Miniaturized and IoT enabled Continuous-flow based Microfluidic PCR Device for DNA Amplification
Abstract : 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 × 72 × 36 mm3 (L x W x H) has a temperature accuracy of ±0.2°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°C and 60°C) on the microfluidic thermal management platform. A PCR mixture of 20 µ 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 µ 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.
? The particular optimized heater spacings were derived by instilling a zero heat flux boundary condition in the y-direction at the leading edges of each of the heaters. Due to the 96 C heater, a negative temperature gradient exists in the positive x direction.
? If a reasonable pressure drop exists in the downstream direction, then it is highly improbable that flow recirculation will occur.
? Oppositely, adverse pressure gradients (backpressure) are much more likely to induce some recirculation regimes near the microchannel wall.
? It was found in the literature that when the angles of the converging/diverging conduits were small, the probability of flow recirculation is minimized vis-à-vis steep profiles.
? In order to solve the above problems, a novel digital PCR system was proposed in the present work.
? The problem of detecting data failures can also serve as a reagent for screening dimers in the assay.
? To avoid PCR inhibition, it is important to prevent the direct interaction between PCR mixtures and device surfaces. However, the use of plugs/segments does not eliminate the adsorption problem even with a surface treatment.
? Therefore early droplet-based microfluidic PCR devices emerged to tackle the contamination problem.
? Future smart microfluidic platform will have a major impact on artificial intelligence and biological computations.
• The proposed device was applicable both in the material science laboratory and in the synthesis industry.
• The proposed device needs other equipment such asmicro-pump stability control makes the process, thus it is costlier and difficult to operatefor minuscule synthesis.
• The proposed method was used for obtaining monodisperse and uniform silver nanoparticles-loaded chitosan microparticles measuring numerous hundred micrometers.
• In addition, together with the benefits of flexible manipulation and largescale integration, microfluidic technology has been hastily developed as one of the most significant platforms in the field of purposeful biomaterial synthesis.
? There is no guarantee that the amplification efficiency will remain constant during the reaction.
? However, there are many factors that affect the amplification efficiency during PCR amplification.
? In other words, the amplification efficiency may be different between the work sample and the standard sample.
? A complementary metal oxide semiconductor (CMOS) camera was used as a detection tool to obtain fluorescence video for the entire loop area or a specified loop area.
? With the development of life science and medical testing, it has become increasingly important that faster and more accurate disease detection devices can be used for more people.
|