基于DNA纳米技术和核酸酶的核酸化学发光传感分析
发布时间：2018-07-17 19:14
【摘要】：随着循环肿瘤DNA、循环microRNA(miRNA)和融合基因等在临床疾病发生和诊疗中作用的逐步揭示,无细胞核酸分析在生物学研究和医学诊疗中变得日趋重要。基于此,本论文拟将整合临床检验诊断学和生物分析化学学科交叉的最新研究成果,以DNA纳米自组装和功能核酸酶为纳米级工具,结合临床检验诊断的需求,构建一系列简单、快速、等温、特异、灵敏、免标记的核酸化学发光传感分析新策略。本研究主要包括以下三个部分:1.基于核酸酶级联指数扩增的化学发光成像用于miRNA传感分析本研究基于功能核酸酶技术和级联指数扩增(exponential amplification reaction,EXPAR)分子机器,构建高灵敏、高特异且可视化的化学发光生物传感器,为循环miRNA提供全新检测新策略。研究中发卡开关能够特异识别靶miRNA并形成杂交双链,从而启动扩增机器I,产生引物链。引物链与模板II探针互补杂交形成双链进级联激活指数扩增机器II,产生大量G四链体辣根过氧化物模拟酶,催化发光底物发光,实现信号的转换输出。发卡结构DNA分子探针的设计提高检测的特异性,级联型的指数扩增提高检测的灵敏度,功能核酸酶免标记技术实现信号转导和可视化的miRNA生物传感分析。本章所构建的化学发光传感方法检测miRNA的线性范围为10 fM到100 pM,最低检测限为2.91 fM,且具有单碱基错配分辨能力。同时,该传感方法具有等温、均相、免标记和可视化的优点,有望为循环mirna分析和临床疾病诊断提供有力的检测工具。2.基于双t型dna纳米开关和核酸酶的化学发光成像用于bcr/abl融合基因传感分析本研究基于dna纳米技术和级联型dna扩增机器构建bcr/abl融合基因化学发光型生物传感器,为融合基因检测提供超灵敏、高特异的化学发光成像传感新策略。利用nupack软件设计功能化分子探针,其能够逻辑性识在bcr/abl融合基因并自组装形成bis-3wj纳米结构(bis-threewayjunctionnanostructure,bis-3wj)。该结构激活级联扩增机器,产生大量g四链体辣根过氧化物模拟酶亚单元。该亚单元能够自组装为完整模拟酶,级联催化化学发光底物实现光信号转换输出和逻辑门操作。本章所构建的化学发光成像检测bcr/abl融合基因的最低检测限23fm,线性范围达7个数量级,且具有逻辑识别能力和单碱基错配分辨能力。该方法在复杂基质中显示好的回收率,具有等温、均相、免标记的优点,为慢性粒细胞白血病诊断提供全新的化学发光成像策略。3.基于发卡开关和原位非线性杂交链反应的电致化学发光用于bcr/abl融合基因传感分析本研究基于发卡探针和原位分支化杂交链反应(hybridizationchainreaction,hcr)构建bcr/abl融合基因的电化学发光型生物传感器,为慢性粒细胞白血病诊断提供操作简便、超灵敏、高特异的传感新策略。首先,将捕获探针固定在金电极。随后,将分支化杂交链反应系统溶液滴加在电极表面,在发卡开关上的触发链DNA触发两种底物链和两种辅助链的级联自组装形成分支状DNA纳米结构。在目标分子存在的情况下,其打开发卡开关并将分支状DNA纳米结构连接在电极表面。这种原位HCR反应形成的DNA纳米结构能够通过其DNA双链凹槽与电致发光指示剂(Ru(phen)32+)间的相互作用将随后加入的指示剂引入到电极表面,从而实现信号的指数形式的输出放大。发卡探针和原位非线性杂交链反应的引入到电致发光传感器设计增强了传感器的特异性和灵敏度。本章所构建的ECL方法检测BCR/ABL融合基因的最低检测限为5.49 fM,线性范围为10 fM至1 nM,且具有与正常BCR基因和正常ABL基因进行逻辑区别的能力。另外,该方法具有无酶,免标记,低成本的优势,有望成为白血病临床诊断中融合基因检测提供新的诊疗策略。
[Abstract]:With the gradual discovery of the role of circulating tumor DNA, circulating microRNA (miRNA) and fusion gene in the occurrence and diagnosis of clinical diseases, acellular nucleic acid analysis becomes more and more important in biological research and medical diagnosis and treatment. Based on this, this paper will integrate the latest research results in the interdisciplinary of clinical diagnostics and bioanalytical chemistry. DNA nano self-assembly and functional nuclease are nanoscale tools, combined with the needs of clinical diagnosis. A series of simple, fast, isothermal, specific, sensitive, and non labeled nucleic acid chemiluminescence sensing analysis strategies are constructed. This study mainly includes the following three parts: 1. based chemiluminescence imaging of the nuclease cascade index for miR NA sensing analysis is based on functional nuclease technology and cascade index amplification (exponential amplification reaction, EXPAR) molecular machines to construct highly sensitive, highly specific and visualized chemiluminescence biosensors, which provide new detection strategies for circulating miRNA. In the study, a hairpin switch can specifically identify the target miRNA and form a hybridization. Double strands, thus initiating the amplification machine I, producing the primer chain. The primer chain and the template II probe complement each other to form a double chain into the cascade activation index to amplify the machine II, producing a large number of G four chain body horseradish peroxidase analog enzymes, catalyzing the luminescence of the luminescent substrate, and realizing the conversion of the signal. The design of the DNA molecular probe of the hairpin structure improves the specificity of the detection. Cascaded exponential amplification improves detection sensitivity, functional nuclease free labeling technique for signal transduction and visual miRNA biosensor analysis. The linear range of miRNA is 10 fM to 100 pM in this chapter, with a minimum detection limit of 2.91 fM and a single base mismatch resolution. The sensing method has the advantages of isothermal, homogeneous, non labeling and visualization. It is expected to provide a powerful detection tool for cyclic miRNA analysis and clinical disease diagnosis.2. based on double T type DNA nanoscale switch and nuclease chemiluminescence imaging for bcr/abl fusion gene sensing analysis based on DNA nanotechnology and cascade DNA amplification machines to construct B Cr/abl fusion gene chemiluminescence biosensor provides a super sensitive, high specific chemiluminescence imaging sensor for fusion gene detection. Using nupack software, a functional molecular probe is designed, which can logically identify the bcr/abl fusion gene and form a bis-3wj nanoscale structure (bis-threewayjunctionnanostructure, bis-3w). J). The structure activates the cascade amplification machine to produce a large number of G four chain horseradish peroxidase subunits. This subunit can self assemble as a complete analog enzyme, cascade catalytic chemiluminescence substrates to realize optical signal conversion output and logic gate operation. The minimum detection limit of bcr/abl fusion gene for detection of chemiluminescence imaging in this chapter is 23 FM, which has a linear range of 7 orders of magnitude, and has the ability to distinguish between logical recognition and single base mismatch. The method shows good recovery in the complex matrix and has the advantages of isothermal, homogeneous and non labeling. It provides a new chemiluminescent imaging strategy for the diagnosis of chronic myelocytic leukemia,.3. based on the hairpin switch and in situ nonlinear hybrid chain. Electrochemiluminescence (electrochemiluminescence) for bcr/abl fusion gene sensing analysis, an electrochemiluminescence biosensor based on the hairpin probe and in situ branched hybrid chain reaction (hybridizationchainreaction, HCR) was used to construct a bcr/abl fusion gene, which provides a simple, sensitive and highly specific sensing for the diagnosis of chronic myelocytic leukemia. First, the capture probe is fixed on the gold electrode. Then, the branched hybrid chain reaction system solution is added to the surface of the electrode. The trigger chain DNA on the card switch triggers two substrate chains and two kinds of auxiliary chains to form a branched DNA nanostructure. The branched DNA nanostructure is connected to the surface of the electrode. The DNA nanostructure formed by the in situ HCR reaction can be introduced into the electrode surface by the interaction between the DNA double chain grooves and the electroluminescent indicator (Ru (phen) 32+), thus realizing the output amplification of the exponential form of the signal, the hairpin probe and the in situ. The introduction of the nonlinear hybrid chain reaction to the electroluminescent sensor design enhanced the specificity and sensitivity of the sensor. The minimum detection limit for detection of BCR/ABL fusion gene by the ECL method in this chapter is 5.49 fM, and the linear range is 10 fM to 1 nM, and it has the ability to distinguish the normal BCR gene and the normal ABL gene. The method has the advantages of no enzyme, label free and low cost, and is expected to provide a new strategy for the detection of fusion genes in clinical diagnosis of leukemia.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R440
本文编号：2130659
[Abstract]:With the gradual discovery of the role of circulating tumor DNA, circulating microRNA (miRNA) and fusion gene in the occurrence and diagnosis of clinical diseases, acellular nucleic acid analysis becomes more and more important in biological research and medical diagnosis and treatment. Based on this, this paper will integrate the latest research results in the interdisciplinary of clinical diagnostics and bioanalytical chemistry. DNA nano self-assembly and functional nuclease are nanoscale tools, combined with the needs of clinical diagnosis. A series of simple, fast, isothermal, specific, sensitive, and non labeled nucleic acid chemiluminescence sensing analysis strategies are constructed. This study mainly includes the following three parts: 1. based chemiluminescence imaging of the nuclease cascade index for miR NA sensing analysis is based on functional nuclease technology and cascade index amplification (exponential amplification reaction, EXPAR) molecular machines to construct highly sensitive, highly specific and visualized chemiluminescence biosensors, which provide new detection strategies for circulating miRNA. In the study, a hairpin switch can specifically identify the target miRNA and form a hybridization. Double strands, thus initiating the amplification machine I, producing the primer chain. The primer chain and the template II probe complement each other to form a double chain into the cascade activation index to amplify the machine II, producing a large number of G four chain body horseradish peroxidase analog enzymes, catalyzing the luminescence of the luminescent substrate, and realizing the conversion of the signal. The design of the DNA molecular probe of the hairpin structure improves the specificity of the detection. Cascaded exponential amplification improves detection sensitivity, functional nuclease free labeling technique for signal transduction and visual miRNA biosensor analysis. The linear range of miRNA is 10 fM to 100 pM in this chapter, with a minimum detection limit of 2.91 fM and a single base mismatch resolution. The sensing method has the advantages of isothermal, homogeneous, non labeling and visualization. It is expected to provide a powerful detection tool for cyclic miRNA analysis and clinical disease diagnosis.2. based on double T type DNA nanoscale switch and nuclease chemiluminescence imaging for bcr/abl fusion gene sensing analysis based on DNA nanotechnology and cascade DNA amplification machines to construct B Cr/abl fusion gene chemiluminescence biosensor provides a super sensitive, high specific chemiluminescence imaging sensor for fusion gene detection. Using nupack software, a functional molecular probe is designed, which can logically identify the bcr/abl fusion gene and form a bis-3wj nanoscale structure (bis-threewayjunctionnanostructure, bis-3w). J). The structure activates the cascade amplification machine to produce a large number of G four chain horseradish peroxidase subunits. This subunit can self assemble as a complete analog enzyme, cascade catalytic chemiluminescence substrates to realize optical signal conversion output and logic gate operation. The minimum detection limit of bcr/abl fusion gene for detection of chemiluminescence imaging in this chapter is 23 FM, which has a linear range of 7 orders of magnitude, and has the ability to distinguish between logical recognition and single base mismatch. The method shows good recovery in the complex matrix and has the advantages of isothermal, homogeneous and non labeling. It provides a new chemiluminescent imaging strategy for the diagnosis of chronic myelocytic leukemia,.3. based on the hairpin switch and in situ nonlinear hybrid chain. Electrochemiluminescence (electrochemiluminescence) for bcr/abl fusion gene sensing analysis, an electrochemiluminescence biosensor based on the hairpin probe and in situ branched hybrid chain reaction (hybridizationchainreaction, HCR) was used to construct a bcr/abl fusion gene, which provides a simple, sensitive and highly specific sensing for the diagnosis of chronic myelocytic leukemia. First, the capture probe is fixed on the gold electrode. Then, the branched hybrid chain reaction system solution is added to the surface of the electrode. The trigger chain DNA on the card switch triggers two substrate chains and two kinds of auxiliary chains to form a branched DNA nanostructure. The branched DNA nanostructure is connected to the surface of the electrode. The DNA nanostructure formed by the in situ HCR reaction can be introduced into the electrode surface by the interaction between the DNA double chain grooves and the electroluminescent indicator (Ru (phen) 32+), thus realizing the output amplification of the exponential form of the signal, the hairpin probe and the in situ. The introduction of the nonlinear hybrid chain reaction to the electroluminescent sensor design enhanced the specificity and sensitivity of the sensor. The minimum detection limit for detection of BCR/ABL fusion gene by the ECL method in this chapter is 5.49 fM, and the linear range is 10 fM to 1 nM, and it has the ability to distinguish the normal BCR gene and the normal ABL gene. The method has the advantages of no enzyme, label free and low cost, and is expected to provide a new strategy for the detection of fusion genes in clinical diagnosis of leukemia.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R440
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