基于核酸适配体的凝血酶化学发光检测新技术

发布时间：2018-01-21 01:31

本文关键词： 核酸适配体磁性微球化学发光凝血酶 96孔板聚苯乙烯微球放大　出处：《复旦大学》2010年硕士论文　论文类型：学位论文

【摘要】： 核酸适配体是近年来发展起来的一类SELEX筛选出的单链寡核苷酸序列,能高效、特异性地结合各种生物目标分子,故它的出现为化学生物学界和生物医学界提供了一种新的高效快速识别的研究平台。目前生物分子检测通常采用抗原抗体特异性识别模式,但由于受到抗体自身稳定性、制备时间较长、对环境敏感等因素的影响,在一定程度上限制了抗体检测技术的广泛应用。相比之下,核酸适配体自身稳定性好、目标分子范围广、特异性和亲和性高、化学合成相对简单、快速、容易获得、易于修饰与标记,且在生物传感器设计中应用灵活等优点,近几年在生物分析检测方面备受关注。目前已经成为临床诊断、环境监测、生物医学、药学研究等许多领域中的研究热点。化学发光(CL)分析法具有不需光源,避免了杂散光的干扰,仪器设备简单、操作简便,具有极高的灵敏度,较宽的检测范围,可实现全自动化等特点,正逐渐成为分析检测中极为有用的工具。随着与其它学科的交叉研究和应用领域的扩展,目前已成功地应用在药学、生物学、分子生物学、临床医学和环境学等诸多领域。在本论文中,我们采用化学发光分析法,利用核酸适配体对目标分子凝血酶的高分辨识别,发展了两种具有创新意义的凝血酶化学发光适配体生物传感器,由以下三部分组成：第一章简要概述了核酸适配体的制备、特点和优势,及其在相关检测领域的应用。第二节中着重介绍了基于核酸适配体的凝血酶生物传感器的研究进展,包括电化学、光学、色谱学和其它检测方法,并列举了多种检测模式和检测技术在近几年的部分典型示例,最后简单介绍了化学发光检测技术基础知识以及本课题的主要研究内容。第二章基于凝血酶的两条核酸适配体与凝血酶的高亲和力,构建了三明治结构、磁性分离的新型化学发光生物传感器,可检测凝血酶的线性范围为0.5-100 nM (I=651.8C+1092.1,R2=0.9850),最低可检测浓度为0.01 nM。该技术采用磁性微球作为生物富集分离载体,具有操作简单、快速、灵敏度高的特点。第三章以96孔板作为载体,基于核酸适配体对凝血酶的高分辨识别,构建了三明治结构的高通量化学发光生物传感器,并进而以490 nin的聚苯乙烯微球作为放大载体,构建了一种超高灵敏度的凝血酶CL检测平台。不放大体系中凝血酶的线性范围为0.9375-30 nM(I=6330.2C+1717.3,R2=0.99035),最低检测浓度为0.46 nM；放大体系中凝血酶的线性范围为7.8-250 pM(I=8711.5C-79898.1,R2=0.9937),最低检测浓度为3.9 pM,检测灵敏度提高了100倍以上。
[Abstract]:Nucleic acid aptamers are a class of single-stranded oligonucleotide sequences screened by SELEX in recent years, which can bind various biological target molecules efficiently and specifically. Therefore, it provides a new research platform for chemical biology and biomedical research. At present, antigen-specific recognition pattern is usually used in biomolecular detection. However, due to the autostability of antibody, long preparation time, sensitive to the environment and other factors, to a certain extent, the wide application of antibody detection technology is limited. In contrast, the aptamer of nucleic acid has good stability. It has many advantages such as wide range of target molecules, high specificity and affinity, relatively simple, rapid, easy to obtain, easy to modify and label, and flexible in biosensor design. In recent years, it has attracted much attention in many fields, such as clinical diagnosis, environmental monitoring, biomedicine, pharmaceutical research and so on. Chemiluminescence CLC analysis has the advantages of no light source, no stray light interference, simple instrument and equipment, simple operation, high sensitivity, wide detection range, full automation and so on. With the development of cross-research and application fields with other disciplines, it has been successfully applied in pharmacy, biology and molecular biology. In this paper, we use chemiluminescence analysis to identify the target molecule thrombin by nucleic acid aptamers. Two novel chemiluminescence biosensors for thrombin have been developed. The biosensors are composed of the following three parts: In the first chapter, the preparation, characteristics and advantages of nucleic acid aptamers and their applications in the field of detection are briefly reviewed. In the second section, the research progress of thrombin biosensors based on nucleic acid aptamers is introduced. It includes electrochemical, optical, chromatographic and other detection methods, and enumerates a number of typical examples of detection patterns and techniques in recent years. Finally, the basic knowledge of chemiluminescence detection technology and the main research contents of this subject are briefly introduced. Based on the high affinity between two nucleic acid aptamers of thrombin and thrombin, a new chemiluminescence biosensor with sandwich structure and magnetic separation was constructed in the second chapter. The linear range of detectable thrombin was 0.5-100nM Igna 651.8C1092.1R2O0.9850). The lowest detectable concentration is 0.01 nm. This technique uses magnetic microspheres as the carrier of bioconcentration separation, and has the advantages of simple operation, fast operation and high sensitivity. In chapter 3, a sandwich structure high throughput chemiluminescence biosensor was constructed based on the high resolution recognition of thrombin by nucleic acid aptamers. And then use 490 nin polystyrene microspheres as the amplification carrier. A high sensitivity detection platform for thrombin CL was constructed. The linear range of thrombin in unamplified system was 0.9375-30 nM(I=6330.2C 17.3. The lowest detection concentration was 0.46 nm. The linear range of thrombin in the amplified system was 7.8-250 pMU 8711.5C-79898.1C, R2O 0.99370.The lowest detection concentration was 3.9 pm. The sensitivity of detection was increased by more than 100 times.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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