| In recent years, every country pays more attention to the food safe accident. The antibiotics residues in milk are relation to the safe of the people's health. Now every country pays more attention to the harm of the antibiotics residues in milk, so it is very important to detect and monitor the antibiotics residues. The SPR detection has the advantage of rapid, non-label, and high detection. So in this thesis we studied the antibiotics residues in milk based the SPR. The details of the work as below:The experiment conditions of the antibiotics detection based on the SPR was studied. And the most suitable condition of the antibiotics detection was analyzed. The study of overseas based on the huge SPR instrument Biocore didn't have a uniform standard for reagents and samples. While the affect factors of regeneration method, inject rates, reagent concentration and PH value were studied in this thesis. And according to the experiment results the optimal condition and the standard approach were established. For example, the ampicllin influence factors was studied, such as reagents , flow rate, and the most suitable measurement condition was established, and the test precision and stability were improved. For instance, the flow rate 10μL/min and regeneration solution 70mM NaOH were the first-rank experiment conditions.The Surface Plasmon Resonance(SPR)-based optical biosensor direct and indirect assays for detecting the ampicilllin residues in milk were developed. The assays were based on the bio-molecular interaction analysis (BIA) and the high sensitivity to the refractive index of the sensor. Combining with the monoclonal antibody anti-ampicillin binding on the sensor surface, the indirect assay could offer the capability of quantitative detection for ampicillin. The measuring system was equipped with miniature integrated SPR sensor. For indirect assay, the method of the preparation of the sensor surface was introduced, and the assay was designed to cover the ampicillin concentration range from 0ng/ml to 100ng/ml. With analyzing the detecting results, the sensorgrams of milk samples with different concentration of were obtained. Both of the methods could detect the sample rapidly, and overcome the disadvantage of the traditional methods. We believe that this assays based on the SPR technology could be applied to the detection of the antibiotics residues in milk. The limit of detection for ampicillin can reach 33.6mg/dL for direct assay and 50ng/mL for indirect assay.The development of an immunoassay for the detection of ampicillin residues in milk using a miniature surface plasmon resonance (SPR) biosensor was reported. The assay was based on a competitive inhibition assay with ampicillin covalently immobilized on the sensor surface. Milk samples with ampicillin residues were incubated with a fixed amount of anti-ampicillin monoclonal antibody(AMA) 3H295 in a first step,whereby ampicillin in the positive samples would bind to AMA. Non-complexed AMA in the sample was then allowed to form a complex with the ampicillin immobilized on the sensor chip. Then the amount of free AMA in the sample was detected in a SPR-based biospecific interaction analysis (BIA). The results were available in 10 min. The limit of detection of the immunoassay was determined as 2.5ng/mL, well below the European maximum residue limit (MRL) of 4ng/mL. Repeatability (or coefficient of variation) between runs was determined as 5.4% at 2.5ng/mL. The results show that this assay is suitable for routine use. |
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