Developing an electrochemical assay to detect and quantify virus particles

Immunoassays are commonly used to sense many analytes and rely on a measureable detection of binding between an antibody and its antigen. The study described in this thesis used electrochemical impedance spectroscopy (EIS) to detect and quantitate viruses, particularly human adenoviruses (Ads), in terms of virus particles per ml. Initial work used bacteriophage MS2 and M13 to construct proof- of-principle sensors, the methodology of which was then applied in sensors built to detect Ads. The number of adenovirus (Ad) infections detected in immunocompromised people has increased due to the number of patients receiving transplants, as we" as the HIV pandemic. Ads cause life-threatening diseases specific to the infected organs of immunocompromised hosts, with discontinuation of immunosuppressive agents necessary to prevent morbidity. Initially, studies focussed on constructing self-assembled monolayer (SAM) based sensors against bacteriophage, after which Ad sensors were made using the same SAM methodology. The construction of sensors was analysed using cyclic voltammetry (CV), quartz crystal microbalance (QCM) and EIS. CV and EIS showed SAM based sensing surfaces collapsed upon virus capture for both bacteriophage and human Ad based sensors. Novel immunosensors were then fabricated using a functionalised conducting copolymer matrix comprising polyaniline and 2-aminobenzylamine onto which half-antibody fragments of polyclonal antibodies raised against a human Ad (AdS) capsid protein were immobilised. Fullv fabricated sensors were incubated with two immunologicallv distinct serotypes of Ad, Ad5 and Ad3, with between 10 and 1012 virus particles per ml prior to sensor interrogation. EIS was used to measure the charge transfer resistance of the sensors across a range of frequencies. They were found to specifically detect, and differentiate between, closely related human Ad serotypes with a limit of detection of 106 virus particles per ml, this was improved to 103 virus particles per ml after optimisation studies. In addition, atomic force microscopy was applied to study the sensor surface nanostructure and viruses and viral proteins were radiolabelled to investigate the binding parameters of the immunosensor and the virus.EThOS - Electronic Theses Online ServiceGBUnited Kingdo