Understanding Bacteria-Dye interactions through Ultrafast Femtosecond Transient Absorption Spectroscopy

More than a quarter of human deaths worldwide are caused by infectious diseases many of which are caused by bacterial infection. Currently, it take up to 72 hours to definitively identify bacterial pathogens. Subsequently, patients are often needlessly treated with broad-spectrum antibiotics, which lead to the buildup of antibiotic resistance of pathogenic bacterial organisms. Using the kinetics of bacterial staining method developed in our lab, we provide a platform for expedient and robust assays for rapid detection and identification of bacteria. While we employ two fluorescent dyes (3,3’-diethylthiacyanine iodide (THIA) and Thioflavin T (ThT)) to collect their kinetics of fluorescence enhancement in presence of bacterial organisms, the mechanisms of dye-bacterial interactions remain unexplored. By employing a series of solvents varying in polarity and density, we conducted pump-probe ultrafast femtosecond transient absorption spectroscopy and lifetime measurements to characterize the physical-chemical properties of THIA and ThT. Preliminary studies showed that these fluorescent dyes exhibit: (1) varying transient peaks with respect to their wavelength and duration; and (2) contrasting lifetimes in the investigated solvents.  Furthermore, we utilized KD measurement to understand the dye uptake into both Gram positive and negative bacteria (Bacillus subtilis and Escherichia coli respectively) in the presence of dye. The results for the KD experiments showed two-fold vs 100-fold decrease in bacterial dye uptake for ThT and THIA respectively in the presence of fetal bovine serum (to mimic bacteremia conditions). The use of ultrafast spectroscopic techniques will provide the basis to facilitate the mechanistic understanding of bacteria-dye interactions.