A High Sensitivity Detection Method for Singlet Oxygen and Superoxide Anion and the Mechanism Investigation

摘要

In this paper, a novel and highly sensitive detection method of singlet oxygen (~1O_2) and superoxide anion (O2~) is reported. The principle of the method is similar to that for a measurement of ATP using luciferin and luciferase. A chemiluminescence analytic reagent, 3,7-dihydro-6-{4-[2-(N'-(5-fluoresceinyl)thioureido)ethoxy]phenyl}-2-methylimidazo{1,2-a}pyrazin-3-one sodium salt (FCLA), can sensitively react with ~1O_2 and O_2~- and emit photons that can be detected with a single photon counting system. The chemiluminence is significantly enhanced, approximately 20 times, when human serum albumin (HSA) is used in company with the FCLA. In order to further confirm the chemiluminance is due to the reactive oxygen species (ROS), we investigated the relationship between the intensity of the chemiluminence and the concentration of dissolved molecular oxygen, the direct transform source for ~1O_2 and O_2~-in the solution. We found that the intensity of the chemiluminence signal decreased linearly to zero, as nitrogen bursting reduced the concentration of the dissolved oxygen. The results indicate that, the FCLA-HSA combination provides a highly sensitive chemilumirence method for detecting reactive oxygen species, ~1O_2 and O_2~-.