双足DNA步行器诱导的双信号比率型电化学传感器构建及其在miRNA液体活检中的应用

MicroRNAs aberrant expression level is closely associated with the occurrence and development of many kinds of diseases, especially cancer. There is a lot of evidence indicate that circulating miRNA molecules exist in human body fluids. Therefore, it has great research significance and application prospect because direct-detection of miRNAs in human body fluids (liquid biopsy) is a non-invasive method. However, there are only a few miRNAs in body fluids. In addition, the presence of protein, ions, and other sample substrates in body fluids can cause signal interference. These two problems bring a huge challenge to the detection of miRNAs in liquid biopsy. On the basis of the electrochemical methods, the project aims to develop dual-signaling ratiometric electrochemical biosensing technology and strive to solve the problem of weak anti-jamming ability of conventional electrochemical detection technology. Additionally, a low cost, rapid response and high sensitivity electrochemical biosensing platform can be achieved by designing a bipedal DNA walker. Furtherly, coupling with isothermal amplification methods, such as catalytic hairpin assembly and entropy-driven catalysis strategy, can achieve the goal of multiple-stage signal amplification for the sensitive detection of miRNAs in liquid biopsy. This study is based on solving the key problems existing in liquid biopsy for miRNAs detection and expected to provide a novel and reliable technical approach for life science, clinical medicine, analytical chemistry.

MicroRNAs的异常表达与人类许多重大疾病（尤其是癌症）的发生与发展密切相关，大量的证据表明人体液中存在循环miRNAs分子。由于直接检测人体液中的miRNAs（液体活检）是一种无创的手段，所以具有重大的研究意义和应用前景。然而体液中miRNAs的含量少，且体液中存在的蛋白、离子等样品基质会带来信号干扰，这对miRNAs的液体活检带来了巨大的挑战。本项目在电化学方法的基础上，发展一种比率型双信号检测手段，力求解决常规电化学检测技术抗干扰能力弱的难题。此外，双足DNA步行器的设计可以实现成本低、响应快、灵敏度高的电化学生物传感平台的构建。进一步与DNA发卡催化自组装以及熵驱动的链取代反应的信号放大策略相结合，实现信号的多级放大，获得体液中miRNAs的灵敏检测。本研究立足解决miRNAs液体活检中存在的关键性难题，有望为生命科学、临床医学、分析化学等领域的发展提供新颖的、可靠的技术方案。

快速、灵敏、准确的痕量生物功能分子分析在生物医学研究、临床诊断和基因治疗评价等领域具有重要的科学意义。本项目以肿瘤标志物、病毒、病原菌等为检测靶标分子，利用DNA双足步行器、CRISPR基因编辑技术、侧流层析技术以及等温扩增技术，发展了一系列高灵敏和特异性的生物传感平台。例如：针对传统单足DNA纳米机器行走速度和范围小以及传感界面组装密度低等问题，发展了3D-DNA双足步行器，实现了对凝血酶的高灵敏检测；利用CRISPR/Cas12和CRISPR/Cas13系统发展了高灵敏的miRNA检测平台；利用CRISPR/Cas9系统特异性的双链核酸识别能力，发展了分别用于单基因和双基因检测的可视化传感平台；通过目标物的多级循环放大策略，发展了超高灵敏的DNA分子检测平台。本项目所发展的光电化学传感平台、荧光分析传感平台、可视化生物传感平台等对于疾病的早期诊断和预警、病毒感染检测和传播链追踪、生物分析和药物研究等方面具有潜在的应用前景。