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The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2(-)) in cell lysates. There was a 2.5- to 4-fold increase in NO2(-) production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2(-) inside a single unstimulated macrophage cell was estimated to be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.

A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.

Microchip electrophoresis (ME) with electrochemical detection was used to monitor nitric oxide (NO) production from diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and 1-(hydroxyl-NNO-azoxy)-L-proline disodium salt (PROLI/NO). NO was generated through acid hydrolysis of these NONOate salts. The products of acid hydrolysis were introduced into a 5-cm separation channel using gated injection. The separation was accomplished using reverse polarity and a background electrolyte consisting of 10 mM boric acid and 2 mM tetradecyltrimethylammonium bromide, pH 11. Electrochemical detection was performed using an isolated potentiostat in an in-channel configuration. Potentials applied to the working electrode, typically higher than +1.0 V vs. Ag/AgCl, allowed the direct detection of nitrite, NO, DEA/NO, and PROLI/NO. Baseline resolution was achieved for the separation of PROLI/NO and NO while resolution between DEA/NO and NO was poor (1.0 ± 0.2). Nitrite was present in all samples tested.