Synthesis Of Protein-imprinted Nanoparticles Via Click Chemistry And Surface-initiated RAFT Polymerization

Molecular imprinting is a separation technology to mimick the specific recognition effect of antigen and antibody in nature and synthesize the molecularly imprinted polymers（MIPs）to choose the predetermined template molecules.MIPs have the advantages of low cost,simple preparation process and high stability.With the development of molecular imprinting technology,the imprinting of proteins and other macromolecules has also attracted much attention.However,there are many difficulties in protein imprinting due to their characteristics such as following:firstly,it’s hard for protein molecules to be eluted and adsorbed because of the mass transfer resistance;secondly,the protein conformation is complex and variable,which unavoidably leads to the difficulties in formation of high-quality imprints.The method of synthesis core-shell protein imprinted nanoparticles（NPs）can effectively reduce the mass transfer resistance of protein;what’s more,MIPs prepared by using reversible addition fragmentation transfer（RAFT）polymerization have the advantages of forming controllable structures and uniform imprinted sites,which is beneficial to the formation of accurate imprinting sites.However,RAFT agent is difficult to be immobilized on the surface of NPs,which often results in insufficient RAFT immobilization;in addition,the dispersion stability of NPs in aqueous solution will be reduced after immobilizing the RAFT agent.To solve the above problems,RAFT agents were immobilized to the surface of SiO₂ nanoparticles by CuAAC click reaction with high efficiency to insure the enough amount of RAFT agent;the carboxyl groups grafted onto the nanoparticles can not only improve the dispersion stability in water but also enrich the template.RAFT residual end groups after RAFT polymerization on the surface can initiate second-step polymerization and post-imprinting functionalization after grafting of PEG on the surface of MIPs can improve selectivity.Therefore,the protein-imprinted nanoparticles with core-shell andcore-shell-corona structures were synthesized controllablyby RAFT polymerization.（1）Surface functionalization of SiO₂ NPsSiO₂ NPs were synthesized by the St（?）ber method,after modification of amino and azide groups,carboxyl was grafted on the surface through condensation reaction of amino and succinic anhydride.EMP-alkyne was attached onto the SiO₂-COOH/N₃NPs via CuAAC click.The amount of grafted RAFT agent was calculated to be 1.53/nm².Attached carboxyl groups can not only ensure the dispersibility of NPs in the aqueous polymeriztion media but also interact with the model print protein,the adsorption capacity reaches 37.4 mg/g.（2）Core-shell Lyz-MIP NPs prepared by surface-initiated RAFT polymerizationLyz-MIP NPs were synthesized above the volume phase transition temperature（VPTT）by using the above functionalized NPs as carriers,lysozyme as template protein,thermoresponsive monomer MEO₂MA as main monomers and ammonium persulfate（APS）as initiator.The shell thickness and recognition properties of Lyz-MIP NPs can be adjusted by controlling the polymerization time.The imprinted factor（IF）was about 2.1 when the optimal polymerization time was 15 h.（3）Post functionalization of imprinted NPs—PMEO₂MA was grafted on the surface of the MIP NPs with using RAFT end groupsPMEO₂MA was grafted from the surface of the core-shell MIP NPs with using RAFT end groups before removal of the template.The results show that the IF is about 9.1 and binding selectivity is improved with reduction of the nonspecific adsorption of NIP NPs as well as the increase of the adsorption capacity of Lyz-MIP NPs.In addition,NPs grafting with PMEO₂MA can reach the saturated adsorption within 10 min;the adsorption isotherm were fitted by Langmuir equation,which showed that the dissociation constant（K_d）of NPs grafting with PMEO₂MA decreased significantly compared with the NPs prepared by conventional free radical polymerization,indicating that RAFT polymerization can improve the affinity to template proteins.