(209a) Investigation of Protein Imprinting Using Miniemulsion Polymerization through the Epitope Approach

Molecular imprinting is a state-of-art technique for the preparation of synthetic materials with pre-determined molecular affinity. To date, some of the most successful illustrations of molecular imprinting had been achieved through the imprinting of smaller ligand molecules like amino acids, drugs, metal ions and hormones whereas that of macromolecules like proteins remains a big challenge. This has mainly to do with the inherently sensitive nature of proteins and their bulkiness, which make them incompatible with the traditional molecular imprinting systems. To answer such limitations of the conventional imprinting methodology for proteins, alternative approaches like surface imprinting and epitope imprinting had been put forward.

We had previously prepared protein surface-imprinted nanoparticles through miniemulsion polymerization. In this work, we aim to investigate the preparation of imprinted nanoparticles with protein affinity through the epitope approach using miniemulsion polymerization. In the epitope approach, a short peptide sequence of the protein (epitope) that is sufficiently significant to represent the whole protein molecules is used as the template for imprinting. For the subsequent protein uptake, only the protein molecules with the template epitope sequence will be recognized and preferentially adsorbed, thus achieving protein selectivity. Importantly, the epitope peptide chosen as templates should be exposed for the ease of recognition and uptake. For some proteins like cytochrome C, the choice is straightforward. The exposed ?tails' of the protein molecules would be good candidates as templates for epitope imprinting. In this work, we have synthesized a 10-residue peptide sequence (AYLKKATNEG) to represent the cytochrome C tail and to be used as template in the miniemulsion polymerization imprinting. In addition to that, other proteins that are globular in shape are also imprinted, such as ribonuclease A (RNase A). For this protein, we chose the exposed, N-terminus 13-residue peptide (KETAAAKFERQHM) as the template peptide for epitope imprinting. The epitope peptides had been prepared through chemical proteolysis of the RNase A molecules and peptide synthesis. In the preliminary batch and competitive rebinding tests, it was found that the imprinted nanoparticles prepared using the RNase A epitope obtained from chemical proteolysis failed to display the expected molecular affinity towards the RNase A molecules in water. This probably signified the non-suitability of the epitope approach for the imprinting of globular proteins. Nevertheless, more investigative works would be required before definite conclusions can be drawn.