Development of a continuous-flow polymerase chain reaction device utilizing a polymer disk with a spiral microchannel of gradually varying width

This paper describes a continuous-flow polymerase chain reaction (CF-PCR) device utilizing a polymer disk, which is equipped with a long spiral microchannel. In the literature, some CF-PCR devices employing a spiral microchannel have been presented to overcome an inherent deployment problem of heaters in CF-PCR of a serpentine microchannel, however, the spiral arrangement also made a problem of non-constant PCR cycle time, which would reduce the PCR efficiency. As a novel solution in this paper, we made the width of the spiral channel decrease gradually in the radial direction in order to keep the cycle time of one round same independent of radial positions and the PCR speed. In the spiral channel of 5.4-m long, through which the PCR reactant is pumped at a constant flow rate, a cycling zone made of thirty spiral rounds corresponding to 30 PCR cycles was placed between a pre-denaturation zone and a post-extension zone, and each zone was designed to have a fixed flow residence time ratio against to the total PCR time. We developed a fast thermal bonding technique minimizing the destruction of the microchannel in the wide polymer disk of 66-mm diameter. A compact heating apparatus was fabricated in order to impose different temperatures at three heating sectors deployed circumferentially in the disk. As a novel trial, the disk is sandwiched by isolated metal plates of constant temperature for stable thermal maintenance. We conducted numerical simulations for the heat transfer to PCR mixture depending on the PCR speed, and discussed on its effect on PCR result. A successful amplification of a human-genome DNA was obtained in less than 10 min. The unique architecture used in this CF-PCR device is understood to be well applied to a field-applicable fast PCR.