Sonochemically fabricated microelectrode arrays for biosensors--part II. Modification with a polysiloxane coating

A polymer-modified sonochemically fabricated glucose oxidase microelectrode array with microelectrode population densities of up to 2.5 x 10(5) microelectrodes per square centimetres is reported. These microelectrode sensors were formed by first depositing an insulating film on commercial screen printed electrodes which was subsequently sonicated to form cavities of regular sizes in the film. Electropolymerisation of aniline at the microelectrode cavities formed polyaniline protrusions containing entrapped glucose oxidase. Chemical deposition of polysiloxane from dichlorodimethysilane was used to deposit a thin protective and diffusion mass transport controlling coating over the electrodes. The physical and electrochemical properties of these films were studied. The performance of the final glucose oxidase based microelectrode sensor array is reported.     

Comparison of three methods for measuring electrical resistances of plant cell membranes

The reliability of two different membrane resistance-measuring methods that use a single intracellular microelectrode was tested against a conventional method that uses two intracellular microelectrodes. The first single-electrode method used a single square current pulse and required a constant microelectrode resistance. This method was unreliable because the electrode resistance changed markedly on cell penetration and changed with time within the cell. The second method used a high frequency square wave for injecting current into the cell and depended upon the membrane having a much longer RC (resistance × capacitance)-time constant than the microelectrode. The resistance values obtained by this latter method were usually different from membrane resistances obtained at the same time on the same cells using two intracellular microelectrodes. Therefore, neither single intracellular microelectrode method was as reliable as the conventional method. All tests were with coleoptile cells of Avena sativa var. Victory.     

Stable membrane potentials and mechanical K+ responses activated by internal Ca2+ in HeLa cells

Stable membrane potentials have been measured with microelectrodes in HeLa cells and their average value (-38 mV) compares well with the Cl- electrochemical potential (-34 mV). A hyperpolarizing response is observed upon microelectrode penetrations and following a mechanical or electrical shock; this response is due to a large increase in the K+ permeability, which can also be triggered by Ca2+ injection or blocked by EGTA injection through the microelectrode. Stable hyperpolarizations are obtained with the Ca2+ ionophore A 23187.     

Resistive artifacts in liquid-ion exchanger microelectrode estimates of Na+ activity in epithelial cells.

In experiments on the rabbit urinary bladder epithelium we have identified an electrical artifact in certain liquid ion-sensitive microelectrodes. This artifact arises from the high electrical resistance of the ion-sensitive resins which in some cases are comparable to the resistance of the microelectrode glass wall. For Na+-sensitive microelectrodes this situation led to shunting of the exchanger potential and consequently artifactually high calculations of intracellular Na+ in the rabbit urinary bladder epithelium. A method for minimizing this shunting effect is described. After reduction of the shunt the frequency response of the Na+-sensitive microelectrode was increased and the estimated ai Na+ was decreased to 7 mM.     

Voltage clamping with a single microelectrode.

A technique is described which allows neurons to be voltage clamped with a single microelectrode, and the advantages of this circuit with respect to conventional bridge techniques are discussed. In this circuit, the single microelectrode is rapidly switched from a current passing to a recording mode. The circuitry consists of: (1) an electronic switch; (2) a high impedance, ultralow input capacity amplifier; (3) a sample-and-hold module; (4) conventional voltage clamping circuitry. The closed electronic switch allows current to flow through the electrode. The switch then opens, and the electrode is in a recording mode. The low input capacity of the preamplifier allows the artifact from the current pulse to rapidly abate, after which time the circuit samples the membrane potential. This cycle is repeated at rates up to 10 kHz. The voltage clamping amplifier senses the output of the sample-and-hold module and adjusts the current pulse amplitude to maintain the desired membrane potential. The system was evaluated in Aplysia neurons by inserting two microelectrodes into a cell. One electrode was used to clamp the cell and the other to independently monitor membrane potential at a remote location in the soma.     

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.     

CMOS microelectrode array for the monitoring of electrogenic cells

Signal degradation and an array size dictated by the number of available interconnects are the two main limitations inherent to standalone microelectrode arrays (MEAs). A new biochip consisting of an array of microelectrodes with fully-integrated analog and digital circuitry realized in an industrial CMOS process addresses these issues. The device is capable of on-chip signal filtering for improved signal-to-noise ratio (SNR), on-chip analog and digital conversion, and multiplexing, thereby facilitating simultaneous stimulation and recording of electrogenic cell activity. The designed electrode pitch of 250 microm significantly limits the space available for circuitry: a repeated unit of circuitry associated with each electrode comprises a stimulation buffer and a bandpass filter for readout. The bandpass filter has corner frequencies of 100 Hz and 50 kHz, and a gain of 1000. Stimulation voltages are generated from an 8-bit digital signal and converted to an analog signal at a frequency of 120 kHz. Functionality of the read-out circuitry is demonstrated by the measurement of cardiomyocyte activity. The microelectrode is realized in a shifted design for flexibility and biocompatibility. Several microelectrode materials (platinum, platinum black and titanium nitride) have been electrically characterized. An equivalent circuit model, where each parameter represents a macroscopic physical quantity contributing to the interface impedance, has been successfully fitted to experimental results.     

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.     

Direct electrochemistry of microperoxidase at Pt microelectrodes modified with carbon nanotubes

Direct electrochemistry of microperoxidase (MP-11) was found at Pt microelectrodes modified with multi-wall carbon nanotubes (MWNTs). The MWNTs used as the immobilization matrix cooperatively promote the bioactivity of MP-11. When MP-11 was immobilized on MWNTs film-modified Pt microelectrodes, a pair of well-defined redox waves was obtained. The resulted stable microelectrode could be used to catalyze the reduction of H2O2 and O2.     

Electrochemical Properties of Hydrated Cation-Selective Glass Membrane: A Model of K+ and Na+ Transport

Electrochemical properties of cation-selective glass microelectrodes made from NAS_27-04 were studied. There was a marked fall in electrical resistance of the microelectrodes stored in 3 M KCl solution (aging). The resistance was in the range of 2 × 10⁷ to 10⁹ Ω, which were much lower than those estimated from the electrical resistivity of dry glass for the equivalent dimensions of microelectrode working tips. This fall in resistance was accompanied by an increase in microelectrode selectivity for K⁺. The low resistance and increased K⁺ selectivity are desirable features that make the microelectrode more suitable for application to biologic studies. The changes in microelectrode resistance and selectivity were interpreted to be due to hydration of the entire thickness of the glass membrane, resulting in a change in the field strength of anionic sites and formation of ionic channels in the glass membrane. Thus, the fall in resistance is explained by decrease in energy barrier, which is equivalent to the activation energy of interaction between the cations and anionic sites in the glass membrane. Some of the microelectrodes showed a transient depolarization that resembled the action potential of a biological membrane. This transient depolarization was associated with the changes in microelectrode resistance and selectivity. The transient depolarizations suggest the temporary development of wide channels in the membrane permitting free movement of hydrated cations according to the bulk electrochemical gradient.     

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.     

Measurement of the Cytoplasmic pH in Nitella translucens: Comparison of Values Obtained by Microelectrode and Weak Acid Methods

A comparison has been made between the use of two types of pH microelectrode and the weak acid method for determining the cytoplasmic pH of Nitella translucens at an external pH of 6. There was good agreement between the value obtained with glass pH microelectrodes (7.54 +/- 0.15 se) and that obtained using the weak acid 5,5-dimethyloxazolidine-2,4-dione (7.42 +/- 0.07 se). Plastic-insulated antimony microelectrodes gave a significantly lower value (6.74 +/- 0.15 se) possibly due to disruption of the insulation by the cell wall. The addition of 1 mM NaN(3) rapidly reduced the pH recorded by the glass pH microelectrodes to about 5.3. A smaller change was observed using the weak acid method. The relevance of this observation to recent work on indoleacetic acid transport is discussed.     

Influence of the first amplifier stage in MEA systems on extracellular signal shapes

Recording of extracellular signals with planar metal microelectrodes (ME) has already been presented more than 30 years ago. To date, microelectrode array (MEA) systems are able to measure extracellular signals at about 64 sites, simultaneously. This enables monitoring of electrical activity of many cells in a large area. The extracellular recording technique has become a widely used method for neurological, toxicological or pharmacological studies. It already proved its potential to supplement the classical methods in electrophysiology. The interpretation of the recorded signal shapes in order to extract electrophysiological meaningful data--however--is still under discussion. In this article, we analyse the preamplifier circuit for extracellular recording of cardiac myocyte signals. We use a circuit model for the cell-electrode contact including the first amplification stage. In test experiments, we observe different signal shapes, when different shunt resistors are introduced at the input of the preamplifier. According to the frequency spectra of the recordings, we evaluate the transfer function between the source signal and the readout signal. As a result of our studies, an optimum readout electronics for originally, preserved extracellular signal shapes is proposed. Our amplifier design will be most valuable, if the use of small microelectrodes with high input impedances for in vitro as well as for in vivo experiments is desired.     

Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.     

Comparative measurements of potassium and chloride with ion-sensitive microelectrodes and x-ray microanalysis in cultured skeletal muscle fibers

Summary Data of the intracellular electrolyte concentration of potassium and chloride in cultured muscle cells measured by x-ray analysis were compared by using the different activity coefficients with intracellular potassium and chloride activities measured with double-barrelled microelectrodes. By using an activity coefficient of 0.6, 95% of the potassium microelectrode measurements are in accordance with the x-ray analysis values, in spite of a scattering of the values. Membrane potential and intracellular potassium values are linearly related. x-ray analysis and ion-sensitive microelectrodes measured the cytoplasmic chloride in the same range. Taking into account known activity coefficients, an error of 25% must be assumed with the intracellular chloride measurements. However, x-ray analysis and ion-sensitive microelectrode investigations are reliable tools to study intracellular potassium and chloride changes, which play an important role in membrane characteristics.     

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.     

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 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 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.     

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.