Oligonucleotides whose 5 ' ends are: linked to mercaptohexyl tether groups (HS-ss-DNA) are attached onto gold regions freshly exposed by reductive desorption of the mercaptopropionic acid (MPA) in a mixed MPA/hexanethiol (HT) or MPA/mercaptohexanol (MCH) self-assembled monolayer (SAM). The size of the bare Au region was shown to be controllable by using various MPA/HT solution compositions during the fabrication of the mixed SAMs. Atomic force microscopy shows that the size of the segregated gold regions has a profound effect on the surface density and orientation of the subsequently immobilized HS-ss-DNA molecules. The DNA surface density begins to increase when the bare gold domain (12-19 nm wide, prepared by stripping MPA off a mixed SAM formed from a solution with an alkanethiol molar ratio of chi (MPA)/chi (HT) = 1/4) approaches the size of the HS-ss-DNA (ca. 5-6 nm for a 17mer). The surface density of the HS-ss-DNA in the mixed HS-ss-DNA/HT SAM or the mixed HS-ss-DNA/MCH SAM is slightly greater than SAMs containing only the HS-ss-DNA, but the orientation of the HS-ss-DNA in the mixed SAM is more favorable for DNA hybridization. The quartz crystal microbalance (QCM) responses for the DNA hybridization at the DNA SAMs and mixed SAMs of HS-ss-DNA and alkanethiol formed using various adsorption methods were compared. The mixed HS-ss-DNA/HT formed using the new method resulted in a QCM signal amplification by almost 1 order of magnitude over SAMs containing only the HS-ss-DNA, with a much improved hybridization efficiency (ca. 96%).