A lysosome-locating and acidic pH-activatable fluorescent probe for visualizing endogenous H₂O₂ in lysosomes

There is increasing evidence indicating that lysosomal H₂O₂ is closely related to autophagy and apoptotic pathways under both physiological and pathological conditions. Therefore, fluorescent probes that can be exploited to visualize H₂O₂ in lysosomes are potential tools for exploring diverse roles of H₂O₂ in cells. However, functional exploration of lysosomal H₂O₂ is limited by the lack of fluorescent probes capable of compatibly sensing H₂O₂ under weak acidic conditions (pH = 4.5) of lysosomes. Lower spatial resolution of the fluorescent visualization of lysosomal H₂O₂ might be caused by the interference of signals from cytosolic and mitochondrial H₂O₂, as well as the non-specific distribution of the probes in cells. In this work, we developed a lysosome-locating and acidic-pH-activatable fluorescent probe for the detection and visualization of H₂O₂ in lysosomes, which consists of a H₂O₂-responsive boronate unit, a lysosome-locating morpholine group, and a pH-activatable benzorhodol fluorophore. The response of the fluorescent probe to H₂O₂ is significantly more pronounced under acidic pH conditions than that under neutral pH conditions. Notably, the present probe enables the fluorescence sensing of endogenous lysosomal H₂O₂ in living cells without external stimulations, with signal interference from the cytoplasm and other intracellular organelles being negligible.