靶向磁性纳米仿酶多功能探针的构建及其食源性致病菌检测新方法研究

Foodborne pathogens greatly threaten food safety and human health, which become prominent public healthcare issues widewide. The pathogens usually exist in complicated food matrixes at very low concentration, so it is important and highly desirable to explore a method that can detect pathogenic bacteria sensitively and specifically. In this project, firstly, we will use bovine serum albumin as biological template to prepare Co3O4 nanoparticles (NPs) in a controlled way under mild conditions. The as-prepared Co3O4 NPs will be biocompatible, monodisperse and uniform in shape and size, as well as possess superb magnetic property and peroxidase-like activity. Meanwhile, the formation mechanism of Co3O4 nanozyme will be revealed. Subsequently, Co3O4 nanozyme will be conjugated with a Staphylococcus aureus (S. aureus)-specific and -affinitive nonapeptide ligand-fused phage coat protein pVIII, which will be isolated from phage monoclone selected from f8/9 landscape phage library against S. aureus in a high-throughput way. Then, by optimizing the experimental conditions, we will design a stable and multi-functional nanoprobe which integrates specific recognition, signal amplification, magnetic enrichment and separation together, aiming at developing a sensitive, selective colorimetric method for S. aureus detection and applied for real food samples detection. This proposed approach will not only illuminate new idea to develop detection method for pathogens, but also play an important role in satisfing demands of food safety monitoring and medical diagnostics.

食源性致病菌严重威胁食品安全和人类健康，成为突出的公共卫生问题。由于食品成分复杂、受污染食品中的致病菌数量通常较少，发展灵敏、特异的检测方法具有重要意义。本项目拟以牛血清白蛋白为模板，在温和条件下可控合成生物相容性好、具有单分散性及均一形态、最优的磁性能和仿酶活性的四氧化三钴纳米酶, 并揭示其形成机理。以金黄色葡萄球菌为靶标，经与f8/9风景噬菌体文库生物淘选和特异性测试，获得亲和性高、特异性好的九肽配体展示的噬菌体单克隆，再经分离纯化获得稳定性好的靶向九肽融合的主要衣壳蛋白pVIII，然后共价修饰到磁性四氧化三钴纳米酶表面。通过优化实验条件，构筑兼具靶向识别、磁性富集分离和信号放大为一体的多功能生物纳米探针，旨在建立灵敏、特异的比色法检测金黄色葡萄球菌，建立标准化实验流程并用于实际样品分析。本项目的实施，将为致病菌检测提供新思路，也为满足国家食品安全监测、医学诊断领域的需求做出贡献。

食源性致病菌严重威胁食品安全和人类健康，成为突出的公共卫生问题。由于食品成分复杂、受污染食品中的致病菌数量通常较少，发展灵敏、特异的检测方法具有重要意义。纳米仿酶因其制备简单、酶活高、稳定性好、价廉等优点而在催化、生物传感、生物纳米医学、环境保护等领域具有广阔的应用。本项目以牛血清白蛋白为模板，在温和条件下可控合成了生物相容性好、单分散性、具有优异仿过氧化酶活性的磁性四氧化三钴纳米花。以金黄色葡萄球菌为靶标，经与f8/9风景噬菌体文库生物淘选和特异性测试，获得亲和性高、特异性好的九肽配体（AQTFLGEQD）展示的噬菌体单克隆，再经分离纯化获得稳定性好的靶向融合主要衣壳蛋白pVIII，然后共价修饰到磁性四氧化三钴纳米酶表面,构筑了兼具靶向识别、磁性富集分离和信号放大的多功能生物纳米探针(Co3O4 MNE@fusion-pVIII)，被识别的金黄色葡萄球菌在外磁场下经磁泳层析法分离富集。优化条件，建立了比色法检测金黄色葡萄球菌，其浓度线形范围为10−10,000 cfu/mL (检出限：8 cfu/mL)。其他菌的存在对金黄色葡萄球菌的测定无干扰。该法用于牛奶样品中金黄色葡萄球菌的分析，结果令人满意。本研究提出了构筑可视化快速检测食源性致病菌的新思路，也为食品安全和临床上致病菌的分析检测提供了新的可靠的方法。