纳米金粒子介导的复合探针的设计合成及其在多环芳烃分析检测中的应用研究

As the important source of drinking, industrial and agricultural water, rivers, reservoirs and lakes are close related to human beings. With the development of industry and agriculure, the pollution of polycyclic aromatic hydrocarbons (PAHs) to the water sources becomes more and more serious. Most of PAHs dissolved in water have great toxicity and are hard to be degraded, which can lead to cancer, abnormality and mutation. The rate of suffering cancer of people who have contacted with PAHs obviously higher than that of people who have not. It is one of the most important content to study the high-throughput detection technique for the determination of PAHs. In this project, acenaphthylene，phenanthrene，pyrene and chrysene would be employed. Firstly, the PAHs are activated by using chemical methods and the activated PAHs will be conjugated with carrier proteins respectively. Then the monoclonal antibodies specific for the PAHs respectively would be produced by cell fusion and immunology techniques. Simultaneously, a versatile and highly sensitive probe would be synthesized by combining goat anti-mice IgG, AuNPs and horseradish peroxidase (HRP). Finally, based on the probe and McAbs, a highly sensitive and high-throughput screening immunoassay for the detection of PAHs would be developed. The practice of the project will provide the theoretics and experiment bases of new techniques and methods for the detection of PAHs in water entironment, and will provide technique support for control and restoring of PAHs pollution.

河流、水库和湖泊作为人类饮用水和工农业用水的重要来源, 与人类生产生活休戚相关。随着工农业的发展，水体受多环芳烃(PAHs)污染日趋严重。水体溶解态PAHs大部分具有较强的毒性、致癌性、致畸性、致突变性和难降解性。接触PAHs的人群癌症发病率明显增高。开展对PAHs高通量检测技术研究是水环境PAHs污染及控制的重要内容之一。本研究拟以二氢苊、菲、芘和屈等为研究材料，首先利用化学方法对其修饰，使其末端带有活性基团，然后与载体蛋白偶联制备完全抗原。采用细胞融合及免疫学技术，制备抗几种PAHs的单克隆抗体(McAb)。同时以纳米金粒子(AuNPs)为载体，介导辣根过氧化物酶(HRP)和羊抗鼠IgG，构建能够同时识别所有鼠源性McAb的集“广谱性”和“高敏感性”于一体的探针(HRP-AuNPs-IgG)。最后基于构建的探针和制备的McAb，建立几种PAHs的灵敏和高通量的免疫学筛检方法。

水体受多环芳烃(PAHs)污染日趋严重。水体溶解态PAHs大部分具有较强的毒性、致癌性、致畸性、致突变性和难降解性。接触PAHs的人群癌症发病率明显增高。开展对PAHs高通量检测技术研究是水环境PAHs污染及控制的重要内容之一。本项目制备了生物素化DNA探针和纳米微球荧光等探针，建立了多环芳烃的IPCR、荧光试纸条和荧光ELISA等高灵敏新型检测技术。.1. 以生物素化的IgG和DNA为探针，将PCR的信号放大功能和ELISA的特异性功能相结合，建立多环芳烃的IPCR是目前报道的最灵敏的技术，最低检测限达450fmol/L [Biosen. Bioelectron., 70 (2015) 42–47. (IF = 9.518)]。.2. 构建了改进型IPCR检测技术，使整个检测过程由传统的5步减少至2步，检测时间缩短2.5小时 [Biosen. Bioelectron.,78 (2016) 194–199. (IF = 9.518)]。.3. 建立了苊、芘等多环芳烃的竞争ELISA [Analytical Biochemistry, 473 (2015) 1-6. (IF = 2.507); Food and Agricultural Immunology, 2017, 28 (5): 1-12. (IF = 2.398)]。.4. 建立的纳米微球荧光试纸条和荧光ELISA，比传统基于纳米金粒子的试纸条和传统ELISA敏感性分别提高30倍和3倍，检测时间分别减少90分钟和100分钟 [Sensors and Actuators B 252 (2017) 633–640; Sensors and Actuators B 262 (2018) 221–227 (IF = 6.393)]。