A mitochondrial suitable ratiometric fluorescent probe for tracking pH change during mitophagy

Intracellular pH is one of the important parameters of cellular metabolism. The function of mitochondria is highly dependent on pH. Developing ratiometric fluorescent probes with suitable alkaline pK_a for the detection of mitochondrial pH is urgently demanded. Herein, a mitochondria-targeted ratiometric pH fluorescent probe SNRF was designed based on 1,6-dihydroxynaphthalene and 4-diethylaminoketo acid. SNRF contains a hydroxyl electron donor and a nitrogen positron acceptor with excellent molecular charge transfer (ICT) performance. The hydroxyl group is a pH-sensitive group, which is prone to be protonated under acidic conditions and deprotonated under alkaline conditions. The ratiometric performance of SNRF can avoid the interference from instrumental factors, concentration of probe and microenvironment around the probe. SNRF exhibits a suitable pK_a (8.30) with a linear variation between pH 7.4–9.0, making it ideal for monitoring mitochondrial pH changes. And, SNRF can be used to detect pH changes in HeLa cells and zebrafish without interference from other biologically active molecules. Furthermore, SNRF showed excellent mitochondrial targeting and was successfully used to track pH changes during rapamycin-induced mitophagy.