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PubMed2000

Perspectives for microelectrode arrays for biosensing and membrane electroporation.

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

Biosensing Techniques↗

PubMed1999

Characterization of rat spinal cord neurons cultured in defined media on microelectrode arrays.

Previous efforts to utilize mammalian spinal cord neurons as biosensor elements have relied on neuronal: glial co-cultures maintained in serum-containing media. We have examined the feasibility of culturing primary spinal cord neurons in serum-free medium, modified for neuronal longevity, on fabricated microelectrode arrays. Embryonic day 15 rat spinal cord cells were plated on trimethoxysilyl-propyldiethylenetriamine coated microelectrode arrays comprised of gold recording sites passivated with silicon nitride. Immunocytochemistry was performed to verify the presence of neurons and quantitatively assess astrocytes using antibodies against glial fibrillary acidic protein on the silicon nitride substrates. Modifications to culture media enabled viable neuronal culture to extend from approximately 14 days in vitro (DIV) to 40 DIV on the arrays containing only 1.1 +/- 0.5% (mean +/- SEM) astrocytes. Extracellular recording revealed tetrodotoxin-sensitive spontaneous electrical activity from the enriched neuronal culture. Threshold detection of extracellular potentials showed an increase in spike rate as a function of glutamate concentration with neurotoxicity at elevated levels. This approach suggests that functional measures related to biosensor applications, pharmacological screening, or the evaluation of neurological disease models can be implemented in a defined culture system.

Action Potentials↗

PubMed1999

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Animals↗

PubMedDate not supplied

Correspondence between visually evoked voltage-sensitive dye signals and synaptic activity recorded in cortical pyramidal cells with intracellular microelectrodes.

Fast, multiple-site optical recording of voltage-sensitive dye (VSD) signals and intracellular microelectrode recordings were combined to characterize visually evoked neuronal responses in the visual cortex of the pond turtle, Pseudemys scripta. By using an in vitro, eye-brain preparation stained with the merocyanine oxazolone voltage-sensitive dye, NK-2495 or a close analog, NK-2761, large VSD signals relatively free of vibrational noise could be recorded in single trials following a stroboscopic light flash to the contralateral eye. VSD signals recorded from the same cortical location in repeated trials exhibited considerable variability in the onset, duration, and amplitude of secondary depolarizations. Because of this variability, secondary depolarizations were largely absent in signal-averaged responses. Superposition of VSD signals with intracellular recordings obtained from cortical pyramidal cells revealed a close correspondence between their signal waveforms. The two signals were virtually identical in their onset, initial rate of rise, and time-to-peak. At later periods (> 500 ms), the correspondence was less close, especially for large cortical depolarizations. Some of this disparity could be attributed to contamination of the VSD signal by a large intrinsic optical response. A second contribution was a failure of the VSD signal to register asynchronous regenerative effects occurring in single pyramidal cells. It is suggested that the close correspondence between the microelectrode and optical recordings in the early phase of the response may reflect the organization of pyramidal cells into clusters that receive virtually identical synaptic inputs.

Animals↗

PubMed1992

Theoretical and experimental investigation of steady-state voltammetry for quasi-reversible heterogeneous electron transfer on a mercury oblate spheroidal microelectrode.

A mercury microelectrode formed by electroreduction of mercury on an inlaid gold microdisk is experimentally shown to be well modeled by oblate spheroidal geometry when the ratio of the semiminor axis to the semimajor axis of the protruding drop is less than 1. The validity of the geometry is established by comparison of the experimentally determined coefficient in the steady-state diffusion current equation with the theoretical value for oblate spheroidal geometry. Spherical cap geometry is also shown to be an equally valid geometric model; however, theoretical treatment for this system is more difficult. The theory of a quasi-reversible electrode process is developed and applied to the determination of the electrode parameters of the RuIII(NH3)6/RuII(NH3)6 electrode reaction on a mercury oblate spheroidal microelectrode. Results agree well with others found in the literature for the same process on a mercury electrode.

Electrons↗

PubMed1993

Dehydrogenase-modified carbon-fiber microelectrodes for the measurement of neurotransmitter dynamics. 2. Covalent modification utilizing avidin-biotin technology.

Dehydrogenase enzymes are immobilized onto carbon-fiber microelectrode surfaces via avidin-biotin technology and a covalently linked hydrophilic tether. The avidin-biotin coupling strategy allows the selectivity of the electrochemical measurement to be easily changed without reoptimizing the immobilization conditions. Optimized derivatization conditions are demonstrated for dimer, tetramer, and hexamer dehydrogenases. Nonelectroactive substrates (ethanol, glucose-6-phosphate, and glutamate) are quantitated through the detection of enzyme-generated NADH by fast scan cyclic staircase voltammetry (100 V/s). The glutamate dehydrogenase-modified microelectrode possesses a 300-ms response time with a detection limit of 0.5 mM glutamate and a 1-60 mM linear concentration range.

Avidin↗

PubMed1994

Characterization of the chemical architecture of carbon-fiber microelectrodes. 3. Effect of charge on the electron-transfer properties of ECL reactions.

Two techniques, cyclic voltammetry and the microscopic imaging of electrochemically generated chemiluminescence (ECL), have been used to evaluate the effectiveness of various electrochemical pretreatments on the electron-transfer properties of carbon-fiber microelectrodes. The surfaces of carbon-fiber microelectrodes were electrochemically treated to produce different levels of surface oxides in the following manner: after normal polishing and cleaning in hot toluene and water, the carbon surface was activated by applying a cyclic potential from -0.2 to 2.0 V at a frequency of 50 Hz for 3 s in solutions of varying pH: 1M HCl, pH 7.4 and pH 12.0 phosphate buffers. Cyclic voltammetry was employed to elucidate the effect of the surface pretreatment on the overall voltammetric properties of the different pretreatment methods. The ECL emission intensity was imaged with resolution on the submicrometer scale with a conventional fluorescence microscope equipped with a cooled, slow-scan CCD camera. In addition to investigating pretreatment effects with luminol, we have also examined the ECL properties of a positively charged species, ruthenium tris (2,2'-bipyridine) dichloride hexahydrate, whose luminescent properties are also well documented. Such information is not only invaluable for the rational design of surface-modified ultramicroelectrodes, but it can also yield considerable information concerning the surface interactions influencing organic electron-transfer reactions at carbon surfaces.

Carbon↗

PubMed2003

A thin-layer amperometric sensor for hydrogen sulfide: the use of microelectrodes to achieve a membrane-independent response for Clark-type sensors.

An electrochemical cell design of the Clark type including a thin layer of electrolyte in contact with a microelectrode has been successfully applied for the determination of sulfide utilizing its electrochemically initiated reaction with aqueous diethyl-p-phenylenediamine. The analytical parameters obtained were independent of the membrane used to separate the inner chamber and the outside sulfide-containing solution. The independence arises since the thickness of the diffusion layer associated with the microelectrode is small and, in contrast to the conventional macroelectrode Clark electrode, does not impinge on the membrane. This provides an improvement in gas sensor design and development as it obviates the need for membrane calibration and should simplify the application of Clark cells for variable-temperature measurements.

Electrochemistry↗

PubMed2005

Microelectrode biosensor for real-time measurement of ATP in biological tissue.

The purines ATP, ADP, and adenosine are important extracellular signaling agents. Analysis of purinergic signaling has been slowed by lack of direct methods for measurement of purine release in real-time during physiological activity. We have previously reported microelectrode biosensors for adenosine, but similar sensors for ATP have remained elusive. We now describe an ATP biosensor formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3-phosphate oxidase. It responds rapidly (10-90% rise time <10 s) and exhibits a linear response to ATP over the physiologically relevant concentrations of 200 nM-50 microM and is very sensitive approximately 250 mA.M(-1).cm(-2). By including phosphocreatine kinase in the biolayer, we can optionally amplify the ATP signal and also make the sensor sensitive to external ADP. We have used our sensors to make the first demonstration that ATP is released from spinal networks in vivo during locomotor activity.

Adenosine Diphosphate↗

PubMed2005

Chlorinated phenol analysis using off-line solid-phase extraction and capillary electrophoresis coupled with amperometric detection and a boron-doped diamond microelectrode.

The analysis of chlorinated phenols (2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in river water was accomplished using off-line solid-phase extraction (SPE) and capillary electrophoresis coupled with electrochemical detection. A key to the sensitive, reproducible, and stable detection of these pollutants was the use of a boron-doped diamond microelectrode in the amperometric detection mode. An off-line SPE procedure was utilized to extract and preconcentrate the pollutants prior to separation and detection, with ENVI-Chrom P, a highly cross-linked styrene-divinylbenzene copolymer, being employed as the sorbent. Pollutant recoveries in the 95-100% range with relative standard deviations of 1-4% were achieved. The diamond microelectrode provided a low and stable background current with low peak-to-peak noise. The oxidative detection of the pollutants was accomplished at +1.05 V vs Ag/AgCl without the need for electrode pretreatment. The method was evaluated in terms of the linear dynamic range, sensitivity, limit of quantitation, response precision, and response stability. A reproducible electrode response was observed during multiple injections of the chlorinated phenol solutions with a relative standard deviation of < or =5.4%. Good electrode response stability was observed over many days of continuous use with no significant electrode deactivation or fouling. The separation efficiencies for all six pollutants were greater than 170,000 plates/m. The minimum concentration detectable for all six ranged from 0.02 to 0.2 ppb (S/N > or = 3) using a 250:1 preconcentration factor.

Boron↗

PubMed2005

Chip-based microelectrodes for detection of single-nucleotide mismatch.

Microelectrode arrays having eight 10-microm-diameter gold microelectrodes arranged on a gold-covered Si chip were designed and characterized. The chips prove useful for the detection of single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy.

Base Pair Mismatch↗

PubMed2006

Controlling the electrochemically active area of carbon fiber microelectrodes by the electrodeposition and selective removal of an insulating photoresist.

A new and simple method permits control of the electrochemically active area of a carbon fiber microelectrode. An electrophoretic photoresist insulates the 10-microm-diameter carbon fiber microelectrodes. Photolysis of the photoresist followed by immersion of the exposed area into a developing solution reveals electroactive carbon fiber surface. The electroactive surface area exposed can be controlled with a good degree of reproducibility.

Carbon↗

PubMed2006

Microelectrode array biochip: tool for in vitro drug screening based on the detection of a drug effect on dopamine release from PC12 cells.

Novel, yet simple detection techniques of drug effect, including the effect of a vesicular monoamine transporter inhibitor (reserpine), a dopamine precursor (L-dopa), and a dopamine transporter inhibitor (nomifensine), on dopamine release from dopaminergic PC12 cells were developed based on a microelectrode array (MEA) biochip. Upon multi-injections of KCl solution into the culture of PC12 cells attached on a MEA biochip, the K+-stimulated dopamine release was temporally and amperometrically recorded by biochip microelectrodes. Two parameters in the recorded amperometric spectra were defined in this study: the peak current of the first KCl injection (Max1), and the steady current after the fourth KCl injection (St4). Statistically significant effects of L-dopa and reserpine were demonstrated by comparing both Max1 and St4 of the second detections in drugs with those of the control without drug treatment. The values of both Max1 and St4 in the first detections were normalized as 1. In contrast, the statistically significant effect of nomifensine was detected by comparing the ratios of St4 to Max1 in the first detections in drug with those of the control. The reason for using different analytical methods for measurements between L-dopa/reserpine and nomifensine lies in the different mechanisms of action on PC12 cells among these drugs. The novel analytical methods developed use the same detection setup and parameters, and the data analysis for the effect of drugs becomes simple. The methods hence may provide a high-throughput in vitro drug screening approach for dopamine-related psychiatric disorders.

Animals↗

PubMed1996

Microelectrodes for the measurement of catecholamines in biological systems.

Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-micron diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that high-pass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo.

Animals↗

PubMed1997

Indium as internal standard in square wave anodic stripping analysis of lead in blood with microelectrode arrays.

The toxic effects of lead on humans, especially children have been well documented. ASV has been an important technique in the analysis of lead in blood of humans. This research has demonstrated that indium(III) can be used as an internal standard in the analysis of lead in blood samples with mercury film microelectrode arrays. Indium is a good choice because of a low endogenous blood concentration and because baseline separation of anodic stripping peaks among Cd, In, and Pb can be achieved under appropriate conditions. The concentration of sodium bromide strongly influences the resolution of these metals and the sensitivity of ASV to In(III) in solution. Square wave anodic stripping voltammetry together with screen-printed microelectrode arrays showed that the ratio of the anodic stripping peak currents of Pb and In varies linearly with the concentration of Pb in blood samples ranging from 1.2 to 30.0 micrograms/dL. The average intraassay precision (rsd) was 6.7%.

Electrochemistry↗

PubMed1998

A laser ablation method for the spatial segregation of enzyme and redox sites on carbon fiber microelectrodes.

A laser-generated interference pattern was used to remove enzyme from micrometer-wide stripes on an enzyme-covered carbon fiber microelectrode surface to create regions of facile electron transfer. Fluorescence microscopy was used to visualize fluorophore-tagged enzyme to indicate where the adsorbed enzyme remained on the surface. The electrochemical kinetics of the carbon fiber surface were examined to see if electron-transfer sites could indeed be segregated from enzyme adsorbed across the entire surface. CCD imaging of the electrochemical luminescence of Ru(bpy)3(2+) was used to verify the segregation between photoablated sites (with facile electron-transfer kinetics) and surfaces with adsorbed enzyme (which exhibit slow electron-transfer kinetics). The laser-ablated surface could also be distinguished from the enzyme-covered carbon surface with atomic force microscopy. Thus, photoablation of the surface of a protein-covered carbon fiber microelectrode with an interference pattern generated by a Nd:YAG laser allows the activation of 1.7-micron-wide bands of the electrode surface (available for facile electron transfer) while leaving 2.6-micron-wide enzyme-modified areas intact, thereby producing electroactive regions directly adjacent to enzyme modified regions of the same surface.

Biosensing Techniques↗

PubMed1999

Myoglobin-containing carbon-paste enzyme microelectrodes for the biosensing of glucose under oxygen-deficit conditions.

The response of first-generation glucose oxidase (GOx) amperometric glucose biosensors is strongly dependent on the concentration of the oxygen cosubstrate. The incorporation of the natural oxygen binder myoglobin into a GOx-containing carbon-paste matrix is shown to satisfy the oxygen demand of the enzymatic reaction and to provide convenient biosensing of glucose in oxygen-free solutions. Such use of myoglobin-containing mineral oil thus offers an attractive alternative to the use of oxygen-rich fluorocarbon pasting liquids. Further improvements are observed upon doping the fluorocarbon oil with myoglobin. Factors affecting the oxygen independence of the new enzyme microelectrodes, including the myoglobin loading or length of the oxygen reservoir, have been optimized. The myoglobin-doped mineral oil or Kel-F-based carbon-paste enzyme microelectrodes display a highly stable glucose response over prolonged (6-7 h) operations in oxygen-free solutions, indicating no depletion of the internal oxygen supply.

Biosensing Techniques↗

PubMedDate not supplied

Two-barrel bile-acids-sensitive microelectrodes based on liquid ion exchanger.

Several liquid membrane microelectrodes sensitive to bile acids (two barrel, tip diameter about 0.5 micron) are described. The results of different liquid ion exchangers such as Aliquat 336/decanol, trioctylmethylammonium/decanol, hexadecyltrimethylammonium/decanol, benzyldimethylhexadecylammonium/decanol, hexadecyltributylammonium/5% hexachlorobenzene + 0.5% bromoacetanilide in o-dichlorobenzene are compared with each other, and the better one among them is the mixture of benzyldimethylhexadecylammonium cholate/decanol with hexadecyltributylammonium taurocholate/5% hexachlorobenzene + 0.5% bromoacetanilide in o-dichlorobenzene because of its quicker response time and low drift. The calibration curves, slopes, test limits, selective coefficients, drifts, and response times of the various bile-acids-sensitive microelectrodes in different calibration solutions were demonstrated and compared with each other.

Bile Acids and Salts↗