磁性高分子微球的制备、表征及应用研究

发布时间：2018-03-20 03:04

本文选题：磁性微球　切入点：免疫PCR　出处：《西北师范大学》2014年硕士论文　论文类型：学位论文

【摘要】：磁性高分子微球（MMS）是指通过一定的方法将磁性材料和化学、生物等高分子材料结合而形成的一种直径在纳米至微米级且具有磁响应性的高分子复合型微球。由于其粒径小，比表面积大，可偶联的生物分子容量大，且能分散在体系中不易沉降，因此，他们被广泛应用在生物技术和生物医药工程方面，比如细胞分离，免疫酶，蛋白分离，，靶向药物和生物化学分析等。本文采用反相聚合包埋技术制备以Fe3O4为磁核，琼脂糖为壳层的琼脂糖磁性微球（AMM），依次用环氧氯丙烷、谷氨酸和精氨酸对其进行表面化学修饰后制得环氧基化磁性微球（EMM）、羧基化磁性微球（CMM）和氨基化磁性微球（AR-CMM）。通过傅里叶变换红外光谱、热重分析和振动样品磁力测定等方法表征磁性微球的结构和性质，采用酶联免疫吸附试验（enzyme linkedimmunosorbent assay，ELISA）检测不同磁性微球对乙肝表面抗原的固定能力。结果表明，琼脂糖磁性微球、环氧基化磁性微球、羧基化磁性微球和氨基化磁性微球的平均粒径分别为20.6、30.3、31.4和33.6μm；磁含量分别为35.33%、50.53%、45.7%和38.9%；环氧基含量为140.19μmol·g1，羧基含量为253.0μmol·g1，氨基含量为59.1μmol·g1；具有良好的单分散性、超顺磁性和生物兼容性；经化学改性后磁性微球对乙肝表面抗原的固定能力均有所增加，其中羧基化磁性微球固定能力最强，其次为环氧基化磁性微球和氨基化磁性微球。另外，我们在羧基化磁性微球的基础上建立了两种检测乙肝表面抗原的新方法：一种方法是磁性微球免疫PCR方法（magnetic microsphere Immuno PCR,MMS-Im-PCR），该方法以羧基化磁性微球作为载体，固定上乙肝表面抗原后加入一抗形成抗原抗体复合物，用单链DNA信号分子偶联抗体形成抗原抗体-DNA复合物，经实时定量PCR（real-time quantitative PCR，RT-PCR）扩增后，将抗原信号传递并放大为核酸信号和电信号，建立了一种利用磁性微球检测乙肝表面抗原的免疫PCR技术体系。另一种方法是核酸信标配基PCR方法（NBA-PCR）。该方法在羧基化磁性微球表面偶联上链霉亲和素，利用生物素和链霉亲和素的高亲和力将生物素修饰的乙肝表面抗原特异性核酸适配体连接在链霉亲和素磁性微球上，代替乙肝表面抗原作为信标配基检测分子，与一抗结合形成信标配基-抗体复合物，再利用信标配基-抗体复合物与DNA信号分子结合形成信标配基-抗体-DNA复合物，经过实时定量PCR扩增将特异性核酸适配体DNA配基信号经抗体传递到DNA信号分子，实现了信标配基信号和抗体信号，核酸信号之间的双向传递并建立了一种利用核酸适配体检测乙肝表面抗原的信标配基PCR技术体系。本实验为磁性微球应用于某种疾病诊断试剂盒的研制和缩短临床检测窗口期方面奠定了坚实的基础。
[Abstract]:Magnetic polymer microspheres (MMSs) is a kind of polymer composite microspheres with the diameter of nanometer to micron and magnetic response, which is formed by combining magnetic materials with chemical, biological and other polymer materials by a certain method. Because of their large specific surface area, large coupling capacity, and the ability to disperse in the system, they are not easy to settle, so they are widely used in biotechnology and biomedical engineering, such as cell separation, immune enzyme, protein separation, Targeted drugs and biochemical analysis. In this paper, agarose magnetic microspheres (AMMs) with Fe3O4 as magnetic nucleus and agarose as shell layer were prepared by reverse phase polymerization entrapment technique, followed by epichlorohydrin. After surface chemical modification with glutamate and arginine, EMMM, CMM (carboxylated magnetic microspheres) and AR-CMM (amino magnetic microspheres) were prepared by Fourier transform infrared spectroscopy (FTIR). The structure and properties of magnetic microspheres were characterized by thermogravimetric analysis and magnetic measurement of vibrating samples. Enzyme linked immunosorbent assay (Elisa) was used to detect the immobilization ability of different magnetic microspheres to hepatitis B surface antigen. The average diameters of epoxy, carboxylated and amino magnetic microspheres were 20.630.3 渭 m and 33.6 渭 m, respectively. The magnetic contents were 35.33 ~ 50.53 and 45.7% and 38.9%, respectively. The epoxy group content was 140.19 渭 mol 路g ~ (-1), carboxyl content was 253.0 渭 mol 路g ~ (-1), amino content was 59.1 渭 mol 路g ~ (-1). Superparamagnetism and biological compatibility; the immobilization ability of chemically modified magnetic microspheres to hepatitis B surface antigen was increased, of which carboxylation magnetic microspheres were the strongest, followed by epoxide magnetic microspheres and amino magnetic microspheres. In addition, we have established two new methods for the detection of hepatitis B surface antigen based on carboxylation magnetic microspheres. One method is magnetic microsphere Immuno PCR method, MMS-Im-PCRN, which uses carboxylated magnetic microspheres as carrier. Hepatitis B surface antigen was immobilized and an antigen-antibody complex was added. The antigen-DNA complex was conjugated with single-stranded DNA signal molecule to form antigen-DNA complex. The antigen antibody was amplified by real-time quantitative PCR(real-time quantitative PCRRT-PCR. The antigen signal is transmitted and amplified into nucleic acid signals and electrical signals. An immune PCR technique system for the detection of hepatitis B surface antigen by magnetic microspheres was established. Another method was the nucleic acid standard ligand PCR method, which coupled Streptavidin on the surface of carboxylated magnetic microspheres. Biotin and streptavidin were used to bind the biotin modified hepatitis B surface antigen specific aptamer to the magnetic microspheres of Streptomycin instead of hepatitis B surface antigen as standard ligand detection molecules. A signal ligand antibody complex is formed by binding with a single antibody, and then a signal ligand antibody antibody complex is formed by combining the signal molecule of DNA with a signal ligand antibody complex. The specific aptamer DNA ligand signal was transmitted to the DNA signal molecule by real-time quantitative PCR amplification, and the standard ligand signal and antibody signal were realized. The bidirectional transmission of nucleic acid signals and the establishment of a standard ligand PCR system for the detection of hepatitis B surface antigen using aptamer of nucleic acid. This experiment has laid a solid foundation for the application of magnetic microspheres in the development of a diagnostic kit and in shortening the window period of clinical detection.
【学位授予单位】：西北师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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