Capillary-driven microfluidic device integrating recombinase polymerase amplification for human papillomavirus detection

Nucleic acid amplification tests (NAATs) have been widely used in clinical diagnosis. However, current NAATs lack of user-friendly operation, require expensive systems and rely on laboratory-based methods. Here, we present low-cost and easy-to-fabricate capillary-driven microfluidic device for detecting DNA targeting of Human papillomavirus (HPV) type 16. The combination of recombinase polymerase amplification (RPA), reagent mixing, and detection of amplified DNA can be completed in a single microfluidic platform. We made the micro fluidic circuit by stacking layers of transparent film and double-sided adhesive film, creating capillary in the gap between two layers of transparent film that drives the flow. The different channel height in each part of microfluidic channel creates push valve that allowed sequential fluid flow to be stopped for RPA reaction for 30 min. Users open the valve by pressing it to transfer RPA product into the detection system and automatically mix with SYBR Green I for fluorometric readout. The microfluidic device could detect HPV 16 DNA within the range of 0.002-1.0 ng/mu L, with a detection limit of 0.24 pg/mu L. The device demonstrated great performance when applied to clinical sample. The innovative device design transforms NAATs development for real-world point-of care use through streamlined single-device operation.