Preparation Of Magnetic Polymer Composite Microspheres,Surface Modification Of Anti-Protein Nonspecific Adsorption And Its Application In Immunoassay

Magnetic microspheres have been widely used in biomedical field due to their large specific surface area,controllable size and easy separation.When magnetic microspheres are used for immunoassay,it is important to reduce the nonspecific adsorption of proteins on the surface of microspheres.At present,there are many products on the market that can be well applied to immunoassay,but domestic research in this area is still relatively little.Therefore,it is necessary to explore the appropriate conditions to obtain magnetic microspheres with excellent performance.There are various preparation methods of magnetic microspheres,mainly by reflux precipitation method,which wraps a polymer layer on the surface of magnetic particles,and then modifies the anti-protein nonspecific adsorption material,finally successfully applies it to immunoassay.Firstly,the factors influencing the synthesis of carboxylated magnetic polymer composite microspheres by reflux precipitation method were investigated,including polymerization temperature,monomer dosage,polymerization time,monomer type and multiple monomer dosage,and the optimal reaction conditions were determined.Magnetic polymer composite microspheres（Fe₃O₄@SiO₂@PMAA）with the size of 400 nm-1100 nm can be obtained by layered precipitation strategy and controlling the number of monomer addition.Then,hyperbranched polyglycerol（HPG）with molecular weight distribution of 500-600was synthesized.HPG is a derivative of polyethylene glycol（PEG）and a classical anti-protein nonspecific adsorption material,and its hyperbranched structure can modify more active sites.The method of grafting HPG onto Fe₃O₄@SiO₂@PMAA surface was explored,and then the active group-COOH was modified on the surface of the microspheres.It was proved that the modified magnetic microspheres Fe₃O₄@SiO₂@PMAA-HPG-COOH could exist stably under complex conditions.Next,the protein nonspecific adsorption capacity of magnetic polymer composite microspheres before and after grafting modification was investigated.The nonspecific adsorption capacity of Fe₃O₄@SiO₂@PMAA-HPG-COOH was much smaller than that of Fe₃O₄@SiO₂@PMAA,both with BSA as model protein and phycoerythrin as model protein.Commercial magnetic beads and magnetic microspheres before and after modification were prepared into immunomagnetic beads to detect cardiac trocalciin I（cTnI）.It was found that the nonspecific adsorption of HPG modified magnetic beads decreased to 23.64%,but was slightly higher than that of commercial magnetic beads,which was 2.08 times of that of JSR-MS150/Streptavidin.The binding amount of specific cTnI antigen was 9.27 times of that before modification,and 5.67 times of that of commercial magnetic beads.It confirmed that the great prospect of Fe₃O₄@SiO₂@PMAA-HPG-COOH in immunoassay.The nonspecific adsorption capacity of magnetic microspheres modified with different molecular weights of PEG was investigated.It was found that the higher the molecular weight was,the less the nonspecific adsorption capacity was.Therefore,higher molecular weight HPG could be synthesized later to graft the carboxyl magnetic beads.Finally,the surface of magnetic microspheres in products is mostly polystyrene matrix,benzene ring not only has no charge,which can reduce the nonspecific adsorption of proteins,but also can take place of benzene ring substitution reaction and modify the required functional groups.So,the synthesis of Fe₃O₄@SiO₂@PSt in different polymerization methods has also been explored.