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

• Published in: Microchemical journal 2024-04, Vol.199, p.109931, Article 109931
• Main Authors: Fan, Yuhang, Peng, Junling, Liu, Jing, Wang, Jianping
• Format: Article
• Language: English
• Subjects: Bioluminescence; Milk; NLuc-TetR; TCs residuces
• ISSN: 0026-265X
• DOI: 10.1016/j.microc.2024.109931

[Display omitted] •NLuc-TetR fusion protein with the bioluminescence of NLuc and the specificity of TetR to nine Tetracyclines was prepared.•A bioluminescence method based on NLuc-TetR was developed after parameter optimization.•The developed method was utilized for the residue detection of nine Tetracyclines in milk, with LODs of 2 to 11 pg mL-1. 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.