Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts

Gunasekara, Dulan B.; Hulvey, Matthew K.; Lunte, Susan M.; Silva, José Alberto Fracassi da

doi:10.1007/s00216-012-5810-4

Cited by 19 publications

(17 citation statements)

References 40 publications

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“…Therefore, this method is especially useful for the detection of chemically labile species since they can be separated and detected before significant degradation occurs. 35 …”

Section: Introductionmentioning

confidence: 99%

“…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. 35 Since nitrate (NO 3 -) is not electroactive, a method using an on-chip Cu 2+ /Cd reductorcan be used to reduce NO 3 - to NO 2 - , which permits the detection of both species by ME-EC. 37 Nitrate and nitrite can also be monitored using ME with combined conductivity and amperometric detection.…”

Section: Introductionmentioning

confidence: 99%

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

Gunasekara

Siegel

Caruso

et al. 2014

Analyst

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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.

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“…Therefore, this method is especially useful for the detection of chemically labile species since they can be separated and detected before significant degradation occurs. 35 …”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Gunasekara

Siegel

Caruso

et al. 2014

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“…The PDMS channels were then conditioned sequentially with 0.1 M NaOH, water, and 10 mM MES pH 6 buffer solutions. Samples were introduced into the separation channel using a gated injection scheme [40,41]. A field strength of 77 V/cm was used for all separations.…”

Section: Fabrication and Operation Of The Hybrid Pdms/pmma Mce-ec Devicementioning

confidence: 99%

Integration of a graphite/poly(methyl‐methacrylate) composite electrode into a poly(methylmethacrylate) substrate for electrochemical detection in microchips

Regel

Lunte

2013

Electrophoresis

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Traditional fabrication methods for polymer microchips, the bonding of two substrates together to form the microchip, can make the integration of carbon electrodes difficult. We have developed a simple and inexpensive method to integrate graphite/PMMA composite electrodes (GPCEs) into a PMMA substrate. These substrates can be bonded to other PMMA layers using a solvent-assisted thermal bonding method. The optimal composition of the GPCEs for electrochemical detection was determined using cyclic voltammetry with dopamine as a test analyte. Using the optimized GPCEs in an all-PMMA flow cell with flow injection analysis, it was possible to detect 50 nM dopamine under the best conditions. These electrodes were also evaluated for the detection of dopamine and catechol following separation by microchip electrophoresis (ME).

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“…Different detection principles have been integrated with MEs [19,20]. Electrochemical (EC) detection schemes [21][22][23][24] are gaining acceptance and maturity, mainly due to their high sensitivity, ease of application and possibility of electrode integration into the chip during the fabrication process, leading to fully integrated microfluidic systems [25][26][27].Currently, polymeric MEs have displaced the more traditional glass devices [17], due to a lower cost of materials and simplicity of fabrication techniques. A wide range of polymeric substrates with different fabrication protocols is reported in the literature [28][29][30].…”

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confidence: 99%

Double-chained cationic surfactant modification of SU-8/Pyrex® microchips for electrochemical sensing of carboxylic ferrocene after reverse electrophoresis

Alonso-Bartolomé

González-López

Fernández‐Abedul

2018

Sensors and Actuators B: Chemical

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Detection of molecules can be approached using an intrinsic property such as e.g., electroactivity, absorbance or fluorescence. In the case of molecules without groups providing these properties, derivatization with a marker allows their detection. Procedures for labeling molecules with chromophores and fluorophores are commonly found in the bibliography [1] but when electroactivity is concerned, examples are scarce. Miniaturized electrochemical sensors and platforms are gaining interest for decentralized analysis and innovative approaches, not only for analytical tools [2,3] but also for instrumentation [4], are being continuously developed.Although some electroactive labels have been studied, especially for selective purposes, either biosensors [5] or separation methodologies [6], more research is needed in order to study possible new markers and establish labeling procedures for developing promising analytical methodologies. Ferrocene, in the form of monocarboxylic acid (FCA) has been employed as mediator of electron transfer reactions, usually as modif ier of the working electrode [7] or more scarcely, as covalent electrochemical label of biomolecules such as e.g., antibodies [8], DNA [9], peptide nucleic acids [10,11] or aptamers [12]. In this context, it is of paramount relevance to have techniques that allow monitoring the bioconjugation process. Then, measurement of the label, either free or conjugated to biomolecules, is required in many cases for following the bioconjugation procedure of for indirect determination of analytes [13].Electrophoresis is a powerful separation technique of biomolecules that is being increasingly adapted to the microchip format (microchip electrophoresis, ME). This is mainly due to recent developments not only in materials [14] but also in surface modifications [15,16] as well as in detection systems and associated instrumentation [17,18]. Different detection principles have been integrated with MEs [19,20]. Electrochemical (EC) detection schemes [21][22][23][24] are gaining acceptance and maturity, mainly due to their high sensitivity, ease of application and possibility of electrode integration into the chip during the fabrication process, leading to fully integrated microfluidic systems [25][26][27].Currently, polymeric MEs have displaced the more traditional glass devices [17], due to a lower cost of materials and simplicity of fabrication techniques. A wide range of polymeric substrates with different fabrication protocols is reported in the literature [28][29][30]. The photoresist EPON SU-8 (SU-8), a negative tone epoxy photopatternable resist, mechanically reliable, optically transparent, chemically resistant and hydrophilic, has been used to fabricate ME devices [18,24,31]. The electroosmotic flow (EOF) in SU-8 is very similar to glass and it does not require any modification to be used with proteins and peptides since no adsorption of biomolecules on the microchannels occurs [32]. In the normal electrophoresis mode, where the microchannel wall is negatively charged, a...

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Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts

Cited by 19 publications

References 40 publications

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

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

Integration of a graphite/poly(methyl‐methacrylate) composite electrode into a poly(methylmethacrylate) substrate for electrochemical detection in microchips

Double-chained cationic surfactant modification of SU-8/Pyrex® microchips for electrochemical sensing of carboxylic ferrocene after reverse electrophoresis

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