Dual-channel fluorescent probes enable synchronous tracking of peroxynitrite and cysteine in mitochondrial redox dynamics.

Peroxynitrite (ONOO-) and cysteine (Cys) are pivotal components of reactive nitrogen and sulfur species that together shape a complex redox regulatory network in living systems. High-resolution, real-time imaging of these species is essential for unraveling their roles in mitochondrial redox dynamics. However, the development of fluorescent probes capable of simultaneously detecting ONOO- and Cys with high fidelity remains limited. In this work, we introduce a single-excitation, dual-response fluorescent probe system (probes A and B) that enables synchronous visualization of ONOO- and Cys. The probe design integrates two orthogonal sensing chemistries: ONOO- is selectively recognized via the oxidation of a naphthylimide-borate unit, while Cys is detected through nucleophilic substitution with a benzothiol-modified tetrahydro-acridine conjugate salt. Incorporation of a long-chain linker effectively suppresses undesired FRET, and the large Stokes shift of the Cys-responsive unit ensures clean spectral separation. As a result, probe B exhibits strong ONOO- emission at 550 nm and distinct Cys emission at 652 nm under a single 460 nm excitation source, with both fluorescence channels operating independently and without spectral crosstalk. Probe B demonstrates high sensitivity, excellent anti-interference capability, and precise mitochondrial localization. Dual-channel imaging in living cells confirms its ability to simultaneously monitor endogenous ONOO- and Cys. Importantly, the probe enables real-time visualization of dynamic redox fluctuations during inflammatory responses and tumor ferroptosis. Overall, this work presents a robust dual-channel fluorescent imaging platform for the simultaneous detection of ONOO- and Cys, offering a powerful tool for dissecting mitochondrial redox dynamics in health and disease.