DCFH₂-DA (2´, 7´-dichlorofluorescin diacetate) is a classical indicator of reactive oxygen species (ROS) formation in vitro [1] and it has been extensively used to monitor oxidation in biological systems. When DCFH₂-DA  is oxidized, it forms a highly fluorescent (l_max = 522 nm) product DCF (2´, 7´-dichlorofluorescein). However, the use of this probe to detect ¹O₂ has been questioned [2]. We aim to clarify the reaction of ¹O₂ with DCFH₂-DA . Methylene blue (MB) is a photosensitizer that releases ROS when irradiated, including ¹O₂ and radicals [3]. In aqueous solution, MB molecules are in monomer/dimer equilibrium. In the presence of sodium dodecyl sulphate (SDS),  MB may be   monomer  (SDS>20mM), which  forms  ¹O₂, or dimer  (SDS<3 mM),   which   forms O₂^·- . In this work, we measured the ROS released by MB in vitro with DCFH₂-DA /DCF fluorescent (solutions of H₂O and D₂O) and ¹O₂ phosphorescent (aqueous in SDS) assays. The solutions were irradiated under visible diode laser light (664 nm, 50 mW) in stirred and air-equilibrated cuvettes, and the fluorescent kinetics were measured. ¹O₂ phosphorescence at 1270 nm was measured with NIR spectrometer by MB excitation.
        The kinetics of DCF formation (at 522 nm) was 3.6 times faster in D₂O than in H₂O. ¹O₂ lifetime is ~7 times longer in D₂O than in H₂O. The ¹O₂ phosphorescence in presence of monomers was 87% greater than in presence of dimers, and 25% greater than in presence of H₂O. These results indicate that ¹O₂ contributes to DCF formation. These results strongly suggest that ¹O₂ does react with DCFH₂-DA . We can conclude that the DCFH₂-DA may suffer oxidation by O₂^·- (a classical response) and ¹O₂ (an unexpected response) actions.
Finantial Support: FAPESP

1- A Gomes, E Fernandes, JLFC Lima. J. Biochem. Biophys. Methods 65:45–80, 2005.
2- P Bilski, AG Belanger, CF Chignell. Free Rad. Biol. Med. 33(7): 938–946, 2002.
3- JP Tardivo, A Del Giglio, CS de Oliveira, DS Gabrielli, HC Junqueira, DB Tada, D Severino, RF Turchiello, MS Baptista. Photodiagnosis and Photodynamic Therapy, 2(3):165-238, 2005.