Rapid and sensitive on-chip microcapillary electrophoresis for discrete SARS-CoV-2 immunocomplex diagnostics

Rapid, sensitive, and quantitative molecular diagnostics remain in sustained demand, driving the development of miniaturized analytical platforms. Conventional rapid antigen tests rely on lateral-flow techniques, requiring capture and detection antibodies, gold nanoparticle labels, and precise binding kinetics to generate a visible signal. Microcapillary electrophoresis (µCE) is well established for genomic DNA separation, yet its extension to protein analysis remains limited. Here we report a µCE method for rapid separation and detection of SARS-CoV-2 immunocomplexes, using a polymethyl methacrylate microfluidic chip filled with 5.5% linear polyacrylamide as the sieving matrix to perform an on-chip immunoassay. Unlike lateral-flow formats, our platform requires only a single fluorescently labeled antibody, with a positive or negative result read directly from the presence or absence of an immunocomplex peak. Fluorescently labeled antibodies were incubated with SARS-CoV-2 nucleocapsid protein (Np) to form immunocomplexes, the bound complex was resolved from free antibody within 30 s, based on the difference in electrophoretic mobility caused by altered charge-to-size ratio. Fluorescence signals were acquired by fluorescence microscopy and processed with a custom Python pipeline for electropherogram analysis and peak profiling. The assay resolved Np antigen across the alpha, beta, and delta variants and produced consistent, reproducible signals from clinical oropharyngeal swab specimens, achieving a detection limit of 5 µg/mL with minimal reagent consumption. Specificity was confirmed against a panel of common respiratory pathogens, respiratory syncytial virus, adenovirus, or influenza virus, with no visible immunocomplex peak indicating negligible cross-reactivity. Its sub-minute separation time and compatibility with automation position this µCE platform as a scalable route to decentralized point-of-care testing and screening for infectious diseases.