Coupling CRISPR/Cas12a and Recombinase Polymerase Amplification on a Stand-Alone Microfluidics Platform for Fast and Parallel Nucleic Acid Detection

Timely identification of human papillomavirus (HPV) infection is crucial for the prevention of cervical cancer. Current HPV detection methods mainly rely on polymerase chain reaction (PCR), which often requires bulky equipment and a long assay time. In this work, we report a heating-membrane-assisted multiplexed microfluidics platform that couples recombinase polymerase amplification (RPA) and CRISPR technology (termed M3-CRISPR) for fast and low-cost detection of multiple HPV subtypes. The heating membrane can provide convenient temperature control for the on-chip RPA and CRISPR assays. This stand-alone system allows simultaneous detection of HPV16 and HPV18 with high specificity and detection sensitivity (0.5 nM and 1 × 10^-18 M for unamplified and amplified plasmids, respectively) in 30 min with a fluorescence-based readout. Furthermore, we introduced an optimized lateral flow dipstick (LFD) into the portable system to allow visualized detection of HPV DNA. The LFD-based readout also reached a detection sensitivity of 1 × 10^-18 M for amplified plasmids and realized successful detection of HPV subtypes in the clinical samples. Finally, we established an automatic microfluidic system that enables the sample-in-answer-out detection of HPV subtypes. We believe that this fast, convenient, and affordable molecular diagnostic platform can serve as a useful tool in point-of-care testing of HPV or other pathogens.