Electrochemical Biosensor for SARS-CoV-2 cDNA Detection Using AuPs-Modified 3D-Printed Graphene Electrodes

Abstract

A low-cost and disposable graphene polylactic (G-PLA) 3D-printed electrode modified with gold particles (AuPs) was explored to detect the cDNA of SARS-CoV-2 and creatinine, a potential biomarker for COVID-19. For that, a simple, non-enzymatic electrochemical sensor, based on a Au-modified G-PLA platform was applied. The AuPs deposited on the electrode were involved in a complexation reaction with creatinine, resulting in a decrease in the analytical response, and thus providing a fast and simple electroanalytical device. Physicochemical characterizations were performed by SEM, EIS, FTIR, and cyclic voltammetry. Square wave voltammetry was employed for the creatinine detection, and the sensor presented a linear response with a detection limit of 0.016 mmol L^-1. Finally, a biosensor for the detection of SARS-CoV-2 was developed based on the immobilization of a capture sequence of the viral cDNA upon the Au-modified 3D-printed electrode. The concentration, immobilization time, and hybridization time were evaluated in presence of the DNA target, resulting in a biosensor with rapid and low-cost analysis, capable of sensing the cDNA of the virus with a good limit of detection (0.30 µmol L^-1), and high sensitivity (0.583 µA µmol^-1 L). Reproducible results were obtained (RSD = 1.14%, n = 3), attesting to the potentiality of 3D-printed platforms for the production of biosensors.

Keywords: 3D printed electrode; AuP modified electrode; SARS-CoV-2; creatinine; electrochemical (bio)sensor.

Scheme 1

Schematic illustration of the production of the biosensor and hybridization step. The production of the biosensor consists of the printing step, chemical treatment of the surface, modification with Au (5.0 mmol L⁻¹), and, finally, modification of the surface with the capture sequence (3.0 µmol L⁻¹ for 1 h). The hybridization step is carried out for 30 min after adding the drop of solution containing the target sequence to the surface of the biosensor.

Figure 1

SEM images of electrode surfaces (a) G-PLA, (b) corresponding element mapping of G-PLA, (c) Au/G-PLA (400 s deposition), (d) corresponding element mapping of Au/G-PLA, (e) Au/G-PLA (250 s deposition), and (f) FT-IR spectra of G-PLA (black) and Au/G-PLA (red) were recorded within the wavenumber range of 600 to 4000 cm⁻¹.

Figure 2

(a) Cyclic voltammograms of blank solution (black line) and (red line) 2.0 mmol L⁻¹ CNN using Au/G-PLA electrode. Supporting electrolyte: 0.5 mol L⁻¹ NaCl. Scan rate: 100 mV s⁻¹, (b) Schematic illustration of the interaction of creatinine with AuPs.

Figure 3

(a) Surface response and (b) level curve obtained for the optimization of the variables: Au³⁺ concentration (mmol L⁻¹) and electrodeposition time (s) as a function of the current in the presence of 3.0 mmol L⁻¹ CNN.

Figure 4

(a) Square wave voltammograms for the addition of eight CNN concentrations (0.05 to 3.2 mmol L⁻¹) in 0.5 mol L⁻¹ NaCl. SWV parameters: −5.0 mV (step potential); 20 mV (modulation amplitude); 10 Hz (frequency), (b) The analytical curve was obtained from the variation of I_peak as a function of CNN concentration.

Figure 5

(a) Cyclic voltammograms and (b) Nyquist plots for each stage of sensor modification and after hybridization, (black line) Au/G-PLA, (red line) Probe/Au/G-PLA, and (blue line) 50.0 µmol L⁻¹ target sequence. All analyses were conducted using 1.0 mmol L⁻¹ ferrocenemethanol in 0.1 mol L⁻¹ KCl; CVs were carried out with a scan rate of 50 mV s⁻¹.

Figure 6

Biosensor calibration curve employing (a) CV and (c) EIS techniques, in the range of 1.0 to 50.0 µmol L⁻¹ and 1.0 to 75.0 µmol L⁻¹ target, respectively. (b) CV calibration curves were obtained between the difference of the biosensor analytical signal in the absence and presence of the target sequence employing 1.0 mmol L⁻¹ ferrocenemethanol in 0.1 mol L⁻¹ KCl. (d) EIS calibration curve was constructed from the obtained R_ct values.

Figure 7

(a) Comparison between target sequences positive (50 µmol L⁻¹) and negative (100 µmol L⁻¹). Cyclic voltammograms obtained in the presence of 1.0 mmol L⁻¹ ferrocenemethanol in 0.1 mol L⁻¹ KCl. The scan rate was 100 mV s⁻¹, (b) Bar column plot.