The Preparation, Characterization And Application Research Of Magnetic Polymer Microspheres

Magnetic polymer microsphere?MMS?refers to a kind of polymer hybridmicrosphere with magnetic response property and diameter ranging from nanometerto micron, which can be formed by combining the magnetic materials with polymericbiomaterials and chemical materials by means of certain method. The magneticpolymer microsphere with its small particle size, large specific surface area, greatcapacity of coupling biomolecules and good suspension in disperse system, which canbe widely used in biotechnology and biomedical engineering, such as cell separation,immobilized enzyme, protein separation, target drug and biochemical assays etc.Agarose magnetic microspheres (AMM), with agarose as shell and Fe3O4asnucleus, which is prepared by reverse phase suspended embedding techniques, istransformed into epoxy-modified magnetic microsphere (EMM), carboxyl magneticmicrospheres (CMM) and amination of magnetic microspheres (AR-CMM) viachemical surface modification of epichlorohydrin, glutamic and arginine successively.The structures and properties of MMS are characterized by Fourier transform infraredspectrometer (FTIR), thermal gravimetric analysis (TGA), vibrating samplemagnetometry (VSM) and so on. The capacity of MMS to fix on hepatitis B surfaceantigen is tested by enzyme-linked immunosorbent assay (ELISA). The resultsindicate that the average particle size ofAMM, EMM, CMM and AR-CMM are20.6?30.3?31.4and33.6?m respectively; the magnetic content are35.33%?50.53%?45.7%and38.9%respectively; the epoxy content is140.19?mol·g1, the carboxylcontent is250.30?mol·g1, the amination content is59.1?mol·g1. The experimentproved that MMS have good monodispersion, superparamagnetism andbiocompatibility. The capacity of MMS to fix on hepatitis B surface antigen hasincreased after chemical modification and this fixation capacity of CMM issignificantly higher than that of CMM and AR-CMM.In addition, we established two kinds of new methods to detect hepatitis Bsurface antigen on the basis of CMM. One is magnetic microsphere Immuno PCR?MMS-Im-PCR?. This method uses the CMM as a carrier, fixing hepatitis B surface antigen on it. By combing with primary antibodies, we get antigen-antibody complex,and then forming antigen-antibody DNA complex by coupling single-stranded DNAsignal molecules. With the help of amplification in real-time quantitative PCR(RT-PCR), the antigen signal is successfully transmitted and amplified into nucleicacid signals and electrical signals. Therefore, we established an immune PCRtechnology system to detect the hepatitis B surface antigen using magneticmicrospheres. Another method is the nucleic acid beacon aptamer PCR?NBA-PCR?.The method of operation should be as follows. Firstly, streptavidin is coupled on thesurface of CMM. By making use of the high affinity of biotin and streptavidin, wecouple the hepatitis B surface antigen specific nucleic acid aptamer which is modifiedby biotin to it so as to replace the hepatitis B surface antigen as beacon aptamermolecular for detecting. Secondly, by using primary antibodies in combination, we getbeacon aptamer-antibody complex, and then combing with DNA signal molecule toform a beacon aptamer-antibody–DNA complex. With the help of amplification inreal-time quantitative PCR, the DNA aptamer signals of specific nucleic acid aptamerare transmitted into the DNA signal molecules via primary antibodies for the purposeof achieving two-way transmission among the beacon aptamer signals, the antibodysignals and nucleic acid signals. In addition, On the basis of the experiment, a beaconaptamer PCR technology system can be established to detect the hepatitis B surfaceantigen by means of nucleic acid aptamer.The experiment, which deals with magnetic microsphere that can be applied todetect some sort of diease, laid a solid foundation for the application of the diseasediagnostic kits and shortening window period of clinical detection.