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Preparation of Ribonuclease Α Surface-Imprinted Nanoparticles with Miniemulsion Polymerization for Protein Recognition in Aqueous Media

Chau Jin Tan and Yen Wah Tong. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117576, Singapore

Molecular recognition is a powerful machinery that ensures the proper functioning of the biological world. Not only it is essential in the biological sense, such recognition property has also been harnessed in the field of bioseparation, like the use of antibody-antigen interaction in affinity liquid chromatography. Despite the high separation efficiency, the thermally sensitive nature of antibody and its high cost of production, however, restrict its popular application in bioseparation. Molecular imprinting has been gaining wide recognition and attention as a feasible way of producing enzyme mimics over the past few decades. Much success has been achieved with small molecules through the traditional method of bulk molecular imprinting. However, such approach, though simple, is not suitable for large molecules like proteins and oligosaccharides due to the inaccessibility of the binding sites to them. In addition, subsequent grinding produces sharp and irregular imprinted polymer and for its poor thermal dispersion, bulk polymerization is not suitable for industrial-scale applications. In this work, redox-initiated mini-emulsion polymerization had been used as an alternative approach for molecular imprinting with Ribonuclease A as the template. Regularly shaped imprinted nano-particles that sized around 40 nm were prepared using methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) as the functional and cross-linker monomer respectively. Extra care had been taken to preserve the template protein integrity in a mini-emulsion. Given the small sizes of the imprinted nano-particles, most imprinted sites were believed to have been created near the particle surface. This relieved the problem of restricted mobility associated with macromolecules. Superior template loading of 750 mg protein/g polymer and favourable binding kinetics had been observed. Previous works on protein imprinting were mostly based on the favourable hydrogen bond formation between the template protein and the functional monomer for imprinting. However, such technique does not work well in an aqueous media due to the interference on hydrogen bond formation by the aqueous environment. In this work, significant recognition property had been achieved in an aqueous media. An imprinting efficiency of 9.21 was observed in the batch-rebinding test while the imprinted nano-particles achieved a separation factor that was almost twice as high as that by the non-imprinted one. Given the viability of mini-emulsion polymerization system as an industrial process, and with the desired properties of the imprinted nano-particles as antibody mimics, this provides a promising way of preparing materials that can be employed in adsorptive bioseparation.

Keywords: Molecular imprinting, Ribonuclease A, nanoparticles, protein, miniemulsion polymerization.