Label-free Sensing of Main Protease Activity of SARS-CoV-2 with an Aerolysin Nanopore

The main protease (M^pro ), which is highly conserved and plays a critical role in the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a natural biomarker for SARS-CoV-2. Accurate assessment of the M^pro activity is crucial for the detection of SARS-CoV-2. Herein, we report a nanopore-based sensing strategy that uses an enzyme-catalyzed cleavage reaction of a peptide substrate to measure the M^pro activity. The peptide was specifically cleaved by the M^pro , thereby releasing the output products that, when translocated through aerolysin, quantitatively produced the signature current events. The proposed method exhibited high sensitivity, allowing the detection of M^pro concentrations as low as 1 nM without the use of any signal amplification techniques. This simple, convenient, and label-free nanopore assay may expand the diagnostic tools for viruses.