K575x sputter coater

The harvested KG1a cells (10⁷ cells) were washed twice with HBSS. The cells were fixed in 2-2.5% glutaraldehyde in 0.1 M Cacodylate buffer (pH = 7.2–7.4) in 4 °C for overnight. The fixed cells were washed three times with the 0.1 M Cacodylate buffer with 15 min submerged in buffer before the next wash and resuspended in the 200 μl of the buffer. Then, 100 μl of the cell suspension was added on a cover slip, which was brushed with polylysine and incubated overnight inside a moisturized chamber before use. The cells were further fixed in 1% osmium tetroxide in 0.1 M Cacodylate buffer for one hour in dark. The cells were washed three times with distilled water, where they were submerged in water for 15 min before the next wash, and dehydrated in gradient ethanol (30, 50, 70, 90, and 100%). Samples were carried further onto the Critical Point Drying apparatus and covered with 100% ethanol to completely submerge the samples. The dehydrated cells were placed on a holder of the scanning electron microscope (SEM) and coated with 4 mm platinum (K575X Sputter Coater, Quorum). The SEM images were recorded using Teneo SEM (Thermo Fischer).

Two sample preparation methods were employed in this work. For imaging the surface morphology of microgels, critical point drying (CPD) was used: first, carrier oil was removed, and photo-cured samples were directly resuspended in 50% ethanol. Microgels were gradually dehydrated consecutively by 75% and 100% ethanol for two days in total before CPD. Microgels were transferred into microporous specimen capsules (78 µm, Agar Scientific) that were placed in a specimen boat filled with 100% ethanol. The specimen boat was inserted into a critical point dryer (E3100, Quorum Technologies) and flushed a minimum of four times with liquid CO₂. Following flushing, the sample was heated to 37 °C at 80 bar pressure to dry. To image the surface morphology of the PAM scaffolds, the sample was lyophilized: briefly, the PAM scaffold was snap-frozen in liquid nitrogen and directly dehydrated in a lyophilizer overnight. For SEM imaging, samples were mounted on aluminum SEM stubs using conductive carbon sticky pads and coated with 15 nm iridium using a K575X sputter coater (Quorum Technologies). A FEI Verios 460 scanning electron microscope was utilized for imaging.

A
thin layer of corundum powder was placed onto a Petri dish and introduced
into the vacuum chamber of a K575X sputter coater (Quorum Technologies).
Palladium was pulverized from a Hauner Metallische Werkstoffe palladium
target with 95% purity. The background vacuum was set to 10^–5 Pa, the deposition was carried with pure argon, and the sputtering
current was maintained at 30 mA. Three sputtering times were chosen
as follows: 30, 90, and 150 s. To determine the amount of Pd deposited
on the powder, the procedures were run under the same conditions but
using a piece of glass. The thickness of the Pd layer on the glass
was calculated from the X-ray reflectometry by the fast Fourier transformation
method. All the samples were dried for 2 h at 120 °C; the samples
were divided in two: one part was calcined at 350 °C for 6 h
and the other one at 600 °C for 6 h; all samples were reduced
in hydrogen, 20 sccm for 2 h at 350 °C. The materials were denoted
as Pds-a-b, where a represents the sputtering time and b the calcination
temperature.

Glass coverslips of 24 × 24 mm and thickness of 0.13–0.16 mm were obtained from Menzel-Glaser. Acetone was utilized for cleaning the coverslips sufficiently to remove the dust and fingerprint available on their surfaces, ensuring that no surface contamination or residual adsorbents were present to impact the accurate experimental measurements and sensor performance. Acetone was chosen since it is volatile, as well as it does not react with the coverslips. Then the glass slip was coated with a gold thin layer using a K575X sputter coater (Quorum Technologies, West Sussex, UK). The sputtering process was run at a current of 20 mA and a voltage of 2.2 kV and lasted 67 s. Then, 0.5 mL of CS solution was applied uniformly to the surface of the gold thin layer on the glass substrate, and spin coater (P-6708D) was employed for 30 s to coat CS sensing layers on the top of the gold thin films (CS/Au) at 2000 rpm as shown in Figure 1.

Particle morphology and internal vesiculation were observed by scanning electron microscopy (SEM), using a FEI Quanta 400FEG ESEM/EDAX Genesis X4M (Hillsboro, Oregon, USA) at Centro de Materiais da Universidade do Porto (CEMUP).
In order to allow visualization of the particle’s interior, dried particles were encapsulated in epoxy resin, and the resulting composite was fractured in liquid nitrogen after hardening overnight. Before being analyzed, samples were sputtered with gold/palladium using a K575X Sputter Coater by Quorum Technologies (Lewes, United Kingdom).

Cells of Virgibacillus sp. strain 21D were filtered onto 0.1 μm polycarbonate Whatman filters (Nucleopore) before fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Filters were washed in 0.1 M cacodylate buffer and subsequently post-fixed in osmium tetroxide. Samples were washed with an ethanol gradient (20–100% ethanol) and subject to critical point drying (Autosamdri-815B, Tousimis). Filters were coated with a 5 nm layer of Au/Pb using a K575X sputter coater (Quorum) and images were acquired using a Quanta 600 FEG (Thermo Scientific) scanning electron microscope at an acceleration voltage of 5 kV.