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.
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