JP2008170350A - Hemoglobin measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hemoglobin measurement method capable of measuring hemoglobin, particularly stabilized hemoglobin A1c which can be used as an indicator for the diagnosis of diabetes, within a short period of time and with high accuracy. <P>SOLUTION: In the method for measuring hemoglobin using an electrophoresis apparatus, the voltage of 100-2,000 V is applied using a microchip having a migration path whose inner surface is coated with hydrophilic polymer, length is 10-100 mm, and width is 10-100 μm, and buffer solution whose pH is 5.0-6.0 and salt concentration is 10-300 mM. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a method for measuring hemoglobin capable of measuring hemoglobin, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time and with high accuracy.

Hemoglobin (hereinafter also referred to as Hb), particularly hemoglobin A1c (hereinafter referred to as HbA1c), which is a kind of glycated hemoglobin, reflects an average blood glucose level in the past 1 to 2 months. It is widely used as a test item for grasping the blood glucose control status of a test or a diabetic patient.
Conventionally, HPLC methods, immunization methods, electrophoresis methods and the like have been used as methods for measuring HbA1c. Among them, the HPLC method widely used in the clinical laboratory field can measure in 1 to 2 minutes per sample, and the CV value of the simultaneous reproducibility test is about 1.0%. It has been realized. This level of performance is required as a measurement method for grasping the blood glucose control state of a diabetic patient.

On the other hand, the electrophoresis method is a technique capable of producing an inexpensive and small system such as microchip electrophoresis because the apparatus configuration is simple, and clinical examination of high-precision measurement technology of HbA1c in electrophoresis method. Application to can expect a very beneficial effect in terms of cost.
Although the measurement of Hbs by electrophoresis has been used for a long time as a method for separating abnormal Hb having an amino acid sequence different from the usual, separation of HbA1c is very difficult. It took more than a minute. As described above, the electrophoresis method has problems in terms of measurement time and measurement accuracy, so that it has hardly been applied to diabetes diagnosis at present.

On the other hand, the capillary electrophoresis method that appeared around 1990 generally enables high separation and high-accuracy measurement. For example, Patent Document 1 discloses a method for separating HbA1c by capillary electrophoresis. Is disclosed.
However, when the method disclosed in Patent Document 1 is used, the problem that the measurement time is long is not solved, and in addition, the pH of the buffer solution used is as high as 9 to 12, and Hb may be denatured. Due to its nature, this method has been difficult to apply to clinical tests.

Further, Patent Document 2 uses a buffer solution containing sulfated polysaccharide by dynamically coating the inner surface of the capillary by passing the cationic polymer through the capillary in capillary electrophoresis. A method is disclosed. According to this method, measurement can be performed in about 10 minutes, and measurement can be performed in a shorter time compared to gel electrophoresis.

However, in this method, since the pH of the buffer solution to be used is as low as 4.7, there is a possibility that Hb as a measurement sample is denatured. In addition, since the salt concentration of the buffer used is as large as about 380 mM and the load voltage is as large as 8.5 kV, the peak in the electropherogram obtained by the generation of Joule heat etc. is deformed and the measurement accuracy is lowered. There was a problem that it was difficult to make a small system.

Furthermore, when making a diagnosis of diabetes, among HbA1c, stable HbA1c, which is an index of diabetes, must be separated to eliminate the influence of modified Hb such as unstable HbA1c, carbamylated Hb, and acetylated Hb. However, the electropherograms obtained by the methods disclosed in Patent Documents 1 and 2 have insufficient separation performance and measurement accuracy, and it is difficult to separate stable HbA1c within the technical scope disclosed in these methods. Met.

In addition, such conditions as buffer solution and voltage load can be applied only to a large and expensive capillary electrophoresis apparatus. Therefore, such a method is applied to a small and inexpensive microchip electrophoresis. It was impossible to apply the method to the measurement.
Japanese National Patent Publication No. 9-510792 Japanese Patent Laid-Open No. 9-105739

An object of this invention is to provide the measuring method of hemoglobin which can perform a highly accurate measurement for a short time at low cost.

The present invention relates to a method for measuring hemoglobin using an electrophoresis method, wherein the inner surface is coated with a hydrophilic polymer, the microchip has a migration path having a length of 10 to 100 mm and a width of 10 to 100 μm; , A method for measuring hemoglobin using a buffer solution having a pH of 5.0 to 6.0 and a salt concentration of 10 to 300 mM and loading a voltage of 100 to 2000 V.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have determined that a microchip having a migration path in which a predetermined size and an inner surface are coated with a hydrophilic polymer, and a buffer solution having a pH and a salt concentration within a predetermined range. As a result, it has been found that hemoglobins can be measured with high accuracy and in a short time at a lower cost than in the past, and the present invention has been completed.

The electrophoresis method used in the method for measuring hemoglobin of the present invention is a method applicable to an electrophoresis apparatus equipped with a microchip. Such an electrophoresis apparatus has a simpler apparatus configuration, can be easily miniaturized, and can be manufactured at a lower cost than the HPLC apparatus and capillary electrophoresis apparatus conventionally used for measuring Hb species.

In this specification, a microchip refers to a plate-like substrate made of an inorganic or organic material and having a size of about several centimeters square. Specifically, for example, a conventionally known chip used in a technique called μ-TAS or Lab-on-a-chip may be used.

The material constituting the microchip is not particularly limited, and a conventionally known material can be used. For example, glass materials such as borosilicate glass and quartz, silica materials such as fused silica and polydimethylsiloxane, and polyacrylic are used. Resin-based materials such as resin, polystyrene resin, polylactic acid resin, polycarbonate resin, and olefin resin are preferable. Of these, glass-based, silica-based, and acrylic resin-based materials are preferable.

The microchip has a migration path.
In the present invention, an electrophoresis path is a channel formed on or in a microchip, and when electrophoresis is performed, a sample moves and / or is separated in the channel. A site (a site detected by the detection unit from the site where the sample is injected).

The migration path has a lower limit of 10 mm and an upper limit of 100 mm. If it is less than 10 mm, the sample may not be sufficiently separated, and accurate measurement may not be possible. If it exceeds 100 mm, the measurement time may be extended or the peak shape may be deformed in the obtained electropherogram, so that accurate measurement may not be possible.

The migration path has a lower limit of 10 μm and an upper limit of 100 μm. If it is less than 10 μm, the optical path length for detection by the detector is small, and the measurement accuracy may be reduced. When it exceeds 100 μm, the peak becomes broad in the electropherogram obtained by diffusing the sample in the migration path, and the measurement accuracy may be lowered.

The shape of the migration path is not particularly limited, and examples thereof include a linear shape, a curved shape having a certain curvature, and a spiral shape. Of these, a linear shape is preferable.
The cross-sectional shape of the migration path is not particularly limited, and examples thereof include a rectangle and a circle.
The microchip may have a single migration path or a plurality of migration paths.

The inner surface of the migration path is coated with a hydrophilic polymer.
The coating layer formed on the inner surface of the migration path by performing the coating may be a single layer or a multilayer made of the same or different materials.
By coating with a hydrophilic polymer in this way, hydrophilicity is imparted to the inner surface of the migration path, and the nonspecificity of each component such as protein and lipid in human blood that is a measurement sample that passes through the interior of the migration path It is possible to suppress adsorption and sufficiently separate and measure hemoglobin as a measurement target. If the hydrophilicity is insufficient, each component in the measurement sample may cause nonspecific adsorption on the inner surface of the migration path, which may adversely affect the separation of each component.
In this specification, the hydrophilic polymer refers to a polymer having a hydrophilic functional group.

It does not specifically limit as a hydrophilic polymer used for the coating of the said migration path, For example, an ionic or nonionic hydrophilic polymer is mentioned.
The ionic hydrophilic polymer is not particularly limited, and examples thereof include poly (meth) acrylic acid, poly (2-acrylamido-2-methylpropanesulfonic acid), poly (2-diethylaminoethyl methacrylate), polyethyleneimine, and polybrene. Ionic synthetic polymers such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan and other sulfate group-containing polysaccharides and salts thereof; alginic acid, hyaluronic acid, pectinic acid and other carboxyl group-containing polysaccharides and salts thereof; cellulose, Anionic polysaccharides such as dextran, agarose, mannan, starch and other neutral polysaccharides and derivatives thereof and anionic polysaccharides such as salts thereof, chitosan derivatives such as chitin and chitosan and salts thereof; aminocellulose, N-methyl N-place such as aminocellulose Cellulose derivatives and salts thereof; neutral polysaccharides such as dextran, agarose, mannan and starch; amino group-introduced products thereof; and cationic polysaccharides such as salts thereof; bovine serum albumin; Examples include proteins such as lactoferrin and skim milk.

The nonionic hydrophilic polymer used for the above coating is not particularly limited, and for example, hydrophilic properties such as polysaccharides such as cellulose, dextran, agarose, mannan and starch, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and the like. Organic obtained by polymerizing or copolymerizing monomers such as polymers, 2-hydroxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, (meth) acrylamide, glycidyl (meth) acrylate, etc. Examples include synthetic polymers.

The method for coating the hydrophilic polymer on the migration path is not particularly limited, and a conventionally known method can be used. For example, by passing a solution containing the hydrophilic polymer into the migration path. Dynamic coating method for coating; Immobilization coating method in which the hydrophilic polymer is brought into contact with the inner surface of the migration path and physically adsorbed and immobilized using hydrophobic or electrostatic interaction, etc. Examples thereof include an immobilization coating method in which the inner surface and the hydrophilic polymer are bonded and immobilized by a covalent bond via a functional group of each substance or other substances. When the above-described immobilization coating method is used, the coating layer does not peel off through a heating step, a drying step, and the like, so that repeated measurement is possible. Specifically, the immobilization coating method includes, for example, passing a solution containing the hydrophilic polymer through the migration path, injecting air into the migration path, and then drying the solution, followed by drying. And the like.

Of these, the immobilization coating method is preferable. By using the fixed coating method, once coating is performed, peeling does not occur, and it is not necessary to perform coating again for each measurement. Therefore, the measurement time can be shortened during continuous measurement or the like.

When immobilizing the hydrophilic polymer in the migration path, another type of layer may be formed between the inner surface of the migration path and the layer made of the hydrophilic polymer. When the different kind of layer is formed, the layer made of the hydrophilic polymer only needs to be formed on the innermost surface of the migration path.

When the hydrophilic polymer is coated on the migration path, the hydrophilic polymer is preferably supplied to the coating as a solution containing the hydrophilic polymer, depending on the type and the coating method.
As a concentration of the hydrophilic polymer solution, a preferable lower limit is 0.01% and a preferable upper limit is 20%. If it is less than 0.01%, the coating may be insufficient. If it exceeds 20%, the layer made of the hydrophilic polymer cannot be formed uniformly and may be peeled off during the measurement of hemoglobin, which may cause a decrease in reproducibility.

In the method for measuring hemoglobin of the present invention, after filling the microchip flow path with a buffer solution, a measurement sample is introduced into the flow path, and voltage is applied to the flow path for electrophoresis. Thus, hemoglobins are separated and measured.
The buffer has a pH lower limit of 5.0 and an upper limit of 6.0. If it is less than 5.0, the hemoglobins are denatured, and the measurement accuracy may be reduced. If it exceeds 6.0, it approaches the isoelectric point of hemoglobin, so the amount of charge of hemoglobin decreases, the electrical difference of each component of hemoglobin decreases, and the separation of each component becomes insufficient. Therefore, the measurement accuracy may be reduced.

The lower limit of the salt concentration of the buffer solution is 10 mM, and the upper limit is 300 mM. If it is less than 10 mM, the electrolytic mass in the buffer solution is small, and the amount of current becomes too small. Therefore, electrophoresis is not performed sufficiently, and the separation performance of each component may deteriorate. If it exceeds 300 mM, the amount of current becomes too large, and the peak shape may be deformed in the chromatogram obtained due to the influence of heat generation or the like, resulting in a decrease in measurement accuracy. A preferred lower limit is 50 mM and a preferred upper limit is 250 mM.
In addition, in this specification, salt concentration means the molar concentration of the buffer composition which has the buffer capacity mentioned later. When a plurality of buffer compositions described later are used, the total molar concentration thereof is used.

The buffer solution used in the present invention contains a conventionally known buffer solution composition having a buffer capacity. Examples of these buffer compositions include organic acids such as citric acid, succinic acid, tartaric acid and malic acid and salts thereof; amino acids such as glycine, taurine and arginine; hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid and boric acid. And inorganic acids such as acetic acid and salts thereof; and derivatives thereof having a buffer capacity. These buffer compositions may be used alone or in combination of two or more.

As long as the pH and salt concentration do not exceed the above ranges, generally used additives such as chaotropic compounds, surfactants, various polymers, and hydrophilic low-molecular compounds are appropriately added to the buffer solution. May be.

It does not specifically limit as said various polymers, For example, an ionic polymer is mentioned.
In this specification, an ionic polymer means a polymer having an ionic functional group in the molecule.
The ionic polymers are roughly classified into anionic polymers and cationic polymers depending on the ionic type. Of these, anionic polymers are preferred.

The anionic polymer is a polymer having an anionic functional group in the molecule.
The anionic functional group is not particularly limited, and examples thereof include a carboxyl group, a phosphoric acid group, and a sulfonic acid group.
The anionic polymer preferably has hydrophilicity.
The anionic polymer having hydrophilicity is not particularly limited, and examples thereof include an anionic group-containing polysaccharide and an anionic group-containing organic synthetic polymer.
Specifically, for example, polysaccharides such as dextran sulfate and chondroitin sulfate; poly (meth) acrylic acid, poly (2-acrylamido-2-methylpropanesulfonic acid), and copolymers thereof, chondroitin sulfate, dextran sulfate , Heparin, heparan, fucoidan and other sulfate group-containing polysaccharides and salts thereof; carboxyl group-containing polysaccharides such as alginic acid, hyaluronic acid and pectinic acid, and salts thereof; neutrality such as cellulose, dextran, agarose, mannan and starch Anionic polysaccharides such as polysaccharides and their derivatives, and their salts, poly (meth) acrylic acid, phosphoric acid group-containing (meth) acrylic acid ester polymer, sulfonic acid group-containing (Meth) acrylic acid polymer, 2-acrylamido-butylsulfonic acid polymer, and Water-soluble anionic group-containing organic synthetic polymers such as those copolymers thereof.

In the method for measuring hemoglobin of the present invention, the lower limit of the applied voltage is 100V, and the upper limit is 2000V. When the voltage is less than 100 V, electrophoresis is not sufficiently performed, so that separation of each component may be insufficient. When it exceeds 2000 V, the heat generation becomes large, and in the obtained electropherogram, problems such as peak deformation may occur. A preferred lower limit is 150V and a preferred upper limit is 1500V.

In the method for measuring hemoglobin of the present invention, the hemoglobin to be measured is not particularly limited, and examples thereof include conventionally known hemoglobins. Specific examples include HbA1a, HbA1b, HbF, unstable HbA1c, stable HbA1c, HbA0, HbA2; modified hemoglobins such as acetylated Hb and carbamylated Hb; and abnormal hemoglobins such as HbS and HbC. Among these, stable HbA1c can be preferably measured.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method which can perform the measurement of hemoglobin, especially stable hemoglobin A1c used as a diagnostic parameter | index of diabetes with high precision in a short time can be provided. Specifically, simultaneous reproducibility can be achieved in a short time of several minutes per sample, particularly in the measurement of stable HbA1c, using a method with a simple and low-cost apparatus configuration called electrophoresis using a microchip. The CV value shown can be measured with high accuracy of about 1.0%. Therefore, it becomes possible to suitably manage the numerical value of HbA1c of diabetic patients by electrophoresis.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Example 1)
(1) Production of microchip An electrophoresis path having a length of 80 mm and a width of 80 μm was formed on a polydimethylsiloxane microchip to produce a cross-shaped electrophoresis microchip. The electrophoresis path was filled with a 0.5% chitosan (hydrophilic polymer) solution and allowed to stand for 20 minutes. Thereafter, air was injected into the migration path to drive off the chitosan solution, and then the film was dried in a dryer at 40 ° C. for 12 hours. Thereafter, a series of steps consisting of injection of chitosan solution, injection of air, and drying was repeated five times to obtain a microchip coated with the migration path. By filling the migration path of the obtained chitosan-coated microchip with 150 mM citrate buffer (pH 5.2) containing 2.0% chondroitin sulfate, a measurement microchip was obtained.

(2) Measurement of blood of healthy persons A citrate buffer solution (pH 6.0) containing 0.05% Triton X-100 (surfactant: manufactured by Wako Pure Chemical Industries, Ltd.) in 70 μL of whole blood of healthy persons obtained by collecting heparin from healthy persons. ) 200 μL was added for hemolysis dilution to obtain a hemolysis sample.
By adding the hemolyzed sample obtained at one end of the migration path of the above-mentioned microchip, performing electrophoresis by applying a voltage of 1000 V to both ends of the migration path, and measuring the change in absorbance at 415 nm visible light The stable HbA1c was measured.
FIG. 1 is a schematic diagram showing an electropherogram obtained by measurement of blood of a healthy person in Example 1. In FIG. 1, peak 1 indicates stable HbA1c, and peak 2 indicates HbA0. The stable HbA1c could be separated with an electrophoresis time of about 60 seconds.

(3) Measurement of samples containing modified Hb Glucose was added to healthy whole blood collected from heparin from healthy individuals so as to be 2500 mg / dL, heated at 37 ° C. for 3 hours, and anxiety as a kind of modified Hb A sample containing a large amount of typical HbA1c was artificially prepared.
Add the hemolyzed sample obtained at one end of the migration path of the obtained microchip, perform electrophoresis by applying a voltage of 1000 V to both ends of the migration path, and measure the change in absorbance at 415 nm visible light. Was used to measure stable HbA1c.
2 is a schematic diagram showing an electropherogram obtained by measuring a sample containing modified Hb in Example 1. FIG. In FIG. 2, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 2, stable HbA1c and unstable HbA1c, which is modified Hb, were well separated.

(Example 2)
(1) Production of microchip An electrophoresis path having a length of 50 mm and a width of 50 μm was formed on a fused silica microchip to produce a cross-shaped electrophoresis microchip. The electrophoresis path was filled with a 0.5% aqueous preprene solution and left for 20 minutes. Thereafter, air was injected into the migration path to drive out the aqueous solution of preprene, followed by drying in a dryer at 40 ° C. for 12 hours. Thereafter, the microchip on which the inner surface of the migration path was coated was obtained by repeating again injection of the aqueous solution of preprene, injection of air, and drying five times. A microchip for measurement was obtained by filling a migration path of the obtained polybrene-coated microchip with 50 mM succinate buffer (pH 5.8) containing 1.5% dextran sulfate.
(2) Measurement of healthy human blood and measurement of sample containing modified Hb The measurement of healthy human blood and the measurement of healthy human blood were carried out in the same manner as in Example 1 except that electrophoresis was performed by applying a voltage of 500 V to both ends of the migration path. Samples containing modified Hb were measured. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 1 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 2 was obtained.

(Example 3)
(1) Production of microchip An electrophoresis path having a length of 90 mm and a width of 30 μm was formed on a methylmethacrylate microchip to produce a microchip for electrophoresis. The electrophoresis path was filled with 0.5% aqueous chondroitin sulfate solution and left for 20 minutes. Thereafter, air was injected into the migration path to drive out the aqueous chondroitin sulfate solution, and then dried in a dryer at 40 ° C. for 12 hours. Thereafter, injection of chondroitin sulfate aqueous solution, air injection, and drying were repeated 5 times to obtain a microchip coated with the inner surface of the migration path. A microchip for measurement was obtained by filling a migration path of the obtained chondroitin sulfate-coated microchip with a 100 mM phosphate buffer solution (pH 5.5) containing 2.0% chondroitin sulfate.

(2) Measurement of healthy human blood and measurement of sample containing modified Hb The measurement of healthy human blood was carried out in the same manner as in Example 1 except that electrophoresis was performed by applying a voltage of 700 V to both ends of the migration path. Samples containing modified Hb were measured. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 1 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 2 was obtained.

Example 4
(1) Production of microchip An electrophoresis path having a length of 60 mm and a width of 60 μm was formed on a methylmethacrylate microchip to produce a microchip for electrophoresis. The electrophoresis path was filled with an aqueous solution of 0.5% poly (2-hydroxyethyl methacrylate) (hereinafter also referred to as poly-2-HEMA) and left for 20 minutes. Thereafter, air was injected into the migration path to drive out the aqueous poly-2-HEMA solution, and then the mixture was dried in a dryer at 40 ° C. for 12 hours. Thereafter, the microchip on which the inner surface of the migration path was coated was obtained by repeating again injection of the aqueous solution of poly-2-HEMA, injection of air, and drying five times. 100 mM malic acid buffer (pH 5.4) containing 2.0% of (2-acrylamido-butylsulfonic acid)-(2-HEMA) copolymer in the migration path of the obtained poly-2-HEMA-coated microchip. By satisfying the above, a measurement microchip was obtained.

(2) Measurement of healthy human blood and measurement of sample containing modified Hb The measurement of healthy human blood and the measurement of healthy human blood were carried out in the same manner as in Example 1 except that electrophoresis was performed by applying a voltage of 400 V to both ends of the migration path. Samples containing modified Hb were measured. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 1 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 2 was obtained.

(Comparative Example 1)
A capillary made of fused silica (GL Science: inner diameter 25 μm × total length 30 cm) was passed through 0.2N-NaOH, ion-exchanged water, 0.5N-HCl in this order to wash the inside of the capillary, and then BSA (cation Malic acid buffer solution containing 0.5% by weight of a functional polymer: bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) was passed for 1 minute to coat the inner surface of the capillary.
The obtained capillary was set in a capillary electrophoresis apparatus (P / ACE MDQ manufactured by Beckman-Couler), and malic acid buffer (pH 4.7) containing 0.2% by weight of chondroitin sulfate (manufactured by Wako Pure Chemical Industries). ) Was filled into the capillary described above.
Using the obtained capillary electrophoresis apparatus for measurement, a sample containing modified Hb was measured by applying a voltage of 10 kV.
FIG. 3 is an electropherogram obtained by measuring a sample containing modified Hb in Comparative Example 1.
In FIG. 3, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 4, in the obtained electropherogram, peak 1 indicating stable HbA1c and peak 3 indicating modified Hb overlap.

(Comparative Example 2)
A microchip for electrophoresis was obtained in the same manner as in Example 1 except that the migration path of the microchip was 120 mm long and 140 μm wide. Furthermore, the blood of a healthy person was measured under the same measurement conditions as in Example 1.
In the obtained electropherogram, only one hemoglobin peak was confirmed, and stable HbA1c could not be separated at all. This result was not improved even when the measurement was performed by changing the electrophoresis conditions such as the buffer composition, the set voltage, and the electrophoresis time.

(Evaluation)
(1) Separation performance test of modified Hb compounds Regarding the measurement conditions of Examples 1 to 4 and Comparative Example 1, a healthy human blood sample and a modified Hb-containing sample prepared based on the same healthy human blood, namely Example 1 (3 ) An unstable HbA1c-containing sample obtained by adding glucose to a healthy human blood prepared in the measurement of a sample containing modified Hb so as to be 2500 mg / dL, and another modified Hb-containing sample, that is, the healthy human blood Samples containing acetylated Hb obtained by adding acetaldehyde to 50 mg / dL and carbamylated Hb containing samples obtained by adding sodium cyanate to 50 mg / dL were prepared. The stable HbA1c value was determined.
The stable HbA1c value of each sample was calculated by determining the ratio (%) of the area of the stable HbA1c peak to the area of all hemoglobin peaks.
A value obtained by subtracting the stable HbA1c value of the obtained healthy human blood sample from the stable HbA1c value of the obtained modified Hb-containing sample was determined.
The results are shown in Table 1.

In the measurement conditions of Examples 1 to 4, there is almost no difference in the measured value of stable HbA1c between the modified Hb-containing sample and the healthy human blood sample not containing modified Hb, and the measured value difference is 0.2% or less. It was found that stable HbA1c can be measured accurately even in the presence of modified Hbs. On the other hand, under the measurement conditions of Comparative Example 1, it was found that the stable HbA1c value greatly fluctuated due to insufficient separation of the modified Hb and the stable HbA1c, and accurate measurement was not possible.

(2) Simultaneous reproducibility test Using the measurement conditions of Examples 1 to 4 and Comparative Example 1, the CV value of the stable HbA1c value obtained by continuously obtaining the same sample of a healthy human blood sample 10 times was calculated.
In Comparative Example 1, when one measurement was completed, 0.2N NaOH, ion-exchanged water, and 0.5N HCl solution were passed in that order to wash the inside of the capillary, and then BSA (cation After conducting dynamic coating by passing 0.5% malic acid buffer solution of functional polymer: bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) for 1 minute, the measurement is repeated by measuring the next sample. A simultaneous reproducibility test was performed.
The results are shown in Table 2.

(3) Durability test Using the measurement conditions of Examples 1 to 4 and Comparative Example 1, the maximum and minimum stable HbA1c values obtained by continuously measuring the same sample of a healthy human blood sample 100 times The difference (R value) from the value was calculated.
The results are shown in Table 2.

As shown in Table 2, in Examples 1 to 4, in the simultaneous reproducibility test, the CV value indicating the degree of variation was around 1%, which was a good result. Further, the variation width at the time of continuous 100 sample measurement in the durability test was also very small at about 0.2%, and it was found that stable HbA1c can be measured with high accuracy even in continuous measurement of a large number of samples.
On the other hand, in Comparative Example 1, the CV value in the simultaneous reproducibility test is as large as 4% or more, and the variation width in the durability test is about 0.5% at the maximum, in managing the HbA1c value of diabetic patients. It was totally inadequate

(4) Influence test of pH and salt concentration Under the measurement conditions of Examples 1 and 2, the pH and salt concentration of the citrate buffer used in Example 1 and the succinate buffer used in Example 2 were changed. The influence of the pH and salt concentration of the buffer solution was measured by performing the above-described simultaneous reproducibility test.
The result when the pH is changed is shown in FIG. 4a, and the result when the salt concentration is changed is shown in FIG. 4b.
In both Examples 1 and 2, it was found that when the pH is 5.0 to 6.0 and the salt concentration is 10 to 300 mM, the CV value becomes small and stable HbA1c can be measured with high accuracy.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method of hemoglobin which can perform the measurement of hemoglobin, especially stable hemoglobin A1c used as a diagnostic index of diabetes with high precision in a short time can be provided.

In Examples 1, 2, 3, and 4, it is the electropherogram obtained by the measurement of the blood of a healthy person. In Examples 1, 2, 3, and 4, it is the electropherogram obtained by the measurement of the sample containing modification Hb. In Comparative example 1, it is the electropherogram obtained by the measurement of the sample containing modification Hb. It is a graph which shows the result of the simultaneous reproducibility test at the time of changing the pH of a buffer solution in the measurement conditions of Example 1 and 2. It is a graph which shows the result of the simultaneous reproducibility test at the time of changing the salt concentration of a buffer solution in the measurement conditions of Example 1 and 2.

Claims (2)

A method for measuring hemoglobin using an electrophoresis method, wherein the inner surface is coated with a hydrophilic polymer, the microchip has an electrophoresis path having a length of 10 to 100 mm and a width of 10 to 100 μm, and a pH of 5 A method for measuring hemoglobin, comprising applying a voltage of 100 to 2000 V using a buffer solution having a salt concentration of 10 to 300 mM in a range of 0.0 to 6.0. The method for measuring hemoglobin according to claim 1, wherein the buffer contains an anionic polymer.

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