Transgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb+) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb-) experienced 30-70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb+ lines did not significantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb- lines were significantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb- line was 2.5-fold higher than that in the Hb+ line. Treatment of transgenic root lines under 40% oxygen with NO resulted in significant declines in the ATP levels and ATP/ADP ratio of an Hb- line and the control line, with no significant change in an Hb+ line. The root cell structure of an Hb- line showed evidence of cell breakdown under hypoxic growth, whereas an Hb+ line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.

Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress.

Methods for the detection and determination of nitrite and nitrate: A review.

Reactive oxygen species and reactive nitrogen species are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity. Because of their ephemeral nature and rapid reactivity, these species are difficult to measure directly with high accuracy and precision. In this statement, we review current methods for measuring these species and the secondary products they generate and suggest approaches for measuring redox status, oxidative stress, and the production of individual reactive oxygen and nitrogen species. We discuss the strengths and limitations of different methods and the relative specificity and suitability of these methods for measuring the concentrations of reactive oxygen and reactive nitrogen species in cells, tissues, and biological fluids. We provide specific guidelines, through expert opinion, for choosing reliable and reproducible assays for different experimental and clinical situations. These guidelines are intended to help investigators and clinical researchers avoid experimental error and ensure high-quality measurements of these important biological species.

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association

Nitric oxide (NO) is the focus of intense research primarily because of its wide-ranging biological and physiological actions. To understand its origin, activity, and regulation, accurate and precise measurement techniques are needed. Unfortunately, analytical assays for monitoring NO are challenged by NO's unique chemical and physical properties, including its reactivity, rapid diffusion, and short half-life. Moreover, NO concentrations may span the picomolar-to-micromolar range in physiological milieus, requiring techniques with wide dynamic response ranges. Despite such challenges, many analytical techniques have emerged for the detection of NO. Herein, we review the most common spectroscopic and electrochemical methods, with a focus on the underlying mechanism of each technique and on approaches that have been coupled with modern analytical measurement tools to create novel NO sensors.

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Analytical Chemistry of Nitric Oxide

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318–1320, 1; Martinez et al., Anal Chem 10:3699–3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606–19611, 3; Lu et al., Electrophoresis 9:1497–1500, 4; Abe et al., Anal Chem 18:6928–6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 μM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 μM) were generated. The time of device fabrication to the time of test results was about 25 min.

Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva

A review of using Griess Reaction method to determine nitric oxide production in biological systems.

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Measurement of Nitric Oxide Production in Biological Systems by Using Griess Reaction Assay

In vivo measurement of nitric oxide (NO) in a biological matrix is very difficult because of its assumed low stability and fugacity, in addition to the complexity of such matrix, limited space and volume of biological samples. Among different NO detection strategies, electrochemical NO sensors are still widely used by NO researchers. Though many kinds of NO sensors are commercial available from World Precision Instruments, Inc. and other companies, the small NO sensors still are needed for the NO detection, especially in single cell levels. In this article a NO-selective ultramicrosensor was developed as an easily applicable tool for real time nitric oxide (NO) detection. The sensor consists of a 7 µm carbon fiber working electrode coated with cation exchanger (Nafion), then covered with NO-selective gas permeable polymeric membranes, and Ag/AgCl micro-reference/counter electrode. Compared with other reported NO sensors, the sensor described herein offers several advantages: i) high selectivity against ascorbate (>104:1), dopamine (>103:1) and nitrite (104:1); ii) detection limit to low nanomolar concentration; iii) rapid, inexpensive and reproducible fabrication; iv) wide linear calibration range from 10 nM to 5 µM with R2=0.995; v) integrated ultramicrosensor eliminating the need of an external reference electrode, accordingly, experiments in small volume are possible with an integrated ultramicrosensor, even at single cell levels.

An Integrated Nitric Oxide Sensor Based on Carbon Fiber Coated with Selective Membranes

Calibration of nitric oxide (NO) sensor is a key step to measurement of NO. Currently there are three methods to calibrate electrochemical NO sensors. However, there are many problems associated with these techniques, especially for calibration of microsensors. This article describes a novel method for calibration of NO microsensors, which has the advantage of being simple, reproducible, wide range and suitable for all kind of NO electrochemical sensors, especially for the commercial available integrated NO microsensors such as ISO-NOP30. This method is based on quantitative release of NO from decomposition of SNAP trigged by Cu+. Using of monovalant copper, as described in the present study, represents a major methodological advancement in the quantitative release of NO from SNAP. The kinetic decomposition of SNAP, effect of Cu+ concentration, pH of the solution, temperature and oxygen concentration in the solution on the release of NO are discussed. Compared with other calibration methods, this method is simple, convenient, reliable, accurate.

Novel Calibration Method for Nitric Oxide Microsensors by Stoichiometrical Generation of Nitric Oxide from SNAP

Nanometer size electrode for nitric oxide and S-nitrosothiols measurement

Since the discovery of nitric oxide (NO) as a vasodilatory messenger, in particular after its identification as an endothelial-derived relaxing factor (EDRF), there has been a mushroom effect in the research of NO in biological systems. Monitoring of NO in biological samples in vivo and in real time is desirable in many fields of NO research. Although several techniques are available for measurement of NO, the electrochemical method is most advantageous because of its speed and sensitivity. Since the first commercially available electrochemical NO detection system, many NO electrodes have been developed with dimensions from μm to cm, and with detection limits in the low nanomolar range. However, there is still a continuing demand for new NO sensors with lower detection limits. An electrochemical sensor to detect nitric oxide gas dissolved in solution as well as in gas phase is described as well as a fabrication method for the electrode. The sensor is based on the selective oxidation of nitric oxide by a multielectrode array of microelectrodes created on activated carbon, which has been deposited on a silicon chip substrate. The array, in turn, is further modified with several layers of cationic ion exchanger then the subsequent addition of NO selective membranes. The microchip NO sensor, is characterized by a linear response to concentrations of NO up to 100 μM, a response time of a few seconds, and a detection limit of less than 0.3 nM. In biological samples the sensor discriminates against substances such as nitrite, dopamine and ascorbic acid. Moreover, compared with present NO sensors, this sensor is significantly less temperature sensitive, resulting in significantly improved sensor detectivity as compared with present electrochemical sensors. Major applications for measurement of NO concentration are in chemical media, biological tissue, cell cultures, or in blood.

Mark Broderick

Papers

Measurement of Nitric Oxide Production in Biological Systems by Using Griess Reaction Assay

An Integrated Nitric Oxide Sensor Based on Carbon Fiber Coated with Selective Membranes

Novel Calibration Method for Nitric Oxide Microsensors by Stoichiometrical Generation of Nitric Oxide from SNAP

Nanometer size electrode for nitric oxide and S-nitrosothiols measurement

A Novel Microchip Nitric Oxide Sensor with sub‐nM Detection Limit