Plastic microfluidic chip for continuous-flow polymerase chain reaction: simulations and experiments.

A continuous flow polymerase chain reaction (CF-PCR) device comprises a single fluidic channel that is heated differentially to create spatial temperature variations such that a sample flowing through it experiences the thermal cycling required to induce amplification. This type of device can provide an effective means to detect the presence of a small amount of nucleic acid in very small sample volumes. CF-PCR is attractive for global health applications due to its less stringent requirements for temperature control than for other designs. For mass production of inexpensive CF-PCR devices, fabrication via thermoplastic molding will likely be necessary. Here we study the optimization of a PCR assay in a polymeric CF-PCR device. Three channel designs, with varying residence time ratios for the three PCR steps (denaturation, annealing, and extension), were modeled, built, and tested. A standardized assay was run on the three different chips, and the PCR yields were compared. The temperature gradient profiles of the three designs and the residence times of simulated DNA molecules flowing through each temperature zone were predicted using computational methods. PCR performance predicted by simulation corresponded to experimental results. The effects of DNA template size and cycle time on PCR yield were also studied. The experiments and simulations presented here guided the CF-PCR chip design and provide a model for predicting the performance of new CF-PCR designs prior to actual chip manufacture, resulting in faster turn around time for new device and assay design. Taken together, this framework of combined simulation and experimental development has greatly reduced assay development time for CF-PCR in our lab.