Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.
Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.
复制标题
使用原子力显微镜测量活细胞中的局部氧化还原酶电子转移。
DOI：
10.1021/ac5015645
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发表时间：
2014
影响因子：
7.4
通讯作者：
Lal,Ratnesh
中科院分区：
文献类型：
--
作者：
Alfonta,Lital;Meckes,Brian;Amir,Liron;Schlesinger,Orr;Ramachandran,Srinivasan;Lal,Ratnesh
细菌系统在能源、传感器和工业化学领域得到了广泛的研究和改进；然而，人们对它们的分子尺度结构和活性了解甚少。设计高效的生物工程细菌需要了解酶的表达和活性。利用原子力显微镜（AFM）检测并分析了e表面表达的氧化还原酶的活性。杆菌。在三电极电化学电池中，采用仅尖端导电的绝缘镀金金属微丝作为AFM悬臂梁和工作电极。对细菌进行工程改造，使其表面显示醇脱氢酶II （ADHII）。醌是一种电子传递介质，共价地附着在显示的ADHII上。AFM探针用于将单个细菌从表面提起，在无氧化还原的缓冲液中进行电化学分析。对乙醇脱氢酶ⅱ中距离NAD+结合位点不同的两个含醌突变体进行了电化学比较。当介质靠近NAD+结合位点时，氧化还原活性蛋白中的电子转移效率提高。这项研究表明，用于单细胞电化学分析的集成导电AFM将允许详细了解酶电子转移到电极的过程，这是创建高效工程生物传感器和生物燃料电池所必需的过程。
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface ofE. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD+binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD+binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.