Nitric Oxide Microsensor Research Articles

Fabrication and Optimization of Poly(ε-caprolactone) Microspheres Loaded with S-Nitroso-N-Acetylpenicillamine for Nitric Oxide Delivery.

Heart disease is one of the leading causes of death in the United States and throughout the world. While there are different techniques for reducing or preventing the impact of heart disease, nitric oxide (NO) is administered as nitroglycerin for reversing angina or chest pain. Unfortunately, due to its gaseous and short-lived half-life, NO can be difficult to study or even administer. Therefore, controlled delivery of NO is desirable for therapeutic use. In the current study, the goal was to fabricate NO-releasing microspheres (MSs) using a donor molecule, S-Nitroso-N-Acetyl penicillamine, (SNAP), and encapsulating it in poly(ε-caprolactone) (PCL) using a single-emulsion technique that can provide sustained delivery of NO to cells over time without posing any toxicity risks. Optimization of the fabrication process was performed by varying the duration of homogenization (5, 10, and 20 min) and its effect on entrapment efficiency and size. The optimized SNAP-MS had an entrapment efficiency of ˃50%. Furthermore, we developed a modified method for NO detection by using NO microsensors to detect the NO release from SNAP-MSs in real time, showing sustained release behavior. The fabricated SNAP-MSs were tested for biocompatibility with HUVECs (human umbilical vein endothelial cells), which were found to be biocompatible. Lastly, we tested the effect of controlled NO delivery to human induced pluripotent stem-derived cardiomyocytes (hiPSC-CMs) via SNAP-MSs, which showed a significant improvement in the electrophysiological parameters and alleviated anoxic stress.

Rapid changes in spectral composition after darkness influences nitric oxide, glucose and hydrogen peroxide production in the Antarctic diatom Fragilariopsis cylindrus

Ice-associated phototrophic taxa contribute significantly to Antarctic primary production and are crucial to ecosystem stability in the Southern Ocean. The quantity and quality of light required for photosynthesis is the single most influential driver of ice-associated algal communities. While the presence of ice and snow greatly reduces the irradiance reaching the ice-water interface, it is also the spectral quality that influences the phototrophy of ice-associated microalgae communities. Here we test the capability of three electrochemical microsensors to detect photosynthetically derived “stress” metabolites produced by the Antarctic diatom F. cylindrus. Following a period of dark incubation, this photo-physiological response differed with respect to the intensity and spectral quality of light during re-illumination. Exposure to blue light resulted in impairment in photosynthetic efficiency of PSII (Fv/Fm) and resulted in the production of nitric oxide (NO), hydrogen peroxide (H2O2) and glucose exudation. A similar trend in metabolite production was observed when subjected to white light, but not during red or green illumination. These results indicate that rapid exposure to light and variation in spectral composition can cause significant stress that can be quantified using H2O2, NO and glucose microsensors. This metabolic overflow was triggered by the disruption of normal photosynthetic electron flow and it is proposed that the detection of extracellular metabolites can be directly attributed to intracellular activity.

Electrochemical Nitric Oxide Microsensors Based on a Fluorinated Xerogel Screening Layer for in Vivo Brain Monitoring.

Nitric oxide (NO) is an important free radical synthesized and released by brain cells. At low (nanomolar) levels, it modulates synaptic transmission and neuronal activity, but at much higher levels mediates neuronal injury through oxidative stress. However, the precise concentrations at which these biological actions are exerted are still poorly defined. Electrochemical detection of NO in vivo requires rigorous exclusion of endogenous redox molecules such as ascorbate or nitrite. A fluorinated xerogel composed of trimethoxymethylsilane and heptadecafluoro-1,1,2,2-tetrahydrodecyl silane has been proposed to create a screening layer around NO sensors, protecting against such chemical interference in vitro. Here we detected NO in the living brain using carbon fiber microelectrodes covered with nickel porphyrin and this fluorinated xerogel. These microsensors were insensitive to interfering redox molecules and surpassed similar microelectrodes coated with a Nafion screening layer. In vivo, in the rat parietal cortex, these electrodes could detect brain NO released by local microinjection of the glutamatergic agonist N-methyl-d-aspartate (NMDA). NMDA-evoked NO release peaked at 1.1 μM and lasted more than 20 min. This fluorinated xerogel screening layer can therefore be applied in vivo, allowing for the fabrication of highly specific microsensors to study NO physio-pathological actions in the brain.

Effects of NOX‐1 on Real‐Time Blood Nitric Oxide and Hydrogen Peroxide in Acute Hyperglycemia

Hyperglycemia has been associated with vascular endothelial dysfunction in part by a reduction in nitric oxide (NO) production and increased oxidative stress (e.g., increased superoxide (SO) and hydrogen peroxide (H2O2). Endothelial‐derived NO can be significantly reduced by increased SO/H2O2 in part by the activation of NADPH oxidase during hyperglycemia. Of the 7 NADPH oxidase isoforms, NOX1 is mainly expressed in the vasculature and may play a major role in hyperglycemia induced oxidative stress and vascular endothelial dysfunction. To test this hypothesis, we measured blood NO and H2O2 levels in real time via NO and H2O2 microsensors inserted into femoral veins of rats. Hyperglycemia (e.g., 200 mg/dl) was maintained by infusion i.v. of 30% glucose solution for 3 hours with or without a selective NOX1 inhibitor, ML171. We found that hyperglycemia for 3 hours significantly increased blood H2O2 levels by 1.88±0.4 μM (n=6) compared to the saline infused control (P<0.05, n=2). By contrast, ML171 (1 and 5 μM) reduced hyperglycemia‐induced H2O2 levels by 1.36±0.61 μM (n=8) and 4.35±1.02 μM (n=5), respectively, at the end of experiment. Meanwhile, hyperglycemia significantly reduced blood NO levels by 82.48±38.12 nM (n=3) compared to the saline control (P<0.05, n=2). By contrast, ML171 (1 μM) attenuated the hyperglycemia induced decrease in blood NO levels and increased blood NO levels by 71.15±24.00 nM (n=5) at the end of experiment. Our preliminary results indicate that NOX1 activation may contribute to hyperglycemia‐induced oxidative stress and NO reduction. Furthermore, inhibition of NOX1 may mitigate the deleterious effects of hyperglycemia.Support or Funding InformationThis study was supported by Division of Research, the Center for Chronic Disorders of Aging, and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

Amperometric Quantification of S-Nitrosoglutathione Using Gold Nanoparticles: A Step toward Determination of S-Nitrosothiols in Plasma.

S-Nitrosothiols (RSNOs) are carriers of nitric oxide (NO) and have important biological activities. We propose here the use of gold nanoparticles (AuNPs) and NO-selective amperometric microsensor for the detection and quantification of S-nitrosoglutathione (GSNO) as a step toward the determination of plasma RSNOs. AuNPs were used to decompose RSNOs with the quantitative release of free NO which was selectively detected with a NO microsensor. The optimal [GSNO]/[AuNPs] ratio was determined, corresponding to an excess of AuNP surface relative to the molar GSNO amount. Moreover, the influence of free plasma thiols on this method was investigated and a protocol based on the blocking of free thiols with iodoacetic acid, forming the carboxymethyl derivative of the cysteine residues, is proposed.

Vasorelaxation induced by a new naphthoquinone-oxime is mediated by NO-sGC-cGMP pathway.

It has been established that oximes cause endothelium-independent relaxation in blood vessels. In the present study, the cardiovascular effects of the new oxime 3-hydroxy-4–(hydroxyimino)-2-(3-methylbut-2-enylnaphtalen-1(4H)-one (OximeS1) derived from lapachol were evaluated. In normotensive rats, administration of Oxime S1 (10, 15, 20 and 30 mg/Kg, i.v.) produced dose-dependent reduction in blood pressure. In isolated aorta and superior mesenteric artery rings, Oxime S1 induced endothelium-independent and concentration-dependent relaxations (10−8 M to 10−4 M). In addition, Oxime S1-induced vasorelaxations were attenuated by hydroxocobalamin or methylene blue in aorta and by PTIO or ODQ in mesenteric artery rings, suggesting a role for the nitric oxide (NO) pathway. Additionally, Oxime S1 (30 and 100 µM) significantly increased NO concentrations (13.9 ± 1.6 nM and 17.9 ± 4.1 nM, respectively) measured by nitric oxide microsensors. Furthermore, pre-contraction with KCl (80 mM) prevented Oxime S1-derived vasorelaxation in endothelium-denuded aortic rings. Of note, combined treatment with potassium channel inhibitors also reduced Oxime S1-mediated vasorelaxation suggesting a role for potassium channels, more precisely Kir, Kv and KATP channels. We observed the involvement of BKCa channels in Oxime S1-induced relaxation in mesenteric artery rings. In conclusion, these data suggest that the Oxime S1 induces hypotension and vasorelaxation via NO pathway by activating soluble guanylate cyclase (sGC) and K+ channels.

Effects of prostaglandin E1 on nitric oxide and oxygen dynamics during rat myocardial ischemia–reperfusion utilizing sol–gel derived microsensors

Prostaglandin E1 (Alprostadil®, PGE1) is often used for patients with critical limb ischemia due to its vasodilator and anti-platelet effects. Although PGE1 is considered to have a cardioprotective effect against myocardial ischemia–reperfusion (IR) injury, its mechanism and correlation with nitric oxide (NO) for cardioprotection are not clear. In this study, we used sol–gel-modified electrochemical NO and O2 microsensors to simultaneously measure the changes in NO level and oxygen tension (pO2) in the myocardium during myocardial IR that were induced by PGE1 pretreatment. Rat hearts were randomly divided into two groups: a control group (n=5) and a PGE1 group (n=5, with PGE1 pretreatment). Myocardium that underwent PGE1 pretreatment (26.0±13.1%, p<0.001) demonstrated more highly reduced endogenous NO production during the ischemic period than that of the control group (86.7±18.5%). After the onset of reperfusion, %NO level in the PGE1 group increased to a maximum of 82.0±6.4 but did not exceed the pre-ischemic basal NO level. However, in the control group, the maximum %NO response (164.9±41.0) on reperfusion was approximately double that of the PGE1 group (p<0.01, n=5). The restoration value of pO2 in the PGE1 group significantly increased and was restored to pre-ischemic level (82.2±21.3%); however, the pO2 of the control group did not increase throughout the reperfusion period (12.3±7.9%, p<0.001). As a result, we suggest that the cardioprotective effect of PGE1 might be attributed to a reduction in excessive NO production during reperfusion. Therefore, simultaneous and real-time monitoring of NO and pO2 dynamics in the myocardium would be effective in evaluating the effects of therapeutic treatments such as drug administration. Also, the reduction in excessive NO release during the early reperfusion period might be helpful as a therapeutic strategy to protect the myocardium from IR injury.

The role of endothelial nitric oxide synthase coupling status during acute hyperglycemia as determined by real‐time measurements of blood nitric oxide and hydrogen peroxide in rat (832.2)

Acute hyperglycemia impairs vascular dilatory function in normal subjects. Normally, vascular endothelial function depends on nitric oxide (NO) production from coupled eNOS in the presence of cofactor 5,6,7,8‐tetrahydrobiopterin (BH4). By contrast, 7,8‐dihydrobiopterin (BH2, oxidized form of BH4) and/or lack of L‐arginine (eNOS substrate) causes eNOS uncoupling to produce superoxide (SO), which can be quickly converted to hydrogen peroxide (H2O2). The role of eNOS coupling status in mediating acute hyperglycemia‐induced vascular dysfunction is not known. We simultaneously measured blood NO and H2O2 during the infusion of saline (control), or 20% glucose with or without BH4 (MW=314 g/mol, 6.5 mg/kg), or L‐arginine (MW=211 g/mol, 600 mg/kg), or BH2 (MW=239 g/mol, 4 mg/kg) by NO or H2O2 microsensors (100 µm, WPI Inc.) from the femoral veins of male Sprague‐Dawley rats. We found that acute hyperglycemia (200 mg/dL, n=6) significantly increased blood H2O2 and reduced blood NO levels compared to the saline group over a 3 hr. period (n=7, p&lt;0.05). By contrast, BH4 (n=6), or L‐arginine (n=5‐6), not BH2 (n=6‐7), significantly reduced blood H2O2 and improved vascular NO levels (p&lt;0.05). This study provides novel evidence indicating that eNOS uncoupling during acute hyperglycemia causes endothelial dysfunction and oxidative stress. Promotion of eNOS coupling may be beneficial to maintain normal vascular function.Grant Funding Source: Supported by Center for Chronic Disorders of Aging and Department of Bio‐Medical Sciences at PCOM

Simultaneous, real-time measurement of nitric oxide and oxygen dynamics during cardiac ischemia–reperfusion of the rat utilizing sol–gel-derived electrochemical microsensors

In this study, we simultaneously measured nitric oxide (NO) and oxygen (O2) dynamics in the myocardium during myocardial ischemia–reperfusion (IR) utilizing sol–gel modified electrochemical NO and O2 microsensors. In addition, we attempted to clarify the correlation between NO release in the ischemic period and O2 restoration in the myocardium after reperfusion, comparing a control heart with a remote ischemic preconditioning (RIPC)-treated heart as an attractive strategy for myocardial protection. Rat hearts were randomly divided into two groups: a control group (n=5) and an RIPC group (n=5, with RIPC treatment). Myocardia that underwent RIPC treatment (182±70nM, p<0.05) released more NO during the ischemic period than those of the control group (63±41nM). The restoration value of oxygen tension (pO2) in the RIPC group significantly increased and was restored to pre-ischemic levels (92.6±36.8%); however, the pO2 of the control group did not increase throughout the reperfusion period (5.7±7.5%, p=0.001). Myocardial infarct size measurements revealed a significant decrease in cell death in the myocardium region of the RIPC group (41.44±6.42%, p=0.001) compared with the control group (60.05±10.91%). As a result, we showed that the cardioprotective effect of RIPC could be attributed to endogenous NO production during the ischemic period, which subsequently promoted reoxygenation in post-ischemic myocardia during early reperfusion. Our results suggest that the promotion of endogenous formation during an ischemic episode might be helpful as a therapeutic strategy for protecting the myocardium from IR injury. Additionally, our NO and O2 perm-selective microsensors could be utilized to evaluate the effect of drug or treatment.

Measurement of Nitric Oxide and Reactive Oxygen Species in the Vascular Wall

Vascular generation of nitric oxide (NO) plays an important role in the regulation of blood flow, and is counterbalanced by the formation of radical oxygen species (ROS). Thus, an imbalance in vascular NO and ROS production contributes to endothelial dysfunction, which is associated with cardiovascular disease. Here we review the main and commonly used methods that have been applied to determine vascular NO and ROS. Only NO microsensors, electron spin resonance (ESR), and NO sensitive fluorescent probes allow real time measurement of NO levels in cell suspensions, cell cultures, and isolated vascular segments in vitro, and of these techniques only NO microsensors have so far been developed for real time detection of NO in vivo. ROS formation has been detected in cell cultures by lucigenin- enhanced fluorescence, fluorescent probes e.g. 2',7'-dichlorodihydrofluorescein and dihydroethidium, by use of ESR, and also by electrochemical detection. The limitations of the electrochemical microsensors include interference from other substances, physical force on the sensors, and influence of temperature and changes in pH that may lead to artifactual changes in current. Therefore, specificity testing and calibration are pivotal, and in case of ROS, application of more than one technique is recommendable. Technical advances have allowed simultaneous detection of both NO and superoxide anion by use of electrochemical microsensors. Miniaturizing the sensors may also allow incorporation of those in lab-on- a-chip and may lead to real time measurements of NO and ROS in the coronary circulation in situ, and hence provide direct evaluation of endothelial and vascular function in cardiovascular disease.

Tetrahydrobiopterin (BH4) attenuates extracorporeal shock wave lithrotripsy (ESWL) induced blood nitric oxide (NO) level reduction in the renal vein

ESWL, a therapy for kidney stone ablation clinically, may lead to hypertension. This may be due to kidney vascular endothelial dysfunction, which is characterized as increased oxidative stress and decreased endothelial NO synthase (eNOS)‐derived NO bioavailability. We hypothesized that ESWL would decrease NO in rat renal veins. By contrast, rats given BH4, the essential cofactor of eNOS, would attenuate ESWL‐induced decrease in NO. Blood NO was directly measured in real‐real time by inserting a NO microsensor (100μm diameter) into the left renal vein in the anesthetized rat. ESWL treatment consisted of 1,000 shocks with a 16kV shock wave for approximately 13 minutes. Saline or BH4 was injected via the jugular vein immediately post‐ ESWL and at the same time point for the non‐ESWL control rats. Our preliminary data showed that saline ESWL‐treated rats (n=3) reduced blood NO by 107–203 nM, which is significantly reduced compared to non‐ESWL controls (n=6, p&lt; 0.05) during the 30 min post‐ESWL. By contrast, blood NO level in rats given BH4 under ESWL conditions (6.5 mg/kg, n=4, p&lt;0.05) only decreased by 15–19 nM post‐ESWL compared to saline ESWL. This data supports that ESWL treatment decreases NO bioavailability, which may be due to decreased BH4 content. Future studies are aimed at evaluating blood oxidative stress levels (i.e. hydrogen peroxide) when given ESWL or ESWL+BH4.

Real time dynamics of nitric oxide during cardiac ischemia-reperfusion of the rat

Nitric oxide (NO) plays important roles in the cardiovascular system by mediating various physiological and pathophysiological processes. However, the role of endogenous NO in ischemia-reperfusion is still controversial. Direct and real-time monitoring of NO is difficult because NO is present only at low concentrations in the body and has a short half-life. Therefore, we utilized a sol–gel derived electrochemical NO microsensor to monitor changes in NO during myocardial ischemia-reperfusion. We also analyzed differences in NO dynamics between intact and hypothermic hearts to investigate the role of endogenous NO formation in the myocardium. A NO sensor was built using a xerogel-modified platinum microsensor and coiled Ag/AgCl reference electrode. Rat hearts were randomly divided into two groups: intact ( n = 6, without hypothermic treatment after harvest) and hypothermic ( n = 6, with hypothermic treatment after harvest) groups. Hearts were placed on a Langendorff apparatus and treated by 40 min of ischemia and 30 min of reperfusion. In the ischemic myocardium, NO showed a time-course dependent change during the 40 min ischemic episode. The restoration concentration of NO was decreased below the pre-ischemic level after myocardial ischemia and early reperfusion. However, myocardium that underwent hypothermic treatment (151 ± 37 nM) released more NO during the ischemic period than that without any treatment (59 ± 15 nM). In addition, the restoration concentration of NO in the hypothermic group (−57 ± 26 nM) was significantly higher than that of the intact group (−170 ± 50 nM, p < 0.05). Endogenous NO formation was observed during ischemic episodes, which is a reaction against ischemia. We inferred that hypothermic treatment for cardioprotection would activate endogenous NO production in the ischemic myocardium. Therefore, promotion of endogenous formation and inhibition of the time course alteration of NO during ischemic episodes might be a helpful therapeutic strategy for protecting the myocardium from ischemic injury.

Opening of Small and Intermediate Calcium-Activated Potassium Channels Induces Relaxation Mainly Mediated by Nitric-Oxide Release in Large Arteries and Endothelium-Derived Hyperpolarizing Factor in Small Arteries from Rat

This study was designed to investigate whether calcium-activated potassium channels of small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SK(Ca) and IK(Ca) channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 μm) and small mesenteric (∼300 μm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both N(G),N(G)-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SK(Ca) and IK(Ca) channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IK(Ca) channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SK(Ca) and IK(Ca) channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SK(Ca) and IK(Ca) channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Simple Fabrication of Amperometric Nitric Oxide Microsensors Based on Electropolymerized Membrane Films

AbstractDemand for highly sensitive, selective, and practically reliable sensors which could be easily fabricated is increasing for various applications in biological and biomedical systems. Thus, here we present a novel and simple amperometric NO microsensor based on electropolymerized polymeric films. The sensor consists of a platinized Pt disk anode (25‐μm diameter) which surface is modified with electropolymerized polymer films and a Ag/AgCl wire cathode coiled around the anode. Three different electropolymerized films prepared from m‐phenylenediamine (m‐PD), 2,3‐diaminonaphthalene (2,3‐DAN), and 5‐amino‐1‐naphthol (5A1N) are compared in terms of their permselectivity for NO over major biological interferents such as anionic nitrite, ascorbic acid, uric acid; neutral acetaminophen; and cationic dopamine. Poly‐5A1N film layer among the three different polymers shows the best anti‐interference characteristics for all the electroactive interferents examined. Indeed, single polymer film of electropolymerized 5A1N without any additional modification as a NO selective membrane is confirmed to be sufficient to reject anionic, neutral, as well as cationic interferents while allowing relatively high permeation of NO through it. Other analytical performance of the NO microsensor fabricated with poly‐5A1N is evaluated: reliable linear dynamic range (a few tens nM to μM); sensitivity of 122.0±2.5 pA/μM; detection limit of &lt;5.8 nM (S/N=3); response time, t90%&lt;5 s, which are excellent when considering the small sensor size. Another sensor design which has both an anode (poly‐5A1N modified platinized Pt) and a cathode (Ag/AgCl disk) embedded in a single sensor body is also presented.

Amperometric Nitric Oxide Microsensor Based on Nanopore-Platinized Platinum: The Application for Imaging NO Concentrations

This paper reports an amperometric nitric oxide (NO) microsensor based on a cone-shaped nanopore-platinized Pt working electrode. The senor was fabricated using the following procedure: (1) a parent nanodisk electrode was prepared by polishing an etched Pt wire (radius = 12.5 microm; dimension of etched tip end point <10 nm) embedded in a glass capillary, (2) the nanodisk Pt was further etched to produce a nanopore (pore opening radius <1 microm; pore depth approximately 30 microm), (3) the Pt base surface in the nanopore electrode was platinized electrochemically to improve the sensor sensitivity, and (4) silanization and further modification with the electropolymerized polymeric film [poly(5-amino-1-naphthol)] on the nanopore-platinized Pt electrode were carried out to obtain the sensor selectivity to NO. The analytical performance of the sensor was characterized. For example, a sensor with a pore opening radius of 797 nm exhibited a decent linear dynamic range (at least for 0.2-1.8 microM), detection limit of < approximately 32 nM, response time (t(90%)) of < approximately 5 s, and sensitivity of 6.5 +/- 0.02 pA/nM. This sensor was used successfully as a NO-selective probe tip in scanning electrochemical microscopy (SECM) to obtain a two-dimensional image of the local NO concentrations for an inlaid NO-emitting microdisk film (radius = 12.5 microm) on a glass substrate.

Fluorinated xerogel-derived microelectrodes for amperometric nitric oxide sensing.

An amperometric fluorinated xerogel-derived nitric oxide (NO) microelectrode is described. A range of fluorine-modified xerogel polymers were synthesized via the cohydrolysis and condensation of alkylalkoxy- and fluoroalkoxysilanes. Such polymers were evaluated as NO sensor membranes to identify the optimum composition for maximizing NO permeability while providing sufficient selectivity for NO in the presence of common interfering species. By taking advantage of both the versatility of sol-gel chemistry and the "poly(tetrafluoroethylene)-like" high NO permselective properties of the xerogels, the performance of the fluorinated xerogel-derived sensors was excellent, surpassing all miniaturized NO sensors reported to date. In contrast to previous electrochemical NO sensor designs, xerogel-based NO microsensors were fabricated using a simple, reliable dip-coating procedure. An optimal permselective membrane was achieved by synthesizing xerogels of methyltrimethoxysilane (MTMOS) and 20% (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilane (17FTMS, balance MTMOS) under acid-catalyzed conditions. The resulting NO microelectrode had a conical tip of approximately 20 microm in diameter and approximately 55 microm in length and exhibited sensitivities of 7.91 pA x nM (-1) from 0.2 to 3.0 nM (R (2) = 0.9947) and 7.60 nA x microM (-1) from 0.5 to 4.0 microM ( R (2) = 0.9999), detection limit of 83 pM (S/ N = 3), response time ( t 95%) of <3 s, and selectivity (log K NO, j (amp)) of -5.74, <-6, <-6, <-6, <-6, -5.84, and -1.33 for j = nitrite, ascorbic acid, uric acid, acetaminophen, dopamine, ammonia/ammonium, and carbon monoxide. In addition, the sensor proved functional up to 20 d, maintaining >or=90% of the sensor's initial sensitivity without serious deterioration in selectivity.

A sensitive nitric oxide microsensor based on PBPB composite film-modified carbon fiber microelectrode

Abstract In this work, a sensitive electrochemical microsensor of nitric oxide (NO) was reported. The microsensor was constructed by coating PBPB composite on carbon fiber microelectrodes (CFME). The NO microsensor displayed excellent electrochemical activity toward the oxidation of NO and have the virtue of good stability, reproducibility and high sensitivity. Under optimal working conditions, the oxidation current of NO at this microsensor exhibited a good linear relationship with NO concentration in the range of 3.6 × 10 −8 to 8.9 × 10 −5 mol/L with a low detection limit of 3.6 × 10 −9 mol/L (S/N = 3). The microsensor was successfully applied to the direct and real-time detection of NO release from biological samples, foreseeing the promising applications of this microsensor in fields like biology and medicine.

Selective Arterial Dilatation by Glyceryl Trinitrate Is Not Associated with Nitric Oxide Formation in vitro

Aims: Glyceryl trinitrate (GTN) is the most commonly used anti-anginal agent, yet its mechanism of action has still to be fully established. Release of nitric oxide (NO) and the selectivity of GTN in the venous system are believed to be crucial to this drug’s anti-anginal action. Methods: Rat superior mesenteric arteries and renal veins were mounted in a wire myograph with an intraluminal NO microsensor. Results: In the superior mesenteric arteries, GTN (1 nM to 10 µM) produced a dose-dependent vasodilatation without NO release, except at concentrations supramaximal for relaxation. GTN was found to be markedly less potent in a wide range of veins tested, and lowering the oxygen concentrations in the myograph to that of the venous system did not improve the venodilator activity of GTN. Conclusion: This is the first time that NO release from GTN has been monitored electrochemically in real time, simultaneously with vasodilatation. Unlike the endothelium-dependent vasodilator carbachol, NO could only be measured at concentrations of GTN that are supramaximal for relaxation. GTN was found to be arterioselective in vitro, even when oxygen levels were lowered to mimic those of the venous system in vivo.

Tonic and stimulus-evoked nitric oxide production in the mouse olfactory bulb

Nitric oxide (NO) has been long assumed to play a key role in mammalian olfaction. This was based largely on circumstantial evidence, i.e. prominent staining for nitric oxide synthase (NOS) and cyclic guanosine 3′,5′-cyclic monophosphate (cGMP) or soluble guanylyl cyclase, an effector enzyme activated by NO, in local interneurons of the olfactory bulb. Here we employ innovative custom-fabricated NO micro-sensors to obtain the first direct, time-resolved measurements of NO signaling in the olfactory bulb. In 400 μm thick mouse olfactory bulb slices, we detected a steady average basal level of 87 nM NO in the extracellular space of mitral or granule cell layers. This NO ‘tone’ was sensitive to NOS substrate manipulation (200 μM l-arginine, 2 mM N G-nitro- l-arginine methyl ester) and Mg 2+ modulation of N-methyl-D-aspartate (NMDA) receptor conductance. Electrical stimulation of olfactory nerve fibers evoked transient (peak at 10 s) increments in NO levels 90–100 nM above baseline. In the anesthetized mouse, NO micro-sensors inserted into the granule cell layer detected NO transients averaging 55 nM in amplitude and peaking at 3.4 s after onset of a 5 s odorant stimulation. These findings suggest dual roles for NO signaling in the olfactory bulb: tonic inhibitory control of principal neurons, and regulation of circuit dynamics during odor information processing.

Effects of Alcohol on Nitric Oxide (NO) Synthesis and Superoxide Production in Human Brain Vascular Cells

Oxidative stress may play an important role in impaired endothelial NO synthase (eNOS)‐dependent dilation of cerebral arterioles during chronic alcohol consumption. The purpose of this study was to examine the effects of acute (3 hours) and chronic (7 days) alcohol on NO synthesis and superoxide production in cultured human brain vascular cells. Primary human brain microvascular endothelial cells (HBMEC) and human brain vascular smooth muscle cells (HBVSMC) were exposed to alcohol (5 and 50 mM) for 3 hours or 7 days. NO synthesis of HBMEC in response to L‐arginine and acetylcholine was measured by a NO microsensor. Superoxide production from HBMEC and HBVSMC was measured using lucigenin‐enhanced chemiluminescence. In addition, protein expression of eNOS in HBMEC was measured by Western blot analysis. Acute and chronic exposure of low dose alcohol did not alter NO synthesis and superoxide production. Acute exposure of high dose alcohol increased NO production in response to L‐arginine, but not acetylcholine. In contrast, chronic exposure of high dose alcohol significantly reduced NO production in response to L‐arginine and acetylcholine. In addition, chronic exposure of high dose alcohol significantly enhanced superoxide production in HBVSMC as well as in HBMEC. Furthermore, both acute and chronic exposure of high dose alcohol increased eNOS protein expression. Our findings suggest that chronic exposure of alcohol may reduce NO bioavailability via enhanced superoxide production in human brain vascular cells.