RESEARCH PAPER Thermal gradient continuous-ﬂow PCR: a guide to design Susan Thomas • Ryan Luis Orozco • Tim Ameel Received: 9 November 2013 / Accepted: 15 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014 Abstract A numerical study has been conducted to determine which of eight fundamental design parameters have the greatest impact on the performance of a realistic, thermal gradient, continuous-ﬂow polymerase chain reac- tion (CF-PCR) device. The 25-pass CF-PCR device con- sidered in this study is practical, effective, easy to fabricate, and circumvents earlier microﬂuidic PCR design challenges. But the question remains: which design parameters have the greatest impact on ﬂuid temperatures and are thus the most critical to device success, and which can be more freely speciﬁed according to manufacturing or other concerns? Parameters investigated in this study include inlet and outlet location, inlet and outlet length, channel spacing, aspect ratio, substrate thickness, substrate material, and temperature gradient, and many practical variations are considered. It is found that substrate material and thickness have the greatest impact on ﬂuid tempera- tures and should always be carefully speciﬁed: glass, for example, is an acceptable substrate in thicknesses of 2, 3, and 4 mm, but likely not 1 mm, and silicon and hybrid devices (glass ? polydimethylsiloxane or PDMS) are also feasible. Other parameters, including fabrication-limited aspect ratio, have negligible impact, and aspect ratios between 0.25 and 4 can be freely speciﬁed for manufac- turing, cost, or other concerns. Channel spacings between 0.6 and 5 mm are also feasible, and increasing channel spacing signiﬁcantly increases uniformity in DNA annealing and denaturing temperatures, a desirable feature. Results of this study, which illuminate chip-wide temper- ature distributions and heat transfer characteristics for 22 total design variations, should assist the thermal gradient CF-PCR designer in making better up-front choices from a range of practical options. Keywords PCR  Microﬂuidics  DNA ampliﬁcation  Continuous-ﬂow  Thermal gradient 1 Introduction Polymerase chain reaction (PCR) is a biological process that has been used to diagnose disease, sequence genetic material, and perform forensic analysis Auroux et al. (2004); Vilkner et al. (2004); Chen et al. (2007). PCR devices replicate DNA by moving a mixture of target DNA, DNA primers, nucleotides, and the enzyme Taq polymerase through three temperature-dependent process steps Erlich and Freeman (1992): In step one, DNA is denatured when hydrogen bonds between base pairs in the double helix break between 90 and 95  C. In step two, primers (short strands of synthetic DNA) bind, or anneal, to single-stranded DNA at temperatures between 40 and 60  C. In step three, the enzyme Taq polymerase extends DNA primers to replicate the original double helix at temperatures between 70 and 75  C. This process of denaturing, annealing, and extending/replicating DNA is repeated 20–40 times to obtain enough DNA for analysis McPherson et al. (1995). Beneﬁts to downsizing this technology include mini- mized process times, energy requirements, and sample volumes, and microscale PCR has accordingly undergone rapid development since the 1990s. There are two device types: well-based PCR, in which a microchip with ﬂuid wells is heated and cooled Northrup et al. (1995), Wilding et al. (1994), Belgrader et al. (1999), and continuous-ﬂow S. Thomas (&)  R. L. Orozco  T. Ameel Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA e-mail: senoritasue@gmail.com 123 Microﬂuid Nanoﬂuid DOI 10.1007/s10404-014-1401-3