Electrochemical DNA-biosensors based on long-range electron transfer: optimization of the amperometric detection in the femtomolar range using two-electrode setup and ultramicroelectrode

We report on the design of electrochemical DNA-biosensors made from 15, 25 and 50 μm 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 FeIII/FeII redox process electro-mediated 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.