JP4854528B2 - Method for measuring hemoglobins - Google Patents

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 (Hb), particularly hemoglobin A1c (hereinafter referred to as HbA1c), which is a kind of glycated hemoglobin, reflects the average sugar concentration in the blood during the past 1 to 2 months. It is widely used as a test item for grasping blood glucose control status of patients with diabetes.
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 a problem in terms of measurement time and measurement accuracy when applied to the clinical laboratory field, 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.

Patent Document 2 discloses a method in which a cationic polymer is dynamically coated on the inner surface of a capillary by passing a cationic polymer through the capillary and a buffer containing sulfated polysaccharide is used in capillary electrophoresis. 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.

In general, when diagnosing diabetes, stable HbA1c as an index of diabetes must be separated from HbA1c 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 was difficult to separate stable HbA1c within the technical scope of these methods. .

Furthermore, conventional methods such as capillary electrophoresis and microchip electrophoresis use a migration path made of a relatively hydrophobic material made of glass or resin, so that components such as proteins in the sample are used. However, the non-specific adsorption on the inner surface of the migration path may adversely affect the measurement accuracy. In addition, when electrophoresis is performed repeatedly using the same migration path, the durability performance of the migration path may be adversely affected.
Special table Hei 09-510792 JP 09-105739 A

An object of the present invention is to provide a method for measuring hemoglobin capable of measuring hemoglobins, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time with high accuracy.

The present invention is a method for measuring hemoglobin using capillary electrophoresis or microchip electrophoresis , and is a method for measuring hemoglobin using an electrophoresis path whose inner surface is treated with ozone.

As a result of diligent study, the present inventors have determined that components such as proteins and lipids in human blood serving as a sample migrate in a method for measuring hemoglobin by electrophoresis by using a migration path whose inner surface is treated with ozone. It has been found that nonspecific adsorption to the road inner surface can be suppressed, and that high-precision hemoglobins, particularly stable HbA1c can be measured in a short time, and the present invention has been completed.
The present invention is described in detail below.

The electrophoresis method used in the method for measuring hemoglobin of the present invention is not particularly limited, and examples thereof include capillary electrophoresis and microchip electrophoresis. Of these, microchip electrophoresis is preferable.

FIG. 1 shows an example of a capillary electrophoresis apparatus used in the method for measuring hemoglobin of the present invention. As shown in FIG. 1, the capillary electrophoresis apparatus 11 includes an anode tank 12, a cathode tank 13, a capillary 14, a high voltage power supply 15, a detector 16, and a pair of electrodes 17 and 18. Both ends of the capillary 14 are immersed in the buffer solution in the anode tank 12 and the cathode tank 13, and the inside of the tubular capillary 14 is filled with the buffer solution. The electrodes 17 and 18 are electrically connected to the high voltage power supply 15.
In the method for measuring hemoglobin of the present invention, the inner surface of the migration path included in the capillary 14 is subjected to ozone treatment. When measuring hemoglobins, a measurement sample moving through the capillary 14 is measured by the detector 16 by injecting a measurement sample from one of the capillaries 14 and applying a predetermined voltage from the high-voltage power supply 15. To do.

The method for measuring hemoglobin of the present invention preferably uses a microchip electrophoresis method.
The microchip electrophoresis is a method of performing electrophoresis using a microchip instead of the capillary in the capillary electrophoresis.
The microchip is made of a conventionally known material such as an inorganic or organic material, has a size of about several centimeters to several tens of centimeters, and is a plate-like substrate on which an electrophoresis path for electrophoresis is formed. For example, a conventionally known chip used in a technique called μ-TAS or Lab-on-a-chip may be used.

In the method for measuring hemoglobin of the present invention, the migration path refers to a site (from the injected site to the site to be detected) where the measurement sample moves and / or separates when electrophoresis is performed. Specifically, for example, in the case of capillary electrophoresis, it means from the part where the sample in the capillary is injected to the part detected by the detector, and in the case of microchip electrophoresis, the microchip groove This means from the portion where the sample is injected to the portion detected by the detector.
The inner surface of the migration path specifically refers to the inner surface of the migration path of the capillary in the case of capillary electrophoresis, for example, and the inner surface of the migration path of the microchip groove in the case of microchip electrophoresis. I mean.

The material constituting the migration path 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, A material such as an acrylic resin, a polystyrene resin, a polylactic acid resin, a polycarbonate resin, and an olefin resin is preferable. Of these, glass-based, silica-based, and acrylic resin-based materials are preferable.

The migration path has a preferred lower limit of length of 10 mm and a preferred upper limit of 300 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 300 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 preferred lower limit of 1 μm width and a preferred upper limit of 100 μm. If it is less than 1 μm, the optical path length for detection by the detector is small, and the measurement accuracy may decrease. 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 migration path may be configured to use a plurality of migration paths at once. That is, in the capillary electrophoresis method, a plurality of capillaries may be used simultaneously, and in the microchip electrophoresis method, a plurality of electrophoresis paths may be formed on one chip.

The migration path may have an inner surface subjected to a conventionally known surface modification treatment.
The surface modification treatment is not particularly limited. For example, chemical treatment such as optical treatment such as ultraviolet irradiation or plasma treatment, chemical treatment such as reaction and bonding with various chemical substances, and coating treatment with various compounds and polymers. And / or physical modification treatment and the like.

Especially, it is preferable to perform the said coating process. By performing the coating treatment, excellent separation performance can be exhibited.
The coating layer formed on the inner surface of the migration path by performing the coating treatment may be a single layer or a multilayer composed of the same or different coating agents.

Although it does not specifically limit as a coating agent used for the said coating process, A hydrophilic polymer is preferable.
By the coating treatment with the hydrophilic polymer, hydrophilicity can be imparted to the inner surface of the migration path, and further, the slight hydrophobic portion in the coating agent can be made hydrophilic by the ozone treatment described later. Become. By such hydrophilicity, nonspecific adsorption of each component such as protein and lipid in human blood that is a sample that passes through the inside of the migration path is suppressed, and the hemoglobins to be measured are sufficiently separated and measured. Is possible. If the hydrophilicity is insufficient, each component such as protein and lipid in human blood as a sample may cause non-specific adsorption on the inner surface of the migration path, which may adversely affect the separation of each component.

It does not specifically limit as a hydrophilic polymer used for the said coating process, For example, the hydrophilic polymer of an ionic polymer or a nonionic polymer is mentioned.
The hydrophilic polymer of the ionic polymer is not particularly limited, and examples thereof include sulfate group-containing polysaccharides such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan, and salts thereof; alginic acid, hyaluronic acid, pectinic acid, etc. Carboxyl group-containing polysaccharides and salts thereof; neutral polysaccharides such as cellulose, dextran, agarose, mannan, starch, and ionic group-introduced products thereof and derivatives thereof, and anionic polysaccharides such as salts thereof, Chitosan derivatives such as chitin and chitosan and salts thereof; derivatives of N-substituted cellulose such as aminocellulose and N-methylaminocellulose; and salts thereof; neutral polysaccharides such as dextran, agarose, mannan and starch; and Such as amino group-introduced products and their salts On-polysaccharide, poly (meth) acrylic acid, phosphoric acid group-containing (meth) acrylic acid ester polymer, sulfonic acid group-containing (meth) acrylic acid polymer, 2-acrylamido-2-methylpropanesulfonic acid polymer, And anionic group-containing organic synthetic polymers such as copolymers thereof, amino group-containing (meth) acrylates such as polyethyleneimine, polybrene, poly (2-diethylaminoethyl (meth) acrylate), and copolymers thereof And cationic group-containing organic synthetic polymers.

The nonionic hydrophilic polymer used in the coating treatment is not particularly limited. For example, polysaccharides such as cellulose, dextran, agarose, mannan, starch, and hydrophilic such as polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone. Organic copolymers, 2-hydroxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, (meth) acrylamide, glycidyl (meth) acrylate and other monomers are polymerized or copolymerized And organic synthetic polymers obtained in the above manner.

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 fixed by a covalent bond via a functional group of each substance or other substances. In the case of using the above-described immobilizing coating method, the coating layer does not peel off through a heating step, a drying step, and the like, and therefore, 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.

Among the coating methods, the immobilization coating method is particularly 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.

The migration path used in the method for measuring hemoglobin of the present invention has an inner surface treated with ozone. By treating the inner surface of the migration path with ozone, nonspecific adsorption of proteins and the like can be prevented, and hemoglobins can be measured with high accuracy in a short time.
The ozone treatment is not particularly limited, and examples thereof include ozone gas treatment and ozone water treatment. It does not specifically limit as a method of performing the said ozone gas process, For example, it can carry out by making ozone gas contact an electrophoresis path. It does not specifically limit as a method of performing the said ozone water process, For example, it can carry out by making ozone water contact an electrophoresis path. Of these, the ozone water treatment is preferable.

In the ozone water used for the ozone water treatment, the concentration of dissolved ozone gas is not particularly limited, but a preferable lower limit is 20 ppm. If it is less than 20 ppm, it may take time for the ozone treatment, or nonspecific adsorption of the measurement target substance or the like may not be sufficiently suppressed. A more preferred lower limit is 50 ppm.

The method for preparing the ozone water used for the ozone water treatment is not particularly limited, and examples thereof include a method in which raw water and ozone gas are brought into contact with each other through an ozone gas permeable film that passes only gas and prevents liquid from passing through. It is done. By using such a method, the ozone water can be adjusted by dissolving the ozone gas into the raw material water through the ozone gas permeable membrane.

The migration path can be further improved in separation performance and the like by coating with a hydrophilic polymer after the ozone treatment. Even when the coating treatment is performed after the ozone treatment, the effect of the ozone treatment performed in advance is maintained.
For the coating treatment, hydrophilic polymers and a coating treatment method used for the coating treatment performed before the ozone treatment can be applied.

The electrophoresis method used in the method for measuring hemoglobin of the present invention is not particularly limited. For example, there is a method in which, after filling the electrophoresis path with a buffer solution, a measurement sample is introduced and a voltage is applied to the migration path. Can be mentioned.

The buffer solution is a buffer solution that fills the inside of the migration path and the flow path during electrophoresis, a buffer solution that fills the anode and cathode tanks installed at both ends of the migration path during the electrophoresis, and the inside of the migration path. Is used as a buffer solution for washing and a hemolysis dilution solution for dissolving and diluting a sample.

The buffer solution is not particularly limited as long as it is a solution containing a conventionally known buffer capacity component having a buffer capacity, and examples thereof include organic acids such as citric acid, succinic acid, tartaric acid, malic acid, and salts thereof; Contains amino acids such as glycine, taurine and arginine; inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid and acetic acid, and salts thereof; or buffering components such as these derivatives having buffering capacity Solution.
These buffer capacity components may be used independently and 2 or more types may be used together.

The buffer solution may be appropriately added with other commonly added substances such as surfactants, chaotropic compounds, various polymers, hydrophilic low-molecular compounds, and the like. Among them, the performance may be improved by adding an ionic polymer as a polymer to be added; in particular, a water-soluble polymer having an anionic group. The water-soluble polymer having an anionic group (anionic polymer) is a water-soluble polymer having a known anionic functional group such as a carboxyl group, a phosphoric acid group, and a sulfonic acid group.

The anionic polymer is not particularly limited, and examples thereof include sulfate group-containing polysaccharides such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan, and salts thereof; carboxyl group-containing polyvalents such as alginic acid, hyaluronic acid, and pectinic acid. Saccharides and salts thereof; Neutral polysaccharides such as cellulose, dextran, agarose, mannan and starch; and ionic group-introduced products of derivatives thereof; and anionic polysaccharides such as salts thereof; poly (meta) Such as acrylic acid, phosphoric acid group-containing (meth) acrylic acid ester polymer, sulfonic acid group-containing (meth) acrylic acid polymer, 2-acrylamido-2-methylpropanesulfonic acid polymer, and copolymers thereof Examples include water-soluble anionic group-containing organic synthetic polymers.

The minimum with preferable content of the said anionic polymer of the said buffer solution is 0.01 weight%, and a preferable upper limit is 10 weight%. If it is less than 0.01% by weight, the effect of adding an anionic polymer is hardly exhibited, and separation or the like may be insufficient in the measurement of hemoglobins. If it exceeds 10% by weight, the measurement time may be prolonged or separation may be caused.

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.

According to the present invention, nonspecific adsorption of components such as proteins and lipids in human blood serving as a sample is suppressed, and hemoglobins, particularly stable HbA1c, exhibit simultaneous reproducibility in a short time of several minutes per sample. Since the CV value can be measured with a high accuracy of about 1.0%, it is possible to suitably manage the HbA1c value by the electrophoresis method for diabetic patients, which was difficult with the prior art. Furthermore, since the electrophoresis method has a simpler apparatus configuration than the HPLC method or the like, an inexpensive method for measuring hemoglobin can be provided.

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) Preparation of migration path A capillary made of fused silica (GL Science, Inc .: inner diameter 25 μm × total length 30 cm) is set in a capillary electrophoresis apparatus (PAC / E MDQ, manufactured by Beckman Coulter). After 2N NaOH aqueous solution, ion-exchange water and 0.5N HCl aqueous solution were passed in this order to wash the inside of the capillary, 0.2N hydrochloric acid solution containing 0.5% by weight chitosan was passed for 20 minutes. . Thereafter, air was injected into the capillary to expel the chitosan solution and dried in a dryer at 40 ° C. for 12 hours. Thereafter, injection of the chitosan solution, injection of air, and drying were repeated 5 times. The obtained capillary was filled with ozone water having a dissolved ozone gas concentration of 100 ppm and allowed to stand. Ten minutes later, ozone water was sucked, the same ozone water was injected again, and the mixture was allowed to stand for 10 minutes to obtain a capillary containing a migration path that had been treated with ozone water. The obtained capillary was filled with 150 mM citrate buffer (pH 5.2) containing 2.0% by weight of chondroitin sulfate.

(2) Measurement of healthy human blood Citrate buffer solution (pH 6) containing 0.05% by weight of Triton X-100 (surfactant, manufactured by Wako Pure Chemical Industries, Ltd.) in 100 μL of healthy human whole blood obtained by collecting heparin from a healthy person. 0.0) 150 μL was added for hemolysis and dilution to obtain a hemolyzed sample of healthy human blood.
By adding the hemolyzed sample obtained at one end of the obtained ozone water-treated capillary, applying a voltage of 25 kV to both ends of the capillary, and measuring the change in absorbance at 415 nm visible light Measurement of stable HbA1c in human blood was performed.
FIG. 2 is an electropherogram obtained in Example 1 by measurement of blood of a healthy person. In FIG. 2, peak 1 indicates stable HbA1c, and peak 2 indicates HbA0. The stable HbA1c could be separated with an electrophoresis time of about 1.0 minutes.

(3) Measurement of a sample containing modified Hb Glucose was added to healthy human whole blood used in the measurement of blood of healthy humans so as to be 2500 mg / dL, heated at 37 ° C. for 3 hours, and a kind of modified Hb A sample containing a large amount of certain unstable HbA1c was artificially prepared. A sample containing the modified Hb was measured using the same method as the above-described measurement of blood of healthy humans.
FIG. 3 is an electropherogram obtained in Example 1 by measurement of a sample containing modified Hb.
In FIG. 3, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 3, stable HbA1c and unstable HbA1c, which is a modified Hb, were well separated.

(Example 2)
(1) Preparation of migration path An electrophoresis path having a length of 75 mm and a width of 60 μm was formed on a polydimethylsiloxane microchip to produce a cross-shaped electrophoresis microchip. The electrophoresis path was filled with a 0.5% by weight aqueous solution of preprene 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, injection of the aqueous solution of preprene, injection of air, and drying were repeated 5 times to obtain an electrophoresis microchip coated with the electrophoresis path. The electrophoresis microchip coated with the obtained migration path was further subjected to ozone water treatment in the same manner as in Example 1 to obtain an ozone water-treated electrophoresis microchip. The migration path of the obtained ozone water-treated electrophoresis microchip was filled with 50 mM succinate buffer (pH 5.8) containing 1.5% by weight of dextran sulfate.

(2) Measurement of healthy human blood and measurement of sample containing modified Hb Electrophoresis was performed by applying a voltage of 500 V to both ends of the electrophoresis path using the obtained ozone water-treated electrophoresis microchip. By measuring the change in absorbance in visible light, the same measurement as (2) measurement of blood of a healthy person and (3) measurement of a sample containing modified Hb in Example 1 was performed. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 3 was obtained.

(Example 3)
(1) Preparation of electrophoresis path An electrophoresis path having a length of 90 mm and a width of 50 μm was formed on a fused silica microchip to produce a microchip for electrophoresis. The electrophoresis path was filled with a 0.2N hydrochloric acid solution containing 0.5% by weight of chitosan and left 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, the injection of the chitosan solution, the injection of air and the drying were repeated 5 times to obtain a microchip coated with the migration path. The obtained electrophoresis microchip coated with the further migration path was further subjected to ozone water treatment in the same manner as in Example 1 to obtain an ozone water-treated electrophoresis microchip. The migration path of the obtained microchip for electrophoresis treated with ozone water was filled with 100 mM phosphate buffer (pH 5.5) containing 2.0 wt% chondroitin sulfate.

(2) Measurement of healthy human blood and measurement of sample containing modified Hb Except that electrophoresis was performed by applying a voltage of 700 V to both ends of the electrophoresis path using the obtained ozone water-treated electrophoresis microchip. In the same manner as in Example 2, measurement of blood of healthy persons and measurement of samples containing modified Hb were performed. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 3 was obtained.

Example 4
(1) Preparation of migration path Dissolve 3.0 g of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 2.0 g of hydroxyethyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) in 50 mL of ion exchange water. To obtain a solution. To the obtained solution, 0.05 g of potassium persulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the temperature was raised to 80 ° C. while stirring in a nitrogen atmosphere to polymerize. After 10 hours of polymerization, the entire content was transferred to a dialysis tube (manufactured by Sanko Junyaku Co., Ltd .: UC C65-50) and dialyzed in ion-exchanged water for 12 hours to obtain a hydrophilic polymer having an anionic group.
An electrophoresis path having a length of 70 mm and a width of 70 μm was formed on a methyl methacrylate microchip to produce an electrophoresis microchip. The electrophoresis path was filled with 0.5% by weight of the above-mentioned hydrophilic polymer aqueous solution and left for 20 minutes. Thereafter, air was injected into the migration path to drive out the hydrophilic polymer aqueous solution described above, followed by drying in a dryer at 40 ° C. for 12 hours. Thereafter, the injection of the hydrophilic polymer aqueous solution, the injection of air and the drying were repeated 5 times to obtain a microchip coated with the migration path. The obtained microchip coated with the migration path was further subjected to ozone water treatment in the same manner as in Example 1 to obtain an electrophoretic microchip subjected to ozone water treatment. The migration path of the obtained ozone water-treated electrophoresis microchip was filled with 100 mM malic acid buffer (pH 5.4) containing 2.0% by weight of the above-mentioned hydrophilic polymer aqueous solution.

(2) Measurement of healthy human blood and measurement of sample containing modified Hb Examples were performed except that electrophoresis was performed using a voltage obtained by applying a voltage of 400 V to both ends of the electrophoresis path using the obtained ozone water-treated microchip. According to the same method as in No. 2, the blood of a healthy person and a sample containing modified Hb were measured. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 3 was obtained.

(Example 5)
(1) Preparation of migration path An electrophoresis path having a length of 85 mm and a width of 55 μm was formed on a fused silica microchip to produce a microchip for electrophoresis. The formed electrophoresis path was subjected to ozone water treatment using the same method as in Example 1 to obtain an electrophoretic microchip with ozone water treatment. The electrophoresis path of the obtained ozone water-treated electrophoresis microchip was filled with a 0.2N hydrochloric acid solution containing 0.5 wt% chitosan and left 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, the injection of the chitosan solution, the injection of air and the drying were repeated 5 times to obtain a microchip coated with the migration path. The migration path of the obtained microchip was filled with a 150 mM malic acid buffer solution (pH 5.5) containing 2.0% by weight of chondroitin sulfate.

(2) Measurement of healthy human blood and measurement of sample containing modified Hb The same as in Example 2 except that electrophoresis was performed by applying a voltage of 750 V to both ends of the electrophoresis path using the obtained microchip. According to the method, blood of a healthy person and a sample containing modified Hb were measured. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 3 was obtained.

(Comparative Examples 1-4)
A capillary electrophoresis apparatus having an electrophoresis path was prepared using the same method as in Example 1 except that the ozone water treatment was not performed, and the sample containing the modified Hb was measured.
A microchip having a migration path formed by using the same method as in Examples 2 to 4 except that the ozone water treatment was not performed, and a sample containing the modified Hb was measured.
In the measurement of the sample containing the modified Hb under the measurement conditions of Comparative Examples 1 to 4, the same electropherogram as in FIG. 3 was obtained.

(Comparative Example 5)
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 A capillary having an inner surface dynamically coated was obtained by passing a 0.5 wt% malic acid buffer solution of a functional polymer, bovine serum albumin, manufactured by Wako Pure Chemical Industries, Ltd.) for 1 minute.
The obtained capillary with the coated inner surface is set in a capillary electrophoresis apparatus (P / ACE MDQ, manufactured by Beckman-Couler), and an apple containing 0.2% by weight of chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) A sample containing modified Hb was measured in the same manner as in Example 1 except that electrophoresis was performed by applying a voltage of 10 kV after filling the acid buffer (pH 4.7) in the capillary. .
FIG. 4 is an electropherogram obtained by measurement of a sample containing modified Hb in Comparative Example 5. In FIG. 4, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 4, peak 1 indicating stable HbA1c overlaps peak 3 indicating modified Hb.

(Evaluation)
(1) Recovery test Under the measurement conditions of Examples 1 to 5 and Comparative Examples 1 to 4, the total peak area values of the electropherogram obtained in the measurement of healthy human blood were calculated. The total peak area value in Example 1 was set to 100, and the relative values of the total peak areas in Examples 2 to 5 and Comparative Examples 1 to 4 with respect to the total peak area value in Example 1 were calculated.
The results are shown in Table 1.

As shown in Table 1, compared with the total peak area values of Examples 1 to 5, the total peak area values of Comparative Examples 1 to 4 were small. This is presumably because part of the hemoglobin component was adsorbed nonspecifically on the inner surface of the migration path under the measurement conditions of Comparative Examples 1 to 4 in which the migration path was not subjected to ozone treatment.

(2) Simultaneous reproducibility test Using the measurement conditions of Examples 1 to 5 and Comparative Examples 1 to 4, the CV value of a stable HbA1c value obtained by continuously obtaining the same sample of a healthy human blood sample 20 times was calculated. did.
The CV value was obtained by calculating (standard deviation / average value).
The results are shown in Table 2.

(3) Durability test Using the measurement conditions of Examples 1 to 5 and Comparative Examples 1 to 4, the maximum stable HbA1c value obtained by continuously measuring the same sample of a healthy human blood sample 200 times The difference (R value) between the value and the minimum value was calculated.
The results are shown in Table 2.

As shown in Table 2, in Examples 1 to 5, 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 200 sample measurement in the durability test was also very small, about 0.2%, and it was found that the stable HbA1c can be measured with high accuracy even in continuous measurement of a large number of samples.
On the other hand, in Comparative Examples 1 to 4, the CV value in the simultaneous reproducibility test is about 3 to 4% and the R value in the durability test is 0.6 to 1.1%, and the HbA1c value of diabetic patients is managed. The value was completely insufficient for

From the above evaluation results, in Comparative Examples 1 to 4 using measurement conditions based on a method that does not perform the ozone treatment, the recovery rate is low (Table 1), and the nonspecific adsorption phenomenon of hemoglobin occurs. Therefore, although the electropherogram at the initial stage of measurement is substantially equivalent to those of Examples 1 to 4 subjected to ozone treatment (FIG. 3), repeated measurements such as simultaneous reproducibility test and durability test are performed. As a result, it was found that the measurement value was not stable due to the influence of the non-specific adsorption described above (Table 2), and the performance did not reach the level for managing the HbA1c value of diabetic patients.

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.

It is a schematic diagram which shows an example of the electrophoresis apparatus used for the measuring method of hemoglobin of this invention. 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 and Comparative Examples 1, 2, 3, and 4, it is the electropherogram obtained by the measurement of the sample containing modification Hb. In Comparative Example 5, an electropherogram obtained by measuring a sample containing modified Hb.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Capillary electrophoresis apparatus 12 Anode tank 13 Cathode tank 14 Capillary 15 High voltage power supply 16 Detector 17 Electrode 18 Electrode 1 Peak of stable HbA1c 2 Peak of HbA0 3 Peak of modified Hb (unstable HbA1c)

Claims (3)

A method for measuring hemoglobin using a capillary electrophoresis method or a microchip electrophoresis method , wherein a hemophoresis path having an inner surface treated with ozone is used. The method for measuring hemoglobin according to claim 1, wherein the migration path is further coated with a hydrophilic polymer after the inner surface is treated with ozone . The method for measuring hemoglobin according to claim 1 or 2, further comprising using a buffer solution containing an anionic polymer that fills the inside of the migration path during electrophoresis .

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