1 Affimer-based impedimetric biosensors: the new analytical platform for biorecognition applications By Pattanapong Thangsunan Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biomedical Sciences, Faculty of Biological Sciences June 2018 2 The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Pattanapong Thangsunan to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. i Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor, Prof. Paul Millner, for his continuous support that he has given me academically and also guidance for everyday life. I really appreciate his patience, motivation and kindness and I could not have come this far without him. I also would like to thank all the previous and current members in the Millner group, especially Ploy, Asif, Shazana, Jack and Kaniz, for the stimulating academic discussions and pleasant moments as we worked together in the lab. All of them made my times in Leeds unforgettable. Additionally, I would like to thank my co-supervisors, Prof. Michael McPherson and Dr. Darren Tomlinson and the members of the BSTG, especially Anna, Christian and Tom, for their assistance regarding Affimers and important knowledge of molecular biology. I also thank Dr. Lars Jeuken, my assessor, for his guidance in electrochemistry and impedance spectroscopy. My deep appreciation also goes to Dr. James Robinson (at St James’s University Hospital) for his assistance with surface plasmon resonance (SPR) and Dr. James Ault and his group for mass spectrometry facility. I gratefully acknowledge the Royal Thai Government and the National Institute of Metrology (Thailand) for the financial support and the opportunity of PhD study. A million thanks go to my lovely friends at the University of Sheffield, particularly Sine and Tee, for being with me during our wonderful and memorable trips around UK and Europe. Special thanks also go to Piyanee for being my buddy when having meals, shopping and sharing our PhD experiences. Last but not the least, for my family, I would like to thank my dad, my mom and my grandma for their immeasurable love and encouragement. Your support always boosts me up and makes me energetic to pursue my dreams. I also thank my ii twin brother, Pan, for always believing in me and lifting my spirits during low points. Throughout my life, you have always been there for me as I will always be there for you as well. As this journey is coming to an end, I cannot wait to start a new journey of my life and hopefully it will be as an awesome experience as I have had here in the UK. Pattanapong Thangsunan June 2018 iii Abstract Thanks to their high sensitivity and specificity, short-processing times, low cost of production, small size, and no requirement for professional users, biosensors have increasingly gained popularity. Electrochemical impedance biosensors have successfully been applied to detect a wide range of target analytes including whole cells, proteins, and small molecules. However, there are some limitations, for example reproducibility and non-specific binding, which still require further development. The main objective of this thesis is to develop impedimetric biosensors using Affimers, novel non-antibody binding proteins, as bioreceptors to detect a small molecule target, dichlorodiphenyltrichloroethane (DDT), and a protein biomarker, fibroblast growth factor receptor 3 (FGFR3). Initial work in this thesis was the selection of Affimers using phage display technology. Affimers were selected from a phage library provided by the BioScreening Technology Group (BSTG) at the University of Leeds. The selected Affimer-encoding sequences were then subcloned into the pET expression vector and expressed in E.coli cells. Prior to using the selected Affimers for biosensor fabrication, specific interaction of the Affimers with their analytes was investigated using ELISA, surface plasmon resonance (SPR) and immunoprecipitation (pull- down) assay. Even though none of the Affimers against DDT succeeded in binding specifically to DDT, some of the Affimers against FGFR3 showed binding to it and were then utilised for biosensor fabrication. Two sensor fabrication methods, the ELISHA “gluing” protocol and NeutrAvidin-biotin linkage, were tested and the latter one was selected for further study. By using the NeutrAvidin-biotin interaction method to functionalise the sensor surfaces, several parameters such as Affimer concentration, NeutrAvidin concentration and blocking agents to minimise non-specific binding were optimised. The fully fabricated Affimer-based biosensors were incubated with the analyte iv (FGFR3) and electrochemical impedance spectroscopy (EIS) was employed to interrogate FGFR3 binding. The data showed that the Affimer-based sensors could detect FGFR3 protein to very low levels. However, further optimisation is still needed in order to minimise non-specific binding effects and make the sensors work consistently. The work presented in this thesis is the first Affimer-based impedimetric biosensor for the detection of FGFR3, a promising biomarker for early diagnosis of bladder cancer. This sensor platform may not only provide an effective tool for bladder cancer surveillance, but also pave the way of designing a new analytical method for monitoring other protein biomarkers of disease. v Abbreviations β2M Beta-2-microglobulin BCA Bicinchoninic acid BLI Biolayer interferometry BSA Bovine serum albumin BSTG The BioScreening Technology Group Cdl Double-layer capacitance CEA Carcinoembryonic antigen CPE Constant phase element CV Cyclic voltammetry DDT Dichlorodiphenyltrichloroethane DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EIS Electrochemical impedance spectroscopy ELISA Enzyme-linked immunosorbent assay FGFR3 Fibroblast growth factor receptor 3 GFP Green fluorescent protein HRP Horseradish peroxidase HSA Human serum albumin Hz Hertz IgG Immunoglobulin G IPTG Isopropyl β-D-1-thiogalactopyranoside ITC Isothermal titration calorimetry KD Dissociation constant koff Dissociation rate constant kon Association rate constant vi LC-MS Liguid chromatography – mass spectrometry PBS Phosphate-buffered saline PBST Phosphate-buffered saline plus 0.05% (v/v) Tween-20 PCR Polymerase chain reaction PEG Polyethylene glycol PMDA Pyromellitic dianhydride Rct Charge transfer resistance Rs Solution resistance SAM Self-assembled monolayer SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error of the mean SPR Surface plasmon resonance Sulfo-SMCC Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate TCEP Tris(2-carboxyethyl)phosphine hydrochloride TMB 3,3’,5,5’-tetramethylbenzidine W Warburg impedance Z’ Real component of impedance -Z” Imaginary component of impedance vii Table of Contents Acknowledgements ...................................................................................... i Abstract ....................................................................................................... iii Abbreviations ............................................................................................... v Table of Contents....................................................................................... vii List of Figures ............................................................................................. xi List of Tables ............................................................................................. xvi Chapter 1 Introduction ................................................................................ 1 1.1 Overview ...................................................................................... 1 1.2 What is biosensor and how is it important? ................................... 2 1.3 Types of biosensors ..................................................................... 5 1.3.1 Optical biosensors ............................................................... 5 1.3.2 Piezoelectric biosensors ...................................................... 7 1.3.3 Electrochemical biosensors ................................................. 8 1.4 Electrochemical impedance spectroscopy (EIS) ......................... 12 1.4.1 The brief principle of electrochemical impedance spectroscopy ...................................................................... 12 1.4.2 Fabrication of impedimetric biosensors .............................. 19 1.4.3 Applications of impedimetric biosensors ............................ 34 1.5 Classification of bioreceptors ...................................................... 37 1.5.1 Types of common bioreceptors used for fabricating electrochemical biosensors ................................................ 37 1.5.2 Antibodies and their limitations .......................................... 42 1.5.3 Antibody-derived fragments ............................................... 51 1.5.4 Non-antibody binding proteins ........................................... 54 1.5.5 Affimers ............................................................................. 61 1.6 Dichlorodiphenyltrichloroethane (DDT) ......................................