Singlet oxygen reacts with 2′,7′-dichlorodihydrofluorescein and contributes to the formation of 2′,7′-dichlorofluorescein

There are controversial reports in the literature concerning the reactivity of singlet oxygen (O-1(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which O-1(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D2O and decreases in sodium azide, proving the direct role of O-1(2) in this process. We have also prepared solutions in which either O-1(2) or dication (MB center dot 2+) and semi-reduced (MB center dot) radicals of the sensitizer and subsequently super-oxide radical (O-2(center dot-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O-2(center dot-), indicating that both O-1(2) and MB center dot 2+ react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB center dot 2+ than in the presence of O-1(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of O-1(2) with DCFH is actually larger than that of MB center dot 2+. We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and O-1(2).