Development Of Molecularly Imprinted Assays Targeting Peptides And Proteins

Many efforts have been made to produce artificial materials with bio-mimetic properties for diagnostics and life science applications. Among these efforts the technique of molecular imprinting has received much attention, however challenges regarding imprinting of biologically relevant analytes are important to overcome if the technology is to reach its full potential. The lack of generality amongst current molecularly imprinted assay formats is a further discouraging factor against the adoption of molecularly imprinted assays over conventional immunoassays. Whilst many elegant methods for specific templates have been devised, a simple, universal format applicable to any analyte of interest would be a far more attractive prospect. The work presented in this thesis aims to address these challenges. A novel screening tool for optimisation of polymerisation mixtures taking advantage of solid-phase imprinting has been developed and applied to both peptides and proteins. The molecularly imprinted polymers (MIPs) generated in this manner have then been applied to a number of assay formats, with the intention of designing a biologic-free assay for target peptides. The potential application of these techniques has then been demonstrated using a model protein, acetylcholinesterase (AChE). A novel epitope mapping approach has been utilised to identify peptide sequences useful as templates for MIP generation for site specific binding to AChE. Changes in structure and enzymatic activity as a result of binding interactions have been investigated, presenting an opportunity for development of novel allosteric MIP based enzyme activators. The site specific binding has been further exploited in the final application in detection of AChE in a novel sandwich assay format. In this way, it has been demonstrated that this process can be used to identify unknown binding sites on a protein, generate high affinity recognition materials for these sites, and then use these in a generic assay format for detection of the original protein of interest.