New Class of Bright, Sharp, Tunable Near-Infrared Fluorophores for Flow Cytometry

Abstract Polychromatic flow cytometry (FC) is one of the most powerful analytical techniques routinely used by both basic research and clinical diagnostics laboratories for the immunological categorization of cells. Dyes used for FC typically exhibit broad fluorescent emission bands with full-width-at-half-maximum (fwhm) values of 50–80 nm. This limits the maximum number of dyes, and thus the number of cell biomarkers, that can be resolved in a given experiment. Compensating for spectral overlap between dyes is currently viewed as a necessary part of experimental design, requiring extensive pre-assay experimentation and mathematical compensation, thereby introducing experimental error and reducing sensitivity. Bacteriochlorins are a unique class of fluorescent dyes that offer a solution for accurate multiplexing with minimal compensation due to their very narrow emission bands (fwhm of 25-35 nm), typically less than half the spectral width of existing dyes. Through chemical modification, they can be tuned to emission wavelengths from the far red through the near-infrared (NIR) spectrum (700–900 nm). In addition, bacteriochlorins share a common excitation band, making possible the development of a full spectrum of NIR dyes excited by a single UV light source. To render bacteriochlorin dyes commercially viable, Phase 2B efforts will be focused on: 1) improving and expanding the bacteriochlorin dye portfolio and synthesis methods; 2) validating dye performance in FC panels with NIRvana Sciences’ collaborators, and 3) developing procedures, methods, and protocols for commercial scale manufacturing. This Phase 2B SBIR proposal is intended to continue the refinement and transfer of bacteriochlorin technology for commercial development, resulting in a bacteriochlorin dye portfolio with high impact potential for polychromatic FC. This enhanced multiplex capability will advance not only basic immunology research, but it will also accelerate novel vaccine and adjuvant discovery for HIV, malaria, tuberculosis, and emerging infectious disease threats. Greater multiplexing also is critical for analyzing reduced volume samples, for single cell studies, and for high throughput, high resolution analyses.Project Narrative/Public Health Relevance: Flow Cytometry is used routinely for both basic research and clinical diagnostic applications for characterizing cells of the immune system. Modern flow cytometers are capable of detecting 12-15 biomarkers per experiment using different fluorescent dye labels or “colors” but the overlapping spectra of most fluorescent dyes limit the number of biomarkers that can currently be detected accurately. NIRvana Sciences proposes to develop a portfolio of novel dyes in the near infrared (NIR) spectral region with low overlap that will enable much greater numbers of biomarkers to be detected simultaneously by flow cytometry, impacting not only basic immunology research, but also accelerating development of immunotherapies and vaccines for HIV and other emerging infectious disease threats.