Scheme 1.

A SARS-CoV-2-specific LFIA-based biosensor using scFv-Fc fusion proteins. (a) Schematic illustration of the development processes of SARS-CoV-2-specific scFv-Fc fusion proteins based on phage display technology. First, the SARS-CoV-2-specific scFv-Fc fusion proteins were screened, and four different scFv-Fcs with high affinity and specificity for the SARS-CoV-2 NP were selected using ELISAs. (b) Schematic showing the scFv-Fc-based LFIA consisting of a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad. The selected scFv-Fc pairs were used as capture and detection antibodies. A test line placed on the NC membrane contains the capture scFv-Fc, and the CNB-conjugated detection scFv-Fc was immobilized on the conjugate pad. In the presence of SARS-CoV-2 NP, a sandwich complex (capture scFv-Fc-SARS-CoV-2 NP-detection scFv-Fc) was formed, and a clear red line appeared on the test line within 20 min. A handheld LFIA reader was used to analyze the line intensity semi-quantitatively. The proposed LFIA has is both sensitive and specific for SARS-CoV-2 NP, with no cross-reactivity with NPs from other coronaviruses such as MERS-CoV and SARS-CoV.