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Microfluidic & nanofluidic technologies for chemistry, physics, biology, and bioengineering



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Paper

Lab Chip, 2008, 8, 2071 - 2078, DOI: 10.1039/b812515e

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics

Ivan K. Dimov, Jose L. Garcia-Cordero, Justin O'Grady, Claus R. Poulsen, Caroline Viguier, Lorcan Kent, Paul Daly, Bryan Lincoln, Majella Maher, Richard O'Kennedy, Terry J. Smith, Antonio J. Ricco and Luke P. Lee

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 µL of crude lysate in under 30 min for the entire process.

Graphical abstract image for this article (ID: b812515e)