Multiplex ALEX-based Test for Protease Activity Profiling in Arthritic Joints

基于多重 ALEX 的关节炎关节蛋白酶活性分析测试

• 批准号: 8525140
• 负责人: Taiho Kim
• 金额: $ 15万
• 依托单位: NESHER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525140
• 关键词: ADAMTS; Adopted; Affinity; Antibodies; Applications Grants; Archives; Area; Arthritis; Basic Science; Binding; Biological Assay; Biophysics; Body Fluids; Botulism; Cathepsins; Clinical; Clinical Trials; Color; Companions; Complex; Computer software; Confocal Microscopy; Custom; DNA; Degenerative Disorder; Detection; Development; Diagnostic; Diagnostic tests; Disease; Disease Progression; Drug Delivery Systems; Dyes; Energy Transfer; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Excision; Exclusion; Extracellular Matrix; Failure; Fluorescence; Fluorescence Spectroscopy; Fluorescent Dyes; Future; Gelatinase A; Goals; Head; Health; Individual; Intellectual Property; Intervention; Joints; Kinetics; Label; Lasers; Legal patent; Licensing; Lubrication; Marketing; Measures; Mediating; Methodology; Microfluidics; Miniaturization; Molecular; Monitor; Neurotoxins; Noise; Nucleic Acids; Pathway interactions; Patients; Peptide Hydrolases; Peptide Synthesis; Peptides; Pharmacologic Substance; Phase; Population; Procedures; Protease Inhibitor; Proteins; Reading; Reagent; Research; Research Design; Sample Size; Sampling; Site; Small Business Innovation Research Grant; Solutions; Sorting - Cell Movement; Spectrum Analysis; Stratification; Subgroup; Synovial Fluid; System; Technology; Testing; Time; Work; articular cartilage; base; collagenase 3; commercialization; cost effective; detector; fluorophore; improved; inhibitor/antagonist; innovation; instrument miniaturization; interest; joint destruction; nanolitre; next generation; operation; point-of-care diagnostics; prototype; response; single molecule; synthetic peptide; tool; user-friendly; virtual

DESCRIPTION (provided by applicant): Arthritis is a heterogeneous disease of the 'whole joint', whereby different combinations of proteolytic enzymes are active in different patient subpopulations leading to progressive joint degeneration. Upfront identification and stratification is critical for appropriate intervention with specific inhibitors, several of which are being evaluated in clinical trials. Nesher Technologies, Inc. (NTI) proposes a next-generation multiplex test for protease activity profiling in minute synovial fluid samples from arthritic joints, using alternating laser excitation (ALEX) single molecule fluorescence spectroscopy. This test should prove highly useful for facilitating treatment (as well as clinical trials) of arthritis with suitale protease inhibitors, or combinations thereof, personalized according to an individual patient's profile. NTI has exclusively licensed the intellectual property for ALEX, 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 and amplification steps). Target recognition molecules are tagged with different color fluorescent dyes (and quenchers). Based on confocal microscopy, it allows ultrasensitive detection of biomolecules in solution, differentiation of numerous targets simultaneously, and direct, amplification-free quantification via molecule counting. NTI recently achieved expansion from 2- color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for detection of proteins and nucleic acids. Furthermore, recent work by our consultants Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization termed "single molecule optofluidics", and ii) enhanced throughput using a multifoci excitation/detection geometry. 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 diagnostic tests requiring minimal sample sizes. For this SBIR Phase I grant application we propose multiplexed detection and quantification of protease activities for MMP- 13 and ADAMTS-4 as proof-of-principle, to be expanded in Phase II to a panel of up to 10 enzymes. Our Specific Aims are: 1. MMP-13 and ADAMTS-4 substrate peptide synthesis and fluorophore/quencher conjugation; 2. Separate as well as multiplexed protease activity detection and quantification using spiked samples; 3. ALEX-based analysis of 42 archived clinical synovial fluid samples (PRoBE study design implementation). Phase II will be dedicated to assay expansion, miniaturization, and instrument prototype development. Sub- sequent technology commercialization will target pharmaceuticals, diagnostics, and basic research markets.

描述（由申请人提供）：关节炎是一种“整个关节”的异质性疾病，其中不同的蛋白水解酶组合在不同的患者亚群中具有活性，导致进行性关节退化。预先识别和分层 对于使用特定抑制剂进行适当干预至关重要，其中一些抑制剂正在临床试验中进行评估。 Nesher Technologies, Inc. (NTI) 提出了一种下一代多重测试，使用交替激光激发 (ALEX) 单分子荧光光谱，对关节炎关节的微小滑液样本中的蛋白酶活性进行分析。该测试对于促进使用合适的蛋白酶抑制剂或其组合（根据个体患者的情况进行个性化治疗）的关节炎治疗（以及临床试验）非常有用。 NTI 独家授权了 ALEX 的知识产权，ALEX 是一种革命性的定量、超灵敏和特异性生物检测技术，由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，具有精湛的单井多重潜力、极低的样品要求、以及极其简化的处理程序（无分离/洗涤和扩增步骤）。目标识别分子用不同颜色的荧光染料（和猝灭剂）标记。它基于共焦显微镜，可以对溶液中的生物分子进行超灵敏检测，同时区分众多目标，并通过分子计数进行直接、无扩增的定量。 NTI 最近实现了从 2 色 (2c) ALEX 到 4 色 (4c) 的扩展，大大扩展了其多重功能，并展示了检测蛋白质和核酸的诊断实用性。此外，我们的顾问教授最近的工作。 Steve Quake 和 Shimon Weiss 展示了 i) 基于微流体的样品处理与 ALEX 光谱的结合，这是一种称为“单分子光流体”的小型化测定的新突破方法，以及 ii) 使用多焦点激发/检测几何结构提高了通量。 NTI 的长期目标是开发快速、高度多重（每个样品可容纳 >100 种分析物）、超灵敏、特异性、定量、经济高效且完全自动化的诊断测试，所需的操作最少 样本大小。对于此 SBIR 第一阶段资助申请，我们建议对 MMP-13 和 ADAMTS-4 的蛋白酶活性进行多重检测和定量，作为原理验证，并在第二阶段扩展到最多包含 10 种酶的组。我们的具体目标是： 1. MMP-13 和 ADAMTS-4 底物肽合成和荧光团/猝灭剂缀合； 2. 使用加标样品进行分离和多重蛋白酶活性检测和定量； 3. 对 42 个存档的临床滑液样本进行基于 ALEX 的分析（PRoBE 研究设计实施）。第二阶段将致力于测定扩展、小型化和仪器原型开发。后续技术商业化将瞄准制药、诊断和基础研究市场。