| 1. |
- Makos, MA, et al.
(författare)
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Development and Characterization of a Voltammetric Carbon-Fiber Microelectrode pH Sensor
- 2010
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 26:12, s. 10386-10391
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Tidskriftsartikel (refereegranskat)abstract
- This work describes the development and characterization of a modified carbon-fiber microelectrode sensor capable of measuring real-time physiological pH changes in biological microenvironments. The reagentless sensor was fabricated under ambient conditions from voltammetric reduction of the diazonium salt Fast Blue RR onto a carbon-fiber surface in aprotic media. Fast-scan cyclic voltammetry was used to probe redox activity of the p-quinone moiety of the surface-bound molecule as a function of pH. In vitro calibration of the sensor in solutions ranging from pH 6.5 to 8.0 resulted in a pH-dependent anodic peak potential response. Flow-injection analysis was used to characterize the modified microelectrode, revealing sensitivity to acidic and basic changes discernible to 0.005 pH units. Furthermore, the modified electrode was used to measure dynamic in vivo pH changes evoked during neurotransmitter release in the central nervous system of the microanalytical model organism Drosophila melanogaster.
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| 2. |
- Omiatek, Donna M, et al.
(författare)
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Hybrid capillary-microfluidic device for the separation, lysis, and electrochemical detection of vesicles.
- 2009
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Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:6, s. 2294-302
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Tidskriftsartikel (refereegranskat)abstract
- 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 approximately 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.
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| 3. |
- Omiatek, Donna M, et al.
(författare)
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Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles
- 2010
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 1:3, s. 234-245
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Tidskriftsartikel (refereegranskat)abstract
- 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, we related electrochemical data quantified from both single cell release experiments and electrochemical cytometry of vesicles to vesicular volume from electron microscopy measurements to investigate the location of intravesicular catecholamine stores retained postfusion.
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