磁性表面增强拉曼基底研制与水产品中致病菌快速检测研究

Faced with complex matrix, low concentration and long consuming time for pathogenic bacteria detection in aquatic food products, it is quite significant to develop rapid, selective and sensitive analytical technology for pathogenic bacteria detection. The project is aim to combine technologies of magnetic enrichment, surface-enhanced Raman spectroscopy (SERS) and external labeling to detection of pathogenic bacteria. Magnetic enrichment is used to reduce matrix interferences and improve analytical selectivity, SERS is used to improve analytical speed, and external labeling is used to improve analytical sensitivity. Magnetic SERS substrate is fabricated to integrate functions of magnetic enrichment and SERS analysis, the labeled SERS probe is fabricated to integrate SERS activity of noble metal nanoparticles, SERS signal of labeled molecules and specificity of aptamers. The characterization, SERS activity and selectivity of the two materials are investigated. A label-free SERS method coupled with magnetic enrichment and principle component analysis (PCA) for rapid and selective differentiation of pathogenic bacteria is developed based on the magnetic SERS substrates, and a magnetic enrichment-assisted labeled SERS method with speediness, high selectivity, high selectivity for pathogenic bacteria detection is developed based on the magnetic SERS substrates and the labeled SERS probes. The two methods are further applied to differentiation and detection of pathogenic bacteria in real aquatic food products, respectively.

针对水产品中致病菌检测存在的基质复杂、待测物含量低、耗时长等问题，开展水产品中致病菌快速、高选择性、高灵敏度的检测新技术研究。本项目拟结合磁富集技术、表面增强拉曼光谱(SERS)技术和标记技术开展致病菌快速检测研究，通过磁富集技术降低基体干扰、提高分析选择性，通过SERS技术提高分析速度，通过标记技术提高分析灵敏度。拟研制集磁富集和SERS检测于一体的磁性SERS基底，研制集贵金属纳米粒子SERS活性、外标分子SERS信号、核酸适配体特异性于一体的SERS标记探针，表征这两种材料的形貌结构，研究其SERS活性和选择性能，基于磁性SERS基底建立快速、高选择性的磁富集-SERS-主成分分析(PCA)联用免标记鉴别致病菌方法，基于磁性SERS基底和SERS标记探针建立快速、高选择性、高灵敏度的磁富集-SERS标记检测致病菌方法，并开展这两种方法在实际水产样品中致病菌鉴别和检测的应用研究。

本项目针对水产品中致病菌检测存在的基质复杂、待测物含量低、耗时长等问题，成功开展了水产品致病菌快速、高选择性、高灵敏度的检测新技术。通过简单、可控、高效的方法制备磁性富集介质和纳米酶活性材料，所制备的磁性材料具有较强的富集作用和选择性分离功能，能有效降低基体干扰，结合纳米酶活性材料的信号转换放大功能，构建了多种快速、高灵敏、高选择性的食源性致病菌检测方法，并成功应用于实际样品分析，具有一定的应用前景。研发了基于磁分离及锌/钴双金属无限配位聚合物催化比色检测金黄色葡萄球菌的新方法，通过磁分离降低了基体干扰，提高了分析物浓度，方法的线性范围为2.2×10^2-2.2×10^7 CFU/mL，检出限为5.8×10^1 CFU/mL，分析过程仅需2.5 h，与不经磁分离的方法相比，灵敏度和检测速度均有明显的提高。研发了基于锌/钴双金属无限配位聚合物固定化酶检测细菌活性的新方法，对不同活性浓度为1.0×10^9 CFU/mL的沙门氏菌悬液进行检测，加标回收率为99.0%-103%，RSD为0.43%-7.5%，与现有方法相比，分析速度、灵敏度均有显著优势；研发了基于适配体介导金属有机骨架材料催化比色检测沙门氏菌的新方法，线性范围为3.63×10^3-3.63×10^7 CFU/mL，检出限为10^3 CFU/mL，该方法成功应用于牛奶、鸡肉、牛肉沙门氏菌的检测，加标回收率为96.8%-108%，RSD为4.6%-7.6%；研发了基于金属有机骨架材料和酶切放大的适配体荧光法检测副溶血性弧菌，线性范围为8.4×10^2-2.0×10^5 CFU/mL，R2为0.9986，检出限为37 CFU/mL，该方法成功应用于虾和八爪鱼中副溶血性弧菌的检测，回收率为88.8%-109%，RSD为3.7%-6.6%，该方法检测速度快、选择性好，对实际样品分析具有一定的潜力。研究成果在Microchimic Acta, Analytical and Bioanalytical Chemistry等杂志上发表，标注基金资助的论文11篇，申请发明专利4件，授权2件。