# Deposition of Cell Culture Coatings Using a Cold Plasma Deposition Method
## 2 Materials and Methods  
### Plasma Deposition of Collagen Film  
Plasma deposition was carried out using a purpose-built deposition system comprising a G2000 High Voltage power supply (Redline, Baesweiler, Germany) connected to a custom build Teflon deposition unit that encased two metal electrodes, as shown schematically in Figure 1. The plasma was operated at an input voltage of 160 V and 20.1 kHz. A pneumatic nebulizer T2100 (Burgener Research, Ontario, Canada) was placed between the electrodes and this was connected to a syringe pump to provide a constant flow of protein solution at 40 uL/min. The liquid was nebulized using a helium flow of approximately 3 slm. In addition, a separate helium flow of 6 slm was provided to the metal electrodes to create the plasma discharge. The plasma discharge was combined with the nebulized droplet spray in an acrylic tube (19 mm inner diameter \(\times\) 35 mmlength) and this is shown in Figures 1 and 2. In order to coat the surface evenly the plasma head was mounted on a computer numerically controlled table and moved in a desired pattern. For the 6-well plates being coated the acrylic chamber, at the bottom of the plasma head, descends into each individual well and circles the well before rising and repeating this on the next well. For coating of flat surfaces, a raster pattern was used (4.5 mm step size, and width dependent on size/no. of samples) over the target surface with a gap height of 2 mm. The applied voltage was measured using a TT-HVP 40 high voltage probe (Testec, Frankfurt, Germany), with a ratio of 1 V per kV, which was directly connected to the high voltage output of the G2000 power supply. Current measurements were made using a Pearson 4100 current monitor placed around the HV cable and readings from both voltage and current were collected on a TDS 2014C digital oscilloscope (Tektronix, Beaverton, OH, USA) and analyzed using Microsoft Excel. Figure 1: Schematic diagram of plasma deposition system. Figure 2: Plasma deposition system treating a 96-well microplate. _Wet Chemical Deposited Film._ A wet chemical deposition approach was used as a direct comparison with the plasma deposited collagen film. The 6-well plates and rat tail collagen protein (Type 1, 3 mg/mL) were purchased from Thermo-Fisher Scientific. Gold-coated silicon electrodes (AmsBio) were prepared by sonicating in acetone for 5 min, rinsing with methanol and deionized water, and drying under nitrogen. An even collagen film was deposited onto both substrates by drop casting 100 uL of the collagen onto the surface with a surface density of 76 ug/cm\({}^{2}\). These were left to dry overnight at room temperature before testing. _Spectroscopy._ Fourier transform infra-red (FTIR) spectroscopy was conducted using a Perkin Elmer Spectrum 2000 instrument (Waltham, MA, USA) operating in single beam mode using 64 scans and a 2 cm\({}^{-1}\) resolution. Optical emission spectroscopy (OES) was carried out using a USB4000 UV/VIS spectrometer (Ocean Optics, Duiven, The Netherlands). The fiber optic and collimating lens were mounted horizontally to the discharge and using a quartz tube to allow light transmission. A minimum of four measurements were carried out under each test regime. _Electrochemistry._ A three-electrode electrochemical cell was used for all electrochemical experiments. The working electrode was a gold-coated silicon slide. The counter electrode was a large area coiled platinum wire and a silver/silver chloride (Ag/AgCl in 3 M KCl) acted as the reference electrode. All electrochemistry experiments were carried out on CHI760E workstation (CH Instruments, Inc., Austin, TX, USA). The three redox active probes that were used to investigate the surface properties were ferrocenemethanol (1 mM), ferri/ferrocyanide (1 mM), and ruthenium hexamine (1 mM). The supporting electrolyte for all redox probes was aqueous 0.1 M LiClO\({}_{4}\).