Activity-Based Fluorescent Molecular Logic Gate Probe for Dynamic Tracking of Mitophagy Induced by Oxidative Stress.

Visualizing and modulating the mitophagy process is essential for understanding the role of mitophagy in cellular homeostasis, physiology, and pathology. To overcome the sensing limitation of available mitophagy probes to only lysosome fusion or degradation, a molecular logic gate probe showing multiple fluorescence responses to different mitophagy stages was proposed in this study to sense the oxidative stress-induced mitophagy via a dual-channel mode. This new fluorescent molecular logic gate probe, Mito-PN, was composed by integrating a peroxynitrite-responsive 1,8-naphthalimide with an acidity-activatable rhodamine spirolactam and possesses the mitochondria-targeting capability due to its triphenylphosphonium group. This probe is able to sense both the mitophagy initiation triggered by peroxynitrite and lysosome fusion at different fluorescence wavelengths. It can be rapidly activated by mitochondrial peroxynitrite to turn on the green fluorescence of naphthalimide, and subsequent lysosome/mitophagosome fusion activates the probe with protons to generate red fluorescence. Moreover, our preliminary results demonstrate that the fluorescence response of Mito-PN to peroxynitrite-induced mitophagy can be discriminated from the mitophagy stimulated by carbonyl cyanide m-chlorophenyl hydrazone, which further proves the specific mitophagy tracking ability of Mito-PN. Overall, this research offers a potentially powerful tool for studying the role played by peroxynitrite in mitophagy and provides a versatile strategy for monitoring oxidative stress-related pathological processes.