Nanobody engineering for enhanced-sensitivity rapid COVID-19 tests

Nanobody-based diagnostics enable rapid and highly specific detection of infectious diseases, supporting early intervention and effective outbreak control. With their small size, high stability, and ease of production, nanobodies serve as promising alternatives to conventional antibodies in various diagnostic platforms, including lateral flow immunoassay (LFIA) for point-of-care testing. In this study, we present a nanobody-based LFIA for the rapid and early detection of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which had a profound global impact, while addressing the limitations of conventional antibody-based LFIAs. Instead of conventional antibodies, we developed nanobodies with high specificity against the SARS-CoV-2 nucleocapsid protein and conjugated them to the surface of gold nanoparticles (GNPs). Various parameters were optimized to effectively incorporate the nanobody-GNPs (nb-GNPs) into the LFIA platform. The resulting nb-GNP-based strip exhibited a lower limit of detection (10^3.15 PFU mL^-1) and higher reliability (<i>R</i>² = 0.9874) compared to existing antibody-based rapid tests, achieving results within 15 minutes and demonstrating strong discriminative power for clinical samples. Moreover, our nb-GNPs-based strip demonstrated high specificity, distinguishing COVID-19 from other respiratory viruses with similar symptoms. Our nanobody-based LFIA exhibited excellent sensitivity and accuracy without the need for additional reagents or target amplification steps. This LFIA approach can serve as an effective alternative to conventional antibody-based LFIAs and play a crucial role in ongoing SARS-CoV-2 surveillance and preparedness for future pandemics.