Magnetron Sputtering of Transition Metals as an Alternative Production Means for Antibacterial Surfaces

Abstract

In the light of the SARS-CoV-2 pandemic and growing numbers of bacteria with resistance to antibiotics, the development of antimicrobial coatings is rising worldwide. Inorganic coatings are attractive because of low environmental leakage and wear resistance. Examples for coatings are hot metal dipping or physical vapor deposition of nanometer coatings. Here, magnetron sputtering of various transition metals, such as gold, ruthenium and tantalum, was investigated. Metal films were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX). We investigated the growth of Pseudomonas aeruginosa isolated from household appliances on different sputter-coated metal surfaces. The fine-grained nanometric structure of these metal coatings was between 14 nm (tantalum) and 26 nm (gold) and the roughness was in a range of 164 pm (ruthenium) to 246 pm (gold). Antibacterial efficacy of metal surfaces followed the order: gold > tantalum > ruthenium. Interestingly, gold had the strongest inhibitory effect on bacterial growth, as analyzed by LIVE/DEAD and CFU assay. High-magnification SEM images showed dead bacteria characterized by shrinkage induced by metal coatings. We conclude that sputtering might be a new application for the development of antimicrobial surfaces on household appliances and or surgical instruments.

Keywords: antibacterial surfaces; biofilm; magnetron sputtering; pseudomonas aeruginosa; transition metals.

Figure 1

Scheme of the experimental design. On the left image, the problem of bacterial contamination in household appliances (such as washing machines or pipes) is depicted. The next step was to develop antibacterial metal surfaces by magnetron sputtering of gold, ruthenium and tantalum on glass slides (top and right images). These surfaces were then analyzed for antibacterial properties by SEM, CLSM and CFU (bottom images). While on glass slides bacterial growth was vital, metal-sputtered surfaces inhibited bacterial growth of Pseudomonas aeruginosa. Scheme was created with BioRender.com (accessed on 10 September 2022).

Figure 2

EDX spectra of three different metal coatings and pure glass, where (A–C) depict the EDX spectra of gold- (A), ruthenium- (B) and tantalum- (C) coated glass and (D) depicts the EDX spectrum of the uncoated glass surface. SEM images of the three different metal coatings, where (E) shows the gold surface, (F) the ruthenium surface and (G) the tantalum surface.

Figure 3

Analysis of surface roughness by atomic force microscopy of four different surfaces. As a control surface we used glass slides (A). Different metal coatings are shown in (B–D). (B) gold, (C) ruthenium and (D) tantalum. The scale bars in the images are 400 nm and the z height is 1.4 nm. In (E) the root mean square roughness for the different surfaces is shown.

Figure 4

SEM images of Pseudomonas aeruginosa grown for 24 h on glass, gold, ruthenium and tantalum surfaces. The first row (A–D) shows an overview of bacterial growth on the different surfaces. On the glass control surface a vital bacterial biofilm is formed (A), whereas on the metal-coated surfaces a clear growth inhibition is visible (C,D). In the second row (E–H) a higher magnification is shown, revealing a healthy horizontal biofilm on glass (E) in contrast to single-spaced-out bacteria on gold (F) and remains of bacterial groups on ruthenium (G) and tantalum (H). High-resolution images are depicted in the last row (I–L). In the vital biofilm on glass a dividing bacterium is highlighted by black arrows (I). Metal coatings induced bacterial death, see white arrows (J–L). A biofilm slime residue is displayed by a white arrowhead on tantalum ((L), white arrowhead).

Figure 5

LIVE/DEAD Assay of Pseudomonas aeruginosa analyzed by CLSM. After cultivation of P. aeruginosa for 24 h on different surfaces bacteria were stained with Syto 9 representing live cells in green and dead cells in purple (propidium iodide) (A–D). Scale Bars = 20 µm. Evaluation of live and dead bacteria in percent (E–H). Note the high amounts of living bacteria on glass (A,E) in contrast to the highest amount of dead bacteria on gold (B,F). Definition of statistical significance: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Figure 6

Colony Forming unit assay of Pseudomonas aeruginosa on different metal coatings and uncoated glass as control. On the left side a scheme of the experimental setup is depicted, showing a cloning cylinder on a glass slide in a Petri dish (A). This setup was used to ensure that the bacterial growth occurs only on the coated surface and in a defined area. On the right side a graph of the CFU assay per ml is presented (B). The first bar (-) is a result of a sterility control with glass incubated in LB medium only. Note that no colonies grow. Uncoated glass shows highly efficient bacterial growth (white-colored bar). Gold coating vastly reduced bacterial growth (medium gray bar). Ruthenium coating showed less antibacterial potential (light gray bar). Tantalum coating reduced bacterial growth nearly as efficiently as gold. Scheme in (A) was created with BioRender.com (accessed on 10 September 2022). Definition of statistical significance: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Figure 7

ROS assay of Pseudomonas aeruginosa grown for 24 h on glass, gold, ruthenium and tantalum surfaces. Reactive oxygen species were measured with the help of 2-7-dichlorofluorescin diacetate assay. Images were obtained by confocal laser scanning microscopy and are presented as false color images, using a fire ice false color coding (A–D). Highest intensity is depicted as yellow white and darkest pixels are shown as black blue. Most pixel values are in the background range (0–63 black blue) (E), while gold shows the highest amount of ROS production (orange and yellow bars).