Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

The direct voltammetry and electrocatalytic properties of catalase, which was adsorbed on the surface of multiwall carbon nanotubes (MWCNTs), was investigated. A pair of well-defined and nearly reversible cyclic voltammetry peaks for Fe(III)/Fe(II) redox couple of catalase adsorbed on the surface of MWCNTs at approximately -0.05 V versus reference electrode in pH 6.5 buffer solution, indicating the direct electron transfer between catalase and electrode. The surface coverage of catalase immobilized on MWCNTs glassy carbon electrode was approximately 2.4x10(-10) molcm-2. The transfer coefficient (alpha) was calculated to be 0.4, and the heterogeneous electron transfer rate constant was 80 s-1 in pH 7, indicating great facilitation of the electron transfer between catalase and MWCNTs adsorbed on the electrode surface. The formal potential of catalase Fe(III)/Fe(II) couple in MWCNTs film had a linear relationship with pH values between 2 and 11 with a slope of 58 mV/pH, showing that the electron transfer is accompanied by single proton transportation. Catalase adsorbed on MWCNTs exhibits a remarkable electrocatalytic activity toward the reduction of oxygen and hydrogen peroxide. The value for calculated Michaelis-Menten constant (1.70 mM) was high, indicating the potential applicability of the films as a new type of reagentless biosensor based on the direct electrochemistry of the catalase enzyme.     

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.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Amperometric determination of sodium nitrite by a microbial sensor

Summary A microbial sensor was prepared to determine sodium nitrite. This microbial sensor consisted of immobilized Nitrobacter sp. and an oxygen electrode. When a sample solution containing sodium nitrite was tested, nitrite was changed to NO₂ gas in the buffer (pH 2.0) and the current of the electrode decreased with time until a steady state was reached. The steady state current was attained within 10 min and the maximum decrease in current was obtained at 30°C and pH 2.0. A linear relationship was observed between the current decrease and the sodium nitrite concentration below 0.59 mM, the minimum sodium nitrite concentration that could be determined was 0.01 mM. The current decrease was reproducible (5% relative error). The current output of the sensor was almost constant for more than 21 days and 400 assays.     

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.     

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

? To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. ? Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). ? Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). ? The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism.     

Activity and adsorption of lipase from Nigella sativa seeds on Celite at different pH values

Lipase from Nigella sativa seeds was immobilized by adsorption on Celite 535 from phosphate buffer solutions varying pH values of 5.0–8.0 at 25?°C. Langmuir isotherms described the adsorption equilibria well for lipase adsorption at all pH range. The saturation capacity for adsorption of lipase increased from 14.5 to 24.3 mg g^?1 Celite as the adsorption pH was reduced from 8 to 5, but the adsorption equilibrium constant remained constant and was determined to be 1.92 × 10⁵ M^?1. The adsorbed enzymes showed different activity values depending on the pH of the adsorption medium. The immobilized enzymes prepared at pH 6 displayed the highest activity values.     

Characterization of the inward-rectifying potassium current in cat ventricular myocytes

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.     

Redox potential of plastoquinone A in spinach chloroplasts

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is ?30 mV per pH between pH 4.0 and 5.7, and ?60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Resistance to Water Uptake in a Mature Citrus Tree

Resistance to water uptake in an irrigated and a water-stressed22-year-old citrus tree was examined during 7 weeks. Frequentirrigation maintained the soil water potential of one citrustree between 0.0 and –0.016 MPa in the upper 0.6 m, whilethe other tree received no water for 44 d. Sap flow in the trunkswas measured once a week from dawn to dusk at hourly intervalswith a calibrated heat pulse technique. At the same time leafwater potential was measured on sunlit and shaded leaves. Sap flow in the stressed tree decreased with time at a higherrate than the water potential difference between soil and leaf,which implies an increase in the resistance of the transportsystem. Diurnal sap flow and diurnal leaf water potential ofthe irrigated and stressed trees were linearly related throughoutthe experimental period. The slope of the linear relationshipbetween flow and potential for both trees remained nearly constant,indicating that the resistance within the tree remained constant.The effect of drying appeared to be a progressive decline ofthe leaf water potential at zero flow (computed by linear regression).This was ascribed to a reduction of the transport of water inthe soil toward the roots. Key words: Transpiration, Leaf water potential, Plant water relations, Soil water stress     

An electrochemical approach to monitor pH change in agar media during plant tissue culture

In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.     

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.     

Interaction of calf thymus dsDNA with anti-tumor drug tamoxifen studied by zero current potentiometry

Since the electrochemical oxidation peaks of both DNA and anti-tumor drug tamoxifen (TAM) overlapped with each other, the known electrochemical methods were limited in the study of the interactions between DNA and TAM. In this paper, zero current potentiometry, a new electrochemical method, was used to study the interaction of calf thymus dsDNA with TAM. The dsDNA was immobilized on the surface of carbon paste (dsDNA/CP). The dsDNA/CP connected in series between the clips of working and counter electrodes of a potentiostat and a reference electrode were immersed in aqueous solution containing TAM, the interaction of dsDNA with TAM produced a change in interfacial potential at the dsDNA/CP/solution interface. When linear sweep potential was applied to the dsDNA/CP and the corresponding I-E curve was recorded, interfacial potential offset applied potential partially, making the I-E curve displace along potential axis. Zero current potential where circuit current I was equal to zero in the I-E curve was measured to check the displacement of the I-E curve. Based on the displacement, the thermodynamic constants of the interaction between dsDNA and TAM were determined. The binding ratio of dsDNA with TAM was found to be 1:1 and the apparent binding constant was (6.85±0.20)×10(6) M(-1). As zero current potentiometry was independent of the changes in redox potential or current of both dsDNA and TAM themselves, the interaction was studied in their natural forms without damage. Moreover, TAM can be determined. The detection limit was 1.1×10(-7) M.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.     

Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine

In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant K_m values of EP-PEI-CCA and EP-EDA-CCA were 22?mM and 30?mM, respectively, which were lower than that of the free enzyme (43?mM), suggesting that the support’s zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.     

Immobilization of hemoglobin on zirconium dioxide nanoparticles for preparation of a novel hydrogen peroxide biosensor

Direct electrochemistry and thermal stability of hemoglobin (Hb) immobilized on a nanometer-sized zirconium dioxide (ZrO2) modified pyrolytic graphite (PG) electrode were studied. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of (7.90 +/- 0.93)s(-1) and a formal potential of -0.361 V (-0.12 V versus NHE) in 0.1M pH 7.0 PBS. Both nanometer-sized ZrO2 and dimethyl sulfoxide (DMSO) could accelerate the electron transfer between Hb and the electrode. Spectroscopy analysis of the Hb/ZrO2/DMSO film showed that the immobilized Hb could retain its natural structure. This modified electrode showed a high thermal stability up to 74 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the aid of an electron mediator. The electrocatalytic response showed a linear dependence on the H2O2 concentration ranging from 1.5 to 30.2 microM with a detection limit of 0.14 microM at 3sigma. The apparent Michaelis-Menten constant KMapp for H2O2 sensor was estimated to be (0.31 +/- 0.02) mM, showing a high affinity.     

Semicontinual synthesis of alkyl galactosides using β-galactosidase entrapped in polyvinylalcohol hydrogel

Fungal β-galactosidase from Aspergillus oryzae was immobilized into polyvinylalcohol (PVA) hydrogel by LentiKats® technology and used for the production of short-chain alkyl glycosides. Ethyl- and propyl-β-d-galactopyranosides were prepared from lactose (100?g/L) and varying initial amounts of alcohol (10–30% v/v) at 40?°C and pH 4.5. The entrapped β-galactosidase preserved 50% of the initial transgalactosylation activity after 25 repeated cycles in the production of ethyl β-d-galactopyranoside. When 5% (v/v) propanol was used as an acceptor, the enzyme activity (30–32?U/g immobilized enzyme) remained constant for 25 repeated batch runs. These findings suggest that entrapped β-galactosidase into LentiKats® has a great potential to be one effective, reusable and easy producible biocatalyst for the production of alkyl glycosides in a large scale.     

Flow kinetics of immobilized beta-glucosidase

The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized on carbon paste electrode by silica sol-gel film

Direct electrochemical and electrocatalytic behaviors of hemoglobin (Hb) immobilized on carbon paste electrode (CPE) by a silica sol-gel film derived from tetraethylorthosilicate (TEOS) were investigated for the first time. Hb/sol-gel film modified electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for Hb Fe(III)/Fe(II) redox couple at about -0.312 V (versus Ag/AgCl) in a pH 7.0 phosphate buffer. The formal potential of Hb heme Fe(III)/Fe(II) couple varied linearly with the increase of pH in the range of 5.0-10.0 with a slope of 49.44 mV pH(-1), which suggests that a proton transfer is accompanied with each electron transfer (ET) in the electrochemical reaction. The immobilized Hb displayed the features of peroxidase and gave excellent electrocatalytic performance to the reduction of O2, NO2(-) and H2O2. The calculated apparent Michaelis-Menten constant was 8.98 x 10(-4)M, which indicated that there was a large catalytic activity of Hb immobilized on CPE by sol-gel film toward H2O2. In comparison with other electrodes, the chemically modified electrodes, used in this direct electrochemical study of Hb, are easy to be fabricated and rather inexpensive. Consequently, the Hb/sol-gel film modified electrode provides a convenient approach to perform electrochemical research on this kind of proteins. It also has potential use in the fabrication of the third generation biosensors and bioreactors.