A novel assay for selective determination of polynucleotides using atomic force microscopy in conjunction with the formation of the probe/target/DNA-gold nanoparticle sandwich structure at a gold surface is described. A 17-mer probe was attached to the surface for subsequent hybridization with a polynucleotide target. Due to the flat orientation of the probe-target hybrid with respect to the surface and the spatial obstruction of the unhybridized probes near the hybrids, the AFM images are not clear. The hybridization efficiency was estimated to be about 1.1% since certain surface features could not be resolved. The utilization of 30-mer-capped gold nanoparticles not only provides another dimension of selectivity, but also reorients the previously formed probe-target hybrid in such a way that the strands of the target become tethered with respect to the surface. This reorientation improves the resolution in imaging the hybridized target molecules and provides an accurate determination of the hybridization efficiency (16%).