PEDOT-AuNPs-based impedimetric immunosensor for the detection of SARS-CoV-2 antibodies

Abstract

Electrochemical sensors and biosensors are useful techniques for fast, inexpensive, sensitive, and easy detection of innumerous specimen. In face of COVID-19 pandemic, it became evident the necessity of a rapid and accurate diagnostic test, so the impedimetric immunosensor approach can be a good alternative to replace the conventional tests due to the specific antibody-antigen binding interaction and the fast response in comparison to traditional methods. In this work, a modified electrode with electrosynthesized PEDOT and gold nanoparticles followed by the immobilization of truncated nucleoprotein (N aa160-406aa) was used for a fast and reliable detection of antibodies against COVID-19 in human serum sample. The method consists in analyzing the charge-transfer resistance (R_CT) variation before and after the modified electrode comes into contact with the positive and negative serum sample for COVID-19, using [Fe(CN)₆]^3-/4- as a probe. The results show a linear and selective response for serum samples diluted in a range of 2.5 × 10³ to 20 × 10³. Also, the electrode material was fully characterized by Raman spectroscopy, transmission electron microscopy and scanning electron microscopy coupled with EDS, indicating that the gold nanoparticles were well distributed around the polymer matrix and the presence of the biological sample was confirmed by EDS analysis. EIS measurements allowed to differentiate the negative and positive samples by the difference in the R_CT magnitude, proving that the material developed here has potential properties to be applied in impedimetric immunosensors for the detection of SARS-CoV-2 antibodies in about 30 min.

Keywords: COVID-19; Gold nanoparticles; Impedance spectroscopy; Impedimetric biosensor; PEDOT; SARS-CoV-2.

Graphical abstract

Fig. 1

(a) Cyclic voltammograms recorded at v = 20 mV s ^− 1 and (b) Nyquist plot. All measurements were performed in 1.0 mmol L ^− 1 K₄Fe(CN)₆/ K₃Fe(CN)₆ in PBS buffer pH 7.2 for the PEDOT/AuNPs modified electrodes (black line), after antigen immobilization (blue line), BSA blocking (green line) and steel mesh/AG/BSA (gray lines in (a)). The solid lines in the Nyquist plots represent the experimental data fitting using the equivalent circuit inserted in the figure.

Fig. 2

Raman spectra obtained for PEDOT, PEDOT/AuNPs and, PEDOT/AuNP's/AG. (a) emphasis in the region around 698 cm⁻¹ highlighting the AuNPs effect on the pristine PEDOT vibrations; (b) range from 1200 – 1600 cm⁻¹ showing the main bands of each material; and (c) the range from 1190 cm⁻¹ to 1360 cm⁻¹, highlighting the antigen influence on the material electrode (c). Radiation incident λ = 514 nm. PEDOT monomeric structure is inserted in the figure. The full spectra are show in the Figure S2 in supplementary material.

Fig. 3

Representative SEM images of (a) PEDOT (b) PEDOT/AuNPs and (c) PEDOT/AuNPs/AG modified electrodes in an increase of 800 and 3500 times, with voltage acceleration of 20 kV. (d-e) Representative TEM images of PEDOT/AuNPs/AG modified electrodes in different magnitude. Some AuNPs is marked with red arrows in the figure.

Fig. 4

(a) Comparative Nyquist plot for positive and negative serum samples for COVID-19 in 2.5 × 10³ dilution factor. The solid lines represent the simulation of the experimental data according to the proposed equivalent circuit, inserted in the graph. (b) Nyquist plot of the PEDOT/AuNPs/AG/BSA modified electrode to different positive serum concentrations. (c) Analytical curves for the evaluated serum samples (n = 3). The solid line represents the linear fit and the respective linear regression is present. The error bars show the standard deviations of three measurements.

Fig. 5

(a) R_CT values for positive and negative serum samples for COVID-19 in different dilution factors (n = 3). (b) Comparison of R_CT increase rate at some dilution factors for the positive serum samples and negative control serum. The values indicate the ratio of increase in R_CT percent response rate. The error bars show the standard deviations of three measurements.