A sensitive pH fluorescent probe with large Stokes shift and narrow transition for lysosome imaging during cell cycle

Intracellular pH plays a crucial role in regulating cellular functions, and its subtle changes can influence normal physiological activities and contribute to various diseases. Although numerous probes have been developed for detecting microenvironmental pH in living cells, their broad dynamic ranges induce blurred imaging boundaries and may generate false-positive results, compromising the accurate detection of subtle pH fluctuations. In response to these issues, we designed and synthesized a novel 1,8-naphthalic anhydride-based pH fluorescent probe (NAM) that demonstrated highly sensitive responses to pH changes with a narrow transition range spanning just 2.4 pH units. The pH responsiveness of NAM originated from the deprotonation of its aniline group, which induced an enhanced intramolecular charge transfer (ICT) process. This mechanism enables NAM to exhibit a remarkable 15-fold fluorescence enhancement at 569 nm with a pKa value of 6.71 and a significant Stokes shift of 112 nm. Furthermore, NAM specifically targets lysosomes and was successfully employed to detect intracellular pH fluctuations induced by lipopolysaccharide (LPS), nutrient deprivation and chloroquine treatment. Significantly, we demonstrate for the first time that NAM achieved pH imaging analysis across different cell cycle phases. During the transition from G0/G1 to S phase, intracellular pH gradually alkalinizes, followed by rapid acidification in the G2/M phase. These observations reflected the dynamic balance between energy storage and consumption during cell cycle progression. These distinctive features position NAM as a promising tool for investigating physiological and pathological processes associated with abnormal pH variations.