Sensitive sandwich-type electrochemical SARS-CoV‑2 nucleocapsid protein immunosensor

Abstract

A sensitive and fast sandwich-type electrochemical SARS-CoV‑2 (COVID-19) nucleocapsid protein immunosensor was prepared based on bismuth tungstate/bismuth sulfide composite (Bi₂WO₆/Bi₂S₃) as electrode platform and graphitic carbon nitride sheet decorated with gold nanoparticles (Au NPs) and tungsten trioxide sphere composite (g-C₃N₄/Au/WO₃) as signal amplification. The electrostatic interactions between capture antibody and Bi₂WO₆/Bi₂S₃ led to immobilization of the capture nucleocapsid antibody. The detection antibody was then conjugated to g-C₃N₄/Au/WO₃ via the affinity of amino-gold. After physicochemically characterization via transmission electron microscopy (TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) analysis were implemented to evaluate the electrochemical performance of the prepared immunosensor. The detection of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP) in a small saliva sample (100.0 µL) took just 30 min and yielded a detection limit (LOD) of 3.00 fg mL^-1, making it an effective tool for point-of-care COVID-19 testing.

Keywords: Bi2WO6/Bi2S3; COVID-19; Electrochemical impedance spectroscopy; Electrochemistry; Immunosensor; Voltammetry; g-C3N4/Au/WO3.

Scheme 1

Schematic illustration of the fabrication procedure of electrochemical SARS-CoV-2 immunosensor

Fig. 1

SEM images of A Bi₂WO₆, B Bi₂S₃, and C Bi₂WO₆/Bi₂S₃; D elemental mapping of Bi₂WO₆/Bi₂S₃ composite

Fig. 2

Raman (A) and B UV–Vis spectra of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃

Fig. 3

A SEM image of g-C₃N₄, TEM images of B and C g-C₃N₄/Au/WO₃, D EDX image of g-C₃N₄/Au/WO₃, and E high-resolution TEM image of g-C₃N₄/Au/WO₃

Fig. 4

A Cyclic voltammograms, B EIS reponses at (a) bare GCE, (b) Bi₂WO₆/GCE, (c) Bi₂S₃/GCE, (d) Bi₂WO₆/Bi₂S₃/GCE, (e) c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, (f) BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, (g) SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, (h) the final immunosensor including c-SARS-CoV-2-Ab₁, SARS-CoV-2 NP, and d-SARS-CoV-2-Ab₂ (scan rate of 50 mV s⁻¹) in 1.0 mM [Fe(CN)₆]³⁻ containing 0.1 M KCl, and C DPV responses of the proposed immunosensors incubated with 0.2000 pg mL⁻¹ SARS-CoV-2 NP using g-C₃N₄/WO₃/d-SARS-CoV-2-Ab₂ (curve b) in presence of 1.0 mM H₂O₂, g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂ (curve c) in presence of 1.0 mM H₂O₂ and in absence of target analyte (curve a)

Fig. 5

Concentration effect (from 0.01 to 1.00 pg mL⁻¹ SARS-CoV-2 NP) on immunosensor signals, Inset: calibration curve for electrochemical SARS-CoV-2 NP immunosensor (potential range is + 0.0/ + 0.4 V; Parameters are frequency of 100 Hz, pulse amplitude of 25 mV, and scan increment of 5 mV)

Fig. 6

A Immunosensor selective responses against the prepared solutions (n = 6): (i) 10.0 pg mL⁻¹ MERS-CoV NP + 10.0 pg mL⁻¹ SARS-CoV NP + 10.0 pg mL⁻¹ H1N1, (ii) 0.2000 pg mL⁻¹ SARS-CoV‑2 NP + 10.0 pg mL⁻¹ MERS-CoV NP, (iii) 0.2000 pg mL⁻¹ SARS-CoV‑2 NP + 10.0 pg mL⁻¹ SARS-CoV NP, (iv) 0.2000 pg mL⁻¹ SARS-CoV‑2 NP + 10.0 pg mL⁻¹ H1N1; B Stability test of electrochemical SARS-CoV-2 NP immunosensor including 0.2000 pg mL⁻¹ SARS-CoV-2 NP (n = 6) at 25.0 °C