Voltammetric-based immunosensor for the detection of SARS-CoV-2 nucleocapsid antigen

Abstract

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL^-1 to 100 ng.mL^-1 and a limit of detection of 0.4 pg.mL^-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.

Keywords: Antibody; Biosensor; Electrochemical detection; Immunosensor; Nucleocapsid protein; Voltammetry.

Scheme 1

Fig. 1

Images of the electrodes by scanning electron microscopy (SEM) for the bare carbon before (a) and after deposition of gold nanoparticles using 10 CV scans (b). The SEM measurements were done using an acceleration voltage of 5 kV, magnification = 50,000× and a working distance of 4.8 mm. c XPS survey spectra of the bare carbon (black) and gold nanoparticles-modified electrodes (red line)

Fig. 2

Cyclic (a) and square wave voltammograms (b) of the bare carbon electrode (black), the gold nanoparticles-modified electrode (red), after functionalization with the 11-mercaptoundecanoic acid (green), after the immobilization of the anti-nucleocapsid antibody (blue) and after blocking with BSA (cyan). The measurements were performed in 5 mM ferro/ferricyanide redox solution in PBS buffer, pH 7.4

Fig. 3

a Square wave voltammograms of the N protein immunosensor before (black curve) and after (red curve) incubation with 1 ng.mL⁻¹ of the N protein solution in PBS buffer pH 4.7 for 15 min. b Binding time optimization of the nucleocapsid protein sensor (a plot of biosensor response as the percentage change in the reduction peak current versus the binding time

Fig. 4

a Square wave voltammograms of the N protein biosensor incubated in different concentrations of the N protein solutions from 1 pg.mL⁻¹ to 100 ng.mL⁻¹ in PBS buffer solution pH 7.4. b Calibration plot of the biosensor (the biosensor response (the percentage of the change in the reduction peak current) is plotted versus the protein concentration). The SWV measurements were carried out in 5 mM ferro/ferricyanide redox couple solution in PBS buffer pH 7.4. The error bars represent the standard deviations of triplicate measurements

Fig. 5

a The response as percentage change in the reduction current of the control sensor (the sensor prepared by immobilization of the MERS-CoV antibody) and the N protein sensor after incubation with 10 ng.mL⁻¹ of the N protein solution in PBS buffer pH 7.4. b The cross reactivity responses of the N protein biosensor towards the binding with 1 ng.mL⁻¹ of N protein, Flu B, Flu A, HCoV and MERS-CoV antigens. c Biosensor response towards binding with 1 ng.mL⁻¹ of the N protein after storage for different number of days. d Immunosensor response obtained for the negative and five patient samples with their corresponding Ct values