Characterization of etched electrochemical detection for electrophoresis in micron inner diameter capillaries

Powell, Paula R.; Woods, Lori A.; Ewing, Andrew G.

doi:10.1002/jssc.200500167

Cited by 9 publications

(11 citation statements)

References 16 publications

(32 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This resulted in an etch with a frustum geometry measuring approximately 40 µm wide at the base, which serves to both ease placement of the electrode at the capillary outlet, as well as to decouple the applied separation voltage from the electrochemical cell (43). …”

Section: Methodsmentioning

confidence: 99%

Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles

Omiatek

Dong

Heien

et al. 2010

ACS Chem. Neurosci.

Self Cite

141

179

View full text Add to dashboard Cite

The primary method for neuronal communication involves the release of chemical messengers that are packaged intracellularly in vesicles. Although experiments measuring release at single cells have classically been thought to assess the entire content of vesicles, there is evidence in the literature that suggests that the total transmitter stored in vesicles is not expelled during exocytosis. In this work, we introduce a novel technology using a microfluidic-based platform to electrochemically probe individual PC12 cell vesicles isolated from the cell environment. We measure the total vesicular content using methodology that circumvents the biophysical processes of the cell associated with exocytosis. Direct comparisons of amperometric data from release experiments at single PC12 cells versus our cell-free model reveal that on average vesicles release only 40% of their total transmitter load. The data support the intriguing hypothesis that the average vesicle does not open all the way during the normal exocytosis process, resulting in incomplete distention of the vesicular contents. In addition, we have shown that vesicular catecholamine levels can be altered with pharmacological manipulation and variances observed from these treatments can be resolved at the single vesicle level in a high-throughput manner, a process that we have termed electrochemical cytometry. Upon establishing that release in exocytotic processes proceeds in an incomplete manner, electrochemical data quantified from both single cell release experiments and electrochemical cytometry of vesicles were related to vesicular volume from electron microscopy measurements to investigate the location of intravesicular catecholamine stores retained post-fusion.

show abstract

Section: Methodsmentioning

confidence: 99%

Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles

Omiatek

Dong

Heien

et al. 2010

ACS Chem. Neurosci.

Self Cite

141

179

View full text Add to dashboard Cite

show abstract

“…The researchers have taken measures, such as modifying the electrodes [32,33] or reducing the size of the microchannel and electrode [34,35] to improve the sensitivity of the CE-ECD system. Ewing and coworkers [31,[34][35][36][37] investigated the influence of the size of the column and the working electrode on the sensitivity of amperometric detection, and the results demonstrated that reducing the size of the column and the working electrode to a certain degree actually enhanced the sensitivity.…”

Section: Introductionmentioning

confidence: 96%

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells

et al. 2007

View full text Add to dashboard Cite

Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.

show abstract

“…A CE system with end-column amperometric detection was built in-house and used as previously described 30. Modifications were made to the system to integrate hydrodynamic flow of lysis buffer into the detection zone.…”

Section: Methodsmentioning

confidence: 99%

Hybrid Capillary-Microfluidic Device for the Separation, Lysis, and Electrochemical Detection of Vesicles

et al. 2009

Self Cite

View full text Add to dashboard Cite

The primary method for neuronal communication involves the extracellular release of small molecules that are packaged in secretory vesicles. We have developed a platform to separate, lyse, and electrochemically measure the contents of single vesicles using a hybrid capillary-microfluidic device. This device incorporates a sheath-flow design at the outlet of the capillary for chemical lysis of vesicles and subsequent electrochemical detection. The effect of sheath-flow on analyte dispersion was characterized using confocal fluorescence microscopy and electrochemical detection. At increased flow rates, dispersion was minimized, leading to higher separation efficiencies, but lower detected amounts. Large unilamellar vesicles (diameter ∼ 200 nm), a model for secretory vesicles, were prepared by extrusion and loaded with an electroactive molecule. They were then separated and detected using the hybrid capillary-microfluidic device. Determination of size from internalized analyte concentration provides a method to characterize the liposomal suspension. These results were compared to an orthogonal size measurement using dynamic light scattering to validate the detection platform.

show abstract

Characterization of etched electrochemical detection for electrophoresis in micron inner diameter capillaries

Cited by 9 publications

References 16 publications

Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles

Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells

Hybrid Capillary-Microfluidic Device for the Separation, Lysis, and Electrochemical Detection of Vesicles

Contact Info

Product

Resources

About