Exploitation of N-Gene of SARS-CoV-2 to Develop a New Rapid Assay by ASOs@AuNPs.

ABSTRACT

A naked-eye (equipment-free), label-free (cost-effective), and RNA extraction-free (to speed up) method for SARS-CoV-2 (as a case study of RNA viruses) detection is developed. Here, the DNA is being used as a template for in situ formation of anisotropic gold nanoparticles (AuNPs) without any chemical modification or DNA labeling. In this study, synthesized AuNPs for the direct detection of N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2 are exploited. To this aim, antisense oligonucleotides (ASOs) with an extra poly guanine tail (G12) were designed. Thus, in the presence of its viral target RNA gene and ASOs@AuNPs-RNA hybridization, there was a red shift in its localized surface plasmon resonance (LSPR), and the intensity of the LSPR peak at 690 nm of throat swab samples was compared to the threshold cycle (Ct) of a reverse-transcriptase real-time polymerase chain reaction (RT-qPCR) (as a gold standard). Results suggested that the plasmonic biosensor can detect a very low amount of SARS-CoV-2 with a detection limit close to RT-qPCR. Simplicity of the new conjugation method with hybridization and annealing without amplification and denaturation steps enabled it to perform in a microfluidic paper-based analytical device.