Isotachophoretic Focusing of Nucleic Acids

Applications

• Microfluidic chips or capillary-based systems for:
  • point-of-care diagnostics
  • biowarfare detection
  • research

Advantages

• Fast:
  • preconcentration, extraction, identification, and separation assays take 10's to 100's of seconds
  • assays can be performed directly from cell lysate (cultured cells, whole blood or other tissue)
• High sensitivity - detection in the 100 amol range
• High volume throughput - can process on the order of 100 ul volumes of DNA in 20 minutes (1 ml volume in 60 min is feasible)
• Small sample volume - can process as little as 1 nL of sample
• High efficiency - between 30 and 100% efficiency
• Low cost - less reagents are needed because there is limited sample preparation and small sample sizes needed for detection
• Selective - can selectively extract 22-base RNA from samples also containing >200-base RNA, >100bp DNA and proteins
• High preconcentration factors - million-fold for simple molecules; 10,000-fold for for proteins or nucleic acids from a complex mixture
• Robust - ITP zones are self-stabilizing and injection protocols are easily conducted with two pipetting dispensions
• Automated - no manual steps are needed between sample on-chip delivery and molecule identification
• Parallelization - miniaturization and automation allows for hugely parallel purification and identification of biomolecules.

Publications

• Bercovici M., Kaigala G.V., Mach, K.E., Han, C.M., Liao, J.C., and Santiago J.G., "Rapid detection of urinary tract infections using isotachophoresis and molecular beacons," in press, Analytical Chemistry, 2011.
• Persat, A. and Santiago, J. G., “microRNA profiling by simultaneous selective isotachophoresis and hybridization with molecular beacons,” 83, 6, pp. 2310-2316, Analytical Chemistry, 2011.
• Persat, A., Chivukula, R., Mendell, J.T. and Santiago, J.G., “Quantification of Global microRNA Abundance by Selective Isotachophoresis” 82, 23, Analytical Chemistry, pp. 9631-9635, 2010.
• Schoch, R.B., M. Ronaghi, and J.G. Santiago, "Rapid and selective extraction, separation, preconcentration, and detection of small RNAs from cell lysate using on-chip isotachophoresis," 9, 15, 2145 – 2152, Lab on a Chip, 2009.
• Persat, A., Marshall, L. and J.G. Santiago, “Purification of Nucleic Acids from Whole Blood Using Isotachophoresis,” 81, Analytical Chemistry, pp. 9507-9511, 2009.
• Bercovici, M., S.K. Lele, and J.G. Santiago, "Open source simulation tool for electrophoretic stacking, focusing, and separation” Journal of Chromatography A, 1216, p. 1008-1018, 2008.
• Khurana, T., and J.G. Santiago, "Sample Zone Dynamics in Peak Mode Isotachophoresis," 80, 16, Analytical Chemistry, p. 6300-6307, 2008.

Published patent application:

US-2010-0224494-A1

Related Links

• Stanford Microfluidics Laboratory
• Utz Lab

Patents

• Published Application: 20100224494
• Issued: 8,846,314 (USA)
• Issued: 9,753,007 (USA)