Biosensor and measurement method using the same

Abstract

The present invention provides a biosensor capable of being manufactured at low cost and simply, and capable of measuring the concentration of a substance to be measured, such as glucose, with high reproducibility, and a measurement method using the biosensor. A biosensor includes an insulating substrate, an electrode pair provided on the insulating substrate, and a reagent having a redox enzyme and an electron transfer substance placed on at least a working electrode among the electrode pair. The biosensor is used to measure a substance to be measured in a sample supplied to the electrode pair. The biosensor is characterized in that the working electrode is made of one or more nickel alloys selected from a nickel-ruthenium alloy, a nickel-tungsten alloy, and a nickel-vanadium alloy.

Description

Technical Field

[0001] The present invention relates to a biosensor and a measurement method using the biosensor. Background Art

[0002] A biosensor generally includes an electrode on which a reagent containing a redox enzyme, an electron transfer substance, etc. is placed. As the electrode material, carbon, gold, palladium, etc. are usually used. Carbon is low in price, but screen printing is required, and there is a problem that it is not easy to control the deviation of the resistance value depending on the manufacturing environment during electrode manufacturing. In addition, although noble metals such as gold and palladium are easy to process electrodes with high precision, they are expensive compared to carbon. Therefore, it is considered to use base metals (metals with an ionization tendency greater than hydrogen) that are low in cost and easy to process as electrodes, but base metals have problems such as a fast deterioration rate and easy dissolution when a voltage is applied.

[0003] Patent Documents 1 to 3 disclose the use of nickel-chromium alloys, nickel-copper alloys, etc. belonging to base metal alloys as electrode materials for biosensors. However, there is still room for improvement in terms of responsiveness to components in reagents such as electron transfer substances and reduction of background current.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2018-503080

[0007] Patent Document 2: Pamphlet of International Publication No. 2015 / 060119

[0008] Patent Document 3: Japanese Patent Application Laid-Open No. 2013-518264 Summary of the Invention

[0009] An object of the present invention is to provide a biosensor that can be manufactured at low cost and simply, and can measure the concentration of a measurement object substance such as glucose with high reproducibility.

[0010] The inventors of the present invention conducted in-depth research to solve the above problems. As a result, it was found that in a biosensor in which a reagent having a redox enzyme and an electron transfer substance is placed at least on a working electrode, by using a specific nickel alloy as the electrode material of the working electrode, a biosensor having a responsiveness to an electron transfer substance close to that of a noble metal electrode can be manufactured at low cost and simply, and thus the present invention was completed.

[0011] One aspect of the present invention relates to a biosensor, which includes an insulating substrate, an electrode pair provided on the insulating substrate, and a reagent having a redox enzyme and an electron transfer substance placed on at least a working electrode in the electrode pair. The biosensor is used to measure a substance to be measured in a sample supplied to the electrode pair. The biosensor is characterized in that the working electrode is made of one or more nickel alloys selected from nickel-vanadium alloy, nickel-tungsten alloy, and nickel-ruthenium alloy.

[0012] Another aspect of the present invention relates to a method for measuring a substance to be measured, which includes the following steps: a step of supplying the sample containing the substance to be measured to the electrode pair of the biosensor; a step of applying a voltage between the electrode pair; a step of measuring the value of the current flowing between the electrode pair; and a step of calculating the amount of the substance to be measured based on the measured current value.

[0013] Effects of the Invention

[0014] According to the present invention, by using a specific nickel alloy in the working electrode of the biosensor, the responsiveness of the electron transfer substance close to that of a noble metal electrode can be obtained. Thereby, a biosensor capable of measuring a substance to be measured such as glucose with high reproducibility can be manufactured at low cost and simply. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a flowchart showing an example of a method for manufacturing a biosensor according to an embodiment of the present invention, and (a) to (f) show schematic views of the biosensor in each step.

[0016] Figure 2 shows Figure 1 a cross-sectional view of the cut portion along line b'-b' in the state of (b) in the biosensor manufacturing step.

[0017] Figure 3 is a schematic view showing one aspect of a measuring device equipped with the biosensor of the present invention.

[0018] Figure 4 is a flowchart showing one aspect of a measurement procedure using a measuring device equipped with the biosensor of the present invention.

[0019] Figure 5This is a graph showing the measurement results of peak current values when performing cyclic voltammetry on a biosensor that uses an electrode pair made of Au, Ni, or various Ni alloys and contains an electron transfer substance in the reagent. Measurements were carried out using electrodes with different concentrations of the electron transfer substance, and the peak current values were plotted. (A) shows the results when using a ruthenium complex as the electron transfer substance, and (B) shows the results when using ferricyanide as the electron transfer substance.

[0020] Figure 6 This is a graph showing the results of measurements using a biosensor with an electrode that uses various buffers (200 mM) in the reagent and investigating the relationship between glucose concentration and current value.

[0021] Figure 7 This is a graph showing the results of investigating Figure 6 the slope in the graph for the low glucose concentration range (0 - 67 mg / dl) and the high glucose concentration range (67 - 134 mg / dl).

[0022] Figure 8 This shows the results of investigating the response current values when changing the thicknesses of the Ni - V alloy and titanium in a biosensor where an electrode is formed of a Ni - V alloy on a contact metal made of titanium. *, ×, △, 〇 respectively indicate that the response current value drops suddenly within 5 seconds, the response current value rises midway within 5 seconds, there is a deviation in the response current value of 20 μA or more at the 5 - second value, and there is no problem. Detailed Description of the Invention

[0023] The present invention will be described below. It should be noted that for a numerical range described as "A - B", it means A or more and B or less.

[0024] (Biosensor)

[0025] The biosensor of the present invention includes a substrate, an electrode pair provided on the substrate, and a reagent placed on at least the working electrode in the electrode pair. This biosensor is used to measure a measurement target substance in a sample supplied to the electrode pair using the reagent, and is characterized in that

[0026] the working electrode is made of one or more nickel alloys selected from nickel - vanadium alloy (Ni - V alloy), nickel - tungsten alloy (Ni - W alloy), and nickel - ruthenium alloy (Ni - Ru alloy) (hereinafter sometimes also referred to as Ni alloy).

[0027] (Electrode Pair)

[0028] The electrode pair in the biosensor of the present invention includes a working electrode and a counter electrode, but the number of electrodes is not limited. For example, the biosensor of the present invention may also be a biosensor of a three-electrode system that includes a reference electrode in addition to the electrode pair including the working electrode and the counter electrode. The biosensor of the present invention may also be a biosensor that, in addition to having the above-mentioned electrode pair including the working electrode and the counter electrode (hereinafter sometimes referred to as the first electrode pair), also has an electrode pair including a working electrode and a counter electrode (hereinafter sometimes referred to as the second electrode pair) for measuring physical values other than the substance to be measured (values of other substances to be measured in the sample, temperature of the sample, etc.).

[0029] A nickel alloy can be used for the working electrode. In the present invention, one or more nickel alloys selected from the group consisting of nickel-vanadium alloys, nickel-tungsten alloys, and nickel-ruthenium alloys are used.

[0030] The ratio of nickel to vanadium in the nickel-vanadium alloy is preferably 80:20 to 95:5, more preferably 85:15 to 95:5, further preferably 90:10 to 94:6, and most preferably 92:8 by weight ratio.

[0031] The ratio of nickel to tungsten in the nickel-tungsten alloy is preferably 70:30 to 90:10, more preferably 75:25 to 85:15, further preferably 80:20 to 82:18, and most preferably 81:19 by weight ratio.

[0032] The ratio of nickel to ruthenium in the nickel-ruthenium alloy is preferably 60:40 to 40:60, more preferably 55:45 to 45:55, further preferably 52:48 to 48:52, and most preferably 50:50 by weight ratio.

[0033] It should be noted that the electrode material of the counter electrode is not particularly limited. For example, metal electrodes such as platinum and carbon electrodes can be used. It should be noted that as the counter electrode, a silver / silver chloride electrode can be used, or the above-mentioned nickel alloy electrode can also be used. From the aspects of low cost and easy fabrication, a nickel alloy the same as the working electrode is preferred.

[0034] In addition, the electrode materials of other electrodes such as the reference electrode are not particularly limited. For example, metal electrodes such as platinum, carbon electrodes, silver / silver chloride electrodes, standard hydrogen electrodes, calomel electrodes, palladium / hydrogen electrodes, etc. can be used. From the aspects of low cost and easy fabrication, a nickel alloy the same as the working electrode is preferably used.

[0035] It should be noted that in the case where the biosensor of the present invention is a biosensor having the above-mentioned first electrode pair and the above-mentioned second electrode pair, the electrode materials of the working electrode and the counter electrode in the second electrode pair can also be the electrode materials for the working electrode and the counter electrode described above respectively. From the aspects of low cost and easy fabrication, it is preferred that the working electrode and the counter electrode in the first electrode pair, and the working electrode and the counter electrode in the second electrode pair are all the same nickel alloy.

[0036] (Substrate)

[0037] The electrode including the electrode pair is provided on the substrate, and an insulating substrate is used as the substrate. The type of the insulating substrate is not particularly limited. For example, it is formed of various thermoplastic resins such as polyetherimide (PEI), polyethylene terephthalate (PET), and polyethylene (PE), various thermosetting resins such as polyimide resin and epoxy resin, insulating materials such as glass, ceramics, and paper. From the aspect of improving the fixing force of the electrode or the contact metal described later on the substrate, a material with an arithmetic mean roughness Ra value of 1 μm or more on the surface is preferred. In the case of a substrate with an Ra value less than 1 μm, since the surface is flat, the adhesiveness of the electrode or the contact metal is reduced. It should be noted that the size and thickness of the electrode and the substrate can be appropriately set, but it is particularly preferred that the thickness of the electrode is 1 μm to 100 μm.

[0038] (Contact metal)

[0039] The electrode layer including the electrode pair is laminated on the substrate, but it is preferred to laminate and provide a contact metal between the substrate and the electrode. When a nickel alloy is provided on an insulating substrate, when a force is applied to the biosensor from the outside or a voltage is applied, the nickel alloy may peel off from the insulating substrate or pores may be generated in the nickel alloy. Therefore, in order to improve the adhesion of the nickel alloy to the insulating substrate, it is preferred to provide a contact metal.

[0040] In the biosensor of the present invention, when only the working electrode is formed of a nickel alloy, it is only necessary to laminate and provide a contact metal between the working electrode and the substrate. When both the working electrode and the counter electrode are formed of a nickel alloy, it is preferred to laminate and provide a contact metal between the working electrode and the substrate and between the counter electrode and the substrate.

[0041] Regarding the type of the contact metal, there is no particular limitation as long as it can firmly fix the nickel alloy constituting the electrode. For example, it is formed of any one of metals such as titanium, molybdenum, tungsten, chromium, and iron, and it is preferably formed of titanium. It should be noted that the thickness of the contact metal can be appropriately set, and it is preferably 5 μm to 100 μm from the aspect of the fixing force. In addition, regarding the total thickness of the nickel alloy and the contact metal, in order not to be affected by the unevenness of the substrate, it is preferably 10 μm to 55 μm.

[0042] When the electrode is a nickel - vanadium alloy and the contact metal is titanium, when the thickness of titanium is x and that of the nickel - vanadium alloy is y, it is preferably within the following ranges.

[0043] 1) 7μm ≤ x ≤ 52μm

[0044] 2) 3μm ≤ y ≤ 25μm

[0045] 3) y = -11 / 16x + 155 / 4

[0046] (Reagent)

[0047] In the biosensor of the present invention, the reagent is placed on at least the working electrode in the pair of electrodes.

[0048] The position where the reagent is placed on the working electrode is not particularly limited, and it is preferably the other end or the vicinity thereof different from the end connected to the power source for applying a voltage to the working electrode.

[0049] It should be noted that the reagent can also be placed on both the working electrode and the counter electrode constituting the pair of electrodes. In addition, it can be further placed on the substrate around the working electrode or the working electrode and the counter electrode, or on the substrate between the working electrode and the counter electrode. In addition, the reagent is preferably continuously placed on the working electrode and is preferably placed in a layer structure with a thickness. It should be noted that in the biosensor of the present invention, in addition to the above - mentioned first pair of electrodes including the working electrode and the counter electrode for measuring the analyte, there is also a second pair of electrodes including the working electrode and the counter electrode for measuring a physical value other than the analyte. In this case, the reagent can be placed on the second pair of electrodes or not. For example, when the physical value other than the analyte is the hematocrit value, it can be configured such that only a part of the reagent is placed on the counter electrode as disclosed in WO 2005 / 103669, or it can be configured not to place the reagent as disclosed in JP 2019 - 035748.

[0050] The reagent contained in the reagent is not particularly limited as long as it is a reagent used in the detection reaction of the analyte, and at least contains an oxidoreductase and an electron transfer substance.

[0051] (Oxidoreductase)

[0052] The oxidoreductase is an enzyme that can oxidize and reduce the analyte using the analyte as a substrate, and examples thereof include oxidoreductases containing at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as a catalytic subunit and a catalytic domain.

[0053] When the substance to be measured is glucose, for example, glucose dehydrogenase or glucose oxidase can be used. As glucose dehydrogenase, specifically, pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) and cytochrome glucose dehydrogenase (CyGDH) having an α subunit containing FAD can be cited. In addition, glucose-3-dehydrogenase derived from Agrobacterium tumefasience can also be cited.

[0054] In addition, the oxidoreductase may include an electron transfer subunit or an electron transfer domain. When the substance to be measured is glucose, as the electron transfer subunit, for example, a subunit having a heme with an electron transfer function can be cited. As the oxidoreductase containing the subunit having heme, an oxidoreductase containing cytochrome can be cited. For example, a glucose dehydrogenase containing cytochrome, a fusion protein of PQQGDH and cytochrome can be applied. It should be noted that the fusion protein of PQQGDH and cytochrome is disclosed, for example, in International Publication No. 2005 / 030807.

[0055] In addition, in the case of a substance to be measured other than glucose, as the oxidoreductase, cholesterol oxidase, quinone heme ethanol dehydrogenase (QHEDH(PQQ Ethanol dh)), sorbitol dehydrogenase (SorbitolDH), D-fructose dehydrogenase (Fructose DH), cellobiose dehydrogenase, lactate dehydrogenase, uricase, etc. can be cited.

[0056] Therefore, the biosensor of the present invention can be used not only as a glucose sensor but also as a cholesterol sensor, an ethanol sensor, a sorbitol sensor, a fructose sensor, a cellobiose sensor, a lactate sensor, a uric acid sensor, etc.

[0057] (Electron transfer substance)

[0058] The electron transfer substance contained in the reagent is a substance that can receive the electrons generated by the reaction of the substance to be measured and the oxidoreductase and then transfer the electrons to the electrode, and is also called a mediator.

[0059] As the electron transfer substance, any compound that can be reduced by receiving electrons from an oxidoreductase, re-oxidized on the electrode, and has no catalytic activity can be used. For example, ruthenium complexes, iron cyanides such as potassium ferrocyanide (also known as hexacyanoferrate(III)), quinone compounds (e.g., 1,4-naphthoquinone, VK3, 9,10-phenanthrenequinone, 1,2-naphthoquinone, dimethyl-p-benzoquinone, methylbenzoquinone, 2,6-dimethylbenzoquinone, sodium 1,2-naphthoquinone-4-sulfonate, 1,4-anthraquinone, tetramethyl-p-benzoquinone, thymolquinone), phenylenediamine compounds (e.g., N,N-dimethyl-1,4-phenylenediamine, N,N,N’,N’-tetramethyl-1,4-phenylenediamine dihydrochloride), 1-methoxy-PMS (1-methoxy-5-methylphenazinium methyl sulfate), coenzyme Q0, azure A chloride, phenosafranine, 6-aminoquinoxaline, tetrathiafulvalene, etc. They can be used alone or in combination of two or more.

[0060] It should be noted that as the ruthenium complex, a ruthenium complex composed of trivalent ruthenium (Ru(III)) and a ligand is preferred, and the following ruthenium ammonia complex is more preferred.

[0061] [Ru(NH 3 ) 5 X] n+

[0062] Here, as X, NH 3 , halide ions, CN, pyridine, nicotinamide, bipyridine or H 2 O, etc. Among these, NH 3 or halide ions (e.g., Cl-, F - , Br - , I - ) are preferred. The n+ in the above chemical formula represents the valence of the oxidized ruthenium(III) complex, which is appropriately determined according to the type of X. It should be noted that the detailed content of the ruthenium complex is disclosed in JP-A-2018-013400.

[0063] The content of the oxidoreductase in the reagent of the biosensor of the present invention can be appropriately determined according to the type of the substance to be measured, but it is necessary to contain a sufficient amount of oxidoreductase relative to the substance to be measured. Therefore, the amount of oxidoreductase per 1 cm 2 surface area of the part of the biosensor on which the reagent is placed is preferably 1 to 10 U, more preferably 1 to 5 U, and particularly preferably 1 to 3 U.

[0064] The content of the electron transfer substance in the reagent is preferably more than the content of the oxidoreductase and can be appropriately determined according to the type of the measurement sample, etc. For example, relative to each 1 cm 2The surface area is preferably 10 mmol to 100 mmol, more preferably 10 mmol to 50 mmol, and particularly preferably 15 mmol to 20 mmol.

[0065] (Other components)

[0066] The reagent contains an oxidoreductase and an electron transfer substance. In addition to these, it may also contain resin binders such as butyral resin-based and polyester resin-based binders, binders such as the layered inorganic compounds disclosed in Japanese Laid-Open No. 2005 / 043146, surfactants, and the like.

[0067] In addition, the reagent may additionally contain additives such as a buffer and a surfactant. As the buffer, amine-based buffers such as Tris, ACES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, TES, tris(hydroxymethyl)methylglycine (Tricine), and ADA can be used, and buffers having a carboxyl group such as a phosphate buffer, a citrate buffer, a phosphate-citrate buffer, an acetic acid-sodium acetate buffer, a malic acid-sodium acetate buffer, a malonic acid-sodium acetate buffer, and a succinic acid-sodium acetate buffer can also be used. The pH of the buffer is preferably 6.8 to 7.2, and more preferably about 7.0.

[0068] Among them, a citrate buffer and a phosphate-citrate buffer are preferably used.

[0069] Regarding the molar concentration of citric acid in the citrate buffer or the phosphate-citrate buffer in the reagent, it is preferably 1.5 μmol / cm 2 to 22 μmol / cm 2 on the part of the working electrode where the reagent is placed, and particularly preferably 7.5 μmol / cm 2 to 15 μmol / cm 2 . When a sample is supplied to the electrode pair of such a biosensor and the reagent is dissolved by the above sample, regarding the concentration of citric acid before diffusion on the working electrode in the mixed solution of the above reagent and the above sample, it is about 20 mM to about 300 mM in the case of a reagent containing citric acid 1.5 μmol / cm 2 to 22 μmol / cm 2 , and it is about 20 mM to about 300 mM in the case of a reagent containing citric acid 7.5 μmol / cm 2 to 15 μmol / cm 2In the case of the reagent, it is about 100 mM to about 200 mM. It should be noted that the mixed solution of the reagent and the sample on the working electrode includes the mixed solution physically present on the working electrode, and is the solution in the region on the working electrode where the reagent reacts with the substance to be measured. In addition, the above-mentioned mixed solution is a fluid and there is also a concentration gradient, so the numerical value of the citric acid concentration mentioned here refers to the average concentration.

[0070] Examples of the surfactant include Triton X-100, sodium dodecyl sulfate, perfluorooctanesulfonic acid or sodium stearate, alkylaminocarboxylic acid (or its salt), carboxybetaine, sulfobetaine, and phosphobetaine.

[0071] It should be noted that in the biosensor, in addition to the substrate, contact metal, electrode pair, and reagent, it may also include a cover covering the electrode provided on the surface side of the substrate having the electrode pair, a spacer provided between the substrate and the cover for forming a predetermined space between the substrate and the cover, a sample supply portion having a predetermined space provided in a region of the reagent placement portion including at least the working electrode in the electrode pair, and the like. The above-mentioned sample supply portion can be formed by processing the above-mentioned cover or spacer.

[0072] It should be noted that the biosensor may also include components other than the above.

[0073] Below, based on Figure 1 An example of the biosensor applicable to the present invention will be described.

[0074] Figure 1 (a) to (f) are perspective views showing a series of steps for manufacturing a biosensor. It should be noted that the biosensor applicable to the present invention is not limited to the following method. For example, a contact metal is used in the following method, but the contact metal is not an essential component in the biosensor of the present invention.

[0075] As Figure 1 (f) shows, the biosensor A includes: a substrate 10; an electrode system composed of a counter electrode 11 and a working electrode 12. The counter electrode 11 has a first end portion, a second end portion connected to a measurement device described later, and a lead portion connecting the first end portion and the second end portion. The working electrode 12 has a first end portion adjacent to the first end portion of the counter electrode 11, a second end portion connected to a measurement device described later, and a lead portion connecting the first end portion and the second end portion ( Figure 1 (b)); a contact metal 19 formed between the substrate 10 and the electrode system ( Figure 1 (a)); an insulating layer 14; a spacer 15 having an opening; and a cover 16 having a through hole 18. As Figure 1As shown in (c), a reagent 13 is placed on the first end portion of the working electrode 12. And, as Figure 1 shown in (d), an insulating layer 14 is laminated on the lead-out portion of the electrode pair on the substrate 10. And, as Figure 1 shown in (e), a spacer 15 is disposed on the insulating layer 14, and an opening is formed in a portion of the spacer 15 corresponding to the first end portion (including the portion with the reagent) of the working electrode 12 and the region including the first end portion of the counter electrode opposed thereto. A cover 16 is further disposed on the spacer 15, and the cover 16 has a through hole 18 in a part corresponding to the above opening ( Figure 1 (f)). In this biosensor, a sample supply portion 17 having a capillary structure is formed in a space portion, which is the space portion of the above opening portion and is sandwiched by the working electrode, the counter electrode, the insulating layer 14, and the cover 16. The sample supply portion 17 is formed as a prescribed space in a region of a reagent placement portion including at least the first end portion of the working electrode in the electrode pair. And, the above through hole 18 serves as an air hole for sucking a sample by capillary action. It should be noted that the following biosensor A may also be used: the insulating layer 14 is not provided, the spacer 15 is formed of an insulating member and has the function of the insulating layer 14, and the space portion sandwiched by the spacer 15 and the cover 16 becomes the sample supply portion 17 having a capillary structure.

[0076] (Method for manufacturing a biosensor)

[0077] The biosensor of the present invention is manufactured, for example, as follows. That is, on one side of an insulating substrate having an arithmetic mean roughness Ra value of about 2 μm on the surface, a contact metal is laminated by sputtering under the conditions of a film formation chamber pressure of 0.1 to 0.5 Pa, an argon flow rate of 160 to 300 Sccm, and 7800 W. Then, a metal layer that functions as a working electrode and a counter electrode is formed by sputtering under the conditions of a film formation chamber pressure of 0.1 to 0.5 Pa, an argon flow rate of 160 to 300 Sccm, and 8000 W, respectively. Then, a reagent preparation solution is applied on the electrode (at least on the first end portion of the working electrode), and dried, whereby the reagent can be placed on the electrode.

[0078] The following shows a more specific method for manufacturing a biosensor, but the method for manufacturing the biosensor of the present invention is not limited to the following.

[0079] First, as Figure 1 shown in (a), a contact metal 19 is applied on the substrate 10 under the above conditions by physical vapor deposition (PVD) based on sputtering. Then, as Figure 1As shown in (b), an electrode system composed of a counter electrode 11 having a first end, a second end, and a lead-out portion and a working electrode 12 having a first end, a second end, and a lead-out portion is deposited on the upper layer of the contact metal 19 under the above conditions by sputtering. The working electrode 12 is formed by depositing the above nickel alloy on the contact metal.

[0080] Figure 2 Shown Figure 1 A cross-sectional view of the cut portion along the line b'-b' in (b). A contact metal 19 is deposited on the substrate 10, and a working electrode 12 and a counter electrode 11 are respectively deposited on the contact metal 19.

[0081] It should be noted that, in addition to sputtering, the contact metal or the electrode material can also be coated on the substrate by means such as film plating, other physical vapor deposition methods, and chemical vapor deposition (CVD) film formation. Then, the electrode system can be formed by performing drilling using a laser or etching using a mask. For example, a contact metal layer can be formed on an insulating substrate by physical vapor deposition, a nickel alloy layer can be formed on the contact metal layer by physical vapor deposition, and the contact metal and the nickel alloy can be partially removed from the insulating substrate, thereby forming an electrode group including an electrode pair.

[0082] In addition, the working electrode 12 can also be formed by printing a nickel alloy on the substrate using screen printing or the like.

[0083] Regarding the counter electrode 11, it can also be formed by simultaneously coating the counter electrode material on the substrate using the same method and performing drilling or etching, or by printing the counter electrode material on the substrate using screen printing or the like. It should be noted that, in the case of having other electrodes such as a reference electrode, they can also be formed together.

[0084] Next, as Figure 1 As shown in (c), a reagent is placed on a part of the working electrode 12, preferably on the first end of the working electrode 12. The method of placing the reagent is not particularly limited. For example, it can be carried out by dropping a solution of the reagent onto a part of the working electrode 12, preferably onto the first end of the working electrode 12 and drying it. More specifically, the reagent 13 can be formed, for example, by preparing a dispersion liquid in which a redox enzyme, an electron transfer substance, and a buffer and a binder if necessary are dispersed, dispensing it onto the working electrode 12 and drying it. As the solvent used in the preparation of the above dispersion liquid, for example, water, buffer alcohol, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), etc. can be used.

[0085] Next, as Figure 1As shown in Fig. (d), an insulating layer 14 is formed on the substrate 10 on which the above-described electrode pair 11, 12 is formed. This insulating layer is formed on the lead-out portions of the electrode pair 11, 12. In other words, it is formed on the substrate 10 except for the reagent-containing portion of the first end of the working electrode 12 and the portion of the counter electrode opposite thereto that includes the first end. The above-described insulating layer 14 can be formed, for example, by printing an insulating paste obtained by dissolving an insulating resin in a solvent on the above-described substrate 10 and then performing heat treatment or ultraviolet treatment thereon. As the insulating resin, for example, polyester, butyral resin, phenolic resin, etc. can be cited, and as the above-described solvent, for example, carbitol acetate, dibasic acid ester-based mixed solvent (DBE solvent), etc. can be cited.

[0086] Next, as Figure 1 shown in Fig. (e), a spacer 15 is disposed on the insulating layer 14. As shown in the figure, the portion of the spacer 15 corresponding to the reagent-containing portion of the first end of the above-described working electrode 12 and the portion of the counter electrode opposite thereto that includes the first end is formed as an opening. As the material of the spacer 15, for example, a resin film, tape, etc. can be used. In addition, if it is a double-sided tape, it can not only be adhered to the above-described insulating layer 14, but also can be easily adhered to the cover 16 described later. In addition to this, the spacer can also be formed by means such as resist printing.

[0087] Next, as Figure 1 shown in Fig. (f), a cover 16 is disposed on the above-described spacer 15. There is no particular limitation on the material of the above-described cover 16. For example, various plastics, etc. can be used, and preferably, transparent resins such as PET can be cited. It should be noted that a hydrophilic agent (such as cinnoline, etc.) can also be coated on a transparent resin such as PET.

[0088] (Use of the biosensor)

[0089] The biosensor of the present invention can be suitably used for the determination of a substance to be measured in a sample.

[0090] (Sample)

[0091] The sample is not particularly limited as long as it is a sample containing a substance to be measured, and preferably a biological sample, and examples thereof include blood, urine, etc.

[0092] (Substance to be measured)

[0093] Examples of the substance to be measured include glucose, cholesterol, ethanol, sorbitol, fructose, cellobiose, lactic acid, uric acid, etc.

[0094] (Measurement principle)

[0095] When a sample containing a substance to be measured is brought into contact with the reagent placement part at the first end of the working electrode of the biosensor of the present invention, the substance to be measured reacts with the reagent. After the reaction, a voltage is applied between the electrode pair, thereby generating a signal based on the reaction. By detecting this signal, the substance to be measured can be measured.

[0096] Specifically, when the reagent of the biosensor contains a redox enzyme and an electron transfer substance, the electrons generated by the oxidation reaction or reduction reaction of the redox enzyme with the substance to be measured are transferred to the electron transfer substance, and the electron transfer substance is reduced. Then, by applying a voltage between the electrode pair, the reduced electron transfer substance is oxidized on the surface of the working electrode, thereby generating an oxidation current depending on the amount of the substance to be measured in the sample. By measuring this current value, the concentration of the substance to be measured in the sample can be measured based on this current value.

[0097] When calculating the concentration of the substance to be measured, voltammetry, amperometry, coulometry, etc. are used. Voltammetry is the following method: the voltage applied to the electrode is changed, the graph of the changing response current value is measured, and the concentration of the substance to be measured is calculated based on the current peak value, etc. in this graph. Amperometry is the following method: a certain voltage is applied to the electrode, the response current value after a certain time from the start of the reaction is obtained, and the concentration of the substance to be measured is calculated based on this response current value. Coulometry is the following method: a certain voltage is applied to the electrode, after almost all of the substance to be measured in the sample has reacted, the cumulative value of the response current value is obtained, and the concentration of the substance to be measured is calculated based on the cumulative value.

[0098] (Method for measuring a substance to be measured)

[0099] The method for measuring a substance to be measured of the present invention includes the following steps:

[0100] A step of supplying the above sample containing the above substance to be measured to the above electrode pair of the biosensor;

[0101] A step of applying a voltage between the electrode pair;

[0102] A step of measuring the value of the current flowing between the electrode pair; and

[0103] A step of calculating the amount of the above substance to be measured based on the measured current value.

[0104] (Sample supply step)

[0105] The supply of the sample to the above electrode pair of the biosensor can be carried out from a container containing the sample using known supply means such as a microchip, a syringe, a capillary tube, etc. The supply of the sample can also be carried out by an automated supply means.

[0106] It should be noted that when the biosensor has the above-described specimen supply unit, by supplying the specimen to the specimen supply unit, the specimen can effectively react with the biosensor.

[0107] (Voltage application step)

[0108] Next, a voltage is applied between the pair of electrodes.

[0109] In the case of amperometry or coulometry, a certain voltage is applied between the pair of electrodes.

[0110] Here, as the voltage applied to the working electrode 12, it suffices that it is a positive voltage with respect to the counter electrode, and it can be appropriately set. For example, it is +50 to +500 mV, preferably +100 to +250 mV, and particularly preferably +200 mV with respect to the counter electrode. When it is in the above voltage range, elution from the base material of the nickel alloy electrode can be suppressed, the stability of the electrode can be maintained, electrode elution during voltage application can be prevented, and thus the background current can be reduced and it can be used for a long time.

[0111] After the specimen is brought into contact with the reagent, the voltage can be applied to the electrode system after maintaining a prescribed time in a state where no voltage is applied, or the voltage can be applied to the electrode system while the above specimen is in contact with the reagent. As the holding time when holding in a state where no voltage is applied, for example, it is 30 seconds or less or 10 seconds or less. On the other hand, in the case of performing cyclic voltammetry measurement, the potential of the working electrode is scanned at a constant speed. The cycle of potential scanning can be multiple cycles.

[0112] It should be noted that when using the biosensor including the first pair of electrodes and the second pair of electrodes as described above, the voltage applied to the working electrode of the second pair of electrodes and the voltage applied to the working electrode of the above first pair of electrodes can be different voltages. For example, when using the biosensor having an electrode pair configuration in which only a part of the reagent is placed on the counter electrode as disclosed in the above WO2005 / 103669, or a biosensor having an electrode pair configuration in which no reagent is placed as disclosed in Japanese Patent Application Laid-Open No. 2019-035748 to measure the hematocrit value, which is a physical value other than the measurement target substance, sometimes a voltage +1.0 to +7.0 V higher than the counter electrode of the second pair of electrodes is applied to the working electrode of the second pair of electrodes.

[0113] (Current measurement step)

[0114] Regarding current measurement, in the case of amperometry, in order to measure the response current value after applying a voltage for a certain period of time, and in the case of coulometry, in order to obtain the integrated value of the response current, the response current value is measured over time. On the other hand, in the case of cyclic voltammetry, in order to obtain the cyclic voltammetry waveform, the current value corresponding to the voltage during scanning is continuously measured. The measurement of the response current value can be performed using a normal ammeter or the like.

[0115] (Concentration calculation step)

[0116] Based on the measured response current value, the concentration of the substance to be measured is calculated.

[0117] In the case of amperometry, the relationship between the response current value and the concentration of the substance to be measured can be obtained in advance using a calibration curve or the like, and the measured response current value is substituted into the calibration curve to calculate the concentration of the substance to be measured.

[0118] In the case of coulometry, the relationship between the integrated value of the response current value and the concentration of the substance to be measured can be obtained in advance using a calibration curve or the like, and the integrated value of the measured response current value is substituted into the calibration curve to calculate the concentration of the substance to be measured.

[0119] In addition, in the case of cyclic voltammetry, the relationship between the peak value in the cyclic voltammetry waveform and the concentration of the substance to be measured can be obtained in advance using a calibration curve or the like, and the measured response current value is substituted into the calibration curve to calculate the concentration of the substance to be measured.

[0120] (Measurement device)

[0121] According to one aspect of the present invention, the biosensor of the present invention is assembled into a measurement device for use. The measurement device may include a control unit 22 for controlling the potential, a potentiostat 25, a detection unit 24 for measuring current, an arithmetic unit 23 based on the current value, an output unit 21 for outputting the measurement result, and two or more connection terminals 26, etc. It may further include a power supply unit for applying a voltage to the electrode pair of the biosensor.

[0122] Next, an aspect of the measurement device using the biosensor of the present invention will be described with reference to the drawings. Here, an aspect of the measurement device equipped with a glucose sensor is illustrated, but the measurement device equipped with the biosensor of the present invention is not limited to the following aspects.

[0123] Figure 3Shows a configuration example of the measurement device B. The measurement device B includes a control computer 20 and a potentiostat 25. When the biosensor A is installed, each connection terminal 26 is electrically connected to the corresponding second end of the working electrode and the counter electrode of the biosensor. The control computer 20 includes a processor such as a CPU (Central Processing Unit) in terms of hardware, a recording medium such as a memory (RAM (Random Access Memory), ROM (Read Only Memory)), and a communication unit. The processor loads the program stored in the recording medium (such as ROM) into the RAM and executes it, thereby functioning as a device having an output unit 21, a control unit 22, an arithmetic unit 23, and a detection unit 24.

[0124] The control unit 22 controls the timing of voltage application, the applied voltage value, etc. The potentiostat 25 is a device that keeps the potential of the working electrode constant relative to the counter electrode, is controlled by the control unit 22, applies a specified voltage (50 mV to 500 mV) between the working electrode and the counter electrode of the biosensor A, measures the response current of the working electrode, and sends the measurement result of the response current to the detection unit 24.

[0125] The arithmetic unit 23 calculates and stores the concentration of the measurement target substance from the detected current value. The output unit 21 performs data communication with a display unit (not shown) and sends the calculation result of the concentration of the measurement target substance obtained by the arithmetic unit 23 to the display unit.

[0126] Next, an example is given where the sample is whole blood, the measurement target substance is glucose, the redox enzyme is glucose dehydrogenase, and the electron transfer substance is a ruthenium (III) compound to illustrate one mode of the usage method of this measurement device.

[0127] Figure 4 Is a flowchart showing an example of the glucose concentration measurement process performed using the measurement device.

[0128] First, the biosensor A is installed in the measurement device. Then, a sample containing the measurement target substance is supplied to the electrode pair of the biosensor A (sample supply step).

[0129] Specifically, for example, a whole blood sample is brought into contact with one end of the sample supply unit 17 of the biosensor A. The sample supply unit 17 is formed into a capillary structure as described above, and an air hole 18 is provided in the cover 16 at a position corresponding to the other end. Therefore, the above-mentioned sample is sucked into the interior by capillary action. The sucked sample reaches the surface of the reagent on the working electrode 12 provided in the detection unit 13. After that, glucose in the sample that reaches the surface preferentially reacts with glucose dehydrogenase and is converted into gluconolactone. By the electrons generated at this time, the ruthenium(III) compound is reduced to generate a ruthenium(II) compound.

[0130] Next, a voltage is applied between the electrode pair (voltage application step).

[0131] Specifically, for example, when the CPU (control unit 22) of the control computer 20 receives an instruction to start glucose concentration measurement, the control unit 22 controls the potentiostat 25 to apply a predetermined voltage to the working electrode and starts the measurement (step S01). It should be noted that it can also be an instruction to start concentration measurement.

[0132] By applying a positive voltage to the counter electrode, electron transfer occurs between the ruthenium(II) compound present in the reagent and the electrode, and a response current flows. Therefore, this current is detected (response current measurement step).

[0133] Specifically, for example, the potentiostat 25 measures the response current obtained by voltage application, that is, the current generated based on the movement of electrons from the measurement target substance (glucose) in the sample to the electrode. For example, it measures the steady-state current 1 to 20 seconds after voltage application and sends it to the detection unit 24 (step S02).

[0134] The value of this oxidation current is proportional to the concentration of glucose in the sample. Therefore, if it is calculated as the glucose concentration by using the above-mentioned calculation means, the glucose concentration in the sample can be calculated (concentration calculation step).

[0135] Specifically, for example, the arithmetic unit 23 performs arithmetic processing based on the current value and calculates the glucose concentration (step S03). For example, the arithmetic unit 23 of the control computer 20 previously stores a calculation formula for glucose concentration or calibration curve data of glucose concentration, and uses these calculation formulas or calibration curves to convert the current value into the glucose concentration and calculates the glucose concentration.

[0136] The output unit 21 sends the calculation result of the glucose concentration to the display unit 25 through the communication link formed between it and the display unit 25 (step S04). In addition, the calculation result can also be saved in the arithmetic unit 23, and the calculation result can be retrieved later, displayed on the display unit and confirmed.

[0137] Examples

[0138] The following examples are given to illustrate the present invention more specifically, but the present invention is not limited to the following embodiments.

[0139] (Example 1)

[0140] To compare the performance of the current responsiveness of various metals of each electrode, a simple biosensor was used which was fabricated through the following process as compared with the Figure 1 biosensor shown. First, as the insulating substrate 10 of the glucose sensor, a PET substrate (length 50 mm, width 6 mm, thickness 250 μm) was prepared, and various metals (gold (Au), nickel (Ni), Ni-V (92:8), Ni-W (81:19), Ni-Ru (50:50)) were sputtered on the entire surface of one of its surfaces, and it was trimmed, thereby forming a working electrode 12 having a first end portion, a second end portion, and a lead-out portion, and a counter electrode 11 having a first end portion, a second end portion, and a lead-out portion.

[0141] Only a ruthenium compound ([Ru(NH 3 ) 6 Cl 3 , manufactured by Dojindo Laboratories Co., Ltd.) or potassium ferricyanide, any one of the electron transfer substances, was placed on the working electrode 12. That is, 0.45 μL of the above electron transfer substance was dispensed on the first end portion of the working electrode 12. Then it was dried at 30 °C, thereby placing the electron transfer substance.

[0142] Next, a spacer was placed on the above electrode pair by means of tape to form a prescribed space. That is, since a capillary structure was formed in the prescribed space of the opening portion of the spacer, it was used as a sample supply portion.

[0143] It should be noted that glucose oxidoreductase can be used as a reagent, and in this case, glucose dehydrogenase can be used. When using glucose dehydrogenase, the preparation solution for preparing the reagent was prepared as follows. First, glucose dehydrogenase (3.0 units / strip) was added to 10 mM or 50 mM ruthenium compound ([Ru(NH 3 ) 6 Cl 3 , manufactured by Dojindo Laboratories Co., Ltd.) or 10 mM or 50 mM potassium ferricyanide, and it was used as the preparation solution for preparing the reagent.

[0144] <Cyclic Voltammetry Measurement>

[0145] For a biosensor fabricated as described above, which contains an electron transfer substance in a reagent and has an electrode (working electrode) made of Au, Ni, or various Ni alloys, the electro-responsiveness of the electron transfer substance is measured by investigating the cyclic voltammetry waveform, and the electrode response characteristics are evaluated. The cyclic voltammetry waveform is investigated as follows: After introducing a sample (100 mM PBS (phosphate buffered saline) pH 7.0) into the sample supply section of the glucose sensor, the scanning speed is set to 20 mV / sec, and the voltage applied to the working electrode is scanned from -400 mV to +400 mV (in the case of ruthenium complex) or from -800 mV to +800 mV (in the case of ferricyanide) with respect to the counter electrode for 3 cycles, and the response current during scanning is measured for investigation. Figure 5 The results show the peak current values measured for the first, second, and third cycles for each biosensor and the average values calculated therefrom.

[0146] The results indicate that when any of the Ni-V electrode, Ni-W electrode, and Ni-Ru electrode is used as the working electrode, although it is inferior to the Au electrode, it still shows better current responsiveness than the Ni electrode. Therefore, according to the present invention, by using any of the Ni-V electrode, Ni-W electrode, and Ni-Ru electrode as the working electrode in the biosensor, the responsiveness of the electron transfer substance close to that of the noble metal electrode can be obtained. Thus, by using the above-mentioned nickel alloy, a biosensor capable of measuring a substance to be measured such as glucose with high reproducibility can be fabricated at low cost and simply.

[0147] (Example 2)

[0148] Next, the influence of the buffer used in the reagent of the electrode on the background was studied. In order to compare the performance of the buffers, a biosensor was used which was simply the same as the biosensor of Example 1 except that the reagent (electron transfer substance and redox enzyme) was not placed on the first end of the working electrode 12 in the detection unit 13. That is, for the Ni-V electrode, Ni-W electrode or Ni electrode, cyclic voltammetry (0 → +1.0 V) was performed using citric acid (final concentration 20 mM, 50 mM, 100 mM or 200 mM) as a buffer in the sample, and the current value at +0.5 V (this voltage is a voltage at which the characteristics are easily grasped) was measured. As a comparative example, phosphoric acid (final concentration 20 mM, 50 mM, 100 mM or 200 mM) was used as a buffer in the sample, and the current value was measured using the same measurement method. In this experiment, only the buffer was present on the electrode in the detection unit 13, so the current value obtained by applying a voltage was only attributable to the current value caused by the solution containing only the buffer. Therefore, the influence of each buffer on the background, that is, the noise component, can be evaluated. The results are shown in Table 1. From the results, it can be seen that the background current value can be reduced when using a citric acid buffer compared to the case of using a phosphoric acid buffer, and this tendency is significant in the Ni-V electrode. One of the reasons for the background current is considered to be the corrosion of the electrode caused by the application of voltage. The reason is that since Ni and V (vanadium) are base metals, they are easily corroded. It is considered that by using a citric acid buffer, citric acid forms a complex with Ni, inhibits the destruction of the passivation film, and reduces the background current value. It should be noted that since it depends on the concentration of citric acid, the same effect can also be obtained in the case of using a phosphoric acid-citric acid buffer. In addition, the higher the concentration, the lower the background current value. Therefore, it can be seen that the same effect can be obtained even when the citric acid concentration is higher than 200 mM, for example, even when it is 200 mM to 300 mM.

[0149] [Table 1] Relationship between citric acid or phosphoric acid concentration and background current value (μA) in various electrodes

[0150] (Example 3)

[0151] Next, the reproducibility in glucose concentration measurement was evaluated. A biosensor was used which was the same as the biosensor of Example 1 except that the electrode was only the Ni-V electrode and the reagent having the composition shown in Table 2 below was placed on the first end of the working electrode (the working electrode has a Ni-V electrode) Figure 1In the biosensor shown, the evaluation of detection sensitivity based on different buffers was implemented in the Ni-V electrode. Phosphoric acid, citric acid, phosphoric acid-citric acid, PIPES, and HEPES (pH 7.0 respectively) were used as buffers, and adjusted to a concentration of approximately 100 mM in the mixed solution of the reagent and the sample on the working electrode when dissolving the reagent with each sample. In the case of citric acid and phosphoric acid-citric acid, the amount of citric acid present in each strip was 17.2 μg. When converted to the amount of substance of citric acid, it corresponded to 90 nmol, and the part of the working electrode where the reagent was placed was 0.006 cm 2 , so the molar concentration of citric acid was equivalent to 15 μmol / cm 2 . Using such a biosensor, with the measuring device shown in Figure 3 , venous whole blood samples were used to make the glucose concentration 0 mg / dL, 67 mg / dL, 134 mg / dL, 336 mg / dL, 600 mg / dL, and 1000 mg / dL for measurement. A voltage of +0.2 V was applied to the working electrode, and the current value was measured 8 seconds after the voltage application. The results are shown in Figure 6 .

[0152] [Table 2] Reagent composition of Example 3

[0153]

[0154] From the results, it can be seen that in the case of using electrodes with citric acid buffer and phosphoric acid-citric acid buffer, high response current values corresponding to the glucose concentration were shown in the same way as in the case of phosphoric acid, and the linearity of the response current value was good. On the other hand, when using PIPES and HEPES, the linearity of the response current value with respect to the glucose concentration deteriorated, so it can be known that the reproducibility in glucose concentration measurement deteriorated significantly. The reason for this phenomenon is that in the case of using reagents of PIPES and HEPES, the solubility in the sample decreased.

[0155] Next, using the same data, the slope of the calibration curve was observed by dividing it into a low glucose concentration range (Glu0 - Glu67) and a medium glucose concentration range (Glu67 - Glu134), and the results are shown in Figure 7 . Figure 7 On the horizontal axis of, the low glucose concentration range and the medium glucose concentration range of phosphoric acid, citric acid, phosphoric acid-citric acid, PIPES, and HEPES buffers were arranged respectively, and on the vertical axis, the slopes of the respective calibration curves were arranged. The greater the slope of the calibration curve, the higher the reproducibility of the glucose concentration conversion value, indicating high detection sensitivity. As shown in Figure 7As shown, it can be seen that in the case of using an electrode containing a citrate buffer and a phosphocitrate buffer in a reagent, in the low concentration range, the slope is greater than that of phosphoric acid, and in the low concentration range and the medium concentration range, the slope is greater than that of PIPES and HEPES. Therefore, it can be known that the electrode containing a citrate buffer and a phosphocitrate buffer in the reagent has high detection sensitivity in the low glucose concentration range.

[0156] In addition, using the same biosensor, samples (whole blood) with glucose concentrations of 67 mg / dl, 134 mg / dl, and 336 mg / dl were measured (n = 10), and the coefficient of variation (CV) was calculated for each. The results are shown in Table 3. From the results, it can be seen that the biosensor with an electrode using a citrate buffer and a phosphocitrate buffer has good reproducibility of the measured values in the low glucose concentration region.

[0157] [Table 3] Relationship between the type of buffer and CV (%)

[0158] Glu67 Glu134 Glu336 Phosphate 2.10 1.87 1.74 PIPES 15.99 16.67 11.97 HEPES 9.46 21.25 10.77 Citric acid 0.93 2.55 2.81 Phosphocitric acid 1.22 1.87 1.94

[0159] It should be noted that in the case of using PIPES and HEPES, it can be confirmed from an experiment similar to Example 2 but not described that the influence of background components is low. From the experiment of Example 3, the slope of the calibration curve is small and the CV value is also large. Therefore, it can be known that high detection sensitivity and measurement reproducibility are specific to citric acid.

[0160] Based on the above, by using a citrate buffer or a phosphocitrate buffer in the reagent, the background current value can be reduced, and the measurement reproducibility can be improved. Thus, the measurement can be performed with high reproducibility and high precision.

[0161] When measuring using the biosensor of the present invention, the following problems were found: Under conditions where the flowing current value is large, specifically, when a high voltage is applied such as a voltage of 1.0 V or more, or when using a sample with a low resistance value (i.e., current easily flows) such as plasma, the electrode peels off from the substrate due to the dissolution of the nickel alloy as a base metal, or pores are generated in the electrode, and a positive response current value cannot be obtained. And it was found that the thicker the thickness of the electrode of the biosensor and / or the thinner the thickness of the contact metal, the more significant this phenomenon occurs. In particular, in a biosensor having a first electrode pair and a second electrode pair both formed of a contact metal and a nickel alloy, and having a configuration in which only a part of the reagent is placed on the counter electrode; or a biosensor having a configuration in which no reagent is placed on either the working electrode or the counter electrode, when a high voltage of +1.0 to +7.0 V is applied to measure the hematocrit value, it was confirmed that this phenomenon occurs significantly.

[0162] Therefore, as described in Example 4 below, in a biosensor in which an electrode system is formed by laminating a contact metal made of titanium on an insulating substrate and laminating a nickel-vanadium alloy on the contact metal, the response current values were evaluated when the thicknesses of the nickel-vanadium alloy and titanium were changed, and in-depth studies were conducted. As a result, it was found that this phenomenon can be prevented when both the electrode and the contact metal are within a specified thickness range and have a certain relationship. That is, at least the following four phenomena were confirmed.

[0163] (1) The thinner the electrode, the more stable the film. However, if it is too thin, the resistance value of the electrode itself becomes too high, or the deviation of the inter-individual difference in the resistance value becomes too large. Therefore, the thickness needs to be above a certain range.

[0164] (2) The purpose of the contact metal is to fix the electrode to the substrate. To make the electrode layer stable, a thickness of more than a certain value is required. However, if it is too thick, it becomes unstable.

[0165] (3) In order not to be affected by the unevenness of the substrate (when it is thin, the surface of the electrode has unevenness and the resistance value increases), the total thickness of the electrode and the contact metal needs to be at least 10 μm or more.

[0166] (4) Up to a certain range, the thicker the contact metal, the thicker the electrode placed on it. However, if it exceeds a certain range, it will affect the measurement value. Therefore, the placed electrode decreases, and the total thickness is 55 μm or less.

[0167] In the case where the electrode is a nickel-vanadium alloy and the contact metal is titanium, as shown in Example 4, it was confirmed that when the thickness of titanium is x and the nickel-vanadium alloy is y, this phenomenon can be prevented by satisfying the following ranges.

[0168] 1) 7 μm ≤ x ≤ 52 μm

[0169] 2) 3 μm ≤ y ≤ 25 μm

[0170] 3) y = -11 / 16x + 155 / 4

[0171] (Example 4)

[0172] A contact metal made of titanium was laminated on an insulating substrate, and a nickel-vanadium alloy was laminated on the contact metal to form an electrode pair, and a biosensor was fabricated (no reagent on the working electrode and counter electrode). Multiple biosensors were prepared by changing the thicknesses of the contact metal and the nickel-vanadium alloy. After adding the following samples, a voltage was applied, and the response current was evaluated.

[0173] <Measurement conditions>

[0174] Voltage: 3.5 V

[0175] Application time: 10 seconds

[0176] Temperature: 40 °C

[0177] Sample: Blood with a hematocrit value of 20%

[0178] Response current value: 100 - 300 μA

[0179] <Judgment criteria for verification results>

[0180] * A sudden drop in the response current value occurs within 5 seconds

[0181] × A mid - rise in the response current value occurs within 5 seconds

[0182] △ The response current value deviation is 20 μA or more at the 5 - second value

[0183] 〇 No problem

[0184] The results are shown in Figure 8 .

[0185] Next, the results of Figure 8 are examined. Before conducting the experiment of Figure 8 , first, the biosensors fabricated under the thickness conditions of each electrode and contact metal are observed, and the unevenness on the electrode surface is visually confirmed. As a result, as described in the above phenomenon (3), when the total thickness of the nickel - vanadium alloy and the contact metal is less than 10 μm, it is affected by the unevenness of the substrate, and it is expected to affect the measurement results, and it is judged unsuitable as a biosensor.

[0186] Thus, multiple biosensors are fabricated with the total thickness of the nickel - vanadium alloy and the contact metal being 10 μm or more so as not to be affected by the unevenness of the substrate. The results obtained under the measurement conditions are Figure 8 . From the Figure 8 results, it is confirmed that under the conditions where the thickness of the nickel - vanadium alloy is 3 μm or more and 25 μm or less, and the thickness of titanium is 7 μm or more and less than 20 μm, even with such thickness conditions, the measurement can be stably performed. Thus, it can be confirmed that, as described in the above phenomena (1) and (2), the electrode and the contact metal have thickness conditions. On the other hand, under the condition where the thickness of titanium is 20 μm or more, it is found that the thicker the titanium, the thinner the thickness of the nickel - vanadium alloy deposited on it. It can be confirmed that when titanium is 50 μm and the nickel - vanadium alloy is 5 μm, the measurement can be carried out, the relationship of the above 3) y = - 11 / 16x + 155 / 4 is found, and the conditions of the above phenomenon (4) are determined.

[0187] Explanation of symbols

[0188] A ··· Biosensor

[0189] 10 ··· Substrate

[0190] 11 ··· Counter electrode

[0191] 12 ··· Working electrode

[0192] 13 ··· Reagent

[0193] 14 ··· Insulating layer

[0194] 15 ··· Spacer

[0195] 16 ··· Cover

[0196] 17 ··· Specimen supply section

[0197] 18 ··· Air hole

[0198] 19 ··· Contact metal

[0199] B ··· Measuring device

[0200] 20 ··· Control computer

[0201] 21 ··· Output section

[0202] 22 ··· Control section

[0203] 23 ··· Arithmetic section

[0204] 24 ··· Detection section

[0205] 25 ··· Potentiostat

[0206] 26 ··· Connection terminal

Claims

1. A biosensor comprising an insulating substrate, an electrode pair provided on the insulating substrate, and a reagent having a redox enzyme and an electron transfer substance disposed on at least a working electrode of the electrode pair, the biosensor being used to measure a substance to be measured in a sample supplied to the electrode pair, the biosensor being characterized in that the working electrode is made of a nickel-vanadium alloy.

2. The biosensor according to claim 1, wherein in the nickel-vanadium alloy, the ratio of nickel to vanadium is 80:20 to 95:5 by weight.

3. The biosensor according to claim 1, wherein in the nickel-vanadium alloy, the ratio of nickel to vanadium is 85:15 to 95:5 by weight.

4. The biosensor according to claim 1, wherein in the nickel-vanadium alloy, the ratio of nickel to vanadium is 90:10 to 94:6 by weight.

5. The biosensor according to any one of claims 1 to 4, wherein the electron transfer substance is a ruthenium complex or a ferrocyanide.

6. The biosensor according to any one of claims 1 to 4, wherein the reagent contains a citrate buffer or a phosphocitrate buffer.

7. The biosensor according to claim 6, wherein On the portion of the working electrode where the reagent is placed, the molar concentration of citric acid contained in the reagent is 1.5 μmol / cm 2 ~22 μmol / cm 2 .

8. The biosensor according to any one of claims 1 to 4, wherein the redox enzyme is glucose dehydrogenase and the substance to be measured is glucose.

9. The biosensor according to any one of claims 1 to 4, wherein a contact metal is laminated on the insulating substrate, and the nickel-vanadium alloy is laminated on the contact metal, whereby the working electrode is formed, and the total thickness of the nickel-vanadium alloy forming the working electrode and the contact metal is 10 μm to 55 μm.

10. The biosensor according to claim 9, wherein the contact metal is titanium, and the titanium and the nickel-vanadium alloy have the following thickness conditions, 1) 7 μm ≤ x ≤ 52 μm 2) 3 μm ≤ y ≤ 25 μm 3) y = -11 / 16x + 155 / 4 X represents the thickness of titanium and y represents the thickness of the nickel-vanadium alloy.

11. The biosensor according to claim 9, wherein the contact metal is laminated on the insulating substrate by physical vapor deposition, the nickel-vanadium alloy is laminated on the contact metal by physical vapor deposition, and an electrode group including the electrode pair is formed by partially removing the contact metal and the nickel-vanadium alloy from the insulating substrate.

12. A method for measuring a substance to be measured, comprising the following steps: a step of supplying the sample containing the substance to be measured to the electrode pair of the biosensor according to any one of claims 1 to 11; a step of applying a voltage between the electrode pair; a step of measuring the value of the current flowing between the electrode pair; and a step of calculating the amount of the substance to be measured based on the measured current value.

13. The method according to claim 12, wherein the voltage applied to the working electrode is a voltage of +50 mV to +500 mV with respect to the counter electrode of the electrode pair.

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