适配体调控纳米磁性探针的食源性致病菌SERS检测及其传感机制研究

As one of the major problems in food safety, foodborne pathogenic bacteria seriously harm human health. Traditional analytical methods cannot meet the demands nowadays, suffering from time-consuming, unable to detect multiple bacteria simultaneously, and low sensitivity. In this proposal, we developed a novel SERS method based on aptamer functional gold nanorod SERS tags and magnetic capture probe for the detection of a variety of pathogenic bacteria in food samples. Based on the combination of aptamer, magnetic nanoprobe with SERS, this method could solve the problems existing in traditional methods for a variety of pathogens detection. One-step method was used to construct the aptamer functioned gold nanorod signal probe and magnetic capture probe respectively, and the regulative rules during the preparation process were revealed by means of characterization techniques. The quantitative sensing method for SERS detection of foodborne pathogenic bacteria was established based on the system of “signal probe-pathogenic bacteria-capture probe” regulated by aptamer, and the SERS response mechanism is further explained by elucidating the binding effect of the assembly and enhancing mechanism of Raman signal. This project will provide a potential method for specific, sensitive and time-efficient detection of pathogenic bacteria in complex food samples, and provide important scientific basis and key theory support for the rapid detection technology and equipment of pathogenic bacteria.

食源性致病菌严重危害人体健康，是食品安全领域的重大问题之一。已有分析法存在耗时费力、灵敏度不高或难以实现多种菌同时检测等问题，无法满足现实需求。针对此问题，本项目将适配体技术、纳米磁性探针和SERS检测的各自优势相结合，创造性提出基于适配体调控“金纳米棒信号探针-致病菌-磁性捕获探针”SERS分析法，并应用于多种致病菌的检测：以常见的大肠杆菌、副溶血弧菌和沙门氏菌等为检测对象，采用“一步法”分别构建适配体诱导的金纳米棒信号探针和磁性捕获探针，通过表征手段揭示其制备过程中的调控规律；建立基于适配体调控的“信号探针-致病菌-捕获探针”的食源性致病菌SERS检测定量传感方法，并从分子结合作用和拉曼信号增强机理两个层面阐明其传感机制。研究结果将为食品复杂基质中致病菌的特异性、灵敏性、时效性检测提供一种具有广阔前景的潜在方案，为致病菌的快速检测技术发展和相应装备研发提供重要科学依据和关键性理论支撑。

本项目针对致病菌污染等造成的食品安全问题，建立了基于适配体调控的“金纳米棒信号探针-致病菌-磁性捕获探针”的食源性致病菌SERS检测体系和定量传感方法，并从分子结合作用、拉曼信号增强机理等层面阐明其传感机制。制备适配体功能化的“金纳米骨”基底，并对其一步合成过程的影响因素和结构稳定性进行着重分析；以适配体功能化的“金纳米骨”为金信号探针，Fe3O4磁性纳米粒子为捕获探针，建立高灵敏性、高稳定性、高特异性检测大肠杆菌O157:H7的SERS传感器。SERS传感器可实现对10-10, 000 CFU/mL浓度大肠杆菌O157:H7的线性检测，检测限3 CFU/mL。该SERS传感器也具有良好的特异性和优越性，且对商业饮用水和生菜实际样品表现出良好的检测适应性。与相关SERS分析平台对比，SERS传感器在线性范围、LOD值、回收率方面优势明显。随后，我们建立了快速、灵敏、同时检测多种致病菌的新型SERS传感器方法。通过不同的适配体和拉曼报告物同时调控金纳米棒生长，构建了八面体裂纹形和环突起形的两种新型的纳米探针，实现了对大肠杆菌O157:H7和沙门氏菌的同时检测，准确度与传统的平板培养法相近，检测时间大大缩短，仅~1小时。该检测模型适用于其它多种致病菌的检测，具有特异性强、快速、灵敏（<10 cfu/mL）的检测效果，有利于食源性致病菌感染的预防和早期控制，可作为国标GB 29921—2013的辅助检测方法。最后，我们建立了一种简便、灵敏的检测和杀灭致病菌的方法。利用DNA调控合成了具有良好催化和抗菌活性的哑铃状Au-Pt纳米颗粒，并将其应用于同时检测和杀灭致病菌。在5分钟内对大肠杆菌O157:H7杀伤效果高达95%，并具有较好的检测效果，线性检测范围为10-107 CFU/mL，检测限为2 CFU/mL。本研究将为食品复杂基质中致病菌的特异性、灵敏性、时效性检测提供一种具有广阔前景的潜在方案，为致病菌的快速检测技术发展和相应装备研发提供重要科学依据和关键性理论支撑。