Imaging of protein layers with an optical microscope for the characterization of peptide microarrays

Solid-phase assays play a crucial role today in biological studies. These assays are based on the immobilization of probe molecules on a surface, which are able to capture specifically soluble receptors. In particular, peptide microarrays have emerged as powerful tools in a variety of applications. In this context, optical techniques that allow imaging of nanometer-thick biomolecular films, and thereby the characterization of microarrays, are of great interest. For this purpose, we used a recently disclosed wide-field optical imaging technique of surface nanostructures called Sarfus, which is based on the use of a standard optical microscope and antireflection substrates. We demonstrate here that this technique allows the imaging of the protein layers that result from the specific capture of antibodies by arrayed peptide probes with a spatial resolution of 0.45 microm. The relationship between the thickness of the antibody layer and peptide or antibody concentration was examined.