Hydroxyl and phenyl co-modified carbon nitride-based ratiometric fluorescent nanoprobe for monitoring mitochondrial pH in live cells and differentiating cell death

Monitoring mitochondrial pH and differentiating live and dead cells are crucial for diagnosing cell status. However, most fluorescent probes suffer from limitations such as high cytotoxicity, photobleaching, unreliability, and an inability to differentiate cell death caused by different inducers. Herein, a ratiometric fluorescent nanoprobe was developed by assembling pH-sensitive hydroxyl- and phenyl-co-modified carbon nitride (HPCN) with pH-insensitive Rhodamine B (RB). HPCN was prepared via thermal condensation of phenylguanidine carbonate using NaOH as the melt. The hydroxyl group modification endowed HPCN with improved water solubility and pH-sensitive characteristics, while the phenyl group modification facilitated mitochondrial targeting and DNA staining via hydrophobic interactions. Based on the fluorescence resonance energy transfer (FRET) from HPCN to RB, the nanoprobe exhibited a linear response in the relative fluorescence intensities at 500 nm and 584 nm over a pH range of 4.5 ∼ 8.5. Benefiting from its low cytotoxicity, excellent reversibility, and outstanding photostability, the nanoprobe was capable of monitoring mitochondrial pH changes in live cells and differentiating live and dead cells, apoptosis and necrosis, and necrosis induced by different agents, regardless of cell type. This work provides a reliable method for diagnosing cell status and cell death induced by various inducers.