基于酶联适配体的四环素检测方法研究

Development of an Enzyme-linked Aptamer-based Assay for the Detection of Tetracycline

摘要:

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8 μg/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0.705 μg/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。

Abstract:

The Mannich method was employed to conjugate the tetracycline (TC) to horseradish peroxidase (HRP). The optimal coating concentration of streptavidin (SA) was 8 μg/mL and aptamer concentration was 20 nmol/L, which were determined by chequerboard titration. The optimized assay conditions were investigated by single-factor experiments. Coating buffer carbonate buffer (CB, 0.05 mol/L, pH 9.6), phosphatic buffer solution (PBS) plus 5 mmol/L MgCl2 and PBS were chosen as coating buffer assay buffer, and standard dilution buffer, respectively.was, The best dilution ratio of TC-HRP and incubation time of aptamer were 1/50 and 1 hour, respectively. An enzyme-linked aptamer assay (ELAA) method was thus established for the determination of TC. The half inhibition concentration (IC50) of the proposed method was 0.705 μg/mL, and the limit of detection (LOD) was 2.5 ng/mL. Compared with other structural analogues (eg. doxycycline, oxytetracycline and chlortetracycline) of TC, there were no conspicuous cross reactions except chlortetracycline (25.9%). This direct competitive ELAA method is specific and highly sensitive that could meet the requirements of quantitative analysis of TC, which is suitable for the rapid detection of TC in variable food samples.