Studies On Adsorption And Surface Imprinting Toward Lysozyme Based On Microsphere HEMA/NVP Grafting Polymerization Of Methacrylic Acid

Homopolymer of N-vinyl pyrrolidone (NVP) and hydroxyethyl methacrylate (HEMA) are polymer materials that both have good biocompatibility and even the blood compatibility, they both have importantly application in the field of biology and medicine. In this study, the functional microspheres PMAA-HEMA/NVP was obtained by grafting of polymethacrylic acid on cross-linked microsphere HEMA/NVP; then its properties and mechanism of adsorption for lysozyme that was the basic protein were explored; finally, the surface imprinting for lysozyme was studied on microsphere HEMA/NVP, and the imprinted materials MIP-HEMA/NVP that has a higher binding capacity and specific recognition properties for lysozyme was obtained. This study has important scientific significance and value in the field of Western blotting and biomedical materials.Firstly , the process conditions were improved about the preparation of cross-linked microsphere HEMA/NVP, The effects of various factors, such as the used amount of redistilled water, the amount of disperser and ratio of oil phase to water phase, on the diameter of the microspheres were examined. The experimental results show that the micron-sized crosslinking microspherers HEMA/NVP with excellent sphericity and with controllable diameter about 90μm can successfully prepared by using inverse suspension polymerization system.Subsequently, the crosslinked microspheres were chemically modified with methacryloyl chloride, and modified microspheres MAO-HEMA/NVP, on which polymerizable double bonds were introduced, were obtained. Finally, the graft polymerization of methacrylic acid (MAA) on the surfaces of microspheres MAO-HEMA/NVP was conducted by adopting"grafting from"method, resulting in the grafted microspheres PMAA-HEMA/NVP. The chemical structures and morphology of the various microspheres were characterized by FTIR and SEM. The effects of the main factors on the graft polymerization of MAA were mainly examined. The experimental results show that the graft polymerization of MAA can favorably carried out on the modified microspheres. During the graft polymerization process, the layer of grafted polymer will form a kinetics barrier to block the graft polymerization. This kinetics barrier leads to no longer varying of the grafting degree of PMAA after 12 h, and leads to that there is a maximum on the relationship curves of thegrafting degree of PMAA vs. temperature and the grafting degree of PMAA vs. the used amount of initiator. For this graft polymerization system, the suitable temperature is 70℃, amount of MAA is 0.3% and a appropriate used amount of the initiator is 0.3% of the monomer mass. the grafted microspheres PMAA-HEMA/NVP that the grafting degree of PMAA was 19.57g/100g can be obtained under the appropriate conditions about.The adsorption property and adsorption mechanism of the grafted microspheres for basic proteins were researched in depth with lysozyme as a model of the basic proteins. The zeta potential of the grafted microspheres was determined, The effects of several factors such as the pH value of the medium, the grafting degree of the microspheres and ionic strength on the adsorption property of the system were examined. The experimental results indicate that in a wider pH range, the zeta potential of the grafted microspheres PMAA-HEMA/NVP is a greater negative value, namely, there is negative charge of high density on the surfaces of the grafted microspheres. By driving of strong electrostatic interaction, the grafted microspheres PMAA-HEMA/NVP exhibit very strong adsorption ability towards lysozyme. The adsorption capacity of the grafted microspheres for lysozyme first increases and then declines with increasing pH value, and a maximum adsorption amount (90mg/g) is produced at pH=9, and it is closed to the isoelectric point of lysozyme. Ionic strength affects the adsorption property of the grafted microspheres greatly. It is found that as pH<9, the adsorption capacity of lysozyme decreases with the increase of NaCl concentration, whereas as pH>9, the adsorption capacity of lysozyme enhances with the increase of NaCl concentration.The modified microspheres MAO-HEMA/NVP was as base microspheres. Then, molecularly imprinted polymers of lysozyme based on the surface-modified microsphere were prepared with methacrylic acid as functional monomer, N,N'-methylene-bis-acrylamide as crosslinker and basic protein lysozyme as a template molecule. In this work, with bovine hemoglobin as contrast substances, both static and dynamic methods were adopted to study the binding properties and recognition character of molecularly imprinted polymers of lysozyme for lysozyme. The experiment results show that molecularly imprinted polymers of lysozyme have specific recognition selectivity and excellent binding affinity for lysozyme, and the maximum adsorption capacity for lysozyme was 216mg/g. In relation to BHb, the selectivity coefficient of molecularly imprinted polymers for lysozyme is 31.070. Besides, molecularly imprinted polymers of lysozyme has excellent elution property, with NaHCO3 solution as eluent, the desorption ratios of lysozyme can reach 96.86%. The optimal preparation conditions of imprinted polymers for lysozyme is determined: pH value is 9 and the ratio of amount of crossing agent and amount of MAA is 11.7:1.