Singlet oxygen quantum yield

Reagents and solvents were obtained commercially and used without further purification. Stock solutions of SOSG (Invitrogen^TM, ThemoFisher) were prepared by dissolving 100 µg in 200 µL of methanol to obtain a stock solution of 825 µM. The solution was stored in the freezer and used within 2 days. Stock solutions of Rose Bengal (RB, Sigma-Aldrich) in PBS (20 µM) and Q2 in DMSO (1 mM) were kept in the dark and stored in the freezer. The solutions of RB and Q2 used for singlet oxygen quantum yield determination were freshly prepared in PBS at pH 7.

The quantum yield of photogeneration of ¹O₂ by Q2 in PBS was investigated using RB as a reference photosensitizer (ΦΔRB=0.76 in water) and SOSG as a fluorescent probe for ¹O₂ probe following the procedure reported by Lin et al. ⁶⁸ The concentrations of RB and Q2 varied in the range 0.25–1 µM and 4–15 µM, respectively. The emission of the singlet oxygen probe (SOSG, 6 μM)⁶⁸ was measured under excitation at 374 nm in the presence of either Q2 or RB to determine the rate of ¹O₂ photogeneration for each concentration. The Xe arc lamp (450 W) of the spectrofluorimeter was used for excitation with the wavelength selected by a monochromator. The excitation slit was fully opened during the measurements. The emission spectra were measured continuously in the 480–620 nm. Each spectrum took 22 s to record. The samples were continuously stirred during measurements and the cuvette was open at the top. Illustrative plots of the increase in SOSG emission under such irradiation conditions are shown in Fig. S23 of the Singlet oxygen quantum yield section of the supplementary information. The ¹O₂ generation quantum yield of Q2 was determined using the Eq. (2):

where C_Q2 and C_RB are the concentrations and ε_Q2 and ε_RB are the molar absorption coefficients at the excitation wavelength for Q2 and RB, respectively. r_Q2 and r_RB are the slopes of the plot of the ¹O₂ generation rate upon photosensitization of Q2 and RB, respectively, as a function of concentration of the photosensitizer, shown in Figure S23. The good linearity of such plots confirms the viability of the procedure used to estimate the ¹O₂ photogeneration yield. We also checked that the emission of the APF ROS probe, used as an indicator of the generation of ROS by Q2 in the cell culture, was increasing upon irradiation of Q2 in solution. Since Q2 solution was prepared from a stock solution of DMSO, the APF probes is only signalling the presence of ¹O₂ giving no additional information about other ROS in solution⁵⁴.