ALEX for fast ultrasensitive multiplexed detection & quantification of microRNAs

描述(申请人提供)：最近，microRNAs(MiRNAs)与包括癌症在内的人类恶性肿瘤的发生和发展有关。体液和活检组织中已识别的表达模式突显了它们作为生物标志物的潜力。由于患者样本有限，而miRNA浓度往往极低，因此进行系列分析是不现实的。因此，只有具有高多重能力的新的、极其灵敏的方法才能显著提高诊断价值，并促进我们对miRNAs生物学作用的理解。Nesher Technologies，Inc.(NTI)已经为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)开发的一种革命性的定量、超灵敏和特定的生物检测技术授予了独家知识产权，该技术具有精致的单井多路复用潜力、最少的样品要求和极其简化的处理程序(无需分离/洗涤和放大步骤)。它基于交替激光激发(Alex)单分子荧光光谱，目标识别分子被不同颜色的荧光染料(和猝灭剂)标记。基于共聚焦显微镜，它可以超灵敏地检测溶液中的生物分子，同时区分多个目标，并通过分子计数直接定量。NTI最近实现了从2色(2c)到4色(4c)Alex的扩展，大大扩展了其多路复用能力，并证明了无需扩增即可在临床相关浓度下进行超灵敏miRNA定量的诊断实用。此外，我们的顾问Steve Quake和Shimon Weiss最近的工作表明，i)将基于微流控技术的样品处理与Alex光谱分析相结合，这是一种称为“单分子光流控”的分析微型化的新突破性方法，以及ii)使用多焦点激发/检测几何结构来提高吞吐量。NTI的长期目标是开发快速、高度多元化(每个样本有100个分析物的能力)、超灵敏和特异、定量、成本效益和全自动化、基于核酸和蛋白质的诊断测试，这些测试需要最小的样本大小。在这个第一阶段的SBIR应用中，我们建议采用4c-Alex来快速、超灵敏地多重检测和定量microRNAs。原则证明将通过对血清样本中癌症特异性miRNAs信号的无扩增表征来建立。随后的技术商业化将瞄准基础研究、制药和诊断市场。我们的具体目标是：1.用4c-Alex单分子荧光光谱分析方法分离检测和定量检测加标样品中的7个肺癌相关miRNAs，并与qPCR方法进行比较。2.这些miRNAs的多重(单井)识别和定量。3.以4C-Alex为基础，对60例癌症患者和40例正常对照的100份临床血清标本进行分析。SBIR第二阶段将致力于分析扩展、小型化和仪器原型开发。 与公共卫生相关：最近，各种组织中microRNAs的异常表达与各种疾病有关，已有研究表明，miRNA签名可用作新的生物标记物，有可能提供比目前用于癌症和其他疾病早期诊断的更敏感和更特异的检测。Nesher Technologies，Inc.打算开发一种基于受专利保护的交替激光激发(Alex)单分子荧光光谱的microRNA签名的创新测试，提供高灵敏度和特异性的早期检测癌症的前景，这将显著提高这种致命疾病的存活率。这将是对Nesher Technologies联邦资助的仪器和试剂开发工作的补充，这些仪器和试剂用于检测早期癌症、传染病和遗传疾病、生物恐怖物质和其他物质，从而将纳米生物技术的尖端创新转化为拯救人类生命和降低医疗成本的社会福利。

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.