Synthesis, Characterization and Antibiofilm Activity Against Pseudomonas Aeruginosa of ZnO-Ag Nanocomposite

The rise of antimicrobial resistance poses a significant challenge to public health worldwide, necessitating the development of novel materials with potent antimicrobial properties. Two of the most prominent antibacterial nanoparticles (NPs) are silver (Ag) and zinc oxide (ZnO). Silver is commonly used in dentistry, where it damages bacteria by releasing silver ions, generating reactive oxygen species (ROS). ZnO NPs, under UV light, enhance ROS generation due to their photocatalytic activity, amplifying their antibacterial activity. Some nano ZnO materials with sharp edges can disrupt the cell membrane. In this work, we synthesized a ZnO-Ag nanocomposite using a calcination-impregnation method adopted from [1]. We analyzed its properties and composition using TEM-EDS, SEM, AFM, FTIR, and XRD. We then tested its antibacterial and antibiofilm activity against <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>), a versatile gram-negative bacterium frequently found in soil, water, and hospital environments. This bacterial strain is known to form biofilm, a cluster of bacteria attached to a surface and/or each other and embedded in a self-produced matrix, being more resistant to antibiotics.<br/>The ZnO-Ag material was synthesized using a simple calcination-impregnation method by initially dissolving Zinc Acetate Dihydrate and Silver Nitrate in deionized water at a 2:1 ratio. The mixed solution was stirred thoroughly to ensure homogeneity and then sonicated, producing a solid precursor. This precursor was then placed in a muffle furnace and calcined at 400°C for 1 hour. During the calcination process, the zinc acetate decomposed to form zinc oxide (ZnO), whereas the silver nitrate decomposed to produce silver nanoparticles (Ag), which enabled them to impregnate the ZnO surface, ensuring a high degree of interaction between ZnO and Ag. The resultant ZnO-Ag composite was allowed to cool to room temperature to be further used. TEM-EDX was performed to check the material’s chemical composition and gain precise images. Furthermore, XRD was performed to analyze the crystal structure and orientation of the nanomaterial. The material’s surface morphology and thickness were analyzed using air topography AFM; the nanocomposite was deposited on a 1cm x 1cm glass slide by spin coating a suspended solution (ZnO-Ag in DI water) and then heating it for 30 minutes. The nanomaterial's thickness was 41 nm, and the surface contained sharp peaks, indicating the nano-composite’s potential to penetrate the bacteria using its sharp edges. Further analysis of the material’s photocatalytic behavior and an FTIR analysis will be performed.<br/>To verify the material's potential as an anti-bacterial agent, it was first tested against <i>P. aeruginosa</i> using inhibition zone assay. LB agar plate was inoculated with <i>P. aeruginosa</i>, then a small amount of the material solution was dispersed on the agar. After incubating the agar plate at 37 C for 24 hours, it was found that the nanocomposite inhibited the growth of the bacteria in the area where the material is dispersed compared to the control sample where DI water was used. Regarding the material’s antibiofilm activity, 1 by 1 cm glass slides were spin coated by 500uL of the suspended nanocomposite, at a concentration of 50 mg/mL . Then, <i>P. aeruginosa</i> was grown on the coated glass slide for 3 days at 27 C. After that, the samples were prepared for SEM by fixation with 2% glutaraldehyde followed by graded ethanol dehydration. Notably, the SEM images showed that the sample coated with nanocomposite inhibited the biofilm growth compared to control sample, which was not coated with nanocomposite. This highlights ZnO-Ag nanocomposite potential use as an anti-biofilm agent.<br/><br/>[1] R. Al-Gaashani <i>et al.</i>, “Antimicrobial activity of ZnO-Ag-MWCNTs nanocomposites prepared by a simple Impregnation–Calcination method,” <i>Scientific Reports</i>, vol. 13, no. 1, Dec. 2023. doi: 10.1038/s41598-023-48831-w