Polymerized Hemin as An Electrocatalytic Platform for Peroxynitrite\u27s Oxidation and Detection

Peroxynitrite (ONOO−) constitutes a major cytotoxic agent, implicated in a host of pathophysiological conditions, thereby stimulating a tremendous interest in evaluating its role as an oxidant in vivo. Some of the detection methods for peroxynitrite include oxidation of fluorescent probes, EPR spectroscopy, chemiluminescence, immunohistochemistry, and probe nitration; however, these are more difficult to apply for real-time quantification due to their inherent complexity. The electrochemical detection of peroxynitrite is a simpler and more convenient technique, but the best of our knowledge there are only few papers to date studying its electrochemical signature, or reporting amperometric microsensors for peroxynitrite. Recently, we have reported the use of layered composite films of poly(3,4-ethylenedioxythiophene) (PEDOT) and hemin (iron protoporphyrin IX) as a platform for amperometric measurement of peroxynitrite. The main goal herein is to investigate the intrinsic catalytic role of hemin electropolymerized thin films on carbon electrodes in oxidative detection of peroxynitrite. The electrocatalytic oxidation of peroxynitrite is characterized by cyclic voltammetry. The catalytic current increased as a function of peroxynitrite\u27s concentration, with a peak potential shifting positively with peroxynitrite\u27s concentration. The catalytic efficiency decreased as the scan rate increased, and the peak potential of the catalytic oxidation was found to depend on pH. We show that optimized hemin-functionalized carbon electrodes can be used as simple platforms for peroxinitrite detection and quantification. We report dose–response amperometry as an electroanalytical determination of this analyte on hemin films and we contrast the intrinsic hemin catalytic role with its performance in the case of the PEDOT–hemin as a composite matrix. Finally, we include some work extending the use of simple hemin films for peroxynitrite determination on carbon microfiber electrodes in a flow system

Manganese Oxide/Hemin-Functionalized Graphene as a Platform for Peroxynitrite Sensing

Peroxynitrite (ONOO−, PON) is a powerful oxidizing agent generated in vivo by the diffusion-limited reaction of nitric oxide (NO) and superoxide (O2˙−) radicals. Under oxidative stress, cumulated peroxynitrite levels are associated with chronic inflammatory disorders and other pathophysiological conditions. The accurate detection of peroxynitrite in biological systems is important, not only to understand the genesis and development of diseases, but also to explore and design potential therapeutics. Herein, a manganese oxide/hemin-modified graphene interface is explored as a platform for peroxynitrite amperometric detection. Hemin-functionalized reduced graphene oxide was further modified with manganese oxide nanoparticles to provide a composite material with catalytic activity toward the electrochemical oxidation of peroxynitrite. The morphology of the composite material was characterized using scanning electron microscopy, energy dispersive X-ray analysis, X-ray photoelectron spectroscopy, and UV-Vis absorption measurements. We investigated the electrocatalytic oxidation of peroxynitrite on graphite electrodes modified with the composite material using cyclic voltammetry and amperometry. The results showed that the incorporation of manganese oxide nanoparticles into graphene/hemin material enhances the catalytic detection of peroxynitrite compared to graphene/hemin alone

P197 – Sensitive and Selective Electrochemical NO Sensors Modified with Nanostructured Catalyst: Towards Probing The Role of NO in Cystic Fibrosis Pathophysiology

Nitric oxide is an important physiologic metabolite implicated in both health and disease states. Accurate determination of NO in tissues and cells is of paramount importance. The major challenges with NO measurement are its short half-life and often low nanomolar concentrations in tissues. Electrochemical tools provide a means to accurately measure this analyte in confined environments using miniaturized probes. In the past, our lab explored electrodeposited alternate layers of ruthenium oxide nanoparticles and a conductive sulfur-containing polymer on carbon microelectrode fibers for catalytic determination of NO. In this work, we explore the performance of combined reference/working electrodes modified with ruthenium oxide in the detection of nitric oxide with the goal to measure nitric oxide at the level of single or collective cultured cells. This is a preliminary work towards preparing a device capable to measure nitric oxide levels in a cystic fibrosis cell line model. It has been found that exhaled NO levels remains unchanged or reduced in cystic fibrosis patients unlike other inflammatory lung diseases like asthma where it increases. However, it is not clear whether the lower NO levels in cystic fibrosis correlate with lowered production of this metabolite in the bronchial epithelium. It was shown that levels of nitrite and nitrate, the primary stable end products of NO metabolism, increases in the breath condensate of patients with cystic fibrosis. We will present preliminary results of our ruthenium oxide modified combined electrodes and how they can be applied to the study of cystic fibrosis at the cellular level

Polymerized Hemin as An Electrocatalytic Platform for Peroxynitrite\u27s Oxidation and Detection

Peroxynitrite (ONOO−) constitutes a major cytotoxic agent, implicated in a host of pathophysiological conditions, thereby stimulating a tremendous interest in evaluating its role as an oxidant in vivo. Some of the detection methods for peroxynitrite include oxidation of fluorescent probes, EPR spectroscopy, chemiluminescence, immunohistochemistry, and probe nitration; however, these are more difficult to apply for real-time quantification due to their inherent complexity. The electrochemical detection of peroxynitrite is a simpler and more convenient technique, but the best of our knowledge there are only few papers to date studying its electrochemical signature, or reporting amperometric microsensors for peroxynitrite. Recently, we have reported the use of layered composite films of poly(3,4-ethylenedioxythiophene) (PEDOT) and hemin (iron protoporphyrin IX) as a platform for amperometric measurement of peroxynitrite. The main goal herein is to investigate the intrinsic catalytic role of hemin electropolymerized thin films on carbon electrodes in oxidative detection of peroxynitrite. The electrocatalytic oxidation of peroxynitrite is characterized by cyclic voltammetry. The catalytic current increased as a function of peroxynitrite\u27s concentration, with a peak potential shifting positively with peroxynitrite\u27s concentration. The catalytic efficiency decreased as the scan rate increased, and the peak potential of the catalytic oxidation was found to depend on pH. We show that optimized hemin-functionalized carbon electrodes can be used as simple platforms for peroxinitrite detection and quantification. We report dose–response amperometry as an electroanalytical determination of this analyte on hemin films and we contrast the intrinsic hemin catalytic role with its performance in the case of the PEDOT–hemin as a composite matrix. Finally, we include some work extending the use of simple hemin films for peroxynitrite determination on carbon microfiber electrodes in a flow system

Manganese Oxide/Hemin-Functionalized Graphene as a Platform for Peroxynitrite Sensing

Peroxynitrite (ONOO−, PON) is a powerful oxidizing agent generated in vivo by the diffusion-limited reaction of nitric oxide (NO) and superoxide (O2˙−) radicals. Under oxidative stress, cumulated peroxynitrite levels are associated with chronic inflammatory disorders and other pathophysiological conditions. The accurate detection of peroxynitrite in biological systems is important, not only to understand the genesis and development of diseases, but also to explore and design potential therapeutics. Herein, a manganese oxide/hemin-modified graphene interface is explored as a platform for peroxynitrite amperometric detection. Hemin-functionalized reduced graphene oxide was further modified with manganese oxide nanoparticles to provide a composite material with catalytic activity toward the electrochemical oxidation of peroxynitrite. The morphology of the composite material was characterized using scanning electron microscopy, energy dispersive X-ray analysis, X-ray photoelectron spectroscopy, and UV-Vis absorption measurements. We investigated the electrocatalytic oxidation of peroxynitrite on graphite electrodes modified with the composite material using cyclic voltammetry and amperometry. The results showed that the incorporation of manganese oxide nanoparticles into graphene/hemin material enhances the catalytic detection of peroxynitrite compared to graphene/hemin alone

Methods of Peroxynitrite Synthesis in the Context of the Development and Validation of Peroxynitrite Sensors

Peroxynitrite has emerged as a major cytotoxic agent implicated in a host of pathophysiological conditions triggered by its multifaceted nitroxidative reactions. Peroxynitrite\u27s very short half-life under physiological conditions reflects both its fast intrinsic decomposition and its notorious reactivity with many cellular targets. Sensitive and accurate measurement of peroxynitrite is crucial to investigations that aim at shedding light on the illusive pathophysiologic roles of this metabolite. The development process of probes and sensors capable of selectively detecting and measuring any analyte depends primarily on the availability of pure authentic amounts of it in order to evaluate sensor performance. There are numerous methods of peroxynitrite synthesis or generation in situ. However, they all come with varying levels of byproducts that may interfere with the characterization of performance of peroxynitrite sensing probes and devices. This chapter gives the background and a brief review of major methods of peroxynitrite synthesis and in situ generation. Particularly, we discuss advantages and drawbacks of the various methods in the context of the development and characterization of peroxynitrite sensors and probes

Investigation of Nitric Oxide Synthase (NOS) Redox and Catalytic Activity in Electrospun Fibers

Background: Nitric oxide (NO) is known to counteract platelet aggregation in mammals, and thus can stop the thrombosis cascade on the surface of blood-contacting medical devices. Nitric oxide synthases (NOSs) are enzymes responsible for catalytic conversion of the substrate L-arginine to NO and L-citrulline. By using NO releasing biomaterial that mimic mammalian tissue, one may be able to solve the issue of thrombosis on the surface of foreign devices implanted or used as part of cardiovascular procedures. In the past, we have tested the use of NOS enzymes in layer-by-layer thin films as a source of in-situ NO generation and release. Our objective in the current work is to use NOS enzymes trapped in electrospun fibers as a biocompatible platform for NO release. Methods and Results: In this project, we investigate the possibility to use nitric oxide synthase (NOS) as NO-making component trapped in aqueous pockets of electrospun biopolymer matrices. The polymers used in this preliminary work are polycaprolactone (PCL) and Polyurethane (PU). A guided stream of polymer solution containing suspended aqueous pockets of enzyme solution is directed towards a collector drum (or an electrode) in strong electric field. In its path of acceleration towards the target, the solvent evaporates and the charged jet thins-out leaving a fibrous membrane, devoid of solvent and containing ‘nodes’ of aqueous pockets with entrapped NOS enzymes. Surface characterizations such as Transmission Electron Microscopic (TEM) and Atomic Force Microscopic (AFM) imaging are carried out on the newly formed NOS-containing electrospun fibers. Further, the NOS-modified membranes are tested electrochemically using a characteristic electrocatalytic reaction mediated by entrapped NOS enzymes. Finally, the NOS-containing electrospun membranes are subject to assays under various conditions to determine the structural integrity of NOS enzymes and their enzymatic activity. Just like the case of our layer-by-layer films reported earlier, we find that the entrapped NOS in electrospun fiber conserves its redox activity and catalytic function. Results on NO flux measurements under physiologically relevant conditions will be presented and discussed

Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes

The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 104 bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼105 bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to β-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h)