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A sub-second fast Fourier transform-adsorptive voltammetric technique for the nano-level determination of guthion at a gold microelectrode in flowing solutions.

This research demonstrates the quick guthion monitoring with the help of a sensitive method called fast Fourier transformation continuous cyclic voltammetry (FFTCV). Fortunately, FFTCV illustrates the benefits of precision, determination speed, cost-effectiveness, accuracy and simplicity, in comparison with formerly reported techniques. In particular, this method was applied to a gold microelectrode in flowing solutions to detect the guthion concentration in its formulations. The effects of several parameters were examined regarding the sensitivity of the method. After a series of experiments, the detection limit of the method was found to be equal to 1.27 pg/mL, when the optimum conditions were imposed, which is a scan rate value of 40V/s, an accumulation time of 0.4s, an accumulation potential of 0mV and a pH value of 2. During the measurements performance, the integration range of currents included all the potential scan ranges, even the oxidation and reduction of the Au surface electrode, for the achievement of a sensitive determination. Then, the potential waveform, consisting of the potential steps for cleaning, accumulation and the step for the potential ramp, was applied to an Au disk microelectrode in a continuous way. It is also important to refer to the positive points, presented only by the use of this technique. Firstly, it is no longer necessary to remove the oxygen from the test solution. Furthermore, the quick determination of any such compound in many chromatographic methods is possible. Thirdly, the corresponding detection limit is of nanomolar level.

Azinphosmethyl↗

Methods for isolating extracellular action potentials and removing stimulus artifacts from microelectrode recordings of neurons requiring minimal operator intervention.

Recent successes in treating neurological disorders with electrical stimulation of the brain have spurred interest in studying the neuronal mechanisms by which such therapies work. However, microelectrode recordings can be confounded by stimulation artifact. Also, large microelectrode arrays now allow recording amounts of data that would otherwise overwhelm current analytic methods that depend heavily on human intervention and interpretation. A set of algorithms is described for automatically removing stimulus artifacts that minimize signal loss with minimum human involvement. Other algorithms automatically differentiate between the extracellular action potentials of individual neurons.

Action Potentials↗

Ion conducting polymer microelectrodes for interfacing with neural networks.

We have examined the stimulation and recording properties of conjugated polymer microelectrode arrays as interfaces with neural networks of dissociated cortical cells. In particular the stimulation properties were investigated as a means of supplying a neural network with information. The stimulation efficiency at low stimulation voltages was evaluated and referenced to bare indium tin oxide (ITO) electrodes. The polymer electrodes were electrochemically polymerized from a blend of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) and ethylenedioxythiophene (EDOT) onto ITO microelectrodes. Dissociated cortical cells were then plated on the electrodes and cultivated to form neural networks. Polymer electrode stimulation evoked a much greater response from the network than stimulation from ITO electrodes. Neural interfaces using polymer electrodes could be maintained for several months.

Action Potentials↗

Spatiotemporal pH dynamics following insertion of neural microelectrode arrays.

Insertion trauma is a critical issue when assessing intracortical electrophysiological and neurochemical recordings. Previous reports document a wide variety of insertion techniques with speeds ranging from 10 microm/s to 10 m/s. We hypothesize that insertion speed has an effect on tissue trauma induced by implantation of a neural probe. In order to monitor the neural interface during and after probe insertion, we have developed a silicon-substrate array with hydrous iridium oxide microelectrodes for potentiometric recording of extracellular pH (pH(e)), a measure of brain homeostasis. Microelectrode sites were sensitive to pH in the super-Nernstian range (-85.9 mV/pH unit) and selective over other analytes including ascorbic acid, Na(+), K(+), Ca(2+), and Mg(2+). Following insertion, arrays recorded either triphasic or biphasic pH(e) responses, with a greater degree of prolonged acidosis for insertions at 50 microm/s than at 0.5 mm/s or 1.0 mm/s (p<0.05). Spatiotemporal analysis of the recordings also revealed micro-scale variability in the pH(e) response along the array, even when using the same insertion technique. Implants with more intense acidosis were often associated histologically with blood along the probe tract. The potentiometric microsensor array has implications not only as a useful tool to measure extracellular pH, but also as a feedback tool for delivery of pharmacological agents to treat surgical brain trauma.

Acidosis↗

Correspondence of microelectrode mapping with magnetic resonance imaging for subthalamic nucleus procedures.

BACKGROUND: Magnetic resonance imaging (MRI) and microelectrode recording (MER) are commonly used to guide stereotactic procedures on the subthalamic nucleus (STN). Little is known about the correlation between the position of the STN as seen on MRI and that as determined by MER mapping. We compared these in 10 patients with Parkinson's disease. METHODS: The position of the STN was determined by intraoperative MER findings and stereotactic axial T2 magnetic resonance images with 2-mm slice thickness. Images were reconstructed in a 3-dimensional workstation. The anterior, posterior, medial, lateral, dorsal, and ventral borders of the STN defined with the MRI were measured relative to the midcommissural point. The location of STN activity during MER was reconstructed relative to the midcommissural point for comparison. RESULTS: Twenty-nine tracks recorded with microelectrodes provided clear spans of STN-like activity in 18 STN nuclei. The coordinates of MER were, in general, within the borders of the STN defined with the MRI. However, when analyzed individually, some of the tracks had STN-like activity outside the borders of the MRI-defined nucleus (mostly <1 mm). Three tracks had STN-like activity recorded between 2 and 3 mm more anterior than the anterior border of the nucleus defined with the MRI. CONCLUSIONS: There was a good correlation between MER and the borders of the STN defined in the MRI, except for the anterior-posterior axis, in which MER indicated that the STN extended more anteriorly than as suggested by MRI. This should be taken into account in STN surgery.

Action Potentials↗

On microelectrode ionophoresis.

An equation for ionophoresis in large tip microelectrodes is derived from Nernst-Planck equations for the general case of a completely dissociated electrolyte. The relation between the release of ions and the applied electric current is mainly determined by two parameters: the transference number of the ions under consideration and the diffusional leak of the microelectrode. Also it is shown how the release of ions is affected by the concentration of the electrolyte within the electrode and that of the external solution. The equation describes the ionophoretic release of polyvalent spermine. In addition, new equations for tip potential and for tip resistance are derived.

Iontophoresis↗

Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes.

Ammonium ion and proton concentration profiles near the surface of a planar bilayer lipid membrane (BLM) generated by an ammonium ion gradient across the BLM are studied by means of microelectrodes. If the concentration of the weak base is small compared with the buffer capacity of the medium, the experimental results are well described by the standard physiological model in which the transmembrane transport is assumed to be limited by diffusion across unstirred layers (USLs) adjacent to the membrane at basic pH values (pH > pKa) and by the permeation across the membrane itself at acidic pH values. In a poorly buffered medium, however, these predictions are not fulfilled. A pH gradient that develops within the USL must be taken into account under these conditions. From the concentration distribution of ammonium ions recorded at both sides of the BLM, the membrane permeability for ammonia is determined for BLMs of different lipid composition (48 x 10(-3) cm/s in the case of diphytanoyl phosphatidylcholine). A theoretical model of weak electrolyte transport that is based on the knowledge of reaction and diffusion rates is found to describe well the experimental profiles under any conditions. The microelectrode technique can be applied for the study of the membrane permeability of other weak acids or bases, even if no microsensor for the substance under study is available, because with the help of the theoretical model the membrane permeability values can be estimated from pH profiles alone. The accuracy of such measurements is limited, however, because small changes in the equilibrium constants, diffusion coefficients, or concentrations used for computations create a systematic error.

Ammonia↗

Two-microelectrode voltage clamp of Xenopus oocytes: voltage errors and compensation for local current flow.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The most common method for their electrophysiological investigation is the two-microelectrode voltage clamp technique. The quality of voltage clamp recordings obtained with this technique is poor when membrane currents are large and when rapid charging of the membrane is desired. Detailed mathematical modeling of the experimental setup shows that the reasons for this weak performance are the electrical properties of the oocytes and the geometry of the setup. We measured the cytosolic conductivity to be approximately 5 times lower than that of the typical bath solution, and the specific membrane capacitance to be approximately 6 times higher than that of a simple lipid bilayer. The diameter of oocytes is typically approximately 1 mm, whereas the penetration depth of the microelectrodes is limited to approximately 100 microm. This eccentric current injection, in combination with the large time constants caused by the low conductivity and the high capacitance, yields large deviations from isopotentiality that decay slowly with time constants of up to 150 micros. The inhomogeneity of the membrane potential can be greatly reduced by introducing an additional, extracellular current-passing electrode. The geometrical and electrical parameters of the setup are optimized and initial experiments show that this method should allow for faster and more uniform control of membrane potential.

Animals↗

Intracellular microelectrode studies of Necturus antral mucosa. Effect of aspirin on cell membrane potentials.

Intracellular microelectrode techniques were used to determine the effects of luminal aspirin on epithelial cell membrane potentials of Necturus antral mucosa. In this tissue, prolonged stable intracellular impalements were obtained with 15-50-M omega microelectrodes filled with 3 M KCl. In acidic mucosal solution (pH 4.0) the addition of aspirin at 5.0 mM resulted in a significant decrease of apical cell membrane potential (Vmc) from -36.8 +/- 2.2 to -22.2 +/- 2.1 mV (p less than 0.001) and basolateral cell membrane potentials (Vcs) from -38.8 +/- 1.7 to -25.3 +/- 2.1 mV (p less than 0.001). Upon removal of aspirin from the mucosal solution, both cell membranes hyperpolarized for a brief period before returning to their original potentials. In neutral mucosal solutions (pH 7.0), addition of aspirin (5.0 mM) resulted in a significant increase in apical cell membrane potential (Vmc) from -40.0 +/- 2.4 to -46.8 +/- 3.3 mV (p less than 0.001) and basolateral cell membrane potential (Vcs) from -41.5 +/- 2.0 to -49.7 +/- 2.5 mV (p less than 0.001). This hyperpolarization of the cell was associated with an increase in transmucosal potential from -1.5 +/- 1.8 to -2.9 +/- 1.8 mV (p less than 0.001) and an increase in the ratio of apical to basolateral membrane resistances from 2.2 +/- 0.4 to 3.1 +/- 0.7 (p less than 0.05). These changes in membrane potentials and the ratio of membrane resistances may be caused by a change in ionic conductance of the cell membranes induced by aspirin.

Animals↗

A technique to prevent dural adhesions to chronically implanted microelectrode arrays.

Minimizing relative movements between neural tissues and arrays of microelectrodes chronically implanted into them is expected to greatly enhance the capacity of the microelectrodes to record from single cortical neurons on a long-term basis. We describe a new surgical technique to minimize the formation of adhesions between the dura and an implanted electrode array using a 12 microm (0.5 mil) thick sheet of Teflon film positioned between the array and the dura. A total of 15 cats were implanted using this technique. Gross examination of 12 implant sites at the time of sacrifice failed to find evidence of adhesions between the arrays and the dura when the Teflon(R) film remained in its initial position. In six implants from which recordings were made, an average of nine of the 11 (81%) connected electrodes in each array recorded evoked neural activity after 180 days post implantation. Further, on average, two separable units were identified on each of the implanted electrodes in these arrays. No significant change was found in the density of cell bodies around implanted electrodes of four of the implanted electrode arrays. However, histological evaluation of the implant sites revealed evidence of meningeal proliferation beneath the arrays. The technique described is shown to be effective at preventing adhesions between implanted electrode arrays and improve the characteristics of chronic recordings obtained with these structures.

Animals↗

Effect of cocaine, nomifensine, GBR 12909 and WIN 35428 on carbon fiber microelectrode sensitivity for voltammetric recording of dopamine.

Electrochemical measurements using voltammetry or amperometry at carbon-fiber microelectrodes have been used in vitro and in vivo to examine regulatory mechanisms for the central dopamine system. In many of these experiments, dopamine efflux concentrations under control conditions are determined followed by their alterations in response to a drug treatment. The present study demonstrates that some drugs can affect dopamine measurements, not only by their expected pharmacological action but also by directly altering the microelectrode responsivity. The commonly used reuptake inhibitors GBR 12909 (10 microM) and nomifensine (5 microM) drastically reduce electrode sensitivity and, in the case of nomifensine, increase the time to reach a plateau in response to dopamine boluses (i.e. reduced 'frequency response'). Cocaine (10 microM) and WIN 35428 (2 microM) have negligible effect on these indices. This decrease in sensitivity was found in both nafion and non-nafion coated electrodes. Further, the reduction in sensitivity seen in non-nafion coated electrodes was not prevented by increasing the reversal potential (from +1.0 to +1.3 V) and voltage scan rate (from 350 to 450 V/s). These data suggest that care must be taken when interpreting data from voltammetric or amporometric experiments using carbon electrodes where GBR 12909 or nomifensine are used, especially at high concentrations. Furthermore, wherever possible, direct effects of a drug on electrode sensitivity and frequency response should be determined.

Animals↗

Carbon fiber microelectrodes with multiple sensing elements for in vivo voltammetry.

Electrically evoked dopamine release was monitored in the striatum of anesthetized rats using voltammetric microelectrode assemblies with two to four separately addressable carbon fiber sensing elements. The sensing elements were disk-shaped, had a diameter of about 1 microm, and were separated from each other by less than 15 microm. The microelectrodes were used to monitor extracellular dopamine at multiple depths beneath the brain surface during electrical stimulation of the medial forebrain bundle. When the sensing elements were 10-15 microm apart, the stimulus responses at each element were distinct, suggesting that each response was representative of a distinct recording site. The possibility of performing measurements at distinct but closely spaced sites provides a potential route to high spatial resolution information about extracellular events. On the other hand, when the individual sensing elements were about 1 microm apart, similarities between the observed stimulus responses suggest that the multiple elements were recording from a single site. The ability to perform multiple chemical measurements at a single site presents several opportunities for new approaches to the in vivo study of neurochemistry.

Animals↗

Rapid coating of glass-capillary microelectrodes for single-electrode voltage-clamp.

The single-electrode voltage-clamp technique requires sharp glass-capillary microelectrodes, whose electrical properties often limit the capabilities of the recording system. Here, we describe a rapid and simple way of coating fine microelectrodes with Dricote and Vaseline that improves their performance during voltage-clamp. The coating prevented clogging of the tips, improved the capacitance compensation of the electrodes, helped to seal the electrode tips into cell membranes and allowed visualization of the tips under saline solution. This new coating method led to greatly improved recordings and better characterization of the transduction and voltage-activated currents in an isolated preparation of spider mechanosensory neurons.

Animals↗

Multi-unit recording from regenerated bullfrog eighth nerve using implantable silicon-substrate microelectrodes.

Multi-microelectrode silicon devices were developed for extracellular recording from multiple axons in regenerated eighth cranial nerves of American bullfrogs. Each includes a photolithographically defined array of holes and adjacent metal microelectrodes. A device is implanted within a transected eighth nerve; regenerating fibers grow through the holes en route to the brainstem. Multiple spike trains were recorded from two animals at up to 21 weeks after implantation. Single units were tracked for over 8 h. Some responded to sound with tuning typical of fibers innervating the amphibian and basilar papillae. Units of vestibular origin also were recorded. Action potentials were 30-140 microV P-P amid noise of 5 10 microV RMS, an adequate signal-to-noise ratio for spike detection and sorting. Histology confirmed that bundles of myelinated fibers grew through holes near electrodes that recorded activity. The implantation success rate was low, due to surgical morbidity, device extrusion, and lack of nerve regeneration through some devices. Future designs will address these issues and incorporate transistor amplifiers on devices to increase signal-to-noise ratios. The potential of implanted silicon devices to simultaneously record from many axons offers an opportunity for multicellular studies of auditor, vestibular and seismic signal processing in the vertebrate inner ear.

Acoustic Stimulation↗

Properties of electrolyte-filled glass microelectrodes: a model analysis.

A novel dynamic mathematical microelectrode model (a model of solvent and solute kinetics in electrolyte-filled microelectrodes) was deduced from experimental observations made on standard (single-barrelled, 3.0 M KCl-filled, approximately 10 M[ohm]) electrodes using (a) electrodiffusion, electro-osmosis, and continuity equations that were placed into the constraints of electrode geometry, and (b) handbook/textbook parameter values, only. The model proved to be able to faithfully reproduce all observed electrochemical and electrical electrode properties, i.e. even those that constituted no part of the model's experimental basis. In theoretical tests, the model shows, for the standard electrode that (a) inside the electrode, any profiles in electrical potential and electrolyte concentration are occurring at the most distal part (approximately 50 microm) of the tip region, (b) asymmetrical shifts in electrolyte concentration just inside the electrode tip opening are the true cause of the electrode's current rectification, and (c) strong transelectrode currents are producing water flows across the electrode orifice that may affect the volume of smaller and medium-sized cells. In further tests, the model shows, among other things, for non-standard electrodes that (a) decreasing the electrode electrolyte concentration will give rise to marked decreases in electrolyte leakage from the electrode, but only very minor changes in tip potential, and (b) increasing the surface charge of the electrode glass (increases in zeta potential) and/or decreasing the electrode electrolyte concentration will produce increases in electro-osmotic water transport, which may be desirable for the intracellular injection of water-soluble (electro-neutral) substances.

Electrochemistry↗

A new technique for measuring the temporal characteristics of the carbon fibre microelectrodes in in vivo voltammetry at millisecond time intervals.

A common approach to test the time-response of carbon fibre microelectrodes entails the use of a flow injection system which allows delivery of a bolus of test compound in the flow stream to a detector electrode. The introduced bolus is distorted from its original form by convective and dispersive forces during the period of transport from the injector to the detector electrode in the connection tubing and couplings. Thus, the flow injection system allows one to change the medium in the vicinity of the electrode in about 100-200 ms. We describe here a simple falling drop system, which is an easy to use for calibration of the carbon fibre microelectrodes with respect to their sensitivity and time-response. This system does not have a loop injector and minimizes problems associated with convective and dispersive bolus distortion. It allows one to change the medium in the vicinity of the electrode in less than 10 ms. With this system, the sensitivity and the temporal resolution of the electrodes may be easily and rapidly estimated in a single experiment. The temporal resolution of the untreated, uncoated carbon fibre cylinder electrode of 300 microns tip length in response to dopamine was estimated as 1.49 +/- 0.1 microM/ms (M +/- SEM, n = 36, rise) and 0.051 +/- 0.003 microM/ms (n = 36, wash-out) on the half of amplitude of electrochemical response which was monitored by constant potential amperometry.

Animals↗

Measurements of net fluxes and extracellular changes of H+, Ca2+, K+, and NH4+ in Escherichia coli using ion-selective microelectrodes.

This study introduced the use of a non-invasive ion-selective microelectrode (MIFE) technique to study membrane-transport processes in bacteria. Net ion fluxes and changes in the extracellular concentrations of H+, Ca2+, K+ and NH4+ in adherent bacteria, isolated from cultures at different growth stages (exponential, late exponential, and stationary phases), were monitored. With the exception of Ca2+, a significant (P=0.05) difference was found in the magnitude of net fluxes of the ions measured from bacterial cells at different stages of the population growth curve. The magnitude of the H+ response was glucose-dependent with maximum changes occurring at the highest concentration. There was a progressive increase in H+ extrusion followed by a gradual return to zero at late stationary phase. Measurements of net ion fluxes crossing the bacterial cytoplasmic membrane, demonstrated here for the first time, may offer insight into underlying mechanisms of ion transport kinetics. Applications of the non-invasive ion-selective microelectrode technique in microbiology are discussed.

Calcium↗

Comparison between the comet assay and the oxygen microelectrode for measurement of tumor hypoxia.

BACKGROUND AND PURPOSE: Hypoxic cells are present in some solid tumours and are known to limit radiocurability. To compare two measures of tumour hypoxia, 25 patients with locally advanced disease and accessible tumours or metastatic nodes were examined using an oxygen microelectrode and the alkaline comet assay. MEASUREMENTS AND METHODS: For the comet assay, fine needle aspirate biopsies were taken immediately following a dose of 5-10 Gy. Single cells were examined for radiation-induced DNA strand breaks, and the percentage of radio-resistant hypoxic cells within the population was calculated from DNA damage histograms. For oxygen tension (pO2) measurements, multiple tracks were made using an Eppendorf oxygen microelectrode. The possibility that application of the first method might influence hypoxic fraction measurement by the second method was examined in a more controlled system by creating four tracks in murine SCC-VII tumours using an oxygen electrode, and measuring hypoxic fraction at subsequent times. RESULTS: For 28 tumours from 25 patients, hypoxic fraction measured by comet assay correlated with the percentage of PO2 values < 5 mmHg (r2 = 0.46, P < 0.001). The mean comet hypoxic fraction was 0.36 for five tumours with a median PO2 < 10 mmHg. For the remaining 23 tumours with a median PO2 > 10 mmHg, the mean hypoxic fraction was 0.09. Advancement of an oxygen electrode through SCCVII tumours had no significant effect on hypoxic fraction measured 5 min to 24 h later using the alkaline comet assay. CONCLUSIONS: Tumours defined as hypoxic based on a median pO2 < 10 mmHg appear to contain more than 20% radio-biologically hypoxic cells as estimated by the comet assay. In an animal tumour model, puncture of the tumour with an oxygen electrode did not influence hypoxic fraction measured using the comet assay, in agreement with the clinical data that the order in which the two methods were performed was not important.

Animals↗