Expressed Peptide Assay for DNA Detection

Innovation in molecular diagnostics ultimately requires the conceptually distinct design of detection architectures. The diagnostic strategies reported thus far (planar/suspension arrays) suffer from either mass transport issues or limitations on the maximum number of targets that can be simultaneously detected. We report herein an expressed peptide assay scheme, by using nanoparticle probes, for detecting DNA hybridization events. The method exploits plasmid-encoded peptide tags as surrogate molecules for the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of target DNA. The binding of target DNA is achieved through its recognition with a gold nanoparticle probe (functionalized with peptide-encoding plasmid and oligonucleotide complementary to part of the target sequence) and a microparticle probe (derivatized with oligonucleotide complementary to the rest of the target sequence). The magnetic separation of the three-component complex and expression of the peptide allows for the target identification by mass spectrometry. The detection of two DNA targets has been demonstrated through the selection of each individual tag for the respective target. Importantly, the modular nature of the probe design, by decoupling molecular binding events from peptide expression processes, should enable the ready extension of the methodology to the analysis of other species. An assay on a protein target has confirmed the efficacy of the conceptual framework proposed herein beyond the detection of DNA. The vast choice of mass tags offered by mass spectrometry provides significant advantages over previously documented assay systems.