WO2018051686A1 - 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2h-tetrazolium salt, biological component concentration measurement reagent containing said salt, and biological component concentration measurement method using said salt - Google Patents

Abstract

The purpose of the present invention is to provide a means that maintains water solubility while making it possible to quantify a biological component with sufficient sensitivity even in the case of a whole blood sample. The present invention is a 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt that is represented by formula (1) (in the formula, R¹ represents a methyl group or any ethyl group, R² and R³ each independently represent a methyl group, an ethyl group, a methoxy group, or an ethoxy group, and X represents a hydrogen atom or an alkali metal).

Description

2-Substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt, reagent for measuring biological component concentration containing the salt, and method for measuring biological component concentration using the salt

The present invention relates to a 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt, a biological component concentration measuring reagent containing the salt, and a biological component concentration measuring method using the salt.

In clinical chemistry tests, there is a method for detecting and quantifying the amount of biological components contained in biological fluids such as blood and urine as the amount of pigment, and the reagent used in this method is called a coloring reagent.

For example, in JP-A-8-53444 (Patent Document 1), the following structure:

Disclosed is a method for quantifying reduced nicotinamide adenine dinucleotide using a water-soluble tetrazolium salt compound having R ¹ represents a hydrogen atom or a methoxy group, and R ² represents a hydrogen atom, a carboxyl group or a sulfonic acid. Has been. According to Japanese Patent Laid-Open No. 8-53444, the use of the water-soluble tetrazolium salt compound improves the water-solubility of the formazan obtained, does not deposit on the measuring instrument, and has a maximum absorption wavelength of 510 to 550 nm. Therefore, it is said that measurement by an automatic analyzer is possible without receiving interference from biological components.

JP-A-8-53444

Formazan produced from the tetrazolium salt disclosed in JP-A-8-53444 has high water solubility, but the color intensity in a wavelength region (over 600 nm) that does not overlap with the main absorption band of hemoglobin is not sufficient. For this reason, the tetrazolium salt disclosed in JP-A-8-53444 cannot achieve sufficient sensitivity for whole blood samples.

Therefore, the present invention has been made in view of the above circumstances, and an object of the present invention is to provide means capable of quantifying a biological component with sufficient sensitivity even for a whole blood sample while maintaining water solubility.

Another object of the present invention is to provide a means capable of stably quantifying a biological component with sufficient sensitivity to a whole blood sample while maintaining water solubility.

As a result of intensive studies to solve the above problems, the present inventor has found that a substituted thiazolyl group at the 2-position and a phenyl group having an alkoxy group and a nitro group (—NO ₂ ) at the 3-position of the tetrazole ring. And a tetrazolium salt having a phenyl group having _two sulfo groups at the 5-position (—SO ₃ ⁻ ; —S (═O) ₂ O ⁻ ; the same shall apply hereinafter) solves the above-mentioned problems. Completed.

That is, the above-mentioned purpose is the following formula (1):

Where R ¹ represents a methyl group or an ethyl group;
R ² and R ³ each independently represents a methyl group, an ethyl group, a methoxy group or an ethoxy group; and X represents a hydrogen atom or an alkali metal,
This can be achieved by the 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt represented by

FIG. 1 is a diagram showing the spectrum of a chelate compound of formazan and Ni ²⁺ produced from tetrazolium compound 1. FIG. 2 is a graph showing the relationship between the glucose concentration and the absorbance of produced formazan for tetrazolium compound 1 and WST-4. FIG. 3 is a diagram showing the results of stability evaluation of the tetrazolium compound 1. FIG. 4 is a plan view schematically showing a blood glucose meter (component measurement device) equipped with the measurement chip according to the present embodiment. 4, 10 represents a blood glucose meter, 12 represents a measurement chip, 14 represents a measurement unit, 18 represents a chip body, 40 represents a housing, 42 represents a control unit, and 44 represents a box. It represents a body part, 46 represents a photometric block, 48 represents a power button, 50 represents an operation button, 52 represents a display, and 54 represents an eject lever. FIG. 5 is an enlarged perspective view showing the measurement chip of FIG. 4 and the photometric block of the apparatus main body. In FIG. 5, 10 represents a blood glucose meter, 12 represents a measurement chip, 14 represents a measurement unit, 16 represents a device body, 18 represents a chip body, 20 represents a cavity, and 20a represents 20b represents the base end, 22 represents the long side, 24 represents the short side, 30 represents the plate piece, 32 represents the spacer, 40 represents the housing, 46 represents photometry. 56 represents an eject pin, 56a represents a bar, 56b represents a receiving portion, 58 represents an insertion port, 58a represents an insertion opening, 59 represents a measurement hole, and 60 represents a tip. 60a represents a wall portion, 68 represents a light emitting element, 70 represents a light emitting element, 72 represents a light receiving element, 74 represents a light receiving element, and 76 represents a coil spring. Represents. FIG. 6 is a side view showing the measurement chip of FIG. In FIG. 6, 12 represents a measuring chip, 18 represents a chip body, 20 represents a cavity, 20a represents a distal end, 20b represents a proximal end, 22 represents a long side, 22a represents the upper long side, 22b represents the lower long side, 24 represents the short side, 24a represents the distal side, 24b represents the proximal side, 26 represents the reagent, and 28 represents the measurement target part. , 30 represents a plate piece, and 32 represents a spacer. FIG. 7A is a first plan view showing the mounting operation of the measurement chip of FIG. 4 and the apparatus main body. In FIG. 7A, 12 represents a measurement chip, 14 represents a measurement unit, 16 represents an apparatus body, 20 represents a cavity, 26 represents a reagent, 30 represents a plate piece, and 32 represents a spacer. 40 represents a housing, 46 represents a photometric block, 46a represents a fixed wall, 56 represents an eject pin, 56a represents a rod portion, 56b represents a receiving portion, 58 represents an insertion slot, 58a represents Represents an insertion opening, 59 represents a measurement hole, 60 represents a chip mounting portion, 60a represents a flange, 62 represents a wall, 64 represents an element housing space, and 66 represents a light guide. 68 represents a light emitting element, 68a represents a first light emitting element, 68b represents a second light emitting element, 70 represents a light emitting part, 72 represents a light receiving element, 74 represents a light receiving part, 76 Represents a coil spring. FIG. 7B is a second plan cross-sectional view showing the mounting operation following FIG. 7A. In FIG. 7B, 12 represents a measurement chip, 14 represents a measurement unit, 16 represents an apparatus main body, 20 represents a hollow portion, 20a represents a tip opening, 26 represents a reagent, and 30 represents a plate. 32 represents a spacer, 32 represents a spacer, 40 represents a housing, 46 represents a photometric block, 46a represents a fixed wall, 56 represents an eject pin, 56a represents a rod portion, 56b represents a receiving portion, 60 Represents a chip mounting part, 60a represents a flange part, 62 represents a wall part, 68 represents a light emitting element, 68a represents a first light emitting element, 68b represents a second light emitting element, and 70 represents a light emitting part. , 72 represents a light receiving element, 74 represents a light receiving portion, and 76 represents a coil spring.

In the first aspect of the present invention, the following formula (1):

2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salts having the structure: In the present specification, the 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt of the above formula (1) is also simply referred to as “tetrazolium salt of the present invention” or “tetrazolium salt”.

The formazan produced from the tetrazolium salt of the present invention or a chelate compound of formazan and a transition metal ion is excellent in water solubility. Moreover, it has a maximum absorption wavelength in a wavelength region (over 600 nm) that does not overlap with the main absorption band of hemoglobin. For this reason, there is little influence of the coloring substance which exists in the blood, and a measurement error can be reduced. Therefore, by using the tetrazolium salt of the present invention, the biological component concentration can be measured with high sensitivity even for a whole blood sample. In the present specification, the formazan produced from the tetrazolium salt of the present invention or a chelate compound of formazan and a transition metal ion is also simply referred to as “formazan according to the present invention” or “formazan compound”.

Formazan produced from the tetrazolium salt described in JP-A-8-53444 has a maximum absorption at 510 to 550 nm (paragraph “0011”). In fact, in Example 3 of the above-mentioned JP-A-8-53444, the NADH concentration is quantified by the absorbance at 550 nm (paragraph “0029”, FIG. 2). On the other hand, when measuring the concentration of a biological component (for example, glucose) using a whole blood sample, it is necessary that the absorbance to be measured be in a wavelength region that does not overlap with the main absorption band of hemoglobin. The wavelength for detecting the concentration of red blood cells in blood is about 510 to 540 nm, and the maximum absorption wavelength of oxygenated hemoglobin is around 550 nm. For this reason, it is preferable that the maximum absorption wavelength of formazan is more than 600 nm (particularly 650 nm or more). As a result, the formazan produced from the tetrazolium salt described in JP-A-8-53444 cannot sufficiently eliminate the influence of blood cells, and it was difficult to measure the concentration of biological components with good sensitivity. . For this reason, it is necessary to shift the maximum absorption wavelength of the tetrazolium salt described in JP-A-8-53444 to the longer wavelength region side. Generally, the maximum absorption wavelength can be shifted to the longer wavelength side by forming formazan with a transition metal ion (for example, nickel ion or cobalt ion) and a chelate compound. However, in Example 3 of JP-A-8-53444, when a transition metal ion (for example, nickel ion) is further added in order to shift to the longer wavelength side, a precipitate is formed. For this reason, it is difficult to say that the tetrazolium salt described in JP-A-8-53444 can be suitably used as a reagent for whole blood samples.

In contrast, the tetrazolium salt of the present invention is soluble in water at a concentration of 200 mM or more, and is excellent in water solubility. In addition, formazan (formazan according to the present invention) produced from the tetrazolium salt of the present invention also has excellent water solubility. Furthermore, the formazan according to the present invention does not form a precipitate even in the presence of a transition metal ion, and has a maximum absorption wavelength in a wavelength region (over 600 nm, particularly 650 nm or more) that does not overlap with the blood absorption band. For this reason, by using the tetrazolium salt of the present invention, the biological component concentration can be measured with high sensitivity even for a whole blood sample. Although the detailed mechanism for achieving the above effect is still unclear, it is considered as follows. The following mechanism is speculative and does not limit the technical scope of the present invention. Specifically, the tetrazolium salt of the present invention has a substituted thiazolyl group bonded to the 2-position of the tetrazole ring, a phenyl group having an alkoxy group and a nitro group (—NO ₂ ) bonded to the 3-position, and the 5-position. It has a structure in which a phenyl group having two sulfo groups (—SO ₃ ⁻ ) is bonded. Here, since it has two sulfo groups (—SO ₃ ⁻ ), the tetrazolium salt and the formazan compound are dissolved in water at a concentration of 200 mM or more, and are excellent in water solubility. Moreover, since a nitro group exists, it is possible to shift the maximum absorption wavelength of formazan (or a chelate compound of formazan and a transition metal ion) generated from a tetrazolium salt to the longer wavelength side (Example 1 and Comparative Example below). Comparison with 2). Further, due to the thiazolyl group present at the 2-position of the tetrazole ring, the formazan according to the present invention forms a chelate compound efficiently and quickly with transition metal ions such as Co ²⁺ and Ni ²⁺ . By forming such a chelate compound, the maximum absorption wavelength is further shifted to the longer wavelength side, that is, the maximum absorption wavelength can be further shifted to a wavelength region (over 600 nm, particularly 650 nm or more) that does not overlap with the blood absorption band. For this reason, by using the tetrazolium salt of the present invention, even a biological component concentration in a whole blood sample can be accurately measured with high sensitivity. Moreover, the tetrazolium salt of the present invention is excellent in stability.

Therefore, by using the tetrazolium salt of the present invention, the biological component concentration can be measured with high sensitivity and promptly. Further, by using the tetrazolium salt of the present invention, the concentration of biological components can be measured with high sensitivity and promptly even after long-term storage.

Hereinafter, embodiments of the present invention will be described in detail.

The tetrazolium salt of the present invention is water-soluble and has a wavelength region (over 600 nm) where the maximum absorption wavelength of the formazan compound, which is a reduced form thereof, does not overlap with the blood absorption band. For this reason, by using the tetrazolium salt of the present invention, the biological component concentration can be measured with high sensitivity even for a whole blood sample. Moreover, the tetrazolium salt of the present invention is excellent in stability.

The tetrazolium salt of the present invention has the following formula (1):

As shown in the figure, a substituted thiazolyl group at the 2-position, a phenyl group having an alkoxy group and a nitro group (—NO ₂ ) at the 3-position, and two sulfo groups (—SO ₃ ) at the 5-position of the tetrazole ring ^-) is a phenyl group having been introduced. In the tetrazolium salt having the above structure, the tetrazole ring is a part that mainly absorbs light.

In the above formula (1), the presence of the substituted thiazolyl group at the 2-position of the tetrazole ring can form a chelate compound efficiently and quickly with the transition metal compound (the maximum absorption wavelength of the formazan compound can be shifted to a long wavelength region). ). Here, R ² and R ³ represent a methyl group, an ethyl group, a methoxy group, or an ethoxy group. Here, R ² and R ³ may be the same or different. Here, when R ² and R ³ are an alkyl group having 3 or more carbon atoms, the resulting tetrazolium salt and formazan produced from the tetrazolium salt are poor in water solubility. Further, when R ² and R ³ are hydrogen atoms, the electron density of the thiazolyl ring is lowered, which is not preferable.

In the above formula (1), a phenyl group having an alkoxy group and a nitro group (—NO ₂ ) exists at the 3-position of the tetrazole ring. Here, due to the presence of the nitro group, the formazan produced from the tetrazolium salt and the tetrazolium salt has a maximum absorption wavelength (λmax) on the long wavelength side exceeding 600 nm (especially 650 nm or more). In addition, the presence of the alkoxy group (—OR ¹ ) improves the stability of the tetrazolium salt. Here, R ¹ represents a methyl group or an ethyl group. When R ¹ is an alkyl group having 3 or more carbon atoms, the resulting tetrazolium salt and formazan produced from the tetrazolium salt are poor in water solubility. Note that the bonding position of the nitro group (—NO ₂ ) and the alkoxy group (—OR ¹ ) to the phenyl group is not particularly limited. From the viewpoint of shifting the maximum absorption wavelength to the longer wavelength side, the nitro group (—NO ₂ ) is preferably present at the 4-position of the phenyl group. In addition, from the viewpoint of further improving the stability, the alkoxy group (—OR ¹ ) is preferably present at the 2-position and 4-position of the phenyl group, and particularly preferably present at the 2-position from the viewpoint of steric hindrance. preferable. That is, in a preferred embodiment of the present invention, in the above formula (1), the phenyl group at the 3-position is a phenyl group in which the alkoxy group (—OR ¹ ) is at the 2-position and the nitro group (—NO ₂ ) is at the 4-position. It is.

In the above formula (1), a phenyl group having two sulfo groups (—SO ₃ ⁻ ) exists at the 5-position of the tetrazole ring. Here, due to the presence of two sulfo groups (—SO ₃ ⁻ ), the formazan produced from the tetrazolium salt and the tetrazolium salt can exhibit high water solubility. In addition, if there is one sulfo group, the formazan produced from the tetrazolium salt and the tetrazolium salt is inferior in water solubility. Here, the bonding position of the sulfo group (—SO ₃ ⁻ ) to the phenyl group is not particularly limited. From the viewpoint of further improving the water solubility, the sulfo group (—SO ₃ ⁻ ) is preferably present at the 2, 4th, 3rd, and 5th positions of the phenyl group, and is located at the 2nd and 4th positions due to water solubility. It is particularly preferred that it is present. That is, in a preferred embodiment of the present invention, in the above formula (1), the phenyl group at the 5-position is a phenyl group in which the sulfo group (—SO ₃ ⁻ ) is present at the 2,4-position or the 3,5-position.

In the above formula (1), X represents a hydrogen atom or an alkali metal. Here, X ⁺ exists to neutralize two anions (sulfo group (—SO ₃ ⁻ )) and one tetrazole ring cation. For this reason, the kind of alkali metal is not particularly limited, and may be any of lithium, sodium, potassium, rubidium, and cesium. Among these, sodium and potassium are preferable from the viewpoint of versatility, and sodium is more preferable.

That is, the preferred structure of the tetrazolium salt of the present invention is as follows. In the following structure, X represents an alkali metal.

The method for producing the tetrazolium salt of the present invention is not particularly limited, and conventionally known methods can be applied in the same manner or appropriately modified. Specifically, the following structure:

A hydrazino-substituted thiazole having the structure:

Is reacted with sodium formylbenzene disulfonate having the following structure:

A hydrazone compound having On the other hand, the following structure:

Hydrochloric acid is added to an alkoxynitrobenzene having an ice-cooling, and a sodium nitrite solution is further added dropwise to form the following structure:

A benzenediazonium chloride compound having The hydrazine compound obtained above and a benzenediazonium chloride compound are reacted under basic conditions (for example, the presence of sodium hydroxide and potassium hydroxide) to form the following structure:

A formazan compound having Subsequently, the formazan compound thus obtained is oxidized in an alcohol solvent (eg, methanol, ethanol) using an oxidizing agent (eg, nitrite such as ethyl nitrite and butyl nitrite), The tetrazolium salt of the present invention is obtained.

The tetrazolium salt of the present invention has high water solubility. Further, formazan produced from the tetrazolium salt, or a chelate compound of formazan and a transition metal ion (formazan or formazan compound according to the present invention) is highly water-soluble by forming a chelate compound alone or with a transition metal compound. And has a maximum absorption wavelength in a wavelength region (over 600 nm, particularly 650 nm or more) that does not overlap with the main absorption band of hemoglobin. For this reason, by using the tetrazolium salt of the present invention, the concentration of biological components can be measured with high sensitivity even for biological samples, particularly whole blood samples. Specifically, the maximum absorption wavelength (λmax) of formazan produced from the tetrazolium salt of the present invention or a chelate compound of formazan and a transition metal ion is preferably more than 600 nm, more preferably 650 nm or more. Formazan having such a maximum absorption wavelength or a chelate compound of formazan and a transition metal ion (hence, a tetrazolium salt capable of generating such formazan) is hardly affected by absorption derived from blood color. By using the tetrazolium salt of the present invention, the concentration of biological components can be measured more accurately and with good sensitivity. Here, the upper limit of the maximum absorption wavelength (λmax) of the formazan produced from the tetrazolium salt of the present invention or the chelate compound of formazan and a transition metal ion is not particularly limited, but is usually 900 nm or less, preferably 800 nm or less. is there. In the present specification, the value measured according to the method described in the examples below is adopted as the maximum absorption wavelength (λmax).

Therefore, in the second aspect of the present invention, there is provided a reagent for measuring the concentration of biological components containing the 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt of the present invention. In the third aspect of the present invention, the 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt, oxidoreductase and transition metal compound of the present invention are added to a biological sample. There is provided a method for measuring the concentration of a biological component, comprising measuring the color development amount and quantifying the concentration of the biological component in the biological sample based on the color development amount.

In the present invention, the biological component measurement target is not particularly limited as long as it includes the target biological component. Specific examples include blood and body fluids such as urine, saliva and interstitial fluid. In addition, the biological component is not particularly limited, and usually, a biological component measured by a colorimetric method or an electrode method can be used in the same manner. Specific examples include glucose, cholesterol, neutral fat, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), uric acid and the like. That is, according to a preferred embodiment of the second aspect of the present invention, the biological component concentration measurement reagent of the present invention comprises glucose, cholesterol, neutral fat, nicotinamide adenine dinucleotide phosphate (NADPH) in blood or body fluid. Used for the determination of nicotinamide adenine dinucleotide (NADH) or uric acid concentration. According to a preferred embodiment of the third aspect of the present invention, the biological component in the biological sample is glucose, cholesterol, neutral fat, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide in blood or body fluid. Adenine dinucleotide (NADH) or uric acid.

The biological component concentration measuring reagent of the present invention essentially contains the tetrazolium salt of the present invention. As described above, in the biological component concentration measuring reagent, the formazan produced by the reduction reaction of the tetrazolium salt can shift the maximum absorption wavelength to the longer wavelength side by producing a transition metal ion and a chelate compound. . For this reason, it is preferable to include a transition metal compound in the biological component concentration measurement reagent, particularly when measuring the biological component concentration in the whole blood sample. That is, in a preferred embodiment of the present invention, the biological component concentration measuring reagent further contains a transition metal compound. According to this form, even when the concentration of a biological component in a whole blood sample is measured, formazan has a maximum absorption wavelength in a wavelength region (over 600 nm, particularly 650 nm or more) that does not overlap with the main absorption band of hemoglobin. For this reason, the measurement sensitivity of the biological component concentration can be further improved for biological samples, particularly whole blood samples. The transition metal compound that can be used when the biological component concentration measuring reagent contains a transition metal compound is not particularly limited. Specifically, compounds capable of generating transition metal ions such as nickel ions (Ni ²⁺ ), cobalt ions (Co ²⁺ ), zinc ions (Zn ²⁺ ), and copper ions (Cu ²⁺ ) ions can be used. With such ions, the maximum absorption wavelength of formazan can be shifted to the longer wavelength side. Of these, nickel ions are preferred. Since nickel ions are less susceptible to oxidation and reduction, measurement errors can be reduced more effectively. That is, according to a preferred embodiment of the present invention, the transition metal compound is a nickel compound. In addition, the compound that generates the transition metal ion is not particularly limited, but it is preferable to generate an ion in an aqueous liquid (for example, water, buffer solution, blood, body fluid). For example, examples of the compound that generates the transition metal ion include organic acid salts of the transition metal such as chloride, bromide, sulfate, and acetate. The transition metal compound may be used alone or in combination of two or more in the biological component concentration measurement reagent. In the present embodiment, the content of the transition metal compound is not particularly limited, but can be appropriately selected according to the desired maximum absorption wavelength of the formazan compound. Specifically, the content of the transition metal compound is such that the transition metal (transition metal ion) is preferably 0.1 to 10 mol, more preferably 0.5 to 4 mol, per 1 mol of the tetrazolium salt. It is an amount like this. With such an amount, the maximum absorption wavelength of the formazan compound can be shifted to a desired wavelength range.

The biological component concentration measuring reagent may further contain other components in addition to the transition metal compound or in place of the transition metal compound. Here, as other components, components that are normally selected according to the type of biological component to be measured and added to measure the concentration of biological components can be used in the same manner. Specific examples include oxidoreductases, electron carriers, pH buffers, surfactants, and the like. Here, the other components contained in the biological component concentration measurement reagent may be used singly or in combination of two or more. Each of the other components may be used alone or in combination of two or more.

Here, the oxidoreductase is not particularly limited, and can be appropriately selected depending on the type of biological component to be measured. Specifically, glucose dehydrogenase (GDH) (for example, glucose dehydrogenase (GDH-PQQ) using pyrroloquinoline quinone (PQQ) as a coenzyme, glucose dehydrogenase (GDH-FAD) using flavin adenine dinucleotide (FAD) as a coenzyme ), Glucose dehydrogenase (GDH-NAD) using nicotinamide adenine dinucleotide (NAD) as a coenzyme and glucose dehydrogenase (GDH-NADP) using nicotine adenine dinucleotide phosphate (NADP) as a coenzyme), glucose oxidase ( GOD), glucose-6-phosphate dehydrogenase, cholesterol dehydrogenase, cholesterol oxidase, glycerophosphate dehydrogenase, glycerophosphate oxidase, Dehydrogenase enzyme (LDH), lactate oxidase, alcohol dehydrogenase, alcohol oxidase, uricase, such as urine dehydrogenase and the like. Here, the oxidoreductase may be used alone or in combination of two or more. For example, when the biological component is glucose, the oxidoreductase is preferably glucose dehydrogenase or glucose oxidase. When the biological component is cholesterol, the oxidoreductase is preferably cholesterol dehydrogenase or cholesterol oxidase. The content of oxidoreductase when the biological component concentration measurement reagent contains oxidoreductase is not particularly limited, and can be appropriately selected according to the amount of tetrazolium salt. For example, the content of the oxidoreductase is preferably 1 to 100% by mass, more preferably 5 to 50% by mass with respect to the tetrazolium salt. With such an amount, the oxidoreductase can sufficiently act on the biological component. For this reason, the measurement sensitivity of the biological component concentration can be further improved.

The electron carrier is not particularly limited, and a known electron carrier may be used. Specifically, diaphorase, phenazine methylsulfate (PMS), 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS or m-PMS), Nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH) and the like can be mentioned. Here, an electron carrier may be used individually by 1 type, or may combine 2 or more types. The content of the electron carrier when the biological component concentration measuring reagent contains an electron carrier is not particularly limited, and can be appropriately selected according to the amount of the tetrazolium salt. For example, the content of the electron carrier is preferably 0.05 to 10% by mass, more preferably 0.1 to 5% by mass with respect to the tetrazolium salt. With such an amount, the reduction reaction can proceed more efficiently. For this reason, the measurement sensitivity of the biological component concentration can be further improved.

Among the above, the biological component concentration measurement reagent preferably further includes a transition metal compound and an oxidoreductase, and more preferably further includes a transition metal compound, an oxidoreductase, and an electron carrier. That is, according to a preferred embodiment of the present invention, the biological component concentration measurement reagent comprises the 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt, transition metal compound and oxidoreductase of the present invention. Including. Further, according to a more preferred form of the present invention, the biological component concentration measuring reagent is a 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt, transition metal compound, oxidoreductase of the present invention. And an electron carrier. Here, for example, the biological component is β-D-glucose, the transition metal compound is nickel chloride, the oxidoreductase is flavin adenine dinucleotide-dependent glucose dehydrogenase (GDH-FAD), and the electron carrier is 1 The case of -methoxy-5-methylphenadium methyl sulfate (m-PMS) will be described. First, β-D-glucose and m-PMS are subjected to the action of GDH-FAD to become gluconic acid and reduced m-PMS. From this reduced m-PMS and tetrazolium salt to m-PMS and formazan. Color. Further, this formazan forms a chelate compound with nickel ions, and the maximum absorption wavelength is shifted to the longer wavelength region side (for example, more than 600 nm). For this reason, since the influence of the main absorption band of hemoglobin at the measurement wavelength to be measured can be reduced by using the biological component concentration measurement reagent of the present invention, it is highly sensitive to biological samples, particularly whole blood samples. Biological component concentration can be measured.

The usage form of the biological component concentration measuring reagent is not particularly limited, and may be either a solid form or a liquid form. In the latter case, the biological component concentration measurement reagent preferably further contains water, a buffer solution, and a surfactant, and more preferably contains water and / or a buffer solution. Here, the buffer solution is not particularly limited, and a buffer agent generally used when measuring the concentration of a biological component can be used in the same manner. Specifically, phosphate buffer, citrate buffer, citrate-phosphate buffer, trishydroxymethylaminomethane-HCl buffer (Tris-HCl buffer), MES buffer (2-morpholinoethane sulfonate buffer) Agent), TES buffer (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer), acetic acid buffer, MOPS buffer (3-morpholinopropanesulfonic acid buffer), MOPS-NaOH buffer, HEPES buffer (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid buffer), GOOD buffer such as HEPES-NaOH buffer, glycine-hydrochloric acid buffer, glycine-NaOH buffer, glycylglycine- Amino acid buffers such as NaOH buffer, glycylglycine-KOH buffer, Tris-borate buffer Borate -NaOH buffer, borate buffer such as borate buffer, or an imidazole buffer are used. Of these, phosphate buffer, citrate buffer, citrate-phosphate buffer, Tris-HCl buffer, MES buffer, acetate buffer, MOPS buffer, and HEPES-NaOH buffer are preferred. Here, the concentration of the buffer is not particularly limited, but is preferably 0.01 to 1.0M. In the present invention, the concentration of the buffering agent refers to the concentration of the buffering agent (M, mol / L) contained in the aqueous solution. Moreover, it is preferable that pH of a buffer solution does not have an effect | action with respect to a biological component. From the above viewpoint, the pH of the buffer solution is preferably near neutral, for example, about 5.0 to 8.0. The concentration of the tetrazolium salt when the reagent for measuring the concentration of biological component is liquid is not particularly limited as long as the concentration of the desired biological component can be measured, but the tetrazolium salt is less than the amount of the desired biological component present. It is preferably contained in a larger amount. In consideration of the above viewpoint and the concentration of the biological component to be normally measured, the concentration of the tetrazolium salt is preferably 0.005 to 0.2 mol / L, more preferably 0.01 to 0.00 in the biological component concentration measuring reagent. 1 mol / L. When preparing a reagent in a dry state, the concentration of the tetrazolium salt is 0.005 to 0.2 mol / L, more preferably 0.01 to 0.1 mol / L in the solution state before being applied to the substrate. It is. In such an amount, the tetrazolium salt depends on the amount of substantially all (for example, 95 mol% or more, preferably 98 mol% or more, particularly preferably 100 mol%) of the biological component contained in the biological sample. React. For this reason, a desired biological component concentration can be quickly measured accurately and with good sensitivity.

By using the biological component concentration measuring reagent of the present invention, the concentration of a specific biological component contained in a biological sample can be measured with good sensitivity. Here, the measurement method is not particularly limited, and can be appropriately selected according to the type of biological component to be measured. For example, in the glucose reductase method in which the biological component is β-D-glucose and the oxidoreductase is glucose dehydrogenase (GDH), glucose is oxidized by GDH to produce gluconic acid. This is based on the reduction of GDH coenzymes or electron mediators. Specifically, as a result, the degree of coloration of the reduced tetrazolium salt (and hence chelate compound of formazan or formazan and transition metal ions) is determined. The method is roughly divided into a method of optical measurement (colorimetric method) and a method of measuring current generated by the oxidation-reduction reaction (electrode method). Among the above methods, the measurement of blood glucose level by the colorimetric method has advantages such as easy correction using a hematocrit value when calculating the blood glucose level, and a simple manufacturing process. Therefore, the biological component concentration measurement reagent can be suitably used for the colorimetric method. In particular, a colorimetric method is preferably used when measuring the glucose concentration in a whole blood sample.

The biological component concentration measuring reagent of the present invention may be used for measuring the biological component concentration as it is, or may be incorporated in the biological component concentration measuring chip. That is, the present invention also provides a biological component concentration measuring chip (hereinafter, also simply referred to as “measuring chip”) including the biological component concentration measuring reagent of the present invention. In addition, the reagent and method for measuring the concentration of biological components of the present invention can be incorporated into an automatic analyzer, a measurement kit, a simple blood glucose meter, etc., and used for daily clinical examinations. It is also possible to incorporate the reagent of the present invention into a commercially available biosensor. When the biological component concentration measuring reagent is incorporated into the biological component concentration measuring chip, the content of the reagent per chip is not particularly limited, and the amount usually used in the field can be similarly adopted. However, it is preferable that the tetrazolium salt is contained in a sufficient amount with respect to the abundance of the desired biological component. Considering the above viewpoint and the concentration of biological components to be normally measured, the amount of tetrazolium salt is preferably 3 to 50 nmol, more preferably 10 to 30 nmol per chip. In such an amount, the tetrazolium salt depends on the amount of substantially all (for example, 95 mol% or more, preferably 98 mol% or more, particularly preferably 100 mol%) of the biological component contained in the biological sample. React. For this reason, a desired biological component concentration can be quickly measured accurately and with good sensitivity.

Hereinafter, the form of the measurement chip (colorimetric blood glucose meter) of the present invention used for measuring the blood glucose level by the colorimetric method will be described with reference to the drawings. However, the present invention is characterized by using the biological component concentration measuring reagent of the present invention, and the chip structure is not particularly limited. For this reason, you may apply the reagent for a biological component density | concentration measurement of this invention to the chip | tip described in gazettes, such as a commercially available measurement chip | tip and WO2014 / 04970, WO2016 / 051930. Similarly, in the following embodiment, a specific form of a chip for measuring blood glucose level will be described. However, the measuring chip is not limited to the application, and may be appropriately or appropriately used for other applications. Applicable with modification.

FIG. 4 is a plan view schematically showing a blood glucose meter used for detection of glucose (blood glucose) using the measurement chip according to the present embodiment.

In FIG. 4, the blood glucose meter 10 is configured as a device for measuring glucose (blood glucose) in a blood sample. This blood glucose meter 10 can be mainly used for personal use operated by a user (subject). The user can also measure blood sugar before meals and manage his blood sugar. In addition, the blood glucose meter 10 can be used by a medical worker to evaluate the health condition of the subject. In this case, the blood glucose meter 10 may be appropriately modified and installed in a medical facility or the like.

The blood glucose meter 10 employs the principle of a colorimetric method for optically measuring the glucose content (blood glucose level) contained in a blood sample. As a measurement method, any of reflected light measurement, transmitted light measurement, and absorbance measurement may be employed. In particular, the blood glucose meter 10 performs blood glucose measurement with a measurement unit 14 for measuring transmitted light, which irradiates the analysis sample (blood) with measurement light having a predetermined wavelength and receives light transmitted through the analysis sample.

The blood glucose meter 10 attaches the measurement chip 12 that has taken in blood, or takes blood into the measurement chip 12 with the measurement chip 12 attached, and detects glucose by the measurement unit 14. The measurement chip 12 may be configured as a disposable type that is discarded every 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 the measurement.

As shown in FIG. 5, the measurement chip 12 includes a chip body 18 formed in a plate shape, and a cavity 20 (liquid cavity) extending in the surface direction of the plate surface inside the chip body 18. Prepare.

As shown in FIG. 5, the chip body 18 has a long side 22 in the insertion and removal direction of the blood glucose meter 10 (the distal end and proximal direction of the blood glucose meter 10) and a short short in the vertical direction, as viewed from the side. It is formed in a rectangular shape having sides 24. For example, the length of the long side 22 of the chip body 18 may be set to a length that is twice or more the short side 24. As a result, a sufficient amount of insertion of the measuring chip 12 into the blood glucose meter 10 is ensured.

Also, the thickness of the chip body 18 is extremely small (thin) compared to the side surface formed in a rectangular shape (in FIG. 5, it is shown to have a sufficient thickness). For example, the thickness of the chip body 18 is preferably set to 1/10 or less of the short side 24 described above. The thickness of the chip body 18 may be appropriately designed according to the shape of the insertion hole 58 of the blood glucose meter 10.

The measuring chip 12 includes the chip body 18 by a pair of plate pieces 30 and a pair of spacers 32 so as to have the cavity 20.

FIG. 6 is a side view showing the measurement chip of FIG. In FIG. 6, the corners of the chip body 18 are pointed, but the corners may be formed as rounded corners, for example. Further, the chip body 18 is not limited to a thin plate shape, and it is needless to say that the shape may be freely designed. For example, the chip body 18 may be formed in a square shape, another polygonal shape, or a circular shape (including an elliptical shape) in a side view.

The cavity 20 provided inside the chip body 18 is located at an intermediate position in the vertical direction of the chip body 18 and is formed linearly over the longitudinal direction of the chip body 18. The hollow portion 20 is connected to the distal end port portion 20 a formed on the distal end side 24 a of the chip body portion 18 and the proximal end port portion 20 b formed on the proximal end side 24 b, and communicates with the outside of the chip body portion 18. Yes. When the cavity 20 receives the user's blood from the distal end 20a, the cavity 20 can flow the blood along the extending direction based on the capillary phenomenon. The amount of blood flowing through the cavity 20 is small, and even if it moves to the proximal end port 20b, leakage is suppressed by tension. It should be noted that an absorption part that absorbs blood (for example, a spacer 32 which will be described later is made porous only on the base end side) may be provided on the base end side 24b side of the chip body part 18.

Moreover, it reacts with glucose (blood glucose) in blood at a predetermined position of the cavity 20 (for example, a position slightly closer to the base end than the intermediate point between the front end opening 20a and the base end opening 20b shown in FIG. 6). Thus, a reagent (coloring reagent) 26 that colors in accordance with the glucose (blood sugar) concentration in the blood is applied, and a measurement target portion 28 to be measured by the blood glucose meter 10 is set. The blood that flows in the proximal direction through the cavity 20 comes into contact with the measurement target portion 28, and reacts with the reagent 26 applied to the measurement target portion 28, thereby causing coloration. It should be noted that the application position of the reagent 26 and the measurement target part 28 may be shifted from each other on the longitudinal direction of the cavity 20. For example, the reaction part to which the reagent 26 is applied is located upstream of the measurement target part 28 in the blood flow direction. It may be provided.

The measuring chip 12 includes the chip main body portion 18 by a pair of plate pieces 30 and a pair of spacers 32 so as to have the cavity 20 described above. The pair of plate pieces 30 are each formed in the rectangular shape described above in a side view and are arranged in the stacking direction. That is, the pair of plate pieces 30 constitutes both side surfaces (left side surface and right side surface) of the chip body 18. The plate thickness of each plate piece 30 is very small, and is preferably set to the same dimension of about 5 to 50 μm, for example. The thicknesses of the two (one set) plate pieces 30 may be different from each other.

The pair of plate pieces 30 has a strength that maintains the plate shape and does not undergo plastic deformation even when a certain amount of pressing force is applied from a direction orthogonal to the surface direction. Each plate piece 30 is configured to be transparent or translucent so that the measurement light can be transmitted. Furthermore, each plate piece 30 is formed on a flat plate surface having appropriate hydrophilicity so that blood can flow in the cavity 20 (or a coating agent is applied to the plate surface). preferable.

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

Also, 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 the plate pieces 30 by a predetermined joining means (adhesive or the like). In other words, each spacer 32 is a member that forms the cavity 20 between the pair of plate pieces 30 and the pair of spacers 32 itself by being disposed between the pair of plate pieces 30 so as to be separated from each other. In this case, one spacer 32 is disposed so as to be in contact with the upper long side 22a of the chip body 18 in FIG. 6 and extend in the distal end and proximal direction along the upper long side 22a. The other spacer 32 is in contact with the lower long side 22b of the chip body 18 in FIG. 6 and is disposed so as to extend in the distal and proximal directions along the lower long side 22b.

The material (base material) constituting the pair of spacers 32 is not particularly limited. For example, styrene, polyolefin, polyurethane, polyester, polyamide, polybutadiene, transpolyisoprene, fluoro rubber And various thermoplastic elastomers such as chlorinated polyethylene. Alternatively, in addition to the thermoplastic elastomer, various elastically deformable materials may be applied, and a structure such as an elastically deformable porous body (for example, a sponge) may be applied. Furthermore, you may apply as the spacer 32 which has the adhesive agent which adhere | attaches the board pieces 30 by becoming a hardening state or a semi-hardened state between a pair of board pieces 30 on one or both surfaces of a base material. Furthermore, the spacer 32 may be configured to elute the reagent 26 into the cavity 20 by containing the reagent 26.

The plate piece 30 and the spacer 32 may be subjected to a hydrophilic treatment. Examples of the hydrophilic treatment method include immersion of an aqueous solution containing a hydrophilic polymer such as polyacrylic acid, polyvinyl pyrrolidone, and polyacrylamide in addition to surfactant, polyethylene glycol, polypropylene glycol, hydroxypropyl cellulose, water-soluble silicone, and the like. Examples thereof include a method of coating by a method or a spray method, a method of plasma irradiation, glow discharge, corona discharge, ultraviolet irradiation (for example, excimer light irradiation), and the like, and these methods may be used alone or in combination.

The measuring chip 12 is configured as described above. Next, the apparatus main body 16 of the blood glucose meter 10 will be described. As shown in FIG. 4, the blood glucose meter 10 has a housing 40 that constitutes the appearance. The housing 40 has a size that is easy for a user to hold and operate, and a box body portion 44 that houses the control unit 42 of the blood glucose meter 10 therein, and protrudes from one side (tip side) of the box body portion 44 in the distal direction to the inside. And a cylindrical photometric block 46 that houses the measuring unit 14 of the optical system. Further, a power button 48, an operation button 50, and a display 52 are provided on the upper surface of the box portion 44, and an eject lever 54 is provided on the upper surface of the photometry block 46.

The power button 48 switches between starting and stopping the blood glucose meter 10 under the operation of the user. The operation button 50 functions as an operation unit for measuring and displaying a blood glucose level based on a user's operation and switching display of measurement results (including past measurement results) in the activated blood glucose meter 10. To do. The display 52 is composed of a liquid crystal, an organic EL, or the like, and displays information to be provided to the user in the measurement operation such as measurement result display or error display.

The eject rod lever 54 is provided so as to be movable in the distal end and proximal direction, and unlocks an eject rod pin (not shown) provided in the photometry block 46 so that the eject rod pin can be advanced in the distal direction.

On the other hand, the photometric block 46 of the apparatus main body 16 extends from the box 44 in the distal direction so as to press the distal end against the user's finger or the like. As shown in FIG. 5, the photometric block 46 is provided with a chip mounting portion 60 having an insertion hole 58 and a measuring portion 14 that optically detects blood glucose (blood glucose).

The tip mounting portion 60 is formed of a material having high rigidity (rigidity) (for example, stainless steel) and has a flange portion 60a that protrudes outward, on the tip side, and is formed in a cylindrical shape having a predetermined length in the axial direction. . The chip mounting portion 60 is positioned and fixed over the tip surface and the axial center portion (center portion) of the photometric block 46 made of a resin material. As shown in FIG. 7A, a fixed wall 46a for firmly fixing the chip mounting portion 60 is formed on the inner surface of the photometric block 46 so as to protrude.

As a material constituting the chip mounting portion 60, for example, a metal such as stainless steel or titanium, anodized aluminum, a liquid crystal polymer, a plastic added with a filler such as glass or mica, or a nickel-plated surface is hardened. Examples thereof include materials such as plastic, carbon fiber, fine ceramic, etc. that are hard and do not easily change in size, and that do not wear easily even when the measurement chip is repeatedly inserted and removed, and that can be processed with high dimensional accuracy. Among these, if a metal material is applied, the insertion hole 58 can be easily formed with high dimensional accuracy when the chip mounting portion 60 is manufactured (injection molding, press molding or the like). In the apparatus main body 16, the chip mounting part 60 may be integrally formed by configuring the photometric block 46 itself from a hard material (for example, a metal material).

The insertion hole 58 is provided in the axial center part of the chip mounting part 60 by being surrounded by the wall part 62 of the chip mounting part 60. The insertion hole 58 is formed in a rectangular shape that is long in the vertical direction and short in the left-right width direction. The insertion hole 58 has a predetermined depth from the distal end surface toward the back (base end direction) in a state where the chip mounting portion 60 is fixed to the photometric block 46.

At the distal end side of the chip mounting portion 60, an insertion opening 58a that is continuous with the insertion hole 58 and communicates with the outside is formed. The vertical dimension of the insertion opening 58a matches the dimension (vertical length) of the short side 24 of the measuring chip 12. Further, the dimension of the insertion opening 58a in the left-right width direction, that is, the distance between the pair of wall portions 62 constituting the side surface of the insertion hole 58 is, as shown in FIG. 7A, the thickness in the stacking direction of the measurement chip 12 (FIG. 7A). Substantially the same as Tall in the middle).

The chip mounting portion 60 forms a pair of element accommodating spaces 64 in cooperation with the fixed wall 46a of the photometry block 46 at a midway position where the insertion hole 58 (measurement hole portion 59) extends. The pair of element housing spaces 64 are a part of the measurement unit 14, are provided at positions facing each other across the insertion hole 58, and each of the measurement hole units is formed via the light guide units 66 formed by the chip mounting unit 60. 59.

The measuring unit 14 configures the light emitting unit 70 by accommodating the light emitting element 68 in one element accommodating space 64, and configures the light receiving unit 74 by accommodating the light receiving element 72 in the other element accommodating space 64. . The light guide part 66 of the chip mounting part 60 plays a role of a so-called aperture by being formed in a circular hole having an appropriate diameter.

The light emitting element 68 of the light emitting unit 70 measures the first light emitting element 68a that irradiates the measuring chip 12 with the measuring light having the first wavelength, and the measuring light having the second wavelength different from the first wavelength. And a second light emitting element 68b that irradiates the chip 12 (not shown in FIG. 5). The first light emitting element 68a and the second light emitting element 68b are arranged side by side at a position facing the light guide portion 66 of the element accommodating space 64.

The light emitting element 68 (first and second light emitting elements 68a and 68b) may be formed of a light emitting diode (LED). The first wavelength is a wavelength for detecting the color density of the reagent 26 according to the blood glucose level, and is, for example, more than 600 nm and 680 nm or less. The second wavelength is a wavelength for detecting the concentration of red blood cells in blood, and is, for example, 510 nm to 540 nm. The control unit 42 in the box unit 44 supplies a drive current to cause the first and second light emitting elements 68a and 68b to emit light at a predetermined timing. In this case, the blood glucose level obtained from the color concentration is corrected using the hematocrit value obtained from the red blood cell concentration, and the blood glucose level is obtained. In addition, you may correct | amend the noise resulting from a blood cell by measuring at another measurement wavelength.

The light receiving part 74 is configured by arranging one light receiving element 72 at a position facing the light guide part 66 of the element accommodating space 64. The light receiving unit 74 receives transmitted light from the measuring chip 12, and may be configured by, for example, a photodiode (PD).

Further, an eject pin 56 (eject portion) connected to the eject lever 54 is provided at the bottom portion (base end surface) of the insertion hole 58. The eject pin 56 includes a rod portion 56a extending along the axial direction of the photometry block 46, and a receiving portion 56b having a large diameter on the radially outer side at the tip portion of the rod portion 56a. The base end side 24b of the measuring chip 12 inserted into the insertion hole 58 is in contact with the receiving portion 56b. A coil spring 76 that surrounds the eject pin 56 in a non-contact manner is provided between the bottom of the insertion hole 58 and the receiving portion 56 b of the eject pin 56. The coil spring 76 elastically supports the receiving portion 56b of the eject pin 56.

When the insertion of the measurement chip 12 is completed, the measurement target part 28 of the measurement chip 12 is arranged at a position overlapping the light guide part 66 as shown in FIG. 7B.

The eject pin 56 is displaced in the proximal direction when the receiving portion 56b is pushed along with the insertion of the measuring chip 12 by the user, and is locked (fixed) by a lock mechanism (not shown) provided in the housing 40. The coil spring 76 is elastically contracted according to the displacement of the receiving portion 56b. When the eject pin 56 is slightly moved by the user's operation of the eject lever 54, the lock mechanism is unlocked and slides in the distal direction by the elastic restoring force of the coil spring 76. As a result, the measuring chip 12 is pushed out to the eject pin 56 and taken out from the insertion hole 58.

Returning to FIG. 4, the control unit 42 of the apparatus main body 16 includes, for example, a control circuit having a calculation unit, a storage unit, and an input / output unit (not shown). The control unit 42 can be a known computer. For example, the control unit 42 drives and controls the measurement unit 14 to detect and calculate blood glucose under the operation of the operation button 50 of the user, and displays the calculated blood glucose level on the display 52.

For example, in the blood glucose meter 10 that measures an analysis object (for example, glucose) by transmitting measurement light to the measurement chip 12, the control unit 42 is based on the Beer-Lambert law expressed by the following equation (A). Calculate the measurement results.

In the above formula (A), l ₀ is the intensity of light before entering the blood sample, l ₁ is the intensity of light after exiting the blood sample, α is the extinction coefficient, and L is the distance through which the measurement light passes (cell Long).

The effect of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples. In the following examples, the operation was performed at room temperature (25 ° C.) unless otherwise specified. Unless otherwise specified, “%” and “part” mean “% by mass” and “part by mass”, respectively.

Example 1: Synthesis of tetrazolium compound 1 A compound having the following structure (tetrazolium compound 1) was synthesized according to the following method.

1. Synthesis of hydrazone compound 1 Disodium 4-formylbenzene-1,3-disulfonate (manufactured by Tokyo Chemical Industry Co., Ltd.) 8.59 g and (4,5-dimethyl-thiazole) -2-yl) hydrazine ((4,5-Dimethyl-thiazol-2-yl) -hydrazine) (manufactured by Fluorochem) was dissolved in 300 mL of RO water. To this was added 1.6 mL of acetic acid, and the mixture was heated and stirred at 60 ° C. for 2 hours in a water bath. After completion of heating and stirring, the solvent was removed. The obtained residue was washed with alcohol, and then the precipitate was filtered off. The obtained precipitate was dried to obtain hydrazone compound 1.

2. Synthesis of formazan compound 1 1.39 g of hydrazone compound 1 was dissolved in a mixed solution of 25 mL of RO water and 25 mL of methanol (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a hydrazone compound 1 solution. 0.437 g of 2-methoxy-4-nitroaniline (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in a mixed solution of 3.44 mL of RO water and 10 mL of acetonitrile. While maintaining this solution at 0 ° C., 560 μL of 9.6N HCl was added, and sodium nitrite solution was added dropwise to perform diazotization. This diazotized solution was kept at −20 ° C. and added dropwise to the hydrazone compound 1 solution. After completion of the dropping, 600 μL of 10N NaOH was added dropwise and stirred for 2 hours at room temperature (25 ° C.) to prepare a solution containing formazan compound 1 (formazan compound 1 solution). The formazan compound 1 solution was adjusted to neutral pH with 9.6N HCl, and the solvent was removed. The obtained residue was washed with isopropanol, and then the precipitate was filtered off. The formazan compound 1 was obtained by drying this precipitate.

3. Purification of formazan compound 1 and synthesis of tetrazolium compound 1 Formazan compound 1 was dissolved in 10 mL of RO water to prepare a formazan compound 1 solution. A disposable column (size: 20 cm × 5 cm) is packed with a packing material for column chromatography (manufactured by Nacalai Tesque Co., Ltd., COSMOSIL 40C _18- PREP), and a column preparative system (manufactured by Nihon Büch, trade name: Sepacore). Set. Formazan compound 1 solution was purified using this column system. After desalting, the solvent of the collected fraction was removed, and 15 mL of methanol, 250 μL of 9.6N HCl, and 5 mL of a 15% ethyl nitrite (CH ₃ CH ₂ NO ₂ ) -ethanol solution were added to the obtained solid components. The mixture was stirred for 72 hours at room temperature (at 25 ° C.) and protected from light.

4). Recovery of tetrazolium compound 1 3. Diethyl ether was added to 5 mL of the reaction solution to precipitate tetrazolium compound 1. This precipitate was dried in a draft to obtain tetrazolium compound 1 (90 mg, yield: 5.0% by mass).

Example 2: Synthesis of tetrazolium compound 2 A compound having the following structure (tetrazolium compound 2) was synthesized according to the following method.

1. Synthesis of hydrazone compound 2 8.59 g and (4,5-dimethyl-thiazole) disodium 4-formylbenzene-1,3-disulfonate (manufactured by Tokyo Chemical Industry Co., Ltd.) -2-yl) hydrazine ((4,5-Dimethyl-thiazol-2-yl) -hydrazine) (manufactured by Fluorochem) was dissolved in 300 mL of reverse osmosis water (RO water). To this was added 1.6 mL of acetic acid, and the mixture was heated and stirred at 60 ° C. for 2 hours in a water bath. After completion of heating and stirring, the solvent was removed. The obtained residue was washed with ethanol, and then the precipitate was filtered off. The hydrazone compound 2 was obtained by drying this precipitate.

2. Synthesis of formazan compound 2 1. A hydrazone compound 2 solution was prepared by dissolving 1.39 g of the hydrazone compound 2 obtained in 1 above in a mixed solution of 25 mL of RO water and 25 mL of methanol. 0.455 g of 2-Ethoxy-4-nitroaniline (manufactured by Goji Chemical Industry Co., Ltd.) was dissolved in a mixed solution of 3.44 mL of RO water and 10 mL of acetonitrile. While maintaining this solution at 0 ° C., 560 μL of 9.6N HCl was added, and sodium nitrite solution was added dropwise to perform diazotization. This diazotized solution was kept at −20 ° C. and added dropwise to the hydrazone compound 2 solution. After completion of the dropwise addition, 600 μL of 10N NaOH was added dropwise and stirred for 2 hours at room temperature (25 ° C.) to prepare a solution containing formazan compound 2 (formazan compound 2 solution). The formazan compound 2 solution was adjusted to neutral pH with 9.6N HCl, and the solvent was removed. The obtained residue was washed with isopropanol, and then the precipitate was filtered off. The formazan compound 2 was obtained by drying the obtained precipitate.

3. Purification of formazan compound 2 and synthesis of tetrazolium compound 2 Formazan compound 2 was dissolved in 10 mL of RO water to prepare a formazan compound 2 solution. A disposable column (size: 20 cm × 5 cm) is packed with a packing material for column chromatography (manufactured by Nacalai Tesque Co., Ltd., COSMOSIL 40C _18- PREP), and a column preparative system (manufactured by Nihon Büch, trade name: Sepacore). Set. Formazan compound 2 solution was purified using this column system. After desalting, the solvent was removed from the collected red fraction with an evaporator, and 15 mL of methanol, 9.6N HCl 250 μL, and 15% ethyl nitrite (CH ₃ CH ₂ NO ₂ ) -ethanol solution 5 mL were obtained. Was added and stirred for 72 hours at room temperature (at 25 ° C.) protected from light.

4). Recovery of tetrazolium compound 2 3. The tetrazolium compound 2 was precipitated by adding 50 mL of diethyl ether to 5 mL of the reaction solution. This was centrifuged, the supernatant was removed, and further washed with diethyl ether. This precipitate was dried in a draft to obtain tetrazolium compound 2 (120 mg, 6.5% by mass).

Examples 1-2, Comparative Examples 1-5
For the tetrazolium compounds 1 to 2 of Examples 1 and 2 and the comparative compounds 1 to 5 having the following structures (Comparative Examples 1 to 5, respectively), water solubility, sensitivity, chelate rate, maximum absorption wavelength (λmax) were determined according to the following methods. The stability was evaluated, and the results are shown in Table 1 below.

(Evaluation of water solubility)
Each compound was added to 100 μL of RO water and 100 μL of 0.5M MOPS solution (pH 7.2) in such an amount that the concentration of the compound in the aqueous solution was 200 mM. After stirring for 5 minutes, the precipitate was visually confirmed. In the case where there was no precipitate in a 200 mM aqueous solution, the solubility was evaluated as ◯. In the case where a precipitate was observed even in a 200 mM aqueous solution, the solubility was evaluated as “x”.

(Evaluation of chelate rate and maximum absorption wavelength (λmax))
A sample was prepared by adding a 10 mM MOPS solution (pH 7.2) so that the final concentration of formazan of each compound was 50 to 200 mM. At this time, each sample developed reddish brown.

10 μL of a nickel aqueous solution containing 1M nickel ions was added to 100 μL of the sample, and the color tone change was observed while stirring rapidly. Measure the time until the color development is visually confirmed after adding the nickel aqueous solution. If the time is within 1 minute, the chelate rate is set to “◯”. If the time exceeds 1 minute, The chelation rate was evaluated as “x”. In Comparative Examples 2 and 4, it was determined that chelate was not formed because the color tone of the sample after the addition of nickel ions did not change toward the long wavelength side, and “-” was displayed in Table 1.

Further, the spectrum of the mixed solution of each compound formazan aqueous solution and nickel aqueous solution was measured with a spectrophotometer (measurement cell length: 10 mm) (n = 1). Based on each spectrum, the maximum absorption wavelength (λmax) (nm) of each compound in formazan was determined. The respective results are shown in Table 1. As an example, a spectrum obtained from the tetrazolium compound 1 is shown in FIG. FIG. 1 shows that the maximum absorption wavelength (λmax) of the chelate compound of formazan and nickel ions produced from compound 1 is 650 nm.

(Evaluation of sensitivity)
Samples were prepared by adding and dissolving 75 μL of RO water and 100 μL of 0.5 M MOPS solution (pH 7.2) to 0.02 mmol of each compound. As a control, 11.6 mg of 2-benzothiazolyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H-tetrazolium (WST-4), 85 μL of RO water and 0 A control sample was prepared by adding and dissolving 100 μL of a 5 M MOPS solution (pH 7.2).

Separately, 100 μL of RO water was added to 7 mg of glucose dehydrogenase (GDH-FAD) (product number: GLD-351, manufactured by Toyobo Co., Ltd.) using flavin adenine dinucleotide (FAD) as a coenzyme, and dissolved to prepare a GDH solution. did. Also, 1-methoxy-5-methylphenadium methyl sulfate (m-PMS) (manufactured by Dojin Chemical Laboratory)
An s-PMS solution was prepared by adding 200 μL of RO water to 3.5 mg and dissolving. 1 mL of RO water was added to 129 mg of nickel chloride and dissolved therein to prepare a nickel solution.

A reaction solution was prepared by adding 10 μL of the GDH solution prepared above, 5 μL of m-PMS solution, and 10 μL of nickel solution to each sample. In addition, 10 μL of the GDH solution prepared above and 5 μL of the m-PMS solution were added to the control sample to prepare a control reaction solution.

To 40 μL of the reaction solution prepared above, 10 μL of glucose solutions having a concentration of 50 mg / dL, 100 mg / dL and 200 mg / dL were added for color development, and the spectrum was measured with a spectrophotometer (measurement cell length: 50 μm) ( n = 1). In each spectrum, the absorbance at the maximum absorption wavelength (λmax) of each compound obtained above was measured, and the glucose concentration and absorbance in the coloring solution were plotted with the horizontal axis and the vertical axis, respectively, and the slope (slope _sample ). Ask for.

To 40 μL of the control reaction solution prepared above, 10 μL of a glucose solution having a concentration of 50 mg / dL and 200 mg / dL was added for color development, and the spectrum was measured with a spectrophotometer (measurement cell length: 50 μm) (n = 1). ). In each spectrum, the absorbance at 650 nm is measured, plotted with the glucose concentration and absorbance as the horizontal axis and the vertical axis, respectively, and the slope (slope _WST-4 ) is obtained.

When the value obtained by dividing the slope _sample obtained above by the slope _WST-4 (slope _sample / slope _WST-4 ) is twice or more, the sensitivity is “◯”, and the slope _sample / slope _WST-4 When the ratio was less than 2 times, the chelate rate was evaluated as “x”. As an example, a plot of tetrazolium compound 1 and WST-4 is shown in FIG. FIG. 2 is a graph showing the relationship between the glucose concentration and the absorbance of produced formazan for tetrazolium compound 1 and WST-4. As can be seen from FIG. 2, since the slopes of Compound 1 and WST-4 are 0.0062 and 0.0026, respectively, the ratio of slope _sample / slope _WST-4 is about 2.4 (sensitivity is ◯). . Although not shown, the ratio of the slope _sample / inclination _WST-4 in Example 2 is 2.4 (sensitivity is ◯), and the ratio of the inclination _sample / inclination _{WST-4 in} Comparative Example 1 is 1.6. (Sensitivity is x), and the ratio of slope _sample / slope _{WST-4 in} Comparative Example 3 was 2.5 (sensitivity was good). Further, Comparative Examples 2 and 4 did not form a chelate, so the maximum absorption wavelength of formazan did not shift to more than 600 nm. For this reason, Comparative Examples 2 and 4 were not evaluated for sensitivity. (The sensitivity result is shown as “−” in Table 1.) The slope is an index of the color intensity of each compound. For this reason, the value obtained by dividing the inclination _sample by the inclination _WST-4 (inclination _sample / inclination _WST-4 ) is more than 1 time, as compared with WST-4, which is a known coloring reagent, color development on the long wavelength side. Means high.

(Evaluation of stability)
Samples were prepared by adding and dissolving 75 μL of RO water and 100 μL of 0.5 M MOPS solution (pH 7.2) to 0.02 mmol of each compound.

To this sample, 10 μL of GDH solution, 5 μL of m-PMS solution and 10 μL of nickel solution prepared in the same manner as described above (sensitivity evaluation) were added to prepare a measurement solution.

The spectrum of the measurement solution prepared above was measured with a spectrophotometer (measurement cell length: 50 μm) at the time of preparation of the measurement solution (0 hour) and 1 hour, 2 hours and 6 hours after the preparation (n = 1). In each spectrum, the absorbance at the maximum absorption wavelength (λmax) of each compound determined above was measured. Absorbances at the time of preparation of the measurement solution (0 hour) and 1 hour, 2 hours, and 6 hours after the preparation are Abs _0h , Abs _1h , Abs _2h, and Abs _6h , respectively. The value obtained by subtracting the absorbance at the time of preparation of the measurement solution (0 hour) from the absorbance at each measurement time divided by the measurement time [(Abs _1h −Abs _0h ) / 1, (Abs _2h −Abs _0h ) / 2 and (Abs _6h -Abs _0h ) / 6] are less than 0.05, the stability is “◯”, and when the above value is more than 0.05 and 0.15 or less, the stability is When “Δ” was given and the above value exceeded 0.15, the stability was evaluated as “x”. As an example, the stability evaluation result of the tetrazolium compound 1 is shown in FIG. FIG. 3 shows that [(Abs _6h -Abs _0h ) / 6] of Compound 1 is about 0.03, indicating that the stability is good. Although not shown, [(Abs6h-Abs0h) / 6] of the compound of Example 2 is about 0.02, indicating that the stability is good. On the other hand, with respect to the examples of the present invention, the values of [(Abs _6h -Abs _0h ) / 6] of the compounds of Comparative Examples 1 and 3 were 0.05 and 0.20, respectively. Since Comparative Examples 2 and 4 did not form a chelate, formazan having a maximum absorption wavelength of more than 600 nm or a chelate compound of formazan and nickel ions could not be obtained. Therefore, the stability of the compounds of Comparative Examples 2 and 4 was not evaluated.

As shown in Table 1, the tetrazolium salt of the present invention exhibits good water solubility. From the above results, it is presumed that the formazan produced from the tetrazolium salt of the present invention and the chelate compound of the formazan and the transition metal ion also show good water solubility, similar to the tetrazolium salt of the present invention.

Also, from the results in Table 1 above, it can be seen that blood sugar levels can be measured quickly with good sensitivity by using the tetrazolium salts of the examples. Moreover, according to the tetrazolium salt of an Example, even if a certain amount of time has passed after the measurement, the same result as the value immediately after the measurement is obtained, and it can be seen that the stability is excellent.

Furthermore, the chelate compound of formazan and nickel ions generated from the tetrazolium salt of the example shows a maximum absorption wavelength (λmax) of 650 nm or more. Therefore, it is considered that the concentration of biological components such as blood glucose level can be accurately measured with high sensitivity even for a whole blood sample.

This application is based on Japanese Patent Application No. 2016-179908 filed on September 14, 2016, the disclosure of which is incorporated by reference in its entirety.

Claims (10)

1. Following formula (1):
   

   

   Where R ¹ represents a methyl group or an ethyl group;
   R ² and R ³ each independently represents a methyl group, an ethyl group, a methoxy group or an ethoxy group; and X represents a hydrogen atom or an alkali metal,
   A 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt represented by
2. In the formula (1), the phenyl group at the 3-position is a phenyl group having an alkoxy group (—OR ¹ ) at the 2-position and a nitro group (—NO ₂ ) at the 4-position. Tetrazolium salt.
3. In the formula (1), 5-position of the phenyl group, a sulfo group (-SO ₃ ^-) is a phenyl group present in 2,4-position or 3,5-position, the tetrazolium salt according to claim 1 or 2 .
4. The following groups:
   

   

   Where X represents an alkali metal,
   2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt having a structure selected from the group consisting of
5. A reagent for measuring a concentration of a biological component comprising the 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt according to any one of claims 1 to 4.
6. The biological component concentration measuring reagent according to claim 5, further comprising a transition metal compound.
7. The biological component concentration measuring reagent according to claim 6, wherein the transition metal compound is a nickel compound.
8. Used for determination of glucose, cholesterol, neutral fat, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH) or uric acid in blood or body fluids 8. The reagent for measuring a biological component concentration according to any one of 7 above.
9. A 2-substituted thiazolyl-3-substituted phenyl-5-sulfonated phenyl-2H-tetrazolium salt, an oxidoreductase and a transition metal compound according to any one of claims 1 to 4 are added to a biological sample, A biological component concentration measurement method comprising measuring a color development amount and quantifying a concentration of a biological component in the biological sample based on the color development amount.
10. The biological component in the biological sample is glucose, cholesterol, neutral fat, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH) or uric acid in blood or body fluid. The method described in 1.

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