Selective Recognition And Separation Of Glycoproteins Based On The Surface Imprinted Nanomaterials And Boronate Affinity

Glycoprotein is one of the most important living matter,which functions critical roles in numerous crucial biological events.Especially,glycoproteins have been frequently employed as clinical biomarkers in clinical diagnostics.However,glycoprotein analysis remains a great challenge,for its tedious pretreatment procedures,inherent low-abundance and severe interference of high-abundance substances in the complex biological components.To this day,multifarious strategies have been used for glycoprotein enrichment.But,these strategies still with several limitations,such as complex operation,high cost and poor stability,furthermore,they could not recognize a specific glycoprotein from its analogs.Boronate affinity has been widely used in the separation of glycoprotein for its special reversible covalent interaction between boronic acid and glycoproteins.Besides,surface imprinting has also been frequently applied to the selective recognition and separation of glycoprotein,which colud improve the mass transfer efficiency.Therefore,considering the advantages of boronate affinity and surface imprinting,great efforts have been dedicated to the integration of surface imprinting and boronate affinity into one system for developing a novel kind of glycoprotein adsorbent,which could be applied to the selective recognition and separation of glycoprotein.In this work,combining the merits of surface imprinting and boronate affinity,three new kinds of boronate affinity-based glycoprotein imprinting materials were prepared and applied to the recognition and separation of glycoprotein.The behavior and mechanism of selective recognition for OVA were further studied.Generally,the main parts of the research were illustrated as follows:（1）Preparation of dendrimeric boronic acid-functionalized surface molecularly imprinted polymers and the study of behavior and mechanism of selective adsorption for glycoproteinFirstly,aldehyde was modified to the surface of amino modified SiO₂nanoparticles,besides,dendriticPEIwasfurthergrafted.Secondly,4-formylphenylboronic acid was successfully grafted onto the SiO₂ through aldimine condensation,respectively.After the adsorption of template glycoprotein OVA,dopamine was added and deposited on the surface of SiO₂.The dendrimeric boronic acid-functionalized surface molecularly imprinted polymers（SiO₂-MIPs）were obtained after removing the OVA.The physical and chemical properties of SiO₂-MIPs were characterized by multiple means,and the results showed that the obtained SiO₂-MIPs were monodisperse and with an obvious core-shell structure.Furthermore,binding experiments demonstrated that SiO₂-MIPs possessed enhanced adsorption capacity,fast kinetics and excellent selectively recognition ability towards OVA,which could reach adsorption equilibrium at 60min and the corresponding maximum adsorption capacity for OVA was 243.4 mg/g.In general,due to the introduction of dendrimeric boronic acid,SiO₂-MIPs have enhanced the binding strength and adsorption selectivity to glycoprotein.（2）Preparation of surface molecularly imprinted polymers with double recognition sites and the study of behavior and mechanism of selective adsorption for glycoproteinFirst,metal ion affinity sites were formed by fixed iminodiacetic acid on the surface of PSG and immobilized Cu²⁺;then the template glycoprotein OVA was immobilizated;third,3-aminophenylboronic acid（APBA）was added,which was both recognition monomer and imprinting monomer;finally,surface molecularly imprinted polymers with double recognition sites（PSG-MIPs）were get after the elution of OVA.The composite PSG-MIPs have effectively solved the tedious preparation of traditional adsorbents.The results of binding experiments demonstrated that PSG-MIPs with high adsorption capacity,highly specific recognition,and rapid binding rate.These materials showed great potential in the biomedical andbiotechnological applications and provided a new strategy for the preparation of boric acid adsorbents.（3）Preparation of magnetic surface imprinted polymers based on the teamed boronate affinity and the study of behavior and mechanism of selective adsorption for OVA under physiological pHIn order to achieve selective separation of glycoprotein at pH=7.4,magnetic molecularly imprinted polymers integrated with low pKa teamed boronate affinity（Fe₃O₄@PGMA-TBA/MIPs）were fabricated.The teamed boronate affinity（TBA）wasformedbyboron-nitrogen（B-N）coordinationbetween1,6-hexamethylenediamine and 3-aminophenylboronic acid,and then was fixed on the surface of magnetic poly（glycidyl methacrylate）（Fe₃O₄@PGMA）through ring-opening reaction.After immobilizing the template glycoproteins（ovalbumin,OVA）,surface imprinting layer was deposited onto Fe₃O₄@PGMA-TBA surface via redox polymerization of aniline,and Fe₃O₄@PGMA-TBA/MIPs with obvious core-shell-shellstructurewerepreparedbyremovingtemplate.Fe₃O₄@PGMA-TBA/MIPs were exhibited superparamagnetic propertyand magnetic stability after multiple regenerations.Additionally,taking advantage of surface imprinting and TBA,as-prepared Fe₃O₄@PGMA-TBA/MIPs posed high binding capacity and fast capture kinetics toward OVA under physiological pH.More importantly,becauseoftheTBAwaselectroneutralatpH=7.4,Fe₃O₄@PGMA-TBA/MIPs not only exhibited superior specific recognition toward OVA,but also maintained the activity of OVA from practical samples analysis.Therefore,this work opened up a universal route for developing intelligent controllability molecular imprinting materials for the specific separation of glycoproteins in biomedical filed under physiological pH.