Development of an impedimetric biosensor using a non-antibody based biological recognition molecule

The molecular recognition layer generally immobilised on the active interface of a biosensor is one of the key factors in governing the biosensor’s performance, and in particular its sensitivity and selectivity. The aim of this thesis was to investigate a novel non-immunoglobulin-based recognition molecule as the capture molecule for electrochemical biosensors with the aim to improve sensitivity and specificity of label-free biosensing. To understand the characteristics of the biomolecular layer of a biosensor formed from the non-immunoglobulin-based recognition molecule, the Adhiron scaffold developed at the University of Leeds was used as the model system. The Adhiron scaffold consists of one α-helix, four β-sheets, and three variable regions. The three variable regions comprise two surface-exposed loops and the N-terminus of the protein. Adhiron-based binders against a well-characterised antibody, the anti-myc tag antibody, were selected using phage-display and used as a model system for this study. The phage-display library was constructed by inserting randomised peptides into the three variable regions of the Adhiron scaffold. The best performing binder, selected from the ten Adhiron myc binders panned from a phage display screen against polyclonal anti-myc tag antibodies, myc binder 2, was chosen as the biological recognition molecules for the development of an electrochemical impedimetric biosensor. Cloning of the Adhiron binders in pET-11(a) expression vector, optimisation of expression and purification of the binders, was carried out and the binders were obtained in soluble form. Adhiron myc binder 2, which showed the best binding against monoclonal anti-myc tag antibodies, showed a high thermal stability of 85º C, with well-defined α-helical and β-sheet structures. This binder was thoroughly characterised further before being used as a recognition molecule of an electrochemical biosensor. An electrochemical Adhiron-based myc binder 2 sensor was fabricated to detect monoclonal anti-myc tag antibodies over a range of concentrations. The Adhiron myc binder 2 based EIS biosensor comprised a highly sensitive insulating layer formed by a self-assembled monolayer of carboxylic acid terminated alkylthiol-PEG onto which Adhiron myc binder 2 was grafted. The sensing mechanism was based on the change in phase of the electrochemical impedance measured at 0.1 Hz observed upon binding of monoclonal anti-myc tag antibodies onto the sensor. Monoclonal anti-myc tag antibodies were detected down to a concentration of less than 100 pM, over a range from 0.1–200 nM anti-myc tag antibodies. These findings demonstrated for the first time the successful use of Adhiron-based antibodymimetics as recognition molecules in label-free biosensors. To improve the sensitivity of the Adhiron-based electrochemical biosensor, and potentially to modulate the sensitivity in situ, the performance of the sensor at different electrochemical DC-biases was investigated. The sensitivity of the sensor was observed to increase with increasing DC bias applied to the sensor surface. This sensitivity modulation was demonstrated to be reversible, therefore opening up a range of opportunities for future label-free biosensor architectures