JP2016042032A - Glucose sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a glucose sensor capable of detecting or quantifying glucose with high accuracy even when maltose, galactose, or xylose are mixed in an aqueous sample that is the object of glucose measurement.SOLUTION: A specific NADGDH (sequence number 1) and/or a mutant thereof, nicotinamide adenine dinucleotide (hereafter described as NAD) and/or nicotinamide adenine dinucleotide phosphate (hereafter described as NADP), and an electron transport mediator are contained in a reaction layer provided on the electrode of a glucose sensor, whereby it is made possible to detect or quantify glucose with high accuracy even when maltose, galactose, or xylose are mixed in an aqueous sample.SELECTED DRAWING: None

Description

  The present invention relates to a glucose sensor. More specifically, the present invention relates to a glucose sensor that can detect or quantify glucose in an aqueous sample with high accuracy. Furthermore, this invention relates to the manufacturing method of the said glucose sensor, and the measuring method of the glucose concentration using the said glucose sensor.

  In recent years, the number of patients with diabetes has been increasing worldwide. According to Non-Patent Document 1, the number of diabetic patients in 2013 is estimated to be 382 million in the world, and is estimated to reach 592 million in 2035. In general, blood glucose control is adopted as a basic policy for the treatment of diabetes, and a self blood glucose measuring device is used for monitoring blood glucose levels and verifying the effects of insulin, oral medicine, and diet therapy. The major autologous blood glucose measuring devices that are currently in circulation are electrode systems that electrochemically detect glucose in the blood. The glucose sensor is composed of an enzyme that catalyzes glucose and an electrode. It consists of a meter that converts current values into blood glucose levels and displays them.

  In the glucose sensor, various enzymes are used to convert glucose into an electrochemical signal. For example, Patent Document 1 discloses a glucose sensor using glucose oxidase (hereinafter also referred to as GOD). However, since GOD uses molecular oxygen as an electron acceptor, the glucose sensor using GOD has a problem that the dissolved oxygen concentration in the measurement sample affects the quantitative value of glucose. As a method for solving this problem, the use of glucose dehydrogenase (hereinafter also referred to as GDH), which cannot convert molecular oxygen into an electron acceptor, has been advanced. Moreover, several types of GDH have been confirmed depending on the required coenzyme, and glucose sensors using various types of GDH have been developed.

  For example, a glucose sensor using pyrroloquinoline quinone-dependent GDH (hereinafter also referred to as PQQGDH) is known. However, unlike QOD, PQQGDH cannot convert molecular oxygen into an electron acceptor, but its substrate specificity is low, and it reacts with maltose, galactose and xylose other than glucose, so it is detected by a glucose sensor using PQQGDH. There are drawbacks in terms of accuracy.

  A glucose sensor using flavin adenine dinucleotide-dependent GDH (hereinafter also referred to as FADGDH) is also known (see, for example, Patent Document 2). However, FADGDH has higher substrate specificity than PQQGDH and low reactivity to maltose and galactose, but has reactivity to xylose. Therefore, even with a glucose sensor using FADGDH, detection accuracy is high. Still not satisfactory.

  Furthermore, a glucose sensor using nicotinamide adenine dinucleotide-dependent GDH (hereinafter also referred to as NADGDH) is also known (see, for example, Patent Document 3). Conventional NADGDH, like FADGDH, has higher substrate specificity than PQQGDH and low reactivity to maltose and galactose, but it has reactivity to xylose. Therefore, even with a glucose sensor using NADGDH, detection accuracy is improved. There is a need for further improvements in this regard. Furthermore, in the conventional glucose sensor using NADGDH, the linearity between the glucose concentration and the observed electrochemical signal is insufficient, and further improvement is required in terms of improvement in detection accuracy.

  Thus, the conventional glucose sensor using GDH does not have sufficient substrate specificity, and development of a glucose sensor capable of measuring glucose with high detection accuracy is required even in the presence of maltose, galactose, and xylose. ing.

  On the other hand, in the manufacture of glucose sensors, generally, a process is employed in which at least a pair of electrodes is formed on an insulating substrate, and a reaction layer including an enzyme, a coenzyme, an electron transfer mediator, etc. is formed on the electrodes. Has been. In general, the reaction layer is formed by spotting a reagent solution obtained by mixing each component into a solution on an electrode and removing the solvent by a drying process. Conventionally, GOD and GDH used in glucose sensors are enzymes isolated from ordinary bacteria that generally grow at 37 ° C. or lower. Therefore, the enzyme is inactivated at the drying temperature during the production of the glucose sensor. It is necessary to set the temperature to below the middle temperature, and the conventional glucose sensor has a disadvantage that the drying time is prolonged. Therefore, development of a glucose sensor manufacturing method capable of drying the reaction layer in a short time and shortening the lead time is also demanded.

Japanese Patent No. 3193721 Japanese Patent No. 5325574 Japanese Patent No. 4373604

IDF (International Diabetes Federation) DIABETES ATLAS Sixth Edition, 2013.

  An object of the present invention is to provide a glucose sensor capable of detecting or quantifying glucose with high accuracy even when maltose, galactose, and xylose are mixed in an aqueous sample to be measured for glucose. Furthermore, the other object of this invention is to provide the manufacturing method of the glucose sensor which can shorten the drying time of the reaction layer provided in a glucose sensor.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor found that a specific NADGDH (SEQ ID NO: 1) and / or a variant thereof and nicotinamide adenine dinucleotide were added to the reaction layer provided on the electrode of the glucose sensor. (Hereinafter also referred to as NAD) and / or nicotinamide adenine dinucleotide phosphate (hereinafter also referred to as NADP) and an electron transfer mediator to contain maltose, galactose, and xylose in an aqueous sample However, it has been found that glucose can be detected or quantified with high accuracy. It has also been found that glucose can be detected or quantified with higher accuracy by further containing diaphorase in the reaction layer. Further, since the specific NADGDH (SEQ ID NO: 1) and / or a variant thereof has heat resistance, the drying temperature of the reaction layer provided in the glucose sensor can be increased, thereby forming the reaction layer. It was also found that the drying time can be shortened. The present invention has been completed by further studies based on these findings.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A glucose sensor for detecting or quantifying glucose contained in an aqueous sample,
An insulating substrate;
An electrode system provided on the insulating substrate and having at least a working electrode and a counter electrode;
A reaction layer provided on the electrode,
The reaction layer is
(i) (i-1) glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, (i-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted Glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, and (i-3) amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, At least one glucose dehydrogenase selected from the group consisting of glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(ii) nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and
(iii) A glucose sensor comprising an electron transfer mediator.
Item 2. Item 2. The glucose sensor according to Item 1, wherein the reaction layer further contains (iv) diaphorase.
Item 3. (Iv) the diaphorase is (iv-1) a diaphorase comprising the amino acid sequence shown in SEQ ID NO: 2, and (iv-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted or added Or a diaphorase having a diaphorase activity equivalent to a diaphorase consisting of the amino acid sequence shown in SEQ ID NO: 2 and (iv-3) an amino acid identity to the amino acid sequence shown in SEQ ID NO: 2 is 80% or more, Item 3. The glucose sensor according to Item 1 or 2, which is at least one diaphorase selected from the group consisting of diaphorases having a diaphorase activity equivalent to the diaphorase having the amino acid sequence shown in SEQ ID NO: 2.
Item 4. (Iii) Electron transfer mediator is potassium ferricyanide, 2-methyl-1,4-naphthoquinone, Meldola blue, p-aminophenol, 1,4-benzoquinone, hydroxymethylferrocene, N, N, N ′, N ′ Item 4. The glucose according to any one of Items 1 to 3, which is at least one compound selected from the group consisting of tetramethyl-1,4-phenylenediamine and 1-methoxy-5-methylphenazinium methyl sulfate. Sensor.
Item 5. A method for producing a glucose sensor for detecting or quantifying glucose contained in an aqueous sample, comprising:
An electrode system provided on an insulating substrate and having at least a working electrode and a counter electrode,
(i) (i-1) glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, (i-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted Glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, and (i-3) amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, At least one glucose dehydrogenase selected from the group consisting of glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(ii) nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and
(iii) A method for producing a glucose sensor, comprising a step of providing a reaction layer containing an electron transfer mediator.
Item 6. (i) (i-1) glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, (i-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted Glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, and (i-3) amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, At least one glucose dehydrogenase selected from the group consisting of glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(ii) nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and
(iii) The glucose sensor according to Item 5, comprising a dropping step of dropping a reaction layer forming solution containing an electron transfer mediator on the working electrode, and a drying step of drying the working electrode to which the reaction layer forming solution has been dropped. Manufacturing method.
Item 7. Item 7. The method for producing a glucose sensor according to Item 6, wherein the drying step is performed under a temperature condition of 60 ° C to 80 ° C.
Item 8. Item 5. A glucose measurement method, wherein the glucose sensor according to any one of Items 1 to 4 is used to detect or quantify glucose contained in an aqueous sample.

  Since the mixture of maltose, galactose and xylose in an aqueous sample containing glucose does not affect the quantitative result of glucose, the glucose sensor of the present invention can be used for more accurate blood glucose measurement, for example. Furthermore, in the glucose sensor of the present invention, the specific NADGDH (SEQ ID NO: 1) and / or a variant thereof contained in the reaction layer exhibits heat resistance, so that the drying temperature during the formation of the reaction layer can be increased. Therefore, the effects of shortening the drying time and simplifying the process in the sensor manufacturing process can be expected.

It is a figure which shows the result of having measured the response current value with respect to glucose of various density | concentrations using the glucose sensor created in Example 5. FIG. In FIG. 1, the horizontal axis represents the glucose concentration (mM), and the vertical axis represents the response current value (μA). In Example 6, it is a figure which shows the result of having measured the response current value with respect to 20 mM glucose of the glucose sensor created by various drying temperature and drying time. In FIG. 2, the horizontal axis represents the drying temperature and the drying time, and the vertical axis represents the response current value (μA).

  The glucose sensor of the present invention is a glucose sensor for detecting or quantifying glucose contained in an aqueous sample, and an insulating substrate, and an electrode system provided on the insulating substrate and having at least a working electrode and a counter electrode, And a reaction layer having a specific composition provided on the electrode system. Hereinafter, a constituent member, a sample to be measured, a measuring method, and the like will be described in detail for the glucose sensor of the present invention.

1. Component [Insulating substrate]
The insulating substrate serves as a member for supporting the electrode system. The material of the insulating substrate is not particularly limited as long as it has insulating properties, and examples thereof include plastic, glass, paper, ceramics, and rubber. Specific examples of the plastic include polyethylene terephthalate, polystyrene, polymethacrylate, polypropylene, polyimide, acrylic resin, and glass epoxy. Specifically, filter paper is exemplified as the paper.

[Electrode system]
The electrode system is provided on the insulating substrate. In the glucose sensor of the present invention, the electrode only needs to include at least a working electrode and a counter electrode, and may be a two-electrode system having only a working electrode and a counter electrode, but there is a three-electrode system in which a reference electrode is further added. Also good.

  Each electrode may be provided with a lead portion for transmitting an electrochemical signal generated by the reaction layer to the electrochemical measuring device.

The material of the electrode is not particularly limited as long as it can be used as an electrode of a glucose sensor, and examples thereof include carbon, platinum, gold, silver, nickel, titanium, and palladium. In the case of a carbon electrode, specifically, the formation method includes a method of screen printing carbon ink on the insulating substrate, a method of coating carbon ink on the insulating substrate, and the like. Further, the carbon ink used for forming the carbon electrode may contain, for example, conductive metal fine particles such as gold and silver. In the case of a metal electrode, specific examples of the formation method include a vapor deposition method, a plating method, a sputtering method, and a metal foil sticking method. The vapor deposition method is, for example, an ion plating method, with a degree of vacuum of 1.33 × 10 ⁻⁴ Pa, an input power of 300 W, a rate of 5 × 10 ⁻¹ nm / second, and a time of 2 minutes, such as polyethylene terephthalate. A metal can be vapor-deposited on the plastic sheet, and a metal foil layer vapor-deposited on the sheet can be cut using a kiss cutting device. Thereby, a cut | interruption part becomes an insulation part and the electrode which consists of a working electrode and a counter electrode can be formed, for example.

  In addition, the reference electrode is not particularly limited, and a common electrode in electrochemical experiments can be applied, and a treatment for bringing silver-silver chloride into contact with the reference electrode may be performed.

[Reaction layer]
The reaction layer is a layer for converting glucose contained in the aqueous sample into an electrochemical signal, and is provided in at least a partial region on the electrode in a form capable of transmitting the electrochemical signal to the electrode. Specifically, the reaction layer may be provided on the working electrode, but may be provided on the reference electrode when the reference electrode is provided.

  The reaction layer includes (i) a specific glucose dehydrogenase, (ii) NAD and / or NADP, and (iii) an electron transfer mediator. Thus, by adopting a reaction layer having a specific composition, glucose can be detected or quantified with high accuracy, and the drying temperature at the time of formation of the reaction layer can be increased. Can be shortened. Hereinafter, each component contained in the reaction layer and a method for forming the reaction layer will be described.

(i) A preferred embodiment of the glucose dehydrogenase used in the specific glucose dehydrogenase reaction layer is glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO: 1 (hereinafter also referred to as glucose dehydrogenase (i-1)). The glucose dehydrogenase (i-1) is classified into EC 1.1.1.147, NADGDH derived from Bacillus subtilis (in the amino acid sequence shown in SEQ ID NO: 1, the 159th cysteine is alanine, the 170th lysine is glutamic acid, The 217th arginine is tyrosine, the 218th leucine is isoleucine, and the 252nd leucine is an amino acid sequence consisting of glutamine. By using the glucose dehydrogenase (i-1), it is possible to detect or quantify glucose with high accuracy even in an aqueous sample in which maltose, galactose, and xylose are mixed. It is also possible to generate an electrochemical signal with high linearity corresponding to the concentration. In addition, the glucose dehydrogenase (i-1) is excellent in thermostability, and unlike general Bacillus genus glucose dehydrogenase, sodium chloride is not required for its thermostability and the presence of sodium chloride. Even if it is treated at 80 ° C. for 30 minutes in an aqueous solution, the glucose dehydrogenase activity of 80% or more can be maintained as compared with that before the heat treatment, so that the drying temperature during the formation of the reaction layer can be increased. The lead time in the manufacture of the glucose sensor can be shortened.

Another preferred embodiment of glucose dehydrogenase used in the reaction layer is a glucose dehydrogenase variant consisting of the amino acid sequence shown in SEQ ID NO: 1. Specific examples of the mutant include the following glucose dehydrogenases (i-2) and (i-3).
(i-2) One or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted, and have the same thermal stability as glucose dehydrogenase (i-1) Glucose dehydrogenase (hereinafter also referred to as glucose dehydrogenase (i-2))
(i-3) A glucose dehydrogenase (hereinafter referred to as glucose dehydrogenase (i-3) having an amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1 of 80% or more and having a thermostability equivalent to that of glucose dehydrogenase (i-1). )

  The amino acid mutation introduced into the glucose dehydrogenase (i-2) may include only one kind of mutation (for example, only substitution) from substitution, addition, insertion, and deletion, or two kinds. The above mutations (for example, substitution and addition) may be included. In the glucose dehydrogenase (i-2), the number of amino acids to be substituted, added, inserted or deleted may be one or several, specifically 1 to 10, preferably 1 to 6, and further Preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2 or 1.

  Further, the “amino acid identity to the amino acid sequence shown in SEQ ID NO: 1” in the glucose dehydrogenase (i-3) may be 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably. 99% or more.

  In the present specification, “amino acid identity” means bl2 seqTraprogramA (BBI PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. (Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999). The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

  In the glucose dehydrogenase (i-2), the site of the mutation (substitution, addition, insertion and / or deletion) introduced into the amino acid sequence shown in SEQ ID NO: 1 is glucose dehydrogenase (i-1) and Although it does not restrict | limit especially as long as it can provide equivalent thermostability, the 159th cysteine in the amino acid sequence shown to sequence number 1, 170th lysine, 217th arginine, 218th leucine, and 252nd leucine An amino acid residue other than is preferable.

  In the glucose dehydrogenases (i-2) and (i-3), the amino acid substitution introduced is preferably a conservative substitution. That is, as the substitution in glucose dehydrogenase (i-2) and (i-3), for example, if the amino acid before substitution is a nonpolar amino acid, substitution with another nonpolar amino acid, Substitution with other uncharged amino acids if charged amino acids, substitution with other acidic amino acids if the amino acid before substitution is an acidic amino acid, and other basicity if the amino acid before substitution is a basic amino acid Examples include substitution with an amino acid.

  In the glucose dehydrogenase (i-2) and (i-3), `` glucose dehydrogenase having the same thermal stability as glucose dehydrogenase (i-1) '' means that when heat treatment is performed under the following conditions, It means that the residual rate of glucose dehydrogenase activity is comparable to the residual rate of glucose dehydrogenase activity of glucose dehydrogenase (i-1) subjected to heat treatment under the same conditions. More specifically, it means that the residual ratio of glucose dehydrogenase activity after heat treatment under the following conditions is 70% or more, preferably 75% or more, and more preferably 80% or more.

  The glucose dehydrogenase (i-1), (i-2) and (i-3) are genes encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or a mutant gene obtained by mutating the gene Can be produced using a known genetic engineering technique. The glucose dehydrogenase (i-1), (i-2) and (i-3) can be produced by referring to International Publication No. 2012/133761.

<Heating conditions>
Glucose dehydrogenase is dissolved in 20 mM potassium phosphate buffer to a concentration of 0.03 mg / ml. This is heated at 80 ° C. for 30 minutes.

<Calculation of residual rate of glucose dehydrogenase activity>
The residual rate of glucose dehydrogenase activity is calculated according to the following formula. The method for measuring glucose dehydrogenase activity is as described later.
Residual rate of glucose dehydrogenase activity (%) = {(glucose dehydrogenase activity after heat treatment) / (glucose dehydrogenase activity before heat treatment)} × 100

  In the reaction layer, one of the glucose dehydrogenases (i-1), (i-2) and (i-3) may be selected and used alone, or two or more may be used in combination. May be.

The amount of glucose dehydrogenase to be applied to the electrode for forming the reaction layer is not particularly limited. For example, 0.001 to 10 U / mm ² , preferably 0.01 to 1.0 U / mm ² on the electrode. More preferably, the amount is 0.1 to 1.0 U / mm ² .

In this specification, the activity unit U of glucose dehydrogenase is a value measured under the following measurement conditions using the following reagents.
(reagent)
100 mM Tris-HCl buffer pH 8.0
100 mM NAD aqueous solution 1M D-glucose aqueous solution enzyme activity measuring reagent: 17.5 mL of the above Tris-hydrochloric acid buffer solution, 0.5 mL of NAD aqueous solution, and 2 mL of glucose aqueous solution are mixed to obtain a reagent for measuring enzyme activity.
Enzyme activity measurement solution: As a solution for diluting glucose dehydrogenase to a desired concentration, 20 mM potassium phosphate buffer (pH 8.0) is used, and glucose dehydrogenase is adjusted so that the following activity value is 5 to 15 U / mL. The stock solution is diluted to make an enzyme activity measurement solution.

(Activity measurement conditions)
0.9 mL of the enzyme activity measurement reagent is put into a spectrophotometer cell (optical path length: 1 cm) and preincubated for 5 minutes or more at 37 ° C. Add 0.005 mL of enzyme activity measurement solution, mix well, record absorbance change at 340 nm for 40 seconds with a spectrophotometer pre-incubated at 37 ° C., and change absorbance after 60 seconds from the start of measurement ( ΔOD) is measured. In the blank, instead of the enzyme activity measurement solution, water is mixed with the enzyme activity measurement reagent, and the change in absorbance (ΔOD blank) per 60 seconds is measured as described above. From these values, glucose dehydrogenase activity is determined according to the following formula.
Glucose dehydrogenase activity (U / mL) = (ΔOD−ΔODblank) × 905 × dilution ratio / (6.22 × 5 × 1.0)
In the above calculation formula, 905 is the amount of the enzyme activity measuring reagent and the enzyme activity measuring solution, 6.22 is the molecular absorption coefficient of NAD (cm ² / micromol) under the present measuring conditions, and 5 is the solution of the enzyme activity measuring solution. The quantity 1.0 indicates the optical path length (cm) of the cell used for enzyme activity measurement.

(ii) NAD and / or NADP
The reaction layer contains NAD and / or NADP required as a coenzyme in the glucose dehydrogenase. The reaction layer may contain only one of NAD or NADP, or both of them. Since NADP is generally more expensive than NAD, it is preferable to use NAD.

The amount of NAD and / or NADP applied to the electrode for forming the reaction layer is not particularly limited. For example, 0.01 to 1,000 nmol / mm ² , preferably 0.1 to 100 nmol on the electrode. / Mm ² , more preferably 1 to 10 nmol / mm ² .

(iii) The electron transfer mediator reaction layer further contains an electron transfer mediator in order to transmit an electrochemical signal generated by the glucose dehydrogenation reaction by the glucose dehydrogenase to the electrode.

  The type of electron transfer mediator used in the present invention is not particularly limited as long as it can reversibly change between oxidized and reduced forms and transfer electrons to the electrode. For example, quinones, cytochromes, viologens Phenazines, phenoxazines, phenothiazines, ferricyanides, ferredoxins, ferrocene and derivatives thereof, osmium complexes, cobalt complexes, ruthenium complexes and the like. More specifically, potassium ferricyanide, 2-methyl-1,4-naphthoquinone, Meldola blue, p-aminophenol, 1,4-benzoquinone, hydroxymethylferrocene, N, N, N ′, N′-tetramethyl -1,4-phenylenediamine, 1-methoxy-5-methylphenazinium methyl sulfate and the like. These electron transfer mediators may be used alone or in combination of two or more.

The coating amount of the electron transfer mediator for forming the reaction layer on the electrode may be appropriately set according to the type of electron transfer mediator to be used. For example, 0.01 to 1,000 nmol / mm ^2, preferably 0.05~500nmol / mm ^2, more preferably include an amount of a 0.1~100nmol / mm ^2.

(iv) The diaphorase reaction layer may contain diaphorase in addition to the above components. By containing diaphorase in the reaction layer, electron transfer from the reduced NAD to the electron transfer mediator can be amplified, and glucose can be detected or quantified with higher accuracy.

  The diaphorase may be NADH-dependent diaphorase if NAD is used as the coenzyme, and NADPH-dependent diaphorase may be used if NADP is used as the coenzyme.

  The origin of diaphorase is not particularly limited, and those derived from various biological species can be used. Examples include diaphorase derived from bacteria, preferably diaphorase derived from the genus Geobacillus, and more preferably diaphorase derived from Geobacillus stearothermophilus. It is done.

  Preferable examples of diaphorase include diaphorase consisting of the amino acid sequence shown in SEQ ID NO: 2 (hereinafter also referred to as glucose dehydrogenase (iv-1)) or a variant thereof. Glucose dehydrogenase (iv-1) is derived from Geobacillus stearothermophilus and is a NADH-dependent diaphorase.

Specific examples of the diaphorase (iv-1) mutant include the following diaphorases (iv-2) and (iv-3).
(iv-2) A diaphorase having one or several amino acids substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and having a diaphorase activity equivalent to that of diaphorase (iv-1) ( (Hereinafter also referred to as diaphorase (iv-2))
(iv-3) A diaphorase (hereinafter also referred to as diaphorase (iv-3)) having an amino acid identity of 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 2 and having diaphorase activity equivalent to that of diaphorase (iv-1) )

  The amino acid mutation introduced into the diaphorase (iv-2) may include only one type of mutation (for example, only substitution) from substitution, addition, insertion, and deletion, or two or more types. Mutations (eg, substitutions and additions). In the diaphorase (iv-2), the number of amino acids to be substituted, added, inserted or deleted may be one or several, specifically 1 to 10, preferably 1 to 6, more preferably Is 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2 or 1.

  The “amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 2” in the diaphorase (iv-3) may be 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably. 99% or more.

  In the diaphorase (iv-2) and (iv-3), the introduced amino acid substitution is preferably a conservative substitution. That is, as the substitution in the diaphorase (iv-2) and (iv-3), for example, if the amino acid before substitution is a nonpolar amino acid, the substitution with another nonpolar amino acid, the amino acid before substitution is uncharged If the amino acid is a basic amino acid, it is replaced with another non-charged amino acid, the amino acid before the substitution is an acidic amino acid, or the amino acid before the substitution is a basic amino acid. The substitution to is mentioned.

  In the diaphorase (iv-2) and (iv-3), “diaphorase having diaphorase activity equivalent to diaphorase (iv-1)” means that the diaphorase activity of diaphorase (iv-1) is 100%. It means that the diaphorase activity in the same amount is 50% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more. The method for measuring diaphorase activity is as described later.

  The diaphorase (iv-1), (iv-2) and (iv-3) are a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or a mutant gene obtained by mutating the gene. And can be produced using known genetic engineering techniques.

The amount of diaphorase applied to the electrode for forming the reaction layer is not particularly limited. For example, 0.001 to 10 U / mm ² on the electrode, preferably 0.01 to 1.0 U / mm ² , More preferably, the amount is 0.1 to 1.0 U / mm ² .

In the present specification, the activity unit U of diaphorase is a value measured under the following measurement conditions using the following reagents.
(reagent)
500 mM Tris-HCl buffer, pH 8.5
13.1 mM NADH aqueous solution 1.2 mM 2,6-dichlorophenolindophenol (DCIP) aqueous solution enzyme activity measuring reagent: 3.00 mL of the above tris-hydrochloric acid buffer solution, 2.28 mL of NADH aqueous solution, 23.22 mL of water, Mix to make a reagent for measuring enzyme activity.
Enzyme activity measurement solution: As a solution for diluting diaphorase to a desired concentration, a 50 mM potassium phosphate buffer (pH 7.5) is used, and the following activity value is 1.0 to 2.0 U / mL. Dilute the stock solution of glucose dehydrogenase to make an enzyme activity measurement solution.
(Measurement condition)
2.85 mL of the enzyme activity measurement reagent is placed in a spectrophotometer cell (optical path length: 1 cm) and preincubated at 30 ° C. for 3 minutes or more. Add 0.15 ml and 0.01 ml enzyme activity measurement solution to DCIP aqueous solution, mix well, record absorbance change at 600 nm for 60 seconds with spectrophotometer pre-incubated at 30 ° C, change absorbance (ΔOD) Measure. In the blank, instead of the enzyme activity measurement solution, water is mixed with the enzyme activity measurement reagent, and the change in absorbance (ΔOD blank) per 60 seconds is measured as described above. From these values, the activity value is determined according to the following formula.
Diaphorase activity (U / mL) = (ΔOD−ΔODblank) × 3.01 × dilution ratio / (19 × 0.01 × 1.0)
In the above calculation formula, 3.01 is the total amount of reaction reagent, 19 is the molecular extinction coefficient (cm ² / micromol) of DCIP under the present measurement conditions, 0.01 is the amount of enzyme activity measurement solution, and 1. 0 shows the optical path length (cm) of the cell used for enzyme activity measurement.

(Other ingredients)
The reaction layer may contain a hydrophilic polymer as necessary in order to improve the coating property when forming the reaction layer, the adhesion of the above components on the electrode, and the like. The hydrophilic polymer is not particularly limited, and examples thereof include starch, carboxymethyl cellulose, methyl cellulose, gelatin, acrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, maleic anhydride polymer, and derivatives thereof. Among these hydrophilic polymers, carboxymethyl cellulose and polyvinyl pyrrolidone are preferable. These hydrophilic polymers may be used individually by 1 type, and may be used in combination of 2 or more type.

The coating amount of the electrode of the hydrophilic polymer for forming the reaction layer is not particularly limited, for example, 0.1 to 1000 / mm ² on the electrode, preferably 1~500μg / mm ^2, further Preferably, the amount is 1 to 100 μg / mm ² .

  The reaction layer may contain various buffering agents. The type of the buffering agent is not particularly limited, but may be any substance that has a buffering capacity and can set the pH of the reaction layer forming solution described later to 5.0 to 9.0, for example. Specific examples of the buffer include phosphates; organic acids such as fumaric acid, maleic acid, glutaric acid, phthalic acid, and citric acid; and good buffers such as MOPS, PIPES, HEPES, MES, and TES.

  Furthermore, the reaction layer contains additives such as proteins, saccharides, amino acids, metal salts, chelating agents, surfactants, etc. for the purpose of increasing the stability of the glucose dehydrogenase and, if necessary, the diaphorase used. You may go out. The type of these additives is not particularly limited as long as it does not act as a substrate for the glucose dehydrogenase and, if necessary, diaphorase. Specifically, proteins such as bovine serum albumin, ovalbumin, sericin; metal salts such as calcium, magnesium, zinc, manganese; chelating agents such as sodium edetate; Triton X-100, deoxycholic acid, cholic acid, Tween 20, Surfactants such as Brij35 and Emulgen can be mentioned.

(Method for forming reaction layer)
The reaction layer may be provided in at least a partial region of the electrode, and the formation method is not particularly limited.

  As a preferred example of the method for forming the reaction layer, an aqueous solution containing each component contained in the reaction layer (hereinafter also referred to as a reaction layer formation solution) is brought into contact with at least a part of the electrode and then dried. A method is mentioned.

  Specific examples of the method of bringing the reaction layer forming solution into contact with the electrode include a method of dropping the reaction layer forming solution onto the electrode using a microsyringe, an air pulse dispenser device, a mechanical dispenser device, or the like.

  Further, the method for drying the reaction layer forming solution dropped on the electrode is not particularly limited, and examples thereof include a method using a heat dryer, a method combining heat drying and ventilation of a dry gas, and the like. Although it does not restrict | limit especially about drying temperature, For example, 20 to 90 degreeC, Preferably it is 20 to 80 degreeC, More preferably, 40 to 80 degreeC is mentioned. In particular, the glucose dehydrogenase contained in the reaction layer has heat resistance and can maintain its activity even when subjected to a drying treatment under high temperature conditions. Therefore, it is possible to shorten the drying time and produce an efficient glucose sensor. It is preferable to dry at 60-80 degreeC from a viewpoint of performing. In addition, what is necessary is just to determine drying time suitably according to a drying method and drying temperature, For example, 1 to 60 minutes, Preferably it is 5 to 30 minutes, More preferably, 30 seconds to 5 minutes are mentioned.

  Another preferred example of the method for forming the reaction layer is a method in which the components to be contained in the reaction layer are immobilized on an electrode using a crosslinking reagent, a polymer matrix, a dialysis membrane, or the like. Typically, a method of blocking glutaraldehyde by immobilizing the component to be contained in the reaction layer on a carbon electrode using glutaraldehyde and then treating with a reagent having an amine group is exemplified.

2. Sample to be measured The glucose sensor of the present invention is used to detect or quantify glucose in an aqueous sample. The aqueous sample to be used for the measurement is not particularly limited as long as it contains water and requires detection or quantification of glucose, as well as an aqueous solution that may contain glucose as a component, blood, body fluid, It may be a biological sample such as urine.

  If glucose in a solid sample is to be detected or quantified, an aqueous sample is prepared by adding the solid sample to a desired amount of water to dissolve the glucose, and detecting the glucose using the aqueous sample. Alternatively, quantitative determination may be performed.

3. Measuring method The detection or quantification of glucose in an aqueous sample using the glucose sensor of the present invention can be performed by the same method as a conventional glucose sensor.

  Specifically, first, the electrode system is connected to an electrochemical measurement device such as a potentiostat / galvanostat, and an aqueous sample to be measured is added on the reaction layer provided on the electrode of the glucose sensor. The aqueous sample to be measured may be added dropwise to the reaction layer on the electrode using a micropipette or the like, and a liquid channel from the opening to the electrode system is provided, and the aqueous sample is electroded through the channel. The liquid may be fed upward.

  Next, a voltage is applied in a state where the reaction layer on the electrode is in contact with the aqueous sample, and a current value as an electrochemical signal obtained is measured. Since this current value depends on the glucose concentration contained in the aqueous sample, the substrate concentration can also be calculated from the current value based on a calibration curve prepared in advance using a standard solution. The strength of the voltage to be applied can be appropriately set according to the oxidation-reduction potential of the electron transfer mediator to be used, and is, for example, 50 to 1000 mV, preferably 100 to 500 mV. In addition, it is desirable that the reaction temperature is kept constant as much as possible in the measurement, but the measurement environment temperature is measured, and the temperature-dependent reaction rate is determined from the correlation equation between the temperature and the electrochemical signal prepared in advance. It is also possible to correct the change.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. Embodiments are also included in the technical scope of the present invention.

  EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples. Glucose dehydrogenase (i-1) (SEQ ID NO: 1) used in the following examples is NADGDH isolated from Bacillus subtilis (in the amino acid sequence shown in SEQ ID NO: 1, the 159th cysteine is alanine, the 170th Lysine is glutamic acid, 217th arginine is tyrosine, 218th leucine is isoleucine, and 252nd leucine is NADGDH consisting of an amino acid sequence). And a mutant NADGDH isolated from a transformant in which the codon corresponding to the 252nd amino acid has been mutated and introduced with the mutated gene (International Publication No. 2012/133376). In addition, the diaphorase (iv-1) (SEQ ID NO: 2) used in the following Examples is a genetic engineering technique using a gene encoding a polypeptide consisting of SEQ ID NO: 2 isolated from Geobacillus stearothermophilus. It was manufactured using.

Example 1
A printed electrode in which a working electrode, a counter electrode, and a reference electrode are arranged on an insulating substrate was obtained from Biodevice Technology Co., Ltd. In the printed electrode, a carbon electrode is printed on an insulating substrate of 12.5 mm × 4.0 mm × 0.3 mm, and the area of the working electrode is 2.64 mm ² .

A predetermined amount of glucose dehydrogenase (i-1) (SEQ ID NO: 1), NAD, diaphorase (iv-1) (SEQ ID NO: 2), and an electron transfer mediator are dissolved in water so that a reaction layer having the following composition can be formed. Thus, a reaction layer forming solution was prepared. The obtained reaction layer forming solution was dropped into a 2.64 mm ² region of the working electrode with a microsyringe.
(Each component amount of reaction layer per 1 mm ^{2 of} working electrode)
Glucose dehydrogenase (i-1) (SEQ ID NO: 1): 0.758 U / mm ²
NAD: 3.788 nmol / mm ²
Diaphorase (iv-1) (SEQ ID NO: 2): 0.758 U / mm ²
Electron transfer mediator ^{# 1} : 0.379 to 75.76 nmol / mm ²
# 1 As an electron transfer mediator, potassium ferricyanide (hereinafter also referred to as FK), 2-methyl-1,4-naphthoquinone (hereinafter also referred to as VK3), or Meldola Blue (hereinafter also referred to as MB) P-aminophenol (hereinafter also referred to as AP), 1,4-benzoquinone (hereinafter also referred to as BQ), or hydroxymethylferrocene (hereinafter also referred to as FMA), or N, N, N ′ , N′-tetramethyl-1,4-phenylenediamine (hereinafter also referred to as TMPD) or 1-methoxy-5-methylphenazinium methyl sulfate (hereinafter also referred to as m-PMS) was used.

  The electrode to which the solution for forming the reaction layer was dropped was put into a dryer preheated to 60 ° C., dried for 5 minutes to remove water, and a reaction layer having the above composition was formed on the surface of the working electrode. A sensor was created.

  The prepared glucose sensor was connected to a potentiostat, and 10 μL of a 20 mM glucose aqueous solution was dropped so as to straddle the working electrode, the counter electrode, and the reference electrode, and a response current value obtained by applying a voltage was measured. The applied voltage is set in consideration of the redox potential of each electron transfer mediator, and is 0.5 V for FK, 0.1 V for VK3, 0.1 V for MB, and 0.4 V for AP. , BQ was 0.4V, FMA was 0.5V, TMPD was 0.4V, and m-PMS was 0.2V.

  The time from dropping the aqueous glucose solution to applying the voltage was 5 seconds, and the time from applying the voltage to obtaining the response current value was 30 seconds.

  Table 1 shows the response current value to glucose in the type of electron transfer mediator and the amount of each added to the working electrode. From this result, a reaction layer containing glucose dehydrogenase (i-1) (SEQ ID NO: 1), NAD, diaphorase (iv-1) (SEQ ID NO: 2), and an electron transfer mediator is formed on the working electrode. It was confirmed that glucose in an aqueous sample can be detected. In addition, when a reaction layer containing no glucose dehydrogenase (i-1) was provided on the working electrode, no response current to glucose was observed, confirming that the background current of the reaction layer layer-electrode system was low. It was. On the other hand, even when a reaction layer not containing an electron transfer mediator was provided on the working electrode, no response current to glucose was observed, so it was confirmed that the use of an electron transfer mediator in the reaction layer was essential.

Example 2
A glucose sensor was prepared under the same conditions as in Example 1 except that a reaction layer forming solution prepared so as to form a reaction layer having the following composition was used and a reaction layer having the following composition was formed on the working electrode. did.
(Each component amount of reaction layer per 1 mm ^{2 of} working electrode)
Glucose dehydrogenase (i-1) (SEQ ID NO: 1): 0.758 U / mm ²
NAD: 3.788 nmol / mm ²
Diaphorase (iv-1) (SEQ ID NO: 2): 0 U / mm ² or 0.758 U / mm ²
Electron transfer mediator shown in Table 2: 0.379 nmol / mm ²
Carboxymethylcellulose: 9.470 μg / mm ²

  Using the prepared glucose sensor, the response current to glucose was measured under the same conditions as in Example 1.

  The obtained results are shown in Table 2. From this result, when an electron transfer mediator other than MB was used, an increase in the response current value was observed by including diaphorase in the reaction layer. This is considered to be because the electron transfer from the reduced NAD to the electron transfer mediator is amplified by the reaction of diaphorase.

Example 3
A glucose sensor was prepared under the same conditions as in Example 1 except that a reaction layer forming solution prepared so as to form a reaction layer having the following composition was used and a reaction layer having the following composition was formed on the working electrode. did.
(Each component amount of reaction layer per 1 mm ^{2 of} working electrode)
Glucose dehydrogenase (i-1) (SEQ ID NO: 1): 0.758 U / mm ²
NAD: 3.788 nmol / mm ²
Diaphorase (iv-1) (SEQ ID NO: 2): 0.758 U / mm ²
Electron transfer mediator shown in Table 3: 0.379 nmol / mm ²
Carboxymethylcellulose: 9.470 μg / mm ²

  Except for using the prepared glucose sensor, except that an aqueous solution containing 20 mM glucose, an aqueous solution containing 20 mM glucose and 20 mM galactose, an aqueous solution containing 20 mM glucose and 20 mM maltose, and an aqueous solution containing 20 mM glucose and 20 mM xylose were used as measurement targets. The response current to glucose was measured under the same conditions as in Example 1.

  The obtained results are shown in Table 3. Table 3 shows the relative value of the response current value of each aqueous solution, where the response current value when measuring an aqueous solution containing 20 mM glucose is 100. From this result, by forming a reaction layer containing glucose dehydrogenase (SEQ ID NO: 1), NAD, diaphorase, and electron transfer mediator on the working electrode, ± 5% even in the presence of galactose, maltose, and xylose It became clear that glucose could be quantified with the accuracy of.

  In general, nicotinamide adenine dinucleotide-dependent glucose dehydrogenase derived from bacteria is known to have high reactivity to xylose, but glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1 used in the present invention is used. The problem was overcome.

Example 4
Using commercially available nicotinamide adenine dinucleotide-dependent glucose dehydrogenase (trade name “GlcDH2”, manufactured by Unitika Ltd .: hereinafter also referred to as GDH2) and glucose dehydrogenase (i-1) (SEQ ID NO: 1), heat resistance A sex comparison was made.

  GDH2 and glucose dehydrogenase (i-1) (SEQ ID NO: 1) were each dissolved in 20 mM potassium phosphate buffer so as to be 0.03 mg / ml, and a GDH solution was prepared. Asked. Subsequently, 0.5 ml of each GDH solution was dispensed and heat-treated at a temperature of 30 ° C. to 80 ° C. for 30 minutes, and then the enzyme activity value was determined by the method described above. The residual activity was calculated when the GDH activity before heat treatment was taken as 100%.

  Table 4 shows the obtained results. From these results, it was confirmed that glucose dehydrogenase (i-1) (SEQ ID NO: 1) had a residual activity of 80% or more even at 80 ° C. and was excellent in heat resistance. That is, it was revealed that by using glucose dehydrogenase (i-1) (SEQ ID NO: 1) in the reaction layer of the glucose sensor, it can be dried at a high temperature when the reaction layer is formed.

Example 5
A glucose sensor was prepared under the same conditions as in Example 1 except that a reaction layer forming solution prepared so as to form a reaction layer having the following composition was used and a reaction layer having the following composition was formed on the working electrode. did. In addition, when GDH2 was used, the drying conditions of the reaction layer were 35 degreeC and 20 minutes.

(Each component amount of reaction layer per 1 mm ^{2 of} working electrode)
Glucose dehydrogenase ^{# 1} : 0.758 U / mm ²
NAD: 3.788 nmol / mm ²
Diaphorase: 0.758 U / mm ²
Potassium ferricyanide: 15.15 nmol / mm ²
Carboxymethylcellulose: 9.470 μg / mm ²
# 1 As a glucose dehydrogenase, a commercially available nicotinamide adenine dinucleotide-dependent glucose dehydrogenase (trade name “GlcDH2”, manufactured by Unitika Ltd .: hereinafter also referred to as GDH2), or glucose dehydrogenase (i-1) (SEQ ID NO: 1) It was used.

  Using the prepared glucose sensor, the response current to glucose was measured under the same conditions as in Example 1 except that an aqueous solution containing 0 mM, 5 mM, 10 mM, 20 mM, and 30 mM glucose was used as a measurement target.

  The obtained results are shown in FIG. From this result, a linear increase in response current was observed for glucose dehydrogenase (i-1) (SEQ ID NO: 1) with respect to 0 to 30 mM glucose, but no linearity was observed for GDH2. Since the same result was obtained even when other than ferricyanide was applied to the mediator, a straight line corresponding to the glucose concentration in the sample was obtained by adding glucose dehydrogenase (i-1) (SEQ ID NO: 1) to the reaction layer. It became clear that a highly reliable response signal was obtained.

Example 6
Using a reaction layer forming solution prepared so that a reaction layer having the following composition can be formed, a reaction layer having the following composition is formed on the working electrode, and the reaction layer is dried at 35 ° C. for 20 minutes at 60 ° C. 5 minutes at 80 ° C., 5 minutes at 100 ° C., and 5 minutes at 120 ° C., and a glucose sensor was prepared under the same conditions as in Example 1.
(Each component amount of reaction layer per 1 mm ^{2 of} working electrode)
Glucose dehydrogenase ^{# 1} : 0.758 U / mm ²
NAD: 3.788 nmol / mm ²
Diaphorase: 0.758 U / mm ²
Potassium ferricyanide: 15.15 nmol / mm ²
Carboxymethylcellulose: 9.470 μg / mm ²
# 1 As a glucose dehydrogenase, a commercially available nicotinamide adenine dinucleotide-dependent glucose dehydrogenase (trade name “GlcDH2”, manufactured by Unitika Ltd .: hereinafter also referred to as GDH2), or glucose dehydrogenase (i-1) (SEQ ID NO: 1) It was used.

  Using the prepared glucose sensor, the response current to glucose was measured under the same conditions as in Example 1.

  The obtained results are shown in FIG. In the case of using GDH2, the response current decreased when the drying temperature of the reaction layer reached 60 ° C. or higher. This is considered to be caused by thermal inactivation of GDH2. On the other hand, when glucose dehydrogenase (i-1) (SEQ ID NO: 1) was used, no decrease in response current was observed even when the reaction layer was dried at 80 ° C. If the formation of the reaction layer can be completed in a short time at a high temperature, it is considered that the thermal inactivation of diaphorase has not occurred.

  Since the same result was obtained even when other than potassium ferricyanide was applied to the mediator, in the present invention, by containing glucose dehydrogenase (i-1) (SEQ ID NO: 1), the drying temperature at the time of forming the reaction layer can be reduced. It has been confirmed that the reaction layer can be formed in a short time because it is possible to raise the temperature higher than in the past.

Claims (8)

A glucose sensor for detecting or quantifying glucose contained in an aqueous sample,
An insulating substrate;
An electrode system provided on the insulating substrate and having at least a working electrode and a counter electrode;
A reaction layer provided on the electrode,
The reaction layer is
(i) (i-1) glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, (i-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted Glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, and (i-3) amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, At least one glucose dehydrogenase selected from the group consisting of glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(ii) nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and
(iii) A glucose sensor comprising an electron transfer mediator.   The glucose sensor according to claim 1, wherein the reaction layer further contains (iv) diaphorase.   (Iv) the diaphorase is (iv-1) a diaphorase comprising the amino acid sequence shown in SEQ ID NO: 2, and (iv-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted or added Or a diaphorase having a diaphorase activity equivalent to a diaphorase consisting of the amino acid sequence shown in SEQ ID NO: 2 and (iv-3) an amino acid identity to the amino acid sequence shown in SEQ ID NO: 2 is 80% or more, The glucose sensor according to claim 1 or 2, which is at least one diaphorase selected from the group consisting of diaphorases having a diaphorase activity equivalent to the diaphorase having the amino acid sequence shown in SEQ ID NO: 2.   (Iii) Electron transfer mediator is potassium ferricyanide, 2-methyl-1,4-naphthoquinone, Meldola blue, p-aminophenol, 1,4-benzoquinone, hydroxymethylferrocene, N, N, N ′, N ′ The compound according to any one of claims 1 to 3, which is at least one compound selected from the group consisting of -tetramethyl-1,4-phenylenediamine and 1-methoxy-5-methylphenazinium methyl sulfate. Glucose sensor. A method for producing a glucose sensor for detecting or quantifying glucose contained in an aqueous sample, comprising:
An electrode system provided on an insulating substrate and having at least a working electrode and a counter electrode,
(i) (i-1) glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, (i-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted Glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, and (i-3) amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, At least one glucose dehydrogenase selected from the group consisting of glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(ii) nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and
(iii) A method for producing a glucose sensor, comprising a step of providing a reaction layer containing an electron transfer mediator. (i) (i-1) glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, (i-2) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, added, or inserted Glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1, and (i-3) amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, At least one glucose dehydrogenase selected from the group consisting of glucose dehydrogenase having the same thermostability as glucose dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(ii) nicotinamide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, and
(iii) The glucose according to claim 5, comprising a dropping step of dropping a reaction layer forming solution containing an electron transfer mediator on the working electrode, and a drying step of drying the working electrode to which the reaction layer forming solution is dropped. Sensor manufacturing method.   The method for producing a glucose sensor according to claim 6, wherein the drying step is performed under a temperature condition of 60 ° C. to 80 ° C.   A method for measuring glucose, wherein the glucose sensor according to claim 1 is used to detect or quantify glucose contained in an aqueous sample.