Development of integrated microdroplet generation device with minimal loss for streamlining ddPCR-based SARS-CoV-2 detection

Abstract

Digital polymerase chain reaction (dPCR) is a promising alternative for achieving absolute quantification in molecular diagnostics. To improve its effectiveness, it is necessary to develop a comprehensive device that not only optimizes the partitioning process but also integrates microdroplet generation and transfer. Herein, we report an advanced microfluidic device for generating uniform gene-encapsulated microdroplets for multiplex droplet-based dPCR (ddPCR). The device consists of eight repeat units, and each unit has three chambers dedicated to reagents, oil, and droplet recovery, respectively. It also has a minimum channel width of 30 μm and can produce approximately 16,000 droplets with a diameter of 100 μm. To verify the practical applicability of the instrument, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected using the carefully designed primer and probe sets for the envelope (E) and spike (S) genes of the virus. The ddPCR shows accurate and quantitative viral analysis results, confirming the mechanical and chemical stability of the integrated device. Furthermore, it is demonstrated that the device-integrated ddPCR system can accurately diagnose 30 clinical samples. We expect that the integrated device will provide a simple, fast, reliable, and reproducible way to encapsulate nucleic acids for precise molecular diagnostics of various infectious and incurable diseases.