Alfa aesar

The C6/36 EV isolates were fixed with a 1:1 mixture of 2.5% glutaraldehyde (Electron Microscopy Sciences [EMS], Hatfield, PA, USA) and 4% paraformaldehyde (Sigma) for 2 h at RT and washed three times (5 min each) with PBS. After fixation, the samples were incubated with 2% osmium tetroxide (Alfa Aesar, Thermo Fisher Scientific) for 90 min at RT. The fixed pellets were washed three times with PBS and dehydrated in an ascending graded series of ethanol (30, 50, 70, 80, 90, and 96%), including three passes (5 min each) in absolute ethanol (J.T.Baker) at RT. Three passes (5 min each) in propylene oxide (Sigma-Aldrich) at RT were then performed. The samples were placed in a 1:1 mixture of propylene oxide/epoxy resin for 18 h at RT and embedded in pure epoxy resin (EMS) at 60 °C for 48 h. Ultrathin sections (40–50 nm thick) were obtained in an ultramicrotome (Leica EM UC7, Leica Microsystems, Buffalo Grove, IL, USA) and mounted on copper grids (EMS) covered with formvar (EMS). The sections were contrasted with uranyl acetate (Merck, Kennerworth Fort, NJ, USA) for 30 min and lead citrate (EMS) for 10 min at RT. The preparations were observed with a transmission electron microscope (JEM1010 model; JEOL, Peabody, MA, USA) operating at 80 kV. The images were captured with a CCD300-RC camera (DAGE-MTI, Michigan City, IN, USA) adapted to the microscope and analyzed with ImageJ software.

To prepare GNP/PDMS surfaces, commercially available GNPs aggregates (Alfa Aesar, Thermo Fisher Scientific, Erlenbachweg, Germany), PDMS elastomer (Sylgard 184 Part A, Dow Corning, Midland, MI, USA), and curing agent (Sylgard 184 Part B, Dow Corning) were used.
The GNP/PDMS composites were produced through a bulk mixing process by incorporation of GNPs into the PDMS base elastomer (Part A) as detailed by Vagos et al. [44 (link)]. The GNPs were first incorporated at different loadings (1, 2, 3, 4, and 5 wt%) to determine the most efficient GNP load to decrease bacterial biofilm formation. To improve the GNP dispersion, the mixture was stirred for 30 min at 500 rpm and then sonicated (Hielscher UP400S, at 200 W and 12 kHz) for 60 min. After that, the composites were kept in an ultrasound bath (Selecta Ultrasons, Lisbon, Tecnilab, Portugal) for 30 min to remove the remaining air bubbles. At that point, the curing agent (Part B) was added to the elastomer/GNP mixture (in an A:B ratio of 10:1) and gently shaken. The GNP composites were placed as a thin layer on top of glass slides (1 x 1 cm, Vidraria Lousada, Lda, Lousada, Portugal) through spin coating (Spin150 PolosTM, Caribbean, The Netherlands) for 1 min with a 500 rpm ramp to 6000 rpm. Likewise, PDMS surfaces were produced as control [45 (link)].

All solvents and reagents were purchased from Sigma-Aldrich, Castle Hill, Sydney, Australia and Alfa-Aesar (Thermo Fisher Scientific Australia Pty. Ltd.), Scoresby, Victoria, Australia and used without any further purification or treatment. Thin layer chromatography was performed on Merck 60F-254 silica gel plates. Flash chromatography was performed on GRACE Reveleris X2 (In Vitro Technologies Pty. Ltd., Noble Park, Victoria, Australia). ¹H and ¹³C-NMR spectra were acquired on a Varian 400MR (Varian Australia Pty. Ltd., Mulgrave, Victoria, Australia) at 400 MHz and 100 MHz, respectively. Coupling constants (J) are in Hertz (Hz), chemical shifts (δ) are expressed in parts per million (ppm) and reported relative to tetramethylsilane (TMS) or relative to residual solvent peaks. Low resolution mass spectra were obtained on a Thermo Scientific TSQ Quantum Access Max LCMS/MS&TLX1 Turboflow Chromatography System (Thermo Fisher Scientific Australia Pty. Ltd.), Scoresby, Victoria, Australia) in positive ion mode.