Optimization of the probe adsorption has a major key in the preparation of electrochemical sensors for the detection of oligonucleotide sequences hybridization. The role of a mixed monolayer of ssDNA sequences and MCH coadsorbed on a gold electrode surface was studied in this work. The working electrode was modified by chemisorption using a solution of thiol-tethered 33-mer DNA probe and mercaptohexanol (MCH), in a concentration range from 2 nM to 20 mu M. The probe surface density was monitored by means of electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and chronocoulometry. From EIS measurements, the charge transfer resistance was obtained as a function of the MCH concentration in the immobilization solution. The time dependence of mixed SAM adsorption was also investigated. The SAM adsorption was characterized regarding the electrode surface coverage with DPV and EIS measurements. Moreover, the probe surface density was investigated with chronocoulometry in Ru(NH3)(6)(3+) solution. Sensor behavior and sensitivity showed significant differences as a function of ssDNA/MCH concentration ratio as hybridization detection efficiency decreases while increasing the MCH concentration. The effect of different probe density in the hybridization detection efficiency was determined. Results demonstrated the effective of the coadsorption of ssDNA and thiols to control the SAM property and the probe density. It was therefore shown the importance to identify the correct density of probes on the electrode, below the saturation value, to ensure both a proper hybridization process and having a high hybridization signal. (c) 2012 Elsevier B.V. All rights reserved.