Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope

Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator.     

Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope

Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator.     

Imaging the stomatal physiology of somatic embryo-derived peanut leaves by scanning electrochemical microscopy

The stomatal physiology, chlorophyll distribution and photosynthetic activity of somatic embryo (SE)- and seedling-derived peanut plants grown in vitro (test tube-grown) and extra vitrum (soil-grown) are investigated using scanning electrochemical microscopy (SECM). This SECM imaging is performed in two different feedback modes, corresponding to oxygen evolution and chlorophyll distribution. More specifically, the oxygen evolution profiles of the in vitro leaves indicate important differences in leaf anatomy between the SE- and seedling-derived leaves. On the other hand, the chlorophyll distribution images show individual stomata of size ca. 27 ± 5μm. Further studies on senescing (aged) leaves reveal interesting voltammograms that vary widely over the stomatal complexes and the surrounding tissues, probably due to the release of electroactive metabolites during chlorophyll breakdown when the leaves turn yellow. Thus, the present investigation could open up new opportunities for characterizing botanical systems using electroanalytical techniques. In addition, it could provide further insights into various areas of current relevance, including signal transduction, cell fate/differentiation and developmental biology. Schematic representation of SECM imaging used in this investigation. The SECM probe is a Pt UME disk (25 μm diameter) embedded in an insulating glass sheath so that the ratio of the diameter of the death to that of the electrode surface (RG) is 7. RE denotes the reference electrode Ag/AgCl, sat. KCl and CE refers to the counter electrode, a Pt wire. Oxygen evolving from the leaf surface during photosynthesis diffuses into the electrolyte (0.1 M KCl) and gets reduced at the Pt UME, biased to a potential of −0.5 V, at a diffusion-limited rate to produce a change in the tip-current     

Recent advances of scanning electrochemical microscopy and scanning ion conductance microscopy for single-cell analysis

Single-cell analysis is important for understanding fundamental biological processes and mechanisms. Scanning electrochemical microscopy and scanning ion conductance microscopy as two kinds of scanning probe microscopy, with high temporal and spatial resolutions as well as in situ and noninvasive characterization capabilities, emerge as strong tools for single-cell analysis. In this review, we introduce the latest advances of scanning electrochemical microscopy and scanning ion conductance microscopy for single-cell analysis, including characterizations of cell morphology dynamics, membrane properties and mechanics, and monitoring cell surface charge, extracellular pH, and intracellular substances.     

The local electrochemical behavior of the AA2098-T351 and surface preparation effects investigated by scanning electrochemical microscopy

In this work, scanning electrochemical microscopy (SECM) measurements were employed to characterize the electrochemical activities on polished and as-received surfaces of the 2098-T351 aluminum alloy (AA2098-T351). The effects of the near surface deformed layer (NSDL) and its removal by polishing on the electrochemical activities of the alloy surface were evaluated and compared by the use of different modes of SECM. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were also employed to characterize the morphology of the surfaces. The surface chemistry was analyzed by X-ray photoelectron spectroscopy (XPS). The surface generation/tip collection (SG/TC) and competition modes of the SECM were used to study hydrogen gas (H₂) evolution and oxygen reduction reactions, respectively. H₂ evolution and oxygen reduction were more pronounced on the polished surfaces. The feedback mode of SECM was adopted to characterize the electrochemical activity of the polished surface that was previously corroded by immersion in a chloride-containing solution, in order to investigate the influence of the products formed on the active/passive domains. The precorroded surface and as-received surfaces revealed lower electrochemical activities compared with the polished surface showing that either the NSDL or corrosion products largely decreased the local electrochemical activities at the AA2098-T351 surfaces.     

On the catalytic activity of palladium clusters generated with the electrochemical scanning tunnelling microscope

Recent experiments have shown that small palladium clusters, deposited on Au(1 1 1) with a scanning tunnelling microscope, are better catalysts for the hydrogen evolution reaction than larger clusters, and may even be more active than bulk palladium. We suggest that the larger clusters contain an admixture of gold, which lowers the density of states at the Fermi level. In addition, there is charge transfer from the gold to the palladium, which induces a high electric field at the surfaces of small clusters, which favours hydrogen evolution.     

Particle analysis for a strengthened safeguards system: Use of a scanning electron microscope equipped with EDXRF and WDXRF spectrometers

The purpose of this study was twofold: the identification of some uranium compounds and a measurement of mixed U/Pu particles with different ratios of these elements. We used a Philips XL-30 scanning electron microscope equipped with an EDAX energy dispersive spectrometer with a Si(Li) detector and a super ultra-thin polymer window and with a Microspec wavelength dispersive spectrometer. A number of WDXRF and EDXRF spectra of U and Pu containing particles were accumulated and evaluated. The software package provided by the manufacturer was used for EDXRF spectra evaluation and calculation of the weight and atomic composition. Eight different U compounds were identified with a different degree of confidence. Several different types of U and Pu particles were measured using the WDXRF spectrometer and the results of the measurements are discussed. The measurement of mixed U-Pu particles showing large differences in the concentration of both elements can best be carried out with the use of WDXRF because the deconvolution of the M lines of U and Pu in the energy dispersive spectra is only possible over a relatively small concentration range. The results of particle analysis are very useful for verifying the absence of undeclared nuclear activities.     

High resolution studies of heterogeneous processes with the scanning electrochemical microscope

The focus of this review is on applications of scanning electrochemical microscopy (SECM) to studies of heterogeneous chemical processes and high resolution characterization of the solid/liquid and liquid/liquid interfaces. Recent advances in SECM imaging of surface topography and chemical reactivity are also surveyed.     

Surface interrogation mode of scanning electrochemical microscopy for oxidation study of adsorbed CO generated from serine on platinum

We report a new method for detection and oxidation of adsorbed carbon monoxide(CO_（ads）) generated from serine on a polycrystalline platinum ultramicroelectrode（UME） by bromine（Br₂） using in situ surface interrogation（SI） mode of scanning electrochemical microscopy（SECM）.In the SI mode,tip and substrate are both Pt UMEs,and CO_（ads） on Pt substrate,generated from serine,can be oxidized by the tip-generated Br₂ giving a positive response.Dosing CO_（ads） from serine instead of purging CO gas expands the newly introduced reaction of Br₂ with CO_（ads） and further enhances the hope to get rid of CO_（ads） on Pt for fuel cells.     

Mapping Cd-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy

Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd²⁺-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd²⁺ in varying concentrations. It is experimentally observed that 50 and 100 μM Cd²⁺ caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd²⁺ concentration. The Cd²⁺ was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd²⁺ stress is realized by the methodology presented.     

Imaging sequential dehydrogenation of methanol on Cu(110) with a scanning tunneling microscope

Adsorption of methanol and its dehydrogenation on Cu(110) were studied by using a scanning tunneling microscope (STM). Upon adsorption at 12 K, methanol preferentially forms clusters on the surface. The STM could induce dehydrogenation of methanol sequentially to methoxy and formaldehyde. This enabled us to study the binding structures of these products in a single-molecule limit. Methoxy was imaged as a pair of protrusion and depression along the [001] direction. This feature is fully consistent with the previous result that it adsorbs on the short-bridge site with the C-O axis tilted along the [001] direction. The axis was induced to flip back and forth by vibrational excitations with the STM. Two configurations were observed for formaldehyde, whose structures were proposed based on their characteristic images and motions.     

Temperature dependent nano indentation of thin polymer films with the scanning force microscope

The scanning force microscope (SFM) was used to investigate the temperature dependent micro mechanical properties of polymethylmethacrylate (PMMA) films with a thickness of 35 nm in the range of the radius of gyration. Force-distance curves were performed in the glass transition range to create permanent nanometric indentations with maximal forces up to 4 μN. Quantitative measurements of the indentation depth during and after application of the force, hysteresis energy and slope of the loading part are carried out as function of sample temperature and applied force. The glass transition of the polymer film can be clearly identified by the change of the mechanical properties of the polymer. Surprisingly, only a small change of elasticity at the glass transition is observed.     

Accurately measuring respiratory activity of single living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current i_p on this first scan curve is composed of i_p1, which corresponds to the cell respiratory activity and i_p2, which corresponds to the cell topography. In order to isolate the i_p2 component, the cell is killed by exposing it to 1.0 × 10⁻³ mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, i_p2 is obtained from the second SECM scan curve. Finally, i_p1 corresponding to the respiratory activity of the living cell can be accurately calculated using i_p1 = i_p − i_p2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN₃ and KCN.     

Immobilization of cytochrome P-450 and electrochemical control of its activity

P-450_cam (camphor-induced cytochrome P-450) was immobilized on an indium tin oxide (ITO) electrode by polypyrrole and its activity was controlled electrochemically. The results showed that P-450_cam was immobilized on the ITO electrode without denaturing and the amount of P-450_cam could be easily controlled. When, the electric potential was swept repeatedly between 0.4 and 0 V, the remarkable decrease of oxygen in the reaction mixture solution was observed only in the presence of camphor. In addition, hydroxycamphor was detected only in the same system by means of gas chromatography/mass spectroscopy. These results suggested that immobilized P-450_cam catalyzed the hydroxylation of camphor by the supply of electron from the electrode. The effect of pH and ionic strength on the activity was examined, and it was found that the high activity expressed at the pH of 6.0 – 7.0 and KCl concentration of 0.1 – 0.2 M . The paper is the first report that P-450 enzyme activity could be controlled artificially. © 1998 John Wiley & Sons, Ltd.     

Covalent connection of individualized, neutral, dendronized polymers on a solid substrate using a scanning force microscope

The synthesis of a neutral, high-molar-mass, acrylamide-based, third-generation dendronized polymer (denpol) with a defined number of azide groups at its periphery is reported. An attach-to route is used in which a first-generation denpol is reacted with second-generation (G2) dendrons. The degree of structure perfection of the resulting denpol is quantified as 99.8 %. This value was obtained after the introduction of a fluorescence label at the sites that remained unaffected by the dendronization. The high coverage was independently confirmed for the dendronization of another first-generation polymer and a closely related G2 dendron. The third-generation denpol resulting from the first dendronization experiment was spin-coated as a sub-monolayer onto highly oriented graphite precoated with an ultrathin layer of C12H25NH2, which was introduced to provide a well-defined substrate for denpol adsorption and manipulation. Scanning force microscopy revealed single denpols, which could be moved across the surface and "welded" by covalent cross-linking induced by photochemical decomposition of the azides into highly reactive nitrenes. The successful formation of covalent bonds between two denpols was confirmed by mechanically challenging the link with the scanning force microscope (SFM) tip. This is the second reported case of a move-connect-prove sequence using polymers and the SFM, which for the first time employs noncharged denpols, thus widening the applicability of this method significantly.     

Identification of respirable asbestos fibres by quantitative x-ray microanalysis with a scanning transmission electron microscope (STEM)

Summary Quantitative X-ray microanalysis of respirable asbestos fibres, based on the ratio method, enables the identification of different asbestos varieties in the submicrometre diameter range. A new identification scheme is proposed and applied for samples from an asbestos-cement factory and from a mining site. STEM analysis is compared with other methods of single particle analysis.

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Identifizierung von lungengängigen Asbestfasern mittels quantitativer Röntgenmikroanalyse mit einem Rastertransmissionselektronenmikroskop

Zusammenfassung Die quantitative Röntgenmikroanalyse von lungengängigen Asbestfasern im Submikrometerbereich nach der Verhältnismethode ermöglicht die Identifizierung der verschiedenen Asbestarten. Ein Schema zur Identifizierung von Asbestfasern wird vorgeschlagen und zur Charakterisierung von zwei Asbestproben angewendet. Vorteile und Nachteile der Methode in Vergleich mit anderen Methoden werden diskutiert.