【作者】 唐点平；

【导师】 袁若； 柴雅琴；

【作者基本信息】 西南师范大学 ， 分析化学， 2005， 硕士

【摘要】 乙型肝炎是由乙肝病毒引起的,是我国当前流行最广泛、危害最严重的一种传染病。传染源主要是患者及乙肝病毒无症状携带者,血液、性接触和生活密切接触是传播的重要方式。白喉是由白喉杆菌引起的急性呼吸道传染病,以咽、喉等处粘膜充血、肿胀并有灰白色伪膜形成为突出临床特征,严重者可引起心肌炎与末梢神经麻痹。目前,用于乙型肝炎及白喉类毒素的诊断方法已有较多报道,但尚远不能满足当前乙型肝炎及白喉类毒素防治工作的需要,由于它们均存在一些不同程度的不足,如灵敏度度低、需用高素质的专业人员、操作繁琐、费时、需要大型贵重仪器、漏检和误检等。因此,开发高灵敏、简便、快速的检测方法对免疫传感器中抗原或抗体的检测具有重要的理论意义和实际应用价值。 电位分析电化学免疫传感器是基于测量电位变化来进行免疫分析的生物传感器。抗原或抗体在敏感膜上的结合或继后的反应引起电极电位或膜电位发生改变,而且这种电位的变化与待测物浓度之间存在对数关系。电位型免疫传感器又分为直接型电位传感器、非酶标记电位型免疫传感器,酶标记型电位免疫传感器。与其它电化学免疫传感技术相比,该方法具有灵敏、快速、成本低、携带方便,操作简单、易数字化、能方便地实现实时输出和检测。本文利用具有强吸附效应的纳米颗粒及复合纳米材料、聚乙烯醇缩丁醛(PVB)的笼效应和微水环境、溶胶-凝胶等技术将抗体(抗原)固定在不同的基体电极上,直接测定抗原与抗体反应前后的电位变化。方法较传统的ELISA和放射性免疫分析(RIA)简单、快速,可望作为一种临床诊断分析的工具。 本论文具体内容包括以下几个方面: 第一部分:绪言—电化学免疫传感器的进展更多还原

【Abstract】 Hepatitis B, an epidemic disease with greatest harms for human health caused by hepatitis B virus, is highly prevalent in our country. Hepatitis B virus, originated from sufferers and infectors, can be spread by blood, sex-contact and closely contact. Diphtheria is an acute bacterial disease that usually affects the tonsils, throat, nose and/or skin, which can lead to breathing problems, heart failure, paralysis and sometimes death. Until now, some diagnostic procedures have been adapted to clinical analyses for hepatitis B and diphtheria. Most methods, unfortunately, require highly qualified personnel, tedious assay time, or sophisticated instrumentation. Therefore, exploring new, simple, and sensitive diagnostic methods with real-time output and a low cost is of considerable interest.The detection of the potentiometric immunoassay is based on the change in the potentiometric response before and after antigen-antibody reaction. Either antibodies or antigens in aqueous solution have a net electrical charge polarity, which is correlated to the isoelectric points of the species and the ionic composition of the solution. If antibodies combine with antigens, the electrical charge of the resulting complex will be different from that of antibody or antigen. If antibody is immobilized on the electrode, the surface charge of the electrode will depend on the net charge of the immobilized antibody. When antigen is present in the solution, the immunochemical reaction will take place at the interface with a resulting change of the surface charge. This change can be measured potentiometrically against the reference electrode immersed in the same solution. The shift of potentiometric response exhibits a linear dependence on the logarithm of the concentration of analytes. In this article, we have constructed several potentiometric immunosensors immobilized antigen/antibody onto substrate electrode based on nanoparticles/compound nanoparticles,polyvinyl butyral, sol-gel as matrixes. Analytical results of clinical samples show that the developed immunoassays are comparable with ELISAs and RIAs, implying a promising alternative approach in the clinical diagnosis. The detailed materials are shown as follows:The first part: Prediction-electrochemical immunosensor.The second part: Highly sensitive, long-term, renewable potentiometric immunosensors based on polyvinyl butyral and nanoparticles as matrixes have been developed. The biocomposite, which needs no additional curing, is directly used to construct the immunosensor at room temperature. The incorporation of BSA into the biocomposite reduces non-specific adsorption of immunosensors to a lower level. (1) In chapter 1, a highly sensitive potentiometric immunosensor for the diagnoses of epidemic diseases has been developed by means of self-assembly to immobilize hepatitis B surface antibody for the detection of hepatitis B surface antigen as a model. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited high sensitivity, wide linear range, rapid potentiometric response (< 3 min) and long-term stability (> 4 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting hepatitis B in the clinical diagnosis. (2) In chapter 2, a novel potentiometric immunosensor for the detection of diphtheria antigen has been developed by means of self-assembly to covalently immobilized diphtheria antibody on a glassy carbon electrode. The modification procedure was electrochemically monitored by characterization of the electrode surface. The performance and factors influencing the performance of the resulting immunosensor were studied in detail. The resulting immunosensor exhibited fast potentiometric response to Diph and the detection limit of the immunosensor was from 24 ng mL~-1 to 600 ngmL~-1 with correlation coefficient of 0.9979 and a detection limit of 5.2 ngmL~-1The third part: A novel potentiometric immunosensor for the detection of hepatitis B surface antigen has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. The self-assembling procedure was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Tests relating更多还原