Peroxynitrite (ONOO−, PON) is a powerful oxidizing agent generated in vivo by the diffusion-limited reaction of nitric oxide (NO) and superoxide (O 2 ˙−) 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. Heminfunctionalized 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.

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Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Nitric oxide (NO), a small free radical molecule, turned out to be pervasive in biology and was shown to have a substantial influence on a range of biological activities, including cell growth and apoptosis. This molecule is involved in signaling and affects a number of physiologic functions. In recent decades, several processes related to cancer, such as angiogenesis, programmed cell death, infiltration, cell cycle progression, and metastasis, have been linked with nitric oxide. In addition, other parallel work showed that NO also has the potential to operate as an anti-cancer agent. As a result, it has gained attention in cancer-related therapeutics. The nitric oxide synthase enzyme family (NOS) is required for the biosynthesis of nitric oxide. It is becoming increasingly popular to develop NO-releasing materials as strong tumoricidal therapies that can deliver sustained high concentrations of nitric oxide to tumor sites. In this paper, we developed NO-releasing materials based on sodium alginate hydrogel. In this regard, alginate hydrogel discs were modified by adsorbing layers of polyethyleneimine and iNOS-oxygenase. These NO-releasing hydrogel discs were prepared using the layer-by-layer film building technique. The iNOS-oxygenase is adsorbed on the positively charged polyethyleneimine (PEI) matrix layer, which was formed on a negatively charged sodium alginate hydrogel. We show that nitric oxide is produced by enzymes contained within the hydrogel material when it is exposed to a solution containing all the components necessary for the NOS reaction. The electrostatic chemical adsorption of the layer-by-layer process was confirmed by FTIR measurements as well as scanning electron microscopy. We then tested the biocompatibility of the resulting modified sodium alginate hydrogel discs. We showed that this NOS-PEI-modified hydrogel is overall compatible with cell growth. We characterized the NOS/hydrogel films and examined their functional features in terms of NO release profiles. However, during the first 24 h of activity, these films show an increase in NO release flux, followed by a gradual drop and then a period of stable NO release. These findings show the inherent potential of using this system as a platform for NO-driven modulation of biological functions, including carcinogenesis.

Peroxynitrite (PON) is a highly reactive oxygen-nitrogen species that facilitates both oxidation and nitration reactions. Early reports have revealed the deleterious effects of PON on DNA, proteins, and lipids. Recent studies have suggested that melanin can act as an antioxidative therapy to scavenge the reactive oxygen-nitrogen species (RO-NS) including PON. Melanin is a natural pigment that has many physiological functions involving the neutralization of highly oxidative species. In this project, the interaction between PON and synthetic melanin has been studied. In addition, the electrochemical characteristics of the polymerized 5,6-dihydroxy indole (DHI) as a model of synthetic melanin were examined using cyclic voltammetry and electrochemical quartz crystal microbalance (EQCM). The ultraviolet-visible (UV-Vis) spectroscopy showed a significant difference in the absorbance of PON alone and in the presence of melanin films. Finally, we report on the possibility of using the DHI-melanin film as a platform for the quantitative detection of PON in solutions. Post-print standardized by MSL Academic Endeavors, the imprint of the