Fully automated sample to result SIMPLE RPA microfluidic chip towards in ovo sexing application

Several European countries have implemented legislations to eliminate day-old male chicks killing. Although embryo sexing (in ovo sexing) is the most promising alternative, no current solution can handle all egg colors with >98% sexing accuracy, low cost and minimal embryo disturbance before day 13 of incubation and processing >20,000 eggs/hour. Recombinase polymerase amplification (RPA) presents a promising alternative to PCR that can be integrated into microfluidic platforms. In this work, we developed fully autonomous microfluidic cartridge (SIMPLE-RPA chip) for female chick-specific synthetic HINTW gene detection in 30 min at 37.7 °C inside an egg incubator. We first optimized off-chip RPA, allowing for highly sensitive DNA detection (1.6 × 10^–5 ng/µL). The SIMPLE-RPA chip was developed to automate the RPA bioassay on-chip, reducing user errors, and contamination risks and maintaining the off-chip LOD while offering low price, small footprint, upscaling compatibility, and easy transfer to other point-of-care applications.