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Three-dimensional on-chip continuous-flow polymerase chain reaction employing a single heater

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Abstract

Multi-step temperature control in a polymerase chain reaction (PCR) is a limiting factor in device miniaturization and portability. In this study, we propose the fabrication of a three-dimensional (3D) microdevice employing a single heater to minimize temperature control required for an on-chip continuous-flow PCR as well as the overall footprint by stacking the device in multi-layers. Two poly(dimethylsiloxane) (PDMS) layers with differing thicknesses are vertically stacked with their microchannel-engraved sides facing down. Through-holes are made in the thicker PDMS layer, which is sandwiched between a glass substrate at the bottom and the thinner PDMS layer at the top. In this way, a fluidic conduit is realized in a 3D configuration. The assembled 3D microdevice is then placed onto a heater glass-side down. The interface of the two PDMS layers displays a relatively lower temperature than that of the PDMS and glass layers due to the low thermal conductivity of the PDMS and its physical distance from the heater. The denaturation temperature can be controlled by adjusting the temperature of the heater, while the annealing/extension temperature can be controlled automatically by molding the thicker bottom PDMS layer into the appropriate thickness calculated using a numerical derivation proposed in this study. In this way, a cumbersome temperature measurement step is eliminated. DNA amplification was successfully carried out using the proposed 3D fluidic microdevice, and the intensity of the resulting amplicon was comparable to that obtained using a thermal cycler. This novel concept of adopting a single heating source greatly simplifies the temperature control issue present in an on-chip continuous-flow PCR. It also allows the use of a commercialized hot plate as a potential heat source, paving the way for device miniaturization and portability in a highly cost-effective manner. In this study, a simple and facile technique to make arrays of through-holes for the fluidic interconnection inside a 3D channel configuration is also addressed.

PCR inside a 3D fluidic conduit and a resulting target amplicon (~230 bp)

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Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0005174). This work was also supported by the R&D Program of MKE/KEIT (10035638, Integrated portable genetic analysis RT-PCR microsystem for ultrafast respiratory infection disease identification) and the Kyungwon University Research Fund in 2009.

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Correspondence to Nae Yoon Lee.

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Wu, W., Lee, N.Y. Three-dimensional on-chip continuous-flow polymerase chain reaction employing a single heater. Anal Bioanal Chem 400, 2053–2060 (2011) doi:10.1007/s00216-011-4947-x

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Keywords

  • On-chip polymerase chain reaction (PCR)
  • Multi-layer microdevice
  • 3D fluidic conduit
  • A single heater
  • DNA amplification