A dual-emission Off-On fluorescent probe with triple reaction sites for the selective detection of peroxynitrite

Peroxynitrite (ONOO⁻) is a crucial physiological and pathological mediator, yet its highly selective detection in complex biological environments remains a demanding analytical task. To address this, we developed DE-P, a triple-reaction-based, dual-emission "Off-On" fluorescent probe designed to achieve high selectivity by integrating naphthalimide and hemicyanine scaffolds. DE-P is initially non-fluorescent. However, upon interaction with ONOO⁻, it undergoes a specific reaction cascade involving three transformations: the cleavage of the trifluoromethanesulfonate group, the oxidative decomposition of the hemicyanine core, and the oxidation of the thiomorpholine moiety. Consequently, distinct dual-emission signals are triggered at 467 nm and 567 nm, providing a broad spectral gap of approximately 100 nm. This triple-reactive design enables sensitive trace-level detection (limits of detection: 0.12 and 0.19 µM) and fluorescence across a wide pH range (3-10), providing high selectivity for ONOO⁻ over competing reactive species (ROS/RNS) and common biological interferents. Elucidated through comprehensive spectroscopic and mass spectrometric analyses, the sensing mechanism of DE-P establishes it as a promising chemical tool for the reliable tracking of ONOO⁻ in biological and environmental samples.