Detection of Nitric Oxide Release from Single Cells by Using Constant‐Distance‐Mode Scanning Electrochemical Microscopy

Işık, Sonnur; Schuhmann, Wolfgang

doi:10.1002/anie.200601708

Cited by 65 publications

(51 citation statements)

References 41 publications

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“…The measurements were performed in Hepes-based saline solution (10 mM Hepes, 150 mM NaCl, 4.2 mM KCl, and 11.2 mM glucose; pH 7.4). In this experiment, we used relatively large electrodes (a ¼ 6.0 μm) to detect neurotransmitter effectively, based on the rationale outlined in earlier work for both NO and neurotransmitter detection (43)(44)(45)(46)(47)(48). The carbon electrode was simply aligned optically over the cell and positioned one radius above the cell surface using the reduction current at a potential of −1.0 V, due to oxygen reduction, which was found to provide reliable distance control.…”

Section: Resultsmentioning

confidence: 99%

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Takahashi

Shevchuk

Novák

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

206

224

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We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.high-resolution imaging | living cell imaging | noninvasive | constant-distance mode S canning electrochemical microscopy (SECM) uses an electrode tip for detecting electroactive chemical species and is an effective tool for the investigation of the localized chemical properties of sample surfaces and interfaces (1). Because SECM has high temporal resolution and can be used under physiological conditions, it is particularly well suited for quantitative measurements of (short-lived) chemicals like neurotransmitters, nitric oxide, reactive oxygen species, and oxygen, which are released/ consumed by living cells. In conventional SECM, the probe is often micrometer scale and the probe vertical position is kept at a constant height, a plane, during probe scanning. If the sample topography is not flat, the electrode-sample separation changes during scanning, complicating the SECM measurement and its analysis.Various methods for SECM electrode miniaturization and control of the electrode-sample separation have been advocated in order to improve the resolution of SECM imaging. The reliable fabrication of nanoelectrodes with a small ratio of electrodeinsulation to active electrode (

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Section: Resultsmentioning

confidence: 99%

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Takahashi

Shevchuk

Novák

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

206

224

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“…The topographic and chemical information about the electrochemical processes taking place at a given site of a single-cell can be obtained from the faradaic currents measured at the working ultramicroelectrode directly or after additional modification using redox mediators. Main requirements for the successful accomplishment of such electrochemical measurements are accurate placement of the tip of microelectrode(s), precise determination of the tip-to-cell distance, and the appropriate selectivity of the ultramicroelectrodes for species that may change in concentration as a result of various cellular actions [58,59].…”

Section: Electrochemistrymentioning

confidence: 99%

Redox regulation of calcium ion channels: Chemical and physiological aspects

et al. 2011

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a b s t r a c tReactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease.Changes in the intracellular free Ca 2+ concentration are key triggers for diverse cellular functions. Ca 2+ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological-and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca 2+ permeable ion channels such as voltage-gated (CaV) Ca 2+ channels, transient receptor potential (TRP) channels and Orai channels.© 2011 Elsevier Ltd. All rights reserved. Basic redox chemistry of oxygenMany chemical processes are linked to the exchange of electrons between two or more molecular entities. The transfer of one (or more) electron(s) is associated with oxidation (loss of electron) and reduction (gain of electron) of the components. Such reactions are also known as redox reactions. The processes of reactive oxygen species (ROS) formation and elimination are exclusively of redox nature.Molecular oxygen is the precursor of all ROS. It contains two unpaired electrons in separate (antibonding) orbitals in its outer electron sphere and is thus, by definition, a radical. Radicals have at least one unpaired electron in their orbital system and usually show high reactivity; transition metal ions also may have unpaired electrons, but are not called radicals. Although having radical character, molecular oxygen is chemically rather inert (luckily!), because high * Corresponding authors at: Institut für Biophysik, Gebäude 58, Universität des Saarlandes, D-66421 Homburg/Saar, Germany. Tel.: +49 6841 1626453; fax: +49 6841 1626060.E-mail addresses: ivan.bogeski@uks.eu (I. Bogeski), barbara.niemeyer@uks.eu (B.A. Niemeyer).

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“…Although SECM has been used over the years for investigating biological systems, 23,24 only recently has attention been given to SECM for single cell studies. [25][26][27][28][29][30][31][32][33][34][35][36][37][38] Mirkin et al 26,27,[32][33][34] have investigated single cells by SECM in order to determine various redox activities across different types of cells, examining the differences between nonmetastatic and metastatic human breast cells. They have identified series of redox mediators that can cross the cell membrane, investigated their distribution between cytosol and nucleus, and found them unable to penetrate the nuclear envelope.…”

Section: Introductionmentioning

confidence: 99%

Glucose and Lactate Biosensors for Scanning Electrochemical Microscopy Imaging of Single Live Cells

et al. 2008

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We have developed glucose and lactate ultramicroelectrode (UME) biosensors based on glucose oxidase and lactate oxidase (with enzymes immobilized onto Pt UMEs by either electropolymerization or casting) for scanning electrochemical microscopy (SECM), and have determined their sensitivity to glucose and lactate, respectively. The results of our evaluations reveal different advantages for sensors constructed by each method: improved sensitivity and shorter manufacturing time for hand-casting, and increased reproducibility for electropolymerization. We have acquired amperometric approach curves (ACs) for each type of manufactured biosensor UME, and these ACs can be used as a means of positioning the UME above a substrate at a known distance. We have used the glucose biosensor UMEs to record profiles of glucose uptake above individual fibroblasts. Likewise, we have employed the lactate biosensor UMEs for recording the lactate production above single cancer cells with the SECM. We also show that oxygen respiration profiles for single cancer cells do not mimic cell topography, but are rather more convoluted, with a higher respiration activity observed at the points where the cell touches the Petri dish. These UME biosensors, along with the application of others already described in the literature, could prove to be powerful tools for mapping metabolic analytes, such as glucose, lactate and oxygen, in single cancer cells.

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Detection of Nitric Oxide Release from Single Cells by Using Constant‐Distance‐Mode Scanning Electrochemical Microscopy

Cited by 65 publications

References 41 publications

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Redox regulation of calcium ion channels: Chemical and physiological aspects

Glucose and Lactate Biosensors for Scanning Electrochemical Microscopy Imaging of Single Live Cells

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