DNA-directed immobilization fluorescent immunoarray for multiplexed antibiotic residue determination in milk.

The presence of antibiotic residues in cow's milk entails high risk for consumers, the dairy industry, and the environment. Therefore, the development of highly specific and sensitive screening tools for the rapid and cost-effective identification of traces of these compounds is urgently needed. A multiplexed screening platform utilizing DNA-directed immobilization (DDI) was developed aiming to detect three classes of antibiotic residues (fluoroquinolones, sulfonamides, and tylosin) prevalently found in milk. Throughout this work, each oligonucleotide sequence was conjugated to a different hapten molecule, while the three complementary strands were immobilized in 24 independent microarray chips on a single glass slide. First, the array was incubated with the pool of hapten-oligonucleotide conjugate site encoded the signal through DNA hybridization. Next, commercial milk samples were incubated with the cocktail of monoclonal antibodies following a secondary fluorophore-labeled antibody which was required for fluorescent readout. Direct sample detection was achieved in milk diluting 20 times in assay buffer. The limits of detection (LODs) reached were 1.43 µg kg