Integrated Antigen-DNA Co-Detection Using Digital Microfluidics for Improving Diagnostic Accuracy in POCT Analysis

Simultaneous detection of protein and nucleic acid (NA) provides complementary information essential for accurate analysis of complex samples. Conventional approaches for the analysis of protein and NA typically rely on separate workflows and instruments, limiting the reliability of point-of-care testing. Here, we present a digital microfluidics-based dual-mode molecular assay for simultaneous detection of both protein and NA from the same microscale sample. The protein is captured by a gold nanoparticle-antibody conjugate, while NA is enriched by magnetic beads. Magnetic manipulation separates the protein-containing fraction from NA, enabling separate analysis of the two molecular classes within an automated workflow. By integrating the analysis of protein and NA, the platform enables more accurate analysis than either modality alone. In 56 clinical samples, the combined analysis of hepatitis B surface antigen and DNA reduces the false-negative rate to 1.79%, compared with 12.5% for HBsAg alone. In food safety assays, simultaneous detection of Staphylococcus aureus-associated toxins and PMA-treated bacterial NA provides information not only on protein-based toxins, but also the presence and viability of bacteria within milk. This automated DMF-based dual-mode molecular assay provides a highly integrated strategy for multimodal POCT and public health risk assessment.