Biosensor

Abstract

Provided is a biosensor, which includes an insulating substrate, a pair of electrodes provided on the insulating substrate, and a reagent placed on at least a working electrode of the pair of electrodes and having an oxidoreductase and an electron transferase, and which is for measuring, by using the reagent, a substance to be measured in a sample supplied to the pair of electrodes, the biosensor being characterized in that the working electrode is formed from a base metal selected from among nickel and vanadium or a metal selected from among alloys thereof, and the reagent further includes one or more amino acids selected from among glycine, lysine, and histidine.

Description

Biorecovery

[0001] The present invention relates to a biosensor for electrochemically measuring biological components such as glucose, and more specifically, to a biosensor using base metals such as nickel and vanadium as electrode materials, which is equipped with a reagent formulation that can mitigate the influence of external environmental factors, such as changes in current value when exposed to a high-humidity environment.

[0002] In biosensors that measure blood components such as blood glucose, a reagent layer containing enzymes and mediators (electron transfer substances) is formed on the electrodes. By supplying blood to the reagent layer, a reaction system is established between the electrodes. The mediator contained in the reagent transfers electrons from blood glucose to the sensor. The amount of current is processed as a signal of glucose level. For example, Patent Document 1 discloses a biosensor using base metals such as nickel as electrodes.

[0003] Mediators are partially reduced by various factors unrelated to the oxidation reaction of glucose, and the current derived from these reduced mediators causes background variability. In particular, base metal materials are more susceptible to oxidation in the atmosphere and reduction of mediators compared to noble metal materials, and this tendency is even stronger in high-humidity environments.

[0004] Japanese Patent Publication No. 2014-215150

[0005] As described above, under high humidity conditions, electron transfer occurs from base metal materials to mediators, which can result in variations in background current. Reducing these variations is therefore required. Accordingly, the present invention aims to provide a biosensor that uses base metal materials as electrodes, which suppresses deviation from initial values ​​while maintaining reproducibility even under high humidity conditions, thereby enabling more accurate measurements.

[0006] The inventors diligently conducted research to solve the aforementioned problems. As a result, they discovered that in a biosensor using a base metal material as an electrode, by adding a specific amino acid to the reagent contained in the electrode, it is possible to reduce the oxidation reaction rate of the base metal electrode while maintaining reproducibility, thereby reducing variations in background current and suppressing deviation from the initial value. This led to the solution of the present invention.

[0007] One aspect of the present invention relates to a biosensor for measuring a target substance in a sample supplied to the electrode pair using the reagent, comprising an insulating substrate, an electrode pair provided on the insulating substrate, and a reagent containing an oxidoreductase and an electron transfer substance, placed on at least the working electrode of the electrode pair, wherein the working electrode is composed of a base metal selected from nickel and vanadium or an alloy thereof, and the reagent further contains an amino acid selected from glycine, lysine, and histidine. In one aspect, the biosensor is a glucose sensor, and the oxidoreductase is glucose oxidoreductase, where glucose oxidoreductase can be glucose dehydrogenase. In one aspect, the amount of one or more amino acids selected from glycine, lysine, and histidine is 50 to 100 mg / cm³ 2 It can be such. In one embodiment, the electron transfer material can be a ruthenium complex or a ferricyanide. Another aspect of the present invention relates to a method for measuring a substance to be measured, comprising the steps of supplying a sample containing the substance to be measured to an electrode pair of a biosensor, applying a voltage between the electrode pair, measuring the value of the current flowing between the electrode pair, and calculating the amount of the substance to be measured based on the measured current value.

[0008] According to the present invention, a biosensor using a base metal material as an electrode can be provided that suppresses deviation from the initial value while maintaining reproducibility even in a high humidity environment, thereby enabling more accurate measurements.

[0009] Figures illustrating examples of biosensors. Comparison of current values ​​under high humidity conditions with and without serine added to the electrodes. Comparison of current values ​​under high humidity conditions with serine and other amino acids (glycine, lysine, histidine) added to the electrodes. Comparison of current values ​​under high humidity conditions with base metal electrodes and precious metal electrodes.

[0010] (Biosensor) The biosensor of the present invention comprises an insulating substrate, an electrode pair provided on the insulating substrate, and a reagent having an oxidoreductase and an electron transfer substance, placed on at least the working electrode of the electrode pair, for measuring a target substance in a sample supplied to the electrode pair using the reagent, wherein the working electrode is composed of a base metal selected from nickel and vanadium or an alloy thereof, and the reagent further contains an amino acid selected from glycine, lysine, and histidine.

[0011] (Electrode Pair) 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 be a three-electrode system biosensor that includes a reference electrode in addition to the electrode pair including a working electrode and a counter electrode.

[0012] Base metals such as nickel and vanadium are used as electrode materials. Nickel alloys can also be used for the working electrode, and one or more nickel alloys selected from the group consisting of nickel-vanadium alloys, nickel-tungsten alloys, and nickel-ruthenium alloys can be used.

[0013] The material of the counter electrode is not particularly limited, but for example, a metal electrode such as platinum or a carbon electrode can be used. Similarly, the material of other electrodes such as the reference electrode is not particularly limited, but for example, a metal electrode such as platinum, a carbon electrode, a silver / silver chloride electrode, a standard hydrogen electrode, a calomel electrode, a palladium-hydrogen electrode, etc. can be used.

[0014] (Substrate) The electrode, including the electrode pair, is provided on a substrate, and an insulating substrate is used as the substrate. The type of insulating substrate is not particularly limited, but for example, it can be made of various thermoplastic resins such as polyetherimide (PEI), polyethylene terephthalate (PET), and polyethylene (PE), various thermosetting resins such as polyimide resin and epoxy resin, or insulating materials such as glass, ceramic, and paper. The size and thickness of the electrode and substrate can be set as appropriate, but the thickness of the electrode is particularly preferably 1 μm to 100 μm.

[0015] (Reagents) In the biosensor of the present invention, reagents are placed on at least the working electrode of the electrode pair. The reagents included are not particularly limited as long as they are reagents used in the detection reaction of the substance to be measured, but they include at least an oxidoreductase and an electron transfer substance.

[0016] (Oxidoreductase) An oxidoreductase is not particularly limited, as long as it is an enzyme that can use the substance to be measured as a substrate and can oxidize or reduce the substance to be measured. Examples include glucose oxidase (GOD), galactose oxidase, bilirubin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine oxidase, L-lactic acid oxidase, ascorbic acid oxidase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, and Examples include glyceraldehyde dehydrogenase, glucose dehydrogenase (GDH), fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, cytochrome oxidoreductase, catalase, peroxidase, and glutathione reductase. Among these, oxidoreductases of sugars are preferred, and examples of oxidoreductases of sugars include glucose oxidase (GOD), galactose oxidase, glucose dehydrogenase (GDH), fructose dehydrogenase, and sorbitol dehydrogenase. Therefore, the biosensor of the present invention can be used as a glucose sensor, cholesterol sensor, ethanol sensor, sorbitol sensor, fructose sensor, cellobiose sensor, lactic acid sensor, uric acid sensor, etc., depending on the type of enzyme.

[0017] When the substance to be measured is glucose, for example, glucose dehydrogenase or glucose oxidase can be used. Specific examples of glucose dehydrogenase include PQQ glucose dehydrogenase (PQQGDH) and cytochrome glucose dehydrogenase (CyGDH) which has an α-subunit containing FAD. Other examples include glucose-3-dehydrogenase derived from Agrobacterium tumesis. Furthermore, oxidoreductases can contain electron transport subunits or electron transport domains. When the substance to be measured is glucose, examples of electron transport subunits include subunits containing heme that have electron transfer function. Examples of oxidoreductases containing this heme-containing subunit include those containing cytochrome, such as glucose dehydrogenase containing cytochrome or a fusion protein of PQQGDH and cytochrome. Furthermore, the fusion protein of PQQGDH and cytochrome is disclosed, for example, in International Publication No. 2005 / 030807.

[0018] (Electron Transfer Substances) Electron transfer substances contained in reagents are substances that can receive electrons produced by the reaction between the substance to be measured and oxidoreductase, and then transfer those electrons to the electrode; they are also called mediators. Any non-catalyzed compound that receives electrons from oxidoreductase, is reduced, and then re-oxidized at the electrode can be used as an electron transfer substance. Examples include ruthenium complexes, ferricyanides such as potassium ferricyanide (also called hexacyanoferrate(III) salts), and quinone compounds (e.g., 1,4-Naphthoquinone, VK3, 9,10-Phenanthrenequinone, 1,2-Naphthoquinone, p-Xyloquinone, Methylbenzoquinone, 2,6-Dimethylbenzoquinone, Sodium). 1,2-Naphthoquinone-4-sulfonate, 1,4-Anthraquinone, Tetramethylbenzoquinone, Thymoquinone), phenylenediamine compounds (for example, N, N-Dimethyl-1,4-phenylenediamine, N, N, N',N'-tetramethyl-1, 4-phenylenediamine dihydrochloride), 1-Methoxy-PMS (1-Methoxy-5-methylphenazinium methylsulfate), CoenzymeQ0, AZURE A Chloride Examples include phenosafranin, 6-aminoquinoxaline, and tetrathiafulvalene, which may be used alone or in combination of two or more.

[0019] Furthermore, the ruthenium complex is preferably a ruthenium complex consisting of trivalent ruthenium (Ru(III)) and a ligand, and the following ruthenium ammonia complex is more preferable: [Ru(NH ３ ) ５ X] ｎ＋ Here, X can be NH3, halogen ions, CN, pyridine, nicotinamide, bipyridine, or H2O, and among these, NH3 or halogen ions (for example, Cl - F -, Br - , I - ) is preferable. In the above chemical formula, n+ represents the valence of the ruthenium(III) complex in the oxidized form, and is appropriately determined according to the type of X. Details of the ruthenium complex are disclosed in JP-A-2018-013400.

[0020] The content of the redox enzyme in the reagent of the biosensor of the present invention can be appropriately determined according to the type of the measurement target substance, but since it is necessary to contain a sufficient amount of the redox enzyme with respect to the measurement target substance, the amount of the redox enzyme is 1 cm of the surface area of the portion on which the reagent of the biosensor is placed ２ per area, 1 to 10 U is preferable, more preferably 1 to 5 U, and particularly preferably 1 to 3 U.

[0021] The content of the electron transfer substance in the reagent is preferably contained more than the redox enzyme, and can be appropriately determined according to the type of the measurement sample and the like. For example, 1 cm of the surface area of the portion on which the reagent of the biosensor is placed ２ per area, 10 mmol to 100 mmol is preferable, more preferably 10 mmol to 50 mmol, and particularly preferably 15 mmol to 20 mmol.

[0022] (Amino acid) In the biosensor of the present invention, in order to suppress the reduction of the electron transfer substance not depending on the oxidation of a substrate (measurement target substance) such as glucose and suppress the variation in the measurement current value, one or more amino acids selected from glycine, lysine and histidine are added to the reagent. The amount of these amino acids is not particularly limited as long as it is sufficient to suppress the reduction of the non-specific electron transfer substance. For example, 1 cm of the surface area of the portion on which the reagent of the biosensor is placed ２ per area, 50 to 100 mg is preferable. When adding two or more selected from glycine, lysine and histidine, the total is preferably within this range. When containing a plurality of types of the above amino acids, the total amount is preferably within the above range.

[0023] (Other components) The reagent contains an oxidoreductase, an electron transfer substance, and the above-mentioned amino acids, but may also contain resin binders such as butyral resins and polyester resins, binders such as the layered inorganic compounds disclosed in Table 2005 / 043146, surfactants, etc.

[0024] Furthermore, the reagent may additionally contain additives such as buffers and surfactants. As buffers, amine-based buffers such as Tris, ACES, CHES, CAPS, TAPS, CAPS, Bis-Tris, TAPSO, TES, Tricinene, and ADA may be used, or buffers having a carboxyl group such as phosphate buffers, citrate buffers, citrate phosphate buffers, sodium acetate-acetate buffers, malic acid-sodium acetate buffers, malonic acid-sodium acetate buffers, and succinic acid-sodium acetate buffers may be used. The pH of the buffer is preferably 6.8 to 7.2, and more preferably about 7.0.

[0025] Examples of surfactants include Triton X-100, sodium dodecyl sulfate, perfluorooctanesulfonic acid or sodium stearate, alkylaminocarboxylic acids (or their salts), carboxybetaine, sulfobetaine, and phosphobetaine.

[0026] In addition to the substrate, electrode pair, and reagent, the biosensor may also include a cover placed on the side of the substrate having the electrode pair and covering the electrodes, a spacer placed between the substrate and the cover to form a predetermined space between the substrate and the cover, and a sample supply section provided on the region including the reagent placement portion of at least the working electrode of the electrode pair and having a predetermined space. The sample supply section may be formed by processing the cover and spacer. The biosensor may also include components other than those described above.

[0027] (Applications of the biosensor) The biosensor of the present invention can be suitably used to measure target substances in a sample. The sample is not particularly limited as long as it contains the target substance, but biological samples are preferred, such as blood and urine. Examples of target substances include glucose, cholesterol, ethanol, sorbitol, fructose, cellobiose, lactic acid, and uric acid.

[0028] (Measurement method using a biosensor) When a sample containing the substance to be measured is brought into contact with the reagent placement area 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 signal based on the reaction is generated by applying a voltage between the electrode pair. By detecting this signal, the substance to be measured can be measured. Specifically, if the reagent of the biosensor contains an oxidoreductase and an electron transfer substance, electrons generated by the oxidation or reduction reaction between the oxidoreductase and 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, and an oxidation current dependent on the amount of the substance to be measured in the sample is generated. By measuring this current value, the concentration of the substance to be measured in the sample can be measured based on the current value.

[0029] When calculating the concentration of a target substance, methods such as voltammetry, amperometry, and coulometry are employed. Voltammetry is a method in which the voltage applied to an electrode is changed, the pattern of the changing response current value is measured, and the concentration of the target substance is calculated based on the current peak value in that pattern. Aperometry is a method in which a constant voltage is applied to an electrode, the response current value is obtained after a certain period of time from the start of the reaction, and the concentration of the target substance is calculated based on this response current value. Coulometry is a method in which a constant voltage is applied to an electrode, almost all of the target substance in the sample is reacted, the integrated value of the response current value is obtained, and the concentration of the target substance is calculated based on the integrated value.

[0030] (Method for measuring a target substance) The method for measuring a target substance of the present invention includes the steps of: supplying a sample containing the target substance to an electrode pair having a biosensor; applying a voltage between the electrode pair; measuring the value of the current flowing between the electrode pair; and calculating the amount of the target substance based on the measured current value.

[0031] (Sample supply process) The sample can be supplied to the electrode pair of the biosensor from a container containing the sample using known supply means such as a microchip, syringe, or capillary. The sample can also be supplied by an automated supply means. When the biosensor has the aforementioned sample supply unit, the sample can be efficiently reacted with the biosensor by supplying the sample to the sample supply unit.

[0032] (Voltage Application Step) Next, a voltage is applied between the electrode pair. When performing amperometry or coulometry, a constant voltage is applied between the electrode pair. Here, the voltage applied to the working electrode can be any voltage that is positive relative to the counter electrode and can be set as appropriate, for example, +50 to +500 mV relative to the counter electrode. After contacting the sample with the reagent, the electrode system may be held without voltage for a predetermined time before the voltage is applied, or the voltage may be applied to the electrode system simultaneously with the contact between the sample and the reagent. When holding without voltage, the holding time may be, for example, 30 seconds or less, or 10 seconds or less. On the other hand, when performing cyclic voltammetry measurement, the potential is swept at a constant speed relative to the working electrode. Multiple cycles of potential sweeping are also possible.

[0033] (Current Measurement Process) In the case of amperometry, the response current value is measured after a certain period of time has passed since the voltage was applied. In the case of coulometry, the response current value is measured over time to obtain the integrated value of the response current. On the other hand, in the case of cyclic voltammetry, the current value corresponding to the voltage during the sweep is continuously measured to obtain the cyclic voltammetry waveform. The response current value can be measured using a standard ammeter or similar device.

[0034] (Concentration Calculation Process) The concentration of the target substance is calculated based on the measured response current value. In the case of amperometry, the relationship between the response current value and the concentration of the target substance can be determined in advance using a calibration curve, and the concentration of the target substance can be calculated by applying the measured response current value to the calibration curve. In the case of coulometry, the relationship between the integrated value of the response current and the concentration of the target substance can be determined in advance using a calibration curve, and the concentration of the target substance can be calculated by applying the integrated value of the measured response current to the calibration curve. In the case of cyclic voltammetry, the relationship between the peak value in the cyclic voltammetry waveform and the concentration of the target substance can be determined in advance using a calibration curve, and the concentration of the target substance can be calculated by applying the measured response current value to the calibration curve.

[0035] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following embodiments.

[0036] Reference Example 1 A base metal layer (nickel vanadium, Ni-V) is formed on a substrate made of polyethylene terephthalate, and an electrode layer consisting of a working electrode 2 and a counter electrode 3 is provided. On this electrode layer, a reagent containing an enzyme, electron carrier, amino acid, buffer, surfactant, and defoamer is prepared in the composition shown below and added to form a reagent layer 1. On top of this, a spacer 4 made of polyethylene terephthalate and a cover 5 made of polyethylene terephthalate with air vents are provided. A biosensor was fabricated in this manner (Figure 1).

[0037] <Composition of Reagent Solution> FAD-dependent Glucose Dehydrogenase: 48 KU / mL Ru(NH3)6Cl3: 626 mM 1-Methoxy PES: 2.27 mM Phosphate Buffer (pH 7.0): 300 mM Serine: 2% by mass CHAPS: 2% by mass Antifoaming agent: 0.04% by mass

[0038] The biosensor prepared as described above and the biosensor prepared by removing the amino acid (serine) from the above were left standing in an environment of 40°C and 80% RH for 10 to 60 minutes. Using these treated biosensors, the sensor response value when blood with a glucose concentration of 0 mg / dL was used as the sample solution was measured. As a result of the measurement, in the Ref condition (with serine), when exposed for 10 minutes or more, the current value increased by 30% from the initial current value. On the other hand, even when exposed for 30 minutes under the condition without serine, the deviation from the initial current value was 6% or less (Figure 2). Although serine is important for improving the reproducibility of the biosensor, it was found that there is a problem that the deviation from the initial value increases.

[0039] Example 1 Among the above formulations, a reagent in which serine was changed to the amino acids glycine (2%), lysine (hydrochloride 3.2%), and histidine (0.64%) was prepared, and a biosensor was fabricated. The biosensor prepared as described above was left standing in an environment of 40°C and 80% RH for 10 to 60 minutes. Using these treated biosensors, the sensor response value when blood with a glucose concentration of 0 mg / dL was used as the sample solution was measured. As a result of the measurement, in the sensor using serine, when exposed for 10 minutes or more, the current value increased by 30% or more from the initial current value. On the other hand, even when exposed for 10 minutes or more under the condition using other amino acids (glycine, lysine, histidine), the deviation from the initial current value was 25% or less (Figure 3). From this, it was found that glycine, lysine, and histidine can improve the reproducibility of the biosensor while suppressing the deviation from the initial value.

[0040] Reference Example 2: In the sensor manufacturing process described above, a biosensor was fabricated in which the base metal layer (nickel vanadium, Ni-V) was replaced with the noble metal ruthenium (Ru). The biosensor, fabricated as described above (under serine conditions), was left to stand for 30 minutes to 24 hours in an environment of 40°C and 80% RH. Using these biosensors, the sensor response values ​​were measured when an aqueous solution with a glucose concentration of 30 mg / dL was used as the sample solution. The measurement results showed that with the Ni-V electrode, the current value increased by 50% from the initial value after 30 minutes of exposure. On the other hand, with the Ru electrode, the deviation from the initial current value was less than 10% even after 24 hours of exposure (Figure 4). Therefore, it was found that the problem of current value deviation under high humidity conditions is specific to base metal electrodes.

[0041] 1... Reagent layer, 2... Working electrode, 3... Counter electrode, 4... Spacer, 5... Cover

Claims

1. A biosensor for measuring a target substance in a sample supplied to the electrode pair using the reagent, comprising an insulating substrate, an electrode pair provided on the insulating substrate, and a reagent having an oxidoreductase and an electron transfer substance placed on at least the working electrode of the electrode pair, wherein the working electrode is composed of a base metal selected from nickel and vanadium or an alloy thereof, and the reagent further contains one or more amino acids selected from glycine, lysine, and histidine.

2. The biosensor according to claim 1, wherein the biosensor is a glucose sensor and the oxidoreductase is glucose oxidoreductase.

3. The biosensor according to claim 2, wherein the glucose oxidoreductase is glucose dehydrogenase.

4. The amount of one or more amino acids selected from glycine, lysine, and histidine is 50–100 mg / cm³. 2 The biosensor according to claim 1.

5. The biosensor according to claim 1, wherein the electron transfer material is a ruthenium complex or a ferricyanide.

6. A method for measuring a target substance, comprising the steps of: supplying a sample containing the target substance to an electrode pair of a biosensor according to any one of claims 1 to 5; applying a voltage between the electrode pair; measuring the value of the current flowing between the electrode pair; and calculating the amount of the target substance based on the measured current value.

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