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A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system

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Abstract

We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 μL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.

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References

  • D. Chen, M. Mauk, X. Qiu, C. Liu, J. Kim, S. Ramprasad, S. Ongagna, W.R. Abrams, D. Malamud, P. Corstjens, H.H. Bau, An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids. Biomed. Microdevices 12, 705–719 (2010)

    Article  Google Scholar 

  • J. Cheng, M.A. Shoffner, G.E. Hvichia, L.J. Kricka, P. Wilding, Chip PCR II: Evaluation of DNA fragment sizing and quantification by the Agilent 2100 Bioanalyzer. Clin. Chem. 46, 1851–1853 (2000)

    Google Scholar 

  • H.J. Crabtee, J. Lauzon, Y.C. Morrissey, B.J. Taylor, T. Liang, R.W. Johnstone, A.J. Stickel, D.P. Manage, A. Atrazhev, C.J. Backhouse, L.M. Pilarski, Inhibition of on-chip PCR using PDMS-glass hybrid microfluidic chips. Biomed. Microdevices 13, 383–398 (2012)

    Google Scholar 

  • M.D. Estes, J. Yang, B. Duane, S. Smith, C. Brooks, A. Nordquist, F. Zenhausern, Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis. Analyst 137, 5710–5719 (2012a)

    Article  Google Scholar 

  • M.D. Estes, J. Yang, B. Duane, S. Smith, C. Brooks, F. Zenhausern, Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis. Analyst 137, 5510–5519 (2012b)

    Article  Google Scholar 

  • T.H. Fang, N. Ramalingam, D. Xian-Dui, T.S. Ngin, Z. Xianting, A.T.L. Kuan, E.Y.P. Huat, G. Hai-Qing, Real-time PCR microfluidic devices with concurrent electrochemical detection. Biosens. Bioelectron. 24, 2131–2136 (2009)

    Article  Google Scholar 

  • O. Frey, S. Bonneick, A. Hierlemann, J. Lichtenberg, Autonomous microfluidic multi-channel hip for real-time PCR with integrated liquid handling. Biomed. Microdevices 9, 711–718 (2007)

    Article  Google Scholar 

  • B.C. Giordano, J. Ferrance, S. Swedberg, A.F. Huhmer, J.P. Landers, Polymerase chain reaction in polymeric microchips: DNA amplification in less than 240 seconds. Anal. Biochem. 291, 124–132 (2001)

    Article  Google Scholar 

  • P.M. Holland, R.D. Abramson, R. Watson, D.H. Gelfland, Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA. Proc. Natl. Acad. Sci. U. S. A. 88, 7276–7280 (1991)

    Article  Google Scholar 

  • A. Hopwood, C. Hurth, J. Yang, Z. Cai et al., Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile. Anal. Chem. 82, 6991–6999 (2010)

    Article  Google Scholar 

  • C. Hurth, S. Smith, A. Nordquist, R. Lenigk, B. Duane et al., An automated instrument for human STR identification: Design, characterization and experimental validation. Electrophoresis 31, 3510–3517 (2010)

    Article  Google Scholar 

  • 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, 2995–3000 (2000)

    Article  Google Scholar 

  • H. Kim, S. Dixit, C.J. Green, G.W. Faris, Nanodroplet real-time PCR system with laser assisted heating. Opt. Express 17, 218–227 (2009)

    Article  Google Scholar 

  • M.U. Kopp, A.J. de Mello, A. Manz, Chemical amplification: continuous-flow PCR on a chip. Science 280, 1046–1048 (1998)

    Article  Google Scholar 

  • E.T. Lagally, I. Medintz, R.A. Mathies, Singlemolecule DNA amplification and analysis in an integrated microfluidic device. Anal. Chem. 73, 565–570 (2001)

    Article  Google Scholar 

  • R.Y. Lai, E.T. Lagally, S.H. Lee, H.T. Soh, K.V. Plaxco, A.J. Heeger, Rapid, sequence-specific detection of unpurified PCR amplicons via a reusable, electrochemical sensor. Proc. Natl. Acad. Sci. U. S. A. 103, 4017–4021 (2006)

    Article  Google Scholar 

  • X.J. Lou, N.J. Panaro, P. Wilding, P. Fortina, L.J. Kricka, Increased amplification efficiency of microchip-based PCR by dynamic surface passivation. Biotechniques 36, 248–251 (2004)

    Google Scholar 

  • I. Pjescic, N. Crews, Genotyping from saliva with a one-step microdevice. Lab Chip 12, 2514–2519 (2012)

    Article  Google Scholar 

  • X. Qiu, M.G. Mauk, D. Chen, C. Liu, H.H. Bau, A large volume, portable, real-time PCR reacto. Lab Chip 10, 3170–3177 (2010)

    Article  Google Scholar 

  • X. Qiu, D. Chen, C. Liu, M.G. Mauk, T. Kientz, H.H. Bau, A portable, integrated analyzer for microfluidic-based molecular analysis. Biomed. Microdevices 13, 809–817 (2011)

    Article  Google Scholar 

  • M.G. Roper, C.J. Easley, L.A. Legendre, J.P. Landers, Infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips. Anal. Chem. 79, 1294–1300 (2007)

    Article  Google Scholar 

  • A.F. Sauer-Budge, P. Mirer, A. Chatterjee, C.M. Klapperich, D. Chargin, A. Sharon, Low cost and manufacturable complete microTAS for detecting bacteria. Lab Chip 9, 2803–2810 (2009)

    Article  Google Scholar 

  • D.C. Saunders, G.L. Holst, C.R. Phaneuf, N. Pak, M. Marchese, N. Sondej, M. McKinnon, C.R. Forest, Rapid, quantitative, reverse transcription PCR in a polymer microfluidic chip. Biosens. Bioelectron. 44, 222–228 (2013)

    Article  Google Scholar 

  • M.A. Shoffner, J. Cheng, G.E. Hvichia, L.J. Kricka, P. Wilding, Chip PCR I: Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Res. 24, 375–379 (1996)

    Article  Google Scholar 

  • P.J. Smith, J. Ballantyne, Simplified Low Copy Number (LCN) DNA analysis by post PCR purification. J. Forensic Sci. 52, 820–829 (2007)

    Article  Google Scholar 

  • D. Trau, T.M. Lee, A.I. Lao, R. Lenigk, I.M. Hsing, M.C. Ip, M.C. Carles, N.J. Sucher, Genotyping on a complimentary metal oxide semiconductor silicon polymerase chain reaction in silicon-based microstructures. Nucleic Acids Res. 74, 3168–3173 (2002)

    Google Scholar 

  • J.H. Wang, L.J. Chien, T.M. Hsieh, C.H. Luo, W.P. Chou, P.H. Chen, P.J. Chen, D.S. Lee, G.B. Lee, A miniaturized quantitative polymerase chain reaction system for DNA amplification and detection. Sensors Actuators B Chem. 141, 329–337 (2009)

    Article  Google Scholar 

  • Q. Xiang, B. Xu, D. Li, Miniature real time PCR on chip with multi-channel fiber optical fluorescence detection module. Biomed. Microdevices 9, 443–449 (2007)

    Article  Google Scholar 

  • J. Yang, C. Brooks, M. Estes, C. Hurth, F. Zenhausern, An integratable microfluidic cartridge for forensic swab samples lysis. Forensic Sci. Int. Genet. 8, 147–158 (2014)

    Article  Google Scholar 

  • Y. Yu, B. Li, C.A. Baker, X. Zhang, M.G. Roper, Quantitative polymerase chain reaction using infrared heating on a microfluidic chip. Anal. Chem. 84, 2825–2829 (2013)

    Article  Google Scholar 

  • C. Zhang, J. Xu, W. Ma, W. Zheng, PCR microfluidic devices for DNA amplification. Biotechnol. Adv. 24, 243–284 (2006)

    Article  Google Scholar 

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Acknowledgments

The authors deeply acknowledge the technical contributions of Glen McCarty and Brett Duane at the Center for Applied Nanobioscience & Medicine as well as helpful discussions with Prof. Tuan Vo-Dinh at the Fitzpatrick Institute for Photonics at Duke University. Funding from the European Community 7th Programme Framework was initially provided for this study.

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Authors and Affiliations

  1. Center for Applied Nanobioscience & Medicine, The University of Arizona College of Medicine, 425 N 5th Street, Phoenix, AZ, 85004, USA

    Cedric Hurth, Jianing Yang, Matthew Barrett, Carla Brooks, Alan Nordquist, Stanley Smith & Frederic Zenhausern

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  1. Cedric Hurth
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  2. Jianing Yang
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  3. Matthew Barrett
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  4. Carla Brooks
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  5. Alan Nordquist
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  6. Stanley Smith
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  7. Frederic Zenhausern
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Correspondence to Cedric Hurth.

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Hurth, C., Yang, J., Barrett, M. et al. A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system. Biomed Microdevices 16, 905–914 (2014). https://doi.org/10.1007/s10544-014-9895-8

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