Acute liver failure (ALF) is a rapidly progressing disease characterized by high mortality rates and a lack of effective real-time diagnostic tools. Peroxynitrite (ONOO-), generated via a diffusion-controlled reaction between nitric oxide (NO) and superoxide anion (O2.-), induces lipid peroxidation and protein nitration, and is closely implicated in the onset and progression of ALF. Here, a COX-2-targeted near-infrared fluorogenic (NIRF) probe, MB-COX, was reported. MB-COX was fabricated by coupling the near-infrared chromophore methylene blue (MB) with the COX-2 inhibitor indomethacin (INM). Owing to disruption of the π-conjugated structure of MB within the molecule, MB-COX exhibited negligible background fluorescence, which was rapidly restored in the presence of ONOO-. MB-COX featured an ultralow detection limit, excellent selectivity, and favorable responsiveness. Using MB-COX, the upregulation of ONOO- levels in LPS/D-GalN-induced ALF cell models was successfully observed, a finding highly consistent with the activation of the NF-κB signaling pathway and the upregulation of iNOS, IL-6, and COX-2. Furthermore, in an ALF mouse model, MB-COX enabled precise real-time visualization of liver ONOO- dynamics and facilitated evaluation of the dose-dependent hepatoprotective effects of the natural flavonoid dihydroquercetin (DHQ). Collectively, this study established ONOO- as a potential biomarker for ALF and provided a real-time, quantitative chemical tool for elucidating pathological mechanisms, enabling precise diagnosis, and evaluating the efficacy of natural medicinal compounds.
