Non-thermal plasma elicits ferrous chloride-catalyzed DMPO-OH

Non-thermal plasma (NTP) induces the generation of reactive oxygen species (ROS) and reactive nitrogen species, such as hydroxyl radicals (^•OH), hydrogen peroxide (H₂O₂), singlet oxygen, superoxide, ozone, and nitric oxide, at near-physiological temperatures. These molecules promote blood coagulation, wound healing, disinfection, and selective cancer cell death. Based on these evidences, clinical trials of NTP have been conducted for treating chronic wounds and head and neck cancers. Although clinical applications have progressed, the stoichiometric quantification of NTP-induced ROS remains unclear in the liquid phase in the presence of FeCl₂ or FeCl₃ in combination with biocompatible reducing agents, which may modulate the final biological effects of NTP. In this study, we employed electron paramagnetic resonance spectroscopy to quantify ROS using spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and H₂O₂, using luminescent probe in the presence of FeCl₂ or FeCl₃. NTP-induced DMPO-OH levels were elevated 10-100 µM FeCl₂ or 500 and 1000 µM FeCl₃. NTP-induced DMPO-OH with 10 µM FeCl₂ or FeCl₃ was significantly scavenged by ascorbate, α-tocopherol, dithiothreitol, reduced glutathione, or oxidized glutathione, whereas dehydroascorbate was ineffective in 2 mM DMPO. NTP-induced H₂O₂ was significantly degraded by 100 µM FeCl₂ and FeCl₃ in an iron-dependent manner. Meanwhile, decomposition of H₂O₂ by catalase decayed DMPO-OH efficiently in the presence of iron, indicating iron causes DMPO-OH production and degradation simultaneously. These results suggest that NTP-induced DMPO-OH is generated by the H₂O₂-consuming, iron-dependent Fenton reaction and ferryl intermediates. The potential iron-mediated ROS production by NTP is also discussed to clarify the interaction between NTP-induced ROS and biomolecules.