An atomic force microscope (AFM) has been used to directly monitor specific interactions between antibodies and antigens employed in an immunoassay system. Results were achieved using AFM probes functionalized with ferritin, and monitoring the adhesive forces between the probe and anti-ferritin antibody-coated substrates. Analysis of the force distribution data suggests a quantization of the forces, with a period of 49 +/- 10 pN. This periodic force may be attributed to single unbinding events between individual antigen and antibody molecules. These results demonstrate that the AFM could be employed as an analytical tool to study the interactions between the molecules involved in biosensor systems. The potential of the technique to provide information relating to the manner in which the antibody molecule binds to its specific antigen is also discussed.

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In this paper, we demonstrate in situ recognition of protein adsorption onto a polymer surface with scanning force microscopy by probe−surface adhesion measurements and topography imaging with protein-coated probes. Albumin-coated probes have been employed in studies of albumin and fibrinogen adsorption to hydrophobic polystyrene surfaces. The adhesion between force microscope probes and sample surfaces were determined using profiles of retract force−distance curves. A large adhesion force profile resulted when the force−distance curves were measured on protein-free polystyrene surfaces. When the measurements were conducted on protein-exposed polystyrene surfaces, the force−distance curves showed negligible adhesion. The same coated probes were also used for in situ topographic imaging. Applications of this novel approach are described:  first, the location of boundaries of preadsorbed protein films and, second, the dynamic detection of protein adsorption onto polystyrene surfaces. Two-dimensional adhesion energy maps were obtained by employing “layered imaging”. We also note that an increase in pressure exerted by the force microscope probe results in penetration of a protein film and contact of the probe with the underlying polystyrene.

Polystyrene microtitre wells are commonly used as supports for the enzyme-linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.