Highly sensitive digital detection of SARS-CoV-2 nucleocapsid protein through single-molecule counting

Area of Science:

• Biotechnology
• Immunology
• Medical Diagnostics

Background:

• SARS-CoV-2 nucleocapsid protein (NP) is a stable, immunogenic biomarker suitable for early COVID-19 detection.
• Existing antigen detection methods often lack the required sensitivity for rapid and accurate NP identification.

Purpose of the Study:

• To develop a rapid and highly sensitive assay for detecting SARS-CoV-2 nucleocapsid protein (NP).
• To establish a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) for improved diagnostic capabilities.

Main Methods:

• Construction of a single-molecule digital FLISA using streptavidin-modified microplates.
• Immobilization of streptavidin and use of biotinylated antibodies and fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as capture and detection probes.
• Detection and counting of individual sandwich immunolabeled complexes of NP via fluorescence microscopy.

Main Results:

• The developed FLISA demonstrated a linear detection range from 0.2 pg/mL to 200 ng/mL for NP.
• Achieved limits of detection (LOD) of 0.73 fg/mL in PBS and 8 fg/mL in spiked nasal swabs.
• Exhibited significantly higher sensitivity compared to commercial antigen detection kits.

Conclusions:

• The novel digital FLISA provides a highly sensitive and rapid method for SARS-CoV-2 NP detection.
• This assay holds significant potential for early clinical diagnosis, environmental monitoring, and other applications.