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Manfred Lindau - One of the best experts on this subject based on the ideXlab platform.
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Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel Amperometry measurements
Pflugers Archiv : European journal of physiology, 2017Co-Authors: Meng Huang, Joannalyn Delacruz, John C. Ruelas, Shailendra S. Rathore, Manfred LindauAbstract:Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell Amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.
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Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na^+ influx through the fusion pore
Nature Cell Biology, 2007Co-Authors: Liang Wei Gong, Guillermo Alvarez De Toledo, Manfred LindauAbstract:Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch Amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that
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Patch Amperometry: High-resolution measurements of single-vesicle fusion and release.
Nature methods, 2005Co-Authors: Gregor Dernick, Liang Wei Gong, Lucia Tabares, Guillermo Alvarez De Toledo, Manfred LindauAbstract:Protocol Published in association with Cold Spring Harbor Laboratory Press Nature Methods - 2, 699 - 708 (2005) doi:10.1038/nmeth0905-699 Patch Amperometry: high-resolution measurements of single-vesicle fusion and release Gregor Dernick1, 3, Liang-Wei Gong1, 3, Lucia Tabares2, Guillermo Alvarez de Toledo2 & Manfred Lindau1 1 School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14850, USA. 2 Department of Physiology and Biophysics, School of Medicine, University of Seville, E-41009 Seville, Spain. 3 Present addresses: F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland (G.D.), and Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA (L.-W.G.). Correspondence should be addressed to Manfred Lindau ml95@cornell.edu Patch Amperometry is a new technique for the observation of single-vesicle exocytosis. Exocytosis of single vesicles as small as 50 nm in diameter can be detected by cell-attached patch-clamp admittance measurements1, 2, 3, 4 indicating fusion of vesicles with the plasma membrane or by Amperometry with a carbon fiber electrode (CFE)5, 6, 7, 8, 9 indicating release of oxidizable molecules such as catecholamines. The admittance measurement provides the membrane capacitance that increases in proportion to the membrane area because of the incorporation of the vesicle into the patch membrane. It also reveals the fusion pore conductance during an exocytotic event, giving an estimate of fusion pore dimensions. Amperometry provides the amount and time course of release of molecules that are readily oxidizable such as dopamine, norepinephrine or serotonin. This technique is capable of detecting as little as a few thousand molecules8, 9. It also resolves the flux of catecholamines through a narrow fusion pore in a so-called foot signal that precedes rapid release indicated by an amperometric spike6. Patch Amperometry combines high-resolution patch capacitance measurements with Amperometry by placing the amperometric detector inside the patch pipet10. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines10, 11, 12, 13, 14. Thus, it is a unique tool to investigate the mechanisms that modulate quantal size and the effect of molecular manipulations affecting the properties of the fusion pore. Here we provide step-by-step instructions for the application of this method
Andrea Versari - One of the best experts on this subject based on the ideXlab platform.
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Fast Analysis of Total Polyphenol Content and Antioxidant Activity in Wines and Oenological Tannins Using a Flow Injection System with Tandem Diode Array and Electrochemical Detections
Food Analytical Methods, 2019Co-Authors: Arianna Ricci, Violeta-ivanova Petropolus, Giuseppina Paola Parpinello, Nemanja Teslic, Andrea VersariAbstract:An analytical method for simultaneous determination of total polyphenol content (TPC) and antioxidant activity (AA) of wines (white and red wines) and oenological tannins, using a flow injection system with sequential diode array and electrochemical Amperometry detectors (DAD-ECD), was proposed. The signal at 280 nm provided aggregate data for TPC. The anodic peak related to wine phenolic oxidation was scanned using pulsed integrated Amperometry over the potential of 800 mV vs. Ag/AgCl, to obtain AA. Serial dilutions avoided the poisoning at the glassy carbon (GC) electrode and the linear response obtained with both detectors was compared with spectrophotometric assays commonly used in oenology laboratory. Intraday and interday analytical repetitions showed a good repeatability and reproducibility (relative standard deviation RSD
Paul W. Bohn - One of the best experts on this subject based on the ideXlab platform.
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Capture of Single Silver Nanoparticles in Nanopore Arrays Detected by Simultaneous Amperometry and Surface-Enhanced Raman Scattering.
Analytical chemistry, 2019Co-Authors: Ju-young Kim, Donghoon Han, Garrison M. Crouch, Seung-ryong Kwon, Paul W. BohnAbstract:The attoliter volumes and confinement abilities of zero-dimensional nanopore-electrode arrays (NEAs) hold considerable promise for examining the behavior of single nanoparticles. In this work, we use surface-enhanced Raman scattering (SERS) in tandem with Amperometry in order to monitor single Ag Raman-sentinel nanoparticles transported to and captured in single nanopores. To that end, highly ordered solid-state NEAs were fabricated to contain periodic arrays of nanopores, each housing a single recessed Au-ring electrode. These were used to electrostatically capture and trap single silver nanoparticles (AgNPs) functionalized with the electrochemically stable Raman reporter, 1,4-bis(2-methylstyryl)benzene (bis-MSB). Transport and capture of the bis-MSB-tagged AgNPs in the nanopores was followed by simultaneous Amperometry and SERS signals characteristic of AgNP oxidation and enhanced Raman scattering by bis-MSB at silver–gold hot spots, respectively. The frequency and magnitude of oxidation-current spikes ...
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Capture of Single Silver Nanoparticles in Nanopore Arrays Detected by Simultaneous Amperometry and Surface-Enhanced Raman Scattering
2019Co-Authors: Ju-young Kim, Donghoon Han, Garrison M. Crouch, Seung-ryong Kwon, Paul W. BohnAbstract:The attoliter volumes and confinement abilities of zero-dimensional nanopore-electrode arrays (NEAs) hold considerable promise for examining the behavior of single nanoparticles. In this work, we use surface-enhanced Raman scattering (SERS) in tandem with Amperometry in order to monitor single Ag Raman-sentinel nanoparticles transported to and captured in single nanopores. To that end, highly ordered solid-state NEAs were fabricated to contain periodic arrays of nanopores, each housing a single recessed Au-ring electrode. These were used to electrostatically capture and trap single silver nanoparticles (AgNPs) functionalized with the electrochemically stable Raman reporter, 1,4-bis(2-methylstyryl)benzene (bis-MSB). Transport and capture of the bis-MSB-tagged AgNPs in the nanopores was followed by simultaneous Amperometry and SERS signals characteristic of AgNP oxidation and enhanced Raman scattering by bis-MSB at silver–gold hot spots, respectively. The frequency and magnitude of oxidation-current spikes increased with stepwise increases in DC voltage, and characteristic bis-MSB SERS spectra were observed. Under AC excitation, on the other hand, two distinctly different types of SERS signals were observed, independent of frequency and amplitude: (1) strong, transient (100 s) monotonically diminishing spectra. We hypothesize that the former behavior results from AgNP aggregates, whereas the latter occurs as a result of multiple incomplete AgNP-oxidation events in succession. These results show that attoliter-volume NEAs are competent for acquiring concurrent SERS spectra and for Amperometry of single nanoparticles and that together these measurements can illuminate the collision dynamics of nanoparticles in confined environments
Ricardo Borges - One of the best experts on this subject based on the ideXlab platform.
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Chromogranins as regulators of exocytosis.
Journal of Neurochemistry, 2010Co-Authors: Ricardo Borges, Jésica Díaz-vera, Natalia Domínguez, María Rosa Arnau, José D. MachadoAbstract:J. Neurochem. (2010) 114, 335–343. Abstract Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca2+. Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like Amperometry, patch-Amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca2+ in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.
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the quantal secretion of catecholamines is impaired by the accumulation of β adrenoceptor antagonists into chromaffin cell vesicles
British Journal of Pharmacology, 2010Co-Authors: Mónica S. Montesinos, David J. Machado, Marcial Camacho, Humberto O Viveros, Beatriz Beltran, Ricardo BorgesAbstract:Background and purpose: The delayed onset of certain effects of antagonists of β-adrenoceptors (β-blockers), such as lowering arterial blood pressure (several days), cannot be explained solely by their effects on β-adrenoceptors, an action that occurs within minutes. Although several mechanisms have been proposed, none of them explain this temporal delay. This work aimed at providing a new explanation based on the interference of these drugs with the functional accumulation of catecholamines within neurosecretory vesicles. Experimental approach: We used the simultaneous on-line monitoring of catecholamine and labetalol release from bovine isolated chromaffin cells and from rat perfused adrenal glands, as well as single cell Amperometry, intracellular electrochemistry, patch Amperometry and HPLC. Key results: Using Amperometry, three β-blockers, labetalol, atenolol and propranolol, reduced the quantal size of secretory events in chromaffin cells, accompanied by a slowing down of exocytosis. By patch Amperometry, we found that treatment with β-blockers also increases the chromaffin vesicle volume, thereby creating a functional dilution of catecholamines. Experiments with intracellular electrochemistry show that vesicles cannot uptake new catecholamines. There was progressive accumulation of labetalol in secretory vesicles of bovine adrenal chromaffin cells, and this β-blocker was co-released with catecholamines from rat and bovine chromaffin tissues. Conclusions and implications: We propose that β-blockers are progressively concentrated into sympathetic secretory vesicles, and interfere with the storage of catecholamines and are co-released with the natural transmitters, resulting in a decrease in the sympathetic tone. This could explain the delayed onset of the hypotensive effects of β-blockers.
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Good practices in single-cell Amperometry.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: David J. Machado, Mónica S. Montesinos, Ricardo BorgesAbstract:Single-cell Amperometry is a powerful tool for the study of the mechanisms underlying secretion from cells that release electrochemically active substances like catecholamines, histamine, or serotonin. Amperometry has changed our view of the secretory process and the quantal release phenomenon. Today, it is a relatively easy technique to set up and affordable for most laboratories. Amperometry can help solve many interesting problems in cell physiology or pharmacology. However, there are a number of issues about the experimental design, data analysis, and result interpretation that need to be considered. Here, we compile some recommendations and advice on how to conduct experiments with Amperometry, covering tissue culture, electrode types and their construction, calibration, equipment, data acquisition, and strategies for electrical noise reduction. We concentrate on cultured chromaffin cells, although most of the information is equally applicable to other cell types.
Pedram Mohseni - One of the best experts on this subject based on the ideXlab platform.
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A Wireless IC for Wide-Range Neurochemical Monitoring Using Amperometry and Fast-Scan Cyclic Voltammetry
IEEE Transactions on Biomedical Circuits and Systems, 2008Co-Authors: M. Roham, David P. Daberkow, Eric S. Ramsson, Dan P. Covey, Srirath Pakdeeronachit, Paul A. Garris, Pedram MohseniAbstract:An integrated circuit for real-time wireless monitoring of neurochemical activity in the nervous system is described. The chip is capable of conducting measurements in both fast-scan cyclic voltammetry (FSCV) and Amperometry modes for a wide input current range. The chip architecture employs a second-order DeltaSigma modulator (DeltaSigmaM) and a frequency-shift-keyed transmitter operating near 433 MHz. It is fabricated using the AMI 0.5-mum double-poly triple-metal n-well CMOS process, and requires only one off-chip component for operation. A measured current resolution of 12 pA at a sampling rate of 100 Hz and 132 pA at a sampling rate of 10 kHz is achieved in Amperometry and 300-V/s FSCV modes, respectively, for any input current in the range of plusmn430 nA. The modulator core and the transmitter draw 22 and 400 muA from a 2.6-V power supply, respectively. The chip has been externally interfaced with a carbon-fiber microelectrode implanted acutely in the caudate-putamen of an anesthetized rat, and, for the first time, extracellular levels of dopamine elicited by electrical stimulation of the medial forebrain bundle have been successfully recorded wirelessly using 300-V/s FSCV.
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a wireless ic for wide range neurochemical monitoring using Amperometry and fast scan cyclic voltammetry
International Symposium on Circuits and Systems, 2007Co-Authors: M. Roham, Pedram MohseniAbstract:An integrated circuit for real-time wireless monitoring of neurochemical activity in the central nervous system (CNS) is described. The chip is capable of conducting measurements in both fast-scan cyclic voltammetry (FSCV) and Amperometry modes for a wide input current range. The chip architecture employs a second-order sigma-delta analog-to-digital converter (SigmaDelta ADC) and a frequency-shift-keyed (FSK) voltage-controlled oscillator (VCO) operating near 433MHz. It is fabricated using the AMI 0.5mum double-poly triple-metal n-well CMOS process, and requires only one off-chip component for operation. In our preliminary measurements, a current resolution of 12pA at a sampling rate of 100Hz and 81pA at a sampling rate of 5kHz is achieved in Amperometry and 150-V/s FSCV modes, respectively, for any input current in the range of plusmn430nA. The ADC core and the VCO consume 22muA and 400muA from a 2.6-V power supply, respectively.
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ISCAS - A Wireless IC for Wide-Range Neurochemical Monitoring Using Amperometry and Fast-Scan Cyclic Voltammetry
2007 IEEE International Symposium on Circuits and Systems, 2007Co-Authors: M. Roham, Pedram MohseniAbstract:An integrated circuit for real-time wireless monitoring of neurochemical activity in the central nervous system (CNS) is described. The chip is capable of conducting measurements in both fast-scan cyclic voltammetry (FSCV) and Amperometry modes for a wide input current range. The chip architecture employs a second-order sigma-delta analog-to-digital converter (SigmaDelta ADC) and a frequency-shift-keyed (FSK) voltage-controlled oscillator (VCO) operating near 433MHz. It is fabricated using the AMI 0.5mum double-poly triple-metal n-well CMOS process, and requires only one off-chip component for operation. In our preliminary measurements, a current resolution of 12pA at a sampling rate of 100Hz and 81pA at a sampling rate of 5kHz is achieved in Amperometry and 150-V/s FSCV modes, respectively, for any input current in the range of plusmn430nA. The ADC core and the VCO consume 22muA and 400muA from a 2.6-V power supply, respectively.
