Potentiometric Biosensors Based on Molecular-Imprinted Self-Assembled Monolayer Films for Rapid Detection of Influenza A Virus and SARS-CoV‑2 Spike Protein

Rapid, yet accurate and sensitive testing has been shown to be critical in the control of spreading pandemic diseases such as COVID-19. Current methods which are highly sensitive and can differentiate different strains are slow and cannot be conveniently applied at the point of care. Rapid tests, meanwhile, require a high titer and are not sufficiently sensitive to discriminate between strains. Here, we report a rapid and facile potentiometric detection method based on nanoscale, three-dimensional molecular imprints of analytes on a self-assembled monolayer (SAM), which can deliver analyte-specific detection of both whole virions and isolated proteins in microliter amounts of bodily fluids within minutes. The detection substrate with nanoscale inverse surface patterns of analytes formed by a SAM identifies a target analyte by recognizing its surface nano- and molecular structures, which can be monitored by temporal measurement of the change in substrate open-circuit potential. The sensor unambiguously detected and differentiated H1N1 and H3N2 influenza A virions as well as the spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle-East respiratory syndrome (MERS) coronavirus in human saliva with limits of detection reaching 200 PFU/mL and 100 pg/mL for the viral particles and spike proteins, respectively. The demonstrated speed and specificity of detection, combined with a low required sample volume, high sensitivity, ease of potentiometric measurement, and simple sample collection and preparation, suggest that the technique can be used as a highly effective point-of-care diagnostic platform for a fast, accurate, and specific detection of various viral pathogens and their variants.