The Competitive Inhibition Assay And Experimental Study On Antibiotics And Melamine Residues In Milk

Surface plasmon resonance (SPR) biosensors were used to detect antibiotics and melamine residues of milk in the thesis. Some key detection techniques in the method are investigated detailedly. Furthermore, the feasibility of detection method is verified through a series of dection experiment of various residues.First of all, the competitive inhibition assay was proposed to detect antibiotics and melamine in milk, considering the analyte is small molecules. Some details of the detection method were discussed, including ligand selection, the immobiliztion of ligand on sensor surface, pretreatment of samples, detecting flow, regeneration method of sensor and so on.Secondly, the influence of some factors related to experiment on the immobiliztion of ligand on sensor surface was analysed, including temperature, flow rate, pH, ionic strength and concentration of ligand. Result of the experiment shows that 25℃, flow rate of 5μL/min, pH 4.5, ionic concentration of 10mM, and ligand concentration of 400μg/mL are optimum experiment conditions, which can immobilize the enough mass of ligand on the sensor surface to guarantee the accurate measurment.Then, water solutions with a series concentration of kanamycin, ampicillin and melamine were detected, using the sonsors immobilized with BSA-kanamycin, BSA-ampicillin, BSA-melamine respectively. The experiment result shows that the limit of detection (LOD) of three residues were 25ng/mL (150 ng/mL for MRL ), 1.7ng/mL (4 ng/mL for MRL), 100ng/mL (1000 ng/mL for MRL ) , which are more lower than the maximum residues limit(MRL). So the detection method is verified very well.Finally, owing to the influence of the non-specific binding(NSB) to detection of antibiotics and melamine, brought by macromolecuLar and protein in milk, the pretreatment of milk is proposed. After a series of processing, milk solutions with kanamycin, ampicillin and melamine were detected respectively. The experiment result shows that LODs were 30ng/mL, 1.8ng /mL, 110ng/mL, which was close to the result of water solution and lower than MRL too.In conclusion, the detection experiments of antibiotics and melamine verified the feasibility of the method very well. The study can be used for the refenrence in the dection fields of antibiotics and other harmful residues in milk using surface plasmon resonance biosensor.