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 pH-sensitive 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 real-time 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.