A Ratiometric Fluorescent Probe with Reversible Redox-Response for Monitoring Dynamic Release of H2S Donors

It is of great importance to monitor H2S levels dynamically over an extended period in living systems. Despite the numerous H2S fluorescent probes that have been reported for biological imaging, only a few are reversible and capable of dynamic tracking. Given the potential impact of extraneous factors on fluorescence intensity, self-calibrating ratiometric probes are the preferred choice. In this study, we present a ratiometric probe named DaCou, which is based on the FRET mechanism. This probe can selectively and reversibly respond to H2S, with the maximal emission shifting between 595 and 476 nm. The FRET process transitions between on and off states during the absence and presence cycles of HS-. Additionally, the absorption band centered at 482 nm undergoes a corresponding cyclic decrement and increment. Mass spectrometry and nuclear magnetic resonance analysis confirmed the hydrogenation reduction of the energy acceptor component in DaCou by HS-. The reduced product, H2DaCou, can be reoxidized to DaCou by O2. The ratiometric and reversible imaging of HS- concentration changes has been successfully achieved in both HeLa cells and zebrafish embryos. Furthermore, DaCou was applied to visualize the release kinetics of slow-releasing H2S donors within cellular environments. Leveraging the advantages of ratiometric signaling, a comparable release kinetics was observed for SFN and GYY4137 at their respective concentrations that inhibit HeLa cells by 50%. This work provides a viable tool for the accurate and dynamic monitoring of H2S levels within living cells, which can assist in predicting the anticancer efficacy of H2S donors.