Establishment of a Point-of-Care Testing Method for Rapid Detection of Multiple Respiratory Virus Antigens Based on a Dual-Drive Microfluidic Chip.

The global impact of pandemics has intensified the demand for rapid, cost-effective, sensitive, and reliable POCT methods. Herein, we developed a multiplex pathogen detection system based on a dual-drive microfluidic chip, enabling simultaneous detection of RSV, FluA, FluB, and SARS-CoV-2 antigens from a single sample via parallel-arranged detection units within microchannels. Monoclonal antibodies targeting conserved viral proteins were cross-paired to identify optimal pairs. The chip's labeling and reference regions were designed, and key parameters were optimized to enhance performance. Cut-off values were established using nasopharyngeal swab samples from healthy donors, and assay performance was rigorously validated. Finally, the concordance between the multiplex chip and GICA and RT-PCR was evaluated. The optimized process ultimately yielded a chip design featuring a labeling region with four antiviral monoclonal antibody-conjugated fluorescent microspheres, four test regions each coated with corresponding antiviral monoclonal antibodies, and a dual-antibody reference region for signal normalization. The developed assay requires only 35 μL of sample and completes detection within 5 min. It demonstrated high repeatability, with coefficients of variation ranging from 9.48% to 13.37%. The detection limits were 0.625 ng/mL for RSV, 2.5 ng/mL for FluA, 5 ng/mL for both FluB and SARS-CoV-2. Validation studies showed perfect agreement with GICA (n = 132, Kappa = 1) and strong consistency with RT-PCR (Kappa = 0.74-0.90, n = 42). A novel POCT method has been successfully established for the simultaneous detection of four respiratory virus antigens. This system shows great potential as a reliable and efficient analytical tool for large-scale epidemic screening.