S21375

Fluorescently labelled bovine serum albumin (A13100, Thermo Fisher Scientific Inc., Waltham, MA, USA) protein with molecular weight of 66 kDa and streptavidin (S21375, Thermo Fisher Scientific Inc., Waltham, MA, USA) were used for the protein adsorption studies of the control, flat and nanostructured Si₃N₄ samples. The BSA and streptavidin were dissolved separately in phosphate buffered saline (PBS, 10 mM, pH 7.4) to a concentration of 2 mM. The substrates were rinsed with PBS to rehydrate the surfaces. All the sample substrates were then immersed in both protein solutions separately and were incubated at 4°C for 24 h. The samples were then removed from the protein solutions, gently washed three times with PBS, and rinsed once with deionized water to remove the PBS salt. Surface protein adsorption was imaged using a Hamamatsu ORCA-Flash4.0 V2 Digital CMOS camera on a Nikon Eclipse 80i fluorescence microscope with a 10X objective. ImageJ/FIJI (https://imagej.nih.gov/ij/) was used to quantify the protein adsorption data on 12 different imaging areas from each sample. All images were converted into binary images with a fixed threshold to enable sample comparison. Statistical methods used to analyse the data were obtained using Prism (GraphPad Software).