A Solid-State Nanopore miRNA Quantification Technology

摘要 micro-RNA是关键的基因表达调控因子，具有作为生物标志物的巨大潜力， 癌症诊断、预后和治疗。对准确的需求很大。 在不久的将来，miRNA谱在癌症研究和临床中的应用。但 目前的技术不能精确、容易、低成本地执行这一非常具有挑战性的任务。 成本和高吞吐量。所有主要检测技术，实时逆转录 PCR（qPCR）、微阵列杂交和下一代测序（NGS） mRNA的挑战。以来 不同的检测技术、协议和 miRNA的提取和纯化方法可能导致不同的结果， 不同研究中的差异表达数据和比较是脆弱的。一 能够对复杂样品中天然miRNA的单个分子进行计数的技术 会更受欢迎 本研究的总体目标是开发一种新型的固态聚合物 纳米孔技术用于快速（1小时）、灵敏（100个分子/样品）和定量 测量癌症患者中差异表达的一小组miRNA。这 该提案将侧重于其初步开发和非临床样本测试。 将原始生物样品加载到集成平台中，该平台将执行样品 在一个芯片中实现了清除和miRNA提取和检测，以最小化样品 损失和协议依赖性偏倚。 在具体目标1中，我们将联合收割机多孔和单孔固态技术与 介电薄膜纳米孔涂层和新发现的纳米孔欧姆加热 现象，以开发用于浓缩的miRNA“捕获、释放和计数”策略， 从复杂的样本中提取特定的miRNA在具体目标2中，我们将整合纳米孔 和离子膜技术的平台，用于快速检测一小组 原始细胞培养物和/或组织裂解物样品中的miRNAS。这样的设计将是 通过将几个纳米孔单元整合到一个 多目标探测平台在Specific Aim 3中，我们将比较miRNA 使用不同的样品制备物在细胞培养物和/或组织裂解物中的测量 方法（SA 3a的离子膜芯片，和用于miRNA的市售试剂盒 提取）结合所提出的纳米孔平台和其他参考 方法（qPCR和NGS）。 我们的长期目标是开发一个强大的，低成本的，便携式的多疾病分析 平台，能够准确定量复杂样品中的大miRNA， 癌症的研究和临床应用。这样的平台将大大加快 从生物标志物发现、验证和监管批准到翻译的过程 进入临床环境。

ABSTRACT Micro-RNAs are key gene expression regulators with great potential as biomarkers for cancer diagnosis, prognosis, and therapeutics. There is a great demand for accurate miRNA profiling in cancer research and in the clinics in the near future. However, the current technologies cannot perform this very challenging task with precision, ease, low cost, and high throughput. All main detection technologies, real-time reverse transcription PCR (qPCR), microarray hybridization, and next-generation sequencing (NGS) face challenges in mRNA profiling. Since different detection technologies, protocols and miRNA extraction and purification methods can lead to different results, interpretation of differential expression data and comparisons across different studies is tenuous. A technology capable of counting individual molecules of native miRNA in a complex sample would be much preferred. The overarching goal of this research is to develop a novel set of solid-state polymeric nanopore technologies for fast (1 hour), sensitive (100 molecules/sample) and quantitative measurement of a small set of miRNAs differentially expressed in cancer patients. This proposal will focus on its preliminary development and testing with non-clinical samples. Raw biological samples will be loaded in an integrated platform, which will perform sample cleanup and miRNA extraction and detection all in one-chip, for minimization of sample loss and protocol-dependent biases. In Specific Aim 1, we will combine multi- and single- pore solid-state technologies with dielectric thin film nanopore coatings and a newly discovered nanopore Ohmic heating phenomenon, to develop a miRNA “capture, release and count” strategy for concentration of a specific miRNA from a complex sample. In Specific Aim 2, we will integrate nanopore and ionic-membrane technologies into a platform for rapid detection of a small panel of miRNAS in raw cell-culture and/or tissue lysate samples. This design will then be expanded to oral cancer- related miRNAs by integrating several nanopore units in a multitarget detection platform. In Specific Aim 3 we will compare results of miRNA measurements in cell culture and/or tissue lysate using different sample preparation methods (the ionic-membrane chip of SA3a, and commercially available kits for miRNA extraction) in conjunction with the proposed nanopore platform and other reference methods (qPCR and NGS). Our long-term goal is to develop a robust, low cost, portable multi-disease profiling platform, capable of accurately quantifying a large miRNAs in complex samples, for research and clinical applications in cancer. Such platform would significantly speed up the process from biomarker discovery, validation, and regulatory approval, to translation into clinical setting.