Peptide Binder with High-Affinity for the SARS-CoV-2 Spike Receptor-Binding Domain.

Rapid antigen detection tests are urgently needed for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The discovery of a binder with high affinity and selectivity for the biomarkers presented by SARS-CoV-2 is crucial to the development of the rapid antigen detection method. We utilized the surface biopanning to identify a peptide binder R1 from a phage-displayed peptide library consisting of 109 independent phage recombinants. The R1 peptide exhibited high-affinity for specific binding with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with a dissociation constant KD of (7.5 ± 1.9) × 10-10 M, which maintained high binding affinity with the RBD derived from Gamma, Lambda, Delta, and Omicron variants. The composition and sequence dependence of binding characteristics in R1-RBD interactions was revealed by the binding affinity fluctuations between RBD and the scrambled sequences or single-site mutants of R1. The R1-functionalized gold nanoparticles possessed concentration-dependent response to RBD and selectivity over bovine serum albumin and human serum albumin. The peptide binder R1 shows the potential to be used for constructing a rapid detection method for the early-stage diagnostics for SARS-CoV-2.

A colorimetric sandwich-type bioassay for SARS-CoV-2 using a hACE2-based affinity peptide pair.

The metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the SARS-CoV-2 receptor required for viral entry based on its specific recognition of the spike protein receptor binding domain (S_RBD) on SARS-CoV-2. We constructed a human ACE2 (hACE2)-based peptide pair by ligating discontinuous key residues involved in the hACE2-S_RBD interaction. We firstly performed in silico simulations to identify a 12-mer and 15-mer peptide pair with capability to bind to the SARS-CoV-2 S_RBD via different binding sites. Then, the bio-layer interferometry validated the specific interactions between the peptides and S_RBD, with affinities at the nanomolar level. Lastly, we developed a colorimetric sandwich-type bioassay based on S_RBD-specific peptide-modified gold nanoparticles and found the colorimetric bioassay offered fast (<30 min), simple, and sensitive detection of S_RBD protein at levels as low as 0.01 nM (0.26 ng mL-1) in SARS-CoV-2. The linear signals ranging from 105 to 107 virus copies mL-1 were achieved in typical types of environmental waters spiked with lysed SARS-CoV-2 pseudovirus. The technology can serve as a beneficial supplement to the routine virus nucleic acid detection in environment media and wastewater treatment.