Selective lysosomal H2O2–ROS imaging with a naphthalimide probe forming hydroxylamine without overoxidation

Abstract

Hydrogen peroxide (H₂O₂) plays a central role in oxidative stress, signaling, and pathophysiology, yet its highly dynamic and compartmentalized distribution in living cells makes precise monitoring a major analytical challenge. Here, we present LysoH₂O₂, a naphthalimide-derived fluorescent probe specifically designed for lysosomal H₂O₂ detection and imaging. The probe exploits a selective transformation of an amine group into a hydroxylamine moiety upon reaction with H₂O₂, leading to a pronounced fluorescence enhancement. This molecular design integrates a lysosome-targeting unit to ensure subcellular specificity, enabling real-time visualization of H₂O₂ fluctuations in the lysosomal microenvironment. Confocal fluorescence microscopy demonstrates the ability of LysoH₂O₂ to track endogenous and exogenous H₂O₂ with high sensitivity and minimal cytotoxicity in living cells. Distinct from conventional boronate-based probes, LysoH₂O₂ employs a novel, biocompatible chemical transformation to achieve sensitive and selective monitoring of H₂O₂ within lysosomes.