A ternary surface consisting of the 5' end 6-mercapto-1-hexane-labeled single-strand DNA (HS-ssDNA) and of thiol alkanes, including 6-mercapto-1-hexanol (MCH) and 11-mercaptoundecanoic acid (MUDA) (abbreviated as HS-ssDNA/MCH/MUDA), was constructed on a polycrystalline gold electrode. Label-free electrochemical sensing of DNA hybridization was performed in the presence of [Fe(CN)(6)](3-) and the double strand-specific intercalator methylene blue (MB). For comparison, other ternary surfaces were constructed using 3-mercaptopropionic acid (MPA) and 11-mercaptoundecanol (MUD) diluents. The observed discrimination effect (ratio of charge transfer resistance, R. between the hybridized and non-complementary hybridized surfaces) for the different ternary surfaces followed the order of HS-ssDNA/MCH/MUDA (1.89) > HS-ssDNA/MPA/MCH (1.37) > HS-ssDNA/MCH/MUD (1.27) > HS-ssDNA/MPA/MUD (0) = HS-ssDNA/MPA/MUDA (0). Differences in the discrimination effects among the ternary surfaces are related to the pinhole behavior of the molecular layer, which is influenced by the chain length and head group functionalities responsible for the intermolecular hydrogen bonding. This criterion was met only when the difference in the chain lengths of the two alkane diluents in the ternary was less than 50%. While the ternary HS-ssDNA/MCH/MUDA surface showed a detection range of 1 x 10(-18) to 1 x 10(-6) M, the ternary HS-ssDNA/MPA/MCH exhibited a range from 1 x 10(-12) to 1 x 10(-6) M. The remarkable increase in sensitivity was attributed to the elimination of non-specific Au-DNA interactions and the proper orientation of HS-ssDNA on the ternary surfaces. The surfaces were characterized using cyclic voltammetry (CV), impedance spectroscopy (IS), chronocoulometry (CC) and Fourier-transform infrared (FTIR) spectroscopy. (C) 2011 Elsevier Ltd. All rights reserved.