethylamine was carried out by following the procedure

described by Kraatz.3 To a cooled solution (0 oC) of ferrocenecarboxaldehyde (2.14 g,

10 mmol) in MeOH (50 mL) in an ice bath (0 oC) was added ammonium acetate (7.7 g,

0.1 mol), and the mixture was stirred for 2 h. NaBH3(CN) (0.46 g, 7.3 mmol) was then

added to the cold solution. After stirring for 12 h, the volume of the solution was

reduced to about 20 mL and water (10 mL) was added. The pH of the solution was

adjusted to pH 2.0 with 1 M HCl, after which the pH was adjusted to 8.0 by adding

solid KOH (about 2 g). The resulting dark-brown solution was extracted with CH2Cl2

(4×50 mL). The organic phases were collected and washed with H2O (3×50 mL), dried

over anhydrous Na2SO4 and then pumped to dryness to yield a brown oily solid (2.0 g).

The resultant material was redissolved in MeOH (10 mL), and 1 M HCl (5 mL) was

54


added and then pumped to dryness again. The crude product was dissolved in a

minimum amount of MeOH and transferred onto an alumina plug (ca. 5 cm long). The

impurity was removed with CH2Cl2 as eluent, and the product was eluted with MeOH.

After evaporation of the solvent, a yellow solid (1.8 g, 72%) was obtained. 1H NMR

(300 MHz, CDCl3): 2.1 (s, 3H), 3.88 (s, 2H), 4.17 (t, 5H), 4.22 (d, J 4.3 Hz, 2H), 4.28

(d, J 8.6 Hz, 2H).

2.2.4 Synthesis of 1,1’-Ferrocenedimethylamine

Synthesis of 1,1’-ferrocenedimethylamine was carried out by following the procedure

by Ossola.4 1,1’-ferrocenedicarboxyaldehyde (0.4 g, 1.65 mmol) was dissolved in EtOH

(8 mL) and the resulting solution was heated at 50 oC. An aqueous solution of

hydroxylamine hydrochloride (0.8 g, 6.76 mmol) and an aqueous solution of sodium

acetate (0.9 g, 11.2 mmol) were added. The resulting mixture was stirred at 50 oC for

4 h and evaporated. The residue was dissolved in diethyl ether, and the resulting

solution was filtered and evaporated to dryness. The reddish solid residue (0.8 g,

2.94 mmol) was dissolved in dry THF (25 mL). A 1 M diethyl ether solution of LiAlH4

(9 mL) was added dropwise to the resulting solution and the mixture was stirred

overnight. Benzene was added (90 mL) and the mixture stirred for 15 min. Ethyl acetate

was added and the mixture stirred for 15 min. Ten drops of 5 M NaOH solution were

added, the mixture stirred for 15 min and filtered. The solution was evaporated, the

residue dissolved in CH2Cl2, the solution filtered and evaporated. The residue was

purified by column chromatography (silica gel, 95% MeOH and 5% NH4OH as eluent)

to give the desired product as a light yellow microcrystalline solid. 1H NMR (300 MHz,

CDCl3): 1.99-1.96 (brs, 6H), 3.58 (s, 2H), 3.82 (s, 2H), 4.08 (s, 8H),

55


2.2.5 Synthesis of Molecular Wires

The synthetic strategy for the MW 5 is depicted in Scheme 2.1 and is based upon

protocols previously published by Tour and coworkers5, 6 with some modification. The

main difference to the MWs synthesised by Tour and coworkers5, 6 is that in this work a

terminal carboxylic acid group was introduced at the distal end of the MW. This is

necessary for the coupling of the proteins to the MW once they were assembled onto the

electrode surface. The detailed synthetic procedures are presented below.

4-Ethynylaniline

Pd(PPh3)2Cl2, CuI, iPr2NEt, THF, rt, 4 h(47%)

Pd/Cu, iPr2NEt, THF, 75 oC, 72 h(81%)

LiOH

MeOH, CH2Cl2, H2O, rt, 72 h(73%)

CH3CN, sulfolane, -40 oC(68%)

1

3

4

2

TMS

K2CO3

MeOH, CH2Cl2, rt, 2 h(95%)

Pd/Cu, iPr2NEt, THF, 60 oC, 24 h(95%)

2

2a

NH2Br

NO2

BrBr

NO2

NH2

NO2

COOMe

MeOOC

NH2

NO2

HOOC

5

N2+BF4

-

NO2

HOOC

IMeOOC TMSMeOOC

MeOOC

NOBF4

Scheme 2.1 The synthetic scheme of molecular wires using the stepwise procedure.

56


2.2.5.1 Synthesis of 1-Bromo-3-nitro-4-(4-aminophenylethynyl)benzene (1)

Compound 1 was synthesised using the method of Kosynkin et al.5 by following the

general procedure for the Pd/Cu-catalysed coupling reaction,7-9 but with substitution of

triethylamine for diisopropylethylamine as the only modification. 1,4-Dibromo-2-

nitrobenzene (2.81 g, 10.0 mmol), bis(triphenylphosphine) palladium(II) dichloride

(0.07 g, 0.10 mmol), copper(I) iodide (0.019 g, 0.10 mmol) were added to an oven-dried

round bottom flask equipped with a magnetic stirrer bar. The vessel was then sealed

with a rubber septum, evacuated and backfilled with nitrogen. Dry THF (5 mL) was

added and followed by diisopropylethylamine (5 mL). After 5 min at room temperature,

4-ethynylaniline (0.590 g, 5.0 mmol) was added and the reaction mixture was stirred at

room temperature for 4 h or until complete reaction was noted by TLC analysis. The

reaction was quenched with water (20 mL). The organic layer was diluted with

dichloromethane (10 mL) and washed with a saturated solution of NaHCO3 (3×50 mL)

and then brine (3×50 mL) until the blue colour of copper complexes could not be seen

in the aqueous phase. The combined aqueous phases were extracted with

dichloromethane (3×100 mL). The combined organic extracts were dried over

anhydrous NaSO4, filtered and the solvent removed in vacuo. The crude red product

was then purified by column chromatography (silica gel, 70% dichloromethane and

30% light petroleum as eluent) to give the desired product 1 as a red solid.

Recrystallization of the major fraction from dichloromethane/light petroleum afforded

the title compound 1 as bright red fine needles (0.75 g, 47%). m.p. 156-158 oC (lit.5

m.p. 147-149 oC). 1H NMR (300MHz, CDCl3): 3.88 (brs, 2H), 6.64 (d, J 8.3 Hz, 2H),

7.40 (d, J 8.7 Hz, 2H), 7.53 (d, J 8.4 Hz, 1H), 7.68 (d, J 1.9 Hz, 1H), 8.21 (d, J 1.9 Hz,

57


1H). 13C NMR (300MHz, CDCl3): 82.8, 100.2, 111.1, 114.3, 114.6, 118.5, 120.6,

127.7, 133.7, 135.1, 135.8, 147.8, 149.2.

2.2.5.2 Synthesis of Methyl 4-(trimethylsilylethynyl)benzoate (2a)

Compound 2a was synthesised previously by Tour et al.6 Methyl 4-iodobenzoate

(5.240 g, 20 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.702 g, 1 mmol),

copper(I) iodide (0.038 g, 2 mmol), trimethylsilylacetylene (4 mL, 26 mmol),

diisopropylethylamine (14 mL, 80 mmol) and dry THF (50 mL) were treated by the

general procedure for the Pd/Cu-catalysed coupling reaction7-9 at 60 C for 24 h to

afford an orange solid. Column chromatography (silica gel, 50 dichloromethane and

50 light petroleum as eluent) afforded the desired product 2a (4.40 g, 95 ) as orange

needles. 1H NMR (300MHz, CDCl3): 0.26 (s, 9H) 3.91 (s, 3H), 7.51 (d, J 8.7 Hz, 2H),

7.96 (d, J 8.3 Hz, 2H). 13C NMR (300MHz, CDCl3): 0.0, 52.1, 97.6, 104.0, 127.7,

129.3, 129.6, 131.8, 166.4.

2.2.5.3 Synthesis of Methyl 4-ethynylbenzoate (2)

Compound 2 was synthesised by following the general procedure of Tour et al. for the

deprotection of trimethylsilyl-protected alkynes.6 The silylated alkyne compound 2a

(0.81 g, 3.5 mmol) was dissolved in MeOH (50 mL) and the cosolvent dichloromethane

(50 mL), and then potassium carbonate (2.42 g, 17.5 mmol) was added. The mixture

was stirred at room temperature for 2 h then added to water (50 mL) to quench the

reaction. The mixture was extracted with diethyl ether (2×50 mL) and then the

combined organic extracts were washed with brine (3×50 mL). The combined organic

extracts were then dried over anhydrous NaSO4, filtered and the solvent was evaporated

58


in vacuo to afford the desired product 2 (0.53 g, 95 ) as brown crystal that was

immediately reacted in the next step without further purification.

2.2.5.4 Synthesis of Methyl 2'-nitro-4, 4 -diphenylethynyl-4 -aminobenzoate (3)

Compound 3 was synthesised in an adaptation of the general procedure for the Pd/Cu-

catalyzed coupling reaction.7-9 Bromide 1 (0.174 g, 0.55 mmol), alkyne 2 (0.12 g, 0.72

mmol), copper(I) iodide (0.002 g, 0.011 mmol), bis(triphenylphosphine)palladium(II)

dichloride (0.016 g, 0.022 mmol), diisopropylethylamine (0.36 mL, 2.08 mmol) and dry

THF (15 mL) were heated at 75 C for 3 days. The crude product was purified by

column chromatography (silica gel, dichloromethane as eluent) to give the product 3 as

red solid (0.176 g, 81 ). 1H NMR (300MHz, DMSO): 3.86 (s, 3H) 6.68 (d, J 8.3 Hz,

2H), 7.24 (d, J 8.7 Hz, 2H), 7.85 (dd, J 8.6, 3.0 Hz, 4H), 8.00 (d, J 8.3 Hz, 2H), 8.28 (s,

1H). 13C NMR (300MHz, DMSO): 54.4, 85.1, 92.0, 93.6, 104.2, 108.7, 115.7, 120.7,

123.1, 128.2, 129.5, 131.5, 131.9, 133.9, 135.5, 136.0, 137.7, 150.6, 152.9, 167.6.

2.2.5.5 Synthesis of Methyl 2 -nitro-4, 4 -diphenylethynyl-4 -amino Benzoic Acid (4)

Carboxylic acid 4 was synthesised by an adaptation of the procedure from Corey et al.10

Ester 3 (0.058 g, 0.15 mmol) was added to a 250 mL round bottom flask equipped with

a magnetic stirring bar along with lithium hydroxide (0.02 g, 0.75 mmol), MeOH

(9 mL), dichloromethane (5 mL) and water (3 mL). The reaction mixture was allowed to

stir at room temperature for 3 days. The reaction was quenched with water (20 mL) and

extracted with dichloromethane (2×20 mL). The combined organic phases were

extracted with water (2×20 mL). The red aqueous extracts were combined and acidified

to pH 3.0 whereupon a red solid precipitated. The solid material was collected on a

fritted funnel, and the filtrate was evaporated in vacuo to afford the desired product 4 as

59


red prisms (0.042 g, 73 ). m.p 300 oC. 1H NMR (300MHz, DMSO): 4.04 (brs, 2H),

6.58 (d, J 8.3 Hz, 2H), 7.24 (d, J 8.3 Hz, 2H), 7.76 (dd, J 8.6, 3.0 Hz, 4H), 7.98 (d, J 8.3

Hz, 2H), 8.28 (s, 1H), 13.16 (brs, 1H). 13C NMR (300MHz, DMSO): 83.1, 90.0, 92.2,

102.5, 107.0, 114.1, 119.8, 122.1, 126.1, 127.8, 130.0, 132.2, 133.8, 134.4, 135.6,

136.1, 149.0, 151.2, 167.0. FTICR HRMS found m/z 405.0858, C23H14 N2O4Na+

requires 405.0846.

2.2.5.6 Synthesis of Methyl 2 -nitro-4, 4 -diphenylethynyl-4 -benzoic acid

Benzenediazonium Tetrafluoroborate (5)

Diazonium salt 5 was synthesised by following the general diazotization procedure of

Tour et al.5 NOBF4 (0.022 g, 0.172 mmol) was weighed out in a glovebox and placed in

a round bottom flask equipped with a magnetic stirring bar and sealed with a septum.

Acetonitrile (1 mL)/sulfolane (0.2 mL) were injected and the resulting suspension was

cooled in a dry ice/acetonitrile bath to -40 C. The solution of the aniline compound 4

(0.060 g, 0.156 mmol) was prepared by adding warm sulfolane (1 mL, 45-50 C) to the

amine under a blanket of nitrogen, sonication for 1 min and subsequent addition of

acetonitrile (0.2 mL). The aniline solution was then added to the nitrosonium salt

suspension over a period of 10 min. The reaction mixture was kept at -40 C for 30 min

and was then allowed to warm to the room temperature. Then the product 5 was

precipitated with diethyl ether (12 mL) and collected by filtration, washed with diethyl

ether and dried over anhydrous NaSO4. Additional purification of the salt 5 was

accomplished by reprecipitation from DMSO (0.5 mL) by dichloromethane (10 mL) to

afford the desired product 5 as a red solid (0.050 g, 68 ). 1H NMR (300MHz, DMSO):

6.56 (d, J 8.3 Hz, 2H), 7.23 (d, J 8.3 Hz, 2H), 7.74 (dd, J 8.6, 3.0 Hz, 4H), 7.96 (d, J

8.3 Hz, 2H), 8.26 (s, 1H). 13C NMR (300MHz, DMSO): 83.1, 90.0, 92.2, 102.5, 107.0,

60


114.1, 119.8, 122.1, 126.1, 127.8, 130.0, 132.2, 133.8, 134.4, 135.6, 136.0, 149.0,

151.2, 167.0. FTICR HRMS found m/z 394.0812, C23H12N3O4+ requires 394.0822.

2.2.6 Synthesis of 4-(2-(2-(2-Methoxyethoxy)ethyl)benzenediazonium

Tetrafluoroborate

The synthetic strategy for the 4-(2-(2-(2-Methoxyethoxy)ethyl)benzenediazonium

tetrafluoroborate (PEG) is depicted in Scheme 2.2 and is based upon protocols

previously published by Tour and coworkers with some modification.11 The detailed

synthetic procedures are presented below.

OO

OOTs

OO

OO NO2

OO

OO NH2

OO

OO N2

+BF4-

OO

OOH TsBr

Pyridine/CH2Cl2, -5 oC, 5 h(98%)

p-Nitrophenol

K2CO3, DMF, 80 oC,16 h(65%)

H2, Pd/C

EtOH/HCl, 60 psi, 70 oC, 12 h(73%)

CH3CN, -40 oC, 0.5 h(61%)

NOBF4

a

b

c

d

Scheme 2.2 The synthetic scheme of poly(ethylene glycol) molecule by the stepwise

procedure.

61


2.2.6.1 Synthesis of 2-(2-(2-Methoxyethoxy)ethoxy)ethyl p-Toluenesulfonate (a)

This step is slightly different from that reported in the literature.11 Tri(ethylene glycol)

monomethyl ether (1.0 g, 6.1 mmol) was added to a mixture of dry pyridine (5 mL) and

dichloromethane (10 mL), which was cooled beforehand to -5 oC. The mixture was then

cooled in an ice bath. A solution of p-toluenesulfonyl bromide (2.04 g, 9.1 mmol) in

dichloromethane (5 mL) was added slowly. The resulting mixture was maintained at –

5 oC with stirring for 5 h, and then moved into a freezer and left there overnight. The

reaction was then quenched with water (60 mL) and extracted with dichloromethane

(4×50 mL). The combined organic extracts were washed successively with 2 M HCl

(2×70 mL), saturated aqueous NaHCO3 (50 mL) and brine (50 mL), before being dried

with Na2SO4. After filtration, the solvent was evaporated in vacuo to afford the desired

pure product a as a colourless liquid (1.52 g, 98 ). 1H NMR (300 MHz, CDCl3): 2.43

(s, 3H), 3.35 (s, 2H), 3.51-3.52 (m, 2H), 3.57-3.59 (m, 6H), 3.65-3.68 (t, J 4.6 Hz, 2H),

4.13-4.16 (t, J 4.8 Hz, 2H), 7.32 (d, J 8.3 Hz, 2H), 7.78 (d, J 8.3 Hz, 2H). 13C NMR

(300 MHz, CDCl3): 21.5, 58.9, 68.2, 68.6, 69.1, 70.5, 70.6, 71.8, 127.9, 129.7, 133.0,

144.7.

2.2.6.2 Synthesis of 4-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)nitrobenzene (b)

Compound a (0.9 g, 3.53 mmol) was dissolved in dimethylformamide (50 mL).

Potassium carbonate (1.18 g, 8.5 mmol) and 4- nitrophenol (0.38 g, 2.75 mmol) were

added. The solution was stirred at 80 oC for 24 h. After cooling to room temperature, the

solution was poured into water (30 mL) with stirring for 5 min and extracted with

dichloromethane (3×30 mL). The combined organic phases were washed with water

(5×50 mL) and then brine (2×50 mL), dried over Na2SO4, and filtered, and the solvent

62


was removed by distillation at reduced pressure to afford the desired product b as a light

brown liquid (0.65 g, 65 ). 1H NMR (300 MHz, CDCl3): 3.36 (s, 3H), 3.53-3.55 (m,

2H), 3.62-3.68 (m, 4H), 3.71-3.74 (m, 2H), 3.89 (t, J 4.6 Hz, 2H), 4.22 (t, J 4.8 Hz, 2H),

6.97 (d, J 9.4 Hz, 2H), 8.17 (d, J 6.2 Hz, 2H). 13C NMR (300 MHz, CDCl3): 58.9,

68.1, 69.3, 70.5, 70.6, 70.8, 71.8, 114.5, 125.7, 141.6, 163.8.

2.2.6.3 Synthesis of 4-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)aniline (c)

Compound c (2.0 g, 7.0 mmol) was dissolved in a mixture of hydrochloric acid (2 mL)

and absolute ethanol (18 mL), and the catalyst 10 palladium on carbon (0.4 g,

0.35 mmol) was added. The mixture was hydrogenated on a Parr apparatus (60 psi,

70 oC) for 12 h with stirring. The mixture was then filtered over Celite and washed with

ethanol (30 mL). Solid sodium bicarbonate (4 g, 48 mmol) was added, and the mixture

was stirred for 2 h and then anhydrous Na2SO4 was added. After filtration, the solvent

was removed by evaporation at reduced pressure. The resulting brown oil was purified

by column chromatography (silica gel, 20 dichloromethane and 80 ethyl acetate as

eluent) to afford the pure compound c (1.3g, 73 ). 1H NMR (300MHz, CDCl3): 3.13

(brs, 2H), 3.37 (s, 3H), 3.55 (t, J 4.8 Hz, 3H), 3.63-3.68 (m, 4H), 3.70-3.71 (m, 2H),

3.81 (t, J 5.9 Hz, 2H), 4.04 (t, J 7.5 Hz, 2H), 6.63 (d, J 8.7 Hz, 2H), 6.75 (d, J 8.7 Hz,

2H). 13C NMR (300MHz, CDCl3): 58.9, 68.1, 69.8, 70.5, 70.6, 70.7, 71.9, 115.8,

116.5, 139.6, 152,1.

2.2.6.4 Synthesis of 4-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)benzenediazonium

Tetrafluoroborate (d)

Nitrosonium tetrafluoroborate (0.64 g, 4.7 mmol) was weighed out in a glovebox and

sealed with septum. After removal from the glovebox, acetonitrile (3 mL mmol-1 of

63


aniline, 12 mL) was added and the solution was cooled to –40 oC with dry

ice/acetonitrile. A solution of c (1.0 g, 3.92 mmol) in acetonitrile (ca. 1 mL mmol-1,

4 mL) was added dropwise for 30 min while stirring in dry ice/acetonitrile bath. After

complete addition, stirring was continued for 30 min, at which time the cold bath was

removed. After stirring for a total of 1 h, the solution was diluted with ether (50 mL)

and stirred to obtain a dark red, sticky residue. The residue was mixed three times with

cold ether, decanting the solvent and dried in vacuo to afford the desired product d

(1.02 g, 61 ) as a dark red, sticky material which was sufficiently pure by 1H NMR. 1H

NMR (300MHz, CDCl3): 3.29 (s, 2H), 3.46 (t, J 5.4 Hz, 2H), 3.58-3.61 (m, 4H), 3.62-

3.66 (m, 2H), 3.87 (t, J 8.7 Hz, 2H), 4.31 (t, J 8.7 Hz, 2H), 7.19 (d, J 9.5 Hz, 2H), 8.42

(d, J 9.5 Hz, 2H). FTICR MS found m/z 267.1344, C13H19O4N2 requires 267.1340.

2.3 Instrumentation

2.3.1 Electrochemical System

The instrument used in this project is mainly an electrochemical device, a potentiostat,

which was used for characterisation and mechanistic studies of redox reactions at

electrodes. Cyclic voltammetry (CV), chronoamperometry (CA), and Osteryoung

Square Wave Voltammetry (OSWV) were performed using a BAS 100B

Electrochemical Analyser (Bioanalytical System Inc., Lafayette, Inc., USA) potentiostat

interfaced with a Bootstrap Dimension Penta 12 computer system. The electrochemical

cell consisted of a three electrode system using a glassy carbon (GC) electrode or gold

electrode as the working electrode, platinum foil as the auxiliary electrode and

Ag/AgCl/3.0 M NaCl electrode as the reference electrode (from Bioanalytical Systems

Inc., USA).

64


CV is one of the most commonly used electrochemical techniques, and is based on a

linear potential waveform; that is the potential is changed linearly as a function of time

between set limits. The rate of change of potential with time is referred to as the scan

rate. The potential can be cycled between the initial potential and final potential for

several cycles before the experiment is ended at the final potential. CA is based on the

constant potential waveform. In CA, the Faradaic current is monitored as a function of

time by applying a constant potential at which the electrochemical reaction of interest

takes place. OSWV is an important variant of anodic stripping voltammetry.12 The

potential waveform for OSWV is the summation of a square wave and a staircase

waveform (Figure 2.1). The Faradaic current is sampled at the end of each half cycle,

so the current is sampled twice during each quare wave. The major advantage of OSWV

over CV is that OSWV incorporates a pulsed waveform. The sensitivity is therefore

enhanced by repeated oxidation and reduction of the same analyte species.

Figure 2.1 A square wave potential waveform applied in OSWV. E: square wave

period; E(sw): the amplitude of each half-cycle; : the cycle-time.

65


2.3.2 Nuclear Magnetic Resonance (NMR) Spectrometer

1H NMR spectra were obtained using a Bruker AC300F (300 MHz) spectrometer or a

Bruker DPX300 (300 MHz) spectrometer. Data were reported as follows: chemical shift

( ) measured in parts per million (ppm) downfield from TMS; multiplicity; proton

count. Multiplicities were reported as singlet (s), broad singlet (brs), doublet (d), triplet

(t), quartet (q), and multiplet (m). 13C spectra were obtained on Bruker AC300F

(300 MHz) spectrometer or a Bruker DPX300 (300 MHz) spectrometer. 13C chemical

shifts ( ) were reported in parts per million (ppm) downfield from TMS and identifiable

signals were given.

2.3.3 X-ray Photoelectron Spectroscopy (XPS)

X-ray photoelectron spectra were collected from glassy carbon plates (Carbon vitreous

foil version 6, Goodfellow Cambridge-Limited England) or gold foil ( 99.99 gold,

Goodfellow, Cambridge, UK) on a VG EscaLab 220-IXL spectrometer with a

monochromated Al K source (1486.6 eV), hemispherical analyzer and multichannel

detector. The spectra were accumulated at a take-off angle of 90 with a 0.79 mm2 spot

size at a pressure of less than 10-8 mbar. The pass energy for the survey scan is 100 eV

and for the narrow scan 20 eV. The step size for the survey scan is 1.0 eV and for the

narrow scan 0.1 eV. Survey spectra (0-1100 eV) were obtained, followed by high

resolution spectra of C1s, O1s, N1s regions. The spectra were generally calibrated on

the C1s Peak (285.0eV) or on the Au4f7/2 peak (84.0 eV). Atomic sensitivity factors

are C1s 1.0, O1s 2.93, S2p 1.68, N1s 1.8, Au4f7/2 9.58. Spectra were analysed using

XPSPEAK41 software. Percentage coverages for the different elements and sub-groups

were estimated from the corresponding fitted areas.

66


2.3.4 Atomic Force Microscopy (AFM)

Atomic force microscopy images were taken using a Digital Instruments Dimension

3100 scanning probe microscope. All images were acquired in tapping mode using

commercial Si cantilevers/tips (Olympus) used at their fundamental resonance

frequencies, which typically varied between 275-320 kHz. AFM images were

performed on GC plates and PPF substrates.

2.3.5 Fourier Transfer Ion Cyclotron Mass Spectrometry (FT-ICR MS)

FT-ICR mass spectra were obtained on a commercial Bruker BioAPEK 70e (Billerica,

MA) Fourier transfer mass spectrometer. This instrument is equipped with a 7 T passive

shielded superconducting magnet, a vacuum system, ion optics voltage supplies, the

external ion sources, a silicon graphics (Unix) workstation and an Infinity Cell.

2.3.6 LEO-Scanning Electron Microscope (LEO-SEM)

All the scanning electron microscope images reported here were taken on a LEO-SEM

(Supra 55VP, Zeiss) using an InLens detector. The images presented were obtained with

an accelerating voltage of 10 KV.

2.4 Procedures

2.4.1 Preparation of Glassy Carbon Electrodes and Calculation of the

Electrochemical Surface Area

Adsorbed impurities on the GC electrode surface may hinder electrochemical processes

and the formation of monolayers. So cleaning electrodes immediately prior to use is

vital. The working GC electrodes (Bioanalytical Systems Inc., USA) were 3 mm

67


diameter rods embodied into epoxy resin. The GC electrodes were hand-polished

successively in 1.0, 0.3, and 0.05 m alumina slurries made from dry Buehler alumina

and Milli-Q water on microcloth pads (Buehler, Lake Bluff, IL, USA). The electrodes

were thoroughly rinsed with Milli-Q water and sonicated in Milli-Q water for 5 min

between polishing steps. Before derivatisation, the electrode was dried with an argon

gas stream.

To ascertain the electrochemical surface area of the GC electrode, the oxidation of

Fe(CN)63-, as K3Fe(CN)6 in 1 M KCl, was studied according to the method described by

Zittel and Miller.13 At 25 oC the peak current (Ip) is related to the scan rate (mV s-1) by

the Randles-Sevick equation 2-1:

CADnI p21

21

23

5 )1069.2( 2-1

where n is the number of electrons in the reaction, A is the working area of the electrode

(m2), C is the concentration of species (mol m-3), D the diffusion coefficient (m2 s-1).

Equation 2-1 predicts that the peak current will grow proportionally to the square root of

the scan rate. The cyclic voltammogram of a GC electrode in 1 mM Fe(CN)63- solution

in phosphate buffer (pH 7.0) at a scan rate of 100 mV s-1 is shown in Figure 2.2, which

shows a well-Faradaic response. The relationship between the peak current and the

square root of the scan rate is recorded in Figure 2.3. It can be concluded that the

measured current is diffusion controlled based on the linear relationship between the

peak current and the square root of the scan rate.

68


-40

-30

-20

-10

0

10

20

30

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

Figure 2.2 Cyclic voltammogram of a bare GC electrode in ferricyanide solution

(1 mM; KCl, 0.05 M; phosphate buffer; pH 7.0).

-100

-80

-60

-40

-20

0

20

40

60

80

0 5 10 15 20 25 30 35

(Scan rate /mV s-1)1/2

Curr

ent

/A

Figure 2.3 Peak current versus the square root of the scan rate for the cyclic

voltammograms.

The actual area of a GC electrode can be calculated according to the equation 2-1 since

all the parameters are known. At room temperature the diffusion coefficient for

ferricyanide is 7.63×10-10 m2 s-1.14 The calculated area was 0.105 0.006 (95

confidence, n 5). The roughness factor of the GC electrodes with a geometrical area of

69


0.07 cm2 used in this thesis can be calculated to be 1.50 0.09 (95 confidence, n 5),

which is very close to the reported typical roughness of the GC electrodes (1.43).15

2.4.2 Derivatisation of Glassy Carbon Electrodes with Aryl Diazonium Salts

The electrochemical modification of the GC electrode was carried out via

electrochemical reductive adsorption reported previously (illustrated in Scheme 2.3).16

Before derivatisation, the freshly polished GC electrodes were dried with an argon gas

stream. Surface derivatisation of GC electrodes was carried out in an acetonitrile

solution containing 1 mM aryl diazonium salts and 0.1 M NBu4BF4 using cyclic

voltammetry at a scan rate of 100 mV s-1 for two cycles between +1.0 V and -1.0 V

versus Ag/AgCl. The aryl diazonium salt solution was deaerated with argon for at least

15 min prior to derivatisation. Then a monolayer was formed on the GC substrate.

R

GC

R

GC GC

e-+

.

R

N2+

Scheme 2.3 Schematic of electrochemical modification of GC substrates by reductive

adsorption of aryl diazonium salts.

The monolayer formed on GC surfaces can be a pure monolayer or a mixed monolayer.

A mixed monolayer can be prepared on a GC substrate by reductive adsorption of a

mixed aryl diazonium salt solution with a total concentration of 1 mM. For similar

alkanethiol molecules, the composition of the alkanethiols on the resulting surface is

70


usually quite similar to that of alkanethiols in solution used for the attachment of self-

assembled monolayers.17 It can be assumed that the composition of the diazonium salts

on the resulting surface is also similar to that of diazonium salts in solution used for

attachment of monolayers. Therefore, the composition of mixed monolayers on GC

electrodes can be adjusted by adjusting the composition of the aryl diazonium salt

mixture in solution.

2.4.3 Preparation of Pyrolysed Photoresist Films

An alternative GC electrode surface referred to as pyrolysed photoresist films (PPF) was

prepared as described previously.18 The whole preparation process is shown in Scheme

2.4. A Si (100) wafer with a protective surface film was precut into 1.4×1.4 cm2

sections, ultrasonically cleaned in successive baths of acetone, methanol, and isopropyl

alcohol to remove the protective film, and dried with nitrogen gas. A small amount of

photoresist (2-3 drops) of AZ4620 (Clariant) was spin-coated onto the wafer with a fast

acceleration rate for 30 s at 6000 rpm. The photoresist-covered wafer was soft-baked at

95 oC for 20 min and cooled to room temperature prior to application of a second layer

of photoresist, giving a final film thickness of 8.2 0.8 µm (95 confidence, n 5) by

AFM.19

The photoresist-coated wafers were placed in a furnace within a silicon glass tube, and a

forming gas atmosphere (95 nitrogen + 5 hydrogen) was flowed through the tube

during the heating and cooling phases of pyrolysis (flow rate 6 L min-1). The furnace

required several hours (ca. 10 h) to reach the maxima temperature of 1100 oC where it

was maintained ( 50 oC for 1 h. After cooling to room temperature, which typically

takes overnight, the samples were removed from the furnace and briefly sonicated (3 s)

71


in successive baths of acetone, methanol, and isopropyl alcohol, dried with nitrogen gas

and stored under a vacuum before use.

Spin coat

Protection film

Cut Wash off

Protection film

Spin coat photoresist

Soft bakePyrolysis at 1100 oC, 1 h

95% N2 + 5% H2

Si wafer

Si

Si

95 oC

7-8 m1.5 m

PPFRMS=0.4-0.6 nm by AFM

Clean

Si

Scheme 2.4 Schematic of the preparation of pyrolysed photoresist films.

2.4.4 Preparation of Gold Electrodes and Calculation of the Electrochemical Surface

Area

Gold electrodes were prepared by sealing 1.0 mm diameter polycrystalline gold wire

( 99.99 , Goodfellow, Cambridge, UK) with EPON Resin 825 and EPI-CURE

3271 curing agent, Shell Chemical Company (Houston, Texas) in glass tubes with

nichrome wires attached for electrical connection. The gold disk electrodes were

polished to a mirror-like finish successively with 1.0 m, 0.3 m and 0.05 m alumina

slurries (Buehler, U.S.A) on microcloth pads (Buehler, U.S.A). After removal of the

trace alumina from the surface by rinsing with water and brief cleaning in an ultrasonic

bath, the electrodes were further cleaned by cycling between –0.3 V and +1.5 V versus

Ag/AgCl in 0.05 M H2SO4 solution at a scan rate of 100 mV s-1 until reproducible scans

were recorded as shown in Figure 2.4 (typically 50 cycles).

72


-13

-10

-7

-4

-1

2

5

-0.5 0 0.5 1 1.5 2

Potential /V

Curr

ent

/A

Oxidation of gold

Reduction of gold oxide

Figure 2.4 The cyclic voltammogram of a clean gold electrode in 0.05 M H2SO4 at a

scan rate of 100 mV s-1.

In this cyclic voltammetry method, a monolayer of gold oxide is first electrochemically

formed and then reduced. Integration of the cathodic wave yields the charge density

passed for reducing the gold oxide layer as shown in Figure 2.4. The real area of the

electrode was determined from the charge density of the reduction of gold oxide by the

method of Oesch et al.20 using a conversion factor of 482 C cm-2. Calculations of the

real surface area, commonly expressed as a roughness factor, are based on the

assumption that a monolayer of chemisorbed oxygen with the gold: oxygen ratio of 1:1

has been formed. The roughness factor of the electrodes was typically 1.3 0.3 (95

confidence, n 5).

2.4.5 Preparation of Homogeneous Pure and Mixed Alkanethiol Self-Assembled

Monolayers on Gold Surfaces

The process for the formation of alkanethiol SAMs is illustrated in Scheme 2.5. Firstly,

an aqueous ethanol solution (75 v/v) containing 1 mM alkanethiols was prepared.

73


After that a clean gold electrode was immersed into the thiol solution for overnight,

followed by rinsing with absolute ethanol and then Milli-Q water, and finally drying the

gold surface under a stream of argon. The alkanethiol molecules self-assembles onto the

gold substrate to give the resultant monolayers.

AuAu

Au Self organisation

Alkanethiolethanolic solution Several minutes later After overnight

AuAuAuAuAu

AuAu Self organisation

Alkanethiolethanolic solution Several minutes later After overnight

Scheme 2.5 Schematic of self-assembling a single component SAM on gold surfaces.

The monolayer formed on gold surfaces can be either a pure monolayer or a mixed

monolayer as illustrated in Scheme 2.6. A mixed monolayer can be prepared by

incubating the gold substrate in a solution of two or more alkanethiols with a total

concentration of 1 mM. The composition of the monolayer can be adjusted by adjusting

the composition of the thiol mixture, since the composition of the thiols on the resulting

surface is usually quite similar to that of the solution used for the attachment.21

Au

a) b)

AuAuAu

a) b)

AuAu

Scheme 2.6 Schematic of self-assembled (a) pure and (b) mixed monolayers of

alkanethiols on gold electrodes.

74


2.4.6 Covalent Attachment of Ferrocene Redox Probes onto Glassy Carbon

Electrodes Modified with Diazonium Salt Monolayers

Covalent attachment of ferrocenemethylamine to monolayers modified GC electrodes

with the terminal carboxylic acid groups was carried out by following the procedures

described by Liu et al.22 The modification of polished GC electrodes with aryl

diazonium salt monolayers with the terminal carboxylic acid groups was firstly carried

out in CH3CN/0.1 M NBu4BF4 solution containing 1 mM aryl diazonium salts by

electrochemical reductive adsorption as described in Section 2.4.2. The terminal

carboxylic acid groups formed on GC surfaces were then activated with 40 mM 1-ethyl-

3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and 20 mM N-

hydroxysuccinimide (NHS) in the 0.1 M N-(2-hydroxyethyl)piperazine-N’-(2-

ethanesulfonic acid) (HEPES) buffer (pH 7.3) solution for 1 h.23-25 The activated GC

electrodes were then taken out from the HEPES buffer solution, rinsed by HEPES

buffer (pH 7.3) followed by copious rinsing with Milli-Q water, and finally dried under

a stream of argon. The ferrocenemethylamine was finally coupled onto the carboxylic

acid groups terminated monolayers on GC electrodes via incubation of the activated GC

electrodes in the HEPES buffer (pH 7.3) solution containing 1 mM

ferrocenemethylamine for 24 h at room temperature. Similarly, another redox probe

ferrocenecarboxylic acid can be covalently attached onto monolayers with terminated

amine groups on GC electrodes using EDC and NHS coupling.

75


2.4.7 Determination of the Surface Coverage of Redox Species

The surface coverage of the redox species was determined electrochemically from a

typical CV of the redox species by calculating the charge passed in the oxidation or

reduction process. The calculation of the coverage is generalised in the equation 2-2:

nFAQ

2-2

where is the coverage of the redox species at the electrode surface, Q is the charge

passed in coulombs, n is the number of electrons transferred, F is Faraday constant, and

A is the electrochemical area of the electrode in cm2.

2.4.8 Calculation of the Rate Constant of Heterogeneous Electron Transfer using

Laviron Theory

A number of electrochemical methods have been used for the calculation of the rate of

electron transfer, namely cyclic voltammetry,26 alternating current impedance

spectroscopy (ACIS),27 square-wave voltammetry,28 and chronoamperometry.29 These

electrochemical methods are useful for determining rate constants in the range 10-2 to

105 s-1. Measurements of rate constants beyond this range require spectroscopic30 or

temperature-jump methods.31 One method developed by Laviron26 uses cyclic

voltammetry to exploit the fact that the difference in potential of the oxidation and

reduction peaks increases with an increase in scan rate. This increase in peak separation

with scan rate can be thought of as the electron transfer keeping pace with the rate of

change of the potential. So the faster the redox reaction the better the electron transfer

can keep up with the change in potential which leads to the smaller potential difference

76


between the peaks. Using this trend, the transfer coefficient and the electron transfer

rate constant can be calculated. The details of the calculation are illustrated below.

Based on Laviron theory, for a redox reaction the electron efficiency can be described

by equation 2-3:

)nv/k)(F/RT(m s 2-3

Where ks is the electron transfer rate constant, is the scan rate, n is the number of

electrons involved in the redox reaction, m is the electron efficiency, F is the Faradic

constant, T is the absolute temperature and R is the gas constant. Equation 2-3 can be

easily rewritten as equation 2-4.

)m/)(n/k)(F/RT(v s 1 2-4

Table 2.2 gives some values of n Ep as a function of 1/m for =0.5 ( is the electron

transfer coefficient; Ep is the difference in potential at current maxima between the

peak for oxidation and that for reduction). Based on Table 2.2, the relationship between

n Ep and 1/m can be illustrated in Figure 2.5. Therefore, the rate constant (ks) of

electron transfer is calculated differently at different conditions. When n Ep<200 mV,

1/m can be obtained from the function in Figure 2.5. Then according to equation 2-4, a

linear relationship is expected between 1/m and scan rate ( ), and ks can be deduced

from the slope of this linear curve.

77


Table 2.2 The relationship between n Ep and 1/m.

m-1 0.5 0.75 1 1.5 2 2.5 3 3.5 4 5

n Ep 18.8 27 34.8 48.8 61.2 72.2 82.4 91.8 100.6 116.2

m-1 6 7 8 9 10 11 12 13 14

n Ep 130 142.4 153.8 164 173.4 182 190 197.6 204.6

y = 9E-07x3 + 9E-06x2 + 0.0313x - 0.1226

0

4

8

12

16

0 50 100 150 200 250

n Ep

1/m

Figure 2.5 The curve between n Ep and 1/m.

When n Ep>200 mV,

]/ln[)/( mnFRTEE jp 2-5

]/)1ln[(])1/([ mnFRTEE jp 2-6

Where Ej is the standard potential of the surface redox reaction. Based on equation 2-5

and equation 2-6, the value can be easily obtained, after which equation 2-7 is used to

calculate k.

RTEnFnFvRTk p 3.2/)1()/log(log)1()1log(log 2.7

78


It is important however to appreciate the limitations of the Laviron method. The Laviron

method assumes the Butler-Volmer theory applies. This is certainly the case with high

reorganisation energy (>2.0 eV). For reorganisation energies below about 2.0 eV,

voltammograms are predicted to be broader and peak potentials are in most cases

predicted to shift further from the formal potential. In this case, the rate constant of

electron transfer can be calculated by the Marcus theory.32 All rates of electron transfer

in this project were calculated by the Laviron method.

2.5 References


6813.

(2) Downard, A.J., Prince, M.J., Langmuir 2001, 17, 5581-5586.

(3) Kraatz, H.B., J. Organomet. Chem. 1999, 579, 222-226.

(4) Ossola, F., Tomasin, P., Benetollo, F., Foresti, E., Vigato, P.A., Inorg. Chim.

Acta 2003, 353, 292-300.

(5) Kosynkin, D.V., Tour, J.M., Org. Lett. 2001, 3, 993-995.



Eur. J. 2001, 7, 5118-5134.

(7) Tohji, K., Goto, T., Takahashi, H., Shinoda, Y., Shimizu, N., Jeyadevan, B.,

Matsuoka, I., Saito, Y., Kasuya, A., Ohsuna, T., Hiraga, H., Nishina, Y., Nature

1996, 383, 679-679.

(8) Suffert, J., Ziessel, R., Tetrahedron Lett. 1991, 32, 757-760.

(9) Stephens, R.D., Castro, C.E., J. Org. Chem. 1963, 28, 3313-3315.

(10) Corey, E.J., Szekely, I., Shiner, C.S., Tetrahedron Lett. 1977, 18, 3529-3533.

79


(11) Bahr, J.L., Yang, J., Kosynkin, D.V., Bronikowski, M.J., Smalley, R.E., Tour,

J.M., J. Am. Chem. Soc. 2001, 123, 6536-6542.

(12) O'Dea, J.J., Osteryoung, J., Osteryoung, R.A., Anal. Chem. 1981, 53, 695-701.

(13) Zittel, H.E., Miller, F.J., Anal. Chem. 1965, 37, 200-203.

(14) Sawyer, D.T., Sobkowiak, A., Roberts, J.L., Experimental Electrochemistry for

Chemists. Wiley: New York, 1995; p 74-75.

(15) Pontikos, N.M., McCreery, R.L., J. Electroanal. Chem. 1992, 324, 229-242.



(17) Gooding, J.J., Mearns, F., Yang, W.R., Liu, J.Q., Electroanalysis 2003, 15, 81-

96.

(18) Ranganathan, S., McCreery, R.L., Anal. Chem. 2001, 73, 893-900.


(20) Oesch, U., Janata, J., Electrochim. Acta 1983, 28, 1237-1246.

(21) Gooding, J.J., In Encyclopedia of Nanoscience and Nanotechnology, Nalwa,

H.S., Ed. American Scientific Publishing: California, 2004; Vol. 1, pp 17-49.


8466.

(23) Yang, W., Jaramillo, D., Gooding, J.J., Hibbert, D.B., Zhang, R., Willett, G.D.,

Fisher, K.J., Chem. Commun. 2001, 1982-1983.

(24) Staros, J.V., Wright, R.W., Swingle, D.M., Anal. Biochem. 1986, 156, 220-2.

(25) Yang, W.R., Gooding, J.J., Hibbert, D.B., Analyst 2001, 126, 1573-1577.

(26) Laviron, E., J. Electroanal. Chem. 1979, 101, 19-28.

(27) Laviron, E., J. Electroanal Chem. 1975, 97, 135-149.

(28) Reeves, J.H., Song, S., Bowden, E.F., Anal. Chem. 1993, 65, 683-689.

80


(29) Chidsey, C.E.D., Science 1991, 251, 919-922.

(30) Ye, S., Yashiro, A., Sato, Y., Uosaki, K., J. Chem. Soc. Faraday Trans. 1996,

92, 3813-1821.

(31) Smalley, J.F., Feldberg, S.W., Chidsey, C.E.D., Linford, M.R., Newton, M.D.,

Liu, Y.P., J. Phys.Chem. 1995, 99, 13141-13149.

(32) Weber, K., Creager, S.E., Anal. Chem. 1994, 66, 3164-3172.

81

Chapter3-Covalent Modification of Electrode Surfaces by Electrochemical Reduction of Aryl Diazonium Salts

Chapter Three

Covalent Modification of Electrode Surfaces by Electrochemical

Reduction of Aryl Diazonium Salts

82

Chapter -Covalent Modification of Electrode Surfaces by Electrochemical Reduction of Aryl Diazonium Salts

3.1 Introduction

In light of the introduction in Chapter One on the advantages of monolayer modification

in electron transfer,1, 2 molecular electronics,3, 4 bioelectronics5, 6 and sensors,7 and the

disadvantages of the commonly used alkanethiol chemistry on gold surfaces, an

alternative monolayer system to gold-thiol chemistry is desirable. This alternative

should overcome some of the disadvantages without severely compromising the

advantages of gold/thiol chemistry. The electrochemical reduction of aryl diazonium

salts is one possible alternative which has been used as a method for the covalent

derivatisation of carbon surfaces.8-10 The reduction reaction results in the loss of N2 and

the formation of a carbon-carbon covalent bond between the adsorbed molecules and

carbon substrates which is strong, stable over both time and temperature, nonpolar and

conjugated.11 The attractiveness of aryl diazonium salts is enhanced further by recent

studies showing that they can also be grafted onto a variety of metal12, 13 and

semiconductor14 surfaces as well as carbon nanotubes.15 This feature raises the exciting

possibility of one monolayer forming system being suitable for a large range of

electrode materials for a diverse range of applications.

The purpose of this chapter is to investigate the strategy for covalent modification of

glassy carbon (GC) and gold substrates with monolayers of aryl diazonium salts by

electrochemical reduction to study the possibility of GC electrodes serving as an

alternative to gold electrodes. The stability of monolayers of aryl diazonium salts on GC

and gold electrode surfaces has been studied. For comparison the stability of self-

83


assembled monolayers (SAMs) of alkanethiols on gold surfaces has been also

investigated.

3.2 Experimental Section

All reagents and materials are listed in Table 2.1 in Chapter Two or prepared according

to the procedures described in Chapter Two. GC and gold electrodes were prepared

according to the method described in Section 2.4. All solutions of monolayers were

prepared as described in Section 2.4.2. All electrochemical measurements were

performed with a BAS-l00B electrochemical analyser. All potentials were quoted

relative to an Ag/AgCl reference at room temperature. All cyclic voltammetry

measurements were performed in phosphate buffer (0.05 M, 0.05 M KCl, pH 7.0).

3.3 Results and Discussion

3.3.1 Influence of Scan Rates on the Capacitance of Bare Glassy Carbon Electrodes

in Aqueous and Nonaqueous Electrolytes

The capacitive current, which is attributed to the charging and the discharging of the

interfacial electrical double layer, provides information regarding the accessibility of the

electrode to ions.16 In the case of monolayer modified electrodes the thickness of the

monolayer and the level of defects will influence the accessibility of ions. Therefore,

measurement of the electrochemical capacitance provides a means of investigating the

surface properties of electrodes.17-19 Double layer capacitances (Cdl) were determined

using cyclic voltammetry20 by scanning the electrode between a potential window in a

supporting electrolyte where the charging current was independent of the applied

voltage (i.e. no Faradaic process occurring). The following equation 3-1 is applied:

84


62

10)2()/(

AIcmFC total

dl 3-1

Where Itotal is the sum of anodic and cathodic current, is the sweep rate (V s-1) and A is

the area of the working electrode (cm2).

The double layer capacitance can be used to give information about the extent of solvent

and electrolyte permeation in a layer and hence the compactness of the surface layer.16

So it plays an important role in electrochemical measurements particularly in the

fabrication of a variety of sensors. Several factors might lead to a change in the double

charge capacitance, such as the composition of the electrode and electrolyte, the

roughness of the electrode surface, the characteristic of the molecules with which the

electrode surface is modified and the density of the surface-bound molecules.

Figure 3.1 shows the capacitance of the freshly polished bare GC in both aqueous and

nonaqueous solutions.

0

100

200

300

400

500

600

700

800

900

0 1 2 3

Scan rate /V s-1

Capacitance /

F c

m-2

4

Figure 3.1 A graph of capacitance of bare GC electrodes against scan rates in

phosphate buffer (pH 7.0) solution (triangle spots) and CH CN/0.1 M NBu4BF4 solution

(square spots).

85


It shows a decrease in capacitance with an increase in scan rates in Figure 3.1. When

the scan rate is lower, there is more time for the ions to diffuse and access the electrode,

which leads to higher capacitance. It was also found that the capacitance in the aqueous

solution is higher than that in the organic electrolyte at low scan rates and that there is

not much difference between them when the scan rate is greater than 2 V s-1. The higher

capacitance in inorganic media is due to the electrode in the organic environment being

more accessible to ions.

The electrode materials can also dramatically influence the double charge capacitance.

Figure 3.2 shows the graph of capacitances of bare gold, GC and Pt electrodes against

scan rates in phosphate buffer solution (pH 7.0). It was found the relationship between

the scan rate and capacitance is similar in trend for bare gold, GC and Pt electrodes in

phosphate buffer (pH 7.0) solution, but with differences in magnitude. When the scan

rate is less than 0.3 V s-1, the capacitance of the bare gold electrode is smaller than that

of the bare GC electrode. The capacitances of the bare gold electrode and bare GC

electrode are almost the same when the scan rate is 0.3 V s-1. When the scan rate is over

0.3 V s-1, the capacitance of the bare gold electrode is slightly larger than that of the

bare GC electrode. The capacitances of the bare gold and bare GC electrode level off at

ca. 200 F cm-2 and 100 F cm-2 respectively. As shown in Figure 3.2 the capacitance

of platinum electrodes is much higher than that of gold and GC electrodes due to the

rougher surface21 which results in a larger working electrode area and more ions

accessible to the electrode surface. These results are therefore consistent with the

literature that the roughness of the electrode, which is caused by the physical property

of the materials and the process of preparing the electrode surface, plays a significant

role in determining the double charge capacitance.22, 23

86


0

500

1000

1500

2000

2500

3000

3500

0 0.2 0.4 0.6 0.8

Scan rate /V s-1

Capacitance /

F c

m-2

Au

GC

Pt

Figure 3.2 The graph of capacitances of bare gold, GC and Pt electrodes against scan

rates in the phosphate buffer solution (pH 7.0).

3.3.2 Modification of Glassy Carbon Electrodes with Aryl Diazonium Salts by

Electrochemical Reductive Adsorption

The electrochemically assisted covalent modification of carbon electrodes has been

explored by a few groups.10, 24-27 The most frequently used methods for the modification

of GC surfaces is the reductive adsorption of aryl diazonium salts. Scheme 3.1 shows

the schematic of modification of 4-carboxyphenyl diazonium salts (CP) on GC

electrodes. In order to achieve this kind of modification, two methods can be used. One

is by poising the electrode at a reducing potential and the other is by repeated cycling.

Downard and Prince28 have reported the surface coverage and the blocking properties of

diazonium–derived surface layers depending on the potential used for film formation. It

is worth investigating the influence of the modification method on the quality of the

monolayers. The commonly used ferricyanide was selected as the redox probe to

achieve this purpose because the blocking properties of monolyers can be tested easily

in ferricyanide. The interfacial capacitance can be used to evaluate the compactness or

87


the extent of solvent and electrolyte permeation in a layer,16 and thereby provides

information on the quality of SAM formation.

+N2 COOH N2COOH

GC.e-

GCGCCOOH

+

Scheme 3.1 Schematic of modification of CP on GC electrodes by reductive adsorption.

Figure 3.3 shows the capacitance of modified GC surfaces in ferricyanide solution

(1 mM, 0.05 M KCl, phosphate buffer, pH 7.0) by using different modification

strategies. When a GC electrode was modified with 1 mM CP in acetonitrile/0.1 M

NBu4BF4 solution by holding at a constant potential for 4 min the capacitance obtained

at the potential of -0.5 V was the smallest of all, indicating that the monolayer has a

more densely packed structure with fewer imperfections due to surface roughness and

shows a larger inhibition effect on the reaction of inorganic ions. Therefore the optimal

potential should be -0.5 V in order to form the best monolayer of CP. However, if the

modification was performed using cyclic voltammetry (CV) with a scan rate of 100 mV

s-1, after two cycles of scanning, the capacitance is similar to that obtained by holding

potential at -0.5 V. Increasing scan cycles did not lead to a change in capacitance, so

two cycles are enough to create a densely packed monolayer. Comparing the two

modification methods (constant potential and CV), it can be concluded that the method

of using CV is superior to that of holding at a fixed potential, which is consistent with

the literature.29, 30 In addition, long deposition time at excessive negative potentials is

prone to form multilayers due to further attachment of active radicals of diazonium

molecules on the previously formed molecules.31, 32 Therefore, all the modifications of

GC electrodes in this thesis were performed by following the method of CV scanning

with a scan rate of 100 mV s-1 for two cycles.

88


0

20

40

60

80

100

120

140

1 2 3 4 5 6 7 8 9 10

Electrode type

Capacitance /

Fcm

-2

0V

-0.2V -0.5V -0.7V -0.9V -1.2V 1cycle2cycles

3cycles 4cycles

Figure 3.3 Capacitance of GC electrodes modified in 1 mM CP, acetonitrile/0.1 M

NBu4BF4 solution after holding electrodes for 4 min at different potentials (1- ) and

sweeping for a different number of cycles between 1.0 V and -1.0 V at the scan rate of

100 mV s-1 (7-10) in 1 mM ferricyanide solution in phosphate buffer (pH 7.0).

The apparent capacitance of the GC electrode before and after modification with CP in

different electrolytes has also been measured electrochemically in background

electrolyte solutions. Table 3.1 shows that the capacitance increases for aqueous media

and decreases for organic media after the modification of CP, which is in agreement

with the literature except that the magnitudes are somewhat different.33 The capacitance

for the bare GC electrode was found to be much larger in aqueous phosphate buffer

solution than that in organic electrolyte acetonitrile/0.1 M NBu4BF4. The smaller

capacitance of the modified electrode in organic media (109 µF cm-2) than that in an

aqueous solution (305 µF cm-2) is due to a less permeable and less solvated film in a

nonaqueous medium.16 The capacitance of GC electrodes decreased slightly in organic

media after modification with the CP. Decreases in electrochemical capacitance were

also observed with gold surfaces modified with alkanethiols.20, 34

89


Table 3.1 The double layer charging capacitance of GC electrodes in phosphate buffer

(pH 7.0) and acetonitrile before and after modification of CP.

Capacitance (µF cm-2) at 100 mV s-1GC electrode

Phosphate buffer solution Acetonitrile solution

Bare

CP Modified

250

305

131

109

3.3.3 Electrochemistry of a Glassy Carbon Electrode Modified with 4-Carboxyphenyl


Cyclic voltammetry was carried out with a freshly polished GC electrode in a 1 mM CP

and 0.1 M NBu4BF4 solution in acetonitrile (Figure 3.4 a). The first sweep gave the

irreversible reduction wave at ca. -0.16 V versus Ag/AgCl, which was attributed to the

formation of the 4-carboxyphenyl radical from the diazonium derivative due to one

electron transfer redox reaction.9, 26, 33 The peak due to the Faradaic process disappeared

completely after the first cycle which implies that the electrode surface has been

occupied by the aryl diazonium salt molecules and the free molecules in the solution can

not access the electrode anymore.9, 26, 33 The surface coverage of the modified layer

could be estimated through integration of the redox peaks of CP in acetonitrile/0.1 M

NBu4BF4 solution. It appears that the surface coverage can be controlled through

controlling the concentration of the aryl diazonium salts, but the surface coverage of CP

reached saturation when the concentration of the aryl diazonium salts was greater than

5 mM (Figure 3.4 b). Based on the area of the reduction peak in Figure 3.4 a, the

coverage of the CP was calculated to be 7.4×10-10 mol cm-2 when the modification was

carried out in a 1 mM CP solution. The reported surface coverage on GC substrates

varies in the range of 4-30×10-10 mol cm-2.35 The theoretical surface coverage of

90


carboxyphenyl groups can be calculated with the Hyperchem Molecular Simulation

program (Autodesk, Inc.) for the case of close packing of adsorbed molecules on a flat

surface. Based on the geometric area of a single diazonium salt molecule, the projection

area of a 4-carboxyphenyl group bonded at the edge of the phenyl ring is 13.9 Å2 or

12×10-10 mol cm-2 for ideal close packing.36 The surface coverage of 7.4×10-10 mol cm-2

indicates the GC electrode was modified with a monolayer or submonolayer rather than

multilayers of aryl groups as has been reported by some groups.31, 32, 37

0

0.5

1

1.5

2

0 2 4 6 8 10 1

Concentration of CP /mM

CP /

nm

ol cm

-2

b

2

-20

-15

-10

-5

0

5

-1 -0.5 0 0.5 1

Potential /V

Curr

ent

/A

1st cycle

2nd cycle

a

Figure 3.4 (a) CVs of a GC electrode in a 1 mM CP, acetonitrile/0.1 M NBu4BF4

solution at a scan rate of 100 mV s-1 and (b) plot of surface coverage of CP as a

function of the concentration of CP in the acetonitrile/0.1 M NBu4BF4 solution.

91


In light of the inhibition of the current during the second sweep of the cyclic

voltammetry experiment of Figure 3.4 a, it is important to evaluate the cyclic

voltammetry behaviour of an electroactive, soluble redox probe couple at CP modified

GC electrodes. CVs were carried out in aqueous and nonaqueous media with three

electroactive redox probes: two outer-sphere inorganic (Fe(CN)63- and Ru(NH3)6

3+), and

one organometallic (ferrocene) redox systems (Figure 3.5). The redox peaks of

Fe(CN)63- observed on bare GC electrodes disappear completely after CP modification

(Figure 3.5 a). And the redox current of the Ru(NH3)63+ in aqueous solution is only

slightly affected by the CP modification as shown in Figure 3.5 b. The blocking

property of CP monolayers was also evaluated in a non-aqueous electrolyte solution

containing ferrocene (Figure 3.5 c). It shows the electrochemical response of ferrocene

is greatly attenuated by the CP monolayers on GC surfaces. The difference in the

blocking behaviour of the CP monolayers in different redox probes studied above can

be explained by considering electrostatic interactions between the modified surface and

the electroactive probe1, 38-41 and electrolyte/solvent effects16, 39, 40 in solution. In a pH

7.0 solution, the 4-carboxyphenyl group is expected to dissociate to some extent if we

assume that its pKa is similar to that of benzoic acid (pKa=4.2). Thus, for a negatively

charged CP monolayer (at pH 7.0), the positively charged Ru(NH3)63+ probe should not

be prevented from reaching the underlying GC electrode surfaces. In this case the

response of Ru(NH3)63+ is almost undistinguishable from that at the bare GC electrode

as shown in Figure 3.5 b. On the contrary, the absence of response of Fe(CN)63- is

attributed to the negative Donnan potential which is established at the film surface as a

result of the high negative charge density of the COO- groups.39, 40, 42 In order to confirm

this hypothesis, CVs for a GC electrode modified with CP were recorded in 1 mM

Fe(CN)63- solutions of various pH values as discussed in the next section.

92


-30

-20

-10

0

10

20

30

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

CP modified GC

a

Bare GC

-30

-20

-10

0

10

20

-0.4 -0.3 -0.2 -0.1 0 0.1

Potential /V

Curr

ent

/A

CP modified GC

b

Bare GC

-30

-20

-10

0

10

20

-0.4 -0.3 -0.2 -0.1 0 0.1

Potential /V

Curr

ent

/A

CP modified GC

c

Bare GC

Figure 3.5 Cyclic voltammograms of bare and CP modified GC electrodes in (a)

Fe(CN) - and (b) Ru(NH ) (1 mM, 0.05 M KCl, 0.05 M phosphate buffer, pH 7.0)

and (c) in ferrocene (1 mM, NBu4BF4, 0.1 M, CH CN) at a scan rate of 100 mV s-1.

93


3.3.4 Effect of Solution pH on the Blocking Property of the 4-Carboxyphenyl Groups

Modified on Glassy Carbon Electrodes

To confirm the electrostatic interaction between the modified surface and electroactive

probe, Fe(CN)63- was used as a redox probe to test the effect of pH of the electrolyte on

its electrochemical response. Figure 3.6 shows that the anodic peak current of CVs

decreases with an increase in pH. As the pH increases, the terminal carboxylic acid

groups on the monolayer surface deprotonated to form a negatively charged interface

which then prevents the access of the negatively charged Fe(CN)63- redox species from

reaching the underlying GC electrode surface. At low pH values, the carboxylic acid

groups will remain in the neutral form and the negatively charged Fe(CN)63- can more

easily penetrate the neutral monolayer surface. These experiments show the carboxylic

acid groups modified on GC surfaces appear either as -COOH or as -COO- depending

on the pH of the solution in which the electrode has been exposed. Similar pH-

modulated electrochemical responses for Fe(CN)63- have been reported in the literature

for alkanethiols with COOH and NH2 terminal groups.1, 39, 40 According to the

literature43, the surface pKa of monolayer can be determined from the current parameter

of CVs in phosphate buffer with different pH. The surface pKa of the CP monolayer on

GC surfaces estimated from the Ipa–pH curve of Figure 3.6 is 2.41. This value is

smaller than that expected for benzoic acid in bulk solution (pKa 4.2).44 It should be

noted that the surfaces’ pKa values are frequently different from that in bulk solution

due to the formation of stable intermolecular hydrogen bonding.43, 45 Hydrogen bond

formation between the atoms of terminal carboxyl causes the protons of carboxyl to be

held more tightly on the monolayer surface. Thus, the deprotonation of the carboxylic

acid groups becomes more difficult, leading to the decrease in the surface pKa.

94


0

2

4

6

8

10

12

14

0 2 4 6 8

pH

Curr

ent

/A

10

Figure 3.6 Plot of the anodic peak current from cyclic voltammograms as a function of

solution pH. CVs were recorded at a CP modified GC electrode in 0.05 M phosphate

buffer solutions with different pH values containing 1 mM Fe(CN) - at a scan rate of

100 mV s-1.

3.3.5 Characterisation of 4-Carboxyphenyl Modified Glassy Carbon Surfaces Using

X-ray Photoelectron Spectroscopy

X-ray Photoelectron Spectroscopy (XPS) measurements were carried out to further

characterise the modified species after modification of the GC surface. The XP survey

spectrum of the bare GC electrode (Figure 3.7 a) showed the peaks expected with small

carbon 1s and oxygen 1s peaks at ~285 and ~532 eV respectively indicating the

presence of organic contaminants such as adventitious hydrocarbons and water.

Importantly, almost no nitrogen 1s peak was observed on the bare GC plate. After the

GC surfaces were modified with parabenzoic acid, the XP survey spectrum showed a

significant increase in the 1s peaks of carbon and oxygen at ~285 and ~532 eV

respectively, but no significant evidence of a nitrogen 1s peak (Figure 3.7 b) indicating

the nitrogens of the diazonium salt are lost in the immobilisation on the GC surface. It

can be seen in Figure 3.7 a (inset) the C1s scan of the bare GC plate is dominated by

95


the graphitic carbon peak at 284.4 eV together with a broader peak on the high binding

energy 285.2 eV due to the presence of low levels of oxidised carbon species on the

electrode surface. After the GC surfaces were modified with parabenzoic acid, the

emergence of a peak at 288.8 eV, which is typical of the carbon of the carboxylic acid

group, in the C1s spectra as shown in Figure 3.7 b (inset) was observed, indicating the

–COOH terminated monolayer has formed on the GC surfaces.9

02004006008001000

Binding energy /eV(b)

O1s

C1s

02004006008001000

Binding energy /eV

O1s

C1s

(a)

279283287291

C1s

279283287291

-COOH

288.8 eV

C1s

Figure 3.7 P survey spectrum and carbon 1s narrow scan (inset) of (a) a bare GC

electrode and (b) a GC electrode modified with 4-carboxyphenyl moieties.

3.3.6 Stability of Carboxyphenyl Monolayers Modified on Glassy Carbon Electrodes

In order to further investigate the stability of aryl diazonium salts modified GC

electrodes, the CVs of CP modified GC electrodes left on a laboratory bench for 2

months and after ultrasonic treatment for 30 min in acetonitrile, were conducted in

ferricyanide solution (1 mM, 0.05 M KCl, phosphate buffer, pH 7.0). It can be seen in

Figure 3.8 monolayers of CP on GC electrodes still can passivate the GC electrodes to

prevent the access of the ferricyanide molecules after air exposure for 2 months. In

addition, the electrodes being exposed to vigorous ultrasonic cleaning in acetonitrile for

96


30 min also displayed very good blocking properties in ferricyanide solution. Therefore,

it can be concluded that the CP monolayers modified on GC electrode are very stable in

air and can resist the ultrosonic treatment, which is consistent with the literature.9

-14

-9

-4

1

6

11

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

Bare GC

Modif ied GC

After air exposure

After sonication

Figure 3.8 CVs at a scan rate of 100 mV s-1 for a GC electrode in ferricyanide (1 mM,

0.05 M KCl, 0.05 M phosphate buffer, pH 7.0) before and after modification of CP

followed by air exposure for 2 months or ultrasonic treatment for 0 min in acetonitrile.

In order to compare the stability of diazonium salts modified monolayers on GC

electrodes to that of alkanethiol monolayers on gold electrodes, similar experiments

were carried out on gold electrodes modified with SAMs of alkanethiols. Figure 3.9

shows CVs of bare and alkanethiol modified gold electrodes left on a laboratory bench

for 2 months and after ultrasonic treatment for 30 min in acetonitrile in ferricyanide

solution (1 mM; KCl, 0.05 M; phosphate buffer; pH 7.0). SAMs of hexadecanthiol

(HDT) on gold electrodes almost completely disappeared after exposure to air for 2

weeks (Figure 3.9 a). Moreover, the capacitance of gold electrodes increased

considerably after ultrasonication and the blocking properties of alkanethiol monolayers

in ferricyanide solution decreased slightly. These results are consistent with the

97


literature46 and the stability with short chain alkanethiols on gold surface is even worse

(Figure 3.9 b). SAMs of 3-mercaptopropionic acid (MPA) on gold electrodes were

completely removed after air exposure and ultrasonication treatment. So diazonium salt

modified monolayers on GC surfaces are more stable than alkanethiol monolayers on

gold surfaces. The higher stability is the consequence of the formation of a covalent

carbon-carbon linkage between organic monolayers and GC surfaces.9

-3.5

-2.5

-1.5

-0.5

0.5

1.5

2.5

-0.2 0 0.2 0.4 0.6

Potential /V

Curr

ent

/A

Bare Au

Modif ied Au

After air exposure

After sonication

b

-1.3

-0.9

-0.5

-0.1

0.3

0.7

1.1

-0.2 0 0.2 0.4 0.6

Potential /V

Curr

ent

/A

Bare Au

Modif ied Au

After air exposure

After sonication

a

Figure 3.9 Cyclic voltammograms at a scan rate of 100 mV s-1 in ferricyanide (1 mM,

0.05 M KCl, 0.05 M phosphate buffer, pH 7.0) for a gold electrode before and after

modification of (a) HDT and (b) MPA followed by air exposure for 2 weeks or

ultrasonic treatment for 0 min in acetonitrile.

98


3.3.7 Electrochemistry of a Glassy Carbon Electrode Modified with Phenyl


Freshly polished GC electrodes were modified with the phenyl diazonium salts using

the same method for modification of CP in section 3.3.3. CVs of a GC electrode in a

1 mM phenyl diazonium tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution are

shown in Figure 3.10. The first sweep gave an irreversible reduction wave at ca. -0.4 V

versus Ag/AgCl. The wave disappeared completely in the second cycle, which is similar

to that for the modification of 4-carboxyphenyl in Section 3.3.3. Based on the area of

the reduction peak during the modification of the GC electrode surface with the

diazonium salt, the coverage of the phenyl moieties was calculated to be

(11.2±0.3)×10 -10 (n=6) mol cm-2, which is larger than that for the 4-carboxyphenyl

group on GC surfaces but lower than the theoretical maximum surface coverage for a

monolayer surface.36 This value is in good agreement with previously reported values

and can be considered to be in the monolayer or submonolayer region.9

-50

-40

-30

-20

-10

0

10

-1.6 -1.2 -0.8 -0.4 0 0.4 0.8

Potential /V

Curr

ent

/A

1st cycle

2nd cycle

Figure 3.10 Cyclic voltammograms of a GC electrode in a 1 mM phenyl diazonium

tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution at a scan rate of 100 mV s-1.

99


In light of the inhibition of the current during the second sweep of the cyclic

voltammetry experiment in Figure 3.10, it is important to evaluate the cyclic

voltammetry behaviour of an electroactive, soluble redox probe couple at phenyl

modified GC electrodes. The passivation of the GC surfaces modified with phenyl was

investigated by scanning the cyclic voltammetry in redox probes such as Fe(CN)63-,

Ru(NH3)63+ and ferrocene as shown in Figure 3.11. After modification of phenyl, the

redox peaks of Fe(CN)63- observed with bare GC electrodes were completely suppressed

(Figure 3.11 a), giving strong evidence that a monolayer of phenyl which blocked

access of redox molecules to the GC electrode had formed on the GC surfaces. The

performance of phenyl monolayer in Ru(NH3)63+ is similar to that in Fe(CN)6

3- (Figure

3.11 b). The phenyl monolayers on GC surfaces can block the access of Ru(NH3)63+

molecules due to the resulted neutral interface, which is different from the performance

of CP monolayers on GC surfaces observed in Figure 3.5 b. The blocking properties of

the phenyl monolayers on GC electrodes were also evaluated in non-aqueous electrolyte

solution containing ferrocene (Figure 3.11 c). The electrochemistry of ferrocene is

almost completely blocked at the phenyl modified electrode, which is in agreement with

the literature.28

100


-30

-20

-10

0

10

20

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

Phenyl modified GC

aBare GC

-6

-4

-2

0

2

4

6

8

-0.4-0.3-0.2-0.100.1

Potential /V

Curr

ent

/A Phenyl modified GC

bBare GC

-125

-75

-25

25

75

00.20.40.60.8

Potential /V

Curr

ent

/A Phenyl modified GC

Bare GCc

Figure 3.11 Cyclic voltammograms of bare and phenyl modified GC electrodes in (a)

Fe(CN) - and (b) Ru(NH ) (1 mM, 0.05 M KCl, phosphate buffer, pH 7.0) and (c)

ferrocene (1 mM, 0.1 M NBu4BF4, acetonitrile) at a scan rate of 100 mV s-1.

101


3.3.8 Modification of Gold Electrodes with Aryl Diazonium Salts

3.3.8.1 Electrochemistry of 4-Carboxyphenyl Modified Gold Electrodes

It has been reported that gold electrodes can also be modified with diazonium salts by

electrochemical reduction.13, 47 For comparison gold electrodes were modified with CP

via reductive adsorption in exactly the same manner as GC electrodes. Cyclic

voltammograms of a gold electrode in a 1 mM 4-carboxyphenyl diazonium

tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution at the scan rate of 100 mV s-1 are

shown in Figure 3.12, which showed similar responses of GC surfaces. The reduction

peak for the attachment of the 4-carboxyphenyl onto the gold electrode, observed in the

first sweep, was shifted anodically relative to that on GC surfaces. Based on the area of

the reduction peak during the modification of the gold electrode surface with the aryl

diazonium salt, the coverage of the 4-carboxyphenyl moieties on the electrode surface

was calculated to be 6.4×10-10 mol cm-2 which was lower than the 7.4×10-10 mol cm-2

observed on GC electrodes and hence lower than the theoretical maximum surface

coverage36 of 12×10-10 mol cm-2 for a monolayer arrangement. The lower surface

coverage could be a reflection of the aryl diazonium salt not sitting normal to the gold

surfaces as suggested by infra-red spectroscopy of Zn and Pt surfaces modified with

diazonium salts.13 Again, the presence of a monolayer or submonolayer of aryl

diazonium salt on the gold electrode is important due to the possibilities of producing

multilayers of diazonium salts as shown for both carbon31, 32, 37 and metal surfaces.13

102


-2

-1.5

-1

-0.5

0

0.5

-0.6 -0.3 0 0.3 0.6

Potential /V

Curr

ent

/A

2nd cycle

1st cycle

Figure 3.12 Cyclic voltammograms of a bare gold electrode in a 1 mM 4-

carboxyphenyl diazonium tetrafuoroborate, acetonitrile/0.1 M NBu4BF4 solution at a


The passivation of the gold surface after the modification with CP was investigated

using potassium ferricyanide as a redox probe. Figure 3.13 shows cyclic

voltammograms of gold electrodes before and after modification with 4-carboxyphenyl

in 1 mM ferricyanide in a 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate

of 100 mV s-1. After modification of a gold electrode with 4-carboxyphenyl,

electrochemistry of 1 mM ferricyanide with a background of 0.05 M phosphate buffer

(pH 7.0, 0.05 M KCl) was suppressed relative to bare gold electrodes, which is similar

to that depicted in Figure 3.5 a for the GC electrode but to a lesser extent, indicating the

formed monolayers on gold surfaces contain some defects. More specifically, the

electron transfer efficiency was reduced as a consequence of the monolayer forming a

barrier to electrode transfer, as is evident from the increase in the Ep value from 80 to

370 mV.

103


-2

-1

0

1

2

-0.2 0 0.2 0.4 0.6

Potential /V

Curr

ent

/A

Bare AuCP modified Au

Figure 3.13 Cyclic voltammograms of bare and CP modified gold electrodes in the

ferricyanide solution (1 mM, 0.05 M KCl, 0.05 M phosphate buffer, pH 7.0) at the scan

rate of 100 mV s-1.

3.3.8.2 Characterisation of 4-Carboxyphenyl Modified Gold Surfaces Using XPS

XPS measurements were carried out to further characterise the modified species after

modification of the gold surface with diazonium salts. The XP survey spectrum of the

unmodified gold electrode (Figure 3.14 a) showed the peaks expected for gold as well

as small carbon 1s and oxygen 1s peaks at ~285 and ~532 eV respectively, indicating

the presence of organic contaminants such as adventitious hydrocarbons and water.

After modification of the gold substrate with 4-carboxyphenyl groups, the XP survey

spectrum showed a significant increase in the 1s peaks of carbon and oxygen at ~285

and ~532 eV respectively but no significant evidence of a nitrogen 1s peak (Figure 3.14

b) indicating the nitrogens of the diazonium salt are lost during the immobilisation on

the gold surface. The carbon 1s envelope (Figure 3.14 b, inset) was fitted with four

peaks at 288.7, 286.2, 284.6 and 283.9 eV assigned to the carboxylic acid moieties, C-O

species, the aromatic carbons of the monolayer and the metal-bonded carbon

104


respectively. The binding energy observed for the carboxylic acid group on gold was

consistent with that observed on the GC surface. The inclusion of the metal carbide

peak is exceedingly tentative as a good fit to the spectra could be obtained without the

presence of this peak. The assignment of the metal carbide peak is based on the

precedence of Pinson and coworkers12, 13, 48 who have previously proposed the existence

of such a peak for the electroreduction of aryl diazonium salts onto metal surfaces. On

iron surfaces the case for a metal-carbide peak is compelling with a very pronounced

shoulder when a high resolution instrument is used48 with the intensity of this shoulder

sensitive to take-off angle indicating it is a surface bound species. However, on copper

electrodes13 and other examples on iron12 the shoulder on the carbon 1s spectra is less

pronounced similar to the observations on gold here.

Binding Energy (eV)

02004006008001000

Counts

(arb

.)

O1s

Au4f

C1s

Au4d

Au4p3/2

281285289293

C1s

Au4p1/2Au4s

(a)

Binding Energy (eV)

02004006008001000

Counts

(arb

.)

O1s

Au4f

Au4p3/2

Au4d

Au4p1/2

Au4s

282286290294

C1s

COOH C-Au

(b)

C1s

Figure 3.14 XP survey spectrum and carbon 1s narrow scan (inset) of (a) and a bare

gold electrode and (b) a gold electrode modified with 4-carboxyphenyl moieties.

3.3.8.3 Robustness of Monolayers Modified on Gold Surfaces

The electrochemistry and XPS data have confirmed the successful modification of gold

surfaces with diazonium salts of CP. The robustness of the formed monolayers on gold

105


electrodes was tested using the method developed by Losic et al.23 The cyclic

voltammogram of a bare gold electrode in aqueous 0.1 M H2SO4 is characterised by

anodic and cathodic peaks that are associated with the formation of gold oxide and its

subsequent removal by reduction, respectively. With a long chain alkanethiol

monolayer modified gold electrode scanning in 0.1 M H2SO4 will exhibit gold oxidation

and reduction peaks are any pin-hole defects in the SAM. Losic et al.23 showed with

repeated scans degradation of the monolayer was observed as evidence from an increase

in the size of the gold oxide reduction peak and used the rate of increase in magnitude

of this peak as a measure of the robustness of a monolayer.

CVs of a CP modified gold electrode in aqueous 0.1 M H2SO4 at a potential of -0.5 to

1.5 V at a scan rate of 100 mV s-1 (Figure 15 a) shows the electrochemistry of the bare

gold surface is still observable after modification of CP monolayers (the charge passed

during the first oxide removal wave is 85% of its value at bare gold), indicating that the

CP monolayer does not provide a very effective barrier to gold reduction. The

significant gold reduction could be a consequence of either the monolayer only covering

a small portion of the gold surface or because the short chain alkanethiol provides an

insufficient barrier to electrons tunneling across the monolayer. The latter possibility

seems more likely as the surface coverage of 6.4×10-10 mol cm-2 is greater than 50% of

the theoretical maximum monolayer coverage and almost as high as the theoretical

maximum monolayer coverage for an alkanethiol SAM49 of 7.6×10-10 mol cm-2 and only

15 % surface coverage is totally inconsistent with the electrochemically determined

surface coverages of SAM.

106


-16

-12

-8

-4

0

4

8

-0.3 0.2 0.7 1.2 1.7

Potential /V

Curr

ent

/A

Bare Au

CP modif ied Au

HDT modif ied Au

a

0

0.2

0.4

0.6

0.8

1

1.2

1 3 5 7 9 11 13

Number of cycles

1-(

I ba

re-I

sca

n,n

)/(I

ba

re-I

sca

n,1

)

15

CP modif ied Au

HDT modif ied Au

MBA modif ied Au

MPA modif ied Au

b

Figure 3.15 (a) Cyclic voltammograms in 0.1 M H2SO4 at the scan rate of 100 mV s-1

for a bare gold electrode, a CP modified gold electrode and an HDT modified gold

electrode during the first cycle, and (b) relative current change from the first cycle for

the CP, HDT, MBA, MPA modified gold electrodes versus number of cycles in 0.1 M

H2SO4 at a scan rate of 100 mV s-1. Ibare, Iscan,1 and Iscan,n refer to the current of bare

gold, current of modified gold in the first scan and current of modified gold in the n

scan (n is the scan number), respectively.

For comparison cyclic voltammetry for different alkanethiols modified gold electrodes

was also carried out in aqueous 0.1 M H2SO4. SAMs of 4-mercaptobenzoic acid (MBA)

107


and MPA on gold electrodes gave similar responses to the CP monolayers in the H2SO4

solution. For MBA and MPA modified gold electrodes the charge passed during the

first oxide removal wave is 91% and 87% of its value at bare gold, respectively. The

amount of gold oxidation is despite the surface coverages determined by

electrochemical desorption being close to ideal again suggesting the monolayers are

providing only a minor tunnelling barrier to electron transfer during gold oxidation. In

contrast, the long chain alkanethiol HDT provides a much more effective tunnelling

barrier and more densely packed monolayer based on the first gold oxide reduction peak

being only 11% of its value at bare gold, Figure 15 a (the still relatively high number of

defects in the SAM is consistent with rough polycrystalline electrodes such as these).23

The robustness of the monolayers was investigated with repeated cycling in sulfuric

acid as depicted in Figure 15 b. To allow for the different amounts of attenuation of

gold oxide formation by the different monolayers in Figure 15 b the y-axis is the ratio

of difference between the gold oxide reduction peak for the bare electrode minus that

for a particular scan relative to the difference in gold oxide reduction peak current for a

bare electrode minus that for the first scan. Using this representation the graph depicts

the relative change in the ability of the monolayer to block gold oxide formation to

allow comparison between the different SAMs. For the CP modified electrodes there is

a significant increase in the gold oxide reduction current with the first two cycles,

presumably due to loss of loosely bound CP whereupon the current does not change

significantly, suggesting that aryl groups are strongly attached to the gold surface and

not perturbed by the formation and removal of gold oxide. This is in contrast with the

data, recorded in similar experimental conditions, for a gold electrode modified with

SAMs of HDT, which with the long aliphatic chains make these very stable alkanethiol

108


SAM. With HDT there is no large initial increase in reduction peak current but there is

a continual gradual increase in the percentage of electrochemically accessible gold on

the repeated cycle.23 A more realistic comparison is with the growth of gold oxide after

modification with short chain alkanethiols. MBA monolayer appeared to exhibit

complete loss of the monolayer after one cycle as indicated by the gold oxide reduction

peak becoming identical to that observed with a bare electrode.50 Similarly, SAMs of

MPA also appeared to be completely removed after just three additional voltammetric

cycles (Figure 15 b). These results indicate the diazonium salt monolayer is more

strongly attached than any of the alkanethiol SAMs. This is the case even with the HDT

SAM where van der Waals forces between the long aliphatic chain help to stabilize the

SAM and thus suggests the robustness of the CP modified gold electrodes is due to the

strength of the bond between the monolayer and the gold electrode.

3.3.8.4 Reductive Desorption of Diazonium Salt Monolayers

Quantitatively reductively desorption of alkanethiols on gold surfaces can be carried out

by scanning cyclic voltammograms in a degassed electrolyte aqueous solution at the

negative potential range, resulting in the thiolate.49, 51 The monolayer desorption

experiment for the CP modified gold surfaces was conducted in a 0.5 M KOH solution

to study the stability of diazonium salt monolayers on gold electrodes (Figure 16 a). No

distinct peak indicative of reductive desorption can be observed for the CP modified

gold electrodes. In contrast, for all the gold electrodes modified with the different

alkanethiols (Figure 16 b-c) distinct reductive desorption peaks at increasingly negative

potentials of -0.66, -0.72 and -1.35 V for MPA, MBA and HDT respectively appear.

This trend in more negative potentials coincides with increasing chain-chain interactions

between the monolayer forming molecules in the SAMs.52

109


-3

-2

-1

0

-1.4 -1.2 -1 -0.8 -0.6 -0.4

Potential /V

Curr

ent

/A

-1.35 V

d

-20

-15

-10

-5

0

5

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2

Potential /V

Curr

ent

/A

a

-6

-5

-4

-3

-2

-1

0

1

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2

Potential /V

Curr

ent

/A

-0.66 V

b

-15

-11

-7

-3

1

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2

Potential /V

Curr

ent

/A

-0.72 V

c

Figure 16 Cyclic voltammograms of a gold electrode modified with (a) CP, (b) MBA, (c)

MPA, and (d) HDT in 0.5 M KOH solution at a scan rate of 100 mV s-1.

In order to clarify if the CP monolayers on gold electrodes have been removed after

sweeping negative to –1.5 V in KOH solution, electrochemistry of SAMs modified gold

electrodes was carried out in redox probe ferricyanide solution (1 mM; 0.05 M KCl;

0.05 M phosphate buffer; pH 7.0) (Figure 16). Monolayers of CP showed similar

restriction of access of ferricyanide to the electrode surface after sweeping potential

negative to -1.5 V in the KOH solution as they did beforehand (Figure 16 a). SAMs of

MPA however showed similar ferricyanide electrochemistry to a bare gold electrode

(Figure 16 b), thus indicating the MPA SAM was completely removed. Blocking

110


properties for SAMs of MBA in ferricyanide is similar to that for SAMs of MPA. These

results further support that a stronger bond is formed by the CP derived monolayers to

the gold surfaces relative to alkanethiol monolayers. As a result aryl diazonium salt

derived monolayers on gold can be used in an extended potential window whereas for

short chain alkanethiols the potential window is significantly reduced due to oxidative

and reductive desorption.

-4

-3

-2

-1

0

1

2

3

-0.2 0 0.2 0.4 0.6

Potential /V

Cu

rre

nt /

A

MPA Modif ied Au

After desorption in KOH

Bare Au

b

-4

-3

-2

-1

0

1

2

3

4

-0.2 0 0.2 0.4 0.6

Potential /V

Curr

ent

/A

CP Modif ied Au

After desorption in KOH

Bare Au

a

Figure 3.16 Cyclic voltammograms in ferricyanide (1 mM; 0.05 M KCl; 0.05 M

phosphate buffer; pH 7.0) at the scan rate of 100 mV s-1 for a bare gold electrode and

(a) a CP and (b) an MPA modified gold electrode before and after sweeping negative to

–1.5 V in 0.5 M KOH.

111


3.4 Conclusions

Glassy carbon and gold electrodes have been successfully modified with aryl diazonium

salt molecules using the two-cycle cyclic voltammetry method to form novel and stable

monolayers. XPS technique has been successfully used to monitor the step-by-step

attachment. The nitrogen groups were found to be lost during the modification of aryl

diazonium salts by reductive adsorption. Monolayers of aryl diazonium salts on GC and

gold electrodes are more stable than the self-assembled monolayers of alkanethiols on

gold electrodes. So the higher stability of diazoniam salt monolayers created on GC

surfaces provides a great potential for GC electrodes to be used as a good alternative to

gold for sensing and other applications.

3.5 References


(2) Adams, D.M., Brus, L., Chidsey, C.E.D., Creager, S., Creutz, C., Kagan, C.R.,

Kamat, P.V., Lieberman, M., Lindsay, S., Marcus, R.A., Metzger, R.M., Michel-

Beyerle, M.E., Miller, J.R., Newton, M.D., Rolison, D.R., Sankey, O., Schanze,

K.S., Yardley, J., Zhu, X.Y., J. Phys. Chem. B 2003, 107, 6668-6697.

(3) Cahen, D., Hodes, G., Adv. Mater. 2002, 14, 789-798.

(4) Salomon, A., Cahen, D., Lindsay, S., Tomfohr, J., Engelkes, V.B., Frisbie, C.D.,

Adv. Mater. 2003, 15, 1881-1890.

(5) Willner, I., Katz, E., Angew. Chem. Int. Edit. 2000, 39, 1181-1218.


(7) Gooding, J.J., Mearns, F., Yang, W.R., Liu, J.Q., Electroanalysis 2003, 15, 81-96.

112







(11) Ranganathan, S., Steidel, I., Anariba, F., McCreery, R.L., Nano Letters 2001, 1,

491-494.


Fagebaume, O., Pinson, J., Podvorica, F., J. Am. Chem. Soc. 2001, 123, 4541-4549.



(14) Stewart, M.P., Maya, F., Kosynkin, D.V., Dirk, S.M., Stapleton, J.J., Mcguiness,

C.L., Allara, D.L., Tour, J.M., J. Am. Chem. Soc. 2004, 126, 370-378.

(15) Bahr, J.L., Yang, J., Kosynkin, D.V., Bronikowski, M.J., Smalley, R.E., Tour, J.M.,

J. Am. Chem. Soc. 2001, 123, 6536-6542.

(16) Anderson, M.R., Evaniak, M.N., Zhang, M., Langmuir 1996, 12, 2327-2331.

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115

Chapter 4-Heterogeneous Electron Transfer Through Organic Monolayers on Carbon and Gold Electrodes

Chapter Four

Heterogeneous Electron Transfer through Organic Monolayers

on Carbon and Gold Electrodes

116


4.1 Introduction

Electron transfer through organic films of nanometer thickness is of fundamental and

practical importance to the area of sensor technologies, anti-corrosion films and

electronic devices. There are phenomenological factors affecting electron transfer at

interfaces, for examples the solvent reorganisation energy,1, 2 the density of electronic

states in the electrode surfaces,3-6 and electronic coupling between the electrode and the

redox couple.7, 8 The development of self-assembled methods for the construction of

monolayer films on electrode surfaces provides a means to control and manipulate the

interfacial characteristics.9 This technology has been exploited to investigate

fundamental issues of electron transfer between an electrode and a redox couple.10

The results in Chapter Three have demonstrated the glassy carbon (GC) electrodes can

be covalently modified with more stable monolayers of aryl diazonium salts by

electrochemical reduction relative to gold. The reduction reaction results in the loss of

N2 and the formation of a carbon-carbon covalent bond between the adsorbed molecules

and carbon substrates. Thus the conjugated carbon network in the glassy carbon (GC)

electrode can be thought of as continuing into the monolayer system rather than the

abrupt change from electrons being in a metallic environment to an organic

environment.11 The continuity of the electrode material into the monolayer has resulted

in the suggestion that GC electrodes modified by aryl diazonium salts have the potential

to reduce the barrier towards electron transfer from the carbon electrode into the

monolayer which is important for all molecular scale devices where communication

with the macroscopic world is achieved through electron transport. However, a rather

117


large tunneling barrier (~2 eV) can be created by the gold/thiol junction based on the

self-assembled monolayers (SAMs) of alkanethiols on gold surfaces.11 McCreery and

coworkers12-15 have extensively studied the effects of surface structure on the

heterogeneous electron transfer kinetics on GC surfaces by examining different redox

probe solutions on polished and modified GC electrodes. It was concluded carbon

surface variables affect sensitively the kinetics of a given system of interest. However,

the heterogeneous electron transfer between redox active molecules and GC electrodes

through aryl diazonium salt derived monolayers has yet to be investigated. Nor has the

notion that the carbon-carbon bond will allow the efficient electron transfer.

The purpose of this chapter is to demonstrate the modification of GC and gold

substrates using mixtures of aryl diazonium salt molecules (introducing phenyl and 4-

carboxyphenyl groups onto the surface) and to compare the kinetics of electron transfer

to GC, pyrolysed photoresist films (PPF) and gold surfaces from the same ferrocene-

based monolayer system. The electron transfer of a similar ferrocene-based system

prepared by using mixed self-assembled monolayers of 4-mercaptobenzoic acid (MBA)

and 1-propanethiol (PT) on gold surfaces has also been studied for further comparison.


All reagents and materials are listed in Table 2.1 and prepared according to the

procedures in Chapter Two. GC, PPF and gold electrodes were prepared according to

the method described in Section 2.4. Mixed monolayers were prepared as described in

Section 2.4.2 and ferrocenemethylamine was covalently attached to monolayers

according to the procedures in Section 2.2.6. All electrochemical measurements were


118


relative to an Ag/AgCl reference at room temperature. All cyclic voltammetry

measurements were performed in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0).



Modified on Glassy Carbon Electrodes Using Ferrocene as the Redox Probe

As studied in Chapter Three, GC electrodes can be covalently modified with

monolayers of 4-carboxyphenyl (CP). The terminated carboxylic acid groups can be

activated with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)

and N-hydroxysuccinimide (NHS) followed by the covalent attachment of

ferrocenemethylamine by forming amide bond as shown in Scheme 4.1.

Diazonium salts

EDC/NHS

FerrocenemethylamineGC GC GC

NHO

Fe

COOH

Scheme 4.1 Schematic of ferrocenemethylamine immobilised covalently on pure

monolayers of 4-carboxyphenyl on GC electrodes.

Cyclic voltammograms of CP modified GC electrodes in the 0.05 M phosphate buffer

(0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1 before and after the immobilisation of

ferrocene are shown in Figure 4.1. The strong redox peaks appeared after the

attachment of ferrocenemethylamine with EDC/NHS activation and showed linear

variation in peak current with scan rates (Figure 4.2), indicating that the ferrocene was

119


surface bound. In the absence of EDC and NHS, such that no covalent coupling of

ferrocene could occur, only very weak redox peaks due to physisorption were observed

in Figure 4.1 c.

-4

-2

0

2

4

6

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A

(c)

(a)(b)

Figure 4.1 Cyclic voltammograms of CP modified GC electrodes in 0.05 M phosphate

buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1 (a) before and after coupling of

ferrocenemethylamine (b) with and (c) without EDC/NHS activation.

-10

-5

0

5

10

0 0.1 0.2 0.3 0.4 0.5 0.6

Scan rate /V s-1

Cur

rent

/A

Figure 4.2 Peak current versus scan rate for cyclic voltammograms of GC electrodes

covalently modified with ferrocenemethylamine.

120


The CVs of the ferrocene covalently coupled to CP monolayers on GC electrodes in

Figure 4.1 show non-ideal behaviour16 with regards to peak separation at slow scan

rates ( Ep=79 mV rather than the ideal Ep=0 mV) and the full width half maximum

(greater than 200 mV rather than the ideal EFWHM= 90.6 mV/n where in this case n=1).

With regards to both peak separation and the EFWHM the non-ideal behaviour has been

attributed to the ferrocene molecules being located in a range of environments with a

range of formal electrode potentials (Eo`).17, 18 Gooding’s group19, 20 and others21 have

noted previously that fabricating redox active SAMs by assembling the self-assembled

monolayers and then attaching the redox molecule, results in broader FWHM than

observed with electrodes where a redox active alkanethiol was attached directly to the

electrode. The reason for modifying electrodes in this step-wise manner where the

monolayer is formed and then the redox active molecule attached, rather than

synthesising a pure redox active self-assembling molecule followed by assembly on the

electrode, is because in applications of interesting in this thesis, bioelectronics, the step-

wise strategy is the only viable approach.

With a monolayer containing only 4-carboxyphenyl moieties the number of redox active

molecules attached to the surface, as determined from the charge passed under the

Faradaic peaks of the ferrocene modified electrode in Figure 4.1 b, is approximately

(0.073±0.012)×10-10 mol cm-2 with a close to unity ratio of anodic to cathodic peak

areas. Comparing the surface coverage of CP (7.4×10-10 mol cm-2 as observed in

Chapter Three) to that of the number of redox centres attached indicates that only

approximately 10% of the CP monolayers had a ferrocene attached. At this surface

coverage the average area per ferrocene molecule, assuming homogeneous distribution,

121


is 2.2 nm2 which suggests there is a high possibility of interaction between redox active

probes.20, 21

Interaction between redox active centres has been reported to decrease the

reorganisation energy and correspondingly increase the electron transfer efficiency,22-24

hence providing an anomalously high measurement of the rate constant for electron

transfer. As a consequence, the number of coupling points within the monolayer that the

ferrocene could couple was reduced by preparing mixed monolayers composed of the 4-

carboxyphenyl diazonium salt and the phenyl diazonium salt (Scheme 4.2).

Mixed aryl diazonium salt

EDC/NHS

Ferrocenemethylamine

COOH NHO

Fe

GC GC GC

Scheme 4.2 Schematic of ferrocenemethylamine covalently modified on mixed

monolayers of 4-carboxyphenyl and phenyl moieties on GC electrode surfaces.

The electrochemical parameters after attachment of ferrocenemethylamine to mixed

monolayers of CP and phenyl (with different molar ratio) modified GC electrodes are

displayed in Table 4.1. The surface coverage initially increased with the spacing of the

coupling points followed by the more expected decrease as the number of coupling

points decreased as shown in Table 4.1. The reason for the initial increase in surface

coverage of ferrocene as the solution composition from which the monolayer forms

changes from entirely 4-carboxyphenyl diazonium salt to a 1:1 ratio of 4-carboxyphenyl

122


to phenyl diazonium salt is unclear. The percentage of carboxyl groups to be activated

using EDC/NHS, as used here, in a SAM composed of entirely carboxylic acids has

been shown to be approximately 50%25 which is equivalent to all the 4-carboxyphenyl

groups being activated in a 1:1 monolayer. Furthermore, the relative surface coverages

of the 4-carboxyphenyl to ferrocene is 10:1 in the entirely 4-carboxyphenyl monolayer

so there should be excess coupling points for the ferrocene to attach. Therefore it is

suggested that the introduction of a second component into the monolayer (the phenyl

diluent) in effect introduces a hydrophobic component into the monolayer. Ferrocene

has been shown previously to adsorb onto the surface of hydrophobic self-assembled

monolayers.19, 26 Therefore it is proposed that more ferrocene is attached when the

phenyl component is introduced into the monolayer because the surface is more

energetically and sterically favourable location for the ferrocene adsorption compared

with an entirely 4-carboxyphenyl monolayer.

Table 4.1 Some parameters of ferrocenemethylamine attached onto GC electrodes

modified with mixed monolayers of 4-carboxyphenyl and phenyl moieties. Ep is

recorded at a scan rate of 100 mV s-1.

[Phenyl]/[4-

Carboxyphenyl]

Eo`

(mV)

Ep

(mV)

EFWHM

(mV) (pmol cm-2)a/ c kapp

(s-1)

0

1

5

10

20

40

264±15

279±13

292±9

298±7

304±17

317±19

79±10

78±14

89±25

93±10

101±21

107±10

241±10

213±19

227±25

262±38

220±29

289±14

72.8±11.6

100.3±10.4

67.4±10.7

48.1±6.9

29.4±3.4

13.3±2.0

0.89±0.07

1.03±0.04

0.94±0.05

0.88±0.11

0.71±0.16

0.77±0.13

17±10

28±10

15±5

16±2

15±10

10±2

123


The rate constant for electron transfer was determined from the variation in peak

position between the anodic and cathodic scans as a function of scan rate. In this thesis

the variation in peak potential over a wide range of scan rates was fitted using the

Laviron27 formalism which relies on Butler-Volmer kinetics and gives rate constants for

electron transfer. Table 4.1 shows that across the spectrum of dilution ratios

investigated the rate constant for electron transfer (kapp) is approximately 15-20 s-1.

4.3.2 Heterogeneous Electron Transfer through Monolayers of Diazonium Salts

Modified on Gold Electrodes Using Ferrocene as the Redox Probe

The results in Chapter Three have demonstrated that gold electrodes can also be

modified with aryl diazonium salts using the same method as that for GC electrodes.

For comparison the rate of electron transfer for the ferrocene and the same diazonium

salts modified gold electrodes was also studied. The electrochemistry of the attachment

of ferrocene to the CP modified gold electrodes is shown in Figure 4.3. The strong

redox peaks appeared after the attachment of ferrocenemethylamine with EDC/NHS

activation and showed linear variation in peak current with scan rates, indicating that the

ferrocene was surface bound. In the absence of EDC and NHS such that no covalent

coupling of the ferrocene could occur, only very weak redox peaks were observed due

to physisorption. The CVs of the ferrocene covalently coupled to 4-carboxyphenyl

monolayers on gold electrodes also show non-ideal behaviour16 with regards to peak

separation at slow scan rates ( Ep=85 mV rather than the ideal Ep=0 mV) and the full

width half maximum (EFWHM=209 mV rather than the ideal EFWHM= 90.6 mV/n where

in this case n=1), suggesting the ferrocene molecules being located in a range of

environments with a range of formal electrode potentials (Eo`).17, 18

124


-400

-200

0

200

400

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6

Potential /V

Cur

rent

/nA

After modificationBefore modification

Figure 4.3 Cyclic voltammograms of 4-carboxyphenyl modified gold electrodes before

and after modification of ferrocenemethylamine in 0.05 M phosphate buffer (0.05 M

KCl, pH 7.0) at the scan rate of 100 mV s-1.

The electrochemical parameters after the attachment of ferrocene to the CP modified

gold electrodes are shown in Table 4.2. The trends were very similar to the GC

modified electrodes with broader than ideal EFWHM and non-ideal Ep at slow scan rates.

The surface coverages of ferrocene with different ratios of diluent to CP were slightly

lower than those on GC in common with the lower coverage in general of the aryl

diazonium salts on gold compared with GC. Most importantly, the rates of electron

transfer measured on the gold modified surface were significantly greater than that

obtained on carbon. Typically rates of more than a hundred s-1 were observed, which

was approximately one order of magnitude higher than for the same monolayer system

on GC electrodes. The values of the rate constants at the low surface coverage of

ferrocene (last three entries in the table) were particularly difficult to determine because

with small redox peaks background subtraction can have a large impact on the peak

positions. As a consequence the rate constants quoted represent the lower limits and

125


therefore we expect the true rate constant is closer to that observed at the 1:5

monolayers.

Table 4.2 Some parameters of ferrocenemethylamine attached onto gold electrodes

modified with mixed monolayers of 4-carboxyphenyl and phenyl moieties. Ep is

recorded at a scan rate of 100 mV s-1.

[Phenyl]/[4-

Carboxyphenyl]

Eo`

(mV)

Ep

(mV)

EFWHM

(mV) (pmol cm-2)a/ c kapp

(s-1)

0

1

5

10

20

40

268±12

277±25

280±18

282±14

292±19

317±21

81±14

85±9

75±10

92±12

89±8

81±16

209±11

191±17

227±8

260±15

272±14

308±10

49.3±7.6

80.6±5.9

53.7±5.4

25.8±4.0

13.3±2.4

7.1±1.0

0.87±0.13

0.86±0.09

0.72±0.14

0.92±0.07

0.76±0.03

0.93±0.02

257±41

530±42

211±23

83±50

69±50

68±50


Modified on Pyrolysed Photoresist Films

4.3.3.1 Pyrolysed Photoresist Films (PPF)

As described in Chapter Two, PPF, which is produced by deposing positive and

negative photoresists on silicon wafers by spin coating and then pyrolysed at

temperature of 600-1100 oC in an inert environment, has attracted particular attention by

electrochemists.28-30 PPF is exceptionally smooth (<0.5 nm rms) comparing to other

carbon electrode surfaces, such as polished GC and vacuum heat-treated GC disks,

whose root-mean-square surface roughness was found by STM to be 4.1 and 4.5 nm,

respectively.29, 31, 32 Ultraflat solid surfaces will be extremely important for future

126


advancements of molecular electronic devices.11 Additionally, curing in a reducing

atmosphere minimises carbon oxidation, leading to low oxygen/carbon atomic (O/C)

ratio that is relatively stable toward air oxidation. The smoothness and low O/C ratio

results in a surface with a low capacitance, contributing to the low background levels

observed. PPF with properties similar to a very smooth version of GC, has a very low

background current and oxygen/carbon atomic ratio compared to conventional GC,31

and can also be chemically modified via aryl diazonium ion reduction to yield a

covalently attached monolayer.29 The low oxygen/carbon atomic ratio, smoother surface

and the relative stability of PPF indicate that PPF surfaces may be a good alternative to

GC substrate for sensing applications. It is worthy studying electron transfer through

ferrocene system modified on PPF to investigate if the roughness of substrate has some

influence to the heterogeneous electron transfer.

4.3.3.2 Heterogeneous Electron Transfer on PPF Surfaces

As studied above, there is huge difference between the rates of electron transfer for the

same ferrocene systems on different electrode materials GC and gold electrodes.

Another carbon substrate PPF with a smoother surface was used for fabrication of the

similar ferrocene system to study the rate of electron transfer. Firstly, the PPF electrodes

were modified with CP by the reductive reduction of aryl diazonium salts. Cyclic

voltammograms of a PPF electrode in the acetonitrile/0.1 M NBu4BF4 solution

containing 1 mM CP at the scan rate of 100 mV s-1 are shown in Figure 4.4, which is

similar to that on the GC surfaces. The first sweep gave the irreversible reduction wave

at ca. -0.16 V versus Ag/AgCl, which is attributed to the formation of the 4-

carboxyphenyl radical from the diazonium derivative. The waves disappeared

completely in the second cycle. The surface coverage of the modified layer was

127


estimated to be (9.8±0.4)×10-10 (n=6) mol cm-2 through the integration of the redox

peaks of 4-carboxyphenyl groups in Figure 4.4. This value is slightly larger than that on

GC surfaces (7.4×10-10 mol cm-2) reported in Section 3.3.3. The aryl diazonium salt

modified PPF surfaces are also very stable as for the GC electrode. No change could be

detected with electrodes left on a laboratory bench for several months or with electrodes

exposed to vigorous ultrasonic cleaning in ethanol, acetone and acetonitrile.

-90

-70

-50

-30

-10

10

30

-1 -0.5 0 0.5 1

Potential /V

Cur

rent

/A

1st cycle

2nd cycle

Figure 4.4 Cyclic voltammogram of the PPF in a 1 mM 4-carboxyphenyl diazonium

tetrafuoroborate, acetonitrile/0.1 M NBu4BF4 solution at a scan rate of 100 mV s-1.

The passivation of the PPF surface after the modification with aryl diazonium salts was

investigated using potassium ferricyanide as a redox probe. Figure 4.5 shows cyclic

voltammograms before and after modification with CP in 1 mM ferricyanide solution in

a 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at the scan rate of 100 mV s-1. After

modification of the PPF surface with the aryl diazonium salts, the redox peaks of

ferricyanide observed with bare PPF surfaces were completely suppressed, indicating

the monolayer formed on the PPF surface can block the access of ferricyanide to the

electrode surfaces. The good passivating ability of the formed CP monolayer on PPF

128


substrate is attributed to the electrostatic repulsion, which is similar to that for the CP

modified GC electrodes studied in Section 3.3.4 of Chapter Three.

-50

-30

-10

10

30

50

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A

4-carboxyphenyl modified PPFBare PPF

Figure 4.5 Cyclic voltammograms of bare and 4-carboxyphenyl modified PPF in 1 mM

ferricyanide (0.05 M KCl, phosphate buffer, pH 7.0) at a scan rate of 100 mV s-1.

Following this, the ferrocenemethylamine was covalently modified to the CP modified

PPF surfaces by the amide bond coupling. The CVs measured in an aqueous solution of

0.05 M phosphate buffer (pH 7.0) at a scan rate of 100 mV s-1 before and after the

immobilisation of ferrocene on the CP modified PPF surfaces are shown in Figure 4.6.

The electrochemical parameters after the attachment of ferrocene to the CP modified

PPF were very similar to the GC modified electrodes with broader than ideal EFWHM

(187 mV) and non-ideal Ep (65 mV) at slow scan rates. It was found the oxidation and

reduction peak currents in the CVs of PPF after attachment of ferrocene (Figure 4.7)

increase linearly with the scan rates, indicating that a surface attachment of ferrocene

has occurred. When the molar ratio of 4-carboxyphenyl and phenyl is 1:1, the rate of

electron transfer is calculated to be 19.7 s-1 using Laviron’s method, which is very close

to that for GC surfaces and still lower than that for gold electrodes. Therefore it can be

129


concluded that the roughness is not the key factor to cause much lower rate of electron

transfer on GC electrodes relative to gold since similar rate of electron transfers were

obtained with both GC and PPF electrodes. It is worth investigating if the electrode

materials play an important role in the rate constant of electron transfer.

-10

-5

0

5

10

15

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A


Figure 4.6 Cyclic voltammograms of 4-carboxyphenl modified PPF before and after the

attachment of ferrocenemethylamine in 0.05 M phosphate buffer (pH 7.0) at a scan rate

of 100 mV s-1.

-14

-9

-4

1

6

11

16

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A

Increasing scan rate

Increasing scan rate

Figure 4.7 Cyclic voltammograms of ferrocenemethylamine attached to 4-

carboxyphenyl modified PPF in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at scan

rates of 0.1, 0.2, 0.3, 0.4, 0.5 V s-1, respectively.

130


4.3.4 Heterogeneous Electron Transfer Through Alkanethiol Monolayers Modified

on Gold Electrodes

The surface materials might be the cause for the difference in rates of electron transfer

between GC and gold substrates. For further comparison with the monolayers formed

by electrochemical reduction of aryl diazonium salts, mixed SAMs of alkanethiols such

as mercaptobenzoic acid (MBA) and propanethiol (PT) on gold electrodes were also

prepared, followed by the covalent attachment of ferrocene moieties as shown in

Scheme 4.3.

Mixed thiol solution S S

COOH

EDC/NHS

Ferrocenemethylamine S

NHO

Fe

Au Au Au

S S S

Scheme 4.3 Schematic of ferrocenemethylamine covalently attached onto mixed

monolayers of MBA and PT on the gold electrode surfaces.

The electrochemistry of the attachment of ferrocene to the mixed SAMs of MBA and

PT modified gold electrodes is shown in Figure 4.8. The strong redox peaks after the

attachment of ferrocene showed linear variation in peak current with scan rate,

indicating that the ferrocene was surface bound. In the absence of EDC and NHS such

that no covalent coupling of the ferrocene could occur, only very weak redox peaks

were observed due to physisorption. The rate constants determined for this equivalent

131


aryl thiol system were in the order of 103 s-1 (at the limits of what can be measured

electrochemically) which was approximately five to ten times the values observed for

the aryl diazonium salt-gold system but two orders of magnitude higher than those

observed for the aryl diazonium salt-glassy carbon system. These observations indicate

that the electrode material has a significant effect on the rate of electron transfer.

-150

-100

-50

0

50

100

150

200

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/nA


Figure 4.8 Cyclic voltammograms of mixed SAMs of MBA and PT modified gold

electrodes before and after the attachment of ferrocenemethylamine in 0.05 M

phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1.

4.3.5 Kinetics of Heterogeneous Electron Transfer Through Organic Monolayers on

Carbon and Gold Electrodes

The rate constants for electron transfer are remarkably slower for the carbon electrodes

relative to the gold electrodes which is contrary to the suggestion that with aryl

diazonium salt modified carbon electrodes the continuity of the conjugated carbon

network from the electrode into the monolayer will result in a lower barrier for electron

transfer than with organic monolayers on metallic electrodes.11 The question that arises

132


is why is there a difference in rate constant of around one order of magnitude for the

same redox active molecule connected to electrodes by the same bridge molecule?

The Marcus-Hush expression for electron transfer between a donor and acceptor

through an organic bridge in solution includes terms for electronic coupling between the

donor and acceptor, the Gibbs free energy for electron transfer (the driving force, GET)

and the nuclear reorganisation energy ( ) of the redox molecule as a consequence of its

change in oxidation state.33 For a given donor and acceptor pair, the rate of electron

transfer decays exponentially with distance according to a proportionality constant, the

value, sometimes called a damping factor. When the organic bridge is anchored to an

electrode such that it can act as the donor and/or acceptor the situation is complicated

somewhat as the electronic properties of the electrode can also play a role in the rate of

electron transfer.34 Equations describing the rate constant for electron transfer now

incorporate terms related to the Fermi levels of the electrode and the effective density of

electronics states near the Fermi level. In this study the only changes between the

monolayer systems studied relate to the electrode material and the bond to the electrode.

Hence the reorganisation energy and the driving force will remain unchanged. The

electronic coupling may be influenced by the electrode material as changes in electrodes

may alter the extent of wave function mixing between the organic molecules and the

substrates, especially when conjugated molecules are involved as in this case. A detailed

discussion of the theory of electron transfer and how different electrode materials will

influence the electron transfer is clearly not within the scope of my expertise. However

some comments on the large difference in the rate of electron transfer within the limited

knowledge of the experimental systems investigated is worthwhile.

133


The first possibility is that there are multilayers on the carbon electrode rather than

monolayers of the bridge molecule as the rate of electron transfer decays exponentially

with distance. However, the surface coverage of the reduced aryl diazonium salts on the

electrode, as determined from the charge passed, was below the maximum theoretical

coverage for a monolayer of reduced aryl diazonium salts, suggesting a monolayer or

submonolayer modification of the carbon electrode. Hence any significant multilayering

can be ruled out and the difference in electron transfer rate must, in someway, be related

to the different electrode surfaces.

The different electrode surfaces could influence the rate of electron transfer due to the

different electronic properties of the surfaces or the nature of the linkage made to each

electrode or both.5 The results support both of these possibilities playing a role. This

conclusion is drawn from the differences in rate constant calculated on the gold surface

for the aryl diazonium salt relative to aryl thiol monolayers and the large difference the

aryl diazonium salt derived monolayers on gold versus the carbon electrodes. The

investigation of the aryl thiol monolayer systems on gold was necessary to draw this

conclusion because of the uncertainty in the nature of the bond formed between the

monolayer and the gold surface during the electroreduction of the aryl diazonium salt.

On carbon electrodes a carbon-carbon covalent bond with little charge transfer is well

established but the existence of a metal-carbon bond much less so. As indicated in the

results section 3.3.8.2, the fitting of the C 1s spectrum with a metal carbide bond is

tentative, as a good fit could also be achieved without including this bond. The

suggestion of a metal-carbon bond is based on the precedence of Pinson and coworkers

35-37 who have provided good evidence for a metal carbon bond on iron and copper

surfaces with very limited charge transfer.36 That is a similar bond to that formed on a

134


carbon electrode. The gold-thiol bond however, although also the subject of some

controversy, is generally accepted as being a pseudocovalent bond with significant ionic

character.38 In my hands the rate constant for the gold-thiol system is at least five that

for the aryl diazonium derived monolayer on gold, despite the extra bond between the

phenyl ring and the electrode, which is the reason for the assertion that the bond to the

electrode is playing some role.

If the bond between the organic monolayer and the electrode is only of minor

importance when considering the large difference in rate constants between the gold and

carbon electrodes, what is it about the electrode materials which cause such a large

difference? For saturated bridge molecules in metal-molecule-metal junctions fabricated

by assembling a monolayer on an electrode surface and contacting the top with a

conducting probe atomic force microscope, Beebe et al.39 have shown that the contact

resistance is increased with increasing work function of the metals in the junction.

Although glassy carbon is a heterogeneous material, the work function for carbon is

approximately 5.0 eV whilst for polycrystalline gold it is 5.1 eV.40 The similarity in the

work functions suggests this is not a dominant factor in the large difference in the rate

constant for electron transfer. As a consequence, it is proposed that the difference in rate

constant is a due to a difference in the electronic coupling between the electrode and the

redox molecule, perhaps as a consequence of the wave function mixing between the

molecule and the electrode.41 Stokbro et al.42 have calculated for dithiol benzene

assembled on a gold molecular break junction that when a gold-thiolate bond is formed

the energies of the HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest

Unoccupied Molecular Orbital) of the dithiol benzene fall below the Fermi level of the

organic molecule. Therefore, the electron density of the metal spills over into the

135


organic molecule and both the HOMO and LUMO are occupied. A similar type of

conclusion is arrived at by Hall et al.43 for a molecular break junction with the same

chemical system. Thus, it is proposed that spill over of electron density into the

resultant monolayer derived from the aryl diazonium salts occurs on gold surfaces to a

far greater extent than carbon surfaces and hence the rate of electron transfer is

significantly greater.

The proposed chemical functionalities at the surface of polished glassy carbon

electrodes include quinones, lactones, ketones, alcohols and carboxylic acids13 as shown

in Figure 4.9 A. The inference of Figure 4.9 A is that the delocalisation of electrons

throughout the carbon network is a consequence of an aromatic network of fused

benzene rings. Radical attack by reduced aryl diazonium salts is expected to occur at

electron rich centres such as carbons in the benzene rings and the carbons ortho to the

alcohols. Therefore after attacking by the aryl radicals the diazonium salts modified GC

surface is expected to look like Figure 4.9 B. The hydrazine species proposed are

observed in the XPS of the aryl diazonium salt modified carbon surfaces whilst no

hydrazine species were observed after modification of the gold electrodes with the aryl

diazonium salts as expected.44 Ignoring the hydrazine, which the XPS suggests is only a

minor component of the surface, the carbon-carbon single bond between the bulk glassy

carbon and the aryl rings from the diazonium salt suggests rather than a continuation of

the aromaticity of the carbon surface, the coupling of the monolayer to this aromatic

network by a single bond actually forms a barrier to the aromatic network. Biphenyl

serves as an analogy to a phenyl diazonium salt coupled to a glassy carbon electrode, as

it is one benzene ring connected to another benzene ring by a carbon-carbon single

bond. Although aromatic, biphenyl represents two effectively isolated aromatic rings

136


with little or no delocalisation of electrons between the rings. It is proposed therefore

that with the carbon surfaces the mixing of delocalised electrons between the glassy

carbon and the monolayer on the surface is unlikely to occur to a significant extent and

hence there is a greater barrier to electron transfer than with the gold electrodes where

electron density can spill over into the organic monolayer.

O

O

OC O

Bulk glassy carbon

Solution

X

N

N

X

X

X X

OH

O

O

OC OOH

OH

OH

A)

B)Solution

Bulk glassy carbon

Figure 4.9 Schematic of A) a GC electrode showing the functional groups typically

found on the electrode surface and B) after modification with an aryl diazonium salt.

137


Evidence to support the notion that there is little mixing of delocalised electrons

between a glass carbon electrode and an aryl diazonium salt derived monolayer comes

from Solak et al.45 Solak et al.45 showed that a biphenyl diazonium salt modified GC

electrode was effectively passivating to outer sphere redox active molecules in

solutions, a similar observation to that in Chapter Three. However, upon poising the

electrode at –0.2 V, in which an electron is injected into the biphenyl ring, the organic

layer deposited onto the GC electrode became conducting. Further layers of diazonium

salts could be deposited and good electron transfer could occur to redox species in

solution. Thus, the situation changed from the monolayer being a layer over the

electrode to part of the electrode. It was proposed that the injection of an electron

resulted in a change in the organization of -bonds with a double bond connecting the

glassy carbon electrode to the monolayer. Thus, the injection of the electron caused a

significant decrease in the HOMO-LUMO gap, a reduction in the barrier to electron

transfer and a higher electronic conductance. Thus, with the data presented in this

chapter, if on gold there is mixing of delocalised electrons between the gold electrode

and the organic monolayer but little or no mixing of delocalised electrons on the GC

electrodes there should be a difference in the amount of electron transfer that can be

achieved with a redox active molecule in solution rather than attached to the monolayer.

The electrochemistry of the monolayer modified GC and gold electrodes with redox

active molecule in solution are shown in Figure 4.10. In both cases the electrode is

modified with a 1:1 molar ratio of the 4-carboxyphenyl and phenyl. The two redox

active molecules are ferricyanide in aqueous solution and ferrocene recorded in

acetonitrile. These were chosen as they are both redox molecules which undergo outer

sphere electron transfer rather than adsorbing onto an electrode surface prior to electron

138


transfer occurring. Ferricyanide is negatively charged and may be repelled from the

electrode surface by the carboxylate species at the electrode surface whilst ferrocene is

neutral.

-30

-20

-10

0

10

20

-0.2 0 0.2 0.4 0.6

Potential /V

Cur

rent

/A

Bare GC

Modified GC

a

-4

-3

-2

-1

0

1

2

3

-0.2 0 0.2 0.4 0.6

Potential /V

Cur

rent

/A

Bare Au

Modified Au

c

-80

-50

-20

10

40

70

100

0 0.2 0.4 0.6 0.8

Potential /VC

urre

nt /

A

Bare GC

Modified GC

b

-10

-5

0

5

10

15

0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A

Bare AuModified Aud

Figure 4.10 Cyclic voltammograms of bare and modified electrodes with an outer

sphere redox active species in solution. The electrodes were all modified with a 1:1

mole fraction ratio of 4-carboxyphenyl:phenyl diazonium salts. The electrodes are a)

GC electrode in a solution of 1 mM ferricyanide (0.05 M potassium phosphate, 0.05 M

KCl, pH 7.0), b) GC electrode in 1 mM ferrocene in acetonitrile/0.1 M

tetrabutylammonium tetrafluoroborate c) gold electrode in a solution of 1mM

ferricyanide (0.05 M potassium phosphate, 0.05 M KCl, pH 7.0) and d) gold electrode

in 1 mM ferrocene in acetonitrile/0.1 M tetrabutylammonium tetrafluoroborate.

139


Figure 4.10 clearly shows diffusion controlled CVs where there is significantly more

electron transfer through the monolayer on gold as distinct from carbon. This is

particularly dramatic with the ferrocene redox couple where on gold the redox

chemistry is identical to when the monolayer is absent. It is important to emphasise the

ferricyanide electrochemistry was performed after the ferrocene measurements,

verifying the monolayer is present. Thus, assuming the packing of the diazonium salt

derived monolayers is not dramatically different, and the similarity in surface coverage

during deposition suggests the coverage of monolayers is similar on each electrode

material, these results provide good evidence that the order of magnitude difference in

electron transfer ability between aryl diazonium salt derived monolayers on gold and the

same monolayers on GC electrodes is due to better mixing of delocalised electrons on

the gold surface.

4.4 Conclusions

Electrochemical reductive adsorption of mixtures of 4-carboxyphenyl and phenyl

diazonium salts on GC, PPF and gold surfaces has yielded stable mixed monolayers to

which ferrocenemethylamine could be covalently attached via activation of the surface

bound 4-carboxyphenyl moieties. The rates of the heterogeneous electron transfer for

immobilised ferrocene through the mixed monolayers were an order of magnitude

higher for the gold electrodes in comparison to the carbon electrodes. Furthermore

mixed aryl diazonium salt derived monolayers on GC and PPF surfaces showed

stronger blocking of electron transfer from redox-couples in solution than the equivalent

monolayers on gold surfaces. These results suggest that the mixing of delocalised

electrons between the electrode material and the monolayer occurs to a greater extent

for gold than for GC allowing more rapid electron transfer for the system on gold than

140


on GC. However, the higher stability of monolayer created on GC and PPF surfaces,

which is very critical for sensing applications, will make GC or PPF surfaces a good

alternative to gold surfaces for the development of electrochemical sensors.

4.5 References

(1) Liu, Y.P., Newton, M.D., J. Phys. Chem. 1994, 98, 7162-7169.

(2) Newton, M.D., J. Electroanal. Chem. 1997, 438, 3-10.

(3) Gosavi, S., Marcus, R.A., J. Phys. Chem. B 2000, 104, 2067-2072.

(4) Forster, R.J., Loughman, P., Keyes, T.E., J. Am. Chem. Soc. 2000, 122, 11948-

11955.

(5) Finklea, H.O., Yoon, K., Chamberlain, E., Allen, J., Haddox, R., J. Phys. Chem. B

2001, 105, 3088-3092.

(6) Liu, G., Liu, J., Böcking, T., Eggers, P., Gooding, J.J., Chem. Phys. 2005, 319, 136-

146.

(7) Marcus, R.A., Ksu, C.P., J. Chem. Phys. 1997, 106, 584-598.

(8) Hsu, C.P., J. Electroanal. Chem. 1997, 438, 27-35.

(9) Ulman, A., An Introduction to Ultrathin Organic Films From Langmuir-Blodgett to

Self-Assembly. Academic Press: London, 1991.

(10) Napper, A.M., Liu, H.Y., Waldeck, D.H., J. Phys. Chem. B 2001, 105, 7699-7707.


491-494.

(12) McCreery, R.L., Carbon Electrodes: Structural Effects on Electron-Transfer

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(13) Chen, P., McCreery, R.L., Anal. Chem. 1996, 68, 3958-3965.

(14) Kuo, T.-C., McCreery, R.L., Anal. Chem. 1999, 71, 1553-1560.

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(15) Yang, H.-H., McCreery, R.L., Anal. Chem. 1999, 71, 4081-4087.


(17) Rowe, G.K., Carter, M.T., Richardson, J.N., Murray, R.W., Langmuir 1995, 11,

1797-1806.

(18) Honeychurch, M.J., Langmuir 1998, 14, 6291-6296.


8466.

(20) Liu, J.Q., Gooding, J.J., Paddon-Row, M.N., Chem. Commun. 2005, 631-633.

(21) Seo, K., Jeon, I.C., Yoo, D.J., Langmuir 2004, 20, 4147-4154.

(22) Finklea, H.O., Hanshew, D.D., J. Am. Chem. Soc. 1992, 114, 3173-81.

(23) Wei, J.J., Liu, H.Y., Khoshtariya, D.E., Yamamoto, H., Dick, A., Waldeck, D.H.,

Angew. Chem. Int. Edit. 2002, 41, 4700-4703.

(24) Salomon, A., Cahen, D., Lindsay, S., Tomfohr, J., Engelkes, V.B., Frisbie, C.D.,

Adv. Mater. 2003, 15, 1881-1890.

(25) Jiang, L., Glidle, A., Griffith, A., McNeil, C.J., Cooper, J.M., Bioelectrochem.

Bioenerg. 1997, 42, 15-23.

(26) Creager, S.E., Rowe, G.K., J. Electroanal. Chem. 1997, 420, 291-299.


(28) Kostecki, R., Schnyder, B., Alliata, D., Song, X., Kinoshita, K., Kotz, R., Thin

Solid Films 2001, 396, 36-43.

(29) Ranganathan, S., McCreery, R.L., Anal. Chem. 2001, 73, 893-900.

(30) Hebert, N.E., Snyder, B., McCreery, R.L., Kuhr, W.G., Brazill, S.A., Anal. Chem.

2003, 75, 4265-4271.

(31) McDermott, M.T., McCreery, R.L., Langmuir 1994, 10, 4307-4314.

(32) Ranganathan, S., McCreery, R.L., J. Electrochem. Soc. 2000, 147, 277-282.

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(33) Paddon-Row, M.N., In Electron Transfer in Chemistry, Balzani, V., Ed. Wiley-

VCH: Weinheim, 2001; Vol. 3, Part 2, Ch. 1, p 179.






Fagebaume, O., Pinson, J., Podvorica, F., J. Am. Chem. Soc. 2001, 123, 4541-4549.




6333-6335.

(38) Ulman, A., Chem. Rev. 1996, 96, 1533-1554.

(39) Beebe, J.M., Engelkes, V.B., Miller, L.L., Frisbie, C.D., J. Am. Chem. Soc. 2002,

124, 11268-11269.

(40) CRC Handbook of Chemistry and Physics. 67 ed.; CRC Press: Boca Raton, Florida,

1986.

(41) Vondrak, T., Wang, H., Winget, P., Cramer, C.J., Zhu, X.-Y., J. Am. Chem. Soc.

2000, 122, 4700-4707.

(42) Stokbro, K., Taylor, J., Brandbyge, M., Mozos, J.L., Ordejon, P., Comp. Mater. Sci.

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143


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144

Chapter 5-Fabrication of Electrochemical Copper Sensors Based on Gly-Gly-His Modified Carbon Electrodes

Chapter Five

Fabrication of Electrochemical Copper Sensors Based on

Gly-Gly-His Modified Carbon Electrodes

145


5.1 Introduction

The development of practical sensors for the detection and quantification of metal ions

in environmental samples is the subject of considerable research owing to the growing

public concern of their toxic effects.1 The majority of this research is based on the

development of electrochemical metal sensors using amino acid and peptides as

recognition elements. There are a number of attractive features of using peptides in the

development of electrochemical metal ion sensors.2-4 The variety of amino acid building

blocks provides a myriad of peptide ligands that will have a broad spectrum of affinities

for different metal ions. The simple, generic chemistry involved in synthesising

different ligands renders peptide ligands a highly attractive and under-exploited class of

ligands for the development of solid-state metal ion sensors.

Self-assembly of alkanethiol monolayers on noble metal surfaces for the fabrication of a

sensing interfaces has received considerable interest owing to the ability to control the

sensing element at the molecular level.5 One of the earlier examples of a self-assembled

monolayer that selectively binds redox metal ions was described by Rubinstein and

coworkers.6 Turyan and Mandler have developed the metal sensors for the detection of

Cd (II) and Cd (VI) in aqueous solution for gold electrodes.7, 8 Flink et al. have extended

the ion recognition to electrochemically inactive cations using impedance

spectroscopy.9 Immobilisation of peptides on self-assembled monolayers (SAMs) of

alkanethiol modified gold surfaces for metal ion sensing has received considerable

recent interest.1 Apart from the selectivity imparted from using different peptide

ligands, an additional level of selectivity can be achieved by exploiting different redox

146


potentials of different metals. Making good use of the diversity of peptides and their

selectivity for different metals, Gooding and coworkers have successfully developed

several highly sensitive and selective metal sensors using amino acids,10, 11

oligopeptides12, 13 and polypeptides14 as the selective recognition elements based on

self-assembled monolayers of alkanethiols on gold surfaces.12, 15-17 However, studies

have revealed that these monolayers are not very stable and that they can be oxidatively

or reductively desorbed.18-20 The problem of instability is worse with the short chain

alkanethiols which are used to bring the peptide closer to the gold electrode surfaces

such that appreciable electrochemistry of the metal or metal ions can be achieved. It is

therefore desirable to develop an alternative sensing interface for metal ion detection,

which is more stable and could overcome the disadvantages of gold-alkanethiol

chemistry without compromising the advantages of the self-assembly system for the

fabrication of chemical sensors.

As introduced in Chapter Three, GC surfaces can be electrochemically modified with

aryl diazonium salts by reductive adsorption to form a stable carbon-carbon covalent

bond on the electrode surface.21 These aryl groups can possess a variety of functional

groups for further modification. So a good candidate methodology to produce more

stable monolayer systems is the modification of GC electrodes with aryl diazonium

salts. GC electrodes are inexpensive and chemically inert, and have a wide potential

window.22 Despite the lower electron transfer kinetics on GC surfaces relative to gold

surfaces, as described in Chapter Four, GC electrodes have better potential for the

fabrication of chemical sensors as the stability of the recognition interface is more

important for sensing than the electron transfer efficiency.

147


The aim of this chapter is to demonstrate the advantages and disadvantages of

developing sensors using GC electrodes modified with diazonium salts relative to gold

electrodes modified with alkanethiols. The model sensing system used to demonstrate

this is Gly-Gly-His modified GC electrodes for detecting copper ions. This system was

chosen because it is well characterised in the laboratory and furthermore it is a system

where the stability in the coupling chemistry is a key problem for the research group.

The GC electrodes were modified by firstly attaching 4-carboxyphenyl groups to the

GC surfaces by the electrochemical reduction of aryl diazonium salts, which led to the

formation of a monolayer on the surface of GC electrode with a carboxylic acid terminal

group. Subsequently the tripeptide could be covalently attached on the carboxylic acid

terminated surface by forming an amide bond. The Gly-Gly-His modified GC electrodes

were characterised by XPS in order to monitor the chemical functionalisation of the

electrode during each step in the modification process. The complexing of copper ions

on the Gly-Gly-His modified GC surface was monitored electrochemically. Furthermore,

the effects of ligand density and roughness of the electrode surface were investigated.

Ligand density was probed by using mixed monolayers formed from 4-carboxyphenyl

and phenyl species followed by the attachment of Gly-Gly-His. Surface roughness

effects were also investigated by using the smoother surfaces photoresist pyrolyzed

films (PPF) as the alternative substrates for the fabrication of Gly-Gly-His modified

surfaces for the detection of copper ions in solutions.


Electrodes modified with Gly-Gly-His were prepared using GC electrodes and PPF. The

electrodes were cleaned and modified as described in Chapter Two. The procedure for

the covalent attachment of Gly-Gly-His on the GC surface is shown in Scheme 5.1.

148


COOHNHO

GC GC GC

Peptide

Aryl diazonium salt EDC/NHS PeptideGly-Gly-His

Scheme 5.1 Schematic of attaching peptide to carboxylate terminated monolayers on a

GC electrode.

GC electrodes were firstly modified with monolayers of 4-carboxyphenyl or mixed

monolayers of 4-carboxyphenyl and phenyl by reductive adsorption. The carboxylic

acid terminated monolayer or a mixed monolayer was activated using 20 mM N-

hydroxysuccinimide (NHS) and 40 mM of 1-ethyl-3-(3-dimethyl aminopropyl)

carbodiimide hydrochloride (EDC) in 2-(N-morpholino) ethanesulfonic acid (MES)

buffer (pH 7.0) to convert the terminated carboxylic acid group to the succinimide ester.

The resultant succinimide ester monolayers were reacted overnight in a solution of Gly-

Gly-His (50 mg mL-1) in 0.1 M MES buffer (pH 7.0) to result in the covalent attachment

of the Gly-Gly-His via nucleophilic attack of the ester by the terminal amino group of

the tripeptide to finally give a modified GC electrode as shown in Scheme 5.2. The

tripeptide Gly-Gly-His is well known as a copper binding peptide because of the

formation of a highly stable complex between peptide sequence Gly-Gly-His and

copper.23, 24 Gly-Gly-His is currently used as a therapeutic for the treatment of Menkes

disease which is a fatal of cross-linked disorder characterised by a widespread defect in

intracellular copper transport.25, 26 Here Gly-Gly-His is attached through the amino

group of the first glycine and terminates with the carboxylic acid of the histidine.

149


Copper ions were accumulated at the Gly-Gly-His modified electrode at open circuit

potential for 10 min in a 0.05 M ammonium acetate buffer solution (pH 7.0) containing

copper nitrate. Cyclic voltammetry (CV) and Osteryoung square wave voltammery

(OSWV) were conducted immediately after preparation.

GC

CO

O

NHH2C

C

O

OHN

CH2

CNH

CH-OOCCH2

N

HN

CON(-)

H2C

CN

Cu2+

N

N

CH

NH

CH2

COO-H2C C

O

(-)(-)

Scheme 5.2 Schematic of Gly-Gly-His modified on GC electrodes before and after

copper binding.


5.3.1 Electrochemistry of Cu2+ Complexed Gly-Gly-His on Glassy Carbon Electrodes

Copper electrochemistry has been reported previously for Cu2+ bound to cysteine

modified gold electrodes.10, 27, 28 Gly-Gly-His modified gold electrodes have been

successfully used for the detection of copper ions in solution with sub-ppt detection

limit.12 However the loss of performance over time and repeated use due to the

degradation of the SAM was observed in such a chemical sensor.16 It is reported SAMs

150


of short chain can be oxidised in the presence of oxygen and light.29-32 So it is necessary

to investigate whether GC can serve as a viable and more stable alternative.

The electrochemistry of the bare GC electrode in the 1 mM Cu2+ in 50 mM ammonium

acetate buffer (pH 7.0) is shown in Figure 5.1. Clear cathodic and anodic waves were

obtained. It is postulated that the cathodic wave is due to the reduction of Cu2+ ions to

copper metal and the anodic wave due to its re-oxidation back to Cu2+. The formal

potential (Eo`) of the Cu2+/0 redox couple was estimated to be +58 mV from the average

of the anodic and cathodic peak potentials which is more negative than the reported

value of +120 mV.33

-9

-7

-5

-3

-1

1

3

-0.4 -0.2 0 0.2 0.4

Potential /V

Cur

rent

/A

Figure 5.1 Cyclic voltammogram of a bare GC electrode in a 0.05 M ammonium

acetate buffer solution (pH 7.0) containing 1 mM Cu2+ at a scan rate of 100 mV s-1.

Figure 5.2 shows a CV of the bare GC electrode before and after incubation in 1 mM

Cu2+ solution for 10 min at a sweep rate of 100 mV s-1. Accumulation of copper ions at

the bare GC electrode was carried out at open circuit potential by dipping the electrode

in a 0.05 M ammonium acetate buffer solution (pH 7.0) containing 1 mM copper nitrate

151


for 10 min. The electrode was removed from the cell and washed thoroughly with water.

The electrochemistry was then conducted immediately in a 0.05 M copper free

ammonium acetate buffer solution. No redox reaction from copper ions was observed in

Figure 5.2, indicating no copper ions had been accumulated onto bare GC surfaces

under this condition.

-1.5

-1

-0.5

0

0.5

1

-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4

Potential /V

Cur

rent

/A

After incubation in Cu2+

Before incubation in Cu2+

Figure 5.2 Cyclic voltammograms of a bare GC electrode before and after exposure to

1 mM Cu2+ in 0.05 M ammonium acetate buffer (pH 7.0) at a scan rate of 100 mV s-1.

Figure 5.3 shows a CV of a freshly prepared 4-carbpxyphenyl/Gly-Gly-His modified

GC electrode before and after incubation in a 500 nM copper solution in 50 mM

ammonium acetate at a scan rate of 100 mV s-1. The voltammogram displays no

electrochemistry between +0.4 V and –0.4 V prior to copper accumulation. After

incubating the electrode in 500 nM Cu2+ for 10 min, copper redox peaks appear at the

formal potential (Eo`) of -60 mV, which is consistent with the standard formal potential

of Cu2+/+ redox couple (-60 mV) and much more negative than the standard formal

potential for the Cu2+/0 couple (+120 mV).34 So the observed redox reaction of copper

for the 4-carboxyphenyl/Gly-Gly-His modified GC electrodes in Figure 5.3 is due to

152


the transition of Cu2+/Cu+ couple but not the Cu2+/0 couple observed on 3-

mercaptopropionic acid/Gly-Gly-His modified gold electrodes which was assigned to

the underpotential deposition (UPD) of Cu.17 UPD has also been identified as the source

of electrochemistry for similar 3-mercaptopropionic acid/peptide modified gold

electrodes for cadmium11 and lead.35 It is unlikely to have the UPD of metals on GC

electrodes due to different natures of the coupling between organic layers and the

electrode surfaces and the surfaces themselves.36

-2

-1

0

1

2

-0.6 -0.4 -0.2 0 0.2 0.4 0.6

Potential /V

Cur

rent

/A

After copper bindingGly-Gly-His initial response

Figure 5.3 Cyclic voltammograms of Gly-Gly-His modified GC electrode before and

after exposure to 5×10-7 M Cu2+ in a 0.05 M ammonium acetate buffer solution

(pH 7.0). In all cases Cu2+ was accumulated at the Gly-Gly-His modified electrode at

open circuit potential for 10 min in a 0.05 M ammonium acetate buffer solution

(pH 7.0) containing 5×10-7 M Cu2+, removed, rinsed and then placed in a copper-free

ammonium acetate buffer solution.

A very stable voltammogram without significant decrease of peak current after multiple

scans was obtained with the Gly-Gly-His modified GC electrodes after accumulation of

Cu2+ ions (Figure 5.3). The stable redox peaks suggest the copper ions are tightly

153


associated with the carboxylic acid terminated peptide modified GC interface. In the

absence of Gly-Gly-His, that is, when the electrode is only modified with 4-

carboxyphenyl groups, there are no redox peaks observed in the same potential window

after incubating the GC electrode in 5×10-7 M copper solution (Figure 5.4). This further

confirms that the electrochemistry observed in Figure 5.3 is a result of copper

complexation to the Gly-Gly-His on GC electrodes.

-5

-4

-3

-2

-1

0

1

2

-0.4 -0.2 0 0.2 0.4

Potential /V

Cur

rent

/A

After incubation in Cu2+

Before incubation in Cu2+

Figure 5.4 Cyclic voltammograms of the 4-carboxyphenyl modified GC electrode

before and after exposure to 5×10-7 M Cu2+ in a 0.05 M ammonium acetate buffer

solution (pH 7.0) at the scan rate of 100 mV s-1.

The CVs of the Cu2+ coupled to the Gly-Gly-His modified GC surface show non-ideal

behaviour37 with regards to peak separation at slow scan rates ( Ep=35 mV rather than

the ideal Ep=0 mV) and the full width half maximum (EFWHM=145 mV rather than the

ideal EFWHM=90.6 mV/n where in this case n=2). The non-ideal behaviour might be

attributed to the copper ions being located in a range of microenvironments and

possessing a range of formal electrode potentials (Eo`).38, 39 One of the possible

microenvironments might be caused by the high density of ligands due to the roughness

154


of the GC surface. The second one might be due to the difference in Cu2+ coupling by

the peptide ligand that is usually variable both in orientation and location. The CVs of a

Gly-Gly-His modified GC electrode after 10 min preconcentration in copper solution as

a function of scan rates are shown in Figure 5.5 a. The linear relationship between the

scan rate and the oxidation and reduction peak currents (Figure 5.5 b) indicates that the

copper ion attachment is a surface binding process.40, 41

-12

-7

-2

3

8

13

0 0.1 0.2 0.3 0.4 0.5 0.6

Scan rate /V s-1

Pea

k cu

rrent

/A

b

-5

-3

-1

1

3

5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6

Potential /V

Cur

rent

/A

Increasing scan ratea

Figure 5.5 (a) Cyclic voltammograms of a 0.1 µM Cu2+ complexed Gly-Gly-His

modified GC electrode in a 0.05 M ammonium acetate buffer solution (pH 7.0) at scan

rates of 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5 V s-1 respectively, and (b) plot of peak current

versus scan rates. Open circles: anodic scan; filled circles: cathodic scan.

155


5.3.2 Characterisation of Gly-Gly-His Modified GC Surfaces Using XPS

X-ray photoelectron spectroscopy (XPS) has proven to be a very useful analytical tool

for studying both the composition of the monolayer and the elemental profile normal to

the surface. The procedures for the fabrication of the peptide modified GC surfaces,

including attachment of aryl diazonium salt, subsequent activation with EDC/NHS and

finally attachment of the tripeptide Gly-Gly-His were characterised using XPS.

Figure 5.6 shows the XP survey spectrum of different steps during the process of Gly-

Gly-His attachment.

Survey spectra of bare GC, before and after modification with aryl diazonium salt, then

being activated by EDC/NHS and finally attached with the tripeptide Gly-Gly-His

showed only the expected chemical components. The electrochemical observations are

entirely consistent with the XPS spectra. The average elemental ratio of oxygen to

carbon on the bare GC surface was 0.126, which lies in the range of reported ratio

values of 0.084-0.221.42-45 The percentage of the surface elemental composition for

nitrogen is only 0.9%, so almost no nitrogen 1s peak was observed on the bare GC

plate. After modification of the surface with 4-carboxyphenyl diazonium

tetrafluoroborate, the XP survey spectrum showed a significant increase in the 1s peaks

of carbon and oxygen at ~285 and ~532 eV respectively but no significant evidence of a

nitrogen 1s peak (1.4%) indicating the nitrogens of the aryl diazonium salt are almost

lost completely during the bond formation with the GC surface. The activation of

benzoic acid modified surfaces with EDC/NHS led to a significant increase in

percentage of nitrogen (7.1%) as expected which increased further after the following

attachment of Gly-Gly-His (8.4%). The steps in the attachment of Gly-Gly-His to GC

156


surfaces were also characterised in details using high resolution scans of the C1s and

N1s regions as follows.

02004006008001000

Binding energy /eV

O1s

N1s

C1sc)

02004006008001000

Binding energy /eV

C1s

N1s

O1s

d)

02004006008001000

Binding energy /eV

b)

O1s

N1s

C1s

02004006008001000

Binding energy /eV

O1s

C1sa)

Figure 5.6 XP survey spectrum of (a) a bare GC plate; (b) a GC plate modified with 4-

carboxyphenyl diazonium tetrafluoroborate; (c) a GC plate modified with 4-

carboxyphenyl monolayer, then activated by EDC/NHS; (d) after further attachment of

Gly-Gly-His. Vertical scale represents the intensity with arbitrary units.

Figure 5.7 shows the C1s signals of the (a) bare GC surface; (b) after modification of

parabenzoic acid; (c) after activation with EDC/NHS and (d) after the attachment of

Gly-Gly-His onto the GC surface. It can be seen from Figure 5.7 a the C1s scan of the

bare GC plate is dominated by the graphitic carbon peak at 284.4 eV together with a

157


broader peak on the high binding energy 285.2 eV due to the presence of low levels of

oxidised carbon species on the electrode surface. After the GC surfaces were modified

with parabenzoic acid, a peak at 288.8 eV, which is typical of the carbon of the

carboxylic acid group, in the C1s spectra as shown in Figure 5.7 b was observed,

indicating the –COOH terminated monolayers have formed on the GC surfaces. This

binding energy is less than that of carboxylate functional groups in polymers

(289.2 eV)46 but is in good agreement with that reported by Allongue and coworkers47.

In addition, the C1s signal at 286.3 eV may be attributed to the organic contaminates

physisorbed on the –COOH terminated monolayer surface, which has been previously

reported by Sieval et al.48

As can be seen from Figure 5.7 c, activation of the carboxylic acid group on the

monolayer with EDC/NHS led to an increase in oxidised carbon species as evidenced

by fitted peaks in the approximate range of 286-289 eV. The emergence of a peak at

287.6 eV can be reasonably assigned to the carbon formed in the NHS ester. This

negative shift in binding energy due to deprotonation of the carboxylic acid group was

also observed on SAMs of 11-mercaptoundecanoic acid (11-MUA) system on gold

electrode49. The corresponding peak area ratio of the newly developed ester carbon peak

and the carboxylic acid group indicates an activation efficiency of 53%. After the

attachment of Gly-Gly-His, no new peak was observed in the C1s spectra as shown in

Figure 5.7 d, and hence this is not useful for providing valuable information for the

attachment of Gly-Gly-His.

158


279281283285287289291

Binding energy /eV

d)

-COOHcarbon288.8 eV

284.8 eV286.3 eV

287.7 eV

279281283285287289291

Binding energy /eV

a)

284.4 eV

285.2 eV

C1s

279281283285287289291

Binding energy /eV

b)

284.4 eV286.3 eV

-COOHcarbon288.8 eV

C1s

279281283285287289291

Binding energy /eV

c)

-COOHcarbon288.9 eV

287.7 eV

286.3 eV284.9 eV

Figure 5.7 C1s core level spectra collected at a take-off angle of 90o of (a) a bare GC

plate; (b) a GC plate modified with 4-carboxyphenyl moieties; (c) a GC plate modified

with 4-carboxyphenyl monolayer, then activated by EDC/NHS; (d) after further

attachment of Gly-Gly-His. Vertical scale represents the intensity with arbitrary units.

Comparing with the C1s, the N1s spectra for the attachment of Gly-Gly-His are more

informative for characterisation. The proposed chemical reactions occurring at the GC

surface were further supported by changes in the N1s spectra. The N1s signal at

399.5 eV as shown in Figure 5.8 a, b was observed for both the bare and the 4-

carboxyphenyl modified GC surface, which was possibly due to contaminants absorbed

from the atmosphere onto the GC surface during the preparation process. In Figure 5.8

159


b, two additional peaks were observed at 400.5 and 402.0 eV (FWHM=1.4 eV). The

former peak can be attributed to the reaction of solution diazonium salt with the already

grafted species such as phenol or naphthol groups on a GC surface to form a hydrazine

attachment to the surface50. The reported N1s value of a hydrazine derivative however is

slightly lower at 400.0 eV51. The latter peak at 402.0 eV might be related to the

presence of tetrabutylammonium groups used in the attachment of the aryl diazonium

salt onto the GC surface, although the peak is slightly higher than the value expected

(401.5 eV) for such cations52.

Figure 5.8 c shows N1s XPS spectra after the activation step of aryl diazonium salt

monolayer with EDC/NHS. Four peaks were fitted to the spectra at 398.7, 400.2

(FWHM=1.4 eV), 401.2 (FWHM=1.4 eV) and 402.5 eV (FWHM=1.4 eV). The peak at

400.2 eV was assigned to the imine and secondary amine of the O-acylisourea

intermediate formed by the reaction of the carboxylic acid group with EDC. The higher

binding energy peak at 401.2 eV was assigned to the protonated tertiary amine of the

EDC intermediate.17, 52-54 The ratio of the areas under the two EDC peaks however is

2.0:1, which is in agreement with the expected value from the EDC structure. The

highest binding energy peak at 402.5 eV was assigned to the NHS ester nitrogen formed

by the reaction of NHS with the O-acylisourea intermediate, since such a positive shift

is indicative of the presence of an electron-withdrawing group attached to the nitrogen,

such as the succinimidyl nitrogen with two neighbouring carbonyl groups.52, 54 In

addition, there is one unidentified peak at 398.7 eV (FWHM=1.4 eV), which might be

related to the nitrogen contamination peak observed in the sample of GC surface

modified with the 4-carboxyphenyl group.

160


395397399401403405407

Binding energy /eV

a)

Contamination nitrogen 399.5 eV

395397399401403405407

Binding energy /eV

c)

NHS ester nitrogen402.5 eV

Protonated tertiaryamine nitrogen (EDC)

401.2 eV

Imine (EDC) and secondary amine (EDC)nitrogen401.2 eV

Unidentifiednitrogen398.7 eV

N1s

395397399401403405407

Binding energy /eV

d)

NHS ester nitrogen402.6 eV

C-N nitrogen(imidazolyl ring)401.6 eV

Amide nitrogen 400.3 eV

C=N nitrogen(imidazolyl ring)398.8 eV

N1s

395397399401403405407

Binding energy /eV

b)

Tetrabutyl ammoniumnitrogen 402.0 eV

Hydrazine nitrogen 400.5 eV

Contaminationnitrogen 399.5 eV

Figure 5.8 N1s core level spectra of (a) a bare GC plate; (b) a GC plate modified with

4-carboxyphenyl moieties; (c) a GC plate modified with 4-carboxyphenyl monolayer,

then activated by EDC/NHS; (d) after further attachment of Gly-Gly-His. Vertical scale

represents the intensity with arbitrary units.

After the attachment of the tripeptide Gly-Gly-His, the N1s core-level photoemission

line was deconvoluted with four components as shown in Figure 5.8 d. The highest

binding energy component at 402.5 eV (FWHM=1.4 eV) can be reasonably assigned to

the unreacted portion of the NHS ester nitrogen. The peak at 398.8 eV (FWHM=1.4 eV)

is attributed to the C=N nitrogen of the imidazole side chain of histidine as well as from

the contaminant. The second highest binding energy component at 401.6 eV

161


(FWHM=1.3 eV) originates from the C-N nitrogen of the imidazolyl ring.55 The peak at

400.3 eV (FWHM=1.3 eV) was assigned to the amide nitrogens of the peptide bonds.17

The peak area ratio of approximately is 1:3:1 for these three components is in good

agreement with the predicted structure of the monolayer of aryl diazonium salt modified

with Gly-Gly-His via the formation of an amide bond. This roughly equals the areas

under the two newly formed imidazolyl nitrogen peaks and that of the unreacted NHS

ester nitrogen, suggesting about 50 % reaction efficiency.

5.3.3 Calibration Curve of Gly-Gly-His Modified Glassy Carbon Electrodes for the

Detection of Cu2+

The attachment of Gly-Gly-His to the 4-carboxyphenyl modified GC surfaces for the

detection of Cu2+ was characterised by electrochemistry and XPS. Subsequently Gly-

Gly-His fabricated GC surfaces were used for the detection of copper ions with different

concentrations. As a more sensitive electroanalytical method than cyclic voltametry,

Osteryoung Square Wave Voltammetry was employed with a Gly-Gly-His modified GC

electrode for the detection of low levels of copper ions. Figure 5.9 shows OSWV peaks

of a Gly-Gly-His modified GC electrode in 0.05 M ammonium acetate buffer solution

(pH 7.0) for a series of copper standards adsorbed for 10 min from copper solution. A

graph of the average peak current density from the OSWV measurements against the

concentration of copper solution is shown in Figure 5.10 for four different electrodes.

The lowest concentration that was detected with this electrode was 0.13 ppb (2 nM).

This lowest detected concentration of Cu2+ is significantly lower than that reported

previously for copper adsorbed on an electrochemically oxidised GC surface (2.5

mM),56 but higher than that for the Gly-Gly-His modified gold electrodes (0.3 nM).57

162


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-0.4 -0.2 0 0.2

Potential /V

Cur

rent

/A

Figure 5.9 OSW voltammograms of a Gly-Gly-His modified GC electrode complexed

with Cu2+ after an accumulation time of 10 min in 0.05 M ammonium acetate buffer (pH

7.0) solutions containing different concentrations of Cu2+. Voltammograms illustrate an

increasing peak height with concentration ([Cu2+]/ppb): 0, 0.32, 3.2, 6.4, 9.8 and 12.8.

0

5

10

15

20

25

30

35

0 30 60 90 120

[Cu2+] /ppb

Cur

rent

den

sity

/A

cm

-2

150

Figure 5.10 Plot of Ioswv versus concentration of copper in the accumulation solution for

four electrodes. In all cases Cu2+ was accumulated at the Gly-Gly-His modified

electrode at open circuit potential for 10 min in a 0.05 M ammonium acetate buffer

solution (pH 7.0) containing copper ions, removed, rinsed and then placed in a copper-

free ammonium acetate buffer solution for electrochemical measurement. Inset is the

linear calibration range.

163


The surprisingly higher detection limit for copper could imply a perturbation in the

thermodynamics of binding. This means inferior accessibility of Cu2+ to the attached

Gly-Gly-His on the GC surfaces relative to gold surfaces. It is hypothesised that two

possible causes of poor accessibility are: 1) the attached Gly-Gly-His ligands are too

close together to allow for efficient binding of Cu2+. This problem can be addressed by

spacing the ligands further apart with mixed monolayers of 4-carboxyphenyl and phenyl

groups; 2) the GC surface may be too rough, such that some of the Gly-Gly-His ligands

are less available to the copper ions. These possibilities are consistent with the fact of

non-ideal Ep and EFWHM due to the coupled Cu2+ being located in different

microenvironments as mentioned in Section 5.3.1. As introduced in Chapter One, PPF

with smoother surfaces might be a good alternative to GC for the fabrication of similar

chemical sensors. The following sections investigate the influence of these two options

(mixed monolayers and PPF) on the detection limit of copper ions.

5.3.4 Attachment of Gly-Gly-His onto Mixed Aryl Diazonium Salts Modified Glassy

Carbon Electrodes for the Detection of Cu2+

Mixed monolayers on gold surfaces have been widely studied, and provide a useful

methodology for incorporating into a monolayer molecular species whose own physical

dimensions would preclude a direct, well-organised assembly.58 A mixed SAM on gold

surfaces formed from two alkanethiols in solution has been regarded as being a

reasonably homogeneous mixture of the alkanethiols59 rather than phase segregation

into “islands” as has been observed in Langmuir-Blodgett films60. However, some phase

segregation has also been reported to occur when the two components are quite different

chemically.61 In fact, whether phase segregation occurs or not depends on the surface

upon which assembly forms, the alkanethiol used and the measurement technique.62 The

164


homogeneity of mixed SAMs with alkanethiols on gold surfaces is attributed to strong

sulfur-substrate interactions. Considering the head group-substrate bond is stronger for

aryl diazonium salts and the two components are very similar, the monolayers with

diazonium salts on GC surfaces are expected to be homogeneous.

Mixed monolayers comprising 4-carboxylphenyl and phenyl moieties were prepared by

immersing the GC electrodes in solutions of mixtures of the corresponding diazonium

salts at a given molar fraction for the electrochemically reductive adsorption. The

tripeptide Gly-Gly-His could be attached to the mixed monolayers modified surfaces as

described previously. After that, the prepared system could be used as a chemical sensor

to detect copper ions in solution. Accumulation of copper ions at this Gly-Gly-His

modified GC electrode was carried out at open circuit potential by dipping the electrode

in a 0.05 M ammonium acetate buffer solution (pH 7.0) containing 0.2 µM copper

nitrate for 10 min. The electrode was removed from the cell and washed thoroughly

with water. The electrochemistry was then conducted immediately in a 0.05 M copper

free ammonium acetate buffer solution. A plot of Cu2+ surface coverage against the

mole ratio of phenyl to 4-carboxyphenyl was obtained as shown in Figure 5.11. It

shows the surface coverage of complexed copper ion decreased with an increasing mole

ratio of phenyl to 4-carboxyphenyl groups as expected. When the Gly-Gly-His was

immobilised on pure 4-carboxyphenyl modified GC surfaces followed by the

complexation of Cu2+, the maximum copper coverage of 7.2×10-11 mol cm-2 was

obtained by integration of the cyclic voltammogram. GC surfaces were modified with

less 4-carboxyphenyl groups when the phenyl diazonium salts were used to dilute the

modification solution of 4-carboxyphenyl diazonium salts, indicating a decrease in

available benzoic acid sites for Gly-Gly-His attachment and the amount of copper ions

165


being complexed decreased correspondingly. Controls show that when the GC surface

was modified with the pure phenyl monolayer, no redox reaction from copper ions was

observed since no Gly-Gly-His could be attached to the pure phenyl modified surface,

indicating Cu2+ could only bind to Gly-Gly-His ligands.

0

20

40

60

80

0 10 20 30 40 5

[phenyl]/[4-carboxyphenyl]

Cop

per c

over

age

/pm

ol c

m-2

0

Figure 5.11 The surface coverage of complexed copper ions versus the mole ratio of

phenyl and 4-carboxyphenyl for Gly-Gly-His immobilised GC electrodes for detection

of Cu2+ with the concentration of 0.2 µM.

A self-assembled monolayer of mixed alkanethiols 3-mercaptopropionic acid (MPA)

and 3-mercaptopropane (MP) for coupling to the synthesised Gly-Gly-His was used by

Gooding and coworkers to investigate influence of the molar fraction of MPA on the

surface coverage of complexed copper ions.63 It was found that the coverage of

complexed copper ions varied with the molar fraction of MPA to MP and the maximum

coverage of 1.2×10-9 mol cm-2 was obtained at a mole ratio of MPA/MP of 1:1, which

implies the density of the ligands on gold surfaces is too high for the maximal

complexation of copper due to steric hindrance when the peptide is immobilised on pure

MPA monolayers. However, the response with dilution on GC surfaces studied here as

shown in Figure 5.11 is different from that on gold surfaces studied by Gooding and

166


coworkers. Using mixed monolayers of 4-carboxyphenyl and phenyl on GC electrodes

did not lead to an increase of copper binding ability, indicating there was no steric

hindrance existing when GC electrodes were modified with pure monolayer of 4-

carboxyphenyl. Comparing the maximum coverage of complexed copper (7.2×10-11 mol

cm-2) on Gly-Gly-His modified GC electrodes with that on gold surfaces, it can be

concluded the density of ligands which can bind Cu2+ on GC surfaces is significantly

lower than that on gold surfaces.

The relationship between the peak current of OSWV and the concentration of copper

ions was studied using the mixed monolayers of 4-carboxyphenyl and phenyl. Cu2+ was

accumulated at Gly-Gly-His modified GC electrodes which was firstly modified with

mixed monolayers of 4-carboxyphenyl and phenyl at a molar ratio of 1:5 at open circuit


various copper ions with different concentrations. The electrode was removed from the

copper solutions and washed thoroughly with water and the electrochemistry was

carried out immediately in the copper-free ammonium acetate buffer solution. The

calibration curves for the pure and mixed monolayer systems are shown in Figure 5.12.

Comparison of the calibration curve obtained from mixed monolayers with that from

pure monolayers for complexing copper ions reveals that there is not much difference

except that the detection limit (0.32 ppb) is higher than that for GC electrodes modified

with pure monolayers of 4-carboxyphenyl. For the detection of the same concentration

of Cu2+, the current density is more sensitive in the pure monolayer system. Thus using

mixed monolayers lower detection limit could not be achieved, indicating that the

higher detection limit for copper ions on GC surfaces relative to gold is not due to the

close packing density of Gly-Gly-His ligands on the substrate.

167


0

5

10

15

20

25

30

35

0 30 60 90 120 1

[Cu2+] /ppb

Cur

rent

den

sity

/A

cm

-2

50

0

5

10

15

20

0 5 10 15

Figure 5.12 Plot of Ioswv versus concentration of Cu2+ in the accumulation solution for

the Gly-Gly-His modified GC electrodes which were firstly modified with pure

monolayer of 4-carboxyphenyl (diamond points) or mixed monolayers of 4-

carboxyphenyl and phenyl with molar ratio of 1:5 (triangle points)). In all cases Cu2+

was accumulated at the Gly-Gly-His modified GC electrodes at open circuit potential

for 10 min in a 0.05 M ammonium acetate buffer solution (pH 7.0) containing copper

ions, removed, rinsed and then placed in a copper-free ammonium acetate buffer

solution. Inset is the linear calibration range.

5.3.5 Electrochemistry of Cu2+ Complexed with Gly-Gly-His Modified on Pyrolysed

Photoresist Films

In order to study whether the roughness of the GC surface was responsible for the

higher detection limit than that on gold, PPF with a smoother surface was used instead

of GC for the preparation of Gly-Gly-His modified electrodes. The PPF is a carbon

surface that can be chemically modified via diazonium ion reduction to yield a

covalently attached 4-carboxyphenyl monolayer. This surface can then be further

modified using Gly-Gly-His for the detection of copper ions. Figure 5.13 shows the

cyclic voltammograms at a sweep rate of 100 mV s-1 of the tripeptide Gly-Gly-His

168


modified PPF before and after incubation in a 5×10-7 M Cu2+ solution. The appearance

of redox peaks with formal potential Eo` of -70 mV due to the reduction of Cu2+ to Cu+

and the oxidation back to Cu2+ suggests the successful complexation of the peptide to

the carboxylic acid terminated PPF interface. The control shows that if the Gly-Gly-His

is absent, such that the electrode is only modified with 4-carboxylphenyl, there is no

redox response in the CV at this copper concentration. The linear relationship between

the scan rates and the oxidation and reduction peak currents gives evidence of a surface

binding process (Figure 5.14).

-14

-11

-8

-5

-2

1

4

7

-0.4 -0.2 0 0.2 0.4

Potential /V

Cur

rent

/A

After copper binding

Gly-Gly-His initial response

After copper removal

Figure 5.13 Cyclic voltammograms of Gly-Gly-His modified PPF before and after

exposure to copper ions in 0.05 M ammonium acetate buffer solution (pH 7.0). In all

cases Cu2+ was accumulated at the Gly-Gly-His modified PPF electrode at open circuit


5×10-7 M copper, removed, rinsed and then placed in a copper-free ammonium acetate

buffer solution. Also shown is the cyclic voltammogram of Gly-Gly-His modified PPF

binding with Cu2+ after regeneration of the PPF surface to Cu2+ free by holding the

working electrode at +0.5 V for 30 s in 0.1 M HClO4. Sweep rate: 100 mV s-1.

169


-20

-15

-10

-5

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6

Scan rate /V s-1

Cur

rent

/A

Figure 5.14 Peak current versus scan rate for the cyclic voltammograms of Cu2+/Gly-

Gly-His complex on the PPF substrate (same conditions as that used in Figure 5.13).

The average peak current versus the concentration of copper solution in which the

electrodes were incubated is shown in Figure 5.15 for four PPF electrodes. The result

shows the lowest concentration detected was 0.13 ppb (2 nM). This detection limit for

Cu2+ is the same as that for GC surfaces, indicating the higher detection limit for the

detection of copper ions on GC surfaces is due to the different surface material between

GC and gold as that studied in Chapter Four. This is a very promising result because it

shows that the PPF can be used as an alternative to the GC electrode. The convenience

of the preparation of low-cost PPF will greatly benefit to the commercialisation of the

chemical sensors fabricated with the carbon substrate.

The same detection limit for Cu2+ on PPF substrates as that achieved using GC surfaces

indicates the roughness is not the key reason for the higher detection limit on GC. The

higher detection limit obtained on GC surfaces relative to gold could be due to a lower

density of ligands problem. As discussed in Section 5.3.5, the maximum coverage of

170


complexed Cu2+ was estimated to be 7.2×10-11 mol cm-2 when the GC surfaces were

modified with only 4-carboxyphenyl monolayers. However, the maximum Cu2+

coverage of 1.2×10-9 mol cm-2 was obtained on gold electrodes at a mole ratio of

MPA/MP of 1:1 by Yang et al.63 indicating the minimum density of ligands on gold

surfaces is 1.2×10-9 mol cm-2. Comparing these two data, it can be concluded the

density of ligands which can bind Cu2+ on GC surfaces is much lower than that on gold

surfaces, such that the fabricated chemical sensor on GC surfaces possesses poorer

copper binding ability relative to gold. Thus the higher detection limit for the detection

of copper ion was obtained on Gly-Gly-His modified GC electrodes relative to gold.

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140

[Cu2+] /ppb

Cur

rent

den

sity

/A

cm

-2

Figure 5.15 Plot of Ioswv versus concentration of copper ions in the accumulation

solution for PPF. In all cases Cu2+ was accumulated at the Gly-Gly-His modified PPF

at open circuit potential for 10 min in a 0.05 M ammonium acetate buffer solution

(pH 7.0) containing copper ions with different concentrations, removed, rinsed and then

placed in a copper-free ammonium acetate buffer solution. Inset is the linear

calibration range.

171


5.3.6 Stability of Gly-Gly-His Modified Glassy Carbon Electrodes for the Detection of

Cu2+

The stability and ability to reuse the fabricated chemical sensor are vital for routine

analysis. Attention was paid to the regeneration and stability of the Gly-Gly-His

modified GC electrodes for the detection of Cu2+. The advantage of Gly-Gly-His

modified electrodes is that it can be regenerated by elimination of Cu2+ from the

electrode by holding the working electrode at +0.5 V for 30 s in 0.1 M HClO4 aqueous

solution. After that, the electrochemistry of Cu2+ completely disappeared as shown in

Figure 5.16. The clean surface of the regenerated electrode can be successfully

restored, and is ready for further detection of copper ions in solution. Experimental

results show that the same Gly-Gly-His modified GC electrode could be regenerated

over 30 times without significant signal deterioration as shown in Figure 5.17.

-1.5

-1

-0.5

0

0.5

1

-0.4 -0.2 0 0.2 0.4

Potential /V

Cur

rent

/A

After copper bindingAfter copper removal

Figure 5.16 Cyclic voltammogram of Gly-Gly-His modified GC electrode after copper

binding and after regeneration of electrodes by holding the working electrode at +0.5 V

for 30 s in 0.1 M HClO4 in 0.05 M ammonium acetate buffer solution (pH 7.0) at the


172


0

1

2

3

4

5

0 4 8 12 16 20 24 28 32

Number of regeneration cycles

Cur

rent

/A

Figure 5.17 Removal/reaccumunation cycles of Cu2+ continuously. Regeneration of

Gly-Gly-His modified GC electrodes to Cu2+ free was achieved by holding the potential

at 0.5 V in 0.1 M HClO4. The electrode was reaccumulated in 10-6 M Cu2+ for 10 min

and the cathodic peak current from the OSWV was measured.

The storage stability of a Gly-Gly-His modified electrode was also assessed by storage

exposure to the air under dry conditions at room temperature. Under this storage

condition the same modified electrode could be used everyday for one month without

loss of performance whereupon the current response began to decline very slightly as

shown in Figure 5.18. It is reported that the response of the Gly-Gly-His modified gold

electrodes for the detection of copper ions decreased quickly after one week usage.16

Comparing the stability of Gly-Gly-His modified gold electrodes with that of GC

electrodes, it can be concluded that Gly-Gly-His modified GC electrodes used for the

detection of copper are more stable. So the GC electrode modified with the tripeptide

Gly-Gly-His is a highly stable and reusable chemical sensor for the detection of copper

ions.

173


0

1

2

3

4

5

0 5 10 15 20 25 30

Days

Cur

rent

/A

Figure 5.18 Removal/reaccumunation cycles of Cu2+ once a day. The experimental

condition is the same as that in Figure 5.17.

5.4 Conclusions

GC electrodes and PPF substrate have been successfully modified with monolayers or

mixed monolayers of 4-carboxyphenyl and phenyl molecules through the formation of a

carbon-carbon covalent bond by the reductive adsorption of aryl diazonium salts using

the two-cycle cyclic voltammetry method. Thus, monolayers of aryl diazonium salts

modified GC surfaces can be covalently attached with Gly-Gly-His peptide through

EDC/NHS coupling. The attachment of the tripeptide sequence Gly-Gly-His onto the

carboxylic acid terminus of the aryl diazonium salt monolayer on the GC surface has

been demonstrated using XPS characterisation technique. The Gly-Gly-His modified

surfaces have been successfully used for the analysis of copper ion solutions. The Cu2+

detection limit was as low as 0.13 ppb using both GC commercial electrode and the PPF

substrate. The surface coverage of complexed copper ions has been systematically

investigated by attachment of Gly-Gly-His to a series of mixed monolayers of 4-

carboxyphenyl and phenyl on GC surfaces. GC electrodes modified with mixed

174


monolayers have a higher detection limit of copper ions than that for GC electrodes

modified with the pure monolayer of 4-carboxyphenyl. The Gly-Gly-His peptide

modified GC electrode for the detection of copper was found to be very stable. After

one month storage and frequent usage, the measured sensitivity did not show much

decay within that period of time. PPF has proved to be a good alternative for the GC

electrode for the fabrication of chemical sensors. The so-fabricated electrode will be a

good example for the fabrication of other chemical sensors built with other polypeptides

for the analysis of other metal ions and also supplies an effective tool for medical

diagnosis.

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179

Chapter 6-An Interface Comprising Molecular Wires and Poly(ethylene glycol) Spacer Units Self-Assembled on Carbon Electrodes for Studies of Protein Electrochemistry

Chapter Six

An Interface Comprising Molecular Wires and Poly(ethylene

glycol) Spacer Units Self-Assembled on Carbon Electrodes for

Studies of Protein Electrochemistry

180


6.1 Introduction

Understanding and manipulating electronic transfer through molecular-scale “wires” is

beginning to attract tremendous attention in the developing fields of biosensors1-3 and

bioelectronics4, 5 as well as molecular electronics.6-9 The attraction of molecular wires

(MWs) in bioelectronics is that good electronic coupling exists between the electron

donor at one end and the acceptor at the other over long distances.10 For biological

systems, there is general agreement that the electronic coupling between electron donors

and acceptors, and hence as a consequence the distance, plays a critical role in

controlling long-range electron transfer (ET) process.11, 12 Numerous studies have

investigated long range ET between redox-active probes and an electrode through self-

assembled monolayers (SAMs) formed on a gold substrate with alkanethiols,13-16

conjugated molecules,2, 3, 17, 18 and DNA1, 19-22 to redox molecules such as ferrocene,15, 16,

23 and ruthenium complex,14, 24-26 and to redox proteins such as cytochrome c,27-30

azurin,31, 32 horseradish peroxidase,33, 34 or laccase.35, 36 Self-assembled monolayers

formed on electrodes are attractive for ET studies in general, and protein

electrochemistry in particular, as surfaces can be designed with a high degree of control

over the molecular architecture.37-39 Such a control allows the preparation of platforms

to immobilise proteins which are compatible with the protein where the spacing

between the protein and the electrode can be precisely manipulated along with the

dielectric constant of the protein environment and the orientation and density of proteins

on the surface; all of which may strongly influence the efficiency of direct ET.27

Both the Waldeck’s group29, 30 and Gray’s group40 have taken the concept of using

181


SAMs for protein electrochemistry a step further than simply being a platform upon

which the protein is immobilised. Both groups incorporated molecular components in

the SAMs which interact with specific sites on the protein and facilitate ET. For

example, recently Gray and coworkers40 have achieved direct ET between the deeply

buried active site of amine oxidase and underlying electrode using a molecular wire.

This study allowed for the first time the interrogation of the electrochemistry of the

active site of the enzyme in its native state. The study also presents the idea of using an

electrode interface with MWs which can penetrate into the protein to facilitate ET. This

same idea of obtaining a more intimate interaction between the protein and the electrode

has also been demonstrated with carbon nanotubes,41-44 nanoparticles45 and other rigid

organic molecules.46 However, in all cases there is little control over whether the redox

proteins adsorb to the ends of the MWs or to other surface sites.

An important issue for electrode constructs where a “molecular wire” penetrates into the

biology is to ensure that the biological molecules interact specifically with the MWs,

rather than the rest of the electrode surface.16 However, the non-specific adsorption of

proteins is a problem that exists with most surfaces when exposed to biological

samples.47 Thus, to achieve a generic surface which ensures specific interactions

between a protein and an electrode requires two important things: 1) MWs that can

interact directly with the protein and have high efficiency of electron transfer and 2)

spacer molecules that are able to resist nonspecific adsorption of proteins. In this

chapter molecular wires derived from oligo(phenyl ethynylene) derivatives were used as

others have shown this class of molecule allows electron transport with high

efficiency.2, 48-56 Similarly, the spacer molecules to be used will be poly(ethylene glycol)

(PEG) derivatives, an important class of molecules with a number of unique properties

182


as introduced in Chapter One, which show considerable promise with regards to

producing surfaces which resist non-specific protein adsorption.47, 57-65

In Chapter Three and Chapter Four, it was demonstrated that the electrochemical

reduction of aryl diazonium salts on glassy carbon (GC) surfaces to form covalent

bonds is a promising alternative monolayer system to the gold-thiol chemistry.66-68 As

introduced in Chapter One, functionalised phenyl films by reductive adsorption of

diazonium salts have been utilised in a number of fundamental studies at carbon

electrodes.6, 49, 66, 69-72 Thus, the modification of carbon electrodes with diazonium salts

promises to be a good system for investigation of protein electrochemistry both from the

perspective of the stability of the monolayer on the GC surface and the rich array of

organic modifying molecules. Hence modified electrodes with tailored molecular

designs can be produced.49, 67

The purpose of this chapter is to demonstrate the modification and utilisation of GC

electrodes with a generic interface compatible with investigating specific protein

electrochemistry. Horseradish peroxidase (HRP) is used as a model protein here to

illustrate the utility of the interface before investigating the electrochemistry of proteins

with redox centres embedded deep within the glycoprotein. The interface consists of a

20 Å long rigid MW as shown in Scheme 6.1, and a PEG molecule with three ethylene

glycol units. The ability of the interface to facilitate efficient electron transfer is

demonstrated using ferrocenemethylamine attached to the end of the MWs whilst the

protein resistance of the interface is studied using protein modified gold nanoparticles

which could be quantified by scanning electron microscopy, changes in electrode

capacitance when exposed to solutions of bovine serum albumin (BSA) and the

183


electrochemical response of the electrode when incubated in HRP. These experiments

showed that the electrode interface resists non-specific interactions of proteins and

therefore only electrochemistry from proteins specifically attached to the MWs is

measured. Such protein electrochemistry is illustrated using HRP attached to the MWs.

The activity of the immobilised HRP is determined from the response of the electrode

interface to hydrogen peroxide.

EnzymeEDC/NHS

GCGC

NO2

COOH

GCGC

NO2

CO

NH

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

Reductiveadsorption

1:20 MW:PEG

GCGC

(a)

N

O2N

COOH-N OO

OO

N-N

(b)

MW: PEG:

20 Å 17.5 Å

Scheme 6.1 Schematic of a) the glassy carbon interface for protein electrochemistry

and b) the structures of molecular wires and poly(ethylene glycol) diluent employed.


6.2.1 Chemicals and Procedures

All the reagents and materials are listed in Table 2.1 of Chapter Two or prepared

according to the procedures described in Chapter Two. GC electrodes were prepared

184


according to the method described in Section 2.4. Ferrocenemethylamine, PEG and MW

were synthesised according to the procedures described in Chapter Two. All pure and

mixed monolayers were prepared as described in Section 2.4.2 and ferrocene was

covalently attached on the monolayers according to Section 2.2.6. All electrochemical

measurements were performed with a BAS-l00B electrochemical analyser. All the

scanning electron microscope (SEM) images reported here were taken in a LEO-SEM

(Supra 55VP, Zeiss) using an InLenes detector. All potentials were quoted relative to an

Ag/AgCl reference at room temperature. All CV measurements were carried out in the

0.05 M phosphate buffer (0.05 M KCl, pH 7.0). All surface coverage measurements are

based on the actual surface area of the GC electrode rather than geometric area.

6.2.2 Bovine Serum Albumin Labelled Au Nanoparticles

Gold nanoparticles (15 nm in diameter) were synthesised according to the literature

method.73 BSA labeled Au nanoparticles were prepared according to the protocols from

the literature.73 To 25 mL of colloidal gold nanoparticles (15 nm) were added 1 mL of

BSA (10 mg mL-1 in Milli-Q water). The BSA-Au conjugates were observed to

sediment after 24 h. Then the fresh polished GC plates were dipped into the prepared

BSA-Au conjugates for 1 h. Before the SEM measurement, the surface was rinsed with

copious amount of water and dried with an argon stream.

6.3 Result and Discussion

6.3.1 Electrochemistry of PEG Modified Glassy Carbon Electrodes

The modification of GC electrodes with the PEG molecule as depicted in Scheme 6.1

was carried out by performing cyclic voltammetry with a GC electrode in a 1 mM PEG

diazonium tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution from +1.0 to -1.5 V

185


(Figure 6.1). The first scan gave an irreversible wave located at +0.11 V versus

Ag/AgCl. The wave disappeared completely by the second cycle.

-13

-10

-7

-4

-1

2

5

8

-1.5 -1 -0.5 0 0.5 1

Potential /V

Curr

ent

/A

1st cycle

2nd cycle

Figure 6.1 Cyclic voltammogram of the GC electrode in 1 mM PEG diazonium

tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution at a scan rate of 100 mV s-1.

The surface coverage of the PEG molecules attached to the GC surface was determined

from the area under the reductive adsorption peak of the CV in Figure 6.1 and the

active working area of the electrode as (5.93±0.5)×10-10 mol cm-2 (n=5). This surface

coverage is consistent with the formation of a monolayer of molecules as distinct from

multilayers since this value is below the theoretical maximum number of diazonium salt

molecules that can be attached to a carbon electrode (12×10-10).74 The ability of the

monolayer of PEG to prevent access of redox active molecules to the GC surfaces was

tested using 1 mM ferricyanide in a 0.05 M phosphate buffer (0.05 M KCl; pH 7.0) at a

scan rate of 100 mV s-1. The electrochemical response of ferricyanide (Figure 6.2) was

completely suppressed after the modification of the GC electrode with PEG molecules

indicating a good blocking layer was produced on the GC surface.

186


-20

-10

0

10

20

-0.1 0.1 0.3 0.5

Potential /V

Curr

ent

/A

PEG modified GC

Bare GC

Figure 6.2 Cyclic voltammograms of GC electrodes before and after modification of

PEG in 1 mM ferricyanide solution (0.05 M KCl; 0.05 M phosphate buffer; pH 7.0) at

the scan rate of 100 mV s-1.

6.3.2 Non-Specific Protein Adsorption on PEG Modified Glassy Carbon Surfaces

The efficiency of the PEG molecules to suppress non-specific adsorption of proteins

was initially studied by measuring changes in the double layer capacitance of electrodes

incubated in solutions of BSA. After incubation in a BSA (3% in water) solution for

1 h, the capacitance of PEG modified GC electrode decreased by 7.3±0.2 % (n=5) from

301 F cm-2 to 280 F cm-2. This reduction in capacitance is significantly less than the

observed capacitance decrease for the bare GC electrodes of 38±5 % (n=5) from

250 F cm-2 to 155 F cm-2 and a benzyl modified GC electrodes of 30±7 % (n=5) from

285 F cm-2 to 199 F cm-2 after incubation in BSA solution for 1 h. None of the

electrodes showed any significant change in capacitance after storing the electrode in a

background solution of water thus indicating the capacitance changes are a consequence

of protein adsorption. The capacitance results indicate a significant reduction in BSA

adsorption on the PEG modified GC surfaces, relative to the bare and phenyl modified

187


GC surfaces but they do not readily allow a determination of the extent of suppression

of the nonspecific adsorption. Therefore to quantify the extent of suppression of non-

specific adsorption, freshly polished GC plates and PEG modified GC plates were

dipped into a 0.25 nM solution of BSA-Au conjugates for 1 h. The surfaces were rinsed

with copious of water, dried with argon and then imaged using SEM. In order to getting

representative SEM images, a few parallel GC samples were prepared and a few

locations were imaged from the same sample. And similar SEM images were obtained

as shown in Figure 6.3. The SEM images show the amount of BSA modified gold

nanoparticles that has adsorbed onto the bare and PEG modified carbon surfaces. For

the freshly polished but unmodified plates (Figure 6.3 a), the number of gold colloids

visible per unit area was 3.2×10-14 mol cm-2. In contrast, the number of colloids per unit

area on the PEG modified surface (Figure 6.3 b) was 8.4×10-15 mol cm-2. Thus the SEM

images indicate a 74% suppression in non-specific protein due to the PEG modification

of the GC plates but illustrates that some protein adsorption is still occurring.

100 nm

a

100 nm

b

Figure 6.3 LEO-SEM images of (a) bare and (b) PEG modified GC plate after exposure

to BSA labelled Au nanoparticles.

To determine whether the extent of protein adsorption is significant with regards to non-

188


specific protein electrochemistry, the non-specific adsorption of HRP was studied on

bare GC electrodes, phenyl modified GC electrodes and PEG modified GC electrodes.

Figure 6.4 shows the cyclic voltammograms in 0.05 M phosphate buffer (0.05 M KCl,

pH 7.0) at the scan rate of 100 mV s-1 for bare GC electrodes, phenyl modified GC

electrodes and PEG modified GC electrodes before and after incubation in 1 mg mL-1

HRP in DMSO solution for 1 h. After incubation of the bare GC electrode in 1 mg mL-1

HRP-phosphate buffer solution, a pair of obvious redox peaks at -0.35 V appeared as

shown in Figure 6.4 a, which is consistent with the result of Guo et al.75 where HRP

adsorbed from DMSO onto GC electrodes had a formal potential in phosphate buffer

(pH 7.0) of -0.365 V. The CVs of phenyl modified GC electrodes after incubation in 1

mg mL-1 HRP solution also gave redox peaks at the same potential position as shown in

Figure 6.4 b. The appearance of redox peaks in both cases indicates that considerable

amount of HRP had adsorbed onto bare and phenyl modified GC electrodes. By

integration of the peak area under the CVs after background subtraction, the amount of

HRP adsorbed on bare and phenyl modified GC surfaces was found to be 2.82×10-11

mol cm-2 and 3.78×10-12 mol cm-2, respectively. So the protein adsorption on bare GC

surfaces is more significant than that on phenyl modified GC surfaces. Importantly,

after incubation of PEG modified GC electrodes in HRP solution under the same

conditions, the CV in Figure 6.4 c does not show any electrochemistry due to adsorbed

HRP. Controls where the bare and PEG modified GC electrodes were soaked in

background solvent DMSO for 1 h showed no redox peaks. It can therefore be

concluded that PEG modified GC surfaces provides sufficient resistance to non-specific

adsorption of protein to confidently allow any electrochemistry observed from an

electrode interface modified with a 1:20 ratio of the MW to PEG molecules to be

unambiguously assigned to protein attached to the end of the MW.

189


-2

-1

0

1

2

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/A

After incubation in HRP

a Before incubation in HRP

-2

-1

0

1

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/A

b


Before incubation in HRP

-3

-2

-1

0

1

2

3

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/A


c

Before incubation in HRP

Figure 6.4 Cyclic voltammograms of (a) bare, (b) phenyl modified and (c) PEG

modified GC electrodes before and after incubation in HRP-DMSO solution for 1 h in

0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at the scan rate of 100 mV s-1.

190


6.3.3 Electrochemistry of Glassy Carbon Electrodes Modified with Mixed Monolayers

of MW and PEG at the Molar Ratio of 1:20

Prior to performing protein electrochemistry experiments it was important to determine

that the MW could be attached to the GC electrode surface and that appreciable electron

transfer through the molecular wire was achieved. Figure 6.5 shows cyclic

voltammogram of GC electrodes in a 1 mM mixed diazonium salts solution of MW and

PEG with the mole ratio of 1:20, acetonitrile/0.1 M NBu4BF4 solution.

-7

-4

-1

2

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6

Potential /V

Curr

ent

/A

1st cycle

2nd cycle

Figure 6.5 Cyclic voltammograms of a GC electrode in a 1 mM MW and PEG mixed

diazonium tetrafluoroborate with the mole ratio of 1:20, acetonitrile/0.1 M NBu4BF4

solution at a scan rate of 100 mV s-1.

As shown in Figure 6.5, the first sweep gave an irreversible reduction wave at ca. +0.12

V versus Ag/AgCl which is indicative of the loss of N2 and the formation of aryl

radicals followed by covalent binding to the carbon surface.76 Subsequent scans showed

no electrochemistry which is indicative of a passivated electrode. Based on the area of

the reduction peak during the modification of the GC electrode with the aryl diazonium

salts, the coverage of the modified layer was estimated to be (4.51±0.12)×10-10 mol cm-2

191


according to the working area of electrodes. The surface coverage is below that of an

ideal close-packed, diazonium salt derived monolayer, on flat GC surfaces of 12×10-10

mol cm-2,74 thus indicating only monolayer coverage of the electrode, as distinct from

multilayer formation as has been observed for some diazonium salts.77-79 The

passivation of the GC surfaces after the modification with aryl diazonium salts was

confirmed using potassium ferricyanide and hexaamineruthenium(III) chloride (RuHex)

as redox probes. Figure 6.6 a shows a cyclic voltammogram before and after

modification of a GC electrode with a mixed monolayer of MW and PEG in 1 mM

ferricyanide in a 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate of 100

mV s-1. After the modification of the surface with the aryl diazonium salts, the redox

peaks of ferricyanide observed with bare GC electrodes were almost completely

suppressed, giving good evidence that a uniform passivating monolayer had formed on

the GC surfaces. The passivation of the electrode could also however be a consequence

of the electrostatic repulsion between the carboxyl terminated MWs and the anionic

ferricyanide. As a consequence, the cationic redox probe RuHex was also used to test

the integrity of mixed monolayers of 1:20 MW:PEG. The mixed monolayer only

partially blocked the RuHex molecules from accessing the GC surfaces as shown by the

minor amount of electrochemistry observable in Figure 6.6 b. In comparison, the

passivation of GC electrodes modified with pure MW and pure PEG was also tested in

RuHex solution. The RuHex molecules can easily penetrate a monolayer of pure MW

modified GC surfaces (Figure 6.6 c) but a monolayer of pure PEG shows a good

blocking ability to the positively charged RuHex molecule (Figure 6.6 d). These results

indicate that the presence of the MW marginally disturbs the passivating ability PEG

spacing molecules to small cationic molecules but not to anionic redox active species

which are far more prevalent in biological systems. What is not clear however, is the

192


distribution of the MW within the PEG monolayer. Attempts to perform STM

measurements to elucidate whether the MW was homogeneously distributed or

clustered in islands on the surface were unsuccessful as the monolayer was too

passivating across the entire surface. The fact that the entire surface was passivating

however suggests that the MW were at least not aggregating into large conducting

clusters but does not preclude the possibility of a small aggregates of MW being present

on the surface.

-20

-10

0

10

20

-0.1 0.1 0.3 0.5

Potential /V

Curr

ent

/A

Bare GC

in Fe(CN)63-a

MW/PEG modified GC

-30

-20

-10

0

10

20

30

-0.4 -0.3 -0.2 -0.1 0 0.1

Potential /V

Curr

ent

/A

MW/PEG modified GC

in Ru3+b

Bare GC

-30

-20

-10

0

10

20

-0.4 -0.3 -0.2 -0.1 0 0.1

Potential /V

Curr

ent

/A

MW modified GC

in Ru3+c

Bare GC

-30

-20

-10

0

10

20

-0.4 -0.3 -0.2 -0.1 0 0.1

Potential /V

Curr

ent

/A

PEG modified GC

in Ru3+d

Bare GC

Figure 6.6 Cyclic voltammograms of GC electrodes before and after (a) modification

with mixed monolayers of MW and PEG in 1 mM ferricyanide solution (0.05 M KCl;

0.05 M phosphate buffer; pH 7.0), (b) modification with mixed monolayers of MW and

PEG (c) modification with MW and (d) modification with PEG in 1 mM Ru3+ solution

(0.05 M KCl; 0.05 M phosphate buffer; pH 7.0) at the scan rate of 100 mV s-1.

193


6.3.4 Electron Transfer Through Monolayers of MW on Glassy Carbon Surfaces

After modification of the GC electrodes with the MW/PEG mixed monolayers the next

step was the attachment of ferrocene to investigate the electron transfer ability of the

modified electrodes. CVs measured in a solution of 0.05 M phosphate buffer (0.05 M

KCl, pH 7.0) at a scan rate of 100 mV s-1 before and after the immobilisation of

ferrocene on the 1:20 MW:PEG modified GC electrodes are shown in Figure 6.7. The

formal potential (Eo`) was 308±17 mV (95% confidence, n=5) and the ratio of the area

of the anodic to cathodic peaks was 0.96±0.07 (95% confidence, n=5). A linear

variation in peak current with scan rate was observed indicating that the ferrocene was

surface bound. The full width half maximum (EFWHM) of the redox peaks was 213 mV,

which is significantly higher than the ideal value (90.6 mV/n in this case n=1). Broader

than ideal EFWHM have previously been observed for electrode interfaces where the

redox active molecule was attached to a preformed SAM.3, 80, 81 The broader than ideal

EFWHM is consistent with some non-specific adsorption of the ferrocene to the electrode

surface, as observed when no EDC/NHS coupling reagents were used, as broader than

ideal EFWHM are indicative of the redox active molecule being located in a range of

environments with a range of formal electrode potentials.82, 83 The surface coverage of

ferrocene molecules was (1.68±0.23)×10-11 mol cm-2 (n =5), suggesting nearly every

MW in the monolayer has a ferrocene attached. This conclusion is based on the

assumption that the CV for the deposition indicated a surface coverage of molecule in

the monolayer of 4.51×10-10 mol cm-2. One twentieth of this coverage is 2.26×10-11

mol cm-2 at number close to the number of ferrocene molecules interrogated

electrochemically. At this surface coverage the area per ferrocene molecule is

approximately 0.99 nm2, assuming a uniform distribution, which is significantly greater

194


than the theoretical size of single ferrocene molecule81 of 0.2 nm2 and hence there

should be little interaction between redox active centers on the surface.13

-3

-2

-1

0

1

2

3

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

After attachment

of ferrocene

Before attachment

of ferrocene

Figure 6.7 Cyclic voltammograms before and after the coupling of

ferrocenemethylamine onto the MW/PEG modified GC electrodes in 0.05 M phosphate

buffer (0.05 M KCl, pH 7.0) at the scan rate of 100 mV s-1.

In order to study the influence of the density of MW on the attachment of ferrocene and

the further influence on the electron transfer rate, mixed monolayers of MW and phenyl,

pure monolayer of MW were also prepared followed by the attachment of

ferrocenemethylamine. Scheme 6.2 shows pictures for the attachment of ferrocene to

GC surfaces modified with different components. The electrochemical performance of

the ferrocenemethylamine modified GC electrodes is summarised in Table 6.1. The

surface coverage of ferrocene is related to the density of MW and the biggest surface

coverage of ferrocene was obtained when the GC electrodes were modified with pure

monolayers of MW (5.55×10-11 mol cm-2). The coverage of ferrocene decreased when

the PEG was used as a diluent on the monolayer surfaces. The surface coverage of

ferrocene further decreased by 0.91×10-11 mol cm-2 when the GC surfaces were

195


modified with mixed monolayers of MW and phenyl with mole ratio of 1:20. The

results are understandable since the coverage of MW on mixed monolayers is smaller

relative to that on pure monolayers of MW, indicating there is less available binding

sites for the attachment of ferrocenemethylamine to occur.

GC

O

O

O

O

O

O

O

O

NO2

NHO

Fe

NO2

NHO

Fe

NO2

NHO

Fe

NO2

NHO

Fe

GC GC

Pure MWMW/PhenylMW/PEG

(a) (b) (c)

Scheme 6.2 Schematic of the coupling of ferrocenemethylamine on GC electrodes

modified with mixed monolayers of (a) MW and PEG, (b) MW and phenyl at the molar

ratio of 1:20, and (c) pure monolayers of MW.

Table 6.1 Some parameters of ferrocenemethylamine immobilised on GC electrodes

modified with MW and other diluents.

Monolayers Eo` (mV) EFWHM (mV) (mol cm-2) a/ c kET (s-1)

Pure MW

MW/PEG

MW/Phenyl

294±15

308±17

314±19

186±10

213±18

227±24

5.55±0.24×10-11

1.68±0.23×10-11

0.91±0.03×10-11

0.89±0.10

0.96±0.07

1.08±0.11

305±28

229±30

113±26

196


The ET rate was calculated using the Laviron’s method, which assumes Butler-Volmer

kinetics and a Langmuir adsorption isotherm.84 The rate of ET was determined by the

relationship between the peak separation and the scan rate as shown in Figure 6.8. The

rate constant was found to be 305±28 s-1 (n=4) when the ferrocene was attached to GC

electrodes modified with pure MW monolayers, which is much smaller than that

through norbornylogous bridge with similar length on gold electrodes (>1000 s-1).85, 86

The rate constant reported by Creager and coworkers was only 6.6 s-1 for ferrocene

attached on the alkanethiol monolayers.87 However rate constants were found to be

extremely high (from ~104 to ~106 s-1) when the ferrocene modified MW with three

phenyleneethynylene units was attached on gold surfaces.2, 3, 53 The huge difference of

electron transfer on similar electron transfer system may be due to two differences

between the system studied here and systems used by Creager’s group and Smalley’s

group. Firstly, electrode materials play an important role in the ET mechanism.88 The

MW used here was modified on GC surfaces, which has been shown to have lower

electron transfer efficiency than gold.89 Secondly, Creager’s group and Smalley’s group

synthesised ferrocene-terminated oligo(phenyleneethynyl)arene-thiol molecule followed

by modification of this target molecule to the gold surface for exploring conductivity of

the oligo(phenyleneethynyl)arene molecular wire that bridges ferrocene complexes and

electrodes. The synthesised whole bridge structures were -conjugated, which can

promote electronic coupling between ferrocene and the underlying electrode, thereby

promote rapid electron transfer over long distances.2 However, this study used step-wise

method to attach ferrocene to MW modified GC surfaces and the resultant MW system

has an aliphatic “tail” of a few bonds length. The electron transfer through this “tail ” is

though - coupling, which can greatly compromise the rate of electron transfer

between ferrocene and GC electrodes.

197


-0.15

-0.1

-0.05

0

0.05

0.1

0.15

-1.5 -1 -0.5 0 0.5 1

Log ( /V s-1)

Ep-E

o`

/V

Figure 6.8 The plot of Ep-Eo` versus the logarithm of scan rate for

ferrocenemethylamine attached on the MW modified GC electrodes

It was also found from Table 6.1 that the rate constant has decreased to 229 s-1 when

ferrocene was attached on the mixed monolayers of MW and PEG, and the rate constant

has even further decreased to 113 s-1 when the ferrocene was attached on the mixed

monolayer of MW and phenyl. As previously studied, the association between the redox

probes might decrease the reorganisation energy, and correspondingly increase the rate

constants.12-14, 90 Thus, the lower rate constant with the mixed monolayers might be due

to the less interaction between the redox species caused by the smaller surface coverage

of redox species. On the other hand, it can be noticed that the diluent also plays an

important role in the ET. Lower rate constant of electron transfer was obtained when

GC electrodes were modified with mixed monolayers of MW and PEG instead of pure

monolayer of MW, which is consistent with the conclusion that the ether linkage in the

diluent decreased the rate of ET relative to the non-ether one as asserted by Waldeck

and coworkers.16 When the PEG was used as the diluent, the rate constant obtained is

bigger than that when phenyl was used as the diluent. The increased electronic coupling

198


between ferrocene and the electrode can be attributed to the hydrogen-bonding network

which might form among the nitro group from the MW and the oxygen atom on the

PEG with the aid of the proton in the electrolyte solution when ferrocene was attached

to the mixed monolayers of MW and PEG.91-93 The destruction of this network, by

introduction of a simple phenyl as the diluent where there is no side chain near MW into

the monolayer, leads to the decrease of the rate of electron transfer through the layer.

6.3.5 Direct Electron Transfer between HRP and MW on Glassy Carbon Surfaces

The results of the ET study on the MW over 20 angstrom are very encouraging.

Considering the ET efficiency and rigidity of the MW, and the protein resistance of

PEG, the interfaces created by MW and PEG together could be uniquely suited for the

protein electrochemistry and bioelectronic applications as firstly specific interactions

between the carboxyl-terminated ends of MW and the protein occur and secondly the

possibility that the MW can penetrate within the glycoprotein shell exists. Here HRP

was used as a model protein to illustrate the ability of the interface to facilitate good ET

to redox proteins. The choice of HRP is partly due to the large body of work on this

protein with regards to ET studies which enables the efficiency of the interface to be

evaluated.33, 34, 43, 75, 94-99

Cyclic voltammograms (Figure 6.9) of the MW/PEG-HRP modified electrodes in

phosphate buffer (pH 7.0) showed oxidation-reduction peak with an Eo` of -0.190 V,

which is characteristic of the Fe /Fe redox couples of HRP.100 The peak current

increased linearly with increasing scan rates, indicating the formation of the surface

bound of redox protein HRP. Cyclic voltammogram peaks of the HRP attached to MW

were stable, and did not decay during repetitive multiple scans. In the absence of EDC

199


and NHS, such that no covalent coupling of the HRP could occur, no redox peaks were

observed. In addition, no electrochemistry was obtained when the pure PEG modified

GC electrode was exposed to HRP. The controls indicate HRP was covalently attached

to the MW/PEG modified interface and direct electron transfer was obtained between

HRP and underlying GC electrode through the MW.

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

-0.6 -0.4 -0.2 0 0.2

Potential /V

Curr

ent

/A

b)

-12

-8

-4

0

4

8

12

-0.6 -0.4 -0.2 0 0.2

Potential /V

Curr

ent

/A

a)

Figure 6.9 Cyclic voltammograms of GC electrodes modified with mixed monolayers of

MW and PEG with the molar ratio of 1:20 after incubation in 1 mg mL-1 HRP solution

(a) and after background subtraction (b) in 0.05 M phosphate buffer (0.05 M KCl, pH

7.0) at the scan rate of 100 mV s-1.

For the background subtracted redox peaks of HRP in Figure 6.9, the full width half

200


maximum of the oxidation wave was 106 mV which is very close to the ideal of 91 mV

and consistent with values for cytochrome c determined by Waldeck and coworkers29

and Gray and workers40 where SAM modified electrodes which interact specifically

with the redox centre of proteins are employed. This almost ideal value of the EFWHM

implies that the HRP molecules that are being electrochemically interrogated are in

close to a uniform environment. The surface concentration of HRP was estimated to be

(6.16±1.29)×10-12 mol cm-2 which corresponds to 27 nm2 per HRP, which is consistent

with the size of a single HRP molecule revealed by X-ray crystallography of 5 nm×4

nm×3 nm101, 102 and is close to the theoretical surfaces coverage for a compact

monolayer (8.5×10-12 mol cm-2) of HRP assuming a projected area of ~20 nm2 per

molecule.103 The calculated coverage of HRP is much lower than the estimate of the

minimum number of MW in the interface determined from the coverage of

ferrocenemethylamine on the GC surfaces (2.41×10-11 mol cm-2). Thus it seems likely

that more than one MW is coupled to each HRP.

The rate constant of electron transfer was calculated to be 13.4±2.3 s-1 (95% confidence,

n=3). The rate constant, despite the HRP being at least 20 Å from the electrode, is faster

than any rate constants reported in the literature using native HRP where the activity of

the enzyme has been demonstrated. For example at an MPA/HRP modified gold

surfaces 0.287 s-1 was reported,34 at nanotube modified electrodes Yu et al.43 measured

a rate of 2.5 s-1 while the rate constant is similar to that reported by Lötzbeyer for

microperoxidase MP-11 covalently attached to a short chain alkanethiol modified gold

electrodes (12 s-1).104 The larger rate constant of electron transfer obtained in this study

suggests either there is some denaturing of the HRP or the MW could be penetrating the

glycoprotein and reducing the distance between the redox centre and the MW. As

201


denaturing of the enzyme will result in loss of catalytic activity, to determine which of

these options is the more likely the activity of the MW/PEG-HRP was investigated.

The MW/PEG-HRP modified GC electrodes were used to detect the H2O2 with the

turning over of the enzyme being achieved by direct electron transfer. The relationship

between the amount of H2O2 consumed by the enzyme and the electrochemical signal at

a MW-HRP modified electrode can be depicted by the following kinetic scheme:95

HRP (Fe3+) + H2O2 Compound I (Fe4+=O) + H2O

Compound I (Fe4+=O) + e- +H+ Compound II

Compound II + e- +H+ HRP (Fe2+)

where Compound I and Compound II are oxygen complexes of HRP. They are reduced

by accepting two electrons from the solvent yielding an enhanced reduction current.

The amperometric responses of the HRP-MW/PEG modified GC electrode to H2O2 are

shown in Figure 6.10 a. After successively adding H2O2 in the stirred phosphate buffer

solution, the current of the HRP-MW/PEG modified GC electrode measured at –0.25 V

increases significantly. Moreover, the catalytic reduction current increases linearly with

increasing H2O2 concentration from 5 to 50 µM as shown in Figure 6.10 b. However,

no increasing of cathodic peak corresponding to the reduction of H2O2 can be observed

at the MW/PEG modified GC electrode or the GOx-MW/PEG modified GC electrode

(Figure 6.10), which was fabricated using the same method as that used for the HRP-

MW/PEG modified GC electrode except replacing HRP with GOx, under the same

conditions. The sensitivity to the change in the concentration of H2O2 was 15 nA/µM,

which is similar to that for H2O2 sensor based on SWNT/HRP system (21 nA/µM).43

202


The relative standard deviation between electrodes was 5.1% (n=6) at a concentration of

50 µM. The lowest detected concentration was 1 µM. All these findings have

demonstrated that the interface comprising mixed monolayers of MW and PEG on GC

electrode surfaces appears ideal for protein chemistry studies.

-800

-600

-400

-200

0

0 1000 2000 3000 4000

Time /s

Curr

ent

/nA

HRP-MW/PEG modif ied GC

GOx-MW/PEG modif ied GC

MW/PEG modif ied GC

a

0

100

200

300

400

500

600

700

0 10 20 30 40 50 60

[H2O2] / M

I /n

A

HRP-MW/PEG modif ied GC

GOx-MW/PEG modif ied GC

MW/PEG modif ied GC

b

Figure 6.10 (a) Amperomeric response at the HRP-MW/PEG modified GC electrode,

GOx-MW/PEG modified GC electrode and MW/PEG modified GC electrode to

successive additions of 5 µM H2O2 in 0.05 M phosphate buffer (pH 7.0). (b) Calibration

plot of H2O2 based on the amperomeric response obtained on (a). The applied potential

was -0.25 V.

203


6.4 Conclusions

The electron transfer through the pure and mixed monolayers of MW to GC electrodes

has been studied. The rate constant of electron transfer of MW on GC electrodes was

found to be much smaller than that through the bridge monolayer on gold electrode, and

also decreased further more when the bridge monolayer was diluted with different

diluents. The PEG modified interface was found to reduce the adsorption of protein

molecules to some degree. With its completely rigid property, easily tailored lengths

and versatile functional groups, the rigid MW could be quite appropriate material for the

fabrication of molecular electronics or nanodevices. Also the interface comprising

mixed monolayers of MW and PEG appears to be a very useful model system in the

biorecognition, biosensors, immunoassays, and enzyme inhibition assays. So the next

chapter is to use this MW to explore flavin adenine dinucleotide (FAD), the deeply

buried active centre of glucose oxidase to achieve direct ET between the enzyme and

the electrode for the purpose of the fabrication of third generation glucose biosensors.

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(104) Lotzbeyer, T., Schuhmann, W., Schmidt, H.L., Sensor. Actuat. B-Chem. 1996,

33, 50-54.

211

Chapter 7-Exploration of Deeply Buried Active Sites of Glucose Oxidase Using Molecular Wires Self-Assembled on Carbon Electrodes

Chapter Seven

Exploration of Deeply Buried Active Sites of Glucose Oxidase

Using Molecular Wires Self-Assembled on Carbon Electrodes

212


7.1 Introduction

Achieving direct electron transfer between the active centre of the enzyme and an

electrode is crucial for the development of novel enzyme biosensors or bioelectronics.1

Direct electron transfer has been observed on small redox proteins such as cytochrome

c,2-7 horseradish peroxidase (HRP),8, 9 laccase,10, 11 and azurin.6 Results in Chapter Six

have demonstrated that rigid molecular wires (MW) have high efficiency of electron

transfer and direct electron transfer has been achieved between HRP and the underlying

glassy carbon (GC) electrodes using MW as the conduit for electron transfer. However,

for most oxidoreductase enzymes, their active centres are embedded deep inside the

glycoproteins. Therefore it is far more difficult to incorporate these proteins into a

biosensor system due to the weak electrical communication to their surrounding

environment.

Understanding the principle of electron transfer (ET) in protein is critical to finding

strategies for facilitating electrical communication between proteins and their

surrounding environment. The most important factor that influences the electron

transfer rate is the distance between the redox centre and the electrode. The rate

constant exponentially decreases with the distance as shown in the expression:

)exp(0 dkkET

where kET is the rate constant, d is the distance between the redox centre and the

electrode surface, is the tunnelling coefficient, and the prefactor k0, represents the

effective rate constant as extrapolated to zero distance. In proteins, the electron-transfer

rates drop by ~104 when the distance between an electron donor and an acceptor is

213


increased from 8 to 17 Å.12 X-ray structure studies13 have shown that the minimal

distance between the redox active centre flavin adenine dinucleotide (FAD) of GOx to

the periphery of the enzyme is 8 Å, and the minimal distance between the periphery of

GOx and the N 7 nitrogen of the isoalloxazine part of FAD, where the redox reaction

occurs, is 13-18 Å. In view of the rapid decay of electron transfer rates with distance it

is not surprising that GOx does not communicate electrically with simple underlying

electrodes. Thus, shortening the distance between the active centre of the enzyme and

the electrode is very important for appreciable electron transfer. Some groups have

achieved direct electron transfer between GOx and an electrode by trying to reduce this

distance.14-20 However, the rate of electron transfer is still very low. The rate of electron

transfer was found to be only 0.026 s-1 after covalently immobilising GOx on a short

self-assembled monolayer on gold surfaces.14 The electron transfer rate of GOx

adsorbed on graphite20 and carbon nanotubes17, 19 was found to be 1.6 s-1. Therefore,

some conduits such as molecular wires for electron transfer are necessary for the

efficient electron transfer communication between the active centre and the surrounding

environment of the enzyme.

Exploration of the amine oxidase enzyme using a rigid molecular wire has been

pioneered by Gray and coworkers21, and they found that the electrical communication

between the gold electrode and the active centre of amine oxidase was greatly enhanced

by a molecular wire with a diethylaniline group. Nevertheless, the activity of the

enzyme was not clearly delineated although the direct electron transfer was reported.

Gooding and coworkers have extended the principle to achieve electron tunnelling

directly to the electrode from the FAD by introducing a norbornylogous bridge as the

electron transfer linker.22 When the apo-GOx was refolded on the FAD modified

214


electrode incorporated with the norbornylogous bridge, the enzyme was found to be

biocatalytically active. However, no biocatalytic event was observed under anaerobic

conditions and it is proposed insignificant direct electron transfer to the enzyme was

achieved due to poor electric coupling between the redox active FAD and the electrode.

The bioactivity of GOx is another crucial issue for a successful GOx biosensor. It has

been reported that GOx is electroactive in some cases and most of these electroactive

enzymes have proven to lose their enzyme activities,19, 20, 23 except that some

researchers obtained direct electron transfer of GOx without significant loss of its

activity.17, 24, 25 Thus, how to achieve significant direct electron transfer to GOx for the

fabrication of third generation biosensors with the biocatalytic activity of the enzyme

retained still needs further work.

It is very important to understand the structure of the GOx before finding a solution to

achieve significant direct electron transfer to GOx for the fabrication of third generation

biosensors. GOx from Aspergillus niger is an oxidoreductase enzyme, which is a

homodimeric protein with a molecular weight of 150 to 180 kDa, containing one tightly

bound FAD molecule per monomer as cofactor (actually two FAD per enzyme).13, 26

Two FAD molecules in GOx are separated by a distance of about 40 Å, a distance

which excludes any electrical communication between them. The dimeric protein has an

ellipsoidal shape with a high content of secondary structure (28% -helix, 18% -

sheets) shown in Figure 7.1 a. The monomeric GOx molecule with the active centre

FAD is shown in Figure 7.1 b. The tertiary structure of the enzyme is characterised by

two separate and distinctly different -sheet systems, which form part of the FAD

binding domain. The second is a large six stranded antiparallel -sheet supported by

four -helices on its back. This -sheet forms one side of the active site. Figure 7.2

215


shows the structure of active centre FAD molecules. The FAD molecule consists of

three parts: isoalloxazine ring, ribotol chain and an adenine. The redox reaction of FAD

occurs at the isoalloxazine moiety.

-helix (28%)-sheet (18%)FAD

-helix (28%)-sheet (18%)FAD

a) b)

Figure 7.1 a) Overall topology of the GOx molecule showing the secondary structure

elements, and b) subunit of the GOx structure with location of the active centre FAD.

N

OH

OHN

OH

NO

O

HN

O

PO

O

OH

PO

O

OH

O

HO

N

OH

N

N

NH2N

isoalloxazine ribotol adenine

Figure 7.2 The structure of active centre FAD.

The enzyme active site is only accessible through a deep pocket, which is funnel shaped

with an opening of 10×10 Å at the enzyme surface and is formed on one side by

residues of the second molecule of the dimer.13 The structure of GOx and one of its

active centres FAD is shown in Figure 7.3. The isoalloxazine end of FAD is located

216


near the bottom of a deep cavity with the minimum distance between the surfaces of the

monomer of 13 Å. The minimum distance from the adenine end of FAD to the enzyme

surface is about 8 Å. So in order to achieve the electrical communication between the

active centre of GOx and the surrounding environment, 13 Å is the minimum distance

between the isoalloxazine part of FAD at which the redox reaction occurs and the

surface of GOx to travel. Thus, if the enzyme is to retain its natural conformation, 13 Å

is the minimum distance an electron must tunnel to observe the electron transfer

between the biological molecule and the underlying electrode. It is possible to explore

the active centre of the GOx using a rigid molecule which is at least 13 Å long and

efficient for the electron transfer.

13Å

8Å

Figure 7.3 The structure of GOx and its active centre FAD.

The monomeric GOx molecule is a compact spheroid with approximate dimensions of

60 Å×52 Å×37 Å. The corresponding overall dimension of the deglycosylated dimer is

determined by X-ray crystallography as 60 Å×52 Å×77 Å.13 Two identical monomers

217


are connected non-covalently via a long but comparatively narrow contact area as

shown in Figure 7.4. There are about 120 contact points between the dimers centred

around 11 residues, which form either salt linkages or hydrogen bonds. The monomer

folds into two structural domains: one of them binds FAD, while the other one contains

the substrate-binding site.

Figure 7.4 The 3-D structure of GOx dimer based on X-ray crystallographic

coordinates and dimensions a = 55 Å b= 70 Å and c= 80 Å.

Scheme 7.1 shows the whole biocatalytical reaction of GOx. The enzyme reaction

occurs through the redox reaction of FAD. In the presence of glucose and under the

appropriate condition, GOx can catalyse glucose into gluconolactone. At the same time

the native GOx-FAD becomes reduced to GOx-FADH2. In the presence of oxygen,

FADH2 is oxidised to regenerate FAD and return GOx to its catalytically active form.

However, under the anaerobic (oxygen free) condition, recycling the reduced form of

FADH2 to FAD can be achieved via direct electron transfer. Scheme 7.2 shows the

mechanism of the redox reaction of the isoalloxazine rings of FAD. Firstly, FAD

accepts a proton and one electron to its lone-electron pair contained nitrogen to form a

semiquinoid free radical, and then another proton and electron can access the other lone-

electron pair contained nitrogen to form FADH2. Thus, the redox reaction of FAD takes

218


place involving two electrons and two protons and finished in two steps. The redox

activity of FAD was first studied by Ball,27 and its formal potential (Eo ) was reported to

be -250 mV at pH 7.8 and 30 oC. Ksenshek and Petriva28 have investigated the

electrochemical properties of FAD in aqueous solutions and claimed equation 7-1 can

be used to express to relationship between the formal potential of FAD and pH value of

the solution.

pHFRTE 3.2 7-1

Therefore, the pH of the surrounding solution can strongly affect the redox reaction of

FAD.29-32 The redox reaction of FAD has been studied both in the free form30, 31, 33 and

within GOx14, 17, 34-37 on various electrode surfaces.

GOx-FAD

GOx-FADH2

H2O2

O2

Glucose

Gluconolactone

Scheme 7.1 Schematic of the biocatalytical reaction of GOx.

N

N

N

O

HN

O

N

N

HN

O

HN

O

N

NH

HN

O

HN

O

H+ + e- H+ + e-

FAD FADH2

Scheme 7.2 Schematic of the redox reaction of FAD.

219


According to the structure of GOx, 13 Å is the minimum distance an electron must

tunnel to observe the electron transfer between the redox active centre FAD of GOx and

the underlying electrode. In Chapter Six, direct electron transfer has been achieved by

covalent attachment of the protein HRP with active sites close to the surface of the

protein onto the GC electrodes modified with the synthesised MW with a length of

20 Å. So the purpose of this chapter is to outline a further attempt to achieve direct

electron transfer between GOx with redox centres embedded deep within the

glycoprotein and GC electrodes using this MW as an electron transfer linker to fabricate

third generation glucose biosensors. The bioactivity of GOx will be discussed.





according to the method described in Section 2.4. All the pure and mixed monolayers

were prepared as described in Section 2.4.2. All electrochemical measurements were


relative to an Ag/AgCl reference at room temperature, and all the cyclic voltammetry

measurements for the electron transfer measurement were carried out in phosphate

buffer (0.05 M KCl, 0.05 M K2HPO4/KH2PO4, pH 7.0).

7.2.2 Direct Attachment of GOx to Glassy Carbon Electrodes Modified with Mixed

Monolayers of 4-Carboxyphenyl and MW

Scheme 7.3 shows a schematic of the strategy employed for the attachment of GOx at a

GC electrode surface modified with mixed monolayers of MW and 4-carboxyphenyl.

220


NO2

COOH

CO CO

FAD

NO2

COOH

COOH COOH

GCGOx EDC/NHSDiazonium salts

NH NHNO2

COOH

COOH COOH

GC

FAD

GCGC

Scheme 7.3 Schematic of attachment of GOx onto GC surfaces modified with mixed

monolayers of MW and 4-carboxyphenyl.

As shown in Scheme 7.3 a GC electrode was firstly modified with mixed monolayers of

MW and 4-carboxyphenyl followed by incubation in N-(2-hydroxyethyl)piperazine-N’-

(2-ethanesulfonic acid) (HEPES) buffer (pH 7.3) containing 3 mM GOx at 4 oC for 24 h

to allow GOx to bind to the MW modified GC surfaces. Then the GOx coupled GC

electrodes were rinsed with copious amount of water and subsequently immersed in 40

mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and 20

mM N-hydroxysuccinimide (NHS) in 0.1 M HEPES buffer for 1 h to activate the

carboxylic acid moieties on the GC surfaces. The GOx was then covalently coupled to

the activated carboxyplic acid moieties on the GC surfaces via the formation of amide

bonds between GOx and 4-carboxyphenyl moieties on GC surfaces.38


7.3.1 Electrochemistry of MW Modified Glassy Carbon Surfaces

Modification of GC surfaces with MW to form monolayers has been presented in

Chapter Six. The electron transfer studies demonstrated that the rigid MW has high

efficiency for electron transfer. Thus, the rigid MW with the length of 20 Å has great

221


potential to be used as a conduit for electron transfer between the redox active centre

FAD of GOx and the underlying electrode.

7.3.2 Exploration of Active Centres of GOx Using MW Modified on Glassy Carbon

Electrodes

7.3.2.1 Electrochemistry of GOx Coupled on Glassy Carbon Electrodes

After incubation in 3 mM GOx solution for 24 h, GC electrodes modified with mixed

monolayers of 4-carboxyphenyl and MW with a molar ratio of 30:1 showed reversible

redox peaks in the potential range from 0 to -650 mV versus Ag/AgCl in phosphate

buffer (pH 7.0) in the absence of oxygen, which were attributed to the reduction and

oxidation of the redox active centre FAD of GOx as shown in Figure 7.5. In addition, a

very stable voltammogram, without significant diminution of peak current after multiple

scans, was obtained. The stable redox peaks suggest that the GOx is tightly associated

with the MW modified GC interface. A pair of significant redox peaks of GOx were

obtained (Figure 7.5 b) after background subtraction of the cyclic voltammogram in

Figure 7.5 a. Controls show that GC electrodes modified with pure monolayers of 4-

carboxyphenyl or pure monolayers of MW gave no response in this potential range after

incubation in the GOx solution under the same conditions as shown in Figure 7.6. In

addition, when the coupling step using EDC and NHS was omitted, mixed monolayers

of 4-carboxyphenyl and MW (with a molar ratio of 30:1) modified GC electrodes gave

no electrochemical signal after incubation in GOx solution. Thus, mixed monolayers

and the addition of EDC and NHS are vital for achieving the electrical communication

between GOx and underlying GC electrodes.

222


-36

-27

-18

-9

0

9

18

27

36

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/nA

b)

-3

-2

-1

0

1

2

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Cu

rre

nt

/A

a)

Figure 7.5 (a) The cyclic voltammogram of GOx immobilised on GC electrodes

modified with mixed monolayers of 4-carboxyphenyl and MW with a molar ratio of 30:1

at a scan rate of 100 mV s-1, and (b) the cyclic voltammogram of GOx after the

background subtraction.

223


-3

-2

-1

0

1

2

-0.8 -0.6 -0.4 -0.2 0

Potential /V

Curr

ent

/A

MW modified GC

4-Carboxyphenyl modified GC

Figure 7.6 Cyclic voltammograms of GOx immobilised on GC electrodes modified with

pure monolayers of 4-carboxyphenyl or pure monolayers of MW at the scan rate of

100 mV s-1.

It was found the formal potential (Eo`) for the GOx redox peaks in Figure 7.5 b is about

-443 mV with Ep of 60 mV and EFWHM of 64 mV (45.3 mV in the ideal case), which is

consistent with the reported formal potential (Eo`=-441 mV) for GOx adsorbed on

nanotubes17 and also identical to previous studies of FAD attached to self-assembled

monolayers.39-42 The experimental EFWHM is close to the ideal EFWHM, indicating that

there is a small amount of denatured or partially open GOx incubated on the GC

surfaces. The ratio of GOx surface coverage from anodic peak to cathodic peak ( a/ c)

is 1.19. The cyclic voltammograms of the GOx incubated GC electrodes with different

scan rates are shown in Figure 7.7 a. The redox peaks after the attachment of GOx

showed linear variation in peak current with scan rates as shown in Figure 7.7 b,

indicating that the adsorbed GOx performed as a surface-confined electrode reaction.

224


-200

-100

0

100

200

0 0.1 0.2 0.3 0.4 0.5 0.6

Scan rate /V s-1

Curr

ent

/nA

b)

-5

-4

-3

-2

-1

0

1

2

3

4

-0.7 -0.55 -0.4 -0.25 -0.1

Potential /V

Curr

ent

/A

a)

Figure 7.7 (a) Cyclic voltammograms of GOx incubated GC electrodes modified with

mixed monolayers of 4-carbpxyphenyl and MW with a molar ratio of 30:1 at scan rates

of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.6 V s-1 from inside cyclic voltammograms to outside

cyclic voltammograms, and (b) plot of peak currents versus scan rates.

As a more sensitive electroanalytical method, Osteryoung Square Wave Voltammetry

(OSWV) was also carried out for mixed monolayers of 4-carboxyphenyl and MW (with

the molar ratio of 30:1) modified GC electrodes after incubation of GOx (Figure 7.8).

225


An obvious redox peak at ca. -450 mV was observed in OSWV, which is consistent

with the redox peaks in the CV as shown in Figure 7.5.

-2.6

-2.4

-2.2

-2

-1.8

-1.6

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/A

Figure 7.8 The OSW voltammogram of GOx immobilised on GC electrodes modified

with mixed monolayers of 4-carboxyphenyl and MW with a molar ratio of 30:1.

7.3.2.2 Effect of Mixed Monolayers on the Redox Response of GOx

Mixed monolayers have a variety of advantages8, 33, 43 in surface fabrication as

introduced in Chapter One. Mixed monolayers can allow either large recognition

elements or nanosacle building blocks to be spaced apart from each other. GOx is a

larger molecule compared to the MW. Therefore, it is necessary to add a spacer

molecule to separate the MW to allow enough room for the coupling of GOx and ensure

a GOx molecule interacts specifically with a single MW. Here mixed monolayers of 4-

carboxyphenyl and MW were adopted to form an interface for the immobilisation of

GOx. In addition, 4-carboxyphenyl as the spacer molecule in mixed monolayers is

required to maintain the adsorbed GOx on the surface via covalent coupling. When

mixed monolayers were prepared by using two components MW and the 4-

carboxyphenyl with different molar ratios, some interesting results from the

226


electrochemistry were obtained after the immobilisation of GOx. Figure 7.9 shows the

peak current from the OSWV of GOx immobilised GC electrodes modified with mixed

monolayers of 4-carboxyphenyl and MW with different molar ratios.

0

0.1

0.2

0.3

0.4

0 20 40 60 8

[4-Carboxyphenyl]/[MW]

Curr

ent

/A

0

Figure 7.9 The peak current from OSWV of GOx immobilised GC electrodes modified

with mixed monolayers of 4-carboxyphenyl and MW at different dilution ratios.

It can be seen in Figure 7.9 no redox current was recorded when the GOx was

immobilised on GC electrodes modified with pure monolayers of MW or pure

monolayers of 4-carboxyphenyl. When the molar ratio of 4-carboxyphenyl and MW

increased, the redox current increased correspondingly until the maximum value was

reached at the molar ratio of 30:1. After that the current decreased when the dilution

ratio was greater than 30:1. The maximal peak current obtained at the dilution ratio of

30:1 was presumably due to the maximal surface coverage of the GOx, indicating that

the distance between the adjacent bridge molecules is more favorable for the

immobilisation of GOx on the interface modified with mixed monolayers of 4-

carboxyphenyl and MW at a molar ratio of 30:1. In this case, it can be hypothesised that

the MW can partially penetrate into the adsorbed GOx as shown in Scheme 7.4, and

227


shorten the distance between the active centre of GOx and the underlying GC electrode,

sufficiently to achieve direct electron transfer between GOx and the underlying GC

electrode. Thus, the use of mixed monolayers, as opposed to pure monolayers, results in

an optimal spacing between GOx molecules and ensures a higher degree of

homogeneity in their environment. Experimental results also show that the strategy of

using mixed monolayers of MW and 4-carboxyphenyl plays an important role in the

surface coverage of GOx and rate constant of electron transfer between the attached

GOx and the underlying GC electrodes as summarised in Table 7.1, which will be

discussed in the following sections.

Scheme 7.4 The scheme of MW entering into the GOx.

228


Table 7.1 Some parameters of GOx immobilised GC electrodes modified with mixed

monolayers of MW and 4-carboxyphenyl at different molar ratios.

[4-Carboxyphenyl]/[MW]

0 10 20 30 40 60

Current (µA) 0 0.16 0.32 0.41 0.28 0.05

GOx (pmol cm-2) - 0.56 1.14 2.41 1.22 0.47

a/ c - 1.22 1.15 1.19 1.13 1.21

7.3.2.3 Surface Coverage of GOx on MW Modified Glassy Carbon Electrodes

The surface coverage of GOx was calculated by integration of the redox peaks in the

cyclic voltammogram of GOx. A value of 2.41×10-12 mol cm2 was found for the 3 mm

diameter circular GC electrodes when the molar ratio of 4-carboxyphenyl and MW was

30:1 (Table 7.1). This surface coverage is very consistent with the coverage of GOx

(2.6×10-12 mol cm2) on 3 mm diameter GC electrodes determined by radioactive 125I

labeling.37 The molecule of GOx is a compact ellipsoid13, 44, 45 and the size of GOx

determined by X-ray crystallography is about 60 Å×52 Å×77 Å.13 Each molecule may

be attached in various orientations; therefore, the Stokes radius, 4.3 nm,44 is a

reasonable estimate for its projection area, i.e. 58 nm2.13, 45 Since the attachment

proceeds randomly ca. 60% of the electrode area can be covered.46, 47 Thus each

molecule of GOx occupies an area of ca. 100 nm2, and the superficial concentration of

enzyme which should correspond to the saturation of a monolayer can be estimated as

1.7×10-12 mol cm2. Comparing the latter figure with the coverage determined by

electrochemistry here would imply that the actual area of the electrode is 1.42 times its

geometrical area, which is very close to the reported typical roughness of the GC

electrodes (1.43).48 Although the above size and surface area estimates are certainly

229


approximate, it can be concluded that the GC electrode surface is covered by a

monolayer of close-packed GOx units. It is understandable when the GOx was

immobilised on pure monolayers of MW, almost no GOx was properly immobilised

onto the MW due to steric hindrance from GOx molecules. On the other hand, when the

enzyme was immobilised on mixed monolayers of the MW and the 4-carboxyphenyl at

a very low molar ratio, the low surface density of the MW will result in fewer sites for

the immobilisation of GOx. Therefore, the results further confirm that a mixed

monolayer of 4-carboxyphenyl and MW with the appropriate molar ratio is essential for

the maximal immobilisation of GOx.

7.3.2.4 The Rate Constant of Electron Transfer between FAD and GC Electrodes

The rate constant of electron transfer between redox centre of GOx and GC electrodes

was calculated using Laviron’s method.49 The rate constants were determined by the

relationship between the peak separation and the scan rate as shown in Figure 7.10.

-40

-30

-20

-10

0

10

20

30

40

-2.5 -2 -1.5 -1 -0.5 0 0.5

Log( /V s-1)

Ep-E

o` /

mV

Figure 7.10 The plot of Ep-Eo` versus the logarithm of scan rates for GOx immobilised

GC electrodes modified with mixed monolayers of 4-carboxyphenyl and MW at a molar

ratio of 30:1.

230


The rate of electron transfer for immobilised GOx on GC surfaces was calculated to be

78 s-1 when the molar ratio of MW and 4-carboxyphenyl was 1:30. This value is similar

to the rate of electron transfer of FAD attached to the end of norbornylogous bridge

with the length of 18.3 Å self-assembled on gold electrodes (100 s-1).22 The rapid rate of

electron transfer through molecular wires is demonstrated by comparison with that for

FAD attached to mercaptoundecanoic acid which is three orders of magnitudes slower

(0.09 s-1).33

7.3.2.5 Measurement of Biocatalytical Activity of GOx

The question remains as to whether the adsorbed GOx retains its glucose-specific

enzyme activity once immobilised at the MW modified GC surface. According to the

biocatalytical reaction of GOx in Scheme 7.1, in the presence of glucose and under the

appropriate condition, GOx can catalyse glucose into gluconolactone and the native

GOx-FAD becomes reduced to GOx-FADH2. Oxygen plays an important role in the

biocatalytical reaction of GOx. So the glucose-specific enzyme activity here was

investigated under aerobic (in the presence of oxygen) and anaerobic (oxygen free)

conditions.

After immobilisation of GOx on GC electrodes modified with mixed monolayers of

MW and 4-carboxyphenyl with a molar ratio of 1:30, the biocatalytical activity of GOx

was firstly investigated under the aerobic condition. Figure 7.11 a shows the cyclic

voltammogram of a GOx/MW electrode obtained in oxygen-free (degassed with argon

for 30 min before measurement) phosphate buffer solution (pH 7.0) at a scan rate of 100

mV s-1. The phosphate buffer was later re-aerated to oxygen-saturation and another

cyclic voltammogram was obtained as shown in Figure 7.11 b. Under these conditions

231


the catalytic reduction peak current for oxygen is clearly observed as in Figure 7.11 b.

After adding 10 mM glucose to the phosphate buffer solution saturated with oxygen, the

cyclic voltammogram (Figure 7.11 c) of a GOx/MW modified GC electrode shifted up

and reached close to the cyclic voltammogram under the oxygen-free condition without

adding glucose. These results are consistent with the redox reaction of GOx in the

presence of glucose under aerobic conditions (Scheme 7.1). In the presence of glucose,

the active centre FAD of GOx is reduced to FADH2, and in the presence of oxygen,

FADH2 is oxidised to regenerate FAD and restore the catalytic form of GOx. Therefore

in this process only the oxygen reduction was observed, resulting in the increase of the

reduction peak current. So the results demonstrate that the molecular oxygen was

consumed at the electrode surface and accordingly confirms that the GOx still

maintained its specific enzyme activity and is sensitive to glucose.

-12

-10

-8

-6

-4

-2

0

2

4

6

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/A

(a)

(b)

(c)

Figure 7.11 Cyclic voltammograms of GOx immobilised GC electrodes modified with

mixed monolayers of benzoic acid and MW at a mole ratio of 30:1 in pH 7.0 phosphate

buffer (a) in the absence of dissolved oxygen and (b) in the presence of dissolved oxygen

with 0 mM and (c) 10 mM glucose at the scan rate of 100 mV s-1.

232


The biocatalytical activity of GOx was also measured under anaerobic conditions

(degassed with argon for 30 min before measurement) by injection of glucose at

different concentrations. The oxidation current in cyclic voltammograms of a GOx/MW

modified GC electrode in oxygen-free buffer solutions increased after addition of

glucose as shown in Figure 7.12. Next, a chronoamperometry experiment at a constant

potential of -400 mV that is close to the oxidation potential of GOx was carried out

under constant stirring by adding glucose with different concentrations. Upon the

addition of glucose, the monitored current of GOx/MW modified GC electrodes

increased as shown in Figure 7.13.

-0.45

-0.3

-0.15

0

0.15

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0

Potential /V

Curr

ent

/A

(a)

(b)

(c)

Figure 7.12 Cyclic voltammograms of GOx immobilised on GC electrodes modified

with mixed monolayers of 4-carboxyphenyl and MW at a mole ratio of 30:1 in 0.05 M

phosphate buffer (oxygen-free, 0.05 M KCl, pH 7.0) with (a) 0 mM, (b) 5 mM, and (c)

10 mM glucose at the scan rate of 5 mV s-1.

233


-100

-90

-80

-70

-60

-50

-40

0 500 1000 1500 2000 2500 3000 3500 4000

Time /sec

Curr

ent

/nA

0 mM 0 mM2 mM 4 mM 6 mM 10 mM 15 mM 20 mM

Figure 7.13 The current record as a function of time for the GOx/MW modified GC

electrodes in 0.05 M phosphate buffer (oxygen-free, 0.05 M KCl, pH 7.0) at a constant

potential of –400 mV after adding glucose at different concentrations.

However, no increase in oxidation current corresponding to the amount of glucose

injected was observed at a GC electrode modified with mixed monolayers of MW and

4-carboxyphenyl alone or a GC electrode modified with mixed monolayers of MW and

4-carboxyphenyl followed by the attachment of another redox enzyme, horseradish

peroxidase (HRP) under the same conditions (Figure 7.14). These controls indicate the

increase of oxidation current in Figure 7.13 is due to the redox reaction of GOx with

glucose. The increase in oxidation current in Figure 7.13 is consistent with the

biocatalytical reaction of GOx in the presence of glucose under anaerobic conditions

(Scheme 7.2). According to the biocatalytical reaction of GOx, in the presence of

glucose GOx can catalyse glucose into gluconolactone and the native GOx-FAD

becomes reduced to GOx-FADH2. Under anaerobic (oxygen free) condition, the only

way to recycle the reduced form of FADH2 to FAD is via direct electron transfer, which

is an oxidation process and will cause the increase of oxidation current correspondingly.

234


So the results have further demonstrated that under both aerobic and anaerobic

conditions, the GOx coupled on MW modified GC surfaces still maintains its specific

enzyme activity and is sensitive to glucose.

-45

-40

-35

-30

-25

-20

-15

0 1000 2000 3000 4000 5000 6000 7000 8000

Time /sec

Curr

ent

/nA

0 mM0 mM

2 mM4 mM

6 mM10 mM

15 mM20 mM

Figure 7.14 The current record as a function of time for mixed monolayers of MW and

4-carboxyphenyl at a mole ratio of 1:30 modified GC electrodes in 0.05 M phosphate

buffer (oxygen-free, 0.05 M KCl, pH 7.0) at a constant potential of –400 mV after

adding glucose at different concentrations.

Based on results of chronoamperometry experiments at a constant potential of -400 mV

for three types of GC electrodes (GOx/MW modified GC, HRP/MW modified GC and

MW modified GC), the relationship between the current and the concentration of

glucose for three types of GC electrodes is shown in Figure 7.15. Both the HRP/MW

modified GC electrodes and mixed monolayers of MW and 4-carboxyphenyl modified

GC electrodes demonstrated almost no current increased after adding glucose. For the

GOx/MW modified GC electrodes, the catalytic oxidation current measured at -400 mV

in the absence of oxygen increased linearly with increasing glucose concentration and

235


saturated at glucose concentrations higher than 10 mM. The saturated current value

corresponds to the highest turnover rate of the biocatalyst.

0

5

10

15

20

25

30

35

0 5 10 15 20 25

[glucose] /mM

Curr

ent

/nA GOx/MW modif ied GC

MW modif ied GC

HRP/MW modif ied GC

Figure 7.15 The increase of the oxidation current versus the concentration of the

glucose injected into the electrochemical cell under anaerobic condition for 3 different

electrodes (GOx/MW modified GC, HRP/MW modified GC and MW modified GC). The

current measured at –400 mV was used for the plot.

The efficient electron transfer turnover rate of the reconstituted enzyme has important

consequences on the properties of the enzyme electrode.25 The saturation current density

(500 nA cm-2) was reached when the concentration of the glucose solution increased to

about 10 mM. From the known surface coverage of the enzyme, and the saturated

current density, it is estimated that the enzyme turnover rate is 1.1 s-1 at room

temperature, which is much smaller than that reported by Willner and coworkers (5000

s-1).25 The slow enzyme turnover rate might be due to the fact that the MW could not

approach the active centre FAD all the way and a gap is possible to exist between the

MW and the active centre FAD, which could result in the weak electrical

communication between the active centre FAD of GOx and the underlying GC

236


electrodes and could compromise the catalytic response. So it is worthwhile to design

another molecular wire which can penetrate into the GOx and access to the active site

FAD to achieve significant electrical communication.

7.4 Conclusions

The conjugated and rigid MW was successfully used as an electron transfer linker for

the exploration of the deeply buried active centre of GOx. The results suggested that the

MW can extend into the interior of the glycoprotein and approach the active centre FAD

of GOx when the GC electrodes was modified with mixed monoalyers of MW and 4-

carboxyphenyl with the molar ratio of 1:30 followed by the coupling of GOx. Direct

electron transfer of GOx from the active centre FAD through the MW to the underlying

GC electrode has been demonstrated. The rate constant of electron transfer was found to

be 78 s-1. In addition, the immobilised GOx still retains its glucose-specific enzyme

activity. For the GOx/MW modified GC electrodes the catalytic oxidation current

measured at -400 mV in the absence of oxygen increased linearly with increasing

glucose concentration and saturates at glucose concentrations higher than 10 mM. So

the GOx/MW GC electrodes can be used as a glucose biosensor. However, the enzyme

turnover rate is only 1.1 s-1 at room temperature due to the weak electrical

communication between FAD and underlying GC electrodes.

7.5 References




237



Am. Chem. Soc. 2002, 124, 9591-9599.

(4) Di Gleria, K., Hill, H.A.O., Lowe, V.J., Page, D.J., J. Electroanal. Chem. 1986,

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(5) Arnold, S., Feng, Z.Q., Kakiuchi, T., Knoll, W., Niki, K., J. Electroanal. Chem.

1997, 438, 91-97.

(6) Ruzgas, T., Wong, L., Gaigalas, A.K., Vilker, V.L., Langmuir 1998, 14, 7298-

7305.

(7) Liu, H., Yamamoto, H., Wei, J.J., Waldeck, D.H., Langmuir 2003, 19, 2378-

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(16) Bourdillon, C., Demaille, C., Moiroux, J., Saveant, J.M., J. Am. Chem. Soc.

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(19) Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., Anal. Sci. 2002, 18, 939-941.

(20) Chi, Q., Zhang, J., Dong, S.J., Electrochim. Acta 1994, 39, 2431-2438.



(22) Liu, J.Q., Gooding, J.J., Paddon-Row, M.N., Chem. Phys. 2006, in press.

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(25) Zayats, M., Katz, E., Willner, I., J. Am. Chem. Soc. 2002, 124, 2120-2121.

(26) Pazur, J.H., Kleppe, K., Biochemistry 1964, 3, 578-583.

(27) Ball, E.G., Cold Spring Harbor Symp. Quant. Biol. 1939, 180, 755.

(28) Ksenzhek, O.S., Petrova, S.A., Bioelectrochem. Bioenerg. 1983, 11, 105.

(29) Lowe, H.J., J. Biol. Chem 1956, 221, 983-989.

(30) Gorton, L., Johansson, G., J. Electroanal. Chem. 1980, 113, 151-158.

(31) Verhagen, M.F.J.M., Hagen, W.R., J. Electroanal. Chem. 1992, 334, 339-350.

(32) Durfor, C.N., Yenser, B.A., Bowers, M.L., J. Electroanal Chem. 1988, 244,

287-295.


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(34) Degani, Y., Heller, A., J. Phys. Chem. 1987, 91, 1285-1289.

(35) Gooding, J.J., Situmorang, M., Erokhin, P., Hibbert, D.B., Anal. Commun. 1999,

36, 225-228.

239


(36) Zayats, M., Raitman, O.A., Chegel, V.I., Kharitonov, A.B., Willner, I., Anal.

Chem. 2002, 74, 4763-4773.

(37) Bourdillon, C., Demaille, C., Gueris, J., Moiroux, J., Saveant, J.-M., J. Am.

Chem. Soc. 1993, 115, 12264-12269.

(38) Staros, J.V., Wright, R.W., Swingle, D.M., Anal. Biochem. 1986, 156, 220-2.

(39) Willner, I., HelegShabtai, V., Blonder, R., Katz, E., Tao, G.L., J. Am. Chem.

Soc. 1996, 118, 10321-10322.


(41) Tam-Chang, S.-W., Mason, J., Iverson, I., Kwang, K.-O., Leonard, C., Chem.

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(42) Cooke, G., Duclairoir, F.M.A., John, P., Polwart, N., Rotello, V.M., Chem.

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(46) Finegold, L., Donnell, J.T., Nature 1979, 278, 443-445.

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240

Chapter 8-Development of a Labe-Freel Immunosensor on Molecular Wire Modified Glassy Carbon Surfaces

Chapter Eight

Development of a Label-Free Immunosensor on Molecular

Wire Modified Glassy Carbon Surfaces

241


8.1 Introduction

The transfer of electrons between an electrode and a redox centre in the presence of a

biological molecule is the basis of behind transduction in many bioelectronic devices

and electrochemical biosensors.1, 2 With enzyme based biosensors this transfer of

electrons is achieved either by the soluble redox active molecules shuttling electrons

between the redox active centres of the enzyme and the electrode3-7 or alternatively

electrons are transferred between the redox active centres and the electrode directly.8-10

In the case of affinity based biosensors, where biorecognition is via antibody-antigen

binding or hybridisation of DNA, the biorecognition event is more difficult to transduce

because of the absence of an obvious redox active centre. The typical solution to this

problem is to add redox labels which exhibit a change in affinity for the biosensing

interface upon the biorecognition event. An example of this approach is the transduction

of DNA hybridisation by long range electron transfer where after DNA hybridisation a

redox active intercalator is added to the solution containing the biosensor.11 When only

a single strand of DNA is present at the interface, the redox reporter has no affinity for

the DNA. However, upon hybridisation to form a duplex, sites for intercalation are

formed, and the redox molecule possesses an affinity for the DNA. The intercalation

then allows the redox reporter to be interrogated electrochemically by electrons

transferring through the DNA. Transduction of the biorecognition event in a typically

electrochemical immunosensor has related elements to the DNA example. The

electrochemical immmunosensors developed by Kuhr and coworkers reflect this

strategy.12 After binding of the antigen to the antibody modified electrode, a second

antigen containing an enzyme label competes with the antigen for binding sites in a

242


competitive assay. Once equilibrium is attained the substrate for the enzyme is added

and a product of the enzyme reaction is detected electrochemically.

Both these examples have the same drawback however, the need to add a reporting

species to the sample solution being analysed at some point during the analysis so that

transduction of the biorecognition event can be achieved. The requirement to add the

reporting molecule necessitates user intervention during the performance of the

analysis. The need for user intervention means the final biosensor requires skills to

operate, contrary to the principles of biosensors that they should be usable by unskilled

operators. One solution to this problem has been to use label free approaches such as

using the quartz crystal microbalance, surface plasmon resonance,13 mechano-acoustical

spectroscopy,14 optical change in liquid crystals15 or a label free electrochemical

immunosensor developed by Tender and co-workers.16 The label free techniques are

however beset by problems of robustness when analysing real complex samples. The

robustness issue comes from non-specific binding at the biosensing interface also giving

a response as well as the specific immunoreaction.

This chapter is to present a new user-intervention-free immunobiosensor where the

electrochemical response is reliant on the change in the amount of antigen bound to the

antibody. The idea stems from the large body of work on direct electron transfer.

Throughout this large body of work there have been examples where the presence of the

protein has an adverse effect on the electrochemistry.4, 17, 18 This is perhaps illustrated

by the refolding of apo-glucose oxidase around its active centre FAD where

electrochemistry is essentially switched off when the protein folds. Willner proposed

this switching off of FAD was due to nonspecific incorporation on the protein.18

243


Gooding expanded on this idea further and suggested that this switch off is a

consequence of poor electronic coupling.17 Support for the ideas of non-specific

adsorption comes from Rubin where glucose oxidase is attached to the gold electrodes

modified with a mixed monolayer of ferrocenylhexadecanethiol and aminoethanethiol

and the redox chemistry of ferrocene was switched off.4 Furthermore, the modulation of

redox electrochemistry by the presence of proteins has been demonstrated where

antibodies bind to an electrode containing a surface attached redox active species,16, 19

which opens the door for the development of immunosensors where the immunobinding

reaction is monitored by the modulation of the electrochemistry.

For the design of an immunosensor, the most vital factors that should be concerned are

reproducibility, specificity, and sensitivity. The design of the biorecognition interface

has the dominant effect on these performance parameters. General strategies for the

immobilisation of immuno-reagents onto solid electrode surfaces include physical

adsorption at a solid surface,20-22 microencapsulation, entrapment,23, 24 cross linking,

covalent bonding,25-27 and the use of biological binding proteins such as protein A or

protein G28, 29 or use of the avidin/biotin system. Furthermore, as mentioned above for

label-free immunosensors, non-specific adsorption of proteins and cross-reactivity of

non-target proteins affect the sensitivity as well as the reliability of the biosensor.

Nonspecific adsorption of proteins is generally minimised by masking the surfaces with

blocking agents such as bovine serum albumin (BSA)30 or a hydrophilic layer such as

poly(ethylene glycol) molecules (PEG).31

Many kinds of electrodes have been used to fabricate immunosensor devices including

carbon paste electrodes,32 screen-printed carbon electrodes,33 GC electrodes34, 35 and

244


gold electrodes.36 Electrochemically reductive modification of GC surfaces with aryl

diazonium salts to form stable monolayers is of interest to electrochemistry37 due to the

potential advantages as introduced in Chapter One. GC electrodes can be covalently

modified with a long completely rigid molecular wire (MW) existing as the aryl

diazonium salts and the interface comprising mixed monolayers of MW and PEG

demonstrated good potential for the protein chemistry in Chapter Six. The modified

MW has demonstrated high efficiency of electron transfer and can be used as the

conduit for electron transfer to explore the active centres of glucose oxidase in Chapter

Seven. So this kind of conjugated aromatic molecule of precise length and composition

has great potential in the development of potential switches, wires, controllers, and

gates of a molecular computer due to the favorable conductivity.38-40

The purpose of this chapter is to present proof-of-concept results for the modulation of

electrochemistry by protein as the basis of an immunosensor. Scheme 8.1 a shows the

schematic of the label-free immunosensor which was fabricated in this study, and

Scheme 8.1 b shows the schematic of using the fabricated immunosensor for the

determination of antibiotin. As shown in Scheme 8.1 a, GC electrodes were firstly

modified with mixed monolayers of MW and PEG. Mixed monolayers on electrode

surfaces are promising as platforms for protein adsorption and immobilisation41 due to

the possibility to control chemical and structural properties of a surface by adjusting the

abundance, type, and spatial (both normal and lateral) distribution of the tail groups.

The redox probe ferrocenedimethylamine was subsequently attached to the carboxylic

acid terminated MW by forming an amide bond followed by the immobilisation of

biotin. After incubation this immunosensor to the antibiotin solution, the antibiotin can

be immobilised to the immunosensor surface as shown in Scheme 8.1 b due to the

245


strong affinity between biomolecular pairs biotin and antibiotin. Electrochemistry was

used to monitor the immobilisation of antibiotin.

O

O

O

O

GC

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

Molecular wireInsulator

NO2

CO

NH

NH

CO

Redox ProbeFe

H2C

H2C

Biotin

Electrochemistry

a)

Electrochemistry

O

O

O

O

GC

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O


NO2

CO

NH

NH

CO

Redox ProbeFe

H2C

H2C

b)

Biotin

Antibiotin

Scheme 8.1 Schematic of (a) the fabricated label-free immunosensor based on MW, and

(b) the antibiotin targeting to the label-free immunosensor.

All components in this immunosensor system play an important role for the

performance of the immunosensors. The MW provides a pathway of electron transfer

between the biomolecular pairs in response to electrochemical reaction at the electrode,

246


and the PEG serves as insulators to resist the non-specific protein adsorption. Thus only

the electrical response from target biomolecules can directly access to the electrode and

the signal caused by interference to the electrode can be excluded. The redox probe

ferrocene can be used to transduce the interactions between bimolecular pairs. Before

the immobilisation of antibiotin, the electrochemistry of redox probe can be observed as

shown in Scheme 8.1 a, but association of the antibiotin to the biotinylation modified

GC electrode blocks the electrochemistry of ferrocene as shown in Scheme 8.1 b. The

extent of the electrode in solution by antibiotin is controlled by the antibiotin

concentration in the sample. Based on the changes in the microenviroments that

influence readily measurable redox properties of redox species, electrochemistry was

used to probe the specific biotin:antibody interactions.






were prepared as described in Section 2.4.6. The procedures for the synthesis of MW,

PEG and ferrocenedimethylamine are shown in Section 2.2. All electrochemical

measurements were performed with a BAS-l00B electrochemical analyser. All

potentials were quoted relative to an Ag/AgCl reference at room temperature. All cyclic

voltammetry measurements for the ferrocene modified electrodes were carried out in

phosphate buffer (0.05 M KCl, 0.05 M K2HPO4/KH2PO4, pH 7.0).

247


8.2.2 Covalent Coupling of Ferrocenedimethylamine to Mixed Monolayers of MW

and PEG Modified Glassy Carbon Electrodes

Covalent attachment of ferrocenedimethylamine to carboxylic acid terminated

monolayers was achieved by dipping the MW and PEG modified GC electrodes into the

absolute ethanol solution containing of 40 mM 1,3-Dicyclohexylcarbodiimide (DCC)

and 5 mM ferrocenedimethylamine for 6 h at room temperature. DCC was used for the

activation of terminal carboxylic acid groups of MW.42

8.2.3 Immobilisation of Biotin and Anti-biotin on Ferrocenedimethylamine Modified

Glassy Carbon Electrode Surfaces

After attachment of ferrocenedimethylamine, the GC substrates covered with amine

terminal groups were immersed into 1 mg mL-1 solution of NHS-biotin in phosphate

buffered saline (PBS, pH 7.3) for 2 h at 4 oC to attach a biotin to the free terminal

amines on the surface bound ferrocenedimethylamine. This formed system was named

as the label-free immunosensor as shown in Scheme 8.1 a. Then the immunosensor was

rinse with copious amount of water and PBS followed by immersion into the PBS

solution containing 0.5 M antibiotin for 20 min at 4 oC, and the immunosensor surface

was immoilised with antibiotin as shown in Scheme 8.1 b.


8.3.1 Electrochemistry of Glassy Carbon Electrodes Modified with Mixed Monolayers

of MW and PEG at a Molar Ratio of 1:20

The electrochemistry of modifying the GC surfaces with mixed monolayers of MW and

PEG on a molar ratio of 1:20 has been presented in Chapter Six. The blocking

248


properties of mixed monolayers of MW and PEG modified GC surfaces in negatively

charged redox probe ferricyanide and in the positively charged redox probe Ru3+ were

also studied in Chapter Six.

8.3.2 Electrochemistry of Ferrocenedimethylamine on Glassy Carbon Electrodes

Modified with Mixed Monolayers of MW and PEG at a Molar Ratio of 1:20

After modification of GC electrodes with mixed monolayers of MW and PEG, the next

step was to covalently attach ferrocenedimethylamine to GC surfaces. The

electrochemistry of bared GC electrodes in ferrocenedimethylamine solution was

studied before investigating the electrochemistry of ferrocenedimethylamine modified

on GC electrodes. Figure 8.1 shows the cyclic voltammograms of bare GC electrodes in

1 mM ferrocenedimethylamine chloride in phosphate buffer (0.05 M; 0.05 M KCl; pH

7.0) at a scan rate of 100 mV s-1. The significant redox peaks from ferrocene molecules

in solution were observed. The formal potential (Eo`) was found to be 349 mV.

-3

-2

-1

0

1

2

3

-0.2 0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

Bare GC in phosphate buffer

Bare GC in phosphate buffer

with ferrocnedimethylamine chloride

Figure 8.1 Cyclic voltammograms of bare GC electrodes in 0.05 M phosphate buffer

(0.05 M KCl, pH 7.0) before and after adding 1 mM ferrocenedimethylamine chloride

at a scan rate of 100 mV s-1.

249


Cyclic voltammogams measured in an aqueous solution of 0.05 M phosphate buffer

(0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1 before and after the attachment of

ferrocene on the mixed monolayer modified GC electrode are shown in Figure 8.2. The

obvious redox peaks with Eo` of 374 mV and EFWHM of 238 mV were observed.

Comparing with the formal potential obtained for bare GC electrodes in the bulk

ferrocenedimethylamine solution (Figure 8.1), the formal potential of ferrocene shifted

25 mV in the positive direction after attaching the ferrocene to the GC surfaces. The

positive potential shift might be due to two reasons. Firstly, ferrocene was attached to

the mixed monolayer modified GC surface by forming an amide bond. The amide

electron-withdrawing groups can cause oxidation to become more difficult, which leads

the redox potential to be shifted more positive than normal ferrocene.43-45 Secondly, it is

postulated that the coadsorbed PEG molecules on the interface created a localised less

polar environment. As the local enviroment of ferrocene becomes less polar, the redox

potential shifts to be more positive because the formation of the ferrocene cation

becomes less energetically favorable.46 The positive shift further confirms that GC

electrodes modified with mixed monolayers of MW and PEG at a molar ratio of 1:20

have been modified with ferrocenedimethylamine. The current of redox peaks versus

scan rates is plotted in Figure 8.3, and this graph shows that both the anodic and

cathodic current are proportional to the scan rates, indicating that

ferrocenedimethylamine was surface bound. In the absence of DCC, such that no

covalent coupling of the ferrocenedimethylamine could occur, only the exceedingly

small peaks due to physisorption were observed.

250


-2

-1

0

1

2

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Curr

ent

/A

After attachment

of ferroceneBefore attachment

of ferrocene

Figure 8.2 Cyclic voltammograms of mixed monolayers of MW and PEG modified GC

electrodes before and after the covalent attachment of ferrocenedimethylamine in

0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1.

-1

-0.5

0

0.5

1

0 0.2 0.4 0.6 0.8

Scan rate /V s-1

Curr

ent

/A

Figure 8.3 Peak current verse the scan rate for the cyclic voltammograms after the

attachment of ferrocenedimethylamine. Triangles: anodic scan, squares: cathodic scan.

Cyclic voltammogams of the ferrocene coupled to the mixed monolayers of MW and

PEG in Figure 8.2 show non-ideal behaviour47 with regards to peak separation at slow

scan rates ( Ep 90 mV rather than the ideal Ep 0 mV) and the full width half

251


maximum (EFWHM 238 mV rather than the ideal EFWHM 90.6 mV/n where in this case

n 1). With regards to both peak separation and the EFWHM the non-ideal behaviour has

been attributed to the ferrocene molecules being located in a range of environments with

a range of formal electrode potentials (Eo`).48, 49 The surface coverage of ferrocene was

calculated to be 1.28 0.11×10-11 (95 confidence, n 5) mol cm-2 by integration of the

area under the redox peaks of ferrocene in the cyclic voltammogram.

Another test was to investigate the change in electrochemistry with time for attachment

of ferrocenedimethylamine. The variations in current density with incubation time are

shown in Figure 8.4. The figure shows the current density continues to increase for at

least 6 h before reaching a saturated current density. So the optimised time for covalent

attachment of ferrocenedimethylamine to mixed monolayers of MW and PEG modified

GC electrodes is about 6 h at room temperature.

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16 18 20 22 24

Time /h

Curr

ent

density

/A

cm

-2

Electrode 1

Electrode 2

Figure 8.4 The plot of current density versus the incubation time for

ferrocenedimethylamine.

252


8.3.3 Heterogeneous Electron Transfer Through Mixed Monolayers of MW and PEG

Modified Glassy Carbon Electrodes Using Ferrocene as the Redox Probe

The rate of electron transfer through mixed monolayers of MW and PEG modified on

GC surfaces by using ferrocenedimethylamine as the redox probe was also studied. The

rate of electron transfer was determined by the relationship between the peak separation

and the scan rate as shown in Figure 8.5 and was found to be ca. 23 5 (95

confidence, n 4) s-1 by Laviron’s method.50 The rate constants of electron transfer

through ferrocene modified MW with three phenyleneethynylene units self-assembled

on gold electrodes have been studied by Creager et al.51-53 The rate constants of electron

transfer were found to be very high ( 350 s-1). The huge difference in the rate of

electron transfer on similar electron transfer system are attributed to two differences

between the system studied here and systems used by Creager’s group. Firstly, electrode

materials play an important role in the electron transfer mechanism.54 The MW used

here was modified on GC surfaces, which has been shown to have lower electron

transfer efficiency than gold.55 Secondly, Creager’s group synthesised the ferrocene-

terminated oligo(phenyleneethynyl)arene-thiol molecule followed by modification of

this target molecule to the gold surface for exploring conductivity of the

oligo(phenyleneethynyl)arene molecular wire. The synthesised whole bridge structures

were -conjugated, which can promote electronic coupling between ferrocene and the

underlying electrode, thereby promote rapid electron transfer over long distances.51

However, this study used a stepwise method to attach ferrocene to MW modified GC

surfaces and the resultant MW system has an aliphatic “tail” of a few bonds length. The

electron transfer through this “tail ” is though - coupling, which can greatly

compromise the rate of electron transfer through this system.

253


-120

-80

-40

0

40

80

120

-2.5 -2 -1.5 -1 -0.5 0 0.5 1

Log /V s-1

Ep-E

o`

/mV

Figure 8.5 The dependence of peak separation (Ep-Eo`) on the scan rate for

ferrocenedimethylamine attached to mixed monolayers of MW and PEG modified GC

electrode surfaces.

8.3.4 Electrochemistry of Ferrocene Modified Glassy Carbon Electrode Surfaces

after Immobilisation of Biotin and Antibiotin

After modification of ferrocenedimethylamine on mixed monolayers of MW and PEG

modified GC electrodes, the ferrocene modified GC surfaces with the terminal amine

groups can be modified with NHS-biotin followed by the immobilisation of antibiotin

through biomolecular affinity. The electrochemistry was used to monitor the step-wise

attachment of biomolecular pairs. Figure 8.6 a shows the cyclic voltammograms of

ferrocene modified GC surfaces before and after incubation of biotin and antibiotin.

After the attachment of biotin, the electrochemistry of GC surfaces showed only minor

change. However, cyclic voltammetry of biotinylated GC surfaces demonstrated

pronounced reductions in peak currents after incubation in antibiotin solution. The OSW

voltammograms, as a more sensitive electrochemical technique, of biotinylated GC

254


surfaces also demonstrated pronounced reductions in peak currents after incubation with

antibiotin as s hown in Figure 8.6 b.

-1.5

-0.5

0.5

1.5

2.5

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Curr

ent /

A

After attachment of Fc

After incubation of biotin

After incubation of

antibiotin

a)

-2.7

-2.6

-2.5

-2.4

-2.3

-2.2

-2.1

-2

0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent /

A


After attachment of biotin

After incubation in

antibiotin

b)

Figure 8.6 (a) Cyclic voltammograms and (b) OSW voltammograms of mixed

monolayers of MW and PEG modified GC surfaces after the step-wise attachment of

ferrocenedimethylamine, biotin and antibiotin in 0.05 M phosphate buffer (0.05 M KCl,

pH 7.0) at a scan rate of 100 mV s-1. Ferrocene was firstly covalent attached to mixed

monolayers of MW and PEG modified GC surfaces followed by the attachment of NHS-

biotin, and antibiotin was finally immobilised by the affinity between biotin and

antibiotin.

255


The results in Figure 8.6 demonstrated that the electrochemistry of biotinylated GC

electrodes decreased after the immobilisation of antibiotin, which is consistent with the

results reported in the literature.16, 19, 56 It is hypothesised that the observed

electrochemistry changes reflect changes in the immediate microenvironment of the

redox probe concomitant with target binding.57, 58 The mechanism resulting in the

observed voltametric changes still remains unclear. One possible explanation is that the

immobilisation of antibiotin results in the formation of a biotin:antibiotin

immunocomplex on electrode surface,59 which covers the modified GC surface like a

cap and can block the ions to access the redox probe and thus decrease the current

correspondingly.

Controls revealed that no redox peaks were observed (Figure 8.7) when the similar

system was fabricated by using ethylenediamine, which is not a redox probe, to replace

ferrocenedimethylamine. The result indicates the obvious redox peaks formed in

Figure 8.6 were caused by the redox probe ferrocenedimethylamine. So the redox probe

is vital for the fabrication of this novel biosensing system. Moreover, in order to

investigate if the observed electrochemistry changes in Figure 8.6 were caused by the

interactions between biomolecular pairs biotin and antibiotin, other controls was carried

out. As shown in Figure 8.8, the cyclic voltammetry showed almost no change in peak

current when the biotin was excluded from the immunosensor system (Scheme 8.1 a).

Similarly, when the biotinylated GC electrodes were incubated in other proteins which

are not specific to biotin, such as BSA, and anti pig IgG under identical conditions

rather than antibiotin, almost no electrochemistry changes were observed as shown in

Figure 8.9.

256


-3.5

-3

-2.5

-2

-1.5

-1

0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A

After attachment of ethylenediamine

After incubation in antibiotin


Figure 8.7 OSWV of mixed monolayers of MW and PEG modified GC surfaces after the

step-wise attachment of ethylenediamine, biotin and antibiotin in 0.05 M phosphate

buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1.

-3.5

-3.4

-3.3

-3.2

-3.1

-3

-2.9

0 0.2 0.4 0.6 0.8

Potential /V

Crr

ent

/A


After attachment of ferrocene


step-wise attachment of ferrocenedimethylamine and antibiotin in 0.05 M phosphate

buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1.

257


-4.5

-4.4

-4.3

-4.2

-4.1

-4

-3.9

-3.8

-3.7

-3.6

0 0.2 0.4 0.6 0.8

Potential /V

Curr

ent

/A



After incubation in anti pig

IgG


step-wise attachment of ferrocenedimethylamine, biotin and anti pig IgG in 0.05 M


The non-specific adsorption of protein antibiotin on modified GC surfaces was also

assessed. This was determined by incubating of BSA and anti pig IgG after incubation

of biotin. Since these two proteins are not specific for NHS-biotin and any current

decrease in this case should be indicative of non-specific adsorption of BSA and anti pig

IgG on the surfaces. When the anti pig IgG at the concentration of 0.5 mg mL-1 was

incubated to the biotinylated interface modified with mixed monolayers of MW and

PEG at the molar ratio of 1:20 (Figure 8.10), only 0.1 current decrease was observed.

Thus, there is only 0.1 non-specific adsorption of anti pig IgG on GC surfaces

modified with mixed monolayers of MW and PEG at a molar ratio of 1:20. However,

the non-specific protein adsorption, which leads to the obvious decrease of current as

shown in Figure 8.10, was observed when the molar ratio of MW and PEG is higher or

lower than 1:20. So the optimised molar ratio of MW to PEG is 1:20 in this fabricated

258


system. Similar results were obtained for the non-specific adsorption of BSA on this

fabricated immunosensor interface (Figure 8.10).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30 40 50

[PEG]/[MW]

Curr

ent

/A

60

After incubation in anti pig IgG

After incubation in BSA

Figure 8.10 Current of biotinylated GC electrodes after incubation in 0.5 mg mL-1 BSA

and 0.5 mg mL-1 anti pig IgG verse the mole ratio of PEG and MW. The initial current

for the biotin modified GC electrode is 0.32 A.

All these controls indicate that GC surfaces modified with mixed monolayers of MW

and PEG at a molar ratio of 1:20 provide an optimised interface for attachment of biotin

and antibiotin. For the fabricated immunosensor, the antibiotin specifically associates

with the biotin sites in the monolayer assembly and only the biospecific interaction can

lead to the change of electrochemistry. Interaction of the monolayer modified electrode

with the complementary antibody results in the antibody association to the monolayer.

The bulky structure of the antibiotin at the electrode surface is believed to perturb the

electrical communication between the redox relay unit ferrocenedimethylamine

assembled in the monolayer and electrode. As a result, the resulting amperometric

signal is inhibited, and the attachment of antibiotin can be monitored by the change of

electrochemistry.

259


It was also observed that the extent of the surface coverage of antibody on biotinylated

GC electrodes could be controlled by the concentration of antibiontin and by the time of

incubating GC electrodes in antibiontin samples (Figure 8.11). As shown in

Figure 8.11 the current density of GC electrodes decreased with the increase of

incubation time to antibiotin, and the surface was saturated with antibiotin after

incubation of biotinylated GC electrodes in 0.5 M antibiotin for 20 min at 4 oC. The

results are consistent with the fact that the biotinylated GC surfaces can be immobilised

with more antibiotin for longer incubation time until the surfaces are fully covered with

antibiotin.

0

1

2

3

4

5

0 20 40 60 80 100 120 140 160 180

Time /min

Curr

ent

density /

A c

m-2

Electrode 1

Electrode 2

Figure 8.11 Current density from the OSWV for the biotinylated GC surfaces after

incubation in 0.5 M antibiotin for different time.

8.3.5 Calibration Curve for the Detection of Antibiotin

The results in section 8.3.4 have demonstrated the attachment of antibiotin can lead to

the decrease of Faradaic current, and this fabricated system can be used as a label-free

immunosensor for the detection of antibiotin with high selectivity. To determine the

immunosensor sensitivity to antibody, the immunosensors were used to detect the target

260


analyte with different concentrations. Experimental results show immersion of resulting

biotinylated GC electrode into phosphate buffer (pH 7.0) and stepwise addition of

antibiotin between 30 and 500 ng mL-1 resulted in a stepwise decrease in OSWV peak

current of the redox probe ferrocenedimethylamine. Figure 8.12 is the calibration curve

for the detection of antibiotin. This immunosensor allows for detecting antibiotin with

the concentration of 5 ng mL-1. This detection limit of the fabricated immunosensor for

the detection antibiotin is lower than that of an amperometric immunosensor for the

detection of rabbit IgG (0.33 g mL-1),60 and also lower than that of an amperometric

immunosensor for the detection of mouse IgG2a (0.02 g mL-1).29 Based on this

calibration curve, the affinity constant between biotin and antibiotin is calculated to be

ca. 109 M-1, which is higher than the affinity constant between rabbit IgG antigen and

anti-rabbit IgG antibody reported in the literature,36 and belongs to the typical affinity

constant for an antigen-antibody reaction (from the order of 108 to 1012 M-1).61

0

0.2

0.4

0.6

0.8

1

0.5 1 1.5 2 2.5 3

Log[Ab /ng mL-1]

Rela

tive c

urr

ent

Electrode 1

Electrode 2

Figure 8.12 Calibration plot of relative current against the logarithm of the antibiotin

concentration. Relative current is obtained by dividing the current before the incubation

of antibiotin with the current after the incubation of antibiotin.

261


Reproducibility is another important factor affecting the performance of the

immunosensors. It was observed the immunosensor response and hence its construction

was quite reproducible (Figure 8.13): 9 immunosensors were prepared by following

identical steps and their response toward attachment of antibiotin led to a relative

standard deviation of only 6 . So this fabricated immunosensors can be used for the

detection of antibiotin with high selectivity, sensitivity and reproducibility.

0

1

2

3

4

0 1 2 3 4 5 6 7 8 9

Electrode type

Curr

ent

density

change /

A c

m-2

Figure 8.13 The stability of fabricated immunosensor system for detection of anti-

biotin. The current density change means the difference between the current density

before and after incubation of antibiotin.

8.3.6 Displacement Immunoassay

As discussed above, immobilisation of antibiotin to the fabricated immunosensor results

in the decrease of current, which is a negative signal. However, achieving a positive

signal is more desirable for a biosensing device. In addition, this project is more

interested in detection of small analytes such as biotin or antigen, not the antibody.

Therefore, it is necessary to develop a displacement immunoassay, which can be used in

the detection of free biotin based on a positive signal. Generally, it is difficult to

262


dissociate the antigen-antibody complex because of the high affinity constants derived

from the strong antigen-antibody reactions (low dissociation constant). However, it has

now been demonstrated that antibody-antigen complexes will dissociate in the presence

of free, unbound antigens,62 which makes it possible to develop a displacement

immunoassay. This possibility was investigated by exposure the system in Scheme 8.1

b to free biotin. The free biotin can compete with the biotin previously modified on GC

electrodes for the antibiotin based on the strong affinity of biotin and antibiotin. The

dissociation of antibiotin from the biotinylated GC surfaces may occur and an

equilibrium system will form due to the competition for antibiotin between biotin and

free biotin (Scheme 8.2).

Electrochemistry

O

O

O

O

GC

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O


NO2

CO

NH

NH

CO

Fe

H2C

H2C

Redox Probe

Biotin

Antibiotin

Free biotin

Scheme 8.2 Schematic of free biotin targeting to the antibiotinylated GC surfaces.

263


Electrochemistry was used to investigate this fabricated displacement immunoassay.

Figure 8.14 shows the change in the current of OSWV after exposure of the

antibiotinylated GC surfaces (Scheme 8.2 b) to 0.3 mg mL-1 of free biotin for different

times. An increase in current density was observed upon increasing the exposure time to

free biotin. It was also found that the increase of the redox peaks proceeds for at least 30

min before reaching a saturated peak current when the concentration of free biotin is

0.3 mg mL-1, indicating the process of dissociation of the antibiotin upon exposure to

0.3 mg mL-1 free biotin takes at least 30 min to complete. The increase in current after

exposure to free biotin reflects the displacement of antibiotin from the monolayer

system. Based on Scheme 8.2, it is hypothesised that the dissociation of antibintin from

the biotinylated GC surfaces will change the immediate microenvironment of the redox

probe ferrocenedimethylamine concomitant with target binding and more ions from the

surrounding environment can communicate with the ferrocene without blocking, which

will result in the increase in electrochemistry of the whole system.

-0.01

0.01

0.03

0.05

0.07

0.09

0.11

0.13

0.15

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Potential /V

Curr

ent

/A

Increasing

incubation time

Figure 8.14 OSWV of the antibiotin modified GC electrode after exposure to free biotin

for different time (0, 2, 4, 8, 10, 15, 20 min from the inner to the outer scan).

264


The variations in relative current versus the exposure time to free biotin with different

concentrations are shown in Figure 8.15. It was found that more antibiotin dissociated

from the biotinylated interface when exposure to free biotin with higher concentration

for a longer time. It took about 1 h to dissociate 50 antibiotin from the surfaces with

exposure to 3 µg mL-1 free biotin. The experimental results also show only 60 of

antibiotin can be dissociated from the interface upon exposure to free biotin

(0.3 mg mL-1) for 30 min for this fabricated immunosensor system. The incomplete

desorption of antibiotin upon exposure to free biotin may be due to the high affinity

constant between biotin and antibiotin on the immunosensor interface. Figure 8.15 also

shows that, after the system was exposed to the PBS background solution, only a

modest increase in current density was observed possibly due to displacement of weakly

bound anti-biotin from the surface.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80 100 120 140

Time /min

Rela

tive c

urr

ent

0 ng/mL

30 ng/mL

300 ng/mL

3 µg/mL

0.3 mg/mL

30 µg/mL

Figure 8.15 Relative current of antibiotinylated GC surfaces after exposure to free

biotin with different concentrations for different time. Relative current is obtained by

dividing the current before exposure of antibiotin with the current after exposure of free

biotin.

265


The calibration curve for the detection of free biotin is shown in Figure 8.16. This

immunosensor allows for detecting free biotin with the concentration between

300 ng mL-1 and 0.3 mg mL-1 with the detection limit of 30 ng mL-1. Based on this

calibration curve, the affinity constant between free biotin and antibiotin was calculated

to be ca. 105 M-1, which is much lower than the affinity constant between biotin and

antibiotin (109 M-1) obtained in section 8.3.5.

0

0.1

0.2

0.3

0.4

0.5

0.6

-1 0 1 2 3 4 5 6 7

Log10([Biotin] /ppb)

Rela

tive c

urr

ent

Electrode 1

Electrode 2

Figure 8.16 Calibration plot of relative current against the logarithm of the free biotin

concentration. The meaning of relative current is the same as that in Figure 8.15.

8.3.7 Electrochemical Stimulation of Antibiotin Dissociation from the Immunosensor

Interface

Results show that it takes a long time to dissociate the antibiotin from the surface, so it

is important to find a way to accelerate the association speed for the displacement assay.

The treatment of antigen-antibody interfaces at high ionic strength, extreme temperature

or acidic pH provides a means to dissociate the antigen-antibody complexes and

eventually allows the regeneration of the sensing surface. However, this regeneration

step is also time-consuming, followed by a significant loss of biospecific activity and

gives irreproducible results.63 Another approach of renewable immunosensors consists

266


of using disposable antibody- or antigen-coated magnetic beads and building up in-situ

the immunosensing surface by localising the immunomagnetic beads on the electrode

area with the aid of a magnet.64-66 This technique seems very attractive, although the

construction of magneto-immunosensors requires sufficient experimental skills, and the

immobilisation of magnetic particles with immunologic material requires a relatively

long period of incubation. Studies into the regeneration of antibody surfaces,67 the

inhibition of antibody-antigen binding36, 68 and the denaturing of DNA duplexes69-71 at

the electrode surfaces all show that a negative electrode potential can cause the

dissociation of an affinity complex involving poly-anionic species. It has also been

demonstrated that application of voltage to SnO2-coated waveguides can affect

desorption of otherwise irreversibly adsorbed proteins.72 Based on these demonstrations

and on the long history of manipulating charged analytes using electric field in

numerous analytical methods, it is speculated that an electric field at an

immunoassay/solution interface induced by application of applied voltage to the

underlying immunoassay substrate may influence charged protein/immunosensor

interactions. The next step for this chapter is to investigate the effect of applied voltage

to GC surfaces on antibiotin binding.

Figure 8.17 shows the change in current of the OSWV with exposure time to

0.3 mg mL-1 free biotin after -0.9 V bias was applied to the fabricated immunoassay

system as shown in Scheme 8.1 b. An increase in peak current was observed upon the

addition of free biotin and the peak current was found to increase with the incubation

time to 0.3 mg mL-1 free biotin, which indicates that the displacement of antibiotin from

the monolayer system occurred.

267


-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 0.1 0.2 0.3 0.4 0.5

Potential /V

Curr

ent

/A

Increasing

incubation time

Figure 8.17 OSWV of the antibiotinylated GC electrodes after application of -0.9 V bias

for 10 min with exposure to free biotin for different time (0, 2, 4, 8, 10, 15, 20 min from

the inner to the outer scan).

The variations in relative current versus the exposure time to 0.3 mg mL-1 free biotin

after application of -0.9 V bias are shown in Figure 8.18. However, no current increase

can be observed when -0.9 V bias was applied without exposure to free biotin. The

control indicates the increase in current after application of potential to the

immunosensor system is caused by the dissociation of antibiotin with the affinity force

from free biotin. It was found the increase of the redox peaks proceeds for 15 min

before reaching a stable peak currents, indicating the desorption of antibiotin upon

exposure to 0.3 mg mL-1 free biotin takes at least 15 min to complete when -0.9 V bias

was applied with exposure to free biotin. This result suggests application of applied

voltage to this fabricated immunosensor system can hasten the dissociation of antibiotin

from the interface.

268


0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140

Time /min

Rela

tive c

urr

ent

-0.9 V applied w ith free biotin

-0.9 V applied w ithout free biotin

Figure 8.18 Relative current of antibiotinylated GC surfaces after application of –0.9 V

bias with exposure of 0.3 mg mL-1 free biotin for different time. The meaning of relative

current is the same as that in Figure 8.15.

8.4 Conclusions

A novel strategy for electrochemically sensing biotin-antibiotin complex formation at

mixed monolayers modified GC electrode surfaces has been developed. The electrodes

were derivatised in a step-wise process. This method includes the assembly of

ferrocenedimethylamine on the mixed monolayers of MW and PEG by amide bond

coupling, which provides an interface effectively resisting the nonspecific adsorption of

protein. The biotin can be further attached to the ferrocene with terminal amine

functional groups. Based on the biotin-antibiotin interaction, this fabricated label-free

immunosensor can be used to detect antibiotin at the concentration between 30 and

500 ng mL-1. The detection limitation is 5 ng mL-1. In addition, the displacement assay

has shown that the free biotin can compete with the attached biotin for binding

antibiotin and the application of applied potential can increase the speed for dissociating

269


the antibiotin from the biotinylated GC surfaces. Thus, this fabricated novel labe-free

immunosensor system can be used for the detection of analytes.

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274

Chapter 9-Towards Fabrication of Immunosensors Using SWNTs as the Conduit for Electron Transfer

Chapter Nine

Towards the Fabrication of Immunosensors Using SWNTs as

the Conduit for Electron Transfer

275


9.1 Introduction

Molecular wires (MW) such as oligo(phenyl-ethynyl)1-4 and norbornylogous5, 6 bridges

have attractive advantages in that they are rigid, give well-defined molecular

architectures and efficient electron transfer. The rigidity allows the molecular wires to

stand free in space above a surface. They do, however, suffer the disadvantage of being

instable under air and difficult to synthesise in large quantities. Thus it is crucial to find

an alternative to these molecular wires. Since their discovery,7-9 carbon nanotubes have

attracted increasing attention due to their unique structural,10 mechanical11 and

electronic12 properties. Carbon nanotubes being small, rigid and simple to produce in

large quantities, and having functional groups at both ends of the tube after being cut

into different lengths, can be metallic13-15 or semi-conducting. Their small size and

conductivity means they can be regarded as the smallest possible electrodes with

diameters of less than one nanometer.16 SWNTs are known to have a number of

carboxylic acid groups at each end of the tubes, which are important for further

fabrication, after being cut in a 3:1 v/v mixture of concentrated H2SO4/HNO3. The

results based on transmission electron microscopy demonstrated that a longer cutting

time results in shorter tubes and most SWNTs possess lengths between 100 and 150 nm

after 4 h of cutting.17

The research in SWNTs is very active due to the good electrochemical properties owned

by SWNTs.17-19 Basic electrochemistry of SWNTs has been studied by a few groups.20-

23 Liu and coworkers24, 25 have successfully immobilised shortened SWNTs on gold

using a surface condensation method. Electrodes modified with carbon nanotubes have

been shown to have outstanding electrochemical properties,26-28 show fast electron

276


transfer properties29 and direct electron transfer to hemoglobin.30 Nanotubes modified

electrodes have been shown to allow direct electron transfer to enzymes with redox

centres close to the enzyme surface.17, 22, 31-33 Thanks to nanotubes being able to

penetrate the protein and get close to the redox centre due to the small size of the tubes

(diameters around 1 nm), Guiseppi-Elie et al.34 achieved the direct electron transfer

between absorbed glucose oxidase (GOx) and an underlying GC electrode. Direct

electron transfer has been observed by Zhao and coworkers35 for GOx adsorbed onto a

carbon nanotube power microelectrode. Gooding and coworkers19 investigated the

electrochemistry of GOx immobilised onto aligned nanotubes electrode arrays formed

by self-assembly with a view to achieving direct electron transfer to this enzyme. Most

protein-nanotube studies described above use electrodes modified with nanotubes

randomly deposited onto an electrode surface. Comparing with the randomly dispersed

nanotubes, the vertical alignment of the cut nanotubes results in electrodes which

possess better electrochemical properties because more of the oxygenated species form

at the ends of nanotubes during acid purification.36 There are many strategies for

aligning the nanotubes vertically from a surface either by growing the tubes vertically

from a surface27, 37, 38 or by self-assembly.17, 19, 21

The studies described above have confirmed that carbon nanotubes have high efficiency

for electron transfer and have great potential for the development of biosensors. The

rigid MW has been successfully used as the conductor for fabrication of immunosensors

in Chapter Eight. This chapter is to study the strategy for modification of SWNTs on

GC electrode surfaces, and to characterise the modified SWNTs on GC substrates using

electrochemistry and AFM. The possibility of replacing MW with SWNTs to fabricate a

label-free immunosensor system (Scheme 9.1) on GC surfaces is then investigated.

277


GC

CO

HN

NH

CO

Fe

H2C

H2C

NHOC

NH

CO

a)

Electrochemistry

Redox Probe

Biotin

SWNTs

GC

CO

HN

NH

CO

Fe

H2C

H2C

NHOC

NH

CO

Redox Probe

Biotin

SWNTs

Antibiotin

b)

Electrochemistry

Scheme 9.1 Schematic of (a) the fabricated label-free immunosensor based on SWNTs,

and (b) the antibiotin targeting to the label-free immunosensor.

Scheme 9.1 a shows the schematic of the label-free immunosensor which was

fabricated in this study, and Scheme 9.1 b shows the schematic of using the fabricated

immunosensor for the determination of antibiotin. As shown in Scheme 9.1 a, GC

electrodes were firstly modified with monolayers of aminophenyl followed by the

covalent attachment of SWNTs. The redox probe ferrocenedimethylamine was

subsequently attached to the terminal carboxylic acid groups on SWNTs by forming an

278


amide bond followed by the attachment of biotin. After incubation this immunosensor

in the antibiotin solution, the antibiotin can be immobilised to the immunosensor

surface as shown in Scheme 9.1 b due to the strong affinity between biomolecular pairs

biotin and antibiotin. Electrochemistry was used to monitor the immobilisation of

antibiotin.






were prepared as described in Section 2.4.6. Ferrocene and SWNTs were covalently

attached on the monolayers according to the procedures described in Chapter Two. All

electrochemical measurements were performed with a BAS-l00B electrochemical

analyser. All potentials were quoted relative to an Ag/AgCl reference at room

temperature. All the cyclic voltammetry measurements for the ferrocene modified

electrodes were carried out in phosphate buffer (0.05 M KCl, 0.05 M K2HPO4 and

adjusted the pH to 7.0 with KH2PO4).

9.2.2 Preparation of the Cut SWNTs

Typically preparation of the cut SWNTs was similar to the procedure of Liu et al.39

10 mg of purified SWNT (from Carbon Nanotechnologies, Inc.) was weighed into a 250

mL flat bottom flask containing 40 mL of a 3:1 v/v solution of concentrated sulfuric

acid (98%) and concentrated nitric acid (70%), and sonicated in a water bath at 35-40

for 4 hours. The resultant suspension was then diluted to 200 mL with water. The cut

279


SWNTs were collected on a 220 nm pore filter membrane (type: Millipore) and washed

with 10 mM NaOH aqueous solution, followed by Milli-Q water until the pH of the

filtrate reached 7.0. The cut SWNTs were dispersed in DMF and formed a very good

suspension without any aid of surfactant.

9.2.3 Fabrication of the Cut SWNTs on the 4-Aminophenyl Modified Glassy Carbon

Electrodes

There are two strategies for modification of 4-aminophenyl modified GC electrodes

with cut SWNTs. The first method is to covalently attach the SWNT to the 4-

aminophenyl modified GC electrodes by dispersing the cut SWNT (0.2 mg) in 1 mL of

dimethylformamide (DMF) with 0.5 mg of dicyclohexyl carbodiimide (DCC). DCC

converts the carboxylic acid group at the end of the shortened SWNTs into an active

carbodiimide ester.25 The 4-aminophenyl modified GC electrode was then placed in the

SWNT solution for 6 hours during which the amine at the terminus of the monolayer on

GC surfaces forms an amide bond with one end of each nanotube. The second method is

to apply droplets of SWNTs (10 µL, 0.1 mg mL-1) dispersed in ethanol onto the 4-

aminophenyl modified GC surface to give a bed of randomly orientated SWNT.


9.3.1 Modification of the Glassy Carbon Electrodes with 4-Nitrophenyl Diazonium

Tetrafluoroborate

GC electrodes can be modified with aryl diazonium salts by electrochemically reductive

adsorption as described in Chapter Three. Scheme 9.2 shows the schematic of

modification GC electrodes with 4-nitrophenyl diazonium tetrafluoroborate. Cyclic

280


voltammograms of a GC electrode in a 1 mM 4-nitrophenyl diazonium

tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution are shown in Figure 9.1.

NO2

GC

NO2

GC GC

e-+ .

NO2

N2+

Scheme 9.2 Schematic of modification of GC electrodes with 4-nitrophenyl diazonium

tetrafluoroborate.

-25

-20

-15

-10

-5

0

5

-2 -1.5 -1 -0.5 0 0.5 1

Potential /V

Cur

rent

/A

The f irst cycle

The second cycle

The tenth cycle

Figure 9.1 Cyclic voltammograms of a bare GC electrode in a 1 mM 4-nitrophenyl

diazonium tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution at a scan rate of

100 mV s-1.

It can be seen in Figure 9.1 that the first scan gave an irreversible wave located at +0.04

V, which was attributed to the formation of the 4-nitrophenyl radical from the aryl

diazonium salts.40 The first wave is followed by the reversible cathodic wave located at

281


-1.2 V corresponding to the reduction of the nitro group to a radical anion.41 The

oxidation wave observed on the return scan is attributed to the oxidation of the radical

anion. On a second scan the first wave at -0.04 V vanished, which indicates an

inhibition of the electron transfer by the 4-nitrophenyl group grafted at the GC surfaces,

and the second wave at -1.2 V still existed and can sustain upon multiple cycles due to

the redox reaction of nitro groups. This observation is consistent with the literature.42, 43

The 4-nitrophenyl modified GC electrodes in a 1mM 4-nitrophenyl diazonium

tetrafluoroborate, acetonitrile/0.1 M NBu4BF4 solution were successively transferred to

a pure supporting electrolyte solution (acetonitrile/0.1 M NBu4BF4), and a single signal

is observed at -1.2 V as that for modification of 4-nitrophenyl groups in Figure 9.1. The

surface coverage of the attached 4-nitrophenyl group was evaluated by integration of the

area under the reductive adsorption peak locating at -0.04 V in the cyclic

voltammogram in Figure 9.1, and a value of (9.0 0.3)×10-10 (n 6) mol cm-2 was found.

This value is in good agreement with previously reported values and can be considered

to be in the monolayer or submonolayer level.44

The blocking properties of the 4-nitrophenyl modified GC electrodes were investigated

in solutions of redox probes such as Fe(CN)63-, Ru(NH3)6

3+ and ferrocene. Figure 9.2

shows the cyclic voltammograms of GC electrodes before and after modification of 4-

nitrophenyl in different redox probe solutions. Figure 9.2 a shows that the

electrochemical response of ferricyanide is completely blocked when a GC electrode is

modified with the 4-nitrophenyl groups, and the reversibility of the Ru(NH3)63+ redox

couple is almost completely suppressed at the 4-nitrophenyl modified GC electrodes

(Figure 9.2 b), which is consistent with the literature.43

282


-20

-10

0

10

20

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6

Potential /V

Cur

rent

/A

4-Nitrophenyl modified GC

a)

Bare GC

-130

-100

-70

-40

-10

20

50

80

00.20.40.60.8

Potential /V

Cur

rent

/A


c)Bare GC

-6

-4

-2

0

2

4

6

8

-0.4-0.3-0.2-0.100.1

Potential /V

Cur

rent

/A

b)


Bare GC

Figure 9.2 Cyclic voltammograms of bare and 4-nitrophenyl modified GC electrodes in

(a) Fe(CN)63- and (b) Ru(NH3)6

3+ (1 mM; 0.05 M KCl; 0.05 M phosphate buffer; pH

7.0) and (c) in ferrocene (1 mM; 0.1 M NBu4BF4; CH3CN) at a scan rate of 100 mV s-1.

283


The suppression of the electrochemical response for hydrophilic species such as

Fe(CN)63- and Ru(NH3)6

3+ at the 4-nitrophenyl modified GC electrode may be due to

the hydrophobicity of the film. It is reported that the electrochemical reduction of

nitroaromatic compounds leads to the formation of phenylhydroxylamine and aniline

depending on the experimental conditions.45 The reduction of nitro to amine can also

occur for the electrochemically generated nitrophenyl layers.42, 44 Thus, although the 4-

nitrophenyl groups are expected to undergo some chemical modification upon cycling

on aqueous media, the results here suggest that the resulting film still retains a

significant hydrophobic character. The blocking properties of the 4-nitrophenyl groups

on GC electrodes were also evaluated in non-aqueous electrolyte. Figure 9.2 c shows

that the electrochemical response of ferrocene is completely blocked at the 4-

nitrophenyl modified GC electrodes. This agrees with the presence of a compact and

dense 4-nitrophenyl monolayers on GC electrode surfaces blocking the access of

ferrocene molecules.

9.3.2 Conversion of the 4-Nitrophenyl Groups on Glassy Carbon Electrode Surfaces

into 4-Aminophenyl Groups

The electrochemistry of nitroaromatic compounds has been widely studied in the past,

and the functionalised aromatic group could be modified by classical chemical reactions

once they are attached to the carbon surfaces.45 So the obtained 4-nitrophenyl groups on

GC electrodes could be reduced electrochemically to the 4-aminophenyl groups.

Scheme 9.3 shows the process for transformation of NO2 groups into NH2 groups. The

electrode is first derivatised by 4-nitrophenyl radicals as described earlier, thus giving

rise to the reversible cyclic voltammetric pattern of the grafted 4-nitrophenyl groups in a

CH3CN/0.1 M NBu4NBF4 solution. It is then transferred to a protic solution (90:10 v/v

284


H2O-EtOH+0.1 M KCl). The cyclic voltammetric wave then becomes irreversible and

the peak height increases by a factor of ca. 6 (Figure 9.3), suggesting that the classical

reduction of NO2 to NH2 occurs within the grafted layer.40 The peak completely

disappeared in the second cycle, indicating that all of the electrochemically access into

nitro groups has been converted to amine groups. The surface coverage of 4-

aminophenyl group is calculated to be (6.3 0.4)×10-10 (n 6) mol cm-2 by integration of

the reduction peak in Figure 9.3, which means only 70 NO2 groups were

electrochemically accessible and were converted into NH2 groups, which is consistent

with the argument that the conversion of NO2 into NH2 is not complete in the

literature.42

GC NO2 + 6e- +6H+ 2H2O+ GC NH2

Scheme 9.3 Schematic of reduction of 4-nitrophenyl groups to the 4-aminophenyl

groups on GC substrates.

-120

-100

-80

-60

-40

-20

0

20

-1.6 -1.2 -0.8 -0.4 0

Potential /V

Cur

rent

/A

1st cycle

2nd cycle

Figure 9.3 Cyclic voltammograms of a GC electrode derivatised by 4-nitrophenyl

radicals in 90:10 H2O-EtOH+0.1 M KCl with a scan rate of 100 mV s-1.

285


The blocking behaviour of the 4-aminophenyl modified GC surfaces was investigated in

the redox probe solution (1 mM ferricyanide in 0.05 M pH 7.0 phosphate buffer with

0.05 M KCl) as shown in Figure 9.4. The redox peaks of ferrocyanide observed with a

bare GC electrode disappeared after the modification of 4-aminophenyl, indicating the

formed 4-aminophenyl monolayers can block the access of the

ferricyanide/ferrocyanide species to GC electrodes.

-30

-20

-10

0

10

20

30

-0.2 0 0.2 0.4 0.6

Potential /V

Cur

rent

/A

4-Aminophenyl modified GCBare GC

Figure 9.4 Cyclic voltammograms of bare and 4-aminophenyl modified GC electrodes

in ferricyanide solution at the scan rate of 100 mV s-1.

9.3.3 Attachment of Ferrocenecarboxylic Acids on 4-Aminophenyl Modified Glassy

Carbon Electrodes

The 4-aminophenyl modified GC electrodes can be covalently attached with

ferrocenecarboxylic acid by forming the amide bond as shown in Scheme 9.4. This was

achieved by dipping the modified GC electrodes in the N-(2-Hydroxyethyl)piperazine-

N'-(2-ethanesulfonic acid) (HEPES) buffer solution (pH 7.3) containing 1 mM

ferrocenecarboxylic acid, 4 mM N-hydroxysuccinimide (NHS) and 20 mM 1-ethyl-3-

286


(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) for 24 h with stirring in

room temperature.

Fe

NO2

GCAryl diazonium salt

NH2

GC GC90:10 H2O-EtOH

NH

GCFc-COOH

C=O

EDC/NHS

Scheme 9.4 Schematic of ferrocenecarboxylic acid attached covalently on the 4-

aminophenyl modified GC surfaces.

Cyclic voltammograms measured in an aqueous solution of 0.05 M phosphate buffer

(0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1 before and after the modification of

ferrocenecarboxylic acid on the 4-aminophenyl modified GC electrode with the

EDC/NHS activation are shown in Figure 9.5 a. The appearance of the reversible redox

peaks with the formal potential (Eo`) of 321 mV indicates that ferrocenecarboxylic acid

has been attached to the electrode surface. The strong redox peaks after the attachment

of ferrocene showed linear variation in peak current with scan rates, indicating that the

ferrocene was surface bound. In the absence of EDC and NHS such that no covalent

coupling of the ferrocene could occur, only very weak and broad redox peaks due to

physisorption were observed (Figure 9.5 b). The control shows that the 4-aminophenyl

modified GC electrodes has been covalently attached with ferrocenecarboxylic acid

moieties.

287


-6

-4

-2

0

2

4

6

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Cur

rent

/A

After modification of ferroceneBefore modification of ferrocene

b)

-2

-1

0

1

2

3

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Cur

rent

/A

a)After modification of ferrocene

Before modification of ferrocene

Figure 9.5 Cyclic voltammograms of a 4-aminophenyl modified GC electrode before

and after modification of ferrocenecarboxylic acid (a) with EDC/NHS activation and

(b) without EDC/NHS activation at a scan rate of 100 mV s-1.

With a monolayer containing only 4-aminophenyl moieties the number of redox active

molecules attached to the surface, as determined from the charge passed under the

Faradaic peaks in the ferrocene modified electrode, is approximately (6.1 0.3)×10-11

(n 5) mol cm-2 with a close to unity ratio of anodic to cathodic peak areas. Comparing

the surface coverage of 4-aminophenyl groups of (6.3 0.4)×10-10 (n 6) mol cm-2, to that

of the number of redox centres attached indicates that only approximately 10 of the 4-

288


aminophenyl monolayers had a ferrocene attached. At this surface coverage the average

area per ferrocene molecule, assuming homogeneous distribution, is 2.7 nm2 which

suggests there is a high possibility of interaction between redox active centres.46, 47

The rate constant of electron transfer for ferrocenecarboxylic acid attached on GC

electrodes modified with 4-aminophenyl was calculated to be 7.9 s-1 using the method

of Laviron,48 which relies on the change in peak potential separation ( Ep) with scan

rate ( ) to obtain the appreciate rate constant of electron transfer (kET). This value is

similar to that studied in Chapter Four for ferrocenemethylamine attached to 4-

carboxyphenyl modified GC electrode surfaces (kET 12 s-1).49

9.3.4 Characterisation of the SWNT Modified Glassy Carbon Surfaces by AFM

The cut SWNTs with the carboxylic acid groups on the end of tubes also can be

covalently attached to the 4-aminophenyl modified GC substrates based on the

procedures described in the experimental section. The modified GC surfaces were

imaged by AFM as shown in Figure 9.6. Comparing with the AFM image of the 4-

aminophenyl modified GC substrates (Figure 9.6 a), the surface of the GC substrates

after modification of SWNT was much rougher as illustrated in Figure 9.6 b indicating

the shortened SWNT aligned normal to the electrode surface. The AFM image also

shows that aligned tubes assemble on the surface not as individual tubes but as bundles,

which is consistent with the literature.17 The assembly of the tubes as bundles is

attributed to the strong hydrophobic attractions between the tubes.24 The observed

lengths of the SWNTs from the AFM image are apparently shorter than those observed

using the TEM image.36 This might be due to the broadening effect of the AFM tip.25

Further evidence that these images represent aligned bundles of nanotubes is that with

289


longer incubation times the surface density of features increases, the same phenomenon

as observed by Liu and coworkers.24

a) b)

Figure 9.6 AFM image of (a) the 4-aminophenyl modified GC plate and (b) self-

assembled SWNTs on the 4-aminophenyl modified GC plates after 24 hours incubation

in SWNTs solution.

9.3.5 Covalent Attachment of Ferrocenemethylamine to SWNT Modified Glassy

Carbon Electrodes

Base on the procedures described in the experimental section, 4-aminophenyl modified

GC electrodes can be attached with aligning and lying down SWNTs. The

ferrocenemethylamine can be further attached to the free ends of the SWNTs by

incubating the standing and lying down SWNT modified electrodes separately in the

1 mM ferrocenemethylamine in N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesulfonic

acid) (HEPES) buffer solution (pH 7.3) containing 10 mM NHS and 40 mM EDC at

room temperature for 6 h. The schematic of ferrocenemethylamine covalently attached

to a GC electrode modified with standing SWNT or a GC electrode modified with lying

down SWNT is shown in Scheme 9.5 a and Scheme 9.5 b, respectively.

290


COOH

C ONH

C ONH

NO2

90:10H2O-EtOH+0.1 M KCl

NH2

GCGCAryl diazonium salt

GC GC

NHO

Fe

Fc-CH2NH2

SWNTs

NO2

90:10H2O-EtOH+0.1 M KCl

NH2

GCAryl diazonium salt Drop down SWNTs

Fc-CH2NH2GC GC GC

NHFe

Fe

CO

NH

CO

NH

CO

OH

CO

HO

a)

b)

Scheme 9.5 Schematic of ferrocenemethylamine attached onto (a) standing SWNT/4-

aminophenyl modified GC electrodes and (b) lying down SWNT/4-aminophenyl

modified GC electrodes.

The cyclic voltammograms measured in an aqueous solution of 0.05 M phosphate

buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1 before and after the

modification of ferrocenemethylamine on the standing and lying down SWNT modified

GC electrodes are shown in Figure 9.7. The strong redox peaks after the attachment of

ferrocene in Figure 9.7 a showed linear variation in peak current with scan rates,

indicating that the ferrocene was surface bound. In the absence of EDC and NHS, such

that no covalent coupling of the ferrocene could occur, only very weak redox peaks due

to physisorption were observed. The cyclic voltammograms of the ferrocene coupled to

291


the SWNT modified GC surfaces show non-ideal behaviour50 with regards to peak

separation at slow scan rates ( Ep 179 mV rather than the ideal Ep 0 mV) and the full

width half maximum (EFWHM 340 mV rather than the ideal EFWHM 90.6 mV/n where

in this case n 1). With regards to both peak separation and the EFWHM the non-ideal

behaviour has been attributed to the ferrocene molecules being located in a range of

environments with a range of formal electrode potentials (Eo`).51, 52 The surface

coverage for ferrocene was calculated to be 2.19 ×10-11 mol cm-2 by integration the area

under the redox peaks in the cyclic voltammogram.

However when the SWNTs were dropped on the 4-aminophenyl modified GC

electrodes, only a pair of weak redox peaks was observed (Figure 9.7 b). The surface

coverage for ferrocene is ca. 1.04×10-11 mol cm-2, which is half of that for ferrocene

attached on the SWNTs which were covalently attached to the GC electrodes in Figure

9.7 a. This result indicates that the SWNTs stand on the 4-aminophenyl modified GC

electrodes when the covalent coupling method is adopted, which is consistent with the

AFM images. Because more SWNTs can be attached to electrodes when they are

covalently modified on telectrodes than that when they are dropped down the electrode

surfaces, and more ferrocenemethylamine can further attached, which contributes to the

larger redox response when the covalent coupling strategy is adopted. The rate constant

of electron transfer was calculated to be 3.8 0.27 s-1 (n 5) using the Laviron’s method48

when the SWNTs were covalently attached onto the 4-aminophenyl modified GC

electrode surfaces. However, with electrodes modified with randomly dispersed

SWNTs, the rate constant of electron transfer was calculated to be 1.3 0.25 s-1 (n 5).

The heterogeneous rate constant for electron transfer has been proposed to be

significantly more rapid from the ends of nanotubes than the walls,17, 53 which is

292


consistent with the edge planes and basal planes of graphite.54 With electrodes modified

with randomly dispersed SWNTs, Compton and coworkers18 have shown that the

electrochemistry is dominated by the ends of tubes. Therefore, it is proposed with the

aligned SWNTs there is a greater presentation of the ends per unit electrode area and

higher rate of electron transfer was obtained correspondingly compared with the

randomly dispersed SWNTs on GC electrodes, which suggests aligned SWNTs have a

potential alternative to MW towards fabricating an immunosensor system.

-3

-2

-1

0

1

2

3

4

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Cur

rent

/A

After modification of ferrocene

Before modificationof ferrocene

b)

-4

-2

0

2

4

6

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Cur

rent

/A

After modification of ferrocenea)

Before modificationof ferrocene

Figure 9.7 Cyclic voltammograms of (a) the standing SWNTs/4-aminophenyl modified

GC electrode and (b) the lying down SWNTs/4-aminophenyl modified GC electrode

before and after modification of ferrocenemethylamine at a scan rate of 100 mV s-1.

293


9.3.6 Fabrication of a Label-Free Immunosensor on SWNTs Modified Glassy

Carbon Substrates

The fabrication of a label-free immunosensor as shown in Scheme 9.1 a is carried out

by stepwise. After the 4-aminophenyl modified GC electrodes were covalently modified

with the cut SWNTs, ferrocenedimethylamine was covalently attached to the open ends

of SWNTs by incubating the SWNT modified GC substrates in an absolute ethanol

solution containing of 40 mM 1,3-Dicyclohexylcarbodiimide (DCC) and 5 mM

ferrocenedimethylamine for 6 h at room temperature. DCC was used for the activation

of terminated carboxylic acid group of SWNTs.17 Cyclic voltammograms measured in

an aqueous solution of 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate of

100 mV s-1 before and after the attachment of ferrocenedimethylamine onto the

SWNTs/4-aminophenyl modified GC electrode are shown in Figure 9.8. The obvious

redox peaks with the formal potential of 349 mV gave strong evidence that ferrocene

was attached to the electrode. The current of the redox peaks show the linear

relationship with the scan rates, indicating that ferrocene was covalently attached on the

SWNTs modified GC surfaces and resulted in a surface bound process. In the absence

of DCC such that no covalent coupling of the ferrocene could occur, only very weak

redox peaks due to physisorption were observed. The cyclic voltammograms of the

ferrocene coupled to SWNTs modified GC surfaces show non-ideal behaviour50 with

regards to peak separation at slow scan rates ( Ep 90 mV rather than the ideal Ep 0

mV) and the full width half maximum (EFWHM 207 mV rather than the ideal

EFWHM 90.6 mV/n where in this case n 1). With regards to both peak separation and

the EFWHM the non-ideal behaviour has been attributed to the ferrocene molecules being

located in a range of environments with a range of formal electrode potentials (Eo`).51, 52

294


-2

-1

0

1

2

3

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Potential /V

Cur

rent

/A

After modification of ferrocene

Before modification of ferrocene

Figure 9.8 Cyclic voltammograms of SWNTs/4-aminophenyl modified GC electrodes

before and after the covalent attachment of ferrocenedimethylamine in 0.05 M


After modification of ferrocenedimethylamine, the GC substrates covered with amine

terminal groups were immersed into 1 mg mL-1 solution of NHS-biotin in 0.1 M

phosphate buffered saline (0.15 m NaCl, pH 7.3) for 2 h at 4 oC to attach a biotin to the

ferrocene modified GC surfaces as shown in Scheme 9.1 a. Then the biotinylated

electrode surfaces were rinsed with copious amount of water and phosphate buffered

saline (PBS) solution followed by immersion into a solution containing 0.5 M

antibiotin dissolved in PBS for 30 min at 4 oC. The biotinylnated GC electrode surfaces

can be immobilised with the antibiotin as shown in Scheme 9.1 b by the strong affinity

between the biotin and antibiotin. Finally, electrochemistry was used to monitor the

attachment of each component.

Figure 9.9 shows the OSW voltammograms of ferrocene/SWNT modified GC surfaces

before and after incubation of biomolecular pairs biotin and antibiotin. After the

attachment of biotin, the current from the OSWV of modified GC surfaces did not show

295


significant change. However, the current from the OSWV of biotinylated GC surfaces

demonstrated pronounced reductions in peak currents after incubation with antibiotin.

These results are consistent with those reported in the literatures55-57 and the studies in

Chapter Eight. Decreased current density upon antibiotin binding is supposed to form an

immunocomplex on electrode surface58 and reflects changes in the interfacial

microenvironment.59 The incubated antibiotin covered the modified GC surface

blocking the ions to access the redox probe and decrease the current correspondingly.

-1.3

-1.2

-1.1

-1

-0.90 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A




Figure 9.9 OSWV of the SWNT/4-aminophenyl modified GC surfaces after the step-wise

attachment of ferrocenedimethylamine, biotin and antibiotin in 0.05 M phosphate buffer

(0.05 M KCl, pH 7.0) at a scan rate of 100 mV s-1.

Controls showed almost no change in peak current when the ferrocene/SWNT modified

GC electrodes were incubated in PBS solution without NHS-biotin followed by the

immobilisation of antibiotin (Figure 9.10), indicating that the decrease of current in

Figure 9.9 is attributed to the biomolecular recognition between biotin and antibiotin.

In addition, almost no decrease in current was observed upon treatment of the

biotinylated GC electrode with other proteins which are not specific to biotin, such as

296


BSA and anti-pig IgG, but not antibiotin under identical conditions. These results

indicate that the antibiotin specifically associates with the biotin sites in the monolayer

assembly and only the biospecific interaction can lead to the change of

electrochemistry. Based on the change of electrochemistry after immobilisation of

antibiotin, this SWNTs fabricated system in Scheme 9.1 a can be used as an

immunosensor system for detecting the antibiotin.

-5

-4.5

-4

-3.50 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A



Figure 9.10 OSWV of the SWNT modified GC electrodes after the stepwise attachment

of ferrocenedimethylamine and antibiotin in 0.05 M phosphate buffer (0.05 M KCl, pH

7.0) at a scan rate of 100 mV s-1.

Figure 9.11 is the calibration curve for the detection of antibiotin with the detection

limit of 10 ng mL-1. This fabricated immunosensor system based on SWNTs can be

used for detecting of antibiotin with the concentration between 50 and 500 ng mL-1.

Based on this calibration curve, the affinity constant between biotin and antibiotin is

calculated to be ca. 108 M-1, which is quite similar to the affinity constant between

biotin and antibiotin obtained in Chapter Eight (109 M-1), and belongs to the typical

affinity constant for an antigen-antibody reaction (from the order of 108 to 1012 M-1).60

297


0

0.2

0.4

0.6

0.8

1

0.5 1 1.5 2 2.5 3

Log[Ab /ng mL-1]

Rel

ativ

e cu

rrent

Electrode 1

Electrode 2

Figure 9.11 Calibration plot of relative current against the logarithm of the antibiotin

concentration. Relative current is obtained by dividing the current before the incubation

of antibiotin with the current after the incubation of antibiotin.

9.4 Conclusions

In summary, this chapter has shown that the 4-aminophenyl modified GC electrode

surfaces can be further modified with chemically cut SWNTs by the strategy of

covalently alignment or randomly dispersion. The electron transfer through SWNTs was

studied by using ferrocenemethylamine as the redox probe. It was found that the rate of

electron transfer for the aligned SWNTs was higher than that for the randomly dispered

SWNTs on GC electrodes because for the aligned SWNTs there is a greater presentation

of the ends per unit electrode area and higher rate of electron transfer was obtained

correspondingly compared with the randomly dispersed SWNTs on GC electrodes. The

aligned SWNTs can act as a good alternative to molecular wires to allow

electrochemical communication between the underlying electrode and biomolecular

pairs biotin and antibiotin attached to the ends of the SWNTs. Another novel

immunosensor system can be fabricated onto GC surfaces by using SWNTs as the

298


conductor. This fabricated immunosensor can be used for the detection of antibiotin

with the detection limit of 10 ng mL-1.

9.5 References



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Yao, Y.X., Rawlett, A.M., Tour, J.M., Bard, A.J., J. Am. Chem. Soc. 2002, 124,

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(5) Paddon-Row, M.N., Aust. J. Chem. 2003, 56, 729-748.

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(8) Iijima, S., Ichihashi, T., Nature 1993, 363, 603-605.

(9) Bethune, D.S., Kiang, C.H., De Vries, M.S., Gorman, G., Savoy, R., Vazquez,

J., Beyers, R., Nature 1993, 363, 605-607.

(10) Ajayan, P.M., Chem. Rev. 1999, 99, 1787-1793.

(11) Salvetat-Delmotte, J.P., Rubio, A., Carbon 2002, 40, 1729-1731.

(12) Colbert, D.T., Smalley, R.E., Trends Biotechnol. 1999, 17, 46-48.

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(13) Tang, Z.K., Zhang, L.Y., Wang, N., Zhang, X.X., Wen, G.H., Li, G.D., Wang,

J.N., Chan, C.T., Sheng, P., Science 2001, 292, 2462-2465.

(14) Ouyang, M., Huang, J.L., Cheung, C.L., Lieber, C.M., Science 2001, 292, 702-

705.

(15) Tans, S.J., Devoret, M.H., Dai, H.J., Thess, A., Smalley, R.E., Geerligs, L.J.,

Dekker, C., Nature 1997, 386, 474-477.

(16) Zhao, Q., Gan, Z.H., Zhuang, Q.K., Electroanalysis 2002, 14, 1609-1613.



(18) Banks, C.E., Moore, R.R., Davies, T.J., Compton, R.G., Chem. Commun. 2004,

1804-1805.



(20) Luo, H.X., Shi, Z.J., Li, N.Q., Gu, Z.N., Zhuang, Q.K., Anal. Chem. 2001, 73,

915-920.

(21) Diao, P., Liu, Z.F., Wu, B., Nan, X.L., Zhang, J., Wei, Z., Chemphyschem 2002,

3, 898-901.

(22) Wang, J.X., Li, M.X., Shi, Z.J., Li, N.Q., Gu, Z.N., Anal. Chem. 2002, 74, 1993-

1997.

(23) Barisci, J.N., Wallace, G.G., Baughman, R.H., J. Electrochem. Soc. 2000, 147,

4580-4583.

(24) Liu, Z.F., Shen, Z.Y., Zhu, T., Hou, S.F., Ying, L.Z., Shi, Z.J., Gu, Z.N.,

Langmuir 2000, 16, 3569-3573.

(25) Nan, X.L., Gu, Z.N., Liu, Z.F., J. Colloid. Interf. Sci. 2002, 245, 311-318.

(26) Wang, J., Musameh, M., Lin, Y.H., J. Am. Chem. Soc. 2003, 125, 2408-2409.

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(27) Gao, M., Dai, L.M., Wallace, G.G., Electroanalysis 2003, 15, 1089-1094.

(28) Valentini, F., Amine, A., Orlanducci, S., Terranova, M.L., Palleschi, G., Anal.

Chem. 2003, 75, 5413-5421.

(29) Nugent, J.M., Santhanam, K.S.V., Rubio, A., Ajayan, P.M., Nano Lett. 2001, 1,

87-91.

(30) Cai, C.X., Chen, J., Anal. Biochem. 2004, 325, 285-292.

(31) Yamamoto, K., Shi, G., Zhou, T., Xu, F., Xu, J., Kato, T., Jin, J., Jin, L., Analyst

2003, 128, 249-254.

(32) Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., Li, S.F.Y., Sens. Actuators B

2002, 87, 168-172.

(33) Yu, X., Chattopadhyay, D., Galeska, I., Papadimitrakopoulos, F., Rusling, J.F.,

Electrochem. Commun. 2003, 5, 408-411.

(34) Guiseppi-Elie, A., Lei, C.H., Baughman, R.H., Nanotechnology 2002, 13, 559-

564.

(35) Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., Anal. Sci. 2002, 18, 939-941.

(36) Chou, A., Bocking, T., Singh, N.K., Gooding, J.J., Chem. Commun. 2005, 842-

844.

(37) Gao, M., Huang, S.M., Dai, L.M., Wallace, G., Gao, R.P., Wang, Z.L., Angew.

Chem. Int. Edit. 2000, 39, 3664-3667.

(38) Koehne, J., Li, J., Cassell, A.M., Chen, H., Ye, Q., Ng, H.T., Han, J.,

Meyyappan, M., J. Mater. Chem. 2004, 14, 676-684.

(39) Liu, J., Rinzler, A.G., Dai, H.J., Hafner, J.H., Bradley, R.K., Boul, P.J., Lu, A.,

Iverson, T., Shelimov, K., Huffman, C.B., Rodriguez-Macias, F., Shon, Y.S.,

Lee, T.R., Colbert, D.T., Smalley, R.E., Science 1998, 280, 1253-1256.

301


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Carbon 1997, 35, 801-807.


5883-5884.




6813.


(45) Cyr, A., Huot, P., Belot, G., Lessard, J., Electrochim. Acta 1990, 35, 147-152.





136-146.




1797-1806.

(53) Li, J., Cassell, A.M., Delzeit, L., Han, J., Meyyappan, M., J. Phys. Chem. B

2002, 106, 9299-9305.


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303

Chapter 10-Conclusions and Future Directions

Chapter Ten

Conclusions and Future Directions

304


10.1 Introduction

This thesis has reported the study of creating more stable and versatile self-assembled

monolayer systems on glassy carbon (GC) substrates relative to gold, by

electrochemically reductive adsorption of aryl diazonium salts for sensors and other

applications. The rate constants of electron transfer through aromatic molecules,

synthesised molecular wires (MW) and single wall carbon nanotubes (SWNTs) on GC

electrodes have been studied. An electrochemical sensor for the detection of copper ions

has been successfully developed on the tripeptide Gly-Gly-His modified GC and

pyrolised photoresist film (PPF) electrode surfaces. The rigid and conjugated MWs can

be used as the efficient conduit for electron transfer to fabricate the mediatorless

glucose biosensor. Moreover, a novel immunosensor system fabricated on an

electrochemical interface comprising mixed monolayers of MW and poly(ethylene

glycol) (PEG) can be used to detect the biomolecular pairs such as biotin and antibiotin.

SWNTs can serve as an alternative to MW for the fabrication of immunosensors.

Despite the fact that summaries and conclusions have been given in each chapter, it is

worthwhile to give a broader summary relating to the whole project followed by the

discussion of the future directions emanating from the current research. It is important

to emphasise that the detailed mechanism of electron transfer, particularly in biological

systems, is far from clear, furthermore the approach towards the fabrication of third

generation glucose biosensors using a conduit for electron transfer, and the development

of novel immunosensors suitable to the detection of analytes are just at the beginning of

their research life.

305


10.2 Brief Summary

The results in Chapter Three have demonstrated that GC, PPF and gold electrodes can

be successfully modified with pure and mixed aryl diazonium salts using the two-cycle

cyclic voltammetry method to form stable and covalently bonded monolayers. The rates

of heterogeneous electron transfer on GC, PPF and gold surfaces have been studied

using ferrocene as the redox probe in Chapter Four. It was concluded the rate of electron

transfer is an order of magnitude higher for gold electrodes in comparison to carbon

electrodes due to the mixing of delocalised electrons between the electrode material and

the monolayer occurring to a greater extent for gold than for GC. However, the higher

stability of monolayer created on GC surface serves as a good alternative for the

development of electrochemical sensors as described in Chapter Five. The tripeptide

Gly-Gly-His modified GC surfaces being characterised by XPS techniques have been

successfully used for the detection of copper ions in solutions. The detection limit could

reach 0.32 ppb using both GC electrodes and PPF substrates. The surface coverage of

complexed copper ions has been systematically investigated by attachment of Gly-Gly-

His to a series of mixed monolayers of 4-carboxyphenyl and phenyl on GC surfaces.

The tripeptide Gly-Gly-His modified GC electrode for the detection of copper was

found to be very stable. After one-month storage and frequent usage, the measured

sensitivity did not show much decay. PPF has proved to be a good alternative to the GC

electrode for the commercialisation of the fabricated electrochemical sensors. The

strategy for fabrication of the copper sensors can serve as a good example for the

fabrication of other electrochemical sensors built with other oligopeptides for the

analysis of other metal ions and also supplies an effective tool for medical diagnosis.

306


The rigid and conjugated MW as the efficient conduit for electron transfer, and a PEG

molecule as an insulator for reducing the non-specific protein adsorption were

successfully synthesised. The ability of the interface comprising mixed monolayers of

MW and PEG to facilitate efficient electron transfer is demonstrated in Chapter Six

using ferrocenemethylamine attached to the end of MW whilst the protein resistance of

the interface is studied using protein modified gold nanoparticles, changes in electrode

capacitance when exposed to solutions of proteins and the electrochemical response of

the electrode when incubated in the enzyme horseradish peroxidase (HRP). Direct

electron transfer to HRP has been achieved by covalent attachment of HRP onto MW

and the activity of the immobilised HRP is determined from the response of the

electrode interface to hydrogen peroxide. As described in Chapter Seven, the rigid MW

was also successfully used as an efficient tool for the exploration of the active centre of

GOx utilising the mixed monolayer technique to achieve direct electron transfer of GOx

from the active centre FAD through the MW to the underlying GC electrode. The

biocatalytical activity was also investigated using glucose as a substrate. Thus, utilising

the rigid molecules incorporating the mixed monolayer technique supplies a feasible

method to explore the active centres of some other redox enzymes, and also has great

potential to be used as a convenient strategy to fabricate third generation biosensor.

A novel label-free immunosensor system has been successfully developed in Chapter

Eight for electrochemical sensing of biotin-antibiotin biomolecular pairs based on

mixed monolayers of MW and PEG modified GC electrode surfaces. The so-prepared

sensor system can be used to detect one of the antigen-antibody pairs with low detection

limitation. In addition, a displacement assay has shown that the free biotin can compete

with the attached biotin for binding antibiotin. As studied in Chapter Nine the SWNTs

307


can be used as an alternative to molecular wire to fabricate another immunosensor

system due to the high efficiency of electron transfer that SWNTs have exhibited.

10.3 Future Directions

10.3.1 Further Investigation of Heterogeneous Electron Transfer within and between

the Enzymes

The heterogeneous electron transfer through the pure and mixed monolayers of organic

molecules modified on GC surfaces has been studied in this thesis. As introduced in

Chapter One the rate constant of electron transfer could be affected by many factors,

such as the electrode materials, the nature of the molecules, the electrolytes and density

of the redox probes. However, because of their diversity and rich behaviour, the kinetics

and mechanism for the electron transfer processes are difficult to identify, a lot of more

issues need to be investigated. Even more complicated is the electron transfer behaviour

between and within the enzymes as pioneered by many groups.1-6 Though the

exploration of the GOx using MW and the fabrication of the glucose biosensor have

given a lot of invaluable information, the molecular basis of the microscopic mechanism

of the electrical communications within and between the enzymes still remains unclear

because of the complexity and inhomogenity of biomolecular systems.

10.3.2 Optimisation of the Fabricated Immunosensors

As discussed in Chapter Eight and Chapter Nine, the fabricated immunosensors using

MWs or SWNTs as the conductor have demonstrated the capability for quantitative

analysis of antibiotin, and the free biotin can also be analysed by the displacement assay.

However, it took half an hour to achieve the quantitative analysis response, which is not

applicable for a real time analysis. Thus, it is valuable to investigate the strategies to

308


speed the detection of biomolecules in order to optimising this fabricated

immunosensors, such as fabricating a more stable sensor interface on GC surfaces or

finding more stable reodox probes. In addition, it is worthwhile using such fabricated

immunosensor system for the detection of the real biological samples.

10.3.3 Development of the Immunosensor Arrays

After optimisation, the MW fabricated immunosensors is supposed to have the ability

for rapid, sensitive and quantitative analysis of biomolecular pairs in real samples. An

additional feature of immunosensor performance becomes more and more important:

the immunosensor should be able to discriminate between multiple analytes in a single

pot of samples. A number of groups have described optical biosensors capable of

simultaneous detection of multiple analytes.7-10 However, demonstration of the ability to

use a single sensor substrate for simultaneous, multi-analyte detection is limited.11-14

The ability to perform multiple analyses on a single sensing surface has a number of

advantages15 over performing multiple parallel analyses on different substrates. i) A

single set of positive and negative controls can be used for all the assays. These controls

can be used to correct for variations in substrate chemistry, illumination, or detector

sensitivity. ii) A series of standards may be analysed at the same time as unknown

samples, allowing construction of standard curves for quantification. iii) Performing

multiple assays simultaneously decreases the assay times compared with sequential

analyses. iv) Fluidic manipulations are less complicated when using a single sensor

substrate than when multiple substrates are used. v) Use of a single substrate provides

more effective (and valid) comparisons of the experimental data and the controls.

309


Considering the performance of biological interface comprising of MWs and PEG

spacer units, a novel immunosensor array might be fabricated as shown in Figure 10.1.

1 2 3 4

Subs

trate

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O


NO2

CO

NH

Redox Probe (DAN)

Antigen 3

Antibody 3

NH

CO

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O


NO2

CO

NH

NH

CO

Redox Probe(Fc)

Fe

H2C

H2C

Antigen 1

Antibody 1

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O


NO2

HN

Redox Probe(PQQ)

Antigen 4

Antibody 4

NH

N

O

OHN

HOOC

CO

OC

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O


NO2

CO

HN

CO

Redox Probe (Ru)

Antigen 2

Antibody 2

NH

N

N

RuH3N

H3N

NH3

NH3

Figure 10.1 Schematic representation of a fabricated immunosensor array.

310


Based on this design described in Figure 10.1, four targets can be detected rapidly and

simultaneously on a single substrate. The preparation of the immunosensor array

generally involves the following steps: i) the flow chamber modules can be made on the

GC substrates by placing a 4-channel poly(dimethylsiloxane) (PDMS) patterning

template in contact with the surface, applying pressure to create a fluid-tight and airtight

seal as shown in Figure 10.2; ii) the patterned channels can be modified with diazonium

salts with different terminal functional groups. Channel 1, channel 2 and channel 3 are

modified with mixed monolayers of PEG and MW with the terminal carboxylic acid

groups by flowing mixed diazonium salt solutions into these channels. And mixed

diazonium salt solutions of PEG and MW with the terminal amine groups are flowed

into channel 4. The solution is incubated for 12 h at 4 oC followed by rinsing the

channels with acetonitrile and water, and dried under a stream of argon; iii) solutions

containing different redox probes are flowed into the channels. Ferrocenedimethylamine

(Fc), Ru(NH3)4(4-aminopyridinyl)2(PF6)3 (Ru3+), 1,5-diaminonaphthalene (DAN) and

pyrrolo quinoline quinone (PQQ) are loaded into channel 1, channel 2, channel 3 and

channel 4, respectively. The solution is left for incubation for 12 h at room temperature

followed by rinsing the channels with water, and dried under a stream of argon; iv)

antigens 1, 2, 3, and 4 are flowed into the channel 1, 2, 3, and 4, respectively. The

antigen solutions are left for incubation in the template for 6 h at 4 oC; v) the PDMS

flow chamber modules are removed and the substrate is rinsed with phosphate buffered

saline (PBS) and water, and dried under a stream of argon. Then the formed

immunosensor array is ready for detection of unknown samples.

311


1 2 3 4Substra

te

PDMS

Figure 10.2 Schematic representation of patterning the GC substrate using 4-channel

PDMS patterning template.

The working mechanism of this immunosensor array is based on the results obtained in

Chapter Eight and the performance of this fabricated immunosensor array can be

monitored electrochemically. It is found the formal potentials for the four redox probes

Fc, Ru3+, DAN, and PQQ are about 0.3 V,16 0 V,17, 18 0.6 V,19, 20 and -0.3 V,21 so the

redox response caused by the different redox probes can be distinguished easily. A

certain antigen can be attached to a specific redox probe by controlling the flowing

solution in Figure 10.2. Based on the results obtained in Chapter Eight, the attachment

of antibodies can result in the decrease of the current response from the redox probes

(Figure 10.3). So when the samples containing different antibodies flow into the

antigens attached template, the target antibody can be attached to the channel fabricated

with the specific antigen by the antigen-antibody affinity. And the attachment of a

certain target antibody can be reflected from the change of peak current at a certain

potential range, and the concentration of specific antibody in the mixture can be

detected from the decrease of peak current at a certain potential. Thus, this fabricated

immunosensor array can be used to simultaneously detect four analytes in one sample.

312


0

0.1

0.2

0.3

0.4

0.5

-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8

Potential /V

Cur

rent

/A

PQQ Ru3+ Fc DAN

ab

OSWV

Figure 10.3 OSW voltammograms of an immunosensor array (a) before and (b) after

the attachment of different antibodies.

10.3.4 Diagnosis and Treatment of Pathogenic Cells Using the Nanoparticle-

Antibody Conjugate

As studied in Chapter Six and Eight, the interface comprising of MW and PEG

derivatives self-assembled on carbon surfaces has demonstrated great potential for

protein chemistry22 and the development of immunosensors in that the MW has the high

efficiency for electron transfer23 and PEG molecules show an obvious effect of

resistance to the non-specific protein absorption.24-26 Gold nanoparticles have attracted

wide attention due to numerous applications.27-29 Furthermore, the bio-recognition

induced by molecular pairs such as antibody-antigen has been extensively investigated

since they are specific30 and very useful in the medical treatment. Therefore, modifying

the surface of the gold nanoparticles with mixed monolayers of PEG and MW which

can be covalently linked to antibody molecules could create an optimal interface

(Figure 10.4) for seeking through bio-recognition to the target antigen on the

pathogenic cells such as tumour cells and parasitic cell.

313


Gold Nanoparticle

PEG

O2N

CONH

O O O O O OH

O O O O O OH

Molecular wire

Antibody

Antigen

Pathogenic cellGold Nanoparticle

PEG

O2N

CONH

O O O O O OH

O O O O O OH

Molecular wire

Antibody

Antigen

Pathogenic cell

Figure 10.4 The self-seeking of the specific antibody-tethered gold nanoparticles on the

pathogenic cell.

MW and PEG molecules in the form of diazonium salts or terminated with thiol groups

can be easily prepared,31-33 and can form the self-assembled monolayers with versatile

functional groups on gold surfaces.23 Mixed SAMs have better functions relative to the

homogeneous one, such as to study the property of a single molecule, to avoid the

interaction between the same molecules, to dilute the immobilised species.34 Thus,

achieving the system as described in Figure 10.4 is applicable and valuable. The gold

nanoparticles can be firstly modified with mixed SAMs of PEG and MW either through

the coadsorption of the mixed thiol-terminated molecules or through reductive

adsorption of mixed diazonium salts. MW serves as a bridge to connect gold

nanoparticles and antibody, and can be replaced by any other alkanethiols or diazonium

salts with the terminal carboxylic acid groups. The length of these alkanethiols should

be comparable to the PEG. After that, the specific antibody can be covalently attached

to the carboxylic acid terminated MW by an amide bonding. This conjugates of gold

nanoparticles and the antibody can be used to automatically seek the specific antigen

surrounding the pathogenic cells by the specific biorecognition. Thus, this methodology

can be used for diagnosis of the specific pathogenic cell if the conjugate is fabricated

314


with the specific antibody, and especially the antibody is labelled with a fluorescent dye

which can be easily monitored using the confocal microscope to obtain the similar

images as shown in Figure 10.5.

a b c

Figure 10.5 The confocal images of the cell surface bonded with nanoparticles labelled

with fluorescent dye. (a) and (b) are the single cell, (c) is a cell cluster.35

Besides the potential application in the medical diagnosis, this methodology can also be

extended to the medical treatment of the pathogenic cells (e.g. cancer cell or parasite

cell) if the gold nanoparticle is replaced by some other nanoparticles such as

nanoshell,36 nanorod,37 nanocap38 that have plasmon adsorption in the near infrared

(NIR) region to fabricate the conjugates. After these conjugates self-attached on the

pathogenic cells (Figure 10.6), the temperature of the conjugates will increase due to

the plasmon adsorption and kill the membrane of the pathogenic cell of parasites when

the laser source with NIR light is applied,39 while the NIR light will mostly pass

through the tissue of alive organisms.

315


Nanoparticleconjugate

Figure 10.6 A pathogenic cell attached with gold-antibody conjugates through

biorecognition.

In conclusion, based on the multidisciplinary research on the modification of carbon

substrates, the heterogeneous electron transfer through organic layers, and the

development of electrochemical sensors, biosensors and immunosensors as described in

this thesis, a lot of other interesting exploration can be extended such as the

development of sensor arrays, nanofabrication for electronics, and fabrication of

nanodevice for medical treatment. It is hoped that the work carried out in this thesis has

laid the foundation for the development of third generation biosensor and

immunosensor via direct electron transfer through the MW and PEG biological

interfaces. It is also hoped that the research presented in this thesis will inspire others to

expand the research in these areas.

10.4 References


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