University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF NATURAL and ENVIRONMENTAL SCIENCES SCHOOL OF CHEMISTRY The Modification of Carbon Electrodes for Biosensor Applications by IZZET KOCAK Thesis for the degree of Doctor of Philosophy June_2013 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL and ENVIRONMENTAL SCIENCES School of Chemistry Thesis for the degree of Doctor of Philosophy THE MODIFICATION OF CARBON ELECTRODES FOR BIOSENSOR APPLICATIONS Izzet Kocak This thesis reports upon the covalent attachment of 6 different types of linkers bearing the Boc protecting group to the surface of glassy carbon electrodes, highly ordered pyrolytic graphite with the edge and basal plane orientation and multi-walled carbon nanotube abrasively immobilised onto a glassy carbon substrate by electrochemical oxidation or reduction of the corresponding diazonium salt. Removal of the Boc group allows redox probes, such as anthraquinone-2-carboxyl acid, 4 nitrobenzoyl chloride and 3 and 4 dihydroxybenzoyl chloride, to be coupled to the surface using solid-phase coupling methods. The surface coverage, scan rate and pH effects, as well as the determination of the kinetic parameters, were evaluated using cyclic voltammetry. It was found that the type of carbon electrode and the choice of linkers had a significant influence on the surface coverage of redox probes and the electron transfer rate. A 4-(N-Boc-aminomethyl) benzene diazonium tetrafluoroborate salt linker (C6H4CH2NH) was also spontaneously grafted onto the CNTs by refluxing in the C6H4CH2NHBoc diazonium salt at 60 oC in an acetonitrile solution. After the removal of the Boc protecting group, the anthraquinone (AQ) and nitrobenzene (NB) groups were attached to the benzyl amine linker by solid-phase amide coupling. The grafted CNTs were characterized using FTIR and cyclic voltammetry techniques; the surface coverage and the stability of the tethered functional groups were also investigated. The oxygen reduction reaction was studied on bare and anthraquinone (AQ) modified edge planes, on a basal plane with highly oriented pyrolytic graphite, and on glassy carbon (GC) and multi-walled carbon nanotube electrodes. The anthraquinone-modified rotating disc GC electrode results show the two electron oxygen reduction reactions with hydrogen peroxide as the final product. The immobilization of laccase (ThL) onto GC electrodes modified with anthraquinone and i anthracene through EDA and C6H4CH2NH– linkers was also achieved by employing high-throughput screening using a multichannel potentiostat for a library of 12 electrodes; the activity of these electrodes to oxygen reduction was examined. The experimental findings demonstrated a successful attachment of laccase to the modified glassy carbon, as well as successful electron transfer between substrate- and enzyme-active sites. DFT calculations were used to investigate the structural properties of the functionalized basal plane of graphene after modification and to discover why the edge site shows higher reactivity to the attachment of linkers than the basal sites and whether the type of linker has an effect on the surface coverage of AQ. On the basis of considering the relationship between binding energy and charge transfer, the root of the effect of the type of linker on surface coverage was investigated to ensure whether it is steric, electronic, or both. ii Contents ABSTRACT i Contents iii List of Figures vii Acronyms xiii DECLARATION OF AUTHORSHIP xv Acknowledgements Chapter 1 INTRODUCTION .................................................................................. 1 1.1. Overview.............................................................................................................. 1 1.2. Carbon-Electrode Materials ................................................................................... 1 1.2.1. Graphene....................................................................................................... 2 1.2.2. Highly Ordered Pyrolytic Graphite.................................................................. 3 1.2.3. Glassy Carbon ............................................................................................... 4 1.2.4. Carbon Nanotubes ......................................................................................... 5 1.3. Electron Transfer Kinetics at Carbon Electrodes...................................................... 6 1.4. Modification of Electrodes................................................................................... 10 1.4.1. The Modification of Electrode Surfaces by Reduction of Diazonium Salts ....... 11 1.4.2. The Modification of Electrode Surfaces by the Oxidation of Amines............... 16 1.4.3. The Characterisation of Modified Surfaces .................................................... 19 1.5. Solid Phase Synthesis Methodology on Modified Carbon Electrodes with Boc- protected Amines ........................................................................................................... 20 1.6. Oxygen Reduction............................................................................................... 21 1.7. Density Functional Theory................................................................................... 24 1.8. Research Aims and Thesis Overview.................................................................... 33 1.9. References .......................................................................................................... 35 Chapter 2 MATERIALS AND METHODS ............................................................ 43 2.1. Reagents............................................................................................................. 48 2.2. Reference and Counter Electrodes ........................................................................ 50 2.3. Construction of Glassy Carbon and Basal and Edge Plane of HOPG Electrodes ...... 50 2.4. Electrode Pre-treatment ....................................................................................... 51 2.5. Electrochemical Measurements ............................................................................ 51 2.6. The Synthesis of 4-(N-Boc-aminomethyl) Benzene Diazonium Tetrafluoroborate Salt. .......................................................................................................................... 52 iii 2.7. Electrochemical Modifications of GC and HOPG and Multiwalled Carbon Nanotube Electrodes...................................................................................................................... 52 2.8. General Procedure for Boc Deprotection of Modified GC and HOPG and MWCNT Electrodes...................................................................................................................... 53 2.9. Coupling Reaction of Anthraquinone-2-carboxylic Acid at the Grafted GC, and HOPG and MWCNT Electrodes...................................................................................... 54 2.10. Coupling Reaction of Acetyl Chloride at the Modified GC, HOPG and MWCNT Electrodes...................................................................................................................... 54 2.11. Bifunctionalization of AQ Modified HOPG Electrodes with Nitrobenzene and Dihidroxybenzene .......................................................................................................... 56 2.12. Abrasive and Wet Casting Immobilisation of MWCNT and to GC surface ......... 56 2.13. Spontaneous Attachment of C6H5CH2NH and Coupling of AQ, NB and di-HB to MWCNT ....................................................................................................................... 57 2.14. Determination of Surface Coverage and Effect of Scan Rate............................... 58 2.15. Oxygen Reduction Studies on the Unmodified and AQ-Modified Carbon Electrodes ....................................................................................................................... 58 2.16. Enzyme Immobilisation onto AQ modified GC electrodes.................................. 60 2.17. Oxygen Reduction on Laccase Immobilzed GC Electrodes Using High-Throughput Screening60 2.18. Computational Details...................................................................................... 62 2.19. References ...................................................................................................... 64 Chapter 3 THE MODIFICATION OF GRAPHITE ELECTRODES ...................... 65 3.1. Overview............................................................................................................ 66 3.2. The Modification of Graphite Electrodes .............................................................