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得能够对生物分子进行多重、超灵敏和高度特异性的检测和定量。尽管它们比整体平均检测方法具有许多优点，但大多数单分子光谱仍然是由训练有素的研究人员专用的工具，因为建立这种设置所需的费用和专业知识。该提案旨在通过创建一种简单、具有成本效益且超稳定的商业单分子光谱读取器来扩大单分子光谱的用户群，该单分子光谱读取器用于诊断应用，其可以由具有很少或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。它将帮助指导医生进行早期疾病检测、健康状况评估和治疗启动，同时还可以监测公众和环境中的疾病爆发、耐药性趋势和潜在的生物恐怖袭击。设想的设备耦合交替激光激发（ALEX）的单井多路复用能力-由加州大学洛杉矶分校的Shimon韦斯教授开创的单分子光谱学-与共聚焦荧光检测的灵敏度。Nesher Technologies，Inc. (NTI)已经建立了商业化这一使能技术，并通过这一建议，打算开发一个原型台式单分子阅读器，通过扩大目前现有的三色ALEX技术，以四个颜色（4c-ALEX）的改进的多路复用能力。以该阅读器为基础，NTI的长期目标是开发各种简单的“混合阅读”核酸和蛋白质测试，可以在单个患者（或环境）样本中同时检测和准确定量多种遗传畸变，生物标志物和/或病原体。试剂开发和概念验证测试目前正在进行中，并通过几项赠款提供资金。本提案的具体目标是：（1）。NTI将建立一个紧凑，低成本（40，000 - 45，000美元）和超稳定的4c-ALEX单分子阅读器（适用于第二阶段的原型开发）。2）。该装置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同时检测和定量单个样品中的多个靶标来证明。3）。将通过将其性能与韦斯教授实验室中现有的非商业3c-ALEX单分子装置进行比较，证明该装置的稳定性和准确度。4）。现有的数据采集和分析软件将被简化，以尽量减少用户输入，并使4c-ALEX的数据采集/分析过程完全自动化。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中快速识别多种可能性中的特定医疗保健相关靶标，从根本上推动了当前技术的极限。通过此授权申请，Nesher Technologies，Inc.提出了一个完全集成的，小足迹，非常创新的光谱生物分子阅读器适合商业化的原型开发的第一个主要步骤。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，用于早期癌症，遗传疾病和生物恐怖剂检测的高度多重，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社会的利益，通过挽救人类生命和降低医疗保健成本。

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.