Formation and characterization of protein monolayers on oxygen-exposing surfaces by multiple-step self-chemisorption Paolo Facci a, * , Dario Alliata a , Laura Andolfi a , Bernhard Schnyder b , R€ udiger K€ otz b a Istituto Nazionale per la Fisica della Materia, c/o Dipartimento di Fisica, Universit  a di Modena e Reggio Emilia, I-41100 Modena, Italy b Department of General Energy Research, Paul Scherrer Institute, CH-5232 Villigen-PSI, Switzerland Received 28 September 2001; accepted for publication 14 December 2001 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 preex- posing these surfaces to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surfaces with glutaric dial- dehyde (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. Ó 2002 Published by Elsevier Science B.V. Keywords: Atomic force microscopy; Electrochemical methods; Ellipsometry; X-ray photoelectron spectroscopy; Chemisorption; Biological molecules – proteins 1. Introduction Research in the fields of molecular and bio- molecular electronics [1], biosensors [2], biocatal- ysis, and biotechnology in general [3], has a very compelling need for methods and procedures yielding high-quality molecular monolayers on Surface Science 504 (2002) 282–292 www.elsevier.com/locate/susc * Corresponding author. E-mail address: p.facci@unimo.it (P. Facci). 0039-6028/02/$ - see front matter Ó 2002 Published by Elsevier Science B.V. PII:S0039-6028(02)01148-2