用于病原菌现场即时并行检测的倏逝波纳米生物传感技术研究

The rapid detection of pathogenic bacteria is greatly required in the field of clinical diagnosis and environmental monitoring. Based on the fusion of evanescent wave, biological affinity reaction, and fluorescence detection principle, the evanescent wave nanobiosensor is developed to achieve on-site real-time detection of pathogenic bacteria by using "quantum dot-biological recognition molecule" nanoprobes with high labeling efficiency and high activity and mesoporous film that self-assemblies on the optical waveguide sensing surface and is suitable for the in-site separation of nanoprobes and bacteria. We are focused on the study of the process and mechanism of the biological molecular recognition assembled on nanomaterials interface, and the self-assembled key factors and regulation methods of the mesoporous film with high orderly structure and proper aperture size. Using the finite element method, the precise optic energy flow coupling model of the evanescent wave nanosensor is established, which is applied to study the evanescent wave distribution in mesoporous film and its interaction with nanoprobes. The key factors improving the performance of evanescent wave nanobiosensor and their mechanisms are explored based on theoretical analysis and experiments. These results are used for optimizing nanosensing platform design to achieve ultrasensitive detection of pathogenic bacteria. Taking Escherichia coli, Salmonella and other typical pathogenic bacteria for example, the on-site real-time detection methods are developed for testing pathogenic bacteria in real clinical and environmental samples. The parallel detection strategy of multiple pathogens is built.

面向临床检验和环境监测等领域亟需病原菌快速检测技术的迫切需求，通过融合倏逝波原理、生物亲和反应原理和荧光检测原理，设计合成高标记效率、高活性的“量子点-生物识别分子”纳米复合探针，在光波导传感表面自组装介孔膜来实现病原菌和游离纳米复合探针的原位分离，运用倏逝波生物传感器实时检测荧光信号，建立病原菌现场即时检测的倏逝波纳米生物传感技术。着重研究抗体/核酸适体在纳米材料界面组装过程及其识别特性、传感表面自组装高度有序和孔径适宜介孔膜的关键因素及调控方法，建立基于有限元法的倏逝波纳米传感精密光能量流耦合模型，阐明介孔膜内倏逝波场分布及其与纳米探针作用机制。从理论和实践上揭示提高倏逝波纳米传感分析性能的关键因素及其机制，指导纳米传感平台优化设计，以实现病原菌超灵敏检测。以大肠杆菌、沙门氏菌等典型病原菌为靶标物，发展适用于临床和环境样品中病原菌现场即时检测的新方法，建立多种病原菌并行检测的新技术。

面向临床检验和环境监测等领域亟需病原菌快速检测技术的迫切需求，通过融合倏逝波原理、生物亲和反应原理和荧光检测原理，创新发展了基于氢氟酸管腐蚀法的介孔膜光纤传感探针，运用“双波长激发光分时激发、双色荧光同步检测”新机制，发展了时间分辨效应的倏逝波双色荧光生物传感平台，建立了病原菌现场即时检测的倏逝波纳米生物传感技术。研制的介孔膜光纤传感探针具有高效筛分和荧光增强能力，可有效实现纳米荧光探针与病原菌的筛分并可将荧光检测灵敏度增强约1倍。发展的倏逝波双色荧光生物传感平台有效克服了传统双色荧光同时检测存在光路复杂和光谱串扰问题，在单光纤传感器中实现双波长倏逝场的完美重叠，利用单光电探测器即可实现双色荧光的同步实时灵敏检测，其检测灵敏度可达50fW。融合倏逝波荧光、微纳尺寸效应和高特异性核酸适体，利用“双波长激发光分时激发、双色荧光同步检测”新机制，建立了基于倏逝波双色荧光生物传感器的病原菌并行快速检测方法。沙门氏菌和大肠杆菌O157:H7的检出限分别达到180 CFU/mL and 340 CFU/mL，检测周期少于40min。对血清、环境和食品实际样品的加标回收实验表明：其加标回收率在80~125%之间，相对标准偏差在10%之内，表明该技术可用于病原菌的现场并行即时检测。项目研究成果可为应对日益复杂、种类繁多病原菌的威胁提供新技术和新方法，以提高对临床病原菌的快速检验和环境污染事故的快速响应能力，保障人民健康安全。