Pushing the Limits of Lateral Flow Immunoassay by Digital SERS for the Ultralow Detection of SARS-CoV-2 Virus

Lateral flow immunoassays (LFIAs) are easy-to-use antigen tests that provide different signal readouts, with colorimetric readouts being the most commonly used. However, these analytical devices have relatively low sensitivity and produce semiquantitative results, limiting their diagnostic applications. Herein, we address these challenges by implementing a digital surface-enhanced Raman spectroscopy (SERS)-based LFIA for the accurate and ultrasensitive quantitative detection of SARS-CoV-2 nucleocapsid (N) protein. Compared with average SERS intensity measurements, the digital approach allowed to overcome fluctuations in Raman scattering signals, thereby increasing the analytical sensitivity of the assay. Our method exhibited a quantification range of the viral protein in nasal swabs from 0.001 to 10 pg mL^-1, and a limit of detection down to 1.9 aM (0.9 fg mL^-1), improving colorimetric LFIAs and conventional-SERS-based LFIAs by several orders of magnitude. Importantly, this approach shows an analytical sensitivity of 0.03 TCID₅₀ mL^-1, which is greater than that reported by other immunoassays. In conclusion, we successfully demonstrate the robust detection and quantification of SARS-CoV-2N protein in nasal swabs at ultralow concentrations. The improvement in the sensitivity of LFIA by digital SERS may pave the way to translate this technology into the diagnostic arena for the ultrasensitive detection of microbes and disease biomarkers.