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

摘要 多色流式细胞术(FC)是最强大的分析技术之一，基本的 用于细胞免疫分类的研究和临床诊断实验室。用于FC的染料 通常表现出宽的荧光发射频带，半最大全宽(FWHM)值为50-80 nm。 这限制了染料的最大数量，从而限制了在给定的 做实验。目前，补偿染料之间的光谱重叠被视为 实验设计，需要广泛的化验前实验和数学补偿，因此 引入实验误差，降低灵敏度。细菌素是一类独特的荧光染料。 这为精确多路复用提供了一种解决方案，由于它们的发射带非常窄，因此补偿最小 (FWHM为25-35 nm)，通常不到现有染料光谱宽度的一半。通过化学修饰， 它们可以通过近红外(NIR)光谱(700-900)调谐到从远红到发射的波长 NM)。此外，细菌素共享一个共同的激发带，使全面发展成为可能 单一紫外光激发下的近红外染料光谱。为了使细菌素染料在商业上可行， 2B阶段的工作将集中在：1)改进和扩大细菌素染料组合和合成 方法；2)与Nirvana Sciences的合作者一起验证FC面板中的染料性能；3)开发 商业规模生产的程序、方法和协议。此阶段2B SBIR建议书是 打算继续提炼和转让细菌素技术用于商业开发， 从而产生了对多色FC具有高影响潜力的细菌素染料组合。这增强了 多重能力不仅将促进基础免疫学研究，而且还将加速新疫苗的研制 以及对艾滋病毒、疟疾、结核病和新出现的传染病威胁的佐剂发现。更大 多路复用对于分析减少体积的样品、对于单细胞研究以及对于高吞吐量也是关键的， 高分辨率分析。

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.