A multi-responsive fluorescent probe for simultaneous detection of mitochondrial esterase and viscosity

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide, with early detection severely hampered by its asymptomatic progression. Carboxylesterases (CEs) are significantly upregulated in HCC cells, making them promising biomarkers for cancer diagnosis. Herein, we report a tri-lock fluorescent probe, CMA, capable of simultaneous detection of mitochondrial CE - an enzyme closely linked to metabolism and drug resistance - and viscosity for early cancer diagnosis. The probe incorporates a CE-cleavable benzoate ester and a molecular rotor formed by an indolium-coumarin conjugated system. Upon hydrolysis of the benzoate ester, the released product displays enhanced fluorescence with red-shifted absorption through strengthened ICT. High viscosity restricts intramolecular rotation and suppresses the TICT pathway, whereas the alkaline mitochondrial pH further modulates the emission via deprotonation of the phenolic hydroxyl group. These three orthogonal stimuli converge to produce a 654-fold fluorescence enhancement under coexistent conditions. Cell imaging confirms that CMA effectively differentiates HCC cells from normal cells and detects drug-induced viscosity changes, suggesting its potential for early-stage liver cancer diagnosis.