| 1. |
- Bergman, Jenny, et al.
(författare)
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Counting the number of enzymes immobilized onto a nanoparticle-coated electrode
- 2018
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 410:6, s. 1775-1783
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Tidskriftsartikel (refereegranskat)abstract
- To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic.
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| 2. |
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| 3. |
- Dunevall, Johan, 1984, et al.
(författare)
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Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision-Adsorption Events at an Electrode
- 2015
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:13, s. 4344-4346
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Tidskriftsartikel (refereegranskat)abstract
- We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 mu m diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.
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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. |
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| 6. |
- Wigström, Joakim, 1971, et al.
(författare)
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Lithographic Microfabrication of a 16-Electrode Array on a Probe Tip for High Spatial Resolution Electrochemical Localization of Exocytosis
- 2016
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:4, s. 2080-2087
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Tidskriftsartikel (refereegranskat)abstract
- We report the lithographic microfabrication of a movable thin film microelectrode array (MEA) probe consisting of 16 platinum band electrodes placed on top of a supporting borosilicate glass substrate. These 1.2 mu m wide electrodes were tightly packed and positioned parallel in two opposite rows within a 20 mu m x 25 mu m square area and with a distance less than 10 mu m from the edge of the glass substrate. We demonstrate the ability to control and place the probe in close proximity to the surface of adherent bovine chromaffin cells and to amperometrically record single exocytosis release events with high spatiotemporal resolution. The two-dimensional position of single exocytotic events occurring in the center gap area separating the two rows of MEA band electrodes and that were codetected by electrodes in both rows was determined by analysis of the fractional detection of catecholamine released between electrodes and exploiting random walk simulations. Hence, two-dimensional electrochemical imaging recording of exocytosis release between the electrodes within this area was achieved. Similarly, by modeling the current spikes codetected by parallel adjacent band electrodes positioned in the same electrode row, a one-dimensional imaging of exocytosis with submicrometer resolution was accomplished within the area. The one- and twodimensional electrochemical imaging using the MEA probe allowed for high spatial resolution of exocytosis activity and revealed heterogeneous release of catecholamine at the chromaffin cell surface.
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| 7. |
- Adams, Kelly L., et al.
(författare)
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Steady-State Electrochemical Determination of Lipidic Nanotube Diameter Utilizing an Artificial Cell Model
- 2010
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:3, s. 1020-1026
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Tidskriftsartikel (refereegranskat)abstract
- By exploiting the capabilities of steady-state electrochemical measurements, we have measured the inner diameter of a lipid nanotube using Fick’s first law of diffusion in conjunction with an imposed linear concentration gradient of electroactive molecules over the length of the nanotube. Fick’s law has been used in this way to provide a direct relationship between the nanotube diameter and the measurable experimental parameters Δi (change in current) and nanotube length. Catechol was used to determine the Δi attributed to its flux out of the nanotube. Comparing the nanotube diameter as a function of nanotube length revealed that membrane elastic energy was playing an important role in determining the size of the nanotube and was different when the tube was connected to either end of two vesicles or to a vesicle on one end and a pipet tip on the other. We assume that repulsive interaction between neck regions can be used to explain the trends observed. This theoretical approach based on elastic energy considerations provides a qualitative description consistent with experimental data.
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| 8. |
- Ali Doosti, Baharan, 1991, et al.
(författare)
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Generation of interconnected vesicles in a liposomal cell model
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca2+ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca2+ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems.
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| 9. |
- Ali Doosti, Baharan, 1991, et al.
(författare)
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Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients
- 2018
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 2018:137
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Tidskriftsartikel (refereegranskat)abstract
- In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.
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| 10. |
- Bergman, Jenny, et al.
(författare)
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Co-detection of dopamine and glucose with high temporal resolution
- 2018
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Ingår i: Catalysts. - : MDPI AG. - 2073-4344. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal activity and brain glucose metabolism are tightly coupled, where triggered neurotransmission leads to a higher demand for glucose. To better understand the regulation of neuronal activity and its relation to high-speed metabolism, development of analytical tools that can temporally resolve the transients of vesicular neurotransmitter release and fluctuations of metabolites such as glucose in the local vicinity of the activated neurons is needed. Here we present an amperometric biosensor design for rapid co-detection of glucose and the neurotransmitter dopamine. The sensor is based on the immobilization of an ultra-thin layer of glucose oxidase on to a gold-nanoparticle-covered carbon fiber microelectrode. Our electrode, by altering the potential applied at the sensor surface, allows for the high-speed recording of both glucose and dopamine. We demonstrate that, even though glucose is electrochemically detected indirectly through the enzymatic product and the electroactive dopamine is sensed directly, when exposing the sensor surface to a mixture of the two analytes, fluctuations in glucose and dopamine concentrations can be visualized with similar speed and at a millisecond time scale. Hence, by minimizing the enzyme coating thickness at the sensor surface, dual detection of glucose and dopamine can be realized at the same sensor surface and at time scales necessary for monitoring fast metabolic alterations during neurotransmission. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
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