《电化学阻抗免疫传感器的研究》由会员分享，可在线阅读，更多相关《电化学阻抗免疫传感器的研究（54页珍藏版）》请在金锄头文库上搜索。
1、湖南大学 硕士学位论文 电化学阻抗免疫传感器的研究 姓名：严八妮 申请学位级别：硕士 专业：分析化学 指导教师：沈国励 20080501 电化学阻抗免疫传感器的研究 II 摘 要 电化学阻抗免疫传感器是通过检测修饰电极的界面特性来检测样品中抗体/抗 原目标物的一种有效方法。电化学阻抗免疫传感技术结合了电化学阻抗的高灵敏 性和免疫反应的高特异性，可响应修饰有生物分子的电极界面的电子转移速率的 信号变化，以简便、快速、灵敏、响应范围广、不用示踪标记物、不需样品纯化、 可进行自动化实时数据输出等优点，突破了以往分析方法的诸多瓶颈，在生化检 验方面显示出诱人的应用前景。本论文一方面利用亲和素生物素放大
2、建立了一种 高灵敏的电化学免疫阻抗传感技术；另一方面结合纳米生物修饰技术，设计一系 列免疫活性材料固定化新方法，探索临床重大疾患干燥综合症B (anti- Sjgren s syndrome B)的传感诊断新技术。具体内容包括： (1) 将巯基苯胺( p- aminobenzenthiol, p- ABT)组装于金电极表面，进而电聚合 形成稳定、致密、有序的 p- ABT 纳米复合膜界面，固定功能化的碳纳米管后，再 通过引入亲和素- 生物素放大系统，将生物素化羊抗人 IgG 固定于电极表面，用以 检测 hIgG，建立了一种高灵敏的电化学免疫阻抗传感器，获得传感器的线性范围 为 0.01 100
3、 ng/mL，检测下限为 0.0075 ng/mL。 (2) 首次在玻碳电极上自组装羟基磷灰石/碳纳米管复合物，制备了检测干燥 综合症 B (anti- Sjgren s syndrome B)抗体的阻抗传感器。结果表明，具有生物相容 性的羟基磷灰石和电子转移载体的碳纳米管的羟基磷灰石/碳纳米管的复合物便于 免疫电化学特别是阻抗的检测。利用本文制作的传感器对抗干燥综合症 B 抗体检 测的线性范围是稀释比 1:800 1:50，检测下限为 1:853，为了检验传感器的实用 性，进行了回收率实验，结果令人满意。该免疫传感方法有望成为的临床自身免 疫疾病中的检测自身抗体新的替代方法。 (3) 采用电
4、聚合羟基磷灰石（Hydroxyapatite）和胶原蛋白表面形成纳米复合 物层用于介导抗体在玻碳电极表面的固定化。以羊抗人 IgG 抗体- 抗原体系为测试 模型，采用该方法固定了羊抗人 IgG 抗体于玻碳电极表面，发展了一种新的电化 学阻抗传感技术检测血清中人 IgG 含量。实验结果表明，该抗体固定法具有传感 界面无需活化、固定抗体的免疫活性高等优点。利用本文制作的传感器对人 IgG 检测的线性范围是 20 500 ng/mL 检测下限 16 ng/mL。该固定化程序可望推广用 于其它各种免疫传感器的敏感界面或平台的设计。 关键词：对巯基苯胺；电聚合；亲和素生物素；羟基磷灰石；碳纳米管；干燥综
5、 合症 B；胶原蛋白；阻抗免疫传感器 电化学阻抗免疫传感器的研究 III Abstract Electrochemical impedance immunosensor has been demonstrated to be an effective method for probing the targets of antigens /antibodies in the samples by monitoring the interfacial properties of the modified electrode. Electrochemical impedance immunosens
6、ing technique, which incorporates high sensitivity of impedancic response and high specificity of antibody- antigen immunoreaction, is sensitive to signal of electronic transfer impendace of the biomolacular on the electrode interface. It presents some outstanding advantages including desirable simp
7、licity, high sensitivity, rapid response, wide detection range, real- time digital output, and free from tracer labeling and sample purification etc. This technique may breakthrough some bottlenecks of the traditional analytical methodologies, showing an attractive future of potential applications i
8、n the biochemical diagnosis of clinical diseases. In this paper, on one hand, based on the biotin- avidin amplified system, a new electrochemical impedance immunosensing technique with high sensitivity has been developed. On the other hand, the electrochemical impedance immunosensors are fabricated
9、by new material bio- molecular modification techniques for the diagnosis of human IgG and some clinical diseases such as anti- Sjgren s syndrome B(SSB). The detailed matericals are shown as follows: (1) p- aminobenzenthiol (p- ABT) was self- assembled on the surface of the gold electrode, then was e
10、lectrochemical polymerizational deposited resulting in a stable, dense and ordered p- ABT nanocomposite film on the interface of the electrode. After the immobility of functionalized carbon nanotubes (CNTs), biotinted- goat antihuman IgG was modified on the electrode surface by introducing the bioti
11、n- avidin system (BAS) to detect human IgG. An electrochemical impedance immunosensor with favorably high sensitivity was founded. The linear range of the resulted immunosensor was observed from 0.01 to 100 ng/mL with a detection limit of 0.0075 ng/mL. (2)An impedance immunosensor has been proposed
12、initially for the determination of anti- SSB antibodies using hydroxyapatite (HA) and CNTs on glass carbon electrode. HA and CNTs were self- assembled on a glass carbon electrode to form HA/CNTs composite film for immobilizing SSB antigens. It was found that the synthesis of the biocompatible HA and
13、 electron- transferring CNTs could facilitate the immunosensor good electrochemical detection performances, especially by way of the impedance spectra. It could detect anit- SSB antibodies in the linear response range of 1:800 to 电化学阻抗免疫传感器的研究 IV 1:50 dilution ratios, with a detection limit of 1:853
14、 dilution ratio. The practical feasibility of the developed immunosensor for detection of anit- SSB antibodies was further verified by recovery tests using the serum- diluted antibody samples. Such an immunosensing method should be promisingly tailored as a new alternative method for the determinati
15、on of auto- antibodies in the clinical diagnosis of autoimmune diseases. (3) A hybrid material formed by electrochemical polymerizational depositing HA and collagen was employed as the interface design of electrochemical impedance immunosensor, on which the antibodies were bound. The detection perfo
16、rmances of the resulting immunosensor were investigated by use of the antibody- antigen model system of goat antihuman IgG and human IgG. It was found that the developed sensing interface have some advantages such as the activation- free of the sensing interface and the relatively high immunological activities of antibodies. The proposed immunosensor could detect human IgG in the linear range of 20 to 500 ng /mL with a detection limit of 16 ng/mL. Such an interface design with the
