The Principles, Development and Application of Microelectrodes for the In Vivo Determination of Nitric Oxide

Nitric oxide (NO) is a hydrophobic, highly labile free radical that is catalytically produced in biological systems from the reduction of l-arginine by nitric oxide synthase (NOS) to form l-citrulline, which produces NO in the process. In biological systems NO has long been known to play various roles in physiology, pathology and pharmacology [1]. In 1987, NO was identified as being responsible for the physiological actions of endothelium-derived relaxing factor (EDRF) [2]. Since that discovery, NO has been shown to be involved in numerous biological processes such as: vasodilatation and molecular messaging [2], penile erection [3], neurotransmission [4,5], inhibition of platelet aggregation [6], blood pressure regulation [7], immune response [8], and as a mediator in a wide range of both anti-tumor and anti-microbial activities [9,10]. In addition, NO has been implicated in a number of diseases including: diabetes [11], Parkinson’s and Alzheimer’s diseases [12]. The importance of NO was confirmed in 1992 when declared NO the “Molecule of the Year” and in 1998, when F. Furchgott, Louis J. Ignarro, and Ferid Murad were awarded the Nobel Prize in Physiology and Medicine for unraveling the complex nature of this simple molecule. Despite the obvious importance of NO in so many biological processes, less than 10% of the thousands of scientific publications over the last decade dedicated to the field of NO research involved its direct measurement. As stated above, NO plays a significant role in a variety of biological processes where its spatial and temporal concentration is of extreme importance. However, the measurement of NO is quite difficult due to its short half-life (~ 5 s) and high reactivity with other biological components such as: superoxide, oxygen, thiols and others. To date, several techniques have been developed for the measurement of NO including: chemiluminescence [13,14], Griess method [15], paramagnetic resonance spectrometry [16], paramagnetic resonance imaging, spectrophotometry [17], and bioassay [18]. Each of these techniques has certain benefits associated with it, but suffer from poor sensitivity and the need for complex and often expensive experimental apparatus. In addition, the above NO sensing techniques are limited when it comes to continuous monitoring of NO concentration in real-time and most importantly in vivo. To date, electrochemical (amperometric) detection of NO is the only available technique sensitive enough to detect relevant concentrations of NO in real-time and in vivo, and suffers minimally from potential interfering species such as: nitrite, nitrate, dopamine, ascorbate, and l-arginine. Also, because electrodes can be made on the micro and nano-scale, these techniques also have the benefit of being able to measure NO concentrations in living systems without any significant effects from electrode insertion. The first amperometric NO electrode used for direct measurement was described in 1990 [19]. In 1992, World Precision Instruments Inc. (WPI) developed the first commercial NO sensor system called the ISO-NO. Over subsequent years a range of highly specialized and sensitive NO electrodes have been developed offering detection limits for NO ranging from below 1 nM up to 100 μM [20]. Most recently, a unique range of high sensitivity NO sensors based on a membrane coated activated carbon microelectrode, with diameters ranging from 200 μm down to 100 nm, have been developed by this lab. These electrodes exhibit superior performance during NO measurement and feature a detection limit of less than 0.5 nM NO.