A New Method Of Epitope Imprinting Based On RAFT Technology

In recent years,molecular imprinting techniques applied to selective protein identification and separation have gradually attracted the attention of researchers in related fields,however,there are still problems such as poor stability of protein templates and difficulty in controlling the formation of imprint recognition layers.To address the above issues,in this paper,three kinds of molecularly imprinted polymers were prepared on silica particles by a reversible addition-fragmentation chain transfer(RAFT)strategy with cytochrome c(Cytc)terminal peptide as a template and 4-cyano-4-(thiobenzoylthio)pentanoic acid(CPCP)as a chain transfer reagent.The specific selectivity,adsorption isotherm,adsorption kinetic process,reusability and application in real samples of the above imprinted polymers(MIPs)were investigated by high performance liquid chromatography(HPLC).Firstly,epitope imprinted polymers,DMIPs,were prepared on silica microspheres using the C-terminal ninepeptide(AE-9)of Cytc as a template and zinc methacrylate(ZDMA)as the main functional monomer.The imprinted polymer had a binding capacity of 1.02 mg/g for AE-9 with an imprinting factor(IF)of 2.82 and 9.96 mg/g for Cytc with an IF of 1.29,and could reach adsorption equilibrium within 120 min.In addition,the MIPs synthesized with ZDMA as the functional monomer showed better recognition of Cytc than those synthesized with copper methacrylate as the functional monomer.On this basis,the epitope-imprinted materials,CPCP-MIPs,were prepared using ethylene glycol dimethacrylate as a cross-linking agent and zinc methacrylate as a monofunctional monomer.The adsorption performance of CPCP-MIPs for Cytc and AE-9was 64.76 and 9.83 mg/g with IF of 7.96 and 6.17,respectively,and equilibrium for Cytc could be reached within 600 min.After six adsorption-desorption cycles,the adsorption of Cytc by CPCP-MIPs remained above 76.5% of the original.Finally,dual-template epitope-imprinted polymers CPCP-EMIPs were prepared using the C-terminal ninepeptide and N-terminal ninepeptide(GI-9)of Cytc as dual templates with binding capacities of 104.64,10.22 and 8.05 mg/g for Cytc,AE-9 and GI-9,respectively,and IF of 5.14,4.44 and 1.70,respectively.The results showed that the adsorption performance of CPCP-EMIPs on AE-9 was higher than that of GI-9,indicating that the template peptide segment AE-9 had the main contribution in the process of imprint site formation.The imprint polymers could reach adsorption equilibrium for Cytc within 600 min.After four adsorption-elution cycles,the adsorption of Cytc by CPCP-MIPs remained above 77.5% of the original.Among the three imprinting materials prepared above,CPCP-EMIPs had the best binding ability to the target protein,but CPCP-MIPs had the highest IF.The imprinting effect of all three imprinting materials was higher than that of the imprinting materials prepared without the RAFT strategy for both the template peptide and the target protein,indicating that the RAFT strategy played an important role in enhancing the imprinting effect.In the competitive recognition of three different proteins,CPCP-MIPs and CPCP-EMIPs showed high selectivity and low non-specific adsorption to the target proteins.In addition,the results of HPLC analysis confirmed the good selective recognition of Cytc in adult bovine serum samples by CPCP-MIPs 和 CPCP-EMIPs,demonstrating their potential application for the recognition of target proteins in more complex biological samples.