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通过Mannich法偶联辣根过氧化物酶(HRP)和四环素(tetracycline,TC)分子制备酶标记物,采用棋盘滴定法确定链霉亲和素(streptavidin,SA)的包被浓度为8μg/mL,适配体稀释浓度为20 nmol/L。通过单因素实验优化了检测条件,碳酸盐缓冲液(CB,0.05 mol/L,pH 9.6)为包被液,适配体稀释液选择含有5 mmol/L Mg2+的磷酸缓冲液PBS,TC-HRP稀释度为1:50,标准品稀释液为PBS溶液,适配体孵育1 h,最终建立了四环素酶联适配体检测(enzyme-linked aptamer assay,ELAA)方法。该法半抑制浓度(IC50)为0.705μg/mL,检测限(LOD)为2.5 ng/mL,与其结构类似物(多西环素、土霉素、金霉素)测试中,发现除与金霉素稍有交叉反应(25.9%)外,与其他药物基本没有明显交叉反应。本研究所建立的直接竞争酶联适配体检测方法特异性强、灵敏度高,能够满足四环素定量分析要求,适用于食品中四环素的快速检测。
Enzyme labels were prepared by Mannich method coupled with horseradish peroxidase (HRP) and tetracycline (TC) molecules, and the coating concentration of streptavidin (SA) was determined to be 8 μg / mL , Aptamer dilution concentration of 20 nmol / L. The detection conditions were optimized by single factor experiments. Carbonate buffer (CB, 0.05 mol / L, pH 9.6) was used as the coating solution. The phosphate buffer PBS containing 5 mmol / L Mg2 + The dilution of HRP was 1:50, the standard dilution was PBS, and the aptamer was incubated for 1 h. Finally, an enzyme-linked aptamer assay (ELAA) was established. The IC50 was 0.705μg / mL and the limit of detection (LOD) was 2.5 ng / mL. Compared with the structural analogue of doxycycline, oxytetracycline and chlortetracycline, There was no significant cross-reaction with other drugs except for a slight cross-reaction (25.9%). The direct competition enzyme-linked aptamer detection method established in this study has strong specificity and high sensitivity, which can meet the requirements of tetracycline quantitative analysis and is suitable for the rapid detection of tetracycline in food.