A Broad-Range Disposable Electrochemical Biosensor Based on Screen-Printed Carbon Electrodes for Detection of Human Noroviruses

Abstract

Human noroviruses (HuNoVs) are the major non-bacterial pathogens causing acute gastroenteritis in people of all ages worldwide. No stable culture system in vitro is available for routing the detection of multiple strains of HuNoVs. A simple and rapid method for detection of HuNoVs is of great significance for preventing and controlling this pathogen. In this work, an electrochemical biosensor for sensitive and fast detection of HuNoVs was constructed based on a screen-printed carbon electrode (SPCE). Gold nanoparticles and protein-A were applied on the SPCE surface for enhancement of the electrical signals and the linkage of antibodies with a fixed orientation, respectively. A monoclonal antibody (MAb) against the S domain protein of the viral capsid (VP1) was further immobilized on the SPCE to bind HuNoVs specifically. The binding of VP1 to the coated MAbs resulted in the reduction of conductivity (current) measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The reduction in the current was correlated to the concentration of VP1/HuNoVs. The detection limitation of Genogroup I.1 (GI.1) VP1 and Genogroup II.4 (GII.4) VP1 was 0.37 ng/ml (≈1.93×10⁷ HuNoVs/mL) and 0.22 ng/ml (≈1.15×10⁷ HuNoVs/mL), respectively. The detection limitation of both GI and GII HuNoVs in clinical fecal samples was 10⁴ genomic copies/mL. The results could be obtained in 1 h. We demonstrated that this disposable electrochemical biosensor was a good candidate for rapid detection of different genogroup and genotype HuNoVs.

Keywords: AuNPs; electrochemical biosensor; human norovirus; protein-A; screen-printed carbon electrode; viral capsid proteins.

FIGURE 1

Profile of the screen-printed carbon electrode (A). Schematic representation of the stepwise modification of electrochemical biosensor (B) and measurement element (C). CE: counter electrode, WE: wording electrode, RE: reference electrode, AuNPs: gold nanoparticles, protein A: Staphylococcal protein-A, H9E: monoclonal antibody generated by recombinant capsid proteins of norovirus, BSA: bovine serum albumin, VP1: major viral capsid protein.

FIGURE 2

Preparation and characterization of the electrochemical biosensor for norovirus detection. Field emission scanning electron microscope images of SPCE (A) and AuNPs/SPCE (B), scale bar is 1.0 μm. The AuNP diameter (B, inset), cyclic voltammetry (C), differential pulse voltammetry (D), and electrochemical impedance spectroscopy (E); measurements of SPCE (a), AuNPs/SPCE (b), protein-A/AuNPs/SPCE (c), H9E/protein-A/AuNPs/SPCE (d), BSA/H9E/protein-A/AuNPs/SPCE (e), and VP1/BSA/H9E/protein-A/AuNPs/SPCE (f). The diagram of the modified Randles equivalent circuit (E, inset). The concentration of GII.4 rVP1 was 10 μg/ml. SPCE: screen-printed carbon electrode, AuNPs: gold nanoparticles, protein A: Staphylococcal protein-A, H9E: monoclonal antibody generated by recombinant capsid proteins of norovirus, BSA: bovine serum albumin, VP1: major viral capsid protein.

FIGURE 3

Cyclic voltammetry performance of VP1/BSA/H9E/protein-A/AuNPs/SPCE in 5.0 mM [Fe(CN)₆] ^3-/4- (1:1) and 0.1 M KNO₃ at different scan rates (20–200 mV s⁻¹) (A). Linear relationship between scan rate^1/2 and peak current (B). The black line and red line are I_pa and I_pc, respectively. Linear regression equation: I_pa (µA) = 54.39 + 30.81v^1/2 ((mV s⁻¹)^1/2; R ² = 0.9988); I_pc (µA) = -27.84–33.72v^1/2 ((mV s⁻¹)^1/2; R ² = 0.9925).

FIGURE 4

Correlation between shifting current percentage and the GI.1 rVP1 concentration (A), GII.4 rVP1 concentration (B), and corresponding calibration curve (C) (D).

FIGURE 5

Reproducibility (A), selectivity (B), and stability (C) of the electrochemical biosensor. VP1, GII.4 rVP1; VP1+MNV, GII.4 rVP1 mixed with murine norovirus lysates; VP1+TV, GII.4 rVP1 mixed with Tulane virus lysates; mixture, GII.4 rVP1 mixed with both murine norovirus and Tulane virus lysates.

FIGURE 6

Correlation between the shifting current percentage and a series of dilutions (log gc/L) of clinical fecal samples. Human norovirus samples (A) GII.4 (1717), (B) GII.4 (1704), and (C) GI.1 (3010). gc, genomic copies.