Binding-Induced Folding of DNA Oligonucleotides Targeted to the Nucleocapsid Gene Enables Electrochemical Sensing of SARS-CoV-2

In the wake of the COVID-19 pandemic, millions of confirmed cases and deaths have been reported around the world. COVID-19 spread can be slowed and eventually stopped by a rapid test to diagnose positive cases of the disease on the spot. It is still important to test for COVID-19 quickly regardless of the availability of the vaccine. Using the binding-induced folding principle, we developed an electrochemical test for detecting SARS-CoV-2 with no RNA extraction or nucleic acid amplification. The test showed high sensitivity with a limit of detection of 2.5 copies/μL. An electrode mounted with a capture probe and a portable potentiostat are used to conduct the test. To target the N-gene of SARS-CoV-2, a highly specific oligo-capturing probe was used. Based on the binding-induced “folding” principle, the sensor detects binding between the oligo and RNA. When the target is absent, the capture probe tends to form a hairpin as a secondary structure, retaining the redox reporter close to the surface. This can be seen as a large anodic and cathodic peak current. When the target RNA is present, the hairpin structure will open to hybridize with its complementary sequence, causing the redox reporter to pull away from the electrode. Consequently, the anodic/cathodic peak currents are reduced, indicating the presence of the SARS-CoV-2 genetic material. Validation of the test performance was performed using 122 COVID-19 clinical samples (55 positives and 67 negatives) and benchmarked to the gold standard reverse transcription-polymerase chain reaction (RT-PCR) test. As a result of our test, the accuracy, sensitivity, and specificity have been measured at 98.4%, 98.2%, and 98.5%, respectively.