Development Of Multi-residue Detection Assays For Tetracyclines And Aminoglycosides

The residues of tetracyclines and aminoglycosides in foods of animal origin are a potential risk to human health.The aim of this thesis is to produce receptors for tetracyclines and aminoglycosides and use them as recognition elements to develope novel detection assays for the residues of these two classes of veterinary drugs in foods of animal origin.First,a photoaffinity labeled activity-based protein profiling probe was synthesized for capturing natural TetR from tetracycline-resistant Escherichia coli in this study.Molecular docking results showed that the binding energies between the protein and 10 TCs were in the range of 7.58-8.56 kcal/mol.They are mainly connected by hydrophobic.The main binding sites of the drugs were on the ring of tetracyclines.Surface plasmon resonance(SPR)results showed that the absolute affinity coefficients were in the range of 17.672-25.110 between the protein and the 10 TCs.The captured receptor was used as a recognition reagent to develop a direct competitive chemiluminescence assay to detect the 10 tetracyclines(TCs)in milk.The results showed that the IC50 were in the range of 0.084-0.32 ng/mL and the limits of detection were in the range of 0.002-0.009 ng/mL,and the recoveries from the standards fortified blank milk samples were in the range of 68.4%-91.3%for the 10 TCs.Then,the investigators performed a targeted mutation of the above natural TetR,and mutated HIS 139 to TRP139.The molecular docking results showed that the binding energies of TetR single site mutant with 10 TCs were in the range of 7.13-9.26 kcal/mol,and they were mainly connected by hydrophobic and Pi-Pi.The main binding sites of the drugs were on the ring of tetracyclines.The SPR results showed that the absolute affinity coefficients were in the range of 23.941-33.327 between the TetR single site mutant and the 10 TCs.The TetR single site mutant was used as a recognition reagent to develop a fluorescence polarization assay to detect the 10 TCs in milk.The results showed that the IC50 were in the range of 15.5-55.2 ng/mL and the limits of detection were in the range of 0.4-1.5 ng/mL,and the recoveries from the standards fortified blank milk samples were in the range of 71.5%-95.4%for the 10 TCs.When SER135 was mutated to PHE135 and LEU 142 was mutated to LYS142 of the above natural TetR,there was a significant enhancement in the stability of the receptor.Molecular docking results showed that the binding energies of multipoint mutant receptor with 10 TCs were in the range of 8.03-9.64 kcal/mol.They were mainly connected by hydrophobic and hydrogen bonds.The main binding sites of the drugs were on the ring of tetracyclines.The multipoint mutant receptor was used as the recognition element to develop a semi-homogeneous competitive fluorescence assay to detect the 10 TCs in milk.The results showed that the IC50 were in the range of 9.3-21.1 ng/mL and the limits of detection were in the range of 0.32-0.94 ng/mL,and the recoveries from the standards fortified blank milk samples were in the range of 74.9%-92.64%for the 10 TCs.In addition,the ribosomal protein S12(RPSL12)of Lysinibacillus sphaericus was expressed in this study.Molecular docking results showed that the binding energies of RPSL12 with 10 aminoglycosides(AGs)were in the range of 5.15-5.84 kcal/mol,and they were mainly connected by hydrophobic.The SPR results showed that the absolute affinity coefficients were in the range of 11.358-26.102 between the RPSL12 and the 10 AGs.The RPSL12 was used as the recognition element to develop a fluorescence polarization assay to detect the 10 AGs in pork.The results showed that the IC50 were in the range of 47.6-72.2 ng/g and the limits of detection were in the range of 2.1-12.1 ng/g,and the recoveries from the standards fortified blank pork samples were in the range of 74.5-97.6%for the 10 AGs.Then,the researchers linked the RPSL12 gene of E.coli with the Renilla luciferase gene and expressed the fusion protein(RPSL12-Rluc).Molecular docking results showed that the binding energies of RPSL12 with 7AGs were in the range of 5.44-6.32 kcal/mol,and they were mainly connected by hydrophobic.The SPR results showed that the absolute affinity coefficients were in the range of 12.502-25.235 between the RPSL12-Rluc and the 7 AGs.The RPSL12-Rluc was used as the recognition element to develop a fluorescence polarization assay to detect the 10 AGs in pork.The results showed that the IC50 were in the range of 43.6-75.5 ng/g and the limits of detection were in the range of 0.51-1.1 ng/g,and the recoveries from the standards fortified blank pork samples were in the range of 73.8%-96.2%for the 10 AGs.Finally,the researchers linked the gene of RPSL12 to the gene of TetR and expressed the fusion protein(RPSL12-TetR).Using it as a recognition element to develop a dual-chemiluminescence assay and a dual-fluorescence assay to detect the 10 TCs and 10 AGs in pork.in the a dual-chemiluminescence assay,the results showed that the IC50 were in the range of 0.0287-0.0427 ng/g and the limits of detection were in the range of 0.0016-0.0064 ng/g,and the recoveries from the standards fortified blank pork samples were in the range of 71%-92%for 10 TCs.The IC50 were in the range of 3.8-17 ng/g and the limits of detection were in the range of 0.21-1.8 ng/g,and the recoveries from the standards fortified blank pork samples were in the range of 70.3%-92%for 10 AGs.In the dual-fluorescence assay,the results showed that the IC50 were in the range of 35.7-54.7 ng/g and the limits of detection were in the range of 2.7-6.4 ng/g,and the recoveries from the standards fortified blank pork samples were in the range of 68.7%-91%for 10 TCs.The IC50 were in the range of 26-106.3 ng/g and the limits of detection were in the range of 1.4-10.2 ng/g,and the recoveries from the standards fortified blank pork samples were in the range of 71.3%-90%for 10 AGs.In summary,seven detection assays for AGs and TCs were established in this thesis using receptors as recognition elements.The results showed that these assays can perform rapid multi-residue detection of AGs and TCs,which is important for safeguarding food safety of animal origin and maintaining consumers’ health.