Rapid Detection of Spike Protein Receptor Binding Region of SARS-CoV-2 and Its Variants Using a Nanosheet Probe

Strategies for the rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are critically needed due to conventional methods' limitations: narrow range, virus mutation-induced failure, time-consuming, and complex operations. Herein, we propose a method utilizing CuInP₂S₆ nanosheet probes and Bio-Layer Interferometry (BLI) technology for the rapid (5-10 min), noninvasive, and broad-spectrum detection of the SARS-CoV-2 spike receptor binding domain (RBD) in human saliva. The nanoprobe exhibits a higher binding affinity to the RBD compared to most saliva proteins, allowing it to be immobilized on BLI sensors for easier detection of protein binding and elution. An eluent buffer containing Tween-20 and salts was employed to separate salivary proteins while retaining the RBD on the probes. This system can detect the RBD across a broad spectrum and a low limit of 25 ng/mL (S/N = 3) in less than 10 min. To validate this system, experiments with pseudoviruses showed accurate identification and binding of the RBD. Molecular dynamics simulations elucidated the mechanism for selective binding of probes to RBD. In conclusion, we propose a conceptual study based on an in situ strategy with inorganic nanoprobes alongside BLI techniques for convenient, noninvasive, and rapid detection of SARS-CoV-2 and its variants. This strategy is anticipated to inspire the design and implementation of nanoprobes for the rapid and selective detection of pathogens in the future.