## 4 Discussion
The thicker or the denser the agarose gel was, the longer RONS took to reach in DI water (Figures 4 and A3). This was also observed in previous UVAS studies using agarose gels [23; 24]. However, the differential changes of each RONS with time were analyzed first in this study. \begin{table}
\begin{tabular}{c c} \hline
**Equation Number** & **Reaction** \\ \hline (1) & OH + OH + M \(\rightarrow\) H\({}_{2}\)O\({}_{2}\) + M \\ (2) & HO\({}_{2}\) + HO\({}_{2}\)\(\rightarrow\) H\({}_{2}\)O\({}_{2}\) +O\({}_{2}\) \\ (3) & H\({}_{2}\)O\({}_{2}\) + O \(\rightarrow\) HO\({}_{2}\) + OH \\ (4) & HO\({}_{2}\) + H\({}_{2}\)O\({}_{2}\)\(\rightarrow\) OH + H\({}_{2}\)O + O\({}_{2}\) \\ (5) & H\({}_{2}\)O\({}_{2}\) + H \(\rightarrow\) OH + H\({}_{2}\)O \\ (6) & NO + OH \(\rightarrow\) HNO\({}_{2}\) \\ (7) & HNO\({}_{2}\) + OH \(\rightarrow\) H\({}_{2}\)O + NO\({}_{2}\) \\ (8) & NO + HO\({}_{2}\)\(\rightarrow\) OH + NO\({}_{2}\) \\ (9) & NO\({}_{2}\) + H \(\rightarrow\) OH + NO \\ (10) & HNO\({}_{3}\) + NH\({}_{2}\)\(\rightarrow\) NH\({}_{3}\) + NO\({}_{3}\) \\ (11) & NH\({}_{3}\) + NO\({}_{3}\)\(\rightarrow\) HNO\({}_{3}\) + NH\({}_{2}\) \\ (12) & NO\({}_{2}\) + OH \(\rightarrow\) HNO\({}_{3}\) \\ (13) & HNO\({}_{3}\) + OH \(\rightarrow\) H\({}_{2}\)O + NO\({}_{3}\) \\ (14) & NO\({}_{2}\)\({}^{-}\) + O\({}_{3}\)\(\rightarrow\) NO\({}_{3}\)\({}^{-}\) + O\({}_{2}\) \\ \hline \end{tabular}
\end{table} 
Table 1: Chemical reactions used for theoretical approaches in this study. Interestingly, the agarose gel barrier effect was different for the three RONSs, which could be attributed to the RONS-integrated chemical reaction occurring in the agarose gel. Reactive species accumulate at the agarose interface and are transferred through agarose by diffusion. We observed that each RONS has different diffusion rate by chemicals (Figures A3 and A4). NO3\({}^{-}\) has the highest diffusion rate, and H2O2 has the lowest rate. However, it could not explain the significant reduction in NO2\({}^{-}\) through an agarose gel. During the slow diffusion process, NO2\({}^{-}\) at the interface between agarose and air has a high possibility of oxidation to NO3\({}^{-}\) by reactive species such as OH radicals and ozone generated from the plasma. Since agarose gels are rich in moisture, reactions with hydroxyl radical may be frequent (chemical reactions proposed in Table 1). These reactions may explain why the nitrite reduction is more substantial than H2O2 and nitrate. We tried to confirm that these reduction processes are caused by plasma-induced radicals. We mixed chemicals containing NO2\({}^{-}\) and H2O2 and measured their diffusions through an agarose gel without plasma treatment (Figure A5). The NO2\({}^{-}\) values were identical with three different concentrations of H2O2. A small amount of nitrate was observed with a high amount of H2O2. However, the concentration was almost in detection limit. Without plasma treatment, the conversion of nitrite to nitrate was not observed. Only three species were studied in this study, but it should be considered that barrier effects for some other species may have different trends [44; 45]. In the case of additional SC layer in Figure 6, it is shown that the SC layer can block even the RONS with a long lifetime. H2O2 was found to be below the detection limit when SC was added. H2O2 is produced from OH radicals, and moisture is the primary variable in its formation. H2O2 reduction is induced because the SC layer blocks the interaction between plasma and water molecules. NO2\({}^{-}\) and NO3\({}^{-}\) have high hydration free energy and difficulty passing through the cell membrane due to absorption by the lipid head [46; 47]. As a result, the absorption reduction under the condition of the addition of the SC layer is due to the decrease in all three reactive species H2O2, NO2\({}^{-}\), and NO3\({}^{-}\). The detection of NO3\({}^{-}\) can predict the reduction of the SC layer's penetration barrier by plasma treatment and hydrophilization by plasma treatment [5; 48].