ALEX for fast ultrasensitive multiplexed detection & quantification of microRNAs

ALEX 用于快速超灵敏多重检测

• 批准号: 8312430
• 负责人: Taiho Kim
• 金额: $ 20万
• 依托单位: NESHER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312430
• 关键词: Affinity; Antibodies; Apoptosis; Applications Grants; Archives; Area; Basic Science; Binding; Biological; Biological Assay; Biological Markers; Biophysics; Biopsy; Body Fluids; Cancer Patient; Cell physiology; Clinical; Code; Color; Communicable Diseases; Complement; Complex; Computer software; Confocal Microscopy; Custom; DNA; Detection; Development; Diagnostic; Diagnostic tests; Discrimination; Disease; Dyes; Early Diagnosis; Energy Transfer; Enzyme-Linked Immunosorbent Assay; Exclusion; Fine-needle biopsy; Fingers; Fluorescence; Fluorescence Spectroscopy; Fluorescent Dyes; Functional RNA; Funding; Gene Expression; Goals; Head; Health Care Costs; Hereditary Disease; Human; Individual; Intellectual Property; Label; Lasers; Legal patent; Length; Licensing; Malignant Neoplasms; Malignant neoplasm of lung; Marketing; Methodology; Methods; MicroRNAs; Microarray Analysis; Microfluidics; Miniaturization; Molecular; Molecular Profiling; Monitor; Noise; Nucleic Acids; Patients; Pattern; Pharmacologic Substance; Phase; Procedures; Process; Proteins; RNA; Reagent; Role; Sample Size; Sampling; Screening for cancer; Sensitivity and Specificity; Serum; Site; Small Business Innovation Research Grant; Societies; Solutions; Sorting - Cell Movement; Spectrum Analysis; Staging; Survival Rate; System; Technology; Testing; Time; Tissues; Translating; Work; base; clinically relevant; commercialization; cost effective; detector; fluorophore; improved; innovation; instrument; instrument miniaturization; interest; nanobiotechnology; nanolitre; novel; operation; prognostic; prospective; prototype; single molecule; tool; tumor; user-friendly; virtual

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.

描述（由申请人提供）：最近，微小RNA（miRNA）与人类恶性肿瘤（包括癌症）的发生和进展有关。在体液和活检中鉴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，因此系列分析不实用。因此，只有具有高多路复用能力的新型、极其敏感的方法才能够显着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)拥有革命性定量、超灵敏和特异性生物检测技术的独家知识产权，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韦斯教授领导）开发，具有精致的单孔多重潜力、最低的样品要求和极其简化的处理程序（无需分离/洗涤和扩增步骤）。它是基于交替激光激发（ALEX）单分子荧光光谱，其中目标识别分子标记不同颜色的荧光染料（和猝灭剂）。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，同时区分许多目标，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，并证明了在临床相关浓度下无需扩增的超灵敏miRNA定量的诊断实用性。此外，我们的顾问Steve Quake和Shimon韦斯最近的工作显示了i）基于微流体的样品处理与ALEX光谱学的组合，这是一种用于测定小型化的新的突破性方法，称为“单分子光流体”，以及ii）使用多焦点激发/检测几何结构提高了通量。NTI的长期目标是开发快速、高度多路复用（每个样本的分析能力>100个）、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，这些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microRNA的快速超灵敏多重检测和定量。将通过血清样品中癌症特异性miRNA特征的无扩增表征来建立原理证明。随后的技术商业化将针对基础研究、制药和诊断市场。我们的具体目标是：1。通过4c-ALEX单分子荧光光谱法单独检测和定量加标样品中的7种肺癌相关miRNA，并与qPCR进行方法比较。 2.这些miRNA的多重（单孔）辨别和定量。 3.基于4c-ALEX的100份存档的临床血清样品（60名癌症患者和40名对照）的分析。 SBIR第二阶段将致力于分析扩展、小型化和仪器原型开发。 公共卫生相关性：最近，各种组织中microRNA的表达失调与多种疾病有关，并且已经表明，miRNA签名可以用作新的生物标志物，可能提供比目前可用于癌症和其他疾病的早期诊断的那些更敏感和特异性的测试。Nesher Technologies，Inc.基于受专利保护的交替激光激发（ALEX）单分子荧光光谱技术，该公司计划开发一种创新的microRNA特征检测方法，为以高灵敏度和特异性在早期阶段检测癌症提供前景，这将显著提高这种致命疾病的存活率。这将补充Nesher Technologies联邦资助的仪器和试剂开发工作，用于检测早期癌症，传染病和遗传病，生物恐怖剂等，从而将纳米生物技术的尖端创新转化为造福社会通过拯救人类生命和降低医疗保健成本。