Effects of Initially Adsorbed Proteins on Substrate Surfaces during Multilayer Heterogeneous Protein Adsorption

When multilayer heterogeneous proteins are adsorbed on substrate surfaces, the effects of the adsorption state of the initially adsorbed proteins may affect subsequent adsorption. In this study, the relationships between the adsorption state of the initially adsorbed proteins and the amount of secondary adsorbed proteins were examined. A carboxylate-terminated self-assembled monolayer was applied to bovine serum albumin (BSA) solutions of different concentrations for 180 min and subsequently applied to phosphate-buffered saline (PBS) for an additional 180 min to remove weakly adsorbed proteins. The amount of adsorbed proteins was measured using a quartz crystal microbalance with dissipation. The obtained BSA adsorption layer was then applied to mucin solution for 60 min. When a 1.7 mg/mL BSA solution was applied to the surface, the amount of adsorbed BSA after 10 min of adsorption and after washing with PBS for 167 min was >5 × 10² ng/cm², representing the saturation amount of monolayer-adsorbed BSA in a side-on orientation. In contrast, the amount of adsorbed BSA after 10 min adsorption was <5 × 10² ng/cm² when a BSA solution with a concentration <0.43 mg/mL was used. The velocity of BSA adsorption plateaued at approximately 0.43 mg/mL, suggesting that the orientation of the adsorbed protein was determined by protein treatment concentration immediately after the proteins were adsorbed. Furthermore, the amount of adsorbed mucin on the BSA adsorption layer decreased as initial BSA treatment concentration increased up to 0.43 mg/mL and plateaued at concentrations above 0.43 mg/mL. These results indicated that the orientation of the initially adsorbed protein was preserved for several hours and affected the subsequent adsorption of mucin.