Chromene-Derived Peroxynitrite Fluorescent Probes: From Environmental Water Monitoring to Oxidative Stress Assessment in Living Cells

Peroxynitrite (ONOO-) is a transient yet potent reactive nitrogen species that participates in both physiological regulation and pathological injury. However, its fleeting lifetime (approximately 10-20 ms) and high chemical reactivity make it notoriously difficult to detect in situ in complex biological and environmental matrices. In this work, we developed LK, an ICT-based probe for selective ONOO- detection, with fluorescence signal reaching equilibrium within less than 4 min. The probe features a D-π-A structure with naphthopyran as the electron donor and cyanobenzothiazole as the acceptor, linked by a C=C bridge. Oxidative cleavage of this C=C bond by ONOO- generates an aldehyde-bearing product, triggering ICT reorganization accompanied by an emission blue-shift from 530 to 505 nm LK showed excellent selectivity and anti-interference performance toward ONOO-, with a sub-4 min response and an LOD of 17.8 nM. The underlying recognition pathway was corroborated by density functional theory (DFT) calculations and 1H NMR analyses. Furthermore, the integration of LK with a smartphone-enabled RGB colorimetric readout afforded a laboratory-scale portable platform for visual semi-quantitative ONOO- monitoring. The proof-of-concept practicability of this method was validated through spiked-recovery tests using real-world water matrices under laboratory conditions. Cell experiments were also conducted, and the results showed that LK exhibits low cytotoxicity and good membrane permeability, enabling endpoint fluorescence imaging of endogenous and inflammation-induced ONOO- in HeLa cells.