干旱区土壤中酞酸酯类环境激素多重信号放大免疫分析新方法研究

The research aims to develop the new methods for fast, sensitive, and multiple Phthalates detection in environmental hormone contaminated soils. Firstly, molecular simulation techniques are adopted to aid designing the general structure of hapten and prepare the broad-spectrum monoclonal antibody. Meanwhile, the quantitative relationship model between structure and activity will be built for design optimization of competition hapten to improve the specificity of antibody. Then, the amino-terminated hyperbranched polymer(HBP) will be prepared, and acts the reducing agent for preparing hyperbranched gold nano complexes, which will be further combined with carbon nanotubes to build nanocomposites modified electrodes. Besides, taking advantage of HBP’s amino groups, many antigens are immobilized by the covalent method to amplify signal. With the thiol-functionalized HBP as the carrier, two types of hyperbranched gold-labelled immune complex are prepared by both immuno-labelling and Au-S bond self-assembly principle, and further research on the rules of signal amplification in electrochemical system. An electrochemical immunosensor with multiple signal amplification will be constructed based on the hyperbranched nano-composites. Finally, a high sensitive method will be established for rapid and multiplex detection of the Phthalates residues in soil. The present study will provide the basic material and technical sopport for the pollution monitoring, residue detection and comprehensive management of Phthalates in soil, guaranteeing the sustainable agricultural development.

本项目以土壤中残留地膜引起酞酸酯类环境激素污染的快速、高灵敏、多残留分析为目标，利用分子模拟技术辅助设计酞酸酯通用结构半抗原，制备群选性单克隆抗体；构建抗原抗体定量构效模型，探索抗原抗体识别机制，模拟优化竞争半抗原设计，改进抗体识别能力的宽谱性；利用不等活性单体对法合成端氨基超支化聚合物，进一步制备超支化金纳米复合物，结合碳纳米管构建新型复合纳米材料修饰电极，利用超支化聚合物的氨基共价偶联大量抗原，实现信号放大；以巯基功能化超支化聚合物作为载体，利用免疫标记和金硫键自组装技术构筑两类超支化金标免疫复合物，作为信号增强探针应用于电化学体系中，研究信号增强规律；基于多种超支化纳米复合物，构建多重信号放大的电化学免疫传感体系并应用于土壤中酞酸酯多残留、高灵敏、快速分析检测。项目任务的完成将为我区土壤中酞酸酯类环境激素的污染调查、残留检测和综合治理提供新的物质基础和技术支撑，保障农业可持续发展。

酞酸酯（PAEs）是土壤有机物污染的必测项目，检测方法的解决是实现环境污染现状调查、预测污染发展趋势的基础，但目前PAEs免疫分析法难以满足同时检测环境中多种PAEs的实际需求。本项目主要研制了PAEs广谱性单克隆抗体并建立了基于纳米免疫复合物的信号放大免疫传感检测方法：（1）基于6个PAEs类化合物的共性结构特征，结合文献设计了5个半抗原分子，基于分子计算模拟结果，综合几何结构、电荷分布和疏水作用因素，选择hapten1和hapten3作为通用结构半抗原并进行合成；（2）制备人工抗原，通过动物免疫和杂交瘤技术筛选得到分泌抗PAEs广谱性单克隆抗体的细胞株并在CGMCC进行了细胞株保藏；建立间接竞争酶联免疫分析法对抗体性能进行评价，抗体亲和常数为2.54×108L/mol，属于高亲和力抗体，抗体特异性和交叉反应测定结果显示，抗体对DMP、DEP、DBP、DOP、DEHP和BBP具有特异性识别反应，优化条件下ic-ELISA方法的IC50值分别为22.19、15.92、9.73、48.04、154.2和170.59ng/mL，具有较宽的识别范围和较好的灵敏度；（3）基于PAEs抗体交叉反应结果，使用CoMFA和CoMSIA 建立了抗原抗体3D-QSAR模型。CoMFA模型的交互验证系数q2=0.734，非交互验证相关系数r2为0.998，CoMSIA模型的交互验证系数q2=0.776，非交互验证相关系数r2为0.987，表明这两个模型均具有较好的预测能力。根据模型抗体活性和PAEs的疏水性有较强相关性，提示在PAEs的双侧链末端为大体积或疏水的官能团和在双侧链为负电性或氢键受体基团将对抗体活性有正贡献，为后续抗体性能提高及竞争原改造提供了理论依据；（4）采用硫堇/聚酰胺胺-金/多壁碳纳米管/玻碳电极为基底，同时利用PAMAM-Au聚集体为载体分别固定抗体和酶标二抗制备增敏探针，建立基于PAMAM-Au免疫复合物的信号放大检测方法，并制备了多重信号放大的电化学免疫传感器用于PAEs多残留分析。结果表明，该增敏策略能够有效放大检测信号，提高检测灵敏度，制备的免疫传感器具有检测限低（0.03ng/mL），识别范围宽，稳定性好的特点。