2.12. Measurements of 1O2 quantum yield of LJ002 by SOSG

Singlet Oxygen Sensor Green (SOSG) is a detection reagent that is highly selective to ¹O₂. In the presence of ¹O₂, SOSG reacts with ¹O₂ and produces SOSG endoperoxides (SOSG-EP), which emit strong green fluorescence with a maximum at 531 nm [37]. Methylene blue (MB) is a phenothiazinium dye with a known singlet oxygen quantum yield of 0.52 [38]. After fixing the concentration of MB in aqueous solution, the reaction rate (r) of SOSG with ¹O₂ can be obtained by using time dependent SOSG fluorescence enhancement data, which are proportional to the concentration of SOSG-EP:

[SOSG-EP] is the concentration of SOSG-EP; [SOSG] and [¹O₂] are the concentrations of SOSG and ¹O₂, respectively; and n is the order of reaction with respect to SOSG. When n = zero, r is dependent on only [¹O₂]. In this case, we define the reaction of SOSG with ¹O₂ as an “n-zero reaction”. As a result, the concentration of ¹O₂ generated by photosensitization of MB can be quantitatively determined from the reaction rate (r).

To determine the minimal concentration of SOSG to meet the n-zero reaction condition, various concentrations of SOSG were used to react with ¹O₂ generated by 1 μM MB photoirradiation in PBS. Furthermore, measurements were performed to explore the feasibility of quantitative measurement of ¹O₂ generation from the photosensitization of various MB concentrations by keeping the SOSG concentration constant. As demonstrated in previous studies, the reaction rate of the ¹O₂ fluorescence probes’ oxidation by ¹O₂ can be determined from the initial region of the kinetic curve, thus allowing for quantification of ¹O₂ generation [39]. Therefore, as an additional test, we estimated the ¹O₂ quantum yield of LJ002 by comparing the reaction rate of a known photosensitizer by using SOSG after photosensitization. In this study, the ¹O₂ quantum yield of LJ002 was determined with respect to MB as a standard photosensitizer as follows [40]:

where r_LJ002 and r_MB are the reaction rates of the SOSG with ¹O₂ generated from photosensitization of LJ002 and MB, respectively. A_LJ002 and A_MB are the absorbance of LJ002 and MB, respectively, and Φ_ΔMB (0.52) is the ¹O₂ quantum yield of MB.