UM-DAE Centre for Excellence in Basic Sciences, University of Mumbai, Kalina, Santacruz (E), Mumbai 400098, India.
Amity Institute of Biotechnology, Amity University, Mumbai-Pune Expressway, Bhatan, Panvel, Mumbai 410206, India.
J Phys Chem B. 2021 Jul 1;125(25):7033-7043. doi: 10.1021/acs.jpcb.1c02127. Epub 2021 Jun 17.
The change in the color of chromophore upon being embedded in a biological tissue is known as metachromasia. Basic Orange 21 (BO21) is a cationic polymethine dye that has been implied as a supravital dye, which produces metachromasia in leukocytes. An improved differential counting of leukocytes has been achieved in the clinical setup based on characteristic metachromatic expressions of BO21 for different types of leukocytes. Although BO21 has been utilized as a chromatic indicator for leukocyte counting, there are limited number of investigations that focus on the factors that may be responsible for the spectral shift in absorption and emission spectra of BO21, which leads to its metachromatic behavior. In this work, we have investigated the effect of a synthetic anionic polyelectrolyte, polystyrene sulfonate (PSS), on the photophysical properties of BO21, using steady-state emission, ground-state absorption, and time-resolved emission measurements, to get an understanding of the factors that may be responsible for the spectral shift of BO21 in the cellular environment. PSS induces aggregation of BO21 molecules with large changes in its photophysical properties; this appears to be most likely the mechanism of spectral shift for BO21 reported in the cellular environment. The employment of external stimulus reveals BO21 aggregates to be significantly responsive toward external stimuli, for example, temperature and presence of salt in the medium, which further strengthens the proposal of aggregate formation. Further, we have also employed fluorescence upconversion spectroscopy with subpicosecond time resolution to estimate the excited-state lifetime of BO21.
发色团在嵌入生物组织后颜色的变化称为变色。碱性橙 21（BO21）是一种阳离子多甲川染料，它被暗示为超活染料，可使白细胞产生变色。基于 BO21 对不同类型白细胞的特征变色表达，在临床环境中已经实现了白细胞的改进差异计数。尽管 BO21 已被用作白细胞计数的显色指示剂，但只有少数研究关注可能导致 BO21 的吸收和发射光谱发生光谱位移的因素，从而导致其变色行为。在这项工作中，我们使用稳态发射、基态吸收和时间分辨发射测量研究了合成阴离子聚电解质聚苯乙烯磺酸盐（PSS）对 BO21 光物理性质的影响，以了解可能导致 BO21 在细胞环境中光谱位移的因素。PSS 诱导 BO21 分子的聚集，其光物理性质发生了很大的变化；这似乎是细胞环境中报道的 BO21 光谱位移的最可能机制。外部刺激的应用表明，BO21 聚集体对外部刺激（例如温度和介质中盐的存在）非常敏感，这进一步加强了聚集体形成的建议。此外，我们还使用具有皮秒时间分辨率的上转换荧光光谱法来估计 BO21 的激发态寿命。
