亚太赫兹纳米生物传感器快速侦检细菌的关键技术研究

For the detection of bacteria at various traits, such as species, survival state, and antimicrobial resistance, it is still unable to avoid time-consuming bacterial culture, unable to directly analyze bacteria in mixed infection samples, or effectively distinguish dead and live bacteria. The sub-terahertz (sub-THz) sensing technologies have physical characteristics and unique advantages to solve above described technical problems. However, it cannot be used to directly detect and analyze bacterial from clinical samples because of its relatively lower sensitivities. Based on our previous works and preliminary experiments, the project plan to firstly further optimize the existing sub-THz nano-biosensor which has integrated the advantages of three innovative technologies, i.e., microfluidic technologies for the separation and preparation of single cell, slot nanoantennas for the colossal amplification of spectroscopic signal, and sub-THz sensing technologies for the directly detect and analyze bacteria without consideration of water interference presented in bacteria cells, clinical samples and environment. And then, using mycobacterium tuberculosis as model system, the macrobiomolecule or cell components having significant different categories, structures and abundance in various traits of bacteria was analyzed and screened using available online database and bioinformatics tools. The bacteria at various traits were directly detect and analyzed by the sub-THz vibration spectroscopy fingerprint characterized by above described macrobiomolecule or cell components using sub-THz nano-biosensors. The sub-THz nano-biosensors technologies prepared in the current project is a real “a novel technique and a novel method to revolutionarily detect and analyze bacteria in minutes”: no need of bacterial culture, no need of label and reagent, real-time, fast, directly qualitative and quantitative detection and analysis. The sub-THz vibration spectroscopy fingerprint provided in the current project would become the novel biomarkers for the bacterial detection and analysis, and could be widely used in the clinical laboratory diagnosis of various infectious diseases. Moreover, it is expected to take the leader position in the formation of research systems, novel academic disciplines and industrial bases of Sub-THz & Laboratory Medicine at both home and abroad.

种属、生存状态和耐药性等不同性状细菌的检测目前仍然无法避免耗时长的细菌培养，或无法直接检测混合细菌和区分死菌与活菌。亚太赫兹(THz)传感技术具有解决上述技术难题的物理特征与独特优势，但其较低的灵敏度无法实现直接检测细菌。本项目在前期工作基础上，将分离和制备单细胞的微流体技术、极端放大光谱信号的狭槽纳米天线技术与亚THz传感技术有机整合，进一步优化现有亚THz纳米生物传感器，筛选不同性状细菌在种类、结构和丰度上存在显著差异的细胞成分，并通过这些细胞差异分子所表征的菌体亚THz振动光谱指纹快速侦检细菌。本项目构建的亚THz纳米生物传感器是一种无需细菌培养、无需标记和试剂、实时快速、直接定性和定量的真正“以分钟计的革命性微生物检测新技术与新方法”，获得的亚THz振动光谱指纹是细菌检测的新指标，可广泛应用于诊治细菌感染性疾病，有望在国内外率先形成亚THz检验医学的研究体系、优势新学科和产业基础。

种属、生存状态等不同性状细菌的检测目前仍然无法避免耗时长的细菌培养，或无法直接检测混合细菌和区分死菌与活菌。太赫兹(THz)技术具有解决上述难题的物理特征与独特优势，但其检测病原菌的研究尚处于起步阶段。本研究通过改进和优化现有的THz光谱和成像技术平台，从病原菌的菌落、菌体和核酸片段三个层次出发，进行临床常见病原菌的THz图谱特征研究和核酸片段的恒温快速检测。我们系统性的开展了病原菌和THz波信号相互作用研究，通过对四种常见感染性病原菌菌体进行THz频谱表征和THz吸收二维模型构建，阐明了病原菌胞内含水量在病原菌THz波信号中的主导作用，实现了病原菌的种属鉴定和生存状态评估。在机理研究的基础上，通过发挥THz波“图谱合一”的技术优势，利用透射式THz成像和THz衰减全反射（THz-ATR）技术进一步开展了培养后菌落的可视化成像检测和培养平板上的菌落原位成像分析。此外，为了进一步提高THz技术检测特异性和灵敏度，我们整合RCA反应和“超材料-纳米颗粒”信号放大系统，以病原菌特异性DNA片段为靶标，从分子水平开展RCA-THz液相传感体系和THz超材料纳米生物传感器的构建，并对其检测临床菌株的性能进行了系统性评估，获得了检测灵敏度和特异性等关键方法学参数。牵头联合全国各地医院，创立了首个以百家医院为网络的THz病原菌样本库，为后续的临床研究、应用和大样本数据库的建立奠定了坚实的基础。通过上述工作，本研究构建了一套病原菌菌落、菌体和核酸水平的THz波无标记检测技术，相对于现有的临床微生物高特异的特点和一定的临床应用价值，为THz波生物医学研究和病原菌检测领域提供了一种全新的研究思路。