# Investigation of Sonosensitizers Based on Phenothiazzinium Photosensitizers
## 3 Results
### Generation and Stability of Singlet Oxygen  
The singlet oxygen yields were checked by tracing the variation of the known singlet oxygen reactor 1,3-diphenyllisobenzofuran (DPBF) with photo/sonosensitizer-generated singlet oxygen. The oxidative rates of DPBF by these compounds in DMSO solvent were investigated under light irradiation/ultrasonic waves. By following the disappearance of the 418 nm absorbance band of DPBF at the initial concentration of 50 uM in the presence of 15 uM of these compounds, it is shown that singlet oxygen generation occurs during the reaction. Methylene blue (MB) is a well-known type II ROS photosensitizer; here, we use MB as the singlet oxygen generation standard. Figure 4a-c shows the changes in the absorption spectra of DPBF in MB, MBOH, and MB6C after light (510 nm long pass, light power 15 mW/cm\({}^{2}\) with variable irradiating times) illumination. All the absorption peaks of DPBF at 418 nm decrease with increasing irradiation time. Meanwhile, the control assay in Figure 4d with their decreasing behavior shows no degradation of the DPBF trap without the addition of the compound. These curves inserted in Figure 4d show that MB exhibits better singlet oxygen generation ability in DMSO solvent.  
Figure 3: Absorption (**left**) and emission (**right**) spectra of 15 uM concentrations of the compounds MB (**a**), MBOH (**b**), and MB6C (**c**) in different solvents. Excitation wavelength emission spectra are depicted as absorption maxima. The partition ratios diagram of octanol/buffer solutions (**d**) and the comparison of the NMR chemical shifts (aromatic region) for these three molecules (**e**).  
A similar trend is also found in the ultrasonicated singlet oxygen generation assay. Figure 5a-c shows that the absorption peaks of DPBF in phenothiazazine derivative-containing DMSO solution decrease with increasing ultrasonic oscillating (using a 42 kHz/35-watt ultrasound source) processing time. It must be stated here that the DPBF will also be quenched when subjected to light irradiation. Thus, to avoid the absorption energy of the DPBF, the absorption peaks of DPBF are quenched by the absorption peaks of DPBF.  
Figure 4: Absorption spectra variations of DPBF mix (**a**) MB, (**b**) MBOH, and (**c**) MB6C in DMSO under an irradiation environment, as described in the experimental section. The control experiment without adding a compound is shown in (**d**), and the insert shows the comparison of DPBF oxidation rates by singlet oxygens of the above experimental results.  
Figure 5: Absorption spectra variations of DPBF mixing (**a**) MB, (**b**) MBOH, and (**c**) MB6C in DMSO under an ultrasound environment. The control experiment without adding a compound is shown in (**d**), and the insert shows the comparison of DPBF oxidation rates by the singlet oxygens of the above experimental results.  
DPBF, we used the light output of the 510 nm long pass as the light source in Figure 4. However, ultrasound cannot avoid this phenomenon. During the experiment, the entire dish was surrounded by ultrasonic energy in the tank, and the free DPBF was also affected (Figure 5d). There are other ways by which the ultrasound mechanism can generate ROS, and it is also possible to consume free DPBF without a photosensitizer (sonosensitizer). Although it was observed that US can also cause free DPBF to disintegrate in the blank experiment, we can judge that the three molecules indeed have the potential to be sonosensitizers, as shown in Figure 5d, and the rate of singlet oxygen generation by these phenothiazazines is as follows: MB > MB6C > MBOH. Nevertheless, we conclude that both MB-OH and MB6C can successfully produce singlet oxygen under luminescence and US conditions.