A novel system combining biocatalytic dephosphorylation of L-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.     

Calibration of a Clark-Type oxygen electrode by tyrosinase-catalyzed oxidation of 4-tert-butylcatechol.

A procedure for calibrating a Clark-type oxygen electrode is described. This method is based on the oxidation of 4-tert-butylcatechol (TBC) by O2 catalyzed by tyrosinase, to yield 4-tert-butyl-o-benzoquinone (TBCQ). This reaction consumes known amounts of oxygen in accordance with the stoichiometry: 2TBC + O2----2TBCQ + 2H2O and can be used to determine the relation between the oxygen concentration and the oxygen electrode response. TBCQ is very stable in the reaction medium for more than 30 min and shows no significant breakdown, which makes the calibration possible. A kinetic study of the oxidation of 3,4-dihydroxyphenylalanine by tyrosinase using the oxygen electrode is shown to confirm the validity of the calibration method.     

Electrochemical detection for high-performance liquid chromatography of ketoconazole in plasma and saliva

Ketoconazole, cis-1-acetyl-4-[4[[2-(2,4-dichlorophenyl)-2-(1H-imidazol- 1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine, a clinically used antifungal agent, is also an inhibitor of steroid hormone biosynthesis. A high-performance liquid chromatographic method is described which resolves ketoconazole with selectivity and high sensitivity provided by the use of electrochemical detection. Ketoconazole can be detected in high-performance liquid chromatography by electrochemical oxidation at a glassy carbon electrode at a potential of +1.0 V. Electrochemical detection offers improved sensitivity and selectivity over ultraviolet absorbance or fluorescence detection after derivatization. The method utilizes a volatile buffer system compatible with postcolumn analyses and an internal standard which is electrochemically active. This technique provides a simple method to assay ketoconazole. Ketoconazole can be detected in human plasma and saliva after a single oral therapeutic dose.     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.     

Electrochemical evaluation of glucose oxidase immobilized by different methods

Glucose oxidase electrodes were constructed on a platinum screen using polyacrylamide gel, glutaraldehyde crosslinking, and glutaraldehyde crosslinking with +0.04 volts dc on the platinum screen as the methods of enzyme immobilization. The electrodes were evaluated in an electrochemical cell for the oxidation of glucose at the enzyme electrode and the reduction of oxygen at a platinum auxiliary electrode, using constant current voltametry or under external load operation. The method of immobilization affected the extrapolated opencircuit potential as well as the half-cell potential and the steady current under external load operation. The charged glutaraldehyde electrode gave the best current performance; however, the small output (microamps) indicated that major problems in electron transfer from an enzyme catalyst to an external circuit must be resolved before such electrodes can be used in practical application.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. I. A study of the "cell-attached" configuration

A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials.     

Polarographic studies in presence of Triton X-100 on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum.

Polarographic studies on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum were carried out at 24 degrees. 1. Using a carbon-paste electrode as the working electrode, polarographic waves characteristic of oxidation-reduction components were observed in the presence, but not in the absence of Triton X-100; these waves were therefore measured in the presence of the detergent. 2. At least two kinds of oxidation-reduction components were detectable, having different half-wave potentials (E1/2); at pH 7, one had an E1/2 value of +275 mV (POC+275) and the other had a value of +60 mV (POC+60). 3. POC+275 was reduced by succinate and by NADH. Both reductions were almost completely inhibited by antimycin A, which hardly affected the reductions of ubiquinone-10 by succinate and by NADH. Most POC+275 molecules were not reduced by the substrates when quinones were extracted from the chromatophores, and the reductions were mostly restored when ubiquinone-10 was re-added. This indicates that POC+275 is functional between ubiquinone-10 and cytochrome c2 in the electron transport system. 4. POC+60 was reduced by succinate, but hardly at all by NADH. The reduction of POC+60 was not influenced either by the addition of antimycin A or by the extraction of quinones. This suggests that POC+60 is functional in the process from succinate dehydrogenase [EC 1.3.99.1] to ubiquinone-10 in the electron transport system. 5. Of the POC+275 reducible by dithionite, approximately 70% could be reduced in the absence of Triton X-100, provided that the potential of the working electrode immersed in chromatophore suspensions was set at potentials of 0 mV or lower and that the electrochemical reaction was carried out at pH 7.5. When the potential of the electrode was set at +50 mV (the same as the E1/2 value of ubiquinone-10 bound with chromatophores), and the suspension was allowed to stand for various lengths in the presence of the detergent, it was found that approximately half of the electrochemically reducible POC+275 was rapidly reduced, followed by a slow reduction. The discrepancy in the oxidation-reduction equilibrium on the basis of the E1/2 values of ubiquinone-10 and POC+275 is discussed.     

Ca2+ measurement with a Ca electrode: artifacts due to some cardiotropic drugs

In order to validate D. M. Bers' method ((1982) Amer. J. Physiol. 242, C404-C408) for the determination of free [Ca] in ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EG-TA)-buffered Ca solution, six cardiotropic drugs were checked for their ability to interfere with Ca2+ measurements with a Ca electrode: verapamil, diltiazem, perhexiline, amrinone, bepridil, and diproteverine. Only amrinone, up to 10(-4) M, showed no effect. With the other compounds, from 5 x 10(-6) to 10(-4) M a gradual increase of the potential was observed. Negligible with 1 mM Ca2+ in the solution, the effects increased while free Ca decreased, regardless of the presence of EGTA. Similar observations were made using a K+ electrode. Relying on the results obtained by three different techniques, potentiometric study, spectrophotometric study, and binding of 45Ca on Chelex 100 resin, it was concluded that the effects of these drugs on the potential measured with an ion-specific electrode are artifacts which are not due to a release of Ca ions from the Ca-EGTA complex. When using Bers' method for the determination of free calcium, interfering drugs should be added to the solutions only after Ca2+ measurements with the Ca electrode.     

Determination of catalase-like activity in plants based on the amperometric monitoring of hydrogen peroxide consumption using a carbon paste electrode modified with ruthenium(IV) Oxide

A carbon paste electrode containing ruthenium(IV) oxide as a modifier was tested as an effective hydrogen peroxide amperometric sensor in bulk measurements (hydrodynamic amperometry). Factors that influence its overall analytical perform ance, such as pH and the applied potential, were examined. The RuO2-modified electrode displayed high sensitivity towards hydrogen peroxide, with detection limits as low as 0.02 mm at pH 7.4 and 0.007 mM at pH 9.0. The method was applied for monitoring the decomposition of hydrogen peroxide (by catalase) in phosphate buffer of pH 7.4. The relative response of the electrode towards ascorbic acid was assessed and it was found that the selectivity of the RuO2-modified electrode towards hydrogen peroxide over ascorbic acid could be significantly improved by electro-polymerizing m-phenylenediamine on its surface prior to measurements. The RuO2-modified electrode was used for the kinetic (fixed time) determination of catalase activity in the range of 4-40 U/mL (detection limit 1.2 U/mL). The method was applied to the determination of catalase-like activity in various plant materials (recov-ery ranged from 93 to 101%, detection limit 480 U/100 g).     

Electrical effects on the proliferation of living HeLa cells cultured on optically transparent electrode surface.

Low d.c. potential application induced changes of cellular morphology and growth of living cells on a potential-controlled electrode. At a potential range higher than +0.7 V (vs. Ag/AgCl), serious electric effects on cell viability, membrane permeability, and cytoskeletal morphology of HeLa cells were observed. On the other hand, at lower than +0.5 V no effect was observed. At the boundary potential range between +0.5 V to +0.7 V, where HeLa cells were cultured on the potential-controlled optically transparent In2O3 electrode (OTE) surface, intriguing effects on HeLa cells appeared. At this potential range, where HeLa cells cultured on a potential-applied OTE, all the cells were alive accompanying morphological change. The morphology of HeLa cells returned to their normal spindle shape, when potential application to the electrode was cut off. At a potential of +0.65 V, cell proliferation ratio of cultured cell on an electrode was about one-fifth of that on a non-controlled electrode. These results suggest that low d.c. electrical effects induce significant change in cellular morphology and function.     

Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B

Polymerization of bovine fibrinogen acted upon by thrombin is accompanied by binding of Ca2+ and a concomitant decrease of the free Ca2+ concentration. The latter can be recorded by a Ca2+-selective electrode as a shift in the electrode potential. The shift shows marked dependence on the initial free Ca2+ concentration, being maximal at about 10(-4.1) M and decreasing sharply on either side of this. Thus, the effect is limited to the 10(-3)-10(-5) M free Ca2+ concentration range. From the initial and the final value of the electrode potential during a clotting experiment, the amount of Ca2+ bound to fibrinogen and fibrin, respectively, can be calculated. The difference between the two, plotted against free Ca2+ concentration, gives a bell-shaped curve. This indicates that the reason for the Ca2+ binding is a shift of the pK of some groups from a lower to higher value. The recordings can be used for evaluation of the kinetics of the Ca2+ uptake. However, they have to be corrected for the effect of the continuous shift in the free Ca2+ concentration during the experiment. The reaction does not follow simple kinetics, showing a lag period. Therefore, rates were estimated from inverse half-reaction times. Half-times of the corrected curves show that the reaction is first order with respect to thrombin. Moreover, the rate of Ca2+ uptake is identical with that of the conformational change seen in differential scanning calorimetry [Donovan, J.W., & Mihalyi, E. '1985) Biochemistry 24, 3434]. The inverse rate and the final corrected Ca2+ uptake increase linearly with the initial fibrinogen concentration. Concomitant estimates of fibrinopeptide A and B release showed that the Ca2+ uptake runs parallel to the release of fibrinopeptide B. Fibrinopeptide A was released largely during the lag period of the Ca2+ uptake. In agreement with this, clotting with Ancrod, an enzyme that liberates only fibrinopeptide A, was not accompanied by binding of Ca2+. Thus, polymerization is not sufficient for the Ca2+ uptake to occur; liberation of fibrinopeptide B seems to be obligatory. Further support for this was obtained with experiments with the polymerization inhibitor Gly-Pro-Arg-Pro. The tetrapeptide inhibits polymerization and also, proportional to this, release of fibrinopeptide B [Hurlet-Jensen, A., Cummins, H.Z., Nossel, H.L., & Liu, C.Y. (1982) Thromb. Res. 27, 419; Lewis, S.D., Shields, P.P., & Shafer, J.A. (1985) J. Biol. Chem. 260, 10192]. Calcium uptake was also depressed by the tetrapeptide in a way similar to its effect upon fibrinopeptide B release.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.     

Redox properties of several bacterial ferredoxins using square wave voltammetry

The equilibrium reduction potential of the 2[4Fe-4S] ferredoxin (Fd) isolated from four different bacterial strains was determined at a methyl viologen-modified gold electrode using square wave voltammetry. The observed reduction potential at pH 8 for Clostridium thermoaceticum Fd was -385 mV; Clostridium pasteurianum, -393 mV; Clostridium thermosaccharolyticum, -408 mV; and Chromatium vinosum, -460 mV versus normal hydrogen electrode at 25 degrees C. The reduction potential of the C. pasteurianum Fd was found to be pH independent from pH 6.4 to 8.7, indicating that the electron transfer mechanism does not involve proton exchange. In contrast, the reduction potential of the C. thermosaccharolyticum Fd was found to be pH dependent from pH 6.4 to 8.7, with pKox approximately 7 and pKred approximately 7.5. The +30 mV change in reduction potential from pH 8.7 to 6.4 was attributed to an electrostatic interaction between the iron-sulfur cluster II and the protonated histidine 2 residue located about 6 A away. The Ch. vinosum Fd interacted reversibly at the methyl viologen-modified gold electrode, and its reduction potential was verified using visible spectroelectrochemistry. The reduction potential of Ch. vinosum Fd was found to be 30 mV more positive than previously reported. The similarities of the bacterial Fd reduction potentials are discussed in terms of the homology of their primary structure as reflected by the similarities in the visible and circular dichroic spectra.     

Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.     

A synthetic analogue for the active site of plant-type ferredoxin: two different coordination isomers by a four-cys-containing [20]-peptide.

The (Fe2S2)2+ complex of an artificial 20-peptide ligand, Ac-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Pro-Leu-Leu-T hr-Cys- Val-NH2, containing an invariant Cys-A-B-C-D-Cys-X-Y-Cys (A, B, C, D, X, Y = amino acid residues) fragment of plant-type ferredoxins was synthesized by a ligand exchange method with [Fe2S2(S-t-Bu)4]2-. 1H-nmr spectroscopic and electrochemical data of the complex indicate the presence of two coordination isomers. One of them having a Cys-X-Y-Cys bridging coordination to the two Fe(III) ions, has the (Fe2S2)2+ core environment similar to those of the denatured plant-type ferredoxins and exhibits a positive shifted redox potential at -0.64 V vs saturated colonel electrode (SCE) in N,N-dimethylformamide (DMF). Another isomer with the Cys-A-B-C-D-Cys bridging coordination shows a negative redox potential at -0.96 V vs SCE in DMF.     

Development of a dimethyl ether (DME) sensor using platinum nanoparticles and thick-film printing

A portable and cost-effective technique to measure the dimethyl ether (DME) concentrations has been developed. It is based on an electrochemical principle measuring the oxidation current of DME at an applied potential of +0.2V versus a Ag/AgCl reference electrode. Thick-film printing technique is used for the fabrication of this DME sensor, and platinum nanoparticles in the crystallite size of 5.5 nm are used for the modification of the working electrode surface. This modification enhances the sensor performance significantly leading to a higher sensitivity of the sensor comparing to bare platinum electrode. Evaluation and characterization of this sensor are carried out over the DME concentration range of 0-7% (v/v), and a linear relationship between sensor outputs and the DME concentrations with an average R(2) of 0.996 exists. The reproducibility of the sensor is also very good. This electrochemically based DME sensor fabricated by thick-film screen printing technique and using the platinum nanoparticles to enhance its performance will be valuable and practical for the estimation of the airway mucosal blood flow.     

Electrochemical profiles of Herba Saussureae Involucratae by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection method has been developed for the determination of the pharmacologically active ingredients, acacetin, rutin, umbelliferone, kaempferol, apigenin, luteolin and quercetin, in Herba Saussureae Involucratae. Under optimum conditions, the seven analytes could be completely separated within 19 min in a 75 cm length capillary at a separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2). A 300 microm diameter carbon disk electrode, positioned opposite the outlet of the capillary in a wall-jet configuration at a potential of +950 mV (vs a saturated calomel electrode) was used as the working electrode. A good linear relationship was established between peak current and concentration of the analytes over two orders of magnitude with detection limits (signal-to-noise ratio = 3) ranging from 1.2 x 10(-7) to 4.1 x 10(-8) g/mL for all analytes. The proposed method has been successfully applied to the analyses of bio-active components of Herba Saussureae Involucratae samples after a relatively simple extraction procedure. The assay results show that the resultant electrochemical profiles are indicative of the content diversity of each electrochemically active ingredient in the various samples, and may also offer some evidence for phytotaxonomy.     

Selective electrode for dibenzyl dimethyl ammonium cation as indicator of the membrane potential in biological systems.

The electrode sensitive to dibenzyl dimethyl ammonium (DDA+), which is considered to be an indicator of the membrane potential, was constructed by using tetraphenyl borone (TPB-) embedded in dichloroethane. Rapid and Nernstian responses were exhibited against DDA+ solutions ranging between 10(-2) and 3 - 10(-6) M in concentration. High selectivity for DDA+ was observed in the presence of various inorganis salts, ADP, ATP, oxidizable substrates and sugars. The electrode developed here was used to measure the DDA+ uptake in Streptococcus faecalis and the results agreed with those reported by Harold, F.M. and Papineau, D. ((1972) J. Membrane Biol. 8, 27-44 and 45-62). While they determined the DDA+ concentration in the medium by measuring the absorbance of the filtrate treated with the ion-exchangers, the electrode can measure directly the DDA+ concentration in the bacterial suspension without any any pretreatment. It was also shown that the electrode can measure the DDA+ uptake in mitochondria during energization.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.