Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers

Abstract

Summary 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 estimatedto 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.

Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence

“…Additional cell lines can also be studied. This method complements our previously reported methods for nitric oxide detection using fluorescence and electrochemical detection [27, 28] as it allows an effective way to study a second reactive species, superoxide, using ME-LIF. The ultimate goal is to develop microanalytical methods that are capable of simultaneously monitoring the production of nitric oxide, superoxide, and peroxynitrite to better evaluate oxidative damage in different biological conditions at bulk and single cell levels.…”

“…To estimate the 2-OH-MitoE + concentration in a single cell, 2-OH-MitoE + concentration for each electropherogram was calculated based on four parameters: the external calibration curve (Online Resource 1), the volume of cell lysis buffer, the estimated volume of an individual macrophage cell (0.05 pL) [28], and the number of cells present in each cell culture flask. Therefore, each SIN-1 incubated cell produced, on average, 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE + , while native cells produced 0.23 ± 0.03 fmol (0.45 ± 0.05 mM) of 2-OH-MitoE + (both treated with DDC).…”

“…Our group has previously reported the separation of other reactive nitrogen and oxygen species, including nitric oxide and nitrite, using ME with fluorescence or electrochemical detection [27, 28]. Since superoxide is difficult to detect electrochemically, the ability to integrate multiple detection platforms into a single ME device could therefore allow for the simultaneous detection of nitric oxide, superoxide, and peroxynitrite.…”

Microchip electrophoresis with electrochemical detection for the determination of analytes in the dopamine metabolic pathway

“…). This electrode alignment has been employed previously in our lab and yields higher efficiency separations than other detection configurations with minimal interference between the separation and detection voltages . All data were analyzed using Origin 8.6 software (OriginLab, Northhampton, MA, USA) after baseline subtraction.…”

Multianalyte Physiological Microanalytical Devices

“…However, noise reduction techniques, such as in-channel detection and an electrically isolated potentiostat, can be implemented (39). ME with amperometric detection (Figure 2d ) has been used to detect redox active biomolecules, interfering species that are electroactive at the same potentials as target molecules (39)(40)(41)(42)(43)(44)(45)(46)(47), and to detect glucose by using a glucose oxidase-modified working electrode (41). Methods have been optimized to observe NO production in macrophages stimulated with lipopolysaccharide by analyzing NO 2 − ions (42) and dopamine (DA) metabolites from rat brain slices (46).…”