A water-soluble fluorescent pH probe and its application for monitoring lysosomal pH changes in living cells

Intracellular pH plays a crucial role in many cellular processes, and abnormal intracellular pH has been linked to common diseases such as cancer and Alzheimer's. To address this issue, a water-soluble fluorescent pH probe was designed based on the protonation/deprotonation of the 4-methylpiperazin-1-yl group, using dicyanoisophorone as the fluorophore. In the neutral form of the probe, fluorescence is quenched due to charge transfer from the 4-methylpiperazin-1-yl group to the fluorophore upon excitation. Under acidic conditions, protonation of the 4-methylpiperazin-1-yl group inhibits the photoinduced electron transfer process, leading to an increase in fluorescence intensity. Density-functional theory calculations also verified the fluorescence OFF-ON mechanism. The probe exhibits high selectivity, photostability, fast response to pH changes, and low cytotoxicity to cells. Additionally, the probe selectively accumulates in lysosomes, with a high Pearson coefficient (0.95) using LysoTracker Green DND-26 as a reference. Notably, the probe can monitor lysosomal pH changes in living cells and track pH changes stimulated by chloroquine. We anticipate that the probe has potential for diagnosing pH-related diseases.