JP6466799B2 - Analysis method and analysis system - Google Patents

Description

  The present invention relates to an analysis method and an analysis system.

  As an index indicating the state of a living body, glycation rates of various proteins are analyzed. Among them, the glycation rate of hemoglobin (Hb) in blood cells, particularly stable HbA1c (hereinafter sometimes abbreviated as “s-HbA1c”) reflects the past history of blood glucose levels in vivo, It is regarded as an important index in the diagnosis and treatment of diabetes. HbA1c is obtained by saccharification of the valine at the N-terminal of the β chain of HbA (α2β2).

  As one of Hb analysis techniques represented by s-HbA1c, electrophoresis is used. Patent Documents 1, 2, 3, 4, and 5 disclose that an additional component is added to the electrophoresis solution for the purpose of optimization of analysis and improvement of accuracy. In particular, Patent Documents 4 and 5 disclose chondroitin sulfate as an example of a component added to the electrophoresis solution. Further, Patent Document 6 describes an analysis method in which a sample is continuously supplied even during sample separation in electrophoresis in order to reduce the size of a chip used for analysis using electrophoresis.

  Blood, which is a representative example of the sample, is a sample derived from a living body. For this reason, for example, when blood collected from a patient is used as a sample, the collected blood can have various properties depending on the patient's medical condition and constitution. It is desirable that accurate analysis is performed using blood of any kind as a sample. However, at present, it has hardly been clarified what kind of analysis-inhibiting factors may be caused by the specific components of the additional components and the analysis methods described above. This is the same even when a sample other than blood is used.

JP 2006-145537 A Japanese National Patent Publication No. 9-510792 Table 2010/010859 JP 2009-109230 A Table 2008/136321 Table 2008/136465

  The present invention has been conceived under the circumstances described above, and an object thereof is to provide an analysis method and an analysis system capable of improving analysis accuracy.

  The analysis method provided by the first aspect of the present invention is a method for analyzing a sample by capillary electrophoresis, wherein a mixed sample is produced by mixing the sample and a cathodic group-containing compound, The mixed sample is continuously supplied in the capillary tube, the step of removing aggregates of the subcomponents other than the analytical component to be analyzed and the cathodic group-containing compound from the mixed sample. And subjecting the complex in which the analysis component and the cathodic group-containing compound are bound to each other in a state of being electrophoresed.

In a preferred embodiment of the present invention, in the removing step, the aggregate is removed by filtration.

  In a preferred embodiment of the present invention, in the removing step, the aggregate is removed by centrifugation.

  In a preferred embodiment of the present invention, the anionic group-containing compound is an anionic group-containing polysaccharide.

  In a preferred embodiment of the present invention, the anionic group-containing polysaccharide is at least one polysaccharide selected from the group consisting of sulfated polysaccharides, carboxylated polysaccharides, sulfonated polysaccharides and phosphorylated polysaccharides. It is.

  In a preferred embodiment of the present invention, the sulfated polysaccharide is chondroitin sulfate.

  In a preferred embodiment of the present invention, the analysis component is hemoglobin.

  In a preferred embodiment of the present invention, the subcomponent is a lipid.

  The analysis system provided by the second aspect of the present invention is a sample analysis system based on capillary electrophoresis, and a mixing unit that generates a mixed sample by mixing the sample and the cathodic group-containing compound; A removal unit that removes an aggregate of the anionic component-containing compound other than the analysis component to be analyzed among the components of the sample from the mixed sample, and a capillary tube, in the capillary tube, And an analysis unit that electrophoreses a complex in which the analysis component and the cathodic group-containing compound are bonded in a state where the mixed sample is continuously supplied.

  In preferable embodiment of this invention, the said removal part has a filtration means.

  In a preferred embodiment of the present invention, the apparatus comprises a disposable analysis chip having the capillary tube, an introduction tank connected to one end of the capillary tube, and a discharge tank connected to the other end of the capillary tube, and the filtering means , Provided in the introduction path to the introduction tank in the analysis chip.

  In a preferred embodiment of the present invention, the mixing unit is a mixing tank formed on the analysis chip.

  In preferable embodiment of this invention, the said removal part has a centrifuge.

  According to the present invention, when electrophoresis is performed in a state where the mixed sample is continuously supplied in a capillary tube by removing aggregates of the subcomponent and the cathodic group-containing compound. In addition, it is possible to suppress the appearance of noise in the measurement result, and the analysis accuracy can be improved.

  Other features and advantages of the present invention will become more apparent from the detailed description given below with reference to the accompanying drawings.

1 is a system schematic diagram showing an analysis system based on a first embodiment of the present invention. It is a top view which shows the analysis chip used for the analysis system of FIG. It is sectional drawing which follows the III-III line of FIG. It is a flowchart which shows the analysis method based on 1st Embodiment of this invention. It is sectional drawing which shows the analysis method of FIG. It is sectional drawing which shows the analysis method of FIG. It is sectional drawing which shows the analysis method of FIG. It is sectional drawing which shows the analysis method of FIG. It is an electropherogram which shows the result by an example of the analysis method of FIG. It is an electropherogram which shows the result by the reference example of an analysis method. It is a system schematic diagram showing an analysis system based on a second embodiment of the present invention. It is a flowchart which shows the analysis method based on 2nd Embodiment of invention. It is an electropherogram which shows the result by the analysis method of FIG.

  Hereinafter, preferred embodiments of the present invention will be specifically described with reference to the drawings.

  FIG. 1 shows an analysis system according to a first embodiment of the present invention. The analysis system A1 according to this embodiment includes an analysis device 1 and an analysis chip 2. The analysis system A1 is a system that executes an analysis method based on electrophoresis for the sample Sa. Although the sample Sa is not particularly limited, in the present embodiment, blood collected from a human body will be described as an example. Of the components contained in the sample Sa, a component to be analyzed is defined as an analysis component.

  Examples of the analysis component include hemoglobin (Hb), albumin (Alb), globulins (α1, α2, β, γ globulins), fibrinogen, and the like. Examples of the hemoglobin include normal hemoglobin (HbA0), glycated hemoglobin, modified hemoglobin, fetal hemoglobin (HbF), and the like. Examples of the glycated hemoglobin include hemoglobin A1a (HbA1a), hemoglobin A1b (HbA1b), hemoglobin A1c (HbA1c), and GHbLys. Examples of the hemoglobin A1c include stable HbA1c (s-HbA1c) and unstable HbA1c. Examples of the modified hemoglobin include carbamylated Hb and acetylated Hb. In the following description, the case where the analysis component is stable HbA1c (s-HbA1c) will be described as an example. s-HbA1c is generally used as an index in the diagnosis and treatment of diabetes.

  The analysis chip 2 holds the sample Sa and provides a place for analysis of the sample Sa in a state of being loaded in the analysis apparatus 1. In this embodiment, the analysis chip 2 is configured as a so-called disposable type analysis chip that is intended to be discarded after one or a specific number of analyses. As shown in FIGS. 2 and 3, the analysis chip 2 includes a main body 21, a mixing tank 22, an introduction tank 23, a filter 24, a discharge tank 25, an electrode tank 26, a capillary tube 27, and a communication channel 28. FIG. 2 is a plan view of the analysis chip 2, and FIG. 3 is a cross-sectional view taken along line III-III in FIG.

  The main body 21 is a base of the analysis chip 2 and the material thereof is not particularly limited, and examples thereof include glass, fused silica, and plastic. In the present embodiment, the main body 21 has a configuration in which the upper portion and the lower portion in FIG. 3 are coupled to each other, but the main body 21 may be integrally formed.

The mixing tank 22 is an example of a mixing unit in which a mixing process for mixing a sample Sa and a diluent Ld described later is performed. The mixing tank 22 is constituted by, for example, a through hole formed in the upper portion of the main body 21. The introduction tank 23 is a tank into which the mixed sample Sm obtained by the mixing process in the mixing tank 22 is introduced. The introduction tank 23 is constituted by, for example, a through hole formed in the upper portion of the main body 21.

  The filter 24 is provided at an opening of the introduction tank 23 which is an example of an introduction path to the introduction tank 23, and is an example of a removing unit (filtering means) referred to in the present invention. The specific configuration of the filter 24 is not limited as long as it can appropriately perform the removal step described later, and a suitable example is a cellulose acetate membrane filter (ADVANTEC, pore size 0.45 μm).

  The discharge tank 25 is a tank located on the downstream side of the electroosmotic flow in the electrophoresis method. The discharge tank 25 is constituted by, for example, a through hole formed in the upper portion of the main body 21. The electrode tank 26 is a tank into which the electrode 31 is inserted in an analysis process by electrophoresis. The electrode tank 26 is constituted by, for example, a through hole formed in the upper portion of the main body 21. The communication channel 28 connects the introduction tank 23 and the electrode tank 26, and constitutes a conduction path between the introduction tank 23 and the electrode tank 26.

  The capillary tube 27 connects the introduction tank 23 and the discharge tank 25 and is a place where an electroosmotic flow is generated in the electrophoresis method. The capillary tube 27 is constituted by, for example, a groove formed in the lower portion of the main body 21. The main body 21 may be formed with a recess or the like for promoting the irradiation of light to the capillary tube 27 and the emission of light transmitted through the capillary tube 27. The size of the capillary tube 27 is not particularly limited. For example, the capillary tube 27 has a width of 25 μm to 100 μm, a depth of 25 μm to 100 μm, and a length of 5 mm to 150 mm. The size of the entire analysis chip 2 is appropriately set according to the size of the capillary tube 27 and the sizes and arrangements of the mixing tank 22, the introduction tank 23, the discharge tank 25, and the electrode tank 26.

  The analysis apparatus 1 performs an analysis process on the sample Sa in a state where the analysis chip 2 on which the sample Sa is spotted is loaded. The analyzer 1 includes electrodes 31 and 32, a light source 41, an optical filter 42, a lens 43, a slit 44, a detector 5, a dispenser 6, a pump 61, a diluting liquid tank 71, an electrophoretic liquid tank 72, and a control unit 8. ing.

  The electrode 31 and the electrode 32 are for applying a predetermined voltage to the capillary tube 27 in the electrophoresis method. The electrode 31 is inserted into the electrode tank 26 of the analysis chip 2, and the electrode 32 is inserted into the discharge tank 25 of the analysis chip 2. Although the voltage applied to the electrode 31 and the electrode 32 is not specifically limited, For example, it is 0.5 kV-20 kV.

  The light source 41 is a part that emits light for measuring absorbance in electrophoresis. The light source 41 includes, for example, an LED chip that emits light in a predetermined wavelength range. The optical filter 42 transmits light of the remaining wavelengths while attenuating light of a predetermined wavelength among the light from the light source 41. The lens 43 is for condensing the light that has passed through the optical filter 42 onto the analysis portion of the capillary tube 27 of the analysis chip 2. The slit 44 is for removing excess light that may cause scattering or the like from the light collected by the optical filter 42.

  The detector 5 receives light that has passed through the capillary tube 27 of the analysis chip 2 and includes, for example, a photodiode or a photo IC.

The dispenser 6 dispenses a desired amount of the diluent Ld, the electrophoresis solution Lm, and the mixed sample Sm, and includes, for example, a nozzle. The dispenser 6 can freely move at a plurality of predetermined positions in the analyzer 1 by a drive mechanism (not shown). The pump 61 is a suction source and a discharge source for the dispenser 6. Further, the pump 61 may be used as a suction source and a discharge source of a port (not shown) provided in the analyzer 1. These ports are used for filling the electrophoresis solution Lm. Further, a dedicated pump different from the pump 61 may be provided.

  The diluent tank 71 is a tank for storing the diluent Ld. The diluent tank 71 may be a tank permanently installed in the analyzer 1 or may be a container in which a predetermined amount of the diluent Ld is filled. The electrophoretic liquid tank 72 is a tank for storing the electrophoretic liquid Lm. The electrophoretic liquid tank 72 may be a tank permanently installed in the analyzer 1 or a container in which a predetermined amount of the electrophoretic liquid Lm is sealed is loaded in the analyzer 1.

  The diluent Ld is for generating the mixed sample Sm by being mixed with the sample Sa. The main agent of the diluent Ld is not particularly limited, and examples thereof include water and physiological saline. Preferred examples include liquids having components similar to the electrophoresis solution Lm described later. Further, the diluent Ld is obtained by adding a cathodic group-containing compound to the main agent. The anionic group-containing compound is, for example, an anionic group-containing polysaccharide. The anionic group-containing polysaccharide is, for example, at least one polysaccharide selected from the group consisting of sulfated polysaccharides, carboxylated polysaccharides, sulfonated polysaccharides and phosphorylated polysaccharides. The carboxylated polysaccharide is preferably alginic acid or a salt thereof (for example, sodium alginate). The sulfated polysaccharide is, for example, chondroitin sulfate. There are seven types of chondroitin sulfate, A, B, C, D, E, H, and K, and any of them may be used. In the following description, the case where the diluted solution Ld is obtained by adding chondroitin sulfate to the main component, which is the same component as the electrophoresis solution Lm, will be described. The concentration of the anionic group-containing compound (chondroitin sulfate) is, for example, in the range of 0.01 to 5% by weight.

  The electrophoretic liquid Lm is a medium that fills the discharge tank 25 and the capillary tube 27 and generates an electroosmotic flow in the electrophoretic method in the analysis step by the electrophoretic method. The electrophoretic liquid Lm is not particularly limited, but is preferably one using an acid. Examples of the acid include citric acid, maleic acid, tartaric acid, succinic acid, fumaric acid, phthalic acid, malonic acid, and malic acid. Moreover, it is preferable that the electrophoresis solution Lm contains a weak base. Examples of the weak base include arginine, lysine, histidine, and tris. The pH of the electrophoresis solution Lm is, for example, in the range of pH 4.5-6. Examples of the buffer of the electrophoresis solution Lm include MES, ADA, ACES, BES, MOPS, TES, and HEPES. In addition, the cathodic group-containing compound described in the description of the diluent Ld is also added to the electrophoresis solution Lm. The concentration of the anionic group-containing compound (chondroitin sulfate) is, for example, in the range of 0.01 to 5% by weight.

  The control unit 8 controls each unit in the analyzer 1. The control unit 8 includes, for example, a CPU, a memory, an interface, and the like.

  Next, the analysis method based on 1st Embodiment of this invention using analysis system A1 is demonstrated below. FIG. 4 is a flowchart showing the analysis method of this embodiment. This analysis method includes a sample collection step S1, a mixing step S2, an electrophoresis liquid filling step S3, an introduction step S4, a removal step S11, and an electrophoresis step S5.

<Sample collection process S1>
First, a sample Sa is prepared. In the present embodiment, the sample Sa is blood collected from a human body. The blood may be whole blood or hemolyzed blood. Then, the analysis chip 2 into which the sample Sa has been dispensed is loaded into the analysis apparatus 1.

<Mixing step S2>
Next, the sample Sa and the diluent Ld are mixed. Specifically, as shown in FIG. 5, a predetermined amount of sample Sa is spotted on the mixing tank 22 of the analysis chip 2. Next, a predetermined amount of the diluent Ld in the diluent tank 71 is sucked by the dispenser 6, and a predetermined amount of the diluent Ld is dispensed into the mixing tank 22 of the analysis chip 2 as shown in FIG. Then, the suction and discharge of the diluent Ld from the dispenser 6 are repeated using the pump 61 as a suction source and a discharge source. Thereby, the sample Sa and the diluent Ld are mixed in the mixing tank 22, and the mixed sample Sm is obtained. Mixing of the sample Sa and the diluent Ld may be performed by a method other than the suction and discharge of the dispenser 6. By this mixing step S2, a complex in which s-HbA1c, which is the analysis component, and chondroitin sulfate are combined is generated.

  In addition, as a result of the inventors' research studies, in the mixing step S2, among the components contained in the blood as the sample Sa, the subcomponent other than the analysis component and the chondroitin sulfate which is an example of the cathodic group-containing compound are aggregated. It has been found that some products may be produced. Moreover, as a specific example of this subcomponent, the knowledge that a lipid was mentioned, for example was acquired.

<Electrophoresis solution filling step S3>
Next, a predetermined amount of the electrophoretic liquid Lm in the electrophoretic liquid tank 72 is sucked by the dispenser 6, and a predetermined amount of the electrophoretic liquid Lm is dispensed into the discharge tank 25 of the analysis chip 2 as shown in FIG. Then, the electrophoretic liquid Lm is filled into the discharge tank 25 and the capillary tube 27 by a technique such as appropriately performing suction and discharge from the port described above.

<Introduction step S4 and removal step S11>
Next, as shown in FIG. 8, a predetermined amount of the mixed sample Sm is collected from the mixing tank 22 by the dispenser 6. Then, a predetermined amount of the mixed sample Sm is introduced from the dispenser 6 into the introduction tank 23. In this introduction, the mixed sample Sm passes through the filter 24 provided at the opening of the introduction tank 23 which is an example of the introduction path to the introduction tank 23. At this time, aggregates of the subcomponent (for example, lipid) and the anionic group-containing compound (in this embodiment, chondroitin sulfate) contained in the mixed sample Sm are removed by the filter 24. This step is the removal step S11. In parallel with the removal step S11, an introduction step S4 that is introduction of the mixed sample Sm into the introduction tank 23 is performed. In the present embodiment, the mixed sample Sm is filled from the introduction tank 23 into the electrode tank 26 through the communication channel 28.

<Electrophoresis step S5>
Next, as shown in FIG. 1, the electrode 31 is inserted into the electrode tank 26, and the electrode 32 is inserted into the discharge tank 25. Subsequently, a voltage is applied to the electrode 31 and the electrode 32 according to an instruction from the control unit 8. This voltage is, for example, 0.5 kV to 20 kV. As a result, an electroosmotic flow is generated, and the mixed sample Sm is gradually moved in the capillary tube 27 from the introduction tank 23 to the discharge tank 25. At this time, since the mixed sample Sm is filled in the introduction tank 23, the analysis component s-HbA1c and the chondroitin sulfate are combined in a state where the mixed sample Sm is continuously supplied in the capillary tube 27. The complex will be electrophoresed. Further, light emission from the light source 41 is started, and absorbance is measured by the detector 5. Then, the relationship between the elapsed time and the absorbance from the start of voltage application from the electrodes 31 and 32 is measured. Here, an absorbance peak corresponding to a component having a relatively fast moving speed in the mixed sample Sm appears when the elapsed time from the start of voltage application is relatively short. On the other hand, an absorbance peak corresponding to a component having a relatively slow moving speed in the mixed sample Sm appears when the elapsed time from the start of voltage application is relatively long. Thereby, analysis (separation measurement) of each component in mixed sample Sm is performed. Furthermore, an electropherogram is created by subjecting the measured absorbance to arithmetic processing (for example, differential processing, differential processing, etc. by the control unit 8). By calculating the peak height and peak area of this electropherogram, the component ratio in the mixed sample Sm and the like are obtained.

  Next, examples of this analysis method will be described.

<Example 1>
As a sample Sa, 95 μL of whole blood collected from a healthy person, for example, a fat emulsion for intravenous injection (Intralipos (Otsuka Pharmaceutical Co., Ltd .: registered trademark)) as a substitute for lipids that can be contained in the blood of hyperlipidemic patients, for example. What added 5 microliters of infusion solutions 20%) was used. As electrophoresis solution Lm, 40 mM citric acid, 1.25% (w / v) chondroitin sulfate C-sodium, 0.1% (w / v) LS-110 (manufactured by Kao Corporation), 0.02% (w / v) v) What was prepared using sodium azide, 0.025% (w / v) Procrine 950 (Sigma Aldrich: registered trademark), and adjusted to pH 5.0 using dimethylaminoethanol (for pH adjustment). Using. As diluent Ld, 40 mM citric acid, 1.0% (w / v) chondroitin sulfate C-sodium, 500 mM NDSB-201 (manufactured by Anatrac), 0.1% (w / v) LS-110 (Kao Corporation) ), 0.02% (w / v) sodium azide, 0.025% (w / v) Procrine 950 (Sigma Aldrich: registered trademark), dimethylaminoethanol (for pH adjustment) What was adjusted to pH 6.0 using was used.

  For the analysis chip 2, an introduction tank 23 having a capacity of 10 μL, a discharge tank 25 having a capacity of 10 μL, and a capillary tube 27 having a width of 40 μm, a depth of 40 μm, and a total length of 30 mm (separation length of 20 mm) were prepared. The inner wall of the capillary tube 27 was coated with polydiallyldimethylammonium chloride (PDADMAC: manufactured by Sigma). As the filter 24, a cellulose acetate membrane filter (manufactured by ADVANTEC, pore size 0.45 μm) was used.

  In the mixing step S2, the sample Sa was diluted 41 times with the diluent Ld to obtain a mixed sample Sm. The filling amount of the electrophoresis solution Lm in the electrophoresis solution filling step S3 is 9 μL. In the introduction step S4 and the removal step S11, the mixed sample Sm was introduced in an amount obtained by adding 9 μL of the volume corresponding to the volume of the electrode tank 26 and the connection channel 28. In the electrophoresis step S5, the voltage of the electrode 31 and the electrode 32 was 0.5 kV to 20 kV, and the current was 76 μA. In the detector 5, the absorbance at a wavelength of 415 nm was measured to obtain the electropherogram. The duration of electrophoresis was 30 seconds. Such measurement was performed 5 times to evaluate simultaneous reproducibility.

<Comparative example>
The same measurement as in Example 1 was performed except that the removal step S11 using the filter 24 was not performed. And this measurement was implemented 5 times and simultaneous reproducibility was evaluated.

  FIG. 9 shows the electropherogram obtained by Example 1, and FIG. 10 shows the electropherogram obtained by the comparative example. In these electropherograms, the horizontal axis represents detection time (unit: seconds), and the vertical axis represents absorbance change rate (unit: mAbs / sec). Table 1 shows the results of simultaneous reproducibility evaluation of the analysis results of s-HbA1c of Example 1 and Comparative Example. The numerical value in each measurement in the table is the ratio (%) of the hemoglobin s-HbA1c concentration to the total hemoglobin concentration. The peak in the vicinity of 21 to 22 seconds in FIG. 9 and the peak in the vicinity of 20 seconds to 21 seconds in the main body in FIG. 10 are absorbance peaks of s-HbA1c, which is an analysis component.

  Next, the operation of the analysis system A1 and the present analysis method will be described.

  As shown in FIG. 9 and FIG. 10, the absorbance peak of s-HbA1c appears in almost the same time zone. The shape of the electropherogram in the time zone before and after the appearance of the absorbance peak of s-HbA1c is significantly smoother than that of the comparative example of FIG. In other words, the electropherogram of FIG. 10 has a shape including a large number of minute irregularities. Further, as shown in Table 1, the analysis result of s-HbA1c in Example 1 is smaller in standard deviation and coefficient of variation (CV) than in the comparative example. From these, it can be said that Example 1 is more reproducible than the comparative example. As a result of examination by the inventors about this difference, in Example 1, the mixed sample Sm passed through the filter 24 in the removal step S11, so that the subcomponent (for example, lipid) contained in the mixed sample Sm and The knowledge that it originates in the aggregate with the chondroitin sulfate as said anionic group containing compound being removed was acquired. That is, the aggregates are recognized in the mixed sample Sm before the removal step S11, whereas the aggregates in the mixed sample Sm are hardly recognized after the removal step S11. It was. Then, the inventors have come to the conclusion that in electrophoresis, the aggregates appear in the electropherogram as minute irregularities in FIG. 10, that is, noise. It is considered that the reproducibility of Example 1 was improved because such aggregates were removed. Moreover, the average value of a measurement result differs in Example 1 and a comparative example, and it is thought that the reliability of the average value of Example 1 is higher about this point. Therefore, according to the analysis system A1 and the present analysis method, the analysis accuracy can be improved.

  In the electrophoresis step S5 of this analysis method, electrophoresis is performed in a state where the mixed sample Sm is continuously supplied. For this reason, for example, it is not necessary to quantify the mixed sample Sm supplied to the capillary tube 27 to a predetermined amount. The analysis method for quantifying the mixed sample Sm is forced to include a relatively complicated mechanism for quantification. In this analysis method and analysis system A1, since it is not necessary to quantify the mixed sample Sm, the analysis chip 2 can be configured to be relatively small. This is advantageous when the analysis chip 2 is used as a disposable type analysis chip.

  By providing the filter 24 at the opening of the introduction tank 23 that is the introduction path of the introduction tank 23, the introduction step S4 and the removal step S11 can be performed in parallel. Further, since the filter 24 is provided in the analysis chip 2 that is a disposable type, the filter 24 is discarded together with the analysis chip 2 when a certain analysis is completed. Thereby, the filter 24 used for the removal of the aggregates does not remain in the analyzer 1, and the analysis method can be performed a plurality of times in a clean and clean state.

  Unlike the above-described configuration, the filter 24 can be appropriately provided in the introduction path to the introduction tank 23. For example, in a configuration in which the mixing tank 22 and the introduction tank 23 are connected by a flow path, a filter 24 is provided in the middle of the flow path or at a connection portion between the flow path and the mixing tank 22 or the introduction tank 23. be able to. When the mixed sample Sm generated in the mixing tank 22 is introduced from the mixing tank 22 into the introducing tank 23 through the flow path, the aggregate is removed by the filter 24.

  In addition, it is assumed that the subcomponent constituting the aggregate is frequently a lipid in blood collected from a hyperlipidemic patient, for example. A specific example of the subcomponent is lipid. It is not limited. The subcomponent may be any component other than the analysis component contained in the sample Sa including blood, and any component that can aggregate with the cathodic group-containing compound.

  11 and 12 show another embodiment of the present invention. In these drawings, the same or similar elements as those in the above embodiment are denoted by the same reference numerals as those in the above embodiment.

  FIG. 11 is a system schematic diagram showing an analysis system according to the second embodiment of the present invention. FIG. 12 is a flowchart showing the analysis method of this embodiment.

  As shown in FIG. 11, the analysis system A2 includes an analysis apparatus 1, an analysis chip 2, a diluent tank 71, a mixing tank Tm, and a centrifuge Cs.

  Unlike the above-described embodiment, the analyzer 1 does not include the diluent tank 71 and the filter 24. Other configurations of the analyzer 1 are the same as those of the analyzer 1 in the analysis system A1 described above.

  In the present embodiment, a diluent tank 71 for storing the diluent Ld is provided outside the analyzer 1. In addition, a mixing tank Tm is provided outside the analyzer 1.

  The centrifuge Cs is for carrying out the removal step S11 in the analysis method of the present embodiment using the analysis system A2, and is an example of the removal unit (centrifugation means) referred to in the present invention. As the centrifuge Cs, for example, a device that performs a centrifugation process on blood is appropriately employed.

  As shown in FIG. 12, in the analysis method of this embodiment, the sample collection step S1, the mixing step S2, the removal step S11, the electrophoresis liquid filling step S3, the introduction step S4, and the electrophoresis step S5 are executed in this order.

  After collecting the sample Sa shown in FIG. 11, a predetermined amount of the diluent Ld is dispensed from the sample Sa and the diluent tank 71 into the mixing tank Tm. A mixing step S2 is performed in which the sample Sa and the diluent Ld are mixed in the mixing tank Tm. Thereby, the mixed sample Sm is obtained. Next, a predetermined amount of the mixed sample Sm is collected in a predetermined container, or loaded into the centrifuge Cs while being stored in the mixing tank Tm. By the centrifugation process by the centrifuge Cs, in the mixed sample Sm, an aggregate of the subcomponent (lipid) and the cathodic group-containing compound (chondroitin sulfate) is precipitated at the bottom (removal step S11). Subsequently, a predetermined amount of the mixed sample Sm is collected from the region separated from the aggregate precipitation region to the liquid surface side of the mixed sample Sm, and introduced into the introduction tank 23 of the analysis chip 2 (introduction step S4). In addition, the electrophoresis solution filling step S3 is performed. Thereafter, the electrophoresis step S5 as described above is performed.

<Example 2>
An example of the present embodiment using the analysis system A2 (for convenience, will be referred to as Example 2) will be described. In the present example, in the removing step S11, the mixed sample Sm was subjected to a centrifugal separation process at 8000 G for 10 minutes using the centrifugal separator Cs. Other conditions are the same as those of Example 1 and the comparative example described above. The electropherogram obtained by Example 2 is shown in FIG. Table 2 shows the simultaneous reproducibility evaluation results of the analysis results of s-HbA1c of Example 2 and the comparative example described above.

  Comparing the electropherogram of Example 2 in FIG. 13 with the electropherogram of the comparative example in FIG. 10, the electropherogram of Example 2 has significantly less noise assumed to be caused by aggregates. It can be seen that it is equivalent to the electropherogram of Example 1. As shown in Table 2, the standard deviation and CV of Example 2 are smaller than those of the comparative example, and it can be said that Example 2 is excellent in reproducibility. In addition, compared with the comparative example, Example 1 and Example 2 are values having a close average value, and both standard deviation and CV are small values.

  The analysis method and analysis system according to the present invention are not limited to the above-described embodiments. The specific configuration of each part of the analysis method and analysis system according to the present invention can be varied in design in various ways.

  The filtering means is not limited to the filter 24 built in the analysis chip 2. For example, instead of Cs in the analysis system A 2, a filtering means provided with a predetermined filter is provided outside the analyzer 1. Also good. Further, the filtration means and the centrifugal separation means are examples of the removing unit in the present invention, and can appropriately remove unnecessary substances such as aggregates that may cause noise in the electropherogram results in the above-described electrophoresis method. If it is a structure, a various mechanism is employable.

A1, A2 Analysis system Cs Centrifuge Sa Sample Sm Mixed sample Ld Diluent Lm Electrophoretic liquid Tm Mixing tank 1 Analyzing device 2 Analytical chip 21 Main body 22 Mixing tank 23 Introducing tank 24 Filter 25 Discharge tank 26 Electrode tank 27 Capillary tube 28 Contact Flow path 31, 32 Electrode 41 Light source 42 Optical filter 43 Lens 44 Slit 5 Detector 6 Dispenser 61 Pump 71 Diluent tank 72 Electrophoretic liquid tank 8 Control unit S1 Sample collection process S2 Mixing process S3 Electrophoretic liquid filling process S4 Introduction process S5 electrophoresis step S11 removal step

Claims (13)

A method for analyzing a sample by capillary electrophoresis,
Producing a mixed sample by mixing the sample and the cathodic group-containing compound;
Removing from the mixed sample aggregates of subcomponents other than the analytical component to be analyzed among the components of the sample and the anionic group-containing compound;
And a step of electrophoresis of a complex in which the analysis component and the cathodic group-containing compound are bound in a capillary tube in a state where the mixed sample is continuously supplied.   The analysis method according to claim 1, wherein in the removing step, the aggregate is removed by filtration.   The analysis method according to claim 1, wherein in the removing step, the aggregate is removed by centrifugation.   The analysis method according to claim 1, wherein the anionic group-containing compound is an anionic group-containing polysaccharide.   The analysis according to claim 4, wherein the anionic group-containing polysaccharide is at least one polysaccharide selected from the group consisting of sulfated polysaccharides, carboxylated polysaccharides, sulfonated polysaccharides and phosphorylated polysaccharides. Method.   The analysis method according to claim 5, wherein the sulfated polysaccharide is chondroitin sulfate.   The analysis method according to claim 1, wherein the analysis component is hemoglobin.   The analysis method according to claim 7, wherein the subcomponent is a lipid. A system for analyzing samples by capillary electrophoresis,
A mixing section for producing a mixed sample by mixing the sample and the anionic group-containing compound;
A removing unit that removes aggregates of subcomponents other than the analysis component to be analyzed among the components of the sample and the anionic group-containing compound from the mixed sample;
An analysis unit that has a capillary tube and electrophores the complex in which the analysis component and the cathodic group-containing compound are bound in a state where the mixed sample is continuously supplied in the capillary tube. , Analysis system.   The analysis system according to claim 9, wherein the removing unit includes a filtering unit. A disposable analysis chip having the capillary tube, an introduction tank connected to one end of the capillary tube, and a discharge tank connected to the other end of the capillary tube;
The analysis system according to claim 10, wherein the filtering means is provided in an introduction path to the introduction tank in the analysis chip.   The analysis system according to claim 11, wherein the mixing unit is a mixing tank formed on the analysis chip.   The analysis system according to claim 9, wherein the removing unit includes a centrifugal separator.