Lu Chunxia, Tang Zonggui, Liu Changbin, Kang Lichao, Sun Fengxia
Analysis and Testing Center, Xinjiang Academy of Agriculture and Reclamation Science, Wuyi Road No. 221, Shihezi, 832000, China,
Anal Bioanal Chem. 2015 May;407(14):4155-63. doi: 10.1007/s00216-015-8632-3. Epub 2015 Apr 9.
This study presents a novel analytical method for the detection of oxytetracycline (OTC) in complex food matrices based on a direct competitive enzyme-linked aptamer assay and magnetic separation technology. In this protocol, free OTC competed with horseradish peroxidase labeled OTC (OTC-HRP) for binding to the OTC aptamer immobilized on magnetic beads. The parameters that can affect the response, such as avidin concentration, aptamer concentration, OTC-HRP concentration, incubation temperature, incubation time, blocking agent, and binding buffer, were optimized. Under the optimal conditions, the linear range for the OTC concentration detection is 0.5-100 ng mL(-1), with a concentration of OTC needed to obtain 50 % of the maximum signal of 14.47 ng mL(-1). The limit of detection and the limit of quantitation were 0.88 and 3.40 ng mL(-1), respectively. There was no obvious cross-reactivity with most of the tetracycline pesticides. The recovery rates ranged from 71.0 to 91.2 % for the food samples, including chicken, milk, and honey, and the relative standard deviation was less than 15.0 %. The proposed method was applied to measure OTC in real samples, and was validated using high-performance liquid chromatography. This method has the advantages of magnetic separation and the concentration effect of magnetic nanoparticles, the specificity of the aptamer, and the high-throughput of microtiter plates; it offers a promising approach for the screening of OTC because it is simple, rapid, highly sensitive, and has low cost.
本研究提出了一种基于直接竞争酶联适配体分析和磁分离技术检测复杂食品基质中土霉素（OTC）的新型分析方法。在此方案中，游离的OTC与辣根过氧化物酶标记的OTC（OTC-HRP）竞争结合固定在磁珠上的OTC适配体。对影响响应的参数，如抗生物素蛋白浓度、适配体浓度、OTC-HRP浓度、孵育温度、孵育时间、封闭剂和结合缓冲液进行了优化。在最佳条件下，OTC浓度检测的线性范围为0.5-100 ng mL(-1)，获得最大信号50%所需的OTC浓度为14.47 ng mL(-1)。检测限和定量限分别为0.88和3.40 ng mL(-1)。与大多数四环素类农药无明显交叉反应。鸡肉、牛奶和蜂蜜等食品样品的回收率在71.0%至91.2%之间，相对标准偏差小于15.0%。该方法应用于实际样品中OTC的测定，并通过高效液相色谱法进行了验证。该方法具有磁分离和磁性纳米颗粒的浓缩效应、适配体的特异性以及微孔板的高通量等优点；它为OTC的筛选提供了一种有前景的方法，因为它简单、快速、高度灵敏且成本低。
