The developed multifunctional fluorescent probe enables the simultaneous detection of chymotrypsin as a model protease and hydrogen peroxide as a representative of reactive oxygen species (ROS) in biologically relevant concentration ranges. The chymotrypsin sensing is based on the cleavage of its selectively recognizable peptide sequence and the consequent disruption of FRET between coumarin (DEAC) and fluorescein (FL). Analogously, the presence of hydrogen peroxide causes the gradual degradation of the H2O2-labile benzopyrylium-coumarin (BC) dye. Considering the fluorescence emission responses of individual chymotrypsin-peroxide probe-attached fluorophores after their excitation at 425 nm, the sole presence of either chymotrypsin (50-1000 ng/mL) or hydrogen peroxide (10-200 μM) in a sample could be unambiguously confirmed or refuted. In addition, reliable simultaneous detection and approximate quantification of both studied species in the concentration ranges of 100-1000 ng/mL and 20-200 μM for chymotrypsin and H2O2, respectively, could be performed as well. The obtained results are summarized and visualized in the graphical models.

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