Quantification of specific immunological reactions by atomic force microscopy

The aim of this work is to demonstrate the ability of atomic force microscopy (AFM) to detect and to quantify specific immunological reactions between antibodies and antigens, with a view to creating a very sensitive biosensor. A monolayer of antiferritin antibodies was adsorbed onto alkyl silane modified silicon oxide substrates, which were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The sensitivity limit for antibody detection was quantified by radioimmunoassay (RIA) and compared to that obtained by enzyme linked immune sorbent assay (ELISA) and by AFM after antibody binding with colloidal gold labeled conjugates. In this latter case, substrate modification after reaction was checked by measuring the surface roughness (R-rms) variations. AFM was found to be more sensitive than RIA, with a detection limit of 0.3 x 10(-3) ng of antibodies per mm(2). Then, the biosensor performance was investigated using ferritin solutions of various concentrations: the antibody/antigen reaction was quantified by directly detecting the antigen and measuring surface roughness modifications. Results were compared to sandwich immunoassay techniques. Up to now, AFM has detected a minimum ferritin concentration of 0.06 mu g/mL.