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Analytical and Bioanalytical Chemistry

, Volume 411, Issue 20, pp 5297–5307 | Cite as

Ultrafast, low-power, PCB manufacturable, continuous-flow microdevice for DNA amplification

  • Georgia D. Kaprou
  • Vasileios Papadopoulos
  • Dimitris P. Papageorgiou
  • Ioanna Kefala
  • George Papadakis
  • Electra Gizeli
  • Stavros Chatzandroulis
  • George KokkorisEmail author
  • Angeliki TserepiEmail author
Research Paper
  • 128 Downloads

Abstract

The design and fabrication of a continuous-flow μPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the μPCR device in the established PCB industry. The μPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented μPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate).

Graphical abstract

Keywords

MicroPCR Continuous-flow PCB substrates Computational fluid dynamics Heat transport Residence time distribution 

Notes

Acknowledgments

The authors would like to thank Drs. S.E. Kakambakos and P.S. Petrou at IPRETEA, NCSR “Demokritos,” for providing access to their roll laminator.

Funding information

This research was financially supported by the (1) FP7 “Love Wave Fully Integrated Lab-on-chip Platform for Food Pathogen Detection”—LOVE FOOD project (Contract No 317742)—and (2) Horizon 2020-EU 2.1.1, Project ID: 68768, “LOVEFOOD2Market—A portable MicroNanoBioSystem and Instrument for ultra-fast analysis of pathogens in food: Innovation from LOVE-FOOD lab prototype to a pre-commercial instrument” (http://lovefood2market.eu/).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1911_MOESM1_ESM.pdf (515 kb)
ESM 1 (PDF 514 kb)
216_2019_1911_MOESM2_ESM.flv (3 mb)
ESM 2 (FLV 3074 kb)

References

  1. 1.
    Arora A, Simone G, Salieb-Beugelaar GB, Kim JT, Manz A. Latest developments in micro total analysis systems. Anal Chem. 2010;82(12):4830–47.Google Scholar
  2. 2.
    Trietsch SJ, Hankemeier T, van der Linden HJ. Lab-on-a-chip technologies for massive parallel data generation in the life sciences: a review. Chemometr Intell Lab. 2011;108(1):64–75.Google Scholar
  3. 3.
    Romao VC, Martins SAM, Germano J, Cardoso FA, Cardoso S, Freitas PP. Lab-on-chip devices: gaining ground losing size. ACS Nano. 2017;11(11):10659–64.Google Scholar
  4. 4.
    Ahmad F, Hashsham SA. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review. Anal Chim Acta. 2012;733:1–15.Google Scholar
  5. 5.
    Chouler J, Di Lorenzo M. Water quality monitoring in developing countries; can microbial fuel cells be the answer? Biosensors. 2015;5(3):450–70.Google Scholar
  6. 6.
    Voetsch AC, Van Gilder TJ, Angulo FJ, Farley MM, Shallow S, Marcus R, et al. FoodNet estimate of the burden of illness caused by nontyphoidal Salmonella infections in the United States. Clin Infect Dis. 2004;38(Supplement_3):S127–S34.Google Scholar
  7. 7.
    Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'Brien SJ, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis. 2010;50(6):882–9.Google Scholar
  8. 8.
    Zhao X, Lin C-W, Wang J, Oh DH. Advances in rapid detection methods for foodborne pathogens. J Microbiol Biotechnol. 2014;24(3):297–312.Google Scholar
  9. 9.
    Pandey CM, Augustine S, Kumar S, Kumar S, Nara S, Srivastava S, et al. Microfluidics based point-of-care diagnostics. Biotechnol Adv. 2018;13(1):1700047.Google Scholar
  10. 10.
    Bruijns B, van Asten A, Tiggelaar R, Gardeniers H. Microfluidic devices for forensic DNA analysis: a review. Biosensors. 2016;6(3):41.Google Scholar
  11. 11.
    Khalid N, Kobayashi I, Nakajima M. Recent lab-on-chip developments for novel drug discovery. Wiley Interdiscip Rev Syst Biol Med. 2017;9(4):e1381.Google Scholar
  12. 12.
    Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H, editors. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction Cold Spring Harbor symposia on quantitative biology. Cold Spring: Harbor Laboratory Press; 1986.Google Scholar
  13. 13.
    Roper MG, Easley CJ, Landers JP. Advances in polymerase chain reaction on microfluidic chips. Anal Chem. 2005;77(12):3887–94.Google Scholar
  14. 14.
    Zhang C, Xu J, Ma W, Zheng W. PCR microfluidic devices for DNA amplification. Biotechnol Adv. 2006;24(3):243–84.Google Scholar
  15. 15.
    Zhang C, Xing D. Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res. 2007;35(13):4223–37.Google Scholar
  16. 16.
    Park S, Zhang Y, Lin S, Wang T-H, Yang S. Advances in microfluidic PCR for point-of-care infectious disease diagnostics. Biotechnol Adv. 2011;29(6):830–9.Google Scholar
  17. 17.
    Zhang Y, Ozdemir P. Microfluidic DNA amplification—a review. Anal Chim Acta. 2009;638(2):115–25.Google Scholar
  18. 18.
    Northrup MA, Gonzalez C, Hadley D, Hills RF, Landre P, Lehew S, et al., editors. A mems-based miniature DNA analysis system. Transducers '95 1995 25-29 June 1995.Google Scholar
  19. 19.
    Xiang Q, Xu B, Fu R, Li D. Real time PCR on disposable PDMS chip with a miniaturized thermal cycler. Biomed Microdevices. 2005;7(4):273–9.Google Scholar
  20. 20.
    Kopp MU, De Mello AJ, Manz A. Chemical amplification: continuous-flow PCR on a chip. Science. 1998;280(5366):1046–8.Google Scholar
  21. 21.
    Wang H, Chen J, Zhu L, Shadpour H, Hupert ML, Soper SA. Continuous flow thermal cycler microchip for DNA cycle sequencing. Anal Chem. 2006;78(17):6223–31.Google Scholar
  22. 22.
    Moschou D, Vourdas N, Kokkoris G, Papadakis G, Parthenios J, Chatzandroulis S, et al. All-plastic, low-power, disposable, continuous-flow PCR chip with integrated microheaters for rapid DNA amplification. Sensors Actuators B Chem. 2014;199:470–8.Google Scholar
  23. 23.
    Sun Y, Kwok Y-C, Foo-Peng Lee P, Nguyen N-T. Rapid amplification of genetically modified organisms using a circular ferrofluid-driven PCR microchip. Anal Bioanal Chem. 2009;394(5):1505–8.Google Scholar
  24. 24.
    Tsung-Min H, Ching-Hsing L, Gwo-Bin L, Chia-Sheng L, Fu-Chun H. A micromachined low-power-consumption portable PCR system. J Med Biol Eng. 2006;26(1):43–9.Google Scholar
  25. 25.
    Papadopoulos VE, Kokkoris G, Kefala IN, Tserepi A. Comparison of continuous-flow and static-chamber μPCR devices through a computational study: the potential of flexible polymeric substrates. Microfluid Nanofluid. 2015;19(4):867–82.Google Scholar
  26. 26.
    Volpatti LR, Yetisen AK. Commercialization of microfluidic devices. Trends Biotechnol. 2014;32(7):347–50.Google Scholar
  27. 27.
    Mohammed MI, Haswell S, Gibson I. Lab-on-a-chip or chip-in-a-lab: challenges of commercialization lost in translation. Proc Technol. 2015;20(Supplement C):54–9.Google Scholar
  28. 28.
    Duchesne L, Lacombe K. Innovative technologies for point-of-care testing of viral hepatitis in low-resource and decentralized settings. J Viral Hepat. 2018;25(2):108–17.Google Scholar
  29. 29.
    Walsh DI, Kong DS, Murthy SK, Carr PA. Enabling microfluidics: from clean rooms to makerspaces. Trends Biotechnol. 2017;35(5):383–92.Google Scholar
  30. 30.
    Merkel T, Graeber M, Pagel L. New technology for fluidic microsystems based on PCB technology. Sens Actuators A Phys. 1999;77(2):98–105.Google Scholar
  31. 31.
    Gaßmann S, Ibendorf I, Pagel L. Realization of a flow injection analysis in PCB technology. Sens Actuators A Phys. 2007;133(1):231–5.Google Scholar
  32. 32.
    Aracil C, Perdigones F, Moreno JM, Luque A, Quero JM. Portable lab-on-PCB platform for autonomous micromixing. Microelectron Eng. 2015;131:13–8.Google Scholar
  33. 33.
    Moschou D, Tserepi A. The lab-on-PCB approach: tackling the μTAS commercial upscaling bottleneck. Lab Chip. 2017;17(8):1388–405.Google Scholar
  34. 34.
    Nguyen N-T, Huang X. Miniature valveless pumps based on printed circuit board technique. Sens Actuators A Phys. 2001;88(2):104–11.Google Scholar
  35. 35.
    Ingle AP, Duran N, Rai M. Bioactivity, mechanism of action, and cytotoxicity of copper-based nanoparticles: a review. Appl Microbiol Biotechnol. 2014;98(3):1001–9.Google Scholar
  36. 36.
    Li J, Wang Y, Dong E, Chen H. USB-driven microfluidic chips on printed circuit boards. Lab Chip. 2014;14(5):860–4.Google Scholar
  37. 37.
    Metz S, Holzer R, Renaud P. Polyimide-based microfluidic devices. Lab Chip. 2001;1(1):29–34.Google Scholar
  38. 38.
    Mavraki E, Moschou D, Kokkoris G, Vourdas N, Chatzandroulis S, Tserepi A. A continuous flow μPCR device with integrated microheaters on a flexible polyimide substrate. Procedia Eng. 2011;25:1245–8.Google Scholar
  39. 39.
    Wangler N, Gutzweiler L, Kalkandjiev K, Müller C, Mayenfels F, Reinecke H, et al. High-resolution permanent photoresist laminate TMMF for sealed microfluidic structures in biological applications. J Micromech Microeng. 2011;21(9):095009.Google Scholar
  40. 40.
    Wu LL, Marshall LA, Babikian S, Han CM, Santiago JG, Bachman M, editors. A printed circuit board based microfluidic system for point-of-care diagnostics applications. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS) 2011.Google Scholar
  41. 41.
    Wu LL, Babikian S, Li GP, Bachman M, editors. Microfluidic printed circuit boards. Proceedings - Electronic Components and Technology Conference 2011.Google Scholar
  42. 42.
    Vasilakis N, Moschou D, Carta D, Morgan H, Prodromakis T. Long-lasting FR-4 surface hydrophilisation towards commercial PCB passive microfluidics. Appl Surf Sci. 2016;368:69–75.Google Scholar
  43. 43.
    Papadopoulos VE, Kefala IN, Kaprou G, Kokkoris G, Moschou D, Papadakis G, et al. A passive micromixer for enzymatic digestion of DNA. Microelectron Eng. 2014;124:42–6.Google Scholar
  44. 44.
    Kefala IN, Papadopoulos VE, Karpou G, Kokkoris G, Papadakis G, Tserepi A. A labyrinth split and merge micromixer for bioanalytical applications. Microfluid Nanofluid. 2015;19(5):1047–59.Google Scholar
  45. 45.
    Kaprou G, Papadakis G, Papageorgiou D, Kokkoris G, Papadopoulos V, Kefala I, et al. Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics. Microsyst Technol. 2016;22(7):1529–34.Google Scholar
  46. 46.
    Temiz Y, Lovchik RD, Kaigala GV, Delamarche E. Lab-on-a-chip devices: how to close and plug the lab? Microelectron Eng. 2015;132:156–75.Google Scholar
  47. 47.
    Becker H, Gärtner C. Polymer microfabrication technologies for microfluidic systems. Anal Bioanal Chem. 2008;390(1):89–111.Google Scholar
  48. 48.
    Kaprou G, Papadakis G, Kokkoris G, Papadopoulos V, Kefala I, Papageorgiou D, et al., editors. Miniaturized devices towards an integrated lab-on-a-chip platform for DNA diagnostics. Progress in Biomedical Optics and Imaging - Proceedings of SPIE; 2015.Google Scholar
  49. 49.
    Cao Q, Kim M-C, Klapperich C. Plastic microfluidic chip for continuous-flow polymerase chain reaction: simulations and experiments. Biotechnol Adv. 2011;6(2):177–84.Google Scholar
  50. 50.
    Ltd E. Technical terms and abbreviations. Available from: https://www.eurocircuits.com/technical-terms-and-abbreviations/.
  51. 51.
    Tserepi A., Chatzandroulis S., Kaprou G., Kokkoris G., Ellinas K., Papageorgiou D., inventorMicrofluidic reactors and process for their production. Greece patent GRA 20170100305 2017 30.06.2017.Google Scholar
  52. 52.
    Tserepi A., Chatzandroulis S., Kaprou G., Kokkoris G., Ellinas K., Papageorgiou D., inventorMicrofluidic reactors and process for their production patent 18386020.4-1101. 2018 29.06.18.Google Scholar
  53. 53.
    Vorkas PA, Christopoulos K, Kroupis C, Lianidou ES. Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis. Clin Biochem. 2010;43(1–2):178–85.Google Scholar
  54. 54.
  55. 55.
    Leonard WF. Yu HY. Thermoelectric power of thin copper films. J Appl Phys. 1973;44(12):5320–3.Google Scholar
  56. 56.
    Kim YS. Microheater-integrated single gas sensor array chip fabricated on flexible polyimide substrate. Sensors Actuators B Chem. 2006;114(1):410–7.Google Scholar
  57. 57.
    Shen K, Chen X, Guo M, Cheng J. A microchip-based PCR device using flexible printed circuit technology. Sensors Actuators B Chem. 2005;105(2):251–8.Google Scholar
  58. 58.
    Wheeler EK, Benett W, Stratton P, Richards J, Chen A, Christian A, et al. Convectively driven polymerase chain reaction thermal cycler. Anal Chem. 2004;76(14):4011–6.Google Scholar
  59. 59.
    Jiang L, Mancuso M, Lu Z, Akar G, Cesarman E, Erickson D. Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics. Sci Rep. 2014;4:4137.Google Scholar
  60. 60.
    Hashimoto M, Chen P-C, Mitchell MW, Nikitopoulos DE, Soper SA, Murphy MC. Rapid PCR in a continuous flow device. Lab Chip. 2004;4(6):638–45.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Georgia D. Kaprou
    • 1
    • 2
  • Vasileios Papadopoulos
    • 1
  • Dimitris P. Papageorgiou
    • 1
    • 3
  • Ioanna Kefala
    • 1
  • George Papadakis
    • 4
  • Electra Gizeli
    • 2
    • 4
  • Stavros Chatzandroulis
    • 1
  • George Kokkoris
    • 1
    Email author
  • Angeliki Tserepi
    • 1
    Email author
  1. 1.Institute of Nanoscience and NanotechnologyNCSR DemokritosAgia ParaskeviGreece
  2. 2.Department of BiologyUniversity of CreteHeraklionGreece
  3. 3.Department of Materials Science and EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.Institute of Molecular Biology and Biotechnology-FORTHHeraklionGreece

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