Mitochondria-Localized Three-Color Fluorescent Sensor for Concurrent Tracking of Mitochondrial H2O2, Hypochlorite and Subcellular Viscosity during Oxidative Stress and Drug-Related Hepatic Damage

Abstract We report a naphthalimide derived mitochondrial targeting fluorescent probe MT for real time simultaneous visualization of mitochondrial hydrogen peroxide (H2O2), hypochlorite (ClO−), and subcellular microviscosity, three key biomarkers closely associated with oxidative stress mediated drug induced liver injury. Free MT shows negligible background fluorescence under physiological conditions, and each biomarker triggers well-separated emission bands: red emission at 680 nm for elevated viscosity, orange emission at 600 nm for H2O2, and green emission at 500 nm for ClO−. MT exhibits low detection limits of 123.24 nM for H2O2 and 6.34 nM for ClO−, enabling spectral crosstalk free triple channel readouts. We validated MT performance in ferroptosis and drug damaged hepatocyte cell models for in situ triple biomarker imaging. In vivo fluorescence imaging dynamically monitored acetaminophen provoked acute liver injury in mice; full three channel signals were only acquired from ex vivo liver slices due to tissue penetration constraints. This sensitive multichannel probe provides a practical tool to dissect liver pathological progressions and quantitatively evaluate hepatoprotective agents in preclinical liver injury models.