Rapid electrochemical detection of coronavirus SARS-CoV-2

Abstract

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

Fig. 1. Overview of the detection platform.

a Detection workflow of SARS-CoV-2 from clinical samples using the electrochemical biosensor with RCA of the N and S genes. b The detection setup for electrochemical analysis by using a portable potentiostat device connected to a laptop.

Fig. 2. Visualization of the silica-redox dye and RCA amplicons.

a SEM image with 1 µm scale bar of silica core, b silica-methylene blue (SiMB), and c silica-acridine orange (SiAO) composite particles with diameter sizes (ϕ) which are presented as mean values ± standard deviation (SD), n = 10. d Target gene, universal capture probe, and gene-specific reporter probe binding regions on the circular DNA template. e Gel representation of RCA amplicons visualized on 0.8% agarose gel electrophoresis (n = 3). Lane M indicates the 1 kb DNA ladder, while lanes 1, 2, and 3 indicate the RCA amplicons of both N and S genes, N gene only, and S gene only, respectively. Source data are provided as a Source Data file.

Fig. 3. Assay performance.

a Comparison of the step-wise (step-by-step) and one-step sandwich hybridization procedures using the N gene as the target. b Step-wise and one-step hybridization strategies produce current signals that are not significantly different (p > 0.05). The two-tailed Student’s t test was used to compare differences between two groups. All current responses are presented as mean values±standard deviation (SD), n = 5. The corresponding data points (as dot plots) are overlaid. c Sensitivity assay for N and S genes shows a positive correlation in current response to the copy number for both genes. All current responses are presented as mean values ±SD, n = 5. d Differential pulse voltammograms showing the increase in the current signal as the concentrations of the N gene increased. Dashed lines show the blank signal. e Differential pulse voltammograms of the S gene showing the increase in the current signal as the concentrations of the S gene increased. Dashed line shows the blank signal. f Multiple sequence alignment of the N and S genes target sequences with mismatch and non-complementary target sequences. Dark shaded areas are the non-complementary sequences to target gene, while the underlined bases are the mismatch bases. g The specificity of the assay with N (blue bar) and S (orange bar) genes, tested with perfect complementary targets (Ng, Sg, Plasmid N, Plasmid S), mismatch DNA target (Mismatch), and non-complementary targets (IAV, IBV) and the corresponding data points (as dot plots) are overlaid. Source data are provided as a Source Data file.

Fig. 4. RCA with rapid electrochemical detection of N and S genes of SARS-CoV-2 in clinical samples.

a The detection of N gene in 51 RNA samples. b The detection of S gene in 51 RNA samples. c The detection of N gene in 55 cDNA samples. d The detection of S gene in 55 cDNA samples. All samples used for the evaluation were prepared from nasopharyngeal swabs. The current signals from electrochemical measurements of the N gene (blue bar graph) and S gene (orange bar graph) are shown compared with the Cq result from qRT-PCR (blue dots for N gene and orange dots for S gene. All current responses are presented as mean values±standard deviation (SD), n = 5, whereas Cq values are from single measurements).