基于高荧光性能碳点的免疫荧光探针构建及其在微生物检测中的应用研究

Fluoroimmunoassay is a popular method for the detection of bacteria, virus and parasite attributed to its high selectivity and sensitivity. However, the use of this method is greatly limited by the fluorescent performance of the labeling dyes. Therefore, construction of low toxic, strong and stable fluorescent labels is critical to the development of fluoroimmunoassay technique. Recently, carbon dot (C-dot), an emerging fluorescent carbon nanomaterial, has drawn tremendous attention owing to its advantages in stable photoluminescence (PL), broad excitation spectra, tunable emission spectra, low cytotoxicity compared with traditional organic dyes. Moreover, its environmental friendly characteristics signify that C-dots are advantageous over toxic metal-based semiconductor quantum dots (QDs) in actual applications. Therefore C-dot is a promising alternative to QDs in energy conversion/storage, bioimaging, drug delivery, sensors and diagnostics. To date, however, there have been few reports on quantitative detction for microorganism using fluorescent probe based on C-dots. In this project, we propose to explore a simple, green and economical route to build C-dots with strong fluorescence and controllable versatile surface groups and will investigate its potential application in immundetection for the first time. We will focus on: (1) controllable preparation of C-dots and (2) application of C-dots in fluoroimmunoassay. (1) Controllable preparation of C-dots: the amino dominated C-dots (Amino-CD) and carboxyl dominated C-dots (Carboxyl-CD) will be synthesized by microwave-assisted pyrolysis of proper organic precursors as carbon source. The structural features of the synthesized C-dots will be analyzed by FTIR, HRTEM, XRD, XPS, Ramman, NMR, DLS, Zeta-potential, fluorometer and laser scanning confocal microscopy to optimize technical conditions for controllable preparation and to illuminate the growth mechanism and PL mechanism of C-dots. (2)Application of C-dots in fluoroimmunoassay: a fluorescent probe for rapid, sensitive and specific determination of microorganism (using the E.coli O157:H7 as a model) will be constructed by covalently binding monoclonal anti-E.coli O157:H7 antibodies to the surface of Amino-CD or Carboxyl-CD through EDC/NHS chemical coupling reaction. The sensitivity and specificity/selectivity of the C-dots based fluorescent probe will be thoroughly investigated and assessed mathematically to build an experimental model with a linear relationship between the concentration of E.coli O157:H7 and fluorescent intensity. The C-dots based fluorescent probe will exhibit great potential in fast detection of microorganism.

研制低毒、强荧光、高稳定性的荧光标记物对于推动荧光免疫技术的发展至关重要。碳点（C-dots）是一种环境友好、生物安全的新型光致发光碳纳米材料，在能量转换、生物医学和高灵敏度检测等领域具有独特的优势。本项目拟在前期研究基础上，采用微波辅助法制备水溶性的氨基化碳点（Amino-CD）与羧基化性碳点(Carboxyl-CD)，系统分析微波功率、微波时间、反应物配比等工艺条件对碳点结构、组成、尺寸的影响规律，揭示碳点生长模型；探索碳点结构、组成、尺寸、表面状态以及金属掺杂物类型与碳点荧光性能之间的内在联系，阐明碳点荧光机理；以大肠杆菌为微生物模型，通过EDC/NHS 化学偶联法将大肠杆菌抗体修饰到碳点表面，构建免疫荧光探针，用于大肠杆菌的特异性识别与检测，为研制具有自主知识产权的新型、高效、低毒、环保荧光标记物奠定基础。

研制低毒、强荧光、高稳定性的荧光标记物对于推动荧光免疫技术的发展至关重要。碳点（C-dots）是一种环境友好、生物安全的新型光致发光碳纳米材料，在能量转换、生物医学和高灵敏度检测等领域具有独特的优势。本项目通过微波加热法制备了高性能荧光碳点，剖析碳点结构、组成、表面状态及金属杂化与碳点荧光性能之间的内在联系；以大肠杆菌为模型，通过EDC/NHS 化学偶联法将碳点与大肠杆菌抗体（E.coli O157：H7抗体）偶联复合，构建了碳点-大肠杆菌抗体（CD-anti-E.coli）免疫荧光探针。利用抗体-抗原特异性结合特性，考察了大肠杆菌样本与探针结合后的荧光性，探索碳点基免疫荧光探针在大肠杆菌检测中的应用。结果表明，碳点粒径分布在1-6 nm之间，平均粒径3 nm；荧光量子产率46.7%，经N、Mg双杂化后荧光量子产率最高可达到83%；荧光性能稳定，且具有典型的多色荧光特性，在紫外光激发下发射蓝色荧光，在蓝色光激发下发射绿色荧光，在绿色光激发下发射红色荧光，因此可用于多色荧光成像。所构建的碳点基免疫荧光探针对大肠杆菌E.coli Ol57：H7具有特异性、专一性识别，检测灵敏度达到103 CFU/mL。通过该项目研究表明，碳点荧光性能可与半导体量子点相媲美，作为新型低毒生物传感器，在理论研究和实际应用中都将具有重要的价值。