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

Area of Science:

• Biochemistry
• Nanotechnology
• Virology

Background:

• Angiotensin-converting enzyme 2 (ACE2) is crucial for SARS-CoV-2 entry by binding the viral spike protein receptor-binding domain (S_{_RBD}).
• Developing rapid and sensitive detection methods for SARS-CoV-2 is essential for environmental monitoring and public health.

Purpose of the Study:

• To engineer novel human ACE2-based peptides that specifically bind the SARS-CoV-2 S_{_RBD}.
• To develop a sensitive and rapid colorimetric bioassay for detecting SARS-CoV-2 S_{_RBD} in environmental samples.

Main Methods:

• In silico simulations were used to design 12-mer and 15-mer peptide pairs targeting the S_{_RBD}.
• Bio-layer interferometry validated peptide-S_{_RBD} interactions with nanomolar affinity.
• A colorimetric sandwich bioassay was created using peptide-modified gold nanoparticles for S_{_RBD} detection.

Main Results:

• The designed peptides demonstrated specific binding to the SARS-CoV-2 S_{_RBD} at nanomolar affinity.
• The colorimetric bioassay detected S_{_RBD} protein with high sensitivity (0.01 nM) in under 30 minutes.
• Linear detection of virus copies (10⁵–10⁷ copies/mL) was achieved in spiked environmental water samples.

Conclusions:

• The developed peptide-based colorimetric bioassay provides a fast, simple, and sensitive method for SARS-CoV-2 detection.
• This technology can supplement existing nucleic acid detection methods for environmental surveillance and wastewater treatment.