Real-time monitoring of the dynamic metabolism and responses of pathogenic bacteria using electroanalytical methods

Microbial infections remain the leading cause of increased morbidity and mortality rates of patients suffering from infectious diseases. While thousands of pathogenic bacteria have been recognized, the majority of healthcare-associated infections are caused by only a few opportunistic pathogens (e.g., Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli), which are associated with increased antibiotic resistance. The rapid detection, reliable identification and real-time monitoring of these pathogens remain not only a scientific problem but also a practical challenge of vast importance, especially in tailoring effective treatment strategies. Various approaches, such as conventional culturing, molecular methods and mass spectrometry techniques, have been employed to identify and quantify pathogenic agents. Yet, these procedures are costly, time-consuming, mostly qualitative, and are indirect detection methods. A great challenge is therefore to develop rapid and quantitative methods for the detection of microbes. As an alternative, electrochemical techniques have been explored as a means for the detection of infection-related biomarkers. This thesis presents the development and application of a robust electrochemical platform using transparent carbon ultramicroelectrode arrays (T-CUAs) for the in vitro detection of bacterial warfare toxin, pyocyanin, and other phenazine metabolites produced by P. aeruginosa. This antibiotic-resistant pathogen is commonly found in chronic wounds and the lungs of cystic fibrosis patients. During early infection stages, P. aeruginosa produces various phenazines as virulence factors, which are highly diffusible signals that are toxic to surrounding host cells and other competing microorganisms. Although phenazines play important roles in cellular functions, very little is known about how their concentrations fluctuate and influence cellular behaviors and population-dependent responses (quorum sensing) during infection and growth. Therefore, quantitative, real-time electrochemical monitoring of distinct redox-active phenazine metabolites from P. aeruginosa in simulated growth media is demonstrated using T-CUAs. Moreover, electrochemical monitoring of the influence of polymicrobial infections on P. aeruginosa phenazine production is presented. In addition to quantifying phenazine concentrations in complex environments, changes in phenazine dynamics are observed in the biosynthetic route for pyocyanin production. Finally, desorption electrospray ionization and nanoelectrospray ionization mass spectrometry are used to identify phenazines observed with our electrochemical devicesChemistr

Fundamentals, Applications, and Future Directions of Bioelectrocatalysis

Bioelectrocatalysis is an interdisciplinary research field combining bio-catalysis and electrocatalysis via the utilization of materials derived from biological systems as catalysts to catalyze the redox reactions occurring at an electrode. Bioelectrocatalysis synergistically couples the merits of both biocatalysis and electrocatalysis. The advantages of biocatalysis include high activity, high selectivity, wide substrate scope, and mild reaction conditions. The advantages of electrocatalysis include the possible utilization of renewable electricity as an electron source and high energy conversion efficiency. These properties are integrated to achieve selective biosensing, efficient energy conversion, and the production of diverse products. This review seeks to systematically and comprehensively detail the fundamentals, analyze the existing problems, summarize the development status and applications, and look toward the future development directions of bioelectrocatalysis. First, the structure, function, and modification of bioelectrocatalysts are discussed. Second, the essentials of bioelectrocatalytic systems, including electron transfer mechanisms, electrode materials, and reaction medium, are described. Third, the application of bioelectrocatalysis in the fields of biosensors, fuel cells, solar cells, catalytic mechanism studies, and bioelectrosyntheses of high-value chemicals are systematically summarized. Finally, future developments and a perspective on bioelectrocatalysis are suggested

Synthesis and Characterization of a Benzoin Type Photoinitiator for Improvement of the Resolution of One-Photon Direct Laser Writing

The use of photoinitiated polymerization is constantly growing due to its large number of applications ranging from 3-D printers to nanotechnology. An interesting application of photoinitiated polymerization reactions is direct laser writing (DLW) lithography, which is a useful way to fabricate microscopic patterns for lab-on-a-chip devices. The goal of this project is to synthesize and characterize a novel photoinitiator for the improvement of the resolution of DLW by using an ultralow one-photon absorption technique. The photoinitiator is a derivative of benzoin methyl ether, where two methyl thioether substituents are introduced. Single-photon polymerization was performed in a liquid acrylate resin with a continuous wave 405 nm diode laser focused to a point using an optical microscope. Since the photoinitiator has an ultralow absorption at 405 nm, the photopolymerization is achieved only at the focusing spot of the microscope objective. Here, we show that this technique enables the fabrication of higher resolution microstructures for lab-on-a-chip devices

Phenazine-Based Compound as a Universal Water-Soluble Anolyte Material for the Redox Flow Batteries

Aqueous organic redox flow batteries (AORFBs) are emerging energy storage technologies due to their high availability, low cost of organic compounds, and the use of eco-friendly water-based supporting electrolytes. In the present work, we demonstrate a unique phenazine-based material that shows redox reversibility in neutral, basic, and acidic conditions with the redox potentials of −0.85 V (1.0 M KOH), −0.67 V (1.0 M NaCl), −0.26 V, and 0.05 V (1.0 M H2SO4) vs. the Ag/AgCl reference electrode and two-electron transfer process at all pH values. High solubility of the phenazine compound in water-based electrolytes up to 1.3 M is achieved by introducing quaternary amonium-based substituents, leading to the outstanding theoretical volumetric capacity of 70 Ah L−1. Laboratory redox flow batteries in neutral and acidic electrolytes presented >100 cycles of stable operation with a capacity loss of 0.25 mAh L−1 and 1.29 mAh L−1 per cycle, respectively. The obtained results demonstrate a material with the potential for not only fundamental understanding but also the practical application of AORFBs in the development of new-generation energy storage technologies

Transparent Carbon Ultramicroelectrode Arrays for the Electrochemical Detection of a Bacterial Warfare Toxin, Pyocyanin

Pyocyanin is a virulence factor produced as a secondary metabolite by the opportunistic human pathogen, <i>Pseudomonas aeruginosa</i>. Fast and direct detection of pyocyanin is of importance as it could provide important insights regarding <i>P. aeruginosa</i>’s virulence mechanisms. Here, we present an electrochemical sensing platform of redox-active pyocyanin using transparent carbon ultramicroelectrode arrays (T-CUAs), which were made using a previously reported simple fabrication process (Duay et al. Anal. Chem. 2015, 87, 10109). Square-wave voltammetry was used to quantify pyocyanin concentrations on T-CUAs with and without chitosan gold nanoparticles (CS/GNP) and planar transparent macroelectrodes (T-Macro). The response time (RT), limit of detection (LOD), and linear dynamic range (LDR) differ for each electrode type due to subtle influences in how the detectable signal varies in relation to the charging time and resistive and capacitive noise. In general lower LODs can be achieved at the consequence of smaller LDRs. The LOD for T-Macro was 0.75 ± 0.09 μM with a LDR of 0.75–25 μM, and the LOD for the CS/GNP 1.54 T-CUA was determined to be 1.6 ± 0.2 μM with a LDR of 1–100 μM, respectively. The LOD for the 1.54T-CUA with a larger LDR of 1–250 μM was 1.0 ± 0.3 μM. These LODs and LDRs fall within the range of PYO concentrations for a variety of <i>in vitro</i> and <i>in vivo</i> cellular environments and offer promise of the application of T-CUAs for the quantitative study of biotoxins, quorum sensing, and pathogenesis. Finally, we demonstrate the successful use of T-CUAs for the electrochemical detection of pyocyanin secreted from <i>P. aeruginosa</i> strains while optically imaging the cells. The secreted pyocyanin levels from two bacterial strains, PA11 and PA14, were measured to have concentrations of 45 ± 5 and 3 ± 2 μM, respectively

Gold Nanoparticle Modified Transparent Carbon Ultramicroelectrode Arrays for the Selective and Sensitive Electroanalytical Detection of Nitric Oxide

Transparent carbon ultramicroelectrode arrays (T-CUAs) were made using a previously reported facile fabrication method (Duay et al. <i>Anal. Chem.</i> <b>2015</b>, <i>87</i>, 10109). Two modifications introduced to the T-CUAs were examined for their analytical response to nitric oxide (NO^•). The first modification was the application of a cellulose acetate (CA) gas permeable membrane. Its selectivity to NO^• was extensively characterized via chronoamperometry, electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). The thickness of the CA membrane was determined to be 100 nm and 88 ± 15 nm using AFM and EIS, respectively. Furthermore, the partition and diffusion coefficients of NO^• within the CA membrane were determined to be 0.0500 and 2.44 × 10^–13 m²/s using EIS measurements. The second modification to the 1.54T-CUA was the introduction of chitosan and gold nanoparticles (CS/GNPs) to enhance its catalytic activity, sensitivity, and limit of detection (LOD) to NO^•. Square wave voltammetry was used to quantify the NO^• concentration at the CA membrane covered 1.54T-CUA with and without CS/GNPs; the LODs were determined to be 0.2 ± 0.1 and 0.44 ± 0.02 μM (<i>S</i>/<i>N</i> = 3), with sensitivities of 9 ± 9 and 1.2 ± 0.4 nA/μM, respectively. Our results indicate that this modification to the arrays results in a significant catalytic enhancement to the electrochemical oxidation of NO^•. Hence, these electrodes allow for the <i>in situ</i> mechanistic and kinetic characterization of electrochemical reactions with high electroanalytical sensitivity