Preparation of N-terminal temperature-sensitive epitope imprinted particles by reversible addition-fragmentation chain transfer strategy for cytochrome c recognition

Abstract N-terminal epitope-imprinted temperature-sensitive polymer on silica particle was prepared using a reversible addition-fragmentation chain transfer (RAFT) strategy, with the N-terminal nonapeptide of cytochrome c as template. 2-Dodecylsulfanylcarbothioylsulfanyl-2-methylpropionic acid (DMP) was used as a chain transfer agent to regulate the polymerization of the imprinted layer, thereby enhancing the recognition performance for the peptide and target protein. After the epitope modification, a surface-imprinted polymer with a controlled imprinted layer was synthesized using monomers and crosslinkers via the RAFT radical polymerization method. As the classic temperature-sensitive functional monomer, the addition of N-isopropylacrylamide imparts temperature-responsive properties to the prepared imprinted polymer. After optimizing the ratio of functional monomers and the recognition temperature, the imprinted particles achieve optimal adsorption capacity and imprinting factor (IF) at a recognition temperature of 45°C. The highest adsorption capacity for GI-9 is 1.50 mg/g, with an IF of 2.33, while the maximum adsorption capacity for Cyt c reaches 8.39 mg/g, with an IF of 2.16, which were clearly higher than the IF of the material without the RAFT strategy. Furthermore, the temperature-sensitive imprinted polymers exhibit strong binding capacity and a high IF for Cyt c, enabling selective recognition within multi-protein mixtures. The protein recognition performance remained at approximately 89.3% after five adsorption-desorption cycles, with almost no decrease of IF. All these results indicate that the imprinted particles exhibit high selectivity, excellent reusability, and sensitivity to recognition temperature conditions. It is expected to be further applied in the selective recognition and controlled release of target proteins.