生物膜中氧气、pH、氧化还原电位检测方案(PH计)

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ENABLING MICROSCALE RESEARCHUnisense A/S-Tueager 1， DK-8200 Aarhus N， Denmark-www.unisense.com Unisense microsensors in biofilm research Oxygen and redox potential in Pseudomonas aeruginosa colony biofilms Figure 1：MicroProfiling setup (left) and microsensor inserted into a P. aeruginosa colony (right).Photo by Jeanyoung Jo. Results and conclusion Figure 2 shows the oxygen concentration and redox potential as a functionof depth in the wild-type P. aeruginosa colony biofilm and a phenazine-null mutant (no phenazine production). The oxygen gradient in the wild-type and mutant biofilms decreased similarly from the surface and downinto the biofilm. The redox potential in the wild-type biofilm decreasedwith depth whereas the redox potential in the mutant remained the samethroughout the biofilm. The decrease of redox potential in the wild typeindicated reduction of the phenazines. The decline in oxygen concentra-tion was seen right from the surface of the wild-type biofilm whereas thedecline in redox potential was mostly pronounced at around 50 um depth.The data suggested that the use of oxygen and phenazines as electronacceptors by the bacteria is depending on the depth in the biofilm and thatoxygen is preferred. The reduction of phenazines in the hypoxic zones of thebiofilm could contribute to survival of the bacteria and may be an importantfinding for the development of new treatment strategies. Figure 2： Oxygen microprofiling data (left) and redox potential microprofil- ing data (right) in the wild type (green dots) and the phenazine-null mutant (red dots) P. aeruginosa PA14 colony biofilm.Data kindly provided by Jeanyoung Jo. For further reading please see the article： Jo et al.(2017)An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growthand virulence. eLife， 6：e30205 Dr. Lars Dietrich is part of the Unisense Ambassador program and you are welcome to contact his group for technical and application based questionsPrimary contacts： Jeanyoung Jo： jj2613@columbia.edu and Krista Cortez： klc2179@columbia.edu. Related publications .Arnaouteli et al. (2017) Biofunctionality of a biofilm matrix protein controlled by redox state. PNAS， vol 114， no 30. Madsen et al. (2015) Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models. Applied and Envi-ronmental Microibology， Vol 81， Number24. Kempes et al.(2014) Morphological optimization for access to dual oxidants in biofilms， PNAS， vol 111， no 1. Cowley et al.(2015) Pediatric cystic fibrosis sputum can be chemically dynamic， anoxic， and extremely reduced due to hydrogen sulfide formation. mBio Volume6，Issue 4. Kolpen et al. (2014) Nitrous oxide production in sputum from cystic fibrosis patients with chronic pseudomonas aeruginosa lung infection. PLOS ONE Vol 9，Issue 1.      在可用作生物还原的氧化剂中，分子氧(O2)提供了最高的自由能收益。条件致病菌-铜绿假单胞菌，是植物和动物宿主的集落体，具有支化的呼吸链，具有将O2还原为水的潜力。生物膜的条件可能与实验室环境非常不同，到目前为止，人们还不知道不同的氧化酶在生物膜群落中起什么作用，以及苯那嗪如何起作用可以补偿低氧水平。为了进一步研究这个问题，研究人员应用unisense微电极研究系统对人工生物膜环境中和线虫宿主对铜绿假单胞菌进行了研究，研究了铜绿假单胞菌都需要末端氧化酶的一个特定部分——一种叫做CcoN4的蛋白质——来实现其最佳生长。而对孤子催化亚基CcoN4在条件致病菌铜绿假单胞菌PA14菌落生物膜发育和呼吸中的独特作用。图1、微剖面分析生物膜实验装置图(左)和微电极插入铜绿假单胞菌菌落中(右)   研究人员应用unisense微电极在琼脂固化培养基上生长的铜绿假单胞菌-PA14菌落生物膜模型中完成了氧气和氧化还原电位的原位分析（图2所示）。生物膜中氧的测试使用了unisense公司生产的前端好直径为25μm的氧微电极（OX-25）。细胞外的氧化还原电势的测量使用了Unisense氧化还原微电极（其尖端直径为25μm (RD-25）和参比电极（REF-RM）。使用SensorTrace 剖面分析软件控制微电极的移动，测量时间为3秒，测量之间的等待时间为5秒。图2、PA14 WT型菌落和突变菌落生物膜的化学梯度和基质分布特征