In Vivo Monitoring of Hepatic Ischemia Using a Dual-Enzyme Activated Near-Infrared Fluorescence Probe

Hepatic ischemia-reperfusion injury (HIRI) is a major complications associated with liver transplantation, contributing to graft rejection and liver dysfunction. Early detection of ischemic damage prior to reperfusion is crucial for improving clinical outcomes. While previous studies have primarily focused on detecting reactive oxygen species (ROS) generated during IRI, specific detection of ischemic injury itself, characterized by hypoxia, remains underexplored. Here, we developed QN-NIR, a near-infrared (NIR) fluorescent probe designed for dual activation by two hypoxia-associated reductases, human NAD(P)H:quinone oxidoreductase 1 (hNQO1) and nitroreductases (NTRs). Both enzymes are upregulated under hypoxic conditions, and their activation of QN-NIR triggers a strong Off–On NIR fluorescence response at 708 nm. This dual-enzyme strategy offers high signal-to-noise (s/n) contrast with minimal false-positive signals under normoxic conditions. QN-NIR enables precise, real-time visualization of hepatic ischemia prior to reperfusion in vivo, thereby supporting the potential of hypoxia as a key and directly targetable feature of ischemic injury. While further studies are needed to fully delineate its clinical applicability, this work highlights QN-NIR as a promising platform for early, noninvasive diagnosis of hepatic ischemia and for distinguishing it from reperfusion-related oxidative stress.