Biological component concentration measurement reagent, measurement method, and sensor

Abstract

To provide means by which changes in coloring characteristics over time can be suppressed in chelate compounds with formazan and transition metal ions generated from tetrazolium salts.SOLUTION: There is provided an organism component concentration measurement reagent containing a tetrazolium salt, a compound capable of producing transition metal ions that can have a tetrahedral coordination structure, and a redox enzyme.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a reagent, a measurement method, and a sensor, and more particularly to a reagent, a measurement method, and a sensor used to measure the concentration of a biological component (analyte) such as glucose contained in a whole blood sample. [Background technology]

[0002] BACKGROUND ART Conventionally, in clinical chemical testing, techniques have been known for measuring an analyte (for example, glucose) contained in a biological sample such as blood or urine by electrochemical means or optical means (colorimetry).

[0003] For example, Patent Document 1 describes a method in which a water-soluble tetrazolium compound is allowed to coexist with a transition metal ion in an enzyme reaction system, the water-soluble formazan, which is a reduced form of the tetrazolium compound, is converted into a colored chelate compound with the metal ion, and the color change is measured. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 60-75470 Summary of the Invention [Problem to be solved by the invention]

[0005] When using the chelate compound of formazan and transition metal ions described in Patent Document 1, the color development characteristics may change over time. For rapid measurement, it is necessary to create a special algorithm and make corrections to accommodate the passage of time.

[0006] Therefore, the present invention has been made in consideration of the above circumstances, and an object of the present invention is to provide a means for suppressing the change over time in color-developing properties of a chelate compound of a formazan produced from a tetrazolium salt and a transition metal ion. [Means for solving the problem]

[0007] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that the above-mentioned problems can be solved by using a tetrazolium salt in combination with a compound capable of generating a transition metal ion that can adopt a specific coordination structure, and thus completed the present invention.

[0008] That is, the above object can be achieved by a reagent for measuring the concentration of a biological constituent, which comprises a tetrazolium salt, a compound capable of generating a transition metal ion capable of adopting a regular tetrahedral coordination structure, and an oxidoreductase. [Effects of the Invention]

[0009] According to the present invention, a chelate compound is formed between a formazan produced from a tetrazolium salt and a transition metal ion, thereby providing a means for suppressing changes in color-developing properties over time. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a plan view schematically showing a blood glucose meter (component measuring device) to which a measurement sensor according to this embodiment is attached. [Figure 2] FIG. 2 is an enlarged perspective view showing the sensor and the photometry unit of the device main body in FIG. [Figure 3] FIG. 3 is a side view showing the measurement sensor of FIG. [Figure 4A] 4A is a first plan view showing the mounting operation of the measurement sensor and the device main body of FIG. 1. FIG. [Figure 4B] FIG. 4B is a second cross-sectional plan view showing the mounting operation subsequent to FIG. 4A. [Figure 5A] FIG. 5A is a schematic diagram of a blood glucose meter sensor used in the examples. [Figure 5B] FIG. 5B is a diagram for explaining the length, width, and thickness of the inner surface of the blood glucose meter sensor of FIG. 5A. [Figure 6]FIG. 6 is a graph showing the relationship between absorbance and wavelength when a blood sample is applied to a blood glucose meter sensor (blood glucose level measuring sensor) using zinc acetate or nickel acetate. [Figure 7] FIG. 7 is a graph showing the relationship between the glucose concentration when an aqueous glucose solution is applied to a blood glucose meter sensor (blood glucose level measuring sensor) using tetrazolium salt 1, and the absorbance of the formazan and Zn2+ chelate compound. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present disclosure will be described, but the present disclosure is not limited to the following embodiments.

[0012] A first aspect of the present disclosure is a reagent for measuring the concentration of a biological constituent, comprising a tetrazolium salt, a compound capable of generating a transition metal ion capable of adopting a regular tetrahedral coordination structure, and an oxidoreductase.

[0013] A second aspect of the present disclosure is a method for measuring the concentration of a biological component, comprising contacting a whole blood sample with a biological component concentration measurement reagent according to the first aspect of the present disclosure, measuring the amount of color development, and quantifying the concentration of the biological component in the whole blood sample based on the amount of color development.

[0014] A third aspect of the present disclosure is a sensor for measuring the concentration of a biological component in a whole blood sample, having a reaction section, wherein the reaction section includes the biological component concentration measurement reagent according to the first aspect of the present disclosure.

[0015] According to the present disclosure, it is possible to suppress changes in color-developing properties over time in a chelate compound of formazan and a transition metal ion.

[0016] In this specification, the reagent for measuring the concentration of a biological constituent is also referred to simply as the "reagent" or the "reagent of the present disclosure."

[0017] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments. Furthermore, the dimensional proportions in the drawings are exaggerated for the sake of explanation and may differ from the actual proportions.

[0018] In this specification, the range "X to Y" includes X and Y and means "X or more and Y or less." "M" means mol / L. Unless otherwise specified, operations and measurements of physical properties are performed at room temperature (20 to 25°C) and a relative humidity of 40 to 50% RH.

[0019] <Biocomponent concentration measurement reagents> A first aspect of the present disclosure is a reagent for measuring the concentration of a biological component, comprising a tetrazolium salt, a compound capable of generating a transition metal ion capable of adopting a regular tetrahedral coordination structure, and an oxidoreductase. Each component of the reagent is described in detail below.

[0020] (tetrazolium salts) The reagent of the present disclosure contains a tetrazolium salt. The tetrazolium salt is a color-developing reagent, and the formazan generated from the tetrazolium salt forms a chelate compound with a transition metal ion described below.

[0021] Examples of tetrazolium salts include 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium, the tetrazolium salts described in WO 2018 / 051822, and 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

[0022] The tetrazolium salts may be used alone or in combination of two or more.

[0023] According to a preferred embodiment of the present disclosure, the tetrazolium salt is at least one selected from the group consisting of 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium (WST-4) or a tetrazolium salt represented by the following formula (1): According to a particularly preferred embodiment of the present disclosure, the tetrazolium salt is a tetrazolium salt represented by the following formula (1):

[0024] [ka]

[0025] In the above formula (1), R 1 is any one selected from the group consisting of a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxy group, and R 2 is a nitro group, -OR 4 and a carboxyl group, 3 are hydrogen atoms, methyl groups, or ethyl groups, at least one of which is a methyl group or an ethyl group, and R 4 is a methyl or ethyl group, and m is a sulfo group (-SO3 - ) is the number of phenyl groups bonded to the 5-position of the tetrazole skeleton, and is 1 or 2; n is R 2 is the number of bonds to the phenyl group at the 3rd position of the tetrazole skeleton, and is an integer of 0 to 2, and p ... - ) is the number of bonds to the phenyl group at the 3-position of the tetrazole skeleton, and is 0 or 1, n+p is 1 or more, q is 1 or 2, and when q is 2, each OR 3 are arranged adjacently, and in this case, each OR 3 may form a ring together, and X represents a hydrogen atom or an alkali metal.

[0026] In the above formula (1), a substituted benzothiazolyl group is present at the 2-position of the tetrazole skeleton. In the above formula (1), the presence of a benzothiazolyl group at the 2-position of the tetrazole ring allows for efficient and rapid formation of a chelate compound with a transition metal compound (the maximum absorption wavelength of the formazan compound can be shifted to a longer wavelength region). Furthermore, by introducing at least one methoxy group or ethoxy group into the benzothiazolyl group at the 2-position of the tetrazole skeleton, the resulting formazan and Zn 2+ When chelated with transition metal ions such as , the maximum absorption wavelength shifts to the longer wavelength side.

[0027] In the above formula (1), q is 1 or 2, preferably 1. When q=1, R 3 R is a methyl group or an ethyl group, and is preferably a methyl group from the viewpoint of water solubility. 3 When is an alkyl group having 3 or more carbon atoms, the tetrazolium salt and the formazan generated from the tetrazolium salt are not preferred because they have poor water solubility.

[0028] In the above formula (1), the -OR of the substituted benzothiazolyl group present at the 2-position of the tetrazole skeleton 3 Preferably, at least one of these is bonded to the 6-position of the benzothiazolyl group. 2+ When chelated with transition metal ions such as benzophenone, the maximum absorption wavelength can be shifted to the longer wavelength side.

[0029] In the above formula (1), when q=1, the substituent of the benzothiazolyl group present at the 2-position of the tetrazole skeleton is —OR 3 The substitution position of -OR is not particularly limited, and may be any of the 4th, 5th, 6th, and 7th positions. 3 The substitution position of is preferably bonded to the 6-position of the benzothiazolyl group. That is, in a preferred embodiment of the present invention, q is 1 and -OR 3 is preferably bonded to the 6-position of the benzothiazolyl group. 2+ When chelated with transition metal ions such as benzophenone, the maximum absorption wavelength can be shifted to the longer wavelength side.

[0030] In the above formula (1), when q=2, R 3 is a hydrogen atom, a methyl group, or an ethyl group, and at least one is a methyl group or an ethyl group. When q is 2, each OR 3 are arranged adjacently, and each OR 3 may form a ring together. In this case, a preferred combination is R 3 is a combination of a hydrogen atom and a methyl group, or a combination of a methyl group and a methyl group. When q=2, -OR is a substituent of the benzothiazolyl group at the 2-position of the tetrazole skeleton. 3 The substitution position is two -OR 3 There are no particular limitations on the positions as long as they are adjacent to each other, and they may be at the 4,5 positions, the 5,6 positions, or the 6,7 positions. 2+ In terms of the effect of the shift of the maximum absorption wavelength to the longer wavelength side when chelated with transition metal ions such as 3 The substitution position of is preferably bonded to the 6-position of the benzothiazolyl group, i.e., two -OR 3 The substitution positions are preferably the 5- and 6-positions or the 6- and 7-positions. Specifically, when q is 2, the substituted benzothiazolyl group at the 2-position of the tetrazole skeleton is preferably any one of the following substituents.

[0031] [ka]

[0032] In the above formula (1), when q is 2, and the substituted benzothiazolyl group at the 2-position of the tetrazole skeleton is any of the above-mentioned substituents, the substituted sulfonated phenyl group at the 3-position of the tetrazole skeleton is a 4-methoxy-5-sulfophenyl group, and Zn 2+ This is preferable in view of the effect of shifting the maximum absorption wavelength to the longer wavelength side when chelated with a transition metal ion such as .

[0033] In a preferred form of the invention, the tetrazolium salt is R 1is any one selected from the group consisting of a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxy group, and R 2 is a nitro group, -OR 4 and a carboxyl group, and at least one R 2 -OR 4 is a group, and R 3 are hydrogen atoms, methyl groups, or ethyl groups, at least one of which is a methyl group or an ethyl group, and R 4 is a methyl or ethyl group, and m is a sulfo group (-SO3 - ) is the number of phenyl groups bonded to the 5-position of the tetrazole skeleton, and is 1 or 2; n is R 2 is the number of phenyl groups bonded to the 3-position of the tetrazole skeleton, and is 1 or 2. p is the sulfo group (-SO3 - ) is the number of bonds to the phenyl group at the 3-position of the tetrazole skeleton, and is 0 or 1, n+p is 1 or more, q is 1 or 2, and when q is 2, each OR 3 are arranged adjacently, and in this case, each OR 3 may form a ring together, and each OR 3 When these groups form a ring together, the substituted benzothiazolyl group at the 2-position of the tetrazole skeleton is any one of the following substituents:

[0034] [ka]

[0035] X represents a hydrogen atom or an alkali metal, and is a tetrazolium salt represented by the above formula (1).

[0036] In the above formula (1), a substituted sulfonated phenyl group is present at the 5-position of the tetrazole skeleton. The substituent R of the sulfonated phenyl group 1 is any one selected from the group consisting of a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxy group. From the viewpoint of improving the water solubility of the tetrazolium salt and the formazan generated from the tetrazolium salt, R 1is preferably a hydrogen atom or a hydroxyl group, and can stably form a chelate with a transition metal ion over a wide pH range. 1 is more preferably a hydrogen atom. 1 When is a hydroxyl group, a methoxy group, or an ethoxy group, the substitution position is not particularly limited, but is preferably the 4-position.

[0037] At the 5th position of the tetrazole skeleton, there is a sulfo group (-SO3 - ) is present (m=1 or 2). This is thought to improve the water solubility of the tetrazolium salt and the formazan produced from the tetrazolium salt. In formula (1), m is a sulfo group (-SO3 - ) is the number of sulfo groups bonded to the phenyl group at the 5-position of the tetrazole skeleton, and is 1 or 2. In particular, when the sulfo group is at the 2- or 4-position, and further when it is at the 2- and 4-positions, further improvement in water solubility can be achieved. Furthermore, when the sulfo group is at the 2- and 4-positions, it is advantageous in that the synthesis of the building blocks for synthesis is easy. In order to obtain high water solubility and to stably form a chelate compound with a transition metal ion in a wide pH range, or to improve water solubility, it is preferable that m=2, and when m=2 and R 1 is more preferably a hydrogen atom.

[0038] In this case, when m=2, a sulfo group (-SO3 - ) bonded to the phenyl group at the 3-position of the tetrazole skeleton, p is preferably 1. By selecting such a number of substituents, the water solubility of the tetrazolium salt and the formazan produced therefrom is further improved.

[0039] From the viewpoint of water solubility, the following (1) to (4) are preferable: (1) m=2 and p=1, (2) m=1 and n=0, (3) in the phenyl group at the 5-position of the tetrazole skeleton, R 1 is a hydroxyl group, and in this case, a sulfo group (SO3 - ) and the hydroxyl groups are at the 2- and 4-positions, or the 4- and 6-positions, (4) p=0 and at least one R2 is a carboxyl group, and (1) m=2 and p=1, or (4) p=0 and at least one R 2 is more preferably a carboxyl group. In addition, in the above (3), a sulfo group (SO3 - ) and hydroxyl groups are not present as adjacent substituents on the benzene ring, so they do not form hydrogen bonds or only a small amount, which is thought to be why both substituents can efficiently contribute to water solubility.

[0040] Here, a sulfo group (-SO3 - The bonding position of the sulfo group (-SO3) is not particularly limited. When m=2, it is preferable to use a sulfo group (-SO3) in order to further improve the water solubility of the tetrazolium salt and the formazan produced from the tetrazolium salt and to shift the maximum absorption wavelength to the longer wavelength side. - ) are preferably present at the 2-, 4-, and 3-, and 5-positions of the phenyl group. It is particularly preferred that sulfo groups be present at the 2- and 4-positions of the phenyl group, and this makes it possible to obtain a tetrazolium salt compound that does not precipitate even in the presence of high concentrations of transition metal ions. In other words, by using this tetrazolium salt as a color-developing reagent, it is possible to prepare a reagent that can quantify even high concentrations of the analyte (biological component). In other words, in a preferred embodiment of the present invention, the phenyl group at the 5-position of the tetrazole skeleton is a sulfo group (-SO3 - ) is preferably a phenyl group located at the 2- and 4-positions.

[0041] In the above formula (1), a substituted phenyl group is present at position 3 of the tetrazole skeleton. Since the phenyl group is necessarily substituted, n+p is 1 or more.

[0042] R as a substituent of the phenyl group at the 3-position of the tetrazole skeleton 2 is a nitro group, -OR 4 and a carboxyl group. 2 From the viewpoint of the chelate formation between formazan and transition metal ions, the nitro group or -OR 4From the viewpoint of water solubility, n is preferably a carboxyl group. 2 is the number of phenyl groups bonded to the 3-position of the tetrazole skeleton, and is an integer of 0 to 2. 2 By introducing R, it is possible to shift the maximum absorption wavelength of the compound to a longer wavelength region and improve the stability of the compound, so it is preferable that n=1 or 2. 2 If there are two, i.e., n=2, then R 2 may be the same or different.

[0043] In the above formula (1), when n=1 or 2, at least one R 2 -OR 4 That is, in a preferred embodiment of the present invention, n is 1 or 2 and at least one R 2 -OR 4 The introduction of an alkoxy group as a substituent of the phenyl group improves the stability of the compound. From the viewpoint of improving the water solubility of the tetrazolium salt and the formazan generated from the tetrazolium salt, -OR 4 Preferably, the group is a methoxy group. 4 R is a methyl group or an ethyl group, and is preferably a methyl group from the viewpoint of water solubility. 4 When is an alkyl group having 3 or more carbon atoms, the tetrazolium salt and the formazan generated from the tetrazolium salt are not preferred because they have poor water solubility.

[0044] R when n is 1 or 2 2 The substitution position of R is not particularly limited, but is preferably the 2nd, 3rd, 4th, 5th or 6th position of the phenyl group at the 3rd position of the tetrazole skeleton. 2 is preferably at position 2 or 4, and more preferably at positions 2 and / or 4. Such a structure improves water solubility and the stability of the tetrazolium salt and the formazan generated from the tetrazolium salt.

[0045] In the above formula (1), p represents a sulfo group (-SO3 - ) is the number of bonds to the phenyl group at the 3-position of the tetrazole skeleton, and is 0 or 1. From the viewpoint of improving the water solubility of the tetrazolium salt and the formazan generated from the tetrazolium salt, p=1 is preferable. When p=1, the sulfo group is an electron-withdrawing group, and the presence of other electron-withdrawing groups (e.g., nitro groups) may destabilize the cationic charge of the nitrogen atom on the tetrazolium ring, thereby reducing the stability of the compound. As mentioned above, the stability of the compound is improved by introducing an alkoxy group as a substituent of the phenyl group, but if a nitro group is introduced at the same time, the improvement in stability due to the introduction of the alkoxy group may not be achieved. Therefore, from the viewpoint of improving stability, when p=1, n is 1 or 2, and preferably n is 1 and R 2 -OR 4 and a carboxyl group, and R 2 HA-OR 4 Alternatively, from the viewpoint of improving the water solubility of the tetrazolium salt and the formazan produced from the tetrazolium salt, it is more preferable that p=0 and R is at least 1. 2 is preferably a carboxyl group. That is, a preferred embodiment is a compound represented by the formula (1) in which p is 1, or p=0 and R is at least 1. 2 is a carboxyl group. More preferably, m=2 and p=1, or p=0 and at least 1 R 2 is a carboxyl group.

[0046] When p=1, the sulfo group (-SO3 - The substitution position of the sulfo group is not particularly limited, but is preferably the 3rd or 5th position. By substituting the sulfo group at this position, the stability of the tetrazolium salt and the formazan generated from the tetrazolium salt can be more effectively improved.

[0047] In the above formula (1), the substituent at the 3-position of the tetrazole skeleton is preferably 4-methoxy-3-sulfophenyl, 2-methoxy-5-sulfophenyl, 2-methoxy-4-nitro-5-sulfophenyl, 2-methoxy-4-nitrophenyl, 4-sulfophenyl, 4-carboxy-2-methoxyphenyl, 5-carboxy-2-methoxyphenyl, 3-carboxy-4-methoxyphenyl, or 4-methoxy-5-sulfophenyl, more preferably 4-methoxy-3-sulfophenyl, 2-methoxy-5-sulfophenyl, 3-carboxy-4-methoxyphenyl, or 4-methoxy-5-sulfophenyl, and particularly preferably 4-methoxy-3-sulfophenyl, 4-methoxy-5-sulfophenyl, or 2-methoxy-5-sulfophenyl. This structure improves color development sensitivity, water solubility, and the stability of the tetrazolium salt and the formazan produced from the tetrazolium salt. Furthermore, it is particularly preferable that the phenyl group at the 3-position of the tetrazole skeleton is a 4-methoxy-3-sulfophenyl group, since this allows the maximum absorption wavelength of the formazan compound itself to be in the longer wavelength region.

[0048] In the above formula (1), X represents a hydrogen atom or an alkali metal. Here, X represents an anion (sulfo group (-SO3 - )) is present to neutralize the alkali metal. Therefore, the type of alkali metal is not particularly limited, and may be any of lithium, sodium, potassium, rubidium, and cesium.

[0049] Preferred examples of tetrazolium salts include those having the following structure: In the following structure, X represents an alkali metal.

[0050] [ka]

[0051] [ka]

[0052] [ka]

[0053] [ka]

[0054] [ka]

[0055] Particularly preferred examples of tetrazolium salts include the following structures: That is, in a preferred embodiment of the present invention, the tetrazolium salt has the following structure:

[0056] [ka]

[0057] Tetrazolium salts may be synthesized or commercially available. For example, 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium, which is preferably used in the present disclosure, is commercially available from Dojindo Molecular Technologies, Inc. under the trade name WST-4. Furthermore, the tetrazolium salt of the above formula (1), which is preferably used in the present disclosure, can be produced, for example, by the method described in International Publication No. 2018 / 051822, or by an appropriate modification of that method, without being limited thereto. For example, a hydrazone is synthesized by dehydration condensation of an aldehyde and hydrazine, and then the corresponding diazonium salt is reacted in an aqueous solvent under basic conditions to obtain a formazan. Here, sodium hydroxide, potassium hydroxide, etc. are used as the basifying agent. The obtained formazan is then oxidized in an alcohol solvent (e.g., methanol, ethanol) using an oxidizing agent such as ethyl nitrite, butyl nitrite, or sodium hypochlorite to obtain the tetrazolium salt of formula (1). In one embodiment, the tetrazolium salt has the following structure:

[0058] [ka]

[0059] and hydrazino-substituted benzothiazoles having the structure:

[0060] [ka]

[0061] with a substituted sulfonated benzaldehyde having the structure:

[0062] [ka]

[0063] On the other hand, a hydrazone compound having the following structure:

[0064] [ka]

[0065] Hydrochloric acid was added to a substituted sulfonated aniline having the following structure while cooling it with ice, and sodium nitrite solution was further added dropwise to the substituted sulfonated aniline having the following structure:

[0066] [ka]

[0067] The hydrazone compound obtained above is reacted with a benzenediazonium chloride compound under basic conditions (for example, in the presence of sodium hydroxide or potassium hydroxide) to obtain a benzenediazonium chloride compound having the following structure:

[0068] [ka]

[0069] The formazan compound thus obtained is then oxidized using an oxidizing agent (e.g., a nitrite ester such as sodium nitrite, ethyl nitrite, or butyl nitrite) in an alcohol solvent (e.g., methanol or ethanol) to obtain the tetrazolium salt of formula (1).

[0070] When the reagent of the present disclosure is in liquid form, the concentration of the tetrazolium salt is not particularly limited as long as it is a concentration that allows the measurement of the desired biological component concentration, but it is preferable that the tetrazolium salt be contained in an amount sufficient for the amount of the desired biological component present. For example, taking into consideration the above-mentioned viewpoints and the biological component concentration that is typically to be measured, the concentration of the tetrazolium salt of formula (1) is, for example, 10 to 200 mmol / L, preferably 30 to 100 mmol / L, and more preferably 40 to 80 mmol / L. At such an amount, the tetrazolium salt of formula (1) reacts in accordance with the amount of substantially all (e.g., 95 mol % or more, preferably 98 mol % or more, and particularly preferably 100 mol %) of the biological component contained in the biological sample. Therefore, the concentration of the desired biological component can be measured accurately and quickly with good sensitivity.

[0071] When the reagent of the present disclosure is a solid (dry reagent), the content of the tetrazolium salt is, for example, 10 to 40 parts by mass, and preferably 18 to 30 parts by mass, relative to 100 parts by mass of the reagent. The content of the tetrazolium salt is substantially the same as the ratio of the amount of the color-developing dye charged when preparing the reagent.

[0072] When the concentration and content of the tetrazolium salt are within the above ranges, the tetrazolium salt can exhibit a sufficient amount of color development. Therefore, the concentration of an analyte (e.g., glucose concentration) in a whole blood sample can be measured with higher accuracy. When the reagent contains two or more tetrazolium salts, the above concentrations and contents of the tetrazolium salts refer to the total amount of the tetrazolium salts.

[0073] The formazan produced from the tetrazolium salt of formula (1) or the chelate compound of formazan and a transition metal ion, either alone or in combination with a transition metal compound, has a wide absorption wavelength band in a wavelength range (600 nm or greater) that does not overlap with the main absorption band of hemoglobin. Furthermore, the tetrazolium salt of formula (1) has high water solubility. Therefore, the use of the tetrazolium salt of formula (1) enables sensitive measurement of the concentration of a biological component even in a whole blood sample. Specifically, the maximum absorbance (in a 200 mg / dL glucose aqueous solution, cell length = 0.045 mm) of the formazan produced from the tetrazolium salt of formula (1) or the chelate compound of formazan and a transition metal ion at 600 nm to 800 nm is 0.3 or greater, preferably 0.5 or greater. Formazan or a chelate compound of formazan and a transition metal ion (and therefore a tetrazolium salt capable of producing such a formazan) having such a maximum absorption wavelength is less susceptible to absorption by blood, allowing the concentration of a biological component to be measured more accurately and with better sensitivity. Unless otherwise specified, the maximum absorption wavelength (λmax) of a chelate compound of formazan and a transition metal ion is used herein as the value measured according to the following method.

[0074] -Evaluation of the maximum absorption wavelength (λmax) of chelate compounds of formazan and transition metal ions- A 10 mM MOPS aqueous solution was added to prepare a sample so that the final concentration of the formazan compound was 50 to 200 mM. Separately, a 1 M aqueous solution of a transition metal (for example, nickel ions) was prepared.

[0075] To 100 μL of the sample, 10 μL of a separately prepared aqueous solution of transition metal ions was added and quickly stirred to prepare a mixed solution. The spectrum of this mixed solution was measured using a spectrophotometer (measurement cell length: 10 mm). Based on the spectrum, the maximum absorption and its wavelength (maximum absorption wavelength (λmax)) above 600 nm were determined. Note that the maximum absorption wavelength (λmax) can be selected at any wavelength within the range that satisfies the maximum absorbance (0.3 or higher) above 600 nm.

[0076] (Compounds that can generate transition metal ions that can take a regular tetrahedral coordination structure) Reagents of the present disclosure include compounds capable of generating transition metal ions capable of adopting tetrahedral coordination geometries.

[0077] Among transition metal ions that can form a regular tetrahedral coordination structure, zinc ions (Zn 2+ ), cobalt ions (Co 2+ ) and the like, and zinc ions are preferred from the viewpoint of further suppressing changes in color-developing properties (chelate structure). In a preferred embodiment of the present invention, the transition metal in the compound capable of generating a transition metal ion capable of adopting a regular tetrahedral coordination structure is zinc.

[0078] The transition metal ions that can form chelate compounds with formazan produced from tetrazolium salts include nickel ions (Ni 2+ ), copper ions (Cu 2+ ) are known. Nickel ions and copper ions mainly have two coordination structures, which is thought to cause the chelate structure to change over time, resulting in a wavelength shift. On the other hand, zinc ions mainly have one coordination structure, which does not cause a wavelength shift due to a change in the chelate structure. Therefore, it is thought that it is possible to suppress changes in the color-developing properties (chelate structure) over time.

[0079] Examples of compounds capable of generating transition metal ions capable of forming a regular tetrahedral coordination structure include zinc acetate, zinc chloride, cobalt chloride, etc. From the viewpoint of being able to more effectively exhibit the effects of the present invention, the compound capable of generating transition metal ions capable of forming a regular tetrahedral coordination structure is zinc acetate.

[0080] When the reagent of the present disclosure is in liquid form, the concentration of the compound capable of generating transition metal ions capable of adopting a regular tetrahedral coordination structure is, for example, 10 to 500 mmol / L, and preferably 50 to 110 mmol / L.

[0081] When the reagent of the present disclosure is a solid (dry reagent), the content (in terms of solid content) of the compound capable of generating transition metal ions capable of adopting a regular tetrahedral coordination structure is preferably 5 to 30 parts by mass, and more preferably 7 to 30 parts by mass, per 100 parts by mass of the dry reagent.

[0082] The concentration of the compound capable of generating transition metal ions capable of forming a regular tetrahedral coordination structure is, for example, 0.1 to 10 mol, preferably 0.5 to 4 mol, and more preferably 1 to 3 mol, relative to 1 mol of the tetrazolium salt.

[0083] The molar ratio of the compound capable of generating a transition metal ion capable of forming a regular tetrahedral coordination structure to the tetrazolium salt is, for example, 100 to 250%, and from the viewpoint of further improving blood spreading, is preferably 116% or more but less than 231%, and more preferably 116 to 145%.

[0084] (oxidoreductase) The reagents of the present disclosure include oxidoreductase enzymes.

[0085] The oxidoreductase is not particularly limited and can be appropriately selected depending on the type of biological component to be measured. Specific examples include glucose dehydrogenases (GDH), such as glucose dehydrogenase (GDH) with pyrroloquinoline quinone (PQQ) as a coenzyme (PQQ-GDH), glucose dehydrogenase with flavin adenine dinucleotide (FAD) as a coenzyme (FAD-GDH), glucose dehydrogenase with nicotinamide adenine dinucleotide (NAD) as a coenzyme (NAD-GDH), and glucose dehydrogenase with nicotine adenine dinucleotide phosphate (NADP) as a coenzyme (NADP-GDH), glucose oxidase (GOD), lactate dehydrogenase (LDH), cholesterol dehydrogenase, cholesterol oxidase, and urate dehydrogenase. Here, the oxidoreductase may be used alone or in combination of two or more. For example, when the analyte (biological component) is glucose, the oxidoreductase is preferably glucose dehydrogenase or glucose oxidase. Furthermore, when the analyte (biological component) is cholesterol, the oxidoreductase is preferably cholesterol dehydrogenase or cholesterol oxidase. The concentration of the oxidoreductase is not particularly limited and can be appropriately selected depending on the amount of the tetrazolium salt.

[0086] (Other ingredients) The reagent of the present disclosure may contain, in addition to a tetrazolium salt, a compound capable of generating a transition metal ion capable of adopting a regular tetrahedral coordination structure, and an oxidoreductase, other components such as a hemolytic agent, nitrous acid or a salt thereof, a disaccharide or a derivative thereof, a sugar alcohol, and a pH buffer.

[0087] When the biological sample containing a biological component is a whole blood sample, the reagent of the present disclosure preferably further comprises a hemolyzing agent, nitrous acid or a salt thereof, and sucralose, and more preferably further comprises a hemolyzing agent, nitrous acid or a salt thereof, sucralose, and a sugar alcohol.

[0088] (hemolytic agent) The reagent of the present disclosure may contain a hemolytic agent. Generally, when measuring the glucose concentration in plasma, the amount of red blood cells (hematocrit value) must be taken into consideration. The amount of red blood cells is usually determined by the amount of hemoglobin, but optical measurement of hemoglobin is difficult. Furthermore, the actual amount of red blood cells and the amount of hemoglobin may not match. This can make correction difficult. Furthermore, light scattering by red blood cells may require a special measurement technique to measure the amount of red blood cells. In contrast, the reagent of the present disclosure hemolyzes red blood cells, allowing for a simpler method to obtain a more accurate hematocrit value. Furthermore, red blood cells typically contain the same concentration of analyte (e.g., glucose) as that contained in plasma. Therefore, the reagent of the present disclosure can measure the concentration of an analyte (biological component) (e.g., glucose) in plasma and blood cells (i.e., a whole blood sample) (i.e., the concentration of the analyte in the whole blood sample can be determined). One type of hemolytic agent may be used alone, or two or more types may be used in combination.

[0089] Examples of hemolytic agents that can be used include nonionic surfactants, amphoteric surfactants, anionic surfactants, etc. Nonionic surfactants are preferred from the viewpoint of not inhibiting the activity of the enzyme.

[0090] The hemolytic agent may be used alone or in combination of two or more.

[0091] The nonionic surfactant preferably has an HLB value of 11 or more and 15 or less (more preferably 12 or more and 14 or less). Examples of such nonionic surfactants include polyoxyethylene alkyl ethers and nonylphenyl polyethylene glycols, in which the average number of moles of oxyethylene groups added is 1 or more and 150 or less and the alkyl group has 1 or more and 18 or less carbon atoms. Such nonionic surfactants may be synthesized or commercially available. Examples of commercially available products include polyoxyethylene (9) octylphenyl ether (octylphenoxypoly(ethyleneoxy)ethanol or octylphenyl-polyethylene glycol) (Sigma-Aldrich, Nonidet).TM Polyoxyethylene pt-octylphenyl ethers (Triton surfactants) such as Triton® P-40, Triton® X-100 (polyoxyethylene (10) octylphenyl ether), and Triton® X-114 (polyoxyethylene (8) octylphenyl ether); polyoxyethylene sorbitan fatty acid esters such as Tween® 85; Dodecyl-β-D-maltose; Octyl-β-D-glucoside; Nonidet® P-40 (octylphenoxypoly(ethyleneoxy)ethanol) and Nonidet® P-40 substitutes; Tergitol® NP-10 Surfactant (Nonylphenol Ethoxylate); IGEPAL® CA-630 (octylphenoxypoly(ethyleneoxy)ethanol); Emulgen® 108 (polyoxyethylene lauryl ether), Emulgen® 109P; Brij® 96 Polyethylene glycol monooleyl ether (n = approximately 2) can be used.

[0092] Amphoteric surfactants and anionic surfactants may also be synthesized or commercially available. Commercially available products include CHAPS (3-(3-cholamidepropyl)dimethylammonio-1-propanesulphonate), alkylpolyaminoethylglycine chloride, and sodium dodecyl sulfate. Saponin may also be used.

[0093] The concentration of the hemolytic agent can be appropriately selected depending on the sample volume (volume of whole blood). When the reagent of the present disclosure is liquid, the concentration of the hemolytic agent is, for example, 2 to 10 mass %, and preferably 3 to 5 mass %, relative to the total mass of the reagent. When the reagent is in the form of a sensor, the content of the hemolytic agent is, for example, 10 to 50 mass parts, preferably 15 to 40 mass parts, and more preferably about 20 to 35 mass parts, relative to 100 mass parts of the reagent. For example, in the case of a sensor for a 1.0 μL whole blood sample, the composition (content) of the hemolytic agent in the reagent (in terms of solid content) is 1 to 10% by volume.

[0094] When the reagent contains two or more kinds of hemolytic agents, the above-mentioned concentration of the hemolytic agents refers to the total amount of the hemolytic agents contained.

[0095] (Nitrite or its salts (nitrite (salt)) When hemolyzed whole blood samples are used, hemoglobin (Fe(II)) is released from red blood cells. Hemoglobin non-enzymatically reduces tetrazolium salts to produce colored formazan (false color development occurs). However, nitrite (salt) does not react with the divalent iron ions (Fe(II)) contained in hemoglobin. 2+ ) to trivalent iron ions (Fe 3+ ), which acts as a methemoglobin-forming agent. Therefore, even when a hemolyzed whole blood sample is measured, nitrite (salt) preferentially accepts electrons from hemoglobin released from red blood cells compared to tetrazolium salts. This suppresses non-enzymatic reduction of tetrazolium salts (false color development).

[0096] Examples of nitrites that can be used include sodium nitrite, potassium nitrite, calcium nitrite, and ammonium nitrite. Among nitrites, sodium nitrite and potassium nitrite are preferred, with sodium nitrite being more preferred, due to their high stability and versatility. Nitrite (salts) may be used singly or in combination of two or more.

[0097] The concentration of nitrite (salt) is, for example, 10 to 200 mmol / L, and is preferably 50 to 80 mmol / L, since this allows the concentration of the analyte (for example, glucose concentration) to be measured with higher accuracy and sensitivity.

[0098] The concentration of nitrous acid (salt) is, for example, 0.2 to 10 mol per mol of tetrazolium salt, and is preferably 1.0 to 3.0 mol since this allows the concentration of the analyte (for example, glucose concentration) to be measured with higher accuracy and sensitivity.

[0099] When the reagent is shaped like a sensor, the content of nitrite (salt) (calculated as sodium nitrite) is, for example, 0.8 to 10 parts by mass, preferably 1.0 to 7.5 parts by mass, and more preferably 1.3 to 5 parts by mass, relative to 100 parts by mass of the reagent. When other salt compounds such as potassium nitrite or nitrite are blended, the content can also be a value calculated as sodium nitrite and converted from these ranges.

[0100] (Other additives) Disaccharides or their derivatives When the reagent of the present disclosure is a dry reagent, the use of a disaccharide or its derivative can suppress the precipitation of compounds capable of generating transition metal ions capable of forming a tetrahedral coordination structure, nitrite (salt), etc., thereby making the reagent transparent. Therefore, dissolution of the dry reagent in the sample is promoted, allowing the concentration of the analyte (biological component) to be measured with higher accuracy.

[0101] In a preferred embodiment of the present invention, the reagent for measuring the concentration of a biological constituent contains a tetrazolium salt (particularly the tetrazolium salt of the above formula (1)) as a color-developing dye, and further contains at least one of sucralose (1',4,6'-trichlorogalactosucrose), sucrose, and trehalose as a disaccharide or derivative thereof. Of these, sucralose is most preferred.

[0102] The concentration of the disaccharide or its derivative is not particularly limited and can be appropriately selected depending on the composition of the reagent, etc. The concentration of the disaccharide or its derivative is, for example, 100 to 200 mmol / L.

[0103] The concentration of the disaccharide or its derivative is, for example, 1 to 10 mol, preferably 2 to 4 mol, relative to 1 mol of the tetrazolium salt.

[0104] When formed into a sensor shape, the content of the disaccharide or its derivative (in terms of solid content) is, for example, 10 to 45 parts by mass, preferably 15 to 40 parts by mass, and more preferably 20 parts by mass or more but less than 40 parts by mass, per 100 parts by mass of the reagent.

[0105] sugar alcohols When the reagent of the present disclosure is a dry reagent, the use of a sugar alcohol can improve wettability. Therefore, the dry reagent can quickly blend with and dissolve in the sample. As a result, the reaction between the dry reagent and the sample proceeds uniformly and without unevenness, allowing for more accurate measurement of the concentration.

[0106] Examples of sugar alcohols include lactitol, erythritol, sorbitol, xylitol, mannitol, maltitol, maltotriose, etc. Among these, from the viewpoint of further improving wettability, erythritol, sorbitol, xylitol, and mannitol are preferred, erythritol, sorbitol, xylitol, and mannitol are more preferred, and sorbitol is particularly preferred.

[0107] The sugar alcohols may be used alone or in combination of two or more.

[0108] The concentration of the sugar alcohol is not particularly limited and can be appropriately selected depending on the composition of the reagent, etc. The concentration of the sugar alcohol is, for example, 5 to 100 mmol / L, and from the viewpoint of further improving wettability, is preferably 15 to 30 mmol / L.

[0109] The concentration of the sugar alcohol is, for example, 0.1 to 3.0 mol, and preferably 0.3 to 2.0 mol, relative to 1 mol of the tetrazolium salt.

[0110] When formed into a sensor, the sugar alcohol content is, for example, 0.1 to 15 parts by mass, preferably 0.5 to 10 parts by mass, and more preferably more than 1 part by mass and less than 8 parts by mass, relative to 100 parts by mass of the reagent.

[0111] When the reagent of the present disclosure contains a disaccharide or a derivative thereof and a sugar alcohol, the content of the sugar alcohol is, for example, 1 mole or more, preferably more than 10 moles, per 100 moles of the disaccharide or a derivative thereof, from the viewpoint of achieving a good balance between the effects of the disaccharide or a derivative thereof and the effects of the sugar alcohol. The content of the sugar alcohol is, for example, more than 0 to 60 moles, preferably more than 0 to less than 25 moles, per 100 moles of the disaccharide or a derivative thereof.

[0112] The reagent may be used in any form, including solid, gel, sol, or liquid. The reagent may further contain water, a buffer (pH buffer), etc. The buffer is not particularly limited, and buffers generally used for measuring the concentration of biological components can be used in the same manner. Specific examples include phosphate buffer, citrate buffer, citrate-phosphate buffer, trishydroxymethylaminomethane-HCl buffer (trishydrochloric acid buffer), MES buffer (2-morpholinoethanesulfonic acid buffer), TES buffer (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer), acetate buffer, MOPS buffer (3-morpholinopropanesulfonic acid buffer), MOPS-NaOH buffer, HEPES buffer, etc. Examples of buffers that can be used include GOOD buffers (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), HEPES-NaOH buffer, and other GOOD buffers; amino acid buffers (glycine-hydrochloric acid buffer, glycine-NaOH buffer, glycylglycine-NaOH buffer, and glycylglycine-KOH buffer); boric acid buffers (Tris-borate buffer, boric acid-NaOH buffer, and boric acid buffer); and imidazole buffers. Among these, phosphate buffer, citrate buffer, citrate-phosphate buffer, Tris-HCl buffer, MES buffer, acetate buffer, MOPS buffer, and HEPES-NaOH buffer are preferred. The concentration of the buffer is not particularly limited, but is preferably 0.01 to 1.0 M. In the present invention, the concentration of the buffer refers to the concentration (M, mol / L) of the buffer contained in the aqueous solution. From the above viewpoint, the pH of the buffer solution is preferably near neutral, for example, about 5.0 to 8.0.

[0113] Preferably, the reagents are used in a solid (dry state), i.e., in a preferred embodiment of the present invention, the reagents are dry reagents.

[0114] The content of each component contained in the dry reagent can be calculated from the concentration of each component contained in the liquid reagent and the volume of the liquid reagent used.

[0115] By using the reagent of the present disclosure, the concentration of a specific biological component contained in a biological sample (a biological component measurement target) can be measured with high accuracy. The biological component measurement target is not particularly limited as long as it contains the target biological component. Specific examples include blood, as well as body fluids such as urine, saliva, and interstitial fluid. In one embodiment, the biological component measurement target is blood, preferably whole blood.

[0116] A second aspect of the present disclosure is a method for measuring the concentration of a biological component, comprising contacting a whole blood sample with a biological component concentration measurement reagent according to the first aspect of the present disclosure, measuring the amount of color development, and quantifying the concentration of the biological component in the whole blood sample based on the amount of color development.

[0117] In a second aspect of the present disclosure, the biological component to be measured is a whole blood sample. The whole blood sample is added to the above-described biological component concentration measurement reagent or a sensor or component measurement device (e.g., a blood glucose meter) containing the reagent, and the whole blood sample is dissolved and mixed with the reagent for measurement. The biological component is not particularly limited, and any biological component typically measured by colorimetry or electrode methods can be used in the same manner. Specific examples include glucose, cholesterol, triglycerides, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), and uric acid. In other words, according to a preferred embodiment of the present disclosure, the reagent of the present disclosure is used to measure the concentrations of glucose, cholesterol, triglycerides, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), or uric acid in whole blood. Furthermore, according to a preferred embodiment of the present invention, the biological component is glucose, cholesterol, neutral fat, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), or uric acid.

[0118] In the present disclosure, the measurement method is not particularly limited and can be selected appropriately depending on the type of biological component to be measured. For example, when the biological component is β-D-glucose and the oxidoreductase is glucose dehydrogenase (GDH), glucose is oxidized by GDH to produce gluconic acid. Taking advantage of the reduction of the GDH coenzyme or electron carrier during this process, specific methods can be broadly classified into two types: a method that optically measures the degree of coloration of the resulting reduced tetrazolium salt (hence, formazan or a chelate compound of formazan and a transition metal ion) (colorimetric method), and a method that measures the current generated by the redox reaction (electrode method). Among these methods, measuring blood glucose levels using colorimetric methods has advantages such as easy correction using hematocrit values ​​when calculating blood glucose levels and a simple manufacturing process. For this reason, reagents for measuring the concentrations of whole blood components are suitable for use in colorimetric methods. Colorimetric methods are particularly preferred when measuring glucose concentrations in whole blood samples.

[0119] The reagent of the present disclosure may be used directly to measure the concentration of a biological component, or may be incorporated into a sensor (sensor for measuring the concentration of a biological component). Specifically, the present invention also provides a sensor (hereinafter simply referred to as "sensor") for measuring the concentration of a biological component in a whole blood sample, the sensor having a reaction unit, the reaction unit containing the biological component concentration measurement reagent of the present disclosure. The reagent and method of the present disclosure can be incorporated into an automatic analyzer, a measurement kit, a simple blood glucose meter, or the like, and used for routine clinical testing. The reagent of the present disclosure can also be incorporated into a commercially available biosensor. When incorporating the reagent of the present disclosure into a sensor, the content of the reagent per sensor is not particularly limited and can be the same as that typically used in the relevant field. However, it is preferable that the tetrazolium salt (particularly the tetrazolium salt of formula (1)) be contained in an amount sufficient for the amount of the desired biological component present. Considering the above viewpoints and the concentration of the biological component to be typically measured, the concentration of the tetrazolium salt per sensor is preferably 3 to 50 nmol, more preferably 10 to 30 nmol. In such an amount, the tetrazolium salt reacts with substantially all of the biological components contained in the whole blood sample in accordance with their amounts, thereby enabling the concentration of the desired biological component to be measured accurately, quickly, and with good sensitivity.

[0120] The following describes the configuration of a measurement sensor (colorimetric blood glucose meter) of the present disclosure used to measure blood glucose levels by colorimetry, with reference to the drawings. However, the present invention is characterized by the use of the biological constituent concentration measurement reagent of the present disclosure, and the structure of the sensor is not particularly limited. Therefore, the biological constituent concentration measurement reagent of the present disclosure may be applied to commercially available measurement sensors or chips, as well as sensors or chips described in publications such as International Publication Nos. 2016 / 051930, 2018 / 51822, and 2020 / 137532. Similarly, in the following embodiments, specific configurations of sensors intended for measuring blood glucose levels are described, but the measurement sensor is not limited to this application and can be applied to other applications in the same manner or with appropriate modifications.

[0121] FIG. 1 is a plan view schematically showing a blood glucose meter used to detect glucose (blood sugar) using a measurement sensor according to this embodiment.

[0122] In FIG. 1, the blood glucose meter 10 is configured as a device for measuring glucose (blood sugar) in a whole blood sample. This blood glucose meter 10 can be mainly used for personal use by a user (subject). A user can also measure their own blood sugar level before meals to manage their own blood sugar. The blood glucose meter 10 can also be used by medical professionals to evaluate the health condition of a subject. In this case, the blood glucose meter 10 may be appropriately modified so that it can be installed in a medical facility or the like.

[0123] The blood glucose meter 10 employs the principle of colorimetry to optically measure the glucose content (blood glucose level) in a whole blood sample. In particular, the blood glucose meter 10 performs blood glucose measurement using a transmission-type measurement unit 14 that irradiates the analysis sample (blood) with measurement light of a predetermined wavelength and receives the light that has passed through the analysis sample.

[0124] The blood glucose meter 10 detects glucose using a measurement unit 14 by attaching a measurement sensor 12 into which blood has been drawn, or by drawing blood into the measurement sensor 12 while the measurement sensor 12 is being worn. The measurement sensor 12 may be configured as a disposable type that is discarded after each measurement. On the other hand, the blood glucose meter 10 is preferably configured as a portable and robust device so that the user can easily repeat measurements.

[0125] As shown in FIG. 2, the measurement sensor 12 includes a sensor body 18 formed in a plate shape, and a cavity 20 (liquid cavity) extending in the planar direction of the plate surface inside the sensor body 18.

[0126] 1 and 2, the sensor main body 18 is formed in a rectangular shape with long sides 22 in the insertion and removal direction of the blood glucose meter 10 (directions toward the tip and base of the blood glucose meter 10, i.e., direction B) and short sides 24 in direction A. For example, the length of the long sides 22 of the sensor main body 18 is preferably set to be at least twice the length of the short sides 24. This ensures that the measurement sensor 12 can be inserted sufficiently far into the blood glucose meter 10.

[0127] The thickness of the sensor main body 18 is formed to be extremely small (thin) compared to the rectangular side surface (in FIG. 2, it is intentionally illustrated as having a sufficient thickness). For example, the thickness of the sensor main body 18 is preferably set to 1 / 10 or less of the above-mentioned short side 24. The thickness of the sensor main body 18 may be designed appropriately according to the shape of the insertion hole 58 of the blood glucose meter 10.

[0128] The measurement sensor 12 has a sensor body 18 formed of a pair of plate pieces 30 and a pair of spacers 32 so as to have a cavity 20 .

[0129] 3 is a top view of the measurement sensor of FIG. 1. In FIG. 3, the corners of sensor main body 18 are sharp, but the corners may be rounded, for example. Furthermore, sensor main body 18 is not limited to a thin plate shape, and its shape may be freely designed. For example, sensor main body 18 may be formed in a square shape, another polygonal shape, or a circle (including an oval shape) when viewed from above.

[0130] The cavity 20 provided inside the sensor main body 18 is located at the center of the sensor main body 18 in the short axis direction and is linearly formed along the longitudinal direction of the sensor main body 18. This cavity 20 is connected to a distal opening 20a formed at the distal edge 24a of the sensor main body 18 and a proximal opening 20b formed at the proximal edge 24b, and is connected to the outside of the sensor main body 18. When the user's blood is taken into the cavity 20 through the distal opening 20a, the blood can flow along the extension direction due to capillary action. The amount of blood flowing through the cavity 20 is small, and even if the blood moves to the proximal opening 20b, leakage is prevented by tension. Note that an absorbent section for absorbing blood (e.g., a spacer 32 described below with only the proximal side porous) may be provided on the proximal edge 24b side of the sensor main body 18.

[0131] Furthermore, a reagent (coloring reagent) 26 that reacts with glucose (blood sugar) in the blood to change color according to the glucose (blood sugar) concentration in the blood is applied to a predetermined position of the cavity 20 (for example, a position slightly closer to the proximal end than the midpoint between the distal opening 20a and the proximal opening 20b shown in FIG. 3), and a measurement object 28 that is measured by the blood glucose meter 10 is set therein. Blood flowing proximally through the cavity 20 comes into contact with the reagent 26 applied to the measurement object 28, and the blood reacts with the reagent 26, changing color. Note that the application position of the reagent 26 and the measurement object 28 may be offset from each other in the longitudinal direction of the cavity 20; for example, the reaction area to which the reagent 26 is applied may be provided upstream of the measurement object 28 in the direction of blood flow.

[0132] The measurement sensor 12 has a sensor body 18 formed of a pair of plate pieces 30 and a pair of spacers 32 so as to have the above-described cavity 20. The pair of plate pieces 30 are each formed in the above-described rectangular shape when viewed from the side, and are arranged relative to each other in the stacking direction. In other words, the pair of plate pieces 30 form both side surfaces (upper and lower surfaces) of the sensor body 18. The plate thickness of each plate piece 30 is very small, and is preferably set to the same dimension of, for example, about 5 to 50 μm. The thicknesses of the two (pair) plate pieces 30 may be different from each other.

[0133] The pair of plate pieces 30 have the strength to maintain their plate shape and not undergo plastic deformation even when a certain amount of pressure is applied from a direction perpendicular to the surface direction. Each plate piece 30 also has a transparent or semi-transparent portion that allows measurement light to pass through. Furthermore, each plate piece 30 is preferably formed with a flat plate surface that has appropriate hydrophilicity to allow blood to flow in the cavity 20.

[0134] The material constituting each plate piece 30 is not particularly limited, but may be a thermoplastic resin material, glass, quartz, etc. Examples of the thermoplastic resin material include polymer materials such as polyolefin (e.g., polyethylene, polypropylene, etc.), cycloolefin polymer, polyester (e.g., polyethylene terephthalate (PET), polyethylene naphthalate, etc.), polyvinyl chloride, polystyrene, ABS resin, acrylic resin, polyamide, fluororesin, etc., or mixtures thereof.

[0135] The pair of spacers 32 are disposed so as to be sandwiched between the pair of plate pieces 30 and are firmly bonded to the opposing surfaces of each plate piece 30 by a predetermined bonding means (such as an adhesive). In other words, each spacer 32 is disposed between the pair of plate pieces 30 so as to separate them, thereby forming a cavity 20 between the pair of plate pieces 30 and the pair of spacers 32 themselves. In this case, one spacer 32 is disposed so as to contact the upper long side 22a of the sensor main body 18 in FIG. 3 and extend along this upper long side 22a in the distal and proximal directions. The other spacer 32 is disposed so as to contact the lower long side 22b of the sensor main body 18 in FIG. 3 and extend along this lower long side 22b in the distal and proximal directions.

[0136] The material (substrate) constituting the pair of spacers 32 is not particularly limited, and examples thereof include various thermoplastic elastomers such as styrene-based, polyolefin-based, polyurethane-based, polyester-based, polyamide-based, polybutadiene-based, trans-polyisoprene-based, fluororubber-based, and chlorinated polyethylene-based. Alternatively, various elastically deformable materials other than thermoplastic elastomers may be used, as well as structures such as elastically deformable porous bodies (e.g., sponges). Furthermore, the spacers 32 may have an adhesive on one or both surfaces of the substrate that adheres the pair of plate pieces 30 together by becoming cured or semi-cured between the plate pieces 30. Furthermore, the spacers 32 may contain a reagent 26, which dissolves into the cavity 20.

[0137] The plate pieces 30 and the spacers 32 may be hydrophilized. Examples of methods for hydrophilization include applying an aqueous solution containing a hydrophilic polymer such as a surfactant, polyethylene glycol, polypropylene glycol, hydroxypropyl cellulose, or water-soluble silicone, as well as polyacrylic acid, polyvinylpyrrolidone, or polyacrylamide, by immersion or spraying, or by plasma irradiation, glow discharge, corona discharge, or ultraviolet irradiation (e.g., excimer light irradiation), and these methods may be used alone or in combination.

[0138] Next, the device main body 16 of the blood glucose meter 10 will be described. As shown in Fig. 1, the blood glucose meter 10 has a housing 40 that forms the exterior. The housing 40 includes a box-shaped portion 44 that is sized to be easily gripped by a user and houses a control portion 42 of the blood glucose meter 10 therein, and a cylindrical photometric portion 46 that protrudes from one side (the tip side) of the box-shaped portion 44 toward the tip and houses the measurement portion 14 of the optical system therein. In addition, a power button 48, an operation button 50, and a display 52 are provided on the top surface of the box-shaped portion 44, and an eject lever 54 is provided on the top surface of the photometric portion 46.

[0139] The power button 48 switches between starting and stopping the blood glucose meter 10 under user operation. The operation button 50 functions as an operation unit that measures and displays blood glucose levels, switches the display of measurement results (including past measurement results), etc., based on user operation in the activated blood glucose meter 10. The display 52 is made up of liquid crystal, organic electroluminescence, etc., and displays information to be provided to the user during measurement operations, such as measurement results and error indications.

[0140] The eject lever 54 is provided so as to be movable in the distal and proximal directions, and unlocks an eject pin (not shown) provided in the photometry unit 46, allowing the eject pin to advance in the distal direction.

[0141] On the other hand, the photometry unit 46 of the device main body 16 extends from the box portion 44 toward the tip so that the tip can be pressed against the user's finger or the like. As shown in Figure 2, this photometry unit 46 is provided with a sensor attachment portion 60 having an insertion hole 58 and a measurement unit 14 that optically detects glucose (blood sugar) in the blood.

[0142] The sensor mounting part 60 is made of a highly hard (rigid) material (e.g., stainless steel) and has a cylindrical shape with a predetermined axial length, with an outwardly protruding flange 60a at its tip. The sensor mounting part 60 is positioned and fixed over the tip surface and axial center (center) of the photometry part 46, which is made of a resin material. As shown in FIG. 4A, a fixing wall 46a is formed on the inner surface of the photometry part 46 to firmly fix the sensor mounting part 60.

[0143] Examples of materials that can be used to form the sensor mounting unit 60 include metals such as stainless steel and titanium, anodized aluminum, liquid crystal polymers, plastics containing fillers such as glass or mica, plastics with hardened surfaces such as nickel plating, carbon fiber, and fine ceramics. These materials are hard and do not easily change dimensions, are resistant to wear even when repeatedly inserting and removing measurement sensors, and can be processed with high dimensional accuracy. Among these, metal materials are preferred, allowing the insertion hole 58 to be easily formed with high dimensional accuracy during the manufacture of the sensor mounting unit 60 (by injection molding, press molding, or the like). The sensor mounting unit 60 may be integrally formed with the device main body 16 by forming the photometric unit 46 itself from a hard material (e.g., a metal material).

[0144] An insertion hole 58 is provided at the axial center of the sensor mounting unit 60, surrounded by a wall 62 of the sensor mounting unit 60. The insertion hole 58 is formed in a rectangular cross section that is long in the insertion direction (direction B) and short in the left-right width direction (direction A). When the sensor mounting unit 60 is fixed to the photometry unit 46, the insertion hole 58 has a predetermined depth from its tip surface toward the back (proximal end).

[0145] An insertion opening 58a that is continuous with the insertion hole 58 and communicates with the outside is formed on the tip side of the sensor attachment part 60. The dimension of this insertion opening 58a in the insertion direction (direction B) matches the dimension of the short side 24 (length in direction A) of the measurement sensor 12. In addition, the dimension of the insertion opening 58a in the left-right width direction, i.e., the distance between a pair of wall portions 62 that form the side surfaces of the insertion hole 58, is substantially the same as the thickness of the measurement sensor 12 in the stacking direction (Tall in FIG. 4A), as shown in FIG.

[0146] The sensor mounting section 60 cooperates with the fixed wall 46a of the photometry section 46 to form a pair of element accommodating spaces 64 at an intermediate position along which the insertion hole 58 (measurement hole section 59) extends. The pair of element accommodating spaces 64 are part of the measurement section 14, are provided at positions facing each other across the insertion hole 58, and communicate with the measurement hole section 59 via the respective light guiding sections 66 formed by the sensor mounting section 60.

[0147] The measurement unit 14 comprises a light-emitting unit 70 by accommodating a light-emitting element 68 in one element accommodating space 64, and a light-receiving unit 74 by accommodating a light-receiving element 72 in the other element accommodating space 64. The light-guiding unit 66 of the sensor mounting unit 60 is formed as a circular hole with an appropriate diameter, and serves as a so-called aperture.

[0148] The light-emitting element 68 of the light-emitting unit 70 includes a first light-emitting element 68a that irradiates the measurement sensor 12 with measurement light having a first wavelength, and a second light-emitting element 68b that irradiates the measurement sensor 12 with measurement light having a second wavelength different from the first wavelength (not shown in FIG. 2). The first light-emitting element 68a and the second light-emitting element 68b are arranged side by side in positions facing the light-guiding unit 66 of the element accommodating space 64.

[0149] The light-emitting element 68 (first and second light-emitting elements 68a, 68b) may be composed of light-emitting diodes (LEDs). The first wavelength is a wavelength for detecting the color density of the reagent 26 corresponding to the blood glucose level, and is, for example, 600 nm to 680 nm. The second wavelength is a wavelength for detecting the red blood cell concentration in the blood, and is, for example, 510 nm to 540 nm. The control unit 42 in the box portion 44 supplies a drive current to cause the first and second light-emitting elements 68a, 68b to emit light at a predetermined timing. In this case, the blood glucose level obtained from the color density is corrected using the hematocrit value obtained from the red blood cell concentration to determine the blood glucose level. Note that noise caused by blood cells may be corrected by further measuring at another measurement wavelength.

[0150] The light receiving section 74 is configured by arranging one light receiving element 72 at a position facing the light guiding section 66 of the element accommodating space 64. This light receiving section 74 receives transmitted light from the measurement sensor 12, and can be configured by, for example, a photodiode (PD).

[0151] An eject pin 56 (eject portion) connected to the eject lever 54 is provided at the bottom (base end surface) of the insertion hole 58. The eject pin 56 includes a rod portion 56a extending along the axial direction of the photometric unit 46 and a receiving portion 56b having a large diameter at the tip of the rod portion 56a and located radially outward. The receiving portion 56b comes into contact with the base end edge 24b of the measurement sensor 12 inserted into the insertion hole 58. A coil spring 76 is provided between the bottom of the insertion hole 58 and the receiving portion 56b of the eject pin 56, surrounding the eject pin 56 without contacting it. The coil spring 76 elastically supports the receiving portion 56b of the eject pin 56.

[0152] When the insertion of the measurement sensor 12 is completed, the measurement target portion 28 of the measurement sensor 12 is positioned so as to overlap the light guide portion 66, as shown in FIG. 4B.

[0153] When the user inserts the measurement sensor 12, the receiving portion 56b is pressed, displacing the eject pin 56 toward the proximal end, and the eject pin 56 is locked (fixed) by a locking mechanism (not shown) provided in the housing 40. The coil spring 76 elastically contracts in accordance with the displacement of the receiving portion 56b. When the user operates the eject lever 54 and the eject pin 56 moves slightly, the locking mechanism is released, and the coil spring 76 slides toward the distal end due to its elastic restoring force. As a result, the measurement sensor 12 is pushed out by the eject pin 56 and removed from the insertion hole 58.

[0154] 1, the control unit 42 of the device main body 16 is configured, for example, by a control circuit having a calculation unit, a memory unit, and an input / output unit (not shown). A well-known computer can be used as this control unit 42. For example, in response to the user's operation of the operation button 50, the control unit 42 drives and controls the measurement unit 14 to detect and calculate glucose in the blood, and displays the calculated blood glucose level on the display 52.

[0155] For example, in a blood glucose meter 10 that measures an analyte (e.g., glucose) by transmitting measurement light through the measurement sensor 12, the control unit 42 calculates the measurement result based on the Beer-Lambert law shown in the following equation (A).

[0156]

number

[0157] In the above formula (A), l0 is the intensity of light before it enters the whole blood sample, l1 is the intensity of light after it leaves the whole blood sample, α is the absorption coefficient, and L is the distance (cell length) that the measurement light passes through. [Example]

[0158] The effects of the present invention will be explained using the following examples and comparative examples. However, the technical scope of the present invention is not limited to the following examples. In the following examples, unless otherwise specified, operations were performed at room temperature (25°C). Furthermore, unless otherwise specified, "%" and "parts" mean "% by mass" and "parts by mass", respectively.

[0159] <Synthesis of tetrazolium salt 1> A compound (tetrazolium salt 1) having the following structure was synthesized according to the following method.

[0160] [ka]

[0161] 1. Synthesis of hydrazone compound 1 1.59 g of disodium 4-formylbenzene-1,3-disulfonate (Tokyo Chemical Industry Co., Ltd.) and 1.0 g of 2-hydrazino-6-methoxy-1,3-benzothiazole (Santa Cruz Biotechnology) were suspended in 60 mL of RO water. This suspension was heated and stirred in an acetic acid-acidic water bath at 60°C for 2 hours. After heating and stirring, the solvent was removed. The residue was washed with isopropanol, and the precipitate was filtered off. The precipitate was dried in a fume hood to obtain hydrazone compound 1.

[0162] 2. Synthesis of formazan compound 1 A hydrazone compound 1 solution was prepared by dissolving 0.76 g of the hydrazone compound 1 described above in a mixture of 10 mL of RO water and 10 mL of DMF. 0.264 g of p-anisidine-3-sulfonic acid (Tokyo Chemical Industry Co., Ltd.) was suspended in 4.09 mL of RO water and dissolved in 130 μL of 10 N NaOH. While maintaining this solution at 0°C, 280 μL of 9.6 N HCl was added, and sodium nitrite solution was added dropwise to carry out diazotization. This diazotized solution was maintained at -20°C and added dropwise to the hydrazone compound 1 solution. After the addition, 300 μL of 10 N NaOH was added dropwise, and the mixture was stirred at room temperature (25°C) for 2 hours to prepare a solution containing formazan compound 1 (formazan compound 1 solution). The pH of this formazan compound 1 solution was adjusted to neutral with 9.6 N HCl, and the solvent was removed. The resulting residue was washed with isopropanol, and the precipitate was filtered off and dried to obtain formazan compound 1.

[0163] 3. Purification of Formazan Compound 1 and Synthesis of Tetrazolium Salt 1 Formazan compound 1 solution was prepared by dissolving the formazan compound 1 from step 2 above in 10 mL of RO water. A disposable column (20 cm x 5 cm) was packed with column chromatography packing material (COSMOSIL 40C18-PREP, manufactured by Nacalai Tesque, Inc.) and placed in a column preparative system (Sepacor, manufactured by Nippon Buchi Co., Ltd.). The formazan compound 1 solution was purified using this column system. The solvent was removed from the collected fraction, and the resulting solid component was mixed with 15 mL of methanol, 250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite (CHCHNO)-ethanol solution and stirred for 72 hours at room temperature (25°C) in the dark.

[0164] 4. Recovery of Tetrazolium Salt 1 Diethyl ether was added to the reaction solution of 3 above to precipitate tetrazolium salt 1. This precipitate was centrifuged, the supernatant was removed, and then washed with diethyl ether. The resulting precipitate was dried in a draft to obtain tetrazolium salt 1. The maximum absorbance (in a 200 mg / dL glucose aqueous solution, cell length = 0.045 mm) of formazan produced from the resulting tetrazolium salt 1 was measured at 600 nm to 800 nm and was found to be 0.5 or greater.

[0165] <Reagent preparation> (Reagent 1: Example) An aqueous solution containing zinc acetate, tetrazolium salt 1 of Synthesis Example 1, glucose dehydrogenase (FAD-GDH) (Toyobo Co., Ltd., trade name: GLD-351 (D-Glucose:(flavine adenine dinucleotide)-dehydrogenase)) as an oxidoreductase, Nonidet P-40 as a hemolyzing agent, sodium nitrite, sucralose, and sorbitol as a sugar alcohol was prepared to have the composition shown in Table 1 below, thereby obtaining Reagent 1.

[0166] Specifically, a 1M zinc acetate aqueous solution (1M = 183.48 g / L) and RO water were mixed to prepare mixed solution 1. Sodium nitrite was then added to mixed solution 1 to prepare mixed solution 2. Sucralose (1M = 397.64 g / L) was then added to mixed solution 2 to prepare mixed solution 3. Sorbitol (1M = 182.17 g / L) was then added to mixed solution 3 to prepare mixed solution 4. Tetrazolium salt 1 (1M = 693 g / L) was then added to mixed solution 4 to prepare mixed solution 5. Reagent 1 was prepared by mixing mixed solution 5, a 30% by mass Nonidet aqueous solution, and FAD-GDH.

[0167] (Reagent 2: Comparative Example) Reagent 2 was prepared in the same manner as Reagent 1, except that nickel acetate was used instead of zinc acetate.

[0168] (Reagents 3 to 16: Examples) Reagents 3 to 16 were prepared in the same manner as Reagent 1, except that the compositions were as shown in Table 1 below.

[0169] [Table 1]

[0170] <Fabrication of Blood Glucose Meter Sensors (Blood Glucose Level Measurement Sensors) 1 to 16> The blood glucose meter sensor (blood glucose measurement sensor) shown in Figure 5 was fabricated as follows. First, 3.15 μL of the coating solution prepared above was applied to a polyethylene terephthalate (PET) film 40 (manufacturer: Toray Industries, Inc., product name: Lumirror T60, thickness: 188 μm, 8 mm × 80 mm) placed on a stage (the "reagent application surface 3" side in Figure 5A) using an inkjet printer (Microjet, Labojet-500Bio) with a patterning accuracy of ±8 μm and a liquid discharge rate of 1 pL to 1000 pL. The PET film 40 was then dried at 25 °C for 10 minutes to form a reagent layer (PET with a reagent layer). This PET with a reagent layer was cut to a specified size (8 mm × 1.6 mm) to prepare film pieces ("film piece 2" in Figure 5A). Each film piece was coated with 0.063 μL of reagent. Each film piece contains the components shown in Table 2 below in the form of a dry reagent. The mass of the reagent per blood glucose sensor (0.063 μL of coating solution) was calculated from the composition (mass) of each reagent component added when preparing the reagent, the final volume of the prepared reagent solution (731.3 μL), and the number of film pieces 2 made from the PET with the reagent layer.

[0171] [Table 2]

[0172] Double-sided tape (manufacturer: Nitto Denko Corporation, product name: 5605BN, thickness: 50 μm) ("double-sided tape 4" in FIG. 5A) was attached to both sides of a hydrophilic polyester film (manufactured by 3M Corporation, product name: Hydrophilic Polyester Film 9901P, thickness: 100 μm) ("polyester film 5" in FIG. 5A) as a second substrate, as a spacer and adhesive (sensor base). The film piece prepared above ("film piece 2" in FIG. 5A) was attached to this sensor base so that the reagent layer ("reagent application surface 3" in FIG. 5A) faced the sensor base and was located at the center of the flow path ("flow path 6" in FIG. 5A). When attaching the film piece, pressure was applied so that the film piece was embedded in the double-sided tape by a predetermined amount. Furthermore, a film piece ("Polyester film 7" in Figure 5A) made of hydrophilic treated polyester film with double-sided tape (manufacturer: 3M, product name: polyester film base, double-sided adhesive tape 9965, thickness: 80 μm) attached was placed over the sensor base with the film piece attached, to produce blood glucose meter sensors (blood glucose level measurement sensors) 1 to 16.

[0173] The fabricated blood glucose meter sensor (blood glucose level measurement sensor) has a flow path section and a measurement section (reagent section). In the flow path section in FIG. 5B, the flow path length ("L1" in FIG. 5B) was 9 mm, the width ("W" in FIG. 5B) was 1.1 mm, and the thickness ("t1" in FIG. 5B) was 0.13 mm. In the measurement section in FIG. 5B, the reagent length ("L2" in FIG. 5B) was 1.6 mm, the width ("W" in FIG. 5B) was 1.1 mm, and the thickness ("t2" in FIG. 5B) was 0.05 mm.

[0174] The length L1 of the flow path section in the direction perpendicular to the sensor thickness direction (longitudinal direction of the flow path) is not particularly limited and can be selected appropriately depending on the purpose, but is preferably 5 to 10 mm. A longer length L1 is advantageous in that it facilitates installation (insertion) into the component measuring device and reduces the intrusion of ambient light into the optical measurement section. A shorter length L1 is advantageous in that it allows for a smaller sample volume. Therefore, the upper and lower limits of length L1 are determined taking into consideration the balance between ease of installation (insertion) into the component measuring device, the influence of ambient light, and the sample volume. The length L2 of the flow path in the reagent section in the direction perpendicular to the sensor thickness direction (longitudinal direction of the flow path) is not particularly limited and can be selected appropriately depending on the purpose, but is preferably 1 to 4 mm. A longer length L2 is advantageous in that it allows for a larger irradiation spot area in the longitudinal direction, thereby enabling more accurate measurement, while a shorter length L2 is advantageous in that it allows for a smaller sample volume. Therefore, the upper and lower limits of length L2 are determined taking into consideration the balance between measurement accuracy and sample volume.

[0175] <Test Example 1> A high-concentration glucose solution (40 g / dL) was added to blood (plasma, hematocrit value (Ht) 0) to prepare a blood sample (BG400) with a glucose concentration (BG) of 400 mg / dL.

[0176] 1 μL of the prepared blood sample was applied to the fabricated blood glucose level measuring sensor 1 or 2, and the absorbance spectrum was measured at a predetermined time from the start of the reaction. The ambient temperature was 25°C. The results are shown in Figure 6.

[0177] As shown in Figure 6, blood glucose sensor 1 containing reagent 1, which uses zinc acetate as a compound capable of generating transition metal ions, can suppress color change over time. However, blood glucose sensor 2 containing reagent 2, which uses nickel acetate as a compound capable of generating transition metal ions, shows that a wavelength shift occurs due to structural changes over time.

[0178] Nickel ions are thought to have two coordination structures, square planar and octahedral, which may cause the chelate structure to change over time, while zinc ions are thought to have a tetrahedral coordination structure, which may suppress color change.

[0179] <Test Example 2> A high-concentration glucose solution (40 g / dL) was added to whole blood (hematocrit (Ht) 40) to prepare blood samples with glucose concentrations (BG) of 100 mg / dL (BG100), 200 mg / dL (BG200), 400 mg / dL (BG400), and 550 mg / dL (BG550). As a control, a glucose-degrading enzyme was added to whole blood (hematocrit (Ht) 40) to prepare a blood sample with a glucose concentration (BG) of 0 mg / dL (BG0).

[0180] BG0, BG100, BG200, BG400 and BG550 were applied to the inlet of the flow channel of the fabricated blood glucose level sensor 3. Nine seconds after each sample was applied to the reagent section, the absorbance at 605 nm was measured using a fiber spectrophotometer. The results are shown in Figure 7. Figure 7 shows the glucose concentration and the formazan and Zn concentrations when each sample was measured using the blood glucose level sensor. 2+ 1 is a graph showing the relationship between the absorbance at the maximum absorption wavelength (605 nm) of the chelate compound of the present invention and the absorbance of the chelate compound of the present invention.

[0181] 7, the absorbance and the glucose concentration show a linear relationship (proportional relationship). From this, it can be considered that the blood glucose level can be accurately measured based on the absorbance by using the sensor of the present disclosure.

[0182] Based on these findings, by applying a hemolyzed whole blood sample to the reagent (blood glucose measurement sensor) of the present disclosure, measuring the amount of color development, and creating a calibration curve based on the amount of color development, the concentration of biological components in the whole blood sample can be accurately calculated.

[0183] <Test Example 3> A blood sample (BG100) with a glucose concentration (BG) of 100 mg / dL was prepared by adding a high-concentration glucose solution (40 g / dL) to high-hematocrit blood (whole blood, hematocrit (Ht) 60), which increases blood viscosity.

[0184] 1 μL of the prepared blood sample was spotted on the prepared blood glucose level measuring sensors 4 to 16, and the blood spreading property was evaluated visually according to the following evaluation criteria. The environmental temperature was 25° C.: Evaluation criteria ◎: The blood sample spread quickly over the entire area ○: It spread quickly to the central measurement range, but took some time to spread to the entire area △: It spread quickly to the central measurement range, but took time to spread to the entire area.

[0185] The results are shown in Table 3.

[0186] [Table 3]

[0187] As shown in Table 3, by setting the molar ratio of zinc acetate to tetrazolium salt 1 to be 116% or more, it is possible to maintain blood spreading within an acceptable range. [Explanation of symbols]

[0188] 1 reagent ribbon, 2 film strips, 3 Reagent application surface, 4 double-sided tape, 5 polyester film, 6 flow paths, 7 polyester film, 10 Blood glucose meter, 12 measuring sensors, 14 Measuring part, 16. Device body, 18 Sensor body, 20 Cavity, 20a Tip opening, 20b proximal opening, 22 long side, 22a upper long side, 22b Lower long side, 24 short side, 24a tip edge, 24b proximal edge, 26 reagents, 28 Measurement target part, 30 board pieces, 32 spacers, 40 enclosures, 42 control section, 44 Box part, 46 Photometry section, 48 Power button, 50 operation buttons, 52 displays, 54 Eject lever, 56 Eject pin, 56a rod part, 56b receiving part, 58 insertion hole, 58a insertion opening, 59 Measuring hole, 60 Sensor attachment part, 60a flange part, 62 wall, 64 element housing space, 66 Light guide section, 68 light-emitting elements, 70 light-emitting part, 72 photodetector, 74 Light receiving section, 76 Coil spring.

Claims

1. A reagent for measuring glucose concentration comprising a tetrazolium salt, a compound capable of generating a transition metal ion capable of forming a regular tetrahedral coordination structure, an oxidoreductase, a hemolytic agent, nitrous acid or a salt thereof, and sucralose.

2. The tetrazolium salt is represented by the following formula (1): 【Chemical 1】 In the above formula (1), R １ is any one selected from the group consisting of a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxy group, and R ２ is a nitro group, -OR ４ and a carboxyl group, and R ３ are hydrogen atoms, methyl groups, or ethyl groups, at least one of which is a methyl group or an ethyl group, and R ４ is a methyl group or an ethyl group, and m is a sulfo group (—SO ３ － ) is the number of phenyl groups bonded to the 5-position of the tetrazole skeleton, and is 1 or 2; n is R ２ is the number of bonds to the phenyl group at the 3-position of the tetrazole skeleton, and is an integer of 0 to 2, and p is a sulfo group (—SO ３ － ) is the number of groups bonded to the phenyl group at the 3-position of the tetrazole skeleton, and is 0 or 1; n+p is 1 or more; q is 1 or 2; when q is 2, each OR ３ are arranged adjacently, and in this case, each OR ３ 2. The reagent for measuring glucose concentration according to claim 1, which is a tetrazolium salt represented by the formula: wherein X represents a hydrogen atom or an alkali metal; and X represents a hydrogen atom or an alkali metal.

3. 3. The reagent for measuring glucose concentration according to claim 1, wherein the transition metal is zinc.

4. 4. The glucose concentration measurement reagent according to claim 1, wherein the molar ratio of the transition metal to the tetrazolium salt is 116% or more and less than 231%.

5. The glucose concentration measurement reagent according to any one of claims 1 to 4, which is a dry reagent.

6. A method for measuring a glucose concentration, comprising: bringing a whole blood sample into contact with the glucose concentration measurement reagent according to any one of claims 1 to 5, measuring the amount of color developed, and quantifying the glucose concentration in the whole blood sample based on the amount of color developed.

7. 1. A sensor for measuring a glucose concentration in a blood sample, the sensor having a reaction portion, 7. A sensor, wherein the reaction section includes the glucose concentration measurement reagent according to claim 1.

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