Development of continuous-flow polymerase chain reaction microchip with integrated electrolytic pump

Abstract

There has been huge demand for point-of-care and on-site nucleic acid analysis. Microfluidic devices or microchips have received considerable attention thanks to their high portability, simple operation, short assay time, and low cost. One important module is deoxyribonucleic acid (DNA) amplification as the amount of nucleic acid sample is usually too little for direct detection. Particularly useful for this task is continuous-flow polymerase chain reaction (CFPCR), which enables high-speed PCR by passing an amplification reaction mixture through a microchannel to achieve the required repeated thermal cycling. Up until now, the fluidic flow had been controlled off-chip by bulky syringe pump, thereby seriously affecting the portability of the microchip device. To address this issue, herein, a CFPCR microchip with integrated on-chip electrolytic pump was developed. The CFPCR microchip comprised an electrolytic pumping chamber, PCR reagent reservoir, oil reservoir, and microchannel for PCR thermal cycling. The chamber, reservoir, and microchannel were made from a polydimethylsiloxane substrate. Platinum electrodes for the electrolytic pump were patterned onto a glass substrate, which was then plasma-bonded to and thus sealed the polydimethylsiloxane substrate. A feedback control system was custom-built to achieve accurate PCR thermal cycling (±1 °C). Gas bubbles generated from the on-chip electrolytic pump with constant voltage control successfully drove PCR reagent through the microchannel. CFPCR could be completed in ~20 min with the PCR product visible by gel electrophoresis. In addition, the effects of other key parameters, which included addition of silicone oil, concentration of Taq DNA polymerase, and dynamic/static passivation with bovine serum albumin on DNA amplification efficiency were studied. In fact, the on-chip electrolytic pump could also be driven by constant current and provide a more stable fluid flow than constant voltage control. This work successfully demonstrated the use of on-chip electrolytic pump to achieve fluidic control for CFPCR. Other functional modules such as sample preparation and product detection can be readily integrated to realize a portable device for decentralized nucleic acid analysis.