DEVELOPMENT OF MOLECULARLY IMPRINTED ASSAYS TARGETING PEPTIDES AND PROTEINS

Many efforts have been made to produce artificial materials with bio-mimetic properties for application to binding assays and biosensors. Among these efforts, the technique of molecular imprinting has received much attention. With increasing industrial interest in the application of molecularly imprinted polymers, challenges regarding biologically relevant analytes such as peptides and proteins are important to overcome if the technology is to reach its full potential.

The lack of generality amongst current molecularly imprinted assay formats is, however, a discouraging factor against the adoption of MIAs 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. Of particular interest are homogeneous assays that do not require separation steps. If these conditions could be met, as well as maintaining the sensitivity and specificity which has already been demonstrated, then there should be little argument against the adoption of MIAs.

The work presented here aims to address these challenges. A novel assay-like format for optimisation of polymerisation mixtures taking advantage of solid-phase imprinting has been developed and applied to both peptides and proteins. The MIPs generated in this manner have then been applied to a number of assay formats, which have been directly compared for their accuracy, sensitivity, selectivity, and ease of development, with the intention of designing a biologic-free homogenous assay capable of analysing any molecule of interest.

The potential application of these techniques has then been demonstrated using a model protein, acetylcholinesterase. MIPs were prepared to map the topography of the surface of the protein and to identify peptide sequences which may be useful for MIP NP preparation using an epitope approach. The identified epitopes were then synthesised and used as templates for polymerisation mixture optimisation and MIP generation. Changes in structure and enzymatic activity of acetylcholinesterase as a result of epitope-imprinted MIP binding have been explored, before their final application in the detection of acetylcholinesterase in the preferred assay format, as well as a sandwich assay, utilising the different epitopes identified.

In this way, we have demonstrated that this process can be used to identify unknown binding sites on a protein, generate high affinity recognition materials for these sites through a rapid optimisation process, and then use these in a generic assay format for detection of the original protein of interest.