JP2023027418A - Method for electrochemical measurement of glycated hemoglobin and measurement kit using the same - Google Patents

Abstract

To provide a method for an electrochemical measurement of glycated hemoglobin that can perform a highly accurate measurement by increasing the sensitivity of detecting the concentration of a total hemoglobin, and a glycated hemoglobin measurement kit using the method.SOLUTION: The method for an electrochemical measurement of glycated hemoglobin includes the steps of preparing a treatment solution including a surface active agent, a pyrazole compound shown by the following general formula (I), and a proteolytic enzyme; preparing a test liquid by bringing a blood sample in contact with the treatment solution; and measuring the concentration of glycated hemoglobin and the concentration of the total hemoglobin in the test liquid. R1, R2, and R3 in the general formula (I) are independently a hydrogen atom or an alkyl group with a carbon number 1 to 4.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to an electrochemical measurement method for glycated hemoglobin and a measurement kit used therefor.

Biosensors are used to measure substances to be detected in biological samples in various fields such as the medical field and the clinical examination field. A biosensor using an electrochemical measurement method is known as an example of a biosensor. In this biosensor, a working electrode, a counter electrode and a reference electrode are formed on an insulating substrate, and an enzyme reaction layer (also referred to as a reagent portion) containing an enzyme and an electron acceptor (hereinafter referred to as a mediator) is in contact with these electrodes. ). According to such a biosensor, in principle, various substances can be measured by selecting an enzyme that uses the substance to be measured as a substrate. For example, by selecting glucose oxidase as the enzyme, a glucose sensor that measures the glucose concentration in a sample liquid has been put into practical use.

On the other hand, in recent years, measurement of glycated proteins such as glycated hemoglobin and glycated albumin has been widely performed as an index for diagnosing diabetes. For example, hemoglobin A1c (hereinafter abbreviated as HbA1c) is one of glycated hemoglobins, and the HbA1c value is a test value that indicates the proportion of hemoglobin bound to sugar among hemoglobin in red blood cells. Since the HbA1c value reflects the average blood sugar level over the past 1 to 2 months, it is less affected by meals before the test than the amount of glucose in the blood, and is an important index for managing diabetes. HbA1c values have been measured by HPLC methods and immunological methods using optical spectroscopic methods. In the measurement, as a pretreatment, a hemolysis treatment is usually performed in which a hemolysis reagent containing a surfactant is allowed to act on a blood sample (eg, Patent Document 1).

JP 2007-163182 A

When measuring glycated hemoglobin, for example HbA1c, using an electrochemical measurement method, a blood sample is reacted with a hemolytic reagent and a proteolytic enzyme to prepare a test solution, and the HbA1c concentration and total hemoglobin concentration of the test solution are determined. is measured electrochemically. However, the present inventors found in the process of earnestly studying that the surfactant used in the hemolytic reagent reduces the detection sensitivity of the total hemoglobin concentration. In the pretreatment, the blood specimen is diluted, for example, about 100-fold, so that the total hemoglobin concentration in the specimen liquid is on the order of μM. In order to measure such a low total hemoglobin concentration, it is necessary to further improve the detection sensitivity of the total hemoglobin concentration.

Accordingly, an object of the present invention is to provide a glycated hemoglobin electrochemical measurement method and a glycated hemoglobin measurement kit for use in the method, which enables highly accurate measurement by improving the detection sensitivity of the total hemoglobin concentration.

In order to solve the above problems, the electrochemical measurement method for glycated hemoglobin of the present invention prepares a pretreatment liquid containing a surfactant, a pyrazole compound represented by the following general formula (I), and a protease. a step of bringing the pretreatment liquid into contact with a blood specimen to prepare a test liquid; and a step of measuring the glycated hemoglobin concentration and the total hemoglobin concentration contained in the test liquid. .

ここで、一般式（Ｉ）中、Ｒ１、Ｒ２、Ｒ３は、それぞれ独立に、水素原子または炭素数１～４のアルキル基である。

Here, in general formula (I), R1, R2 and R3 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

Further, the glycated hemoglobin measurement kit of the present invention provides a step of preparing a pretreatment liquid containing a surfactant and a protease, a step of contacting a blood sample with the pretreatment liquid to prepare a test liquid, A measurement kit for glycated hemoglobin used in an electrochemical measurement method for glycated hemoglobin, comprising a step of measuring the concentration of glycated hemoglobin and the concentration of total hemoglobin contained in the test solution, wherein as an additive to the pretreatment solution, It is characterized by containing a pyrazole compound represented by the following general formula (I).

ここで、一般式（Ｉ）中、Ｒ１、Ｒ２、Ｒ３は、それぞれ独立に、水素原子または炭素数１～４のアルキル基である。

Here, in general formula (I), R1, R2 and R3 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

According to the electrochemical measurement method of the present invention, glycated hemoglobin can be measured with higher accuracy by improving the detection sensitivity of the total hemoglobin concentration.

4 is an example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is another example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 1 of the present invention. 4 is an example of a graph showing the relationship between total hemoglobin concentration and measured current value in Example 2 of the present invention. FIG. 10 is an example of a graph showing the relationship between total hemoglobin concentration and measured current value with and without surfactant present; FIG. 1 is a perspective view showing an example of the structure of an electrochemical biosensor A used in Embodiment 1. FIG. FIG. 14 is an exploded perspective view of the electrochemical biosensor A of FIG. 13;

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

Embodiment 1
The present embodiment relates to an electrochemical measurement method for glycated hemoglobin, the electrochemical measurement method including a surfactant, a pyrazole compound represented by the following general formula (I), and a protease. a step of preparing a pretreatment liquid; a step of bringing the pretreatment liquid into contact with a blood specimen to prepare a test liquid; and a step of measuring glycated hemoglobin concentration and total hemoglobin concentration contained in the test liquid. It is characterized by comprising

ここで、一般式（Ｉ）中、Ｒ１、Ｒ２、Ｒ３は、それぞれ独立に、水素原子または炭素数１～４のアルキル基である。

Here, in general formula (I), R1, R2 and R3 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

An example of glycated hemoglobin to be measured by the electrochemical measurement method according to the present embodiment is HbA1c.

The pretreatment liquid used in the electrochemical measurement method according to this embodiment contains a surfactant, a pyrazole compound represented by the following general formula (I), and a protease. A surfactant is a hemolytic reagent that hemolyzes a blood sample and elutes hemoglobin from red blood cells. Also, the proteolytic enzyme decomposes the eluted hemoglobin.

Surfactants are not particularly limited, and cationic surfactants, anionic surfactants, amphoteric surfactants and nonionic surfactants can be used. Examples of cationic surfactants include alkylamine salt types and quaternary ammonium salt types. The quaternary ammonium salt type includes benzyldodecyldimethylammonium dihydrate, decyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, and the like. Examples of anionic surfactants include polyoxyethylene alkyl ether carboxylates, N-acyl amino acid salts, sulfonate types such as alkanesulfonates, sodium dodecyl sulfate, and polyoxyethylene alkyl ether sulfates. Examples include alkyl sulfate ester types such as alkyl phosphates, and phosphate ester types such as polyoxyethylene alkyl ether phosphates. Examples of amphoteric surfactants include sulfobetaine types such as dodecyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt and 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate. . Examples of nonionic surfactants include ester types such as glycerin fatty acid esters, and polyoxyethylene alkylphenyl ethers such as 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100). ether type containing polyoxyethylene sorbitan monoralate (Tween), ester ether type containing aliphatic polyoxyethylene sorbitan such as polyoxyethylene sorbitan monoralate (Tween), alkanolamide type, glucoside type such as dodecyl maltoside. Although not particularly limited, a preferred surfactant in combination with the pyrazole compound represented by the general formula (I) is an N-acylamino acid salt. Also, the concentration of the surfactant in the pretreatment liquid is 1 to 10% by weight, preferably 2.5 to 5% by weight. Here, the acyl group of the N-acylamino acid salt (also referred to as an alkanoylamino acid salt) is a linear or branched aliphatic acyl group having 8 to 24 carbon atoms, such as octanoyl group, decanoyl group, dodecanoyl group, Myristoyl group, palmitoyl group, stearoyl group and the like can be mentioned. In addition, examples of the amino group of N-acylamino acid salts include glycine, sarcosine, alanine, glutamic acid and the like. Examples of salts of N-acylamino acid salts include alkali metal salts such as sodium salts and potassium salts. Preferred N-acyl amino acid salts include alkanoyl sarcosine salts. Specific examples of alkanoylsarcosine salts include sodium N-dodecanoylsarcosinate and sodium N-myristoylsarcosinate.

The protease is not particularly limited as long as it has proteolytic activity or peptidolytic activity, but is preferably one that efficiently releases glycated dipeptides. Examples of such proteolytic enzymes include protease, trypsin, papain, and the like. For example, actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) and the like can be mentioned. The concentration of the proteolytic enzyme used varies depending on the amount of glycosylated hemoglobin in the blood sample and the treatment conditions.

The pH of the pretreatment solution is not particularly limited as long as it is suitable for proteolytic enzymes, but is preferably adjusted to pH 5.5-10. Phosphate buffer (pH 6.5), for example, can be used as the buffer. The concentration of the buffer solution is also not particularly limited, but is preferably 5 to 300 mM.

Examples of the pyrazole compound represented by the general formula (I) include 3-ethylpyrazole, 4-methylpyrazole, 3,5-dimethylpyrazole, and 3,4,5-trimethylpyrazole. Pyrazole is preferred. The concentration of the pyrazole compound in the hemolytic reagent is 10-80% by weight, preferably 40-80% by weight. If it is less than 10% by weight, the effect of improving the detection sensitivity of the total hemoglobin concentration is not sufficient, and if it is more than 80% by weight, the detection sensitivity is not so improved. Here, in the present invention, the detection sensitivity of the total hemoglobin concentration is defined by the magnitude of the slope of the regression line obtained from the current value detected by the electrochemical biosensor described later and the total hemoglobin concentration, and the greater the slope, the higher the detection sensitivity. be high.

Also, the glycated hemoglobin concentration and the total hemoglobin concentration contained in the test liquid can be measured using an electrochemical biosensor. As an electrochemical biosensor, for example, at least an insulating substrate, a first electrode pair formed on the insulating substrate and comprising a pair of working electrodes and a counter electrode, and a second electrode pair comprising a pair of working electrodes and a counter electrode. two electrode pairs, a first reagent portion disposed in contact with or near the first electrode pair and containing a detection enzyme, and a second reagent portion disposed in contact with or near the second electrode pair and containing a mediator and can be used.

FIG. 13 is a perspective view showing an example of an electrochemical biosensor used in this embodiment, and FIG. 14 is an exploded perspective view thereof. The electrochemical biosensor A has an insulating substrate 1 having a conductive portion 4 and a cover 2 laminated with a spacer 3 interposed therebetween. FIG. 13 shows an example having a long rectangular piece shape with the X direction as the longitudinal direction and the Y direction as the width direction.

A conductive portion 4 is formed on one main surface of an insulating base material 1 having a pair of opposing main surfaces. The conductive portion 4 is a conductive portion, and includes a first electrode pair 41 and a second electrode pair 42, a terminal portion 43 formed at one end of the insulating substrate 1, the first electrode pair 41 and the second electrode pair 42. and a lead portion 44 for connecting the terminal portion 43 . The first electrode pair 41 includes a pair of working electrodes 41a, 41a that are arranged in the longitudinal direction and are electrically connected to each other, and a pair of working electrodes 41a that face each working electrode 41a via a minute gap so as to sandwich each working electrode 41a in plan view. It has two pairs of counter electrodes 41b, 41b. In addition, the second electrode pair 42 faces the working electrode 42a arranged in the longitudinal direction so as to be separated from the first electrode pair 41, and the working electrode 42a is opposed to the working electrode 42a via a minute gap so as to sandwich the working electrode 42a in plan view. It has a pair of counter electrodes 42b, 42b. A pair of working electrodes 41a and 41a are connected to terminal 43a through lead 44a, two pairs of counter electrodes 41b and 41b are connected to terminal 43d through lead 44d, and working electrode 42a is connected to terminal 43b through lead 44b. A pair of counter electrodes 42b, 42b are connected to a terminal 43c via a lead 44c. Although FIG. 14 shows the structure excluding the first reagent section and the second reagent section, the first reagent section is arranged in contact with or in the vicinity of the first electrode pair, and the second reagent section is arranged in the vicinity of the first electrode pair. It is placed in contact with or in the vicinity of the two electrode pairs. Two electrode pairs, the first electrode pair 41 and the second electrode pair 42, can be used when measuring two different types of substances to be detected in the sample liquid. The case of measuring two types of substances to be detected is, for example, the case of measuring the HbA1c value, where the first electrode pair 41 measures the HbA1c concentration and the second electrode pair 42 measures the Hb concentration. can be done.

The spacer 3 should have at least one opening, but the electrochemical biosensor A has two openings 3a and 3b spaced apart from each other along the longitudinal direction. Moreover, the spacer 3 is made shorter than the insulating base material 1 so that the terminal portion 43 is exposed. The cover 2 has an opening 2a at one end in the longitudinal direction, an opening 2c at the other end, and an opening 2b at an intermediate portion between the openings 2a and 2c. As with the spacer 3, the cover 2 is shorter than the insulating base 1 so that the terminal portions 43 are exposed. Here, the opening of the spacer 3 and the opening of the cover 2 are such that when the spacer 3 and the cover 2 are stacked, the opening 2b at the center of the cover 2 is the center side edge of the opening 3a of the spacer 3. and the central side edge of the opening 3b, and the opening 2a at one end of the cover 2 at least partially overlaps the side edge of the opening 3a of the spacer 3, and the cover 2 The opening 2c at the other end of the spacer 3 is arranged so as to at least partially overlap the side edge of the opening 3b of the spacer 3. As shown in FIG. The opening 2b in the central portion of the cover 2 can be used as an introduction opening for the test liquid. When the test liquid is dropped into the opening 2b, the test liquid comes into contact with the first reagent part and the second reagent part, and the reaction between the test liquid and the first reagent part and the second reagent part progresses.

In the pretreatment, hemoglobin is degraded by a proteolytic enzyme, at which time the saccharified portion of saccharified hemoglobin is cleaved to produce a saccharified dipeptide. The working electrode of the first electrode pair carries a detection enzyme that specifically reacts with the glycated dipeptide, and the glycated dipeptide is oxidized by the detection enzyme to generate hydrogen peroxide. When a predetermined voltage is applied between the working electrode and the counter electrode of the first electrode pair, a current value corresponding to the concentration of hydrogen peroxide is detected. Since the concentration of hydrogen peroxide correlates with the concentration of glycated dipeptide, the concentration of glycated hemoglobin can be measured by detecting the current value corresponding to the concentration of hydrogen peroxide. On the other hand, the second electrode pair carries a mediator that reacts with hemoglobin. The mediator is a redox body. By oxidizing hemoglobin, the mediator itself becomes a reductant. When a predetermined voltage is applied between the working electrode and the counter electrode of the second electrode pair, a current value corresponding to the concentration of the reductant is generated. detected. Since the reductant concentration correlates with the total hemoglobin concentration, the total hemoglobin concentration can be measured by detecting the current value corresponding to the reductant concentration.

As the detection enzyme, one that oxidizes a glycated dipeptide (eg, fructosylvalyl histidine) to produce hydrogen peroxide can be used. Specifically, fructosyl peptide oxidase can be mentioned. The amount of detection enzyme is, for example, 0.01 to 100 U, preferably 0.05 to 10 U, per sensor or per measurement.

Mediators include, but are not limited to, metal complexes (e.g., osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (e.g., benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, derivatives thereof, etc.). ), phenazine compounds, viologen compounds, phenothiazine compounds, and phenolic compounds. More specifically potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfone acid, 9,10-phenanthrenequinone-2-sulfonate, 9,10-phenanthrenequinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonate , 1-methoxy-5-methylphenazinium methylsulfate, methylviologen, benzylviologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol One or more selected types are used. The amount of the mediator compounded is not particularly limited, and is, for example, 0.1 pmol to 100 μmol, preferably 10 pmol to 10 μmol, more preferably 50 pmol per measurement or per biological substance detection sensor. ~1 μmol.

According to this embodiment, even if the pretreatment liquid contains a surfactant, the detection sensitivity of the total hemoglobin concentration can be improved. This enables more accurate measurement of saccharified hemoglobin.

Embodiment 2
The present embodiment relates to a glycated hemoglobin measurement kit used in the electrochemical measurement method for glycated hemoglobin according to Embodiment 1, and includes steps of preparing a pretreatment liquid containing a surfactant and a protease; Used in an electrochemical measurement method for glycated hemoglobin, comprising the steps of contacting a sample with the pretreatment liquid to prepare a test liquid, and measuring the glycated hemoglobin concentration and the total hemoglobin concentration contained in the test liquid. The glycated hemoglobin measurement kit is characterized by containing a pyrazole compound represented by the following general formula (I) as an additive to the pretreatment liquid.

ここで、一般式（Ｉ）中、Ｒ１、Ｒ２、Ｒ３は、それぞれ独立に、水素原子または炭素数１～４のアルキル基である。

Here, in general formula (I), R1, R2 and R3 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

Examples of the configuration of this measurement kit include an electrochemical biosensor, a buffer solution, a proteolytic enzyme, a surfactant, a pyrazole compound represented by general formula (I), and the like.

According to the present embodiment, since the measurement kit contains the pyrazole compound, it is possible to improve the detection sensitivity of the total hemoglobin concentration compared to the case where the pyrazole compound is not contained. This enables more accurate measurement of saccharified hemoglobin.

EXAMPLES The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

Example 1
In this example, using an electrochemical biosensor, the effect of a pyrazole compound on total hemoglobin concentration detection sensitivity in the presence of various surfactants was investigated.

(experimental method)
<Composition of the second reagent part of the electrochemical biosensor>
Potassium ferricyanide 2% by weight
Taurine 1% by weight
Acupana 0.06% by weight
MVE/MVA 0.02% by weight
Iodine 180 μM
Dodecylmaltoside 0.005% by weight
Here, potassium ferricyanide (manufactured by Wako Pure Chemical Industries, Ltd.), taurine (manufactured by Wako Pure Chemical Industries, Ltd.), Acpana partially neutralized polyacrylic acid (manufactured by Sumitomo Seika Co., Ltd.), MVE/MVA (manufactured by Sansho) ), iodine (manufactured by Tokyo Chemical Industry Co., Ltd.), and dodecyl maltoside is a nonionic surfactant (manufactured by Dojindo Kagaku Co., Ltd.).

<Composition of pretreatment liquid>
Phosphate buffer (PB) (pH 6.5) 5 mM
Actinase E 1200U/mL
Surfactant 2.5% by weight
pyrazole 40 mM

Pyrazole (manufactured by Tokyo Chemical Industry Co., Ltd.) was used. In addition, actinase E used was manufactured by Kaken Pharmaceutical Co., Ltd. The following surfactants were used.
・ Anionic surfactant sodium dodecyl sulfate (abbreviation: SDS) (manufactured by Wako Pure Chemical Industries, Ltd.)
N-lauroyl sarcosine (abbreviation: NDDS) (manufactured by Wako Pure Chemical Industries, Ltd.)
・Cationic surfactant benzyldodecyldimethylammonium dihydrate (manufactured by Tokyo Chemical Industry Co., Ltd.)
Decyltrimethylammonium chloride (manufactured by Tokyo Kasei Co., Ltd.)
Tetradecyltrimethylammonium bromide (abbreviation: TTAB) (manufactured by Tokyo Chemical Industry Co., Ltd.)
・ Nonionic surfactant 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (TritonX-100) (manufactured by Wako Pure Chemical Industries, Ltd.)
Polyoxyethylene sorbitan monolate (Tween) (manufactured by Wako Pure Chemical Industries, Ltd.)
N-dodecyl-β-D-maltoside (manufactured by Dojindo)
・ Amphoteric surfactant dodecyldimethyl (3-sulfopropyl) ammonium hydroxide inner salt (manufactured by Tokyo Chemical Industry Co., Ltd.)
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (abbreviation: CHAPS) (manufactured by Dojin Kagaku Co., Ltd.)

A test solution was prepared by mixing a blood sample with a pretreatment liquid so that the blood sample was diluted 25 times. A test solution was spotted on an electrochemical biosensor, and an electrochemical measurement was performed. In the electrochemical measurement, the electrochemical biosensor is connected to a BAS potentiostat, a voltage of 0.4 V is applied between the working electrode and the counter electrode of the second electrode pair, and the current value after 15 seconds was measured. Total hemoglobin concentration was varied from 0 to 120 μM. The slope of the regression line (hereinafter referred to as the calibration curve) between the total hemoglobin concentration and the measured current value was calculated, and if it was greater than the slope of the calibration curve in the absence of pyrazole, it was determined that the detection sensitivity of the total hemoglobin concentration was improved. .

(result)
FIG. 12 is an example of a calibration curve with and without SDS. When SDS was present in the pretreatment liquid, the slope of the calibration curve was smaller than when SDS was not present, and the detection sensitivity was lowered. The total hemoglobin concentration was measured in the range of 0-2782.95 μM. On the other hand, FIGS. 1 to 10 show the measurement results of calibration curves when the pretreatment liquid contains pyrazole. The presence of pyrazole in the pretreatment solution improved the detection sensitivity for all surfactants used. Table 1 below shows the slope values of the calibration curve.

Example 2
In this example, the effect of total hemoglobin concentration on detection sensitivity was examined by varying the pyrazole concentration. The composition of the pretreatment liquid used is as follows.

<Composition of pretreatment liquid>
Phosphate buffer (PB) (pH 6.5) 135 mM
Actinase E 1200U/mL
Surfactant NDDS 1% by weight
Sodium glutamate 300 mM
Pyrazole 10, 40, 80, 120, 200 mM

(result)
FIG. 11 shows the measurement results of the calibration curve when the pyrazole concentration was changed. The detection sensitivity improved as the pyrazole concentration increased, and became almost constant at 80 mM or higher.

Comparative example 1
In this comparative example, instead of pyrazole, a five-membered heterocyclic compound other than pyrazole represented by general formula (I) (hereinafter referred to as a comparative additive) was used. The composition of the pretreatment liquid used is as follows.

<Composition of pretreatment liquid>
Phosphate buffer (PB) (pH 6.5) 135 mM
Actinase E 1200U/mL
Surfactant NDDS 1% by weight
Sodium glutamate 300 mM
Sodium 9,10-phenanthrenequinone-2-sulfonate (PQSA) 12 mM
Comparative additive 10, 30 mM

The following additives were used for comparison.
・Imidazoles Imidazole (manufactured by Tokyo Chemical Industry Co., Ltd.)
Ethylimidazole (manufactured by Tokyo Chemical Industry Co., Ltd.)
1-Butylimidazole (manufactured by Tokyo Chemical Industry Co., Ltd.)
・Aminomethylpyrazole (manufactured by Sigma-Aldrich)
・Triazole (manufactured by Tokyo Kasei Co., Ltd.)
・ 4-amino-1,2,4-triazole (manufactured by Sigma-Aldrich)
・ 1H-tetrazole (manufactured by Sigma-Aldrich)
The concentrations of aminomethylpyrazole and triazole are 10 and 40 mM. Also, for 4-amino-1,2,4-triazole and 1H-tetrazole, the concentration was 40 mM and no PQSA was added.

(result)
In the case of any of the comparative additives used, the slope of the calibration curve hardly changed compared to the case where these comparative additives were not added, and no improvement in detection sensitivity was observed.

Reference Signs List 1 insulating substrate 2 cover 2a, 2b, 2c cover opening 3 spacer 3a, 3b spacer opening 4 conductive portion 41 first electrode pair 41a working electrode 41b counter electrode 42 second electrode pair 42a working electrode 42b counter electrode 43 terminal portion 44 Lead part

Claims (4)

A step of preparing a pretreatment solution containing a surfactant, a pyrazole compound represented by the following general formula (I), and a protease;
a step of bringing the pretreatment liquid into contact with a blood specimen to prepare a test liquid;
A method for electrochemically measuring glycated hemoglobin, comprising a step of measuring a glycated hemoglobin concentration and a total hemoglobin concentration contained in the test solution.

Here, in general formula (I), R1, R2 and R3 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. 2. The electrochemical measurement method according to claim 1, wherein said surfactant is an N-acylamino acid salt. 3. The electrochemical measurement method according to claim 1, wherein said proteolytic enzyme is actinase E. a step of preparing a pretreatment liquid containing a surfactant and a proteolytic enzyme; a step of contacting the pretreatment liquid with a blood specimen to prepare a test liquid; A glycated hemoglobin measurement kit for use in an electrochemical measurement method for glycated hemoglobin, comprising:
A measurement kit for glycated hemoglobin, containing a pyrazole compound represented by the following general formula (I) as an additive to the pretreatment liquid.

Here, in general formula (I), R1, R2 and R3 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

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