【作者】 李芳；

【导师】 肖波； 胡涌刚；

【作者基本信息】 华中科技大学 ， 环境工程， 2012， 博士

【摘要】 以磁珠为载体的电化学免疫传感技术是当前的一个研究热点。如何有效地将携带了大量检测信息的磁珠固定在工作电极的检测表面,并对其进行直接电化学测定是制约该技术发展的一个关键性问题。使用磁性工作电极可有效解决此困难。本论文将围绕磁性工作电极的制作,基于纳米信号放大的以磁珠为载体的电化学免疫检测新方法的构建及其在动物疾病诊断中的应用,开展以下几方面的工作：(1)磁性工作电极的制作分别建立了磁性金工作电极和磁性玻碳电极的制作方法,且制作方法简单、易行,成本低廉,易于推广。此外,我们对磁性工作电极的性能进行了测试,结果表明,它们能有效捕获磁珠进行直接电化学测定,为构建以磁珠为载体的电化学免疫分析新方法奠定了基础。(2)基于碳纳米管掺杂以磁珠为载体的电化学免疫新方法在日本乙型脑炎病毒检测中的应用以磁珠为载体,掺杂碳纳米管构建了一种新的电化学免疫分析方法,并将其应用于日本乙型脑炎病毒的检测中。选用金磁纳米粒子为载体,辣根过氧化物酶为示踪标记物。免疫反应后,掺杂碳纳米管。利用磁性金工作电极将碳纳米管掺杂的金磁复合物捕获到电极检测表面上进行电化学测定。掺杂碳纳米管可有效改善电极表面的导电性,得到高灵敏的检测结果。采用日本乙型脑炎病毒作为目标分析物。在最佳检测条件下,日本乙型脑炎病毒的检测限为2.0×103 PFU/mL,比胶体金免疫试纸条法低两个数量级,与RT-PCR法的基本类似。分别用本方法和RT-PCR法分析60份临床样品,结果显示本方法与RT-PCR法的符合率高达95%,表明本方法在日本乙型脑炎病毒的临床诊断中具有较好的应用前景。(3)基于纳米金标记的以磁珠为载体的电化学免疫新方法在伪狂犬病毒抗体检测中的应用采用纳米金为电活性探针,构建了一种新的以磁珠为载体的电化学免疫分析方法,并将其应用到伪狂犬病毒抗体的检测中。选用羧基修饰的磁珠为载体。免疫反应后,利用磁性玻碳电极将磁珠捕获到电极检测表面上,采用电化学氧化法使得纳米金生成AuC14-,通过测定AuC14-的信号对检测物进行分析。采用伪狂犬病毒抗体作为目标分析物。在最佳检测条件下,可检测到稀释了1：1000的伪狂犬病毒抗体标准阳性血清,其检测限低于传统的ELISA检测法。分别用本方法和ELISA法分析52份猪的血清样本,结果表明本方法与ELISA法具有很好的符合率。(4)基于纳米金催化银沉积的以磁珠为载体的电化学免疫新方法在猪圆环病毒2型抗体检测中的应用结合纳米金催化银沉积信号放大技术,构建了一种新的以磁珠为载体的电化学免疫分析方法,并将其应用于猪圆环病毒2型抗体的检测。选用异硫氰根修饰磁珠为载体,纳米金为标记物。免疫反应后,加入银染液进行银放大,固定在磁珠表面的纳米金可催化溶液中的银离子以纳米金为核进行沉积。利用磁性玻碳电极将磁珠捕获到电极的检测表面上,通过测定银单质的溶出信号对分析物进行检测。以猪圆环病毒2型抗体为目标分析物。在最佳检测条件下,可检测到稀释了1：4096的猪圆环病毒2型抗体标准阳性血清,该检测限明显低于传统的ELISA检测法。分别用本方法和ELISA法去分析猪的血清样本,结果表明本方法与ELISA法的符合率高。更多还原

【Abstract】 Special attention has been paid in magnetic beads-based electrochemical immunosensor in the recent years. However, one major obstacle for magnetic beads-based electrochemical immunoassay is how to confine the magnetic beads on the detection surface for the direct electrochemical sensing. The use of magnetic electrodes for magnetic beads manipulation is an effective solution. Focusing on the preparation of magnetic electrodes, and the construction of the novel magnetic beads-based electrochemical immunoassay with nanoparticles amplification for animal diseases diagnosis, this thesis carried out some researches as follows.(1) The fabrication of magnetic electrodesThe simple and low cost methods were developed to fabricate the magnetic gold electrode and magnetic glass carbon electrode. The properties of the magnetic electrodes were carefully examined. The results showed that magnetic electrode could effectively confine the magnetic beads on their detection surfaces for the direct sensing. It is telling that the developed magnetic electrodes can be applied in the magnetic beads-based electrochemical immunoassay.(2) Magnetic beads-based electrochemical immunoassay for the detection of Japanese encephalitis virus using multiwalled carbon nanotubes s as dopantA novel magnetic beads-based electrochemical immunoassay strategy using multiwalled carbon nanotubes as dopant has been developed for the detection of Japanese encephalitis virus. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. After immunological reaction, multiwalled carbon nanotubes were introduced to improve electric conductivity and the sensitivity of the assay. With the help of the magnetic gold electrode, magnetic beads mixed with multiwalled carbon nanotubes were confined on the detection surface for the direct sensing. The Japanese encephalitis virus was selected as the model. Under the optimal conditions, the detection limit of Japanese encephalitis virus was 2.0×103 PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. Finally, the proposed method was applied in the clinical diagnosis of Japanese encephalitis virus, and had a good diagnostic agreement (95%) with the results from RT-PCR. This is telling that the present method has a great promise for the clinical diagnosis of Japanese encephalitis virus.(3) Magnetic beads-based electrochemical immunoassay for the detection of pseudorabies virus antibody using gold nanoparticles as tagsA novel magnetic beads-based electrochemical immunoassay has been developed for the detection of pseudorabies virus antibody using gold nanoparticles as electroactive labels. COOH-coated magnetic beads were employed as the platforms. After immunological reaction, the magnetic beads were captured and focused on the detection surface with the help of magnetic glass carbon electrode. The gold nanoparticles were first oxidized electrochemically to produce AuC14-, and then DPV was employed for the determination of AuC14-. Pseudorabies virus antibody was chosen as the model. Under the optimal conditions, the detection limit of pseudorabies virus antibody was 1:1000 (standard positive serum), which was lower than that obtained from ELISA. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.(4) Magnetic beads-based electrochemical immunoassay with silver amplification for the detection of antibody against porcine circovirus type 2A novel magnetic beads-based electrochemical immunoassay with silver amplification has been developed for the detection of antibody against porcine circovirus type 2. ITC-coated magnetic beads were employed as the platforms, and gold nanoparticles were selected as labels. After immunological reaction, the silver deposition solution was introduced to deposit silver on the gold nanoparticle tags. Magnetic beads were then confined on the detection surface with help of magnetic glass carbon electrode. The deposited silver was measured by anodic stripping analysis. Antibody against porcine circovirus type 2 was employed as model analytes. Under the optimal conditions, the detection limit of antibody against porcine circovirus type 2 was 1:4096, which was lower than that obtained from ELISA. Finally, this developed immunoassay method was successfully applied in the detection of antibody against porcine circovirus type 2 in swine serum, and had a good diagnostic accordance in comparison with ELISA.更多还原