Beyond immune escape in SARS-CoV-2: an ACE2-functionalized gold nanoparticle vertical flow assay for saliva-based detection

Abstract

Rapid and accurate detection of SARS-CoV-2 is crucial as the spread of COVID-19 has led to more than 5.4 million deaths worldwide, resulting in a pandemic. SARS-CoV-2, being an RNA virus, is prone to mutations over time. A point mutation in RNA can lead to structural variations in the spike protein, the primary target of vaccines and immune responses. This can make the virus less recognizable to antibodies generated from previous infections or vaccinations. In this study, we present a novel flow-through assay for the detection of SARS-CoV-2 in saliva targeting conserved regions of the viral spike protein. The designed assay includes angiotensin-converting enzyme 2 (ACE 2)-functionalized gold nanoparticles (AuNPs) for viral capture and signaling. ACE 2 is the key receptor for the spike protein, offering a precise binding mechanism. Also, the use of saliva makes the test more user-friendly, preventing false results arising from incorrect and inadequate sample collection. The viral capture by ACE2 leads to changes in the optical properties of the AuNPs, causing a visible color change. The characterization of the specialized nanoparticles was done by UV spectroscopy, dynamic light scattering, zeta potential analysis, and transmission electron microscopy. This assay demonstrates high sensitivity and specificity of 96.67% and 95%, respectively, with an area under the curve of 0.99. The LOD of 0.02 pg μl⁻¹ is comparable to standard PCR tests with significantly reduced processing time. Moreover, the kit outperformed the saliva-based marketed LFA kit when tested with 30 spiked samples. The simplicity of the developed flow-through design enables point-of-care applicability, preventing future outbreaks.