Abstract
Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL–5 μL), amplification speed (100–400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of PCR in 1 μL chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from λ-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple, relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
K.B. Mullis, F.A. Faloona, Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction (ETATS-UNIS: Elsevier, San Diego, 1987)
T.M. Rose, J.G. Henikoff, S. Henikoff, CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 31, 3763–3766 (2003)
Y.H. Kim, I. Yang, Y.S. Bae, S.R. Park, Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Biotechniques 44, 495–505 (2008)
I. Yang, Y.H. Kim, J.Y. Byun, S.R. Park, Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling. Anal. Biochem. 338(2), 192–200 (2005)
C.T. Wittwer, K.M. Ririe, R.V. Andrew, D.A. David, R.A. Gundry, U.J. Balis, The LightCycler(TM) a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22(1), 176–181 (1997)
J. Khandurina, T.E. McKnight, S.C. Jacobson, L.C. Waters, R.S. Foote, J.M. Ramsey, Integrated system for rapid PCR-based DNA analysis in microfluidic devices. Anal. Chem. 72(13), 2995–3000 (2000)
Y. Matsubara, K. Kerman, M. Kobayashi, S. Yamamura, Y. Morita, E. Tamiya, Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes. Biosens. Bioelectron. 20(8), 1482–1490 (2005)
H. Nagai, Y. Murakami, K. Yokoyama, E. Tamiya, High-throughput PCR in silicon based microchamber array. Biosens. Bioelectron. 16(9–12), 1015–1019 (2001)
M. Slyadnev, M. Lavrova, M. Erkin, V. Kazakov, A. Ganeev, Development of a multireactor microfluidic system for the determination of DNA using real-time polymerase chain reaction. J. Anal. Chem. 63(2), 192–198 (2008)
C.-S. Liao, G.-B. Lee, H.-S. Liu, T.-M. Hsieh, C.-H. Luo, Miniature RT-PCR system for diagnosis of RNAbased viruses. Nucleic Acids Res. 33(18), e156 (2005)
K.-Y. Lien, W.-C. Lee, H.-Y. Lei, G.-B. Lee, Integrated reverse transcription polymerase chain reaction systems for virus detection. Biosens. Bioelectron. 22(8), 1739–1748 (2007)
P. Liu, T.S. Seo, N. Beyor, K.-J. Shin, J.R. Scherer, R.A. Mathies, Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing. Anal. Chem. 79(5), 1881–1889 (2007)
B. Minqiang et al., Design and theoretical evaluation of a novel microfluidic device to be used for PCR. J. Micromech. Microeng. 13(4), S125 (2003)
P. Neuzil, C. Zhang, J. Pipper, S. Oh, L. Zhuo, Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes. Nucleic Acids Res. 34(11), e77 (2006)
M.A. Northrup, B. Benett, D. Hadley, P. Landre, S. Lehew, J. Richards et al., A miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers. Anal. Chem. 70(5), 918–922 (1998)
Z. Wang, A. Sekulovic, J.P. Kutter, D.D. Bang, A. Wolff, Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR. Electrophoresis 27(24), 5051–5058 (2006)
A.T. Woolley, D. Hadley, P. Landre, A.J. de Mello, R.A. Mathies, M.A. Northrup, Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem. 68(23), 4081–4086 (1996)
Q. Xiang, B. Xu, R. Fu, D. Li, Real time PCR on disposable PDMS chip with a miniaturized thermal cycler. Biomed. Microdevice 7(4), 273–279 (2005)
Q.B. Zou, U. Sridhar, Y. Chen, J. Singh, Miniaturized, independently controllable multichamber thermal cycler. IEEE Sensors J. 3(6), 774–780 (2003)
M. Focke, F. Stumpf, G. Roth, R. Zengerle, F. von Stetten, Centrifugal microfluidic system for primary amplification and secondary real-time PCR. Lab Chip (2010)
E.K. Wheeler, W. Benett, P. Stratton, J. Richards, A. Chen, A. Christian et al., Convectively driven polymerase chain reaction thermal cycler. Anal. Chem. 76(14), 4011–4016 (2004)
C.T. Wittwer, G.C. Fillmore, D.J. Garling, Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal. Biochem. 186(2), 328–331 (1990)
C.J. Easley, J.M. Karlinsey, J.M. Bienvenue, L.A. Legendre, M.G. Roper, S.H. Feldman, J.P. Landers, A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability. Proc. Natl. Acad. Sci. 103(51), 19272–19277 (2006)
A.F.R. Hühmer, J.P. Landers, Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes. Anal. Chem. 72(21), 5507–5512 (2000)
R.P. Oda, M.A. Strausbauch, A.F.R. Huhmer, N. Borson, S.R. Jurrens, J. Craighead et al., Infrared mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA. Anal. Chem. 70(20), 4361–4368 (1998)
M. Slyandev, Y. Tanaka, M. Tokeshi, T. Kitamori, Photothermal temperature control of a chemical reaction on a microchip using an infrared diode laser. Anal. Chem. 73, 4037–4044 (2001)
H. Kim, S. Vishniakou, G.W. Faris, Petri dish PCR: laser-heated reactions in nanoliter droplet arrays. Lab Chip 9(9), 1230–1235 (2009)
H. Terazono, A. Hattori, H. Takei, K. Takeda, K. Yasuda, Development of 1480 nm photothermal highspeed real-time polymerase chain reaction system for rapid nucleotide recognition. Jpn. J. Appl. Phys. 47, 5212 (2008)
C.R. Phaneuf, N. Pak, C.R. Forest, Modeling radiative heating of liquids in microchip reaction chambers. Sens. Actuators, A 167, 531–536 (2011)
A.A. Sodemann, J.R. Mayor, Parametric investigation of precision in tool-workpiece conductivity touch-off method in micromilling. Trans. N. Am. Manuf. Res. Inst. SME 37, 565–572 (2009)
D.S. Lee, S.H. Park, H. Yang, K.H. Chung, T.H. Yoon, S.J. Kim, K. Kim, Y.T. Kim, Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption. Lab Chip 4, 401–407 (2004)
C. Zhang, J. Xu, W. Ma, W. Zheng, PCR microfluidic devices for DNA amplification. Biotechnol. Adv. 24(3), 243–284 (2006)
Author information
Authors and Affiliations
Corresponding author
Additional information
Nikita Pak and D. Curtis Saunders contributed equally to this work.
Rights and permissions
About this article
Cite this article
Pak, N., Saunders, D.C., Phaneuf, C.R. et al. Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip. Biomed Microdevices 14, 427–433 (2012). https://doi.org/10.1007/s10544-011-9619-2
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10544-011-9619-2