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

• 批准号: 7956780
• 负责人: JOHN P NOLAN
• 金额: $ 3.22万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956780
• 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

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 抽象的 国家流式小细胞运动资源和生物工程研究伙伴关系（BRP）开发用于诊断和药物发现的拉曼流式细胞仪，均旨在开发生物医学科学的新型流动仪器。这两个项目具有许多协同作用，在该领域将建立合作以加快这两个项目的进度。 背景 对生物医学研究的许多领域进行定量，高吞吐量分子测量的能力是至关重要的。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开发一个强大的新分析平台，以基于拉曼流式细胞仪，用于高吞吐量筛查和选择。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具有独特的光学特性的纳米结构材料，用于敏感的报告和编码。这项新技术将对流量的单个颗粒进行拉曼光谱，以实现敏感多路复用检测，药物发现和诊断的新应用。拉曼流式细胞仪仪器和应用将由涉及学术界，政府和工业的工程师，生物学家和化学家的合作伙伴关系开发。我们已经修改了一个商业粒子分选项（COPAS），以检测来自单个颗粒的单个拉曼振动带，并根据其光学特征对这些颗粒进行排序。我们还在开发从单个颗粒收集和分析完整拉曼光谱的能力（1）。同时，该合作伙伴关系开发了新的编码和报告策略，用于多重分子分析和分离。这种拉曼流式细胞仪技术将应用于针对细菌病原体及其毒素的疗法和诊断的开发。拉曼流式细胞仪将是一个重要且一般的新分析和分离能力，它将影响基本和应用生物医学研究的许多领域。 方法 上面讨论的BRP将开发用于流式细胞术的新拉曼分析能力。该BRP将在NFCR的所有研究项目上进行合作。首先，由于灵敏度对拉曼分析至关重要，因此NFCR将与BRP一起使用，以提供高灵敏度测量值，而不会同时损失粒子分析速率。其次，可以合成肽库以特异性结合许多不同蛋白质。 Nolan博士的生物工程研究伙伴关系正在开发技术，以快速选择结合毒素蛋白的肽。 BRP正在合成大颗粒（> 50微米）上的许多肽文库，该肽将结合荧光蛋白靶标。 BRP计划的方法是通过流式细胞仪分析通过流式细胞仪分析同时提供与荧光报告基因结合的拉曼分析，这将允许通过其Raman微球上的Raman Barcode高速解码，用于与结合事件呈阳性的微球的Raman条形码（2，3）。提供大型粒子分类技术向该项目提供两种方式：可以重新分析排序的颗粒，以确认在线流量分析和绑定荧光记者的稀有颗粒的高速分类，然后确定置换的Raman显微镜技术[2，3]，以在Raman signation上进行解码，以识别Raman的选择。库。我们将使用这些文库作为示范方法，以鉴定荧光毒素粘合剂到具有微球的肽。将通过分类的微球的质谱或通过拉曼显微镜确定的微球内的整体拉曼特征来鉴定肽的身份。最后，BRP和NFCR正在开发用于正交目的和不同方法的光谱仪器。 NFCR和BRP将在频谱流式细胞仪的许多技术方面进行合作。 具体而言，NFCR将与BRP一起在光谱流式细胞仪，数据系统，并行分析和大粒子分类上使用。我们将使用荧光技术对BRP提供的大型粒子文库进行排序。在本提案的范围内，我们将提供将使用其系统实施的大型粒子分类技术。我们还将在BRP光谱系统上实现ORCA，并为它们提供线路驱动的流动池，以最大程度地提高BRPS光谱系统的灵敏度。 通过Nolan博士的BRP将提供大粒子微球库和蛋白质靶标，可以使用荧光技术筛选。我们将专注于演示，即我们可以检测荧光标记的蛋白质与产生的肽库的结合，这些蛋白具有与多种毒素相关的生成。 Nolan博士的实验室将作为数据系统，线路驱动器和新的高速平行分析技术和分类器的Beta测试设施，并将与NFCR通信以优化仪器性能。