AFM STUDIES OF PROTEIN ADSORPTION .2. CHARACTERIZATION OF IMMUNOGLOBULIN-G ADSORPTION BY DETERGENT WASHING

The adsorption of immunoglobulin G (IgG) on chemically modified mica s urfaces via physical and covalent immobilisation and the effect of det ergent washing have been studied using atomic force microscopy (AFM). The chemical modification was performed by introducing amino groups to the mica surface via chemical vapor deposition of 3-aminopropyltrieth oxy silane. The covalent and physical immobilization of IgG on the sil anized mica surfaces were carried out with and without the use of a co upling reagent, glutaraldehyde, in three different buffer solutions of pH 7.3, 6.5, and 5.0 and at concentrations of 25 and 100 mu g . ml(-1 ). The samples were subsequently subjected to sodium dodecyl sulfate d etergent washing. The IgG molecules either covalently attached to the glutaraldehyde/silanized mica surfaces or physically adsorbed onto the silanized mica surfaces before and after the detergent washing were c haracterized qualitatively and quantitatively using AFM in the corresp onding buffer solutions, in terms of surface topography, surface rough ness, layer thickness, and adhesive forces. The experimental results s how that detergent washing removes loosely adsorbed IgG molecules from the surfaces, reduces the surface roughness and the adhesive force, a nd, most importantly, improves the resolution of AFM images obtained. Remarkable differences in the AFM topography and data were observed, c learly indicating that (1) nonspecifically adsorbed IgG molecules on t he silanized mica form a partial mono- or multilayer of flat orientati on and with characteristic strand-like structures and (2) specifically bound IgG molecules build a compact monolayer of random orientations and with representative granular structures. This study demonstrates t hat AFM has the capability of characterizing protein adsorption qualit atively and quantitatively. (C) 1996 Academic Press, Inc.