Xiaoying Ye, Stanislav Rubakhin and Jonathan Sweedler of the University of Illinois, Urbana, US, describe fluorescent separation and electrochemical methods for detecting a crucial, but tiny, biomolecule
First described in the 1980s, the cell-signalling molecule nitric oxide (NO) has garnered tremendous interest in the scientific community. Investigations have revealed a wide variety of biological functions for NO, which regulates the immune, cardiovascular and central nervous systems. Pharmaceutical treatments, such as Viagra for impotence and BiDil for heart disease, quickly followed these discoveries.
NO, in contrast to most physiologically-active biomolecules, is surprisingly small. Interestingly, most cells are affected by NO, but only a few actually produce it. Nonetheless, the endogenous generation of this simple molecule is complex. Organisms have evolved an elaborate biochemical system that employs several variants of nitric oxide synthase and six cofactors - Ca2+, calmodulin, NADPH, FAD, FMN, and tetrahydrobiopterin. Furthermore, other enzymes, such as argininosuccinate synthase and lyase, are required to complete the NO cycle. This sophisticated system exerts precise control over NO production: the right amount at the right time and in the correct location is pivotal for NO function. Reliable measurements of NO production and the levels of NO-related metabolites are key to understanding NO function in normal and diseased states, and important in both research and clinical settings.
Tissue homogenates are often used in functional assays. Unfortunately, results represent an average from many cells and can therefore be misleading, especially for inherently heterogeneous systems such as the brain. The low abundance of cells that release NO and their scattered distribution make single cell analysis important. Why? Because bulk assays of whole tissues or pooled samples may not accurately reflect the presence of NO and particularly, NO cycle metabolite levels. Furthermore, NO is generated at nanomolar to low micromolar levels and subject to relatively fast deactivation. These factors combine to present significant analytical challenges.
"Fluorescence-based detection, combines high sensitivity with high spatial resolution"
There are a variety of methods for single cell NO measurement. One option, fluorescence-based detection, combines high sensitivity with high spatial resolution. Since NO does not luminesce significantly under typical experimental conditions, a variety of fluorescence indicators have been introduced that interact with NO in a fast, sensitive and selective manner. Advances in nanofabrication and NO-binding compounds, including fluorescent dyes, now allow the creation of biologically inert nanostructures that can be protected from inactivation and targeted to specific cells. Alternatively, proteins that interact with NO can be genetically encoded in cells to become a new class of NO indicator. One example, Piccell, exhibits exceptional sensitivity and real-time monitoring of changes in NO levels in vivo
Although fluorescence indicators offer impressive performance, absolute quantitation and the potential effects of the probes themselves on NO levels must also be considered. Electrochemical detection, especially useful for extracellular monitoring, provides another strategy for single cell NO measurement. NO is a highly reactive molecule and can be rapidly oxidised under in vivo conditions. NO-selective electrodes provide fast, quantitative measurement of small fluctuations in NO concentration; they offer significant advantages when monitoring local concentrations of NO.
"NO-selective electrodes offer significant advantages when monitoring local concentrations of NO"
Another concern during NO measurement is that the complex and dynamic cellular environment can interfere with the measurement process, possibly leading to false-positive or -negative responses. Capillary-scale separations of the intracellular or extracellular environment provide chemically-rich information on NO cofactors and catabolites. Thus, by coupling capillary electrophoresis separations with laser induced fluorescence electrochemical detection, NO measurements from single identified neurons have been accomplished.
The spectrum of tools available for probing NO at the single cell level has contributed significantly toward revealing the secrets of this surprisingly enigmatic, cell-cell signaling molecule.
Read more in Jonathan Sweedler et al's critical review 'Detection of nitric oxide in single cells' in issue 4 of The Analyst.