基于酶循环放大反应荧光检测生物小分子及DNA的研究

发布时间：2018-08-20 14:39

【摘要】：生物传感器是将目标识别转换为物理可检测的信号(例如光学,电子质量或磁信号)的装置。通常,生物传感器包含两个基本上功能的组分:目标识别和信号转导。理论上,识别组件是传感器性能的关键。理想的识别组件应具有高灵敏度,令人羡慕的选择性,快速响应,强大的性能和各种目标的通用性等特点。随着生化分析领域的不断发展,单一的生物传感器已经无法满足我们的需要。由于生物小分子无法直接进行扩增、待测物浓度太低等各种各样的局限性,因此大力发展信号放大技术已经成为了生物化学分析领域的工作要点。本文主要介绍了利用DNA酶循环放大方法和DNA自组装扩增等方法,实现了DNA和生物小分子的高灵敏度、高特异性、高选择性、快速直接的检测。主要包括以下三个方面:1.DNA甲基化和MTase活性的分析在癌症的早期临床诊断中非常重要,目的是提供对基因阻遏机制的洞察和开发治疗甲基化相关疾病的新药物。结合树枝状滚环扩增和DNA酶特异性识别作用,构建了DNA甲基转移酶检测的新的荧光方法。在DNA甲基转移酶(MTase)的存在下,双链DNA探针被甲基化,生成限制性内切核酸酶DpnI的特异性识别位点。然后切割的杂交DNA探针作为信号引物起作用,以启动树状滚环扩增反应。随后,滚环扩增产生与Mg2+依赖性DNA酶的互补序列,在Mg2+存在下释放荧光信号。该方法对DNA甲基化酶的检测限达0.36 U/m L,线性范围为1.0?10 U/mL。此外,利用该方法可以评估和筛选MTase的抑制剂,这可能有助于发现抗癌药物。2.基于三通交叉结构和自组装循环放大技术荧光检测DNA,利用目标DNA触发三通(3-WJ)结构以及DNA酶的聚合剪切作用,结合DNA自组装技术(CHA)对检测信号进行二次放大,设计了一种高度特异性、灵敏性、快速的DNA检测方法。CHA作为一种酶信号扩增技术,已被多次使用在设催化扩增反应的方案中。目标DNA与3-WJ引物和3-WJ模板紧密结合形成稳定的3-WJ结构,在DNA聚合酶和剪切酶作用下形成引物链,这些引物链包含多段催化发卡探针自组装的催化序列,可以通过互补杂交的方式打开发夹探针H1和H2,引发自组装反应,对信号进行二次放大。反应后释放荧光探针,通过两步循环放大技术,实现了对目标DNA的高灵敏度、高选择性检测。3.基于酪胺酶信号放大新型免疫传感器,用于高度灵敏的检测凝血酶。本体系利用凝血酶及其两条适体的特异性结合将磁性微球与金纳米粒子相连,形成一个磁性微球-凝血酶-金纳米粒子的复合结构。同时,将酪胺与辣根过氧化物酶(HRP)结合,与上一复合物相连,形成信号放大。HRP催化反应底物过氧化氢氧以及对羟基苯乙酸。通过对荧光信号的检测,实现对肿瘤标志物凝血酶的灵敏度检测。
[Abstract]:Biosensors are devices that convert target recognition into physically detectable signals, such as optical, electronic mass or magnetic signals. Typically, biosensors contain two basic functional components: target recognition and signal transduction. In theory, the identification component is the key to sensor performance. The ideal identification module should be characterized by high sensitivity, enviable selectivity, rapid response, strong performance and versatility of various targets. With the development of biochemical analysis, a single biosensor can not meet our needs. Because the biological small molecules can not be amplified directly and the concentration of the samples is too low, the development of signal amplification technology has become the key point in the field of biochemistry analysis. In this paper, we mainly introduce the methods of DNA enzyme cycle amplification and DNA self-assembly amplification to realize the high sensitivity, high specificity, high selectivity and rapid and direct detection of DNA and biological small molecules. The analysis of DNA methylation and MTase activity is very important in the early clinical diagnosis of cancer. The aim is to provide insight into the mechanism of gene repression and to develop new drugs for the treatment of methylation-related diseases. A new fluorescence method for the detection of DNA methyltransferase was constructed by combining the dendritic rolling ring amplification and the specific recognition of DNA. In the presence of DNA methyltransferase (MTase), the double-stranded DNA probe was methylated to form a specific recognition site for restriction endonuclease DpnI. Then the cut hybrid DNA probe acted as a signal primer to initiate the dendritic rolling ring amplification reaction. Subsequently, the rolling amplification produced complementary sequences with Mg2 dependent DNA enzymes and released fluorescence signals in the presence of Mg2. The detection limit of DNA methylase was 0.36 UmL, and the linear range was 1.0 渭 10 UmL. In addition, the method can be used to evaluate and screen inhibitors of MTase, which may be helpful in the detection of anticancer drugs. 2. 2. Based on the three-way cross structure and self-assembly cyclic amplification technique, the detection signal was re-amplified by using the target DNA triggered three-way (3-WJ) structure and the polymeric shear action of DNA enzyme, combined with the DNA self-assembly technique (CHA). As an enzyme signal amplification technique, a highly specific, sensitive and rapid DNA detection method, cha, has been used in the catalytic amplification reaction for many times. The target DNA tightly binds with 3-WJ primers and 3-WJ templates to form a stable 3-WJ structure, forming primer chains under the action of DNA polymerase and shearing enzyme. These primer chains contain self-assembled catalytic sequences of multi-segment catalytic hairpin probes. The hairpin probes H1 and H2 can be opened by complementary hybridization to initiate a self-assembly reaction and the signal can be re-amplified. The fluorescence probe was released after reaction and the high sensitivity and selectivity of detection of target DNA was achieved by two-step cyclic amplification technique. A novel immunosensor based on tyramine signal amplification is used to detect thrombin with high sensitivity. In this system, the magnetic microspheres are connected with gold nanoparticles by the specific binding of thrombin and two aptamers to form a composite structure of magnetic microspheres, thrombin and gold nanoparticles. At the same time, tyramine was combined with horseradish peroxidase (HRP) to form signal amplification. HRP-catalyzed substrates, hydrogen peroxide oxygen and p-hydroxyphenylacetic acid, were synthesized by the combination of tyramine and horseradish peroxidase (horseradish peroxidase). The sensitivity of tumor marker thrombin was detected by fluorescence signal detection.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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