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

说明(申请人提供)：肉毒杆菌神经毒素(BoNTs)是已知的毒性最大的物质，被归类为A类生物治疗药物。BoNTs是一种锌金属蛋白酶，可以切割和失活与突触囊泡融合和乙酰胆碱释放有关的蛋白。多年来，肉毒杆菌毒素的治疗应用稳步增加，需要新的可靠的分析方法来评估BONT产品的效力和稳定性。Nesher Technologies，Inc.(NTI)已经为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)开发的一种革命性的定量、超灵敏和特定的生物检测技术授予了独家知识产权，该技术具有精致的单井多路复用潜力、最少的样品要求和极其简化的处理程序(无需分离/洗涤步骤)。它基于交替激光激发(Alex)单分子荧光光谱学，即用不同颜色的荧光染料标记两个(或更多)目标识别分子。两种(或更多)颜色的重合共焦检测构成了积极的目标检测事件，允许识别溶液中的生物分子并同时检测多个目标。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光谱的成功融合，用于定量酶活性，为检测微型化和增加吞吐量提供了一种新的突破性方法，称为“单分子光流体”。NTI的长期目标是开发快速、高度多样化(每个样本有100个分析物的能力)、超灵敏和特异、定量、成本效益和全自动化、基于蛋白质和核酸的综合症检测，这些检测需要最少的患者样本。在这一阶段的SBIR应用中，我们建议采用基于Alex的单分子光流体来开发一种创新的、完全基于溶液的体外内肽酶分析方法，克服了基于动物和细胞的分析方法的局限性，通过测量荧光团标记的底物切割来定量其蛋白酶活性，从而确定BoNT/A的效力和稳定性。具体目标是：1.底物多肽的合成和荧光基团/猝灭剂的偶联。2.基于亚历克斯基单分子光流控技术的BONT/A检测与定量3.特异性、敏感性(D100 FM)、定量的线性范围(e4对数阶)和时间(1小时)的测定。第二阶段将致力于分析优化、自动化、原型开发，如果需要，还将扩展到包括其他BONT底物，以进行快速多路(单孔)识别和定量。将开发适用于完全集成的基于Alex的测试系统的用户友好操作的软件，用于对BONT产品进行常规商业发布测试。它可以作为未来开发FDA批准的用于BONT和其他传染病试剂检测的诊断系统的基础。 与公共卫生相关：Nesher Technologies，Inc.打算开发一种基于交替激光激发(Alex)单分子荧光光谱的创新替代方法，以替代目前使用的小鼠腹膜注射检测方法，该方法测量小鼠单位(MU)的活动，将1MU的活动定义为LD50剂量(50%的小鼠在72-96小时内死亡)，以便对不同制造商生产的肉毒神经毒素产品的效力单位进行标准化。这将极大地减少动物的使用，也将补充Nesher Technologies联邦资助的仪器和试剂开发工作，用于生物恐怖因子、传染病和遗传疾病的测试，以及早期癌症检测，从而将纳米生物技术的尖端创新转化为拯救人类生命、监测人口生物恐怖袭击和疾病爆发以及降低医疗成本的社会福利。

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.