An Electrochemical Impedance Spectroscopy-Based Aptasensor for the Determination of SARS-CoV-2-RBD Using a Carbon Nanofiber-Gold Nanocomposite Modified Screen-Printed Electrode

Abstract

Worldwide, human health is affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, the fabrication of the biosensors to diagnose SARS-CoV-2 is critical. In this paper, we report an electrochemical impedance spectroscopy (EIS)-based aptasensor for the determination of the SARS-CoV-2 receptor-binding domain (SARS-CoV-2-RBD). For this purpose, the carbon nanofibers (CNFs) were first decorated with gold nanoparticles (AuNPs). Then, the surface of the carbon-based screen-printed electrode (CSPE) was modified with the CNF-AuNP nanocomposite (CSPE/CNF-AuNP). After that, the thiol-terminal aptamer probe was immobilized on the surface of the CSPE/CNF-AuNP. The surface coverage of the aptamer was calculated to be 52.8 pmol·cm^-2. The CSPE/CNF-AuNP/Aptamer was then used for the measurement of SARS-CoV-2-RBD by using the EIS method. The obtained results indicate that the signal had a linear-logarithmic relationship in the range of 0.01-64 nM with a limit of detection of 7.0 pM. The proposed aptasensor had a good selectivity to SARS-CoV-2-RBD in the presence of human serum albumin; human immunoglobulins G, A, and M, hemagglutinin, and neuraminidase. The analytical performance of the aptasensor was studied in human saliva samples. The present study indicates a practical application of the CSPE/CNF-AuNP/Aptamer for the determination of SARS-CoV-2-RBD in human saliva samples with high sensitivity and accuracy.

Keywords: CNF–AuNP; SARS-CoV-2-RBD; aptasensor; electrochemical impedance spectroscopy.

Figure 1

(A,B) SEM images of the CSPE and (C–F) CSPE/CNFs–AuNPs.

Figure 2

(A) EDS of the CSPE/CNFs–AuNPs (a) and the CSPE/CNF–AuNP/Aptamer (b). (B) ATR spectrum of the CSPE/CNF–AuNP/Aptamer.

Figure 3

(A) EIS and (B) CVs and of the CSPE (a) the CSPE/CNFs–AuNPs (b), the CSPE/CNF–AuNP/Aptamer (c), and the CSPE/CNF–AuNP/Aptamer/SARS-CoV-2-RBD (d) at 0.1 M 5.0 mM Fe(CN)₆^3−/4− solution (0.1 M PBS, pH 7.4). The equivalent electric circuit is compatible with the Nyquist diagrams. R_s: Solution resistance; R_ct: charge transfer resistance; C_dl: Double layer capacitance; Z_w: Warburg impedance. The AC amplitude voltage was 10 mV, DC voltage was 0.13 V, and frequency range was 100 kHz−0.1 Hz. The solid black lines indicate fitting curves. CVs were recorded at the scan rate of 0.05 V·s⁻¹.

Figure 4

(A) The EIS of the CSPE/CNF–AuNP/Aptamer at the optimum operating conditions for different concentration of SARS-CoV-2-RBD (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2, 4, 8, 16, 32, and 64 nM) at 5.0 mM Fe(CN)₆^3−/4− solution (0.1 M PBS, pH 7.4). (B) The linear-linear and (C) linear-logarithmic calibration curve plots. (D) The plot of the Langmuir binding isotherm model. The equivalent electric circuit is compatible with the Nyquist diagrams. R_s: Solution resistance; R_ct: charge transfer resistance; C_dl: Double layer capacitance; Z_w: Warburg impedance. The AC amplitude voltage was 10 mV, DC voltage was 0.13 V, and the frequency range was 100 kHz–0.1 Hz. The error bars were obtained by using four different aptasensors.

Figure 5

(A) EIS of the CSPE/CNF–AuNP/Aptamer to 2 nM SARS-CoV-2 RBD in the absence (black curve) and presence of 200 nM of HIgG, HIgA, HIgM, and HSA (red curve). (B) The EIS of the CSPE/CNF–AuNP/Aptamer to 2 nM of SARS-CoV-2 RBD in the absence (red curve) and presence of 200 nM of HA, and N from an influenza A virus (blue curve). (C) The stability of the CSPE/CNF–AuNP/Aptamer on the different days (First day: green curve; Seventh day: black curve; Fourteenth day: red curve). EIS spectrums were recorded in 5.0 mM Fe(CN)₆^3−/4− solution (0.1M PBS, pH 7.4). The equivalent electric circuit is compatible with the Nyquist diagrams. R_s: Solution resistance; R_ct: Charge transfer resistance; C_dl: Double layer capacitance; Z_w: Warburg impedance. The AC amplitude was 10 mV, DC potential was 0.13 V, and the frequency range was 100 kHz−0.1 Hz.