Resumen de Novel Specific Receptor?based Techniques for Antibiotic Residue Analysis
The indiscriminate and/or improper use of veterinary drugs is the cause of potential adverse health effects due to the risk of entering into the food chain and the appearance of residues in food products of animal origin. Moreover, in the case of antibiotics, this fact has been identified as one of the causes for the appearance of antimicrobial resistance mechanisms in bacteria causing human diseases, which is the cause of a big concern within the health authorities, distinct governmental agencies and the society in general. There is an increased need to ensure safety and quality of the foodstuff and customers have also started to become more exigent forcing the industry to introduce consumer worries in their daily procedures, in terms of getting more natural, ecological and healthy products with a clear traceable origin of the ingredients. Nowadays, food control is performed on centralized laboratories that in most cases use very reliable procedures, but involving expensive equipment, specialized personnel and time consuming sample treatment procedures. In order to drastically improve this situation, the European Commission (EC) and the member state agencies are strongly supporting research activities aimed to increase the efficiency of the actual analytical methods. An strategy is to take advantage of the latest bio-micro-nanotechnological advances and of the complementary skills of multidisciplinary research teams to develop more rapid, sensitive and specific methodologies capable of detecting a wide variety of chemical, biological or any other health risk associated to the agrofood industry and along to the whole food chain. This thesis describes the research performed in respect to the possibility to develop improved alternatives for food residue analysis based on the combination of selective receptors and novel micro/nanotechnologies. Particularly, the final objective of this thesis was addressed to the development of a multiplexed sensor device to detect inappropriate farm practices and or the contamination of food products by antibiotic residues, mainly milk. In this respect, production of selective receptors with a broad recognition of the most important antibiotic families used in the veterinary field has been one of the most important aims of this research work. Thus, we report here the investigation made regarding development of synthetic receptors for sulfonamide antibiotics (SAs), particularly molecular imprinted polymers (MIPs), and their evaluation of a rational approach. Moreover, production of generic (or class-selective) antibodies for SAs and tetracycline antibiotics (TCs) has also been approached through the rational design and synthesis of appropriate haptens. Evaluation of the features of the antibodies produced has been accomplished through the development of microplate-based ELISAs (enzymelinked immunosorbent assays). The results show that although it has been possible to obtain class-selective antibodies for SAs (up to 11 congeners can be detected), the approach followed for the case of the TCs afforded antibodies with a high selectivity versus TCs possessing an alkyl/alkene group at position 6 of the C-ring, but lacking the hydroxyl group at this position. The necessary protocols to apply these immunochemical procedures to the analysis of milk and hair samples have been established showing that determination of these antibiotics according to the EC regulations was possible for the case of milk. For the case of hair, no regulations do exist at the moment. However this matrix holds great value regarding their potential use to trace inappropriate treatments through the life of the farm animals. The immunoreagents and immunochemical procedures established have been implemented on an optical sensor device developed by the Centre Suisse for Electronics and Microelectronics Inc. (CSEM, Neuchâtel - Switzerland). This device is based on the evanescent wave principle using a particular technological design based on waveguide grating couplers and it is very sensitive to the changes in the refractive index produced at the surface of the transducer. Moreover, the chip developed by CSEM has 24 sensing pads which allow multiple measurements with the same transducer. As a consequence of this collaboration it has been possible to develop a biosensor device able to detect SA residues in milk samples in compliance with the EC regulations. Further investigation, has led to the development of a multiplexed biosensor device by combining immunoreagents (SAs and fluoroquinolone antibiotics (FQs)) produced at the Applied Molecular Receptors group (AMRg) of the CSIC, with bioreceptors (ß lactam antibiotics (BLs) and TCs) provided by UNISENSOR S.A. (Liege - Belgium). The results obtained were very good. About 34 antibiotics from four different antibiotic families can be detected in milk samples following all the EC regulations with the device developed. Before, a multianalyte microplate-based ELISA had been developed combining the same bioreagents to evaluate performance and to establish the most appropriate immunochemical procedures. The immunochemical methods developed within this thesis, including immunoassays and immunosensors, have been preliminarily evaluated in collaboration with the Nestlé Research Centre (NRC), at Lausanne in Switzerland, in order to demonstrate performance in real milk samples.
