RAMAN FLOW CYTOMETRY FOR DRUG DISCOVERY AND DIAGNOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要 国家流式细胞仪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术都旨在开发用于生物医学科学的新型流动仪器。这两个项目有许多协同领域，将在这些领域建立协作，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许多生物医学研究领域的关键需求。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术，旨在开发一个基于拉曼流式细胞术的强大的新分析平台，用于高通量筛选和选择。这一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性能的纳米结构材料。这项新技术将对流动中的单个颗粒进行拉曼光谱，以实现在敏感的多路检测、药物发现和诊断方面的新应用。拉曼流式细胞仪仪器和应用将由来自学术界、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开发。我们已经改进了商用粒子分选器(COPAS)，以从单个粒子中检测单个拉曼振动带，并根据这些粒子的光学特征对这些粒子进行分类。我们还在开发收集和分析单个粒子的完整拉曼光谱的能力(1)。同时，该伙伴关系还为多重分子分析和分离制定了新的编码和报告战略。这项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发展。拉曼流式细胞术将是一种重要的、通用的新的分析和分离能力，将对生物医学基础和应用研究的许多领域产生影响。 方法 以上讨论的BRP将为流式细胞术开发新的拉曼分析能力。该BRP将在NFCR的所有研究项目上进行合作。首先，由于灵敏度是拉曼分析的关键，NFCR将与BRP合作，为高灵敏度测量提供声学聚焦流动池，而不会同时损失颗粒分析率。其次，可以合成多肽库来特异性地结合一些不同的蛋白质。诺兰博士的生物工程研究伙伴关系正在开发技术，以快速选择与毒素蛋白结合的多肽。BRP正在大颗粒(50微米)上合成许多多肽库，这些颗粒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在结合荧光报告的同时对微球进行拉曼分析，这将允许通过其对结合事件(2，3)呈阳性的微球的拉曼条形码对拉曼微球上的化合物进行高速解码。为该项目提供大颗粒分选技术，将从两个方面帮助该项目的进展：分选颗粒可以重新分析，以确认结合荧光记者的稀有颗粒的在线流动分析和高速分选，随后建立的拉曼显微镜技术[2，3]对识别微球上化合物的拉曼信号进行解码，可以为在拉曼微球库上合成的多肽提供替代路线选择。我们将使用这些文库作为演示方法来鉴定含肽微球的荧光毒素结合。将通过已分类微球的质谱仪或通过拉曼显微镜鉴定的微球内的积分拉曼信号来鉴定多肽的身份。最后，BRP和NFCR正在开发用于正交目的的光谱仪器，并采用不同的方法。NFCR和BRP将在光谱流式细胞术的许多技术方面进行合作。 具体地说，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类方面进行合作。我们将使用荧光技术对BRP提供的大粒子库进行排序。在这项提议的未来几年里，我们将提供大颗粒分选技术，并与他们的系统一起实施。我们还将在BRP光谱系统上实现ORCA，并为它们提供线驱动流动单元，以最大限度地提高BRPS光谱系统的灵敏度。 通过诺兰博士的BRP将提供可用荧光技术筛选的大颗粒微球库和蛋白质靶标。我们将重点展示我们可以检测到荧光标记的蛋白质与多肽文库的结合，这些多肽文库产生的多肽与各种毒素具有亲和力。诺兰博士的实验室将作为数据系统、直线驱动器和新的高速并行分析技术和分类器的测试版测试设施，并将与NFCR进行通信，以优化仪器性能。

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.