A glucose oxidase inmobilized electrode based on modified graphite

Glucose oxidase (E. C. 1.1.3.4) was immobilized on electrochemically modified graphite to obtain an enzyme electrode. The working surface of the electrode was coated with gelatine to prevent desorption of the enzyme. In substrate (glucose) solutions the amperometric signal of the enzyme electrode was due to the electroreduction of H202 generated in the enzyme layer. The linearity of the electrode response was found up to a substrate concentration of 300 microM at a working potential of 0 mV (vs. Ag/AgCl). It was shown that the electrode did not respond to L-ascorbic and uric acid at that working potential. The response time was about 2 min. The enzyme electrode keeps about 50% of its initial activity after a one-week storage at 4 degrees C.     

Magnetic enzyme membranes as active elements of electrochemical sensors: specific amino acid enzyme elctrodes.

The basic principle of the described magnetic enzyme electrodes is a kinetic accumulation of CO2 at the active layer electrode interface. The local pCO2 level is linked to three simultaneous phenomena: substrate diffusion in, enzyme reaction CO2 diffusion out. After a transient state there is a stationary state between the quantity of CO2 produced by the enzyme reaction and the CO2 diffusing from the active membrane to the bulk solution. Continuous determination of free amino acids in biological media is useful in biological processing, fermentation, medicine, pharmaceutical industries and biological research. No methods are presently available for any specific continuous measurement of lysine which is of nutritional importance in protein industrial syntheses; of phenylalanine and tyrosine which have to be monitored in several inborn diseases (phenylketonuria being the most important of them); of arginine and histidine which play a still imperfectly understood part in neurochemistry. The use of decarboxylase bearing membranes as sensors in such measurements could offer several novel advantages: (a) a simple device made of a currently manufactured electrode slightly modified by the use of an enzyme membrane; (b) The absence of any enzymic consumption due to the immobilization and the negligible consumption of substrate during the measurements; (c) The sensitivity which can be sharpened by a systematic study of the membrane parameters; (d) the continuous response of the electrode as long as it is in contact with the substrate solution; (e) the further feasibility as a miniature sensor. The magnetic device introduced allows obviously a convenient use of the enzyme electrode, the active part can be removed and replaced without disturbance for the pCO2 electrode itself. The enzyme electrodes are not only useful at the applied point of view but also at the fundamental point of view by allowing a direct measurement of an intra membrane concentration. The influence of simple structures on enzyme kinetics was studied with enzyme electrodes by our group, in the case of memory and oscillations obtained with enzyme systems.     

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 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.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

A rapid competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) test strip for microcystin-LR (MCLR) determination

A competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) is described for the determination of microcystin-LR (MCLR) using a double-sided microporous gold electrode in cartridge-type cells. A gold film sputtered on one side of porous nylon membrane constitutes a working electrode, while another gold film formed on the opposite side serves as a pseudo reference electrode. After immobilizing MCLR antibody on working electrode by physical adsorption, the double-sided electrode was placed simply in a diffusion U-type or within a dry strip-type cell with a conjugate pad pre-loaded with a glucose oxidase labeled MCLR (GOx-MCLR) on working electrode side. Assays were performed in two steps: an MCLR-containing sample mixed with a known amount of GOx-MCLR conjugate either in buffer solution or in pre-loaded dry pad was incubated for an appropriate period (about 10 min) to induce competitive reaction with an immobilized anti-MCLR antibody on working electrode, and a fixed concentration of glucose solution (substrate) was then added to the backside of the working electrode. Due to the competitive nature of the assay, enzymatically generated product, hydrogen peroxide (H2O2), was detected at the working gold electrode (at +800 mV versus Au) by oxidation, and the magnitude of amperometric current was inversely proportional to the concentration of MCLR in the sample. The response time after substrate addition was about 30s. Mean recovery of MCLR added to tap water was 93.5%, with a coefficient of variation (CV) of 6.6%. The proposed competitive NEEIA system is in general comparable to existing heterogeneous enzyme immunoassays with a similar detection limit (100 pg/mL MCLR), and suitable for developing a disposable type biosensor for on-site monitoring of environment.     

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at l00-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO₂, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated matix after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.     

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.     

A glucose oxidase electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant

This study investigated a new glucose sensor prepared by electrochemical polymerization of pyrrole with polyanion/poly(ethylene glycol) (PEG)/glucose oxidase (GOD) conjugate dopants. GOD was coupled to a strong polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid) (AMPS) via PEG spacer to effectively and reproducibly immobilize GOD within a polypyrrole matrix onto a Pt electrode surface. PEGs with four different chain lengths (1000, 2000, 3000, and 4000) were used as spacers to study the spacer length effect on enzyme immobilization and electrode function. After conjugation, more than 90% of the GOD bioactivity was preserved and the bioactivity of the conjugated GOD increased with longer PEG spacers. The resulting polyanion/PEG/GOD conjugate was used as a dopant for electropolymerizing pyrrole. The activity of the immobilized enzyme on the electrode ranged from 119 to 209 mU cm(-2) and the bioactivity increased with the use of longer PEG spacers. The amperometric response of the enzyme electrode was linear up to 20 mM glucose concentration with a sensitivity ranging from 180 to 270 nA mM(-1) cm(-2). The kinetic parameters Michaelis-Menten constant (K(M)(app)) and maximum current density (j(max)) depended on the amount of active enzyme, level of substrate diffusion, and PEG spacer length. An increase in the electrical charge passed during polymerization (thus, increasing polypyrrole thickness) to 255 mC cm(-2) increased the sensitivity of the enzyme electrode because of the greater amount of incorporated enzyme. However, although the amount of incorporated GOD continued to increase when the charge increased above 255 mC cm(-2), the sensitivity began to decline gradually. The condition for preparing the enzyme electrode was optimized at 800 mV potential with a dopant concentration of 1 mg ml(-1).     

Bionanotechnology approach for FAD-dependent enzymes with nanomaterials sensor

We have reported the modification of biomolecule with nanomaterials. In this paper, the electrochemical response of different FAD-dependent enzymes at carbon nanomaterials modified electrode. The modified electrode also exhibits a promising enhanced electrocatalytic activity toward the oxidation of substrate. Different methods were used for fabrication of modified electrode. The presence of nanomaterials enhances the enzyme loading and stability. Cyclic voltammograms (CVs) were used for the determination of substrate and the apparent coefficient values for these compounds at different electrodes. Finally, we have studied the surface morphology of the modified electrode using scanning electron microscopy (SEM), which revealed that enzyme is coated on nanomaterials.     

An amperometric xanthine oxidase enzyme electrode based on hydrogen peroxide electroreduction

A xanthine oxidase enzyme electrode (xanthine oxidase immobilized on electrochemically modified graphite and conveniently coated with gelatine electrode working surface) for quantitative analysis of xanthine is proposed. The detection of thus developed electrochemical system is based on the electroreduction of hydrogen peroxide generated in enzyme layer and offered L-ascorbic and uric acid reducing interference effect on the substrate determination. At a working potential -50 mV (vs. Ag/AgCl) the detection limit of 4.5 microM and the linearity of the amperometric signal up to substrate concentration of about 40 microM were found. At that working potential, the electrode is practically inert towards L-ascorbic- and uric acid present. The response time did not exceed 2 min.     

Hydrogen peroxide production from reactive liposomes encapsulating enzymes.

Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating dimethyldioctadecylammonium bromide and DMPC incorporating phosphatidylinositol, respectively. The liposomes were prepared by the vesicle extrusion technique and had the enzymes glucose oxidase (GO) encapsulated in combination with horseradish peroxidase (HRP) or lactoperoxidase (LPO). The generation of hydrogen peroxide from the liposomes in response to externally added D-glucose substrate was monitored using a Rank electrode system polarised to +650 mV, relative to a standard silver-silver chloride electrode. The effects of encapsulated enzyme concentration, enzyme combinations (GO+HRP, GO+LPO), substrate concentration, electron donor and temperature on the production of hydrogen peroxide have been investigated. The electrode signal (peroxide production) was found to increase linearly with GO incorporation, was reduced on addition of HRP and an electron donor (o-dianisidine) and showed a maximum at the lipid chain-melting temperature from the anionic liposomes containing no cholesterol. To aid interpretation of the results, the permeability of the non-reactive substrate (methyl glucoside) across the bilayer membranes was measured. It was found that the encapsulation of the enzymes effected the permeability coefficients of methyl glucoside, increasing them in the case of anionic liposomes and decreasing them in the case of cationic liposomes. These observations are discussed in terms of enzyme bilayer interactions.     

The electrode adsorption method for determination of enzyme activity: a study of substrate requirements

The electrode adsorption method for the determination of enzyme activity requires substrates that, besides having good kinetics constants for the enzyme, also show good adsorption/desorption kinetics to the electrode surface and adsorb in such a way that they change the double-layer capacitance of the electrode. A series of peptide substrates containing one to three aromatic groups has been synthesized. Our results show that the aromatic groups are of crucial importance for the capacitance change caused by the adsorbing/desorbing substrate. Thus, the tripeptide substrate, Bz-Phe(NO2)-Val-Arg-pNA, with three aromatic groups is superior to the other synthesized substrates containing only one or two aromatic groups. Our desorption experiments show that several factors determine the rate of capacitance increase observed when thrombin is added to a substrate solution in equilibrium with a substrate-covered electrode. The kinetic constants of the substrate determine how the substrate concentration in the solution decreases and, consequently, determine the spontaneous desorption measured as capacitance increase. Thrombin does not seem to split adsorbed substrate molecules but it adsorbs to the substrate-covered surface and in that way causes a capacitance decrease counteracting the change caused by desorption of substrate.     

A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

Electronically modulated biological functions of molecular interfaced enzymes and living cells

Conducting polymer molecular interfaces have been implemented to modulate biological functions of fructose dehydrogenase, pyruvate oxidase and Saccharomyces cerevisiae at the electrode surface by adjustment of electrode potential. The enzyme activity of the polypyrrole-interfaced fructose dehydrogenase was electronically modulated by means of electron transfer between the enzyme and the electrode surface. The enzyme activity of polypyrrole-interfaced pyruvate oxidase was modulated by an electronically driven change of substrate concentration. The gene expression in polypyrrole-interfaced Saccharomyces cerevisiae was electronically induced by a change in the phosphate concentration.     

A facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode

A kind of solid substrate, glassy carbon (GC) electrode, was selected to support self-assembled lipid layer membranes. On the surface of GC electrode, we made layers of dimyristoylphosphatidylcholine (DMPG, a kind of lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We immobilized horseradish peroxidase (HRP) into the supported bilayer lipid membranes (s-BLM) to develop a kind of mediator-free biosensor for H2O2. The biosensor exhibited fine electrochemical response, stability and reproducibility due to the presence of the s-BLM. As a model of biological membrane, s-BLM could supply a biological environment for enzyme and maintain its activity. So s-BLM is an ideal choice to immobilize enzyme for constructing the mediator-free biosensor based on GC electrode.     

Glycosylated proline-rich peptide of human parotid saliva. Circular dichroism study.

The direct monitoring of sugars such as lactose, maltose, saccharose is not only useful at the applied point of view but also at the fundamental point of view for studying enzymology, especially in microbiology and fermentation. Benzyme systems were extensively used in solution for analytical applications in industry and medicine. The progress in the field of immobilization of bienzyme systems [1-3], especially within membranes [4-5], makes possible the production of new analytical devices. From the studies dealing with concentration profiles in artificial enzyme membranes [14], evidence was obtained for a well defined relationship between the local concentration of a metabolite and concentration of the first substrate in the bulk solution. In the described systems a substrate is transformed into glucose within a membrane, the glucose is then transformed in gluconic acid with a local oxygen consumption. The local pO2 level is linked to the glucose oxidase velocity, which is only linked to the glucose production, that is to say to the concentration of the first substrate. The enzyme electrode is based on the transformation of kinetic phenomena (reaction rates) into absolute values (local concentrations) through the diffusion-reaction coupling process. The manufacture of magnetic enzyme electrodes [6] allows convenient use of the active sensors. The pO2 electrode has some adventages, namely the specificity based on the selectivity of the gas permeable membrane and the linear relationship between the oxygen and the output of the electrode. pCO2, pH, ion electrodes give a logarithmic response as a function of the concentration. The grafting of a multienzyme system on a sensor allows a study of sequential systems in a defined context with a measurement of the local concentration of the metabolites. The tool is useful for both kinetics [4] and regulation studies [5].     

Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode

A new amperometric biosensor, based on adsorption of glucose oxidase (GOD) at the platinum nanoparticle-modified carbon nanotube (CNT) electrode, is presented in this article. CNTs were grown directly on the graphite substrate. The resulting GOD/Pt/CNT electrode was covered by a thin layer of Nafion to avoid the loss of GOD in determination and to improve the anti-interferent ability. The morphologies and electrochemical performance of the CNT, Pt/CNT, and Nafion/GOD/Pt/CNT electrodes have been investigated by scanning electron microscopy, cyclic voltammetry, and amperometric methods. The excellent electrocatalytic activity and special three-dimensional structure of the enzyme electrode result in good characteristics such as a large determination range (0.1-13.5mM), a short response time (within 5s), a large current density (1.176 mA cm(-2)), and high sensitivity (91mA M(-1)cm(-2)) and stability (73.5% remains after 22 days). In addition, effects of pH value, applied potential, electrode construction, and electroactive interferents on the amperometric response of the sensor were investigated and discussed. The reproducibility and applicability to whole blood analysis of the enzyme electrode were also evaluated.     

Polyvinylferrocenium immobilized enzyme electrode for choline analysis

The response characteristics of a new enzyme electrode for determining choline are reported. The enzyme electrode consists of a polyvinylferrocenium perchlorate coated Pt surface onto which the enzyme, choline oxidase, is attached. Choline oxidase catalyzes the oxidation of choline to betaine, producing H₂O₂. Current due to H₂O₂ oxidation catalyzed by polyvinylferrocenium centers was measured. The effects of choline concentration, the amount of enzyme immobilized and the operating pH and temperature on the response of the enzyme electrode were studied. The effects of interferents were also investigated. The response time was found to be 60–70 s and the upper limit of the linear working portion was found to be 1.2 mM choline concentration. The minimum substrate concentration that produced detectable current was 4.0×10⁻⁶ M choline concentration. The steady-state current of this enzyme electrode was reproducible within ±4.6% of relative error. The apparent Michaelis–Menten constant (K_Mapp) and the activation energy, E_a, of this immobilized enzyme system were found to be 2.32 mM and 38.91 kJ/mol, respectively.