Preparation of dual-epitopes imprinted particles with γ-cyclodextrin host-guest interaction and reversible addition-fragmentation chain transfer strategy for cytochrome c collaborative recognition

A dual-epitopes imprinted strategy for cytochrome c selective recognition assisted with γ-cyclodextrin host-guest interaction via N-terminal and C-terminal epitope's simultaneous imprinting and reversible addition-fragmentation chain transfer (RAFT) polymerization was developed. N-terminal and C-terminal nonapeptides of Cyt c (GI-9 and AE-9) were used simultaneously as the epitope to achieve collaborative recognition for cytochrome c. As a supramolecule, γ-cyclodextrin can encapsulate the aromatic functional groups of amino acid residues to capture the peptide and improve the corresponding spatial orientation for epitope or cytochrome c recognition by host-guest interaction. After the γ-cyclodextrin modification and dual-epitopes immobilization, the imprinted polymer was synthesized by RAFT polymerization with 4-cyano-4-(phenyl-carbonothioylthio) pentanoic acid as a chain transfer agent. After the template removal, the obtained dual-epitopes imprinted particles showed well binding ability to AE-9 (26.50 mg·g⁻¹, IF= 4.13), GI-9 (7.36 mg·g⁻¹, IF= 2.18) and cytochrome c (79.56 mg·g⁻¹, IF= 3.27). With the successive addition of RAFT agent, the imprinting factor rising of epitope peptide and cytochrome c further illustrated the regulation of imprinted polymer chains. The imprinted particles had the advantage for cytochrome c recognition compared to other proteins and good reusability with 82.60 % repeated reproduction rate after six cycles of adsorption and desorption. Furthermore, the selective recognition for cytochrome c in bovine serum proved its potentiality to be applied in complex biological samples. It indicated that the combination of dual-templates epitope imprinting, γ-CD host-guest interaction and RAFT polymerization provided an efficient method for collaborative protein recognition with well selectivity, reusability and stability.