Do Hyein, Kwon Seung-Ryong, Baek Seol, Madukoma Chinedu S, Smiley Marina K, Dietrich Lars E, Shrout Joshua D, Bohn Paul W
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Analyst. 2021 Feb 21;146(4):1346-1354. doi: 10.1039/d0an02022b. Epub 2021 Jan 4.
The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa, which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal-insulator-metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM 〈n〉∼ 0.082 per nanopore - a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. The NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities.
机会致病菌铜绿假单胞菌会产生几种具有氧化还原活性的吩嗪代谢产物，包括绿脓菌素（PYO）和吩嗪 - 1 - 甲酰胺（PCN），它们是电子载体分子，也有助于毒力。特别是，PYO是铜绿假单胞菌产生的一种独特代谢产物，它在医院获得性感染中作为一种毒力因子，因此是识别该病原体早期定植的良好生物标志物。在此，我们描述了使用具有金属 - 绝缘体 - 金属环形电极结构的纳米孔电极阵列（NEA）来增强对这些吩嗪代谢产物的检测。纳米孔的尺寸允许吩嗪代谢产物自由扩散到内部并接触工作电极，而细菌则被排除在外。因此，NEA中高效的氧化还原循环反应可以通过不受细菌存在阻碍的自由扩散来实现。这种策略产生了低检测限，即PYO和PCN的检测限分别为10.5和20.7 nM，这些值远低于单分子孔占有率，例如在10.5 nM时，每个纳米孔的〈n〉约为0.082 - 这一极限反映了NEA中非凡的信号放大。此外，比较基本培养基和丰富培养基结果的实验表明，尽管这两种培养基中的生长速率和吩嗪产生模式不同，但铜绿假单胞菌产生的吩嗪代谢产物类型相同。这里开发的NEA测量策略一般应可作为病原体诊断工具，并有助于理解临床重要微生物群落中的代谢情况。
