抗生素广谱适配体识别机制及牛奶中抗生素多残留适配体传感检测新方法研究

The existing rapid detection methods for antibiotic residues cannot meet the requirements for rapid, timely and efficient detection of a large number of samples.Research and development of detection techniques that can detect several or even dozens of antibiotic residues at the same time is a focus of current research. Aptamer sensor analysis has become a hot topic for antibiotic residue detection.However, the identification mechanism between the broad-spectrum aptamers and the antibiotic and the interference mechanism between milk samples components and aptamer sensors are not clear, which restricts the development of multi-residue detection technology. This project intends to use SELEX technology to select broad-spectrum antibiotic aptamers, and then the structure-activity relationship between aptamers and antibiotics will be established by spectral characterization and Cerius2 software. Meanwhile, the molecular recognition model will be established by a Swiss-model tool. Based on the above methods, the identification mechanism between aptamers and antibiotics will be clarified, and then high specificity and affinity of broad-spectrum aptamers will be obtained. The electrochemical response mechanism of aptamer sensors will be clarified, and the target recycling amplification technology and special elements mark technology will be used to construct a sensing method based on broad-spectrum aptamers. The main interference factors for aptamer sensors from milk components will be elucidated, and the influence laws of different interference factors on the sensor signal will be revealed, which will provide a theoretical guidance for the processing of the milk sample. The studies will obtain a theoretical foundation for the development of high specificity, high sensitivity antibiotic multi-residues detection method.

现有的抗生素残留检测方法不能满足大量样本快速、及时、高效要求。研究同时检测几种甚至几十种抗生素残留的检测技术是当前研究重点。适配体传感分析方法已成为抗生素残留检测的热点。但适配体与抗生素识别机制、牛奶样品成分对传感器干扰机制不明晰，制约了多残留检测技术的发展。本项目拟采用SELEX技术筛选抗生素广谱适配体，通过谱学表征，运用Cerius2软件，建立适配体与抗生素的构效关系，用Swiss-model工具建立适配体和抗生素的分子识别模型，揭示广谱适配体和抗生素的识别机制，确定高特异性和亲和力广谱适配体；明确适配体传感器检测抗生素的电化学响应机制，利用目标循环放大技术和特殊元素标记适配体，构建基于广谱适配体的传感方法。阐明牛奶成分对传感器造成干扰的主要因子，揭示不同干扰因子对传感器的影响规律，为牛奶样品处理方法提供理论指导。以上研究为建立高特异性、高灵敏度的抗生素多残留检测技术发展奠定理论基础。

通常采用多种抗生素协同治疗奶牛养殖中出现的疾病，因而一种牛奶样品中检测出多种抗生素超标现象时有发生。建立针对同一类抗生素的同步检测方法，是实现样品快速高效检测的关键。适配体传感器多残留检测技术的突破有赖于获得抗生素广谱适配体。本项目利用SELEX技术筛选获得氨基糖苷类、青霉素类和四环素类3类抗生素的广谱适配体，分别识别10种氨基糖苷类、4种青霉素类和4种四环素类抗生素，解离常数分别为94.44~185.94 nM，8.24~17.59 nM和4.27~7.20 nM。采用分子对接模拟确定了广谱适配体参与结合口袋形成的关键碱基识别位点，两者以空间结构互补的方式，在适配体的磷酸基团与抗生素共有结构之间的氢键和静电盐桥作用下形成稳定的复合物。以筛选的适配体为识别元件，采用OMC@Ti3C2 MXene等纳米材料与Exo III酶辅助循环放大技术分别构建了适配体传感分析方法用于检测这3类抗生素，检测限分别为35.5 pM、3.33 nM和0.33 nM。研究发现牛奶样品中蛋白质、脂肪、乳糖、Na+、Ca2+对传感器中适配体与抗生素的结合造成干扰，确定了乙腈有机溶剂萃取结合MMIPs磁吸附分离的牛奶样品处理方法，回收率为97.19%~98.70%。建立了基于广谱适配体识别的抗生素检测方法，为牛奶中抗生素多残留同步检测技术的发展提供了重要理论参考和技术指导。