A sequentially activatable fluorescent probe for discriminating drug-resistant cancer cells through azoreductase and viscosity integration

Multidrug resistance (MDR) involves complex biochemical and biophysical changes in cancer cells, whereas most fluorescent probes respond to a single intracellular feature. Herein, we developed IN-AZO , a sequentially activatable fluorescent probe integrating reductive activation with viscosity-mediated fluorescence amplification for MDR phenotyping. Reductive cleavage of the azo linkage converts weakly emissive IN-AZO into the molecular-rotor fluorophore IN-NH 2 , whose fluorescence is enhanced by viscosity-dependent restriction of intramolecular rotation. IN-NH 2 exhibited a good Förster–Hoffmann relationship, and HPLC-MS confirmed the reductive conversion of IN-AZO to IN-NH 2 . In A549 cells with graded doxorubicin resistance, IN-AZO fluorescence increased progressively with resistance and showed greater discrimination than single-responsive probes. Moreover, its fluorescence decreased after tariquidar-mediated resistance reversal. These results demonstrate the feasibility of a sequential chemical–physical sensing strategy for fluorescence phenotyping of MDR-associated cellular states.