Tailing-Actuated Interfacial Liquid Mixing (TAIL-Mix) Enables Efficient Nucleic Acid Extraction and Integrated HPV Genotyping on a Digital Microfluidics Platform

Abstract High-risk human papillomavirus (HPV) genotyping is essential for risk-stratified cervical cancer screening. However, current laboratory workflows remain multistep, instrument-intensive, and difficult to scale up for high-throughput multiplex testing. Here we present an active-matrix digital microfluidic (AM-DMF) platform for high-throughput HPV genotyping, utilizing a tailing-actuated interfacial liquid mixing (TAIL-Mix) strategy to enable highly efficient nucleic acid extraction and robust downstream amplification. The AM-DMF chip features a 921,600-electrode array that uses programmable, time-sequenced electrode actuation to stretch and retract droplets. This mechanism generates TAIL-Mix, significantly enhancing mixing efficiency under low-Reynolds-number conditions. Through combined COMSOL simulations and OpenCV-based image analysis, we determined that optimal TAIL-Mix parameters (a tailing distance of 7–8 pixels; 1.5 steps s–1) achieve a homogeneous state (mixing index ≥ 0.9) within 2 s, superior to traditional schemes. This enhanced mixing proved to be critical for high-performance processing, enabling magnetic-bead DNA extraction with 75% DNA recovery while reducing the protocol time to 26 min and the sample input to 1.5 μL of sample (100-fold reduction compared with conventional protocols). By integrating TAIL-Mix-enhanced extraction, thermal cycling, and 21-plex end point fluorescence readout on a single chip, the platform successfully performs parallel 21-genotype PCR analysis for two samples. In a clinical cohort of 20 cervical samples, the AM-DMF assay shows 100% sensitivity and 100% specificity relative to qPCR. Ultimately, this TAIL-Mix-empowered AM-DMF system offers a robust solution for low-reagent consumption, high-throughput clinical diagnostics.