A buffering fluorogenic probe for real-time lysosomal pH monitoring

Real-time monitoring of lysosomal pH is critical for understanding lysosome-related biological processes and diseases, but few lysosome probes can achieve this response effectively. Therefore, it is essential to develop pHsensitive probes with better performance. In this study, we designed and synthesized a lysosome-targeting "buffering fluorogenic probe" (Lyso-BFP) by introducing a salicylamide group to naphthalimide for buffering strategy and a hydroxyethyl piperazine unit to target lysosome as well as specific pH response. Lyso-BFP demonstrated excellent photostability, pH specificity, and responsiveness to lysosomal acidification in living cells, thanks to its well-suit pKa. Density functional theory calculation revealed that Lyso-BFP's excellent pH sensing performance resulted from the inhibition of the photo-induced electron transfer (PET) process from hydroxyethyl piperazine to the naphthalimide moiety after the piperazine ring's protonation at low pH. Remarkably, Lyso-BFP allowed for wash-free imaging and long-term real-time monitoring of lysosome pH changes based on its off-on fluorescence behavior and buffer strategy. With its superior pH selectivity and realtime pH monitoring capability, Lyso-BFP holds great potential in the diagnosis of lysosome-related disease, and the design concept of this fluorogenic probe provides a new perspective for real-time tracking of cell organelles.