牛乳中糖肽类抗生素的倏逝波荧光生物传感器快速检测技术研究

Food safety problems caused by antibiotics had been one focus of attention. The glycopeptide antibiotics, with vancomycin as the representative, were explicitly included in the list of non-food substances by National Health and Family Planning Commission (NHFPC), but their corresponding rapid detection method was not yet mature. In this study, the typical glycopeptide antibiotics (vancomycin, norvancomycin) in milk were used as the targets to design and synthesize immunogen molecules which could maximally present the structure of glycopeptides, to prepare high-quality antibody suitable for glycopeptides, to analyze evolution law of glycopeptide antibiotics aptamer, to elucidate the molecular recognition basis of aptamer, and to screen the aptamer with excellent performance. On this basis, the influencing factors and change rules of biosensor modification process and biological recognition molecule immobilization process were researched, and the evanescent wave fluorescence biosensor with high biological recognition molecule activity, large load, good uniformity and consistency, weak non-specific adsorption and good reproduction performance was manufactured to realize rapid determination of glycopeptide antibiotics in milk. This paper has important theoretical and methodological innovation in the aspects of improving specific identification and rapid and sensitive detection of antibiotics in food, and supplies new ideas and methods for detection and analysis of harmful substances in food, so as to promote the development and progress of testing technology in food safety.

抗生素引发的食品安全问题一直是人们所关注的热点，以万古霉素为代表的糖肽类抗生素被卫计委明确列入非食用物质清单，但其相应的快速检测方法还不成熟。本研究以牛乳中典型糖肽类抗生素（万古霉素、去甲万古霉素）为研究靶标，设计合成能最大程度突出糖肽类抗生素结构的免疫原分子，淘选制备适合糖肽类抗生素的高质量抗体；分析筛选过程中糖肽类抗生素核酸适体的进化规律，阐明核酸适体的分子识别基础，筛选具备优良性能的核酸适体；在此基础上，研究生物传感元件修饰过程和生物识别分子固定过程的影响因素及变化规律，制备生物识别分子活性高、载量大、均匀性和一致性好、非特异性吸附弱、具有良好再生性能的糖肽类抗生素倏逝波荧光生物传感器，实现牛乳中糖肽类抗生素的快速测定。在提高对食品中抗生素特异性识别和快速、灵敏检测等方面具有重要的理论和方法创新，为食品中有害物质的检测分析提供了新的思路和方法，不断推进食品安全检测技术的发展进步。

抗生素引发的食品安全问题一直是人们所关注的热点，以万古霉素为代表的糖肽类抗生素被卫计委明确列入非食用物质清单，但其相应的快速检测方法还不成熟。本研究以牛乳中典型糖肽类抗生素（万古霉素、去甲万古霉素）为研究靶标，设计合成能最大程度突出糖肽类抗生素结构的免疫原分子，淘选制备适合糖肽类抗生素的高质量抗体；分析筛选过程中糖肽类抗生素核酸适体的进化规律，阐明核酸适体的分子识别基础，筛选具备优良性能的核酸适体；在此基础上，研究生物传感元件修饰过程和生物识别分子固定过程的影响因素及变化规律，制备生物识别分子活性高、载量大、均匀性和一致性好、非特异性吸附弱、具有良好再生性能的糖肽类抗生素倏逝波荧光生物传感器，实现牛乳中糖肽类抗生素的快速测定。通过项目的实施，获得了万古霉素特异性强、亲和力高的多克隆抗体和单克隆抗体，经ELISA实验验证性能符合要求；获得两条亲和力较高的万古霉素核酸适配体，分别为：①GTCAGTCATTCTAGTCTAGTCGTAGTCGTAGTCGTAGTCGTGTAGTCACGTGTAGTCAGTCGTAGTCGTGTA；②GTCAGTCATTCTAGTCTAGGTCAGTCGTAGTCGTAGTCGTGTACGTGTCAGTGTCAGTCGTCAGTCGTAGTCGTGTA；研制了万古霉素的免疫荧光倏逝波生物传感器，并建立了基于生物传感器的牛乳中万古霉素快速检测方法。方法检测结果与质谱方法一致，检测时间15分钟，可以满足万古霉素快速检测的需要；建立了糖肽类抗生素（万古霉素、去甲万古霉素和替考拉宁）的液相色谱-串联质谱确证检测方法。上述研究结果的获得为牛乳中糖肽类抗生素残留检测后续的研究及推广应用奠定了基础，在提高对食品中抗生素特异性识别和快速、灵敏检测等方面具有重要的理论和方法创新，为食品中有害物质的检测分析提供了新的思路和方法，不断推进食品安全检测技术的发展进步。