WO1992012254A1 - Electrodes a enzyme - Google Patents

Electrodes a enzyme Download PDF

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Publication number
WO1992012254A1
WO1992012254A1 PCT/US1991/000201 US9100201W WO9212254A1 WO 1992012254 A1 WO1992012254 A1 WO 1992012254A1 US 9100201 W US9100201 W US 9100201W WO 9212254 A1 WO9212254 A1 WO 9212254A1
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redox
enzyme
polymer
crosslinking agent
electrode
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Brian A. Gregg
Adam Heller
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes

Definitions

  • This invention relates to electrodes that can selectively oxidize or reduce a biochemical in a solution. More particularly, it relates to electrodes that can translate the concentration of a biochemical to an electrical current, or can utilize an electrical current to selectively convert one biochemical to another.
  • Enzyme based biosensors i.e., electrochemical sensors capable of detecting the concentration of a single biochemical species in a medium containing a diverse mixture of other compounds
  • Amperometric enzyme electrodes typically require some form of electrical communication between the electrode and the active site of the redox enzyme that is reduced or oxidized by the substrate.
  • the electrooxidation of a reduced site or the electroreduction of an oxidized site is complicated by the fact that the active site is often located deep inside an insulating protein shell.
  • redox enzymes such as glucose oxidase do not directly exchange electrons with simple metal electrodes.
  • the first mediator employed for FAD-enzyme electrodes was the natural substrate of the flavin-linked oxidases, 0 2 .
  • the reaction of glucose oxidase (GO) is GO-FAD + glucose GO-FADH 2 + gluconolactone (l)
  • the first commercial ampero ⁇ tetric glucose sensors measured either the decrease in 0 2 concentration at an oxygen electrode, or the increase in H 2 0 2 concentration at a platinum electrode.
  • the H 2 0 2 formed degraded the enzyme. Nature alleviates this problem through the use of a second enzyme, usually catalase, which is present in high concentrations in cells and catalyses the disproportionation of the H 2 0 2 ; (2) the electrode current depended on the concentration of both the enzyme substrates, i.e., both glucose and 0 2 ; (3) measurement of the H 2 0 2 concentration required both a highly catalytic electrode (e.g., Pt) and a potential (ca. 0.7 V vs. SCE) substantially positive of the reversible potential for the FAD/FADH 2 couple (E° is approximately equal to -0.4 V vs. SCE) . This resulted in large spurious currents due to a number of easily oxidized species in the system to be measured. Because of (2) and (3) , the amperometric biosensors were not adequately substance-specific.
  • a second enzyme usually catalase
  • Enzyme electrodes such as those just described generally require that the enzyme and shuttle be confined to the proximity of the electrode surface.
  • the small shuttles commonly employed can, however, readily diffuse through the membranes that are needed to contain the enzyme, but permit the passage of the enzyme's substrate, e.g., glucose.
  • a polymeric redox "wire” based on the poly(vinyl-pyridine) (PVP) complex of Os(bpy) 2 Cl (abbreviated POs + ; the bpy of the complex is 2,2'- bipyridine) has been introduced which electrically connects the enzyme to the electrode yet, by virtue of its molecular size, remains confined behind the enzyme-containing membrane.
  • This polycationic redox polymer forms electrostatic complexes with the polyanionic glucose oxidase in a manner mimicking the natural attraction of some redox proteins for enzymes, e.g., cytochrome c for cytochrome c oxidase.
  • Enzyme electrodes now in use are of several different types.
  • One type of electrode amperometrically measures the oxygen content of gas streams entering and leaving a reactor containing the substrate and its enzyme. If oxygen is involved in the substrate's enzymatic oxidation, its level is depleted and the substrate concentration can be deduced from the decrease in the oxygen content of the gas.
  • a second type of enzyme electrode a natural electroreactive product of the enzyme-catalyzed reaction is amperometrically monitored.
  • the enzymatic reaction of substrates like glucose or lactate with oxygen, catalyzed by some oxidases produces hydrogen peroxide. Hydrogen peroxide can be electrooxidized and thereby the substrate concentration over a certain range can be translated into a current.
  • a non-natural redox couple mediates electron transfer from the substrate- reduced enzyme to the electrode.
  • the enzyme is reduced by its natural substrate at a given rate; the reduced enzyme is in turn, rapidly oxidized by a non- natural oxidizing component of a redox couple that diffuses into the enzyme, is reduced, diffuses out and eventually diffuses to an electrode where it is oxidized.
  • the oxidation current can be related to the concentration of the substrate.
  • a specific example of such a redox mediator is the ferricinium carboxylate/ferrocene carboxylate couple that diffusionally mediates electron transfer from glucose reduced glucose oxidase to a carbon electrode.
  • the current produced at a given substrate level can depend on the concentration of the active enzyme molecules. It has been shown that natural reaction products, like hydrogen peroxide, deactivate the enzyme. Enzymes are also continuously denatured. It has been shown that the denaturing of enzymes can be retarded by embedding the enzyme in a rigid three-dimensional polymer structure. It has been suggested that such embedding fixes the protein structure of the enzyme, preventing conformational changes that result in its eventual denaturing. For example, chymotrypsin has been stabilized by embedding it in crosslinked poly(methyl methacrylate) .
  • the invention relates to materials (and films formed from such materials) which include at least two components that can combine to form a three dimensional molecular structure. At least one of the components comprises a redox compound, and at least one other component comprises an oxidoreductase (hereinafter referred to as a redox enzyme) .
  • a redox enzyme an oxidoreductase
  • the three dimensional molecular structure provides electrical contact between that surface and the redox enzyme.
  • sigma bonds dominate the polymer's backbone, wherefore electron delocalization is limited.
  • three dimensional molecular structure means a structure in which covalent chemical bonds extend in three dimensions.
  • the term is not meant to include a three dimensional structure formed by mere physical bonding of molecules, for example through Van der Waals forces.
  • redox compound is used herein to mean a compound that can be oxidized and reduced.
  • the redox compound may have one or more functions that are reducible and oxidizable.
  • redox compound means a compound which contains one or more redox centers, "redox center” meaning a chemical function that accepts and transfers electrons.
  • a material comprising a redox enzyme, a crosslinking agent, and a crosslinkable compound capable of reaching with the crosslinking agent and the redox enzyme. Either the crosslinkable compound or the crosslinking agent, or both, have one or more redox centers.
  • a material is provided comprising a redox enzyme and a redox compound having two or more functional groups capable of reacting with the enzyme (i.e. a redox compound capable of crosslinking with the enzyme) .
  • the redox enzyme itself is used as the crosslinking agent to crosslink the redox compound into a three dimensional molecular structure.
  • Most (if not all) enzymes have multiple (more than two) functions that can react. Examples of such enzyme functions are amine, phenol, tryptophane, thiol, and imidazole functions. -7-
  • the redox enzyme is contained or incorporated within the crosslinked polymer structure in such a manner that the enzyme will not tend to diffuse out of the structure.
  • the enzyme may be chemically (covalently) bonded, electrostatically bonded, or hydrogen bonded to the polymer, or simply physically bound or trapped within cavities of the polymer surface.
  • crosslinkable compound is used herein to mean a compound containing at least two groups (i.e., a bi- or-multifunctional compound) capable of reacting with itself or another bi-or-multifunctional compound, resulting in a macromolecule.
  • crosslinking agent is used herein to mean a compound containing at least two functional groups capable of reacting with and crosslinking other compounds, i.e. it is the substance that crosslinks the crosslinkable compound.
  • Biosensors can be made in accordance with this invention to selectively sense numerous chemicals, including glucose, lactates, glycerol-3-phosphate, L-amino acids, D-amino acids, and nitrates.
  • an electrode having a surface coated with a film of a material of the class described above.
  • the term "film” is used broadly to include any coating or layer of the material regardless of thickness or method of application.
  • the present invention provides for the construction of enzyme electrodes employing this class of materials. This process may involve the mixture of the enzyme and the various polymer components in a common solution followed by the application of the solution to an electrode surface. Various application methods may be used, including (1) addition of drops of the solution onto the electrode surface; (2) dipcoating; (3) spincoating, or (4) spraying the solution onto the electrode surface. The application step is followed by a curing step such as drying in air or vacuum.
  • the process may involve the addition of the enzyme and polymer components in separate solutions to the surface of the electrode, mixing, and then curing in air or vacuum.
  • the preferred crosslinkable compounds for use in this invention are hydrophilic, containing chemical groups such as alcohols, carboxylic acids, amines, amides, sulfonates, sulfates, phosphates and phosphonates. Such groups tend to promote the solubility of the components in water which facilitates contact with the water soluble enzymes. Such groups may also improve the stability of the immobilized enzyme against denaturation.
  • the redox compounds (or redox centers contained within compounds) used in this invention may be either organic or inorganic. Transition metal complexes with organic ligands such as bipyridine or cyclopentadiene are often preferred as redox centers because of their chemical stability in various oxidation states and their facile electron transfer kinetics. Typical examples of such complexes are the polypyridine complexes of di-or trivalent osmium ions and the various derivatives of ferrocene (bis-cyclopentadienyl iron) or cobaltocene (bis-cyclopentadienyl cobalt) . However, a number of organic redox centers may also be employed. The various derivatives of viologen (N,N'-bis alkyl-4,4'-bipyridine) constitute typical examples of this class.
  • the preferred crosslinking agents are water soluble compounds that react under conditions where most enzymes are stable, that is around pH 7 and room temperature. Included in this category of crosslinking agents are multifunctional epoxides, aldehydes, imidoesters, N- hydroxysuccinimide esters and carbodiimides. A number of reagents with limited solubility in water may also be used by dissolving them in a water- iscible organic solvent such as acetone, methanol, acetonitrile or dimethylformamide. Included in this category are reagents such as cyanuric chloride, tetrachlorobenzoquinone, benzoquinone and tetracyanoquinodimethane. These reagents may react with one or more types of functions including amines, alcohols, thiols and carboxylic acids which may be present on the surface of enzymes and may also be included in the structure of the redox compound.
  • the electrodes to which the crosslinked redox polymer is applied can be made of any of a number of metals, semi- metals, or semiconductors. For example, gold, platinum, glassy carbon, or graphite electrodes may be used.
  • osmium bis(2,2'- bipyridine) dichloride is coordinated to a poly(vinyl- pyridine) chain forming approximately one osmium bis(bipyridine) vinylpyridine chloride complex per five vinylpyridine units.
  • the remaining vinylpyridines are quaternized with bromoethylamine hydrobromide, leading to a very hydrophilic redox polymer containing pendant ethylamine groups.
  • This polymer may be dissolved in an aqueous solution containing the enzyme and a water soluble diepoxide, such as poly(ethylene glycol diglycidyl ether) .
  • the epoxide may react with both the ethylamine pendant groups of the redox polymer and the surface lysine residues of the enzyme. This results in an enzyme-containing crosslinked redox polymer film on the electrode surface.
  • the method of operation of such an enzyme electrode may be illustrated using a glucose electrode as an example.
  • the glucose diffuses into the film where it may react with the glucose oxidase enzyme forming gluconolactone and the reduced form of the enzyme.
  • the reduced enzyme may then be oxidized by the osmium complex- containing polymer. Electrons are subsequently transferred through the polymer to the electrode. Thus, an electrical current proportional to the concentration of the enzyme substrate is achieved.
  • Electrons from a substrate-reduced enzyme can be transferred either to the enzyme's natural re-oxidizer (oxygen in the case of glucose oxidase, lactate oxidase and other flavoenzymes) or, via the redox-centers of the polymer to the electrode. Only the latter process contributes to the current. Thus, it is desirable to make the latter process fast relative to the first. This can be accomplished by (a) increasing the concentration of the redox centers (e.g. the number of osmium complexes) in the film, or (b) assuring that these centers are fast, i.e. that they are rapidly oxidized and reduced. It is also desirable to make the redox centers oxidizing with respect to the reduced enzyme. This often increases the rate of transfer of electrons to the electrode.
  • the enzyme's natural re-oxidizer oxygen in the case of glucose oxidase, lactate oxidase and other flavoenzymes
  • FIG. 1 is a schematic drawing of a crosslinked redox polymer-enzyme electrode as provided by the present invention.
  • FIG. 2 shows several examples of redox centers bound to multifunctional compounds capable of forming crosslinked polymers when reacted with crosslinking agents, including enzymes or other multifunctional compounds, in accordance with the present invention.
  • FIG. 3 shows several examples of crosslinking agents used by the present invention and some of the typical reactions which they undergo.
  • FIG. 4 shows a synthetic scheme for one of the preferred crosslinkable redox polymers as provided by the present invention.
  • FIG. 5 shows a number of cyclic voltam ograms of a crosslinked redox polymer film containing glucose oxidase prepared according to the present invention. There is no glucose in solution. Scan rates (mV/s) (a) 10, (b) 20, (c)
  • FIG. 6 shows a cyclic voltammogram of the film used in FIG. 5 after addition of 40 mM glucose. Scan rate 5 mV/s.
  • FIG. 7 shows a typical response curve (current density versus substrate concentration) for a glucose electrode prepared in accordance with the present invention.
  • the materials and processes provided by the present invention, the crosslinked redox polymers and the incorporation of redox enzymes in them, have particularly important applications in the manufacture of enzyme electrodes of the type illustrated in FIG. 1. These electrodes may be used in such applications as amperometric biosensors and the electrosynthesis of biochemicals.
  • an enzyme electrode system based on a crosslinked redox polymer there are several advantages to an enzyme electrode system based on a crosslinked redox polymer.
  • the use of crosslinked films on the electrode surface eliminates the requirement for a membrane which is often required in conventional systems to confine the enzyme to a small volume close to the electrode surface.
  • the use of crosslinked redox films tends to simplify the design and the manufacture of the enzyme electrode.
  • the process by which the electrodes are produced is relatively simple, reproducible and can be easily automated.
  • the enzyme may be stabilized by its interaction with the polymer matrix, thus retarding thermal denaturation. Also, it may be physically protected from attack by proteases in solution which are too large to diffuse through the polymer film.
  • the versatility of these materials allows the tailoring of properties for specific applications.
  • the redox potential, the hydrophilicity and the charge on the polymer may be adjusted as may the crosslinking method.
  • Sixth, the resulting electrodes are in general mechanically rugged and typically exhibit excellent stability during storage.
  • the water soluble crosslinking agent polyethylene glycol diglycidylether (PEG-DGE, FIG. 3) is used to react with redox compounds with amine functions and with amine functions of the lysine groups of the enzyme.
  • PEG-DGE polyethylene glycol diglycidylether
  • the reaction between epoxides and amines is particularly advantageous since the reaction (1) releases no low molecular weight species; (2) does not greatly change the local pH; (3) does not greatly change the charge on either the redox compound or the enzyme; and (4) is compatible with a number of different enzymes.
  • PEG- DGE is also commercially available in a number of chain lengths. The reaction between PEG-DGE and amines proceeds very slowly in dilute aqueous solution.
  • all the reactants may be combined in a single solution before the application step which greatly simplifies the manufacture of the electrodes.
  • the crosslinking reaction may then proceed to completion when the solution is dried on the surface of the electrode.
  • the cure time for the film is 24 to 48 hours at room temperature.
  • FIG. 1 An enzyme electrode as provided by the present invention is shown schematically in FIG. 1.
  • the electrode 10 has a surface 12 which is coated with a crosslinked redox polymer film 14.
  • a redox enzyme 16 is bound to the polymer 14.
  • the polymer 14 electrically connects the electrode 10 to the enzyme 16.
  • Various preferred crosslinkable compounds containing redox active centers are shown in FIG. 2.
  • Polymer A and Polymer F are representative of that class of compounds which require only the addition of enzymes to form crosslinked films, i.e. the enzyme is the only required crosslinking agent.
  • the other compounds are representative of that class of compounds which do not react directly with chemical functions on the enzyme. They therefore require a separate crosslinking agent such as those illustrated in FIG. 3.
  • FIG. 3 shows three representative classes of crosslinking agents, and their reactions with a typical organic compound having an amine group, represented as RNH 2 .
  • the crosslinking agents shown are an epoxide (e.g. PEG- DGE) , cyanuric chloride, and an N-Hydroxysuccinimide.
  • FIG. 6 shows a cyclic voltammogram of the same film as FIG. 5 after the addition of glucose to a final concentration of 40 mM.
  • a catalytic oxidation is exhibited as the electrons are transferred from the glucose-reduced enzyme to the redox polymer and from the redox polymer to the electrode.
  • FIG. 7 A typical response curve of a Polymer C-glucose oxidase-PEG-DGE film is shown in FIG. 7. As the glucose concentration is increased the current response follows the characteristic Michaelis-Menten behavior of the enzyme.
  • Solution 1 contained 10 mg/ml
  • Polymer C Solution 2 contained 5 mg/ml glucose oxidase Solution 3 contained 2.7 mg/ml PEG-DGE
  • the enzyme containing solution was made up fresh every day; the other two solutions were stable for at least one month. 15 microliters of solution 1, 15 microliters of solution 2 and 5 microliters of solution 3 were thoroughly mixed in a vial and 3 microliters of the mixture was deposited onto a glassy carbon disk electrode (4.5 mm in diameter). The electrode was then placed in .a vacuum dessicator for 24 hours. Upon exposure to solutions containing high concentrations of glucose (>. 60 mM) , such electrodes commonly exhibited current densities of 400 -1100 microA/cm 2 at a potential in the 0.35 -0.45 volt range measured relative to the potential of the Standard Calomel Electrode (SCE) . In the absence of glucose, the current density was approximately 1 microA/cm 2 .
  • SCE Standard Calomel Electrode
  • Example 1 The procedure of Example 1 was repeated but cyanuric chloride was used as the crosslinking agent in place of PEG-DGE.
  • the polymer and enzyme were made up in 100 mM phosphate buffer solution at pH 7.1. 2 microliters each of the polymer and enzyme solution were mixed on the electrode surface with 0.5 microliters of an acetonitrile solution of cyanuric chloride (20 mM) .
  • This crosslinking reaction is quite fast and the electrode films required a curing time of only about 30 minutes in air or vacuum.
  • bromoacetyl chloride was dissolved in 120 ml of methylene chloride and cooled to 0"C under nitrogen. 13.4 gram N-hydroxysuccinimide and 11.8 gram triethylamine were dissolved in 50 ml of methylene chloride and slowly dripped into the cold solution of acid chloride over 30 minutes. The solution was stirred for an additional 20 minutes. Then ice water was added, the phases were separated, the organic phase was washed two more times with ice water, once with saturated sodium chloride solution and dried over magnesium sulfate. The solution was concentrated under vacuum until crystals started to appear. Then hexane was added and the solution was cooled to 0 ⁇ C. The crystals of bromoacetoxysuccinimide were filtered and dried in a vacuum dessicator.
  • Example 3 The synthetic procedure of Example 3 was repeated with the substitution of 3-bromopropionyl chloride for bromoacetyl chloride.
  • the resulting polymer containing esters of hydroxysuccinimide was dispersed in DMF and a large excess of ethanolamine was added. The mixture was stirred overnight at room temperature, filtered and poured into stirred tetrahydrofuran (THF) . The precipitate was filtered and dried. This procedure led to a polymer whose approximate structure is shown in FIG. 2 (Polymer B) .
  • Solution 1 contained 10 mg/ml Polymer B
  • Solution 2 contained 8 mg/ml glycerol-3- phosphate oxidase
  • Solution 3 contained 4 mg/ml cyanuric chloride in acetonitrile 5 microliters each of solutions 1 and 2 were mixed on the surface of a glassy carbon disk electrode with 2 microliters of solution 3.
  • the electrode was dried in vacuum for 50 minutes.
  • this electrode In the presence of 10 mM L-alpha- glycerophosphate this electrode exhibited a current density of 30 microA/cm 2 when held at a potential of 0.45 volts relative to the SCE reference.
  • the current density was l.l microA/cm 2 at the same potential.
  • N-methyl-4,4 '-bipyridinium iodide (monoquat) wa ⁇ synthesized by a standard technique. 1.13 gram monoquat was dissolved in 70 mis. acetonitrile and 25 mis. DMF.
  • Solution 1 was 5 mg/ml
  • Polymer D Solution 2 was about 5 mg/ml nitrate reductase Solution 3 was 2.7 mg/ml PEG-DGE 25 microliters of solutions 1 and 2 were thoroughly mixed with 10 microliters of solution 3. 4 microliters of this mixture was applied to the surface of a 3 mm diameter glassy carbon disk electrode and cured overnight in a vacuum at room temperature. Upon exposure of this electrode to a deaerated solution containing 25 mM nitrate, a reduction current density of 22.6 microA/cm 2 was recorded at a potential of -0.8 volts relative to the SCE reference. Under the same conditions in the absence of nitrate ion the background current density was 7.0 microA/cm 2 .
  • 4'-Methy1,4 '-(4-bromobutyl) bipyridine made from the monolithium salt of dimethylbipyridine and 1,4- dibromobutane, was used as a starting material, l.ll gram of this was dissolved in 50 mis. of ethylene diamine and warmed to about 80"C for 2.5 hours. The solvent was then removed under vacuum, the residue was dissolved in ethyl acetate and the product was extracted into aqueous solution at pH 5.1. The aqueous solution was washed with methylene chloride. It was then made basic and the product was extracted into methylene chloride, washed with water, dried and evaporated.
  • a 3mm glassy carbon disk electrode was made by applying 3 microliters of 5 mg/ml glucose oxidase in 10 mM HEPES buffer pH 8.1, 1 microliter of 2.7 mg/ml PEG-DGE in the same buffer and 3 microliters of 10 mg/ml Polymer G in acetonitrile. The electrode was cured overnight in vacuum. Upon exposure to a solution containing a high concentration of glucose (> 60 mM) , this electrode exhibited a current density of 2.1 microA/cm 2 when held at a potential of 0.15 V relative to the SCE reference. The background current density in the absence of glucose was 0.84 microA/cm 2 at the same potential.

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Abstract

Electrodes a enzyme dont une surface est recouverte d'une pellicule. Ladite pellicule est formée à partir de matériaux dans lesquels une enzyme redox est liée par covalence à une structure moléculaire tridimensionnelle. La structure moléculaire est du type comprenant des centres redox multiples, par exemple un polymère redox réticulé.
PCT/US1991/000201 1991-01-10 1991-01-10 Electrodes a enzyme Ceased WO1992012254A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2699170A1 (fr) * 1992-12-15 1994-06-17 Asulab Sa Complexes d'un métal de transition à ligands 2,2'-bipyridine substitués par au moins un radical ammonium alkyle, leur procédé de fabrication et leur application comme médiateur redox.
EP0639268A4 (fr) * 1992-05-08 1995-04-12 Heller E & Co Electrodes enzymatiques ameliorees.
GB2296773A (en) * 1994-12-26 1996-07-10 Agency Ind Science Techn Enzyme electrode
EP1199357A1 (fr) * 2000-09-20 2002-04-24 Randox Laboratories Ltd. Stabilisation d'enzymes pendant la congélation
USRE44522E1 (en) 2001-09-14 2013-10-08 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
WO2020019786A1 (fr) * 2018-07-27 2020-01-30 三诺生物传感股份有限公司 Méthode de préparation d'un film de biodétection, film de biodétection et dispositif de surveillance
WO2022037439A1 (fr) * 2020-08-18 2022-02-24 微泰医疗器械(杭州)有限公司 Capteur électrochimique de glucose et son procédé de préparation

Citations (1)

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JPS56126757A (en) * 1980-03-12 1981-10-05 Toshiba Corp Manufacture of fixed enzyme film for enzyme electrode

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS56126757A (en) * 1980-03-12 1981-10-05 Toshiba Corp Manufacture of fixed enzyme film for enzyme electrode

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL CHEMISTRY vol. 62, 1990, WASHINGTON DC USA pages 258 - 263; B.A. GREGG ET AL.: 'Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications.' See whole article. *
M.P. COUGHLAN. 'Molybdenum-containing enzymes.' 1980 , PERGAMON PRESS , OXFORD UK Chapter 5: Concepts and approaches to the understanding of electron transfer processes in enzymes containing multiple redox centers. G. Palmer et al. see page 187 - page 220 *
PATENT ABSTRACTS OF JAPAN vol. 6, no. 2 (P-96)(880) 8 January 1982 & JP,A,56 126 757 ( TOKYO SHIBAURA DENKI KK ) 5 October 1981 see abstract *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0639268A4 (fr) * 1992-05-08 1995-04-12 Heller E & Co Electrodes enzymatiques ameliorees.
FR2699170A1 (fr) * 1992-12-15 1994-06-17 Asulab Sa Complexes d'un métal de transition à ligands 2,2'-bipyridine substitués par au moins un radical ammonium alkyle, leur procédé de fabrication et leur application comme médiateur redox.
EP0602488A1 (fr) * 1992-12-15 1994-06-22 Asulab S.A. Complexes d'un métal de transition à ligands 2,2-bipyridine substitués par au moins un radical ammonium alkyle, leur procédé de fabrication et leur application comme médiateur redox
US5410059A (en) * 1992-12-15 1995-04-25 Asulab S.A. Transition metal complexes having 2,2'-bipyridine ligands substituted by at least one ammonium alkyl radical
GB2296773A (en) * 1994-12-26 1996-07-10 Agency Ind Science Techn Enzyme electrode
GB2296773B (en) * 1994-12-26 1998-12-09 Agency Ind Science Techn Enzyme electrode and method of manufacturing same
EP1199357A1 (fr) * 2000-09-20 2002-04-24 Randox Laboratories Ltd. Stabilisation d'enzymes pendant la congélation
USRE44522E1 (en) 2001-09-14 2013-10-08 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
USRE45764E1 (en) 2001-09-14 2015-10-20 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
WO2020019786A1 (fr) * 2018-07-27 2020-01-30 三诺生物传感股份有限公司 Méthode de préparation d'un film de biodétection, film de biodétection et dispositif de surveillance
JP2021516764A (ja) * 2018-07-27 2021-07-08 三諾生物伝感股▲フン▼有限公司Ｓｉｎｏｃａｒｅ Ｉｎｃ． バイオセンシングフィルムの製造方法、バイオセンシングフィルム及び監視装置
WO2022037439A1 (fr) * 2020-08-18 2022-02-24 微泰医疗器械(杭州)有限公司 Capteur électrochimique de glucose et son procédé de préparation

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