Development of a portable multi-step microfluidic device for point-of-care nucleic acid diagnostics

Abstract

BackgroundThe COVID-19 pandemic has significantly affected global health, economies, and societies, and highlighted the urgent need for rapid, sensitive, affordable, and portable diagnostic devices for respiratory diseases, especially in areas with limited resources. In recent years, there has been rapid development in integrated equipments using microfluidic chips and biochemical detection technologies. However, these devices are expensive and complex to operate, showing limited feasibility for in point of care tests (PoCTs). This study aims to develop a cost-effective, portable, and practical microfluidic nucleic acid PoCT device for rapid virus diagnosis. ResultsHere, we developed a device based on freeze-dried reverse transcription loop-mediated isothermal amplification (RT-LAMP) reagents for rapid nucleic acid diagnostics. Homebrew RT-LAMP reagents were optimized to eliminate non-specific amplification. The multi-step device combines nucleic acid extraction, RT-LAMP and fluorescence detection in an integrated microfluidic chip, enabling sample-in, result-out diagnosis. This device showed satisfactory sensitivity in detecting SARS-CoV-2 and Trichomonas vaginalis RNA samples, with a limit of detection (LOD) of 400 copies/μL and 80 copies/μL respectively in 45 minutes. The LOD of cultured Trichomonas vaginalis samples were 0.32 cells/μL in 50 minutes. Additionally, the freeze-dried homebrew RT-LAMP can be stored at 4 °C for up to 30 days while still maintaining high sensitivity and detection capabilities. The cost of diagnosis was reduced to as low as 0.45 $ per reaction. Significance and NoveltyOverall, by integrating freeze-dried homebrew RT-LAMP and microfluidic chip, the device achieves ready-to-use, laboratory-free, quick, resource-independent and cost-effective nucleic acid detection, and provides a feasible alternative to complex equipments. The device shows potentials for point-of-care testing of SARS-CoV-2 and other respiratory diseases in remote or resource-limited areas with proper implementation.