The Preparation Of Functional Magnetic Materials With Boric Acid And The Application Of Specific Separation Of Glycoprotein

As one of the important post-translational modifications of proteins,glycosylation of proteins is widely found in more than 50% of proteins and affects the structure and function of proteins,not only plays a regulatory role in important life processes of cells,but also some physiological processes of the occurrence and development of some diseases are related to protein glycosylation.Therefore,the study of glycoproteins has important biological significance and clinical value.However,the low abundance of glycoproteins in biological samples and the microscopic heterogeneity of sugar chains,so the separation and enrichment of glycoproteins is an important prerequisite for research.At present,there have been many reports on the separation and enrichment of glycoproteins,among which the method of carrying phenylboronic acid on the surface of magnetic nanomaterials as an affinity ligand has been widely used.However,there are some problems in the application of phenylboronic acid functionalized magnetic materials,such as the target proteins after binding need to be eluted under acidic conditions,but the conditions of acid elution are inconsistent,and the conditions of overacidity will also affect the glycoprotein structure to some extent.On the other hand,the application of sensitive materials will be limited,besides,the coprecipitation of non-glycoproteins in acid elution condition give rise to false positive,cause the waste of time and economic on follow-up experiments.To solve the above problems,in this study,the magnetic nanomaterials were firstly coated with carbon,which not only improved the hydrophilicity of the materials to resist the problem of non-specific adsorption,but also provided an active site for further modification.Then a functional magnetic material of phenylboronic acid connected by disulfide bond was prepared?Fe₃O₄@C-AC-SS-MPBA?,By means of the reduction sensitivity of disulfide bond under mild conditions,the glycoprotein on the surface of the material was specifically released.In order to distinguish false positive,another functional boric acid material was prepared?Fe₃O₄@C-AC-APBA?,which has the same phenylboronic acid shell skeleton as Fe₃O₄@C-AC-SS-MPB,but there is no reduction sensitivity chemical bond.The above two kinds of boric acid functionalized magnetic materials are used for the precise separation and identification of glycoproteins.First,preparation and characterization of two functional magnetic materials of boric acid,that is,Fe₃O₄ magnetic nuclei were synthesized by solvent heat method,The magnetic cores were then coated with a glucose solution containing acrylic acid as a carbon source,forming Fe₃O₄@C-AC magnetic nanoparticles of core-shell structure,and then we decorate it based on that,two kinds of magnetic materials functionalized with boric acid?Fe₃O₄@C-AC-SS-MPBA and Fe₃O₄@C-AC-APBA?were synthesized,a series of methods such as IR?XPS?TGA were used to characterization analysis,the reduction sensitivity of disulfide bond was tested by ultraviolet spectrophotometer,the result provide the good reduction sensitive bond breaking effect.Then the specific binding ability of two magnetic nanomaterials functionalized with boric acid to glycoproteins was verified by protein adsorption experiment and protein adsorption kinetics experiment,Subsequently,FITC-labeled glycoprotein OVA was used to test the protein release by broken bonds from Fe₃O₄@CAC-SS-MPBA material surface,the reliability of Fe₃O₄@C-AC-APBA as control group was also verified.Finally,the system was used to separation and identify the glycoprotein from two biological samples of egg serum and human serum,the results show that the system is efficient and accurate for glycoprotein identification.