Separation and Detection of Peroxynitrite Using Microchip Electrophoresis with Amperometric Detection

Hulvey, Matthew K.; Frankenfeld, Celeste; Lunte, Susan M.

doi:10.1021/ac902821v

Cited by 66 publications

(45 citation statements)

References 26 publications

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“…ME is a technique that is able to generate very fast, highly efficient separations in a small and potentially portable format [3-6]. The combination of amperometric detection with ME provides a powerful approach to the determination of a variety of biologically important compounds including reactive oxygen species (ROS) [7], reactive nitrogen species (RNS) [8], catecholamines [9, 10], thiols [11, 12], and carbohydrates [13-15]. This technique has been used in a variety of applications including biomedical [16], agricultural [17], forensics [18, 19], and environmental analyses [20].…”

Section: Introductionmentioning

confidence: 99%

In‐channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

2011

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The combination of microchip electrophoresis (ME) with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of microchip electrophoresis and electrochemical detection (ME-EC) with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (TTAB, as a buffer modifier) for the separation of nitrite (NO2-), glutathione (GSH), ascorbic acid (AA), and tyrosine (Tyr). A half-wave potential (E½) shift of approximately negative 500 mV was observed for NO2- and H2O2 standards in the in-channel configuration compared to end channel. Higher separation efficiencies were observed for both NO2- and H2O2 with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.

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Section: Introductionmentioning

confidence: 99%

In‐channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

2011

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“…SIN-1 decomposition to SIN-1C is triggered by the addition of NaOH, producing both superoxide and nitric oxide (Online Resource 2) [32, 33]. These two molecules can then react with each other to form peroxynitrite.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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Superoxide is a naturally produced reactive oxygen species (ROS) in the human body and is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE+) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE+ per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE+ signal from possible interferences.

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“…The newly etched device was placed in an oven to evaporate the residual isophorone at 75°C for 2 hours. Channel depths were measured using a profilometer (Dektak 3 ST, Veeco Instruments, Woodbury, NY, USA). Ink printed channel patterns were printed onto commercially available Shrinky-Dinks© (SD) using a laser jet printer (HP Laser Jet P1102w, Hewlett-Packard Development Company, LP, Palo Alto, CA).…”

Section: Methodsmentioning

confidence: 99%

Fabrication and characterization of all-polystyrene microfluidic devices with integrated electrodes and tubing

Pentecost

Martin

2015

Anal. Methods

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A new method of fabricating all-polystyrene devices with integrated electrodes and fluidic tubing is described. As opposed to expensive polystyrene (PS) fabrication techniques that use hot embossing and bonding with a heated lab press, this approach involves solvent-based etching of channels and lamination-based bonding of a PS cover, all of which do not need to occur in a clean room. PS has been studied as an alternative microchip substrate to PDMS, as it is more hydrophilic, biologically compatible in terms of cell adhesion, and less prone to absorption of hydrophobic molecules. The etching/lamination-based method described here results in a variety of all-PS devices, with or without electrodes and tubing. To characterize the devices, micrographs of etched channels (straight and intersected channels) were taken using confocal and scanning electron microscopy. Microchip-based electrophoresis with repetitive injections of fluorescein was conducted using a three-sided PS (etched pinched, twin-tee channel) and one-sided PDMS device. Microchip-based flow injection analysis, with dopamine and NO as analytes, was used to characterize the performance of all-PS devices with embedded tubing and electrodes. Limits of detection for dopamine and NO were 130 nM and 1.8 μM, respectively. Cell immobilization studies were also conducted to assess all-PS devices for cellular analysis. This paper demonstrates that these easy to fabricate devices can be attractive alternative to other PS fabrication methods for a wide variety of analytical and cell culture applications.

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Separation and Detection of Peroxynitrite Using Microchip Electrophoresis with Amperometric Detection

Cited by 66 publications

References 26 publications

In‐channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

In‐channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

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

Fabrication and characterization of all-polystyrene microfluidic devices with integrated electrodes and tubing

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