Automated Multiplexed Electrochemical Immunosensing Platform for Antibiotic Residue Analysis in Milk Samples

Abstract

Increasingly research efforts are focused on the development of multi-analyte residue analysis using portable devices for the on-site evaluation of foodquality, and specifically in milk. According to the directive 37/2010/EU, the EU establishes maximum residue limits (MRL) for each family of antibiotics (ATB) used in the veterinary field. The risk of ATB overuse in animals raises serious concerns for human health and the threat of antibiotic-resistant microorganisms. In this regard, we developed an automatized electrochemical platform for the multiplexed determination of the most commonly used antibiotics families in cow milk. The different immunoreagents involved in the determination of the targeted antibiotics (fluoroquinolones, streptomycin, sulfonamides and chloramphenicol) were characterized by indirect competitive enzyme-linked immunosorbent assay (ELISA). Each assay was based on the immobilization of haptenized proteins and the use of broad specificity antibodies, following a previous strategy developed by our group. Once the parameters of the assay were optimized, the matrix effect of full-fat commercial milk was assessed at different dilutions factors, ranging from 1:1 to 1:5. In all cases, the ELISAs reached a IC50 values lower than the corresponding EU MRL. Afterwards, the same immunoreagents were implemented in an automated amperometric immunosensing platform provided of gold screen-printed electrode arrays formed by eight 3-electrode electrochemical cells. The device worked using a cocktail of antibodies for the simultaneous determination of all targeted antibiotics. The LOD and IC50 values achieved are in the same range as the microplate ELISA. Matrix effect studies demonstrated that samples could be measured without any pretreatment. Moreover, a regeneration protocol was established for the consecutive measurements of real samples using the same electrodes. Altogether, our results suggest that the proposed system has a great potential for the on-site semicontinous analysis of a broad range of antibiotics residues in milk samples