New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen

In the present study, a new fluorescence microplate screening assay for evaluating scavenging activity against singlet oxygen (O-1(2)) was implemented. The chemical generation of O-1(2) was promoted using the thermodissociable endoperoxide of disodium 3,3'-(1,4-naphthalene)bispropionate (NDPO2). The detection of O-1(2) was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a O-1(2)-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the O-1(2) scavenging activities of ascorbic acid, penicillamine, cysteine, N-acetylcysteine (NAC), methionine, reduced glutathione (GSH), dihydrolipoic acid, lipoic acid, and sodium azide. All of these antioxidants exhibited concentration-dependent O-1(2) scavenging capacities. They could be ranked according to observed activity: ascorbic acid > cysteine > penicillamine > dihydrolipoic acid > GSH > NAC > sodium azide > lipoic acid (IC50 values of 3.0 +/- 0.2, 8.0 +/- 0.7, 10.9 +/- 0.8, 25.2 +/- 4.5, 57.4 +/- 5.9, 138 +/- 13, 1124 +/- 128, 2775 +/- 359 mu M, mean +/- SEM, respectively) > methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO2 as a selective generator for O-1(2) and its fluorescence detection by the highly sensitive probe DHR is shown to be a reliable and resourceful analytical alternative means to implement a microplate screening assay for scavenging activity against O-1(2).