| 靶标介导DNA自组装及催化电流放大的microRNA电化学检测研究 |
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| 基金项目:国家自然科学基金项目（82260702，82060647，21974037）；广西自然科学基金项目（2021GXNSFFA220003，2021JJD120048）；广西研究生教育创新计划项目（YCSW2021142） |
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| 中文摘要:该研究报道了一种靶标介导的DNA自组装及催化信号放大免标记电化学传感器定量检测microRNA－21的分析方法。根据靶标序列，设计一条末端标记巯基且具有茎环结构的捕获探针以及两条与捕获探针和靶标部分互补的DNA单链，通过金－硫键作用将捕获探针固定在金电极表面。当靶标（microRNA－21）存在时，自组装形成一种H结构的DNA复合结构；利用核酸链中磷酸骨架静电吸附电解液中的钌氨离子（［Ru（NH3）6］3+，RuHex）以及DNA电子传递作用产生电化学信号；当无靶标时，不能形成DNA复合结构，电化学信号较弱。进一步利用铁氰根离子（［Fe（CN）6］3-）能够氧化电化学还原产物（［Ru（NH3）6］2+），产生电化学－化学偶联，从而实现催化电流信号放大。采用电化学阻抗谱确证DNA复合结构的形成，采用计时电量法考察捕获探针密度对电化学信号的影响，并优化探针浓度、比例以及自组装时间，采用差示脉冲伏安法进行定量分析。结果显示，在0.1 fmol/L ~ 0.1 nmol/L范围内，峰电流与microRNA－21浓度具有良好的线性关系，检出限为12.8 amol/L。方法能有效区分其他microRNA以及单碱基错配核酸序列，成功用于多种细胞中microRNA－21的定量检测。该电化学传感器具有灵敏度高、选择性好、线性范围宽等优点，无需繁琐的电化学探针标记以及费时费力的PCR扩增、滚环扩增、链置换反应等分析策略，简化了操作流程，提高了方法的实用性。 |
| 中文关键词:核酸自组装 电化学传感器 催化电流 microRNA |
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| Detection of microRNA Using an Electrochemical Sensor Based on Target-mediated DNA Self-assembly and Catalytic Current Amplification |
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| Abstract:In this work，a label-free electrochemical sensor for microRNA－21 detection was reported，based on target-mediated self-assembly of DNA nanostructure and catalytic signal amplification．A thiol-tethered and stem-loop structured DNA capture probe（CP） and two sequence-specific DNA strands that were partly complementary to the CP and the target（microRNA－21） were used．Initially，the CP formed self-assembly monolayer on the surface of gold electrode vis Au－S chemistry，which further evolved to a H-shaped DNA－RNA complex in the presence of the target．The electroactive ions－［Ru（NH3）6］3+ （RuHex） electrostatically adsorbed to the negatively charged phosphate moieties in the nucleic acid skeleton could generate strong electrolytic current through DNA-mediated electron transfer mechanism．By contrast，the current signal was rather weak in the absence of the target，due to the inexistence of the DNA－RNA complex．Furthermore，another electroactive specie，［Fe（CN）6］3- ions were ultilized to chemically oxidize the electroreduction product－［Ru（NH3）6］2+ ions，thereby establishing an electrochemistry－chemistry cascade reaction and consequently realizing current signal amplification．Electrochemical impedance spectroscopy（EIS） was used to confirm the as-assembled DNA－RNA complex，and chronocoulometry technique was employed to calculate the capture probe density that may affect the electrochemical response．In addition，capture probe concentration，ratio and self-assembly time were optimized．The peak currents measured from differential pulse voltammetry（DPV） were proportional to the target concentration in the range of 0.1 fmol/L－0.1 nmol/L，with a detection limit down to 12.8 amol/L．Particularly，it could effectively distinguish other microRNAs and even single-base mismatch analogs，enabling to the detection of microRNA－21 in diverse cell lines．With the advantages of high sensitivity，favorable specificity and broad linearity，the reported sensor could work without the need of electrochemical probe labeling，and circumvents currently prevailing strategies such as PCR，rolling circle amplification and strand displacement reaction that are typically time-consuming and labor-intensive，sthus simplifying the analytical procedures and improving the practicality. |
| Key Words:nucleic acid self-assembly electrochemical sensor catalytic current microRNA |
| 引用本文：李鳗亭，韦颖怡，杨帆，李新春.靶标介导DNA自组装及催化电流放大的microRNA电化学检测研究[J].分析测试学报,2022,41(11):1584-1590. |
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