Evolution of a natural TetR protein and development of a Fe₃O₄ assisted semi-homogeneous fluorescent method for determination of tetracyclines in milk

Compared with antibody, the recognition spectrum of a receptor is broader, and its recognition ability can be improved using simple mutagenesis technique. Compared with conventional immunoassay, the magnetic bead based immunoassay is simpler and can be recycled. Compared with colorimetric and luminescent immunoassays, fluoroimmunoassay is simpler because it does not require a substrate. So a method combines these merits is desirable. In this study, two amino acids in the binding pocket of a natural Escherichia coli TetR protein were mutated to produce a mutant, and the molecular docking showed the binding energies and the numbers of contact acid for 10 tetracyclines all increased. The mutant was coupled with Fe₃O₄ to synthesize a magnetic complex, and a fluorescent tracer was synthesized by coupling quantum dot and minocycline with bovine serum albumin. Under the assistance of 96-well bottom magnet, a semi-homogeneous method based on the two materials was developed on conventional microplate for determination of the 10 tetracyclines in milk. Results showed once assay was finished within 20 min, the limits of detection (drug concentration showing 10% inhibition) for the 10 drugs were in the range of 0.32-0.94 ng/mL, and the magnetic complex could be regenerated for 6 times. Furthermore, the sensitivities were improved for 4-6 folds in comparison with the use of natural TetR. Therefore, this method is simple, sensitive, time-saving and recyclable, and it can be used for routine screening of the 10 tetracyclines in milk.