Gold nanoparticles functionalized with thiol-oligonucleotides are ideal platforms for detection of specific DNA sequences. Here we evaluate the effect of single base mismatches in hybridization efficiency according to the position of the mismatch, base pairing combination and thiol-oligonucleotide density in terms of specificity and efficiency of target recognition. Hybridization efficiency and single-nucleotide polymorphism discrimination at room temperature is maximized at a density of 83+/-4 thiol-oligonucleotides per 13.5 nm gold nanoparticle (24 pmol/cm(2)), and when the mismatch is localized at the 3'-end of the Au-nanoprobe, i.e. away from the gold nanoparticle surface.