Correlation between Surface Morphology and Surface Forces of Protein A Adsorbed on Mica

Abstract

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves.

Destabilization and removal of immobilized enzymes adsorbed onto polyethersulfone ultrafiltration membranes by salt solutions

“…On the other hand, it is relevant to underline that the convective transport to the membrane enhances protein overlapping with gel formation. These results are in agreement with those obtained by Ohnishi et al [37], who demonstrated protein layer was denser at the highest concentration tested, as it occurs in our work.…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…There are several reports about force curves taken between protein layers [488][489][490][491] or on biological membranes [466,492,493]. In most cases no short-range repulsive forces could unambiguously be identified.…”

Adsorption and Bioactivity of Protein A on Silicon Surfaces Studied by AFM and XPS

“…The immunoreaction that consists of binding immunoglobulin G (IgG) to protein A is one of the most employed immunoassays, due to its simplicity and its specific reaction. Protein A actually has a specific high affinity for IgG and a considerable stability over a wide range of temperatures and pH and in the presence of denaturing agents [7]. It has therefore been employed as a simple biochemical model for the sensing pad development of integrated optical immunosensors [8].…”