Gold nanoparticle-modified screen-printed carbon electrodes for label-free detection of SARS-CoV-2 RNA using drop casting and spray coating methods

Abstract

Background and purpose: This study aimed to explore the modification of screen-printed carbon electrode (SPCE) to produce an extensive conductive surface with gold nanoparticles (AuNPs) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA).

Experimental approach: The experiment was carried out using drop casting (DC) and spray coating (SC) methods. Au-S covalent interactions were formed between thiolated single-stranded DNA (ssDNA) and Au surface, which further hybridized with the target RNA to be detected using differential pulse voltammetry (DPV). Optimization of experimental conditions was performed using Box-Behnken design (BBD) on probe ssDNA concentration, probe ssDNA immobilization time, and target hybridization time. The morphology of the modified electrode was characterized using a scanning electron microscope, while the electrochemical behaviour was determined with DPV and electron impedance spectroscopy.

Key results: The results showed that SPCE modification with AuNPs by DC produced a higher peak current height of 12.267 μA with an R _ct value of 2.534 kΩ, while SC improved the distribution of AuNPs in the electrode surface. The optimum experimental conditions obtained using BBD were 0.5 μg mL^-1 ssDNA-probe concentration, an immobilization time of 22 minutes, and a hybridization time of 12 minutes. The limit of SARS-CoV-2 RNA detection at a concentration range of 0.5 to 10 μg mL^-1 was 0.1664 and 0.694 μg mL^-1 for DC and SC, respectively. The T-test results for both methods show that the current response of target RNA with SPCE/AuNP by DC does not show the same result, indicating a significant difference in the current response between those two methods.

Conclusion: SPCE/AuNP by DC is better than SPCE/AuNP by SC for immobilizing inosine-substituted ssDNA, which subsequently hybridizes with viral RNA, enabling label-free detection of guanine from SARS-CoV-2 RNA.

Keywords: Electrochemistry; SARS-CoV-2; biosensor; drop casting; gold nanoparticles; spray coating.

Figure 1.

Illustration of SPCE modification with AuNPs using DC and SC techniques of electrochemical DNA biosensor for detection RNA SARS-CoV-2

Figure 2.

Images of morphological surface observation using SEM for (a) bare SPCE; (b) SPCE/AuNP by DC; and (c) SPCE/AuNP by SC

Figure 3.

(a) DPV voltammogram of SPCE in scan rate of 0.008 V s^-1 in the potential range of -0.8 to 0.8 V with a scan rate of 0.008 V s^-1, E_step 0.005 V, E_pulse 0.05 V, and t_pulse 0.05 s and (b) Nyquist plot of SPCE in a frequency of 0.1 to 1000000 Hz at anodic peak current potential of 0.01 V using 10 mM K₃[Fe(CN)₆] 10 mM in 100 mM KCl solution

Figure 4.

The difference of guanine oxidation current peak in DPV voltammograms for the DNA probe-target RNA hybridization, DNA probe-non target hybridization, and DNA probe on the SPCE/AuNPs by (a) DC and (b) SC. The solution was 0.01 M PBS with a scan rate of 0.008 V s^-1 in the potential range of -0.8 to 1.2 V with a scan rate of 0.008 V s^-1, E_step 0.005 V, E_pulse 0.05 V, and t_pulse 0.05 s

Figure 5.

DPV voltammogram of guanine oxidation against variations in synthetic target RNA concentration on SPCE/AuNPs by (a) DC and (b) SC. Calibration curve of guanine oxidation signal against variation in target RNA concentration (0.5; 1; 1.5; 5; 10 μg mL^-1) on SPCE/AuNP by (c) DC and (d) SC, measured using DPV with a scan rate of 0.008 V.s^-1 over a potential range of -0.8 to +1.2 V in 0.01 M PBS solution