Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes

We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. We show that this sensing strategy can directly detect spike (S) and nucleocapsid (N) proteins in unprocessed human saliva. Moreover, our approach enables the identification of RNA fragments from patient samples using nasal/throat swabs, enabling the identification of critical mutations such as D614G, G446S, or Y144del among viral variants. In particular, it can detect and discriminate between SARS-CoV-2 lineages of wild-type B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.1.539 (Omicron) within a single measurement without the need for nucleic acid sequencing. The sensing strategy of the molecular probes is easily adaptable to other viral targets and diseases and can be expanded depending on the application required.

Automated summary

We present a single-molecule nanopore sensing approach combined with position-encoded DNA molecular probes, which can simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. This strategy enables direct detection of spike (S) and nucleocapsid (N) proteins in unprocessed human saliva as well as the identification of RNA fragments from patient samples using nasal/throat swabs, allowing for the detection of critical mutations among viral variants. The sensing strategy can detect and distinguish between different SARS-CoV-2 lineages without the need for nucleic acid sequencing.