Structural characterization of protein microsensors arrays by means of optical profilometry and AFM

A microarray is a matrix containing from hundreds to thousands of microscopic sensory elements or spots printed on a flat functionalized surface which allows a specific interaction of multiple biomolecules including proteins. Some examples of reading this technology include the surface plasmon resonance and variable wavelength scanners used to determine the superficial density of biomolecules interacting with the microsensors. Nevertheless, none of these techniques provides the information relative to the structure of the interaction of the microsites fabricated for the biosensing. As a result, in this work we propose the combined use of the Atomic Force Microscopy (AFM) and optical profilometry to determine the structure and density of the interaction of microsite lines of bovine serum albumin (0.1, 0.5, 0.75 and 1.0 mg/ml) fabricated on the previously functionalized gold/glass substrate. For this purpose, we utilized solutions of bovine serum albumin (1.0 mg/ml) as the analytes during the protein-protein interaction. The negative control microsites corresponded to a line of white solutions of fibrinogen of human serum (1.0 mg/ml) which proved that the surface density (molecules/area) of the not-washed BSA spots is correlated to their thicknesses: 957.9 nm (1.0 mg/ml), 636.6 nm (0.75 mg/ml), 639.7 nm (0.5 mg/ml), and 490.4 nm (0.1 nm), whereas after the interaction with anti-BSA (1.0 mg/ml) they corresponded to 508.6, 218.0, 170.7, and 130.8 nm, respectively. In this way we proved that, before and after the protein interaction, the average spot roughness decreased with the concentration of the protein used for the fabrication of microsensors.