We report on the design of electrochemical DNA-biosensors made from 15, 25 and 50 mm diameter gold ultramicroelectrodes (UMEs) in a two-electrode setup. The biosensors were prepared by chemical adsorption of 22-base thiol-labeled DNA-probes in a self-assembled monolayer configuration onto the surface of the gold UME. Hybridization events were transduced by Fe-III/Fe-II redox process electromediated by DNA-redox intercalated mediators. The optimization of the DNA-biosensors was achieved by using different FeIII/FeII ligands (cyanide, cefadroxil, deferoxamine) and intercalators (methylene blue, proflavine). Optimized biosensors allowed direct and selective amperometric detection in the femtomolar range and of single mismatch within a 35 minutes protocol. (C) 2016 Elsevier Ltd. All rights reserved.