Feasibility Study on Quantitative Measurements of Singlet Oxygen Generation Using Singlet Oxygen Sensor Green

The purpose of this study is to investigate the feasibility for quantitative measurement of singlet oxygen (O-1(2)) generation by using a newly developed O-1(2)-specific fluorescence probe Singlet Oxygen Sensor Green reagent (SOSG). O-1(2) generation from photoirradiation of a model photosensitizer Rose Bengal (RB), in initially air-statured phosphate buffered saline (PBS) was indirectly monitored with SOSG. In the presence of O-1(2), SOSG can react with O-1(2) to produce SOSG endoperoxides (SOSG-EP) that emit strong green fluorescence with the maximum at 531 nm. The green fluorescence of SOSG-EP is mainly dependent on the initial concentrations of RB and SOSG, and the photoirradiation time for O-1(2) generation. Furthermore, kinetic analysis of the RB-sensitized photooxidation of SOSG is performed that, for the first time, allows quantitative measurement of O-1(2) generation directly from the determination of reaction rate. In addition, the obtained O-1(2) quantum yield of porphyrin-based photosensitizer hematoporphyrin monomethyl ether (HMME) in PBS by using SOSG is in good agreement with the value that independently determined by using direct measurement of O-1(2) luminescence. The results of this study clearly demonstrate that the quantitative measurement of O-1(2) generation using SOSG can be achieved by determining the reaction rate with an appropriate measurement protocol.