A Solid-State Nanopore miRNA Quantification Technology

固态纳米孔 miRNA 定量技术

• 批准号: 9147175
• 负责人: Hsueh-Chia Chang
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-07 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9147175
• 关键词: Affinity; Aluminum Oxide; Biological; Biological Markers; Cancer Diagnostics; Cancer Patient; Cell Culture Techniques; Characteristics; Clinic; Clinical; Complex; Data; Detection; Development; Diagnosis; Diagnostic Neoplasm Staging; Disease; Disease Management; Face; Film; Future; Gene Expression; Generic Drugs; Goals; Heating; Hour; Human; Human Papillomavirus; Individual; Institutes; Ions; Lead; Length; Malignant Neoplasms; Measurement; Membrane; Messenger RNA; Methods; MicroRNAs; Modification; Nucleic Acids; Nucleotides; Performance; Preparation; Process; Proteins; Protocols documentation; Relaxation; Research; Resolution; Resources; Reverse Transcription; Role; Sampling; Speed; Stress; Surface; Technology; Temperature; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Translations; Validation; Variant; Work; anticancer research; biochip; biomarker discovery; cancer diagnosis; clinical application; cost; design; differential expression; malignant mouth neoplasm; malignant oropharynx neoplasm; melting; microRNA biomarkers; nanopore; next generation sequencing; novel; outcome forecast; point of care; rapid detection; response; saliva diagnostic; single molecule; solid state; tumor progression

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.

摘要 Micro-RNAs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。对精确度的需求很大 在不久的将来癌症研究和临床中的miRNA图谱。然而， 目前的技术无法精确、轻松、低成本地完成这项极具挑战性的任务 成本和高吞吐量。所有主要检测技术，实时反转录 聚合酶链式反应、微阵列杂交和下一代测序(NGS) 信使核糖核酸谱的挑战。自.以来 不同的检测技术、协议和 MiRNA的提取和纯化方法可能会导致不同的结果，解释 不同研究之间的差异表达数据和比较很少。一个 能够对复杂样品中的单个天然miRNA分子进行计数的技术 会更受欢迎。 本研究首要目标是开发一套新型固态聚合物 用于快速(1小时)、灵敏(100个分子/样品)和定量的纳米孔技术 检测癌症患者中差异表达的一小部分miRNAs。这 Proposal将专注于其初步开发和非临床样本测试。 原始生物样本将被装载到一个综合平台上，该平台将进行采样 清理、miRNA提取和检测都在一个芯片上，最大限度地减少了样品 丢失和依赖于协议的偏见。 在具体目标1中，我们将多孔和单孔固态技术与 介电薄膜纳米孔涂层及新发现的纳米孔欧姆加热 现象，以制定一种miRNA“捕获、释放和计数”的浓缩策略 从复杂样本中提取特定的miRNA。在具体目标2中，我们将整合纳米孔 和离子膜技术集成到一个平台上，用于快速检测一小块 原始细胞培养和/或组织裂解物样品中的miRNAs。然后，这个设计将被 通过将几个纳米孔单元整合到一个 多目标检测平台。在特定的目标3中，我们将比较miRNA的结果 不同制样方法在细胞培养和/或组织裂解液中的测量 方法(SA3a离子膜芯片和商用miRNA试剂盒 提取)结合建议的纳米孔平台和其他参考 方法定量聚合酶链式反应(QPCR)和非聚合酶链式反应(NGS)。 我们的长期目标是开发一种健壮、低成本、便携的多种疾病简档 平台，能够准确定量复杂样本中的大量miRNAs，用于 癌症的研究和临床应用。这样平台将显著提高速度 从生物标记物的发现、验证和监管批准到翻译的过程 进入临床环境。