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).
Subject(s)
Biosensing Techniques/methods , Clenbuterol/isolation & purification , Food Analysis/methods , Graphite/chemistry , Animal Feed/analysis , Animals , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Carbon/chemistry , Clenbuterol/toxicity , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Horseradish Peroxidase/chemistry , Humans , Limit of Detection , Polymers/chemistryABSTRACT
Development of an immunoassay for clenbuterol (CLB) detection required an anti-CLB antibody as an important bioreceptor. In this study, we report our work on production and purification of a rabbit-derived polyclonal anti-CLB antibody. The antibody was then purified by nProtein A Sepharose affinity column and the antibody purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The activities of purified antibody were evaluated based on high antibody titer determined from enzyme-linked immunosorbent assay (ELISA). The sensitivity and selectivity of this antibody was evaluated and exhibits negligible cross-reactivity to antibiotics other than ß-agonist families. Evaluation of the antibody as bioreceptor in immunoassay was performed using direct competitive ELISA and exhibited linear calibration plot (R² = 0.9484). The antibody was used to detect the content of CLB in spiked milk samples and the recovery of more than 92% indicating significant performance as bioreceptor for the development of a rapid and simple immunoassay.
Subject(s)
Antibodies/chemistry , Clenbuterol/analysis , Animals , Antibodies/immunology , Enzyme-Linked Immunosorbent Assay/methods , RabbitsABSTRACT
Clenbuterol (CLB) is an illegal antibiotic for livestock, which is misused as a growth promoter drug. In this study, an immunosensor modified with poly(3,4-ethylenedioxythiophene) (PEDOT), multi-walled carbon nanotubes (MWCNT) and anti-clenbuterol antibody (Ab) was developed for the detection of CLB. A screen-printed carbon electrode (SPCE) was modified with PEDOT/MWCNT as a sensor platform before immobilizing Ab for specific CLB binding through a competitive-type immunoassay. Free CLB in the sample solution competed with clenbuterol-horseradish peroxide (CLB-HRP) to bind with Ab. A high current signal was obtained after optimization of the electrochemical immunoassay conditions (pH, incubation temperature, antigen (Ag) incubation time and % blocking) using the response surface methodology/central composite design (RSM/CCD). The developed immunosensor is highly reproducible and sensitive with good storage stability, which are necessary for practical application. In real sample application, this immunosensor produces comparable results with liquid chromatography-mass spectrometry; thus, it is useful for CLB screening and monitoring in real meat samples.
