Deoxygenation Increases Photoluminescence Lifetime of Protein-Responsive Organic Probes with Triplet-Singlet Resonant Energy Transfer

Cells and bodily fluids possess strong nanosecond-lifetime autofluorescence, therefore photoluminescent probes with microsecond-scale luminescence decay time would be useful for analysis of biological samples, as they allow the performance of measurements in time-resolved (TR) format in a time gate (time window) where the nonspecific background fluorescence has ceased. We have previously disclosed binding responsive luminescent probes for protein kinases (PKs), ARC-Lum(Fluo) probes. High brightness of the probes is achieved through intramolecular Forster-type resonant energy transfer (FRET) from excited triplet state of a thiophene- or selenophene-comprising phosphor (D-3*) to singlet acceptor dye ((1)A) leading to amplified emission from the dye. Here, we determined quantum yields (QYs) and oxygen sensitivity of separate phosphorescent donor and fluorescent acceptor and compared these with those of the corresponding ARC-Lum(Fluo) probes both in nonbound and PK-bound states. The microsecond-scale luminescence of free and of PK-bound probes was quenched with different efficiency by molecular oxygen and the luminescence intensity of the probes was substantially increased upon deoxygenation. The brightness of an ARC-Lum(Fluo) probe in PK-bound state was more than 50-fold higher than that of the phosphorescent donor alone. The findings of the study can be used for the construction of bright long-lifetime organic tandem probes.