Abstract
Mitochondria, powerhouses of cells, possess a weakly alkaline environment. Various stress stimulations may lead to mitophagy, which further gives a rise to mitochondrial acidification and disfunction. Therefore, monitoring mitochondrial pH alterations is of great importance to better elucidate their role in the cellular metabolism. Toward this end, a number of mitochondrial fluorescent pH probes have been proposed, but most of them are based on electrostatic attraction and readily leak out from the mitochondria during mitophagy with decreased membrane potential, thus failing to accurately measure the pH changes. In this work, we report a mitochondria-immobilized ratiometric fluorescent pH probe, which allows the quantitative measurements of mitochondrial pH. The probe was designed and prepared by introducing a reactive benzyl chloride into a positively charged near-infrared hydroxyl-hemicyanine. The cationic property facilitates the probe to be quickly enriched into mitochondria, the hydroxyl group is responsible for producing a reversible ratiometric fluorescence signal, and benzyl chloride is used to react with nucleophiles for immobilizing the probe in mitochondria. Taking these advantages of the probe, the mitochondrial pH variations during mitophagy caused by rapamycin and hypoxia have been determined quantitatively for the first time. The observed severe acidification of mitochondria under these stimulations, together with the rationally designed probe, may be useful for studying the detailed function of mitochondria in some bioprocesses.