Development of a Peptide Aptamer-Based TRFIA for the Quantitive Detection of SARS-CoV-2 Nucleocapsid Protein

This study, we employed computer-aided design to develop 27 peptide aptamers, and initially screened 11 candidates with binding energies less than - 6.0 kcal/mol. Subsequently, three peptide aptamers with strong specificity were selected using a direct time-resolved fluorescence immunoassay (TRFIA). Peptide aptamer B3, in combination with monoclonal antibody R001, was used to construct a TRFIA-based sandwich assay system. The performance of this system was evaluated concerning sensitivity, specificity, and reproducibility, and was further tested using 20 simulated pharyngeal swab samples. The dominant peptide aptamer, B3(RGQGVPI), was identified as the most promising candidate. This peptide demonstrated high specificity for the SARS-CoV-2 nucleocapsid (N) protein, with the lowest detectable concentration of 203.78 pg/mL. Importantly, the peptide had no cross-reactions with other potential interferents, including RSV, Flu A, and MP, confirming its specificity. The assay also exhibited good reproducibility, with coefficients of variation of 11.33% and 8.00% at different concentrations in pharyngeal swab samples. Additionally, the LOB and LOD in clinical samples were 31.59 pg/mL and 243.70 pg/mL and the correlation coefficient R² was 0.9784, which indicated that the method had good resistance to clinical interference. In this study, we successfully developed a sandwich TRFIA method based on the peptide aptamer B3-R001, which can be used to quantitatively measure the SARS-CoV-2 N protein level in pharyngeal swabs. Compared to antibodies, peptide aptamers are easily synthesized, inexpensive, and show great potential for clinical applications. This method provides a novel approach for rapidly detecting viral pathogens, contributing to the advancement of diagnostic tools.