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 (1O2) was implemented. The chemical generation of 1O2 was promoted using the thermodissociable endoperoxide of disodium 3,3'-(1,4-naphthalene)bispropionate (NDPO2). The detection of 1O2 was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a 1O2-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the 1O2 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 1O2 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 microM, mean+/-SEM, respectively)>methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO2 as a selective generator for 1O2 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 1O2.
Costa
David
D
REQUIMTE, Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal.
Fernandes
Eduarda
E
Santos
João L M
JL
Pinto
Diana C G A
DC
Silva
Artur M S
AM
Lima
José L F C
JL
D000975:Antioxidants / Q000737:chemistry; D002021:Buffers; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D010084:Oxidation-Reduction; D020112:Rhodamine 123; D026082:Singlet Oxygen / Q000032:analysis* / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D014867:Water
D000975:Antioxidants; D002021:Buffers; D012996:Solutions; D014867:Water; D026082:Singlet Oxygen; D020112:Rhodamine 123; D006639:Histidine