AM‐DMF‐ddRPA: an All‐in‐One Digital Microfluidic Platform for Rapid and Automatic Digital Nucleic Acid Analysis

Accurate nucleic acid quantification analysis (NQA) is crucial for disease treatment and prevention. However, existing digital NQA methods often lack sufficient automation and speed. In this study, we introduce a novel rapid and automated active-matrix digital microfluidics-based droplet digital recombinase polymerase amplification method (AM-DMF-ddRPA). This method can quantify influenza A and B viruses (IAV and IBV) within a mere 60 min, covering the entire workflow from nucleic acid extraction to digital amplification, fluorescence detection, and statistical analysis. We fabricated an AM-DMF chip equipped with high-throughput electrodes and optimized its control logic. Leveraging an artificial intelligence droplet navigation algorithm, we successfully achieved motion and mixing operations of high-throughput droplets (4608 droplets, each with a volume of 0.9 nL). A dual-target RPA system was designed to detect IAV and IBV, and its detection performance was fine-tuned. Experiments conducted with clinical samples revealed that this method exhibits ultra-high sensitivity for single-molecule detection, with a detection limit of 1.69 copies per reaction, and it achieved 100% concordance with qPCR results. This groundbreaking quantification technology holds tremendous promise for advancing disease diagnosis, treatment, and fundamental research.