A new multi-purpose FRET fluorescent probe for the simultaneous detection of proteases

A new methodology for the detection of three model proteases using a multi-purpose peptide probe equipped with three selectively cleavable sites and four fluorophores was developed and studied. The probe was designed as a single-excitation, triple-emission system, allowing for the monitoring of characteristic real-time changes in the fluorescence emission responses of individual fluorophores during enzymatic cleavage. It was labelled with diethylaminocoumarin (DEAC), fluorescein (FL) and Rhodamine B (RhB), forming the DEAC → FL → RhB FRET-cascade, with estimated Förster distances of 3.08 ± 0.02 nm or 3.02 ± 0.02 nm for the DEAC → FL FRET-pair (depending on the DEAC labeling site) and 6.04 ± 0.05 nm for the FL → RhB FRET-pair. Although spectroscopic analyses indicate that photophysical processes other than FRET are involved in the probe, the huge changes in its fluorescence intensities (<i>e.g.</i>, the fluorescence intensity of the probe for the excitation/emission wavelengths 421 nm/476 nm is 50-fold lower compared to that of the DEAC-only labeled construct, or it is 200-fold lower for 480 nm/521 nm compared to that of the FL-only labeled construct) enable the easy detection of protease activities. In a single-enzyme mode, trypsin, chymotrypsin, and thrombin can be determined according to the ratiometric graphical model at the lowest detectable concentrations of 0.0625 ng mL^-1, 0.125 μg mL^-1, and 0.0125 U mL^-1, respectively. The prepared probe, in combination with selective inhibitors (Kunitz, trypsin inhibitor; chymostatin, chymotrypsin inhibitor; and dabigatran, thrombin inhibitor), was also successfully used for the simultaneous detection of individual proteases in their two-enzyme mixtures. Finally, the probe was studied as a tool for three-protease screening using two selected inhibitors. Unfortunately, a universal model capable of unambiguous confirmation of the three studied enzymes in various combinations of their quantities has not been found.