Sc7620 sputter coater

To examine the effect of free fatty acids on the structure of fibrin, 7.5 μM fibrinogen was clotted with 20 nM thrombin for 3 hours at 37°C in the presence of 40–400 μM sodium oleate or 50–500 μM sodium stearate. The fibrin clots were fixed in 1%(v/v) glutaraldehyde in 100 mM sodium cacodylate, pH 7.2, dehydrated in a series of ethanol dilutions, ethanol/acetone and pure acetone followed by critical point drying with CO₂ in an E3000 Critical Point Drying Apparatus (Quorum Technologies, Newhaven, UK). The specimens were mounted on adhesive carbon discs, sputter-coated with gold in an SC7620 Sputter Coater (Quorum Technologies), and images were taken with a scanning electron microscope (SEM) EVO40 (Carl Zeiss GmbH, Oberkochen, Germany). Fibrin fiber diameters were measured and data distributions were analyzed using Kuiper’s test and Monte Carlo simulation procedures running under Matlab R2016a [31 (link),32 ].

SC7620 sputter coater (Quorum Technologies Ltd., Laughton, East Sussex, UK) was used to sputter gold electrodes (7 mm in diameter) onto both sides of the sample disks (~1 mm thick). An impedance analyzer (Novocontrol Alpha-AN dielectric spectrometer, Novocontrol Technologies GmbH & Co. KG, Hundsangen, Germany) was used to collect the complex impedance measurements from 0.01 Hz to 1 MHz. The temperature range was from 303 to 483 K and the temperature accuracy was ±0.5 K.

Fibrin clots of 50 μl volume were prepared in duplicate: fibrinogen (at concentration in the range 1.5 – 6 μM) in 10 mM HEPES buffer pH 7.4 containing 150 mM NaCl and the additives (arginine or CPB) was clotted with 20 nM thrombin at 37 °C for 30 min. Thereafter clots were placed into 10 mL 100 mM Na-cacodylate pH 7.2 buffer for 24 h at 4 °C. Following repeated washes with the same buffer, samples were fixed in 1%(v/v) glutaraldehyde for 16 h. The fixed samples were dehydrated in a series of ethanol dilutions (20 – 96%(v/v)), 1:1 mixture of 96%(v/v) ethanol/acetone and pure acetone followed by critical point drying with CO₂ in E3000 Critical Point Drying Apparatus (Quorum Technologies, Newhaven, UK). The specimens were mounted on adhesive carbon discs, sputter coated with gold in SC7620 Sputter Coater (Quorum Technologies, Newhaven, UK) and images were taken with scanning electron microscope EVO40 (Carl Zeiss GmbH, Oberkochen, Germany). The SEM images were analyzed to determine the diameter of the fibrin fibers using self-designed program functions running under the Image Processing Toolbox v. 8.2 of Matlab 8.1.0.604 (R2013a) (The Mathworks, Natick, MA) as previously described [12] (link), [31] (link).

Prior to analysis on the SEM, each polymer sample was mounted on double-sided carbon adhesive tape on a metal stub and sputter-coated with a thin coating of gold/palladium (80:20) for 60 s using a Quorum SC7620 Sputter Coater under vacuum. A Jeol JSM-6060CV SEM operating at 20 kV was used to obtain the electron micrographs. Different magnifications were taken to aid the study of particle morphology.

Scanning electron microscopy was carried out using a German Zeiss SEM field emission scanning electron microscope GeminiSEM 300 for microstructure observation. A 10 mg substrate, with a particle size of 0.075 mm, was glued to the conductive adhesive, then 45 s gold spraying with 10 mA using a Quorum SC7620 sputter coater. Working distance is 8.5 mm. The acceleration voltage was set to 3 kV and a magnification of 2000X for morphology filming. The microstructures of sludge, cotton straw, and corn stover were observed and analysed before and after blending.