Compact ALEX-based Reader for Highly Multiplexed Single Biomolecule Detection

基于 ALEX 的紧凑型阅读器，用于高度多重单一生物分子检测

• 批准号: 8147381
• 负责人: Taiho Kim
• 金额: $ 3.93万
• 依托单位: NESHER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8147381
• 关键词: AFP gene; Applications Grants; Attention; Benchmarking; Biological Markers; Biomedical Research; Bioterrorism; Bontoxilysin; CA-125 Antigen; CA-19-9 Antigen; Color; Complement; Computer software; Couples; Coupling; DNA; Detection; Development; Devices; Diagnostic; Diagnostic tests; Disease; Disease Outbreaks; Drug resistance; Dyes; Electrons; Elements; Environment; Fiber; Fiber Optics; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Foundations; Funding; Genetic; Goals; Grant; Health Care Costs; Health Status; Healthcare; Hereditary Disease; Hospitals; Human; Human Resources; Hybrids; Immersion Investigative Technique; Individual; Label; Lasers; Life; Ligase Chain Reaction; Medical; Methods; Mind; Monitor; Nanotechnology; Nucleic Acids; Oils; Oligonucleotides; Optics; Pathway interactions; Patients; Performance; Phase; Photons; Physicians; Process; Proteins; Pump; Reader; Reading; Reagent; Reporter; Research; Research Personnel; Resolution; Sampling; Scheme; Serotyping; Societies; Source; Spectrum Analysis; Spottings; System; Technology; Testing; Training; Translating; Tumor Markers; United States National Institutes of Health; Water; base; cancer genetics; charge coupled device camera; commercialization; cost; data acquisition; design; detector; experience; improved; innovation; pathogen; prototype; public health relevance; single molecule; software development; solid state; tool; trend

DESCRIPTION (provided by applicant): Recent advances in the field of single molecule spectroscopy have enabled multiplexed, ultra-sensitive, and highly specific detection and quantification of biomolecules. Despite their many advantages over ensemble-averaged detection methods, most single molecule spectroscopies remain tools which are used exclusively by highly trained researchers due to the expense and expertise required to build such setups. This proposal seeks to expand the user-base of single molecule spectroscopies by creating a simple, cost-effective, and ultra-stable commercial single molecule spectroscopic reader for diagnostic applications which can be operated by personnel with little to no experience in optics. The device will find numerous uses in hospitals and diagnostic labs as well as in biomedical research. It will help guide physicians in early disease detection, assessment of health status, and initiation of treatment, while it concomitantly allows monitoring the public and environment for disease outbreaks, drug resistance trends, and potential bioterrorism attacks. The envisioned device couples the single-well multiplexing capabilities of Alternating Laser Excitation (ALEX) -a single molecule spectroscopy pioneered by Prof. Shimon Weiss at UCLA- with the exquisite sensitivity of confocal fluorescence detection. Nesher Technologies, Inc. (NTI) has been established to commercialize this enabling technology and through this proposal intends to develop a prototype table-top single molecule reader with improved multiplexing capabilities by expanding the currently existing three-color ALEX technology to four colors (4c-ALEX). With this reader as its foundation, NTI's long-term goal is to develop a variety of simple "mix-and-read" nucleic acid- and protein-based tests which can simultaneously detect and accurately quantify multiple genetic aberrations, biomarkers, and/or pathogens, in a single patient (or environmental) sample. Reagent development and proof-of-concept testing is currently ongoing and funded through several grants. The Specific Aims of the present proposal are: 1). NTI will build a compact, low-cost ($40,000-$45,000), and ultra-stable 4c-ALEX single molecule reader (suitable for prototype development in Phase II). 2). The device's single-well multiplexing capabilities will be demonstrated by the simultaneous detection and quantification of multiple targets in a single sample using dye-labeled nucleic acid and protein reporter molecules. 3). The device's stability and accuracy will be demonstrated by comparing its performance to an existing non- commercial 3c-ALEX single molecule setup in Prof. Weiss' lab. 4). Existing data acquisition and analysis software will be streamlined to minimize user input and fully automate the data-acquisition/analysis process for 4c-ALEX. PUBLIC HEALTH RELEVANCE: Development of a highly multiplexed, ultrasensitive and -specific, quantitative, low-cost automated medical diagnostic testing system, capable of quickly identifying specific healthcare-associated targets among the multitude of possibilities from a single patient sample, radically pushes the limits of current technologies. With this grant application Nesher Technologies, Inc. proposes a first major step towards prototype development of a fully integrated, small-footprint, very innovative spectroscopic biomolecule reader suitable for commercialization. It will complement Nesher Technologies' ongoing, federally-funded efforts of reagent development for highly multiplexed, ultrasensitive and -specific, quantitative, fully automated, and cost-effective tests for early cancer, genetic diseases, and bioterror agent detection, thereby translating cutting-edge innovations in nanotechnology into benefits for the society at large by saving human lives and reducing healthcare costs.

描述（由申请人提供）：单分子光谱领域的最新进展使生物分子的多路、超灵敏和高度特异性检测和定量成为可能。尽管它们比整体平均检测方法有许多优点，但由于建立这种装置所需的费用和专业知识，大多数单分子光谱仍然是专门由训练有素的研究人员使用的工具。该提案旨在通过创建一种简单、经济、超稳定的商用单分子光谱读取器来扩大单分子光谱的用户基础，该读取器用于诊断应用，可以由几乎没有光学经验的人员操作。该设备将在医院、诊断实验室以及生物医学研究中得到广泛应用。它将有助于指导医生早期发现疾病、评估健康状况和开始治疗，同时它还可以监测公众和环境，以了解疾病爆发、耐药性趋势和潜在的生物恐怖主义袭击。设想的设备将交替激光激发（ALEX）的单孔复用能力（ALEX是由加州大学洛杉矶分校的Shimon Weiss教授首创的单分子光谱学）与共聚焦荧光检测的精致灵敏度耦合在一起。Nesher Technologies, Inc. （NTI）已经建立了商业化这项使能技术，并通过该提案打算通过将目前现有的三色ALEX技术扩展到四色（4c-ALEX）来开发具有改进多路复用能力的台式单分子阅读器原型。以这个阅读器为基础，NTI的长期目标是开发各种简单的“混合和读取”核酸和蛋白质为基础的测试，可以同时检测和准确量化多种遗传畸变，生物标志物，和/或病原体，在单个患者（或环境）样品。试剂开发和概念验证测试目前正在进行中，并通过几笔赠款提供资金。本建议的具体目标是：1)。NTI将打造一款紧凑、低成本（4万至4.5万美元）、超稳定的4c-ALEX单分子阅读器（适用于II期原型开发）。该设备的单孔多路复用能力将通过使用染料标记的核酸和蛋白质报告分子在单个样品中同时检测和定量多个目标来证明。3）. 该装置的稳定性和准确性将通过将其性能与Weiss教授实验室现有的非商业3c-ALEX单分子装置进行比较来证明。4)。现有的数据采集和分析软件将被精简，以尽量减少用户输入，并完全自动化4c-ALEX的数据采集/分析过程。公共卫生相关性：开发一种高度多路复用、超灵敏和特异性、定量、低成本的自动化医疗诊断测试系统，能够从单个患者样本的众多可能性中快速识别特定的医疗保健相关目标，从根本上推动了当前技术的极限。通过这项拨款申请，Nesher科技公司向完全集成、占地面积小、非常创新的光谱生物分子读取器的原型开发迈出了重要的第一步，适合商业化。它将补充Nesher科技公司正在进行的，由联邦政府资助的试剂开发工作，用于早期癌症，遗传疾病和生物恐怖剂检测的高度多路复用，超灵敏和特异性，定量，全自动和具有成本效益的测试，从而将纳米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效益。