Miniaturized continuous-flow polymerase chain reaction microfluidic chip system

Abstract

A miniaturized continuous-flow polymerase chain reaction (PCR) microfluidic chip system was developed to perform DNA amplification. This system consists of a 20-cycle continuous-flow PCR microfluidic chip, an electrical heating system and a miniature air pressure-vacuum pump. The chip was ablated with excimer laser direct-writing micromachining technique on a polymethyl methacrylate (PMMA) sheet. The ablated microchannel was inverse trapezoidal with a depth of 70 µm, top width of 200 µm and bottom width of 120 µm. Its surface roughness Ra was 1.42 µm after being treated with excimer laser polishing. The substrate sheet ablated with the microchannel was bonded with other cover sheets using hot-press bonding method to form a closed structure. The electrical heating system consisted of three groups of heating membranes, Pt100 sensors, copper blocks and PID temperature digital controllers. It could provide three distinct maintained temperature zones and a uniform temperature distribution in each zone. PCR amplification of a 170 base pair (bp) DNA fragment was carried out to validate the system’s feasibility. The PCR temperatures were set as 94°C for denaturation, 55°C for primer annealing and 72°C for extension. The flow rate in the microchannel was 40 nL/s and the total time for the completion of a 20-cycle amplification of 20 µL reagent was 15 min.

Keywords

continuous-flow / polymerase chain reaction (PCR) / microfluidic chip / excimer laser / micromachining