Electrochemical immunosensors using electrodeposited gold nanostructures for detecting the S proteins from SARS-CoV and SARS-CoV-2

Abstract

This paper reports the development of a low-cost (< US$ 0.03 per device) immunosensor based on gold-modified screen-printed carbon electrodes (SPCEs). As a proof of concept, the immunosensor was tested for a fast and sensitive determination of S proteins from both SARS-CoV and SARS-CoV-2, by a single disposable device. Gold nanoparticles were electrochemically deposited via direct reduction of gold ions on the electrode using amperometry. Capture antibodies from spike (S) protein were covalently immobilized on carboxylic groups of self-assembled monolayers (SAM) of mercaptoacetic acid (MAA) attached to the gold nanoparticles. Label-free detection of S proteins from both SARS-CoV and SARS-CoV-2 was performed with electrochemical impedance spectroscopy (EIS). The immunosensor fabricated with 9 s gold deposition had a high performance in terms of selectivity, sensitivity, and low limit of detection (LOD) (3.16 pmol L^-1), thus permitting the direct determination of the target proteins in spiked saliva samples. The complete analysis can be carried out within 35 min using a simple one-step assay protocol with small sample volumes (10 µL). With such features, the immunoplatform presented here can be deployed for mass testing in point-of-care settings.

Keywords: Diagnosis; Gold nanoparticles; Immunosensor; S protein; SARS-CoV; SARS-CoV-2.

Fig. 1

Step-by-step immunosensor fabrication. a Fabrication steps of the screen-printed electrodes. b Electrochemical treatments, Au deposition, and modification of working electrodes with antibodies

Fig. 2

a SPE before and after electrodeposition of gold for 9, 30, and 90 s. b Elemental mapping of an SPE after electrodeposition of gold for 9 s. Pink and yellow shades, respectively, represent carbon and gold. c Bare SPE. SPE modified with gold nanostructures electrodeposited during d 9 s, e 30 s, and f 90 s. g Cyclic voltammograms in 0.1 mol L⁻¹ H₂SO₄ for bare SPE (black), SPE modified with gold nanostructures electrodeposited during 9 s (red), 30 s (blue), and 90 s (yellow) at − 4.0 V in a 5.0 mmol L⁻¹ hydrogen tetrachloroaurate (III) solution containing 0.5 mol L⁻¹ sulfuric acid. Scan rate 100 mV s⁻¹. The insert shows a zoomed view of the CV of SPE. h Nyquist plots for bare SPE (black), SPE modified with gold nanostructures electrodeposited during 9 s (red), 30 s (blue), and 90 s (yellow) in 0.1 mol L⁻¹ PBS containing 4.0 mmol L⁻¹ [Fe(CN)₆]^4−/3− from 10 kHz to 100 mHz. i CVs for bare SPE (black), SPE modified with gold microstructures electrodeposited during 9 s (red), 30 s (blue), and 90 s (yellow). CV conditions: 0.1 mol L⁻¹ PBS containing 4.0 mmol L⁻¹ [Fe(CN)₆]^4−/3− at a scan rate of 100 mV s⁻¹.

Fig. 3

a Nyquist plots in 0.1 mol L^–1 phosphate buffer containing 5.0 mmol L^–1 [Fe(CN)₆]³⁻/⁴⁻ using: (i) bare SPCE/AuNP (inverted triangle, black), (ii) SPCE/AuNP/MAA (diamond, red), (iii) SPCE/AuNP/MAA/EDC-NHS (triangle, yellow), (iv) SPCE/AuNP/MAA/EDC-NHS/antibody (circle, green), (v) SPCE/AuNP/MAA/EDC-NHS/antibody/BSA (left-pointing triangle, blue), (vi) and SPCE/AuNP/MAA/EDC-NHS/antibody/BSA/antigen (square, purple). Conditions: 0.1–100,000 Hz frequency range with pulse amplitude 10 mV. Inset: equivalent circuit to fit the experimental data. The insert brings R_ct values (Ω) after each functionalization step calculated using [R_s(CPE[R_ctZ_W])] as an equivalent circuit. Fluorescence images of b MAA/EDC-NHS/Ab functionalized electrode, and c functionalized electrode after detection of fluorescein-labeled protein S from SARS-CoV. Amplification 20 ×

Fig. 4

Calibration curves of S protein and BSA for different Au deposition times: 9 (a), 30 (b), and 90 s (c). d Sensitivities for S protein and BSA for each Au deposition condition. EIS conditions: 0.1 mol L⁻¹ PBS containing 4.0 mmol L⁻¹ [Fe(CN)₆]^4−/3− at OCP. e R_ct signals from SARS-CoV, SARS-CoV-2, and BSA at 10⁻⁹ mol L⁻¹ in PBS

Fig. 5

Application of the proposed immunosensor in biological samples. a Nyquist plots after incubation of S protein from SARS-CoV from 10⁻¹¹ to 10⁻⁷ mol L⁻¹ in saliva samples. b Calibration curves for S protein from SARS-CoV and BSA in saliva. c Nyquist plot for detection of inactivated SARS-CoV-2 viruses in saliva. The inset shows the R_ct obtained for negative (in the absence of the virus) and positive (in the presence of 10⁶ PFU mL⁻¹ of the virus) samples