Miniaturized and IoT Enabled Continuous-Flow-Based Microfluidic PCR Device for DNA Amplification

Herein, a continuous-flow driven microfluidic device has been designed and fabricated using the CO2 laser ablation method for polymerase chain reaction (PCR). The device consists of a polymethyl methacrylate (PMMA) microfluidic channel with 30 serpentine thermal cycles, an arduino board, two custom-made cartridge heaters, and thermocouple sensors. The portable thermal management system, with aluminium blocks placed on a wooden substrate, working on the PID controller principle, is low-cost, battery-powered, automated, integrated, and IoT-enabled. The device with dimensions 80 x 72 x 36 mm(3) (L x W x H) has a temperature accuracy of +/- 0.2 degrees C. The IoT module enables accessing and storage of real-time temperature values directly onto the smartphone through ThingSpeak analytics. It was developed to achieve desirable accurate temperature at two thermal zones, denaturation and annealing (95 degrees C and 60 degrees C) on the microfluidic thermal management platform. A PCR mixture of 20 mu l was infused into the serpentine-based microchannel using a syringe pump. Amplification of DNA template with 594-base pair (bp) fragment of the rat GAPDH gene was successfully performed on the miniaturized thermal management system. The total time required for a complete PCR reaction was 32 min at an optimum flow rate of 5 mu l/min. The amplified sample of the target DNA obtained from the PCR microchannel was then separated by agarose gel electrophoresis and was further analyzed using a gel-doc system. Finally, the obtained results were compared to the conventional PCR instrument showing excellent performance.

Automated summary

In this study, a continuous-flow driven microfluidic device for polymerase chain reaction (PCR) was designed and fabricated using the CO2 laser ablation method. The device, with low-cost, battery-powered, automated, integrated, and IoT-enabled features, achieved accurate temperature control for PCR reactions in two thermal zones. The device demonstrated excellent performance in amplification of DNA template and was compared to the conventional PCR instrument.