CONCLUSION

In summary, we reported a unimolecular dual-reporter probe, DHU-AKI-3, with crosstalk-free multiplexed output for precise and portable diagnosis of early AKI. DHU-AKI-3 showed a renal enrichment effect and could be specifically activated by ROS, thus ensuring high sensitivity for early AKI diagnosis. Its rapid response facilitates PA signal accumulation to a detectable threshold within a short time, allowing for effective long-term in situ monitoring. The ‘turn-on’ NIRF signal (MB) of DHU-AKI-3 after being stimulated by ROS reduced the interference of other reactive species. The integration of in situ and in vitro detection benefited from the distinct metabolic characteristics of crosstalk-free multiplexed signals. In particular, by directly linking the urine signal to the same activation event occurring in the injured kidney, this unimolecular dual-reporter probe established a physiological bridge between urine readout and renal pathology and provided an accurate and portable tool for the diagnosis of early-stage AKI, offering a practical route for point-of-care screening, bedside monitoring, and longitudinal follow-up in high-risk patients (e.g. ICU, transplantation, nephrotoxic chemotherapy). As a result, this work offers a versatile two-pronged strategy for rationally designing probes that not only allow for the long-term monitoring of diseases in situ and portable diagnosis in vitro but also make simultaneous visual readout of the AKI state possible, thus holding potential for further point-of-care testing.