Research And Application Of Boronic Acid Affinity Based Glycoprotein Imprinted Polymers

Glycoproteins are important component in the human body and are marker of many diseases.However,in biological samples with complex content and many interfering substances,lower levels of glycoproteins often fail to meet the requirements of disease diagnosis,sensing,and bioimaging,so the separation and enrichment of glycoproteins in biological samples appears extremely important.So,this paper selected three glycoproteins（ovalbumin,OVA;transferrin,TRF;horseradish peroxidase,HRP）based on the advantages of simple preparation,strong recognition selectivity,and reusability of molecularly imprinted materials.as the target,and magnetic nanomaterials were used as carriers to simplify the subsequent separation process.Based on the reversible affinity between boronic acid and the adjacent diol groups on glycoprotein sugar groups,the selectivity of the materials was improved.The polymerization reaction at room temperature and in aqueous solution was used.In order to reduce the chance of protein denaturation during the preparation process,molecularly imprinted materials of these three glycoproteins were prepared,and the morphology and structure of the materials were characterized,and the adsorption properties of the materials were studied.The specific research contents are as follows:（1）Ovalbumin-imprinted magnetic nanomaterials based on boric acid affinity and dopamine polymerization.Ovalbumin（OVA）was selected as the glycoprotein model,phenylboronic acid-modified magnetic Fe₃O₄ nanomaterials were used as the carrier,and dopamine was used as the polymerized monomer.After optimizing the preparation conditions,including the amount of template molecules and monomers,prepared at room temperature An molecularly imprinted material for imprinting ovalbumin（Fe₃O₄-PDA MIPs）based on phenylboronic acid affinity,and the structure and morphology of the material were characterized.Through adsorption experiments,it was found that due to the introduction of phenylboronic acid affinity and the application of surface imprinting technology,Fe₃O₄-PDA MIPs had high equilibrium adsorption capacity,fast adsorption equilibrium speed and good adsorption selectivity,and the equilibrium adsorption capacity was 110.6 mg/g,the imprinting factor reached 1.76;while its adsorption capacity for other glycoproteins（such as human transferrin,TRF;horseradish peroxidase,HRP）and non-glycoproteins（bovine serum albumin,BSA）was only 68.9,72.4 and 46.2 mg/g,the imprinting factor was only 1.24,1.44 and 1.31.In addition,because the dopamine that can self-polymerize at room temperature is introduced as the polymerization monomer,the preparation process is simplified;because the magnetic nanomaterial is introduced as the carrier,the subsequent separation process is simplified.（2）Research on the preparation of magnetic imprinting materials for imprinting transfrrin based on boric acid affinity and microemulsion polymerization.In order to separate and enrich transferrin（TRP）in biological fluids,2,3-difluoro-4-formylphenylboronic acid（DFFPBA）,a phenylboronic acid derivative with smaller p Ka,was selected as the affinity ligand for TRP.,so that the use p H of the prepared TRP molecularly imprinted polymer is within the physiological p H range.Then,magnetic nanoparticles Co Fe₂O₄@Si O₂ were prepared by solvothermal reaction and emulsion polymerization as a carrier,and then DFFPBA was modified on the surface of Co Fe₂O₄@Si O₂,and then TRP was immobilized on the surface of TRP by reacting phenylboronic acid with the o-dihydroxyl group on the sugar group of TRP.On the carrier,Molecularly imprinted polymer for imprinting TRF Co Fe₂O₄@Si O₂-TRF-MIPs based on the affinity of phenylboronic acid was prepared at room temperature using tetraethyl orthosilicate（TEOS）as the polymerized monomer.Under the optimized experimental conditions,the adsorption of TRF by the molecularly imprinted polymer was based on monolayer chemisorption.),horseradish peroxidase（HRP）and bovine serum albumin（BSA）,the adsorption of TRP has high recognition selectivity and has a good application prospect.（3）Magnetically Imprinted Nanomaterials of horseradish peroxidase based on boric acid affinity.In order to obtain the separation and enrichment material with simple,rapid,high selectivity and high adsorption capacity for horseradish peroxidase（HRP）,HRP was selected as the template molecule,N-isopropylacrylamide（NIPAM）and 4-Vinylbenzene boronic acid（VPBA）was used as the monomer,and N,N’-methylenebisacrylamide（MBA）was used as the cross-linking agent.HRP was immobilized on its surface by affinity,and polymerized at room temperature in the presence of monomer NIPAM and cross-linker MBA to obtain horseradish peroxidase-imprinted magnetic material Fe3O4@NIPAM-HRP-MIPs.The adsorption of HRP by this material depends on the monolayer chemisorption of HRP and the imprinted sites on the surface of the material.The adsorption of HRP has a fast equilibrium speed,the equilibrium adsorption amount is 120.3 mg/g,and the imprinting factor IF reaches 2.37.Moreover,compared with ovalbumin（OVA）,transferrin（TRF）and bovine serum albumin（BSA）,it has higher adsorption selectivity for HRP,which has the prospect of enriching and separating HRP in practical samples.