## 4 Discussion  
H\({}_{2}\)O\({}_{2}\) is widely used as a biocide, particularly in applications where its decomposition into non-toxic byproducts is important. It is currently believed that the Fenton reaction generating OH- radicals is the leading mechanism in H\({}_{2}\)O\({}_{2}\)-induced cytotoxicity [1]. Many studies have reported the oxidation of DNA and proteins rather than cell membrane by H\({}_{2}\)O\({}_{2}\). Similarly, the main mechanism of CAP is known as the oxidation of molecules by ROS/RNS originated from CAP [18]. Differently from H\({}_{2}\)O\({}_{2}\), CAP has complex reactive molecules, charged particles, and UV light at one time. Those reactive species diffuse from the CAP device into cells with damaging the outermost cytoplasmic membrane first. The shrinkage and destruction of cells due to the membrane damage is widely accepted as a main cause of bacterial cell death by CAP [19]. In this study, we tried to understand the underlying mechanism of the synergistic effects of the combination of these two modalities. There have been trials to combine these two modalities, especially in sterilizing devices. Most trials used H\({}_{2}\)O\({}_{2}\) vapor, which passes through the plasma device to generate highly reactive species directly [20; 21]. In dentistry, this combination method has been applied to sterilize surgical tools or to conduct tooth whitening [22; 23]. The OH- radical has been reported as a key player in those applications with the help of other reactive species such as atomic O. The mechanism for the synergistic bacterial killing in liquid seems a little different from the vaporized H\({}_{2}\)O\({}_{2}\) discharge in CAP. Our results show that H\({}_{2}\)O\({}_{2}\) was dissociated by CAP treatment, but OH- radical concentrations measured in DI water and H\({}_{2}\)O\({}_{2}\) solution after CAP treatment are almost the same (Figure 1c). The OH- radical scavenger does not reduce the bacterial cell death in the combination treatment of H\({}_{2}\)O\({}_{2}\) and CAP (Figure 2b). Instead, we observed that a certain amount of H\({}_{2}\)O\({}_{2}\) disappeared when cells were in the solution (Figure 2a). We can assume that co-treatment of H\({}_{2}\)O\({}_{2}\) and CAP may enhance the bactericidal effects not by generating more OH- radicals but by other cellular interaction with H\({}_{2}\)O\({}_{2}\). It is presumed to be the same for DBD CAP. Results in Figure 4 provide us some information about cellular responses to the combination therapy. The extracellular pH, the lipid peroxidation (MDA), and the amounts of intracellular ROS (H2 DCFDA) were more significantly affected by CAP (Figure 4a-c). On the contrary, the membrane permeability, the membrane potential depolarization, and the intracellular DNA oxidation were more significantly affected by H\({}_{2}\)O\({}_{2}\) (Figure 4d-f). An anionic dye PI transferred more through the membrane when cells were incubated in H\({}_{2}\)O\({}_{2}\), though lipids were less peroxid oxidized than by CAP. The higher intensity of Flipr dye in H\({}_{2}\)O\({}_{2}\) treatment may be more strongly related to the membrane permeability. The high Flipr intensity is known to be related to the penetrations of charged particles [24]. Based on these measurements, we can assume that H\({}_{2}\)O\({}_{2}\) can enhance transmembrane transportation of reactive species from CAP, which are important in bactericidal effects. In addition, we can guess that the H\({}_{2}\)O\({}_{2}\) reactions such as damaging of membrane-bound proteins such as glutathione (GSH) or catalase should be prerequisite for synergistic biocidal effects [25]. Though our experimental results do not elucidate exact pathways of molecular interactions, these support that the combination treatment of the two modalities can amplify bactericidal effects through the oxidation of intracellular molecules. There are cases where Jet type CAP is useful for such combination treatment.