Fast accurate ultrasensitive in vitro assay to determine BoNT potency & stability

描述（由申请人提供）：肉毒杆菌神经毒素（BoNTs）是已知的毒性最强的物质，被列为A类生物威胁剂。bont是一种锌金属蛋白酶，可切割和灭活参与突触囊泡融合和乙酰胆碱释放的蛋白质。多年来，肉毒杆菌毒素的治疗应用稳步增加，需要新的和可靠的分析来评估BoNT产品的效力和稳定性。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，该技术由加州大学洛杉矶分校（UCLA）单分子生物物理实验室（由Shimon Weiss教授领导）开发，具有精致的单孔复用潜力，最小的样品要求，以及极其简化的处理程序（无分离/洗涤步骤）。它基于交替激光激发（ALEX）单分子荧光光谱，其中两个（或更多）目标识别分子被不同颜色的荧光染料标记。两种（或两种以上）颜色的同步共聚焦检测构成了一个积极的目标检测事件，可以同时识别溶液中的生物分子和检测多个目标。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。此外，我们的顾问教授最近的工作。Stephen Quake和Shimon Weiss展示了基于微流体的样品处理与2c-ALEX光谱的成功结合，用于定量酶活性，这是一种新的突破性方法，用于分析小型化和提高吞吐量，称为“单分子光流体”。NTI的长期目标是开发快速、高度多路复用（每个样品可分析100个分析物）、超灵敏和特异性、定量、成本效益高、全自动、基于蛋白质和核酸的综合征驱动检测，所需的患者样本最少。在这个I期SBIR应用中，我们建议采用基于alex单分子光流体技术来开发一种创新的、完全基于溶液的体外内肽酶测定方法，克服基于动物和细胞的测定方法的局限性，通过测量荧光团标记的底物切割来量化BoNT/A的蛋白酶活性，从而确定BoNT/A的效力和稳定性。具体目标是：1。底物肽合成和荧光团/猝灭剂偶联。2. 基于alex的单分子光流体BoNT/A检测与定量。3. 测定特异性、灵敏度（d100 fM）、定量线性范围（e4对数阶）和时间（1小时）。第二阶段将致力于分析优化、自动化、原型开发，如果认为需要的话，还将扩展到包括其他BoNT底物，用于快速多路（单孔）鉴别和定量。将开发适合用户友好操作的完全集成的基于alexa的测试系统，用于BoNT产品的常规商业发布测试。它可以作为fda批准的BoNT和其他传染病试剂检测诊断系统未来发展的基础。

DESCRIPTION (provided by applicant): Botulinum Neurotoxins (BoNTs) are the most toxic substances known and classified as Category A biothreat agents. BoNTs are zinc metalloproteases that cleave and inactivate proteins involved in synaptic vesicle fusion and acetylcholine release. Therapeutic applications of botulinum toxins have increased steadily over the years requiring new and reliable assays to assess the potency and stability of BoNT products. 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 steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby two (or more) target recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two (or more) colors constitutes a positive target detection event, allowing identification of biomolecules in solution and detection of numerous targets simultaneously. NTI recently achieved expansion from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power. Furthermore, recent work by our consultants Profs. Stephen Quake and Shimon Weiss demonstrates successful merging of microfluidics-based sample handling with 2c-ALEX spectroscopy for quantification of enzymatic activity, a new breakthrough approach for assay miniaturization and increased throughput termed "single molecule optofluidics". 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, protein- and nucleic acid-based syndrome-driven tests that require minimal patient samples. In this Phase I SBIR application, we propose to adapt ALEX-based single molecule optofluidics for development of an innovative, completely solution-based in vitro endopeptidase assay, overcoming limitations of animal- and cell-based assays, to determine BoNT/A potency and stability based on quantifying its protease activity by measuring fluorophore-labeled substrate cleavage. Specific Aims are: 1. Substrate peptide synthesis and fluorophore/quencher conjugation. 2. BoNT/A detection and quantification using ALEX-based single molecule optofluidics. 3. Determination of specificity, sensitivity (d100 fM), linear range of quantification (e4 log orders), & time (1 hr). Phase II will be dedicated to assay optimization, automation, prototype development, and, if considered desirable, expansion to include other BoNT substrates for rapid multiplexed (single-well) discrimination and quantification. Software will be developed suitable for user-friendly operation of a fully integrated ALEX-based testing system for routine commercial release testing of BoNT products. It may serve as basis for future development of an FDA-approved diagnostic system for BoNT and other infectious diseases agents testing. PUBLIC HEALTH RELEVANCE: Nesher Technologies, Inc. intends to develop an innovative alternative approach, based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, to the currently used mouse intraperitoneal injection assay which measures activity in Mouse Units (MU), with 1 MU of activity defined as the LD50 dose (death within 72 to 96 hours in 50% of the mice), in order to allow for standardization of potency units for Botulinum Neurotoxin products made by different manufacturers. This will greatly reduce use of animals and will also complement Nesher Technologies' federally-funded efforts of instrument and reagent development for tests for bioterror agents, infectious and genetic diseases, and early cancer detection, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives, monitoring the population for bioterror attacks and disease outbreaks, and reducing healthcare costs.