Molecular diagnostics lies at the heart of modern-day chemistry and biology. The identification of genetic and proteomic targets and patterns relies on the proper choice of detection format, a combination of biological recognition architecture and signal readout scheme. Biochip technology, [1] by placing multiple probes on a single flat surface, allows for the storage of tremendous amount of information and massively parallel detection of target species. However, the convenience of use associated with this strategy usually comes at the cost of complexity in the fabrication of surface-bound recognition elements because of limitations in the signal readout.[2] The serial generation of spatially resolved spot arrays, either photolithographically or through a microarrayer, is typically a labor-intensive procedure. Mass spectrometry (MS), [3] a tool with inherent multiplexing capability, offers a potentially attractive alternative by providing massresolved signatures simultaneously for multiple analytes, thus obviating the necessity to use patterned arrays. However, for molecules with low ionization efficiency and facile fragmentation such as DNA, [4] the implementation of such a technique reproducibly in chip-based biodiagnostics is extremely challenging. Thus far, only a couple of attempts have been documented and none have been elaborated into routine practice.[5] Moreover, these methods suffer from restrictions on measurable DNA sizes, which pose significant obstacles for real clinical samples. Sequence-specific detection of DNA is crucial to the diagnosis of genetic and pathogenic diseases and central to the biological and medical research. A diagnostic platform that takes advantage of massive parallelization offered by gene chips while at the same time eliminates the detection bottleneck associated with MS, which is broadly applicable for DNA of arbitrary length, can provide a powerful technology for numerous fields. Herein, we report on the utility of barcoded nanoparticles for on-chip DNA hybridization assay, by employing surrogate molecules as mass tags with amplified viable signals coupled with laser desorption/ionization time-of-flight (LDI-TOF) MS for readout. The research is part of our ongoing efforts to exploit nanoparticles functionalized with probe and MS tagging elements for multiplexed analysis of biomolecules.A three-component sandwich assay, composed of surfacebound capture strands, target strands to be detected, and probe-strand-capped, monolayer-barcoded nanoparticles (MBNs), was designed for DNA hybridization (Scheme 1).Key to the success of this method is the preparation of MBNs, which, by drawing on well-established monolayer chemistry, [6] could be carried out with a one-pot, two-step strategy (Scheme 1). The first step involves functionalization of nanoparticles with appropriately modified oligonucleotide structures. Subsequent derivatization with mass-tag molecules for barcoding purposes, through ligand-exchange process, affords MBNs that simultaneously incorporate hybridization and readou...