Development of Highly Sensitive Immunosensor for Clenbuterol Detection by Using Poly(3,4-ethylenedioxythiophene)/Graphene Oxide Modified Screen-Printed Carbon Electrode

Abstract

Clenbuterol (CLB) is an antibiotic and illegal growth promoter drug that has a long half-life and easily remains as residue and contaminates the animal-based food product that leads to various health problems. In this work, electrochemical immunosensor based on poly(3,4-ethylenedioxythiophene)/graphene oxide (PEDOT/GO) modified screen-printed carbon electrode (SPCE) for CLB detection was developed for antibiotic monitoring in a food product. The modification of SPCE with PEDOT/GO as a sensor platform was performed through electropolymerization, while the electrochemical assay was accomplished while using direct competitive format in which the free CLB and clenbuterol-horseradish peroxidase (CLB-HRP) in the solution will compete to form binding with the polyclonal anti-clenbuterol antibody (Ab) immobilized onto the modified electrode surface. A linear standard CLB calibration curve with R² = 0.9619 and low limit of detection (0.196 ng mL^-1) was reported. Analysis of milk samples indicated that this immunosensor was able to detect CLB in real samples and the results that were obtained were comparable with enzyme-linked immunosorbent assays (ELISA).

Keywords: clenbuterol; electrochemical; graphene oxide; immunosensor; poly(3,4-ethylenedioxythiophene).

Figure 1

Schematic diagram of fabrication of clenbuterol hydrochloride (CLB) immunosensor. (a) Electrochemical immunosensor format used for CLB detection; (b) Indirect electron transfer for TMB redox shown by complex TMB/HRP/H₂O₂ enzyme reaction on modified SPCE for reduction current formation.

Figure 2

(a) Cyclic voltammetrys (CVs) of (i) bare screen-printed carbon electrode (SPCE); (ii) screen-printed carbon electrode poly(3,4-ethylenedioxythiophene)/graphene oxide (SPCE/ PEDOT/GO); (iii) SPCE/PEDOT/GO/Ab; (iv) SPCE/PEDOT/GO/Ab-CLB in 1mM K₃[Fe(CN)₆] and 0.1 M KCl; (b) electrochemical impedance spectroscopy (EIS) of (i) bare SPCE; (ii) SPCE/PEDOT/GO; (iii) SPCE/PEDOT/GO/Ab; (iv) SPCE/PEDOT/GO/Ab-CLB in 5 mM K₃[Fe(CN)₆], 5 mM K₄[Fe(CN)₆], and 0.1 M KCl.

Figure 3

Field emission scanning electron microscope (FESEM) images of (a) bare SPCE; (b) SPCE/PEDOT/GO; (c) SPCE/PEDOT/GO/Ab; and, (d) SPCE/PEDOT/GO/Ab-CLB.

Figure 4

(a) Plot signal to background (S/B) for each step potential from −0.6 to 0.6 V with chronoamperometry measurement at various concentrations (0, 75, 150 and 250 ng mL⁻¹); (b) Enzyme-linked immunosorbent assays (ELISA) titer of Ab activity at various concentrations from 10⁰ to 10⁻⁷ mg mL⁻¹ (the black points represent Ab titer before immunization, while the red points represent Ab titer after immunization); (c) Standard immunosensor CLB calibration curve.

Figure 5

(a) The measured currents are fitted to a sigmoidal curve for estimation of limit of detection (LOD) (solid line); (b) Immunosensor selectivity against other antibiotics from β-agonist family. Structures of some representative of β–agonist families; (c) clenbuterol; (d) salbutamol; (e) mabuterol; (f) ractopamine; and (g) terbutaline.