Protecting Consumer Health By Developing a Multiplex Amperometric Immunosensor for the Detection of Banned Antibiotic Residues in Milk
Authors: Gaudin Valerie , Hedou Celine , Soumet Christophe , Verdon Eric 
Published: May 2020

Introduction

Chemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer’s market.
Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach.

Electrochemical immunosensor

An innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in milk as a proof of concept. The matrix effect (milk samples) was high and so sample preparation has to be improved to reduce matrix effects.
The objective is to develop an amperometric bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk.

Method

Antibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogene peroxide (H2O2) to the solution, an amperometric signal is produced, due to the enzymatic activity of HRP and measured. The amperometric signal is inversely proportional to the antibiotic concentration in the sample.

Results and Conclusions

Screening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantage sand drawbacks of amperometric biosensors for the screening of antibiotic residues in food products.

References

1. Commission Decision (EC) N° 2002/657 of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. 2002: Official Journal of European Communities. p. 8-36.
2. Commission Regulation (EC) N° 470/2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin 2009, The European parliement and the Council: Official Journal of the European Union p. 11-22.
3. Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014. Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors. Anal. Chim. Acta. 820:32-38.
4. CRL, Guideline for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). 2010: Available from:<http://ec.europa.eu/food/food/chemicalsafety/residues/lab_analysis_en.htm>: Guideline_Validation_Screening_en.pdf. p. 1-18.