title: Isotachophoresis with ionic spacer and two-stage separation for high sensitivity DNA hybridization assay.
abstract: We present an on-chip electrophoretic assay for rapid and high sensitivity nucleic acid (NA) detection. The assay uses isotachophoresis (ITP) to enhance NA hybridization and an ionic spacer molecule to subsequently separate reaction products. In the first stage, the probe and target focus and mix rapidly in free solution under ITP. The reaction mixture then enters a region containing a sieving matrix, which allows the spacer ion to overtake and separate the slower probe-target complex from free, unhybridized probes. This results in the formation of two focused ITP peaks corresponding to probe and probe-target complex signals. For a 149 nt DNA target, we achieve a 220 fM limit of detection (LOD) within 10 min, with a 3.5 decade dynamic range. This LOD constitutes a 12× improvement over previous ITP-based hybridization assays. The technique offers an alternative to traditional DNA hybridization assays, and can be multiplexed and extended to detect other biomolecules.
authors.affiliation: Department of Mechanical Engineering, Stanford University, 440 Escondido Mall, Bldg 530, room 225, Stanford, CA 94305, USA.
authors.forename: Charbel
authors.initials: C
authors.lastname: Eid
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Garcia-Schwarz
authors.forename: Juan G
authors.initials: JG
authors.lastname: Santiago
mesh_terms: D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods*; D046208:Microchip Analytical Procedures; D009693:Nucleic Acid Hybridization / Q000379:methods*; D059629:Signal-To-Noise Ratio; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; D004247:DNA