A sensitive fluorescent probe (E)-4-(3-(benzo[d]thiazol-2-yl)-4-hydroxy-5-methylstyryl)-1-methylpyridin-1-ium iodide (HBTMP) for the monitoring of pH in mitochondria was rationally exploited. This novel probe exhibited remarkable pH-dependent behavior in the linear range of 5.5–8.0, along with a pKa value of 6.829 ± 0.02627. A large Stokes shift of 205 nm was obtained. This fluorescent probe demonstrated good biocompatibility and high sensitivity for detecting the dynamic changes in mitochondrial pH in living cells and zebrafish. The results of the CCCP (m-chlorophenyl hydrazone) treatment experiment indicated that the probe can effectively monitor changes in mitochondrial pH caused by cell damage.
Guli Li,Bei Zhang,Xinbo Song,Ying Xia,Haibo Yu,Xinfu Zhang,Yi Xiao,Youtao Song
Mitochondrial pH plays a pivotal role in the regulation of physiological process. Developing ratiometric fluorescent probes for real-time detection of mitochondrial pH fluctuation is still highly demanded yet challenging. Herein, we present a novel strategy to design a ratiometric probe (FDI) by broadening absorption spectrum (360–700 nm) of fluorescein fluorophore. Unsaturated dialkene/indole quaternary ammonium moiety has broadened absorption and emission spectrum of fluorescein fluorophore to provide ratiometric detection by CLSM in dual excitation/dual emission mode, as well as directionally accumulated in mitochondria in living cells. Superior to commercially mitochondrial tracker, Carboxy-SNARF, probe FDI exhibits excellent and fast cell-membrane permeability and mitochondrial-targetability. It has been demonstrated that FDI could permit real-time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Owing to nondestructive process and reversible ratiometric response to pH, image acquisition can be repeated frequently to trace and monitor the time course of mitochondrial pH responses. It is clearly confirmed that FDI would be a promising probe for real-time tracking of mitochondrial pH changes in the biomedical and biological fields.
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.
Bo Lin,Li Fan,Jinyin Ge,Wenjia Zhang,Caihong Zhang,Chuan Dong,Shaomin Shuang
A mitochondrial-targeted pH fluorescent probe 4-(2-(6-hydroxynaphthalen-2-yl)vinyl)-1-methylpyridin-1-ium was facilely synthesized via the carbon–carbon double bond bridging of 6-hydroxy-2-naphthaldehyde and 1,4-dimethylpyridinium iodide salt. The probe exhibited remarkable pH-dependent behavior in the linear range of 7.60–10.00, with a pKa value of 8.85 ± 0.04 near mitochondrial pH. A significantly large Stokes shift of 196 nm was obtained, which reduces the interference of excitation light. Application of the probe in live HepG2 cells indicated that the probe had excellent mitochondrial targeting ability and was successfully used to visualize mitochondrial pH fluctuations in live cells.
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