MERCAPTOBENZOTHIAZOLE-ON-GOLD BIOSENSOR SYSTEMS FOR ORGANOPHOSPHATE AND CARBAMATE PESTICIDE COMPOUNDS (b) (1) (A) Vernon Sydwill Somerset (b) (1) (A) (b) (1) (A) Summary Organophosphate and carbamate pesticides are powerful neurotoxins that impede the activity of cholinesterase enzyme leading to severe health effects. This study firstly reports the development, characterisation, and application of thick-film acetylcholinesterase (AChE) biosensors based on a gold electrode modified with a mercaptobenzothiazole (MBT) self-assembled monolayer and either poly(o- methoxyaniline) (POMA) or poly(2,5-dimethoxyaniline) (PDMA) in the presence of polystyrene(4-sulphonic acid) (PSSA). The Au/MBT/POMA-PSSA/AChE and Au/MBT/PDMA-PSSA/AChE biosensors were then applied to successfully detect standard organophosphorous and carbamate pesticides in a 0.1 M phosphate buffer, 0.1 M KCl (pH 7.2) solution. Secondly, it reports the construction of the Au/MBT/PANI/AChE/PVAc thick-film biosensor for the determination of certain organophosphate and carbamate pesticide solutions in selected aqueous organic solvent solutions. The Au/MBT/PANI/AChE/PVAc electrocatalytic biosensor device was constructed by encapsulating acetylcholinesterase (AChE) enzyme in the PANI polymer composite, followed by the coating of poly(vinyl acetate) (PVAc) on top to secure the biosensor film from falling off. The electroactive substrate called acetylthiocholine (ATCh) was also chosen to replace acetylcholine (ACh) as substrate, since ATCh has better redox activity and can both be oxidised and reduced to provide better movement of electrons in the amperometric biosensor. The voltammetric results have shown that the current shifts more anodically as the iii Au/MBT/PANI/AChE/PVAc biosensor responded to successive acetylthiocholine (ATCh) substrate addition under anaerobic conditions in 0.1 M phosphate buffer, KCl (pH 7.2) solution. For the Au/MBT/PANI/AChE/PVAc biosensor, various performance and stability parameters were evaluated. These factors include the optimal enzyme loading, effect of pH, long-term stability of the biosensor, temperature stability of the biosensor, the effect of polar organic solvents, and the effect of non-polar organic solvents on the amperometric behaviour of the biosensor. The Au/MBT/PANI/AChE/PVAc biosensor was then applied to detect a series of 5 organophosphorous and carbamate standard pesticide solutions. The organophosphorous pesticides studied were diazinon, fenthion, parathion-methyl, malathion and chlorpyrifos; while the carbamate pesticides included dioxacarb, aldicarb, carbaryl, carbofuran and methomyl. Very good detection limits were obtained for the standard pesticide solutions and they were within the nanomolar range. The detection limit values for the individual pesticides were 0.137 nM (diazinon), 0.826 nM (fenthion), 1.332 nM (parathion-methyl), 0.189 nM (malathion), 0.018 nM (chlorpyrifos), 1.572 nM (dioxacarb), 1.209 nM (aldicarb), 0.880 nM (carbaryl), 0.249 nM (carbofuran) and 0.111 nM (methomyl). The detection limit results showed that the thick-film Au/MBT/PANI/AChE/PVAc biosensor was more sensitive to organophosphorous and carbamate pesticides compared to other biosensor results found in the literature. iv Dedication To my son, Enver Stuart and daughter, Emma Marion. “So that others may learn, experiment and report.” v Acknowledgements I wish to express my sincerest gratitude to my supervisors, Prof Emmanuel Iwuoha and Dr Priscilla Baker, for their guidance, valuable advice, encouragement and support that enabled me to complete this research. I would also like to thank my fellow researchers in the Sensor Research Laboratory, Michael Klink, Richard Akinyeye, Immaculate Michira, Nicolette Hendricks, Joseph Owino, Everlyne Songa, M. Sarah Maoela, R. Fanelwe Ngece, Lawrence Ticha, Luvuyo Sixaba, Neil Benjamin and Munkombwe Muchindu for their sharing of ideas, advice, general concern, and laughter during those stressful times. The help and support of the post-doctoral fellows are also appreciated: they are Drs Mantoa Sekota, Amir Al-Ahmed, Raju Khan, Anna Ignaszak, and Tesfaye Waryo. My warm thanks also to fellow students in the Chemistry department, especially Dr Mugera Wilson Gitari for his encouragement and support. A special thanks also to my good friend and former co-researcher, N. Gretta Mathebe for her support and encouragement. A special thanks also to Dr Aoife Morrin of Dublin City University, Dublin, Ireland for her guidance and advice throughout my research and help in acquiring some journal reference material. A big thanks to you to Mss E. Davids and M. Benn (Senior Library Assistants) at the Inter-library Loan Department (UWC) for your assistance in obtaining journal reference materials. Thank you also Dr Barbara Floris of University of Rome, Italy for the journal article you sent me on your research work. A big thank you also to the Department of Agriculture (DoA), South African Agricultural Food, Quarantine & Inspection Services (SAAFQIS) – Analytical Services South in Stellenbosch, for the supply of some of the initial pesticide standards used in this study. A big thank you to my previous employer, Denel Land Systems Western Cape (ex Somchem), for the granting of study leave that enabled me to attend several national and international conferences during my studies. Warm thanks also to my present employer, CSIR in Stellenbosch, and colleagues for the assistance provided during the final stages of the thesis preparation. A special thank you to the NRF, Chemistry Department at UWC, Senate and Faculty of Sciences Research Committees for funding and financial support provided to perform this study and attend Chemistry conferences. A warm and sincere thank you to my wife, Lorencia, and son, Enver for their love, support, and encouragement that enabled me to accomplish this milestone in my career. A big thank you also to my parents, parents-in-law, the rest of the family and friends for all their love, support, and encouragement. Finally, yet importantly, I want to thank my Heavenly Father for giving me the strength and support throughout my studies and especially during those hard and trying times when courage dwindled. vi Table of Contents MERCAPTOBENZOTHIAZOLE-ON-GOLD BIOSENSOR SYSTEMS FOR ORGANOPHOSPHATE AND CARBAMATE PESTICIDE COMPOUNDS ............i Vernon Sydwill Somerset ......................................................................................i DOCTOR OF PHILOSOPHY ...............................................................................i Summary......................................................................................................................iii Dedication .....................................................................................................................v Acknowledgements......................................................................................................vi Table of Contents........................................................................................................vii List of Figures ............................................................................................................xiv List of Tables ...........................................................................................................xxvi Acronyms and Abbreviations...................................................................................xxix List of Publications and Presentations ....................................................................xxxii Papers xxxii Oral and Poster Presentations at Conferences....................................................xxxiii CHAPTER 1 ...............................................................................................................37 Introduction.................................................................................................................37 1.1 Introduction.....................................................................................................37 1.2 Biosensors .......................................................................................................37 1.3 Self-Assembling Monolayers (SAMs)............................................................39 1.4 Conducting Polymers......................................................................................41 1.5 Thick film electrodes ......................................................................................42 1.6 Pesticides.........................................................................................................43 1.7 Objectives of the Study...................................................................................45 1.8 Layout of the Thesis........................................................................................50 CHAPTER 2 ...............................................................................................................52 Literature Review........................................................................................................52 2.1 Introduction.....................................................................................................52 2.2. Sensors ............................................................................................................54 2.2.1 Introduction to Chemical Sensors...........................................................54 2.2.2 Introduction to Biosensors ......................................................................55 2.2.3 Components of Biosensors......................................................................56