【作者】 钟桐生；

【导师】 黄杉生；

【作者基本信息】 湖南大学 ， 分析化学， 2001， 硕士

【摘要】 安培分析电化学免疫传感器是一种耦联电化学分析技术和传统的酶联免疫技术的高灵敏度传感装置，与其它电化学免疫传感技术相比，更加灵敏、快速，而且装置便宜，操作简单，易数字化，能方便地实现实时输出和监测。影响免疫传感器实际应用的最大障碍是传感器的可重复使用性，可更新的安培分析免疫传感器为解决这一难题提供了一种可能的途径。将抗原(抗体)与石墨或者碳固定在载体材料中，在一个竞争性的或者夹心式的免疫反应后，将酶标抗原(抗体)键合在传感器表面，通过一个酶催化反应来确定待测抗原(抗体)的浓度。传感器在一次免疫分析后，经过一个简单的抛光就可得到一个新的免疫组分层用于下一次免疫分析。方法较传统的ELISA和放射性免疫分析(RIA)简单、快速，可望作为一种新的安培分析临床诊断分析的工具。本论文报导了一种基于溶胶-凝胶技术的可更新的安培分析免疫传感器的制备和特性，分别制备了用于人血清中补体第3成份的测定和日本血吸虫抗体的测定。 本论文具体内容包括以下几个方面： 第一部分：以溶胶-凝胶作为载体材料，制备了一种可重复使用的安培分析电化学免疫传感器。生物组分在室温下快速制备且无需额外的培养，在生物组分中加入一定量的牛血清白蛋白使免疫电极的非特异性吸附降低到一个很低的水平。以辣根过氧化酶作为标记物，邻-氨基酚作为酶催化反应的底物，采用一个竞争性免疫分析来确定待测抗原(或抗体)的浓度。同时还研究了1-O-烯丙基-4-羟基蒽醌的电化学行为以及2-氨基吡啶聚合膜修饰玻碳电极的制备及其电化学性质。(1)在第一章中报导了采用该技术，在低温下将补体第3成份抗血清固定在生物组分中。制备了可重复使用的补体3抗血清 O 湖南人学分析化学专业硕卜学位论文 钟桐生2001年10月 亿。＊el石墨安培分析免疫传感器，可对人血清中浓度为！．门～35．5阻9M－’ 范围内的补体第3成份进行定量测量。结果与临床诊断用的ELISA方法相比 较，令人满意八2）在第二章中报导了制备可重复使用的日本血吸虫电化学免 疫传感器，对 R本血吸虫抗体（SjAb）的实验条件进行了研究和优化。在 0．32～ 19．00 ug’ml”’浓度范围内对R本血吸虫抗体进行了定量检测／3）在第 3章报 导了在l－O－烯丙基－4－羟基葱酮（OAHAQ）水溶液中加入BSA，OMAQ的循 环伏安行为的变化情况，并可用来探测这些物种之间的相互作用关系以及用．＿，、、，、＿＿』＿＿＿。。、＿＿。＿＿＿＿＿＿＿＿＿．．＿＿。＿。。＿＿．＿＿．＿。L回 电化学法测定BSA。O）在第一章研究了2－氨基毗哇聚合膜修饰玻碳电极的 制备及其电化学性质，在出．7 BR缓冲溶液中，以 150 mV为工作电位，抗 坏血酸（AA）在修饰电极上的响应电流与 AA的浓度在4xl06～10”3 mol·L－’ 范围内呈良好的线性关系，在此条件下，多巴胺（DA）对 AA无干扰，电极 重现性良好，可用于实际样品AA的测定。 第二部分：主要研究了酞苔钮、4，4’，4”，4”’－四硝基酞誓耙、PVP－甲壳糖接 枝共聚物、珐酸正丙酯以及丙酸酯的合成／0在第5章研究了采用改进的缩 合法合成了酞青铅和对称取代四硝基衍生物4，4’，4”，4’“－四硝基酞菩铂，经元 素分析和红外光谱分析确定了这两种贵金属酞营化合物的组成与结构，并探 讨了酞青铂类化合物结构特点。（2）在第6章利用PVP优良的生物相容性， 将其接枝到甲壳糖分子链，以改善甲壳糖作为组织工程材料应用的不足之处。 为此，分别考察了不同引发剂及用量对PVP－甲壳糖接枝共聚物的反应性能、 接枝分子量、接枝效率以及产物的基本性状等的影响。结果表明PVP在Ce”” 引发、适当水含量的存在下可以接枝到甲壳糖的分子链上，反应时间、甲壳A 糖与N－乙烯基毗烙烷酮（NVP）单体比例、引发剂浓度等条件均影响接枝反 应的进行及接枝产物性质。该共聚物材料具有优良的亲水性、生物可降解性 和加工性能，可供作为组织工程支架材料。m 在第7章讨论了利用十二水 合硫酸铁按催化合成丙酸正丙酯的条件，并利用它催化合成了甲酸正丙酯、 乙酸正丙酯、丙酸正丙酯、丁酸正丙酯和异戊酸正丙酯八4）在第8章讨论了 用一水硫酸氢钠为催化剂，丙酸和不同醇的酯化作用及催化剂的重复使用性 能。表明，该酯化作用中硫酸氢钠活性良好，反应温和，无腐蚀和无废酸排 放等优点。更多还原

【Abstract】 Amperometric immunosensors are highly sensitive to multiple electrochemical analytical technology and conventional ELISA method. Compared other electrochemical sensing technologies, they are advantage of low cost, simplicity to use, high sensitivity, ease to digitize and real-time output. One of the obstacles retarding the practical application of immunosensors is their reusability. The development of renewable amperometric is a possible way to circumvent the problem. Here, antigen (antibody) is immobilized with graphite (carbon) and carrier on a transducer, the analyze is measured through on enzyme catalytic reaction after sandwitch or competitive immunoreaction. The surface of immuno-sensor can be renewed by used in a new immunoassay. Compared to the ELISA and radioimmunoassay (RIA), the proposed method is simply, speedy and provides a promising alternative to the diagnostic method in clinical laboratory. This thesis describes the preparation and’ characterization of a renewable amperometric immunosensor based on sol-gel technology. Renewable amperometric immunosensors have been developed for the complement 3 in human serum and Schistosoma-Japonium antibody, respectively. The detailed materials are showed as follows:This first part: Renewable amperometric immunosensors have been developed with sol-gel as carrier. 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 nonspecific adsorption of immunosensors to a lower level. Using o-aminophenol as a substrate, HRP as label, a competitive immunoassay is used to determine the concentration of the analyte.We also reported the cyclic voltammetric behavior of 1 -O-allyl-4-hdroxy-anthraquinone (OAHAQ) and the preparation and electrochemical characteristics of electrode modified by 2-pyridinamine. (1) In chapter 1, renewable amperometric immunosensor based on sol-gel technique has been developed by dispersing graphite, complement 3 antiserum and sol-gel at low temperature for the determination of complement 3 in human serum. Using €3 labeled with horseradish peroxidase, and o-AP as the substrate, an amperometric detection at -150 mV (vs SCE) results in a linear detection range of 1.17-35.5 ug-ml"1. Serum samples have been assayed and the results demonstrate feasibility of the proposed immunosensor for clinical analysis. (2) In chapter 2, a renewable amperometric immunosensor has been proposed for the determination of Schistosoma-Japoniumantibody (SjAb) in rabbit serum. The experimental conditions of SjAb determination were studied and optimized. The SjAb can be determined in the rang of 0.32-19.00 ng-ml"1. (3) In chapter 3, we reported that the cyclic voltammetric behavior of l-O-allyl-4-hdroxy-anthraquinone (OAHAQ) in an aqueous medium and electrochemical characteristic of complex of OAHAQ and BSA. The complex reaction can be used to probe the interaction between these species and to electrochemical determine BSA. (4) In chapter 4, the preparation and electrochemical characteristics of electrode modified by 2-pyridinamine are described. A sensitive amperometric response for ascorbic acid was obtained covering a linear range from 4.0* 10"6 to 1.0X10"3 mol-L"1. The modified electrode showed good stability and reproducibility, and used to the determination of ascorbic acid in vitamin C tablet and tomato. The method showed promising results compared with conventional methods.The second part: We mainly reported the synthesis of some chemical reagents such as palladiumphthalocyanine, palladium 4,4’,4",4’"-tetranitrophthalo-cyanine, PVP-chitosan grafting copolymer, n-propyl carboxylate, propionic esters. (1) In chapter 5, palladiumphthalocyanine and palladium 4,4’,4",4’"-tetranitrophthalo-cyanine were synthesized according to condensation method with high yield and high product purity. The structures of products were characterized by elementanalysis and IR spectra. (2) In chapter 6, to obtain a synthes更多还原