Anionic Polyelectrolyte-Induced Aggregation of Basic Orange 21: A Clue toward Metachromasia.

Abstract

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.