基于Toehold介导的DNA链置换构建电化学传感MicroRNAs与凝血酶的应用研究

发布时间：2018-03-04 05:25

  本文选题：电化学生物传感器　切入点：Toehold　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着基因诊断与治疗、药物筛选与作用机理探究、环境污染与食品安全监测等诸多研究领域蓬勃迅速的发展,实现对病原蛋白和特定序列microRNA(miRNA)的方便、耗时少、高灵敏且高特异性分析检测的意义是非常重要的。电化学DNA生物传感器(DNA electrochemical biosensor)因为其自身构造简单、易实现便携性、成本低廉、高灵敏且特异性强等优点而备受关注。目前,对于研究者们来说,开发电化学DNA生物传感器在生物和医学科学领域已成为具有吸引力和极具挑战的前沿性课题。粘性末端(Toehold)介导的链置换反应(Toehold-mediated strand displacement reaction,TSDR)是构建动态DNA纳米机器的基础反应。本文结合TSDR辅助目标物循环放大技术构建了三种电化学DNA生物传感器,实现了对mi RNA和凝血酶的超灵敏检测,具体研究工作如下:1.基于DNA分子机器实现无酶目标物循环放大和最小化背景噪声的电化学检测癌细胞中的MicroRNAMicroRNA(miRNA)表达水平的变化是可以作为诊断不同癌症的有用生物标记物。在本项工作中,基于miRNA引发无酶目标物循环信号放大分子机器,我们开发了一个简单新颖的电化学DNA生物传感器实现高灵敏地检测人乳腺癌细胞中的miRNA-21。相应传感器的制备是由3条单链DNA组成的双链探针通过自组装在金电极表面上,目标物miRNA-21结合到双链探针的末端第一个toehold区域,将其中一条短DNA链置换下来,并暴露出第二个toehold区域用于随后与亚甲蓝(MB)修饰的燃料MB-DNA链进行杂交反应,MB-DNA链进一步置换出miRNA-21和另一短链DNA以激活分子机器的操作。结果,目标miRNA-21循环重复使用,导致许多MB-DNA燃料链连接到传感器表面产生明显放大的电流响应,实现对miRNA-21的高灵敏检测,检测限低至1.4 fmol/L。我们开发的传感器对目标物表现出很强的序列特异性,同时可用于在癌细胞样品中检测miRNA-21。此外,不仅该传感器的构建有使用目标物循环放大技术避免了任何酶参与的优点,而且还有高度最小化减少背景噪声的优点。使得该方法可以方便地监测不同miRNA生物标志物,具有对各种癌症的早期诊断中有巨大潜力。2.基于级联链置换无酶介导目标物循环放大和免标记电化学检测肿瘤细胞的MicroRNA痕量检测miRNA在肿瘤细胞中的表达水平对于癌症诊断具有非常重要意义。本项工作中,基于两个级联toehold介导的链置换反应(TSDRs),我们设计了一个免标记和无酶辅助目标物循环放大的电化学方法检测人乳腺癌细胞中的miRNA-21。含有被锁定的G-四链体序列的“Y”型DNA探针通过自组装在传感器表面上。当目标物miRNA-21存在的时候,引发第一个TSDR使“Y”型DNA探针解体的同时释放活性G-四链体序列。随后,DNA燃料链触发第二个TSDR,使得miRNA-21循环再利用。通过级联TSDR,传感器表面上产生许多活性G-四链体序列,其与hemin结合以产生显着放大的电流响应,实现灵敏检测miRNA-21低至1.15 fmol/L。该传感器表现出具有很强的选择性,并且可以用于检测来自人乳腺癌细胞样品中的miRNA-21。3.基于结合目标物催化发夹自组装(CHA)和TdT催化DNA聚合双信号放大策略电化学检测人血清中的凝血酶对蛋白质生物标志物的特异和灵敏性检测在生物医学和生物分析应用中具有非常重要的意义。该项工作中,实现了基于集成催化发夹自组装(CHA)和末端脱氧核苷酸转移酶(TdT)原位DNA聚合的双放大信号放大技术,高灵敏和免标记的电化学方法检测人体血清中的凝血酶。当目标物凝血酶的存在情况下,传感器电极上捕获的具有游离3'-OH末端的发夹DNA信号探针。随后,在dGTP与dATP摩尔比为6:4的情况下,TdT可以催化信号探针的游离3'-OH末端延伸形成许多重复的G-四链体序列,G-四链体序列结合hemin并产生显着放大的电流响应,实现超灵敏并且完全无标记的方式检测凝血酶,灵敏度达到0.12 pmol/L。此外,我们开发的传感器对凝血酶与其他干扰蛋白表现出具有良好的选择性,并能在人血清样品中检测凝血酶。
[Abstract]:Along with the development of gene diagnosis and treatment research, drug screening and mechanism, the development of many research fields of environmental pollution and food safety monitoring is rapid, the realization of microRNA on pathogenic protein and specific sequence (miRNA) is convenient, less time-consuming, high sensitivity and high specificity of detection and analysis of the significance is very important. The electrochemical DNA biosensor (DNA electrochemical biosensor) because of its simple structure, easy portability, low cost, high sensitivity and high specificity is attracting more and more attention. At present, for researchers, the development of electrochemical DNA biosensors have become a frontier topic attractive and challenging in biological and medical sciences. Sticky ends (Toehold) chain replacement reaction mediated (Toehold-mediated strand displacement reaction, TSDR) is the basic reaction to construct the dynamic DNA nano machine. This paper combined with T SDR auxiliary target cycle amplification technology and constructs three kinds of electrochemical DNA biosensor, the ultra sensitive detection of MI RNA and thrombin, the main research work is as follows: 1. DNA molecular machine to achieve the target of non enzymatic electrochemical detection cycle amplification and minimization of background noise measurement in cancer cells based on MicroRNAMicroRNA (miRNA) expression level change can be used as a useful biomarker for the diagnosis of different cancers. In this work, based on the miRNA lead to non target enzyme signal amplification cycle molecular machine, we developed a simple and novel electrochemical DNA biosensor has high sensitive detection of miRNA-21. sensor in human breast cancer cells are prepared by double chain the probe consists of 3 single stranded DNA by self-assembly on gold electrode surface, the target miRNA-21 binding to the end of the first double chain probe toehold region, the In a short DNA chain replacement down, and expose the second toehold region for subsequent and methylene blue (MB) modified MB-DNA hybrid fuel chain reaction, MB-DNA chain further replacement of miRNA-21 and another short chain DNA to activate the molecular machine operation. As a result, the target miRNA-21 cycle of repeated use, resulting in many MB-DNA fuel chain connected to the sensor surface significantly enlarge the current response, high sensitive detection of miRNA-21 sensor, low detection limit to 1.4 fmol/L. we developed on the target sequence showed strong specificity, but also can be used in cancer cell samples for detection of miRNA-21. in addition, the sensor is used not only to build target circulating amplification technique avoids any enzymes involved in the advantages, but also highly minimize background noise. The method can conveniently monitor different miRNA biomarkers ,鍏锋湁瀵瑰悇绉嶇檶鐥囩殑鏃╂湡璇婃柇涓�鏈夊法澶ф綔鍔�.2.鍩轰簬绾ц仈閾剧疆鎹㈡棤閰朵粙瀵肩洰鏍囩墿寰�鐜�鏀惧ぇ鍜屽厤鏍囪�扮數鍖栧�︽��娴嬭偪鐦ょ粏鑳炵殑MicroRNA鐥曢噺妫，

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