ALEX for fast ultrasensitive multiplexed detection & quantification of microRNAs

ALEX 用于快速超灵敏多重检测

• 批准号: 8458519
• 负责人: Taiho Kim
• 金额: $ 9.61万
• 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 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8458519
• 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.

描述（由申请人提供）：最近，微小RNA（miRNA）与包括癌症在内的人类恶性肿瘤的发生和进展有关。体液和活检中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​​，连续分析并不实用。因此，只有具有高多重能力的新的、极其敏感的方法才能显着提高诊断价值并增进我们对 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，该技术是在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，具有精湛的单井多重分析潜力、最少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）进行标记。它基于共焦显微镜，可以对溶液中的生物分子进行超灵敏检测，同时区分众多目标，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，大大扩展了其多重能力，并证明了在临床相关浓度下超灵敏 miRNA 定量的诊断实用性，无需扩增。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 光谱的结合，这是一种称为“单分子光流体”的小型化测定的新突破方法，以及 ii) 使用多焦点激发/增强通量检测几何。 NTI 的长期目标是开发快速、高度多重（每个样品可容纳 >100 种分析物）、超灵敏、特异性、定量、经济高效、全自动、基于核酸和蛋白质的诊断测试，这些测试需要最小样本量。在此 I 期 SBIR 应用中，我们建议采用 4c-ALEX 进行快速超灵敏多重检测和 microRNA 定量。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原理验证。后续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. 通过 4c-ALEX 单分子荧光光谱法对加标样品中的 7 种肺癌相关 miRNA 进行单独检测和定量，并与 qPCR 进行方法比较。 2. 这些 miRNA 的多重（单孔）区分和定量。 3. 对 100 份存档的临床血清样本（60 名癌症患者和 40 名对照者）进行基于 4c-ALEX 的分析。 SBIR 第二阶段将致力于测定扩展、小型化和仪器原型开发。