Quenching of long lifetime emitting fluorophores with paramagnetic molecules.

In this work, we have studied quenching of the fluorescence of two well-known oxygen probes, 1-pyrene butyric acid (PBA) and tris(2,2'-bipyridine)ruthenium ([Ru(bpy)(3)](2+)) by reactive oxygen species (superoxide anion, nitric oxide derivative, hydrogen peroxide) and by the O(2) molecule. Both, time-resolved and steady state fluorescence measurements were performed in solution (ethanol, dimethyl sufoxide, water) and in micelles of Sodium Dodecyl Sulfate that serve as a model for membrane-containing biological structures. We have found that only the free radicals and O(2) can actively quench for the two probes, but not the diamagnetic H(2)O(2). Our data correspond to the classical Stern-Volmer equation. H(2)O(2) has an effect only at high molar concentrations (>0.1 M). In contrast, effective concentrations of free radicals and O(2) that lead to quenching are in millimolar range. In conclusion, our methods allows for detecting global ROS that are small free radicals without interference from the reactive hydroxyl radical. Our data suggest that the method can be used for the quantification of ROS in individual living cells based on the measurement of fluorescence lifetime of those probes.