Controllable Preparation And Recognition Mechanism Of Surface Protein Imprinted Magnetic Polymer Microspheres

With the development of the studies of biochemistry and molecular biology for biological phenomena in molecular level,protein structure analysis and protein drugs have been developed rapidly and applied widely.At the same time,the requirements of high purity,high yield and low cost have been put forward for the upstream protein products.Many advanced materials and technologies have been developed and used in the separation and purification of biological macromolecules such as proteins.The imprinted polymer based on molecular imprinting technique has attracted much attention because of the advantages of high stability,low cost,easy preparation and specific recognition.Among them,the magnetic imprinted materials become a hot research topic due to the characteristic of easy separation and simple operation.But so far,the research progress of protein and other biological macromolecules imprinted polymers is still slow.The recognition mechanism is not mature.Moreover,the regeneration of protein imprinted polymer is difficult.The effect of carrier surface property on imprinted site is not clear.The non-specific adsorption capacity of imprinted polymer surface will decrease the recognition performance.These scientific issues have yet to be further studied.Based on this background,using the magnetic microsphere coated with polymer as the imprinted carrier,the structure,regulation and recognition performance of magnetic imprinted materials are studied systematically for protein separation and enrichment.And the surface protein imprinted magnetic microspheres with high adsorption capacity,high selectivity and strong regeneration performance are prepared.The main topics of this thesis are as follows.In order to solve the problem that the elution and regeneration of protein imprinted polymers were difficult,the surface imprinting and temperature-sensitivity were combined together and a novel bovine serum albumin?BSA?surface imprinted thermosensitive magnetic microsphere?Fe₃O₄@SiO₂@BSA-MIP?was prepared in this section.The adsorption model of template protein was established,and the recognition mechanism was elucidated.The results showed that the Fe₃O₄@SiO₂@BSA-MIP microsphere possessed excellent temperature-sensitivity.The adsorption and desorption of Fe₃O₄@SiO₂@BSA-MIP to BSA could be controlled by external temperature.The adsorption capacity and the imprinting factor of Fe₃O₄@SiO₂@BSA-MIP to BSA decreased with the decrease of temperature.When the temperature was 21?,the desorption rate after once elution could be up to 90.81%.The studies of adsorption kinetics and adsorption isotherms showed that the adsorption recognition process of Fe₃O₄@SiO₂@BSA-MIP to BSA was divided into three stages:BSA closing to the surface of Fe₃O₄@SiO₂@BSA-MIP,BSA permeating in the imprinted layer;imprinted site capturing BSA.The adsorption process was in accordance with the Langmuir adsorption model.In the respect of imprinting recognition mechanism,the shape memory effect,the size of proteins and the multiple noncovalence interactions provided by functional co-monomers with proteins were the major factors affecting the imprinting formation and template recognition.Thermosensitive imprinted layer could improve the elution and regeneration performance of imprinted materials effectively.However,the introduction of thermosensitive monomer would decrease the strength of polymer matrix and affect the selectivity of imprinted site to template molecule.Therefore,the structure of thermosensitive imprinted layer was optimized by controlling the thickness of imprinted layer and the degree of cross linking.The research results showed that too thin or too thick imprinted layer were not conducive to the imprinting effect.When the imprinted layer was too thin,the obtained imprinted site was not complete.When the imprinted layer was too thick,the effective utilization of imprinted site decreased.Too high or too low degree of crosslinking were also not conducive to the imprinting effect.The optimal imprinted layer thickness for recognition of BSA was determined to be 17nm.Meanwhile,the optimal crosslinking degree was also obtained and the numerical value was 20%.Under such conditions,the adsorption capacity and the imprinting factor could reach 42.01 mg/g and 3.41,respectively.The elution efficiency after once washing process was up to 78.60%.Furthermore,the strategy was also applied to another template protein?Lyz?.In summary,on the premise of guaranteeing excellent temperature sensitivity,the control of crosslinking degree provided an effective means for improving the recognition performance of thermosensitive MIPs.In order to study the effect of functional group on the surface of carrier on imprinted site,the Fe₃O₄ particles coated by hydroxyethyl acrylate layer modified with maleic anhydride were used as the carrier and the surface protein imprinted magnetic microspheres with controllable carboxyl group density at core-shell interface?Fe₃O₄@HEA@protein-MIP?were designed and prepared.The effect of carboxyl group density at core-shell interface on the imprinting efficiency of different template proteins and the adsorption kinetics were studied,and the relationship between the critical thickness of imprinted layer and the size of template protein was illustrated.The results showed that increasing carboxyl group density at the interface could improve the adsorption rate,adsorption capacity and recognition performance of the imprinted site to template protein.Meanwhile,the increase of carboxyl group density at the interface was more conducive to the improvement of imprinting efficiency for the proteins with high isoelectric point in pH 7.0 solution.At last,the effect of carboxyl group at the core-shell interface on imprinting efficiency was restricted by the thickness of imprinted layer.That is,the thickness of imprinted layer should be smaller than a critical value,and the critical value was proportional to the size of template protein.For reducing the nonspecific adsorption on the surface of imprinted polymers to further improve recognition performance,a novel BSA surface-imprinted magnetic microsphere with2-methacryloyloxyethyl phosphorylcholine?MPC?in the imprinted layer was fabricated.The adsorption kinetics,thermodynamics and selectivity of the magnetic imprinted microspheres containing MPC were studied systematically.The results indicated that the introduction of MPC could significantly inhibit the non-specific adsorption of BSA on the surface of the imprinted polymer.When the content of MPC was 10 mol%,the imprinting factor?8.32?of imprinted microsphere to BSA was the highest,which was far higher than the one?1.90?of the imprinted microsphere without MPC.However,the adsorption capacity was only 21.79mg/g.Compared with the one of the imprinted microsphere without MPC?32.11 mg/g?,it decreased significantly.Meanwhile,the longer adsorption equilibrium time indicated that the presence of MPC polymer in imprinted layer might slightly hinder the combination of template proteins with imprinted sites.Even if the introduction of MPC would lead to the bad phenomenon,it could significantly increase the recognition specificity of the BSA-imprinted microsphere.At last,this strategy had an excellent versatility and was also suitable for another template,lysozyme.To give consideration to both selectivity and adsorption capacity,the AGET-ATRP method was used to replace the copolymerization method in this section.Then,the surface BSA imprinted magnetic microspheres?Fe₃O₄@SiO₂@Pdop-MIPs@PMPC?with anti-protein nonspecific adsorption were prepared by grafting MPC onto the surface of imprinted layer.The effect of the length of MPC chain segment on the imprinting efficiency and nonspecific adsorption were studied.The results showed that when the MPC chain segments were too short,the protein resistance effect would be very weak and it could not reduce the non-specific adsorption of the imprinted layer for proteins effectively.On the other hand,when the MPC chain segments were too long,the protein resistance effect and rocker effect would be very strong.These characteristics easily hindered the binding of imprinted sites and template proteins.Thus,MPC/Fe₃O₄@SiO₂@Pdop-MIPs-Br mass ratio=12/1 was most appropriate.The imprinted factor and adsorption capacity of the optimized imprinted microspheres for BSA were 5.74 and 8.26 mg/g,respectively.Compared with the adsorption capacity of Fe₃O₄@SiO₂@Pdop-MIPs without MPC?8.87 mg/g?,the adsorption capacity of Fe₃O₄@SiO₂@Pdop-MIPs@PMPC with MPC for BSA only decreased 6.88%,which was much smaller than the one?32.14%?of copolymerization method.This phenomenon indicated that the introduction of MPC by AGET-ATRP method possessed advantages in improving the using performance of the materials.