Compact ALEX-based Reader for Highly Multiplexed Single Biomolecule Detection

描述(由申请人提供)：单分子光谱学领域的最新进展使生物分子的多重、超灵敏和高度特异的检测和量化成为可能。尽管大多数单分子光谱仪与集合平均检测方法相比有许多优势，但由于建立这种装置所需的费用和专业知识，大多数单分子光谱仪仍然是专门由训练有素的研究人员使用的工具。这项建议旨在通过创建一种简单、经济高效和超稳定的商用单分子光谱读取器来扩大单分子光谱仪的用户基础，用于诊断应用，可由几乎没有光学经验的人员操作。该设备将在医院和诊断实验室以及生物医学研究中找到许多用途。它将帮助指导医生及早发现疾病、评估健康状况并开始治疗，同时还允许监测公众和环境中的疾病暴发、耐药性趋势和潜在的生物恐怖主义袭击。这种设想的设备将交替激光激发(Alex)的单井多路复用能力与共聚焦荧光检测的精致灵敏度结合在一起。Nesher Technologies，Inc.(NTI)已经成立，将这种使能技术商业化，并通过这项提案打算开发一种原型桌面单分子阅读器，通过将目前的三色Alex技术扩展到四种颜色(4c-Alex)，具有改进的多路复用能力。以此阅读器为基础，NTI的长期目标是开发各种简单的基于核酸和蛋白质的混合阅读测试，可以同时检测和准确量化单个患者(或环境)样本中的多个遗传异常、生物标记物和/或病原体。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。本提案的具体目标是：1)。NTI将制造一款紧凑、低成本(40,000-45,000美元)、超稳定的4C-Alex单分子阅读器(适合第二阶段的原型开发)。2)。该设备的单井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同时检测和量化单个样本中的多个目标来展示。3)。该装置的稳定性和准确性将通过与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得到精简，以最大限度地减少用户输入，并使4c-Alex的数据采集/分析过程完全自动化。与公共健康相关：开发一种高度多元化、超灵敏和特异、定量、低成本的自动化医疗诊断测试系统，能够从单一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，从根本上突破了当前技术的极限。通过这项赠款申请，Nesher Technologies，Inc.向完全集成的、占地面积小、非常创新的、适合商业化的光谱生物分子读取器的原型开发迈出了重要的第一步。它将补充Nesher Technologies正在进行的、由联邦政府资助的试剂开发工作，用于针对早期癌症、遗传疾病和生物恐怖制剂检测的高度多元化、超灵敏和特异、定量、完全自动化和具有成本效益的测试，从而通过拯救人类生命和降低医疗成本，将纳米技术的尖端创新转化为对整个社会的好处。

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.