Bioscreening specific peptide-expressing phage and its application in sensitive dual-mode immunoassay of SARS-CoV-2 spike antigen

Biorecognition components with high affinity and selectivity are vital in bioassay to diagnose and treat epidemic disease. Herein a phage display strategy of combining single-amplification-panning with non-amplificationpanning was developed, by which a phage displaying cyclic heptapeptide ACLDWLFNSC (peptide J4) with good affinity and specificity to SARS-CoV-2 spike protein (SP) was identified. Molecular docking suggests that peptide J4 binds to S2 subunit by hydrogen bonding and hydrophobic interaction. Then the J4-phage was used as the capture antibody to establish phage-based chemiluminescence immunoassay (CLIA) and electrochemical impedance spectroscopy (EIS) analytical systems. The as-proposed dual-modal immunoassay platform exhibited good sensitivity and reliability in SARS-CoV-2 SP and pseudovirus assay. The limit of detection for SARS-CoV-2 SP by EIS immunoassay is 0.152 pg/mL, which is dramatically lower than that of 42 pg/mL for J4-phage based CLIA. Further, low to 40 transducing units (TU)/mL, 10 TU/mL SARS-CoV-2 pseudoviruses can be detected by the proposed J4-phage based CLIA and electrochemical immunosensor, respectively. Therefore, the as-developed dual mode immunoassays are potential methods to detect SARS-CoV-2. It is also expected to explore various phages with specific peptides to different targets for bioanalysis.