磁性微球的制备及其用于免疫层析检测中的研究

发布时间：2018-05-24 11:58

本文选题：磁性微球 + 共价偶联　；参考：《东南大学》2016年硕士论文

【摘要】：在信息时代,人们对健康的关注达到了前所未有的高度,对健康数据的获得方式和处理过程也提出了新的要求。免疫层析作为体外诊断技术的一种,因其廉价、快速、简易等优势,在互联网时代迎来了发展的新机。但是基于胶体金和胶乳微球探针的免疫层析技术普遍存在制备过程繁琐,可重复性差、灵敏度低等问题。因此,开发出新类型的标记探针,对于免疫层析技术具有革命性的意义。另一方面,磁性微球凭借其在外加磁场下的磁响应特性,广泛应用于生物分子的分离纯化和体内药物运输等方面。本研究致力于将磁性微球作为标记探针,用于免疫层析检测当中,取得了以下结果：(1)实验采用溶剂热法制备了磁性微球,通过场发射扫描电镜、红外光谱仪、振动样品磁强计、动态光散射测试对其进行了表征,结果表明制备的磁性微球平均粒径在250nm,单分散性良好,红外光谱显示磁性微球表面带有羧基,具有良好的水溶性和单分散性。磁性微球呈超顺磁性,比饱和磁化强度为56.15emu/g。实验还测试了反应铁浓度和反应时间对磁性微球的影响,发现随着铁浓度的增加,磁性微球整体大小随之增加,随着反应时间的延长,组成磁性微球的小颗粒尺寸随之增加。(2)采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)/N-羟基琥珀酰亚胺(NHS)法将单克隆抗体蛋白偶联在磁性微球表面,制备出免疫磁性微球。采用BCA试剂盒进行单因素分析,得到偶联的最佳条件是：活化过程中,Fe、EDC、NHS之间的质量比为1：0.5：0.3,反应体系为纯水；偶联过程中缓冲体系是离子强度为0.02 M,pH=6.6的硼酸缓冲溶液,偶联1h,采用胎牛血清白蛋白封闭1h。通过红外光谱仪、热分析仪确定单克隆抗体蛋白已经偶联在磁性微球表面。动态光散射测试表明,偶联过后磁性微球依然维持良好的水溶性和稳定性。(3)初步验证了免疫磁性微球作为免疫层析标记探针的性能,制备出检测人绒毛膜促性腺激素(hCG)免疫层析和心肌肌钙蛋白I (cTnl)试纸条。实验研究了不同层析速度的硝酸纤维素膜对试纸条的灵敏度的影响,并且创新性的采用普鲁士蓝染色对检测结果进行放大。实验结果表明,层析速度慢的层析膜灵敏度较高,hCG试纸条检测线性范围为0～1000 IU/L, cTnl检测线性范围为0～8 ng/mL。并且采用普鲁士蓝染色能够进一步放大检测信号,使试纸条达到能够用肉眼观察的程度。通过以上研究实验确定,采用溶剂热法制备的磁性微球能够作为免疫层析检测的标记探针,并且极大的简化了试纸条的制备过程,采用普鲁士蓝染色能够有效放大检测信号,且制备流程和工艺具备一般性和可重复性。
[Abstract]:In the information age, people pay more attention to health than ever before. Immunochromatography as an in vitro diagnostic technology, because of its cheap, fast, simple and other advantages, in the Internet era ushered in a new development of the machine. However, immunochromatography based on colloidal gold and latex microsphere probes has many problems, such as tedious preparation, poor repeatability and low sensitivity. Therefore, the development of a new type of labeled probes has revolutionary significance for immunochromatographic techniques. On the other hand, magnetic microspheres are widely used in the separation and purification of biomolecules and drug transport in vivo. In this study, magnetic microspheres were used as labeling probes for immunochromatographic detection. The following results were obtained: (1) the magnetic microspheres were prepared by solvothermal method. The magnetic microspheres were prepared by field emission scanning electron microscopy, infrared spectrometer, vibrating sample magnetometer. The results showed that the average diameter of the prepared magnetic microspheres was 250 nm, the monodispersity was good, and the surface of the magnetic microspheres had carboxyl groups and good water solubility and monodispersity. The magnetic microspheres are superparamagnetic with a specific saturation magnetization of 56.15 emu / g. The effects of reaction iron concentration and reaction time on magnetic microspheres were also tested. It was found that with the increase of iron concentration, the overall size of magnetic microspheres increased and the reaction time increased. The size of the small particles of the magnetic microspheres increased with the increase. 2) Immunomagnetic microspheres were prepared by coupling monoclonal antibody proteins to the surface of the magnetic microspheres by the method of 1-D _ 3-dimethylamino-propyl -3-ethylcarbodiimide (EDCN / N-hydroxysuccinimide). Single factor analysis using BCA kit showed that the optimum conditions for coupling were as follows: the mass ratio of EDCHs during activation was 1: 0.5: 0.3, the reaction system was pure water, and the buffer system was boric acid buffer solution with ion strength of 0.02 M0. 2, pH = 6. 6 during the coupling process, and the optimum conditions were as follows: 1: 0. 5: 0. 3 in the activation process, and 1: 0. 5: 0. 3 in the reaction system. Fetal bovine serum albumin (FSA) was used to block the coupling for 1 h. The monoclonal antibody protein was confirmed to be coupled to the surface of magnetic microspheres by IR spectrometer and thermal analyzer. Dynamic light scattering (DLS) test showed that the magnetic microspheres maintained good water solubility and stability after coupling.) the performance of immunomagnetic microspheres as immunochromatographic probe was preliminarily verified. A test strip for detecting human chorionic gonadotropin (hCG) and cardiac troponin I (cTnl) was prepared. The effect of nitrocellulose membrane at different chromatographic speeds on the sensitivity of the test strip was studied, and Prussian blue staining was used to amplify the results. The experimental results show that the sensitivity of the membrane with low chromatography speed is higher than that of the hCG strip, and the linear range of cTnl detection is 0 ~ (8) ng 路mL ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) 渭 m 路L ~ (-1). Prussian blue staining can further amplify the detection signal and make the test strip visible with the naked eye. The results show that the magnetic microspheres prepared by solvothermal method can be used as a labeling probe for immunochromatographic detection, and the preparation process of the test strip is greatly simplified. Prussian blue staining can effectively amplify the detection signal. The preparation process and process are general and reproducible.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.6

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