Unimolecular near-infrared chemiluminescent reporter for cascaded multiplex imaging of ischemia-reperfusion injury in the liver-kidney axis

Acute kidney injury (AKI) frequently arises as a complication of hepatic ischemia-reperfusion injury (HIRI), yet simultaneous optical imaging of both remains challenging due to the lack of unimolecular dual-responsive probes. Herein, we report a library of hemicyanine-based chemiluminophores (HCLs) with tunable emission to second near-infrared window (725 - 1025 nm) achieved by integrating bicyclic dioxetane onto hemicyanine skeletons to develop multiple-responsive chemiluminescent probes. HCL1 and HCL5 respectively emitting at 725 nm and 1025 nm are selected to construct a cascaded activatable reporter CAR for crosstalk-free duplex chemiluminescence imaging of interlinked biomarkers. Following systemic injection to male mice, CAR preferentially accumulates in the liver and reports HIRI-associated superoxide anion (O₂^•-), which initiates self-fragmentation and liberates the secondary reporter KIR into kidneys to report AKI-associated N-acetyl-β-D-glucosaminidase (NAG). Such mechanism allows CAR to serve as a reservoir for gradual release of AKI reporters, providing a significantly prolonged imaging window compared to co-administering separate probes. CAR further permits remote detection of HIRI-induced AKI via urinalysis. This study not only offers a powerful tool for simultaneous detection of HIRI and HIRI-induced AKI, but also highlights a unimolecular probe design for ultrasensitive detection of deeply-seated intercorrelated diseases.