Electrochemiluminescence Arrays for Studies of Metabolite‐related Toxicity

Bano, Kiran; Rusling, James F.

doi:10.1002/elan.201600207

Cited by 5 publications

(2 citation statements)

References 44 publications

(76 reference statements)

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“…In previous works, we explored the use of NOS enzymes, including NOS oxygenases (NOSoxy), immobilized in thin films formed by the layer-by-layer (LbL) electrostatic adsorption method, as the source of catalytic NO synthesis . Charge-based LbL electrostatic adsorption of thin-film components and their biocompatible nature have been studied previously and continue to find widespread applications. − In principle, the LbL method could be used to immobilize multiple numbers of alternating layers of iNOSoxy as a negatively charged species and polyethylenimine (PEI) as the positively charge counterpart for sustained NO release. In this work, we show that the pH of the solution from which the NOS enzyme is adsorbed modulates the amount of enzyme loading on the PEI layers.…”

Section: Introductionmentioning

confidence: 99%

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity

Gunasekera

Diwan

Altawallbeh

et al. 2018

ACS Appl. Mater. Interfaces

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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.

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Section: Introductionmentioning

confidence: 99%

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity

Gunasekera

Diwan

Altawallbeh

et al. 2018

ACS Appl. Mater. Interfaces

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“…A high-throughput ECL chip with 64 microwells capable of holding 1 lL droplets were integrated into a microfluidic system for measuring DNA damage caused by metabolites of benzo[a]pyrene [95]. In addition, a low-cost microfluidic paper electrochemical device (lPED) equipped with RuPVP/DNA/enzyme spots was used to screening the genotoxic compounds in water, food, and smoke [96,97]. Recently, the oxidation of guanosines in DNA from the reactive oxygen species (ROS) was evaluated by using a new [Os(bpy) 2 (phen-benz-COOH)] 2?…”

Section: Genotoxicity Screeningmentioning

confidence: 99%

Imaging Analysis Based on Electrogenerated Chemiluminescence

et al. 2017

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Electrochemiluminescence (ECL), also called electrogenerated chemiluminescence, is luminescence that is produced by chemical reactions triggered by electrochemical method. ECL imaging is a novel imaging technique, which can simultaneously provide two kinds of signals of both electrochemistry and optical image. Over the past two decades, ECL imaging has been not only a powerful tool for investigating the fundamental scientific questions such as ECL mechanisms and reaction kinetics, but also a versatile analytical technique for detection of a wide range of analytes including small molecules, DNA, proteins, and cells. In the first part of this review, we briefly describe the reaction mechanisms of ECL generation. Then the review focuses on the research progress on the ECL imaging approach. It is basically introduced from the following five aspects: (i) visualization of electroactive sites on surfaces, (ii) imaging analysis at the single-bead and single-cell levels, (iii) array bioanalysis, (iv) multi-color ECL imaging, and (v) paper chip based on ECL imaging. Finally, some perspectives and future directions in this active research area are presented.

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