Electrochemical detection in microcolumn separations.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
A G Ewing
Publications and source records attributed to A G Ewing.
Capillary electrophoresis of single cells: observation of two compartments of neurotransmitter vesicles.
Capillary electrophoresis has been used to directly identify and measure the neurotransmitter, dopamine, in two vesicular compartments in a single nerve cell of Planorbis corneus. Dopamine in the cytoplasm and in easily released transmitter vesicles was separated from dopamine in what are apparently non-functional storage vesicles. In this method, the two peaks in the electropherogram attributed to dopamine were differentiated based on cell lyse time in a non-physiological buffer. The measurements presented here suggest that of the total dopamine present in the cell: 24% is in the cytoplasmic and easily released compartments and 76% is more centrally located, perhaps in a reserve compartment, in the cell. This methodology provides the means to determine molecular species in subcellular compartments and should allow kinetic parameters associated with membrane lysing to be evaluated at single nerve cells.
Animals↗
Direct observation of the effect of autoreceptors on stimulated release of catecholamines from adrenal cells.
The direct effect of alpha 2-autoreceptors was studied by measuring the effects of piperoxan, an alpha 2-autoreceptor antagonist, and clonidine, an agonist on catecholamine exocytosis, from single bovine chromaffin cells in culture. Catecholamine release was elicited by stimulation with 100 microM nicotine and was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. These electrodes allowed the number of exocytotic release events to be monitored and reported as total charge for release following a specific stimulus. Repeated stimulation with 100 microM nicotine showed that total release caused by the second exposure to nicotine was 32% of the first, and release caused by the third exposure to nicotine was 80% of the second. Total release of catecholamine increased significantly after application of 20 microM piperoxan relative to a control application of balanced salt solution. Application of 20 microM piperoxan alone did not cause release. After the cells were incubated in culture medium containing 20 microM clonidine, a significant decrease in nicotine-stimulated catecholamine release was observed. These results confirm that there are autoreceptors on chromaffin cells and, when relatively high levels of catecholamine are released, the catecholamine stimulates the alpha 2-autoreceptors, which inhibits subsequent release through a negative feedback mechanism. In addition to piperoxan, the sympathomimetic drug amphetamine also increases quantal release after application of nicotine. Amphetamine increases the extracellular concentration of catecholamine, and these data appear to indicate that at least part of the pharmacology of amphetamine might involve blocking catecholamine autoreceptors.
Adrenal Medulla↗
Continuous electrophoretic separations in narrow channels coupled to small-bore capillaries.
Continuous zone electrophoretic separations in channels have been demonstrated. This new technique has the potential to continuously sample and separate analytes from volume-limited microenvironments. A small-bore capillary is used to electrophoretically sample, but not separate, a mixture of dansylated amino acids. The capillary is coupled to a quartz channel structure in a manner which allows continuous injection of the sampled material into the channel. The channel functions to continuously separate the sampled material via electrophoresis. A laser-induced fluorescence detection scheme, which involves two fiber optic arrays situated at the channel exit, monitors eluting analytes. A continuous separation of dansylated amino acids on the time scale of a few minutes demonstrates the utility of the technique. Sampling has been performed continuously up to 400 s, and initial detection limits are approximately 30 microM.
Dansyl Compounds↗
Electroosmotic flow control and surface conductance in capillary zone electrophoresis.
Electroosmotic flow within capillary zone electrophoresis can be altered electronically through a mechanism modeled as surface conductance. Experimentally, a small zone of conductive silver on the outer surface of the capillary is shown to permit good control of electroosmotic flow with an applied external voltage. This control is modeled as capacitance across the capillary wall and conductance along the double layer on the inner surface. Experimental results presented agree with theory developed from this model. Surface conductance in capillary zone electrophoresis may significantly simplify electronic control of electroosmotic flow and lead to a better understanding of the effects of chemical modifications to the inner surface of the capillary.
Electrochemistry↗
Effects of buffer pH on electroosmotic flow control by an applied radial voltage for capillary zone electrophoresis.
Electroosmotic flow has been shown to be controlled via an applied radial voltage. Many factors determine the effectiveness of this control, and one major factor is buffer pH. In this study the effectiveness of the applied radial voltage for controlling electroosmotic flow while varying buffer pH is examined. Previously developed theory is applied and compared to experimental results for a pH range from 1.4 to 6.32. Analysis time is dramatically reduced by applying a radial voltage for separation of a peptide mixture at pH 1.4. Theory predicts laminar flow profiles under some conditions when applying this technique. However, experimental evidence at pH 6.32 and 1.4 shows no evidence of band broadening from a laminar flow profile. Theoretical and experimental results indicate the largest range of effective electroosmotic flow control via an applied radial voltage occurs at low ph. Furthermore, a sigmoidal relationship between electroosmotic flow and applied radial voltage is clearly apparent under these conditions. In contrast, at high buffer pH ( > 6) the relationship appears to be linear and is only over a limited range of flow velocities.
Electrophoresis↗
Microcolumn separations of single nerve cell components.
Miniaturization of separation techniques is providing new methods to examine neurochemistry and neurophysiology at the single cell level. Open tubular liquid chromatography and capillary electrophoresis have been scaled down to levels where picoliter and femtoliter volume samples can be separated. This provides the means to profile the chemistry of single whole nerve cells and to sample subcellular regions.
Animals↗
Characterization of glucose microsensors for intracellular measurements.
Ultrasmall glucose sensors have been constructed by using platinum-deposited carbon ring microelectrodes with glucose oxidase. Response times as low as 270 ms have been obtained with these sensors. Moreover, there is a linear relationship between sensor tip diameter and response times. The use of these sensors has been demonstrated in the detection of glucose in single-cell cytoplasm of the large dopamine cell of the pond snail Planorbis corneus. Current responses obtained at these sensors implanted into a cell increase following injection of 2 pL of glucose solution (3 M) into the cell. Results obtained from these experiments show that these sensors are suitable for glucose monitoring in ultrasmall environments. In addition, characterizations of these sensors have been investigated under different O2 concentrations. At atmospheric oxygen concentrations, glucose levels in the submillimolar range can be measured without oxygen interference; however, oxygen interference can be substantial at low oxygen concentrations.
Animals↗
Voltammetric measurement of oxygen in single neurons using platinized carbon ring electrodes.
Naflon-coated ultrasmall platinum ring electrodes have been implanted in the giant dopamine neuron of the pond snail Planorbis corneus, and the oxygen concentration inside these single neurons has been estimated. Experimental data suggest that the intracellular oxygen level in the identified dopamine neuron of P. corneus is approximately 0.032 mM. The oxygen concentration immediately outside the cell (ca. 10 microns away from the cell) is 0.041 mM. Furthermore, staircase voltammetry can be used to monitor dynamic changes in oxygen concentration inside the cell after bathing with Ringer's solution saturated with air/oxygen. Data obtained for intracellular oxygen concentrations suggest that intracellular oxygen consumption is increased following potassium chloride-induced stimulation of these cells.
Animals↗
Electroosmotic flow control and monitoring with an applied radial voltage for capillary zone electrophoresis.
The control of electroosmotic flow by electronic means for capillary zone electrophoresis is presented. This is accomplished by the application of a radial voltage field with a rugged and flexible conductive polymer sheath. Fundamental theory for effect of the applied radial voltage on electroosmotic flow is developed. In addition, the effects of surface pretreatments are examined and compared to trends predicted by the theory.
Electric Conductivity↗
End-column detection for capillary zone electrophoresis.
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3,4-Dihydroxyphenylacetic Acid↗
Capillary electrophoresis in 2 and 5 microns diameter capillaries: application to cytoplasmic analysis.
Capillary electrophoresis with electrochemical detection in 2- and 5-micron capillaries has been developed to study ultrasmall biological environments. Sample volumes as low as 270 fL have been injected into the electrophoresis capillary with subattomole detection limits for easily oxidized species. We have applied this method to the analysis of single cell cytoplasm. Sampling of the cytoplasm is accomplished by inserting one end of the electrophoresis capillary directly into a single nerve cell. The high-voltage end of the electrophoresis capillary has been etched with hydrofluoric acid to form a microinjector. This injection scheme represents an improvement over those previously used for similar applications. The excellent selectivity of this method is demonstrated for catecholamine and indolamine neurotransmitters and their metabolites found in the invertebrate system, the pond snail Planorbis corneus.
Animals↗
Capillary electrophoresis with indirect amperometric detection.
The use of indirect amperometric detection with capillary electrophoresis is demonstrated. The system consists of a porous glass coupler which allows amperometric detection at a carbon fiber electrode placed in the end of the capillary. 3,4-Dihydroxybenzylamine is added to the buffer system as a continuously eluting electrophore. Indirect amperometric detection in 9-mumol I.D. capillaries provides detection limits as low as 380 attomole for the amino acid arginine. Finally, both direct and indirect amperometric detection can be accomplished simultaneously.
Amino Acids↗
Dopamine concentration in the cytoplasmic compartment of single neurons determined by capillary electrophoresis.
It has long been a goal of the biochemist to study the chemistry of single cells. This is particularly important in neuroscience, since each cell can maintain a specific function and identity. Investigations into neurotransmitter compartmentalization within the cell body of neurons should lead to an understanding of the role of the cell body in metabolism, uptake and storage of neurotransmitters. Using capillary electrophoresis, it has been possible to separate and detect attomole levels of neurotransmitter in picoliter volumes of cytoplasm withdrawn from single neurons of the pond snail, Planorbis corneus. This work demonstrates, for the first time, the direct determination of the cytoplasmic concentration of dopamine in single, intact neurons.
Cytoplasm↗
Estimation of free dopamine in the cytoplasm of the giant dopamine cell of Planorbis corneus by voltammetry and capillary electrophoresis.
Voltammetric electrodes having a tip diameter of 2-12 microns and microscale capillary zone electrophoresis have been used for dynamic and static monitoring of dopamine in the somal cytoplasm of the giant dopamine neuron of Planorbis corneus. Intracellular dopamine levels can be altered by extracellular application of dopamine or ethanol. The latter experiment provides a means to estimate total stores of endogenous dopamine, and the evidence presented suggests that at least 98% of these stores are bound and not directly accessible to the cytoplasm.
Animals↗
Capillary electrophoresis.
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Electrophoresis↗
Separation of serotonin from catechols by capillary zone electrophoresis with electrochemical detection.
Capillary zone electrophoresis with electrochemical detection is demonstrated with columns having only 9-microns inner diameter. Amperometric detection limits of 0.7 amol are reported for serotonin. The difficult problem of resolving serotonin and dopamine--two neurotransmitters of interest having similar electrophoretic mobilities--is addressed by chemical means to improve selectivity. These include buffer modification with 2-propanol and a system employing borate complexation of the catechol in combination with sodium dodecyl sulfate micelles.
Catecholamines↗
Capillary electrophoresis.
The combined techniques that comprise capillary electrophoresis are under rapid development for a wide range of molecules and sample types. The capillary format provides fast, highly efficient separations and makes automation possible. At present, systematic approaches to separation methodology are still in the development stages and better detection schemes need to be developed. There is no doubt that electrophoresis in a capillary tube will take its place as a substitute for HPLC in specific applications. This is especially apparent when analysis time, efficiency, or simplicity is of greatest concern. Capillary electrophoresis will prove extremely useful for separations of nucleotides and proteins for analytical purposes in biotechnology. Finally, the ability to use ultrasmall-volume samples makes capillary electrophoresis ideal for the development of separations-based sensors for the analysis of microenvironments.
Electrophoresis↗