Separation of a weak fluorescence signal of 3-hydroxyflavone using dynamic quenching

The spectral and time characteristics of the dual fluorescence of a 3-hydroxyflavone probe in a solution with a fluorescence quencher are studied in the temperature range of 20–80°C. At room temperature, the fluorescence consists of two bands that belong to the normal and tautomeric forms of the luminophore, while heating of the solution results in the appearance of an additional fluorescence band belonging to the anionic form of the luminophore. The additional band is strongly overlapped with the band of the normal form, and its intensity rapidly increases with temperature to exceed the intensity of the normal band. The introduction of the TEMPO spin quencher of excited states into the solution completely quenches the fluorescence of the anionic form, which allows one to record the pure dual fluorescence of 3-hydroxyflavone in the entire temperature range studied. The detection of probe signals in the pure form is important for applications of proton transfer molecular probes using the intensity ratio of the fluorescence bands as the main sensitive parameter.