Affimer-based impedimetric biosensors: the new analytical platform for biorecognition applications

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 (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.