Evaluation Of Singlet Oxygen Detection In Biologically Relevant Media For Sonodynamic Therapy

Sonodynamic therapy (SDT) is a promising non-invasive cancer treatment that combines a sensitizer, low-intensity ultrasound, and molecular oxygen to generate reactive oxygen species (ROS) through acoustic cavitation and sonochemical processes. Among these species, singlet oxygen (¹O₂) has been reported and may contribute to oxidative cellular damage, however, its generation strongly depends on the sensitizer microenvironment. Therefore, detecting ¹O₂ is important for understanding the oxidative mechanisms underlying SDT. This study aimed to evaluate ¹O₂ generation using indirect detection with Singlet Oxygen Sensor Green (SOSG), a probe commonly regarded as specific for ¹O₂, in biologically relevant media. SOSG (10 μM), which becomes fluorescent upon reaction with ¹O₂, was prepared in ultrapure water, phosphate-buffered saline (PBS), 1% Triton X-100 and 10% fetal bovine serum (FBS). A second set of experiments was performed using the same SOSG concentration with the addition of 2.5 μM Photogem (PG). Samples were irradiated with ultrasound at 1 MHz, 50% duty cycle, 100 Hz and 1.5 W/cm² in a custom setup using degassed water as the coupling medium. Fluorescence spectra were recorded every 5 minutes of sonication (ꟛ_exc = 488 nm). An increase in SOSG fluorescence was observed during ultrasound irradiation in all evaluated media, including in the absence of PG. The fluorescence increase rates of samples without PG (min^-1) were: k_water = (4.7 ± 0.8)x10^-3, k_PBS = (2.2 ± 0.9)x10^-3, k_Triton = (0.78 ± 0.45)x10^-3 and k_FBS = (2.63 ± 0.15)x10^-3. In the presence of PG, the corresponding rates (min^-1) were: n_water = (5.4 ± 0.8)x10^-3, n_PBS = (3.1 ± 1.1)x10^-7, n_Triton = (11 ± 10)x10^-3 and n_FBS = (3.3 ± 0.6)x10^-3. This response may be associated with the activation of SOSG by hydroxyl radicals (·OH) generated via acoustic cavitation, indicating that the probe response is not solely attributable to ¹O₂ generation under sonication. However, the increase in fluorescence was higher in the presence of PG, suggesting an additional contribution from ¹O₂ generation. These results highlight the influence of the surrounding medium on SOSG response and demonstrate the importance of considering ultrasound-induced ·OH formation when using chemical probes for indirect ¹O₂ detection.