Amperometric Detection Of Nitric Oxide Research Articles

A 3D-printed, multi-modal microfluidic device for measuring nitric oxide and ATP release from flowing red blood cells.

In this paper, a 3D-printed multi-modal device was designed and fabricated to simultaneously detect nitric oxide (NO) and adenosine triphosphate (ATP) in red blood cell suspensions prepared from whole blood. Once a sample was injected into the device, NO was first detected (via amperometry) using a three-electrode, dual-opposed, electrode configuration with a platinum-black/Nafion coated gold working electrode. After in-line amperometric detection of NO, ATP was detected via a chemiluminescence reaction, with a luciferin/luciferase solution continuously pumped into an integrated mixing T and the resulting light being measured with a PMT underneath the channel. The device was optimized for mixing/reaction conditions, limits of detection (40 nM for NO and 30 nM for ATP), and sensitivity. This device was used to determine the basal (normoxic) levels of NO and ATP in red blood cells, as well as an increase in concentration of both analytes under hypoxic conditions. Finally, the effect of storing red blood cells in a commonly used storage solution was also investigated by monitoring the production of NO and ATP over a three-week storage time.

Cover Picture: Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization, Direct RBC Contact, and Amperometric Detection of Nitric Oxide (Electroanalysis 8/2019)

Cover Picture: Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization, Direct RBC Contact, and Amperometric Detection of Nitric Oxide (Electroanalysis 8/2019)

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization, Direct RBC Contact, and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC-derived ATP. These studies incorporate on-chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC-EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum-black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8 ± 0.2 nM NO per endothelial cell (average ± SEM, n = 8). Pretreatment of RBCs with glybenclamide inhibited treprostinil-induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92 ± 0.1 nM NO per endothelial cell, n = 7). In the future, this device can be used to study interactions between many other cell types (both adherent and non-adherent cell lines) and incorporate other detection schemes.

Amperometric detection of nitric oxide using a glassy carbon electrode modified with gold nanoparticles incorporated into a nanohybrid composed of reduced graphene oxide and Nafion

The authors show that the electrocatalytic performance toward the detection of nitric oxide (NO) can be enhanced by making use of gold nanoparticles (AuNP) in a matrix consisting of reduced graphene oxide and Nafion (rGO-Nf). The rGO-Nf@Au nanohybrid was synthesized via a hydrothermal method. The spherical AuNP have diameters in the range from 50 to 200 nm as proven by field emission scanning electron microscopy (FESEM). A glassy carbon electrode (GCE) modified with the nanohybrid displays excellent electrocatalytic activity towards NO oxidation compared to other kinds of modified electrodes. Best operated at a voltage of +0.8 V (vs. SCE), the amperometric response was linear in the 1 μM to 0.16 mM nitrite concentration range, with 0.5 μM detection limit (at an S/N ratio of 3). The high surface area of the AuNP along with the synergistic effect of AuNP and rGO-Nf film on the signal current is believed to cause the enhanced electrocatalytic activity of the nanohybrid. The sensor is not interfered by dopamine (DA), ascorbic acid (AA), uric acid (UA), glucose, urea, and NaCl even in 5-fold higher concentrations. In our perception, the rGO-Nf@Au modified electrode is a promising tool for highly sensitive and selective amperometric sensing of NO.

Amperometric Nitric Oxide Sensor Based on Nanoporous Platinum Phthalocyanine Modified Electrodes

This article describes the fabrication of electropolymerized Metallo 4', 4″, 4‴, 4'''' tetra-amine phthalocyanine (poly-MTAPc) modified electrodes for the detection of nitric oxide (NO) in phosphate-buffered saline (PBS) at pH 7.4. A two-step synthetic protocol using a laboratory microwave reactor was adopted to provide three MTAPc complexes bearing different metal centers (M = Cu(2+): CuTAPc, M = Zn(2+): ZnTAPc, and M = Pt(2+): PtTAPc). The MTAPc complexes and the intermediates were characterized by MALDI-TOF mass spectrometry, UV-vis spectroscopy, (1)H NMR spectroscopy, and elemental analysis. The MTAPc products were separately electropolymerized either onto a glassy carbon (GC) electrode as a thin-film or within the pores of Anodisc nanoporous alumina membrane as a densely packed array of poly-MTAPc nanotubes to produce two electrode systems. In the latter system, the surface area enhancement provided by the nanotube-arrayed morphology of the poly-MTAPc enabled a high faradaic (signal) to capacitative (background) current during NO electro-oxidation. Amperometric detection of NO using these two electrode systems shows that the sensitivity and linear ranges were insensitive to the metal centers (M = Cu(2+), Zn(2+), and Pt(2+)) of the poly-MTAPc material.

Visible Photolysis and Amperometric Detection of S-Nitrosothiols

The concentration of S-nitrosothiols (RSNOs), endogenous transporters of the signaling molecule nitric oxide (NO), fluctuate greatly in physiology often as a function of disease state. RSNOs may be measured indirectly by cleaving the S-N bond and monitoring the liberated NO. While ultraviolet photolysis and reductive-based cleavage both decompose RSNOs to NO, poor selectivity and the need for additional reagents preclude their utility clinically. Herein, we report the coupling of visible photolysis (i.e., 500-550 nm) and amperometric NO detection to quantify RSNOs with greater selectivity and sensitivity. Enhanced sensitivity (up to 1.56 nA μM(-1)) and lowered theoretical detection limits (down to 30 nM) were achieved for low molecular weight RSNOs (i.e., S-nitrosoglutathione, S-nitrosocysteine) by tuning the irradiation exposure. Detection of nitrosated proteins (i.e., S-nitrosoalbumin) was also possible, albeit at a decreased sensitivity (0.11 nA μM(-1)). This detection scheme was used to measure RSNOs in plasma and illustrate the potential of this method for future physiological studies.

On-chip multi-electrochemical sensor array platform for simultaneous screening of nitric oxide and peroxynitrite

In this work we report on the design, microfabrication and analytical performances of a new electrochemical sensor array (ESA) which allows for the first time the simultaneous amperometric detection of nitric oxide (NO) and peroxynitrite (ONOO(-)), two biologically relevant molecules. The on-chip device includes individually addressable sets of gold ultramicroelectrodes (UMEs) of 50 µm diameter, Ag/AgCl reference electrode and gold counter electrode. The electrodes are separated into two groups; each has one reference electrode, one counter electrode and 110 UMEs specifically tailored to detect a specific analyte. The ESA is incorporated on a custom interface with a cell culture well and spring contact pins that can be easily interconnected to an external multichannel potentiostat. Each UME of the network dedicated to the detection of NO is electrochemically modified by electrodepositing thin layers of poly(eugenol) and poly(phenol). The detection of NO is performed amperometrically at 0.8 V vs. Ag/AgCl in phosphate buffer solution (PBS, pH = 7.4) and other buffers adapted to biological cell culture, using a NO-donor. The network of UMEs dedicated to the detection of ONOO(-) is used without further chemical modification of the surface and the uncoated gold electrodes operate at -0.1 V vs. Ag/AgCl to detect the reduction of ONOOH in PBS. The selectivity issue of both sensors against major biologically relevant interfering analytes is examined. Simultaneous detection of NO and ONOO(-) in PBS is also achieved.

Amperometric Detection of Nitric Oxide with Microsensor in the Medium of Seawater and Its Applications

Amperometric detection of nitric oxide with ISO-NOPMC NO microsensors(WPI) is systemically studied in the six media including seawater. The linear range of themicrosensor for NO was from 10-6 to 10-9 mol/l and the detection limit was4.2×10-10 mol/L(S/N=2). With this method, we provide preliminary evidence that NOproduction could be a general attribute of marine alga (Haeterosigma akashiwo).Experiment conducted with inhibitor of uncoupler 2,4-DNP (2,4-dinitropheno) revealed thatNR (nitrate reductase) activity is responsible for NO formation.

Electrochemical activation of electrodes for amperometric detection of nitric oxide

An open question in the literature of nitric oxide detection was investigated: does electrochemical activation account for the enhanced properties of certain presumed chemically-modified electrodes? Uniform electrodes of graphite, iridium, palladium, platinum, and ruthenium were exposed to potential cycling and then tested for amperometric response to nitric oxide to identify principles that govern electrochemical activation of nitric oxide electrodes. These electrodes were compared to similar electrodes that were not cycled. Only cycled graphite and ruthenium showed significantly increased responses. Graphite demonstrated enhanced performance after exposure to cycling potentials at which oxygen, CO 2, and soluble carbonates form, suggesting that erosion of the electrode enhanced its response by increasing the surface area accessible to nitric oxide. This may explain the performance of carbon fibers cycled to the same potentials in solutions containing metalloporphyrins. The response of ruthenium was enhanced after cycling to less extreme potentials at which soluble species do not form and at which a metallic conductive oxide, RuO 2, could lay down a stable, adherent layer on the electrode surface. Cycled ruthenium also exhibited a much greater increase in capacitance after cycling, consistent with the formation of a conductive surface layer.

Cytochrome c′ from Chromatium vinosum on gold electrodes

Cytochrome c′ from Chromatium vinosum (CVCP) was immobilized at a surface-modified gold electrode. Characterization of the CVCP electrode showed a quasi-reversible, diffusionless electrochemical redox behavior of the surface adsorbed protein with a formal potential of −128±5 mV vs. Ag/AgCl. The heterogeneous electron transfer rate constant of adsorbed CVCP was determined to be about 50 s −1. Different immobilization strategies were compared. The interaction of the immobilized CVCP with nitric oxide (NO) was investigated and applied for a primary amperometric detection of NO in solution.

In vivo nitric oxide sensor using non-conducting polymer-modified carbon fiber

Nitric oxide (NO) is emerging as a very important and ubiquitous gaseous messenger in the body. The response characteristics of NO sensors made of non-conducting polymer modified carbon fiber electrodes are investigated to determine their selectivity, sensitivity, and stability for in vivo use. A composite polymer, comprising Nafion, m-phenylenediamine, and resorcinol, showed the best selectivity and stability to amperometric NO detection. The non-conducting, self-limiting polymer film protects the electrode from interference and fouling by other biochemicals. Although the relative sensitivity to NO of the modified sensor is lower than that of the unmodified carbon fiber electrodes (less than 6%), the composite polymer electrode showed high selectivity against ascorbic acid (>2000:1), nitrite (>600:1), and dopamine (>200:1). The stability of the NO sensor was maintained for at least 1 week. The NO sensitivity after in vivo experiments ( n=8) is 88.1±5.6% of initial sensitivity data obtained before in vivo experiments. Preliminary in vivo experiments done with this electrode are shown to capture elevated NO levels in brain following an ischemic injury.