Real-Time Electrochemical Detection of Pseudomonas aeruginosa Phenazine Metabolites Using Transparent Carbon Ultramicroelectrode Arrays
Real-Time Electrochemical Detection of Pseudomonas aeruginosa Phenazine Metabolites Using Transparent Carbon Ultramicroelectrode Arrays
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DOI：
10.1021/acssensors.8b01152
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发表时间：
2019-01-01
期刊：
影响因子：
8.9
通讯作者：
Stevenson, Keith J.
中科院分区：
文献类型：
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作者：
Simoska, Olja;Sans, Marta;Stevenson, Keith J.
在这里，我们使用一个最近开发的电化学传感平台的透明碳超微电极阵列（T-CUAs）的吩嗪代谢物的体外检测从人类机会致病菌铜绿假单胞菌。具体地，氧化还原活性代谢物绿脓菌素（PYO）、5-甲基吩嗪-1-羧酸（5-MCA）和1-羟基吩嗪（OHPHZ）由铜绿假单胞菌产生，其通常在慢性伤口感染和囊性纤维化患者的肺中发现。作为高度扩散的化学物质，PYO和其他代谢产物对周围宿主细胞和其他竞争微生物具有极强的毒性，因此它们的检测非常重要，因为它可以提供有关铜绿假单胞菌毒力机制的见解。已知吩嗪代谢物在细胞功能中发挥重要作用;然而，关于它们的浓度如何波动并影响感染和生长过程中的细胞行为知之甚少。在这里，我们报告了使用易于组装，低成本的电化学传感器，提供快速的响应时间，增强的灵敏度和高再现性。因此，这些T-CUA能够实时电化学监测PYO和另一种极具反应性和不同的氧化还原活性的吩嗪代谢物5-甲基吩嗪-1-羧酸（5-MCA），这些代谢物来自高毒力的实验室铜绿假单胞菌菌株PA 14。除了定量吩嗪代谢物浓度外，还在生产PYO的生物合成途径中观察到吩嗪动力学的变化。我们的定量结果，在48小时内，显示增加PYO浓度在细菌生长的第一个21小时，之后PYO水平平台，然后略有下降。此外，我们探讨吩嗪动力学和PYO浓度在两种生长培养基，胰蛋白酶大豆肉汤（TSB）和溶原肉汤（LB）的环境影响。细胞PYO的最大浓度在TSB和LB中分别测定为190 +/- 5 μ M和150 +/-μ M。最后，使用解吸电喷雾离子化（DESI）和纳米电喷雾离子化（nano-ESI）质谱，我们确认检测和鉴定反应性吩嗪代谢物。
Here, we use a recently developed electrochemical sensing platform of transparent carbon ultramicroelectrode arrays (T-CUAs) for the in vitro detection of phenazine metabolites from the opportunistic human pathogen Pseudomonas aeruginosa. Specifically, redox-active metabolites pyocyanin (PYO), 5-methylphenazine-1-carboxylic acid (5-MCA), and 1-hydroxyphenazine (OHPHZ) are produced by P. aeruginosa, which is commonly found in chronic wound infections and in the lungs of cystic fibrosis patients. As highly diffusible chemicals, PYO and other metabolites are extremely toxic to surrounding host cells and other competing microorganisms, thus their detection is of great importance as it could provide insights regarding P. aeruginosa virulence mechanisms. Phenazine metabolites are known to play important roles in cellular functions; however, very little is known about how their concentrations fluctuate and influence cellular behaviors over the course of infection and growth. Herein we report the use of easily assembled, low-cost electrochemical sensors that provide rapid response times, enhanced sensitivity, and high reproducibility. As such, these T-CUAs enable real-time electrochemical monitoring of PYO and another extremely reactive and distinct redox-active phenazine metabolite, 5-methylphenazine-1-carboxylic acid (5-MCA), from a highly virulent laboratory P. aeruginosa strain, PA14. In addition to quantifying phenazine metabolite concentrations, changes in phenazine dynamics are observed in the biosynthetic route for the production of PYO. Our quantitative results, over a 48-h period, show increasing PYO concentrations during the first 21 h of bacterial growth, after which PYO levels plateau and then slightly decrease. Additionally, we explore environmental effects on phenazine dynamics and PYO concentrations in two growth media, tryptic soy broth (TSB) and lysogeny broth (LB). The maximum concentrations of cellular PYO were determined to be 190 +/- 5 mu M and 150 +/- mu M in TSB and LB, respectively. Finally, using desorption electrospray ionization (DESI) and nanoelectrospray ionization (nano-ESI) mass spectrometry we confirm the detection and identification of reactive phenazine metabolites.