基于解旋酶酶促信号扩增的氧化石墨烯生物传感平台检测microRNA的研究

发布时间：2018-05-08 18:25

本文选题：解旋酶 + 生物传感平台　；参考：《太原理工大学》2017年硕士论文

【摘要】：目前,分子诊断生物学研究正不断地对人类疾病诊断产生变革性的影响,它可以帮助医生更好地了解每个病人的临床状况,如当前疾病状态、预测疾病状态、药物反应和治疗预后。分子诊断的本质在于灵敏检测疾病生物标志物,如蛋白质、碳水化合物或核酸。MicroRNAs(miRs)作为一组内源性非编码RNA(18-25核苷酸),在多种疾病细胞过程中发挥着重要的作用。值得注意的是,miRs的频繁异常表达将这些小细胞分子成分加入到癌症的潜在生物标志物行列。因此,敏感和有效的检测方法对于更好地理解这些相对较小的核酸分子和进一步验证其在临床分子诊断中的功能具有重要的意义。然而,由于它们具有小尺寸和高度同源等特性,敏感和经济的miRs检测方法仍然具有很大的挑战性。近年来,各种以检测灵敏度和特异性为研究重点的新方法处于不断开发中,如纳米颗粒复合探针、等温扩增方法和电化学方法等。其中,大部分等温扩增方法所依靠的链置换、链杂交和链延伸等循环扩增原理都与核酸酶密切相关,如聚合酶、核酸内切酶或切口酶、双链特异性核酸酶等。解旋酶是一类重要的核酸酶,它可以利用ATP水解的能量沿着核酸磷酸骨架定向移动,分离两条退火的核酸链(即dna链、rna链或rna-dna杂交链)。解旋酶对核酸分子探针内氢键的水解活性,使其在相应的分子诊断系统中具有巨大的潜在应用性,特别是以杂交反应为检测原理的传感系统。本文通过pet24a-recqe重组质粒诱导表达获得大肠杆菌recq解旋酶(recqe),系统研究了recqe在不同mir氧化石墨烯(go)传感平台中的作用机理以及酶学活性。分析了recqe在以1:1杂交反应以及杂交链反应(hcr)为检测原理的传感平台中的分子作用机制,构建了recqe辅助go传感平台和高灵敏recqe酶促hcr/go传感平台,并对这两个传感平台做了进一步的研究,主要内容如下:(1)开发了一种以1:1杂交反应为检测原理的recqe酶辅助go传感平台,用于系统的研究解旋酶对传感平台中杂交反应的分子作用机制。此传感平台通过简单的dna/rna杂交反应,实现dna探针(fdna)所标记荧光基团在go表面的荧光猝灭与恢复,通过荧光信号的变化直观地分析recqe对杂交反应的分子作用机制。recqe参与的检测系统荧光信号强度增强高达22.56%,这表明recqe对go表面的杂交反应具有明显的促进作用。recqe酶辅go传感平台检测let-7a的灵敏度实验与特异性实验的结果表明,此传感平台能够高特异性的区分各种碱基错配的mirs并能准确检测系统相对应的靶目标,能够灵敏的定量检测mir至0.18nm。这说明,此go传感平台并没有因recqe的引入而影响其检测灵敏度以及特异性,验证了recqe对此go传感平台的优良兼容性。此外,fdna与阻断dna(BDNA)组成的FDNA-BDNA复合探针可以有效阻止RecQE以及其他小分子蛋白与FDNA的非特异性结合,降低外界干扰所引起的系统背景荧光强度变化。(2)利用RecQE在FDNA-BDNA-GO传感平台当中的作用机理,我们进一步开发了一种基于RecQE酶促杂交链反应(HCR)信号扩增的灵敏检测miR的GO传感平台。相比于无酶HCR/GO传感平台,RecQE酶促HCR/GO传感平台荧光信号强度表现出高达244.3%信号增强,并将检测时间从4 h缩短至50 min。条件优化后的RecQE酶促HCR/GO传感平台应用于检测靶目标let-7a的灵敏度实验与特异性实验当中,结果表明此传感平台对10 fM到2000 fM之间的目标let-7a具有良好的检测线性关系,能够灵敏的检测低至4.2 fM靶目标的荧光信号,比无酶HCR/GO传感平台的检测灵敏度高出两个数量级。同时,此传感平台能够高特异性的区分各种碱基错配的miRs并能准确检测系统相对应的靶目标。本实验首次成功的将解旋酶应用于高灵敏度检测mi R的GO传感平台当中,改善了无酶HCR/GO传感平台灵敏度低和效率低等不足,为生物医学研究和临床早期诊断提供了又一可靠的、灵敏的miR定量方法。
[Abstract]:At present, molecular diagnostic biology is constantly changing the diagnosis of human disease. It can help doctors better understand the clinical status of each patient, such as the status of the current disease, the state of the disease, the drug reaction and the prognosis. The molecular diagnosis of molecular diagnosis is the sensitive detection of biomarkers, such as protein, Carbohydrates or nucleic acid.MicroRNAs (miRs), as a group of endogenous non coded RNA (18-25 nucleotides), plays an important role in a variety of disease cell processes. It is noteworthy that the frequent abnormal expression of miRs adds these small cell components to the potential biomarkers of cancer. Therefore, sensitive and effective detection parties The method is of great significance to better understand these relatively small nucleic acids and to further verify their function in the diagnosis of clinical molecules. However, because of their small size and high homology, the sensitive and economical miRs detection methods are still very challenging. In recent years, various sensitivity and specificity have been detected. The new methods for the study of heterosexual focus are in continuous development, such as nano particle composite probes, isothermal amplification methods and electrochemical methods. Among them, the principle of chain replacement, chain hybridization and chain extension, which most of the isothermal amplification methods rely on, is closely related to the nuclease, such as polymerase, endonuclease or incisiase, double stranded. Heteronuclease, a kind of important nuclease, is an important class of nuclease, which can use the energy of ATP hydrolysis to move along the nucleic acid skeleton of nucleic acid to separate two annealed nucleic acid chains (DNA chain, RNA chain or RNA-DNA hybrid chain). The hydrolytic activity of the hydrolytic enzyme to the hydrogen bond in the nucleic acid probe makes it have a huge amount in the corresponding molecular diagnostic system. Large potential application, especially the sensing system of hybridization reaction as detection principle. In this paper, Escherichia coli RecQ helicase (recqe) was induced by pet24a-recqe recombinant plasmid, and the mechanism of action and enzyme activity of recqe in different Mir oxidation Shi Moxi (go) sensing platform were systematically studied. The reaction of recqe in 1:1 hybridization reaction was analyzed. And the molecular mechanism of the hybridization chain reaction (HCR) as the sensing platform, a recqe assisted go sensing platform and a highly sensitive recqe hcr/go sensing platform were constructed, and the two sensing platforms were further studied. The main contents were as follows: (1) a recqe enzyme assisted go with the detection principle of 1:1 hybridization reaction was developed. The sensing platform is used to systematically study the molecular mechanism of the interaction between the helicase on the sensing platform. The sensing platform realizes the fluorescence quenching and recovery of the fluorescent groups marked by the DNA probe (FDNA) on the surface of the go through a simple dna/rna hybridization reaction. By the changes of the fluorescence signal, the molecular composition of the hybridization reaction is intuitively analyzed by the changes of the fluorescence signal. The intensity enhancement of the fluorescence signal of the detection system with the mechanism.Recqe is up to 22.56%, which indicates that recqe has an obvious promoting effect on the hybridization reaction on the go surface. The results of sensitivity and specificity of the.Recqe enzyme assisted go sensing platform to detect let-7a show that the sensing platform can distinguish a variety of base mismatched Mirs with high specificity. It can accurately detect the target target of the system, and can detect Mir to 0.18nm. sensitively. The go sensor platform does not affect its detection sensitivity and specificity because of the introduction of recqe, and validates the good compatibility of recqe go sensing platform. In addition, FDNA and FDNA-BDNA compound probe composed of blocking DNA (BDNA) can be used. In order to effectively prevent the non specific binding of RecQE and other small molecular proteins with FDNA, the changes of system background fluorescence intensity caused by external interference are reduced. (2) we have further developed a sensitive detection miR based on the amplification of RecQE enzyme catalyzed hybridization chain reaction (HCR) using the mechanism of RecQE in the FDNA-BDNA-GO sensing platform. GO sensing platform. Compared with the non enzyme HCR/GO sensing platform, the fluorescence signal intensity of the RecQE HCR/GO sensing platform is up to 244.3% signal intensities, and the RecQE enzyme promoting HCR/GO sensing platform after the optimization of the detection time from 4 h to 50 min. is applied to the sensitivity test and specific experiment of detecting target target let-7a. It shows that the sensing platform has a good linear relationship with the target let-7a between 10 fM and 2000 fM, and can detect the fluorescence signal of the target with a low to 4.2 fM target sensitively. It is two orders of magnitude higher than the detection sensitivity of the non enzyme HCR/GO sensing platform. At the same time, the sensing platform can distinguish a variety of base mismatched miRs with high specificity and can be highly specific. The target target of the system is detected accurately. In this experiment, the first successful application of the helicase to the GO sensing platform of high sensitivity detection of MI R has improved the low sensitivity and low efficiency of the non enzyme HCR/GO sensing platform, which provides a reliable and sensitive miR quantitative method for biomedical research and early clinical diagnosis.

【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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