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Abstract
Introduction
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Chemistry

通过活生物膜表征电子传输

Published: June 01, 2018
doi:
10.3791/54671
, , , , , , , ,
1Center for Bio/Molecular Science and Engineering,Naval Research Laboratory, 2George Mason University, 3Chemistry Division,Naval Research Laboratory, 4Departments of Physics, Biological Sciences, and Chemistry,University of Southern California, 5Department of Chemical Engineering and Materials Science,Michigan State University

Summary

提出了一种在生理相关条件下测量活体微生物生物膜电导率的方法。

Abstract

在这里, 我们演示了电化学浇口的方法, 用于表征电极生长的微生物生物膜在生理相关条件下的电导率。1这些测量是用在交叉电极 (IDA) 阵列的专门配置中, 在玻璃表面上图案的源和漏电极对水介质中活生物膜进行的。在连接源和排泄的缝隙中生长的生物膜。电位被应用到电极 (eS和 eD) 上, 通过电极之间的生物膜生成源漏电流 (ISD)。电导率对栅极电位的依赖性 (源和漏电位的平均值, EG = [eD + ES]/2) 是通过系统地改变栅极电位和测量产生的源漏来确定的。当前。电导率对栅极电位的依赖性提供了有关在调查的特定生物膜电导率基础上的胞外电子传输过程的机械信息。此处描述的电化学浇口测量方法直接基于 Wrighton2、3和同事和刻录默里4、5、6和同事在1980是研究薄膜导电聚合物。

Introduction

胞外电子输运 (EET) 是一种使某些微生物在细胞内代谢过程和不溶性电子受体之间传输电子的过程, 它是由天然矿物质到电极。在某些情况下, EET 使微生物在电极表面形成导电的多细胞厚生物膜, 而与电极不直接接触的细胞仍然可以利用它作为代谢电子受体或捐献者。在诸如微生物合成、污染物传感/去除、远程能源生成和存储等各种应用的电极催化剂等生物膜中, 有相当大的兴趣,7,8,9 、10、11、12、13、14由于微生物所执行的新陈代谢过程多种多样, 微生物生物膜的耐久性比较以酶为基础的 bioelectrodes。15,16此外, EET 通路可能会被用来电控或信号改变自然发生或基因工程微生物代谢过程中涉及, 例如, 在生产所需的产品或检测目标分析或刺激的。电催化生物膜的电导率与其他生物材料不同, 是其催化性能的一个核心方面, 但对于电极环境下的底层 EET 过程却知之甚少,众所周知的是高度争议的。17,18,19,20,21,22,23,24

这里描述的是一个2电极法测量电导率通过生活, 电极生长生物膜使用交叉电极阵列 (IDAs)。IDAs 包括在平板玻璃表面上图案的平行矩形电极, 使每一个其它波段连接在阵列的另一侧, 导致2电极 (源和漏)。对 IDA 的仔细检查 (例如, 参见 #1 的图 6.12b) 揭示了分隔相邻带的缝隙也以这样的方式连接在一起, 形成一个单独的缝隙, 在分离两个电极的阵列之间来回编织。结果是一个长而窄的缝隙, 分离源和排泄电极, 产生非常高的源漏电流, 当导电材料形成, 铸造, 聚合, 或种植 (在这里所考虑的生物膜类型的情况下) 阵列。此外, 由于电容充电和导电材料的氧化状态随栅极电位的变化而改变, 电极的小尺寸导致了小背景电流, 因为电导率所需的材料量使用 IDAs 的测量是如此之小。本文介绍了基于 IDA 的电化学门控技术, 开发了用于表征薄膜导电聚合物,2,3,4,25最近才被应用于生活系统。18另一种用于测量活生物膜电导率的技术使用了大格式的拆分源和排泄电极和源表来设置栅极电位。26,27但是, 对这些方法的关注以前已经详细说明过。18

下面的协议封装了我们对活体地杆菌能 sulfurreducens 和 biocathode MCL 生物膜进行电导率测量的经验。G. sulfurreducens是一种能够使用不溶性材料 (包括电极) 作为唯一代谢电子受体的模型电极还原有机体。此外, 它形成厚的生物膜, 能够传输电子在多个细胞长度, 使它成为一个理想的模型有机体研究阳极远距离细胞外电子转移。我们还包括详细的研究 biocathode MCL, 一个有氧, 自养混合社区生物膜分离的底栖微生物燃料电池的阴极。Biocathode MCL (命名为三主要成分– Marinobacter、 Chromatiaceaea和Labrenzia) 能够将电极氧化为其唯一的电子供体, 并在多个细胞长度上传输电子, 使它是一个有趣的阴极系统来研究。此外, biocathode MCL 有最高的报告电导率的生活系统迄今使用这些方法。在本议定书中列入这些不同的活性生物膜, 是为了强调这项技术适用于通过任何能够与电极电相互作用的生物生物膜来测量电子的传输。

Protocol

1. 交叉微电极阵列 (IDA) 的制备 获取在导电基板上图案的商用 IDA 电极, 或者使用标准平版方法合成它们。28注: IDA 尺寸和/或材料可以根据所需条件进行不同的实验。这里使用的 IDAs 是商业获得的, 包括两个交叉金电极图案上的玻璃基板连接到大电极垫在阵列的两端。电极被暴露, 而连接电极到大接触板的母线被涂上薄薄的绝缘材料。IDAs 在这里使用的是由两套10µm 和2毫?…

Representative Results

IDAs 进行了有线、绝缘和测试, 以确保两个电极彼此电隔离 (图 1)。反应器组装, 接种与G. sulfurreducens, 并孵化, 直到生物膜弥合电极之间的差距。可以直观地看到sulfurreducens生物膜覆盖阵列。其他生物膜可能要求研究员做电化学门控测量, 以查看两个电极是否已电连接。显微镜也应用于验证阵列电极之间的连接。电化学浇注实验是为了确定<…

Discussion

在 IDA 的安装过程中, 测试源和漏油是否在电化学浇口测量之前没有短路是至关重要的, 因为这将改变 ISD与 EG曲线, 并可能导致错误的结果和解释。选择 vsd和 v (例如, 当前线性依赖于 vsd并且独立于五个) 也是至关重要的。如果不是这种情况, 那么上面描述的方程不能用来计算电导率。

至少必须考虑两个背景电流, 并从进行的电流测量中移除。第?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

博士, s.m., L.M.T. 承认海军研究办公室 (奖 #N0001415WX01038 和 N0001415WX00195), 海军研究实验室, 海军研究实验室 Nanosciences 研究所;M.Y.E.-N。得到美国能源部赠款 DE-FG02-13ER16415 的支持。

Materials

IDAs CH Instruments 012125 Manufactured by ALS-Japan; sold by CH Instruments
Wire Digikey W7-ND
Conductive silver epoxy Electron microscopy sciences 12670-EE
Insulating material 3M 2131-B Scotchast flame retardant compound
15 mL conical centrifuge tube VWR 89004-368
21g needle VWR BD-305165
5 mL pipette tips VWR 82018-842
5 mL pipettor VWR 89079-976
Freshwater medium components Sigma Aldrich All standard laboratory chemicals
    Ammonium chloride
    Sodium phosphate monobasic
    Sodium bicarbonate
Artificial seawater medium components Sigma Aldrich All standard laboratory chemicals
    Sodium chloride
    Magnesium chloride hexahydrate
    Magnesium sulfate heptahydrate
    Potassium chloride
    Sodium bicarbonate
    Calcium chloride dihydrate
    Ammonium chloride
    Potassium phosphate dibasic
Ag/AgCl reference electrode Basi MF-2079
Graphite rod counter electrode Electron microscopy sciences 70230
Recirculating water bath Thermo Scientific 152-5256
Bipotentiostat Pine Instruments WD-20 http://www.voltammetry.net/pine/aftermath/user
Stir bars VWR 58947-114
G. sulfurreducens culture ATCC 51573
Jacketed reactor Pine Instruments RRPG085
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Tags

Keywords: BiofilmElectron TransportMicrobial ElectrochemistryInterdigitated ElectrodesExtracellular Electron TransportConductive Polymer FilmsBipotentiostatSilver EpoxyInsulating MaterialPolyurethane Resins

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Yates, M., Strycharz-Glaven, S., Golden, J., Roy, J., Tsoi, S., Erickson, J., El-Naggar, M., Calabrese Barton, S., Tender, L. Characterizing Electron Transport through Living Biofilms. J. Vis. Exp. (136), e54671, doi:10.3791/54671 (2018).
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