Thermal gradient continuous-flow PCR: a guide to design

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 reaction (CF-PCR) device. The 25-pass CF-PCR device considered 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 temperatures 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 manufacturing, 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 temperature 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.