JP4854525B2 - Method for measuring hemoglobins - Google Patents

Description

The present invention relates to an electrophoretic device, a hemoglobin measuring method, a hemoglobin, and a glucose measuring device capable of measuring hemoglobins, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time with high accuracy. And a method for simultaneous measurement of hemoglobins and glucose.

Hemoglobin (hereinafter also referred to as Hb), particularly hemoglobin A1c (hereinafter also 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 screening test and a test item for grasping the blood glucose control status of diabetic patients.
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.

Patent Document 2 uses a buffer containing a 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.

When performing diabetes diagnosis by these methods, stable HbA1c that is an index of diabetes must be separated from HbA1c, and the influence of modified Hb such as unstable HbA1c, carbamylated Hb, and acetylated Hb must be excluded. Don't be. 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.

Further, since the conditions such as the buffer solution and the voltage load disclosed in Patent Documents 1 and 2 can be applied only to a large and expensive capillary electrophoresis apparatus, such a method is small. It was impossible to perform measurement by applying to an inexpensive microchip electrophoresis method.
Japanese National Patent Publication No. 9-510792 Japanese Patent Laid-Open No. 9-105739

The present invention relates to an electrophoretic device, a hemoglobin measuring method, a hemoglobin, and a glucose measuring device capable of measuring hemoglobins, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time with high accuracy. And it aims at providing the simultaneous measuring method of hemoglobin and glucose.

The present invention is a method for measuring hemoglobin using an electrophoretic apparatus comprising an electrode and a microchip having an electrophoresis path whose inner surface is coated with a hydrophilic polymer, a power supply unit, and a detection unit. In the detection unit, hemoglobins are measured by simultaneously measuring two absorbances at a main wavelength selected from 400 to 430 nm and a sub wavelength selected from 450 to 600 nm .
The present invention is described in detail below.

As a result of intensive studies, the present inventors have determined that a measurement unit comprising a microchip having an electrophoresis path whose inner surface is coated with a hydrophilic polymer, a power supply unit for applying a voltage to the measurement unit, and a separated Hb It has been found that by using an electrophoresis apparatus comprising a detection unit capable of detecting a component, hemoglobin can be measured with high accuracy and in a short time at a lower cost than in the past. The invention has been completed.

FIG. 1 shows an example of the electrophoresis apparatus of the present invention. FIG. 1a is a schematic view showing a top view of the electrophoresis apparatus of the present invention. As shown in FIG. 1a, the electrophoresis apparatus 1 has a measurement unit 2 having a flow path including a reservoir 21, an electrode 22 installed in the reservoir, and an electrophoresis path 23 through which a measurement sample moves and separates during electrophoresis. . The reservoir 21 is a liquid reservoir for storing a buffer solution.
FIG. 1a shows an electrophoresis according to the present invention in which the flow path including the migration path 23 is configured in a cross shape, four reservoirs 21 are provided at each end of the migration path 23, and electrodes 22 are installed in each reservoir 21. It is an example of an apparatus. Each electrode 22 is connected to a power supply unit 3 that is a high-voltage power supply via a voltage supply cable 31.

FIG. 1 b is a schematic diagram showing a cross-sectional view of the electrophoresis apparatus of the present invention. As shown in FIG. 1 b, the electrophoresis apparatus 1 has a detection unit 4. The detection unit 4 includes a light source 41 and a light receiving unit 42. In the detection unit 4, the light source 41 and the light receiving unit 42 are located on the opposite sides via the measurement unit 2. The light source 41 generates light of a specific wavelength at a predetermined location of the migration path 23 formed in the measurement unit 2. The light receiving unit 42 measures the absorbance of the measurement target component separated in the migration path 23, and calculates a stable HbA1c value based on the absorbance data.

The electrophoretic device of the present invention has a measuring unit composed of an electrode and a microchip. Such an electrophoresis apparatus is smaller and cheaper than the HPLC and capillary electrophoresis apparatuses conventionally used for measuring Hb species.

In this specification, the microchip refers to a plate-like substrate made of an inorganic or organic material and having a size of about 150 mm square or less. 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.
However, a material that does not absorb light having a specific wavelength, which will be described later, is preferable.

In the electrophoresis apparatus of the present invention, the measurement unit may be composed of a plurality of microchips. In such a case, the respective microchips may have the same specification or different specifications.

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. This refers to a site (a site detected by the detection unit from the site where the sample is injected). For example, in FIG. 1, it refers to a portion from the intersection 24 of the flow channel to a position on the flow channel where light is irradiated by the light source 41.
The shape of the entire flow path including the migration path is not particularly limited, and may be any shape that can ensure the shape and size of the migration path described later, such as a cross shape, as well as a linear shape.

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 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.
The hydrophilic polymer is preferably one that does not absorb light of a specific wavelength (main wavelength and sub wavelength) described later.

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; carboxyl group-containing polysaccharides such as alginic acid, hyaluronic acid and pectinic acid and salts thereof; cellulose; Anionic polysaccharides, such as dextran, agarose, mannan, starch, and the like, and anionic polysaccharides such as salts thereof, chitosan derivatives such as chitin and chitosan, and salts thereof; aminocellulose, N- N- such as methylaminocellulose Derivatives of modified cellulose and salts thereof; Neutral polysaccharides such as dextran, agarose, mannan, starch, and amino group-introduced products thereof, and cationic polysaccharides such as salts thereof; Bovine serum albumin, lactoferrin, skim milk, etc. Examples include proteins.

The nonionic hydrophilic polymer used for the coating is not particularly limited, and examples thereof include polysaccharides such as cellulose, dextran, agarose, mannan and starch, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and poly (2-hydroxyethyl). Methacrylate), hydrophilic polymers such as polyvinylpyrrolidone, monomers such as 2-hydroxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, (meth) acrylamide, and glycidyl (meth) acrylate Examples thereof include organic synthetic polymers obtained by polymerization or copolymerization.

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.

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.

The measurement unit includes the electrode.
The electrode is in contact with a buffer solution, which will be described later, in the flow path in the measurement unit, and is connected to a power source unit, which will be described later. Since it has such a configuration, electrophoresis can be performed by supplying a voltage from the application unit and applying a voltage to the sample via a buffer solution filled in the flow path in the measurement unit.

The installation position of the electrode is not particularly limited as long as the electrode can be brought into contact with the buffer solution in the flow channel in the measurement unit. For example, the electrode may be fixed to the measurement unit, or the lid of the measurement unit or You may fix to the support stand (chip holder) etc. which fix the said measurement part.
The mode of bringing the electrode into contact with the buffer solution is not particularly limited as long as it is on the flow channel sandwiching the migration path. For example, the electrode is preferably in contact with the buffer solution in a reservoir described later.

It does not specifically limit as a raw material of the said electrode, A conventionally well-known raw material can be used, For example, electroconductive metals, such as platinum, etc. are mentioned.

The measurement unit preferably has a reservoir.
The reservoir is formed at the end of the migration path and serves as a buffer solution used for electrophoresis, a supply port for a measurement sample, a discharge port, and an electrode insertion portion.
The shape of the reservoir is not particularly limited, and a conventionally known shape can be used. The size of the reservoir is not particularly limited, and a conventionally known size can be used.
The reservoir may be connected to the bottom or upper part for supply and drainage as necessary for the supply and drainage of buffer solution, measurement sample and the like.

The electrophoresis apparatus of the present invention has a power supply unit.
The power supply unit serves to supply a voltage for electrophoresis.

A preferable lower limit of the voltage applied to the power supply unit is 100V, and a preferable upper limit is 3000V. When the voltage is less than 100 V, there is a possibility that the component to be measured is not sufficiently moved by electrophoresis. If it exceeds 3000 V, the measurement accuracy may be impaired due to the occurrence of peak deformation or the like in the electropherogram obtained by the generation of Joule heat or the like.

The power supply unit serves to supply a voltage to the buffer solution in the measurement unit by being connected to the electrode installed in a reservoir or the like in the measurement unit.
A method for connecting the power supply unit and the electrode is not particularly limited, and examples thereof include a method using a conventionally known cord.

The electrophoresis apparatus of the present invention has a detection unit.
The detection unit is a mechanism for optically detecting a measurement sample component separated by electrophoresis. The principle of such optical detection is not particularly limited as long as it can detect hemoglobin as a measurement sample, but from the viewpoint of simplicity, a method for measuring absorbance of visible light in the maximum absorption wavelength region of hemoglobin. Is preferred.
Specifically, the absorbance of visible light is, for example, by receiving light including visible light from a light source at a predetermined position on the migration path, so that the light received on the opposite side across the migration path. The absorbance in the visible light of various components of hemoglobin moving in the migration path can be measured.

The detection unit preferably includes a light source and a light receiver.
In the detection unit, the installation mode of the light source and the light receiver is not particularly limited. For example, the detection unit may be installed in an upper part and a lower part of the measurement unit across the migration path. It may be an aspect that is installed on the side portion of the measurement unit with a gap in between.
In the detection unit, the light emitted from the light source to the light receiving unit may be emitted from the upper part to the lower part or from the lower part to the upper part depending on the installation mode of the light source and the light receiver. The light may be irradiated from the side surface to the side surface, or may be irradiated obliquely with a predetermined angle.

In the detection unit, it is preferable to measure the absorbance of the main wavelength and the absorbance of the sub wavelength at the same time, with one wavelength selected from 400 to 430 nm as the main wavelength and one wavelength selected from 450 to 600 nm as the sub wavelength. By using such a specific wavelength, it is possible to perform high-accuracy measurement in a short time at low cost.
A method for measuring hemoglobin using the electrophoresis apparatus of the present invention, wherein hemoglobins measure two absorbances at a main wavelength selected from 400 to 430 nm and a subwavelength selected from 450 to 600 nm in the detection unit. This measuring method is also one aspect of the present invention.
A preferable range of the main wavelength is 410 to 420 nm, and a preferable range of the sub wavelength is 500 to 550 nm.

In the method for measuring hemoglobin of the present invention, the absorbance at the primary wavelength and the absorbance at the secondary wavelength may be measured by irradiating only visible light in the primary wavelength range and the secondary wavelength range. After all the multi-wavelength absorbances including the range and the sub-wavelength range are measured, only the main-wavelength absorbance and the sub-wavelength absorbance may be selected. Thus, in either case, the peak of the target hemoglobin is obtained by obtaining an electropherogram from the difference between the absorbance at the main wavelength and the absorbance at the sub-wavelength at the same time (absorbance at the main wavelength minus the absorbance at the sub-wavelength). The area can be calculated.

The method for measuring the absorbance at the main wavelength and the absorbance at the sub wavelength is not particularly limited, and conventionally known detection techniques such as a wavelength filter, a spectroscope, various mirrors, and various lenses can be used.

The electrophoresis apparatus of the present invention may have other accessory parts in addition to the basic configuration including the measurement unit, the power supply unit, the detection unit, and the like. By having such an accessory, electrophoresis can be performed more efficiently.
The accessory part is not particularly limited, and for example, a control mechanism for controlling the voltage, polarity, and load time of the power supply unit, or executing a series of automation programs, the reservoir, the electrophoresis as necessary. Road, supply and drainage mechanism for supplying and draining cleaning fluid for cleaning the electrodes, etc., data for creating electropherograms from measured absorbance, and calculating and printing stable HbA1c values Examples include a processing mechanism, an automatic dilution of a measurement sample, an automatic supply mechanism to a measurement unit, a mechanism for cleaning a sample container, a dilution tank and a channel, erection of a sample container, and supply.

When measuring hemoglobin by electrophoresis using the electrophoresis apparatus of the present invention, after filling the buffer in the flow path of the measurement unit, the sample is introduced into the flow path, and the flow path It can be implemented by loading a voltage inside.
The buffer solution is not particularly limited as long as it is a solution containing a conventionally known buffer solution composition having a buffer capacity, and examples thereof include organic acids such as citric acid, succinic acid, tartaric acid, malic acid, and salts thereof. And the like; amino acids such as glycine, taurine and arginine; and solutions containing inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid and acetic acid, and salts thereof.

Commonly used additives such as chaotropic compounds, surfactants, various polymers, and hydrophilic low molecular compounds may be appropriately added to the buffer solution.

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; alginic acid, hyaluronic acid, pectinic acid and other carboxyl group-containing polysaccharides 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 synthetic organic polymers such as those copolymers thereof.

In the electrophoresis apparatus of the present invention, it is preferable that the measurement unit is further provided with a mechanism for measuring glucose. Since the electrophoretic device of the present invention plays a role as a mechanism for measuring hemoglobins, a mechanism for measuring glucose is further provided in the measurement unit of the electrophoretic device of the present invention, thereby providing an index for diagnosis of diabetes. It is possible to simultaneously measure stable HbA1c and glucose.
An apparatus for measuring hemoglobin and glucose comprising the electrophoresis apparatus of the present invention and a mechanism for measuring glucose provided in the measurement unit of the electrophoresis apparatus is also one aspect of the present invention.

The mechanism for measuring glucose is not particularly limited, and a conventionally known measurement mechanism can be used. For example, a mechanism using a reduction method using the reducing action of glucose in an alkaline solution; glucose and aromatic Examples include a mechanism using a condensation method using a condensation reaction with an amine; a mechanism using an enzyme method using a reaction between glucose and an enzyme such as glucose dehydrogenase, glucose oxidase, and hexokinase. Among these, a mechanism using an enzymatic method is preferable.

The mechanism for measuring glucose is preferably provided on the microchip in the measurement unit.
A mechanism for measuring glucose, for example, a mechanism using an enzyme method is not particularly limited as a microchip. For example, a platinum thin film electrode or the like is formed on the microchip by sputtering or the like, and the In addition, a method for preparing an enzyme sensor on a microchip by immobilizing glucose dehydrogenase, glucose oxidase and the like can be mentioned.

In the hemoglobin and glucose measuring device of the present invention, the mechanism for measuring the hemoglobin and the mechanism for measuring the glucose may be formed on different microchips, respectively. It may be formed on a chip.
When the mechanism for measuring the hemoglobin and the mechanism for measuring the glucose are formed on the same microchip, the flow path for measuring the hemoglobin and the glucose are measured. These channels may be formed independently, and a mechanism capable of measuring the hemoglobins and the glucose in the same channel may be formed.

FIG. 5 is a schematic view showing an example of a hemoglobin and glucose measuring device of the present invention.
FIG. 5a shows a mode in which the hemoglobin measurement sample introduction port 25 and the glucose measurement sample introduction port 51 are installed independently of each other.
In such an embodiment, hemoglobins are measured by introducing a sample into the hemoglobin measurement sample inlet 25 and then applying a voltage to the electrode 22 for electrophoresis to separate the components into the electrophoresis path 23. Then, optical detection is performed in the detection unit 4. The glucose is measured by the enzyme electrode 52 with respect to a sample separately introduced into the glucose measurement sample inlet 51.
By such an aspect, hemoglobins and glucose can be measured individually. In such a case, when one of hemoglobin and glucose is measured, it is not necessary to operate the other mechanism, so that consumption of reagents and the like can be saved.

FIG. 5b shows a mode in which the sample inlet for measuring hemoglobin and the sample inlet for measuring glucose are common.
In such an embodiment, after the sample is introduced into the common sample introduction port 60, each sample is guided to the hemoglobin measurement reservoir 26 and the glucose measurement reservoir 53. The hemoglobins are subjected to electrophoresis by applying a voltage between the hemoglobin measurement reservoir 26 and the reservoir 21, separated into components in the migration path 23, and optical detection is performed in the detection unit 4. On the other hand, glucose is measured by the enzyme electrode 52 of the glucose measurement reservoir 53.
According to such an embodiment, hemoglobins and glucose can be measured simultaneously or individually.

FIG. 5 c shows a mode in which the sample inlet for measuring hemoglobin and the sample inlet for measuring glucose are common.
In such an embodiment, for example, after the sample is introduced into the common sample introduction port 60, first, glucose is measured by the enzyme electrode 52 in the common sample introduction port 60, and then the common sample introduction port 60, the reservoir 21, Electrophoresis is performed by applying a voltage to the electrode 22 installed in the electrode, and the hemoglobins can be measured by separating each component in the electrophoresis path 23 and performing optical detection in the detection unit 4.
In such an embodiment, the order of measurement is not particularly limited, and after measuring glucose, hemoglobin may be measured, or after measuring hemoglobin, glucose may be measured. Good.
According to such an embodiment, hemoglobins and glucose can be measured simultaneously or individually.

By using such an electrophoresis apparatus, it becomes possible to perform high-accuracy measurement in a short time using an apparatus capable of producing the hemoglobins and glucose simultaneously or individually at a low cost. .
A method for simultaneously measuring hemoglobins and glucose using the hemoglobin and glucose measuring device of the present invention is also one aspect of the present invention.

The present invention uses the principle of electrophoresis that can be instrumented with a simple configuration, and further uses a microchip that can be easily miniaturized, so that high-accuracy measurement can be performed at low cost in a short time. An electrophoretic device, a method for measuring hemoglobin, a device for measuring hemoglobin and glucose, and a method for simultaneously measuring hemoglobin and glucose can be provided. Specifically, while using a simple and low-cost method called electrophoresis, the CV value indicating simultaneous reproducibility is about 1.0% in a short time of several minutes per specimen, particularly in the measurement of stable HbA1c. Can be measured with high accuracy. Therefore, it becomes possible to suitably manage the stable HbA1c value of diabetic patients by electrophoresis. In addition, a mechanism for measuring glucose can be easily incorporated into the measurement unit of the electrophoresis apparatus, and a measurement apparatus for hemoglobins and glucose can be obtained. In this way, it is possible to simultaneously measure two items of the diabetes index of stable HbA1c and glucose without enlarging the entire apparatus, that is, without significantly increasing the cost.

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 electrophoresis apparatus 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 solution 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 chitosan-coated microchip was filled with a 150 mM citrate buffer solution (pH 5.2) containing 2.0% chondroitin sulfate to obtain a measurement microchip. The obtained microchip for measurement is placed on a support base having a platinum electrode (diameter 1 mm × length 5 mm), the electrode is inserted into a reservoir on the microchip, and the electrode and a power source (manufactured by Labo Smith) are connected. Connect using a voltage supply cord, and install a halogen lamp light source (MHF-V501, manufactured by Moritex Corp.), a spectroscope (B & W Tech, BTC112), and a detector having a data processing personal computer on the microchip. Thus, an electrophoresis apparatus was prepared.

(2) Measurement of healthy human blood Citrate buffer containing 0.05% Triton X-100 (surfactant, manufactured by Wako Pure Chemical Industries, Ltd.) 200 μL of pH 6.0) was diluted by hemolysis, and a hemolyzed sample of healthy human blood was obtained.
The above-mentioned hemolyzed sample is added to a reservoir formed at one end of the migration path of the obtained microchip, and electrophoresis is performed by applying a voltage of 1000 V to both ends of the migration path. By measuring the change in absorbance in visible light at 500 nm, stable HbA1c in healthy human blood was measured.
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 60 seconds.

(3) Measurement of a sample containing modified Hb Glucose was added to healthy whole blood collected from sodium fluoride from a healthy person 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. The modified Hb-containing hemolyzed sample was obtained in the same manner as in the above (2) measurement of healthy human blood.
The above-mentioned modified Hb-containing hemolyzed sample is added to a reservoir formed at one end of the electrophoresis path of the microchip obtained by the preparation of the above-described (1) electrophoresis apparatus, and a voltage of 1000 V is applied to both ends of the electrophoresis path. The stable HbA1c in the modified Hb-containing sample was measured by measuring the change in absorbance in visible light having a main wavelength of 415 nm and a sub-wavelength of 500 nm.
FIG. 3 is a schematic diagram showing an electropherogram obtained by measurement of a sample containing modified Hb in 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. 2, stable HbA1c and unstable HbA1c, which is modified Hb, were well separated.

(Example 2)
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, injection of the aqueous solution of preprene, injection of air, and drying were repeated 5 times to obtain a microchip coated with the migration path. The migration path of the obtained polybrene-coated microchip was filled with a 50 mM succinate buffer solution (pH 5.8) containing 1.5% dextran sulfate to obtain a measurement microchip. A healthy human blood was measured and a sample containing modified Hb was measured by the same method as in Example 1 except that the obtained measurement microchip was used. 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)
An electrophoresis path having a length of 90 mm and a width of 30 μm was formed on a methyl methacrylate 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 migration path. The migration path of the obtained chondroitin sulfate-coated microchip was filled with a 100 mM phosphate buffer solution (pH 5.5) containing 2.0% chondroitin sulfate to obtain a measurement microchip. A healthy human blood was measured and a sample containing modified Hb was measured by the same method as in Example 1 except that the obtained measurement microchip was used. 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
An electrophoresis path having a length of 60 mm and a width of 60 μm was formed on a methyl methacrylate 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 injection of the poly 2-HEMA aqueous solution, the injection of air, and the drying were repeated again 5 times to obtain a microchip coated with the migration path. 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. The measurement microchip was obtained. A healthy human blood was measured and a sample containing modified Hb was measured by the same method as in Example 1 except that the obtained measurement microchip was used. 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 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 A 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 through for 1 minute for coating.
The obtained capillary was set in a capillary electrophoresis apparatus (P / ACE MDQ manufactured by Beckman-Couler) and malic acid buffer (pH 4.) containing 0.2 wt% chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd.). 7) was filled in the above-mentioned capillary to obtain a capillary electrophoresis apparatus for measurement.
A sample containing modified Hb was measured by applying a voltage of 10 kV.
FIG. 4 is a schematic diagram showing an electropherogram obtained by measurement of a sample containing modified Hb in Comparative Example 1.
In FIG. 4, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 5, in the obtained electropherogram, peak 1 indicating stable HbA1c and peak 3 indicating modified Hb overlap.

(Comparative Example 2)
Healthy human blood was measured using the same method as in Example 1 except that the microchip migration path was not coated.
No peak was detected in the obtained electropherogram. This is presumably because the hemoglobins in the measurement sample caused nonspecific adsorption on the inner surface of the migration path that was not coated. It was found that hemoglobins could not be measured when using a migration path that was not coated with a hydrophilic polymer.

(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. Moreover, the variation width at the time of continuous 100 sample measurement in the durability test was as very small as about 0.2%, and it was found that stable HbA1c can be measured with high accuracy even in continuous measurement of a large amount of specimens.
On the other hand, in Comparative Example 1, the CV value in the simultaneous reproducibility test is as large as 3% 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. The value was completely inadequate.

(4) Influence of measurement wavelength test Under the measurement conditions of Examples 1 to 4, the measurement wavelength used (main wavelength and subwavelength) was changed to perform measurement. Specifically, the value obtained by subtracting the absorbance at the sub-wavelength from the absorbance at the main wavelength at the same time was taken as the absorbance at the time, and the stable HbA1c value was calculated by obtaining an electropherogram. Next, the influence of the measurement wavelength was measured by performing the above-described simultaneous reproducibility test.

When the stable HbA1c value was calculated using only the absorbance at 415 nm, which is the main wavelength, without taking the subwavelength, the CV value was around 3% in each of Examples 1 to 4. On the other hand, when 500 nm and 550 nm are selected as the sub-wavelengths, the absorbance is measured, and when the stable HbA1c value is calculated from the difference from the absorbance at 415 nm, which is the main wavelength, the CV value is about 1% and more accurate measurement is possible. It was possible.

(5) Simultaneous measurement of stable HbA1c and glucose On the same microchip as used in Examples 1 to 3, channels (length 80 mm, width 80 μm) having the same configuration as in FIG. 5 c were formed. .
The enzyme electrode for measuring glucose (manufactured by Oji Scientific Instruments, BF-6M) has an enzyme-immobilized membrane (thickness) in which glucose oxidase (GOD) is immobilized on the surface of a platinum hydrogen peroxide electrode. 0.6 mm), and the glucose was measured by electrolyzing the hydrogen peroxide generated by the reaction of the GOD in the immobilized membrane with the glucose in the measurement sample using a hydrogen peroxide electrode.
After introducing a measurement sample from the sample introduction port and measuring glucose by the above-described method, electrophoresis was performed under the same electrophoresis conditions as in Examples 1 to 3 to measure stable HbA1c.
The results are shown in Table 4.

Good measurement results were obtained for the stable HbA1c value and the CV value of the glucose value when blood of a healthy person was continuously measured 10 times.
Furthermore, even if the same microchip as used in Examples 1 to 3 is formed with a flow path similar to that shown in FIG. 5a or FIG. 5b to simultaneously measure stable HbA1c and glucose, the same good Performance was obtained.

According to the present invention, an electrophoretic device, a method for measuring hemoglobin, a hemoglobin and a glucose that can measure hemoglobins, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time and with high accuracy. A measuring apparatus and a method for simultaneously measuring hemoglobins and glucose can be provided.

It is a schematic diagram which shows an example of the electrophoresis apparatus for hemoglobin measurement of this invention. It is a schematic diagram which shows an example of the electrophoresis apparatus for hemoglobin measurement 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, 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 schematic diagram which shows an example of the measuring apparatus of hemoglobin and glucose of this invention. It is a schematic diagram which shows an example of the measuring apparatus of hemoglobin and glucose of this invention. It is a schematic diagram which shows an example of the measuring apparatus of hemoglobin and glucose of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Electrophoresis apparatus 2 Measurement part 21 Reservoir 22 Electrode 23 Electrophoretic path 24 Intersection 25 Sample introduction port 26 for hemoglobin measurement 26 Reservoir 3 for hemoglobin measurement 3 Power supply part
31 Voltage supply code 4 detector
41 Light source unit 42 Light receiving unit 51 Glucose measurement sample introduction port 52 Enzyme electrode 53 Glucose measurement reservoir 60 Common sample introduction port

Claims (1)

A method for measuring hemoglobin using an electrophoretic device comprising a microchip having an electrophoresis path whose inner surface is coated with an electrode and a hydrophilic polymer, a power supply unit, and a detection unit. The method for measuring hemoglobin, wherein two absorbances at a main wavelength selected from 400 to 430 nm and a subwavelength selected from 450 to 600 nm are simultaneously measured.

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