# Repetitive Bacterial Disinfection of Respiratory by Polydopamine Coating
## 3 Results and Discussion
The bacterial level decreased significantly in PP-PDA1 (0.54 \(\times\) 10\({}^{9}\)\(\pm\) 0.08 CFU/mL-cm\({}^{2}\), \(n\) = 5) compared to the initial bacterial concentration in the control sample (bare PP, 0.81 \(\times\) 10\({}^{9}\)\(\pm\) 0.09 CFU/mL-cm\({}^{2}\), \(n\) = 15). Figure 3: (**a**) Illustration of the device for investigating antimicrobial activity and H2O2 production in polydopamine-coated polypropylene. (**b**) Bacterial concentration and representative images used for counting of _E. coli_ colonies from each sample: control (bare polypropylene) and polydopamine-coated polypropylene after 9 h of incubation. (**c**) Relative cell viability from in vitro cytotoxicity test (i.e., CCK-8 assay) of polydopamine-coated polypropylene (PP-PDA1). Asterisks denote the level of statistical significance (_p_-value). A single asterisk refers to _p_-value less than 0.05. Four asterisks indicate \(p\) < 0.0001. To demonstrate the correlation between H\({}_{2}\)O\({}_{2}\) generation and the antimicrobial properties of the mask, the amount of H\({}_{2}\)O\({}_{2}\) in each sample was quantified. For this, the samples were submerged in PBS solution with a pH of 7.4, with gentle shaking (50 rpm), as described in a previous study [26], and the ferrous oxidation-xylenol orange (FOX) assay was utilized to quantify the exact amount of generated H\({}_{2}\)O\({}_{2}\) (see the Supplementary Materials for the detailed procedures). It was confirmed that the PP-PDA1 produced 18.11 \(\pm\) 0.74 \(\upmu\)M/cm\({}^{2}\) (\(n\) = 5) of H\({}_{2}\)O\({}_{2}\) after 9 h of incubation. This suggests that the antibacterial properties of PP-PDA1 originate from H\({}_{2}\)O\({}_{2}\) production. An in vitro cell viability test was conducted using the CCK-8 assay to demonstrate that the PDA-coated mask does not have a toxic effect. A blank medium was used as a negative control (NC), and DMSO was used as a positive control (PC). The relative cell viability was calculated based on the NC. The cell viability results from the PC (14.02% \(\pm\) 5.88%, \(n\) = 5) demonstrated that the test results were reliable. According to the ISO 10993-5 method which we performed, a relative cell viability of \(>\)80% is defined as the condition with no cytotoxicity [29]. The relative cell viability in PP-PDA1 was 84.11% \(\pm\) 11.34% (\(n\) = 5), indicating that the cytotoxicity of PP-PDA1 was low. According to the results, it is believed that PDA-coated PP should be a non-cytotoxic and safe material for use in masks. H\({}_{2}\)O\({}_{2}\) production in PDA is not permanent and has a saturation point [26]. The advantage of the developed system is that the H\({}_{2}\)O\({}_{2}\) source (catechols) can be easily refilled by additional PDA dip-coating cycles. Thus, the antibacterial activity of the mask can be maintained by continuous PDA coatings, which are virtually unlimited, as illustrated in Figure 4a. To test this hypothesis, additional bacterial growth experiments were performed by utilizing PDA-coated PP meshes with 1, 2, and 3 coating cycles, and the corresponding samples are designated as PP-PDA1, PP-PDA2, and PP-PDA3, respectively (Figure 4b). The bacterial concentration in each substrate after 9 h of incubation was similarly diminished from 0.81 \(\times\) 10\({}^{9}\)\(\pm\) 0.09 CFU/mL\(\cdot\)cm\({}^{2}\) (control, \(n\) = 15) to 0.54 \(\times\) 10\({}^{9}\)\(\pm\) 0.08 CFU/mL\(\cdot\)cm\({}^{2}\) (\(n\) = 5, PP-PDA1), 0.69 \(\times\) 10\({}^{9}\)\(\pm\) 0.17 CFU/mL\(\cdot\)cm\({}^{2}\) (\(n\) = 5, PP-PDA2), and 0.52 \(\times\) 10\({}^{9}\)\(\pm\) 0.12 CFU/mL\(\cdot\)cm\({}^{2}\) (\(n\) = 5, PP-PDA3), respectively. Thus, the antibacterial ability can be constantly sustained in every single substrate regardless of the coating cycles, demonstrating antibacterial recyclability. Figure 4: (**a**) Schematic of the proposed method for reusing masks by simple additional polydopamine coatings. (**b**) Effect of polydopamine coatings on the concentration of _E.