基于细胞色素P450酶代谢的内分泌干扰物体外筛检电化学平台构建与应用

With the emergent of environmental chemicals, there are urgent needs for the development of low-cost, convenient and accurate methods of in vitro screening endocrine disrupting chemicals (EDCs). To assess the potential risks of EDCs, it is of great importance to perform the metabolic reaction of cytochrome P450 enzyme (CYP450) in the process of in vitro screening EDCs. In this project, the electrode-driven or light-driven CYP450 electrochemical reactors will be constructed by using the functionalized nanocarbon materials with excellent electrical or optical performance (such as graphene, nitrogen-doped carbon nanotubes and nanocarbon balls). By studying the electron transfer, energy transfer and reaction performance of the immobilized enzyme, the method to improve the biological activity and the catalytic efficiency of the immobilized enzyme will be explored. Furthermore, the electrochemical method, LC-MS and other means of detection will be applied to study the metabolic behavior and dynamic mechanism of EDCs in the CYP450 electrochemical reactors. Based on the research above, the detection system of the CYP450 electrochemical reactor in series with the receptor binding assay will be constructed by the dual working electrode system or chromatographic separation technique. In the system, the endocrine disrupting activity of EDCs will be evaluated according to the changes of the optical or electrochemical responses caused by the completive binding with the receptors between EDCs metabolites and probe molecules. After that, the new principles and methods of in vitro screening EDCs based on the CYP450 electrochemical reactor will be established.

随着环境化学品的不断涌现，发展廉价、方便、准确的内分泌干扰物(EDCs)体外筛检方法迫在眉睫，而在EDCs体外筛检中进行细胞色素P450酶(CYP450)代谢反应，对评估EDCs的潜在风险尤为重要。本项目拟利用功能化纳米碳材料(如石墨烯、掺氮碳纳米管、纳米碳球等)优异的光电性能，构建基于电驱动或光驱动的CYP450电化学代谢反应器。通过研究固定化酶的电子转移、能量传递和反应性能，探索提高固定化酶的生物活性以及催化效率的方法；采用电化学、LC-MS等检测手段考察CYP450电化学代谢反应器对EDCs的代谢行为及动力学机制；借助双工作电极系统或色谱分离技术，构建CYP450电化学代谢反应器和受体结合试验相串联的检测系统，通过代谢产物与探针分子竞争性结合受体前后造成的电或光响应信号的改变，评估EDCs的内分泌干扰活性，进而提出基于CYP450电化学代谢反应器的EDCs体外筛检新原理和新方法。

本项目基于多巴胺/石墨烯/金纳米粒子（Au@PDA-RGO）仿生复合材料以及功能化石墨烯泡沫的优异性能，分别构建了酪氨酸酶（Try）、人肝微粒体（HLM）以及CYP3A4电化学代谢反应器，应用于生物传感及类固醇激素代谢研究。研究发现设计具有良好生物相容性、优异电子传递性能的酶固定界面，是构建高催化效率P450电化学代谢反应器的关键。首先，设计合成的Au@PDA-RGO仿生复合材料为固定化酶提供了理想的生物微环境，构建的Try/Au@PDA-RGO电化学生物传感器对双酚A（BPA）具有良好的分析性能，其灵敏度高、线性范围宽、检出限低，成功应用于塑料制品中BPA的检测。进一步，以PDA/Au@RGO为载体，构建了HLM电化学生物传感器，成功实现了HLM在电极表面的直接电化学以及固定化HLM对固醇类激素的代谢行为。HPLC-MS检测结果表明HLM电化学反应器催化生成的代谢产物与实际体内相一致。此外，我们设计合成了孔径可控的聚多巴胺功能化石墨烯泡沫（PNGFs）用于CYP3A4纳米反应器的构建，研究发现PNGFs孔的大小会影响CYP3A4的负载量、酶构型和传质过程，进而影响酶的活性和反应速率。当孔径与酶尺寸相匹配时，固定化CYP3A4对底物的亲和力最高。更有意思的是，当构建的P450电化学反应器应用于睾酮、孕酮、雌酮的代谢研究时，求得的Kmapp值的大小顺序与文献报道的结果相一致。该研究结果表明，P450电化学代谢反应器在内分泌干扰物筛选和安全性评价领域具有潜在的应用前景。项目实施过程中，发表SCI论文7篇，会议论文2篇，申请发明专利1项，培养硕士研究生3名。