Protein adsorption on crosslinked polydimethylsiloxane using total internal reflection fluorescence

Abstract

Total internal reflection fluorescence (TIRF) is used to examine the adsorption of bovine serum albumin (BSA) and bovine fibrinogen on crosslinked polydimethylsiloxane (silicone rubber) surfaces from flowing solutions. By comparing experimentally observed adsorption rates with the predictions of a convection/diffusion model, it is shown that the adsorption of both BSA and fibrinogen on silicone rubber is diffusion-controlled over the range of solution concentrations (0.01 < C o < 1000 mg% for BSA, and 1.0 < C 0 < 500 mg% for fibrinogen) and the wall shear rate (γ = 58 sec −1) used. The isotherm for BSA adsorbed on silicone rubber is best described by a model that incorporates both irreversibly and reversibly adsorbed protein. The presence of reversibly adsorbed protein was demonstrated by exchanging fluorescently labeled adsorbed protein with unlabeled protein. Silicone rubber becomes saturated with BSA at a surface concentration of 0.14 μg/cm 2 for BSA solution concentrations higher than 2 mg%. The fibrinogen isotherm does not show a saturation plateau within the range of concentrations used. At 500 mg%, the highest solution concentration examined, the surface concentration of fibrinogen on silicone rubber is 0.40 μg/cm 2. Competitive adsorption of BSA and fibrinogen (100 mg% BSA, 8 mg% fibrinogen, γ = 58 sec −1) showed that BSA adsorbed initially with rates comparable to pure BSA diffusion-limited adsorption rates, fibrinogen adsorption followed, on surfaces now partially covered with BSA, at rates slower than fibrinogen adsorbing alone. Fibrinogen adsorption continued long after BSA adsorption had equilibrated at 0.12 μg/cm 2, after 6 hr, the fibrinogen surface concentration was 0.06 μg/cm 2. This continued fibrinogen adsorption may be due to the presence of different preferred sites for BSA and fibrinogen on the surface, or to multilayer adsorption. The partitioning between surface adsorbed and solution proteins shows that fibrinogen is preferentially adsorbed over BSA.