Mixed layers were obtained after one step deposition by reduction of a diazonium salts mixture. The layers are constituted of electroactive 5-hydroxy-1,4-naphthoquinone derivative (JUG), and 4-aminobenzoic acid (ABA) which allows subsequent grafting of DNA probes. Each functionalizaton step was characterized by IRRAS and XPS. The redox properties of JUG were used to quantify this compound in the film. The combination of surface analysis and voltammetry leads to a quantitative characterization of all the components at each step. The resulting layers are composed of distinct JUG and ABA domains despite the one step deposition procedure. The developed approach allowed rationalization of experimental conditions to design direct electrochemical DNA biosensor with a detection limit of 10 pM. (C) 2014 Elsevier Ltd. All rights reserved.