Development of biosensors using novel bioreceptors; Investigation and optimisation of fundamental parameters at the nanoscale

Point of care diagnostics is hailed as a potential revolution which could lower the significant cost of diagnosis, lead to earlier interventions and lower the mortality and morbidity of a variety of diseases. In spite of the early promises made and notable breakthroughs such as the glucose biosensor, the field of biosensors has yet to achieve the commercial and societal gains it promises. One of the primary reasons for this is the cost of testing a remaining obstacle in biosensor development. The work in this thesis aims to address different approaches to address this which may help accelerate the development of impedimetric immunosensors and enhance their adoption in diagnostic and field applications. Initial work in this thesis has focussed on the development of a biosensor which could be regenerated, permitting repeated use. This work was done using a previously demonstrated biosensor where the signal behaviour was known and the process of regeneration could be studied in isolation. This proof-of-concept work it was discovered that regeneration and therefore re-use of impedimetric immunosensors was possible The biosensors throughout this thesis were constructed using electropolymer to which proteins were attached before interrogating the sensor using electrochemical impedance spectroscopy (EIS). The fully constructed sensors were then incubated with analyte of increasing concentrations before repeating interrogations. EIS was used to monitor receptor - analyte binding and provide a method of sensor calibration. Later work in this thesis explored the role of the bioreceptor in signal generation in EIS. The recent move towards the use of antibody mimetic receptors may have profound implications for biosensor development. There is however, limited demonstration of their use in biosensors and even less so in EIS based sensors. In this thesis nanobodies have been used to fabricate biosensors. They have also been re-engineered to include oriented peptide spacer arms with terminal cysteines to allow both oriented conjugation onto the transducer surface and precise positioning above it. This work has highlighted the importance of spacing and physical constraints at the nanoscale which may be important for determining signal generation in reagentless impedimetric immunosensors.