Electrochemical biosensor for nucleic acid amplification-free and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA via CRISPR/Cas13a trans-cleavage reaction.

The outbreak of the COVID-19 pandemic has led to millions of fatalities worldwide. For preventing epidemic transmission, rapid and accurate virus detection methods to early identify infected people are urgently needed in the current situation. Therefore, an electrochemical biosensor based on the trans-cleavage activity of CRISPR/Cas13a was developed in this study for rapid, sensitive, and nucleic-acid-amplification-free detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a redox probe conjugated with ssRNA is immobilized on the electrode surface modified with a nanocomposite (NC) and gold nanoflower (AuNF) for enhancing the sensing performance. The SARS-CoV-2 RNA is captured by the Cas13a-crRNA complex, which triggers the RNase function of Cas13a. The enzymatically activated Cas13a-crRNA complex is subsequently introduced to the reRNA-conjugated electrochemical sensor, and consequently cleaves the reRNA. A change in current occurs due to the release of the redox molecule labeled on the reRNA, which is trans-cleaved from the Cas13a-crRNA complex. The biosensor can detect as low as 4.4 × 10 fg/mL and 8.1 × 10 fg/mL of ORF and S genes, respectively, over a wide dynamic range (1.0 × 10 to 1.0 × 10 fg/mL). Moreover, the biosensor was evaluated by measuring SARS-CoV-2 RNA spiked in artificial saliva. The recovery of the developed sensor was found to be in an agreeable range of 96.54-101.21%. The designed biosensor lays the groundwork for pre-amplification-free detection of ultra-low concentrations of SARS-CoV-2 RNA and on-site and rapid diagnostic testing for COVID-19.