Rapid and Reversible Reaction-Based Ratiometric Fluorescent Probe for Imaging of Different Glutathione Levels in Living Cells

Glutathione (GSH) serves many cellular functions, within the range of 1-10 mM in mammalian cells, abnormal levels of which have been related to many pathological processes. Until now, many researches have been devoted to qualitative sensing of intracellular GSH. However, it is still quite difficult to determine the dynamic GSH levels in living cells. To solve this problem, we designed and synthesized a ratiometric fluorescent probe RP-1 for the detection of different GSH levels based on a reversible Michael addition reaction. The Michael addition product RP-GSH was characterized by ESI-MS. The reversibility and kinetics of the reaction of RP-1 with GSH were studied by spectroscopic methods. According to the concentration dependence of the ratio F-405/F-488 (F-405 and F-488 are the fluorescence intensities at 505 and 571 nm when RP-1 in the presence of GSH was excited at 405 and 488 nm, respectively), GSH in the range of 0.1-50 mM could be detected by a very low concentration of 2.5 mu M RP-1 in the PBS buffer, indicating that the interference of RP-1 to the biological system in living cell imaging would be minimized. Simultaneously, the dissociation constant (K-d) was obtained as 4.89 mM, suggesting its capability to accurately quantify the GSH concentration in the range of 1-10 mM in cells. Moreover, to improve the cell permeability, we converted RP-1 with a carboxylic acid group into RP-ester with an acetoxymethyl ester group. It is noteworthy that the rapid cell uptake rate of the RP-ester made it very promising because the living cells were efficiently stained with high brightness after incubation with RP-ester for only 1 min. Furthermore, the successful ratio imaging (merged image of 405 channel and 488 channel) of different GSH levels by RP-ester in living cells demonstrated the further development and utilization in the future.