The design and fabrication of various nanoscale architectures for the effective MALDI-TOF MS analysis of surface-confined DNA are reported. The nanoscale architectures are derived from layer-by-layer assemblies of negatively charged nanoparticles and positively charged polymers. An important finding is that porous structures comprising bottom SiO(2) nanoparticle layers and top Au nanoparticle layers enable the observation of both covalently bound (with Au-S bond) and hybridized oligonucleotides. The achievement of this unprecedented detection capability is attributed to the unique combination of a nanoporous dielectric film and discrete noble metal nanoparticles. The conclusion is supported by control experiments performed on a variety of single and multilayered Au nanoparticle structures, where only the hybridized oligonucleotides in a two- or three-strand system could be detected.