Multiplexed Protein & miRNA Biomarker-based Next-gen Test for Alzheimers Disease

描述：为了最大限度地从单个小患者样本(例如脑脊液-脑脊液)中获取临床信息内容，单分子检测(SMD)技术是最有希望发展下一代医疗诊断的技术。最近，microRNAs(MiRNAs)被认为与包括神经退行性疾病在内的人类恶性肿瘤有关，这为将传统的蛋白质标记物与新的miRNA候选标记物相结合提供了令人兴奋的前景。但由于患者样本有限，标记物浓度往往很低，连续分析是不现实的。因此，只有高度敏感的方法 多路复用能力将能够最大限度地提高诊断价值，并适用于改进的测试，以早期发现疾病。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)开发的一项革命性、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工作流程(无需分离/洗涤和放大步骤)。它基于交替激光激发(Alex)单分子荧光光谱，目标识别分子被不同颜色的荧光染料(和猝灭剂)标记，允许在同一平台上超灵敏地检测体液中的蛋白质和核酸(包括miRNAs)。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(特别是在涉及FRET的情况下)，并证明了对超敏感蛋白质的诊断作用以及在临床相关浓度下无需扩增的miRNA定量。此外，我们的顾问教授最近的工作。Steve Quake和Shimon Weiss展示了i)基于微流控技术的样品处理与Alex光谱学的结合，这是一种称为“单分子光学流体”的分析小型化的新突破性方法，以及ii)使用多焦点激发/检测几何结构来提高吞吐量。NTI的长期目标是开发快速、高度多样化(每个样本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诊断测试，这些测试需要最小的样本量。在这里，我们建议改进早期阿尔茨海默病(AD)诊断的检测开发和临床验证，实施一组候选蛋白质和miRNA生物标记物。我们的具体目标是：1.基于多重蛋白质和microRNA生物标记物的下一代AD测试试剂的开发2.使用添加的样本进行分离和多重生物标记物的检测和量化3.基于Alex的对108名患者的存档临床样本的分析(探针研究设计实施)SBIR第二阶段将建议扩大检测范围、小型化，并开发一个通用的、用户友好的诊断系统，作为早期检测AD和其他神经退行性疾病的有用工具。

DESCRIPTION: To maximize clinical information content obtainable from a single small patient sample (e.g. cerebrospinal fluid-CSF), single molecule detection (SMD) technologies are the most promising for development of next- generation medical diagnostics. Recently, microRNAs (miRNAs) have been implicated in human malignancies including neurodegenerative disorders, offering the exciting prospect to combine traditional protein markers with novel miRNA candidate markers in panels. But as patient samples are limited and marker concentrations often very low, serial analysis is not practical. Thus, only very sensitive methodologies with high multiplexing power will be able to maximize diagnostic value and be suitable for improved tests for early disease detection. 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 workflows (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers), allowing, on the same platform, ultrasensitive detection of both proteins and nucleic acids (including miRNAs) in body fluids. NTI recently achieved extension from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power (particularly when involving FRET), and demonstrated diagnostic utility for ultrasensitive protein as well as miRNA quantification at clinically relevant concentrations without amplification. 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 opt fluidics", and ii) enhanced throughput using a multifocal 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, protein- and nucleic acid- based diagnostic tests that require minimal sample sizes. Here we propose assay development and clinical validation for improved early-stage Alzheimer's disease (AD) diagnosis, implementing a panel of candidate protein and miRNA biomarkers. Our Specific Aims are: 1. Reagent development for a multiplex protein & microRNA biomarker-based next-generation AD test 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of archived clinical samples from 108 patients (PRoBE study design implementation) SBIR Phase II will propose assay expansion, miniaturization, and development of a versatile, user-friendly, diagnostic system as useful tool for early detection of AD and other neurodegenerative disorders.