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

产品说明：为了最大化可从单个小患者样品（例如，脑脊液-CSF）获得的临床信息内容，单分子检测（SMD）技术对于下一代医学诊断的开发是最有前景的。近年来，microRNAs（miRNAs）被发现与包括神经退行性疾病在内的人类恶性肿瘤有关，将传统的蛋白质标记物与新的miRNAs候选标记物联合收割机进行组合，具有令人振奋的前景。但由于患者样本有限，标记物浓度通常很低，因此系列分析不实用。因此，只有非常敏感的方法， 多路复用能力将能够最大化诊断价值，并适用于早期疾病检测的改进测试。 Nesher Technologies，Inc. (NTI)独家授权了一项革命性的、定量的、超灵敏的和特异性的生物检测技术的知识产权，该技术是在加州大学洛杉矶分校单分子生物物理实验室（由Shimon韦斯教授领导）开发的，具有精致的单孔多路复用潜力，最小的样品要求和极其简化的工作流程（没有分离/洗涤和扩增步骤）。它基于交替激光激发（ALEX）单分子荧光光谱，其中目标识别分子用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miRNA）。 NTI最近实现了从2色（2c）到4色（4c）ALEX的扩展，大大扩展了其多重能力（特别是当涉及FRET时），并证明了超灵敏蛋白质的诊断实用性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韦斯展示了i）基于微流体的样品处理与ALEX光谱学的组合，这是一种用于测定小型化的新的突破性方法，称为“单分子光学流体学”，和ii）使用多焦点激发/检测几何结构增强的通量。NTI的长期目标是开发快速、高度多路复用（每个样本的分析能力>100个）、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诊断测试，这些测试需要最小的样本量。在这里，我们提出了改进早期阿尔茨海默病（AD）诊断的检测方法开发和临床验证，实施了一组候选蛋白质和miRNA生物标志物。我们的具体目标是：1。基于多重蛋白质和microRNA生物标记的下一代AD检测试剂开发2. 使用加标样品进行分离以及多重生物标志物检测和定量3. SBIR第二阶段将提出分析扩展、小型化和开发通用、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.