荧光相关光谱均相免疫新方法研究

发布时间：2018-02-10 07:56

本文关键词： 银纳米颗粒荧光相关光谱荧光增强均相免疫分析甲胎蛋白　出处：《上海交通大学》2010年硕士论文　论文类型：学位论文

【摘要】： 免疫分析是利用抗原与抗体之间高特异性的反应实现对抗体、抗原或相关物质进行检测的分析方法。免疫分析是一种非常重要的生物分析方法,它被广泛地应用于临床诊断,食品和环境分析,生物及医学研究等领域。目前免疫分析主要以微孔板为实验平台的异相分析模式。这种方法需要包埋、冲洗等多步操作。因此,这种方法操作繁琐,分析时间长,不能满足某些快速检测和诊断的要求。近年来,均相免疫分析以其快速便捷的操作和低廉的成本,受到越来越多的关注。然而,由于这种方法的灵敏度较低,不能满足高背景噪音的临床检测要求。本文旨在利用贵金属纳米颗粒合成生物探针,并结合荧光相关光谱,发展了一种高灵敏的均相免疫分析新方法。主要研究工作包括如下两个方面: 1、银纳米颗粒(SNP)具有很强的共振散射光,并对荧光染料有信号增强的效果,是性能优良的标记分子。但是其在均匀性与稳定性上的劣势阻碍了它的应用。本文采用巯基十一烷基酸(MUA)对银纳米颗粒进行了表面修饰,有效地改善了它的均匀性与稳定。并通过控制抗体与银纳米颗粒的物质的量比例(2:1与10:1),实现了银纳米颗粒的生物连接,合成了低抗体-银纳米结合物与高抗体-银纳米结合物。同时,本文还利用共振光散射相关光谱(RLSCS),对银纳米颗粒及其表面修饰和生物连接过程进行了表征研究,并优化了实验条件。通过与其他表征结果的比较,证明RLSCS是一种表征银纳米颗粒的有效的单分子检测手段。 2、本文选择甲胎蛋白抗原(AFP)与其抗体(AFP1A6)作为实验模型进行均相免疫研究,并利用Alexa Fluor 647标记的AFP作为均相竞争免疫的示踪抗原。低抗体-银纳米结合物和高抗体-银纳米结合物分别与抗原进行免疫反应而成的免疫复合物,同自由的染料标记抗原相比,扩散时间分别增加了60倍(低抗体银纳米探针)与120倍(高抗体银纳米探针)。同时,这些免疫产物的荧光强度分别增加了18倍与49倍,信噪比也增加了10倍与20倍。基于银纳米颗粒在荧光增强与扩散时间增加上的作用,本文结合双组分荧光相关光谱,发展了纳米银增强荧光相关光谱均相竞争免疫分析方法。在优化的条件下,这种方法具有良好的灵敏度,其检测限为1.5 pM,线性范围从6 pM至60 pM(R0.99)。在人体血清AFP水平的测试中,检测的相对标准偏差(RSD)约为5%,回收率大于90%。从而实现了均相免疫在实体生物样品中应用的目标。综上所述,利用纳米银增强荧光相关光谱方法进行的免疫分析,不仅能够提高抗原与免疫结合物的区分度,而且能够显著的增加免疫分析的灵敏度。这一技术必将在临床诊断、食品与环境分析以及生物和生物医药等研究领域展现其巨大的发展潜力。
[Abstract]:Immunoassay is a method to detect antibodies, antigens or related substances by using highly specific reactions between antigens and antibodies. Immunoassay is a very important biological analysis method, and it is widely used in clinical diagnosis. Food and environment analysis, biological and medical research and other fields. At present, immunoassay is mainly based on microporous plate as the experimental platform of heterogeneous analysis model. This method needs to be embedded, flushing and other multi-step operation. Therefore, the operation of this method is cumbersome. In recent years, homogeneous immunoassay has attracted more and more attention due to its rapid and convenient operation and low cost. However, the sensitivity of this method is low. Can not meet the high background noise clinical detection requirements. The aim of this paper is to synthesize biological probes using noble metal nanoparticles and develop a highly sensitive homogeneous immunoassay method combined with fluorescence correlation spectroscopy. The main research work includes the following two aspects:. 1. Silver nanoparticles SNPs have strong resonance scattering light and signal enhancement effect on fluorescent dyes. In this paper, silver nanoparticles were modified by mercaptodecyl acid MUAs. The homogeneity and stability of the silver nanoparticles are improved effectively, and the biological connection of silver nanoparticles is realized by controlling the ratio of antibody to substance of silver nanoparticles in the ratio of 2: 1 to 10: 1. The low antibody silver nanoconjugate and the high antibody silver nano bond were synthesized. At the same time, the silver nanoparticles, their surface modification and biological bonding processes were characterized by resonance light scattering correlation spectroscopy (RLS). The experimental conditions were optimized and compared with other characterization results, it was proved that RLSCS is an effective single molecular detection method for the characterization of silver nanoparticles. 2. AFP6 and AFP1A6) were selected as the experimental model to study the homogenous immunity of alpha-fetoprotein antigen (AFP) and its antibody (AFP1A6). AFP labeled by Alexa Fluor 647 was used as a tracer antigen for homogeneous competition immunity. The low antibody silver nanoconjugate and the high antibody silver nanoconjugate were used to react with the antigen respectively. Compared with free dye labeled antigens, the diffusion time was increased by 60 times (low antibody silver nanoprobes) and 120 times (high antibody silver nanoprobes), respectively. Meanwhile, the fluorescence intensity of these immune products increased by 18 and 49 times, respectively. The SNR is also increased by 10 and 20 times. Based on the effect of silver nanoparticles on fluorescence enhancement and diffusion time, two component fluorescence correlation spectra are combined in this paper. A homogeneous competitive immunoassay method for silver enhanced fluorescence correlation spectroscopy has been developed. Under the optimized conditions, this method has a good sensitivity with a detection limit of 1.5 pm and a linear range from 6 pm to 60 pm R0.99. In the test of human serum AFP level, the relative standard deviation (RSD) is about 5, and the recovery rate is more than 90. Thus, the goal of applying homogeneous immunization in solid biological samples is achieved. Immunoassay using silver nanoparticles enhanced fluorescence correlation spectroscopy can not only improve the differentiation of antigens from immune conjugates, but also significantly increase the sensitivity of immunoassay. This technique is bound to be used in clinical diagnosis. Research fields such as food and environment analysis and biology and biomedicine show great potential for development.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

【共引文献】