*3.1. Plasma Characterization*

The temperature of He-based CAPJ was measured at the distance of 10 mm from the nozzle, and it rose continually for the first 5 min and then kept constant for at least 30 min under pure He gas flow rate of 5 slm, as shown in Figure 2. The temperatures increased from 34.5, 35.5, 36.5 to 38.5 ◦C when the applying voltages were increased from 6.5, 7.5, 8.5 to 9.5 kHz, respectively.

**Figure 2.** CAPJ temperature keeps steady for at least 30 min. Temperature changes of CAPJs operated at different applied voltages ranging from 6.5 to 9.5 kV under a fixed He gas flow rate of 5 slm. The temperature was detected at the position of 1 cm below the downstream of CAPJ.

The compositions of ROS generated by CAPJ are mainly dependent on both the working gas and the atmospheric air. The compositions, therefore, can be controlled by the working gas mixture. Figure 3a–c illustrate the excited and radiant species in optical emission spectroscopy (OES) spectra under the mixtures of various Ar gas flow rates of 0, 200, and 500 sccm into a fix He gas flow rate of 5 slm with the application voltage of 6.5 kV. The emission lines between 300 and 400 nm, 450 and 700 nm and 700 and 900 nm were dominated by the nitrogen (N), He, and Ar atoms, respectively. Both hydroxyl radical (OH) emission at 309 nm and nitrogen monoxide (NO) emission at 283 nm are of particular interest because they might play an effective role for bacterial growth inhibition [1,30,31]. The intensities of OH radicals remarkably increased with increasing Ar gas flow rate but were unaffected at various application voltages, as observed in Figure 3d. It is also important to point out that the temperature of each Ar added CAPJ test was below 38.5 ◦C.

**Figure 3.** *Cont.*

**Figure 3.** CAPJ plasma characterizes by OES. The OES spectra of the CAPJs operated at a fixed applied voltage of 6.5 kV and fixed He gas flow rate of 5 slm and (**a**) without Ar gas inlet, (**b**) with 200 sccm Ar gas and (**c**) with 500 sccm Ar gas, and (**d**) the dependence of Ar flow rate on the intensity of OH radicals @ 309 nm.

#### *3.2. CAPJ Possess Bactericidal Activity*

The bactericidal activity was investigated under various plasma parameters designed using the Taguchi method shown in Figure 4 and summarized in Table 4. Comparing with the untreated control sample S0, the bactericidal activities against *E coli* by CAPJ treated under S1 to S9 were 45.7%, 31.3%, 90.6%, 92.8%, 53.2%, 37.7%, 85.9%, 100%, and 22.6%, respectively.

**Figure 4.** Bacterial colonies evaluate the bactericidal activity of CAPJ operating parameters. Photos of bacterial colonies of control S0 and CAPJ treated samples (**S1**) to (**S9**) with a combination of different Taguchi experimental parameters. The visualized colonies grew on the LB agar plates and were counted and represented in CFU as tabulated in Table 4.

**Table 4.** The bactericidal activity of *E. coli* by nine different CAPJ treatments.


#### *3.3. Analysis of the Bactericidal Activity by Using Taguchi Method*

The four important operating parameters of CAPJ to achieve desired performance considered in this study were application voltages, CAPJ-sample distance, Ar gas flow rate, and CAPJ treatment time, which are noted as A, B, C, and D, respectively, in Table 1, and each was given three levels. The greater S/N value corresponded to the better performance regardless of the category of the performance characteristics [26]. Therefore, parameters with high S/N value and the better efficiency were selected to define the optimal level of the operating parameters. The average S/N ratios for each level of each parameter, in terms of bactericidal activity, were shown in Figure 5. The horizontal red line was the overall mean of the S/N values. The best combination of process parameters corresponding to the bactericidal activity was found to be A3B2C3D3, which correlates to application voltage of 8.5 kV, CAPJ-sample distance of 10 mm, Ar gas flow rate of 500 sccm, and CAPJ treatment time of 300 s. Furthermore, the CAPJ treatment time was discovered as the most impactful factor for bactericidal activity because of the greatest range of outcomes between the three experimental levels.

**Figure 5.** Taguchi analysis determines the best set of parameter combination. The effect diagrams for antimicrobial of CAPJ are based on the higher S/N and the better efficiency. Four factors include A: applying voltage (kV), B: CAPJ-sample distance (mm), C: Ar gas flow rate (sccm), and D: CAPJ treated time.

The relative significance of each parameter was investigated by ANOVA to estimate their contributions. The ANOVA results of the control factors, A, B, C, and D were calculated for the bactericidal activity and the degree of freedom, sum of squares, variance, and percentage contributions are shown in Table 5. Higher percentage contribution correlated to more significant influence of the overall process. Factor D, the CAPJ treatment time, was the most impactful with the highest variance of 4.70 and a percentage contribution of 76.5%. This was followed by factor C, Ar gas flow rate, and factor B, CAPJ-sample distance, with contributions of 12.1% and 11.2%, respectively. Meanwhile, the contributions of 0.2% for factor A, application voltage, is obtained implying its insignificant role.


**Table 5.** Summary of the ANOVA results for the bactericidal activity of *E. coli* by CAPJ.

*3.4. Validation of Bactericidal Activity Conferred by CAPJ Treated under S10*

Based on the optimal condition deduced from the Taguchi method, the CAPJ condition with the application voltage of 8.5 kV, CAPJ-sample distance of 10 mm, He/Ar gas flow of 5 slm/500 sccm, and CAPJ treated time of 300 s was assigned as S10, and a confirmation test was performed to validate the conclusions on the previous discovery. As compared with the control S0, the bactericidal activity of S10 was up to 100% in Figure 6.

**Figure 6.** S10 CAPJ induces bacterial death. Photos of bacterial colonies of control S0 and confirmation test of CAPJ treated under S10, fluorescence live/dead bacterial viability assay images of *E. coli* without CAPJ (S0) and with CAPJ treated under S10.

To further verify whether the S10 CAPJ possesses potent bactericidal activity, a LIVE/DEAD Bacterial Viability assay was employed. As shown in Figure 6, dead bacteria, appearing with red fluorescence, were rarely observed in the control group, but those bacteria treated with S10 CAPJ had significantly more dead bacteria presented in the lower panel of Figure 6. These results confirmed that the S10 CAPJ had the most effective bactericidal activity in the treatment of bacterial pathogens and indicated that the Taguchi method is handy to find the optimal parameters on the bactericidal activity by CAPJ.

#### *3.5. CAPJ Treatment Induced DNA Double-Strand Breaks (DSB) and Disruption of Cell Wall Integrity*

To further explore the mechanism of the bactericidal activity by CAPJ treatment, we conducted a DNA electrophoresis assay. As shown in Figure 7, the untreated plasmid DNA (S0) was intact and exhibited the supercoiled and circular forms. In contrast, clear smear DNA fragments were shown in the plasmid DNA treated with S10 CAPJ, indicating that CAPJ contributed to induce DSB in bacteria. To strengthen our findings, the morphologies of *E. coli* were visualized by FE-SEM after bacteria was treated by S10 CAPJ. In the control group without CAPJ treatment, the bacterial shape and cell wall showed intact morphologies (Figure 8a,b). By contrast, FE-SEM images of the bacteria treated with S10 CAPJ exhibited a shriveled and burst appearance on the bacterial surface (Figure 8c,d). Taken together, these results demonstrated that CAPJ treated under S10 sustainably inhibited bacterial growth by inducing DSB and disrupting cell wall integrity.

**Figure 7.** S10 CAPJ induces DNA double-strand breaks (DSB). The pGL3 (2 μg/μL) was untreated (S0) or treated with S10 CAPJ. The plasmid DNA was loaded on 1.0% agarose gel for electrophoresis. Ethidium bromide-stained DNA was visualized under UV light. The positions of the size markers are shown at left of the image. M, DNA marker.

**Figure 8.** S10 CAPJ disrupts bacterial cell wall integrity. The FE-SEM images of the bacterial morphologies before CAPJ treatment (**<sup>a</sup>**,**b**) and after S10 CAPJ treatment (**<sup>c</sup>**,**d**).

#### *3.6. In Vivo Evaluation*

Bactericidal activity in vivo evaluation by CAPJ treatment was presented in Figure 9. SD rates were used to create wound exposure and analyze the bacterial infection as described in the Experimental Section. The wounds were either untreated (S0) or treated with S10 CAPJ, and wound exudates were collected on days 0 and 4. Our results showed that S10 CAPJ treatment remarkably decreased bacterial load on day 0 when compared to S0. Most importantly, this effect is still seen on day 4, which showed that the bacterial infection continued to be reduced in rat treated with S10 CAPJ.

**Figure 9.** S10 CAPJ reduces bacterial infection in the wound. Rat wound was untreated or treated with S10 CAPJ, and the bacterial loads in the wound were counted on days 0 and 4. Viable bacteria were represented as colony forming units (CFUs).
