Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide.

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (●OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide (O2●-), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify ●OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); 1O2 using a fluorescent probe; and O2●- and H2O2 using luminescent probes in the presence of thiols or tempol. NTP-induced ●OH was significantly scavenged by dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) in 2 or 5 mM DMPO. NTP-induced O2●- was significantly scavenged by 10 μM DTT and GSH, while 1O2 was not efficiently scavenged by these compounds. GSSG degraded H2O2 more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with H2O2. In the presence of 1-50 mM DMPO, NTP-induced H2O2 quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on H2O2 production was observed in 1 and 10 mM DMPO, whereas it became ineffective in 50 mM DMPO. Furthermore, DMPO-OH did not interact with tempol. These results suggest that DMPO and tempol react competitively with O2●-. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.