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

摘要 多色流式细胞术（FC）是基础免疫学和免疫学常规使用的最强大的分析技术之一。 用于细胞免疫学分类的研究和临床诊断实验室。用于FC的染料 典型地表现出具有50-80 nm的半峰全宽（FWHM）值的宽荧光发射带。 这限制了染料的最大数量，并且因此限制了在给定的时间内可以分辨的细胞生物标志物的数量。 实验补偿染料之间的光谱重叠目前被认为是染料选择的必要部分。 实验设计，需要大量的预分析实验和数学补偿，从而 引入实验误差并降低灵敏度。细菌绿素是一类独特的荧光染料 其提供了用于精确多路复用的解决方案， (fwhm 25-35 nm），通常小于现有染料光谱宽度的一半。通过化学修饰， 它们可以被调谐到从远红到近红外（NIR）光谱（700-900 nm）。此外，菌绿素具有共同的激发带，使得完全的 由单个UV光源激发的NIR染料的光谱。为了使菌绿素染料具有商业可行性， 第2B阶段的工作将集中在：1）改进和扩大菌绿素染料组合和合成 方法; 2）与NIRCNOSciences的合作者验证FC面板中的染料性能，以及3）开发 用于商业规模生产的程序、方法和方案。此2B期SBIR提案是 打算继续改进和转让菌绿素技术用于商业开发， 从而产生对多色FC具有高影响潜力的菌绿素染料组合。这种增强 多重能力不仅将促进基础免疫学研究，而且还将促进新疫苗的开发。 以及艾滋病毒、疟疾、结核病和新出现的传染病威胁的辅助发现。更大 多路复用对于分析减少体积的样品、对于单细胞研究，以及对于高通量， 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.