A miniaturized platform based on reciprocating-flowing immunobinding for ultrafast, highly-sensitive and on-site immunoassay.

Immunoassays are considered important methods for detecting disease biomarkers for biomedical diagnosis. Particularly, enzyme-linked immunosorbent assay (ELISA) is a commonly-used immunoassay method due to its high reliability and low cost. Nowadays, automatic analyzers integrating all steps of ELISA can achieve detection without manual operation to save labor, increase detection efficiency and avoid manual error. However, most ELISA analyzers require more than 30 min for one detection, which are not fast enough for on-site point-of-care testing (POCT). Herein, based on the reciprocating-flowing immunobinding strategy, we developed a miniaturized reciprocating-flowing POCT platform (RF-POCT platform) for rapid immunodetection. RF-POCT platform, which included an electronic control module, a fluid control module, a mechanical control module and a detection module, enabled fast immunobinding of antigen and antibody. Taking ELISA as an example of immunoassay methods for RF-POCT platform, we developed a miniaturized reciprocating-flowing ELISA analyzer (RF-ELISA analyzer) to automatically detect SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 N protein) and dust mite-specific immunoglobulin E (DM-IgE), with the LOD of 5.2–6.6 pg/mL and the CV less than 3 %. The detection required only 3.5 min. Afterwards, we detected the SARS-CoV-2 N protein in nasal swab samples and the DM-IgE in serum samples; the specificities were 85.7–100 %, the sensitivities were 96.3–100 %. The quantitative results from RF-ELISA analyzer were generally-consistent with those from large ELISA analyzers in hospitals. Therefore, RF-ELISA analyzer demonstrated excellent performance on ultrafast, highly-sensitive, and on-site immunoassay, with potential to support on-site POCT for primary hospitals and community clinics. • A miniaturized point-of-care testing (POCT) platform enables fast immunobinding of antigen and antibody. • A miniaturized ELISA analyzer based on this platform achieves automatic detection of multiple disease biomarkers. • This ELISA analyzer requires only 3.5 min for a detection, may be the fastest ELISA analyzer so far. • Compared to existing miniaturized ELISA analyzers, the LODs of this ELISA analyzer are much lower. • This ELISA analyzer could strongly support the on-site POCT for primary hospitals and community clinics. [ABSTRACT FROM AUTHOR]