新型全细胞生物电化学传感器的构建原理及应用研究

Whole-cell electrochemical biosensor is expected to achieve the efficient, convenient, low-cost detection method of oncometabolite. It has important significance for the early diagnosis and treatment of cancer. While the electron transfer mechanism involved in the biosensor is unclear, it is difficult for rational design and effective regulation. Its research and development is hindered for this reason. In our early research, we chose Shewanella oneidensis MR-1, one of the most famous exoelectrogenic microorganisms, as our research object and noticed that the bacteria was able to transfer exogenous electrons by its own electronic delivery system.. Therefore, in this project, we select the well-known oncometabolie D-2- hydroxyglutarate as research model. From the start of transcriptome alignments, we will further systematically investigate the electron transfer mechanisms in the biosensor. Modularized design is introduced to the design of biosensor strains. Firstly, strengthen internal and extracellular electron transfer process to construct the signal conversion module. Secondly, construct the specific receptor module by the location expression of D-2-hydroxyglutarate dehydrogenase gene. The further regulatory mechanism of the biosensor will be revealed by coupling cells and electrode materials, artificial biofilm strategies, and so on. The highly efficient controlled generation of biosensor will be obtained and used for detecting oncometabolite. . This project will provide new sight into the molecular mechanism of biosensor electron transfer, and develop new construction principle of novel whole-cell electrochemical biosensor and new oncometabolite bioassay method. This work will show new ideas for the design of biosensors. This study is also helpful for promoting the attention of microbial electrochemical systems and raise awareness of microbial electronic transfer. It will have great significance in energy-efficient analysis and environmental protection.

全细胞电化学传感器有望实现癌代谢物的高效、便捷、低成本检测，对癌症的早期诊治具有重要意义。而其电子传递机制尚不清楚，难以进行理性设计和有效调控，阻碍了其研究和发展。前期以产电模式菌希瓦氏菌为研究对象，注意到菌体自身的电子传递系统能实现对外源电子的传递，本项目以著名癌代谢物D-2羟基戊二酸的检测为研究模型，从转录组比对入手进一步系统研究传感器中电子传递过程的途径机制；以模块化思路设计传感器菌株，强化胞内外的电子传递过程，构建信号转换模块；定位表达D-2-羟基戊二酸脱氢酶，构建专一性的接收器模块；通过耦合细胞与电极、构建人工导电生物膜等方法进一步揭示传感器的调控机制；获得高效可控的新一代传感器，用于癌代谢检测。该研究有助于揭示传感器中的电子传递机制，研究所获得的全细胞生物电化学传感器构建新理论与癌代谢物生物检测新方法，将为生物传感器的设计提供新理论，为微生物电化学系统的应用提供新思路和新方法。

项目获得资助以来，我们根据申请书中提出的技术路线和研究方法，取得了较好的研究成果主要开展了以下几方面的研究工作：1）对全细胞电化学生物传感器的宿主菌株的设计、功能模块的构建原理研究，研究希瓦氏菌的转化方法，并分别利用自身的膜蛋白和冰核蛋白的信号肽序列引导目标蛋白锚定，构建了用于全细胞电化学生物传感器的信号接收器模块；2）全细胞电化学生物传感器的调控机制研究，研究了菌体状态、接种密度、静置时间、电极材料对全细胞电化学生物传感器的传感性能的影响，采用新型电极提高菌体与电极之间的电子传递效果，并对新型材料进行材料和电化学表征；3）全细胞电化学生物传感器的应用，构建全细胞电化学生物传感器，通过对传感器的线性范围、重复性、稳定性、特异性的考察证明了全细胞电化学传感器具有良好的分析性能；4）对全细胞电化学生物传感器的构建原理进行总结。伴随项目的开展，我们共发表了3篇期刊论文，申请了2项国家发明专利，编写了1篇学术专著章节。