ABSTRACT 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.
Yang et al. (Thu,) studied this question.