Steric exclusion forces and protein imaging using the atomic force microscope

The atomic force microscope (AFM) was evaluated for its potential to image proteins adsorbing to surfaces from solution. Imaging of immunoglobulin M (IgM) with forces as low as 4 nN produced manipulation of the protein on mica. The IgM adsorbed to the surface was swept into aggregates that would line up to form strands on mica. Extent of manipulation depended on the magnitude of applied force and duration of scanning. Manipulation of the adsorbed proteins made it difficult to observe individual molecules of IgM on the mica surface. Although seemingly detrimental, manipulation may be useful after refinement. Manipulation of an adsorbed layer of protein into predetermined patterns was demonstrated provided the protein had sufficient affinity for the surface.;The usefulness of the AFM to image proteins is limited by its resolution capabilities even if protein manipulation could be eliminated. Steric exclusion imaging with PEO did not eliminate manipulation and produced images with poor resolution. Better derivatization is required to evaluate the performance of PEG modified tips.;AFM tips were modified with 200 kDa or 900 kDa polyethylene oxide (PEO) by physical adsorption or with 2 kDa and 5 kDa polyethylene glycol (PEG) by chemical binding. This modification allowed imaging of surfaces using steric exclusion forces. Steric exclusion forces were expected to be more forgiving to adsorbed proteins and diminish the extent of protein manipulation. Force plots obtained with PEO physisorbed tips demonstrated long-range steric exclusion forces. Multiple compression cycles, however, caused the physisorbed PEO to be exuded from the contact area producing large adhesion forces. 200 kDa PEO modified tips were used to image a gold diffraction grating with steric exclusion forces. Image resolution was poor with small applied forces but improved with increasing applied force. Imaging of adsorbed IgM with 200 kDa PEO tips did not prevent manipulation of the protein on mica. Since the steric exclusion force for the chemically bound PEG is much shorter-ranged, steric exclusion was demonstrated by the disappearance of the repulsive force upon addition of a poor solvent. Chemical coupling of the PEG with 3-aminopropyltriethoxysilane produced multilayers preventing comparison of the force plots to force-distance profiles calculated from self-consistent mean field theory.