Generation of free radicals and electrochemiluminescence from simple aromatic molecules in aqueous solutions.

Oxide-covered aluminium electrodes were used to demonstrate that aromatic compounds, such as the simple derivatives of benzene, can be electrochemically excited at cathodically pulse-polarized conductor/insulator/electrolyte (C/I/E) tunnel junction electrodes (e.g. oxide-covered aluminium electrodes). The primary cathodic process at these electrodes was a tunnel emission of hot electrons into an aqueous electrolyte solution. Fluorescence (FL) and electrochemiluminescence (ECL) spectra were compared and the dependence of the electrochemiluminescence on the concentrations of benzene, toluene, phenol, p-cresol and aniline were measured and detailed mechanisms for the present electrochemiluminescence are proposed.     

The influence of acidity on microbial fuel cells containing Shewanella oneidensis

Microbial fuel cells (MFCs) traditionally operate at pH values between 6 and 8. However, the effect of pH on the growth and electron transfer abilities of Shewanella oneidensis MR-1 (wild-type) and DSP10 (spontaneous mutant), bacteria commonly used in MFCs, to electrodes has not been examined. Miniature MFCs using bare graphite felt electrodes and nanoporous polycarbonate membranes with MR-1 or DSP10 cultures generated >8W/m(3) and approximately 400muA between pH 6-7. The DSP10 strain significantly outperformed MR-1 at neutral pH but underperformed at pH 5. Higher concentrations of DSP10 were sustained at pH 7 relative to that of MR-1, whereas at pH 5 this trend was reversed indicating that cell count was not solely responsible for the observed differences in current. S. oneidensis MR-1 was determined to be more suitable than DSP10 for MFCs with elevated acidity levels. The concentration of riboflavin in the bacterial cultures was reduced significantly at pH 5 for DSP10, as determined by high performance liquid chromatography (HPLC) of the filter sterilized growth media. In addition, these results suggest that mediator biosynthesis and not solely bacterial concentration plays a significant role in current output from S. oneidensis containing MFCs.     

An all-solid-state amperometric ethanol sensor

An all-solid-state pellet electrode for ethanol determination has been developed and compared with the membrane-layered sensor. It is found that the new pellet electrodes have reliable responses (current vs. concentration) to ethanol from 0.1 to 10 m with response times of about 2 min. The current response decreases about 10% h⁻¹ during continuous operation, compared with a drop of 50% h⁻¹ with normal membrane electrodes. Fresh electrodes can be stored in a freezer for 2 weeks without apparent activity loss. The measurement procedure is convenient and it is probable that reproducible, disposable electrodes can be made at low cost. This format is general and can easily be extended to many other systems with β-NAD⁺/NADH as coenzyme.     

Evaluation of different strategies for the development of amperometric biosensors for L-lactate

Two strategies were investigated for the development of lactate biosensors based on sol-gel matrixes and polysulfone composite films, both containing L-lactate dehydrogenase (LDH). Firstly, reagentless disposable screen-printed electrodes (SPE's) with Meldola's Blue (MB) and the cofactor NAD(+) inside a sol-gel matrix were prepared. These showed relatively low sensitivities (260 microA/M). Secondly, mediator-modified-polysulfone-graphite composite films deposited over both cylindrical epoxy-graphite and SPE's. These electrodes showed enhanced performance characteristics: improved sensitivity (80 mA/M), detection limit (0.87 microM) and reproducibility (2%). Reagentless electrodes, incorporating NAD(+) in the polysulfone film, had a decreased sensitivity, although better than that achieved by the sol-gel electrodes. While sol-gel electrodes showed a linear range between 1.25 x 10(-4) and 2.48 x 10(-3)M, the epoxy-graphite composite electrodes based on polysulfone composite films allowed the detection of lactate at a linear range of lower concentrations from 1 x 10(-6) to 1.2 x 10(-5)M. Finally, the performance of the LDH-MB-polysulfone-composite film-based SPE's in a flow system was studied. Short response times were obtained (t<30s). Furthermore, repeatability and reproducibility values were notably improved, especially when working with electrodes covered with a polyamide layer prepared with N-(2-aminoethyl)-piperazine.     

Amperometric enzyme electrode for determination of theophylline in serum.

This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxidation of theophylline by the haem-containing enzyme theophylline oxidase. Results are presented for two approaches. First, ferrocene monocarboxylic acid was used as a mediator. The second-order rate constant was 1.1 x 10(3) 1 mol-1 s-1. Secondly, the organic conducting salt NMP.TCNQ was used to construct enzyme electrodes. These electrodes were employed for the rapid (60 s) measurement of theophylline in serum at a working potential of +100 mV versus Ag/AgCl. Linear calibration curves were obtained over the clinically relevant range (y = 0.13x + 0.22, n = 8). Caffeine, theobromine and 3-methylxanthine at levels up to 100 mg l-1 do not interfere and 1-methylxanthine shows cross-reactivity at concentrations greater than 50 mg l-1.     

Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes

A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. The steady-state current of these enzyme electrodes were reproducible within +/-5.0% of the relative error. Response time was found to be 30-50s and upper limit of the linear working portions was found to be 1.2mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 x 10(-6)M substrate concentrations. The apparent Michaelis-Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.9 kJ mol(-1), respectively. The effects of interferents and stability of the enzyme electrodes were also investigated.     

Measurement of intracellular calcium ion activity with neutral exchanger ion sensitive microelectrodes

Micropipettes filled with the neutral liquid ion exchanger ETH 1001 can be used to make microelectrodes that are sensitive to cytoplasmic levels of Ca2+. They are high resistance electrodes, so that care is required in order to record the low current signal. The electrodes often yield 10-15 mV change between intracellular Ca2+ activities of 10(-6) and 10(-7) M, according to a log relation. The microelectrodes are non-destructive, even in rather small cells, and can be used to monitor Ca2+ changes during experimental interventions.     

Reduction of electrode polarization capacitance in low-frequency impedance spectroscopy by using mesh electrodes

Dielectric measurements of biological samples are obscured by electrode polarization, which at low frequencies dominates over the actual sample response. Reduction of this artifact is especially necessary in studying interactions of electric field with biological systems in the α-dispersion range. We developed a method to reduce the influence of electrode polarization by employing mesh instead of solid electrodes as sensing probes, thereby reducing the area of the double layer. The design decreases the electrode-electrolyte contact area by almost 40% while keeping the bulk sample capacitance the same. Interrogation electric fields away from the electrode surface and sensitivity are unaffected. Electrodes were microfabricated (600μm×50μm, spacing of 100μm) with and without mesh holes 7.5μm×7.5μm in size. Simulations of electric field performed using Comsol Multiphysics showed non-uniformity of the electric field within less than 1.5μm from the electrode surface, which encompasses the double layer region, but at greater distance the solid and mesh electrodes gave the same results. Mesh electrodes reduced capacitance measurements for water and KCl solutions of different concentrations at low frequencies (<10kHz), while higher frequency capacitance remained the same for both electrode types, confirming our hypothesis that this design leaves the electric field mainly unaffected. Impedance measurements at low frequencies for water and mice heart mitochondrial suspension were lower for mesh than for solid electrodes. Comsol simulations confirmed these results by showing that mesh electrodes have a greater charge density than solid electrodes, which affects conductance. These electrodes are being used for mitochondrial membrane potential studies.     

Electrical immunosensor based on a submicron-gap interdigitated electrode and gold enhancement

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1μm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations).     

Fabrication of comb interdigitated electrodes array (IDA) for a microbead-based electrochemical assay system

This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase. Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA.     

Detection of secretion from pancreatic islets using chemically modified electrodes

Secretion of insulin from pancreatic islets was monitored indirectly by detecting zinc. Anodic stripping voltammetric measurements of zinc were done on a bismuth-modified electrode. Comparison of the performance of bismuth-modified electrodes and mercury film electrodes showed that bismuth is an appropriate alternative for Zn detection. The bismuth-coated electrode was used to detect zinc in insulin samples and insulin secreted from pancreatic islets upon stimulation with high concentrations of K(+). Detection of zinc released from pancreatic islets was done in the culture medium without any further cleanup. This detection method can be used to monitor secretion from pancreatic islets in their native environment.     

A new technique for the selective recording of extensor carpi radialis longus and brevis EMG.

Tennis Elbow or Lateral Epicondylalgia is manifested by pain over the region of the lateral epicondyle of the humerus, related to use of the wrist extensor muscles. Extensor carpi radialis longus (ECRL) and brevis (ECRB) have been implicated in the dysfunction associated with Lateral Epicondylalgia. For muscles in the human forearm, particularly those in close proximity, selective recordings are nearly impossible without the use of fine wire, indwelling electrodes. These can be inserted in precise locations and have small recording areas. Standard electromyography texts indicate, however, that the activity of ECRL and ECRB cannot be distinguished, even with intramuscular electrodes. We present a new technique for determining the most appropriate sites at which to insert intramuscular electrodes for selective recordings of ECRB and ECRL. The location of ECRB and ECRL was measured on 10 cadaver specimens, 5 right arms and 5 left arms. The distance from the muscle origin to (1) insertion, (2) largest portion of the muscle belly, (3) most proximal fibres and (4) most distal fibres were measured and expressed relative to forearm length. The mean distance and 95% confidence interval was calculated for each of the four measures. These data indicated a significant separation of the belly of each muscle along the length of the forearm. These relative distances were used to mark electrode insertion points on three volunteers. Fine wire electrodes were used to record the electromyogram in three participants. Each participant was required to perform isometric contractions to produce (1) wrist extension torque, (2) radial deviation torque, (3) elbow flexion torque and (4) finger extension. The electromyographic recordings show clear differentiation of ECRB and ECRL with the relative activation patterns reflecting the underlying anatomical organisation of the two muscles. This technique provides an important objective method that can be used in conjunction with manual muscle testing to provide a means of ensuring accurate intramuscular electromyographic recording from these two muscles.     

Vitamin analysis with use of a yeast electrode

A vitamin B₁ (thiamin)-sensitive electrode has been devised by combining an oxygen electrode with a yeast-containing membrane. The assembly was used for assaying thiamin at concentrations down to 10^?11 gl^?1. The analytical procedure developed should allow the measurement of 10–20 samples per hour. The performance of the yeast electrode was improved when alginate membranes reinforced with a nylon network were used. An apparatus for preparing such membranes is described together with a magnetic membrane holder facilitating handling of membranes in combination with electrodes.     

Amperometric protein sensor - fabricated as a polypyrrole, poly-aminophenylboronic acid bilayer

An approach to the design of electrodes for the production of sensors, which show significant changes to the passage of current in response to the concentration of target protein molecules, is presented. Screen-printed platinum electrodes, modified with two separately applied conducting polymer layers, have been developed as a potential route to forming cheap disposable protein sensors. To achieve a heightened response for the target molecules, an initial layer of polypyrrole was formed on the electrode's surface by electro-deposition. This composite was then employed as a substrate for the subsequent electro-deposition of a relatively thin 'sensing layer' of poly-aminophenylboronic acid. Cyclic voltammetry (CV) of the prepared films revealed an excursion in the current versus potential curve in the anodic phase at approximately 0.0 to +0.2V. It was clearly shown that the introduction of proteins into the CV cell resulted in a measurable decrease in the passage of current in buffered aqueous media. Measured current reductions observed on introducing lysozyme (10ppm) into the test solution were 2.3x10(-6)A for an electrode formed with a poly-aminophenylboronic acid layer on platinum, and 1.75x10(-5)A for a composite electrode formed with poly-aminophenylboronic acid on a polypyrrole coated platinum substrate. The introduction of the competing analytes, dl adrenaline or dopamine, at concentrations typically found in human urine, had little effect on the sensor's response. Additionally, the sensing system was able to maintain a response to added target proteins with as much as 2vol.% urine in the test solution. Using the electrodes in high concentrations of competing physiological analytes, they were able to respond to protein concentrations as low as 0.5ppm in buffered solutions containing urea at a concentration representative of human urine (17,000ppm), which additionally contained glucose (1000ppm).     

From Oscillatory Transcranial Current Stimulation to Scalp EEG Changes: A Biophysical and Physiological Modeling Study

Both biophysical and neurophysiological aspects need to be considered to assess the impact of electric fields induced by transcranial current stimulation (tCS) on the cerebral cortex and the subsequent effects occurring on scalp EEG. The objective of this work was to elaborate a global model allowing for the simulation of scalp EEG signals under tCS. In our integrated modeling approach, realistic meshes of the head tissues and of the stimulation electrodes were first built to map the generated electric field distribution on the cortical surface. Secondly, source activities at various cortical macro-regions were generated by means of a computational model of neuronal populations. The model parameters were adjusted so that populations generated an oscillating activity around 10 Hz resembling typical EEG alpha activity. In order to account for tCS effects and following current biophysical models, the calculated component of the electric field normal to the cortex was used to locally influence the activity of neuronal populations. Lastly, EEG under both spontaneous and tACS-stimulated (transcranial sinunoidal tCS from 4 to 16 Hz) brain activity was simulated at the level of scalp electrodes by solving the forward problem in the aforementioned realistic head model. Under the 10 Hz-tACS condition, a significant increase in alpha power occurred in simulated scalp EEG signals as compared to the no-stimulation condition. This increase involved most channels bilaterally, was more pronounced on posterior electrodes and was only significant for tACS frequencies from 8 to 12 Hz. The immediate effects of tACS in the model agreed with the post-tACS results previously reported in real subjects. Moreover, additional information was also brought by the model at other electrode positions or stimulation frequency. This suggests that our modeling approach can be used to compare, interpret and predict changes occurring on EEG with respect to parameters used in specific stimulation configurations.     

EMG muscle scanning: comparison to attached surface electrodes.

Electromyographic (EMG) muscle scanning measures brief samples of integrated muscle action potentials from individual muscles using a hand-held scanner with post-style electrodes. This "scanning" technique is widely used by biofeedback practitioners to quickly assess muscle activity in the diagnosis of musculoskeletal disorders. In an effort to compare muscle scanning with the established technique using attached surface electrodes, ten healthy subjects (25-35 years old) were scanned using 2-second sampling at five bilateral muscle sites while simultaneously monitoring the same sites with surface electrodes. This was repeated using 10-second scanning samplings. Pearson's product-moment correlations between scanning for 2 seconds and prolonged surface recording at all sites were 0.54-0.89. Scanning for 10 seconds improved the correlations to 0.68-0.91. EMG scanning for 2 seconds compares favorably with attached surface electrode recording. Comparisons are further improved by 10-second scans.     

Multiply osmium-labeled reporter probes for electrochemical DNA hybridization assays: detection of trinucleotide repeats

In electrochemical DNA hybridization assays target or probe DNAs end-labeled with electroactive compounds have been frequently used. We show that multiple osmium labels yielding faradaic (at carbon or mercury electrodes) and catalytic signals (at mercury electrodes) can be easily covalently bound to DNA molecules. We use (GAA)(7) (T)(n) oligodeoxynucleotides (ODNs) with n ranging between 5 and 50. (T)(n) tails are selectively modified with osmium tetroxide,2,2'-bipyridine leaving the (GAA)(7) repeat intact for the DNA hybridization. These ODNs are applied as reporter probes (RP's) in DNA hybridization double-surface (DS) assay using magnetic beads for the DNA hybridization and pyrolytic graphite (PGE) or hanging mercury drop (HMDE) electrodes for the electrochemical detection. We show that in difference to the usual single-surface methods (where the RP has to be bound to target DNA near to the surface to communicate with the electrode) in the DS assay the RP can be bound to DNA regardless of its position and can used for the determination of the length of DNA repetitive sequences. Several fmols or about a hundred of amol of a RP with osmium-labeled (T)(50) tail can be detected at PGE and HMDE, respectively, at 1-2 min accumulation time.     

Labeless AC impedimetric antibody-based sensors with pgml(-1) sensitivities for point-of-care biomedical applications

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.     

A polyvinylchloride-membrane based anion selective electrode for continuous registration of delta pH (interior alkaline) with salicylate as the indicator probe

An anion sensitive electrode has been constructed with the use of the lipid soluble cation benzyl-dimethyl-hexadecylammonium analogous to the procedure described for tetraphenylphosphonium-sensitive electrodes [Shinbo, T., Kamo, N., Kurihara, K. and Kobatake, Y. (1978) Arch. Biochem. Biophys. 187, 414-422]. The anion electrode responds to salicylate concentrations above 400 microM with a Nernstian sensitivity. Less lipid soluble anions like chloride and phosphate do not interfere. Below 400 microM salicylate the response of the electrode decreases gradually so that the sensitivity of the electrode is less than 10 mV per decade change at concentrations of the anion of 50 microM. A computer program has been developed to fit the electrode response curve with a polynomal function of the fourth power. Additional software-allows calculation of changes in the concentration of the salicylate anion, also under conditions where the sensitivity of the electrode for the anion is not constant. In this way the electrode can be used to measure changes in salicylate concentration that occur in a suspension of bacteria when, upon energization, a pH gradient is generated. 31P nuclear magnetic resonance measurements demonstrated that the pH gradient measured with the salicylate-sensitive electrode in the phototrophic bacterium Rhodopseudomonas sphaeroides is quantitatively correct. The response time of the electrode decreases from 1 min at 20 microM salicylate to 10 s at concentrations greater than or equal to 200 microM.     

Metabolic control analysis of an enzymatic biofuel cell

Metabolic control analysis (MCA) is an analytical technique that aims to quantify the distribution of control that enzymes exhibit over the steady‐state fluxes through a metabolic network. In an enzymatic biofuel cell, the flux of interest is the electrical current generated by the system. Regardless of transport limitations and other constraints, kinetic limitations can become potential bottlenecks in the operation of a biofuel cell. We have used an indirect approach to MCA to investigate a common osmium‐mediated glucose oxidase/laccase enzymatic biofuel cell. The results of the analysis show that the control of the electron flux strongly depends on the total mediator concentrations and the extent of polarization of the individual electrodes. The effect of varying oxygen concentrations is also examined, as oxygen is required for the cathode, but it participates in a non‐productive reaction at the anode. Under normal operating conditions the electrodes will be highly polarized and will both contain high mediator concentrations. This configuration will result in a dominant FCC at the anode, and the conditions that are needed for balanced flux control between the anode and cathode are explored. As increasingly complex bioelectrocatalytic systems and architectures are envisioned, MCA will be a valuable framework to facilitate their design and subsequent operation. Biotechnol. Bioeng. 2009;102: 1624–1635. © 2008 Wiley Periodicals, Inc.