A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel

The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.

Automated summary

This study proposes a nanobioassay called OVER-SARS-CoV-2, which combines SERS-read and CRISPR/Cas technology to enable fast, highly sensitive, accurate, and portable detection of SARS-CoV-2 in a single vessel without amplification and interference. Gold nanoparticles are combined with Raman reporting molecules and single-stranded DNA probes to convert nucleic acid signals into SERS signals. By customizing a vessel, the steps of reverse transcription, Cas12a trans-cleavage, and SERS nanoprobes crosslinking are integrated. The nanobioassay is able to detect SARS-CoV-2 as low as 200 copies/mL within 45 minutes without pre-amplification, and has been validated with clinical samples and complex environmental and food samples.