Preparation And Adsorption Properties Of Magnetic-thermal Dual Responsive Poly N-isopropylacrylamide Based Lysozyme Molecularly Imprinted Materials

The efficient selective identification,separation and purification of proteins is extremely important for genetic engineering,biomedicine,life sciences and other researches.The development of new protein separation technologies and the development of protein selective identification materials have important application value.Lysozyme is an antibacterial active substance and a component of the body’s immune defense mechanism.Studies in recent years have shown that lysozyme can be used for clinical diagnosis of acute leukemia,renal tubular damage,meningitis,central nervous system tumors and other diseases.Its demand is increasing year by year,and it is of great significance to achieve efficient and rapid separation and purification.Protein imprinted materials can specifically recognize the template protein,is simple to prepare,has stable properties,and can be reused;The lysozyme molecularly imprinted material has great research and application value.In this paper,thermo responsive materials for protein affinity adsorption were synthesized and studied from the chemical engineering unit operation perspective.PNIPAM is used as the main raw material and lysozyme as the model molecule.The adsorption capacity,temperature-controlled adsorption/desorption,adsorption kinetics were studied.A mathematical model for adsorption kinetics was constructed.The main contents and conclusions of this dissertation are as follows:（1）Synthesis of Lysozyme affinity adsorption microgels.Affinity adsorption microgels are synthesized using N-isopropylacrylamide and N-Vinylcaprolactam as main raw materials.Monomers containing complementary function groups of protein amino acid residue are introduced.Factors that influences the adsorption properties,affinity precipitation and LCST of the microgels are studied.Results indicate that the LCST of the microgels gradually increased with the increase of the MAA content.And the LCST of the polymer disappeares when MAA content is greater than 10.00%.TBAM decreased the LCST of the microgels.MAA can improve the adsorption capacity for Lysozyme,TBAM has little effect on the adsorption capacity.MBA content for best adsorption capacity is 2.00%.PNVCL-based microgels have better self-flocculation separation performance than PNIPAM.The precipitation rate is close to 90.00%,while PNIPAM-based microgels do not self-flocculate.（2）Improved separation performance of the gels by grafting from Si O₂ microspheres.A series of Si O₂ with different sizes are synthesized by the improved St（?）ber method.The composites are prepared by grafting PNIPAM-based molecular imprinting polymer from Si O₂ surface by ATRP.The adsorption capacity,temperature-responsive adsorption-desorption and adsorption kinetics of Lysozyme are studied.Results indicate that the average adsorption capacity of the composite nanospheres is 70.37 mg·g^-1 when the temperature is higher than LCST,and the average adsorption capacity is 70.37 mg·g^-1 when the temperature is lower than LCST.The adsorption capacity is 14.63 mg·g^-1,and 79.20%of the adsorption capacity can achieve temperature-controlled adsorption-desorption;after 7 cycles,the adsorption capacity does not decrease significantly,and the composite nano-microspheres have good stability;isothermal adsorption and kinetic adsorption The model fitting indicates that the adsorption of Lysozyme by the composite nanospheres is Langmuir EXT1 isothermal adsorption and pseudo-first-order kinetic adsorption.（3）Superparamagnetic Fe₃O₄ enhanced separation of the gels.ATRP of PNIPAM is induced on the surface of Fe₃O₄@Si O₂ to prepare magnetic-temperature dual-responsive Lysozyme-imprinted nanospheres.The microscopic morphology of the nanospheres is observed by SEM and DLS,and the chemical composition of the nanospheres is characterized by FT-IR,¹HNMR and XRD.The thermal stability and surface grafting rate of the nanospheres analyzed by TGA.The lowest critical solution temperature（LCST）of the nanospheres is determined by UV-Vis.The selective adsorption of the nanospheres is analyzed by SDS-PAGE.Results shown that the adsorption of Lysozyme can be controlled by temperature.Lysozyme is adsorbed when the temperature is higher than LCST and is desorbed when the temperature is lower than LCST.The fitting of kinetics showed that the model of adsorption is Langmuir EXT1 isotherm adsorption and follows a pseudo-first-order kinetic adsorption.The equilibrium adsorption capacity was 117.12 mg·g^-1.The adsorption reached equilibrium in 50 min.Magnetic separation is achieved in 3 min.SDS-PAGE indicates that the nanospheres are selective for Lysozyme.Compared with non-imprinted polymer,the adsorption capacity of Lysozyme increased by 33.74%.This material is suitable for the affinity adsorption of Lysozyme.