Singlet Oxygen Reacts with 2′,7′‐Dichlorodihydrofluorescein and Contributes to the Formation of 2′,7′‐Dichlorofluorescein

Abstract There are controversial reports in the literature concerning the reactivity of singlet oxygen ( 1 O 2 ) with the redox probe 2′,7′‐dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which 1 O 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 D 2 O and decreases in sodium azide, proving the direct role of 1 O 2 in this process. We have also prepared solutions in which either 1 O 2 or dication (MB •2+ ) and semi‐reduced (MB • ) radicals of the sensitizer and subsequently super‐oxide radical (O 2 • − ) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O 2 •− , indicating that both 1 O 2 and MB • 2+ react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB • 2+ than in the presence of 1 O 2 , considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of 1 O 2 with DCFH is actually larger than that of MB • 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 1 O 2 .