Biomedical Microdevices

, Volume 12, Issue 2, pp 333–343 | Cite as

Glass-composite prototyping for flow PCR with in situ DNA analysis

  • Ilija Pješčić
  • Collin Tranter
  • Patrick L. Hindmarsh
  • Niel D. Crews


In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 µl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 µl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (ΦX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.


Microfluidics Continuous-flow PCR DNA melting analysis Xurography Thermal gradient Rapid prototyping 



Authors credit funding by Louisiana Tech University and the Louisiana Space Consortium (LaSPACE). Authors thank Jimmy Cook for machine shop support, Debbie Wood, Dee Tatum, and the rest of the technical staff at the Institute for Micromanufacturing for their support on this work. Special thanks is also given to Dr. Rastko Selmic for his role in the formation of this research team, and Dr. Eric Gilbeau for his mentorship and assistance with the buildup of the research laboratory.

Supplementary material

10544_2009_9389_MOESM1_ESM.pdf (174 kb)
Supplementary Material (PDF 173 kb)


  1. D.A. Bartholomeusz, R.W. Boutte, J.D. Andrade, Xurography: Rapid prototyping of microstructures using a cutting plotter. J. Microelectromech. Syst. 14(6), 1364–1374 (2005)CrossRefGoogle Scholar
  2. H. Becker, C. Gartner, Polymer microfabrication technologies for microfluidic systems. Anal. Bioanal. Chem. 390(1), 89–111 (2008)CrossRefGoogle Scholar
  3. N. Crews, T. Ameel, C.T. Wittwer, B. Gale, Flow Induced thermal effects in spatial DNA melting. Lab Chip 8, 1922–1929 (2008a)CrossRefGoogle Scholar
  4. N. Crews, C.T. Wittwer, B. Gale, Continuous-flow thermal gradient PCR. Biomed. Microdevices 10(2), 187–195 (2008b)CrossRefGoogle Scholar
  5. N. Crews, C.T. Wittwer, R. Palais, B. Gale, Product differentiation during continuous-flow thermal gradient PCR. Lab Chip 8, 919–924 (2008c)CrossRefGoogle Scholar
  6. N. Crews, C.T. Wittwer, J. Montgomery, R. Pryor, B. Gale, Spatial DNA melting analysis for genotyping and variant scanning. Anal. Chem. 81(6), 2053–2058 (2009)CrossRefGoogle Scholar
  7. I. Erill, S. Campoy, N. Erill, J. Barbé, J. Aguiló, Biochemical analysis and optimization of inhibition and adsorption phenomena in glass-silicon PCR-chips. Sens. Actuators, B 96(3), 685–692 (2003)CrossRefGoogle Scholar
  8. J. Greer, S.O. Sundberg, C.T. Wittwer, B.K. Gale, Comparison of glass etching to xurography prototyping of microfluidic channels for DNA melting analysis. J Micromech Microeng 17(12), 2407–2413 (2007)CrossRefGoogle Scholar
  9. M. Hashimoto, P.C. Chen, M.W. Mitchell, D.E. Nikitopoulos, S.A. Soper, M.C. Murphy, Rapid PCR in a continuous flow device. Lab Chip 4(6), 638–645 (2004)CrossRefGoogle Scholar
  10. M.G. Herrmann, J.D. Durtschi, L.K. Bromley, C.T. Wittwer, K.V. Voelkerding, Amplicon DNA melting analysis for mutation scanning and genotyping: Cross-platform comparison of instruments and dyes. Clin. Chem. 52(3), 494–503 (2006)CrossRefGoogle Scholar
  11. M.U. Kopp, A.J. de Mello, A. Manz, Chemical amplification: continuous-flow PCR on a chip. Science 280(5366), 1046–1048 (1998)CrossRefGoogle Scholar
  12. T. Nakayama, Y. Kurosawa, S. Furui, K. Kerman, M. Kobayashi, S.R. Rao, Y. Yonezawa, K. Nakano, A. Hino, S. Yamamura, Y. Takamura, E. Tamiya, Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications. Anal. Bioanal. Chem. 386(5), 1327–1333 (2006)CrossRefGoogle Scholar
  13. P.J. Obeid, T.K. Christopoulos, H.J. Crabtree, C.J. Backhouse, Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection. Anal. Chem. 75(2), 288–295 (2003)CrossRefGoogle Scholar
  14. Y. Schaerli, R.C. Wootton, T. Robinson, V. Stein, C. Dunsby, M.A.A. Neil, P.M.W. French, A.J. deMello, C. Abell, F. Hollfelder, Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets. Anal. Chem. 81(1), 302–306 (2008)CrossRefGoogle Scholar
  15. I. Schneegass, R. Brautigam, J.M. Kohler, Miniaturized flow-through PCR with different template types in a silicon chip thermocycler. Lab Chip 1(1), 42–49 (2001)CrossRefGoogle Scholar
  16. S. Sundberg, C. Wittwer, J. Greer, R. Pryor, O. Elenitoba-Johnson, B. Gale, Solution-phase DNA mutation scanning and SNP genotyping by nanoliter melting analysis. Biomed. Microdevices 9, 159–166 (2007)CrossRefGoogle Scholar
  17. C.T. Wittwer, M.G. Hermann, Rapid thermal cycling and PCR kinetics, in PCR applications: Protocols for functional genomics, ed. by M.A. Innis, D.H. Gelfand, J.J. Sninsky, 1st edn. (Academic, San Diego, 1999), pp. 211–229Google Scholar
  18. L. Zhou, A.N. Myers, J.G. Vandersteen, L. Wang, C.T. Wittwer, Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin. Chem. 50(8), 1328–1335 (2004)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ilija Pješčić
    • 1
  • Collin Tranter
    • 1
  • Patrick L. Hindmarsh
    • 2
  • Niel D. Crews
    • 1
  1. 1.Institute for MicromanufacturingLouisiana Tech UniversityRustonUSA
  2. 2.School of Biological SciencesLouisiana Tech UniversityRustonUSA

Personalised recommendations