Digital Hybridization Human Papillomavirus Assay with Attomolar Sensitivity without Amplification

Detection of nucleic acid without amplification can avoid problems associated with thermal cycling such as labor-intensiveness and aerosol pollution. Here we develop a droplet-based digital microfluidic hybridization assay for nucleic acid detection with attomolar sensitivity. This assay provides a clinically useful sensitivity for detecting human papillomavirus (HPV) without amplification. The sensitivity is accomplished using femtoliter-sized droplet microfluidics for concentrating enzyme-catalyzed fluorescent products into a detectable signal and magnetic beads for accelerating reaction time. Meanwhile, using magnetic beads and droplet microfluidic chips, we can improve the sampling efficiency over conventional methods. We characterized the sensitivity, selectivity, detection range, stability, and accuracy of our assay. Our assay is 50-fold more sensitive than the traditional hybrid capture assay. The assay without amplification avoids problems of complex handling procedures and aerosol pollution. The direct and sensitive detection of nucleic acid using a droplet microfluidic system provides an early disease diagnosis tool.