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 10(5) to 10(7) 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.