Abstract
Polymerase chain reaction (PCR) is commonly used to amplify and quantify the nucleic acid segments typically using a benchtop thermocycler. To automate, integrate, and miniaturize the PCR process, several strategies have been studied in a microfluidic environment. Among them, continuous-flow-based microfluidic PCR allows fast thermal cycling using minuscule volume, in a minimal reaction time with multiplexing. The objective was to develop a microfluidic device with a soft-lithographically fabricated continuous-flow serpentine microchannel for DNA amplification executed on a separately designed portable, easy-to-use, low-cost, automated, and miniaturized temperature controller platform (TCP). Direct laser writer (DLW) was used for developing a master on glass using a dry-film photoresist (DFR). Further, a PDMS-based microfluidic device, with dimensions 30 (L) × 0.32 mm2 (W) × 35 µm (H), was developed which was bonded on glass using oxygen plasma. The portable device exhibits key features of live data streaming using an IoT platform enabling easy data accessing, monitoring and storage onto the smartphone. The temperature sensitivity of the device was ± 0.5 °C and the maximum achievable temperature was 300 °C. The microfluidic device was placed on TCP. A 20 µL of reaction volume was introduced using an automated syringe pump at various flow rates. As a proof-of-concept, the rat GAPDH gene of the 594-base pair was successfully amplified on the proposed platform which was validated using the gel electrophoresis method. Finally, the results obtained from the proposed device were compared with the conventional thermocycler which showed promising performance and novelty exists in the significant reduction of required amplification time with good device efficiency and low-power consumption.
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Acknowledgements
The authors are thankful to the Centre for Human Disease Research (CHDR), BITS-Pilani, Hyderabad Campus for providing financial support during this work. Madhusudan B Kulkarni is thankful to Tata Consultancy Services (TCS) for providing scholarship during this project. The authors are thankful to Mr. Srashti Gopal Goyal, Department of Pharmacy, BITS-Pilani, Hyderabad campus, for providing the samples and helping in the experimental setup.
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Kulkarni, M.B., Goel, S. Miniaturized DNA amplification platform with soft-lithographically fabricated continuous-flow PCR microfluidic device on a portable temperature controller. Microfluid Nanofluid 25, 69 (2021). https://doi.org/10.1007/s10404-021-02473-4
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DOI: https://doi.org/10.1007/s10404-021-02473-4