Nanopore single-molecule dielectrophoresis and biomedical detection

 描述（由申请人提供）：本项目的目的是阐明一种新的纳米孔单分子效应“载体引导纳米孔介电电泳”，并探索其在临床样本中癌症来源microRNA生物标志物的选择性捕获和无噪声检测中的应用。肺癌的早期发现迫切需要非侵入性和成本效益的验证性测试。肺癌来源的循环microRNA正在成为肺癌诊断和预后的潜在生物标志物。我们开发了一种纳米孔单分子“计数器”，用于microRNA的电检测，为疾病筛查和诊断提供了一种潜在的非侵入性工具。然而，由于临床样品（血浆核酸提取物）的复杂性，将概念性纳米孔传感器转化为临床可用的技术面临着巨大的挑战，这导致严重影响靶miRNA测定的强烈的“污染信号”，并且不能通过任何现有方法消除。我们发现了一种新的纳米孔效应，载体引导的纳米孔介电电泳（CND）。利用这种效应，我们首次设计了一种非常有用的方法，用于临床样品中的无噪声核酸检测。该方法利用载体探针结合靶miRNA。在纳米孔入口外的高度不均匀电场下，仅miRNA* 载体-探针偶极通过介电泳力被捕获在纳米孔中，而没有载体-探针结合的所有非靶核酸被排斥静电力排斥。因此，只能识别miRNA* 载体-探针复合物的特征;任何来自非靶物种的干扰信号都被完全消除。这一发现为复杂混合物中任何目标核酸的选择性捕获和无噪声检测开辟了一条新途径，代表了向纳米孔临床应用迈出的实质性一步。本课题的主要目的是：1）全面阐明CND效应，探索靶点多样性，提高灵敏度; 2）建立纳米孔介电电泳单核苷酸识别方法和条形码检测方法; 3）利用纳米孔介电电泳机制检测肺癌患者样本中的miRNAs生物标志物。该项目的成功将为纳米科学和生物技术做出重大贡献，并为纳米孔在医学诊断、生物标志物发现和病原体检测以及需要快速基因检测的植物科学和食品工程中的应用开辟了一条道路。长期愿景是将一个强大的，灵活的纳米孔设备应用于解决生命科学问题。

 DESCRIPTION (provided by applicant): The objective of this project is to elucidate a new nanopore single-molecule effect of "carrier-guided nanopore dielectrophoresis" and explore its application in selective capture and noise-free detection of cancer-derived microRNA biomarkers in clinical samples. Lung cancer early detection urgently needs non-invasive and cost-effective confirmatory tests. Lung cancer-derived circulating microRNAs are becoming potential biomarkers for lung cancer diagnosis and prognosis. We have developed a nanopore single molecule "Counter" for electric detection of microRNAs, offering a potential non-invasive tool for disease screening and diagnostics. However, translating a conceptual nanopore sensor into a clinically usable technology faces a big challenge due to the complexity of clinical samples (plasma nucleic acids extracts), which cause intensive "contaminative signals" that severely influence the target miRNA determination and cannot be eliminated by any current methods. We discovered a new nanopore effect, carrier-guided nanopore dielectrophoresis (CND). Using this effect, we for the first time devise an extremely useful approach for noise-free nucleic acids detection in clinical samples. This approach utilizes a carrier-probe to bind the target miRNA. Under a highly non- uniform electric field outside the nanopore entrance, only the miRNA*carrier-probe dipole is captured in the nanopore by a dielectrophoresis force, whereas all non-target nucleic acids without carrier-probe binding are rejected by the repulsive electrostatic force. Consequently only the signatures for the miRNA*carrier-probe complex can be identified; any interference signal originating from non-target species is completely eliminated. This discovery opens a new route to the selective capture and noise-free detection of any target nuclic acids in the complex mixture, representing a substantial step toward the nanopore clinical applications. In this project, we are motivated to 1) completely elucidate the CND effect, explore the target diversity, enhance the sensitivity; 2) establish a nanopore dielectrophoresis approach for single-nucleotide discrimination and a barcode approach for multiplex miRNA detection; 3) utilize nanopore dielectrophoresis mechanism to detect lung cancer-derived miRNAs biomarkers in patient samples. The success of this project will be a substantial contribution to nanoscience and biotechnology, and open an avenue to nanopore applications in medical diagnostics, biomarkers discovery and pathogen detection, as well as plant science and food engineering where rapid genetic detection is required. The long term vision is applying a robust, flexible nanopore device in solving life science problems.