The influence of substrate and ethanol treatment on properties and quantitative analysis of retention of recombinant spider silk coatings

Silk coatings have significant application potential in medical materials due to their biocompatibility. Through processing of silk materials and functionalization, new coating properties can be gained. Following initial protein adsorption on a surface, an ethanol post-treatment can be used to enhance properties. Ethanol is thought to induce changes in protein structure, but very little is known on its effect on the binding stability and retention of coatings. Here, we study the effect of ethanol post-treatment and substrate material on recombinant silk coating adhesion, stability, structural organization, and wettability. For critical information on binding, we use tritium labeling to accurately quantify the bound silk layer. Further, scanning electron microscopy, optical microscopy, spectroscopy techniques, and static contact angle measurements are used for detailed analysis of surface-bound protein. Ethanol is shown to affect properties widely throughout the entire silk coating. It significantly increases binding stability on all tested substrates and improves adhesion to the substrate. Ethanol also renders otherwise hydrophilic coatings more hydrophobic. Differences in the morphology of silk sublayer organization due to substrate material are also observed. Interestingly, no significant change in secondary structure is observed to explain the changes in properties observed. The presence of salt complicates the coating arrangement due to the appearance of salt crystals but enables even higher retention and larger changes in wettability. Our work complements existing knowledge of ethanol post-treatment of silk coatings with important quantification of binding, which offers new insight and provides steps for improved durability of coatings in medical applications.