Single-Molecule Dual-Channel NIR Fluorescence Probe for Simultaneous Monitoring ATP and ONOO – during Ferroptosis and Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion injury (HIRI) is a common and severe complication following liver transplantation and resection. Its core pathological mechanisms involve oxidative stress and disrupted energy metabolism, with ATP depletion and peroxynitrite (ONOO – ) accumulation being key biomarkers. In this study, we designed a fluorescent probe, ATP-ON, capable of dual real-time monitoring of ATP and ONOO – in vivo. The probe exhibits high sensitivity for detecting ATP and ONOO – (detection limit for ONOO – is 88 nM, for ATP is 0.83 μM), rapid response, high selectivity, and no spectral crosstalk. In vivo experiments showed that ATP-ON is capable of imaging both endogenous and exogenous ATP and ONOO –, with the probe predominantly localized in lysosomes. It successfully enabled real-time, in situ bioimaging of ATP and ONOO – during ferroptosis in lysosomes. By establishing HIRI cell and animal models, ATP-ON successfully assessed the levels of ONOO – and ATP depletion during HIRI, and was able to monitor the efficacy of therapeutic interventions such as N-acetylcysteine. This study provides a solid technical foundation for the use of ATP-ON in the dynamic monitoring of HIRI and other oxidative stress-related diseases.