Thermal analysis of a novel continuous flow multi layered polymerase chain reaction device

In this paper are presented the results of a thermal analysis on a high throughput droplet based, nucleic acid amplifier. Initial data of successful amplification of DNA is also presented. The advent of microfluidics offers many opportunities to integrate all functional steps of DNA analysis onto a single platform allowing for reduced analysis times, reduced sample volume requirements and higher throughput. There are many technical challenges facing this goal, thermal control being pivotal. This paper involves fundamental theoretical and experimental characterization of the core element of such a platform; a polymerise chain reaction (PCR) thermal cycler. The PCR process can be either a two-step or three-step temperature cycling procedure, depending on the chemistry involved. Presented in this paper is a thermal analysis of four, two-step thermocyclers arrayed in parallel. Two step PCR requires samples to be cycled through temperature ranges of 92-95°C and 60°C, the preciseness of these temperatures again depending on the chemistry involved. For optimum efficiency, fast heating and cooling between steps, and uniformity within each step is crucial. Our thermocycler design comprises a flow conduit in a serpentine pattern, machined in two segmented aluminium blocks, in which the conduit extends through the denaturation (92-95°C) and the annealing and extension (60°C) zones. Circular tubing, in which the samples are passed through to ensure biocompatibility for the reaction, is embedded in the machined channel which results in high heat transfer from the block to the sample. The device is then positioned in the vertical plane and an array of thermocyclers are formed by combining multiple planar systems. Thermofoil heaters are attached to the underside of the upper blocks creating the denaturation temperature, and by adjusting the air gap, passive thermal control is used to create the temperature required for the annealing and extension zone, thereby reducing the number of heaters and thus the power required. Theoretical thermal analysis of the device is performed, in conjunction with CFD simulations, experimental testing, and thermal imaging. Finally successful PCR of the gene ABL1 was performed.