Miniaturized Thermal Monitoring Module With CO₂ Laser Ablated Microfluidic Device for Electrochemically Validated DNA Amplification

Polymerase chain reaction (PCR) is the gold standard method to amplify deoxyribonucleic acid (DNA) fragments. Several methods have been used to miniaturize and integrate PCR to execute in a microfluidic platform. A continuous-flow-based microfluidic PCR device permits the required rapid thermal cycling and multiplexing, which are hard to realize in a benchtop PCR or microchamber PCR. This work aims to develop a microfluidic PCR platform using a custom-made Internet of Things (IoT)-enabled portable thermal monitoring module. The microfluidic device was fabricated on polymethyl methacrylate (PMMA) using a CO₂ laser. Apart from 30 serpentine cycles, the PCR microchannel also had initial denaturation and final elongation regions to enhance the overall amplification. As a proof-of-application, rat GAPDH gene, with a fragment size of 594 base pair, has been amplified on the miniaturized platform. Furthermore, the agarose gel electrophoresis technique was used to separate the DNA molecules validated using a gel-doc system. Subsequently, the acquired results were quantitatively compared with the conventional thermocycler using ImageJ software. Furthermore, this was also validated with electrochemical DNA detection using differential pulse voltammetry (DPV). The results from the smart low-power device showed promising performance for the nucleic acid amplification process while reducing the overall amplification time and achieving benchmarked efficiency.