Graphene Oxide Nanofluidic Ion Channels with Two-Gene Rolling Circle Amplification for Ultrasensitive and Specific Detection of SARS-CoV-2

Rapid, sensitive, and specific detection of SARS-CoV-2 remains a critical challenge for early diagnosis and effective outbreak control. Here, we present a solid-state nanochannel biosensor integrated with two-gene rolling circle amplification (RCA) for the ultrasensitive and specific detection of SARS-CoV-2. In this system, target gene fragments initiate RCA to generate highly charged, three-dimensional DNA nanoflowers, which can effectively hybridize with the capture probes functionalized on the surface of the graphene oxide membranes. The accumulation of these nanostructures modulates the surface charge, significantly enhancing ion current signals through the nanochannels for highly sensitive electrical detection. The biosensor achieves detection limits of 0.3 fM for the S gene DNA fragment and 0.1 fM for the N gene DNA fragment. When applied to pseudovirus samples, the dual-gene RCA dramatically improves sensitivity and can detect SARS-CoV-2 pseudovirus at as low as a 10,000-fold dilution, corresponding to just 0.3 copies/μL of the S gene and 0.4 copies/μL of the N gene, which represents a 10-fold improvement over single-gene assays. By integrating nanofluidic sensing with programmable nucleic acid amplification, this label-free platform offers robust and specific SARS-CoV-2 detection with promising adaptability for diagnosing other pathogens.