Isotachophoresis with ionic spacer and two-stage separation for high sensitivity DNA hybridization assay
| Autor: | Giancarlo Garcia-Schwarz, Charbel Eid, Juan G. Santiago |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
DNA
Bacterial Electrophoresis Detection limit chemistry.chemical_classification Time Factors Chromatography Biomolecule DNA–DNA hybridization Nucleic Acid Hybridization DNA Signal-To-Noise Ratio Biochemistry Analytical Chemistry Matrix (chemical analysis) chemistry.chemical_compound Spectrometry Fluorescence chemistry Microchip Analytical Procedures Electrochemistry Nucleic acid Environmental Chemistry Isotachophoresis Spectroscopy |
| Zdroj: | The Analyst. 138:3117 |
| ISSN: | 1364-5528 0003-2654 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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