**2. Experimental Section**

#### *2.1. Plasma Experimental Setup*

Figure 1 depicts the setup of the CAPJ used. A quartz tube with 4 mm inner diameter was covered with two parallel cylindrical pure copper electrodes, which were connected with a high voltage pulsed direct current (DC) power supply for generating the plasma jet [24]. The distance between two Cu electrode was 15 mm. The frequency and the duty cycle of the power supply were fixed at 10,000 Hz and 50%, respectively. In this work, the He gas flow rate was kept at 5 slm (standard liter per min), and the argon gas flow rate was changed from 0 to 500 sccm (standard cubic centimeter per min). Taguchi method with L9 experiment was applied to discover the optimal sterilization e ffect. The CAPJs were generated under four di fferent processing parameters in three various conditions as listed in Table 1, including the applying voltage of 6.5, 7.5, and 8.5 kV; the distance between CAPJ downstream and sample surface of 10, 20, and 30 mm; the CAPJ treating time of 60, 180, and 300 s; and the Ar gas flow rates of 0, 200, and 500 sccm. The radical compositions at the CAPJ downstream of each experiment was detected by an optical emission spectrometer (OES, AvaSpec ULS2048L, Avantes, Louisville, CO, USA) in the wavelength ranging from 200 to 1100 nm. The integration time for the OES detection was 80 ms.

**Figure 1.** The setup of a cold atmospheric plasma jet (CAPJ).


**Table 1.** Factors and levels of CAPJ parameters for the antimicrobial of *E. coli.*

#### *2.2. Bacterial Strain and Culture*

*E. coli* (ATCC 25922TM) was used as a reference strain, which was described elsewhere [25]. The bacteria were routinely cultured in Luria–Bertani (LB) broth (Becton Dickinson, Franklin Lakes, NJ, USA) at 37 ◦C for 24 h to reach the logarithmic phase and performed in the following experiments.

#### *2.3. In Vitro Growth Inhibition Assay*

*E. coli* was cultured in LB broth at 37 ◦C for 24 h. The bacterial suspension was adjusted to an optical density of 1.0 at 600 nm (OD600), which corresponded to 1 × 10<sup>5</sup> colony-forming units (CFUs)/mL. The bacteria were untreated by CAPJ under various combinations of experimental parameters assigned as S1–S9 revealed in Table 2. The untreated *E. coli* was assigned as S0 (control). The bacteria were serial diluted in phosphate buffer saline (PBS) and plated onto LB agar plates. After incubation at 37 ◦C for 24 h, the viable CFUs were counted. The bactericidal activity was represented as a percentage of CAPJ treated one divided by the untreated control group (S0). The results were expressed as the means of three independent experiments performed in duplicate.

#### The bactericidal activity = (Nx/N0) × 100%

where N0 and Nx are the number of the viable bacteria on an untreated control S0 and CAPJ treated Sx (x = 1–9) experiment after incubation at 37 ◦C for 24 h, respectively.


**Table 2.** Antimicrobial conditions of *E. coli* using the Taguchi L9 orthogonal array table. Symbols and numbers of control factors reflect the parameters and levels in Table 1.
