Pushbutton-activated DNA extraction and droplet generation in a microfluidic device for point-of-care molecular diagnostics

Abstract

In the light of the COVID-19 pandemic, the development of sensitive, specific, and cost-effective diagnostic tools has been highlighted. Among various diagnostic methods, molecular diagnostics including real-time polymerase chain reaction (qPCR) is considered to be the golden standard in the current state of the art due to its high sensitivity, specificity, and quantitation. Despite the advantages of qPCR, it is vulnerable to PCR inhibitors and relies on relative quantitation based on a standard curve, leading to false-negative diagnostic results. As an alternative, droplet digital PCR (ddPCR) has been highlighted for its higher sensitivity compared to qPCR and absolute quantitation that does not require a standard curve for each diagnostic run. In spite of the strengths of ddPCR, the high cost of ddPCR equipment and the complex and cumbersome sample pretreatment procedures significantly hinder its direct application to diagnostics. To overcome such limitations, a number of microfluidic devices have been introduced to perform ddPCR in microfluidic chips. Although several microfluidic devices have demonstrated comparable performances to that of the conventional benchtop ddPCR, their complicated operation principles such as centrifuge and external energy requirement and long sample preparation process still need to be improved. In this study, we developed a push-button activated microfluidic device based on polydimethylsiloxane using indirect pressurization for sample preparation in ddPCR. We have adopted a previously developed pushbutton-activated microfluidic device for nucleic acid extraction and droplet generation into a single microfluidic device to efficiently perform integrated sample preparation steps. A conventional solid-phase extraction method was adopted for nucleic acid extraction to capture and release DNA from silica microbeads. A reciprocating flow was generated inside a microfluidic device to increase the chance of DNA binding to silica beads. The generated reciprocating flow was further characterized to confirm that no fluidic loss occurred during the oscillation of the fluid. Subsequently, the extracted DNA was compartmentalized into droplets based on a conventional T-junction channel. The size of droplets was controllable by adjusting the dimension of the droplet generation channels. To examine the validity of monodisperse droplet generation, droplets from various geometries of the droplet generation channel were generated and the size distribution of droplets from each geometry was calculated. The proposed device significantly reduced the time for sample preparation by 75% compared with the time for sample preparation in the conventional benchtop method. To check the validity of the integrated device, hepatitis B virus standard DNA was first extracted, mixed with digitized PCR mixtures, and compartmentalized into droplets in a single device. The generated droplets underwent a thermal cycle for their fluorescence reading by the conventional fluorescence signal analyzer. We believe that the proposed device has the potential in simplifying and reducing the cost of sample preparation steps in ddPCR.