Preparation Of Boronate-affinity Functionalized Adsorbent And Its Application In Ovalbumin Analysis

Protein glycosylation is one of the most important post-translational modifications of proteins.Protein glycosylation affects the structure and function of proteins,not only plays a regulatory role in the important life process of cells,but also is related to the occurrence and development of some diseases,and is the target of many clinical biomarkers and treatments.Therefore,the study of separation and enrichment of glycoprotein has important biological significance and clinical value.Boronate-affinity technology is a commonly used technology to isolate glycoproteins,which means that a reversible covalent interaction between a boric acid group and a compound containing cis-dihydroxy structure can be formed.In this work,two novel boron affinity nanomaterials were prepared based on boron affinity technology,and the selective recognition and separation mechanism of the two materials for glycoprotein-OVA was studied.The specific research contents include the following two parts:Firstly,a boron affinity molecularly imprinted adsorbent was prepared.In this study,MCM-48 was used as the matrix,POSS as the auxiliary monomer,VPBA as the affinity functional monomer of boric acid,OVA as the template molecule,dopamine as the self-polymerization reagent.Synthesis of novel cage-type semisiloxane functionalized borate affinity molecularly imprinted（MCM-48@P-B-MIPs）materials.The material was used as solid phase extraction sorbent for selective enrichment of OVA.Due to its rich boric acid groups and large specific surface area,MCM-48@P-B-MIPs shows excellent adsorbent properties for OVA.The target protein OVA can be selectively distinguished from interfering proteins such as HRP,BHb and Lyz using the specific imprinting sites of molecularly imprinted polymers.MCM-48@P-B-MIPs had good selective adsorption capacity for OVA,with adsorption capacity of 222.1 mg g^-1,adsorption time of 27 min and imprinting factor of 2.1.The adsorbent conforms to the quasi-second-order adsorption kinetics and Langmuir isothermal adsorption model.The method was successfully used for selective enrichment of OVA in egg white samples.Secondly,a metal-organic framework modified boronate affinity adsorbent was prepared.A novel boronate affinity material（Zr-MOF@S-S@B）was prepared using Zr-MOF as substrate,disulfide covalent bond could be eluted at physiological p H and4-mercaptophenylboric acid as boronate affinity monomer.It was used for selective enrichment and successful elution of glycoprotein OVA at physiological p H.The morphology and particle size of the adsorbent were determined by SEM and DLS.Meanwhile,the characteristic structure,thermal stability and elements of the adsorbent were analyzed by FT-IR,TGA and XPS.The conditions of solid phase extraction and the selectivity of adsorbent were optimized and discussed.The affinity between the boric acid group and the cis dihydroxyl group makes the adsorption capacity of OVA reached 625.5 mg g^-1.At the same time,Zr-MOF@S-S@B has good reusability and large specific surface area.In addition,the substance contains disulfide bonds that specifically release surface-bound OVA at physiological p H,effectively avoiding a reduction in OVA activity.The method was successfully applied to selective enrichment of OVA in egg white samples.