Microelectrode System Research Articles

An integrated electrochemical capillary liquid chromatography-dual microelectrode system for bromine based reaction detection.

Amperometric reaction detection of model peptides containing either a thiol or disulfide was successfully achieved with a novel coupled capillary liquid chromatography-dual microelectrode system. Detection is facilitated via dual platinum wire electrodes fabricated directly on a liquid chromatography capillary column. The detection strategy is based on the electrogeneration of bromine at the upstream working electrode followed by reduction of the bromine to bromide at the downstream working electrode. When bromine reacts with analyte eluting from the capillary column, the amount of bromine that reaches the downstream electrode decreases. As a result, the current at the downstream electrode will decrease in proportion to the amount of analyte eluting from the capillary column. Glutathione, glutathione disulfide, alpha-TGF and oxytocin were used to characterize the system and to determine system parameters. The feasibility of the determination of disulfide containing peptides is demonstrated with glutathione disulfide, alpha-TGF and oxytocin.

Trapping in AC octode field cages

Three-dimensional micro-electrode systems, fabricated by IC technology, were used for the trapping and aggregation of suspended microscopic dielectric particles. To achieve negative dielectrophoresis (repelling forces) in aqueous solution, phase-shifted RF electric fields were applied to the micro-electrodes. Reliable dielectrophoretic trapping in a liquid stream usually requires dipole forces (lowest order contribution). Due to symmetry, AC (two-phase) field cages are not closed in dipole approximation. We introduce a new AC drive of octode cages that allows efficient trapping against streaming at much lower expense than rotating (four-phase) electrode excitation. This is confirmed analytically, numerically and by experiments using latex beads.

Perspectives for microelectrode arrays for biosensing and membrane electroporation

Electrochemical microelectrode devices are among the great challenges for bioelectrochemistry, cell biology and recently also for biomedical research and new clinical electrotherapies. Two representative cases in cell biology and medical research for new trends in the technical devices are selected, heading at new diagnostic and therapeutic clinical applications. One example is from the field of biosensing cholinergic neurotransmitter substances by the nicotinic acetylcholine receptor (AcChoR) in solid-supported lipid bilayer membrane and the other one refers to new developments of electrode systems for the electrochemical delivery of drugs and genes to biological cell aggregates and tissue by the powerful method of membrane electroporation. In both cases addressed to, the new developments include the use of electrical feedback control of electrode arrays for biosensing processes as well as for the extent and duration of tissue electroporation. In line with the impressive advances in medical microsurgery, where increasingly smaller organ targets become accessible, microelectrode systems have become a continuous technical challenge for bioanalytical purposes and, as discussed here in some detail, for the new field of the electroporative delivery of effector substances like drugs and genes, using miniaturized electrochemical electrode arrays.

Electrical Properties of Metal Microelectrodes

ABSTRACT.As metal microelectrode use increases, especially in intraoperative procedures, there is a need for greater understanding of the intrinsic signal and noise properties of these electrodes. In this communication we explore some intrinsic noise levels in the electrode-amplifier system and its frequency response function. Noise levels were studied in the presence of a grounded Faraday cage, an ungrounded Faraday cage, and an open electrode-amplifier system, In the 100-2000 Hz frequency range, total noise power in the grounded Faraday cage was 44 μV, compared to 168 μV for the ungrounded Faraday cage and 525 μV without any shielding. The Faraday cage appeared to reduce most of the low frequency noise and grounding reduced mostly line noise. This noise was still in the region of 1000 times greater than the noise in other electrode-amplifier systems. The frequency response of the metal microelectrode system is fairly consistent above 1000 Hz but decreases sharply below 1000 Hz, showing approximately −15 dB between 100 and 1000 Hz. Even though these measured results are fairly mediocre compared to other electrode-amplifier systems, in practice they are still acceptable because single-cell recording amplitudes (intraoperatively, with human subjects) are on the order of 100 to 10,000 times greater than other recordings such as evoked potentials and EEG.

Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra grevilleana

We have adapted the self-referencing microelectrode technique to allow sensitive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a selective microelectrode through the gradient next to the cell wall or membrane. The electrode is moved at a known frequency and between known points. The differential electrode output values are converted into a directional measurement of flux by the Fick equation. By coupling the newly developed oxygen-selective self-referencing electrochemical microelectrode (SREM-O2) system with self-referencing ionselective proton measurements (SRIS-H+) we have characterized oxygen and proton fluxes from a single cell of the filamentous green algaSpirogyra gre illeana (Hass.). Oxygen showed a net efflux and protons showed a net influx when the cell was illuminated. These photosynthesis-dependent fluxes were found to be spatially associated with the chloroplasts and were sensitive to treatment with dichlorophenyldimethylurea. In the dark the directions of oxygen and proton fluxes were reversed. This oxygen influx was associated with mitochondrial respiration and was reduced by 78% when the cells was treated with 0.5 mM KCN. The residual cyanide-resistant respiration was inhibited by the application of 5 mM salicylhydroxamic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 μM NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O2 system to measure and characterize metabolic fluxes at a level of sensitivity that allows for subcellular resolution. These measurements, in conjunction with SERIS-H+ measurements, have led to new insights in our understanding of basic cellular physiology in plant cells.

Design of a Silicon Based Amperometric Microbiosensor Involving NAD-Dependent Dehydrogenase

An electrochemical microbiosensor involving NAD-dependent dehydrogenase has been designed and developed using silicon technology. The sensor is based on a weakly polarized two-gold microelectrode system without any reference electrode. The process allowed mass production of miniaturized devices. An enzymatic solution including diaphorase and NAD-dependent dehydrogenase was confined in the microstructure by means of a semi-permeable membrane. The biocompatibility of the various materials was verified and the gold electrodes were electrochemically characterized. The assay of D-lactic acid was taken as an example using D-lactate dehydrogenase. The sensitivity and detection limit of the microbiosensor were 8 μA mM–1 cm–2 and 0.01 mM respectively. Its lifetime was 3 weeks.

Recessed microelectrode array for a micro flow-through system allowing on-line multianalyte determination in vivo

Using CMOS-compatible processes, a microelectrode system for use in a micro flow-through cell was manufactured. The electrode was specially designed to enable multianalyte determination with immobilized oxidase enzymes and combines minimal flow dependency with a very small dead volume (< 1 μL) of the cell. This allows biomedical applications like measurements of glucose and lactate in interstitial fluid, which can be collected by ultrafiltration. Besides a 3-electrode system with 4 individually addressable platinum working electrodes, the sensor contains 2 electrodes that measure the conductivity of the sample as well as a Pt thermoresistor to measure the temperature. The temperature dependence in enzyme reactions can thus be controlled during on-line measurements. The 4 working electrodes comprise multielectrode arrays, each comprising 192 micro-holes with a diameter of 3.6 μm. They are arranged symmetrically around the central counter electrode, which is surrounded by a circular Ag/AgCl reference electrode. Between the array and the reference electrode are the loops of the Pt thermoresistor. The thermoresistor is electrically insulated from the measurement solution by a Si3N4 layer. A method for the pretreatment of platinum thin-film electrodes that increases the reversibility of the electrode process is described. The chemical modification of the working electrodes by electropolymerization of a resorcinol/1,3-diaminobenzene mixture enables interference-free measurement in blood and plasma as well as protection against electrode fouling.

A 3-D microelectrode system for handling and caging single cells and particles

We have designed and constructed several designs of 3-D microelectrode systems consisting of two layers of electrode structures separated by a 40 μm thick polymer spacer forming a flow channel. The electrode elements such as funnel, aligner, cage and switch are driven by alternating current (AC) and/or rotating electric fields. They are designed to focus, trap and separate eukaryotic cells (Jurkat) or latex particles with a diameter of 10–30 μm using negative dielectrophoresis (nDEP). The electrode width is ∼10 μm and active electrode surfaces have been minimised in order to reduce heating of the solution. A more flexible employment of the different electrode elements was realised by working with up to three generators, and/or a laboratory-made distribution box. The electrodes of the funnel, aligner or switch were operated with 5–11 V at 5–15 MHz, efficient handling of particles could be achieved with flow rates up to 3500 μm/s. Cells could be aligned effectively at flow rates up to 300 μm/s in PBS. Latex particles could be retained within the dielectric field cages (DFC) or aligner against a laminar flow of 40–200 μm/s using an amplitude of 8 V at 5–15 MHz. Cells could be held at flow rates up to 50 μm/s. Numerical calculations for dielectric forces and the induced membrane potential in field cages are given for solutions of different conductivities at different applied frequencies.

マイクロ電極システムを用いた生体材料の局所機能探索と応用

マイクロ電極システムを用いた生体材料の局所機能探索と応用

Electrochemical detection in capillary electrophoresis with dual-parallel on-capillary electrodes.

A new approach for dual electrode electrochemical detection in capillary electrophoresis (CEEC) is described. In this approach, two identical capillaries, each containing an on-capillary electrode incorporated permanently onto its tip, were paired together for simultaneous sample injection and detection. This procedure permitted dual-parallel detection to be performed without the need for painstaking alignment of the electrodes with respect to one another and to the capillary outlet as is required for the off-capillary microelectrode systems usually employed in CEEC. As a result, independent detection at two electrodes held at different potentials or at two electrodes of different composition or structure could be performed simply and with wide flexibility. Fabrication of on-capillary electrodes was carried out by sputter-coating the exit end of the capillaries with a thin layer of Au or Pt. Dual electrode system performance was demonstrated by separation and analysis of phenol and catechol samples. In addition, the detection system was coupled with glucose oxidase for the selective detection of glucose.

CO2 uptake mechanism in Eremosphaera viridis

Electrophysiological measurements of the acidophilic alga Eremosphaera viridis De Bary explored the effects of low CO2 levels on both membrane potential and resistance. This procedure incorporates a double-barreled microelectrode and suction pipette system, coupled with an approximately CO2-free environment. A key requirement is an artificial pond water perfusion media that has been purged of dissolved inorganic carbon by being boiled and bubbled with nitrogen gas. Both membrane potential and resistance were measured at pH5 in both low-CO2 conditions (2µM) and high-CO2 conditions (14µM) in both light, where CO2 transport is known to be active, and dark, where CO2 transport is not active. To avoid dissolved inorganic carbon contamination of the perfusion media, a special chamber was constructed, featuring a laminar flow of nitrogen gas over the solution, which allowed for the manipulation of cells while preventing any contamination by CO2 from the air. Results indicate that the uptake of CO2 by the alga is electrically silent and, therefore, not the result of a symport or antiport cotransport system that would "drive" CO2 uptake by coupling it to the electrochemical gradient of ions such as protons or sodium. The uptake is most likely facilitated by a transporter directly coupled with ATP hydrolysis.Key words: Eremosphaera viridis, dissolved inorganic carbon, CO2-ATPase, electrophysiology.

Use of a Microelectrode System as a Tool of the Quantitative Application of Wound to the Cell-to-Cell Junctional Membrane of Cultured Tobacco Cells

Use of a Microelectrode System as a Tool of the Quantitative Application of Wound to the Cell-to-Cell Junctional Membrane of Cultured Tobacco Cells

Aluminum resistance in the Arabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH.

A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H+ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H+ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H+ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H+ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.

Greatly increased mucosal nitric oxide in ulcerative colitis determined in situ by a novel nitric oxide-selective microelectrode.

Although current nitric oxide (NO) electrodes are simple, selective and sensitive, they are fragile and hard to use in clinical studies of patients. By preparing an improved NO electroneedle that overcomes these defects, we directly measured mucosal NO concentrations in 11 patients (six male, five female; mean 26.0 years old) with ulcerative colitis (UC) and five normal volunteers (three male, two female; mean 28.3 years old) in situ. An electroneedle was inserted into colonic mucosa through a biopsy channel during colonoscopy. The information concerning the concentration of NO generated and the appearances of the colonic mucosa at the same site were obtained simultaneously. In the ulcerative colitis patients, NO concentrations were significantly increased at all 24 mucosal sites tested. These included sites where: there was an absence of visible inflammation (five sites); the mucosa was mildly inflamed (eight sites); the mucosa was moderately inflamed (five sites); or severely inflamed (six sites). The NO concentrations in ulcerative colitis patients were 12-72 times higher than the NO levels in normal controls (10 sites). At the same 10 sites in four ulcerative colitis patients, the high NO concentrations were decreased by 53% after glucocorticoid treatment. These data are consistent with those of previous studies utilizing different NO electrodes. Excess mucosal NO is generated from inducible NO synthase in the inflamed mucosa itself and the invading inflammatory cells. Our results suggested that mucosal NO could be a marker for the extent of inflammation and its various actions correlated with the pathogenesis, natural history and prognosis of UC. Using the NO microelectrode system reported here, the concentration of NO generated can be monitored in real-time while observing the mucosal condition at the same site during endoscopy. This novel NO electrode may contribute to understanding the role of NO in colonic mucosal inflammation.

In vivo measurement of the electrical impedance of cell membranes of tobacco cultured cells with a multifunctional microelectrode system

The effects of electric field on living cells are mostly concentrated on cell membranes. In order to evaluate these effects quantitatively, it is necessary to estimate the electric potential intensity actually working on the cell membrane concerned. In this study, a high-performance impedance measuring system has been developed using a multifunctional microelectrode (MME) system and applied to the in vivo analysis of electrical impedance of cell membranes of tobacco cultured cells, line BY-2. A cell membrane in contact with the external medium (CM EXT), and an intercellular membrane (CM INT) were analysed according to the linear circuit models composed of ohmic resistances and capacitances. The ohmic resistances of MMEs, CM EXT and CM INT were estimated as 300 MΩ, 1.7 GΩ, 4–760 MΩ, respectively. On the other hand, respective capacitances were 27 pF, 68 pF, and 80 pF. The ohmic resistance of CM INT was smaller and less stable than that of CM EXT. These results indicate promising properties of the present system as an experimental tool for the in vivo analysis of electric field intensity actually working on the cell membranes.

Manipulation and characterization of living cells using microelectrode systems

Manipulation and characterization of living cells using microelectrode systems

Recent advances in heterogeneous TiO2 photocatalysis

Current progress in the area of photocatalysis is presented, particularly regarding technological applications. Highly efficient TiO2 films on different substrates such as tile and glass have been developed for indoor environmental clean-up. TiO2 films coated on SiO2-precoated soda lime glass showed about 80% transparency and high photocatalytic activity towards the decomposition of thin oil films. A novel phenomenon, superhydrophilicity, has been observed on these transparent TiO2 coatings. In addition, we have made use of a microelectrode system to monitor oxidation and reduction products separately. The mechanistic and kinetic aspects of TiO2 photocatalysis are discussed.

Theoretical calculation of collection efficiencies for collector-generator microelectrode systems

Abstract Steady-state mass transport is considered in a ‘collector-generator’ system of microelectrodes, in which an electroactive species is generated at the surface of one microelectrode (the ‘generator’) and is transported by diffusion to the surface of another (the ‘collector’), where it reacts. Following its generation, the species may also be consumed by a homogeneous first-order reaction. Using a form of Green's theorem, the total current at the collector is obtained as an integral over the surface of the generator, in which the integrand contains the surface current density, multiplied by a weighting function that can be determined by solving a reaction-diffusion problem for the collector microelectrode alone. Therefore, the collection efficiency may be calculated without solving the detailed transport problem in the presence of both microelectrodes. As an illustration of these ideas, the collection efficiency is calculated numerically as a function of the rate constant of the homogeneous reaction, in the mathematically simplest case, where the current density on the surface of the generator is uniform. Three geometrical configurations are considered: (i) both collector and generator are circular disc microelectrodes of equal radii; (ii) the collector is a disc and the generator a concentric thin ring and (iii) the collector is a thin ring and the generator a concentric disc. Comparison with asymptotic approximations suggests that the collection efficiency is fairly insensitive to the distribution of current at the generator. Similar results are therefore to be expected in the more general situation of a non-uniform current density at the generator. This remains true if generation is enhanced by species produced at the collector or by the homogeneous reaction.

Micro-extraction spectrometer structures for voltage contrast in the scanning electron microscope

This work aims at the achievement of efficient extraction of secondary electrons from the surface of the specimen, with the least possible corruption of the information which they contain. The method adopted is to use a system of microelectrodes very close to the conductor under investigation to extract the electrons and to act as a retarding-field spectrometer system. In the present case, the first extraction electrode structure was added to the integrated circuit specimen at an appropriate point by the use of focused ion beam technology.

Micro-extraction spectrometers for voltage contrast in the SEM

This work was directed at the achievement of efficient extraction of all secondary electrons from the surface of the specimen, with the least possible corruption of the information which they contain. The method adopted is to use a system of microelectrodes very close to the conductor under investigation to extract the electrons and to act as a retarding-field spectrometer system. It is applicable both to conventional SEMs and those where the specimen is immersed in the magnetic field of the final lens.