JP2006189401A - Device for measurement and device and method for measuring hemoglobins - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring device capable of easily and precisely measuring optically detectable components and further biological material components derived from living bodies by a compact measuring device with a current HPLC measurement precision and provide a device and a method for measuring hemoglobins capable of easily using each measuring section of the measuring device to a maximum extent and highly precisely analyzing hemoglobins by a portable and compact device in a short time without having to require a wide space for installation. <P>SOLUTION: The measuring device for measuring components to be measured in an analyte containing the components to be measured is constituted of a prefilter part capable of removing foreign matter in a specimen sample; a separation part for separating the components in the specimen sample; and a detection part for optically detecting the components in the specimen sample. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a measuring device and is not particularly limited as long as it is a component capable of detecting light. Furthermore, the present invention relates to a measuring device capable of analyzing biological sample components and hemoglobins contained in blood, plasma, and serum, and a method for measuring hemoglobin using the same.

In recent years, a microreactor or μTAS (micro / miniaturized total analysis system) technology has been actively studied. This technology uses microfabrication technology to produce fine grooves and liquid reservoirs on a substrate such as silicon and use them as flow channels and reaction tanks for liquid delivery, thereby allowing various chemical reactions on the substrate. Is to implement. The analysis method using a microreactor or μTAS has advantages that the reaction temperature is easily controlled because the reaction is performed in a very small space, the reaction efficiency is high, and the reaction time can be shortened.
However, as in the prior art, an analysis site related to measurement, for example, (1) a prefilter unit for removing foreign matter in a measurement sample, (2) a column unit for separating components in a measurement sample, (3) When using a measurement device in which a detection unit for detecting a component in a measurement sample separated by a column unit is mounted on the same substrate, the following problems do not occur when measuring a small number of samples. However, in the case of continuous measurement or when measuring a large amount of samples with the same measurement device, there is a disadvantage that only the prefilter part has an abnormality and must be discarded even if the column part and the detection part are still usable. . In addition, when the number of samples used in the prefilter unit, column unit, and detection unit mounted on the same substrate is different, the sample that can actually be measured must be matched to the region where the number of samples used is the least. This point is very detrimental for users and manufacturers.

It was studied to analyze proteins using the microreactor or μTAS shown above. It was considered to combine a spectrophotometer as an analyzer that can be miniaturized with relatively high accuracy. However, it is difficult to secure a sufficient optical path length with a conventional microreactor or μTAS. Could not be analyzed with high accuracy.
As a similar technique in that a minute flow path is used, there is a flow injection analysis method that is an analysis technique using a capillary (capillary). For example, Z-type as described in Non-Patent Document 1 An optical path length is secured by using a flow path. However, even if this technology is applied to a microreactor, for example, when detection light is introduced from the inflow side flow path, the detection light is output to the outflow side flow path while being reflected by the flow path wall surface in order to lengthen the optical path length. come. In this case, there is a problem that if there is a slight distortion on the side surface of the flow path between the inflow side flow path and the outflow side flow path, the detection light is disturbed and noise increases. In order to eliminate the slight distortion generated on the side surface of the flow path between the inflow side flow path and the outflow side flow path, (1) the yield deteriorates in industrial production. (2) Quality control is difficult, (3) It is not preferable in terms of production, such as having to use a specific flow path material.

"Prior art of hemoglobin A1c measurement"
As one of the measurements by liquid chromatography, glycated hemoglobin in blood, particularly hemoglobin A1c (hereinafter referred to as HbA1c), is widely measured because it serves as an index for diagnosis of diabetes. Glycated hemoglobin is produced by combining sugar in blood with hemoglobin. The glycated hemoglobin in the hemolyzed sample reflects the average sugar concentration in the blood during the past 1 to 2 months, so that the glycated hemoglobin in the hemolyzed blood, particularly in recent years, stable A1c has been useful in diagnosing diabetes. It is widely known that it is the most important index (marker), and it is required to measure this stable A1c accurately in a short time. The reason for this is, for example, a “pre-clinical examination” in which a stable A1c measurement of a patient is performed before medical treatment in a hospital, and a doctor decides the treatment policy of the patient based on the stable A1c measurement result and performs treatment. This is because there is a growing need.

  The measurement of HbA1c by liquid chromatography is mainly performed by cation exchange liquid chromatography (Japanese Patent Publication No. 8-7198). When a hemolyzed sample is separated by cation exchange liquid chromatography, usually, hemoglobin A1a (hereinafter referred to as HbA1a) and hemoglobin A1b (hereinafter referred to as HbA1b), hemoglobin F (hereinafter referred to as HbF), unstable HbA1c, stable HbA1c, and It can be fractionated into peaks such as hemoglobin A0 (hereinafter referred to as HbA0). HbA1c used as an index for diagnosis of diabetes is stable HbA1c, and is determined as a ratio (%) of the area of stable HbA1c peak to the total hemoglobin peak area.

  Measurement of this HbA1c by the liquid chromatography method uses a column packed with a cation exchange liquid chromatography filler (for example, a filler having a carboxyl group or a sulfonic acid group as a functional group), pH 5.0 to Performed using 9.0 eluent.

"Disadvantages of the conventional HPLC method for stable A1c measurement"
As described above, the conventional stable A1c measurement method by the HPLC method (FIG. 1) can measure the stable A1c with high accuracy, but requires a slightly larger HPLC measurement apparatus. In these stable A1c measurements by HPLC, the sample to be measured diffuses in a relatively large column (inner diameter 4.6 mm × length 35 mm) and the detector cell, or is detected from the sample injection to the column and detection cell. Because of the long pipe length, the sample to be diffused further spreads out the peaks of each hemoglobin, and even if the measurement time is further shortened, the peak separation of each hemoglobin becomes insufficient. The stable A1c cannot be measured with high accuracy.
In addition, these HPLC measuring instruments for stable A1c measurement are also being miniaturized, but the equipment is still too large to be placed in a medical field, especially a small hospital such as a practitioner, and the following There is a problem. (1) It is necessary to secure a space for installation. (2) Too heavy to carry. (3) There is a problem such as diffusion of the measurement sample.

Anal. Chem. 1983, 65, 3454-3459 Japanese Patent Publication No.8-7198

  An object of the present invention is to provide a measuring device that can easily and accurately measure a component capable of detecting light, and further a biological sample component derived from a living body, with a smaller measuring device. In addition, it is a portable device that can carry hemoglobin while maintaining the current HPLC measurement accuracy, and does not require a wide space for installation. It is an object of the present invention to provide a hemoglobin measuring device and a hemoglobin measuring method capable of analyzing hemoglobin with high accuracy, wherein the site can be used easily and maximally.

  The device for measuring photodetectable components, biological sample components derived from living organisms, and hemoglobins according to the present invention is a microreactor that has been actively studied in recent years, or microfabrication represented by μTAS (micro / miniaturized Total Analysis System). The technology is applied to the pre-filter part, the column part, and the flow path connecting them. It is smaller than conventional immunoassay systems and HPLC measurement systems, and the diffusion of the analyte is minimized. For this reason, it has become possible to perform high-resolution measurements in a further short time that cannot be realized with conventional immunoassay systems and HPLC measurement systems.

  Further, in the measurement device for measuring a measurement component derived from a living body of the present invention, a cation exchange group, an anion exchange group, a prefilter part that can remove foreign matters in the measurement sample, and a component in the measurement sample are separated. Consists of a octyldodecyl group or size exclusion, a column part formed by combining at least one of a group of hollow columns, or a combination of a plurality, and a detection part for performing photodetection of components in the measurement sample This is a measuring device.

Claim 1 of the present invention will be described in detail below.
The type of the measurement sample of the measurement device of the present invention is not particularly limited as long as it is a component that can be detected by light. Furthermore, any biologically-derived component may be used, for example, a virus, cell, tissue, organ or animal or plant, microorganism, etc., a biological sample such as a cell, tissue, organ lysate or Body fluids such as blood, plasma, serum, urine, tears, stool, sputum, gastric juice; organic molecules of biological origin such as sterolide, amino acids, nucleotides, sugars, polynucleotides, polynucleotides, complex carbohydrates, lipids; RNA, Nucleic acids such as DNA can be raised.
In addition, known measurement items currently measured in clinical tests can also be measured. For example, (1) Biochemical tests: proteins such as albumin, low molecular nitrogen compounds such as uric acid, creatinine and amino acids; enzymes such as γ-GTP, GOT, GPT and glutamate dehydrogenase; isozymes such as LDH isozyme and amylase isoisozyme Glycoproteins and organic acids such as hemoglobin A1c, fructosamine, 1.5AG and glycol alcohol; lipids such as LDL, HDL and riboprotein; vitamins; electrolytes and trace metals; biological pigments such as benzylamine; hippuric acid, pesticides; Toxic substances such as organic solvents and industrial hygiene related; antiepileptics, hypnotics, psychotropic drugs, bronchodilators, antibiotics, immunosuppressants, diuretics, anticancer drugs, etc., tumor markers, catecholamines and other hormones Kind,
Immunoglobulins (allergy tests) such as IgE and IgG, autoimmune factors such as rheumatoid factors, hepatitis virus antibodies / antigens, influenza viruses, T-detail systems, B cell systems, osteocytic cells, NK cell systems, and other blood cells Examples include immune cell classification, lymphoid subset test, and cytokines.
As the prefilter part according to claim 1 of the present invention, a known prefilter; stainless fiber, filter paper, non-woven fabric, or membrane filter can be used as long as it can remove foreign substances in the measurement sample.

Further, the column part for separating the components in the measurement sample according to claim 1 of the present invention is a cation exchange group such as a carboxyl group or a sulfonic acid group, an anion exchange group such as an amino group, or a hydrophobic group such as an octyldodecyl group. Functional groups with sexual interaction, or size exclusion that separates by molecular size, hollow columns that do not interact with measurement components, such as antigen-antibody reaction, color reaction, luminescence reaction, latex agglutination reaction, etc. Provides a reaction field for
The column part of the present invention is a column part formed by combining at least one or a combination of the above cation exchange group, anion exchange group, functional group that performs hydrophobic interaction, size exclusion, and hollow column part. Can be used.
Moreover, the detection part for performing the light detection of the component in the measurement sample of Claim 1 of this invention will not be specifically limited if it is well-known light detection, For example, a light absorbency, color development, latex aggregation, etc. are mentioned. It is done.

Further, in the device for measuring hemoglobin according to claim 2 of the present invention, a prefilter part capable of removing foreign substances in the measurement sample, a column part having a cation exchange group for separating components in the measurement sample, measurement Since at least one or a plurality of parts in the group of detection parts for detecting the absorbance of the components in the sample are configured in a unit form so that they can be appropriately replaced, the prefilter part and the column By replacing each unit part of the part where the number of specimens used in the head part and the detection part are different or where the failure has occurred, the pre-filter part, the column part and the detection part are mounted on the same substrate as in the past. This is a device for measuring hemoglobin and a method for measuring hemoglobin, which can measure hemoglobin more efficiently.
The above unit type of the present invention includes, for example, the meaning of a cartridge type in which parts (units) having functions such as each prefilter part, column part, and detection part can be freely replaced.

Hereinafter, the second aspect of the present invention will be described in detail.
In this specification, the column portion means a specific region of a measuring device having a function capable of separating hemoglobin as a component in a measurement sample.
In the column part, hemoglobins, which are components contained in the measurement sample, can be separated quickly and easily, and stable measurement of stable A1c is also possible.

The column part in the present invention will be described in detail below.
The column part of the device for measuring hemoglobin of the present invention may be in the form of particles, cylinders, capillaries, meshes, hollow fibers, porous membranes, and monoliths as long as they have cation exchange groups. It ’s fine. Preferably, in consideration of a decrease in pressure loss and contact efficiency with a fluid such as a specimen flowing inside, a granular shape, a porous membrane shape, and a monolith shape are suitable.
(1) The particles having a cation exchange group will be described below.
The filler for cation exchange liquid chromatography in the method for measuring hemoglobin of the present invention is composed of particles having at least one cation exchange group. For example, a cation exchange group is introduced into polymer particles. It is obtained by doing.

  The cation exchange group may be a known one and is not particularly limited. Examples thereof include cation exchange groups such as carboxyl group, sulfonic acid group, and phosphoric acid group. A plurality of these cation exchange groups may be introduced.

The diameter of the particles is preferably 0.5 to 20 μm, more preferably 1 to 10 μm.
The particle size distribution is preferably 40% or less, more preferably 30% or less, as a coefficient of variation (CV value) (standard deviation of particle size ÷ average diameter × 100).

Examples of the polymer particles include inorganic particles such as silica and zirconia; natural polymer particles such as cellulose, polyamino acid and chitosan; and synthetic polymer particles such as polystyrene and polyacrylate.
In the polymer particles, components other than the ion exchange group to be introduced are preferably more hydrophilic. Further, those having a high degree of crosslinking are preferred from the viewpoint of pressure resistance and swelling resistance.

  The introduction of the cation exchange group into the polymer particle can be performed by a known method. For example, after preparing the polymer particle, the functional group of the particle (hydroxyl group, amino group, carboxyl group, epoxy group, etc.) Further, it can be carried out by a method of introducing a cation exchange group into the particles by a chemical reaction.

  The cation exchange filler can also be prepared by a method of preparing polymer particles by polymerizing a monomer having a cation exchange group. Examples thereof include a method in which a cation exchange group-containing monomer and a crosslinkable monomer are mixed and polymerized in the presence of a polymerization initiator.

  In addition, a polymerizable cation exchange group-containing ester such as methyl (meth) acrylate and ethyl (meth) acrylate is mixed with a crosslinkable monomer and polymerized in the presence of a polymerization initiator. It may be hydrolyzed to convert the ester into a cation exchange group.

  Furthermore, as described in JP-B-8-7197, after preparing crosslinked polymer particles, a monomer having a cation exchange group is added, and the monomer is placed near the surface of the polymer particles. It may be polymerized.

"Filling method"
The above packing material may be directly packed in the column portion formed in the measuring device of the present invention.
Moreover, in order to prevent the degradation of the separation performance due to repeated measurement, for example, it is necessary to pack the column densely with the packing material. In order to achieve this, for example, the filler slurryed by the slurry filling method can be pressed into the column by a liquid feed pump or the like. Thereafter, a method of fitting the column filled with the filler into the column portion formed in the measuring device of the present invention can be mentioned. As a method for packing the packing material into the column part or the column on the measuring device of the present invention, there is a method in which the filler particles are slurried with an aqueous solution containing a buffering agent, but there are other known methods. However, any method can be used as long as it can fill the column part with the packing material. The packing pressure is appropriately selected in the range of 0.1 MPa to 50 MPa according to the column portion on the measuring device of the present invention, or the column material, the type of packing material, and the separation performance.

The column material used in the present invention is not particularly limited, such as known stainless steel, glass, and resin (PEEK, polypropylene, Teflon (registered trademark), polyethylene). The column part on the microreactor of the present invention, or the size of the column, is preferably a cylindrical shape having an inner diameter of 0.01 to 3.0 mm and a length of 0.1 to 50 mm, and an inner diameter of 0.05 to 2 mm and a length. A columnar shape of 0.5 to 30 mm is more preferable.
Further, the column part or column shape of the measuring device of the present invention may be a known shape that does not adversely affect the separation performance of hemoglobins such as a quadrangular column and a triangular column other than the columnar shape. Moreover, even if the column part of the measuring device of the present invention or the shape of the column is other than the columnar shape, the column part of the measuring device of the present invention or the cross-sectional area and the length of the column are the above cylindrical shape. A size corresponding to the cross-sectional area and length of the column is preferred.

  For analysis of trace samples, the usual methods such as liquid chromatography have a large dead volume of columns, detectors, piping, etc., so the trace sample diffuses and the peak of each measurement component becomes broad, and the detection sensitivity is also high. High accuracy measurement was extremely difficult because of its lowness. However, in the measurement device of the present invention, the flow path for measuring the absorbance is a small volume, and the flow path connecting the column section and the flow path for measuring the absorbance is also a small volume. Even minute amounts can be analyzed. This can reduce the amount of sample and improve detection sensitivity and high separation even with a small amount of sample.

“The measuring device of the present invention has a cell portion for measuring absorbance. (FIGS. 2 to 6)”
In this specification, a cell part means the site | part through which the light ray for a light absorbency measurement passes.
The cell part is composed of a channel formed in a straight line. By using such a flow path as the cell portion, it is possible to measure the absorbance even for a very small amount of sample.

  The cell portion preferably has an optical path length of 1 to 20 mm. Here, the optical path length means the length of the liquid through which the light beam for absorbance measurement passes, and is substantially equal to the length of the channel formed in a straight line in the cell portion. If it is less than 1 mm, the optical path length is too short and the detection sensitivity may be greatly reduced. If it exceeds 20 mm, absorption by the substance whose absorbance is to be measured and absorption by other substances, that is, background and May be difficult to distinguish.

  There may be a plurality of the cell portions. By separating different target components or the next sample to be measured in a plurality of column parts, and performing analysis by providing a corresponding cell part for each column part, various substances or large quantities of samples can be simultaneously obtained. Separation and analysis are possible.

  The measuring device of the present invention is configured so that the cell portion is inserted into the linearly formed flow path so that the optical axis of the light beam for absorbance measurement is in the flow direction of the linearly formed flow path. It can be connected to the absorbance measuring device so that the incident angle and the outgoing angle of the light beam are 0 °. By making the cell part connectable to the absorbance measuring device in this way, the measuring device of the present invention is a problem of the flat microreactor, which lacks the sensitivity due to the short optical path length in the absorbance measurement. Can be prevented.

  The measurement device of the present invention is connected to an absorbance measurement apparatus, and the light transmittance at an absorbance measurement wavelength measured in the absence of a specimen is 50 of the light transmittance when air is measured at the same optical path length and absorbance measurement wavelength. % Or more is preferable. If it is less than 50%, the sensitivity of absorbance measurement may decrease, or the error may increase.

In order to make it possible to connect the cell part of the measuring device of the present invention to the absorbance measuring apparatus under the above-described conditions and to ensure the above-mentioned sufficient light transmittance, the material of the cell part is as transparent as possible. While selecting a high thing, it is preferable to make the structure of a cell part as short as possible so that the light ray for a light absorbency measurement may pass the material which comprises a cell part.
An example of a preferable structure of the cell portion is shown in FIGS.
In the cell portion shown in FIGS. 18A to 18D, the cell portion is connected to the connection channel in a U-shape or L-shape at the inflow portion and / or the outflow portion. By connecting in this way, the cell part can be protruded from the substrate so that it can be connected to the absorbance measuring device, and the distance through which the light beam for absorbance measurement passes through the material constituting the cell part becomes as short as possible. Can be.

  Further, when mass-producing the measuring device of the present invention, it is considered that production by molding processing of plastics occupies a large weight. In particular, production by injection molding technology is considered important because of its productivity. However, when the measurement device of the present invention is to be produced with the same technology, the cell part is connected to the connection flow path in a U shape or L shape at the inflow part and / or the outflow part as described above. Then, molding defects such as sagging are likely to occur at the connection portion, and the yield may be extremely deteriorated. Therefore, it is preferable to form a protruding flow path at the connection portion between the cell portion and the connection flow path. By forming such a protruding flow path, sagging at the connection portion is less likely to occur, and as a result, the productivity of the measuring device of the present invention is improved.

  A preferable lower limit of the length of the jumping channel is 0.01 mm, and a preferable upper limit is 2 mm. When the thickness is less than 0.01 mm, the sagging prevention effect may not be obtained. When the thickness exceeds 2 mm, the absorbance measurement may be adversely affected.

  An example of a preferable structure of the cell portion when the pop-out flow path is formed is shown in FIGS. 18-C and 18-D. In the cell part shown in FIG. 18-C, a protruding flow path is provided at the connection part with the connection flow path in the inflow part and / or the outflow part of the cell part, and the E-shaped flow path is formed as a whole. Forming. In the cell portion shown in FIG. 18-D, a protruding flow path is provided at the connection portion with the connection flow path at the inflow portion of the cell section, and a T-shaped flow path is formed as a whole.

  The absorbance measuring apparatus to which the measuring device of the present invention is connected is not particularly limited, and for example, a commercially available portable small spectrophotometer can be used. By using such a spectrophotometer, ultraviolet absorption, infrared absorption, visible light absorption, etc. can be easily measured. Examples of commercially available portable small-sized spectrophotometers include a USB2000 spectrophotometer manufactured by Ocean Optics.

The column part and the cell part are connected to each other by a connection channel. The cross-sectional area of the connection channel is preferably 1 mm ² or less. If it exceeds 1 mm ² , components such as hemoglobin separated in the column part or the like diffuse in the flow path, and the detection sensitivity may be lowered. More preferably, it is 0.09 mm ² or less.
Moreover, the length of the flow path for connection which connects the said column part and cell part is 0.1-30 mm. Preferably, it is 0.2 mm-20 mm. If the length of the connecting channel is shorter than 0.1 mm, it is difficult to manufacture the measuring device of the present invention. Further, when the length of the connecting channel is longer than 30 mm, there is a problem that the separated components diffuse and the separation performance is deteriorated.

  The connection flow path connecting the column part and the cell part has a flow path cross-sectional area of 2 times or more or 0.5 times the flow path cross-sectional area immediately before the flow path length within 1 mm. It is preferable to own at least one such part. By having at least one such part, bubbles are mixed in the flow path when injecting a specimen into the measuring device of the present invention or when injecting various eluents into the measuring device of the present invention. Can be temporarily suppressed, and measurement errors in absorbance due to bubbles can be suppressed.

  The measuring device of the present invention may further include a flow path for flowing an eluent for separating and eluting hemoglobin, a hemolytic dilution of blood, and the like in addition to the connection flow path for connecting the respective parts. Moreover, you may have the storage part for storing an eluent, a hemolysis dilution liquid, etc. in the unit form which can be removed.

The measuring device of the present invention may further have a pump for feeding liquid therein.
Although it does not specifically limit as said pump, For example, the micropump whose total volume of a drive part which consists of mechanisms, such as a peristaltic pump, a rotary pump, a syringe pump, a diaphragm pump, is 1 cm < ³ > or less can be used. As such a micro pump, for example, a diaphragm pump in which a diaphragm structure described in Japanese Patent Laid-Open No. 2001-132646 is miniaturized as it is by a MEMS processing technique; intermittent by a micro piston described in Japanese Patent Laid-Open No. 2002-021715 Examples of a structure for automatically feeding liquid include a pump for feeding a liquid feeding medium by a method for generating an electroosmotic flow on a fine flow path described in JP-A-10-010088.

  The measurement device of the present invention preferably further has a microvalve. As the microvalve, for example, a known valve such as a microvalve or an electromagnetic valve that uses a gas action to control the movement of fluid described in Japanese Patent Application Laid-Open No. 2000-120617 can be used.

Although it does not specifically limit as a magnitude | size of the measuring device of this invention, When handling property is considered, it is preferable that it is 400 cm < ² > or less, More preferably, it is 100 cm < ² > or less.

  It does not specifically limit as a material which comprises the board | substrate of the measuring device of this invention, For example, inorganic substances, such as glass, fluorescent glass, ceramics; Organic compounds, such as plastics; Metal etc. are mentioned. When considering recyclability, glass, thermoplastics, or metal is suitable. On the other hand, when considering economy on the premise of processability and disposal, various plastics are preferred. Further, when various plastics are used as the material constituting the substrate of the measuring device of the present invention, it is inexpensive and light, and thus has excellent portability and workability.

It does not specifically limit as said plastic, Both a thermoplastic resin and a thermosetting resin can be used. Among them, although the thermosetting resin does not have the advantage of being easily plasticized by heating and surface processing, by introducing a precursor liquid mixed with a curing agent in advance into a transfer mold and curing it, It is possible to shape the resin surface. In this case, since the precursor liquid is liquid, the shape of the transfer mold can be transferred more faithfully. In general, a statically cured resin is also useful because it exhibits a low linear expansion coefficient and a low molding shrinkage ratio.
Although it does not specifically limit as said thermosetting resin, An epoxy resin is suitable from the point of cost or easy handleability.

In the measurement of hemoglobins, an eluent having a pH of 5 to 9 is used as an eluent for separating and eluting hemoglobins. In this case, a polyolefin resin, acrylic resin, PEEK resin having acid resistance and alkali resistance is used. Etc. are suitable. The surface of these resins may be modified with various reagents for the purpose of improving chemical resistance, reducing pressure loss, fluid control, and the like.
In addition, since the cell portion is particularly required to have light transmissivity, a plastic that is different from other parts and that is particularly excellent in light transmissibility may be used. Examples of such plastics excellent in light transmission include acrylic resins, polycarbonate resins, polystyrene resins, and the like.

The method for producing the measuring device of the present invention is not particularly limited. For example, various machining such as laser processing and cutting; etching, atomic beam etching; the flow path portion becomes a through hole or a through groove by laser processing or the like. Examples of the method include a method of forming a flow path and a reservoir using a conventionally known method such as injection molding.
In addition, the substrate on which the flow path and the storage part are formed should be sealed except for the reagent introduction part and the connection place of the external pump by bonding it to another substrate by various adhesives, anodic bonding, etc. Is preferred. In addition, although there is a limit to the materials that can be used, it is possible to form a reactor in which the sealing process is omitted by using stereolithography technology.
In addition, when using different materials for the cell part and other parts, a method of adhering the separately produced cell part to other parts with various adhesives, coupling agents, etc., a molding method by coextrusion unit type ( Each part can be manufactured by screw connection) or the like.

“Surface treatment of the site where hemoglobin contacts with the measuring device of the present invention”
Although the materials of the measurement device of the present invention are shown above, when the hemoglobins that are measurement specimens come into contact with the measurement device of the present invention, the hemoglobins are adsorbed and correct measurement values of each hemoglobin may not be obtained. .
In order to suppress the adsorption of hemoglobin to the measurement device, it is preferable to perform a known protein adsorption reduction treatment at least on the surface of the measurement sample where the hemoglobin contacts the measurement device.
The protein adsorption reduction treatment is preferably performed by a protein treatment such as blocking treatment (physical adsorption treatment, chemical crosslinking treatment), silicone treatment, water repellency treatment, chemical hydrophilization treatment, plasma treatment or the like.

"Air bleeding structure"
The measurement device of the present invention can be provided with an air vent structure that can easily remove air (air) that has entered the flow path of the measurement device as shown in FIGS. The air vent structure can be installed in each flow path of the measuring device, and the installation location and the number of installation are not particularly limited as long as the air in the flow path can be efficiently and completely removed.
As long as the air bleeding structure of the present invention has a function capable of opening and closing each flow path of the measuring device, a known technique such as a valve type, a valve type, a screw type, or the like can be used. 7 to 8 show a screw type as an example of air bleeding of the measuring device of the present invention.

Moreover, the material similar to the raw material of the measuring device of this invention can be used for the raw material of the air bleeding structure of this invention. However, it is necessary to select a material suitable for each of which the eluent does not leak due to the material of the measurement device of the present invention and the air vent structure material. Basically, it is preferable to use a soft material, for example, a resin such as Teflon (registered trademark) as the air vent of the present invention rather than the material of the measuring device. The size of the air bleed structure is not particularly limited as long as the air can be reliably evacuated from each flow path, but a preferable hole diameter for air bleed is about 0.01 mm to 3 mm.
In addition, when air enters the column for separating and measuring hemoglobin, the flow of the eluent for separating hemoglobin is disturbed, so that the separation performance of hemoglobin is reduced and each peak of hemoglobin becomes broad. For example, there is a problem that the stable A1c value cannot be measured accurately.

"Filter structure"
The filter structure used in the present invention includes (1) a screw type as shown in FIGS. 9 to 10, (2) a type installed in a measuring device (FIG. 2), and (3) a Fritz type.
The filter of the present invention has a role of about 1 which is present in a large amount of blood cell-derived cell membrane fragments, plasma proteins, lipids and the like present in a hemoglobin measurement sample prepared by hemolyzing and diluting whole blood with a hemolysis reagent. There is a role to prevent the column portion from deteriorating by removing foreign matter of about ˜5 μm, entering these column portions and repeating the sample measurement.
The filter of the present invention is capable of removing foreign substances of about 1 to 5 μm that are present in large amounts such as cell membrane fragments derived from blood cells, plasma proteins, lipids, etc. in the above-described measurement specimen, and has little adsorption of hemoglobins, A known filter can be used as long as the filter pressure does not increase easily even when blood samples are continuously measured.
Materials that can be used as the filter of the present invention are (1) metal filters such as titanium and stainless steel, (2) polyethylene, PEEK, polypropylene, polyethylene terephthalate, nylon, rayon, acrylic, vinylidene chloride, Teflon (registered trademark), etc. (3) Other materials: coconut fiber, cotton, wool, hemp, glass fiber and the like.
If it is necessary to further suppress the adsorption of hemoglobin with the above material, it is preferable to perform surface treatment such as silicone treatment, blocking treatment, chemical treatment (hydrophilization treatment), plasma treatment or the like.

  In the filter of the present invention, a filter in which the filter pressure does not easily increase even when blood samples are continuously measured is more preferable. As the filter structure, it is necessary to increase the filter porosity. The method includes (1) a filter with a high porosity (stainless fiber filter, non-woven fabric), (2) a filter with a large pore size (non-woven fabric), and (3) a filter with a high porosity and a large pore size. There is a method of using a (nonwoven fabric) and providing a filter (stainless fiber filter, filter paper) capable of removing 1 to 5 μm of foreign matters in blood in the subsequent stage.

  As the filter of the present invention, (1) silicone-treated stainless fiber filter, (2) non-woven fabric + filter paper, (3) non-woven fabric + membrane filter, and (4) filter paper + membrane filter are preferable. Furthermore, (2) non-woven fabric + filter paper is inexpensive, can remove foreign matters of about 1 to 5 μm in blood, and has a very small increase in filter pressure even when continuously measured.

Hereinafter, “nonwoven fabric + filter paper” which is one of the filters of the present invention will be described in detail.
Further, if necessary, in order to reduce the adsorption of biological sample components, the surface of the non-woven fabric is at least a cellulose resin, a fluorine resin, a sulfone resin, polyethylene, a polymer of an ethylene alkyl derivative, an acrylic resin. , Nylon, carbide ceramic, nitride ceramic, silicide ceramic, boride ceramic, at least one selected from the group consisting of silylated silicon dioxide, glass and titanium on the surface, or a combination of these May be processed.
The nonwoven fabric has a thickness of 0.05 mm or more in order to suppress an increase in pressure due to clogging of the biological sample.
The thickness of the nonwoven fabric is preferably 10 mm or less in order to suppress the diffusion of the measurement sample.
The thickness of the nonwoven fabric used in the present invention is more preferably 0.1 mm to 5 mm in order to suppress the increase in pressure due to clogging of the biological sample and the diffusion of the measurement sample.

  The porosity of the nonwoven fabric used in the present invention is 40% to 95%. If the porosity of the nonwoven fabric is less than 40%, there is a drawback that the pressure increase of the filter becomes very high due to clogging by foreign substances derived from biological components. Further, when the porosity of the nonwoven fabric is 95% or more, the filter strength is weakened, and accordingly, the filter may be broken by an increase in pressure.

The filter paper used in the present invention is a known filter paper, and the material of the filter paper is not particularly limited.
Specific examples of the filter paper material include cellulose, glass fiber, fluororesin, and silica fiber.

Also, a filter paper that is a known filter paper and that has been subjected to special processing for realizing the strength of the filter paper, suppression of adsorption of a biological sample, and the like can be used. For example, filter paper (wet strength filter paper manufactured by Advantech Toyo Co., Ltd.) with increased wet strength can be used.
The retained particle diameter of the filter paper used in the present invention is 5 μm or less in order to remove foreign substances in the biological sample. In order to more reliably remove foreign matter in the biological sample, the retained particle diameter of the filter paper of the present invention is preferably 3 μm or less, and in order to more reliably remove foreign matter derived from the biological sample, The retained particle diameter is preferably 1.5 μm or less.
When the retained particle diameter of the filter paper is larger than 5 μm, foreign substances in the biological sample cannot be removed, and these foreign substances migrate to the column, and there is a problem of deteriorating column performance (decrease in resolution and increase in column pressure).

In order to increase the effective filtration area of the nonwoven fabric and the filter paper, the filter of the present invention is configured as “support + nonwoven fabric + filter paper + support” or “nonwoven fabric + filter paper + support” from the side away from the column. Can do.
The shape of the support is not particularly limited as long as the effective filtration area of the nonwoven fabric and the filter paper can be increased, but a mesh shape is preferable.

Examples of the support material used in the present invention include polypropylene, polyethylene, nylon, polyethylene terephthalate, and metal mesh (stainless steel, titanium).
The filter of the present invention may be silicone-coated if necessary.
In the filter of the present invention, it is preferable that the liquid chromatography filter is further subjected to a blocking treatment with a blocking reagent. Examples of the blocking reagent include proteins such as bovine serum albumin, casein, gelatin, hemoglobin, and myoglobin; polar lipids such as phospholipids; and SDS, polyethylene glycol mono-4-octylphenyl ether (Triton X-100). ) And the like.

  The shape of the filter of the present invention is not particularly limited as long as it does not disturb the liquid flow. For example, as shown in FIGS. 10 to 11, (a) a columnar shape, (b) a conical shape, (c) a truncated cone shape, and (d) a shape where two conical surfaces are in contact with each other. 10 to 11, either the liquid inflow side or the liquid outflow side may be used, or the both sides described above may be used.

  The size of the filter of the present invention is preferably from 0.1 to 10 mm, and more preferably from 0.5 to 8 mm. The thickness is preferably 0.1 to 5 mm, and more preferably 0.2 to 3 mm. The filtration pore diameter may be a known pore diameter, preferably 0.1 to 5 μm, and more preferably 0.2 to 3 μm.

"Method for measuring hemoglobin"
The eluent used in the present invention contains an inorganic acid, an organic acid and / or a salt thereof containing chaotropic ions and having a buffering capacity at pH 4.0 to 6.8.

  The chaotropic ion is an ion generated by dissociation when a compound is dissolved in an aqueous solution, which destroys the structure of water and suppresses the decrease in entropy of water that occurs when the hydrophobic substance and water come into contact with each other. .

  Examples of the anionic chaotropic ion include tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, chloride ion, acetate ion and the like. Examples of the cation chaotropic ion include barium ion, calcium ion, lithium ion, cesium ion, potassium ion, magnesium ion, and guanidine ion.

  Among the chaotropic ions, tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, etc. as cation, barium ion, It is preferable to use calcium ion, magnesium ion, lithium ion, cesium ion, guanidine ion or the like. More preferably, thiocyanate ion, perchlorate ion, nitrate ion, guanidine ion or the like is used.

  When the concentration of chaotropic ions in the eluent is lower than 0.1 mM, the separation effect may be reduced in the measurement of hemoglobins. When the concentration is higher than 3000 mM, the separation effect of hemoglobins is not further improved. Therefore, 0.1 mM to 3000 mM is preferable, 1 mM to 1000 mM is more preferable, and 10 mM to 500 mM is more preferable.

A plurality of chaotropic ions may be used as a mixture.
The chaotropic ion may be added to a liquid that comes into contact with the measurement sample, for example, a hemolytic reagent, a sample diluent, or the like.

  In the present invention, an inorganic acid, an organic acid, or a salt thereof is included as a substance having a buffer capacity used in the eluent. Examples of the inorganic acid include carbonic acid and phosphoric acid. Examples of the organic acid include carboxylic acid, dicarboxylic acid, carboxylic acid derivative, hydroxycarboxylic acid, amino acid, cacodylic acid, pyrophosphoric acid and the like.

  Examples of the carboxylic acid include acetic acid and propionic acid. Examples of the dicarboxylic acid include malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, and phthalic acid. Examples of the carboxylic acid derivative include β, β-dimethylglutaric acid, barbituric acid, aminobutyric acid, and the like. Examples of the hydroxycarboxylic acid include citric acid, tartaric acid, and lactic acid. Examples of the amino acid include aspartic acid and asparagine.

  The inorganic acid or organic acid salt may be a known salt, and examples thereof include a sodium salt and a potassium salt.

  The inorganic acid, organic acid or salt thereof may be used as a mixture of two or more kinds, or a mixture of inorganic acid and organic acid may be used.

  The concentration of the above-mentioned inorganic acid, organic acid and / or salt thereof in the eluent, and when a plurality of types are used, the total concentration of the plurality of types has a buffering effect to make the pH of the eluent 4.0-6.8. It may be sufficient, 1-1000 mM is preferable and 10-500 mM is especially preferable.

  In the present invention, the pH of the eluent is limited to 4.0 to 6.8, preferably 4.5 to 5.8. If the pH of the eluent is less than 4, hemoglobins may be denatured. If the pH exceeds 6.8, the positive charge of hemoglobins decreases and it becomes difficult to be retained by the cation exchange group. The separation of becomes worse.

The following substances may be added to the eluent.
(1) Inorganic salts (such as sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, and sodium phosphate) may be added. The concentration of these salts is not particularly limited, but is preferably 1 to 1500 mM.

  (2) A known acid or base may be added as a pH regulator. Examples of the acid include hydrochloric acid, phosphoric acid, nitric acid, and sulfuric acid, and examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, and calcium hydroxide. The concentration of these acids and bases is not particularly limited, but is preferably 0.001 to 500 mM.

  (3) A water-soluble organic solvent such as methanol, ethanol, acetonitrile, or acetone may be mixed. The concentration of these organic solvents is not particularly limited, but is preferably 0 to 80% (v / v), and is preferably used to such an extent that chaotropic ions, inorganic acids, organic acids, salts thereof, and the like do not precipitate.

  (4) Preservatives such as sodium azide, thymol, ethylenediaminetetraacetic acid (EDTA), 2-phenoxyethanol, sodium propionate and sodium salicylate may be added.

  (5) As a stabilizer for hemoglobin, a known stabilizer, for example, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), a reducing agent such as glutathione or sodium azide, an antioxidant, or the like may be added.

The eluent of the present invention contains the above-mentioned chaotropic ion and contains a buffer having an acid dissociation constant (pKa) in the range of 2.15 to 6.39 and in the range of 6.40 to 10.50. It is preferable to use an eluent that
As the buffer, those having an acid dissociation constant (pKa) in the range of 2.15 to 6.39 and 6.40 to 10.50 are used. That is, a single substance having at least one pKa in the range of 2.15 to 6.39 and 6.40 to 10.50 may be used as the buffer, or 2.15 to 6.5. A substance having at least one pKa in the range of 39 and a substance having at least one pKa in the range of 6.40 to 10.50 may be used in combination as a buffering agent. Moreover, you may use combining the said buffering agent in multiple numbers.

  The pKa range of the above-mentioned buffering agent is 2.61 to 6.39 and 6.40 to be able to exhibit a better buffering capacity in the vicinity of the pH of the eluent appropriate for separating the peak for measurement. The range of 10.50 is preferable, and the range of 2.80 to 6.35 and 6.80 to 10.00 is more preferable. More preferably, it is the range of 3.50-6.25 and 7.00-9.50.

  Examples of the buffer include inorganic substances such as phosphoric acid, boric acid, carbonic acid, carboxylic acid, dicarboxylic acid, carboxylic acid derivatives, hydroxycarboxylic acid, aniline or aniline derivatives, amino acids, amines, imidazoles, and alcohols. Organic substances such as In addition, ethylenediaminetetraacetic acid, pyrophosphoric acid, pyridine, cacodylic acid, glycerol phosphoric acid, 2,4,6-collidine, N-ethylmorpholine, morpholine, 4-aminopyridine, ammonia, ephedrine, hydroxyproline, peridine, tris (hydroxy Organic substances such as methyl) aminomethane and glycylglycine may also be used.

Examples of the carboxylic acid include acetic acid, propionic acid, benzoic acid, and the like.
Examples of the dicarboxylic acid include malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, phthalic acid, and fumaric acid.

Examples of the carboxylic acid derivative include β, β′-dimethylglutaric acid, barbituric acid, 5,5-diethylbarbituric acid, γ-aminobutyric acid, pyruvic acid, furancarboxylic acid, and ε-aminocaproic acid. It is done.
Examples of the hydroxycarboxylic acid include tartaric acid, citric acid, lactic acid, malic acid and the like.

Examples of the aniline or aniline derivative include aniline and dimethylaniline.
Examples of the amino acid include aspartic acid, asparagine, glycine, α-alanine, β-alanine, histidine, serine, leucine and the like.

  Examples of the amines include ethylenediamine, ethanolamine, trimethylamine, and diethanolamine. Examples of the imidazoles include imidazole, 5 (4) -hydroxyimidazole, 5 (4) -methylimidazole, and 2,5 (4) -dimethylimidazole.

  Examples of the alcohols include 2-amino-2-methyl-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, and 2-amino-2-methyl-1-propanol. Is mentioned.

  Examples of the buffer include 2- (N-morpholino) ethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris), N- (2-acetamido) imidodiacetic acid ( ADA), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 1,3-bis (tris (hydroxymethyl) -methylamino) propane (Bistrispropane), N- (acetamido) -2- Aminoethanesulfonic acid (ACES), 3- (N-morpholine) propanesulfonic acid (MOPS), N, N′-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N-tris ( Hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxy Tilpiperazine-N′-ethanesulfonic acid (HEPES), N-2-hydroxyethylpiperazine-N′-propanesulfonic acid (HEPPS), N-tris (hydroxymethyl) methylglycine (Tricine), tris (hydroxymethyl) aminoethane (Tris), N, N′-bis (2-hydroxyethyl) glycine (Bicine), glycylglycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), glycine, cyclohexylaminopropanesulfone Substances that make up what is commonly referred to as Good's buffer, such as acids (CAPS), can also be used. The pKa values of these substances are shown in Tables 1 and 2 (cited literature: Takeichi Horio and Nihei Yamashita Protein and enzyme basic experiment method, Nanedo 1985).

  The buffer concentration in the eluent may be in a range having a buffering effect, and is preferably 1 to 1000 mM, more preferably 10 to 500 mM. Moreover, the said buffer may be used individually or in mixture of multiple, for example, you may mix and use an organic substance and an inorganic substance.

Further, in order to further reduce the baseline fluctuation, it is more preferable to use an eluent having the same buffer concentration as the eluent used for separating the peak for measurement.
A plurality of eluents used in the present invention may be sent by a gradient elution method or a step elution method.

  Examples of the hemoglobin in the present invention include HbA1a, HbA1b, HbF, unstable HbA1c, stable HbA1c, AHb, CHb, HbA0, HbA2, HbS, HbC and the like.

  If the pH of the eluent for separating hemoglobins eluted before HbA0 is less than 4.0, hemoglobins may be denatured, and if it exceeds 6.8, the positive charge of hemoglobin decreases. In addition, since it becomes difficult to be retained by the cation exchange group and the resolution is lowered, 4.0 to 6.8 is preferable, and 4.5 to 5.8 is more preferable.

In the present invention, there is the following method for eluting HbA0.
(1) As an eluent for eluting HbA0, an eluent whose pH at the time of flowing into the column is equal to the isoelectric point of hemoglobin or is on the alkali side from the isoelectric point is used. More preferably, the eluent contains chaotropic ions. The chaotropic ions and their concentrations are as described above.
(2) As an eluent for eluting HbA0, the salt concentration is preferably as high as 200 mM to 3000 mM, and the pH of the eluent is preferably 5.0 to 9.0.
(3) Eluent obtained by combining the above (1) and (2): salt concentration as high as 200 mM to 3000 mM. In addition, as the pH of the eluent, an eluent is used in which the pH when flowing into the column is equal to the isoelectric point of hemoglobin, or becomes more alkaline than the isoelectric point.

  When the pH of the hemoglobin changes from the acidic side to the alkaline side from the isoelectric point, the total charge changes from positive to negative, so that the HbA0 component can be eluted with the cation exchange group of the filler. .

  In addition, as described in Rikagaku Dictionary (4th edition, September 1987, edited by Iwanami Shoten, Ryogo Kubo et al.), Page 1178, the isoelectric point of hemoglobin is pH 6.8 to 7.0. Therefore, in order to elute the HbA0 component, the pH of the eluent when flowing into the column is more preferably 6.8 or more.

  In order to satisfy this condition, it is necessary that at least one of the eluents used for the measurement has a pH of 6.8 or higher. The pH of the eluent is desirably 7.0 to 12.0, more preferably 7.5 to 11.0, and even more preferably 8.0 to 9.5. When the pH of the eluent becomes less than 6.8, the elution of the HbA0 component becomes insufficient, so that the elution of the HbA0 component can be sufficiently performed by increasing the salt concentration. Further, the pH of the eluent may be set in a range in which the used filler does not decompose.

  Examples of an eluent suitably used for elution of the HbA0 component and having a buffer capacity at a pH of 6.8 or more include inorganic acids such as phosphoric acid, boric acid and carbonic acid, or salts thereof; hydroxycarboxylic acids such as citric acid; Examples include acids, carboxylic acid derivatives such as β, β-dimethylglutaric acid, dicarboxylic acids such as maleic acid, organic acids such as cacodylic acid, and buffer solutions thereof. In addition, 2- (N-morpholino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris) ), Tris, ADA, PIPES, Bistrispropane, ACES, MOPS, BES, TES, HEPES, HEPPS, Tricine, Bicine, glycylglycine, TAPS, CAPS, and the like, which are generally referred to as good buffer solutions, can also be used. Britton and Robinson buffer; GTA buffer can also be used. Organic substances such as imidazoles such as imidazole; amines such as ethylenediamine, methylamine, ethylamine, triethylamine, diethanolamine, and triethanolamine; amino acids such as glycine, β-alanine, aspartic acid, and asparagine;

  In addition, an inorganic acid; an organic acid; a salt of an inorganic acid or an organic acid; and a plurality of organic substances may be used as a mixture, or an organic acid, an inorganic acid, and an organic substance may be mixed.

In order to elute the HbA0 component more effectively, it is preferable to add chaotropic ions to the eluent. The chaotropic ions to be added are as described above.
The concentration of chaotropic ions to be added is 1 to 3000 mM, preferably 10 to 1000 mM, and more preferably 50 to 500 mM.

  For elution of hemoglobins eluted before HbA0, one type or two or more types of eluents having a pH of 4.0 to 6.8 can be used. In order to elute the measurement target peak such as stable HbA1c sharply, two or more kinds of eluents can be used and the target peak can be sharply eluted by the salt concentration gradient method, the pH gradient method, or a combination of the two.

  In the present invention, at least two or more kinds of eluents are used for elution of hemoglobins (HbA1a and b, HbF, unstable HbA1c, stable HbA1c) eluted before HbA0, and have the highest elution power. A weak eluent can be run first.

  In the hemoglobin measurement method of the present invention, preferably, when the eluent is sent by gradient elution or step elution, and the target hemoglobin peak is separated and measured, the elution power of the eluate is reduced during the measurement. Sometimes it is desirable.

  The gradient elution method is called “gradient elution”. That is, using a plurality of liquid feed pumps, the liquid feed ratios of a plurality of eluents having different dissolution powers are continuously changed and fed.

  The step elution method is called “stepwise elution”. In this method, one liquid feed pump is connected to a plurality of eluents via a solenoid valve or the like. Then, by switching the electromagnetic valve, the eluent having a low melt output is switched to the eluent having a high melt output, and the liquid is sent.

  However, if the properties of each component to be eluted are similar or if it is required to elute in a short time, the conventional gradient elution method or step elution method causes peaks between components having similar properties, There was a risk that the degree of separation would decrease.

  In contrast, in the present invention, when the eluent is sent by the gradient elution method or the step elution method, a plurality of eluents are sequentially switched and sent in the middle, that is, the gradient elution method or the step elution method. In the middle, the elution power of the eluent can be temporarily reduced so that the separation target peak or peak-to-peak elution timing is taken into consideration so that the separation target peak or peak separation state is improved. Specifically, in the case of the step elution method, after switching from an eluent having a weak dissolution power to an eluent having a high dissolution power, switching to an eluent having a weak dissolution power, and after a while, eluting with a strong dissolution power. Switch to liquid and feed.

  In the cation exchange LC, in order to decrease the elution power of the eluent, there are a method of lowering the salt concentration of the eluent, a method of lowering the pH, or a method of combining the two.

  The case where hemoglobins are separated by a method in which the solution is fed by the gradient elution method or the step elution method as described above and the elution power of the eluate is reduced in the middle thereof will be described more specifically.

  For the separation of hemoglobins, a column packed with a cation exchange packing is used, and the eluent is sent by a gradient elution method or a step elution method in a salt concentration range of 20 to 1000 mM and pH 4 to 9, and elution is performed in the middle. Separation is carried out by lowering the elution capacity of the eluent by lowering the salt concentration of the liquid in the range of 5 to 500 mM and pH in the range of 0.1 to 3.

  A configuration example of the apparatus when the method of the present invention is performed by the step elution method is shown in FIG. The eluents A, B, C, and D have different dissolution outputs (for example, different in salt concentration, pH, polarity, etc.) and are configured to be switched to each eluent at a set time by the solenoid valve 1. ing. The eluent is introduced to the column 4 by the liquid feed pump 2 together with the sample introduced from the sample injection unit 3, and each component is detected by the detector 5. The area, height, etc. of each peak are calculated by the integrator 6.

  When measuring a blood sample containing hemoglobin components such as HbA2, HbS, and HbC that may adversely affect the measurement of stable HbA1c, HbA is mainly eluted as an HbA0 component (peak), and thereafter HbA2, HbS In some cases, a measurement method for eluting HbC or the like is set. Thereby, since hemoglobin components other than HbA can be removed from the HbA0 peak, a more accurate stable HbA1c (%) can be calculated.

  When setting a measurement method for eluting HbA2, HbS, HbC, etc., the “eluent for eluting HbA0” in the above-described method using three types of eluents having at least different elution powers is mainly composed of HbA. It means the eluent that elutes the HbA0 peak. At this time, in order to elute HbA2, HbS, HbC, etc. after the “eluent for eluting HbA0”, it is necessary to send an eluent with stronger dissolution power.

  Since the measuring device of the present invention is smaller in size than other experimental devices, the internal environment can be easily controlled and the experimental accuracy can be improved. In addition, since various operations are performed in the measuring device of the present invention, sample loss is small. As a result, it is possible to clear the problem of minute samples that often become a problem in biochemical experiments, and it can be suitably used particularly for the separation measurement of hemoglobins.

  Examples of the specimen used for the measurement device of the present invention include blood containing hemoglobins, but are not particularly limited. The hemoglobins include hemoglobin A1a + b, hemoglobin F, hemoglobin A1c (stable A1c), hemoglobin A, hemoglobin A2, abnormal hemoglobin (hemoglobin S, hemoglobin C), modified hemoglobin (acetylated hemoglobin, carbamylated hemoglobin, anxiety Regular hemoglobin).

"Peripheral equipment of the measurement device of the present invention: HPLC measurement system"
The LC device used in the present invention may be a well-known device and includes, for example, a liquid feed pump, a sample injection device (sampler), a column, a detector, and the like. Further, other attached devices (such as a column thermostat or an eluent degassing device) may be added as appropriate.
In the measurement device of the present invention, in order to achieve high separation of the measurement components, semi-micro high performance liquid chromatography (for example, manufactured by Shiseido Co., Ltd.) that realizes reduction of the diffusion of the measurement components with the following extremely low dead volume structure. NANOSPACE SI-2) It is preferable to use each accessory as appropriate.
(1) Autosampler that reduces the diffusion of a small amount of sample to the utmost, has excellent injection accuracy, and is free from contamination (for example, Autosampler 3023 manufactured by Shiseido Co., Ltd.)
(2) Pump with reduced pulsation / pulsation (double plunger pump is more preferred)
(For example, inert pump 3001 manufactured by Shiseido)
(3) Rotary valve that can reduce contamination of multiple eluents used for measurement to a minimum (4) UV-VIS detector (for example, Shiseido) that is compatible with semi-micro columns, has a long optical path length, and is equipped with a low diffusion flow cell UV-VIS detector 3002)
(5) Eluent degassing device (eg, Shiseido degassing device 3009/3010)

  Other measurement conditions in the above measurement method may be known conditions, and the flow rate of the eluent is preferably 0.001 to 5 mL / min, more preferably 0.005 to 3 mL / min. The detection of hemoglobin is preferably visible light of 415 nm, but is not limited to this. As the measurement sample, a solution obtained by diluting hemolyzed blood with a solution containing a substance having hemolytic activity such as a surfactant is usually used. The sample injection amount varies depending on the dilution rate of the blood sample, but is preferably about 0.01 to 100 μL.

In the method for measuring hemoglobin using the measuring device of the present invention, it is preferable that at least a part of each step is automated. Automatable processes include, for example, a process in which a blood sample and a hemolyzed diluted sample are mixed and hemolyzed, a step in which a fixed amount of hemolyzed sample is injected into the column, and each eluent used for hemoglobin separation measurement The process of switching and feeding according to the measurement sequence, the process of feeding each eluent at the liquid feed speed set by the liquid feed pump, the process of replacing the prefilter, the process of replacing the column section, and the separation measurement result of hemoglobins More specifically, there are a step of calculating the measured value of each hemoglobin by data processing, a step of printing out the measurement result in the form of a chromatogram, and the like.
The automation can perform each process without human work if the liquid feeding speed and capacity, time, etc. of the liquid feeding pump and suction pump used in each process are programmed in advance. Moreover, you may use a valve | bulb as needed and may control the operation | movement by a program.

  According to the device for measuring hemoglobins of the present invention, a prefilter part capable of removing foreign substances in a measurement sample, a column part having a cation exchange group for separating the components in the measurement sample, and the absorbance of the components in the measurement sample Use of the prefilter unit, column unit, and detection unit because at least one or a plurality of portions of the detection unit group for detection are configured in a unit form so that they can be replaced as appropriate. Each unit part of the part where the number of specimens is different or where a failure has occurred can be easily replaced. In addition, it is possible to provide a hemoglobin measuring device and a hemoglobin measuring method capable of measuring hemoglobin more efficiently than a conventional substrate in which a prefilter unit, a column unit, and a detection unit are mounted.

  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 measurement specimens The following samples were prepared from whole blood specimens obtained by collecting sodium fluoride from healthy human blood. As a hemolysis reagent, a phosphate buffer solution (pH 7.0) containing 0.1 wt% polyethylene glycol mono-4-octylphenyl ether (Triton X-100) (manufactured by Tokyo Chemical Industry Co., Ltd.) was used.
a) Glucose-loaded blood: A 500 mg / dL aqueous glucose solution was added to the whole blood sample, reacted at 37 ° C. for 2 hours, then hemolyzed with the above hemolytic reagent, and diluted 50 times to give sample a.
b) AHb-containing sample: To 10 mL of whole blood sample, add 1 mL of 0.3 wt% acetaldehyde physiological saline solution, react at 37 ° C. for 2 hours, and then hemolyze with the above hemolytic reagent and dilute 50 times. Sample c was designated.
c) CHb-containing sample: Add 10 mL of 0.3% by weight sodium cyanate physiological saline solution to 10 mL of whole blood sample, react at 37 ° C. for 2 hours, and then hemolyze with the above hemolytic reagent and dilute 50 times Sample b was obtained.

(2) Construction of Measurement Device and Analysis System A measurement device having the structure shown in FIG. 2 was made of acrylic resin, and this cell portion was connected to a spectrophotometer to constitute an analysis system for hemoglobins. Further, all the channels in the measurement device shown in FIG. 2 have a width of 200 μm and a depth of 200 μm.
The pre-filter part is composed of "nonwoven fabric + filter paper + support" from the specimen-in side. Body (Advantech Toyo Co., Ltd., mesh sheet, polyethylene material), the size was 3mm width x 1mm depth x 1mm thickness.
The cell part has a width of 200 μm, a depth of 200 μm, and a length of 7 mm.
The column part has a width of 1000 μm, a depth of 330 μm, and a length of 10 mm. A filler prepared by the method described below was press-fitted and filled. Further, a spectrophotometer (Ocean Optics, USB2000) and a light source (Ocean Optics, LS-1) were used as detection devices.

(Preparation of filler)
1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a mixture of 400 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 150 g of 2-acrylamido-2-methylpropanesulfonic acid. This was dispersed in 2500 mL of a 4% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical), heated to 75 ° C. in a nitrogen atmosphere with stirring, and polymerized for 8 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle diameter of 6 μm.

(3) (Measurement conditions)
System: Liquid pump: Inert pump 3001 (manufactured by Shiseido)
Autosampler: Autosampler 3023 (manufactured by Shiseido)
Detector: Spectrophotometer (Ocean Optics, USB2000 type)
Light source (manufactured by Ocean Optics, LS-1)
Eluent: Eluent A: Contains 40 mM perchloric acid
50 mM phosphate buffer (pH 5.3)
Eluent C: Contains 50 mM perchloric acid
50 mM phosphate buffer (pH 5.3)
Eluent B: Contains 200 mM perchloric acid
50 mM phosphate buffer (pH 8.0)
The pKa of phosphoric acid is as shown in Table 1. The eluent C was sent for 0 to 30 seconds from the start of measurement, the eluent B was sent for 30 to 40 seconds, and the eluent A was sent for 40 to 60 seconds.
Flow rate: 0.035 mL / min Detection wavelength: 415 nm
Sample injection volume: 2 μL

(Measurement result)
Chromatograms obtained by measuring samples under the above measurement conditions are shown in FIGS. FIG. 12 shows the result of measuring the sample a, FIG. 13 shows the result of measuring the sample b, and FIG. 14 shows the result of measuring the sample c. Peak 1 is HbA1a and HbA1b, Peak 2 is HbF, Peak 3 is unstable HbA1c, Peak 4 is stable HbA1c, Peak 5 is HbA0, Peak 6 is AHb, and Peak 7 is CHb.
In FIG. 12, peaks 3 and 4 are well separated. Further, peak 6 (AHb) in FIG. 13 and peak 7 (CHb) in FIG. 14 are well separated from peak 4 (stable HbA1c).

(Example 2)
Except for the creation of the column in the unit format shown below, the same as in Example 1.
The PEEK resin was processed to prepare a column having an inner diameter of 0.65 mm and a length of 10 mm, and the packing material of Example 1 was slurry-filled at 30 MPa using an HPLC phamp. The packed column is assembled with a screw type (male, female) to a measuring device having a pre-filter (same as in Example 1) and a detection cell (cell width 200 μm, depth 200 μm, length 7 mm), and eluent The unit-type measuring device of Example 2 of the present invention shown in FIG. 3 was prepared.

(Example 3)
Example 2 is the same as Example 2 except for the pre-filter creation in the unit format shown below.
As shown in FIG. 9, a pre-filter built-in screw was made using PEEK resin. The pre-filter built-in screw (male / female: each with a taper of 150 degrees) has an inner diameter of 200 μm, and the filter material used was a stainless fiber filter (silicone treatment) inner diameter of 2 mm shown in FIG. The measuring device of Example 3 is shown in FIG.

Example 4
The same as Example 3 except the creation of the unit format column shown below.
As shown in FIG. 5, a PEEK resin was processed to prepare a column having an inner diameter of 0.65 mm × length of 10 mm. The column was sandwiched between the upper and lower lids made of acrylic resin shown in FIG. In such a manner, a Teflon (registered trademark) resin was sandwiched between the column, the upper lid, and the lower lid, and adhered with a UV curable adhesive to produce the measurement device of Example 4.
The measurement device of Example 4 is shown in FIG.

(Example 5)
Except for the liquid feeding system using the syringe type pump shown below, the same as Example 3.
As a syringe pump, Muromachi Kikai Co., Ltd. KDS250 was used, and the syringe was the same as Examples 1-4 except that a Terumo 10 mL syringe was used in the eluent ABC.
Using each measurement device created in Examples 1 to 5 above, each hemoglobin was measured and evaluated by the same method as Example 1, and the results are shown in FIGS.

(Comparative Example 1)
Preparation of filler 1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a mixture of 400 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 150 g of 2-acrylamido-2-methylpropanesulfonic acid. This was dispersed in 2500 mL of a 4% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical), heated to 75 ° C. in a nitrogen atmosphere with stirring, and polymerized for 8 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle diameter of 6 μm.

Packing of the packing material into the column The obtained particles were packed into the column as follows. 0.7 g of the particles were dispersed in 30 mL of 50 mM phosphate buffer (pH 5.8), sonicated for 5 minutes, and then stirred well. The whole amount was injected into a packer (Umeya Seiki Co., Ltd.) connected with a stainless steel empty column (inner diameter 4.6 × 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 30 MPa.

Measurement of hemoglobins Using the obtained column, hemoglobins were measured under the following measurement conditions.
(Measurement condition)
System: Liquid pump: Inert pump 3001 (manufactured by Shiseido)
Autosampler: Autosampler 3023 (manufactured by Shiseido)
Detector: UV-VIS detector (manufactured by Shiseido)
Eluent: Eluent A: Contains 40 mM perchloric acid
50 mM phosphate buffer (pH 5.3)
Eluent C: Contains 50 mM perchloric acid
50 mM phosphate buffer (pH 5.3)
Eluent B: Contains 200 mM perchloric acid
50 mM phosphate buffer (pH 8.0)
The pKa of phosphoric acid is as shown in Table 1. The eluent C was sent for 0 to 30 seconds from the start of measurement, the eluent B was sent for 30 to 40 seconds, and the eluent A was sent for 40 to 60 seconds.
Flow rate: 2.0 mL / min Detection wavelength: 415 nm
Sample injection volume: 2 μL

(Measurement sample)
As the measurement sample, one prepared in the same manner as in Example 1 was used.

(Measurement result)
Chromatograms obtained by measuring samples under the above measurement conditions are shown in FIGS. 15 shows the result of measuring the sample a, FIG. 16 shows the result of measuring the sample b, and FIG. 17 shows the result of measuring the sample c. Peak 1 is HbA1a and HbA1b, Peak 2 is HbF, Peak 3 is unstable HbA1c, Peak 4 is stable HbA1c, Peak 5 is HbA0, Peak 6 is AHb, and Peak 7 is CHb.
In FIG. 15, peaks 3 and 4 are well separated. In FIG. 17, peak 6 (AHb) is well separated from peak 4 in FIG.

  As described above, in Comparative Example 1, the result of performing stable A1c separation by attaching a conventional column (inner diameter 4.6 mm × length 35 mm) to a semi-micro compatible HPLC measurement system is shown in FIGS. As shown in FIG. 3, the peak of each hemoglobin was broader than that of Examples 1 to 5, and the separation performance of the modified hemoglobin was also worse than that of the example.

On the other hand, in Examples 1 to 5, as a result of performing stable A1c separation by a minute column portion provided in the measurement device of the present invention, as shown in FIGS. The peak of each hemoglobin was much sharper than that of Comparative Example 1, and the separation performance of the modified hemoglobin was very good. Although the measurement time was 1 minute, it was revealed that the peak of each hemoglobin was very sharp, so that the measurement could be further performed in a short time.
The measuring device of the present invention used in Example 1 (size: width 10 cm × length 10 cm × width 3 cm) includes a column portion and a cell portion. Obviously, if other pumps, specimen injection devices, detectors, etc. are made smaller, they can be made smaller than the current HPLC measurement system, and the installation location is not too wide, and it is also portable. became.

  According to the present invention, a biological sample component measuring device, a hemoglobin measuring device, and hemoglobin capable of analyzing a photodetectable component, a biologically derived measuring component, and hemoglobin quickly, easily, and with high accuracy. Class of measurement methods can be provided.

Conventional HPLC: HPLC for measuring hemoglobins It is a schematic diagram which shows the measuring device produced in the Example. The unit format is shown in FIG. It is a schematic diagram which shows the measuring device produced in the Example. The unit format is shown in FIG. It is a schematic diagram which shows the measuring device produced in the Example. The unit format is shown in FIG. It is a schematic diagram which shows the measuring device produced in the Example. The unit format is shown in FIG. It is a schematic diagram which shows the measuring device produced in the Example. The unit format is shown in FIG. It is a schematic diagram which shows the air bleeding structure of this invention. It is a schematic diagram which shows the air bleeding structure of this invention. It is a schematic diagram which shows the filter shape of this invention, and the structure of a pre filter built-in screw. It is a schematic diagram which shows the filter shape of this invention. It is a schematic diagram which shows the filter shape of this invention. The chromatogram of the Example (normal human blood) of this invention is shown. The chromatogram of the Example (acetylated hemoglobin) of this invention is shown. The chromatogram of the Example (carbamylated hemoglobin) of this invention is shown. The chromatogram of the comparative example (normal human blood) of this invention is shown. The chromatogram of the comparative example (acetylated hemoglobin) of this invention is shown. The chromatogram of the comparative example (carbamylated hemoglobin) of this invention is shown. It is a schematic diagram which shows an example of the preferable structure of the cell part of the measuring device of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell part 2 Connection flow path 3 Connection part of a cell part and a connection flow path 4 Board | substrate 5 Waste liquid tank 6 Waste liquid port (connection with an external tank)
7 Jumping flow path

Claims (8)

    In a measurement device that measures the component to be measured in a sample including the component to be measured, a prefilter unit that can remove foreign matter in the sample sample, a separation unit for separating the component in the sample sample, and a component in the measurement sample A measurement device comprising a detection unit for performing light detection.   The measurement device according to claim 1, wherein a prefilter part capable of removing foreign substances in the measurement sample, a column part having a cation exchange group for separating the components in the measurement sample, and absorbance detection of the components in the measurement sample are performed. A device for measuring hemoglobin, characterized in that at least one or a plurality of parts in a group of detection units for measuring the structure are configured in a unit form.   The hemoglobin measuring device according to any one of claims 1 to 2, wherein an air vent structure is provided at least in a front channel of the column.   The device for measuring hemoglobin according to any one of claims 1 to 3, wherein the cell portion of the detection unit is formed of a linear channel, and the cell portion is a light beam for measuring absorbance. So that the optical axis is in the flow direction of the linearly formed flow path, and the incident angle and the outgoing angle of the light beam into the linearly formed flow path are 0 ° A device for measuring hemoglobin, which is connectable to an absorbance measuring apparatus.   The measurement device for measuring hemoglobin according to any one of claims 1 to 4, wherein the light transmittance at an absorbance measurement wavelength measured in a state where there is no specimen connected to an absorbance measurement device has the same optical path length and A device for measuring hemoglobin, which is 50% or more of light transmittance when air is measured at an absorbance measurement wavelength.   The measurement device for measuring hemoglobin according to any one of claims 1 to 5, wherein the cell portion is connected in a U-shape or L-shape with the connection channel at the inflow portion and / or the outflow portion. A device for measuring hemoglobin, characterized by comprising:   The measurement device for measuring hemoglobin according to any one of claims 1 to 6, wherein the surface of one or a plurality of parts in a group consisting of a prefilter part, a column part, a cell part, and a flow path part, A device for measuring hemoglobin, which is subjected to a surface treatment method for suppressing adsorption of biological components.   The method for measuring hemoglobin according to any one of claims 1 to 7, wherein tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion , An inorganic acid containing at least one chaotropic ion selected from the group consisting of barium ion, cesium ion and guanidine ion and 0.001% to 0.1% azide ion and having a buffer capacity at pH 4.0 to 6.8 A method for measuring hemoglobin, which comprises using a liquid that flows over a substrate containing an organic acid and / or a salt thereof and the above-described measurement device.

Images