A dual-cascade-activatable molecular probe with microenvironment-adapted performance for accurate differentiation of hepatopathy

Fluorescence imaging utilizing biomarker-activatable fluorescent probes has emerged as a powerful tool for the precise and early diagnosis of hepatopathy. However, the development of effective molecular probes remains challenging due to limitations, such as single-stimulus responsiveness and incompatible with microenvironment characteristic of hepatopathy. These limitations often result in a lower signal-to-noise ratio, false positives and ultimately reduced diagnostic accuracy. In this study, we developed a novel dual-lock-controlled fluorescent probe (H_dual) based on basic blue 3 dye. This probe was designed to be sequentially activated by two potential hepatopathy biomarkers, leucine aminopeptidase (LAP) and monoamine oxidase (MAO), through a cascade mechanism. Moreover, after addition LAP and MAO, H_dual exhibited a linear fluorescence change within a pH range of 6.2–6.8, ensuring high compatibility with the weakly acidic microenvironment characteristic of hepatopathy. The dual-cascade-activatable design, combined with the probe's microenvironment-adapted property, enabled H_dual to achieve a significantly higher target-to-noise ratio (T/N) of 2.40 in in vivo imaging for drug-induced liver injury, compared to “single-locked” probe (T/N < 0.79). Notably, H_dual demonstrated the ability to differentiate between cirrhotic and hepatitis B samples by analyzing patient blood samples through both fluorescent imaging and a distinct colorimetric change, observable either visually or via smartphone-based color analysis. These findings highlight H_dual's high specificity and accuracy in fluorescence imaging-based detection, underscoring its potential to improve the early diagnosis of hepatopathy.