【作者】 郭秋平；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2011， 博士

【摘要】 多年来,人们一直致力于发展和完善生物大分子的研究和分析方法,使其更加灵敏、精确、便捷、经济,以满足各个领域对其越来越高的要求。生物分子信号放大检测技术是近年来发展起来的一种利用各种工具酶和纳米颗粒等手段,并结合电化学、光学、压电等技术的高灵敏检测方法,可实现对生物分子的直接高灵敏检测,是生物分析科学领域中的一个研究重点和热点。如何利用现有分子生物学技术和手段将生物分子的含量、相互作用等信息实时、灵敏、特异地转化为易于检测的物理量并拓展其在分子生物学及生物医学中的应用,如何设计新的信号转换手段和建立新的生物分子信号放大检测技术平台来解决生命研究过程中的新问题,仍然是生物医学和分析化学工作者所面临的重大科学问题。本论文以上述科学问题为目标,以核酸和蛋白质这两类组成生命的最主要生物大分子为研究对象,利用多种核酸工具酶和荧光核酸探针技术,建立了一系列简便、快速的生物大分子荧光信号放大检测技术平台,在核酸定量分析、点突变(point mutation)检测、多重单核苷酸多态性(Single Nucleotide Polymorphisms, SNPs)基因分型和蛋白质的的分析方面得到重要应用。主要内容归纳如下：1、基于接近连接效应的滚环扩增(Rolling Chain amplification, RCA)方法的建立及用于点突变的高灵敏检测。滚环扩增技术由于具有高灵敏、高特异性和等温操作等诸多优点,在生物化学分析和临床医学诊断中有很好的应用前景,但同时也存在着用于扩增的模板-挂锁探针的合成难的问题,限制了滚环扩增技术的广泛应用。本部分在普通的滚环扩增技术基础上,利用接近连接效应原理,建立了一种滚环扩增模板-挂锁探针合成的新方法。该方法不但解决了普通滚环检测技术中存在的长片段DNA合成难和成本高的问题,而且提高了线性片段成环的效率,为滚环扩增的进一步广泛应用提供了帮助,并利用该方法合成的挂锁探针进行滚环扩增实现了对基因点突变的高灵敏检测。2、基于分子信标的等温循环链置换聚合酶反应的荧光信号放大技术用于核酸的检测。利用特殊设计的长臂分子信标作为聚合酶反应的模板和检测探针,在靶核酸分子与分子信标杂交中引发引物与分子信标臂部退火,在DNA聚合酶存在下产生循环的链置换聚合酶反应,从而实现了对靶核酸的荧光信号循环放大检测。该技术以靶DNA诱发链置换聚合酶反应的循环发生来实现信号放大,而无需直接扩增靶DNA分子,避免了反应产物污染,并且设计简单、操作简便快捷、在等温条件下反应,灵敏度高,检测下限可达6.4fmol/L,为核酸定量分析和病毒检测提供了一种简单、快速、高灵敏的荧光信号放大分析方法,有望在病毒检测分析等方面得到广泛的应用。3、基于切刻酶介导的循环链置换聚合反应用于多重SNPs的荧光信号放大检测。癌症、心脑血管疾病和精神性疾病等复杂疾病均由多个SNPs相互作用引起,因而在关联分析研究中同时对多个SNPs进行基因分型和检测已成为多基因复杂性状疾病的遗传易感性研究的重要技术手段。目前基于杂交稳定性的差异或点突变引起的酶反应速度差异的多重SNPs基因分型和检测方法具有分析点突变差异不显著的缺点,并同时很难将多个SNPs等位基因完全有效地分开,且没有信号放大检测的过程。本部分设计合成了分别带信号识别序列、酶切位点和引物识别序列的等位基因核酸探针,通过连接酶反应,巧妙结合切刻内切酶和聚合酶反应,建立了一种等温、快速、高灵敏和高特异的等温循环荧光信号放大的多重SNPs检测技术平台。该方法同时将多重SNPs基因的点突变信号准确转换为了多种具有显著差异、并可循环生成的单链DNA信号识别序列分子,并与带不同荧光标记的分子信标识别,获得不同的荧光信号,从而准确实现了对多重SNPs的信号放大检测。该检测平台克服了现有检测方法的缺陷,操作简单,等温反应,既能准确判断SNPs,又能将检测信号进行放大,还能对多个SNPs等位基因同时进行分析,为相关疾病筛查和临床诊断、防治和手术预后监测提供了一种新的强有力手段。4、利用双链荧光核酸适体探针建立了简便快速的蛋白质的荧光分析方法。核酸适体因能高效、特异地结合各种配体和具有易合成、易修饰等特点,在蛋白质检测方面具有广泛的应用前景。本部分基于结构转换信号核酸适体探针的设计,发展了一种新的基于双链荧光核酸适体探针的蛋白质检测方法。该方法对蛋白质的线性响应范围为6.0～100.0nmol/L,检测下限为6.0nmol/L。该方法对核酸适体探针的空间结构和与靶标的结合位点没有特殊的限制和要求,设计简单,操作方便,有望为基于核酸适体探针的靶物质检测提供一种简便、快速的通用检测平台5、基于发夹型核酸适体探针和聚合酶反应的荧光信号放大检测技术用于蛋白质检测。核酸分子由于可借助各种工具酶来检测,因而其检测方法远比传统的蛋白质检测方法灵敏和特异。通过检测核酸来定量蛋白质,一直是人们研究的热点和重点。本部分设计了一种新型的发夹型核酸适体探针,利用其与靶蛋白反应后构象发生变化的特点,建立了一种基于聚合酶反应的荧光信号放大的蛋白质检测新方法。该发夹型核酸适体被设计成蛋白质配体和聚合酶反应模板,当蛋白质与核酸适体特异性结合后,探针的构象由发夹型转变为线型,成为聚合酶反应的模板,在引物和聚合酶作用下启动聚合酶反应,聚合酶反应的进程被荧光染料实时转换为荧光信号,实现了在未直接标记核酸适体的情况下检测蛋白质,同时由于聚合酶的链置换活性,使诱导发夹型核酸适体构象变化的靶蛋白能在聚合酶反应中被置换出来,重新诱导下一轮的聚合酶反应,从而可实现对靶蛋白的循环放大检测,对蛋白质的线性响应范围为0.5～8.0nmol/L,检测下限为0.5nmol/L。该方法无须直接标记核酸适体探针、对核酸适体的空间结构无特殊要求、且只需一个核酸适体靶位点,因而可广泛应用于蛋白质的高灵敏检测。更多还原

【Abstract】 With the development of Genome Project and Protein Project, more sensitive, accurate, convenient and economic detection methods for biological macromolecules have been the focus of biology, medicine and chemistry. In recent years, signal amplification detection technologies based on electrochemical, optical, piezoelectric technologies and the use of various enzymes and nanoparticles, have realized highly sensitive detections of biological macromolecules. How to utilize the existing molecular biology techniques and tools to convert the information of biological molecules to detectable signals with high sensitivity and specificity, how to further expand the use of signal amplifying methods in molecular biology and biomedical application, and how to design novel signal conversion means and develop novel signal amplification detection technology platform to address the research process to life in the new issue, are still significant topics for researches in biomedical and analysis chemical areas.In this thesis, a series of fluorescence signal amplifying detection technology platforms based on a variety of nucleic acid enzymes and fluorescent nucleic acid probes have been developed for quantitative analysis of DNA, point mutation detection, multiple single nucleotide polymorphisms (SNPs) genotyping and protein detection. The main researches included in this dissertation are presented as following:1. The construction of a padlock probe for rolling chain amplification based on proximity-ligation and detection of point mutation. Rolling circle amplification (RCA) has a wide range of applications in the biochemical analysis and clinical diagnosis due to its high sensitivity and specificity, isothermal operation and many other advantages. But this method is still difficult to construct padlock probes, which has restricted the RCA to be used widely. In this part, we developed a novel method for construction of padlock probe based on proximity-ligation. This method avoids to syntheses cost long DNA fragment and improves the construction efficiency of padlock probes. Here, point mutant genes have been detected sensitively using this method.2. Fluorescence signal amplifying detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction with molecular beacon. In this method, the specific long-stem molecular beacon (MB) is designed as a template of polymerization reaction and fluorescence signal carrier and the target is designed as a trigger of polymerization reaction. Upon recognition and hybridization with the target ssDNA, the stem of the MB is opened. The opened MB anneals with the primer and triggers the polymerization reaction circle-after-circle, which results to amplification of fluorescence signal. This design of method is simple and its operation is easy in isothermal reaction conditions. The detecting limit is6.4×10-15mol/L. It is expected to provide a simple, fast and sensitive platform for detection and subsequent analysis of nucleic acids, and potential to be widely used in virus detection.3. Multiple fluorescence signals amplifying detection of SNPs based on nicking enzyme mediated circular strand-displacement polymerase reaction. In this part, based on nicking enzyme mediated circular strand-displacement polymerase reaction, an isothermal fluorescence signals amplifying method for multiple SNPs detection platform,is established through the use of multiplex allele-specific probes containing signal discriminating sequences, nicking enzyme sites and primer annealing site. The trick of this method is to divert the allele-specific discriminations to multiplex fluorescence signals. This method is simple and easy to operation. Furthermore, this assay could not only determine the sites of multiplex SNPs, but also identify them accurately. This approach would be a promising technology for multiple SNPs genotyping.4. Development of a simple method for protein detection based on double-stranded fluorescent aptamer probe. Aptamer has a wide range of applications for protein detection due to its easy to synthesis and label, high and specific affinity with its target. In this part, a novel method with a double-stranded fluorescent probe for protein detection has been developed. A liner range of6.0-100.0nmol/L of protein detection is achieved. The method has a detection limit of6.0nmol/L. This strategy is easy to generalize for any aptamer without prior knowledge of its secondary tertiary structure, and would be used as a simple and general tool for protein detection.5. Fluorescence signal amplifying detection of protein based on hairpin aptamer probe and strand-displacement polymerase reaction. In this part, a novel fluorescent method of protein detection has was developed using hairpin aptamer based on polymerase reaction. The hairpin aptamer was designed to be employed as the protein ligand and template of the polymerase reaction. When the aptamer was bound to the protein, it would change to a liner strand and induce the strand-displacement polymerase reaction. Then protein detection was carried out by monitoring the polymerase reaction without directly labeling with the aptamer. Then the displaced protein could bind to the aptamer again and trigger a next strand-displacement polymerase reaction, leading to fluorescence signal amplifying detection of target protein. The result showed the method with a liner range of0.5～8.0nmol/L and detection limit of0.5nmol/L. This proposed method has the potential to design other protein probe with complex structure aptamer and be used as a simple and general tool for protein detection.更多还原