Despite the fact that cytochrome P450 2C19 (CYP2C19) is an essential xenobiotic-metabolizing enzyme in many drug metabolisms, the real-time and dynamic monitoring of its activity remains challenging due to the lack of highly specific activity-based probes with translational applicability. Herein, we report a library of O-alkylated hemicyanine derivatives and screen the optimal probe (CyB) with favorable sensitivity and specificity toward CYP2C19. CyB was subsequently selected to construct a dual-responsive nanoprobe (CyBPPD) that could be activated by hepatic CYP2C19 and glutathione, triggering nanostructure disassembly into renal clearance fragments that can be excreted in urine. The cascaded activation thus turned on near-infrared fluorescence (NIRF) for in vivo imaging and in vitro point-of-care urinalysis. CyBPPD was successfully proven to have ideal selectivity and sensitivity in the real-time detection of CYP2C19 in Cyp2c19-knockdown living cells and inhibitor-treated mice. In age-related decrement models, CyBPPD-based urinalysis enables discrimination of the changes in expression and function of hepatic CYP2C19 in different age rats, ranging from suckling to adult and aged, providing a level of sensitivity comparable to that of immunoblotting-based expression analysis. In summary, isoform-specific optical nanoprobes have been developed to measure CYP2C19 activity in diverse biological systems, providing a convenient and effective approach to characterizing CYP2C19-related individual differences, drug–drug interactions, and modulatory effects, with an added advantage of highly sensitive urinalysis.
