Single-unit pH-sensitive double-barreled microelectrodes for extracellular use

The purpose of this study is to systematically describe the construction of pH-sensitive double-barreled microelectrodes for extracellular use. The most important advantages of these microelectrodes are as follows: the reference and the pH barrels are next to each other, and therefore the measured pH is not affected by asymmetric or slowly spreading direct current potential. The diameter of the tip of the microelectrodes is between 7 and 35 micron. These pH-sensitive microelectrodes are generally stable and Nernstian. They can be used repeatedly both in vivo and in vitro to measure tissue extracellular fluid pH. Some applications are described.     

Gold nanograin microelectrodes for neuroelectronic interfaces

Neuroelectronic interfaces are imperative in investigating neural tissues as electrical signals are the main information carriers in the nervous system and metal microelectrodes have been widely used for recording and stimulation of nerve cells. For high performance microelectrodes, low tissue-electrode interfacial impedance and high charge injection limits are essential and nanoscale surface engineering has been utilized to meet the requirements for microelectrodes. We report a single-cell sized microelectrode, which has unique gold nanograin structures, using a simple electrochemical deposition method. The fabricated microelectrode had a sunflower shape with 1–5 (m of micropetals along the circumference of the microelectrode and 500 nm nanograins at the center. The nanograin electrodes had 69-fold decrease of impedance and 10-fold increase in electrical stimulation capability compared to unmodified flat gold microelectrodes. The recording and stimulation performance of nanograin electrodes was tested using dissociated rat hippocampal neuronal cultures. Noise levels were extremely low (2.89 μV_rms) resulting in high signal-to-noise ratio for low-amplitude action potentials (18.6–315 μV). Small biphasic current pulses (20–60 μA) could evoke action potentials from neurons nearby electrodes. This new nanostructured neural electrode may be applicable for the development of cell-based biosensors or clinical neural prosthetic devices.     

Neutral carrier Na+- and Ca2+-selective microelectrodes for intracellular application.

Na+- and Ca2+-selective microelectrodes were made with Simon's neutral carrier ETH 227 and ETH 1001, respectively, and their properties were studied for intracellular application. The kNaK (selectivity coefficient for Na+ with respect to K+) values of the Na+-selective microelectrodes were in the range of 0.01-0.02, which is comparable to those of recessed-tip Na+-selective glass microelectrodes. The kNaMg values of the microelectrodes were approximately 0.005 so that the interference by intracellular Mg2+ levels could be negligible. The kNaCa values were approximately 2 and the Na+-selective microelectrodes were more selective to Ca2+ than Na+. This indicates that their intracellular application requires special care to handle Ca2+ interference under certain conditions. The kNaK, kNaMg, and kNaCa values did not depend significantly on the methods used for their determination or on the ion activity levels tested. The Nicolsky equation described well the microelectrode potentials in the mixed solutions of NaCl (1-100 mM) and KCl. Potential and resistance of the microelectrodes were stable for a long period and their response time was fast. The results indicate that the Na+-selective microlectrodes are suitable for measurements of intracellular Na ion activities. Ca2+-selective microelectrode potentials at Ca2+ concentrations lower than 10(-4) M changed significantly for the first 2-3 h and then became fairly stable. The rate of the potential change was dependent on the column length of the Ca2+-selective liquid filled. Potentials of the microelectrodes varied from 10-20 mV for Ca2+ between 10(-7) and 10(-6) M concentrations, which may be the cytosolic free-Ca2+ range. With the Ca2+ concentrations greater than 10(-6) M, the microelectrodes had potential changes of approximately 30 mV or greater for a tenfold change in Ca2+ concentration. The kCaK and kCaNa values were in the ranges of 10(-5)-10(-6) and 10(-4)-10(-5), respectively. The kCaMg values were approximately 10(-7). The results show that the Ca2+-selective microelectrodes can be used for measurements of cytosolic Ca ion activities.     

Procedures for manufacturing double-barrelled ion-sensitive microelectrodes employing liquid sensors

Liquid ion-sensitive/selective sensors are available for most inorganic ions of physiological and biochemical importance. In order to measure intracellular ionic activities in relatively small cells, it is advisable to manufacture and use double-barrelled microelectrodes. Procedures for making two types of double-barrelled ion-sensitive microelectrode are described in detail. Such microelectrodes have been used successfully to measure intracellular K+, Cl- and Na+ activities in retinal horizontal cells of fish and body-wall muscles of insect larvae.     

Electrochemical properties of Na+- and K+-selective glass microelectrodes.

Electrochemical properties of Na+-selective glass microelectrodes were studied and compared with those of K+-selective glass microelectrodes. The selectivity of Na+-selective glass microelectrodes depended on the ion concentration of test solutions. With aging, resistance of Na+-selective microelectrodes increased and their selectivity for Na over K decreased. Na+-selective microelectrodes potential measured in NaCl solution remained constant with aging, while the potential measured in KCl solution decreased and became more positive. The changes in resistance and potential of Na+-selective microelectrodes may be due to the effects of the less mobile cation, i.e., H+ or K+ on the Na ion exchange in the Na-sensing region. The results indicate that Na+-selective microelectrodes must be used as soon after filling as possible. The selectivity of Na+-selective microelectrodes increased with increase of the sensitive exposed-tip length, whereas their response time became slow due to a large recessed volume, indicating requirement of an optimum exposed-tip length for intracellular applications. The changes in the properties of Na+-selective glass microelectrodes with aging contrasted with those of K+-selective glass microelectrodes in which resistance decreased and K+-selectivity increased. The K+-selective microelectrodes required aging before use for a high selectivity and low resistance. The K+-selective microelectrodes with low resistance after sufficient aging can be used without insulation to measure K+ and Na+ activities in aqueous solutions. The different properties between Na+- and K+-selective microelectrodes are understandable, because hydration of N+-selective glass is much less extensive than that of K+-selective glass.     

Design of ionophores for ion-selective microsensors

Requirements for a reliable use of liquid membrane microelectrodes are discussed in terms of stability, response time, and lifetime on the basis of membrane technological considerations. The selectivity of H+, Li+, Na+, K+, Mg2+, Ca2+, and Cl- microelectrodes is critically evaluated using the Nikolskii-Eisenman formalism. Recent progress in the design of new ionophores is presented. A novel neutral carrier-based Ca2+-selective microelectrode with a detection limit of about 5 X 10(-10) M Ca2+ at a background of 125 mM K+ has been realized. An neutral carrier-based microelectrode for H+ with extended pH range of the sample solution is now available. Promising developments in the field of Li+-, Mg2+-, and Cl--selective ionophores are discussed.     

A thin film microelectrode array for monitoring extracellular neuronal activity in vitro

A planar array of microelectrodes has been developed for monitoring the electrical activity of neurons in cell culture. The microelectrode array was tested and characterized using impedance measurements and SEM. To verify the spatial sensitivity of the microelectrodes we used a specially developed simulation device.     

An improved method for constructing and selectively silanizing double-barreled,neutral liquid-carrier,ion-selective microelectrodes

We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ∼150 μm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix.     

Flexible polyimide probes with microelectrodes and embedded microfluidic channels for simultaneous drug delivery and multi-channel monitoring of bioelectric activity

The study of intracellular communication requires devices that can not only monitor the bioelectric activity, but also control and observe the biochemical environment at the cellular level. This paper reports on the development and characterisation of implantable polyimide microprobes that allow simultaneous, selective chemical delivery/probing and multi-channel recording/stimulation of bioelectric activity. The key component of the system is a flexible polyimide substrate with embedded microchannels that is batch-fabricated combining polyimide micromachining and a lamination technique. The devices provide platinum microelectrodes on both sides of the polyimide substrate with an active surface between 20 microm x 20 microm and 50 microm x 50 microm. The embedded microchannels permit highly localised drug delivery or probing at the tip of the device via channel outlets adjacent to the microelectrodes. The microelectrodes were characterised by electrical impedance spectroscopy and the microchannels were studied in microflow experiments. Two different fluid delivery schemes were explored in two different designs. The first device type consists of a simple combination of microchannels and microelectrodes on one substrate. Liquids are ejected at the tip of the device by pressure injection techniques. The second device was inspired by the so-called U-tube concept allowing for highly localised delivery of controlled amounts of liquids in the picoliters range. Thus, the influence of chemical compounds on the electrical activity of cells can be studied with high temporal and spatial resolution. The flexible, implantable devices can be used for studying the chemical and electrical information exchange and communication of cells in in vivo and in vitro experiments.     

Carbon Nanofiber Electrode for Neurochemical Monitoring

The ability to rapidly detect neurotransmitter release has broad implications in the study of a variety of neurodegenerative diseases. Electrochemical detection methods using carbon nanofiber nanoelectrodes integrated into the Wireless Instantaneous Neurotransmitter Concentration Sensing System (WINCS) offer many important advantages including biocompatibility, selectivity, sensitivity, and rapid adsorption kinetics. Carbon nanofiber nanoelectrodes exhibit greater selectivity and sensitivity in the electrochemical detection of neurotransmitters compared to macroelectrodes and are able to resolve a ternary mixture of dopamine (DA), serotonin (5-HT), and ascorbic acid as well as to detect individual neurotransmitters in concentrations as low as 50 nM for DA and 100 nM for 5-HT using differential pulse voltammetry. Adsorption kinetics studies and isopropyl alcohol treatments modeled on previous studies on carbon fiber microelectrodes were conducted to investigate the analogous properties on carbon nanofiber electrodes using fast-scan cyclic voltammetry with WINCS and showed analogous results in carbon nanofiber electrodes compared with carbon fiber microelectrodes.     

An improved Na+-selective microelectrode for intracellular measurements in plant cells.

The high background K+ concentration in plant cells is a problem for intracellular measurements of Na+ using ion-selective microelectrodes. The discrimination between Na+ and K+ of the microelectrode ionophore molecule limits the usefulness of this technique. A new Na+-selective microelectrode with an ionophore incorporating a tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene has been developed. Microelectrodes made with this new sensor have superior selectivity for Na+ over K+ resulting in a lower limit of detection when compared with microelectrodes made using a commercially available ionophore (ETH227). Both types of microelectrodes were insensitive to changes in ionic strength and physiological ranges of pH, but only the calixarene-based electrodes showed no protein interference. To test the suitability of the calixarene-based microelectrodes for measurements in plants, they were used to measure Na+ in epidermal cells in the zone 10-20 mm from the root apex of barley (Hordeum vulgare L.). Seedlings were grown in a nutrient solution containing 200 mM NaCl for 1-6 d. The range of intracellular Na+ activity (a(Na)) measured varied from < or =0.1 mM (limit of detection) to over 100 mM, and these values increased significantly with time. The membrane potential (E(m)) of these cells was variable, but the values became significantly more negative with time, although there was no significant correlation between E(m) and a(Na). These intracellular measurements could not be separated into distinct populations that might be representative of subcellular compartments.     

A rapid and non leaky way for preparation of the sharp intracellular recording microelectrodes

To fill microelectrodes using backfilling method needs excessive time approximately 4–6  h. It is often difficult to fill microelectrodes without damage or leakage. A main problem is bubble formation in microelectrodes which has an impact on the electrical properties of the electrode and thus it influences the quality of the recording. Based on Archimede's principle there is a force within a solution which pushes insoluble material with a lower specific gravity upward and outside of the solution. Centrifugation can increase the force to eliminate the bubbles.

We designed a microelectrode holder to protect microelectrode sensitive tips from mechanical damage due to the gravity tensions; it can help to eliminate the bubbles easily and simultaneously in 10  min or less.

The tests were performed for 2000, 4000, and 8000  rpm centrifugation each one for 3, 6 and 12  min duration respectively, it was found that the bubbles were completely eliminated at 8000  rpm for 6–12  min and there were no significant differences for resistance, and the number of leaky or damaged electrodes between the two methods.

In the new design of devices, the materials used and the design of the holder are simple and the approach is applicable to many laboratories worldwide.     

Thin-film IrOx pH microelectrode for microfluidic-based microsystems

Microsensors are valuable tools to monitor cell metabolism in cell culture volumes. The present research describes the fabrication and characterization of on-chip thin-film iridium oxide pH microsensors with dimensions of 20 microm x 20 microm and 20 microm x 40 microm suitable to be incorporated into nl volumes. IrOx thin films were formed on platinum microelectrodes by electrochemical deposition in galvanostatic mode. Anodically grown iridium oxide films showed a near super-Nernstian response with a slope of -77.6+/-2 mV/pH at 22 degrees C, and linear responses within the pH range of 4-11. Freshly deposited electrodes showed response times as low as 6s. Long-term studies showed a baseline drift of 2-3 mV/month, which could easily be compensated by calibration. This work demonstrated for the first time the use of planar IrOx pH microelectrodes to measure the acidification rate of CHO and fibroblast cells in an on chip cell culture volume of 25 nl with microfluidic control.     

Microelectrodes for studying neurobiology

Microelectrodes have emerged as an important tool used by scientists to study biological changes in the brain and in single cells. This review briefly summarizes the ways in which microelectrodes as chemical sensors have furthered the field of neurobiology by reporting on changes that occur on the subsecond time scale. Microelectrodes have been used in a variety of fields including their use by electrophysiologists to characterize neuronal action potentials and develop neural prosthetics. Here we restrict our review to microelectrodes that have been used as chemical sensors. They have played a major role in many important neurobiological findings.     

A method for marking the position of nichrome microelectrode tips in nervous tissue

A method for revealing nickel deposits from nichrome microelectrodes in the mammalian central nervous system is described. These deposits are stained red by dimethylglyoxime and can be observed directly or in Nissl stained sections. This method allows one to identify the exact position of a nichrome electrode in a microelectrode bundle chronically implanted in the brain.     

A Method for Marking the Position of Nichrome Microelectrode Tips in Nervous Tissue

A method for revealing nickel deposits from nichrome microelectrodes in the mammalian central nervous system is described. These deposits are stained red by dimethylglyoxime and can be observed directly or in Nissl stained sections. This method allows one to identify the exact position of a nichrome electrode in a microelectrode bundle chronically implanted in the brain.     

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called “concentric” ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K⁺]_o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na⁺]_o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.     

Printed circuit microelectrodes and their application to honeybee brain.

The application of printed circuit technology to the production of a new type of multi-channel microelectrode is described. 2. Recordings have been obtained from the the protocerebrum of the honeybee Apis mellifera L. using printed circuit microelectrodes in both restrained and free-moving preparations. 3. These recordings are compared with those previously obtained from conventional probe microelectrodes and are found to have similar characteristics of transient voltage changes superimposed on an undifferentiated high frequency background. 4. A wide range of development possibilities for the printed circuit microelectrode are discussed.     

Detection of extracellular action potentials in noise for the control of microelectrode advancement

A digital computer was programmed to detect impulses in the presence of noise, rather than identify or classify impulse activity from microelectrodes. The analog signal was abstracted into a sequential series of voltage time vectors that measured peak-to-peak activity. The amplitude and time difference between a peak-positive potential and the next peak-negative potential defined one vector. The amplitude and time difference between that negative peak and the next positive peak defined the next vector, and so on. An algorithm determined if each successive vector was part of a signal pattern by comparing the properties of the vector to those in a stored list. The algorithm was designed for future application with minimum computer systems and multiple-tip microelectrodes.     

Simultaneous Measurement of Intracellular pH and K+ or NO3- in Barley Root Cells Using Triple-Barreled,Ion-Selective Microelectrodes

The manufacture and use of triple-barreled microelectrodes, which are capable of simultaneous in vivo measurement of intracellular pH and the activities of K+ or NO3- and cell membrane potential (Em), are described. Scanning electron micrographs showed that the three tips were aligned and that the overall tip diameter was approximately 0.8 [mu]m. When filled with 100 mM KCl, all three barrels simultaneously reported identical transmembrane potentials, showing that all three tips were located in the same subcellular compartment. Intracellular estimates of Em in barley (Hordeum vulgare L. cv Klaxon) root epidermal cells obtained with these triple-barreled microelectrodes were indistinguishable from those obtained using single- or double-barreled microelectrodes. Measurements made with triple-barreled K+ and pH-selective microelectrodes in root cells of 7-d-old barley plants grown in a nutrient solution containing 0.5 mM K+ yielded cytosolic and vacuolar populations having mean K+ activity values of 71.3 and 68.7 mM, respectively. The associated mean pH values ([plus or minus]SE) were 7.26 [plus or minus] 0.06 (cytosol) and 5.18 [plus or minus] 0.08 (vacuole). Analysis of whole-tissue digests confirmed the microelectrode measurements. Measurements made using triple-barreled pH- and nitrate-selective microelectrodes confirmed earlier double-barreled measurements of pH and nitrate in barley root epidermal cells growing in 10 mM nitrate.