Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection

Abstract

Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively.

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

“…36 This method was limited to the determination of NO and could not be used to detect any other RNOS related species. NO can also be detected by amperometric detection, and we also recently reported a ME-EC method for the detection of NO and NO 2 - produced by NONOate salts.…”

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

“…The fabrication of the PDMS/glass microfluidic has been previously described by our group [27]. First, a SU-8 10 photoresist (Silicon, Inc., Boise, ID, USA) mold containing the microdevice design was fabricated using soft photolithography.…”

“…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.…”

“…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. Moreover, microfluidic devices are also the most suitable platform to perform single cell analysis because it allows for on-line integration of cell sample preparation and analysis [27]. …”

Development and evaluation of a calibration free exhaustive coulometric detection system for remote sensing.

“…DNA analysis in particular has benefitted from adapting this technique in miniaturized microfluidic devices [41]. More recently, Mainz et al used a LIF detection technique to determine the concentration of nitrous oxide (NO) on a microfluidic platform [42]. This study quantified NO production in control and externally stimulated Jurkat cells, whose subsequently lysed cell contents were sent through the microchip capillary electrophoresis device.…”