Formation and characterization of protein monolayers on oxygen-exposing surfaces by multiple-step self-chemisorption

Abstract

Extended protein monolayers were formed by multiple-step self-chemisorption and characterized by scanning force microscopy (SFM), X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry, and cyclic voltammetry. These uniform protein monolayers were deposited on oxygen-exposing surfaces (SiO 2, mica, glass, etc.) after preexposing these surfaces to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surfaces with glutaric dialdehyde (GD). The samples appear flat and robust under SFM imaging conditions, both at low and molecular resolution. The thickness of the three-layer structure measured by engraving the layer with the SFM tips (4.5 nm) matches fairly well the value expected for the supramolecular architecture. XPS performed after each of the three different stages of sample preparation confirmed the presence of the elements expected (such as Cu and S for the azurin layer) and allowed film growth to be followed. Similar results have been obtained for the thicknesses of the different layers by spectroscopic ellipsometry. Optical absorption spectroscopy has provided data consistent with a 75% surface coverage by a protein (sub)monolayer. Redox proteins immobilized with linkers involving a similar chemistry but providing groups binding to gold (2-mercaptoethylamine and GD) rather than to oxygen-exposing surfaces made it possible to record cyclic voltammograms of single monolayers. These electrochemical experiments have confirmed retention of the protein's redox activity upon surface immobilization. The chemisorption approach reported in the present paper appears to be applicable to all kinds of proteins.