Study On Preparation And Recognition Properties Of Molecularly Imprinted Polymer Microspheres For Glutathione

The research of molecularly imprinting technology(MIT)is mainly focused on the small organic molecules,while rarely cares for the biomacromolecules,especially for proteins and peptides,which is due to the fact that the biomacromolecules have unfavorable factors such as flexible conformation,complex structures,more binding sites and insoluble in most organic solvents.Conventional molecularly imprinted polymers(MIPs)were prepared mainly through traditional free radical polymerization(FRP)with the characteristics of slow initiation,fast growth and quick termination,which may cause the uncontrollably internal cross-linked structure of MIPs,leading to dispersible recognition sits,low binding ability and poor selectivity.Besides,the recognition sites of FRP-MIPs were embedded in the polymeric matrix,which would lead to the elution and mass transfer of the template with difficulty.Moreover,the FRP-MIPs must be grinded and sieved,respectively,before their usage for recognizing target molecules,which would destroy the effective recognition sites of FRP-MIPs.There is no doubt that the development and application of molecularly imprinted polymer materials for biomolecules will be limited.Therefore,it is a promising subject to explore novel preparation methods for the effective imprinting and accurate recognition.The purpose of this research is to effectively and efficiently solve technical problems that exist in the current molecularly imprinting field of biomolecules.In this work,we have proposed a new method of combinating controlled/?living? radical polymerization(CRP)with traditional precipitation polymerization,surface imprinting,template immobilization and miniemulsion polymerization,respectively,for the biological peptide imprinting and recognition.The research was carried out as follows:(1)Monodisperse molecularly imprinted polymers(CRPP-MIPs)microspheres were prepared via CRP precipitation polymerization using glutathione(GSH)as the template.Moreover,the effects of the monomer loading,initiator concentration,molar ratio of cross-linker to functional monomer on the structure and recognition properties of the MIPs microspheres were investigated.In addition,the application of CRP technology in biological peptide molecularly imprinting systems was discussed in detail.The results showed that cross-linked structure of the imprinted polymerscould be controlled by the CRP process effectively.By comparing the FRPP-MIPs prepared via FRP precipitation polymerization,the relationship between cross-linked structure and recognition property was confirmed.The results of batch adsorption tests suggested that the saturated adsorption capacity and imprinting factor of CRPP-MIPscould reach 13.76 mg·g-1 and 2.20,respectively.The selective analysis demonstrated that the specific recognition ability of CRPP-MIPs towards GSH was superior to that of FRPP-MIPs.In addition,the hydrophilic of CRPP-MIPs prepared by surface iniferter polymerization was also investigated,suggesting that the non-specific adsorption of CRPP-MIPs was reduced effectively and the selectivity of CRPP-MIPs towards GSH was improved significantly.(2)Porous imprinted polymers(O-MIPs@PCS)microspheres were fabricated via surface iniferter polymerization using the immobilized living initiator polymers as substrate and a mixed organic solvent as porogen.Following that,the structure,morphology and specific surface area of O-MIPs@PCS were fully characterized.The results revealed that the thickness of imprinted layer of O-MIPs@PCS with porous surface structure was approximately 30 nm and O-MIPs@PCS possessed higher selectivity toward GSH than its analogs.Moreover,the Langmuir isotherm model was fitted to the equilibrium data,and the adsorption equilibrium constant of O-MIPs@PCS towards GSH was 3.57 m L·mg-1 at 25 °C.The kinetic properties of O-MIPs@PCS towards GSH were described by the pseudo-second-order equation,suggesting O-MIPs@PCS have fast adsorption rate.(3)The particle substrate with surface-bound dithiocarbamyl groups and benzyl groups(PVC)was first synthesized by dispersion polymerization and grafting polymerization,respectively,and then the particle with tetraalkylammonium groups(TMA@PVC)were obtained by quaternization reaction.Then the well-ordered GSH molecules were immobilized on the surface of TMA@PVC through anion exchange reaction,and the surface GSH imprinted polymer(MIPs-TMA@PVC)microspheres with uniform recognition sites were synthesized by surface iniferter polymerization.The immobilization template process was fully tracked,which verified that GSH molecules were immobilized on the surface of TMA@PVC through ionic bonding force and the combination between residue groups of GSH and functional monomer by hydrogen bonding interaction.Moreover,the immobilization template process was monitored by Zeta potential analyzer,and the immobilization capacity was 35.25 mg·g-1.In the polymerization process,the thickness of imprinted polymer layer kept a linear relation with the irradiation time and the optimal thickness of 15 nm could be obtained.The results of batch adsorption tests showed that the saturated adsorption capacity and imprinting factor of MIPs-TMA@PVC was 27.28 mg·g-1and 3.68,respectively.Besides,the selective adsorption tests demonstrated a high affinity and selectivity of MIPs-TMA@PVC towards GSH.(4)The GSH imprinted polymers(ILs-MIPs)microspheres with large specific surface area and high selectivity were controllably synthesized via surface iniferter miniemulsion polymerization using the long-chain alkyl ionic liquids(1-dodecyl-3-methyl imidazole bromide)as solubilizer of template and co-stabilizer of continuous phase.The effects of the ionic liquids on the stability of the template-functional monomer complex were investigated by UV-vis spectroscopy,1H-NMR spectroscopy and fluorescence spectroscopy,suggesting that the template-functional monomer complex has a good stability in the miniemulsion polymerization systems.In addition,the effects of the ionic liquids on the apparent morphology and recognition property of ILs-MIPs were investigated in detail.The N2 adsorption tests manifested that the ILs-MIPs have larger specific surface area of 34.58 m2?g-1,well-distributed pore size and good hydrophilicity for improvingthe surface biocompatibility of ILs-MIPs.The results of batch adsorption tests indicated that the saturated adsorption capacity and imprinting factor of ILs-MIPs was 68.24 mg·g-1 and 3.77,respectively.Moreover,the recognition performance of ILs-MIPs was superior to that of MIPs(2.02)without ionic liquids as addition.Besides,the experimental data of ILs-MIPs was fitted to Langmuir isotherm model and the adsorption of ILs-MIPs towards GSH follows the pseudo-second-order kinetic model,indicating that the adsorption process is chemical in nature.Subsequently,the dynamic adsorption tests demonstrated that the ILs-MIPs have excellent selectivity towards the template GSH.