Distinguishing splanchnic nerve and chromaffin cell stimulation in mouse adrenal slices with fast-scan cyclic voltammetry

Electrical stimulation is an indispensible tool in studying electrically excitable tissues in neurobiology and neuroendocrinology. In this work, the consequences of high-intensity electrical stimulation on the release of catecholamines from adrenal gland slices were examined with fast-scan cyclic voltammetry at carbon fiber microelectrodes. A biphasic signal, consisting of a fast and slow phase, was observed when electrical stimulations typically used in tissue slices (10 Hz, 350 μA biphasic, 2.0 ms/phase pulse width) were applied to bipolar tungsten-stimulating electrodes. This signal was found to be stimulation dependent, and the slow phase of the signal was abolished when smaller (≤250 μA) and shorter (1 ms/phase) stimulations were used. The slow phase of the biphasic signal was found to be tetrodotoxin and hexamethonium independent, while the fast phase was greatly reduced using these pharmacological agents. Two different types of calcium responses were observed, where the fast phase was abolished by perfusion with a low-calcium buffer while both the fast and slow phases could be modulated when Ca2(+) was completely excluded from the solution using EGTA. Perfusion with nifedipine resulted in the reduction of the slow catecholamine release to 29% of the original signal, while the fast phase was only decreased to 74% of predrug values. From these results, it was determined that high-intensity stimulations of the adrenal medulla result in depolarizing not only the splanchnic nerves, but also the chromaffin cells themselves resulting in a biphasic catecholamine release.     

In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry

Brain norepinephrine and dopamine regulate a variety of critical behaviors such as stress, learning, memory, and drug addiction. In this study, we demonstrate differences in the regulation of in vivo neurotransmission for dopamine in the anterior nucleus accumbens (NAc) and norepinephrine in the ventral bed nucleus of the stria terminalis (vBNST) of the anesthetized rat. Release of the two catecholamines was measured simultaneously using fast-scan cyclic voltammetry at two different carbon-fiber microelectrodes, each implanted in the brain region of interest. Simultaneous dopamine and norepinephrine release was evoked by electrical stimulation of a region where the ventral noradrenergic bundle, the pathway of noradrenergic neurons, courses through the ventral tegmental area/substantia nigra, the origin of dopaminergic cell bodies. The release and uptake of norepinephrine in the vBNST were both significantly slower than for dopamine in the NAc. Pharmacological manipulations in the same animal demonstrated that the two catecholamines are differently regulated. The combination of a dopamine autoreceptor antagonist and amphetamine significantly increased basal extracellular dopamine whereas a norepinephrine autoreceptor antagonist and amphetamine did not change basal norepinephrine concentration. α-Methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, decreased electrically evoked dopamine release faster than norepinephrine. The dual-microelectrode fast-scan cyclic voltammetry technique along with anatomical and pharmacological evidence confirms that dopamine in the NAc and norepinephrine in the vBNST can be monitored selectively and simultaneously in the same animal. The high temporal and spatial resolution of the technique enabled us to examine differences in the dynamics of extracellular norepinephrine and dopamine concurrently in two different limbic structures.     

The use of cyclic voltammetry to detect biofilms formed by Pseudomonas fluorescens on platinum electrodes

The development of an electrochemical detector to monitor the in situ formation of biofilms is described. The detector consisted of an electrochemical cell containing three electrodes, whose response to the application of a potential profile to the working electrode was sensitive to the amount of biofilm present on the surface. The electrochemical technique used was repetitive cyclic voltammetry. Differences between the response of the uncolonised electrode and after Pseudomonas fluorescens biofilms of different ages were grown on its surface were determined. The results show that cyclic voltammetry applied to platinum electrodes can be used to detect young biofilms. The development of the shape of the voltammogram as the potential is cycled may constitute a means of providing information on the coverage of the surface. Observation of the platinum electrodes before and after the electrochemical measurements showed that even after 30 min of recycling, most of the cells were still adhered to the surface, although some may have lost viability.     

Molybdenocene-oligonucleotide binding study at physiological pH using NMR spectroscopy and cyclic voltammetry

The self-complementary oligonucleotide CGCATATATGCG was used as a model to establish the binding interactions of antitumor molybdenocene dichloride and DNA. The free dodecamer was first characterized using ¹H, NOESY, and DQF-COSY NMR experiments, which enable to pinpoint the guanines and adenines as well as the cytosines and thymines signals in the aromatic region. Molybdenocene dichloride was characterized in saline and buffer solutions as function of pH by ¹H NMR spectroscopy. In 10 mM NaCl/D₂O solution at pH of 6.5 and above, Cp₂Mo(OD)(D₂O)⁺ is in equilibrium with its dimeric species, [Cp₂Mo(μ-OH)₂MoCp₂]²⁺. In 25 mM Tris/4 mM NaCl/D₂O at physiological pH, a new stable species is formed, coordinated by the buffer, Tris(hydroxymethyl)aminomethane. The interactions of molybdenocene dichloride species with CGCATATATGCG were studied at different pH. At pH 6.5, in 4 mM NaCl/D₂O solution, ¹H NMR spectra of CGCATATATGCG exhibit downfield shifts in the signals associated mainly to adenines and guanines, upon addition of molybdenocene dichloride. At pH 7.4, in 25 mM Tris/4 mM NaCl/D₂O, molybdenocene species causes broadening and small downfield shifts to the purines and pyrimidine signals, suggesting that molybdenocene dichloride can get engaged in binding interactions with the oligonucleotide in a weak manner. ³¹P NMR spectra of these interactions at pH 7.4 showed no changes associated to Mo(IV)-OP coordination, indicating that molybdenocene–oligonucleotide binding interactions are centered, most likely, on the bases. Cyclic voltammetry titration showed a 4.9% of molybdenocene–oligonucleotide interaction. This implicates that possible binding interactions with DNA are weak.     

Study of thermal and acid denaturation of DNA by means of voltammetry at graphite electrodes

Conformational changes in guanine–cytosine (G·C) and adenine–thymine (A·T) pairs in DNA were investigated by means of differential pulse voltammetry at a pyrolytic graphite electrode (PGE). As a monitor of these conformational changes, two separated voltammetric peaks, G and A, which correspond to electrochemical oxidation at the PGE of guanine and adenine residues, respectively, were used. It was found that peak A was first increased in the course of thermal denaturation of DNA. This indicates that, on heating a native DNA sample, regions rich in A·T pairs melt first. In the course of acid denaturation of a native DNA sample, the height of peak A was changed just before the denaturation. It is suggested that protonation of adenine residues in DNA regions rich in A·T pairs was responsible for these changes.     

Characterization of plant phenolic compounds by cyclic voltammetry

Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (E_ap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The E_ap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (E_1/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one-and two-electron oxidation are proposed.     

Characterization of plant phenolic compounds by cyclic voltammetry

Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (Eap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The Eap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (Ep/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one- and two-electron oxidation are proposed.     

Ion-exchange voltammetry and electrocatalytic sensing capabilities of cytochrome c at polyestersulfonated ionomer coated glassy carbon electrodes

The incorporation and electrochemical behaviour of cytochrome c (Cyt C) at glassy carbon electrodes modified with the polyestersulfonated ionomer Eastman AQ 55 are examined. The presence of the polyelectrolytic coating allows the preconcentration of the protein within the polymer and the observation of the direct electrochemistry of Cyt C at the modified electrode without addition of promoters or mediators in the solutions. The dependence of voltammetric signals on typical parameters such as solution pH and nature or concentration of the supporting electrolyte supports the ion-exchange nature of the incorporation process. The relevant role of the permselectivity of the polymeric modifier is highlighted also by the study of electrocatalytic processes which take place at the modified electrode loaded with Cyt C. No electrocatalytic effect is observed when the electrogenerated dication (ferricenylmethyl)trimethylammonium is present as possible oxidant. On the contrary, electrocatalytic current enhancements are observed for anionic substrates such as Fe(CN)(6)(3-) (oxidant) and ascorbate (reductant). Catalytic currents increase with the substrate concentration, with higher sensitivity for Fe(CN)(6)(3-). Due to ionic repulsion, the reaction with anions occurs at the polymer-solution interface. In the case of chemically unstable substrates, such as superoxide anion, ionic repulsion slow down the approach rate of the substrate so that spontaneous decomposition can prevail over the reaction with Cyt C incorporated in the coating.     

Hyper-Rayleigh scattering of protein-modified gold nanoparticles

The nonlinear optical properties of protein-modified gold nanoparticles has been studied by the hyper-Rayleigh scattering (HRS) technique. HRS signals from the nanoparticles coated with goat-anti-human IgG have been obtained when pumped with a laser pulse with a wavelength of 1064 nm. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles. This can be explained by a noncentrosymmetric effect. It was also found that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added due to gold nanoparticle aggregation. Our experiment found that the HRS method could produce a measurable signal with 10 microg/ml antigen added, while the colorimetric method using UV spectrum detection required 100 microg/ml of added antigen. The results show that the HRS measurement of immunogold nanoparticles could become a potential immunoassay in determining small levels of antigen in aqueous samples.     

The use of cyclic voltammetry for the evaluation of antioxidant capacity

Low-molecular weight antioxidants (LMWAs) play a major role in protecting biological systems against reactive oxygen-derived species and reflect the antioxidant capacity of the system. Cyclic voltammetry (CV), shown to be convenient methodology, has been validated for quantitation of the LMWA capacity of blood plasma, tissue homogenates, and plant extracts. Analysis of the CV tracing yields the values of (i) the biological oxidation potential, E and E(1/2), which relate to the nature of the specific molecule(s); (ii) the intensity (Ia) of the anodic current; and (iii) the area of the anodic wave (S). Both Ia and S relate to the concentration of the molecule(s). LMWA components of human plasma and animal tissues were identified and further validated by reconstruction of the CV tracing and by high-performance liquid chromatography-electrochemical detection. To reflect the oxidative stress status, the use of an additional parameter, R, has been proposed. R represents the level (%) of oxidized ascorbate (compared with total ascorbate) and is measured by high-performance liquid chromatography-electrochemical detection. All these parameters were monitored in healthy human subjects as well as in chronic (diabetes mellitus) and acute care patients (subjected to total body irradiation before bone marrow transplantation). The electroanalytical methodologies presented here could be widely employed for rapid evaluation of the status of subjects (in health and disease) for monitoring of their response to treatment and/or nutritional supplementation as well as for screening of specific populations.     

The synthesis and cyclic voltammetry of select ferrocene piano-stool isocyanide complexes

Ferrocene piano-stool isocyanide complexes ([CpFeL₃]⁺, Cp = η⁵-C₅H₅, L = tert-butyl isocyanide (1), cyclohexyl isocyanide (2), and 2,6-dimethylphenyl isocyanide (3)) are formed by chemical oxidation of ferrocene in the presence of a stoichiometric amount of isocyanide ligand (1:3). The complexes are characterized by elemental analysis, routine spectroscopic methods (IR, ¹H NMR, ¹³C NMR, and UV-Vis), and cyclic voltammetry.     

Discrete but simultaneous release of adenine nucleotides and serotonin from mouse megakaryocytes as detected with patch- and carbon-fiber electrodes

Using patch- and carbon-fiber electrodes, we studied release phenomena of adenine nucleotides and serotonin from megakaryocytes isolated from the bone marrow of the mouse. Megakaryocytes express ionotropic purinergic receptors on their surfaces. Under the condition of whole cell recording, the cells showed spikelike spontaneous inward currents. The spontaneous currents were carried by cations and had amplitudes of 30–800 pA at –43 mV and durations of 0.1–0.3 s. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 100 µM) and suramin (100 µM), purinoceptor-blocking agents, depressed the currents reversibly. It is thought that the receptor involved was the P_2X1 subtype on the cell and that the currents were due to activation of the P_2X1 receptor by adenine nucleotides released from the cell. The currents showed a skewed amplitude distribution, suggesting variation of vesicular contents and/or distinct localization or varied density of receptors on the cell. Frequency of the spontaneous inward currents was enhanced by external application of platelet-activating substances, thrombin (0.4 U/ml), phorbol ester (100 nM), and ADP (2 µM), at low concentrations. With a carbon-fiber electrode, which can detect oxidizable substances including serotonin, spikelike oxidation currents from the external surface of the megakaryocyte were detected. The frequency of the oxidation currents increased remarkably after the application of thrombin (10 U/ml). The majority of the oxidation currents coincided with the rising phase of the whole cell currents, suggesting corelease of serotonin and adenine nucleotide from the same vesicle. We concluded that megakaryocytes store adenine nucleotides and serotonin in the same vesicle and release them simultaneously in a discrete manner. ADP; ATP; serotonin; patch clamp; P_2X1 receptor     

Direct electrochemistry of spinach plastocyanin at a lipid bilayer-modified electrode: cyclic voltammetry as a probe of membrane-protein interactions.

The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.     

Redox potential of the active bleomycin-Fe(III) complex by cyclic voltammetry

The redox potential of the active Fe(III) complex of bleomycin (BLM), which is a DNA cleaving species, was measured by cyclic voltammetry at 25 °C under a hydrogen atmosphere. The cyclic voltammogram showed the reversible one-electron Fe(III)/Fe(II) coupled redox reaction at −0.225 V versus SCE. Under the same conditions the redox potentials of the iso-BLM—Fe(III) complex and the deglyco-BLM—Fe(III) complex were also observed, but the cyclic voltammogram for the inactive Fe(III) complex of BLM could not be obtained.     

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.     

Determination of total antioxidant capacity in plasma by cyclic voltammetry. Two case reports

The cyclic voltammetry (CV) was used for the measurement of the plasma total antioxidant capacity from two types of patients. The first one consisted of 29 volunteers (men aged 18-21 years) who were administered placebo or silymarin at a dose of 858 mg/day. After two months of silymarine administration, CV revealed a statistically significant increase in total antioxidant capacity compared to placebo. No statistically significant changes in TBARS, SH-groups, creatininin, urea, and uric acid concentrations were found. The second group under study comprised 49 patients with chronic renal disease during dialysis therapy. After dialysis, CV revealed a decrease of total antioxidant capacity in the plasma, which was equivalent to a decrease in creatinine, urea and uric acid. CV was performed using a system consisting of a working glassy carbon electrode, an auxiliary platinum electrode, and a reference saturated calomel electrode; a linear change of voltage of 200 mV/s was applied. CV is a simple and relatively reliable method for assessment of body antioxidant status. It is also time and cost effective.     

固体电极上L－半胱氨酸的电合成

固体电极上Ｌ－半胱氨酸的电合成ＥｌｅｃｔｒｏｓｙｎｔｈｅｓｉｓｏｆＬ－ｃｙｓｔｅｉｎｅａｔｓｏｌｉｄｅｌｅｃｔｒｏｄｅｓ￥／／（英）Ｒａｌｐｈ,ＴＲ．,Ｈｉｔｃｈｍａｎ,ＭＬ,Ｍｉｌｌｉｎｇｔｏｎ,ＪＰ等著张关永摘译陆兆锷校华东理工大学化学系上海２０...     

Detection of human leucocytes by cyclic voltammetry and its application to monitoring of allergic reaction

Cyclic voltammetry was applied to the detection of human leucocytes and the monitoring of allergic reactions. A basal plane pyrolytic graphite electrode with attached leucocytes on a porous nitrocellulose membrane filter was employed as a working electrode. An anodic peak current appeared at 0.33 V versus the saturated calomel electrode (SCE) when the potential of the working electrode was scanned in the range of 0-1.0 V versus SCE. This peak current was attributed to the electrochemical oxidation of serotonin. When egg white was added to leucocytes obtained from patients who were allergic to egg, the peak current decreased owing to degranulation of leucocytes leading to serotonin release. The peak current decreased with increasing allergen concentration in the range of 5-50 micrograms ml-1. Leucocytes did not respond to other allergens such as soybean, milk and dinitrophenylated bovine serum albumin (DNP-BSA).