JP4854577B2 - 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 (hereinafter also referred to as Hb), particularly hemoglobin A1c (hereinafter also referred to as HbA1c), which is a kind of glycated hemoglobin, reflects the average sugar concentration in the blood for the past 1 to 2 months. Therefore, it is widely used as a screening item for diabetes and a test item for grasping the blood glucose control state of a diabetic patient.

Conventionally, HbA1c has been measured by HPLC, immunization, electrophoresis, and the like.
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 has a simple apparatus configuration, and thus is a technique capable of producing an inexpensive and small system such as microchip electrophoresis, and a high-precision measurement technique for HbA1c in the electrophoresis method. The application to clinical testing can be expected to have 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 a capillary surface by passing a cationic polymer through the capillary in a capillary electrophoresis method, and a buffer containing sulfated polysaccharide is used. 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 such a dynamic coating method, it is necessary to perform coating again at the time of measuring the next specimen after the measurement is completed. Therefore, in order to obtain the same coating state at every measurement, it is necessary to remove the coating layer once by a cleaning operation and perform the coating operation again. That is, when performing continuous measurement, it is necessary to insert cleaning and coating operations between measurements, leading to an increase in measurement time. Also, these cleaning and coating operations can cause measurement errors. In addition, since it is necessary to provide a coating reagent at the time of measurement, it is disadvantageous from the viewpoint of cost. Even when continuous measurement is not performed, the measurement time of about 10 minutes is very long compared with the HPLC method, which is insufficient for application to clinical tests.

Furthermore, in general, when diagnosing diabetes, stable HbA1c, which is an index of diabetes, can be separated from HbA1c to eliminate the effects of modified Hb such as unstable HbA1c, carbamylated Hb, and acetylated Hb. I must. 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. .

In order to solve such dynamic coating problems, a method of immobilizing and coating various polymers on the capillary surface has been studied, and the electroosmotic flow phenomenon and the non-specific adsorption phenomenon of sample components are suppressed, and the separation performance, etc. Many attempts have been made to improve performance. For example, Patent Document 3 discloses a method of immobilizing coating on an ionic capillary surface by electrostatically bonding a polymer having a charge opposite to that of the surface. Patent Document 4 discloses a method of alternately and repeatedly coating polymers having opposite charges on the surface of a capillary.
However, in these fixed coating methods, since the coating polymers are coated by a physical adsorption method such as electrostatic force, the bonding force is relatively weak and cannot be used for a long time. There was a drawback.

As a method for solving such a drawback of the immobilizing coating, a technique for strongly bonding a polymer as a coating agent to the capillary surface by a covalent bond is disclosed. For example, in Patent Document 5, a silane coupling agent having a nucleophilic reactive functional group is covalently bonded to a capillary surface, and further, a protein is covalently bonded to the nucleophilic reactive functional group, thereby coating with proteins. A method of performing is disclosed. In this method, a silane coupling reaction that has been known for a long time is used, and a protein as a coating agent is covalently bonded to the capillary surface through a silane coupling agent. As described above, many techniques for indirectly coating a capillary surface with polymers via a silane coupling agent have been disclosed.
However, in such a method, when used for a long period of time, the polymer itself as a coating agent changes, so that the migration time and area value of each peak in the obtained electropherogram may vary, which is high. In the measurement of hemoglobins such as stable HbA1c, which requires measurement accuracy, no significant improvement in separation performance or the like was observed.

Further, as a method using a silane coupling agent, in addition to a method using a silane coupling agent as an intermediary between a capillary and a coating agent, a method of introducing a functional group of the silane coupling agent to the capillary surface is also disclosed. Has been. For example, Patent Document 6 discloses a method of introducing an alkyl group to the capillary surface by covalently bonding a silane coupling agent having an alkyl group to the capillary surface.
However, such a method has a problem in that non-specific adsorption reaction occurs with proteins such as hemoglobin on the material constituting the hydrophobic capillary surface, and measurement cannot be performed. .
Special table Hei 09-510792 JP 09-105739 A Special table hei 05-503989 JP-A-10-221305 Japanese Patent Laid-Open No. 02-1212756 JP-A 63-210767

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.

As a result of intensive studies, the present inventors have found that highly accurate hemoglobins can be measured by covalently bonding a hydrophilic compound having a predetermined low molecular weight to the outermost surface of the migration path. The present invention has been completed.

The present invention is a method for measuring hemoglobin using capillary electrophoresis or microchip electrophoresis, wherein a hydrophilic compound having a molecular weight of 20 to 800 is provided with a spacer on the outermost surface in contact with the electrolyte filled in the migration path. This is a method for measuring hemoglobin using a migration path that is directly or covalently bound without intervention .
The present invention is a method for measuring hemoglobin using capillary electrophoresis or microchip electrophoresis, and a hydrophilic compound is indirectly attached to the outermost surface in contact with the electrolyte filled in the migration path via a spacer. This is a method for measuring hemoglobin, which uses a migration path bonded by a covalent bond, and the total of the molecular weight of the hydrophilic compound and the molecular weight of the spacer is 20 to 800.
The present invention is described in detail below.

The electrophoresis apparatus to which the method for measuring hemoglobin of the present invention can be applied is not particularly limited as long as it has an electrophoresis path having a length, a width, a shape, and the like, which will be described later. An example is a chip electrophoresis apparatus.

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, a hydrophilic compound having a molecular weight of 20 to 800 or less is covalently bonded to the outermost surface of the capillary 14 serving as the migration path.
When measuring hemoglobin, a sample is injected from one of the capillaries 14 and a predetermined voltage is applied from the high-voltage power supply 15 so that the target component moving in the capillaries 14 is measured by the detector 16. .

When the method for measuring hemoglobin of the present invention is applied to a microchip electrophoresis apparatus, a microchip provided with portions corresponding to the capillary 14, the anode tank 12, and the cathode tank 13 in the capillary electrophoresis apparatus is used.

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

The migration path refers to a site (from a site where the measurement sample is injected to a site where the measurement sample is detected) where the measurement sample moves and / or separates by electrophoresis in a channel where electrophoresis is performed. Specifically, for example, in the case of capillary electrophoresis, it means from the site where the sample in the capillary is injected to the site detected by the detector, and in the case of microchip electrophoresis, the microchip This means from the part where the sample in the microchip groove in the type electrophoresis apparatus is injected to the part detected by the detector.
Specifically, the surface of the migration path means, for example, the surface of the capillary migration path in the case of capillary electrophoresis, and the microchip electrophoresis apparatus in the case of microchip electrophoresis. The surface of the migration path of the microchip groove in FIG.

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, silica, and acrylic resin materials are preferable.

A preferable lower limit of the length of the migration path is 10 mm, and a preferable upper limit is 500 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 500 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.

A preferable lower limit of the width of the migration path is 1 μm, and a preferable upper limit is 200 μm. If it is less than 10 μm, the optical path length for detecting the sample component is small, and the measurement accuracy may decrease. If it exceeds 200 μm, the sample diffuses inside the migration path, and the peak in the obtained electropherogram becomes broad, and the measurement accuracy may decrease.

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. Especially, it is preferable that it is linear.
The cross-sectional shape of the migration path is not particularly limited, and examples thereof include a rectangle and a circle.

In the method for measuring hemoglobin of the present invention, a single migration path may be used, or a plurality of migration paths may be used. That is, when a capillary electrophoresis apparatus is used, a plurality of electrophoresis paths may be used simultaneously as capillaries. When a microchip electrophoresis apparatus is used, a plurality of electrophoresis paths are provided on one chip. May be formed.

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

The surface of the migration path has a structure (hereinafter also referred to as “covalent bond structure”) that can be bonded to a hydrophilic compound described later by a covalent bond reaction.
By having the above-described covalent bond structure, a covalent bond is formed with the covalent bond structure of a hydrophilic compound having a molecular weight of 20 to 800, which will be described later, and the hydrophilic compound having a molecular weight of 20 to 800 is converted into the migration path. It is possible to firmly bond to the outermost surface.

The covalent bond structure is not particularly limited as long as a covalent bond can be generated by a conventionally known chemical reaction. For example, a hydroxyl group, a carboxyl group, a sulfonic acid group, an amino group, an ester group, an ether group, Examples include structures or substances having a functional group such as an epoxy group, a halogen group, an imino group, an imidazole group, a silanol group, and a trialkoxysilyl group.

In the migration path, the material itself constituting the migration path may have the covalent structure, and by performing the surface modification treatment, the covalent structure is formed on the surface of the migration path. It may be granted. Especially, it is preferable that the raw material which comprises the said migration path has the said covalent bond structure.

A hydrophilic compound having a molecular weight of 20 to 800 (hereinafter also simply referred to as a hydrophilic compound) is bonded to the outermost surface of the migration path through a covalent bond. Since the hydrophilic compound is covalently bonded to the outermost surface in contact with the electrolyte filled in the migration path, nonspecific adsorption or the like of the measurement component can be suppressed and measurement can be performed with high accuracy.

The hydrophilic compound means a compound having a hydrophilic functional group in the structure.
The hydrophilic functional group is not particularly limited, and examples thereof include a hydroxyl group, a glycol group, an amino group, a carboxyl group, a carbonyl group, a phosphoric acid group, a sulfonic acid group, a carbonyl group, and an amide group.

The hydrophilic compound has a molecular weight lower limit of 20 and an upper limit of 800. When the molecular weight is less than 20, the hydrophilic effect of the hydrophilic compound is small, and it may be difficult to suppress nonspecific adsorption. When the molecular weight exceeds 800, when it binds to the surface of the migration path, the separation performance of hemoglobin may be insufficient, and the measurement accuracy may be deteriorated. It may change and accurate measurement may not be possible. A preferred lower limit is 30, and a preferred upper limit is 500.
The hydrophilic compound may have a repeating structure such as a dimer and a trimer as long as the molecular weight is 800 or less.

The hydrophilic compound has a covalent bond structure.
By having the said covalent bond structure, a covalent bond is produced between the covalent bond structure which the surface of the said migration path has, and the said hydrophilic compound can couple | bond with the surface of the said migration path firmly.

The covalent bond structure is not particularly limited, and examples thereof include a structure capable of generating a covalent bond by a conventionally known chemical reaction. Specifically, the structure similar to the covalent bond structure which the surface of the said migration path has is mentioned.

The combination of the covalent bond structure possessed by the hydrophilic compound and the covalent bond structure possessed by the surface of the migration path is not particularly limited as long as a covalent bond reaction can be performed. The covalent bond structure possessed by and the covalent bond structure possessed by the surface of the migration path may be the same structure or different structures.

The method for bonding the hydrophilic compound to the surface of the migration path by covalent bonding is not particularly limited, and may be performed under a conventionally known reaction condition depending on the type of the hydrophilic compound. Specifically, for example, there is a method in which a solution containing the hydrophilic compound is passed through the inside of the migration path, the hydrophilic compound is brought into contact with the surface of the migration path, and reacted under appropriate reaction conditions. Can be mentioned.
Here, functional group activation treatment may be appropriately performed, and heat treatment, drying treatment, or the like may be performed during or after the reaction.

When the solution containing the hydrophilic compound is passed through the inside of the migration path and reacted by bringing the hydrophilic compound into contact with the surface of the migration path, the concentration of the solution containing the hydrophilic compound is preferably a lower limit. Is 0.1% by weight, and a preferable upper limit is 30% by weight. If it is 0.1% or less, the concentration is insufficient, and the effect of binding the hydrophilic compound by a covalent bond may be difficult to appear. If it is 30% by weight or more, it may be difficult to handle depending on the kind of the hydrophilic compound, and problems such as clogging of reaction products in the migration path may occur.

The hydrophilic compound may have other functional groups having other functions in addition to the covalent bond structure.
Although it does not specifically limit as said other functional group, It is preferable that it is a cationic group.
Although it does not specifically limit as said cationic group, For example, the functional group which has conventionally well-known cationic properties, such as onium groups, such as a 1-3 primary amino group, a quaternary ammonium group, an imide group, a guanidino group, and a phosphonium group, is mentioned. It is done. Especially, it is more preferable that it is a 1-3 primary amino group, and it is still more preferable that it is a 1-2 secondary amino group.

Examples of the other functional group also include a functional group having a structure that can be easily converted into a cationic group by a conventionally known chemical reaction such as a hydrolysis reaction, a reduction reaction, or a transfer reaction. Specific examples include compounds having a functional group such as an amide group, an imine group, and an isocyanate group. These functional groups can be converted into a cationic group such as an amino group by binding a compound having such a functional group to the surface of the migration path and then performing a hydrolysis reaction or the like.

Among the hydrophilic compounds, a hydrophilic compound of a preferred embodiment, that is, a molecular weight of 20 to 800, and a hydrophilic compound having a covalent bond structure and a cationic group is not particularly limited, and examples thereof include urea. Urea derivatives, salts, substituents, polymers, etc .; methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, isopropylamine, diisopropylamine, tributylamine, methylaminopropylamine, dimethylamino Aminoalkanes such as propylamine, glycidylaminopropylamine, diethanolaminopropylamine and their derivatives, salts, substituents, polymers, etc .; ethylenediamine, propylenediamine, tetramethylenediamine, 1,3-pro Diaminoalkanes such as diamine, hexamethylenediamine, hydrazino group propanediamine and their derivatives, salts, substituents, polymers, etc .; monoethanolamine, diethanolamine, trietalamine, monoisopropanolamine, diisopropanolamine, triisopropanolamine , Alkanolamines such as dimethylmethanolamine and diethylethanolamine and derivatives thereof, salts, substituents, polymers, etc .; amino acids such as lysine, arginine, glutamine, ornithine, γ-aminobutyric acid, 3-aminoalanine and the like Derivatives, salts, substituents, polymers, etc .; imines and imides such as guanidine, aminoguanidine, biguanide, trimethyleneimine and derivatives thereof, salts, substituents, polymers, etc .; 3-aminopropyltri Amino group-containing silane couplings such as ethoxysilane, 3- (2-aminoethyl) aminopropyltrimethoxysilane, 3- (2-aminoethyl) aminopropylmethyldimethoxysilane, aminosilane, 3-aminopropyltrimethoxysilane Agents and derivatives thereof, salts, substituents, polymers and the like. Of these, aminoalkanes, diaminoalkanes, and silane coupling agents are more preferable.

The covalent structure on the surface of the migration path and the covalent structure of the hydrophilic compound preferably form a covalent bond by direct reaction. However, the covalent structure of the hydrophilic compound and the hydrophilic structure The hydrophilic compound and the surface of the migration path are bonded via a compound having a functional group capable of forming a covalent bond by reacting with a covalent structure on the surface of the migration path (hereinafter also referred to as a spacer). You may couple | bond together indirectly.

The spacer has at least a functional group capable of binding to the covalent structure of the hydrophilic compound through a covalent reaction, and a function capable of binding to the covalent structure on the surface of the migration path through a covalent reaction. Has a group. That is, the spacer may be a substance having two or more covalent bonds in one molecule. In the spacer, the two or more covalent bonding structures may be the same type of structure or different structures.

Specific examples of the spacer include carbodiimides having an imide group; aminocarboxylic acids having an amino group and a carboxyl group; alkylmethylene diamines having an amino group; epihalohydrins such as epichlorohydrin having a halogen group; Diglycidyl ether; diepoxides such as diepoxide and the like.

In the method for measuring hemoglobin of the present invention, when the hydrophilic compound and the surface of the migration path are bound via the spacer, the molecular weight of the spacer and the molecular weight of the hydrophilic compound are summed. 20-800.

The hydrophilic compound only needs to be covalently bonded to the outermost surface of the migration path, and may be bound only to the outermost surface of the migration path, or the outermost surface of the migration path and the migration path. You may couple | bond with other flow-path outermost surfaces other than.

In the method for measuring hemoglobin of the present invention, the inside of the migration path is filled with an electrolyte, the hydrophilic compound is covalently bonded to the surface of the migration path, and a measurement sample is introduced into the migration path. Then, it can be performed by applying a voltage to both ends of the migration path.

The electrolyte is not particularly limited, but a buffer solution is preferable.
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 addition of an ionic polymer as the polymer to be added; in particular, a water-soluble polymer having an anionic group is preferable because the performance may be improved.

The anionic group is not particularly limited, and examples thereof include conventionally known anionic functional groups such as a carboxyl group, a phosphoric acid group, and a sulfonic acid group. Among these, it is preferable to have a sulfonic acid group.
The water-soluble polymer having an anionic group may have a plurality of the anionic groups in the molecule or may have different types of the anionic groups. Although the water-soluble polymer which has the said anionic group should just be the state melt | dissolved completely in the said buffer solution at the time of use, the preferable minimum of the solubility with respect to water is 1 g / L. If it is less than 1 g / L, it can be used only at a low concentration of the water-soluble polymer having an anionic group, so that the effect is hardly exhibited and the measurement accuracy may be insufficient. A more preferred lower limit is 5 g / L.

The water-soluble polymer having an anionic group is not particularly limited, and specifically, for example, a polysaccharide having an anionic group and a water-soluble organic synthetic polymer having an anionic group are preferable.

The polysaccharide having an anionic group is not particularly limited, and examples thereof include sulfonic acid group-containing polysaccharides such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan, and salts thereof; carboxyl groups such as alginic acid and pectinic acid. -Containing polysaccharides and salts thereof; neutral polysaccharides such as cellulose, dextran, agarose, mannan and starch; anionic group-introduced products thereof; and polysaccharides having known anionic groups such as salts thereof Etc.

Examples of the organic synthetic polymer having an anionic group include known water-soluble organic synthetic polymers containing an anionic functional group, particularly an acrylic polymer; that is, acrylic acid or methacrylic acid and derivatives thereof. Polymers mainly composed of alcohols and esters are preferred.
Specifically, for example, an acrylic polymer obtained by polymerizing a monomer having a carboxyl group such as (meth) acrylic acid and 2- (meth) acryloyloxyethyl succinic acid, ((meth) acryloyloxyethyl) acid phosphate Acrylic polymer obtained by polymerizing a monomer having a phosphate group such as (2- (meth) acryloyloxyethyl) acid phosphate, (3- (meth) acryloyloxypropyl) acid phosphate, 2- (meth) acrylamide Acrylic polymers obtained by polymerizing monomers having a sulfonic acid group such as 2-methylpropanesulfonic acid, (meth) acrylamidepropanesulfonic acid, sulfopropyl (meth) acrylate, (meth) acryloyloxynaphthalenesulfonic acid, etc. Can be mentioned.

The acrylic polymer may be a copolymer of a (meth) acryl monomer having an anionic group and a (meth) acryl monomer having no anionic group.
The (meth) acrylic monomer having no anionic group is not particularly limited as long as it is a (meth) acrylic monomer copolymerizable with the (meth) acrylic monomer having the anionic group. The (meth) acrylic acid esters having a hydrophilic property are preferably used. Specifically, for example, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, glycerol (meth) acrylate; glycidyl (meth) acrylate, polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate Etc.

The preferable lower limit of the content of the water-soluble polymer having an anionic group in the buffer solution is 0.01% by weight, and the preferable upper limit is 10% by weight. If it is less than 0.01% by weight, the effect of adding a water-soluble 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.

According to the method of the present invention, CV value showing simultaneous reproducibility in a short time of several minutes per sample for measurement of stable HbA1c, which is an index for diagnosis of diabetes, by suppressing non-specific adsorption or the like of measurement components Can be measured with high accuracy of about 1.0%. Moreover, even if it repeats a measurement, since the stability of measurement accuracy is not impaired, high-precision measurement can be performed at low cost. Furthermore, since the stable HbA1c value of a diabetic patient can be suitably managed by electrophoresis, which is a simpler measurement principle, an inexpensive and small measurement system such as a microchip electrophoresis apparatus should be constructed. Is possible.

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 fused silica capillary (GL Science, inner diameter 25 μm × total length 30 cm) was set in a capillary electrophoresis apparatus (Beckman Coulter, PAC / E MDQ). After 0.2N NaOH solution, ion-exchanged water, and 0.5N HCl solution were passed in this order to wash the inside of the capillary, 1.0% by weight of 3-aminopropyltriethoxysilane (hydrophilic compound, An aqueous solution containing a molecular weight 221 (manufactured by Shin-Etsu Silicone) was passed through for 60 minutes. Thereafter, air was injected into the capillary to drive out the above aqueous solution, followed by drying in a dryer at 40 ° C. for 12 hours to obtain a capillary having a hydrophilic compound covalently bonded to the outermost surface.

(2) Preparation of measurement sample As a measurement sample, citrate buffer containing 0.01% by weight of Triton X-100 (manufactured by Wako Pure Chemical Industries, Ltd.) in 70 μL of healthy whole blood obtained by collecting EDTA blood from a healthy person A solution obtained by adding 200 μL of solution (pH 6.0) and hemolyzing and diluting it was used as a blood sample of a healthy human.
Also, a kind of modified Hb obtained by adding glucose to 2500 μg / dL to 70 μL of healthy whole blood obtained by collecting EDTA blood from the same healthy person and heating at 37 ° C. for 3 hours. A measurement sample containing a large amount of unstable HbA1c was artificially prepared and used as a modified Hb-containing sample.

(3) Measurement by electrophoresis Using the capillary in which a hydrophilic compound is covalently bonded to the above-described surface, the above-described healthy human sample and the modified Hb-containing sample were measured. As a buffer for electrophoresis, a 240 mM citrate buffer (pH 5.0) containing 2.0% by weight of chondroitin sulfate (anionic polymer, manufactured by Wako Pure Chemical Industries, Ltd.) was used. A sample or a modified Hb-containing sample was injected, electrophoresis was performed at a voltage of 20 kV, and absorbance was measured at 415 nm.

FIG. 2 is an electropherogram obtained in Example 1 by measurement of a healthy human blood sample. In FIG. 2, peak 1 indicates stable HbA1c, and peak 2 indicates HbA0. As shown in FIG. 2, the stable HbA1c peak was well separated.
FIG. 3 is an electropherogram obtained in Example 1 by measurement of a modified Hb-containing sample. 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 A PVA-coated capillary (manufactured by Agilent, inner diameter 75 μm × total length 30 cm) was set in a capillary electrophoresis apparatus (manufactured by Beckman Coulter, PAC / E MDQ). Ion exchange water and 0.1N HCl solution were passed in this order to clean the inside of the capillary, and then 1.0 wt% aqueous epichlorohydrin (spacer, molecular weight 93, manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution was passed for 5 minutes. Then, the capillaries were sealed at both ends of the capillary while being filled with this aqueous solution, and then allowed to stand in a thermostat at 50 ° C. for 5 hours. Thereafter, a 2.0% by weight aqueous solution of diethanolamine (hydrophilic compound, molecular weight 105, manufactured by Wako Pure Chemical Industries, Ltd.) was passed through for 5 minutes, and the caps were sealed at both ends of the capillary while filling the aqueous solution. After standing for a time, a capillary having a hydrophilic compound covalently bonded to the outermost surface was obtained.

(2) Preparation of measurement sample A healthy human blood sample and a modified Hb-containing sample were prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis Using the capillary in which a hydrophilic compound is covalently bonded to the above-described surface, the above-described healthy human sample and the modified Hb-containing sample were measured. As the electrophoresis buffer, a 200 mM citrate buffer (pH 5.0) containing 2.0% by weight of dextran sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The contained sample was injected, electrophoresis was performed at a voltage of 22 kV, and absorbance at 415 nm was measured.
In the measurement of a healthy human blood sample, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the modified Hb-containing sample, an electropherogram similar to that shown in FIG. 3 was obtained.

(Example 3)
(1) Preparation of migration path A double T-type electrophoresis channel having a width of 100 μm was formed on a polydimethylsiloxane microchip (7 cm × 5 cm). A 0.1N NaOH solution, ion-exchanged water, and a 0.2N HCl solution were passed through the formed channel in this order to wash the inside of the channel, and then 1.0% by weight of 3-aminopropyltrimethoxy. An aqueous solution containing silane (hydrophilic compound, molecular weight 179, manufactured by Shin-Etsu Silicone) was passed through for 30 minutes. Thereafter, air was injected into the capillary to expel the above-mentioned aqueous solution, followed by drying in a dryer at 40 ° C. for 12 hours to obtain a flow path having a hydrophilic compound covalently bonded to the outermost surface.

(2) Measurement sample A healthy human blood sample and a modified Hb-containing sample were prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis The above-mentioned healthy human sample and modified Hb-containing sample were measured using a flow path in which a hydrophilic compound was covalently bonded to the above-described surface. As the electrophoresis buffer, a 250 mM succinic acid buffer (pH 5.2) containing 2.0% by weight of chondroitin sulfate (anionic polymer, manufactured by Wako Pure Chemical Industries, Ltd.) was used. A human sample or a modified Hb-containing sample was injected, electrophoresis was performed at a voltage of 0.8 kV, and absorbance was measured at 415 nm.
In the measurement of a healthy human blood sample, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the modified Hb-containing sample, an electropherogram similar to that shown in FIG. 3 was obtained.

Example 4
(1) Preparation of migration path A double T-type flow path for electrophoresis having a width of 80 μm was formed on a microchip (7 cm × 5 cm) made of a copolymer of methyl methacrylate and glycidyl methacrylate. A 0.01N NaOH solution, ion-exchanged water, and a 0.01N HCl solution were passed through the formed channel in this order to wash the channel, and then 20 wt% ethylenediamine (hydrophilic compound, molecular weight 60). (Made by Wako Pure Chemical Industries, Ltd.) An aqueous solution was passed through for 5 minutes, and both ends of the flow channel were sealed with the aqueous solution filled, and then left in a thermostatic bath at 50 ° C. for 3 hours. Thereafter, the inside of the flow path was washed with ion-exchanged water, 0.05N sulfuric acid was passed through the flow path for 3 minutes, and the capillaries were sealed at both ends of the capillary while the aqueous solution was filled. It left still in a thermostat at 5 degreeC for 5 hours. Thereafter, the inside of the above-described channel was washed with ion-exchanged water to obtain a channel having a hydrophilic compound covalently bonded to the outermost surface.

(2) Preparation of measurement sample A healthy human blood sample and a modified Hb-containing sample were prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis The above-mentioned healthy human sample and modified Hb-containing sample were measured using a flow path in which a hydrophilic compound was covalently bonded to the above-described surface. As an electrophoresis buffer, a 300 mM malic acid buffer (pH 5.2) containing 1.7% by weight of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) copolymer was used. The above-mentioned healthy human sample or modified Hb-containing sample was injected from one side of the path, electrophoresed at a voltage of 1.0 kV, and absorbance at 415 nm was measured.
In the measurement of a healthy human blood sample, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the modified Hb-containing sample, an electropherogram similar to that shown in FIG. 3 was obtained.

(Comparative Example 1)
(1) Preparation of migration path A fused silica capillary (made by GL Science, inner diameter 25 μm × total length 30 cm) is set in a capillary electrophoresis apparatus, 0.2 N NaOH solution, ion exchange water, 0.5 N HCl solution. In this order, the inside of the capillary was washed, and then a 240 mM malic acid buffer solution (pH 4.7) containing 0.5% by weight of bovine serum albumin (molecular weight: about 67,000, manufactured by Wako Pure Chemical Industries, Ltd.) was added. The mixture was passed for 0.5 minutes to obtain a capillary whose surface was dynamically coated with bovine serum albumin.

(2) Measurement sample A modified Hb-containing sample was prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis The above-mentioned modified Hb-containing sample was measured using a capillary whose surface was dynamically coated with bovine serum albumin. As the electrophoresis buffer, 350 mM citrate buffer (pH 4.7) containing 0.2 wt% chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and the above-mentioned modified Hb-containing sample was injected from one of the capillaries. Then, electrophoresis was performed at a voltage of 8.5 kV, and absorbance at 415 nm was measured.
FIG. 4 is an electropherogram obtained by measurement of a modified Hb-containing sample in Comparative Example 1. In FIG. 4, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c).
In FIG. 4, stable HbA1c and modified Hb were not sufficiently separated.

(Comparative Example 2)
(1) Preparation of migration path A fused silica capillary (GL Science, inner diameter 75 μm × total length 50 cm) was set in a capillary electrophoresis apparatus (Beckman Coulter, PAC / E MDQ). After 0.2 N NaOH solution, ion-exchanged water and 0.5 N HCl solution were passed in this order to wash the inside of the capillary, 5.0 wt% polybrene (molecular weight 5,000 to 10,000, Nacalai) Tesque) aqueous solution was passed for 20 minutes. Thereafter, air was injected into the capillary to expel the above-mentioned polybrene aqueous solution, and then, it was dried in a dryer at 40 ° C. for 12 hours. Thereafter, the injection of the polybrene aqueous solution, the injection of air, and the drying were repeated again five times to obtain a capillary having a polybrene fixed coating on the surface.

(2) Preparation of measurement sample A modified Hb-containing sample was prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis The above-mentioned modified Hb-containing sample was measured using a capillary having polybrene immobilized on the surface. A 350 mM citrate buffer solution (pH 4.7) containing 0.2% by weight of dextran sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) is used as an electrophoresis buffer solution, and the above-mentioned modified Hb-containing sample is injected from one of the capillaries. Then, electrophoresis was performed at a voltage of 18 kV, and absorbance at 415 nm was measured.
In the measurement of the modified Hb-containing sample, an electropherogram similar to that shown in FIG. 3 was obtained.

(Comparative Example 3)
(1) Preparation of migration path A double T-type electrophoresis channel having a width of 100 μm was formed on a polydimethylsiloxane microchip (7 cm × 5 cm). A 0.1N NaOH solution, ion-exchanged water, and a 0.2N HCl solution were passed through the formed channel in this order to wash the inside of the channel, and then 1.0% by weight of 3-aminopropyltrimethoxy. An aqueous solution containing silane (manufactured by Shin-Etsu Silicone) was passed for 30 minutes. Thereafter, air was injected into the capillary to drive out the aqueous solution, and then dried in a dryer at 40 ° C. for 12 hours.
Next, a phosphate buffer containing 5.0% by weight of glutaraldehyde was passed through the flow path for 60 minutes, and then 100 mM phosphate buffer was passed through for 60 minutes for washing. Further, a 100 mM phosphate buffer containing 0.2 mM bovine serum albumin (molecular weight: 67,000, manufactured by Wako Pure Chemical Industries, Ltd.) was passed through the flow path for 5 minutes, and then allowed to stand at 4 ° C. for 120 minutes. As a result, a flow channel having a surface coated with bovine serum albumin was obtained.

(2) Preparation of measurement sample A modified Hb-containing sample was prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis The above-mentioned modified Hb-containing sample was measured using a flow channel having the above-mentioned surface coated with bovine serum albumin. As the electrophoresis buffer, a 200 mM malic acid buffer (pH 5.0) containing 0.2% by weight of chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was used. After injection, electrophoresis was performed at a voltage of 0.9 kV, and absorbance at 415 nm was measured.
In the measurement of the modified Hb-containing sample, an electropherogram similar to that shown in FIG. 3 was obtained.

(Comparative Example 4)
(1) Preparation of migration path According to the same method as in Example 4, except that 0.1% by weight of polylysine (molecular weight: 1,000 to 4,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used as the hydrophilic compound. A channel having a hydrophilic compound covalently bonded to the outermost surface was obtained.

(2) Preparation of measurement sample A modified Hb-containing sample was prepared in the same manner as in Example 1.

(3) Measurement by electrophoresis The modified Hb-containing sample was measured by the same method as in Example 4 except that the obtained flow path was used.
In the measurement of the modified Hb-containing sample, an electropherogram similar to that shown in FIG. 3 was obtained. In the obtained electropherogram, all the peaks were well separated.

(Evaluation)
(1) Confirmation of Separation Performance of Modified Hb Regarding the measurement conditions of Examples 1-4 and Comparative Examples 1-4, a healthy human blood sample and a modified Hb-containing sample prepared based on the healthy human blood, namely Example 1 And a sample containing unstable HbA1c obtained by adding glucose to healthy human blood to 2500 mg / dL, and another modified Hb-containing sample, ie, 50 mg / dL of acetaldehyde in the healthy human blood. An acetylated Hb-containing sample obtained by adding so as to obtain a carbamylated Hb-containing sample obtained by adding sodium cyanate to 50 mg / dL, and the stable HbA1c value in each sample was prepared. Asked.
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 (ΔHbA1c value) obtained by subtracting the stable HbA1c value of the obtained healthy human blood sample from the stable HbA1c value of each of the obtained modified Hb-containing samples was determined.
The results are shown in Table 1.

Under 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. Under the measurement conditions of Comparative Examples 2 to 4, the modified Hb and stable HbA1c were better separated than those of Comparative Example 1, but there were samples in which the measured values fluctuated when compared with Examples 1 to 4. .

(2) Simultaneous reproducibility test Using the measurement conditions of Examples 1 to 4 and Comparative Examples 1 to 4, the same healthy human blood sample was continuously measured 10 times, and the CV value of the obtained stable HbA1c value Was calculated.
Further, in Comparative Example 1, after one measurement was completed, a 0.2N NaOH solution was passed for 0.3 minutes, a buffer solution was passed for 1.8 minutes, and the inside of the flow path was washed. Then, again, a malic acid buffer solution containing 0.5% by weight bovine serum albumin as a coating agent was passed through for 0.5 minutes to apply a dynamic coating to the surface of the flow path, and then injected the next measurement sample, A simultaneous reproducibility test was performed by repeating the measurement.
The results are shown in Table 2.

(3) Durability test Using the measurement conditions of Examples 1 to 4 and Comparative Examples 1 to 4, the maximum value of the stable HbA1c value obtained by continuously measuring the same healthy human blood sample 100 times and The difference (R value) from the minimum 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 about 5%, and the variation width in the durability test is about 1%, which is quite insufficient for managing the HbA1c value of diabetic patients. It was value. Moreover, about Comparative Examples 2-4, although isolation | separation was possible about some modification Hb, a measured value changed gradually by performing a repeated measurement, and finally showed the big CV value and the variation width. Therefore, it was found that stable measurement cannot be performed.

As described above, in Examples 1 to 4, stable HbA1c can be separated with high accuracy in a short time, and stable performance was exhibited even when repeatedly measured. The numerical value indicated by the evaluation results of Examples 1 to 4 was a level that can be sufficiently used to manage the HbA1c value of diabetic patients.
On the other hand, in Comparative Example 1, since the separation performance was insufficient, the measurement could not be performed with high accuracy. In Comparative Examples 2 and 3, a part of the modified Hb can be separated, but when it was repeatedly measured, the performance was severely degraded and stable measurement could not be performed. Furthermore, as shown in Comparative Example 4, even when an electrophoresis path to which a hydrophilic compound deviating from a predetermined molecular weight range was used, the performance during repeated measurement was poor. The numerical values indicated by the evaluation results of Comparative Examples 1 to 4 were levels that were not applicable 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 a capillary electrophoresis apparatus. In Examples 1-4, it is an electropherogram obtained by the measurement of the blood of a healthy person. In Examples 1-4 and Comparative Examples 2-4, 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.

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 (5)

A method for measuring hemoglobin using a capillary electrophoresis method or a microchip electrophoresis method , wherein a hydrophilic compound having a molecular weight of 20 to 800 is directly applied to the outermost surface in contact with an electrolyte filled in the migration path without using a spacer. A method for measuring hemoglobin, characterized by using a migration path that is covalently bonded. A method for measuring hemoglobin using capillary electrophoresis or microchip electrophoresis, in which a hydrophilic compound is indirectly covalently bonded to the outermost surface in contact with the electrolyte filled in the migration path via a spacer. A method for measuring hemoglobin, wherein the total of the molecular weight of the hydrophilic compound and the molecular weight of the spacer is 20 to 800. The method for measuring hemoglobin according to claim 1 or 2, wherein the hydrophilic compound has a cationic group. Measurement method according to claim 1, 2 or 3 hemoglobin of, wherein the hydrophilic compound is a silane coupling agent. The method for measuring hemoglobin according to claim 1, 2, 3, or 4, wherein a buffer solution containing a water-soluble polymer having an anionic group that is filled in the electrophoresis path during electrophoresis is used.

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