An electrochemical immunosensor using SARS-CoV-2 spike protein-nickel hydroxide nanoparticles bio-conjugate modified SPCE for ultrasensitive detection of SARS-CoV-2 antibodies

Abstract

As a promising approach for serological tests, the present study aimed at designing a robust electrochemical biosensor for selective and quantitative analysis of SARS-CoV-2-specific viral antibodies. In our proposed strategy, recombinant SARS-CoV-2 spike protein antigen (spike protein) was used as a specific receptor to detect SARS-CoV-2-specific viral antibodies. In this sense, with a layer of nickel hydroxide nanoparticles (Ni(OH)₂ NPs), the screen-printed carbon electrode (SPCE) surface was directly electrodeposited to ensure better loading of spike protein on the surface of SPCE. The differential pulse voltammetry (DPV) showed signals which were inversely proportional to the concentrations of the antibody (from 1 fg mL^-1 L to 1 µg mL^-1) via a specific and stable binding reaction. The assay was performed in 20 min with a low detection limit of 0.3 fg mL^-1. This biodevice had high sensitivity and specificity as compared to non-specific antibodies. Moreover, it can be regarded as a highly sensitive immunological diagnostic method for SARS-CoV-2 antibody in which no labeling is required. The fabricated hand-held biodevice showed an average satisfactory recovery rate of ~99-103% for the determination of antibodies in real blood serum samples with the possibility of being widely used in individual serological qualitative monitoring. Also, the biodevice was tested using real patients and healthy people samples, where the results are already confirmed using the enzyme-linked immunosorbent assay (ELISA) procedure, and showed satisfactory results.

Keywords: Biodevice; Nickel hydroxide nanoparticles; SARS‐CoV‐2; SARS‐CoV‐2-specific viral antibody; Screen-printed carbon electrode; Spike protein.

Graphical abstract

Scheme 1

Schematic of the step-by-step preparation of biodevice. a) bare SPCE; b) electrodeposition of Ni(OH)₂ NPs on the SPCE surface; c) Immobilization of spike protein on the Ni(OH)₂ NPs/SPCE surface; d) Immobilization of BSA on the spike protein/Ni(OH)₂ NPs/SPCE surface and e) Immobilization of IgG or IgM on the BSA/spike protein/Ni(OH)₂ NPs/SPCE surface.

Fig. 1

The FESEM images of SPCE (a and b), Ni(OH)₂ NPs@SPCE (c and d), EDS mapping of the Ni(OH)₂ NPs@SPCE (e and f).

Fig. 2

The FESEM images of spike protein/Ni(OH)₂ NPs@SPCE (a and b), EDS mapping of the spike protein/Ni(OH)₂ NPs@SPCE (c-f).

Fig. 3

The FTIR spectra of Ni(OH)₂ NPs@SPCE (a) and The FTIR spectra of spike protein/Ni(OH)₂ NPs@SPCE (b).

Fig. 4

The resulted from EIS (A) and CV (B) from various stages of fabrication of the modified electrode in the probe solution containing 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1:1) and 0.1 M KCl with a scan rate of 100 mV S⁻¹: SPCE (a), Ni(OH)₂ NPs@SPCE (b), spike protein/Ni(OH)₂ NPs@SPCE (c), BSA/spike protein/Ni(OH)₂ NPs@SPCE (d) and IgM or IgG/BSA/spike protein/Ni(OH)₂ NPs@SPCE (e).

Fig. 5

Optimization of analytical conditions. (A) Optimization of spike protein antigen concentration from 1 µg mL⁻¹ to 20 µg mL⁻¹, (B) Effect of binding time between spike protein antigen and Ni(OH)₂ NPs from 15 min to 65 min, (C) Effect of binding time between antibody and immobilized antigen from 5 min to 30 min, (each measurement was performed 3 times and the RSD averaged 1.5%).

Fig. 6

DPV responses of the designed biodevice after incubation with different of the IgM/IgG solutions with concentrations of 0, 1 fg mL⁻¹, 10 fg mL⁻¹, 100 fg mL⁻¹, 1 pg mL⁻¹, 10 pg mL⁻¹, 100 pg mL⁻¹, 1 ng mL⁻¹, 10 ng mL⁻¹, 100 ng mL⁻¹, 1 µg mL⁻¹, 1.2 µg mL⁻¹ (out of the linear range) and 1.5 µg mL⁻¹ (out of the linear range) (from top to bottom) n = 3. Calibration curve of DPV signal vs log C _IgM/IgG (fg mL⁻¹).

Fig. 7

(A) The histogram and DPV response of reproducibility investigation of the biodevice for 100 pg mL⁻¹ of IgG/IgM on the five different modified electrodes, (B) the histogram of the repeatability investigation of the biodevice for five repeated measurements of 100 pg mL⁻¹ of IgG/IgM, (C) the histogram and DPV response of then biodevice after incubation with IgM/IgG and some off-target species such SARS-CoV influenza A and B antibodies and a mixture of them, (D) the histogram and DPV response of the control electrode, (E) the recorded CVs of the 2th and 100th cycles related to the IgM or IgG/BSA/spike protein/Ni(OH)₂ NPs@SPCE in electrolyte solution with scan rate of 100 mV s⁻¹ and (F) the histogram of the long-term stability of the biodevice in present of 100 pg mL⁻¹ of IgG/IgM.