Integrated microfluidic tmrna purification and real-time nasba device for molecular diagnostics
Dimov, Ivan K.
Garcia-Cordero, Jose L.
Poulsen, Claus R.
Smith, Terry J.
Ricco, Antonio J.
Lee, Luke P.
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Dimov, Ivan K. Garcia-Cordero, Jose L.; O'Grady, Justin; Poulsen, Claus R.; Viguier, Caroline; Kent, Lorcan; Daly, Paul; Lincoln, Bryan; Maher, Majella; O'Kennedy, Richard; Smith, Terry J.; Ricco, Antonio J.; Lee, Luke P. (2008). Integrated microfluidic tmrna purification and real-time nasba device for molecular diagnostics. Lab on a Chip 8 (12), 2071-2078
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 &lt; 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 mu L of crude lysate in under 30 min for the entire process.