(19)
(11)EP 3 242 129 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
01.04.2020 Bulletin 2020/14

(21)Application number: 15875765.8

(22)Date of filing:  31.12.2015
(51)International Patent Classification (IPC): 
G01N 27/327(2006.01)
C07D 401/04(2006.01)
C12M 1/40(2006.01)
G01N 33/66(2006.01)
(86)International application number:
PCT/KR2015/014582
(87)International publication number:
WO 2016/108671 (07.07.2016 Gazette  2016/27)

(54)

ELECTROCHEMICAL BIOSENSOR

ELEKTROCHEMISCHER BIOSENSOR

BIOCAPTEUR ÉLECTROCHIMIQUE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 31.12.2014 KR 20140195173

(43)Date of publication of application:
08.11.2017 Bulletin 2017/45

(73)Proprietor: I-sens, Inc.
Seoul 06646 (KR)

(72)Inventors:
  • YOON, In Jun
    Seoul 08735 (KR)
  • SHIN, Jae Ho
    Seoul 01405 (KR)
  • JUNG, Yeon Ho
    Namyangju-si Gyeonggi-do 12078 (KR)
  • CHA, Geun Sig
    Seoul 03610 (KR)
  • NAM, Hakhyun
    Seoul 01227 (KR)

(74)Representative: Duxbury, Stephen et al
Arnold & Siedsma Bavariaring 17
80336 München
80336 München (DE)


(56)References cited: : 
WO-A2-2008/007277
JP-A- 2014 089 096
KR-B1- 101 239 381
JP-A- 2003 503 728
KR-A- 20120 023 208
US-A1- 2012 065 614
  
  • NIEH CHI-HUA ET AL: "Electrostatic and steric interaction between redox polymers and some flavoenzymes in mediated bioelectrocatalysis", JOURNAL OF ELECTROANALYTICAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 689, 29 November 2012 (2012-11-29), pages 26-30, XP028991334, ISSN: 1572-6657, DOI: 10.1016/J.JELECHEM.2012.11.023
  • JENKINS, PETER ET AL.: 'A Mediated Glucose/oxygen Enzymatic Fuel Cell based on Printed Carbon Inks Containing Aldose Dehydrogenase and Laccase as Anode and Cathode' ENZYME AND MICROBIAL TECHNOLOGY vol. 50, no. 3, 10 March 2012, pages 181 - 187, XP055460886 DOI: 10.1016/J.ENZMICTEC.2011.12.002
  • JENKINS, PETER A. ET AL.: 'Evaluation of Performance and Stability of Biocatalytic Redox Films Constructed with Different Copper Oxygenases and Osmium-Based Redox Polymers' BIOELECTROCHEMISTRY vol. 76, no. 1-2, 01 September 2009, pages 162 - 168, XP026470119 DOI: 10.1016/J.BIOELECHEM.2009.04.008
  • CONGHAILE, PETER O. ET AL.: 'Mediated Glucose Enzyme Electrodes by Cross-linking Films of Osmium Redox Complexes And Glucose Oxidase On Electrodes' ANALYTICAL AND BIOANALYTICAL CHEMISTRY vol. 405, no. 11, 01 April 2013, pages 3807 - 3812, XP055460895 DOI: 10.1007/S00216-012-6628-9
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Technical field



[0001] The present invention relates to an osmium complex which may not be affected by oxygen partial pressure and which stably retains its redox state for a long time, and an electron transfer mediator including an osmium complex, and an electrochemical biosensor comprising the electron transfer mediator and an oxidoreductase.

Related Art



[0002] Recently, the development of biosensors has been interested for the quantitative and qualitative analysis of target analyte from the medical field to the environment and foods. In particular, a biosensor using an enzyme is a chemical sensor used for selectively detecting and measuring a chemical substance in a sample using a biological sensing function in which an organism such as a microorganism or a functional substance in an organism reacts sensitively with a specific substance. The biosensor has been developed mainly for medical measurement such as blood sugar sensor, and active research is also being done in the fields of food engineering and environmental measurement.

[0003] Periodic measurement of blood glucose is very important in diabetes management, and a wide variety of electrochemical biosensors with accuracy and precision are widely used for measurement of blood glucose. An electrochemical biosensor for measuring blood glucose is prepared by coating a reagent prepared by mixing an enzyme, an electron transfer mediator, various stabilizers and a dispersing agent, to a working electrode and drying it. The kind of the enzyme and the property of electron transport mediators used are most important factor for influencing the characteristics of the biosensor.

[0004] For example, FAD-GOx (flavin adenine dinucleotide-glucose oxidase), a glucose oxidase reductase used in most commercial electrochemical sensors, is thermally stable and has excellent reaction selectivity to oxidize only glucose. However, because of FAD-GOx reacts with oxygen dissolved in blood, sensors adopting FAD-GOx enzymes can produce very different measurement result depending on the type of blood sample, such as venous, arterial, or capillary blood.

[0005] Guibault and Lubrano et al. proposed a method of measuring the generated hydrogen peroxide by the current method. When an oxidation potential of about +600 mV (vs. Ag / AgCl) is introduced into the electrode, the current value is obtained in the next step. However, the glucose sensor adopting this method is highly dependent on the amount of dissolved oxygen in the sample. By applying a high oxidation potential when measuring the oxidation current of hydrogen peroxide, the drug and metabolites (ascorbic acid, uric acid, acetaminophen, dopamine, and etc.) being capable of oxidized in a low potential are oxidized together at the electrodes, thereby causing severe measurement errors.

[0006] As one of the methods to overcome the oxygen dependency of the glucose sensor, an electron transfer mediator instead of oxygen was introduced to facilitate the electron transfer between the electrode surface and the enzyme active site on the electrode surface, and organometallic electron transfer mediator such as Ferrocene derivatives having a relatively low potential was introduced to minimizing the interference effect of other oxidative substances (ascorbic acid, uric acid, acetaminophen, dopamine, and etc.)

[0007] The development trend of blood glucose sensors is changed into the use of GDH requiring no oxygen in the enzymatic reaction, in order to block changes in measured values due to differences in oxygen partial pressure (pO2), instead of GOx including oxygen involved in the enzymatic reaction with the blood glucose. In addition, as an electron transfer mediators, organic compounds such as quinone derivatives (phenanthroline quinone, Quineonediimine etc.) and organometallic compound such as Ru complexes (ruthenium hexamine, etc.) or osmium complexes replace the Ferricyanide having low stability to temperature and humidity.

[0008] The most electron transfer medium used commonly is potassium Ferricyanide [K3Fe(CN)6]. Because it is inexpensive and has a good reactivity, it can be useful for all sensors using FAD-GOx, PQQ-GDH or FAD-GDH. However, the sensor using the above-mentioned electron transfer mediator has a measurement error caused by the interfering substance such as uric acid or gentisic acid in the blood and is easily deteriorated due to temperature and humidity. Thus, it must be carefully prepared and stored. It has a difficulty to accurately detect glucose at a low concentration due to a change in background current after long storage.

[0009] The hexamine ruthenium chloride [Ru(NH3)6Cl3] has higher redox stability than the Ferricyanide. The biosensor using hexamine ruthenium chloride as an electron transfer medium has advantages in manufacturing and storage and has stability due to small change of background current even when it is stored for a long time. However, it cannot match the reactivity of FAD-GDH, when it is used with FAD-GDH, and thus, it cannot be manufactured as a commercial product. Also, this electron transfer medium has a problem in that the accuracy of the sensor strip is affected by the oxygen partial pressure.

[0010] Therefore, there is still a need to develop a reagent for reduction-oxidation reaction, specifically for electrochemical biosensor that is no influenced by oxygen, has little change in performance due to temperature and humidity, has little change in performance even after storage for a long period of time, can measure a wide range of concentration, and is suitable for mass production.

DETAILED DESCRIPTION


Technical Problem



[0011] An object of the present invention is to provide an osmium complex, its salt compound and a preparation method thereof, where the compound or its salt is excellent as an electron transfer mediator for an electrochemical biosensor, because the compound or its salt has a stable oxidation-reduction form for a long time and a capacity to react with oxidoreductase without an effect of oxygen partial pressure.

[0012] It is still another object of the present invention to provide a reagent composition for redox reaction comprising an electron transfer mediator comprising the osmium complex or a salt thereof.

[0013] It is still another object of the present invention to provide an electrochemical biosensor comprising an electron transfer mediator comprising an osmium complex or a salt thereof.

Technical Solution



[0014] The present invention relates to an osmium complex or a salt thereof, an electron transfer mediator comprising the osmium complex or a salt thereof, a reagent composition for redox reaction, an electrochemical biosensor, for example glucose sensor, because the compound or its salt has a stable oxidation-reduction form for a long time and a capacity to react with oxidoreductase being capable of performing the redox reaction of the analytes in the biological sample without an effect of oxygen partial pressure.

[0015] Specifically, the present invention relates to an osmium complex or a salt thereof, and a preparation method thereof, where the osmium complex or a salt thereof has a stable oxidation-reduction form for a long time and a capacity to react with oxidoreductase being capable of performing the redox reaction of the analyte in the biological sample without an effect of oxygen partial pressure.

[0016] In an embodiment, the present invention relates to an electron transfer mediator comprising the osmium complex or its salt which has a stable oxidation-reduction form for a long time and a capacity to react with oxidoreductase being capable of performing the redox reaction of the analytes in the biological sample without an effect of oxygen partial pressure. The osmium complex is represented by the following Chemical Formula 1.

        [Chemical Formula 1]     Os(A)mXn

Wherein,

A is a compound represented by the following Chemical Formula 2,

X is independently halogen, for examples at least one selected from the group consisting of F, Cl, Br and I,

m is an integer of 1 to 3, n is an integer of 0 to 4, and a sum of m and n is an integer of 3 to 5.



[0017] The reagent for reduction-oxidation reaction, specifically for electrochemical biosensor has no effect of oxygen partial pressure, little change in performance due to temperature and humidity, and little change in performance even after storage for a long period of time, can measure a wide range of concentration, and is suitable for mass production.

[0018] An embodiment of present invention relates to an electrochemical biosensor, which is prepared by immobilizing an osmium complex or its salt represented by Chemical Formula 1 and an enzyme performing the redox reaction of analytes in liquid biological sample on at least two electrodes. The examples of the electrodes are a working electrode and an auxiliary electrode, for example, where the osmium complex or its salt and the enzyme are immobilized on the working electrode.

[0019] In an embodiment, although a biosensor for measuring glucose is presented as an example of the applicable electrochemical biosensor, the present invention can be applied to a biosensor for quantitative determination of various substances such as cholesterol, lactate, creatinine, hydrogen peroxide, alcohol, amino acid and glutamate, by changing the types of enzymes contained in the reagent composition of the present invention.

[0020] The long-term stability was tested for a glucose strip sensor including the osmium complex in an oxidative state, with applied voltage 0.3V under the storage condition. As a result, the background signal (0.3 µA), response slope (45 nA/(mg/dL)) and error percentage (%) depending on the oxygen partial pressure (at most 4%) are stably maintained for 8 weeks or longer. On the basis of the result of calibrating in 20 mM to 200 mM, the error percentage (%) calculated by using the current value of 70 mg/dL glucose and 100 mg/dL glucose over time shows a significant result of 10% or lower. The effect of oxygen partial pressure which is most serious problem of conventional sensors was evaluated as 4% or lower.

[0021] Hereinafter, the present invention will be described in more detail.

[0022] An embodiment of the present invention relates to an electrochemical biosensor which is prepared by immobilizing an osmium complex or its salt represented by Chemical Formula 1 and an enzyme performing the redox reaction of analytes in liquid biological sample on at least two electrodes.

        [Chemical Formula 1]     Os(A)mXn

Wherein,

A is a compound represented by Chemical Formula 2, that is, 4,4'-dicarboxy-2,2'-bipyridine (dcbpy),

X is independently selected from the group consisting of halogen, for examples, one selected from the group consisting of F, Cl, Br and I,

m is an integer of 1 to 3, n is an integer of 0 to 4, a sum of m and n is an integer of 3 to 5.



[0023] The examples of the osmium complexes in accordance with the present invention include the compounds of Chemical Formula 3, Chemical Formula 4 and Chemical Formula 5.









[0024] The osmium complex having the structure represented by the formula (1) according to the present invention may include a trivalent osmium complex and a divalent osmium complex, the osmium complex having the structure represented by the formula (1) may preferably be an oxidized compound (trivalent Os compound). In addition, when the osmium complex is a mixture containing both an oxidized state and a reduced state compound as an osmium complex represented by the general formula (1), the osmium complex in an oxidized state may be provided by the oxidative treatment, providing the oxidized osmium complex, or adding an oxidant to the reagent composition.

[0025] The osmium complex according to the present invention may be in a salt form, and it is more preferable since the salt compound has a high solubility. The salt compound may be a salt compound of at least one alkali metal selected from the group consisting of Li salt, Na salt, K salt, Rb salt, Cs salt and Fr salt.

[0026] Therefore, the compound or its salts of present invention may be an osmium complex having the structure of Formula 1 or a salt thereof, and the osmium contained in the osmium complex or its salt is preferably in oxidation state, i.e., trivalent osmium. Specifically, after preparing an osmium complex or preparing a salt of an osmium complex, an osmium complex or its salt is treated by an oxidant to produce an osmium complex in an oxidized state, followed by changing the an osmium complex in an oxidized state into its salt.

[0027] In one embodiment of the present invention, the osmium complex may be synthesized using a compound having the Chemical formula (2) and a compound represented by the following formula (6).



        [Chemical formula 6]     YpOsXq

wherein,

Y is K, Na or NH4, X is a halogen,

p is an integer of 1 to 2, and q is an integer of 1 to 6.



[0028] In a specific embodiment, the osmium compound of present invention may be synthesized by using 4,4'-dicarboxy-2,2'-bipyridine (dcbpy) and the compound of Chemical Formula 6, for examples K2OsCl6 or (NH4)2[OsCl6] as a starting material. An example of preparing method of osmium compound, the procedure using dcbpy and K2OsCl6 is shown in Reaction Scheme 1.



[0029] The synthesized osmium complex may be the osmium complex in the oxidized state (trivalent Os compound) obtained by performing oxidation treatment on the synthesized osmium complex using various oxidizing agents. The oxidizing agent used in the oxidation treatment of the present invention is not particularly limited, but specific examples are NaOCl, H2O2, O2, O3, PbO2, MnO2, KMnO4, ClO2, F2, Cl2, H2CrO4, K2Cr2O7, N2O, Ag2O, OsO4, H2S2O8, pyridinium chlorochromate, and 2,2'-Dipyridyldisulfide. According to an embodiment of the present invention, the compound in oxidation state can be prepared according to the following Reaction Scheme 2.



[0030] In accordance with one embodiment of the present invention, the salt form of the osmium complex is more preferred because of the increased solubility. As an example of preparing a salt of an osmium complex, a process for preparing a salt compound of an osmium complex using NaOH is shown in the following reaction scheme (3). The salt compound of the osmium complex may be a salt of at least one alkali metal selected from the group consisting of Li salt, Na salt, K salt, Rb salt, Cs salt and Fr salt, but is not limited thereto.



[0031] An example of the present invention relates to an electron transfer mediator comprising an osmium complex which reacts with GDH, has no effect of oxygen partial pressure and stably maintains a redox state for a long time. The osmium complex according to the present invention is represented by Chemical Formula 1, and the osmium complex is as described above.

[0032] The electrochemical biosensor according to the present invention may include an enzyme capable of oxidizing and reducing an analyte in a liquid biological sample, and an electron transfer mediator. The electron transfer mediator may include an osmium complex or a salt thereof as a single component, or as a main component. Further, in the electrochemical biosensor according to the present invention, it is preferable that the electron transfer mediator comprises an osmium complex or its salt as a main component, and preferably does not contain a metal complex other than an osmium complex or its salt.

[0033] A further embodiment of present invention relates to a reagent for redox reaction, preferably an electrochemical biosensor, comprising an electron transfer mediator comprising an osmium complex and an oxidoreductase.

[0034] In another aspect, there is provided a method of preparing a reagent for a redox reaction with improved stability, comprising mixing an oxidoreductase and an electron transfer mediator. In an embodiment, the reagent composition for redox reaction may be applied to an electrochemical biosensor, and thus, in another example, a method of preparing a reagent for electrochemical biosensor with improved stability, comprising a step of mixing an oxidoreductase and an electron transfer mediator.

[0035] Another embodiment provides an electrochemical biosensor comprising the reagent composition for an electrochemical biosensor with improved stability.

[0036] In an electron transfer mediator, a reagent composition for redox reaction, and a electrochemical biosensor according to the present invention, the electron transfer mediator is reduced by performing the redox reaction with the reduced enzyme which is produced by the metabolite, and then can produce the current on the surface of electrode applied by the oxidative potential. In an electron transfer mediator, a reagent composition for redox reaction, and an electrochemical biosensor according to the present invention, the osmium complex or its salts can be used as a single component, or in combination with at least one of second electron transfer mediator.

[0037] In an electron transfer mediator, a reagent composition for redox reaction, and an electrochemical biosensor according to the present invention, the osmium complex may be used as an osmium complex itself, or a salt of an osmium complex, an oxidized compound of an osmium complex, or oxidized compound of its salt.

[0038] According to an embodiment of the present invention, in the reagent composition for redox reaction and the electrochemical biosensor, (a) an oxidoreductase and (b) an osmium complex, a salt compound of an osmium complex, an oxidized compound of an osmium complex, or a compound obtained by oxidizing a salt compound of the osmium complex, and (c) an oxidizing agent. When the oxidizing agent (c) is further contained, the component (b) is an osmium complex or a salt compound thereof which is not oxidized. The oxidizing agent is not particularly limited, but specific example may be at least one selected from the group consisting of NaOCl, H2O2, O2, O3, PbO2, MnO2, KMnO4, ClO2, F2, Cl2, H2CrO4, K2Cr2O7, N2O, Ag2O, OsO4, H2S2O8, pyridinium chlorochromate and 2,2'-Dipyridyldisulfide.

[0039] The amount of the oxidizing agent added to the reagent composition for redox reaction according to the present invention is not particularly limited as long as it provides the oxidation state of the osmium complex. For example, the amount of the oxidizing agent according to the present invention is 0.1 to 10 molar ratios on the basis of 1 mole of the osmium complex.

[0040] According to an embodiment of the present invention, the use of the osmium complex in combination of the second electron transfer mediator can significantly increase the glucose detection performance and minimize the effect of various interfering substances for glucose detection.

[0041] The reagent composition according to the present invention may contain 20 to 700 parts by weight, for example, 60 to 700 parts by weight or 30 to 340 parts by weight of the osmium complex based on 100 parts by weight of the redox enzyme. The content of the osmium complex can be appropriately adjusted according to the activity of the oxidoreductase. If the activity of the oxidoreductase contained in the reagent composition is high, the reagent composition can exhibit the desired effect, even if the content of the metal complex is low. Thus, as the activity of the oxidoreductase is higher, the content of the metal-containing complex can be adjusted to the relatively low content.

[0042] Unlike the conventional ruthenium complex, the osmium complex alone is used along in the reagent compositions of the present invention; it can function as an electron transfer mediator sufficiently. The present invention can additionally include a second electron transfer mediator other than the metal complex.

[0043] The second electron transfer mediator is selected from the group consisting of 1-Methoxy-5-methylphenazinium methylsulfate(1-methoxyPMS), 3-amino-7-(2,3,4,5,6-pentahydroxy hexanamido)-5-phenothiazinium, 1-Methoxy-5-methylphenazinium, Azure C, Azure A, Methylene Blue, Toluidine Blue and derivatives thereof.

[0044] When the electron transfer mediator is a mixture of an osmium complex and thionine or a derivative thereof, and/or a mixture of an osmium complex with 1-methoxy PMS or a derivative thereof, the molar ratio of thionine or 1-methoxy PMS to osmium complex (mole of thionine or 1-methoxy PMS: mole of osmium complex) can be from 1: 1 to 20, specifically from 1: 1 to 10.

[0045] The oxidoreductase refers to an enzyme that catalyzes the oxidation-reduction reaction of a living body. In the present invention, the oxidoreductase means an enzyme that is reduced by reacting with the target substance to be measured, such as the metabolite to be measured in the biosensor. The reduced enzyme may react with the electron transfer mediator and generate signal such as current change and the metabolite is quantified by measuring the signal such as the current change occurring at this time. The oxidoreductase used in the present invention may be at least one selected from the group consisting of various dehydrogenases, oxidases, esterases, and the like. Depending on the redox reaction or target substance, an enzyme adopting the substrate as a target substance can be selected and used among the enzymes belonging to the enzyme group.

[0046] More specifically, the oxidoreductase can be at least one selected from the group consisting of glucose dehydrogenase, glutamate dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase and bilirubin oxidase.

[0047] Meanwhile, the oxidoreductase may include a cofactor for storing hydrogen taken from a target substance (for example, a metabolite) to be measured. For examples, the cofactors may be at least one selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and pyrroloquinoline quinone (PQQ).

[0048] Meanwhile, the oxidoreductase may be contained in combination with a cofactor for storing hydrogen taken from a target substance (for example, a metabolite) to be measured. For examples, when measuring the blood glucose concentration by using glucose dehydrogenase (GDH) as an oxidoreductase, the combination of cofactors and the oxidoreductase may include flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) and/or nicotinamide adenine dinucleotide-glucose dehydrogenase containing FAD.

[0049] In an embodiment, the available oxidoreductase may be at least one selected from the group consisting of FAD-GDH (e.g. EC 1.1.99.10 etc.), NAD-GDH (e.g. EC 1.1.1.47 etc.), PQQ- GDH (e.g. EC1.1.5.2 etc.), Cholesterol oxidase (for example, EC 1.1.3.6 and the like), cholesterol esterase (for example, EC 3.1.3.2 and the like), glutamate dehydrogenase (for example, EC 1.4.1.2 and the like), glucose oxidase 1.13), lactate oxidase (for example, EC 1.1.3.2 etc.), ascorbic acid oxidase (for example EC 1.10.3.3 etc.), alcohol oxidase (for example EC 1.1.3.13 etc.), alcohol dehydrogenase , EC 1.1.1.1 etc.), bilirubin oxidase (EC 1.3.3.5 etc.), and the like.

[0050] Meanwhile, the reagent composition according to the present invention may contain at least one additive selected from the group consisting of a surfactant, a water-soluble polymer, a quaternary ammonium salt, a fatty acid, a thickening agent, etc. as a dispersant for dissolving a reagent, or a, adhesive agent for preparing a reagent, a stabilizer for long-term storage, and the like.

[0051] The surfactant may make the reagent spread evenly over the electrode, so as to dispense the reagent at a uniform thickness, when the reagent is dispensed. The surfactant may be at least one selected from the group consisting of Triton X-100, sodium dodecyl sulfate, perfluorooctane sulfonate, sodium stearate, and the like. The reagent composition of present invention may contain the surfactant at an amount of 3 to 25 parts by weight, for example 10 to 25 parts by weight based on 100 parts by weight of oxidoreductase, in order that the surfactant makes the reagent spread evenly over the electrode and be dispensed at a uniform thickness. For example, when the used oxidoreductase has an activity of 700 U/mg, the amount of surfactant may be used at 10 to 25 parts by weight based on 100 parts by weight of oxidoreductase. If the activity of the oxidoreductase is higher than the activity, the content of the metal-containing complex can be adjusted to the relatively low content.

[0052] The water-soluble polymer may serve as a polymer support in the reagent composition to help stabilize and disperse the enzyme. Examples of the water-soluble polymer include at least one selected from the group consisting of polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), perfluorosulfonate, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), cellulose acetate, polyamide, and the like. The reagent composition according to the present invention may contain 10 to 70 parts by weight, for example 30 to 70 parts by weight of the water-soluble polymer, based on 100 parts by weight of the oxidoreductase, in order to help stabilize and disperse the enzyme sufficiently. For example, when an oxidoreductase having an activity of 700 U / mg is used, it may contain 30 to 70 parts by weight of the water-soluble polymer based on 100 parts by weight of the oxidoreductase. When the activity of the oxidoreductase is higher than the activity, the content of water-soluble polymer can be adjusted lower than the range.

[0053] The water-soluble polymer may have a weight average molecular weight of about 2,500 to 3,000,000, for example, about 5,000 to 1,000,000 in order to effectively help stabilization and dispersion of the polymer support and the enzyme.

[0054] The quaternary ammonium salt may serve to reduce the measurement error which depends on the amount of hematocrit. The quaternary ammonium salt may be at least one selected from the group consisting of ecyltrimethyl ammonium, myristyltrimethyl ammonium, cetyltrimethyl ammonium, octadecyltrimethyl ammonium, tetrahexyl ammonium, and the like. The reagent composition according to the present invention may contain the quaternary ammonium salt at an amount of 20 to 130 parts by weight, for example 70 to 130 parts by weight, based on 100 parts by weight of the oxidoreductase enzyme, in order to efficiently reduce the measurement error according to the hematocrit. For example, when an oxidoreductase having an activity of 700 U / mg is used, it may contain 70 to 130 parts by weight of a quaternary ammonium salt based on 100 parts by weight of the oxidoreductase. When the activity of the oxidoreductase is higher than that, the content of quaternary ammonium salt can be adjusted to a lower level.

[0055] The fatty acid serves to reduce the measurement error according to the amount of hematocrit, as the quaternary ammonium salt described above and to expand the linear dynamic range of the biosensor in the high concentration region. The fatty acid may be at least one selected from the group consisting of a fatty acid having a C4 to C20 carbon chain and a fatty acid salt thereof, preferably a fatty acid having an alkyl carbon chain of C6 to C12 or a fatty acid salt thereof. Examples of the fatty acid include at least one selected from the group consisting of caproic acid, heptanoic acid, caprylic acid, octanoic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid heptadecanoic acid, stearic acid, nonadecanoic acid, arachidonic acid, salts of fatty acids, and the like. The reagent composition according to the present invention may contain 10 to 70 parts by weight, for example 30 to 70 pars by weight of the fatty acid with respect to 100 parts by weight of the oxidoreductase, in order to appropriately obtain the reduced measurement error depending on the hematocrit and the expansion effect of a linear dynamic range of the biosensor in a high concentration region. For example, when an oxidoreductase having an activity of 700 U / mg is used, it may contain 30 to 70 parts by weight of fatty acid based on 100 parts by weight of the oxidoreductase. When the activity of the oxidoreductase is higher than that, the content of fatty acid can be adjusted to lower level.

[0056] The thickening agent serves to firmly attach the reagent to the electrode. The thickening agent may be one or more selected from the group consisting of Natrozol, diethylaminoethyl-Dextran hydrochloride (DEAE-Dextran hydrochloride), and the like. The reagent composition according to the present invention may contain the thickening agent in an amount of 10 to 90 parts by weight, for example 30 to 90 parts by weight, based on 100 parts by weight of the oxidoreductase, so that the reagent is firmly attached to the electrode. For example, when an oxidoreductase having an activity of 700 U / mg is used, it may contain 30 to 90 parts by weight of the oxidoreductase, based on 100 parts by weight of the oxidoreductase. When the activity of the oxidoreductase is higher than that, the amount can be adjusted lower than this.

[0057] In addition, the present invention provides an electrochemical biosensor containing the reagent composition. In one embodiment, the electrochemical biosensor can be prepared by coating an osmium complex or its salt compound having the following formula (1) and an enzyme capable of oxidizing and reducing an analyte in a liquid biological sample on a substrate having at least two electrodes and drying it. For example, an electrochemical biosensor is provided with a working electrode and an auxiliary electrode formed on one plane, where the reagent composition according to the present invention is contained on the working electrode.

[0058] According to another embodiment, in the electrochemical biosensor, the working electrode and the auxiliary electrode are provided so as to face each other on different planes, and the reagent composition according to the present invention is contained on the working electrode.

[0059] The form of the reagent composition according to the present invention applied to the biosensor is not particularly limited, but may be specifically coated on the surface of the working electrode, but is not limited thereto.

[0060] The planar-type and face-to-face type electrochemical biosensors according to the present invention can be manufactured according to the method disclosed in the prior arts, for example, KR10-2004-0105429A, KR10-2006-0089464A, KR10-0854389B, KR10-2008-0080841A, KR10-2008-0084030A, KR10-2008-0088028A and the like.

[0061] Hereinafter, the structures of the planar and face-type electrochemical biosensors will be described with reference to FIG. 1 and FIG. 2.

[0062] The planar-type electrochemical biosensor in Fig. 1 includes a working electrode and an auxiliary electrode located on the same plane. From the top to the bottom direction, upper plate 11 equipped with air-discharging air outlet 10 for making the blood spread into the sensor; middle plate 9 adhering the upper plate and the lower plate by using the adhesive layers on both sides and making blood spread into the electrode due to the blood capillarity; a reagent composition 8 of present invention contained in the working electrode and auxiliary electrode (counter electrode), for example by coating; an insulating plate 7 equipped the channel part for defining the areas of working electrode and auxiliary electrode; the working electrode 2 and the auxiliary electrode on the lower plate 3; and a lower plate 1 where the working electrode and the auxiliary electrode are formed, are sequentially stacked structure.

[0063] The face-to-face type electrochemical biosensor in Fig. 2 includes a working electrode and an auxiliary electrode on the different plane. From the top to the bottom direction, a upper plate 11 equipped with air outlet 10 for making the blood spread into the sensor and an auxiliary electrode printed thereon; an auxiliary electrode 3 printed on the upper plate; middle plate 9 adhering the upper plate and the lower plate by using the adhesive layers on both sides and making blood spread into the electrode due to the blood capillarity; a reagent composition 8 of present invention contained in the working; an insulating plate 7 equipped the channel part for defining the areas of working electrode and auxiliary electrode; working electrode 2 printed on the lower plate; a lead 4 of auxiliary electrode; flow-sensing electrode 6 for detecting the blood input rate; and a lower plant 1 where the working electrode, the lead of auxiliary electrode and the flow-sensing electrode are sequentially stacked structure.

[0064] The electron transfer mediator of the present invention has advantages of reacting with GDH, no effect of oxygen partial pressure, stable maintenance of reduced and oxidized state for a long time, and thus an reagent for redox reaction and an electrochemical biosensor including the electron transfer mediator can minimize the measurement error caused by the oxygen partial pressure and be used stably for a long time.

Brief Description of Drawings



[0065] 

Fig. 1 is an exploded skew drawing of planar-type biosensor in accordance with an embodiment of the present invention.

Fig. 2 is an exploded skew drawing of face-to-face (sandwich-type) biosensor in accordance with an embodiment of the present invention.

Fig. 3a and Fig. 3b are the result of 1H NMR spectrum analysis for the osmium compound obtained in Example 1.

Fig. 4a and Fig. 4b are grapes showing the measurement current according to the change in the glucose concentration in Example 1.

Fig. 5a to Fig. 5c are the comparison of change in UV-visible light spectrum according to various electron transfer mediators in an aqueous solution with time change; Fig. 5a for osmium complex, Fig. 5b for ruthenium hexamine and Fig. 5c for Ferricyanide.

Fig. 6 shows the change in the characteristics of the osmium complex-based glucose strip sensor over time in the storage conditions; (a) background current and (b) response slope.

Fig. 7 is a comparison of interference result caused by oxygen partial pressure for the Os-based glucose strip sensor over time, depending on the glucose concentration.

Fig. 8 shows the change in the characteristics of the osmium complex-based glucose strip sensor over time in the storage conditions, after the addition of oxidizing agent (NaOCl); (a) background current and (b) response slope.

Fig. 9 shows the change in the characteristics of the osmium complex-based glucose strip sensor over time in the storage conditions, after the addition of oxidizing agent (H2O2).

Fig. 10 shows the change in the characteristics of the osmium complex-based glucose strip sensor over time in the storage conditions, where the osmium complex (oxidized state) was obtained by oxidizing with oxidizing agent (H2O2).

Fig. 11 shows the change in the characteristics of the osmium complex -based glucose strip sensor over time in the storage conditions, where the osmium complex (oxidized state) was obtained by oxidizing with oxidizing agent (NaOCl).

Fig. 12 is a comparison of interference result caused by oxygen partial pressure for the osmium complex-based glucose strip sensor over time, depending on the glucose concentration, where the osmium complex (oxidized state) was obtained by oxidizing with oxidizing agent (NaOCl).

Fig. 13a and Fig. 13b are shows the change in the characteristics of the osmium complex -based glucose strip sensor depending on the applied voltage, where the osmium complex (oxidized state) was obtained by oxidizing with oxidizing agent (NaOCl).

Fig. 14a and Fig. 14b show the change in the characteristics of the osmium complex (oxidized state)-based glucose strip sensor over time in the storage conditions.

Fig. 15a and Fig. 15b show the change in the error % of the osmium complex (oxidized state)-based glucose strip sensor over time in the storage conditions, depending on the glucose concentration.

Fig. 16 is a comparison of interference result caused by oxygen partial pressure for the osmium complex (oxidized state)-based glucose strip sensor over time, depending on the glucose concentration.


Mode for Invention



[0066] Hereinafter, the present invention will be described referring to the following examples. However, these examples are merely illustrative of the present invention, the scope of which shall not be limited thereto.

<Example 1> Preparation of osmium complex


1-1. synthesis of osmium complex



[0067] 4,4'-dicarboxy-2,2'-bipyridine (dcbpy) and K2OsCl6 were used as starting materials to synthesize Os(dcbpy)2Cl2 (osmium complex).

[0068] K2OsCl6 0.481 g (1 mM) and dcbpy 0.488 g (2 mM) were poured to 500 mL three-neck round-bottom flask, and dissolved with agitation for 1 hour by addition of dimethylformamide (DMF) 40 mL. Then, the mixture was incubated in oil bath at 180 °C for 2 hours at the atmosphere of N2 with reflux. After the solution was completely reacted, the solvent was removed using a rotary evaporator, the product was filtered under reduced pressure while washing with distilled water. The filtered product was dried at 50 °C for 12 hours, to produce Os(dcbpy)2Cl2 (osmium complex) at yield of 60%.

1-2. Spectroscopic characterization



[0069] To test the successful synthesis of Os(dcbpy)2Cl2 (osmium complex) by using dcbpy and K2OsCl6, 1H NMR (400 MHz, DMSO-d6) of the product was measured. 1H NMR spectrum of dcbpy (a) and osmium complex (b) is shown in Fig. 3.

[0070] As shown in Fig. 3, as the dcbpy ligand is coordinated to the central metal Os, the band width of H peak of -COOH substituted in the aromatic ring (Fig. 3 (b) 1) was still wider than that measured before the coordination to the central metal Os (Fig. 3 (a) 1). It is generally known that when the organic compound is coordinated to the metal, the band width of the peak tends to widen. The 1H NMR spectrum of the reaction shows that the reaction has proceeded successfully.

1-3. Electrochemical characterization



[0071] To verify the electrochemical properties of osmium complex, cyclic voltammetry was performed. Carbon electrode as working electrode, platinum as auxiliary electrode, and Ag/AgCl (sat. KCl) electrode as reference electrode were used, and the current was measured by changing the glucose concentration in a mixed solution including FAD-glucose dehydrogenase (FAD-GDH) 10 mg/mL, osmium complex 30 mmol and 0.1 M PBS (pH 7.4). The current was measured in the ranges of -0.2 V ∼ 0.3 V at scan rate 10 mV/sec. the measurement result was shown in Fig. 4.

[0072] As shown in Fig. 4, the response slope of 77.1 nA/(mg/dL) and relatively excellent linearity were shown in the ranges of 90 mg/dL to 540 mg/dL of glucose concentration. The spectroscopic result of Fig. 3 and the electrochemical result of Fig. 4 confirmed that the osmium complex was synthesized successfully.

<Example 2> Preparation of salt of osmium complex



[0073] The structure of the synthesized osmium complex is an organometallic compound where dcbpy ligand having a bypyridine structure with a carboxy group (COOH) is coordinated to osmium (Os) as a central metal, and has low solubility in water. The solubility was increased by converting -COOH group of the osmium complex to the salt form by substituting with -COO-Na+. First, the equivalence ratio was determined by titration to convert to the salt form, and the proper equivalence ratio was 1: 3. In subsequent examples, osmium complex sodium salt was used by substituting the osmium complex with NaOH at 1: 3 equivalent ratios.

<Example 3> Oxidative treatment of osmium complex



[0074] 0.75 g (1 mM) of the synthesized osmium complex was poured to 500 mL one-neck round-bottom flask and was dissolved in 100 mL of distilled water. After 0.037 g / 0.034 g (1 mM) of oxidizing agent (NaOCl / H2O2) was added, the reaction was carried out for 2 hours with agitation. After the reaction was completed, the solvent was removed using a rotary evaporator, and the product was filtered under reduced pressure while washing with ethyl ether. The filtered product was dried at 50 °C for 12 hours or more to obtain 90% osmium complex (oxidation state, Os(ox)complex).

<Example 4> Stability test of osmium complex



[0075] In order to confirm the stability of the osmium complex as an organometallic compound, the UV-Visible spectrum change over time in aqueous solution was compared with that of ruthenium hexamine and Ferricyanide, which were commercially used as electron transfer media in glucose strip sensors.

[0076] Specifically, the UV-Vis spectrum of an aqueous solution of 1 mg of each osmium complex, ruthenium hexamine, and Ferricyanide dissolved in 1 mL of 0.1 M PBS (pH 7.4) was measured and stored at room temperature for 11 days. The experimental results are shown in Figs. 5A to 5C.

[0077] As shown in FIG. 5b, no change in the UV-Visible spectrum was observed over time in the case of ruthenium hexamine (FIG. 5b), indicating that the oxidized Ru (III) was very stable. In the case of the osmium complex (FIG. 5a) synthesized in Example 1, the absorbance of absorption peaks at 400 nm and 520 mm decreased with time. At initial stage, the oxidation state Os (III) and the reduced state Os (II) were mixed and the reduced state Os (II) were changed into the oxidation state Os (III) with time. The result indicates that the oxidation state Os (III) is more stable than reduced state Os (II) in an aqueous solution at room temperature. In case of Ferricyanide, the absorption peaks of 300 nm and 420 mm decreased with time, and the absorbance at 260 nm absorption peak increased, indicating that the oxidized Fe (III) was changed into reduced state Fe (II). These results show that the reduced Fe (II) is more stable than the reduced Fe (III).

<Example 5> Biosensor Manufacture


5-1.Manufacture of biosensor



[0078] Os-based glucose strip sensor was manufactured by using carbon as a working electrode, Ag/AgCl face-to-face sensor, osmium complex30 mM, FAD-GDH 10 mg/mL, surfactant, and 0.1 M PBS (pH 7.4) of background electrolyte. The electrochemical properties such as response activity, stability, effect of oxygen partial pressure of the strip sensor was measured and compared with those of three type glucose strip sensors using CareSens N (Ru + GOx), CareSens Pro (Fe + GDH) or VetMate (Ru + GDH + Thionin). The response activity was measured with multi-channel biosensor system at the applied voltage of 0.2V.

5-2. Property changes over time in storage conditions



[0079] The properties such as background current and response slope for Os-based glucose strip sensor over time in storage conditions were analyzed. 30 mM Os-based glucose strip sensor, and three glucose strip sensors using CareSens N, CareSens Pro and VetMate were stored at 23 °C, RH 20% or lower, and tested for property changes over time in storage conditions.

[0080] Three glucose strip sensors using CareSens N, CareSens Pro and VetMate maintained the background current and response slope, but in case of Os-based glucose strip sensor under the general storage conditions, the background current was changed from 3.2 µA to 2.5 µA over time (Fig. 6). The experimental result was in accord with that of UV-Visible spectrum which was performed for osmium complex under room temperature in Example 4.

5-3. Comparison of effect of oxygen partial pressure



[0081] In order to remove the difference in the measured values of capillary blood and venous blood GOx caused by the different oxygen partial pressure (pO2), the research is being carried out to substitute with GDH having no effect of oxygen partial pressure, but its application is restricted due to the electron transfer mediator affected by the oxygen partial pressure.

[0082] The effect of oxygen partial pressure was tested for 30 mM Os-based strip, and three strips using CareSens N, CareSens Pro and VetMate.

[0083] The oxygen partial pressure was obtained by measuring the glucose concentrations of low (90 mg/dL), middle (200 mg/dL) and high (400 mg/dL) at saturated state (160 mmHg) and low-pressure state (40 mmHg) applied by deoxidation process. The % error caused by the oxygen partial pressure was calculated by using the following formula.



[0084] The Ru-based strip (CareSens N, VetMate) showed about 8% error, but 30 mM Os-based strip showed about 3% or lower (Fig. 7).

<Example 6> Test of strip sensor properties using the osmium complex with the addition of oxidizing agent or the osmium complex treated with oxidizing agent


6-1. Test of strip sensor properties using the osmium complex with the addition of oxidizing agent



[0085] On the basis of the experiment described above, Os-based strip sensor was manufactured by adding an oxidizing agent and tested for the background current and the change of the background current with the addition of oxidizing agent over time.

A.Addition of NaOCl



[0086] Os-based glucose strip sensors were manufactured by using carbon as a working electrode, Ag/AgCl face-to-face sensor, and two kinds of base compositions including 20 mM of osmium complex or 30 mM of osmium complex, FAD-GDH 10 mg/mL, surfactant, and 0.1 M PBS (pH 7.4) of background electrolyte, or two composition including the base compositions and H2O2. The electrochemical properties such as response activity, stability, effect of oxygen partial pressure of the strip sensor was measured and compared with those of three type glucose strip sensors using CareSens N and CareSens Pro2. The response activity was measured with multi-channel biosensor system at the applied voltage of 0.2V.

[0087] Four kinds of Os-based strip sensor, CareSens N and CareSens Pro 2 were stored at 23 °C, RH 20% or lower, and tested for property changes over time in storage conditions.

[0088] When being compared with the conventional strip sensor having no addition of NaOCl, Os-based strip including the NaOCI showed the change in background current from about 2 µA to about 0.2 µA, and showed comparatively stable background current over time under storage conditions (Fig. 8).

B. Addition of H2O2



[0089] Os-based glucose strip sensors were manufactured by using carbon as a working electrode, Ag/AgCl face-to-face sensor, and two kinds of base composition including 20 mM of osmium complex or 30 mM of osmium complex, FAD-GDH 10 mg/mL, surfactant, and 0.1 M PBS (pH 7.4) of background electrolyte, two composition including the base compositions and H2O2, one composition with two-fold amount of GDH and 30 mM of osmium complex.

[0090] For the five kinds of Os-based strip sensor, the electrochemical properties such as response activity, stability, effect of oxygen partial pressure of the strip sensor was measured and compared with those of two glucose strip sensors using CareSens N and CareSens Pro2.

[0091] The response activity was measured with multi-channel biosensor system at the applied voltage of 0.2V.

[0092] Five kinds of Os-based strip sensor, CareSens N and CareSens Pro 2 were stored at 23 °C, RH 20% or lower, and tested for property changes over time in storage conditions.

[0093] As shown in Fig. 9, Os-strip sensor with the addition of H2O2 showed about 0.2 µA of background current and response slope of 20 nA/(mg/dL) or higher, compared with that of Os-strip sensor with the addition of NaOCl

6-2. Test of strip sensor properties using the osmium complex treated with oxidizing agent


A. Oxidative Treatment with H2O2



[0094] Os-based glucose strip sensors were manufactured by using carbon as a working electrode, Ag/AgCl face-to-face sensor, and two kinds of base compositions including 20 mM of osmium complex or 30 mM of osmium complex, FAD-GDH 10 mg/mL, surfactant, and 0.1 M PBS (pH 7.4) of background electrolyte, two compositions including the osmium complex treated with H2O2 (Os(ox)) and two compositions including the salt of oxidized osmium complex (Os(ox)salt). For six kinds of Os-based strip sensor, the electrochemical properties such as response activity, stability, effect of oxygen partial pressure of the strip sensor was measured and compared with those of two glucose strip sensors using CareSens N and CareSens Pro2.

[0095] The response activity was measured with multi-channel biosensor system at the applied voltage of 0.2V.

[0096] Six kinds of Os-based strip sensors including 6 kinds of compositions, CareSens N and CareSens Pro 2 were stored at 23 °C, RH 20% or lower, and tested for property changes over time in storage conditions.

[0097] Os(ox)complex-based strip sensor including the Os complex treated with H2O2 showed about 0.2 µA of background current, which was similar to that of Os complex-based strip sensor with the addition of H2O2. Os(ox) salt-based strip sensor showed higher background current than that of Os(ox) complex-based strip sensor. Four kinds of Os(ox)complex-based strip sensor including the Os complex treated with H2O2 or Os(ox) salt showed the increased response slop, compared to the Os complex-based strip sensor with addition of H2O2 (Fig. 10).

B. Oxidative Treatment with NaOCl



[0098] Os-based glucose strip sensors were manufactured by using carbon as a working electrode, Ag/AgCl face-to-face sensor, and two kinds of base compositions including 20 mM of osmium complex or 30 mM of osmium complex, FAD-GDH 10 mg/mL, surfactant, and 0.1 M PBS (pH 7.4) of background electrolyte, two compositions including the osmium complex treated with NaOCl (Os(ox)) and two compositions including the salt of oxidized osmium complex (Os(ox)salt). For six kinds of Os-based strip sensor, the electrochemical properties such as response activity, stability, effect of oxygen partial pressure of the strip sensor was measured and compared with those of two glucose strip sensors using CareSens N and CareSens Pro2.

[0099] The response activity was measured with multi-channel biosensor system at the applied voltage of 0.2V.

[0100] Six kinds of Os-based strip sensors including 6 kinds of compositions, CareSens N and CareSens Pro 2 were stored at 23 °C, RH 20% or lower, and tested for property changes over time in storage conditions.

[0101] Os(ox)complex-based strip sensor including the Os complex treated with NaOCl showed most excellent electrochemical response properties such as low background current, response slope, stability with time, and decreased effect of oxygen partial pressure, among the Os-based strip sensor which has been studied until now (Fig. 11 and Fig. 12).

[0102] To investigate the response properties of Os(ox) complex strip sensor according to the applied voltage, Os-based glucose strip sensors were manufactured by using carbon as a working electrode, Ag/AgCl face-to-face sensor, and two kinds of base compositions including 20 mM of osmium complex or 30 mM of osmium complex, FAD-GDH 10 mg/mL, surfactant, and 0.1 M PBS (pH 7.4) of background electrolyte. The electrochemical properties were analyzed according to the applied voltage.

[0103] The response properties were measured at applied voltage of 0.1 V ∼ 0.4 V, at 0.05 V of interval, by using multi-channel biosensor system which the applied voltage was easily changed.

[0104] FIG. 13 showed the changes in background current and response slope depending on the applied voltage. The background current showed a gradual increase from 0.2 V to 0.35 V and a sharp increase after 0.35 V, and the response slope increased rapidly to 0.3 V and then gradually increased after 0.3 V. When the applied voltage increased by 0.1 V to 0.3 V, the background current increased by 0.05 µA, while the response slope improved about two times, that is, by about 45 nA / (mg / dL).

<Example 7> Long-term stability of strip sensor using the osmium complex treated with NaOCl



[0105] The strip sensor using the osmium complex treated with NaOCl was tested for the background current, response slope and effect of oxygen partial pressure over time under the storage conditions.

[0106] The response activity was measured with multi-channel biosensor system at the applied voltage of 0.3V.

[0107] As shown in FIG. 14, the strip sensor including Os (ox) complex oxidized with NaOCl maintained a stable background current and response slope for more than 8 weeks under storage conditions. As a result of calculating % Error using the current values of 70 mg / dL glucose and 100 mg / dL glucose over time, followed by calibrating in 20 to 200 mM range with the measurement result for 1 day, as shown in FIG. 15, the overall result was significantly valid by 10% or less of % error.

[0108] It was also found that the effect of oxygen partial pressure was also 4% or less (Fig. 16).

[0109] The strip sensor including FAD-GDH enzyme and Os (ox) complex oxidized with NaOCl stably maintained the effect of the background current, the response slope and the change in effect of oxygen partial pressure for 8 weeks under the storage conditions at room temperature.

[Explanation of reference numerals of drawings]



[0110] 
1:
substrate
2:
working electrode
3:
auxiliary electrode
4:
lead of auxiliary electrode
5:
circuit connection ground
6:
flow sensing electrode
7:
Insulating plate
8:
reagent composition for redox reaction
9:
Fitting plate
10:
vent
11:
upper plate



Claims

1. An electrochemical biosensor, comprising
an electrode substrate, and
an osmium complex or its salts represented by Chemical formula 1, and an enzyme being capable of oxidizing and reducing a target substance in a liquid biological sample, which are immobilized on the electrode substrate:

        [Chemical formula 1]     Os(A)mXn

Wherein, A is represented by Chemical formula 2,

X is a halogen, m is an integer of 1 to 3, n is an integer of 0 to 4, and a sum of m and n is an integer of 3 to 5.


 
2. The electrochemical biosensor according to claim 1, wherein the salt is a salt of at least one alkali metal selected from the group consisting of Li salt, Na salt, K salt, Rb salt, Cs salt and Fr salt.
 
3. The electrochemical biosensor according to claim 1, wherein the osmium complex comprises trivalent osmium (III).
 
4. The electrochemical biosensor according to claim 1, wherein the osmium complex comprises trivalent osmium (III) and divalent osmium (II).
 
5. The electrochemical biosensor according to claim 1, wherein the osmium complex is obtained by using the compound represented by Chemical formula 2, and the compound represented by Chemical formula 6:

        [Chemical formula 6]     YpOsXq

Wherein, Y is K, Na or NH4, X is a halogen, p is an integer of 1 to 2, and q is an integer of 1 to 6.
 
6. The electrochemical biosensor according to claim 1, further comprising an oxidizing agent.
 
7. The electrochemical biosensor according to claim 6 , wherein the oxidizing agent is contained in the molar ratio of 0.1 to 10, based on 1 mole of osmium complex or its salt.
 
8. The electrochemical biosensor according to claim 6, wherein the oxidizing agent is at least one selected from the group consisting of NaOCl, H2O2, O2, O3, PbO2, MnO2, KMnO4, ClO2, F2, Cl2, H2CrO4, K2Cr2O7, N2O, Ag2O, OsO4, H2S2O8, pyridinium chlorochromate and 2,2'-Dipyridyldisulfide.
 
9. The electrochemical biosensor according to claim 1 which does not comprises an organometallic compound other than the osmium complex or its salt as an electron transfer mediator.
 
10. The electrochemical biosensor according to claim 1, wherein the osmium complex or its salt is contained in the amount of 20 to 700 parts by weight based on 100 parts by weight of enzyme.
 
11. The electrochemical biosensor according to claim 1, wherein the biosensor further comprises a second electron transfer mediator, which is at least one selected from the group consisting of thionine, 1-Methoxy-5-methylphenazinium methylsulfate, 3-amino-7-(2,3,4,5,6-pentahydroxy hexanamido)-5-phenothiazinium, 1-Methoxy-5-methylphenazinium, Azure C, Azure A, Methylene Blue, Toluidine Blue, and derivatives thereof.
 
12. The electrochemical biosensor according to claim 11, wherein the enzyme is at least an oxidoreductase selected from the group consisting of dehydrogenase, oxidase and esterase; or at least an oxidoreductase selected from the group consisting of dehydrogenase, oxidase and esterase, in combination with at least a cofactor selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and Pyrroloquinoline quinone (PQQ).
 
13. The electrochemical biosensor according to claim 12, wherein the at least an oxidoreductase selected from the group consisting of dehydrogenase, oxidase and esterase is at least one selected from the group consisting of glucose dehydrogenase, glutamate dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase and bilirubin oxidase.
 
14. The electrochemical biosensor according to claim 12, wherein the enzyme is at least one selected from the group consisting of flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH), and nicotinamide adenine dinucleotide-glucose dehydrogenase.
 
15. The electrochemical biosensor according to claim 1, further comprising at least one selected from the group consisting of surfactants, water-soluble polymers, quaternary ammonium salts, fatty acids, and thickening agents.
 
16. The electrochemical biosensor according to claim 15, wherein the surfactant is at least one selected from the group consisting of Triton X-100, sodium dodecyl sulfate, perfluorooctane sulfonate and sodium stearate;
the water-soluble polymer is at least one selected from the group consisting of polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), perfluorosulfonate, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), cellulose acetate and polyamide;
the quaternary ammonium salt is at least one selected from the group consisting of ecyltrimethylammonium, myristyltrimethylammonium, cetyltrimethylammonium, octadecyltrimethylammonium and tetrahexylammonium;
the fatty acid is at least one selected from the group consisting of a fatty acid having a C4 to C20 carbon chain and a fatty acid salt thereof, or
the thickening agent may be one or more selected from the group consisting of Natrozol and diethylaminoethyl-Dextran hydrochloride (DEAE-Dextran hydrochloride.
 
17. The use of the electrochemical biosensor according to any of the preceding claims for analyzing blood.
 


Ansprüche

1. Elektrochemischer Biosensor, der Folgendes umfasst:

ein Elektrodensubstrat, und

einen Osmiumkomplex oder dessen Salze, dargestellt durch die chemische Formel 1, und ein Enzym, das in der Lage ist, eine Zielsubstanz in einer flüssigen biologischen Probe zu oxidieren und zu reduzieren, die auf dem Elektrodensubstrat immobilisiert sind:

        [Chemische Formel 1]     Os(A)mXn

wobei A durch die chemische Formel 2 dargestellt wird,

wobei es sich bei X um ein Halogen handelt, bei m um eine ganze Zahl von 1 bis 3, bei n um eine ganze Zahl von 0 bis 4 und wobei es sich bei einer Summe von m und n um eine ganze Zahl von 3 bis 5 handelt.


 
2. Elektrochemischer Biosensor nach Anspruch 1, wobei es sich bei dem Salz um ein Salz von mindestens einem Alkalimetall handelt, das aus der Gruppe ausgewählt ist, die aus Li-Salz, Na-Salz, K-Salz, Rb-Salz, Cs-Salz und Fr-Salz besteht.
 
3. Elektrochemischer Biosensor nach Anspruch 1, wobei der Osmiumkomplex dreiwertiges Osmium (III) umfasst.
 
4. Elektrochemischer Biosensor nach Anspruch 1, wobei der Osmiumkomplex dreiwertiges Osmium (III) und zweiwertiges Osmium (II) umfasst.
 
5. Elektrochemischer Biosensor nach Anspruch 1, wobei der Osmiumkomplex unter Verwendung der durch die chemische Formel 2 dargestellten Verbindung und der durch die chemische Formel 6 dargestellten Verbindung erhalten wird:

        [Chemische Formel 6]     YpOsXq

wobei es sich bei Y um K, Na oder NH4 handelt, bei X um ein Halogen, bei p um eine ganze Zahl von 1 bis 2, und wobei es sich bei q um eine ganze Zahl von 1 bis 6 handelt.
 
6. Elektrochemischer Biosensor nach Anspruch 1, der weiterhin ein Oxidationsmittel umfasst.
 
7. Elektrochemischer Biosensor nach Anspruch 6, wobei das Oxidationsmittel in einem Molverhältnis von 0,1 bis 10, bezogen auf 1 Mol Osmiumkomplex oder dessen Salz, enthalten ist.
 
8. Elektrochemischer Biosensor nach Anspruch 6, wobei es sich bei dem Oxidationsmittel um wenigstens eines handelt, das ausgewählt wird aus der Gruppe bestehend aus NaOCl, H2O2, O2, O3, PbO2, MnO2, KMnO4, ClO2, F2, Cl2, H2CrO4, K2Cr2O7, N2O, Ag2O, OsO4, H2S2O8, Pyridiniumchlorochromat und 2,2'-Dipyridyldisulfid.
 
9. Elektrochemischer Biosensor nach Anspruch 1, der keine andere metallorganische Verbindung als den Osmiumkomplex oder dessen Salz als Elektronenübertragungsvermittler umfasst.
 
10. Elektrochemischer Biosensor nach Anspruch 1, wobei der Osmiumkomplex oder dessen Salz in einer Menge von 20 bis 700 Gewichtsteilen, bezogen auf 100 Gewichtsteile Enzym, enthalten ist.
 
11. Elektrochemischer Biosensor nach Anspruch 1, wobei der Biosensor weiterhin einen zweiten Elektronenübertragungsvermittler umfasst, bei dem es sich um wenigstens einen handelt, der ausgewählt wird aus der Gruppe bestehend aus Thionin, 1-Methoxy-5-methylphenaziniummethylsulfat, 3-Amino-7-(2,3,4,5,6-pentahydroxyhexanamido)-5-phenothiazinium, 1-Methoxy-5-methylphenazinium, Azure C, Azure A, Methylenblau, Toluidinblau und Derivate davon.
 
12. Elektrochemischer Biosensor nach Anspruch 11, wobei es sich bei dem Enzym um wenigstens eine Oxidoreduktase handelt, die ausgewählt wird aus der Gruppe bestehend aus Dehydrogenase, Oxidase und Esterase; oder um wenigstens eine Oxidoreduktase handelt, die ausgewählt wird aus der Gruppe bestehend aus Dehydrogenase, Oxidase und Esterase in Kombination mit mindestens einem Cofaktor ausgewählt aus der Gruppe bestehend aus Flavinadenindinukleotid (FAD), Nicotinamidadenindinukleotid (NAD) und Pyrrolochinolinchinon (PQQ).
 
13. Elektrochemischer Biosensor nach Anspruch 12, wobei es sich bei der wenigstens einen Oxidoreduktase, die ausgewählt wird aus der Gruppe bestehend aus Dehydrogenase, Oxidase und Esterase, um wenigstens eine handelt, die ausgewählt wird aus der Gruppe bestehend aus Glucosedehydrogenase, Glutamatdehydrogenase, Glucoseoxidase, Cholesterinoxidase, Cholesterinesterase, Lactatoxidase, Ascorbinsäureoxidase, Alkoholoxidase, Alkoholdehydrogenase und Bilirubinoxidase.
 
14. Elektrochemischer Biosensor nach Anspruch 12, wobei es sich bei dem Enzym um wenigstens eines handelt, das ausgewählt wird aus der Gruppe bestehend aus Flavinadenindinukleotid-Glucosedehydrogenase (FAD-GDH) und Nicotinamidadenindinukleotid-Glucosedehydrogenase.
 
15. Elektrochemischer Biosensor nach Anspruch 1, der wenigstens einen Bestandteil umfasst, der ausgewählt wird aus der Gruppe bestehend aus Tensiden, wasserlöslichen Polymeren, quaternären Ammoniumsalzen, Fettsäuren und Verdickungsmitteln.
 
16. Elektrochemischer Biosensor nach Anspruch 15, wobei es sich bei dem Tensid um wenigstens eines handelt, das ausgewählt wird aus der Gruppe bestehend aus Triton X-100, Natriumdodecylsulfat, Perfluoroctansulfonat und Natriumstearat;
wobei es sich bei dem wasserlöslichen Polymer um wenigstens eines handelt, das ausgewählt wird aus der Gruppe bestehend aus Polyvinylpyrrolidon (PVP), Polyvinylalkohol (PVA), Perfluorsulfonat, Hydroxyethylcellulose (HEC), Hydroxypropylcellulose (HPC), Carboxymethylcellulose (CMC), Celluloseacetat und Polyamid;
wobei es sich bei dem quaternären Ammoniumsalz um wenigstens eines handelt, das ausgewählt wird aus der Gruppe bestehend aus Ecyltrimethylammonium, Myristyltrimethylammonium, Cetyltrimethylammonium, Octadecyltrimethylammonium und Tetrahexylammonium;
wobei es sich bei der Fettsäure um wenigstens eine handelt, die ausgewählt wird aus der Gruppe bestehend aus einer Fettsäure mit einer C4- bis C20-Kohlenstoffkette und einem Fettsäuresalz davon, oder
wobei es sich bei dem Verdickungsmittel um wenigstens eines handelt, das ausgewählt wird aus der Gruppe bestehend aus Natrozol und Diethylaminoethyl-Dextranhydrochlorid (DEAE-Dextranhydrochlorid).
 
17. Verwendung des elektrochemischen Biosensors nach einem der vorhergehenden Ansprüche zur Analyse von Blut.
 


Revendications

1. Biocapteur électrochimique, comprenant
un substrat d'électrode, et
un complexe d'osmium ou ses sels représentés par la formule chimique 1, et une enzyme apte à oxyder et réduire une substance cible dans un échantillon biologique liquide, lesquels sont immobilisés sur le substrat d'électrode :

        [Formule chimique 1]     Os(A)mXn

dans laquelle A est représenté par la formule chimique 2,

dans laquelle X représente un halogène, m est un nombre entier valant de 1 à 3, n est un nombre entier valant de 0 à 4, et la somme de m et n est un nombre entier valant de 3 à 5.


 
2. Biocapteur électrochimique selon la revendication 1, dans lequel le sel est un sel d'au moins un métal alcalin, choisi dans le groupe constitué par le sel de Li, le sel de Na, le sel de K, le sel de Rb, le sel de Cs et le sel de Fr.
 
3. Biocapteur électrochimique selon la revendication 1, dans lequel le complexe d'osmium comprend de l'osmium trivalent (III).
 
4. Biocapteur électrochimique selon la revendication 1, dans lequel le complexe d'osmium comprend de l'osmium trivalent (III) et de l'osmium divalent (II).
 
5. Biocapteur électrochimique selon la revendication 1, dans lequel le complexe d'osmium s'obtient au moyen du composé représenté par la formule chimique 2 et du composé représenté par la formule chimique 6 :

        [Formule chimique 6]     YpOsXq

dans laquelle Y représente K, Na ou NH4, X représente un halogène, p est un nombre entier valant 1 ou 2, et q est un nombre entier valant de 1 à 6.
 
6. Biocapteur électrochimique selon la revendication 1, comprenant en outre un agent oxydant.
 
7. Biocapteur électrochimique selon la revendication 6, dans lequel l'agent oxydant est présent selon un rapport molaire allant de 0,1 à 10, par rapport à 1 mole de complexe d'osmium ou son sel.
 
8. Biocapteur électrochimique selon la revendication 6, dans lequel l'agent oxydant est au moins un agent choisi dans le groupe constitué par NaOCl, H2O2, O2, O3, PbO2, MnO2, KMnO4, ClO2, F2, Cl2, H2CrO4, K2Cr2O7, N2O, Ag2O, OsO4, H2S2O8, chlorochromate de pyridinium et disulfure de 2,2'-dipyridyle.
 
9. Biocapteur électrochimique selon la revendication 1, lequel est exempt de composé organométallique autre que ledit complexe d'osmium ou son sel à titre de médiateur de transfert d'électrons.
 
10. Biocapteur électrochimique selon la revendication 1, dans lequel le complexe d'osmium ou son sel est présent dans une quantité allant de 20 à 700 parties en poids par rapport à 100 parties en poids d'enzyme.
 
11. Biocapteur électrochimique selon la revendication 1, dans lequel le biocapteur comprend en outre un second médiateur de transfert d'électrons, consistant en au moins un médiateur choisi dans le groupe constitué par : thionine, méthylsulfate de 1-méthoxy-5-méthylphénazinium, 3-amino-7-(2,3,4,5,6-pentahydroxy hexanamido)-5-phénothiazinium, 1-méthoxy-5-méthylphénazinium, Azure C, Azure A, bleu de méthylène, bleu de toluidine et leurs dérivés.
 
12. Biocapteur électrochimique selon la revendication 11, dans lequel l'enzyme est au moins une oxydoréductase choisie dans le groupe constitué par : déshydrogénase, oxydase et estérase ; ou au moins une oxydoréductase choisie dans le groupe constitué par : déshydrogénase, oxydase et estérase en combinaison avec au moins un cofacteur choisi dans le groupe constitué par : flavine adénine dinucléotide (FAD), nicotinamide adénine dinucléotide (NAD) et pyrroloquinoline quinone (PQQ).
 
13. Biocapteur électrochimique selon la revendication 12, dans lequel l'au moins une oxydoréductase choisie dans le groupe constitué par déshydrogénase, oxydase et estérase est au moins une oxydoréductase choisie dans le groupe constitué par : glucose déshydrogénase, glutamate déshydrogénase, glucose oxydase, cholestérol oxydase, cholestérol estérase, lactate oxydase, acide ascorbique oxydase, alcool oxydase, alcool déshydrogénase et bilirubine oxydase.
 
14. Biocapteur électrochimique selon la revendication 12, dans lequel l'enzyme est au moins une enzyme choisie dans le groupe constitué par : flavine adénine dinucléotide-glucose déshydrogénase (FAD-GDH), et nicotinamide adénine dinucléotide-glucose déshydrogénase.
 
15. Biocapteur électrochimique selon la revendication 1, comprenant en outre au moins un composant choisi dans le groupe constitué par : tensioactifs, polymères hydrosolubles, sels d'ammonium quaternaire, acides gras et agents épaississants.
 
16. Biocapteur électrochimique selon la revendication 15, dans lequel le tensioactif est au moins un tensioactif choisi dans le groupe constitué par: Triton X-100, dodécylsulfate de sodium, sulfonate de perfluorooctane et stéarate de sodium ;
le polymère hydrosoluble est au moins un polymère hydrosoluble choisi dans le groupe constitué par : polyvinylpyrrolidone (PVP), alcool polyvinylique (PVA), perfluorosulfonate, hydroxyéthylcellulose (HEC), hydroxypropylcellulose (HPC), carboxyméthylcellulose (CMC), acétate de cellulose et polyamide ;
le sel d'ammonium quaternaire est au moins un sel d'ammonium quaternaire choisi dans le groupe constitué par : écyltriméthylammomium, myristyltriméthylammonium, cétyltriméthylammonium, octadécyltriméthylammonium et tétrahexylammonium ;
l'acide gras est au moins un acide gras choisi dans le groupe constitué par : un acide gras ayant une chaîne carbonée de C4 à C20 et un sel d'acide gras de celui-ci, ou
l'agent épaississant peut consister en un ou plusieurs acides gras choisis dans le groupe constitué par : Natrozol et diéthylaminoéthyl-dextrane chlorhydratée (DEAE-dextrane chlorhydratée).
 
17. Utilisation du biocapteur électrochimique selon l'une quelconque des revendications précédentes pour analyser le sang.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description