基于磁分子印迹纳米粒子偶合SPR传感器的食品中抗生素实时快速检测研究

For the contradiction between detection speed, accuracy and sensitivity, and problem of the short life and high cost of the biosensor, a new method of the sample pretreatment coupling real time, high sensitive SPR detection is presented based on the characteristics of the enrichment separation and enhancing SPR signal of magnetic surface molecular imprinting nanoparticle. The preparation methods and conditions of the different size (< 40 nm) of magnetic surface molecular imprinting nanoparticle were investigated Application of sol-gel method, graft copolymerization method and atom transfer radical polymerization technique. The recognition mechanism and the acting site between the antibiotic and polymers were investigated from space structure according to the research of adsorption dynamics, thermodynamics, scatechard analysis and spectral characterization results. The nanopartical of best size were screened based on the high selectivity, adsorption capacity and ability of enhancing SPR signal. Organics is selected to modify SPR chip according to the interaction with molecular imprinting nanoparticle. The mechanism of enhancing SPR signal for magnetic nanoparticles of different particle size and the recognition mechanism of the modified layer are revealed.The SPR detection method with real-time, accuracy and high sensitivity is established and applied for antibiotic detection in food. The successful development of sensor will provide important technical support for food safety and the international trade and technology barrier.

针对复杂基质中小分子检测速度、准确度和灵敏度之间的矛盾，生物芯片寿命短、成本高的问题。本项目提出利用磁表面分子印迹纳米粒子的选择性、富集分离和增强SPR信号的特性，实现样品提取净化与SPR实时高灵敏检测一体化的新思路。应用溶胶-凝胶法、接枝共聚法和原子转移自由基聚合技术，制备多种粒径（＜40 nm）的四环素类抗生素磁表面分子印迹纳米粒子，利用吸附动力学、热力学、Scatechard分析，光谱等分析该纳米粒子的表面结构、形态、吸附等性能，揭示抗生素与聚合物的作用位点、识别机理；研究不同粒径磁纳米粒子增强SPR信号的作用机制，筛选高选择性、吸附量大和增强SPR信号最强的纳米粒子的粒径，探索与其相互作用的化合物修饰SPR芯片；建立抗生素实时快速、特异性强、准确、高灵敏的SPR检测新方法。开展其在食品中抗生素检测的应用评价研究，为保障我国食品安全，应对国际贸易技术壁垒提供技术支撑。

鉴于现有检测技术存在复杂基质中小分子检测速度、准确度和灵敏度之间的矛盾，生物芯片寿命短、成本高问题。本项目以四环素，诺氟沙星等抗生素为例，研发了三种磁性表面分子印迹纳米粒子的制备方法；基于磁分子印迹纳米粒子即可特异性捕获和分离待测物，又可提高SPR信号特性，建立了一系列快速、高准确、高灵敏检测食品中抗生素的方法。. 分别制备了表面羧基化和氨基化的四氧化三铁磁纳米粒子。以其为载体，优化模板分子、功能单体等条件，采用表面印迹法，成功制备了三种磁分子印迹纳米粒子，其吸附容量范围：37.63mg/g—14.17mg/g，印迹因子范围为：2.03—2.88，可重复使用6-8次；确定了最佳吸附条件，1h内（常温），吸附率均可达到100％。. 分别选用巯基丙酸、巯基乙胺等作为SPR芯片修饰层，优化组装和检测条件后，将捕获待测物的磁分子印迹纳米粒子注入SPR传感器，直接进行待测物的检测，三种待测物的低检测限分别为0.98pg/mL、8.73pg/mL和2.79pg/mL；且所建方法均具有良好的特异性。在牛奶、猪肉等样品的回收率均符合要求。与国家标准、市售ELISA试剂盒检测方法相比较，具有更好的灵敏度。三种方法对实际样品的检测结果一致，证明所建方法具有实用性。. 本项目研发的磁性分子印迹纳米粒子，即可作为食品中待测物高效富集分离的吸附剂，也可联合其他技术检测抗生素；所建检测方法，大大节省了样品前处理的时间和成本，提高了检测的准确性和灵敏性，实现了样品前处理和直接检测的完美结合，为促进我国食品进出口，保护国民的生命健康，提供了重要的技术保证和支撑。此外，该项目为发展高效富集分离、检测食品中小分子的研究领域，奠定了理论基础，具有重要的科学意义和学术价值。