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Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor.

ABSTRACT: Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/μL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.

SUBMITTER: Kashefi-Kheyrabadi L

PROVIDER: S-EPMC8447555 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 ?L-1)-1 and limit of detection of 6.9 copies/?L 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.

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Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor.

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