Characterization of Fast-Scan Cyclic Voltammetric Electrodes Using Paraffin as an Effective Sealant with In Vitro and In Vivo Applications

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffin embedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants.     

A spectroelectrochemical cell designed for low temperature electron paramagnetic resonance titration of oxygen-sensitive proteins

In this paper we describe an anaerobic titrator made virtually from glass with a small amount of high vacuum epoxy mounted directly to a quartz EPR tube. A complete titration may be carried out with as little as 600 microliters of sample. This cell features the anaerobic manipulation of an electrochemically poised solution from an electrochemical pouch to an EPR tube. The cell uses a gold foil working electrode and Ag/AgCl reference and counter electrodes. The reference and counter electrodes are isolated from the sample by leached Vycor glass. In the work reported here, we used this cell to determine the equilibrium redox potential of methyl viologen in an EPR titration. With methyl viologen as an indicator we found that the cell has a residual oxygen level of 1.5 microM with a leak rate of 0.005 nmol/min. After moving the solution into the EPR tube, freezing, performing EPR, and thawing, the potential of the methyl viologen solution drifted only 2 mV. During the titration, the poised potentials were stable, drifting only 1 mV/min. Formal potentials as low as -630 mV in a vitamin B12-type protein have been determined with this cell (S. R. Harder, W.-P. Lu, B. A. Feinberg, and S. W. Ragsdale (1989) Biochemistry, in press).     

Demonstration of spontaneous and stretch induced urinary bladder EMG in the living rabbit

Spontaneous bladder EMG was recorded in the living rabbit from an isovolumetric bladder without chemical or electrical stimulation. Mechanical intervention, either by lifting the bladder out of the abdomen or by rapid filling, resulted in stretch induced bladder EMG. A self made epoxy resin electrode device that embedded 32 EMG recording electrodes in a matrix like pattern, each electrode Ag/AgCl, d = 0.6 mm with an interdistance of 2.3 mm, was used for registration. The recorder used a common average reference technique and a sample frequency of 400 Hz. A signal bandwidth of 0.05 to 108 Hz was available for analysis. Spontaneous EMG consisted of single spikes and bursts (2-20 spikes), but not of continuous activity. The shape of spikes was triphasic. Single spikes appeared with and without burst activity. Small (2-5 spikes) and large bursts (6-20 spikes) were discerned; small bursts not necessarily propagated across electrodes, large bursts did and were able to organize, suggesting that they were under short neuron system control. Spontaneous EMG was probably related to both contraction and relaxation. Stretch induced EMG was characterised by continuous activity on all electrodes, spikes that followed each other immediately, slowly fading away. The spikes had an elongated third phase when compared to the shape of spontaneous activity. Highest activity and amplitudes were found after lifting the bladder out of the abdomen and placing it on the electrode device. A concept is put forward in which the continuous activity is not unequivocally related to muscle shortening, but where the current stress and strain situation of the bladder tissue can cause a muscle fibre elongation upon the appearance of electrical activity. The EMG activity found was in many aspects similar to results of a previous study using mortalized rabbits. Artifact sources like the heart, respiration, or local movement between electrode and bladder could easily be identified due to the new experimental methodology used.     

A pyrene fluorescence technique and microchamber for measurement of oxygen consumption of single isolated axons

Pyrene fluorescence is quenched by oxygen in an inverse and linear manner related to the partial pressure of O2 in solution. We have developed a microchamber for measuring QO2 of a single isolated axon, monitoring the change in fluorescence of a pyrene probe. The probe consists of a Spectra/Por dialysis hollow fiber filled with 2.5 mM pyrene in paraffin oil. The probe is inserted into a 1-mm-i.d. 2-cm-long quartz capillary tube with a freshly isolated crayfish medial giant axon. The capillary is mounted in an apparatus that forms an air- and water-tight seal except for a 0.2-mm-i.d. stainless steel tube at both ends permitting the exchange of solutions. An Olympus inverted microscope, equipped with epifluorescence optics and a 150-W xenon lamp, is used to view the preparation, generate the excitation light, and monitor the emitted fluorescence with a photomultiplier tube placed in the microscope TV port. A dichroic filter unit is utilized to select an excitation wavelength of 350 nm and collect emitted light above 420 nm. The signal is amplified with a Keithley 480 picoammeter and recorded on a strip chart. QO2 of isolated axons was 552 +/- 70 X 10(-6) mol O2/liter tissue X min. Following sequential treatment with 2 mM ouabain and 2 mM NaCN, QO2 decreased by 22 and 82%, respectively. These data are consistent with QO2 measurements of whole nerve cord made with a Clark electrode O2 monitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

ELECTRIC IMPEDANCE OF SINGLE MARINE EGGS

Alternating current impedance measurements have been made on several single marine eggs over the frequency range from 1 to 2500 kilocycles per second. The eggs were placed in the center of a short capillary made by heating the end of a 2 mm. thin walled glass tube until it nearly closed, and electrodes were placed in the sea water on each side of the egg. When it is assumed that the membrane conductance is negligible, the membrane capacity and internal resistances of unfertilized and fertilized Arbacia eggs agree with the values obtained from suspensions. Preliminary data on centrifugally separated half Arbacia eggs, and whole Cumingia and Chaetopterus eggs are given.     

Measurement of histamine in individual rat peritoneal mast cells by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector with a carbon fiber microdisk bundle electrode. In this method, individual mast cells and then 0.02 mol/l NaOH as a lysing solution are injected into the front end of the separation capillary by electromigration with an aid of a inverted microscope. A cell injector was constructed. Using it, the cell suspension was static, when a voltage for injecting single cells was applied. Histamine in single rat peritoneal mast cells have been identified. Quantitation has been accomplished through the use of calibration curves. The mean amount of histamine for nine cells is 95.8 fmol, which is consistent with the literature value.     

Recent progress in the diagnostic use of surface EMG for neurological diseases.

The different techniques to measure and analyze surface EMG are summarized with an emphasis on the clinician's point of view. The application of surface EMG in neurological disease is hampered by many inherent problems, especially the difficulties in extracting features of single motor units. However, the evolution of surface EMG from single bipolar recordings via a linear array of multiple electrodes to densely packed, multi-channel electrode arrays could in principle solve this problem. The added value of using multiple channels (up to 128) with an interelectrode distance of a few millimetres to obtain more spatial information is emphasized. At least for some muscles it is now possible to extract information from the surface EMG, conventionally thought to belong to the domain of needle EMG (for example the "electrical size" of motor units). The use of analysis techniques such as the estimation of muscle fiber conduction velocity has already proven to be of diagnostic value in several myopathies characterized by a disturbed membrane function and in metabolic myopathies with abnormal fatigue profiles. Future research should be directed at the development of analysis techniques enabling the extraction of more relevant motor unit variables from surface EMG signals.     

Impedance analysis of bio-fuel cell electrodes

To determine the criteria for the selection of an electrode suitable for a bio-fuel cell (BFC), five electrodes, i.e. silver, aluminum, nickel, stainless steel and carbon fiber cloth were investigated. The performance of the BFC according to the electrode material, including the generated voltage, current density and power density was observed. These results show that the materials used for constructing the electrodes affect the performance of the BFC. An impedance analysis was used to describe the characteristics of the electrodes in the solution. Equivalent circuits of each component such as solution, electrodes-solution interface and electrode were determined from the impedance data. The constant-phase element (CPE) model was applied for data analyzing. It was found that stainless steel, nickel and aluminum behaved like a polarized electrode which has a high electrode-solution interfacial impedance, while carbon fiber cloth and silver had a low impedance like a non-polarized electrode. The impedance data indicated that a higher interfacial impedance will result in a higher loading effect. The results can be summarized that the carbon fiber cloth electrode offers a good electron transfer in the system and thus supplies higher power to the external load.     

Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR

Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5′-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.     

Influence of cell adhesion and spreading on impedance characteristics of cell-based sensors

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.     

Progress and Prospects in Symmetrical Solid Oxide Fuel Cells with Two Identical Electrodes

Symmetrical solid oxide fuel cells (SOFCs) have attracted increasing attention due to their potential for improved thermomechanical compatibility of the electrolyte and the electrodes, reduced fabrication cost, and enhanced immunity to coking and sulfur poisoning. While the electrode materials of symmetrical SOFCs are initially limited to those with stable phase structures under both reducing and oxidizing atmospheres, many novel electrode materials are currently being developed and investigated that may undergo a beneficial phase transition or reduction in a reducing atmosphere, although the same material may be used initially for the construction of both anode and cathode. Here, the advances made in the development of electrode materials and structures for symmetrical SOFCs are summarized, including single‐phase electrodes, multi‐phase (composite) electrodes, and those that are reducible upon exposure to a reducing atmosphere. The electrical conductivity, thermomechanical properties, and redox behavior of these electrode materials, together with their performance and stability in different SOFCs, are discussed and analyzed. The problems associated with different types of symmetrical SOFCs are outlined and the materials that show promise as symmetrical electrodes are highlighted, offering critical insights and useful guidelines for knowledge‐based rational design of better electrodes for commercially viable symmetrical SOFCs.     

The automated multiparameter analyzer for cells (AMAC) IIA, a true bridge circuit Coulter-type electronic cell volume transducer.

A true bridge Coulter effect (electronic cell volume) transducer has been developed. All resistances of this bridge are now the result of current flow through saline channels. Contamination by electrode products including gas bubbles has been completely eliminated since both power electrodes are now remote from the flow chamber. Since the orifice is in series with an approximately 10 K ohm resistance generated by a gel-filled capillary and a displacement rheostat, it floats electrically, at virtual ground. The other side of the bridge consists of a fluid side-wire. Removing the power electrode from the orifice outlet makes possible downward flow and the use of a single outer sheath, and eliminates noise generated by gas bubbles which could possibly be trapped. It should now be possible to combine this design with that of the AMAC III square orifice, to produce an electro-optical sorter where all parameters are measured simultaneously. This true bridge circuit possesses the further advantage that noise due both to the power supply and to overvoltage at the power electrodes is common-mode rejected, and any drift due to changes in electrode polarization is eliminated. Preliminary experiments confirm results with the AMAC II that hemoglobinopathies can be recognized by the increased coefficient of variation (CV) of the erythrocyte spectra.     

Determination of lactate dehydrogenase isoenzymes in single rat glioma cells by capillary electrophoresis with electrochemical detection

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD(+)) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.     

Secretion of Catecholamines from Individual Adrenal Medullary Chromaffin Cells

Abstract: Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cationexchange polymer. Catecholamine secretion is induced by nicotine (10–500 μ M ), carbamylcholine (1 m M ), and K⁺ (60 m M ). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K⁺ is inhibited by Cd²⁺ (0.5 m M ), and hexamethonium selectively blocks the nicotineinduced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.     

Reconstruction of cell-electrode-adjacencies on multielectrode arrays

The multichannel recordings of signals of many cells cultivated on a multielectrode array (MEA) impose some challenging problems. A meanwhile classic problem is the separation of the recordings of a single electrode into classes of recordings where each class is caused by a single cell. This is the well-known spike sorting. A “dual” problem is the determination of the set of electrodes that record signals of a single cell. This set is called the neighborhood of the cell and has often more than one element if the MEA has a large number of electrodes with high density. A method for the reconstruction of the neighborhoods from the multichannel recordings is presented. Special effort is directed to a precise peak detection. For the evaluation of the algorithm, artificial data, obtained from an appropriate model of MEA recordings, are used. Because the artificial data provide a ground truth, an evaluation of the accuracy of the algorithm is possible. The algorithm works well for realistic parameters.     

A method for measuring bacterial chemotaxis parameters in a microcapillary

A new method was developed which enables chemotaxis parameters to be measured at a single-cell level inside a capillary for the first time. The chemotaxis chamber consists of two reservoirs communicating through a capillary tube 50 mum in diameter. Chemotaxis parameters are measured inside the capillary using image analysis, after a nearly linear attractant concentration gradient has been generated along the capillary by diffusion. Compared to previously published techniques, this method provides a well-characterized chemoattractant concentration profile in addition to allowing single-cell parameters to be measured inside a fine capillary. This procedure was used to measure the single-cell chemotaxis parameters for Escherichia coli K12, and the results are compared to published data on single E. coli cells chemotaxing in bulk. (c) 1996 John Wiley & Sons, Inc.     

Measurement of ascorbic acid in single rat peritoneal mast cells using capillary electrophoresis with electrochemical detection

In this paper, the amount of ascorbic acid (AA) in single rat peritoneal mast cell was determined by the method of capillary electrophoresis (CE) with electrochemical detection (ED) at a carbon fiber microdisk bundle electrode. The CE–ED system and the single-cell injection system were rearranged to make the operation more convenient and efficient. In the experiment, a self-made holder made of foam was used to keep the capillary from swing, which kept the stability of the baseline of the electropherogram. The single cell was lysed completely within 5 s using the 0.1% sodium dodecylsulfate (SDS) as the cell lysis solution together with the lysis voltage of 2 kV. The quantitation analysis was accomplished by the use of calibration curves, and the amount of AA in single rat peritoneal mast cell was from 2.4 to 7.1 fmol.     

Development of a novel, multi-analyte biosensor system for assaying cell division: identification of cell proliferation/death precursor events

A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.     

Observation and Investigation of “Reverse Breakdown” in a Discharge Tube

A discharge operating in a 80-cm-long discharge tube with an inner diameter of 15 mm, filled with a 3 : 1 neon–argon mixture at a pressure of 1 Torr, was investigated experimentally. Square voltage pulses with a period of 1 s were supplied to one of the tube electrodes, the second electrode being ungrounded. The initial stage of breakdown—the primary breakdown between the high-voltage (active) electrode and the tube wall, accompanied by the propagation of the prebreakdown ionization wave—was the same as in the conventional scheme with a grounded low-voltage electrode. Since the discharge gap was not closed, the discharge was not ignited. An essentially new effect was observed after the end of the voltage pulse. After a certain time interval, voltage spikes of opposite polarity, the amplitude and shape of which were close to those observed during the primary breakdown, appeared in the voltage and current waveforms of the active electrode. Simultaneously, a radiation pulse from the region adjacent to the active electrode was observed and an ionization wave began to propagate toward the second electrode. This work is dedicated to investigating this effect (which was named “reverse breakdown”) and analyzing its mechanism. A conclusion is made on the similarity of this phenomenon to the processes occurring in atmospheric-pressure dielectric barrier discharges.     

Expression of antisense to integrin subunit beta 3 inhibits microvascular endothelial cell capillary tube formation in fibrin

alpha(v)beta(3) antagonists are potent angiogenesis inhibitors, and several different classes of inhibitors have been developed, including monoclonal antibodies, synthetic peptides, and small organic molecules. However, each class of inhibitor works by the same principal, by blocking the binding of ligands to alpha(v)beta(3). In an effort to develop an alpha(v)beta(3) inhibitor that down-regulates the actual level of alpha(v)beta(3), we developed an antisense strategy to inhibit alpha(v)beta(3) expression in vitro. beta(3) antisense expressed in endothelial cells specifically down-regulated alpha(v)beta(3) and inhibited capillary tube formation, with the extent of down-regulation correlating with the extent of tube formation inhibition. This inhibition was matrix-specific, since tube formation was not inhibited in Matrigel. These findings support the notion that alpha(v)beta(3) is required for an essential step of angiogenesis in fibrin, namely capillary tube formation. These results suggest that pseudogenetic inhibition of beta(3) integrins using antisense techniques may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.