Study On The Methodology Of Separation Of Polysaccharide Receptors On Cell Membrane Using The Constructed Polysaccharide Magnetic Nanoprobe

Polysaccharides have a wide range of biological activities,and binding to the polysaccharide receptor protein on the cell membrane is the key to their physiological functions.Therefore,the study of polysaccharide receptor is of great significance to elucidate the biological activity and molecular mechanism of polysaccharides,and the study of polysaccharide receptor on the cell membrane has become a research hotspot.At present,most of the research methods of polysaccharide receptors are based on the known receptor types to screen the polysaccharide drug receptors,but there is a lack of effective identification and separation methods for the unknown receptor proteins of some polysaccharide drugs on the membrane.Although the structure of polyhydroxyl benzene ring of polysaccharides is complex and diverse,there are few groups that can be used for recognition or chemical labeling,and UV does not have characteristic absorption.Therefore,there are some difficulties in the study of polysaccharides and proteoglycans,which also leads to the study of polysaccharides far behind the study of protein.Iron oxide nanoparticles have shown good application prospects in various biomedical fields.In particular,magnetite Fe₃O₄ has been successfully used in biological separation due to its advantages of good biocompatibility,chemical stability,harmlessness,high saturation magnetization value and low cost.Compared with traditional affinity column chromatography,the magnetic separation method has advantages in biological specificity,enrichment recovery,simplicity of operation and cost.At present,there are few reports on the separation of proteins on cell membranes by Fe₃O₄ magnetic nanoprobe functionalized by surface polysaccharides.The conditions for coupling magnetic nanoparticles with polysaccharide biomolecules need to be explored further without affecting polysaccharide activity.In this paper,polysaccharides were modified on the surface of magnetic nanoparticles to construct a polysaccharide magnetic nanoprobe,and the methodology of ligand-receptor binding and magnetic separation of polysaccharide receptors was studied.The research contents of this paper are as follows:1.Using Fe₃O₄ MNPs as magnetic separator,two kinds of magnetic nanoparticles,Fe₃O₄@SiO₂-NH₂ and Fe₃O₄@SiO₂-COOH,were synthesized by forming SiO₂ core-shell coating structure on the surface of Fe₃O₄ MNPs,and then treated with amino and carboxyl groups on the surface.The structure and properties of the magnetic nanoparticles were analyzed by means of laser particle size analyzer and Zeta potential analyzer,X-ray crystal diffraction,infrared spectroscopy and magnetic property detection.The results showed that the two magnetic nanoparticles were successfully synthesized with good water solubility,high stability and superparamagnetism.2.A functionalized Fe₃O₄@SiO₂-NH₂-?-D-Glucan magnetic nanoprobe was synthesized by coupling Fe₃O₄@SiO₂-NH₂ MNPs with?-D-Dlucan by reductive amination reaction.The results showed that Fe₃O₄@SiO₂-NH₂-?-D-Glucan magnetic polysaccharide nanoprobe was successfully synthesized by infrared spectroscopy,laser particle size analyzer and Zeta potential analyzer.3.Using the condition that the proteoglycan SBT of Songshan ganoderma lucidum contains 10-20%protein,using EDC/NHS combination,Fe₃O₄@SiO₂-NH₂and Fe₃O₄@SiO₂-COOH MNPs were conjugated with the proteoglycan SBT,respectively.To try to synthesize functionalized Fe₃O₄@SiO₂-NH₂-SBT and Fe₃O₄@SiO₂-COOH-SBT magnetic nanoprobes.The experimental results showed that the construction of Fe₃O₄@SiO₂-NH₂-SBT polysaccharide magnetic nanoprobe was successfully realized.However,no new bonds were found in the infrared spectroscopic analysis of Fe₃O₄@SiO₂-COOH-SBT.In order to further analyze the reasons for the failure of Fe₃O₄@SiO₂-COOH-SBT construction,bovine serum protein was selected to conjugate with Fe₃O₄@SiO₂-COOH MNPs.The results of infrared spectroscopy and SDS-PAGE gel electrophoresis showed that Fe₃O₄@SiO₂-COOH-BSA MNPS was successfully constructed.It is speculated that although proteoglycan SBT contains protein,the coupling between SBT and Fe₃O₄@SiO₂-NH₂ is mainly achieved by the reductive end of polysaccharide due to the connection mode of polysaccharide and protein and the low protein content.4.To compare and explore the methods of polysaccharide receptor protein separation with polysaccharide magnetic nanoprobe.Method 1:The probe was co-incubated with cells.Firstly,the polysaccharide magnetic nanoprobe was recognized by the receptor on the cell membrane,and then the cells were collected and broken,and the polysaccharide receptor protein was magnetically separated under the action of external magnetic field.Method 2:First,the total protein of cells was extracted,and the magnetic polysaccharide nanoprobe was mixed into the total protein at 4?,and the polysaccharide receptor protein was magnetically separated under the action of external magnetic field.The receptor proteins were characterized by SDS-PAGE gel electrophoresis or Western Blot.The experimental results showed that both of the above methods could enrich and recover receptor proteins.It was found in the experiment of using Fe₃O₄@SiO₂-NH₂-?-D-Glucan probe to separate the receptor that more receptor protein could be obtained in the first method.In the experiment of using Fe₃O₄@SiO₂-NH₂-SBT probe to isolate the receptor,it was found that the second method obtained more receptor proteins.This indicates that for different receptors,the methods of separation are different,and screening is needed.The construction of magnetic nanoprobe to isolate polysaccharide receptor protein is a new method to study polysaccharide receptor,and many details need to be further screened and studied.The successful construction of polysaccharide magnetic nanoprobe in this experiment will also provide a new means for the application of polysaccharides in the field of biomedicine.