Novel Insights into the Electrochemical Detection of Nitric Oxide in Biological Systems ( nitric oxide / electrochemical detector / biological systems / carbon fibre )

In recent years, microsensor technologies have made a rapid expansion into different fields of physical sciences, engineering, and biomedicine. For analyses of various biomolecules, novel sensors and detection platforms in the electrochemical field have been reported recently. The most important applications based on microelectromechanical systems dramatically reduce the need of manipulation steps with samples, while improving data quality and quantitative capabilities. This is also the case of a special class of electrochemical sensors that allow direct, real-time and non-invasive measurements of nitric oxide, whose determination is crucial for the purposes of basic research, as well as of preclinical and clinical studies. Therefore, this minireview will focus on the description of recent discoveries in the electrochemical determination of nitric oxide, released in different in vitro systems. Nitric oxide as an important regulatory molecule in the organism Among the molecules that are constantly in the focus of scientific interest is nitric oxide (NO), whose biological and clinical importance lies in its fundamental role in the regulation of vascular and immune functions (Bogdan 2001; Fostermann et al., 2006; Lundberg et al., Received June 27, 2014. Accepted July 21, 2014. This work was supported by the project of the Ministry of Education, Youth and Sports of the Czech Republic (CZ.1.07/2.3.00/ 30.0030) and by the Czech Science Foundation (13-40882P). Corresponding author: Michaela Pekarová, Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Královopolská 135, 612 65 Brno, Czech Republic. E-mail: pekarovam@ibp.cz Abbreviations: ACh – acetylcholine, CaI – calcium ionophore, eNOS – endothelial NOS, HUVEC – human umbilical vein endothelial cell, iNOS – inducible NOS, LPS – lipopolysaccharide, nNOS – neuronal NOS, NO – nitric oxide, NOS – nitric oxide synthase, ROS – reactive oxygen species. 2008; Pacher et al., 2007). Importantly, serious human diseases, e.g. systemic and pulmonary hypertension, hypercholesterolaemia, diabetes, and heart failure are accompanied by significant reduction of NO bioavailability in the organism, which probably results from the multifactorial alteration of immune and vascular endothelial functions. NO plays an important role in many diverse proces ses, including vasodilatation, immune responses, neurotransmission, and adhesion of platelets and leucocytes to the endothelium (Bogdan 2001; Fostermann et al., 2006). It is synthesized by the enzyme nitric oxide synthase (NOS). There are three different types of NOS: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Endothelial and neuronal NOS are constitutively expressed at low levels in a variety of cell types and their activities are dynamically regulated by Ca2+ and calmodulin. On the other hand, iNOS is highly inducible in macrophages by bacterial endotoxin and/or Th1 cytokines (e.g. interferon γ), but once expressed it is constitutively active (Mayer and Hemmens, 1997; Bogdan 2001; Krejcova et al., 2009). As it was mentioned above, down-regulation of NO-dependent processes occurs early in the course of human vascular disease and it is a contributing factor to defective endothelial vasodilator function. Generally, it is accepted that the majority of functional defects of vascular endothelium are associated with impaired eNOS and iNOS activity, which is followed by reduced total NO generation. This reduction of NO formation is associated with increased production of circulating levels of reactive oxygen species (ROS) and impaired endothelial uptake of L-arginine (the only known substrate for NO production) (Mayer and Hemmens, 1997). It is believed that these events could represent the step in the process of endothelial dysfunction in small vessels that evolves through large vessel disease to organ dysfunction and premature cardiovascular morbidity and mortality (Bogdan, 2001; Fostermann et al., 2006; Mayer and Hemmens, 1997; Pacher et al., 2007). Therefore, in order to evaluate the cellular function and bioavailability of NO, direct measurements of its concentration