Electrochemical Immunosensors Based on Screen-Printed Gold and Glassy Carbon Electrodes: Comparison of Performance for Respiratory Syncytial Virus Detection

Abstract

This paper presents the development and comparison of label-free electrochemical immunosensors based on screen-printed gold and glassy carbon (GC) disc electrodes for efficient and rapid detection of respiratory syncytial virus (RSV). Briefly, the antibody specific to the F protein of RSV was successfully immobilized on modified electrodes. Antibody coupling on the Au surface was conducted via 4-aminothiophenol (4-ATP) and glutaraldehyde (GA). The GC surface was modified with poly-L-lysine (PLL) for direct anti-RSV conjugation after EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide) activation. Electrochemical characterizations of the immunosensors were carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). GC-based immunosensors show a dynamic range of antigen detection from 1.0 × 10⁵ PFU/mL to 1.5×10⁷ PFU/mL, more than 1.0 × 10⁵ PFU/mL to 1.0 × 10⁷ PFU/mL for the Au-based sensor. However, the GC platform is less sensitive and shows a higher detection limit (LOD) for RSV. The limit of detection of the Au immunosensor is 1.1 × 10³ PFU/mL, three orders of magnitude lower than 2.85 × 10⁶ PFU/mL for GC. Thus, the Au-based immunosensor has better analytical performance for virus detection than a carbon-based platform due to high sensitivity and very low RSV detection, obtained with good reproducibility.

Keywords: cyclic voltammetry; electrochemical impedance spectroscopy; glassy carbon; gold electrode; respiratory syncytial virus; sensor.

Figure 1

Mechanisms of different electrode modifications with anti-respiratory syncytial virus (RSV) antibodies and RSV detection: (A) gold electrode; (B) glassy carbon (GC) electrode.

Figure 2

Example of HeLa cells with stained nuclei (blue) and RSV F protein (green) after 24 h of infection.

Figure 3

Measurements of biosensor fabrication steps: (A) cyclic voltammograms for the bare and modified Au electrode, (B) electrochemical impedance spectra for the bare and modified Au electrode, (C) cyclic voltammograms for the bare and modified GC electrode, (D) electrochemical impedance spectra for bare and modified GC electrode. Registered in 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]/0.01 M PBS. Insets represent electric equivalent circuit (EEQC) utilized for fitting and data analysis.

Figure 4

The impedance spectra registered for Au modified with anti‑RSV-bovine serum albumin (BSA), incubated for different periods (1–10 min) with RSV, registered in 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]/0.01 M PBS.

Figure 5

The relation between the immunosensor response expressed as R_ct change (ΔR_ct) and the virus protein concentration for: (A) Au/4-ATP/anti-RSV/BSA, (B) GC/PLL/anti-RSV/BSA. Registered in 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]/0.01 M PBS. Error bars denote confidence interval (α = 0.05, n = 3).

Figure 6

Impedance spectra recorded after incubation with RSV and interfering pathogens. Registered in 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]/0.01 M PBS, the incubation time was 5 min. Error bars denote confidence interval (α = 0.05, n = 3).