基于纳米结构硼掺杂金刚石电极的微生物燃料电池生物传感器及其药敏试验研究

The emergence of bacterial strains with antibiotic resistance is a serious public health problem. However, the current antimicrobial susceptibility testing (AST) methods are time-consumed and incapable of monitoring in real time. Herein, a novel pathogen-based microbial fuel cell (MFC) biosensor is developed for antimicrobial susceptibility test. Firstly, pathogens are used as bio-recognition elements, high-density and uniform BDD nanowire, nanopillar and nanocone arrays electrodes are fabricated and utilized as transducers, the effects of structure and morphology of nanostructured BDD electrodes on the extracellular electron transport of pathogens are studied for the development of a sensitive nanostructured BDD electrode-based MFC biosensor. Secondly, the relationship between current response of biosensor and antimicrobial susceptibility of pathogen is studied, and the current signal is monitored in real time for further research on the interaction between pathogens and antibiotics. Then the AST result is compared with the traditional AST method, and the sensitivity, repeatability as well as stability of this method are evaluated. The purpose of this research is to develop a rapid, real-time, accurate and sensitive AST method and provide new insight into research on the interaction between bacteria and antibiotics. Furthermore, a nanostructured BDD electrode-based microarray MFC biosensor is developed for the purpose of a high-throughput AST method in clinical application.

耐药细菌的出现对人类公共卫生安全提出了严峻的挑战。针对现有药敏试验方法耗时长且不能实时监测的问题，本项目提出基于病原菌的微生物燃料电池（MFC）生物传感器的药敏试验新方法。首先利用病原菌作为生物检测元件，制备高密度且大小均一的纳米须/柱/锥硼掺杂金刚石（BDD）薄膜电极并作为信号转换器，通过研究BDD电极表面纳米结构形貌与病原菌胞外电子传递的关系，构建高灵敏的基于纳米结构BDD电极的MFC生物传感器。再研究传感器电流信号和病原菌药物敏感性的关系，并通过实时监测电流信号进一步研究病原菌和抗生素的相互作用。然后将传感器的药敏试验结果和传统药敏试验方法进行比较，并对该方法的灵敏度、重复性、稳定性等方面进行评价，建立一种快速、实时、准确和灵敏的药敏试验新方法，并为细菌和抗生素相互作用研究提供新思路。最后构建基于纳米结构BDD电极的微阵列MFC生物传感器，建立高通量的有望用于临床的药敏试验新方法。

本文首先研究不同底物对S. loihica PV-4产电特性及生长情况的影响。然后利用层层自组装方法分别制备不同层数CNTs修饰ITO电极和不同层数CNTs/PANI复合物修饰ITO电极，研究不同修饰层数导电聚合物对MFC产电性能的影响。利用优选的12层CNTs/PANI修饰ITO电极作为信号转换器，E. coli 25922为生物检测元件，分别构建三电极单室MFC生物传感器和多通道双室MFC生物传感器检测庆大霉素。此外，利用纳米草结构BDD电极作为信号转换器，S. loihica PV-4为生物检测元件，构建三电极单室MFC生物传感器检测妥布霉素。最后，我们利用从废水中分离获得的混合菌，构建了基于碳布电极的MFC生物传感器检测妥布霉素。.底物影响结果表明，乳酸、甲酸和环糊精有利于有氧环境下S. loihica PV-4的生长，但不利于厌氧环境下S. loihica PV-4的产电和悬浮细胞的生长。而D-半乳糖、L-阿拉伯糖和α-D-葡萄糖不利于有氧环境下S. loihica PV-4的生长，但有利于厌氧环境下S. loihica PV-4的产电和悬浮细胞的生长。不同纳米结构修饰电极结果表明，9层CNTs修饰ITO电极和12层CNTs/PANI修饰ITO电极分别产生最优产电性能。其中，利用12层CNTs/PANI修饰ITO电极构建的双室MFC获得最大输出功率密度值34.5 mW m-2，较空白ITO电极的输出功率密度提升了14.8%。基于12层CNTs/PANI修饰ITO电极的MFC生物传感器检测庆大霉素结果表明，三电极单室MFC生物传感器和多通道双室MFC生物传感器检测结果具有良好一致性，庆大霉素的最低检测限均为0.25 mM。而基于纳米草结构BDD电极的MFC生物传感器检测妥布霉素的最低检测限为1.0 μg mL-1。基于碳布电极的MFC生物传感器检测妥布霉素结果表明，该传感器具有良好的稳定性。当妥布霉素的浓度增大至6.0 mM时，被显著抑制的电流经历6个周期后仍然恢复到一个稳定的电流水平。加入妥布霉素后，微生物燃料电池中的生物膜群落发生改变，并随着抗生素浓度的增大产生耐药细菌。.本研究建立了一种新型的药敏试验方法，为细菌和抗生素相互作用研究提供新思路。构建了基于纳米结构电极的多通道MFC 生物传感器，有潜力在临床高通量药敏试验中高通量药敏试验中发挥积极的作用。