On the Route to Quantitative Detection and Real-Time Monitoring of Glutathione in Living Cells by Reversible Fluorescent Probes

In the last few decades, growing numbers of fluorescent probes have been developed to detect intracellular GSH. However, the majority of probes for GSH were irreversible without monitoring the changes of intracellular GSH concentration. Therefore, recently, fluorescent probes for monitoring concentrations of GSH in real-time in living cells have come into being to address this challenge. This Perspective aimed at the development of reversible probes for GSH was organized by structural features, chemical reactions, and physicochemical properties. The reversible probes designed by a coumarin skeleton as a read-out fluorophore and the Michael addition reaction as a response mechanism accounted for most of the reported reversible probes. The performances of reversible fluorescent probes based on Michael addition could be roughly predicted by fundamental laws of kinetics and thermodynamics in physical chemistry. Essentially, the design principles included a highly reactive site for GSH, a small thermodynamic driving force, a desirable Kd of 1–10 mM, and excellent cell membrane permeability. Prospectively, the development of various mechanisms and fluorophores will be effective measures to enrich the types of reversible probes for GSH.