Preparation And Performance Evaluation Of N-terminal Epitope Imprinting Materials

In recent years,the research on high selective recognition and efficient separation of proteins by molecular imprinting technique had attracted more and more attention of researchers in related fields.However,there were still some difficulties,such as poor stability of protein templates and uncontrollable formation of imprinting layers.To solve the problem of poor stability of the protein template,the epitope of the protein was used to replace the whole protein as the template,and the polymerization process of forming imprinted recognition layer was regulated by reversible addition-fragmentation chain transfer(RAFT)strategy.In this work,the template N-terminal nonapeptide epitope of Cytochrome c was immobilized on the surface of silica spheres through covalent interaction using silica microspheres as the matrix.Methacrylic acid and its derivatives were used as functional monomers,and methylene bisacrylamide was used as crosslinking agent.Under the regulation of trisulfide,the core-shell epitope molecular imprinted polymer(EMIP)was obtained by thermally initiated RAFT radical polymerization.The binding capacity of EMIP to the template N-terminal epitope peptide(GI-9)reached 1.88 mg/g with imprinting factor(IF)as 2.42 and the adsorption capacity of the target protein Cytochrome c(Cyt c)reached 8.89 mg/g with IF as 1.71,which could reach the adsorption equilibrium within240 minutes.Among the competitive recognition of three proteins with different molecular weights and isoelectric points,EMIP had the highest selectivity and low nonspecific adsorption to Cyt c.The above recognition performance was obviously better than that of the imprinted materials prepared without RAFT strategy for GI-9 and Cyt c.After five cycles of adsorption and desorption,the protein recognition performance of EMIP could still maintain more than 90% of the first recognition.On the basis of the above work,the thermosensitive epitope-imprinted polymer(TS-EMIP)was prepared by RAFT strategy using the thermosensitive monomer N-isopropylacrylamide as the main functional monomer.The adsorption and elution of peptides and proteins were achieved at 45℃ and 25℃,respectively.The binding capacity of TS-EMIP to GI-9 was 1.50 mg/g with the imprinting factor(IF)as 2.33 and the adsorption capacity of Cyt c reached 8.39 mg/g with IF as 2.16.The adsorption equilibrium was achieved within 240 minutes.It had the highest selectivity and lower non-specific adsorption to Cyt c among the competitive recognition of three proteins with different molecular weights and isoelectric points.The imprinted material prepared by RAFT strategy showed significantly better imprinting factor for Cyt c than the imprinted material prepared without RAFT strategy.After five times of repeated adsorption and desorption,it still maintained relatively good stability and could be reused for many times.The above results indicated that the identification of the target protein was achieved by using the N-terminal epitope peptide of the protein to replace the whole protein as a template for imprinting.The polymerization process of the imprinted recognition layer was regulated by the RAFT strategy,which realized the higher recognition ability,recognition efficiency and recognition selectivity of the imprinted material for the target protein.This method was expected to be applied to the selective recognition and separation of target proteins in biological samples.