Real-Time Electrochemical Detection of Pseudomonas aeruginosa Phenazine Metabolites Using Transparent Carbon Ultramicroelectrode Arrays

Abstract

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 1hydroxyphenazine (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 μM and 150 ± 1 μM 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.

Electrochemical Detection of Pyocyanin as a Biomarker for Pseudomonas aeruginosa: A Focused Review

“…Although only pyocyanin is exclusively produced by PA, the simultaneous detection of several metabolites is advantageous both for bacterial research purposes, but also for using the presence of the compounds as a molecular signature for PA. Several studies have conducted multiple molecule detection using electrochemical measurements. These studies have mapped the potential values belonging to the aforementioned metabolites and do not overlap, which allows the simultaneous detection of the compounds in one measurement, e.g., through DPV or SWV in the relative potential range of −0.7 to 1.5 V [ 35 , 41 , 48 , 49 , 50 , 51 , 52 ].…”

Emerging Technologies for the Electrochemical Detection of Bacteria

“…Reproduced with permission. [ 78 ] Copyright 2019, American Chemical Society. D) Schematic representation of the lab‐on‐a‐glove design with the screen‐printed set up of the device (C‐based WE and CE vs an Ag/AgCl ink RE).…”

“…[ 77 ] In addition to their high reproducibility, TCUAs have proven to exhibit high sensitivity to PA‐associated markers (including PYO, also 5‐methylphenazine‐1‐carboxylic acid (5‐MCA), OHPHZ) when tested in vitro using TSB and LB broths (Figure 8C). [ 78 ] Only two TCUA configurations have been reported so far in the tracing of PYO, including a carbon‐based model generated via microsphere lithography [ 78 ] and a TCUA model with chitosan (biocompatible polymer) and Au nanoparticle. [ 78b ] The carbon‐based model generated via microsphere lithography [ 78 ] has been used for the real‐time measurement of cellular concentrations of PYO and 5‐MCA during the first 48 h of bacterial growth.…”

“…[ 78 ] Only two TCUA configurations have been reported so far in the tracing of PYO, including a carbon‐based model generated via microsphere lithography [ 78 ] and a TCUA model with chitosan (biocompatible polymer) and Au nanoparticle. [ 78b ] The carbon‐based model generated via microsphere lithography [ 78 ] has been used for the real‐time measurement of cellular concentrations of PYO and 5‐MCA during the first 48 h of bacterial growth. The peaks observed at −0.256 and −0.115 V are attributed to PYO and 5‐MCA redox activity, respectively (Figure 8C) and using SWV, the concentrations of cellularly secreted PYO have been determined.…”

Laser-Induced Carbon Electrodes in a Three-Dimensionally Printed Flow Reactor for Detecting Lead Ions

“…This process is called the stripping process. 16 , 17 Compared with the direct voltammetric analysis of the original solution, the biggest advantage of this method is that the analyte can be pre-enriched on the electrode, so the dissolution current is not affected by the charging current and the impurity residual current.…”