| 1. |
- Lovric, Jelena, 1980, et al.
(författare)
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Analysis of liposome model systems by time-of-flight secondary ion mass spectrometry
- 2014
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Ingår i: Surface and Interface Analysis: 19th International Conference on Secondary Ion Mass Spectrometry (SIMS). - : Wiley. - 0142-2421 .- 1096-9918.
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Konferensbidrag (refereegranskat)abstract
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is an important technique for studying chemical composition of micrometer scale objects because of its high spatial resolution imaging capabilities and chemical specificity. In this work, we focus on the application of ToF-SIMS to gain insight into the chemistry of micrometer size liposomes as a potential model for neurotransmitter vesicles. Two models of giant liposomes were analyzed: histamine and aqueous two-phase system-containing liposomes. Characterization of the internal structure of single fixed liposomes was carried out both with the Bi-3(+) and C-60(+) ion sources. The depth profiling capability of ToF-SIMS was used to investigate the liposome interior.
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| 2. |
- Berglund, E Carina, et al.
(författare)
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Oral administration of methylphenidate blocks the effect of cocaine on uptake at the Drosophila dopamine transporter.
- 2013
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Ingår i: ACS chemical neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 4:4, s. 566-74
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Tidskriftsartikel (refereegranskat)abstract
- Although our understanding of the actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in mammalian central nervous systems similar to cocaine, but it is thought to elicit fewer addictive and reinforcing effects owing to slower pharmacokinetics for different routes of administration between the drugs. This study utilizes the fruit fly model system to quantify the effects of oral methylphenidate on dopamine uptake during direct cocaine exposure to the fly CNS. The effect of methylphenidate on the dopamine transporter has been explored by measuring the uptake of exogenously applied dopamine. The data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter and the inhibition is concentration dependent. The peak height increased to 150% of control when cocaine was used to block the dopamine transporter for untreated flies but only to 110% for methylphenidate-treated flies. Thus, the dopamine transporter is mostly inhibited for the methylphenidate-fed flies before the addition of cocaine. The same is true for the rate of the clearance of dopamine measured by amperometry. For untreated flies the rate of clearance changes 40% when the dopamine transporter is inhibited with cocaine, and for treated flies the rate changes only 10%. The results were correlated to the in vivo concentration of methylphenidate determined by CE-MS. Our data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter for cocaine uptake, and the inhibition is concentration dependent.
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| 3. |
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| 4. |
- Keighron, Jacqueline, 1982, et al.
(författare)
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Amperometric Detection of Single Vesicle Acetylcholine Release Events from an Artificial Cell
- 2015
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 6:1, s. 181-188
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Tidskriftsartikel (refereegranskat)abstract
- Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.
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| 5. |
- Keighron, Jacqueline, 1982, et al.
(författare)
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Analytical tools to monitor exocytosis: A focus on new fluorescent probes and methods
- 2012
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Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 137:8, s. 1755-1763
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Forskningsöversikt (refereegranskat)abstract
- A great deal of research has been focused on unraveling the processes governing the exocytotic pathway and the extent of release during the process. Arguments abound for and against both the occurrence and significance of full release during exocytosis and partial release including kiss-and-run events. Several optical methods to directly observe the exocytosis process have been developed and here we focus on fluorescence methods and probes for this work. Although fluorescence imaging has been used for cell experiments for decades, in the last two decades a plethora of new approaches have arrived on the scene. These include application of new microscopy techniques, like total internal reflectance and stimulated emission depletion that are offering new ways to circumvent the limits of far field microscopy with a diffraction limit of 200 nm, and allow tracking of single synaptic vesicles. For selective imaging of synaptic vesicles the introduction of methods to stain the vesicular compartment has involved developing probes of the vesicular membrane and intravesicular solution, nanoparticle quantum dots that can be observed during exocytosis but not via the fusion pore, and fluorescent false neurotransmitters.
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| 6. |
- Keighron, Jacqueline, 1982, et al.
(författare)
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Coimmobilization of Acetylcholinesterase and Choline Oxidase on Gold Nanoparticles: Stoichiometry, Activity, and Reaction Efficiency
- 2014
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 30:38, s. 11348-11355
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Tidskriftsartikel (refereegranskat)abstract
- Hybrid structures constructed from biomolecules and nanomaterials have been used in catalysis and bioanalytical applications. In the design of many chemically selective biosensors, enzymes conjugated to nanoparticles or carbon nanotubes have been used in functionalization of the sensor surface for enhancement of the biosensor functionality and sensitivity. The conditions for the enzyme:nanomaterial conjugation should be optimized to retain maximal enzyme activity, and biosensor effectiveness. This is important as the tertiary structure of the enzyme is often altered when immobilized and can significantly alter the enzyme catalytic activity. Here we show that characterization of a two-enzyme:gold nanoparticle (AuNP) conjugate stoichiometry and activity can be used to gauge the effectiveness of acetylcholine detection by acetylcholine esterase (AChE) and choline oxidase (ChO). This was done by using an analytical approach to quantify the number of enzymes bound per AuNP and monitor the retained enzyme activity after the enzyme:AuNP synthesis. We found that the amount of immobilized enzymes differs from what would be expected from bulk solution chemistry. This analysis was further used to determine the optimal ratio of AChE:ChO added at synthesis to achieve optimum sequential enzyme activity for the enzyme:AuNP conjugates, and reaction efficiencies of greater than 70%. We here show that the knowledge of the conjugate stoichiometry and retained enzyme activity can lead to more efficient detection of acetylcholine by controlling the AChE:ChO ratio bound to the gold nanoparticle material. This approach of optimizing enzyme gold nanoparticle conjugates should be of great importance in the architecture of enzyme nanoparticle based biosensors to retain optimal sensor sensitivity.
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| 7. |
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| 8. |
- Keighron, Jacqueline, 1982, et al.
(författare)
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Electrochemistry of Single-Vesicle Events
- 2020
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Ingår i: Annual Review of Analytical Chemistry. - : Annual Reviews. - 1936-1327 .- 1936-1335. ; 13, s. 159-181
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Forskningsöversikt (refereegranskat)abstract
- Neuronal transmission relies on electrical signals and the transfer of chemical signals from one neuron to another. Chemical messages are transmitted from presynaptic neurons to neighboring neurons through the triggered fusion of neurotransmitter-filled vesicles with the cell plasma membrane. This process, known as exocytosis, involves the rapid release of neurotransmitter solutions that are detected with high affinity by the postsynaptic neuron. The type and number of neurotransmitters released and the frequency of vesicular events govern brain functions such as cognition, decision making, learning, and memory. Therefore, to understand neurotransmitters and neuronal function, analytical tools capable of quantitative and chemically selective detection of neurotransmitters with high spatiotemporal resolution are needed. Electrochemistry offers powerful techniques that are sufficiently rapid to allow for the detection of exocytosis activity and provides quantitative measurements of vesicle neurotransmitter content and neurotransmitter release from individual vesicle events. In this review, we provide an overview of the most commonly used electrochemical methods for monitoring single-vesicle events, including recent developments and what is needed for future research.
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| 9. |
- Lin, Y. Q., et al.
(författare)
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Carbon-Ring Microelectrode Arrays for Electrochemical Imaging of Single Cell Exocytosis: Fabrication and Characterization
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:6, s. 2949-2954
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Tidskriftsartikel (refereegranskat)abstract
- Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10-50 mu m, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) were successfully detected with the CRMAs.
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| 10. |
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