Rapid, Ultrasensitive, and Quantitative Detection of SARS-CoV‑2 Using Antisense Oligonucleotides Directed Electrochemical Biosensor Chip

• Published in: ACS nano Vol. 14; no. 12; pp. 17028 - 17045
• Main Authors: Alafeef, Maha, Dighe, Ketan, Moitra, Parikshit, Pan, Dipanjan
• Format: Journal Article
• Language: English
• Published: American Chemical Society 22.12.2020
• Subjects: COVID-19; SARS-CoV-2; electrochemical sensor; gold nanoparticles; antisense oligonucleotide
• ISSN: 1936-0851; 1936-086X; 1936-086X
• DOI: 10.1021/acsnano.0c06392

A large-scale diagnosis of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is essential to downregulate its spread within as well as across communities and mitigate the current outbreak of the pandemic novel coronavirus disease 2019 (COVID-19). Herein, we report the development of a rapid (less than 5 min), low-cost, easy-to-implement, and quantitative paper-based electrochemical sensor chip to enable the digital detection of SARS-CoV-2 genetic material. The biosensor uses gold nanoparticles (AuNPs), capped with highly specific antisense oligonucleotides (ssDNA) targeting viral nucleocapsid phosphoprotein (N-gene). The sensing probes are immobilized on a paper-based electrochemical platform to yield a nucleic-acid-testing device with a readout that can be recorded with a simple hand-held reader. The biosensor chip has been tested using samples collected from Vero cells infected with SARS-CoV-2 virus and clinical samples. The sensor provides a significant improvement in output signal only in the presence of its targetSARS-CoV-2 RNAwithin less than 5 min of incubation time, with a sensitivity of 231 (copies μL–1)−1 and limit of detection of 6.9 copies/μL without the need for any further amplification. The sensor chip performance has been tested using clinical samples from 22 COVID-19 positive patients and 26 healthy asymptomatic subjects confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit. The sensor successfully distinguishes the positive COVID-19 samples from the negative ones with almost 100% accuracy, sensitivity, and specificity and exhibits an insignificant change in output signal for the samples lacking a SARS-CoV-2 viral target segment (e.g., SARS-CoV, MERS-CoV, or negative COVID-19 samples collected from healthy subjects). The feasibility of the sensor even during the genomic mutation of the virus is also ensured from the design of the ssDNA-conjugated AuNPs that simultaneously target two separate regions of the same SARS-CoV-2 N-gene.