基于表面增强拉曼散射的免疫分析方法和免疫层析技术的研究与应用

发布时间：2018-07-03 03:00

本文选题：表面增强拉曼光谱(SERS) + 免疫分析方法(IA)　；参考：《苏州大学》2016年硕士论文

【摘要】：表面增强拉曼光谱(Surface-Enhanced Raman Scattering,SERS)因其高灵敏度等优势被广泛应用于化学和生物的检测分析中,而基于抗原抗体特异性反应的免疫分析方法(Immunoassay,IA),其特异性强。SERS结合IA作为一种新型的检测手段,兼具了SERS的高灵敏度和免疫分析方法的高特异性,在最近几年被广泛应用于食品安全检测及环境监测等领域。在SERS-IA中,用于支撑免疫反应的基底研究一直以来都是人们研究的热点,基底材料各有千秋。本研究的一个主要目的旨在探索出一种能很好支撑免疫反应、信号均匀、操作简便、成本较低的固相基底材料。在几经尝试之后,发现硝酸纤维素膜(即NC膜)兼具这些优点。在此基础上,建立了基于NC膜的多通道SERS-IA,并应用于肉样中一种新型β-激动剂(即瘦肉精)溴布特罗(Brombuterol)的检测。另外,本论文也对以GNPs为免疫探针基底的表面增强拉曼光谱结合免疫层析技术(SERS-ICT)进行了深入研究,建立了测定Hg~(2+)的SERS-ICT,并对加标水样(河水、湖水、自来水)、尿样、血样中的Hg~(2+)进行了检测,取得了令人满意的结果。本研究主要内容为以下三个部分:一.介绍论文的研究背景及意义。阐述了表面增强拉曼光谱的相关概念以及应用,说明了免疫分析的基本理论,着重介绍了SERS的基底材料,SERS-IA以及SERS-ICT。另外,也叙述了一直以来人们关注的环境和食品安全问题。二.在探索以银膜、银纳米颗粒、金纳米颗粒作为免疫反应的支撑物时,发现这些材料存在前处理和制备过程复杂繁琐,与蛋白结合不牢固,而且重现性差,信号不均匀等种种问题。而以NC膜为免疫反应的固相支撑物,不仅与蛋白的交联作用好,而且快速、方便。与此同时制备了一种基于新型核壳型GNPs@Ag纳米粒子结合抗体的具有拉曼活性的免疫探针,建立了基于SERS和NC膜的免疫分析方法(SERS-IA),并对溴布特罗进行检测,IC50和检出限分别为0.02 ng m L~(-1)和0.16 pg m L~(-1),显示出了高的灵敏性。由于溴布特罗与盐酸克伦特罗的分子结构相近,SERS-IA与盐酸克伦特罗存在40.8%的交叉反应。SERS-IA与其他β-激动剂无交叉反应。将建立的SERS-IA应用于加标肉样中溴布特罗的检测,结果令人满意。三.本论文对表面增强拉曼光谱结合免疫层析技术(SERS-ICT)进行了深入研究。制备了以Au NPs为基底的SERS免疫探针,建立了测定Hg~(2+)的SERS-ICT,对加标水样(河水、湖水、自来水)、尿样、血样中的Hg~(2+)进行了检测。在最优的实验条件下,该SERS-ICT有着低于ng g~(-1)级的高灵敏度,IC50和检出限LOD分别为0.12 ng m L~(-1)和0.45 pg m L~(-1)。与19种阴、阳离子的交叉反应率均小于0.01%。另外,对与汞接触的职业病患者实际样品中Hg~(2+)进行了检测,并与其他方法进行了比较,取得了令人满意的结果。
[Abstract]:Surface-enhanced Raman scattering spectroscopy (SERS) is widely used in chemical and biological analysis because of its high sensitivity. As a new detection method, Immunoassay based on specific reaction of antigen and antibody (Immunoassay IA) has both the high sensitivity of SERS and the high specificity of immunoassay. In recent years, it has been widely used in food safety detection and environmental monitoring. In SERS-IA, substrate research for supporting immune response has been a hot topic, and substrate materials have their own merits. One of the main purposes of this study is to explore a solid phase substrate material which can support immune response well, signal is uniform, operation is simple and cost is low. After several attempts, it was found that the nitrocellulose membrane (NC membrane) had these advantages. On this basis, a multichannel SERS-IA-based NC membrane was established and applied to the detection of brombuterol, a new 尾 -agonist (lean meatine) in meat samples. In addition, the surface enhanced Raman spectroscopy combined with immunochromatography (SERS-ICT) based on GNPs was also studied in this paper. The SERS-ICTs for the determination of Hg2 were established, and the standard water samples (river, lake, tap water), urine samples were also studied. The detection of Hg ~ (2) in blood samples has been carried out and satisfactory results have been obtained. The main contents of this study are as follows: 1. This paper introduces the research background and significance. The related concepts and applications of surface-enhanced Raman spectroscopy are described. The basic theory of immunoassay is explained. The substrate materials of SERS, SERS-IA and SERS-ICTare emphatically introduced. In addition, it also describes the environmental and food safety issues that people have been concerned about all along. II. When exploring silver film, silver nanoparticles and gold nanoparticles as the support of immune reaction, it was found that the pretreatment and preparation of these materials were complicated, the binding to proteins was not firm, and the reproducibility of these materials was poor. A variety of problems such as uneven signals. The solid phase support with NC membrane not only crosslinked with protein, but also was fast and convenient. At the same time, a novel Raman immunosurgery probe based on novel core-shell GNPs @ Ag nanoparticles binding antibody was prepared. An immunoassay (SERS-IA) based on SERS and NC membranes was established. The detection limits of IC50 and detection limits for bromobuterol were 0.02 ng mL ~ (-1) and 0.16 PG mL ~ (-1), respectively, showing a high sensitivity. Because the molecular structure of bromobuterol and clenbuterol hydrochloride is similar, there is 40.8% cross reaction between SERS-IA and clenbuterol hydrochloride. There is no cross reaction between SERS-IA and other 尾 -agonists. The established SERS-IA was applied to the determination of bromobuterol in standard meat samples with satisfactory results. III. Surface enhanced Raman spectroscopy combined with immunochromatography (SERS-ICT) has been studied in this paper. A SERS immunoprobe based on au NPs was prepared and a SERS-ICT-based method was established for the determination of Hg2 in water (river, lake, tap water), urine and blood samples. Under the optimal experimental conditions, the SERS-ICT has a high sensitivity IC50 and a detection limit of 0.12 ng mL ~ (-1) and 0.45 PG mL ~ (-1), respectively, which are lower than ng g ~ (-1). The cross reaction rate with 19 kinds of anions and cations was less than 0.01. In addition, Hg ~ (2) in the actual samples of occupational disease patients exposed to mercury were detected and compared with other methods, and satisfactory results were obtained.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.37

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