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Characterisation, Quantification and Modelling the EDC/NHS Activation

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Characterisation, Quantification and Modelling the EDC/NHS Activation IMPEDANCE-BASED ANALYSIS AND MATHEMATICAL MODELLING OF IMMUNOSENSOR BIOLAYERS ANDREW PETER HENDERSON Doctor of Philosophy (PhD) Teesside University October 2010 This research project was carried out as part of an Engineering and Physical Sciences Research Council (EPSRC) CASE studentship and was done in collaboration with Immunodiagnostic Systems (IDS) Ltd. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without proper acknowledgement. 1 Teesside University in Middlesbrough Impedance-based Analysis and Mathematical Modelling of Immunosensor Biolayers ANDREW PETER HENDERSON Doctor of Philosophy October 2010 Abstract A study to optimise an IgG based immunosensor is presented, that has been carried out by absorbing monolayers to a gold transducer surface at varying immersion times and temperatures. The theory and kinetics of monolayer adsorption are analysed and discussed. Existing mathematical models are reviewed and experimentally researched, to highlight gaps in knowledge that would facilitate high quality, cost effective immunosensor production. The creation of two mathematical models to predict monolayer adsorption kinetics and optimal immersion times are discussed. Details are provided of how the new mathematical models may be advanced, and how the production of immunosensors may be further improved. The first novel mathematical model (PTCS) has been created to model the presence of two sequentially forming structures on the surface of a substrate. It gives an insight into the percentages of each structure on the surface, along with the actual adsorption process. This model provides a good fit to all applicable experimental data and has allowed the deduction of optimum immersion times. The second novel model (PIF) provides a greater insight than existing models into the individual contributions to surface coverage by both random and island growth. This allows an insight into how the monolayer surface is covered, which is critical to determine the optimum conditions for adsorption. This model also provides a good fit to the isotherm data it has been applied to. To provide a thorough understanding of the bulk properties of monolayer formation over the gold transducer, and how these properties vary with immersion time and temperature, various measurement techniques have been employed. Electrochemical Impedance Spectroscopy (EIS) has been the principle measurement technique used to measure the bulk properties, but confirmation studies have also been carried out including, Contact angle measurements, FTIR microscopy with BSA molecular labels, Fluorescence microscopy for small adsorbed molecules and AFM for layers assembled from macromolecules. The data generated from the different techniques show consistency with the arguments discussed in each instance. Two different IgG adsorption processes have been compared. These include direct IgG addition and a multilayered streptavidin-based process. The results indicate that IgG molecules adsorbed via the streptavidin based multilayer process are more vertically orientated and have a higher packing density of IgG molecules. Keywords: Self Assembled Monolayer, impedance-based immunoassay, Streptavidin, biotinylated IgG, mathematical adsorption modelling. 2 “Any intelligent fool can make things bigger; more complex and more violent. It takes a touch of genius - and a lot of courage-to move in the opposite direction.” Albert Einstein 3 Acknowledgements First and foremost, I would like to thank my wife Sarah for the inspiration, understanding, devotion and support she has afforded me over the duration of my PhD project. I would also like to thank: fellow PhD students for their co-operation, the Teesside University members of academic staff that supported me (in particular, Dr Paul Russell for choosing to formally become the second supervisor of my PhD project during my final year of study and for his help up until that point and my Director of Studies, Prof. Zulfiqur Ali for providing me with the opportunity of studying for this PhD and allowing me freedom to work autonomously). I would also like to thank the Teesside University technical staff who provided me with significant help, the Teesside University administrative staff for helping to progress my PhD and providing me with the opportunity to develop my transferable skills and Dr David Pritchard for his support. I would like to thank the EPSRC and Immunodiagnostic Systems (IDS) Ltd for funding to carry out this research project. Finally, I would like to thank my Mother, Father and Brother, Julie, Peter and Matthew, for their love and support. 4 Author‟s Declaration I confirm that all the novel material presented in this thesis is solely the work of the author: Andrew Peter Henderson. Signature………………………………………………………….. 5 Contents Abstract ........................................................................................................................ 2 Acknowledgements………………………………………………… .............. ………4 List of Figures……………………………………………………………………... 14 List of Tables…………………………………………………………….................. 24 Chapter 1 Introduction…………………………………………………………….. 27 1.1 Project brief……………………………………………………………… .. 29 Chapter 2 Measurement and Electrochemical Modeling Techniques for Individual Monolayer Formation………..................................................................................... 30 2.1 Introduction…………………………………………………………… .. …30 2.2 Surface Profiling techniques…………………………………… ... ……….30 2.2.1 Photon-based Spectroscopic techniques…………………………… .. ……30 2.2.1.1 Auger Electron Spectroscopy (AES)………………………………. …..30 2.2.1.2 X-ray Photoelectron Spectroscopy (XPS)…………………………… .. .31 2.2.1.3 Secondary Ion Mass Spectroscopy (SIMS)…………………………… . 31 2.2.1.4 Scanning Electron Microscopy (SEM)…………………………… . …..31 2.2.1.5 Infra-Red Spectroscopy……………………………………………… …33 2.2.1.6 Raman Spectroscopy…………………………………………… . ……..33 2.2.2 Scanning Probe Microscope Techniques (SPM)………………………... .. 34 2.2.3 Quartz Crystal Microbalance (QCM).……………………………… . ……34 2.2.4 Ellipsometry…………………………………………………………… .... .35 2.2.5 Surface Plasmon Resonance (SPR)…………………………………… ….35 2.2.6 Contact Angle Measurement…………………………………………….. ..36 2.2.7 Electrochemical techniques…………………………………………… . …36 2.3 Selection of the measuring techniques to be used in this study……. ......... .37 2.4 Electrochemical Impedance Spectroscopy………………………………. 38 2.4.1 Introduction…. ............................................................................................. 38 2.4.2 The EIS method… ....................................................................................... 38 2.4.3 Electrochemical theory…… ........................................................................ 39 2.4.3.1 Equivalent Circuits…. ............................................................................... 39 2.4.3.1.1 Resistance Elements…. ...................................................................... 41 2.4.3.1.2 Capacitance Elements… ..................................................................... 41 2.4.3.1.3 Gouy-Chapman theory…. .................................................................. 42 6 2.4.3.1.4 Constant Phase Element (CPE)… ...................................................... 43 2.4.3.1.5 Warburg Impedance Elements….. ..................................................... 44 2.4.3.2 Simulated Equivalent Circuit Elements in ―ZView2‖ by Scribner Associates, Inc. ..................................................................................... 45 2.4.4 Adapting the standard EIS measurements for use in surface adsorption studies.. ........................................................................................................ 45 2.5 Fourier Transform Infra Red (FTIR) Microscopy… ................................... 46 2.6 Fluorophore labeling and imaging.. ............................................................. 47 2.7 Atomic Force Microscope (AFM)… ........................................................... 47 2.8 Summary.. .................................................................................................... 49 Chapter 3 Mathematical Modeling of Monolayer Adsorption Processes… .............. 50 3.1 Introduction.. ................................................................................................ 50 3.2 Extant Adsorption Models.. ......................................................................... 50 3.2.1 The Freudlich Adsorption Isotherm.. ........................................................... 50 3.2.2 The Largergren Adsorption Isotherm.. ........................................................ 51 3.2.3 The Langmuir Adsorption Isotherm.. .......................................................... 51 3.2.4 Kisliuk Adsorption Isotherm........................................................................ 53 3.2.5 RSA Adsorption Isotherms… ...................................................................... 56 3.2.5.1 Monte Carlo Algorithms.. ..................................................................... 58 3.2.5.2 Adsorption of ―Real‖ Anisotropic molecules… ................................... 58 3.3 New Adsorption Isotherms… ...................................................................... 60 3.3.1 Proposed Island Formation (PIF) Adsorption Isotherm.. ........................ 60 3.3.2 Proposed Two Component Sequential
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