【作者】 李玲；

【导师】 王海燕；

【作者基本信息】 安徽师范大学 ， 分析化学， 2010， 硕士

【摘要】 近年来,对疾病相关标志物的灵敏检测引起了现代生物化学和生物医学研究的广泛兴趣。基于抗原与抗体之间特异性结合建立的免疫分析方法成为定量检测疾病标志物的最重要的分析方法之一,因为该方法中免疫反应分子之间高的特异结合性,能大大减小干扰。与放射免疫、荧光、化学发光等免疫分析法相比,电化学免疫分析方法有很多固有的优势,如成本低,灵敏度高,携带方便等,因此出现了大量各种各样的电化学免疫传感器。但是,在免疫传感器的发展过程中,人们发现抗原-抗体免疫复合物本身所形成的信号很弱,灵敏度有限,而临床中对早期超灵敏的生物标志物筛查癌症的需求不断增加。为此,出现了很多的信号放大策略。其中,将脂质体用于免疫传感器的信号放大引起了人们的极大兴趣,因为它具有制备简单,比表面积大,内部能包裹大量的信号分子等优点。本论文基于把电化学发光技术的优势和脂质体的特点结合起来,构建了电化学发光免疫传感器。为检验其性能,将其用于人免疫球蛋白G的检测,结果表明这种免疫传感器可以实现现代免疫传感器所要求的高灵敏度、高选择性、稳定及可再生等方面的研究目标。具体的研究工作包括以下几个方面:(1)以联吡啶钌为例,系统地研究了电化学发光脂质体的合成条件,建立了一种新的测定脂质体包封率的方法,另外还考察了脂质体的后处理如超声、挤压、渗析等对其包封率的影响;(2)基于碳纳米管修饰的玻碳电极以层层组装的方法,构建脂质体电化学发光免疫传感器,以抗体标记的吡啶钌脂质体为二抗,采用三明治型模式检测人IgG;(3)基于金纳米粒子和脂质体的信号放大作用,建立了脂质体电化学发光免疫传感器,实现了对人IgG双重信号放大的超灵敏检测,该传感器同时具有高灵敏度、高选择性、长期稳定性及易于再生等性能;(4)对联吡啶钌脂质体电化学发光免疫传感器在竞争检测模式中的应用进行了初步探讨,该检测模型可以实现对只有单一抗体的抗原的检测。本文为超灵敏免疫传感器的构建提供了一种新思路,在临床诊断中有潜在的应用价值。更多还原

【Abstract】 Recently, sensitive detection of disease-related proteins is critical to many areas such as modern biomedical research and clinical diagnosis. Immunoassay is one of the most important analytical techniques in the quantitative detection of disease markers with selectivity due to the highly specific molecular recognition between antigen and antibody. In comparison with other immunological methods such as radioimmunoassay, fluorescence immunoassay and chemiluminescence immunoassay, electrochemiluminescence immunoassay (ECLIA) possesses intrinsic advantages of low cost, high sensitivity, and good portability and has attracted considerable interest. Various ECL immunosensors have been developed for the determination of tumor markers. However, the sensitivity of the immunosensors is low, thus restricts their wide applications. At the same time, the demand for early and ultrasensitive screening of cancer biomarkers is increasing, therefore lots of signal amplification strategies arised. Among them, liposomes as the signal amplification of immunosensors aroused great interest because of its simple preparation, high specific surface area and large internal volume to contain a great number of signal molecules.In this dissertation, tris(2,2′-bipyridyl)ruthenium (Ru(bpy)32+)- encapsulated liposomes were prepared and ultrasensitive ECL immunosensors were constructed by combining the advantages of ECL technique and signal amplification function of liposomes. Four main work is involved: (1) Ru(bpy)32+-encapsulated liposomes was synthesized and the effect of ratio of ingredients, post-treatments such as ultrasound-treatment, extrusion and dialysis on the encapsulation efficiency (EE) and stability of the liposomes were investigated. Moreover, a new method based on ECL for the determination of EE of liposome was established; (2) A simple and sensitive electrochemiluminescent immunosensor was fabricated by immobilizing goat-anti-human immunoglobulin G (hIgG) antibody on a multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode. Using antibody-tagged Ru(bpy)32+-encapsulated liposome as a second antibody, The hIgG antigen and antibody-tagged liposome can be sequentially bound on the immunosensor through antigen-antibody specific interaction, forming a sandwich-type immunocomplex. The ECL intensity of the immunosensor increased linearly with the hIgG concentration from 0.01 to 0.8 ng/mL with a detection limit of 0.004 ng/mL (S/N=3). In addition, it was successfully applied for the detection of hIgG in human serum; (3) Ru(bpy)32+-encapsulated, antibody-tagged liposomes (liposomal biolabels) was used as the label for the ECL immunosensor based on an electrodeposited gold nanoparticles (NPs) modified electrode. The use of liposomal biolabels with large internal volume to carry a great number of reporter molecules, together with the gold NPs with large surface for high antibody loading, could lead to enormous dual signal amplification, thus result in high sensitivity. With a sandwich type immunoassay format, a linear response to hIgG from 0.1 to 25 pg/mL (R=0.992) with a detection limit of 0.01 pg/mL (S/N=3) was obtained, which is five orders magnitude lower than that in the previous reports. In addition, the immunosensor showed some good performance such as high sensitivity, high selectivity, long-term stability and could be regenerated easily; (4) Preliminary studies on the the application of Ru(bpy)32+-encapsulated liposome in competitive ECL immunoassay were carried out. The assay was based on the competition between the antigen and antigen-tagged liposomes. This detection model could be used for the determination of antigen with a single antibody. The developed protocols provide new ways of designing immunosensors with high sensitivity, which may find potentially broad applications in clinical diagnosis.更多还原