基于石墨烯量子点和石墨烯的荧光开关传免疫感器在环境雌激素检测中的应用研究

Water pollution from estrogens has been a world wide problem, which not only seriously influences on the wild animals and plants, but also human been indirectly. Vitellogenin(Vtg) is a substance with estrogenic activity. Zebra fish was recommended as a kind of model fish for screening estrogen by United States Environmental Protection Agency (USEPA) and Organization for Economic Co-operation and Development (OECD). Zebra fish was wildly used in the research of environmental estrogens. Vtg usually exists only in the vitellogenic stage of female fish. Because male fish or escrod have Vtg gene, they can also produce Vtg for induction of environmental estrogens. So estrogenic effects from polluted matter in water can be estimated by testing the concentration of Vtg in male fish or escrod. The present methods for measuring Vtg exist some shortcoming, for example, tedious sample treatment and time-consuming, poor stability from enzymes, and unsatisfied specificity caused by polyclonal antibodies. So it is necessary to develop a new non-enzymed and immuno method to test Vtg quickly. In this project, immune analysis and fluorescence resonance energy transfer (FRET) between graphene quantum dots (GQDs）and graphene(Gr) will be combined to establish a new fluorescence-switching immunosensor for Vtg measurement. This sensor will overcome the shortcoming in traditional methods and will supply new testing method for environmental estrogens. This project has important probing meaning and potential application merits.

水体雌激素污染已经成为世界性的环境问题，它们不仅严重影响野生动植物，而且也间接地影响人类。卵黄原蛋白（Vtg）是具有雌激素活性的化学物质，斑马鱼被美国环境保护署与经济合作与发展组织推荐为筛选雌激素类物质的模式鱼种，被广泛地应用于环境雌激素研究。Vtg通常只存在于卵黄生成期的雌鱼体内，雄鱼和幼鱼因含有Vtg基因，在环境雌激素的诱导下也能合成。因此，通过检测雄鱼或幼鱼体内的Vtg含量可以评价水体污染物的雌激素效应。目前Vtg的测量方法主要缺点是样品处理繁琐、耗时长；使用酶导致稳定性差；多克隆抗体的使用导致特异性差。因而急需发展基于单克隆抗体的非酶连免疫且快速准确检测Vtg的方法。本课题拟采用石墨烯和石墨烯量子点的荧光共振能量转移, 并结合免疫分析，构建一种测量Vtg的新型荧光开关免疫传感器,以克服传统测量方法的缺点，为环境雌激素检测提供新方法。课题具有重要的探究价值和潜在实用价值。

环境雌激素（EEs）通过干扰内分泌、免疫、神经等系统，导致人或动物内分泌紊乱、代谢紊乱和生殖系统损伤等，对野生动物和人类造成很大危害。水体脊椎或无脊椎动物的卵黄原蛋白（Vtg）被推荐作为EEs生物标志物。卵黄原蛋白或卵黄脂磷蛋白（Lv）的浓度反应了EEs的含量。免疫传感器具有固有的高特异性和灵敏度，有望在复杂环境中快速检测特定物质而备受关注。石墨烯量子点(GQDs)和碳量子点（CQDs）是强荧光物质，具有绿色、无毒、易于修饰、良好的水溶性和生物相容性等特点，成为最有前途的生物荧光纳米材料。.本研究发展了三种荧光免疫传感器和两种电化学免疫传感器，用于定量检测EEs生物标志物。（1）以氧化石墨烯（GO）为前体，首次采用可见光Fenton法制备了GQDs。基于GQDs与还原氧化石墨烯（rGO）之间的荧光共振能量转移（FRET），建立了一种检测Lv的超灵敏荧光免疫传感器。褐牙鲆Lv浓度检测的线性范围：1.0 pg/mL到1500 ng/mL, 最低检测限（LOD）0.9 pg/mL，灵敏度26,407.8 CPS/(ng/mL)，检测在一刻钟内完成，且抗干扰性强。（2）采用一步超声水热法制备了氮掺杂荧光碳量子点（N-CQDs），发展了一种对海洋青鳉Vtg具有高特异性和敏感性的无标记免疫传感器。通过对rGO修饰聚二烯丙基二甲基氯化铵(PDDA), 改变了rGO的Zeta电位，提高了FRET效率。最终得到的免疫传感器线性范围：0.1 ng/mL到3000 ng/mL，LOD：0.038 ng/mL，灵敏度：28833.63 CPS/(ng/mL)，并具有优异的选择性和重现性。（3）采用一步水热法制备了氮掺杂的强荧光光GQDs，避免了氨基化步骤。以此建立的荧光免疫传感器具有宽线性测试范围（0.001-2000 ng/mL）、高灵敏度（9910.67 CPS/ng/mL）、较低的LOD（0.9 pg/mL），以及优异的选择性和再现性, 对于含有1 ng/mL Lv的海水和自来水，测量误差分别为10.27%和1.65%。（4）基于Cu2O_BSA修饰玻碳电极的电化学免疫传感器线性范围0.128 pg/mL 到 50 ng/mL，LOD 0.09 pg/mL，低于先前报道的Vtg探测技术。本研究建立的非标记荧光/电化学免疫传感器有望用于估计海洋雌激素污染物；制备的N-GQDs及N-CQ