RAMAN FLOW CYTOMETRY FOR DRUG DISCOVERY AND DIAGNOSIS

用于药物发现和诊断的拉曼流式细胞术

基本信息

批准号：
8169398
负责人：
JOHN P NOLAN
金额：
$ 3.34万
依托单位：
LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8169398
关键词：
Academia Affinity Area Binding Biomedical Engineering Biomedical Research Cells Collaborations Computer Retrieval of Information on Scientific Projects Database Detection Diagnosis Diagnostic Engineering Event Flow Cytometry Fluorescence Funding Government Grant Individual Industry Information Systems Institution Label Libraries Measurement Microscopy Microspheres Molecular Molecular Analysis Optics Peptide Library Peptides Performance Plant Resins Polymers Property Proteins Raman Spectrum Analysis Reporter Reporting Research Research Personnel Research Project Grants Resources Route Science Screening procedure Sorting - Cell Movement Source Speed System Techniques Technology Testing Toxin United States National Institutes of Health Work abstracting base biological systems chemical synthesis drug discovery high throughput screening instrument instrumentation nanostructured new technology novel particle pathogen therapeutic development

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract The National Flow Cytoemtry Resource and the Bioengineering Research Partnership (BRP) to develop Raman Flow Cytometry for Diagnostics and Drug Discovery both aim to develop novel flow instrumentation for biomedical science. The two projects have many areas of synergy where collaborations will be established to speed the progress of both projects. Background The ability to make quantitative, high throughput molecular measurements of biological systems is a critical need for many areas of biomedical research. The Bioengineering Research Partnership (BRP) to develop Raman Flow Cytometry for Diagnostics and Drug Discovery aims to develop a powerful new analytical platform for high throughput screening and selection based on Raman Flow Cytometry. This Partnership will develop new analytical instrumentation, optically encoded polymer resins for chemical synthesis and screening, and nanostructured materials with unique optically properties for sensitive reporting and encoding. The new technology will perform Raman spectroscopy on single particles in flow to enable new applications in sensitive multiplexed detection, drug discovery, and diagnostics. The Raman Flow Cytometry instrumentation and applications will be developed by a Partnership involving engineers, biologists, and chemists from academia, government and industry. We have modified a commercial particle sorter (the COPAS) to detect individual Raman vibrational bands from single particles and sorted these particles based on their optical signature. We are also developing the ability to collect and analyze complete Raman spectra from single particles (1). In parallel, the Partnership has developed new encoding and reporting strategies for multiplexed molecular analysis and separation. This Raman Flow Cytometry technology will be applied to the development of therapeutics and diagnostics for bacterial pathogens and their toxins. Raman Flow Cytometry will be an important and general new analytical and separation capability that will impact many areas of basic and applied biomedical research. Approach The BRP discussed above will develop new Raman analysis capabilities for flow cytometry. This BRP will collaborate on all of the research projects of the NFCR. First, as sensitivity is critical to Raman analysis, the NFCR will work with the BRP to provide acoustically focused flow cells for high sensitivity measurements without a concurrent loss of particle analysis rate. Second, Peptide libraries can be synthesized to specifically bind a number of different proteins. Dr. Nolan's Bioengineering Research Partnership is developing technologies to rapidly select peptides that bind toxin proteins. The BRP is synthesizing many peptide libraries on large particles (>50 microns) that will bind fluorescent protein targets. The approach planned by the BRP has been to provide Raman analysis of the microspheres concurrently with fluorescent reporter binding via flow cytometry analysis, which will allow high speed decoding of the compound on the Raman microspheres via its Raman barcode for microspheres that are positive for binding events (2, 3). Provision of large particle sorting technology to this project, will aid its progression in two ways: sorted particles could be re-analyzed to confirm the online flow analysis and high speed sorting of the rare particles that bind the fluorescent reporters followed by established Raman microscopy technologies [2, 3] to decode the Raman signature that identifies the compound on the microsphere could provide an alternate route selection of peptides synthesized on Raman microsphere libraries. We will use these libraries as demonstration approach to identify fluorescent toxin binders to peptide bearing microspheres. The identity of the peptide will be identified via mass-spec of sorted microspheres or by integral Raman signatures within the microsphere identified via Raman microscopy. Finally, the BRP and the NFCR are developing spectral instrumentation for orthogonal purposes and with different approaches. The NFCR and the BRP will collaborate on many technical aspects of spectral flow cytometry. Specifically, he NFCR will work with the BRP on spectral flow cytometry, data systems, parallel analysis and large particle sorting. We will sort large particle libraries provided by the BRP using fluorescence techniques. In the out years of this proposal we will provide the large particle sorting technology to be implemented with their systems. We will also implement ORCA on the BRP spectral systems and provide them with line driven flow cells to maximize the sensitivity of the BRPs spectral systems. The BRP through Dr. Nolan will provide large particle microsphere libraries and protein targets that can be screened using fluorescence techniques. We will focus on demonstration that we can detect the binding of fluorescently labeled proteins to peptide libraries generated to have affinities for a variety of toxins. Dr. Nolan's lab will serve as a beta testing facility for data systems, line drives and new high speed parallel analysis technologies and sorters and will communicate with the NFCR to optimize instrument performance.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。抽象的国家流式细胞术资源和生物工程研究合作伙伴关系 (BRP) 开发用于诊断和药物发现的拉曼流式细胞术，旨在为生物医学科学开发新型流式仪器。这两个项目在许多领域具有协同作用，将建立合作以加快两个项目的进展。背景对生物系统进行定量、高通量分子测量的能力是生物医学研究许多领域的关键需求。开发用于诊断和药物发现的拉曼流式细胞术的生物工程研究合作伙伴关系 (BRP) 旨在开发一个强大的新分析平台，用于基于拉曼流式细胞术的高通量筛选和选择。该合作伙伴关系将开发新的分析仪器、用于化学合成和筛选的光学编码聚合物树脂，以及用于敏感报告和编码的具有独特光学特性的纳米结构材料。这项新技术将对流动中的单个颗粒进行拉曼光谱分析，以实现灵敏的多重检测、药物发现和诊断方面的新应用。拉曼流式细胞术仪器和应用程序将由来自学术界、政府和工业界的工程师、生物学家和化学家组成的合作伙伴关系开发。我们改进了商用颗粒分选机（COPAS）来检测单个颗粒的单个拉曼振动带，并根据它们的光学特征对这些颗粒进行分类。我们还正在开发从单个粒子收集和分析完整拉曼光谱的能力 (1)。与此同时，该伙伴关系还为多重分子分析和分离开发了新的编码和报告策略。这种拉曼流式细胞术技术将应用于细菌病原体及其毒素的治疗和诊断的开发。拉曼流式细胞术将成为一种重要且通用的新型分析和分离能力，将影响基础和应用生物医学研究的许多领域。方法上述 BRP 将为流式细胞仪开发新的拉曼分析功能。该 BRP 将就 NFCR 的所有研究项目进行合作。首先，由于灵敏度对于拉曼分析至关重要，NFCR 将与 BRP 配合提供声学聚焦流动池以进行高灵敏度测量，而不会同时损失颗粒分析率。其次，可以合成肽库来特异性结合许多不同的蛋白质。诺兰博士的生物工程研究合作伙伴正在开发快速选择与毒素蛋白结合的肽的技术。 BRP 正在大颗粒（>50 微米）上合成许多肽库，这些肽库将结合荧光蛋白靶标。 BRP 计划的方法是通过流式细胞术分析同时提供微球的拉曼分析和荧光报告分子结合，这将允许通过其拉曼条形码对拉曼微球上的化合物进行高速解码，以获取对结合事件呈阳性的微球(2, 3)。为该项目提供大颗粒分选技术，将通过两种方式帮助其进展：可以重新分析分选的颗粒，以确认结合荧光报告基因的稀有颗粒的在线流分析和高速分选，然后采用成熟的拉曼显微镜技术[2, 3] 解码识别微球上化合物的拉曼特征可以为拉曼微球文库上合成的肽提供替代途径选择。我们将使用这些库作为示范方法来识别带有肽的微球的荧光毒素结合剂。肽的身份将通过分选微球的质谱或通过拉曼显微镜鉴定的微球内的整体拉曼特征来鉴定。最后，BRP 和 NFCR 正在开发用于正交目的并采用不同方法的光谱仪器。 NFCR 和 BRP 将在光谱流式细胞术的许多技术方面进行合作。具体来说，NFCR 将与 BRP 在光谱流式细胞术、数据系统、并行分析和大颗粒分选方面开展合作。我们将使用荧光技术对 BRP 提供的大颗粒库进行分类。在本提案的最后几年，我们将提供大颗粒分选技术，并在他们的系统中实施。我们还将在 BRP 光谱系统上实施 ORCA，并为其提供线驱动流动池，以最大限度地提高 BRP 光谱系统的灵敏度。 Nolan 博士的 BRP 将提供可使用荧光技术筛选的大颗粒微球文库和蛋白质靶标。我们将重点演示我们可以检测荧光标记蛋白与生成的对多种毒素具有亲和力的肽库的结合。 Nolan 博士的实验室将作为数据系统、线路驱动器以及新型高速并行分析技术和分类器的 Beta 测试设施，并将与 NFCR 进行通信以优化仪器性能。