Multiplex and Ultrasensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 and Influenza A/B Nucleocapsid Proteins Based on Nanometer-Sized Core–Shell Superparamagnetic Antifouling Probes

Infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A (Flu A), and influenza B (Flu B) show similar clinical symptoms, such as cough, fever, and dyspnea, but patients infected by these viruses should be treated differently. The rapid and accurate diagnosis of infections caused by SARS-CoV-2, Flu A or Flu B is critical during the influenza season. Herein, we synthesized core–shell magnetic particles (MNPs) with excellent antifouling properties and applied them in the MNP-based immunochromatographic test (MICT) for simultaneous detection of SARS-CoV-2, Flu A, and Flu B nucleocapsid(N) proteins in 20 min. Two kinds of carboxyl-modified MNPs, MNP@pMBAA and MNP@Si-SA, were prepared and evaluated as probes in the MICT. Among them, the MNP@pMBAA showed lower nonspecific adsorption of proteins and low background noise in the application in MICTs. Particularly, the MNP@pMBAA50 bead-based MICT strip exhibited the highest signal-to-noise ratio for SARS-CoV-2 N protein detection with a limit of detection (LOD) of 0.072 ng/mL. Moreover, the proposed MICT strip demonstrated a minimal cross-reactivity and a broad linear dynamic detection range under a magnetic assay reader in the simultaneous detection of SARS-CoV-2, Flu A, and Flu B N proteins with relative LOD values of 0.0086, 0.012, and 0.018 ng/mL, respectively. The results demonstrated that the synthesized MNPs showed great potential for use as MICT probes for sensitive and multiplex detection of biomarkers in the development of point-of-care testing systems.