基于硫醌氧化还原酶表达的微生物传感器构建及其对环境污染物硫化物检测研究

恶臭污染物硫化物引起的环境污染已成为影响人类生存与健康的重大问题。基于目前硫化物有效检测方法缺失及对微生物硫化物代谢中关键酶的研究，本项目拟在硫醌氧化还原酶对硫化物特异催化氧化及前期硫醌氧化还原酶克隆表达研究基础上，深入开展基于硫醌氧化还原酶表达的大肠杆菌全细胞传感器构建及其对硫化物检测研究。选择具有独特理化性能的纳米多孔金作为固定化载体，构建基于硫醌氧化还原酶表达的大肠杆菌全细胞传感器；探索大肠杆菌细胞在电极表面的电化学行为及界面电化学反应机理；进行传感器对硫化物的电催化及电化学响应分析，确定检测范围、检测限、灵敏度及再生条件；在此基础上，考察水环境和血液中硫化物的电化学响应，并对传感器的抗干扰能力、重复利用及稳定性进行评价。该申请项目研究将建立硫化物直接电化学检测技术，为硫化物污染环境治理、硫化物生物氧化机理研究和内源性硫化物作为临床诊断指标的相关研究提供一定理论和技术基础。

Environmental pollution caused by sulfide has become a major issue for human survival and health. However, there is no efficient detection method towards sulfide for environment detection. Sulfide:quinone oxidoreductase, as a key enzyme in the sulfide metabolism in microbial cells, has specific catalytic oxidation of sulfide. Moreover, the cloning and expression of sulfide:quinone oxidoreductase in Escherichia coli BL21 has been completed in our previous work. In this project, further study will be carried out to explore the construction of a microbial biosensor and its direct electrochemical catalysis towards sulfide according to the specific oxidation catalyzed by sulfide:quinone oxidoreductase. First, nanoporous gold with unique physical and chemical properties is selected for E. coli BL21 immobilization and microbial biosensor construction. Second, the electrochemical behavior and interfacial electrochemical reaction mechanism of E. coli BL21 on electrode surface will be explored. Third, the electrochemical performance of the microbial biosensor will be evaluated, and the optimal working and regeneration condition will be confirmed. Additionally, the detection range of microbial biosensor will be determined along with detection limit and sensitivity. Fourth, the practical performance of the microbial biosensor will be evaluated by detecting sulfide in water environment and blood. Besides, the anti-interference ability, stability, and reproducibility of the microbial biosensor will be measured, respectively. This project will establish an efficient direct electrochemical detection method towards sulfide. It will provide a theoretical and technical foundation for the environmental pollution control caused by sulfide, the mechanism of biological oxidation of sulfide, and the research on endogenous sulfides as diagnostic indicator.