# Infection Control with Antimicrobial Solid-State ZnO Nanoparticles on Silk Fiborin Gauze
## 2 Materials and Methods
### In Vitro Assay  
Antibacterial properties of ZnO NP gauze against _Pseudomonas aeruginosa_ (ATCC(r) 15442(tm)) and _Staphylococcus aureus_ (ATCC(r) 29213(tm)) were assessed on three samples for each strain. Cultures were carried out overnight in Luria-Bertani (LB) broth media (Thermo Fisher Scientific, Toronto, ON, Canada), then diluted to 1 \(\times\) 10\({}^{6}\) CFU/mL with LB broth [27]. Segments measuring 1 cm\({}^{2}\) of ZnO-SF gauze, SF gauze (control), or commercial non-woven surgical gauze (positive control) (Fisherbrand, 22028558, Thermo Fisher Scientific, Toronto, ON, Canada) were incubated in 5 mL of bacterial solution. Live (negative) and dead (positive) controls were utilized to normalize sample data during analysis; both controls consist of a bacterial solution without any sample, with the dead one being processed with ethanol during the assay to kill the bacteria. Samples were maintained in an orbital incubator at 37 \({}^{\circ}\)C throughout the experiment until reaching the 24 h, 48 h, or 72 h timepoints. After incubating for 24 h, 48 h, and 72 h, the samples were centrifuged at 10,000\(\times\)\(g\) for 10 min and resuspended in 2 mL of Abcam bacterial viability assay kit wash buffer. A 1 mL suspension from each bacterial sample was then diluted in 5 mL of wash buffer. This process of pelleting, resuspending, and diluting was repeated before staining with the LIVE/DEAD Abcam bacterial viability assay kit (ab189818, Abcam, Shanghai, China). After staining, gauzes were incubated for 1 h in total darkness and then transferred in triplicate to a 96-well plate for fluorescence assessment at 490 nm and 536 nm, and 525 nm and 617 nm, as excitation and emission filters, respectively. The fraction of dead bacteria in each sample was calculated from the readings. We followed the protocol provided with the LIVE/DEAD Abcam Bacterial Viability assay kit (ab189818). To assess the biocompatibility, Chinese hamster ovary (CHO) cells (ATCC(r) CLL-61(tm)) were seeded onto ZnO-SF gauze, SF gauze, and commercial non-woven surgical gauze in Dulbecco's Modified Eagle Medium (Gibco, Burlington, ON, Canada) with 10% fetal bovine serum (Gibco, New York, NY, USA), 10 mM HEPES buffer, and 1% penicillin-streptomycin (Gibco) at 37 \({}^{\circ}\)C under 5% CO\({}_{2}\). After quantification using Trypan Blue, when they reached 80% confluency, cells containing fewer than 8 passages were plated in a 24-well plate at a density of 1 \(\times\) 10\({}^{5}\) cells/mL over sterilized materials. A standard curve from 1 \(\times\) 10\({}^{3}\) to 1 \(\times\) 10\({}^{6}\) cells/mL and a blank control were prepared. Cell viability was measured using the alamarBlue(r) Cell Viability Reagent (Invitrogen, 1025) after 24, 28, and 72 h of exposure to 1 cm\({}^{2}\) gauze segments. At each time point, the media was replaced with a fresh solution of complete media with 10% alamarBlue(r) and incubated for 4 h at 37 \({}^{\circ}\)C, protected from light. Following incubation, triplicate samples (\(n\) = 3) were transferred to a 96-well plate to measure absorbance at 570 nm and 600 nm. A best-fit line from the OD readings of standards represented cell density. The percent difference in alamarBlue(r) reduction between exposed and control cells was calculated as per the manufacturer's protocol. To unravel the antibacterial effect of the ZnO NPs-gauze, ROSs and zinc release were measured. ROSs production was assessed after 24, 48, and 72 h following [PERSON] et al. 's protocol [PERSON] et al. (2017). Release of all ROSs was evaluated by immersing the 1 cm\({}^{2}\) films into 15 mL vials filled with 4 mL of PBS using a chemically reduced form of fluorescein H2 DCFDA as an ROSs indicator. A 25 \(\upmu\)M reagent solution of H2 DCFDA was produced by dilution with 99.9% ethanol and used as a probe to measure ROS. 200 \(\upmu\)L of the sample solution was added into a black microplate (Corning, 3915), and 5 \(\upmu\)L of reagent solution was then added and incubated for 10 min wrapped in aluminum foil at room temperature.