ALEX for fast ultrasensitive multiplexed detection & quantification of microRNAs

描述（由申请人提供）：最近，microRNAs （miRNAs）被认为与包括癌症在内的人类恶性肿瘤的发生和发展有关。体液和活检中已确定的表达模式强调了它们作为生物标志物的潜力。由于患者样本有限且miRNA浓度通常极低，因此串行分析并不实用。因此，只有新的、高度敏感的、具有高复用能力的方法才能显著提高诊断价值，并促进我们对mirna生物学作用的理解。Nesher Technologies, Inc. （NTI）独家授权了一项革命性的定量，超灵敏和特异性生物检测技术的知识产权，该技术由加州大学洛杉矶分校单分子生物物理实验室（由Shimon Weiss教授领导）开发，具有精致的单孔复用潜力，最小的样品要求，以及极其简化的处理程序（无分离/洗涤和扩增步骤）。它基于交替激光激发（ALEX）单分子荧光光谱，其中目标识别分子用不同颜色的荧光染料（和猝灭剂）标记。基于共聚焦显微镜，它可以对溶液中的生物分子进行超灵敏检测，同时对众多靶标进行分化，并通过分子计数直接定量。NTI最近实现了从2色（2c）扩展到4色（4c） ALEX，大大扩展了其多路复用能力，并证明了在临床相关浓度下无扩增的超灵敏miRNA定量诊断的实用性。此外，我们的顾问Steve Quake和Shimon Weiss最近的工作表明，i)基于微流体的样品处理与ALEX光谱相结合，这是一种新的突破性方法，用于分析小型化，称为“单分子光流体”，ii)使用多焦点激发/检测几何结构提高了吞吐量。NTI的长期目标是开发快速，高度多路复用（每个样品的容量为100个分析物），超灵敏和特异性，定量，成本效益高，全自动，基于核酸和蛋白质的诊断测试，需要最小的样本量。在这个I期SBIR应用中，我们建议将4c-ALEX用于快速超灵敏的多路检测和定量microrna。原理验证将通过血清样本中癌症特异性mirna特征的无扩增表征来建立。随后的技术商业化将针对基础研究、药品和诊断市场。我们的具体目标是：1；4c-ALEX单分子荧光光谱分离检测和定量加标样品中7种肺癌相关mirna，并与qPCR方法比较。2. 这些mirna的多路（单孔）鉴别和定量。3. 100份存档的临床血清样本（60例癌症患者和40例对照）的4c- alexs分析。SBIR二期将致力于分析扩展、小型化和仪器原型开发。

DESCRIPTION (provided by applicant): Recently, microRNAs (miRNAs) have been implicated in initiation and progression of human malignancies including cancer. Identified expression patterns in body fluids and biopsies underscore their potential as biomarkers. As patient samples are limited and miRNA concentrations often extremely low, serial analysis is not practical. Thus only new, extremely sensitive methodologies with high multiplexing power will be able to significantly boost diagnostic value and advance our understanding of the biological roles of miRNAs. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified handling procedures (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers). Based on confocal microscopy, it allows ultrasensitive detection of biomolecules in solution, differentiation of numerous targets simultaneously, and direct quantification via molecule counting. NTI recently achieved expansion from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for ultrasensitive miRNA quantification at clinically relevant concentrations without amplification. Furthermore, recent work by our consultants Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization termed "single molecule optofluidics", and ii) enhanced throughput using a multifoci excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of >100 analytes per sample), ultrasensitive and -specific, quantitative, cost- effective, and fully automated, nucleic acid- and protein-based diagnostic tests that require minimal sample sizes. In this Phase I SBIR application, we propose to adapt 4c-ALEX for fast ultrasensitive multiplexed detection and quantification of microRNAs. Proof-of-principle will be established via amplification-free characterization of cancer-specific miRNAs signatures in serum samples. Subsequent technology commercialization will target basic research, pharmaceuticals, and diagnostics markets. Our Specific Aims are: 1. Separate detection and quantification of seven lung cancer-related miRNAs in spiked samples by 4c-ALEX single molecule fluorescence spectroscopy and method comparison to qPCR. 2. Multiplexed (single-well) discrimination and quantification of these miRNAs. 3. 4c-ALEX-based analysis of 100 archived clinical serum samples (60 cancer patients and 40 controls). SBIR Phase II will be dedicated to assay expansion, miniaturization, and instrument prototype development. PUBLIC HEALTH RELEVANCE: Dysregulated expression of microRNAs in various tissues has recently been associated with a variety of diseases and it has been shown that miRNA signatures can be used as novel biomarkers, potentially offering more sensitive and specific tests than those currently available for early diagnosis of cancer and other diseases. Nesher Technologies, Inc. intends to develop an innovative test for microRNA signatures based on patent-protected alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, offering the prospect to detect cancers at an early stage with high sensitivity and specificity which would significantly improve survival rates of this deadly disease. This will complement Nesher Technologies' federally-funded efforts of instrument and reagent development for tests for detection of early cancer, infectious and genetic diseases, bioterror agents, and others, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives and reducing healthcare costs.