LT4605B - Enzymatic glucose sensor and process for producing thereof - Google Patents

Enzymatic glucose sensor and process for producing thereof Download PDF

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Publication number
LT4605B
LT4605B LT99-032A LT99032A LT4605B LT 4605 B LT4605 B LT 4605B LT 99032 A LT99032 A LT 99032A LT 4605 B LT4605 B LT 4605B
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Lithuania
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enzyme
glucose
electrode
biosensor
dehydrogenase
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LT99-032A
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Lithuanian (lt)
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LT99032A (en
Inventor
Valdas-Stanislovas Laurinavičius
Bogumila Kurtinaitienė
Klemensas Bernotas
Rolandas Meškys
Remigijus Šimkus
Virginijus Liauksminas
Terje Skotheim
Leonid Boguslavsky
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Biochemijos Institutas
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Publication of LT4605B publication Critical patent/LT4605B/en

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Abstract

This invention includes a ferment biosensor for determining glucose, its composition, polymer for biosensor selection and production. Glucose sensor may be used in medicine for purposes of diagnostics, determining the amount of sugar in blood, for food quality controls, determining the amount of glucose in food products, the microbiology industry, determining the amount of glucose in microbiologic environments or for scientific research, determining glucose concentration in various biologic liquids and microbiological media, and for controlling production processes. The invention is implemented by the sensor structure, made of a carbonic electrode, ferment dehydrogenises and electrochemically active redox polymers. The polymers described are original electrochemically active substances at the same time performing several functions: mediation, ferment immobilizing structure and diffusion controlling layer on the sensor's surface. They are obtained by affecting certain monomers through lactase ferment. Using the sensor proposed in the invention, it is possible to measure glucose concentration from 0.1 mmoles/l to 22 mmoles/l, and the oxygen concentration changes during glucose determination do not influence the sensor indications.

Description

1

The invention relates to the analysis of materials by means of enzymes, in which electrochemical parameters are determined. The invention relates to an enzyme, a biosensor for glucose detection, its creation, polymers for the production of a biosensor and their production. The glucose sensor can be used in medicine for diagnostic purposes (eg for determining blood sugar levels), for food quality control (eg for determining glucose in food), for microbiological industry (eg for determination of glucose in microbiological media) or for research for glucose determination. concentrations in various biological fluids and microbiological media as well as in the control of manufacturing processes.

The method of glucose enzyme detection is based on the reaction: glucose + Oox - - gluconic acid + Ored 1 where E (enzyme) is a catalyst that selectively recognizes β-D-glucose and catalyzes its oxidation, and the reduced form of the oxidizer (Orecj) is recorded in some way: eg electrochemically or spectrophotometrically. In the solution, these reactions are generally carried out using glucose oxidase or β-nicotinamide adenine dinucleotide (NAD) containing glucose dehydrogenase and water-soluble, often colored oxidant.

Using a glucose oxidation-catalyzing enzyme in immobilized state and assembling a catalytic layer with an appropriate oxidant on the surface of the electrochemical sensors, enzymatic electrodes (sensors) are obtained.

Electrochemical Bio-Sensors Containing Various Biological Materials - A Rapidly Developing Research Area [J. Davis, D.H. Vaughan, M.F. Cardosi Enzyme and Microbial Technology, 1995, 17, 1030-1035] The operating principles of these biosensors are similar, but the biosensor structures have a wide variety. This diversity can be interpreted on the basis of some of the electrochemical sensor design elements: the sensory-enzyme, the polymers that are used to immobilize the sensory material on the electrode surface and the artificial mediators that carry the electrons between 2

LT 4605 B enzyme and electrode. The latter are necessary because it is very difficult to directly carry out the electron exchange between the enzyme's active center and the electrode surface. Due to the high NADH superconductivity mediators are necessary for the construction of biosensors containing dehydrogenases belonging to the cofactor of β-nicotinamide adenine dinucleotide (NADH) [P. Leduc, D. Thevenot Bioelectrochem. Bioenerg., 1974, 1, 96-107. J. Moiroux, P.J. Elving Anai. Chem., 1979, 51, 346-350]. Other known coenzymes, such as flavidinenindinucleotide (FAD) and pyrolochinolinquinone (PQQ), are within the globule of the protein, which further complicates electron transport [R. Vilson, A.P.F. Turner Biosensor & Bioelectronics 1992,7,165-185]. When constructing multi-use electrochemical biosensors and biosensors used in vivo, it is necessary to use water-insoluble mediators or immobilize them on the electrode surface. Reliable mediator immobilization techniques can be considered to be their covalent attachment to the polymer matrix or to the enzyme itself. [J. Davis, D.H. Vaughan, M.F. Cardosi Enzyme and Microbial Technology, 1995, 17, 1030-1035] Theoretically, covalent binding of mediators to a polymer matrix and / or enzyme would be an appropriate solution to the problem, but in practice the binding of mediators to polymers and the synthesis of suitable polymers for these purposes is difficult to solve.

Variants of enzyme sensory structures with oxidase and dehydrogenase for glucose detection in various biological fluids are widely described in the patent literature. Most often, they are complex membrane systems that contain large molecular substances either as a mediator or as an enzyme immobilizing agent, for example, as described in WO 90/12889, EP-A-0194 578, EP-A-0 206 471. U.S. Pat. 545, 382 and EP-A-0 078 636 describe sensors for in vivo glucose detection using glucose oxidase and glucose dehydrogenase consisting of a metal or carbon rod, a mediator layer and an enzyme (oxidase or dehydrogenase), a retaining gel layer, and a protective layer. waterproof and glucose membrane. 3

LT 4605 B

Polymeric materials, such as polyvinylenes, polyvinylferrocene, are described in the construction, and act as a mediator. The construction and design of this sensor is complicated from a technological point of view, since several polymers with different functions have to be used. EP-A-01770743 describes a membrane electrode for blood glucose and other biological fluids in which the enzyme is partially immobilized or applied to a substrate (binding agent) which may be co-impregnated with electron mediators. Polyacetylenes, polypyrols, polyphenylsulfides and other polymers are used as the binder of the invention. The sensor is coated with another, semi-conductive, polymeric membrane. Various large molecular materials used in the development of known electrodes (sensors) for different functions have led to the presentation of sophisticated design sensors obtained using sophisticated technologies in the patent literature. Therefore, the acquisition of simple and quick-to-produce sensors, where the redox polymer simultaneously performs the function of a mediator, an enzyme immobilizing agent and a protective layer, is a topical, complex and unresolved task. The object of the invention is to provide an electrochemical sensor for glucose detection, in which redox polymers perform all of the above-mentioned functions. Another object of the present invention is to obtain electroactive redox polymers suitable for use in the construction of a biosensor. The invention relates to a sensor structure consisting of a carbon electrode, an enzyme-dehydrogenase and an electroactive redox polymer. The inventive sensor can be used to measure glucose concentration from 0.1 mmol / l to 22 mmol / l. These values include the entire physiological range of glucose concentrations. No additional reagents should be added during glucose detection, and the analysis is performed by immersing the sensor in the liquid to be analyzed or by dropping the test liquid onto the surface of the working electrode. 4

LT 4605 B

The sensor is obtained by forming a redox polymer film on the surface of the carbon electrode. The construction of a glucose sensor can be accomplished in several ways: 1) on the surface of the carbon electrode by forming separate layers consisting of an enzyme dehydrogenase and a polymer having redox properties; 2) the carbon electrode is coated with a film formed from an enzyme and a redox polymer; electrode coating with polymer film.

The sensor design uses NADH, a FAD cofactor-containing dehydrogenase from Sigma. According to the best embodiment of the sensor construction, dehydrogenase containing cofactor pyrolochinolinquinone (PQQ) isolated from Erwinios 34-1 is used, described in the Institute of Biochemistry [Biotechnol Lett t 21, 187-192, 1999]. The strain Erwinios 34-1 for isolating the PQQ dehydrogenase of the invention is cultured in a liquid medium. The medium may be comprised of conventional carbon sources such as glucose, glycerol, mannitol, arabinose, fructose, galactose, mannose, sucrose, xylose, trehalose, molasses. The source of nitrogen can be peptone, yeast extract, meat extract, corn extract, inorganic compounds, ammonium salts, inorganic magnesium salts, phosphates, iron sulphate. PQQ-dependent dehydrogenase, which could be used in industrial glucose oxidation processes, to create a biosensor produced by conventional enzyme purification techniques [G. Jagow, H. Schagger A Practical Guide to membrane protein purification. 1994, Academic press, New York]

The newly isolated PQQ cofactor-containing dehydrogenase has the following characteristics: optimal pH 7.5 - 8.5; an optimum temperature of 20 to 60 ° C; stable at 40 ° C, molecular weight determined by electrophoresis of polyacrylamide gel sodium dodecyl sulfate and equal to 88 kDa, Michael constant O-maltose, L-arabinose, D-glucose, 2-deoxyglucose equivalent to 5.77, 2.46, 0.185, 0.69 mmol / l, respectively . The dependence of enzyme activity and stability on pH and temperature stability is given in FIG. 1-4. These dehydrogenases are catalytic 5

The properties of LT 4605 B are independent of oxygen concentration in the test liquid, so changes in oxygen concentration do not affect sensor readings.

In the sensor construction, electro-polyphenic derivatives such as poly-3,4-dihydroxybenzaldehyde, polyhydroquinone, as well as polymeric ferrocene-containing derivatives, such as poly-ferrocenyloxyethylene, are used as redox polymers. These materials simultaneously perform the functions of an electron mediator, an enzyme immobilizing structure, and a diffusion control layer. The electrochemical activity of the described polymers, determining their mediating properties, has been demonstrated by cyclic voltamperometry. From cyclic voltamperogram data (see Fig. 7,13,16), it follows that the polymers exhibit reverse oxidation-reduction surfaces. Immobilization of the enzyme occurs mechanically by insertion into the polymeric layer, or by forming a separate polymer layer on the electrode that retains the globules of the enzyme on the electrode surface. The polymeric film used in the sensor design allows the glucose to diffuse to the immobilized enzyme and to catalytic reaction, and at the same time limits glucose diffusion to the enzyme. These signs extend the linear range of the biosensor signal dependence on glucose concentration. The effect of the diffusion control layer is shown in FIG. 20. The synthesis of the polychinonic polymers of the invention is carried out by chemo-enzymatic or organic synthesis techniques. It is known that the enzyme lactase oxidizes and polymerizes many phenols and aromatic amines [R. B Brovvn. Oxidative coupling of phenols. New York, 1967, 167] In many cases, electrochemically active quinones are formed, as well as oligomers and polymers containing quinone groups. Thus, it is expected that the synthesis of redox oligomers and polymers suitable for biosensors will be possible from some of the lactase substrates. The present invention provides a method for obtaining polymers selected for the realization of this sensor, wherein phenolic groups containing monomers act as an electrochemically active polymer having a mediator and diffusion limiting function. Redox polymers with these properties can also be obtained with the ferrocene group

LT 4605 B by organic electrochemical coupling of electrochemically active components to oligomers, e.g. Αοκ / iaAbi PAH, 1996, N ° 12]. The invention is illustrated by graphs and experimental examples.

FIG. 1 PQQ-dependent glucose dehydrogenase activity dependence on the nature and pH of the buffer.

FIG. 2 PQQ-dependent glucose dehydrogenase activity dependence on pH.

FIG. 3 Temperature stability of PQQ-dependent glucose dehydrogenase.

FIG. 4 Temperature dependence of PQQ-dependent glucose dehydrogenase activity.

FIG. 5. The spectrum of the poly-dihydroxybenzaldehyde saturated solution NMR 1H (90 MHz) in methanol.

FIG. 6. (A) Polymer-3,4-dihydroxybenzaldehyde, synthesized by lactase IR spectrum in KBr tablet. (B) IR spectrum of poly-benzoquinone, synthesized by lactase.

FIG. 7. A - Carbon Voltage Modified Poly-DBA Cyclic Voltage Curve in 0.05 mol / l phosphate buffer with 0.1 mol / l KCl, pH 6.7. Spreading speed 10 mV / s. B - Dependence of pH equilibrium potential of carbon electrode, modified poly-DBA. 7

LT 4605 B

FIG. 8. Carbon electrode modified poly-DBA anode current dependence on NADH concentration. 1, 2 - two calibration curves of the same electrode.

FIG. 9. Scheme of poly-hydroquinone synthesis.

FIG. 10. Changes in UV and Visible Arbutin Spectrum (100 µmol / L) by oxidation of the lactase (25 ng / ml). Spectroscopy without lacase (1) and addition of lactase every 8 min (2-6), after 2, 4 and 24 hours (7 - 9, respectively).

FIG. 11. IR spectra of polyhydroquinone obtained by hydrolyzing polarbutine (A) and hydroquinone (B) in KBr tablets.

FIG. 12. Hydroquinone (1) and polyhydroquinone obtained by hydrolyzing the synthesized polarbutine (2) with UV and visible area spectra recorded in methanol (10 mg / l).

FIG. 13. Cyclic voltammetric curve (1) of a carbon electrode modified by polyhydroionionone. 2 - CV curve of the same electrode plus 12 mmol / l NADH. Spreading boom 10 mV / s, medium - phosphate buffer, pH 7.0.

FIG. 14. Dependence of pH equilibrium potential of a carbon electrode modified by polyhydroionionone.

FIG. 15. A carbon electrode modified with polyhydroxyionone in response to NADH in 0.05 mol / phosphate buffer with 0.1 mol / l KCl, pH 7.0 under potentiostatic conditions E = 200 mV, Ag / AgCl. 8

LT 4605 B

FIG. 16. Cyclic voltammetric curve of a carbon electrode coated with a poly-Fc layer of a 12 mg / ml solution in cyclohexanone. Measurements were made in 0.05 mol / l phosphate buffer with 0.1 mol / l KCl, pH 7.0. Spreading speed 10 mV / s.

FIG. 17. The response of the carbon electrode modified to poly-DBA and PQQ-GDH to glucose. Electrode potential 300 mV with respect to Ag / AgCI electrode. 0.05 mol / l Glycine buffer, pH 7.0 with 0.1 mol / l KCl. 1 and 2 are two sequential calibration curves with the same electrode.

FIG. 18. The reaction of a carbon electrode modified with polyhydroxyionone to glucose. Measured in 0.05 mol / l phosphate buffer, pH 7.0, under potenxiostatic conditions, E = 550 mV, Ag / AgCl.

FIG. A carbon electrode coated with a poly-Fc layer to respond to glucose. Measurements were made in 0.05 mol / l phosphate buffer with 0.1 mol / l KCl, pH 7.0 in solution 1.5. PQQ-GDH. Electrode potential 550 mV. A-layer is formed from concentrated (120 mg / ml) poly-Fc solution cyclohexanone; The B-polymer layer was formed from a diluted (24 mg / ml) poly-Fc solution cyclohexanone.

FIG. 20. Carbon electrodes with surface-coated poly-Fc and adsorbed PQQ-GDH (0.06 units) calibration curves. 1 is an electrode film coated with a concentrated polymer solution (120 mg / ml) cyclohexanone; 2 - an electrode on which a film of diluted poly-Fc solution (60 mg / ml) is formed. 9

LT 4605 B

Experimental Part 1 Example. Production of poly-3,4-dihydroxybenzaldehyde (poly-DBA) by lactase and product characteristics

CHO

Poly-3,4-dihydroxybenzaldehyde is obtained from the purchased reagent 3,4-dihydroxybenzaldehyde (product of Sigma) under the action of an aqueous solution of the lactase. An aqueous solution of 690 mg of substrate and 1 mg of lactase was stirred for 24 hours in an open container. The dark brown reaction mixture is spread on 1 m2 of celluloid film and further dried slowly in the air. The product, when dried, dissolved in water, was used in studies.

The spectral characteristics of the poly 3,4-dihydroxybenzaldehyde are given in FIG. 5 and FIG. 6. In the methanol-dissolved poly-DBA BMR 1H spectrum (Fig. 5), signals are observed in the aromatic and / or double bond protons (7.24.6.84 ppm) and signal in the aliphatic protons region (3.48 ppm).

The C13 NMR spectrum of the polymer dissolved in D-dimethylsulfoxide with a chemical impulse of 138.2 and 126 ppm, assigned to the aromatic group, and a signal with a chemical shift of 67.55 ppm assigned to the aliphatic group.

The poly-DBA IR spectrum in KBr tablet is shown in FIG. 6, curve A. Pikas in the spectrum at 1200 cm -1 corresponds to the absorption band of the phenolic hydroxyl group. The electrochemical properties of poly-3,4-dihydroxybenzaldehyde were analyzed by cyclic voltamperometry. The cyclic voltamperogram data shown in FIG. 7A shows that the polymer exhibits a reversible oxidation-reduction surface having a balance potential of pH 185 at 185 mV. The dependence of equilibrium potential on pH is shown in FIG. B. Bending angle (- 10

LT 4605 B 51 mV / pH) shows that 2 protons and 2 electrons are involved in the electrode process.

On the carbon electrode, a poly-DBA layer was formed by evaporating a solution of 3 µl of ethanol polymer. The carbon electrode with a poly-DBA layer, polarized with 200 mV Ag / AgCI reference electrode, responds to low concentrations of the reducer - nicotinamide adenindinucleotide (NADH). Anode current increase is recorded with the help of a recorder. The calibration graph is shown in FIG. 8, curve 1. The linear part of the graph is 1.5 mM NADH. The polymer oxidant stays stable on the electrode surface and the redox properties are not lost by washing and reusing the electrode. FIG. Curves 8, 2 show that for the second time measuring NADH at the same electrode, the signals are similar to the first curves. This indicates that the redox polymer oxidizes the -NADH in the solution to the electrode surface and can transmit electrons to the electrode. Example 2. Synthesis and Characteristics of Polyhydroquinone

OH

The synthesis of the material was performed according to the modified methodology [J.S. Dordick et al., J. Am. Chem. Soc. 1995, 117, 12885-12886] from purchased arbutin (Sigma, USA). The synthesis is carried out in two stages: first, by polymerizing arbutin in phosphate buffer pH 5.25 (the lactase is a catalyst for this process) and by hydrolyzing the resulting polarbutine in an acid medium. The reaction scheme is shown in FIG. 9. Product obtained - Insoluble water-brown precipitate is washed on the filter paper with distilled water.

FIG. Figure 10 illustrates spectrum changes in 0.1 mM arbutin solution during lactase-catalyzed oxidation. It is seen that the process is accompanied by UV absorption 11

LT 4605 B expires after 24 h. Polarbutin is soluble in water, brown, absorbing light at 400 nm.

The poly (hydroquinone IR spectrum of the resulting final product (KBr tablet) is shown in FIG. 6, curve B and FIG. 11, A. The spectrum of the polymer is observed in a band of similar frequencies as in the monomer spectrum shown in FIG. 11, B.

The Monomer (Hydroquinone) and Polymer UV and Visible Spectrum Spectra are shown in FIG. 12. In the UV field, the observed band in the polymer case (curve 2) is shifted to the side of the long waves, which is typical of the polymers.

The electrochemical properties of the polymer were investigated in aqueous buffer solutions by analyzing the cyclic voltamperometric curves of the carbon electrodes of modified polymer layers. A typical such curve in the phosphate buffer is shown in FIG. 13. It is seen that the polymeric material has a reversible oxidation-reductive surface (curve 1) with a equilibrium potential at pH 7.0 of 57 mV. The equilibrium potential dependence on pH is the straight line shown in FIG. 14. The angle of the linear inclination (-56 mV / pH) indicates that 2 protons and 2 electrons are involved in the electrode process.

The ability of the polyhydroquinone to oxidise the reduced nicotinamide adenindinucleotide (NADH) was reflected in an anodic current increase by adding 12 mM NADFI (Fig. 14, curve 2). Under potential potential at 200 mV, the modified poly-hydroquinone carbon electrode response to NADH is linear up to 5 mM NADH (Fig. 15). Example 3. Poly-ferrocenyloxyethylene (poly-Fc) synthesis and polymer characteristics

The synthesis of the material was performed according to the methodology [/ 1 EorycnaBCKuPi n Ap; AoKnaąbi PAH, 1996, N ° 12], by attaching ferrocenylmethane to ethylene glycol vinylglycidyl diester at 75 ° C with azobisizosic acid dinitrile.

Poli-Fc spectral characteristics, showing the structure of the material, coincide with the characteristics indicated in the literature. 12

LT 4605 B

Materials The IR spectra in KBr tablets showed the bands characteristic of the ferrocene ring (double peak 480 and 500 cm'1). The vinyl oxide-specific bands (1605, 1625 cm'1) disappeared during the synthesis.

Structured oligomer pepper:

(CH-o-ch,) - CH,

The electrochemical properties of poly-ferrocenyloxyethylene (poly-Fc) have been studied by simply evaporating a 2 μΙ cyclohexanone polymer solution on the surface of the carbon electrode. The poly-Fc cyclic voltamper curve is shown in FIG. 16. An observable single-electron oxidation-reduction process with an equilibrium potential of about 450 mV relative to Ag / AgCl comparator electrode is observed.

The operation of enzyme electrodes produced using the polymers described above is described in the following examples. Example 4. Glucose sensor with poly-DBA and PQQ-GDH.

Coated on a carbon electrode surface, a layer of 0.05 pcs. enzyme PQQ-glucose dehydrogenase and evaporated 3 μΙ saturated ethanol poly-DBA solution.

Such an electrode responds to the addition of glucose to the buffer solution under potent static conditions (300 mV) (Fig. 17). The sensitivity of the sensor is about 0.2 μΑ / mM. The sensitivity remained unchanged when measured for the second time with the same electrode (curve 2), indicating that the enzyme and redox polymer properties were unchanged and immobilization was effective. 13

EN 4605 B 5 Example. Production of glucose electrode with PQQ-GDH and polyhydroquinone.

The enzyme electrode is produced by forming two layers on a carbon electrode: an adsorbed layer of enzyme from 0.06 PQQ enzyme glucose dehydrogenase and polymer film. The film is formed by evaporation on the electrode surface of dimethylsulfoxide 2 µl polymer solution.

Glucose measurements are performed in a 0.4 ml volume cell with 0.05 M phosphate buffer pH 7.0 by polarizing the prepared electrode with 550 mV Ag / AgCl for the reference electrode. Adding glucose to the solution records the changes in the anodic current. The results reflecting the signal dependence on glucose concentration are shown in FIG. 18. Electrode sensitivity - 0.08 μΑ / mM glucose. Example 6. Production of glucose electrode with poly - Fc

A polymer film was formed on the surface of the carbon electrode by drying cyclohexanone of 3 μΙ poly-Fc solution. The electrode with a polymer layer is placed in a 0.2 ml cell with 1.5 pcs. The PQQ-glucose dehydrogenase buffer solution (pH = 6) and the background electrode current are determined by polarizing it with a 550 mV Ag / AgCl reference electrode. The cell is loaded with 2 μΙ of 0.1 M glucose solution and the anodic current changes recorded. Glucose measurements using a poly-Fc-coated electrode when the poliferocene layer serves as a system oxidizer and the enzyme is in solution are illustrated in FIG. 19. Glucose susceptibility of the electrode having a film formed of 120 mg / ml poly-Fc solution was 0.04 μΑ / mmoH and the linear portion of the calibration graph was up to 8 mmol / l (Fig. 21, curve A). The sensitivity of the electrode having a surface film formed of 24 mg / ml polyFc solution was 0.25 µΑ / mmol l and the linearity of the calibration graph was 6 mmol / l (Fig. 19, curve B). The results show that the electrode susceptibility to glucose is 5 times the polymer content.

LT 4605 B is 6 times larger, but such a Biosensor has a shorter linear portion of the calibration graph. Example 7. Production of glucose sensor with poly-Fc and PQQ GDH.

The polymer was used to immobilize the enzyme on the surface of the electrode. To do this, we built a two-layered enzyme electrode. The inner layer contained the enzyme PQQ-GDH (0.06 pcs) adsorbed on the surface of the carbon electrode. The outer layer consisted of a polymer film of cyclohexanone of 2 µl poly-Fc solution. Such a design has several advantages. First, the polymeric film, being slightly water-soluble, protects the enzyme from leaching into solution. Secondly, the film causes diffuse encumbering of glucose from the solution to the enzyme layer, resulting in prolonged linear portion of the calibration graph. 0.008 ml of 0.1 mol / l glucose solution is added to the prepared 0.4 ml volume cell and mixed. The final glucose concentration in the cell is 2 mmol / l. Measurements are performed in a cell with a 0.05 M phosphate buffer solution pH 7.0, polarizing the prepared electrode 550 mV with respect to Ag / AgCI reference electrode. Record the anode current changes six times in succession by adding the enzyme electrode to the same 2 mmol / l glucose solution and after each measurement wash the electrode with distilled water. The stability of the enzyme layer and the survival of the catalytic activity after multiple application of the electrode are illustrated by these results. The recorded signals were: 0.16; 0.25; 0.15; 0.15 0.14 and 0.24 μΑ. Mean values x = 0.18 μΑ ,. The coefficient of variation CV = 11.6% indicates that the values obtained are within the error limits of the experiment.

The susceptibility to glucose of an electrode having a two-layer surface modified by PQQ-GDH and polyFc is shown in FIG. 20. From curve 1, it appears that, in the case of a polymeric film formed from a 120 mg / ml polyFc solution, the electrode sensitivity was 62 nA mmol'11, and when the polymer film was obtained from a double-lower poly-Fc amount (Fig. 20, curve 2) , the electrode sensitivity to glucose was higher and was about 98 nA / mmol I. This suggests that a thinner layer is present at a lower polymer content.

The LT 4605 B ferrocene derivative has greater mobility and can interact more effectively with the active enzyme center. The results also confirm that the polymer film complicates glucose diffusion in the catalytic layer because the linear portion of the calibration graph extends to 24 mmol / l.

Claims (10)

16 EN 4605 B DEFINITION DEFINITION 1. A biosensor (enzyme electrode) for the determination of glucose in biological fluids, comprising a carbon electrode, an enzyme dehydrogenase and a polymeric film which simultaneously carries out the function of an enzyme immobilizing structure and a diffusion control layer.
2. The biosensor of claim 1, wherein the enzyme dehydrogenase comprises α-nicotinamide adenine dinucleotide (NADH), flavidinen indinucleotide (FAD), or dehydrogenase-containing dehydrogenase of pyrolochinolinquinone (PQQ).
The biosensor, wherein the dehydrogenase enzyme is a dehydrogenase of the pyrolochinolinquinone (PQQ) cofactor according to claim 2, characterized in that the pyrolochinolinquinone (PQQ) cofactor-containing dehydrogenase is secreted from the microorganism strain by Erwinia sp 34-1.
4. The biosensor of claim 1, wherein the polymeric film contained therein is redox polymers of a polychinon structure.
5. Biosensor according to claim 1 and 4, characterized in that the redox polymers contained in the polychinon structure receive the quinone structure monomers by polymerization with the enzyme lactase.
6. The biosensor according to claim 1, wherein the polymeric film contained therein is a ferrocene-containing derivative.
7. A method of producing a biosensor according to claim 1 by coating an enzyme and a redox polymer film on an electrode surface, characterized in that the enzyme is immobilized by noncovalent insertion into a polymeric layer.
8. A method of producing a biosensor according to claim 7, characterized in that the polymer is coated on the electrode by forming a separate, enzyme-retaining layer.
9. A method of producing a biosensor according to claim 7, characterized in that the electrode is coated with a film formed from an enzyme and a redox polymer.
10. A method of producing a biosensor according to claim 7, wherein the enzyme is immobilized after the electrode coating polymer.
LT99-032A 1999-04-07 1999-04-07 Enzymatic glucose sensor and process for producing thereof LT4605B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0078636A1 (en) 1981-10-23 1983-05-11 MediSense, Inc. Sensor for components of a liquid mixture
EP0194578A2 (en) 1985-03-13 1986-09-17 Miles Inc. Proteins immobilised on polyamides or cellulose hydrate and the use thereof for the preparation of biocatalysts, test strips or chromatography materials
EP0206471A2 (en) 1985-04-24 1986-12-30 Mitsubishi Gas Chemical Company, Inc. Process for preparation of pyrrolo-quinoline quinone
WO1990012889A1 (en) 1989-04-25 1990-11-01 Migrata Uk Ltd Method of analysis, reagent composition and use thereof for glucose determination
EP1770743A2 (en) 2005-09-30 2007-04-04 Schott AG Composite system, method for manufacturing a composite system and luminous body

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0078636A1 (en) 1981-10-23 1983-05-11 MediSense, Inc. Sensor for components of a liquid mixture
US4545382A (en) 1981-10-23 1985-10-08 Genetics International, Inc. Sensor for components of a liquid mixture
EP0194578A2 (en) 1985-03-13 1986-09-17 Miles Inc. Proteins immobilised on polyamides or cellulose hydrate and the use thereof for the preparation of biocatalysts, test strips or chromatography materials
EP0206471A2 (en) 1985-04-24 1986-12-30 Mitsubishi Gas Chemical Company, Inc. Process for preparation of pyrrolo-quinoline quinone
WO1990012889A1 (en) 1989-04-25 1990-11-01 Migrata Uk Ltd Method of analysis, reagent composition and use thereof for glucose determination
EP1770743A2 (en) 2005-09-30 2007-04-04 Schott AG Composite system, method for manufacturing a composite system and luminous body

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JAMES DAVIS, D HUW VAUGHAN, MARCO F CARDOSI: "Elements of biosensor construction", ENZYME AND MICROBIAL TECHNOLOGY, 1995, pages 1030 - 1035, XP055283224, DOI: doi:10.1016/0141-0229(95)00013-5
P. LEDUC, D. THEVENOT: "Chemical and electrochemical oxidation of aqueous solutions of NADH and model compounds", BIOELECTROCHEMISTRY AND BIOENERGETICS, 1974, pages 96 - 107, XP026713699, DOI: doi:10.1016/0302-4598(74)85011-7
R.B. BROWN: "Oxidative coupling of phenols", pages: 167

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