Displacement induced off-on fluorescent biosensor targeting IDO1 activity in live cells.

We show how the macrocyclic host cucurbit[8]uril (CB[8]) and a fluorescent dye form a biosensing ensemble while its cavity simultaneously traps tryptophan, the upstream substrate of IDO1 enzymes, therefore providing a label-free method to monitor the activity of IDO1 in real time. Incubation of malignant HeLa and HepG2 cells overexpressing IDOL with the associative biosensor resulted in its spontaneous uptake and a fluorescence switch-on response in situ, which can be traced to the displacement of tryptophan from CB[8] upon IDO1-catalyzed oxidation. The results, for the first time, establish a supra-molecular sensing concept for the detection of intracellular enzymatic activity in live cells, thus allowing direct cell-based analysis and inhibitor screening compatible with commercial instruments including microplate reader, fluorescent microscopy, and flow cytometry.