Development and Analytical Evaluation of a Point-of-Care Electrochemical Biosensor for Rapid and Accurate SARS-CoV-2 Detection

Abstract

The COVID-19 pandemic has underscored the critical need for rapid and accurate screening and diagnostic methods for potential respiratory viruses. Existing COVID-19 diagnostic approaches face limitations either in terms of turnaround time or accuracy. In this study, we present an electrochemical biosensor that offers nearly instantaneous and precise SARS-CoV-2 detection, suitable for point-of-care and environmental monitoring applications. The biosensor employs a stapled hACE-2 N-terminal alpha helix peptide to functionalize an in situ grown polypyrrole conductive polymer on a nitrocellulose membrane backbone through a chemical process. We assessed the biosensor's analytical performance using heat-inactivated omicron and delta variants of the SARS-CoV-2 virus in artificial saliva (AS) and nasal swab (NS) samples diluted in a strong ionic solution, as well as clinical specimens with known Ct values. Virus identification was achieved through electrochemical impedance spectroscopy (EIS) and frequency analyses. The assay demonstrated a limit of detection (LoD) of 40 TCID₅₀/mL, with 95% sensitivity and 100% specificity. Notably, the biosensor exhibited no cross-reactivity when tested against the influenza virus. The entire testing process using the biosensor takes less than a minute. In summary, our biosensor exhibits promising potential in the battle against pandemic respiratory viruses, offering a platform for the development of rapid, compact, portable, and point-of-care devices capable of multiplexing various viruses. The biosensor has the capacity to significantly bolster our readiness and response to future viral outbreaks.

Keywords: SARS-CoV-2; electrochemical biosensor; impedance spectroscopy; lactam-stapled peptide.

Figure 3

Characterization of biosensor and detection of SARS-CoV-2 virus using electrochemical impedance spectroscopy. (a) Characteristic impedance measurement at different stages of sensor development across a range of frequencies. Blue lines: after coating with polypyrrole, red lines: after GA linker addition, purple lines: after hACE2 peptide attachment, and green lines: after blocking with skimmed milk protein. (b) Sensor response in terms of relative impedance change (|dZ|/Z) for different virus concentrations and media control. Green lines are media control; purple, grey, and red lines are virus in artificial saliva at concentrations of 20, 40, and 1000 TCID₅₀/mL, respectively. (c) Heatmap for optimization of separation factor between virus and control class. Frequency band corresponds to the optimum separation factor (sf) between media control and different virus concentrations. The greener shades indicate higher sf values while the reddish shades indicate lower sf values. The rows are sorted in descending order based on the sf values of viral RNA copies 20 TCID₅₀/mL. (d) Sensor response in terms of relative impedance change (|dZ|/Z) for different virus concentrations and media control (10 representative samples from 20 replicates) separated by a threshold line. Green bars are media control; purple, grey, and red bars are viral RNA copies (20 TCID₅₀/mL, 40 TCID₅₀/mL, and 1000 TCID₅₀/mL, respectively). A dashed black threshold line is drawn at 3 standard deviations below the mean of the control data set.

Figure 4

Classification of virus detection: (a) Relative impedance change (|dZ|/Z) for different virus concentrations and media control. The colors indicate virus concentrations: green (media control), purple (20 TCID₅₀/mL), grey (40 TCID₅₀/mL), blue (100 TCID₅₀/mL), yellow (200 TCID₅₀/mL), orange (500 TCID₅₀/mL), and red (1000 TCID₅₀/mL). sf values are annotated for each box. (b) Limit of detection validation at 40 TCID₅₀/mL. Y-axis: relative impedance value (|dZ|/Z). Green and grey bars represent media control and virus, respectively. (c) Comparable sensitivity of virus spiked in nasal swabs in 0.45 M KCl buffer. Y-axis: relative impedance value (|dZ|/Z). Green and grey bars represent media control and virus, respectively. (d) Relative impedance changes for influenza vaccine and media control. y-axis: relative impedance value (|dZ|/Z). Green and red bars represent media control and influenza, respectively. (e) Evaluation of frozen clinical specimens. X-axis represents Ct values from RT-PCR experiments. Each sample is tested in five replicates. The colors of boxes correspond to the different Ct values. sf values are annotated for each box. Threshold lines are denoted by black dashed lines for all figures and represent 3 standard deviations below the mean of control data’s relative impedance change.

Figure 1

Schematic presentation of biosensor development. (a) Nitrocellulose membrane (NC) (1 mm × 10 mm in dimension) as the base of the sensor substrate, (b) polymerization of conducting polymer (polypyrrole) on NC membrane, (c) covalent attachment of organic linker (glutaraldehyde), (d) functionalization with lactam stapled SARS-CoV-2 specific peptide, (e) blocking with skim milk protein, and (f) interaction of the SARS-CoV-2 virus with the receptor peptide.

Figure 2

Selective binding of SARS-CoV-2 to a lactam-based stapled hACE-2 functionalized on Ppy substrate on glass slide. The Ppy-coated and glutaraldehyde-linked glass slides were treated with hACE-2 peptide and blocked with skim milk protein before addition of virus or controls. Alexa Fluor 488 fluorophore-tagged peptide was then used to probe virus binding. (a) Artificial saliva without virus spike-in was used as a media control. (b) Heat-attenuated influenza vaccine containing a mix of Influenza A (H1N1, H3N2) and B viruses. (c) SARS-CoV-2 delta variant at concentration of 10⁵ virus copies/µL. (d) SARS-CoV-2 omicron variant at concentration of 10⁵ virus copies/µL.