A bioluminescence method based on NLuc-TetR for the detection of nine Tetracyclines in milk

Trace antibiotic residues in animal foodstuff may lead to resistance and hazard human health, so simple and rapid detection methods have aroused multiple attention. Here, a novel bioluminescence pseudo-ELISA for the trace detection of multiple tetracyclines (TCs) in milk was designed and prepared based on a new fusion protein NLuc-TetR. The N-terminal of the bio-recognition motif TetR was linked to the C-terminal of the reporting motif nano-luciferase (NLuc) through SOE-PCR. Biolumescence signal showed that NLuc-TetR was specificity to nine TCs. And the acting pattern of substrate, NLuc-TetR, and TCs ternary complex was analyzed by molecular docking. With the optimal parameters, the half inhibition concentrations (IC50) of the developed bioluminescence pseudo-ELISA for nine TCs ranged from 0.39 to 1.44 ng mL−1, and the detection limits (LODs) ranged from 2 to 11 pg mL−1. The results demonstrated that the bioluminescence method based on NLuc-TetR is a promising method for routine screening measures for the residues of TCs in milk.