## 3 Results and Discussion
### Plasma-Activated Water and Plasma-Induced Intracellular ROS  
When the soft plasma jet was introduced into the aqueous solution, additional reactive species could be induced from the interaction between plasma radicals and water molecules [4; 12]. Among the reactive oxygen species derived from the interaction of plasma radicals with the aqueous solution, \(H_{2}\)O\({}_{2}\) has the highest stability and the lowest reactivity [21]. \(H_{2}\)O\({}_{2}\) can induce the modification of cellular proteins [23]. Thus, \(H_{2}\)O\({}_{2}\) is an important factor deciding fungal spore inactivation. The concentrations of \(H_{2}\)O\({}_{2}\) in the plasma-activated water were measured using a QuantiChrom(tm) Peroxide Assay Kit (_DIOX_-250). By comparing the absorption spectrum of the 5-fold-diluted plasma-activated water with that of standard 30 uM \(H_{2}\)O\({}_{2}\), the concentration of \(H_{2}\)O\({}_{2}\) in the plasma-activated water treated for 3 min by the soft plasma jet was measured to be 137 uM, as shown in Figure 3a. The concentration of plasma-induced \(H_{2}\)O\({}_{2}\) increased with increasing plasma treatment time. Next, we investigated the \(OH\) radical concentration in the plasma-activated water treated by the soft plasma jet. Although the \(OH\) concentration of 0.2 uM was found in the plasma-activated water treated for 3 min in the 48-well plate with a diameter of 11 mm, it was too low in the plasma-activated water treated for 3 min in the 6-well plate with a diameter of 35 mm. These results might be attributed to the fact that \(OH\) radicals are a short-lived reactive species with a lifetime of microseconds order in aqueous solution. Finally, plasma-induced reactive nitrogen species can also attack the spore cell wall and spore DNA [24]. The concentrations of \(NO_{x}\) (\(NO_{2}^{-}\)_and_\(NO_{3}^{-}\)) in the plasma-activated water were measured using QuantiChrom\({}^{\text{TM}}\) Nitric Oxide Assay Kit (_DIOX_-100). By comparing the absorption spectrum of the 28-fold-diluted plasma-activated water with that of standard 100 \(\upmu\)M \(NO_{x}\), the concentration of \(NO_{x}\) in the plasma-activated water treated for 3 min by the soft plasma jet was measured to be 3.14 mM, as shown in Figure 3b. The plasma-induced \(NO_{x}\) concentration increased with increasing plasma treatment time. The long-lived reactive species (\(H_{2}O_{2}\) and \(NO_{x}\)) may contribute to the inactivation of _C. pruinosa_ spores. Consequently, plasma-generated reactive species can affect the intracellular ROS of _C. pruinosa_ spores. To detect intracellular ROS generated inside the spore cell by the soft plasma jet, the fluorescence intensity of \(H_{2}\)_DCF-DA_-stained _C. pruinosa_ spores was measured using a flow cytometer. The black-colored curve in Figure 4 indicates that the control spores contained ROS at some level. This result indicates that the fungal cells could produce ROS internally. In fungi, intracellular ROS enhancement is accompanied by cessation of growth, involving not only changes of cellular morphology to adapt to  
Figure 3: (**a**) Absorption spectra of 5-fold-diluted plasma-activated water and standard 30 \(\upmu\)M \(H_{2}O_{2}\) solutions mixed with a QuantiChromTM Peroxide Assay Kit (_DIOX_-250). (**b**) Absorption spectra of 28-fold-diluted plasma-activated water and standard 100 \(\upmu\)M \(NO_{x}\) solutions mixed with a QuantiChromTM Nitric Oxide Assay Kit (_D2 NO_-100). The plasma-activated water was obtained by treating sterile de-ionized water for 3 min with the soft plasma jet. the changed conditions, but also a decrease in intracellular oxidant concentration [25]. Fluorescence intensity increased with increasing plasma treatment time, indicating that the amount of intracellular ROS was clearly enhanced in the plasma-treated spores. The enhanced intracellular ROS in the fungal cell can, therefore, be attributed to the penetration of plasma-generated ROS into the cell, and/or ROS reactively forming via interactions between plasma-generated reactive species and the spore cell.