基于液相体系的microRNA电化学检测新方法及其响应机制研究

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the activity of specific mRNA targets and play crucial role in a wide variety of biological processes, including gene expression, development, proliferation, differentiation and apoptosis. Implicated in human diseases, miRNA expression signatures will become an emerging class of molecular biomarkers. Though many methods for miRNA analysis have been reported at present, most of them are time-consuming and expensive, which greatly limit their application in clinical laboraory. In this project, we aim to discuss and validate the feasibility of a liquid-phase detection strategy for miRNA using Fe3O4/Au nanoparticle as a class of microcarrier. In our early study, we have demonstrated that the Fe3O4/Au nanoparticle we synthesized shows great advantages of biocompatibility, conductive ability, and strong superparamagnetism. We are going to increase the sensitivity analysis in amplification current signal and electrode electronic conduction respectively by constructing nucleic acid multienzyme marking system and Nafion-graphene modified screen printing working electrode based on previous results. Besides we intend to elucidate the response mechanism by a variety of methods of characterization and establish the mathematical model in quantitative analysis by optimization analysis conditions. Finally we verify the detection efficiency of this method by detecting miRNA expression of tumor cell and clinical serum sample, aiming to construct an economic and simple electrochemical miRNA detection technology with high sensitivity and speed for future clinical application.

作为一种重要的转录后基因调节分子，微小核糖核酸（miRNA）表达水平变化与多种疾病密切相关，有望成为一类新型的生物标志物。目前常用的miRNA检测方法多需要复杂操作、昂贵设备，很大程度限制了其临床普及。本项目在前期已合成出具备生物相容性好、导电能力强、超顺磁性强等优点的金磁复合纳米材料的基础上，探讨并验证利用其作为miRNA液相检测微载体的可行性。在此基础上拟通过构建核酸多酶标记体系、制备Nafion-石墨烯修饰丝网印刷工作电极的手段，分别从放大电流信号以及增加电极电子传导能力两方面增加分析的灵敏度。研究将通过多种表征手段阐明其响应机制，探讨并优化分析条件，建立定量分析的数学模型，最后通过检测肿瘤细胞株与临床血清样本miRNA以验证该方法的检测效能，旨在为构建一种高敏、快速、经济、简便的电化学miRNA检测技术提供实验基础与理论依据。

微小核糖核酸是一类由19～23核苷酸组成的内源性单链非编码RNA，被认为是具有广阔应用前景的新型生物标志物，有望用于疾病的诊断、治疗及预后判断等。本项目探究并解决了miRNA及液态活检中稀有肿瘤细胞、外泌体提取与检测的相关问题。首先，本项目采用新材料和新技术，建立了两种miRNA检测新方法，一方面，利用磁性纳米粒子作为液相检测的微载体，通过循环探针打开-延伸反应-酶促反应多级信号放大过程，建立了一种新型miRNA电化学传感检测新技术。另一方面，利用DNA/2-OMe-RNA这一独特的嵌合探针作为检测信号的传递轴，创建了一种双链特异性核酸酶介导的双重循环酶切信号放大的miRNA荧光检测新体系。两种方法均具有快速、经济的特点，灵敏度可达到飞摩尔水平，具有单碱基错配识别能力，并在实际样品检测中证实了具有临床应用潜能。同时，本项目根据血液miRNA的特点，对传统RNA提取方法TRIzol法改良用于血液miRNA提取，并分别通过将其与传统TRIzol法及两种常见的代表性方法进行比对证明其具有更高的提取效率。此外，本项目设计并建立双向催化发夹自组装介导的电化学传感器，以肿瘤细胞为模型进行了优化及验证，性能优良，经济有效，可实现液态活检中miRNA、稀有肿瘤细胞等的POCT化检测。并对比了几种常用的外泌体及外泌体RNA提取方法，总结了各方法的特点并分析了各方法的适用范围；建立了尿液细胞外囊泡液压透析分离方法，筛选出具有诊断效能的囊泡miRNA。本项目所建立的方法均具有一定优越性，具有广阔的应用前景，为miRNA定量检测提供新方法，提升miRNA的临床应用价值，并为其他稀有核酸、细胞等靶物质检测提供新思路，奠定研究基础。

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