Considering the limited sensitivity and accuracy of single-signal assay strategies, the multi-signal assay strategy has sparked significant excitement in recent years. In this study, for the first time, we reported a one-pot method in situ synthesis of carbon-containing nanoparticles (CNPs) via p-aminophenol (AP) and diethylenetriamine (DETA). The CNP solution exhibits yellow and light blue fluorescence under UV-light. Moreover, the CNPs exhibited excellent photoluminescence stability even under extreme conditions. Inspired by the alkaline phosphatase (ALP)-triggered specific catalytic reaction, we constructed an ultrasensitive fluorescence and colorimetric two-channel strategy for monitoring the ALP activity. By optimizing the detection parameters, the detection limits for both fluorometric and colorimetric were 0.05 mU mL. Moreover, the strategy showed high specificity and was successfully applied to monitor the ALP activity level in human serum samples. The analytical strategy opened a new window for the detection of the ALP activity, screening of the ALP inhibitor, and disease diagnosis.
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