Smartblock

Dispersions
of CaCO₃/AgNP hybrids in Milli-Q water, with and without
PVP, were prepared by mixing all of the components at 2000 rpm for
5 min in a ThermoMixer C with a SmartBlock (Eppendorf, Germany), followed
by sonication at 30 Hz, power 30%, for 5 min in a Fisherbrand Transsonic
TI-H-10 ultrasonic bath (Fisher Scientific, UK) and a final mixing
for 3 min. After that, 60 or 90 μL of the dispersions were poured
on the wells of a 96-well microplate and dried in the oven (Heratherm
oven, Thermo Scientific) at 50 and 37 °C for 2 and 20 h, respectively,
or at 80 °C for 2 h. The concentrations of CaCO₃/AgNP
hybrids and PVP in the coating dispersions were 6 and 8 mg·mL^–1, respectively. After drying the coatings, the adhesion
to the wells was tested by scraping the formed coatings with a spatula.
The coatings were also analyzed under the microscope (Life Technologies
EVOS FL, Invitrogen) to study the effect of the coating production
steps on the polymorphism of the hybrids.

Individual FFR coupons were placed within the wells of 24-well plates and inoculated with either (i) 5 µL of the SARS-CoV-2 NZ/Queenstown/01 strain (10⁷ TCID₅₀/mL) or (ii) 5 μL of S. aureus, A. baumannii, or P. aeruginosa from bacteria culture (1 × 10⁶ CFU) or biofilm (one colony representing 3 × 10⁷ to 5 × 10⁸ CFU). Following a 10 min incubation, the 24-well plate with the FFR coupons was placed on a pre-heated Eppendorf ThermoMixer^® C with a SmartBlock™ (Eppendorf, Hamburg, Germany) and covered with the ThermoTop^® (Eppendorf) to guarantee a constant thermal profile over the entire plate, at different temperatures (22 to 80 °C) and times (15 to 120 min). Finally, we also performed a 24 h stand-down experiment where bacterial biofilms were prepared as above but left for 24 h at room temperature prior to starting the experiment. Control experiments to determine the number of bacteria within the biofilm were determined by the selection of one colony resuspended in 1 mL PBS, diluted and plated onto the respective selective media for each strain (five colonies each), incubated overnight, and colonies counted.