JP6846324B2 - Analysis method and analysis system - Google Patents

Description

The present invention relates to analytical methods and systems.

Various proteins have been analyzed as indicators of the state of the living body. Among them, regarding hemoglobin (Hb) in blood cells, there are a plurality of hemoglobin species, and in addition to normal hemoglobin (HbA), there are a plurality of mutant hemoglobins (HbC, HbD, HbE, HbS, etc.).

In component analysis of a sample (for example, liquid chromatography), for example, by measuring a sample whose composition is known in advance, confirm the detection time and peak shape at which the component to be detected is detected, and compare it with the analysis result of the subject. (See, for example, Patent Document 1).

In the analysis of hemoglobin species (components) of blood (sample), the detection time differs depending on the components, so separation analysis using this is performed. In the separation analysis, considering the variation for each measurement (for example, the difference in detection time due to column deterioration, eluent storage state, etc.), for example, if the detection time of a certain component is within the predetermined detection time range, the component is concerned. Is identified as.

Electrophoresis is used as one of the methods for analyzing hemoglobin (Hb) (see, for example, Patent Document 2). The analysis of the sample by the electrophoresis method is performed with the analysis chip loaded in the analyzer. The analysis chip holds a sample and provides a place for analysis of the sample. As this analysis chip, there is a disposable type that is intended to be discarded after completing only one analysis. A method for analyzing hemoglobin using such a disposable type analysis chip is disclosed in, for example, Patent Document 3 below.

The waveform obtained by the analysis has a peak or the like corresponding to a specific component. Which peak corresponds to which specific component is identified in consideration of the elapsed time from the time point of analysis reference to the time point when the peak is obtained, the shape of the peak, and the like. At this time, the identification of the specific component may be inaccurate depending on whether or not the reference time point reflects the start time point of electrophoresis. In particular, when a disposable type analysis chip is used, there is a possibility that an error may occur in the elapsed time from the reference time to the time when the peak of a specific component is obtained due to the difference between lots for each analysis chip and the individual difference between the same lots. There is. In particular, conventionally, it is common to set the time point at which the application of the voltage is started as the reference time point (t = 0). In this case, there is a problem that the error due to individual differences between the analysis chips becomes large in the disposable type analysis chip with respect to the elapsed time from the time of t = 0 to the time when the specific component is generated.

As another conventional technique for dealing with such a problem, there is a method based on a known waveform. As one method of identifying a known waveform, a reference solution having a known waveform is introduced into the same analysis chip before the sample is introduced, and the measured waveform is used to obtain an error due to individual differences in the chip. There is a method of calculating and using the information to determine the waveform of a sample introduced using the same analysis chip. However, it is necessary to add a reference solution, which poses a problem in terms of time and cost. Further, this method is not possible in the first place with a disposable type analysis chip that is discarded after each measurement.

In addition, as another conventional technique for dealing with the problem, the peak value of a component having a typical and characteristic waveform contained in the introduced sample and the peak value of the reference substance added to the sample are used as representatives. Was being done. However, if the specific component contained in the sample to be introduced is unknown, it is difficult to identify the peak of the specific component because the peak of the specific component occurs, the waveform shifts, or it overlaps with another component. In some cases. In particular, when the specific component is hemoglobin, for example, the types of hemoglobin differ greatly between HbA and HbS, and problems such as overlapping with the reference component and interaction with the reference substance may occur. In addition, the method of adding a reference substance requires a high cost of the reference substance and also requires consideration of the influence of interaction with another component, which may make it difficult to commercialize the product.

Japanese Unexamined Patent Publication No. 60-73458 Japanese Unexamined Patent Publication No. 11-337521 Japanese Unexamined Patent Publication No. 2016-57289

The present invention has been devised under the above circumstances, and an object of the present invention is to provide an analysis method and an analysis system capable of more accurate analysis.

The analysis method provided by the first aspect of the present invention applies a voltage to the sample solution introduced into the microchannel, separates and analyzes the components contained in the sample solution, and corresponds to the elapsed time after the start of measurement. This is a method for analyzing a sample by capillary electrophoresis that measures the measured optical values, and is based on the optical measured values when the interface between the sample solution and the running solution reaches a predetermined measurement position in the microchannel. The step of determining the time point of reaching the interface and the step of identifying the component contained in the sample solution using the optical measurement value in the elapsed time elapsed from the time of reaching the interface are included.

Here, the "predetermined measurement position in the microchannel" means a position through which the measurement light for measuring the optical measurement value passes. The optical measurement value related to this interface is preferably measured at the same position as the optical measurement value related to the component to be identified, but may be measured at a different position.

The sample solution in the above analysis method is a solution containing a sample to be analyzed, and the sample itself occupies 100%, or the sample is appropriately diluted. , Both are included as a concept. The sample solution is not particularly limited as long as the time point at which the interface is reached can be specified and the sample solution has the properties of a liquid. The liquid may be, for example, a diluted solution in which the solid as a sample is suspended, dispersed or dissolved in a liquid medium. When the sample is a liquid, for example, the stock solution of the sample may be used as it is as a sample solution, or when the concentration is too high, the stock solution is suspended, dispersed or dispersed in a liquid medium, for example. The dissolved diluted solution may be used as a sample solution. The liquid medium is not particularly limited as long as it can suspend, disperse or dissolve the sample, and examples thereof include water and a buffer solution. Examples of the sample include a biological sample, an environmental sample, a metal, a chemical substance, a pharmaceutical product, and the like. The biological sample is not particularly limited, and examples thereof include urine, blood, hair, saliva, sweat, and nails. Examples of the blood sample include red blood cells, whole blood, serum, plasma and the like. Examples of the living body include humans, non-human animals, plants and the like, and examples of the non-human animals include mammals other than humans, amphibian reptiles, fish and shellfish, insects and the like. The sample derived from the environment is not particularly limited, and examples thereof include food, water, soil, air, and air. Examples of the food include fresh foods and processed foods. Examples of the water include drinking water, groundwater, river water, seawater, domestic wastewater and the like.

In a preferred embodiment of the present invention, the sample solution is a solution containing blood as a sample, and the component is hemoglobin.

In a preferred embodiment of the present invention, in the step of determining the interface arrival time point, the interface arrival time point is determined based on the change of the optical measurement value when the interface reaches the predetermined measurement position. ..

In a preferred embodiment of the present invention, the optically measured value is the absorbance of the sample solution, and a waveform relating to the absorbance corresponding to the elapsed time after the start of measurement is formed prior to the step of determining the time point of reaching the interface. The change in the optical measurement value in the step of having a step and determining the time point of reaching the interface is a change occurring in the waveform.

In a preferred embodiment of the present invention, the step of forming the waveform is a step of forming a differential waveform in which the differential value obtained by time-differentiating the waveform related to the absorbance is represented as a waveform corresponding to the elapsed time. Including, the differential waveform is used in the step of determining the time point of reaching the interface.

In a preferred embodiment of the present invention, the step of determining the interface arrival time point includes a step of determining a reference value determined based on the differential waveform within a predetermined search time and the reference within the predetermined search time. The step of determining the first specific point and the second specific point based on the degree of separation from the value, and the first specific point and the first specific point located between the first specific point and the second specific point on the time axis. It includes a step of specifying an average value point that takes an average value of the differential values of the second specific point, and a step of determining the time point of the average value point as the interface arrival time point.

In the step of determining the first specific point and the second specific point, the point that takes the value most distant from the reference value in the negative direction along the differential value axis within the predetermined search time is set as the first feature point. A step, a step in which a point having a value farthest from the first feature point on the negative direction side along the time axis along the differential value axis as a second feature point within the predetermined search time is set as a second feature point, and the predetermined search. A step in which a point having a value farthest from the first feature point on the positive direction side along the time axis along the differential value axis as the third feature point in time is set as a third feature point, and a step on the time axis within the predetermined search time. A step in which the point that takes the value farthest from the third feature point on the positive direction side along the third feature point is set as the fourth feature point, and the first feature point, the second feature point, the third feature point, and the fourth feature point. The step includes selecting the two points most distant from the feature points along the differential value axis as the first and second specific points.

In a preferred embodiment of the present invention, a disposable type analysis chip in which the fine flow path is formed is used.

The analysis system provided by the second aspect of the present invention includes a measuring unit that measures an optical measurement value of a liquid in a fine flow path and a control unit that performs an analysis process using the measurement results of the measuring unit. It is a sample analysis system by a separation analysis method, which comprises applying a voltage to a sample solution introduced into the microchannel, separating and analyzing the components contained in the sample solution, and starting measurement. A sample is analyzed by a capillary electrophoresis method for measuring the optical measurement value corresponding to a later elapsed time, and the control unit has a predetermined interface between the sample solution and the running solution in the microchannel. The components contained in the sample solution are identified by using the means for determining the interface arrival time point based on the optical measurement value when the measurement position is reached and the optical measurement value in the elapsed time elapsed from the interface arrival time point. Means, including.

In a preferred embodiment of the present invention, the sample solution is a solution containing blood as a sample, and the component is hemoglobin.

In a preferred embodiment of the present invention, the means for determining the interface arrival time point determines the interface arrival time point based on the change in the optical measurement value when the interface reaches the predetermined measurement position.

In a preferred embodiment of the present invention, the optically measured value is the absorbance of the sample solution, has means for forming a waveform relating to the absorbance corresponding to the elapsed time after the start of measurement, and determines the time point at which the interface is reached. The means for determining the time point at which the interface is reached is determined based on the change that occurs in the waveform as the change in the optical measurement value.

In a preferred embodiment of the present invention, a disposable type analysis chip in which the fine flow path is formed is used.

According to one aspect of the present invention, more accurate analysis is possible.

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

It is a system schematic diagram which shows an example of the analysis system which concerns on this 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 line III-III of FIG. It is a flow chart which shows the analysis method which concerns on this invention. It is a flow chart which shows the procedure of the preparation process. It is sectional drawing which shows one step of the preparation process of FIG. It is sectional drawing which shows one step of the preparation process of FIG. It is sectional drawing which shows one step of the preparation process of FIG. It is sectional drawing which shows one step of the preparation process of FIG. It is a flow chart which shows the procedure of an analysis process. It is a graph which shows an example of the waveform data formed by the waveform formation process. It is a histogram for finding a reference value. It is a graph which shows the determination of the most separated point. It is a graph which shows the determination of the 1st feature point. It is a graph which shows the determination of the 2nd feature point. It is a graph which shows the determination of the 3rd feature point. It is a graph which shows the determination of the 4th feature point. It is a graph which shows the determination of the average value point. It is a graph which shows another example of waveform data. It is a graph which shows another example of waveform data. It is a graph which shows another example of waveform data. It is a graph which shows another example of waveform data. It is a graph which shows another example of waveform data. It is a graph which shows another example of waveform data. It is a graph which shows the result of the component identification process in this invention. It is a graph which shows the result of the component identification process in a comparative example.

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

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

Examples of the analysis component include hemoglobin (Hb), albumin (Alb), globulin (α1, α2, β, γ globulin), fibrinogen and the like. Examples of the hemoglobin include a plurality of hemoglobin species such as normal hemoglobin (HbA), mutant hemoglobin (HbC, HbD, HbE, HbS, etc.), and fetal hemoglobin (HbF). Mutant hemoglobin is known to cause various diseases and pathological conditions (for example, HbS causes sickle cell anemia), and by identifying the hemoglobin species, it is possible to diagnose diseases and pathological conditions. It is expected to be useful for treatment. In the following description, a case where the analysis component is hemoglobin (particularly a mutant hemoglobin species) will be described as an example. Healthy adult hemoglobin contains the majority of HbA and a small amount of HbF and HbA2. Hemoglobin is a tetramer, for example HbA is composed of two α chains and two β chains. If any mutation occurs in the gene sequence that controls the production of this α chain or β chain, a chain with an unusual amino acid sequence is produced or the production of that chain is suppressed, resulting in an unusual hemoglobin species. Occurs. Generally, such a hemoglobin species is called a mutant hemoglobin. The genotype of hemoglobin in healthy subjects is HbA / HbA homozygotes, but the genotype of mutant hemoglobin carriers is HbA / HbV heterozygotes (HbV is a mutant hemoglobin other than HbA) or HbV / HbV. Homozygotes (heterozygotes if HbVs are different). In the clinical testing industry, blood samples derived from healthy individuals are HbAA samples, blood samples derived from humans with heterozygotes of HbA / HbV are HbAV samples (V is an arbitrary mutation), and homozygotes of HbV / HbV (HbV). When they are different from each other, a blood sample derived from a person having a heteroconjugate) is called an HbVV sample (V is an arbitrary mutation). Thus, for example, an HbAS sample contains the majority of HbA and HbS and a small amount of HbF and HbA2. For example, HbSS specimens contain the majority of HbS and a small amount of HbF and HbA2.

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 analyzer 1. In the present embodiment, the analysis chip 2 is configured as a so-called disposable type analysis chip intended to be discarded after one analysis is completed. 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 pipe 27, and a communication flow path 28. FIG. 2 is a plan view of the analysis chip 2, and FIG. 3 is a cross-sectional view taken along the line III-III of FIG. The analysis chip 2 is not limited to the disposable type, and may be used for a plurality of analyzes. Further, the analysis system according to the present invention is not limited to a configuration including a separate analysis chip 2 loaded in the analysis device 1, and a functional portion having the same function as the analysis chip 2 is incorporated in the analysis device 1. It may be a configuration.

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, molten silica, and plastic. In the present embodiment, the main body 21 has a configuration in which the upper portion 2A and the lower portion 2B in FIG. 3 are formed separately, and these are connected to each other. Not limited to this, for example, the main body 21 may be integrally formed.

The mixing tank 22 is an example of a place where a mixing step of mixing the sample Sa and the diluent Ld, which will be described later, is performed. The mixing tank 22 is configured as a recess opened upward by, for example, a through hole formed in the upper portion 2A of the main body 21. The introduction tank 23 is a tank into which the mixed sample Sm as the sample solution obtained by the mixing step in the mixing tank 22 is introduced. The introduction tank 23 is configured as a recess opened upward by, for example, a through hole formed in the upper portion 2A of the main body 21.

The filter 24 is provided at the opening of the introduction tank 23, which is an example of the introduction path to the introduction tank 23. The specific configuration of the filter 24 is not limited, and a preferable example is a cellulose acetate membrane filter (manufactured by ADVANTEC, pore diameter 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 configured as a recess opened upward by, for example, a through hole formed in the upper portion 2A of the main body 21. The electrode tank 26 is a tank into which the electrode 31 is inserted in the analysis step by the electrophoresis method. The electrode tank 26 is configured as a recess opened upward by, for example, a through hole formed in the upper portion 2A of the main body 21. The connecting flow path 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 is a fine flow path connecting the introduction tank 23 and the discharge tank 25, and is a place where an electroosmotic flow (EOF, electro-osmotic flow) in an electrophoresis method is generated. The capillary pipe 27 is configured as, for example, a groove formed in the lower portion 2B of the main body 21. The main body 21 may be appropriately formed with recesses or the like for promoting the irradiation of light on 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, and for example, the width thereof is 25 μm to 100 μm, the depth thereof is 25 μm to 100 μm, and the length thereof is 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 size and arrangement of the mixing tank 22, the introduction tank 23, the discharge tank 25, and the electrode tank 26.

The analysis chip 2 having the above configuration is an example, and an analysis chip having a configuration capable of analysis by electrophoresis can be appropriately adopted.

The analyzer 1 performs an analysis process on the sample Sa with the analysis chip 2 on which the sample Sa is instilled is loaded. The analyzer 1 includes electrodes 31, 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 diluent tank 71, a running fluid tank 72, and a control unit 8. ing. The light source 41, the optical filter 42, the lens 43, and the detector 5 constitute an example of the measuring unit according to the present invention.

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. The voltage applied to the electrode 31 and the electrode 32 is not particularly limited, but is, for example, 0.5 kV to 20 kV.

The light source 41 is a portion that emits light for measuring the absorbance as an optical measurement value in the electrophoresis method. The light source 41 includes, for example, an LED chip that emits light in a predetermined wavelength range. The optical filter 42 attenuates the light having a predetermined wavelength among the light from the light source 41 and transmits the light having the other wavelength. The lens 43 is for condensing the light transmitted through the optical filter 42 to the analysis point 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 lens 43.

The detector 5 receives light from the light source 41 that has passed through the capillary tube 27 of the analysis chip 2, and is configured to include, for example, a photodiode or a photo IC.

In this way, the path through which the light emitted from the light source 41 reaches the detector 5 is the optical path. Then, the optical measurement value is measured for the solution flowing through the capillary tube 27 at the position where the optical path intersects the capillary tube 27 (that is, either the sample solution or the running solution or a mixed solution thereof). That is, the position where the optical path from the light source 41 to the detector 5 intersects in the capillary tube 27 is the measurement unit of the optical measurement value. Examples of this optical measurement value include absorbance. The absorbance represents the degree to which the light in the optical path is absorbed by the solution flowing through the capillary tube 27, and represents the absolute value of the common logarithm value of the ratio of the incident light intensity to the transmitted light intensity. In this case, a general-purpose spectrophotometer can be used as the detector 5. Even if the absorbance is not used, any optical measurement value such as the transmitted light intensity value itself can be used in the present invention. In the following, a case where absorbance is used as the optical measurement value will be described as an example.

The dispenser 6 dispenses a desired amount of the diluted solution Ld, the running solution Lm, and the mixed sample Sm, and includes, for example, a nozzle. The dispenser 6 can freely move 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 running solution Lm and the like. Further, a dedicated pump other than 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 a container in which a predetermined amount of the diluent Ld is sealed may be loaded in the analyzer 1. The running solution tank 72 is a tank for storing the running solution Lm. The running solution tank 72 may be a tank permanently installed in the analyzer 1, or a container in which a predetermined amount of the running solution Lm is sealed may be loaded in the analyzer 1.

The diluent Ld is for producing a mixed sample Sm as a sample solution by mixing with the sample Sa. The main agent of the diluted solution Ld is not particularly limited, and examples thereof include water and physiological saline, and preferred examples thereof include a liquid having a component similar to that of the running solution Lm described later. Further, in the diluted solution Ld, an additive may be added as needed in addition to the above-mentioned main agent.

The running solution Lm is a medium that is filled in the discharge tank 25 and the capillary tube 27 in the analysis step by the electrophoresis method to generate an electroosmotic flow in the electrophoresis method. The running solution Lm is not particularly limited, but one using an acid is desirable. Examples of the acid include citric acid, maleic acid, tartaric acid, succinic acid, fumaric acid, phthalic acid, malonic acid, and malic acid. Further, the running solution Lm preferably contains a weak base. Examples of the weak base include arginine, lysine, histidine, tris and the like. The pH of the running solution Lm is, for example, in the range of pH 4.5 to 6. The type of buffer of the running solution Lm includes MES, ADA, ACES, BES, MOPS, TES, HEPES and the like. Further, an additive may be added to the running solution Lm as needed, as described in the description of the diluted solution Ld.

The running solution Lm, the diluted solution Ld, and the mixed sample Sm exemplify the following, but at the time of reaching the interface described later, the change in the optically measured value due to the arrival of the interface between the sample solution (mixed sample Sm) and the running solution Lm. Any combination can be selected as long as it causes.

(Electrophoretic solution Lm)
The running solution Lm contains, for example, the following components.
Citric acid: 40 mM
C sodium chondroitin sulfate: 1.25% w / v
Piperazine: 20 mM
Polyoxyalkylene alkyl ether (trade name: Emargen LS-110, manufactured by Kao Corporation): 0.1% w / v
Sodium azide: 0.02% w / v
Procrine 300: 0.025% w / v
In addition to the above components, dimethylaminoethanol for pH adjustment was added dropwise to adjust the pH to 5.0.

(Diluted solution Ld)
The diluent Ld contains, for example, the following components.
Citric acid: 38 mM
C sodium chondroitin sulfate: 0.95% w / v
1- (3-sulfopropyl) pyridinium hydroxide intramolecular salt (NDSB-201): 475 mM
2-Morpholine ethanesulfonic acid (MES): 19 mM
Polyoxyalkylene alkyl ether (trade name: Emargen LS-110, manufactured by Kao Corporation): 0.4% w / v
Sodium azide: 0.02% w / v
Procrine 300 0.025% w / v
In addition to the above components, dimethylaminoethanol for pH adjustment was added dropwise to adjust the pH to 6.0.

(Mixed sample Sm)
1.5 μL of sample Sa was added to 60 μL of the diluted solution Ld to prepare a mixed sample Sm.

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. A program and various data for performing the analysis method according to the present invention, which will be described later, are appropriately stored in the memory.

Next, an example of the analysis method according to the present invention performed using the analysis system A1 will be described below. FIG. 4 is a flow chart showing the analysis method of the present embodiment. This analysis method includes a preparation step S1, an electrophoresis step S2, and an analysis step S3.

<Preparation step S1>
FIG. 5 is a flow chart showing a specific procedure in the preparation step S1. In the present embodiment, as shown in the figure, the preparation step S1 includes a sampling step S11, a mixing step S12, a running solution filling step S13, and an introduction step S14.

<Sampling step S11>
First, sample Sa is prepared. In this embodiment, the sample Sa is blood collected from the human body. The blood may be whole blood, component-separated blood, hemolyzed blood, or the like. Then, the analysis chip 2 to which the sample Sa is dispensed is loaded into the analyzer 1.

<Mixing step S12>
Next, the sample Sa and the diluent Ld are mixed. Specifically, as shown in FIG. 6, 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 of the diluent tank 71 is sucked by the dispenser 6, and as shown in FIG. 7, the predetermined amount of the diluent Ld is dispensed into the mixing tank 22 of the analysis chip 2. Then, using the pump 61 as a suction source and a discharge source, the suction and discharge of the diluent Ld from the dispenser 6 are repeated. As a result, the sample Sa and the diluent Ld are mixed in the mixing tank 22, and a mixed sample Sm as a sample solution is obtained. The sample Sa and the diluent Ld may be mixed by a method other than suction and discharge of the dispenser 6.

<Electrophoretic solution filling step S13>
Next, a predetermined amount of the running solution Lm in the running solution tank 72 is sucked by the dispenser 6, and a predetermined amount of the running solution Lm is dispensed into the discharge tank 25 of the analysis chip 2 as shown in FIG. Then, the discharge tank 25 and the capillary tube 27 are filled with the running solution Lm by a method such as appropriately performing suction or discharge from the port described above.

<Introduction step S14>
Next, as shown in FIG. 9, a predetermined amount of 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. Further, in the present embodiment, the mixed sample Sm is filled from the introduction tank 23 into the electrode tank 26 through the connecting flow path 28. At this time, the mixed sample Sm flows from the introduction tank 23 to the electrode tank 26 via the connecting flow path 28, but the flow from the introduction tank 23 to the connecting flow path 28 is relative to the longitudinal direction of the capillary tube 27. The mixed sample Sm flows in a direction substantially orthogonal to each other (see FIG. 2). On the other hand, the running solution Lm of the capillary tube 27 has hardly moved at this stage. As a result, a shear flow is generated at the connection portion (see FIG. 3) between the introduction tank 23 and the capillary tube 27, so that a clear interface between the mixed sample Sm and the running solution Lm is formed. If the method is such that an interface between the mixed solution Sm and the running solution Lm is formed, a movable filter may be provided at the boundary between the introduction tank 23 and the capillary tube 27, or the flow method may be changed in a controlled manner. Any means can be adopted.

<Electrophoresis step S2>
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 the 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 from the introduction tank 23 to the discharge tank 25 in the capillary pipe 27. At this time, since the introduction tank 23 is filled with the mixed sample Sm, the hemoglobin (Hb), which is the analysis component, is electrophoresed while the mixed sample Sm is continuously supplied in the capillary tube 27. Become. At this time, the mixed sample Sm runs through the capillary tube 27 while pushing the running solution Lm in the downstream direction while maintaining the above-mentioned interface between the mixed sample Sm and the running solution Lm. Further, the light emission from the light source 41 is started, and the absorbance is measured by the detector 5. Then, the relationship between the elapsed time from the start of voltage application from the electrode 31 and the electrode 32 and the absorbance is measured.

<Analysis step S3>
Here, the absorbance peak corresponding to the component having a relatively high moving speed in the mixed sample Sm appears at a time when the elapsed time from the start of applying the voltage is relatively short. On the other hand, the absorbance peak corresponding to the component having a relatively slow moving speed in the mixed sample Sm appears at a time when the elapsed time from the start of applying the voltage is relatively long. Utilizing this, the analysis (separation measurement) of the components in the mixed sample Sm is performed. In the present embodiment, hemoglobin (particularly mutant hemoglobin) contained in blood is analyzed, and hemoglobin (mutant hemoglobin) that can be contained in the mixed sample Sm (blood) is an analytical component. Based on the measured absorbance, the analysis step S3 is executed under the control of the control unit 8. The analysis step S3 of the present embodiment includes a waveform forming step S31, an interface arrival time point determination step S32, and a component identification step S33.

<Waveform forming step S31>
In this step, an electropherogram is prepared by arithmetically processing the measured absorbance by the control unit 8. Here, with the start of voltage application as the start of measurement, a measurement waveform relating to absorbance representing a change in the optical measurement value corresponding to the elapsed time after the start of the measurement is formed. The waveform forming step S31 of the present embodiment includes the differential waveform forming step S311. The differential waveform forming step S311 forms a waveform of the differentiated value by time-differentiating the measured absorbance. FIG. 11 shows an example of the differential waveform formed by the differential waveform forming step S311. The x-axis in the figure is the time axis, and the y-axis is the differential value axis. In the following figures and explanations, the negative direction side along the time axis x is the direction x1 side and the positive direction side is the direction x2 side, and the negative direction side along the differential value axis y is the direction y1 side and the positive direction side. The direction is on the y2 side.

<Step of determining interface arrival time point S32>
The interface arrival time point determination step S32 is a step of determining the interface arrival time point as the time point at which the interface between the mixed sample Sm and the running solution Lm reaches the above-mentioned measuring unit (specifically, the optical path) after the voltage is applied. .. If a measuring unit (optical path) for interface measurement is provided separately from the measuring unit and the optical path, it may be determined at that position. In this analysis method and analysis system, as the reference time point of time in the component identification step S33 described later, not the time point of voltage application but the time point when the interface between the mixed sample Sm and the running solution Lm reaches the above-mentioned measurement unit is adopted. To do. The time when the interface reaches the measurement unit depends on the configuration of the capillary tube 27 of the analysis chip 2 and the analysis conditions such as the voltage applied by the electrode 31 and the electrode 32, and how long after the voltage is applied to the electrode 31 and the electrode 32. It is possible to grasp by empirical rule or pre-test whether it is the time point after the lapse of. Further, since the interface is the interface between the mixed sample Sm and the running solution Lm whose compositions are not the same as each other, it can optically perform a function similar to that of a lens. Therefore, when the interface reaches the measurement unit, some change is observed in the optically measured value (for example, absorbance). This change is due to the components expected to be contained in the sample Sa, the properties of the diluent Ld and the running solution Lm, and the basic configuration of the analysis chip 2, and a plurality of typical differential waveforms (FIG. It appears as a change in the differential value that occurs in 13, FIG. 19, FIG. 21 and FIG. 23). That is, the change generated in this waveform is due to the arrival of the interface between the mixed sample Sm as the sample solution and the running solution Lm. The present analysis method and the analysis system A1 determine the time point at which the interface is reached based on the change in the waveform as the change in the optical measurement value. The change caused by the arrival of the interface between the sample solution and the running solution is not based on the change generated in such a waveform, but is the transmitted light intensity value itself or its change, the absorbance value or its change, or the change. It is possible to detect even if a means for determining the time change rate such as a change in the differential value is adopted instead of the differential waveform.

The interface arrival time point determination step S32 of the present embodiment includes the reference value determination step S321, the farthest point determination step S322, the first to fourth feature point determination steps S323, the first and second specific point selection steps S324, and the average value point. The specific step S325 is included.

<Reference value determination step S321>
The reference value determination step S321 is a step of determining a reference value that serves as a reference for the waveform value (differential value) in the time range (for example, several seconds) estimated to include the interface arrival time point. FIG. 11 shows the waveform in the time range. In the reference value determination step S321 of the present embodiment, as shown in FIG. 12, a histogram is created with the frequency of appearance of the differential value of the differential waveform as the vertical axis. Then, the most frequent bin, bin Bn1, is specified. Next, of the two bins adjacent to bin Bn1, the most frequent bin Bn2 is identified. Then, for example, the reference value Ls of the differential value is determined by the weighted average using the frequencies of bin Bn1 and bin Bn2 and the differential value. For example, as shown in FIG. 13, this reference value Ls roughly corresponds to a horizontal line portion extending along the left and right sides in the figure. Strictly speaking, the reference value Ls may be a value slightly different from the horizontal portion, but in the following description, it is assumed that the error is negligible, and a case where the horizontal portion corresponds to the reference value Ls will be described as an example. ..

<Most distance point determination step S322>
Next, the farthest distance point determination step S322 is performed. In this step, as shown in FIG. 13, the point where the differential value is most distant from the reference value Ls determined in the reference value determination step S321 in the above-mentioned time range is determined. In the illustrated example, the point separated from the reference value Ls in the direction y2 is determined as the most separated point PL.

<Steps 1 to 4 for determining feature points S323>
Next, the first to fourth feature point determination steps S323 are performed. First, as shown in FIG. 14, the point that takes the most value on the direction y1 side within a predetermined search time is searched with reference to the farthest point PL (in other words, with reference value Ls as a reference). This predetermined search time is set in a time range in which the waveform can change due to the presence of the interface, and is appropriately set according to the analysis method and the analysis system. In the present embodiment, for example, 0.3 seconds. It takes about 1.5 seconds. Such a first feature point P1 is a point that can exist on either the direction x1 side or the direction x2 side with respect to the farthest point PL, and in the illustrated example, the direction with respect to the farthest point PL. A point located on the x1 side and located on the direction y1 side of the reference value Ls is determined as the first feature point P1.

Next, as shown in FIG. 15, a point that takes the value on the direction y2 side most on the direction x1 side within a predetermined search time is searched with reference to the first feature point P1. In the illustrated example, the differential value generally increases from the first feature point P1 toward the direction x1 and reaches the reference value Ls. As a result, a point that takes a value near the first reference value Ls when the waveform is traced from the first feature point P1 in the direction x1 is determined as the second feature point P2.

Next, as shown in FIG. 16, the point having the value on the direction y2 side most on the direction x2 side within the predetermined search time is searched with reference to the first feature point P1. In the illustrated example, the point determined as the farthest point PL corresponds to this point, and is determined as the third feature point P3. Next, as shown in FIG. 17, a point that takes the value on the direction y1 side most on the direction x2 side within a predetermined search time is searched with reference to the third feature point P3. In this example, a point having substantially the same value as the reference value Ls is determined as the fourth feature point P4, and the bending point of the differential waveform is selected for convenience.

<First and second specific point selection steps S324>
Next, the first and second specific point selection steps S324 are performed. In this step, as shown in FIG. 18, the first specific point and the second specific point are selected from the first feature point P1 to the fourth feature point P4. The selection of the first specific point and the second specific point is, for example, a combination of the first feature point P1 to the fourth feature point P4 in which the difference between the differential values (values on the differential value axis y) is the largest. The basic policy is to select the following two points. An example of the concrete method is given. First, the values of the second feature point P2 and the third feature point P3 are compared. In this example, the value of the third feature point P3 is larger than the value of the second feature point P2. Therefore, the third feature point P3 is selected as the first specific point. Next, it is determined whether or not the value of the first feature point P1 is sufficiently smaller than the reference value Ls. This judgment is made, for example, between the difference between the reference value Ls and the value of the first feature point P1 which is lower than the reference value Ls and the value of 5% of the difference between the value of the third feature point P3 and the reference value Ls. Perform by comparison. When the difference between the reference value Ls and the first feature point P1 is larger than the value of 5% of the difference between the value of the third feature point P3 and the reference value Ls, the first feature point P1 is the reference value. It is determined that it is sufficiently smaller than Ls, and that the graph forms a clear valley at the first feature point P1. As a result, the first feature point P1 is selected as the second specific point.

<Mean value point identification step S325>
Next, the mean value point specifying step S325 is performed. In this step, a point that takes the average value of the differential values of the first specific point, the third characteristic point P3 as the second specific point, and the first characteristic point P1 is searched. In the present embodiment, the differential waveform is traced starting from the point located on the direction x2 side on the time axis x among the first specific point and the second specific point, and the first specific point and the average of the second specific points are first averaged. Search for points that take a value. As a result, in the illustrated example, the point that is approximately the midpoint between the first feature point P1 and the third feature point P3 is determined as the mean value point PA. That is, the difference between the differential values of the third feature point P3 and the mean value point PA and the difference between the differential values of the mean value point PA and the first feature point P1 are both equal values dy.

In the interface arrival time point determination step S32, when the average value point PA is determined, the time point of the average value point PA is determined as the interface arrival time point. The control unit 8 performs processing such as appropriately storing the interface arrival time point in a memory or the like, and uses the interface arrival time point in the subsequent analysis steps.

19 to 24 show an example in which the interface arrival time point determination step S32 is performed on the other differential waveforms formed by the differential waveform forming step S311 of the waveform forming step S31.

In the waveform shown in FIG. 19, the reference value Ls determined in the reference value determination step S321 is used as a reference (in other words, the reference value Ls is used as a reference), and the point most distant from the reference value Ls in the farthest point determination step S322. , The farthest distance point PL located on the direction y1 side is determined. Next, in the first to fourth feature point determination steps S323, the point located closest to the direction y1 within the predetermined search time with respect to the farthest point PL is determined as the first feature point P1. In this example, the same point as the farthest point PL is determined as the first feature point P1. Next, a point located on the direction x1 side with respect to the first feature point P1 and on the most direction y2 side is determined as the second feature point P2. In this example, the second feature point P2 is a point that takes substantially the same value as the reference value Ls, and the bending point of the differential waveform is selected for convenience. Next, the point located on the direction x2 side with respect to the first feature point P1 and on the most direction y2 side is determined as the third feature point P3. In this example, the third feature point P3 is a point that takes substantially the same value as the reference value Ls, and in the illustrated example, the bending point of the differential waveform is selected for convenience. Next, the point located on the direction x2 side with respect to the third feature point P3 and on the most direction y1 side is determined as the fourth feature point P4. In this example, for convenience, the same point as the third feature point P3 is determined as the fourth feature point P4.

Next, as shown in FIG. 20, in the first and second specific point selection steps S324, the first specific point and the second specific point are selected. Similar to the above-mentioned example, first, the values of the second feature point P2 and the third feature point P3 are compared. In this example, the value of the second feature point P2 and the value of the third feature point P3 are substantially the same. In this case, for convenience, the third feature point P3 is selected as the first specific point. Next, it is determined whether or not the value of the first feature point P1 is sufficiently smaller than the reference value Ls. This judgment is made, for example, between the difference between the reference value Ls and the value of the first feature point P1 which is lower than the reference value Ls and the value of 5% of the difference between the value of the third feature point P3 and the reference value Ls. Perform by comparison. When the difference between the reference value Ls and the first feature point P1 is larger than the value of 5% of the difference between the value of the third feature point P3 and the reference value Ls, the first feature point P1 is the reference value. It is determined that it is sufficiently smaller than Ls, and that the graph forms a clear valley at the first feature point P1. As a result, the first feature point P1 is selected as the second specific point. Then, in the mean value point specifying step S325, the differential waveform is traced starting from the third feature point P3 located on the direction x2 side of the first feature point P1 and the third feature point P3 on the time axis x, and first the first. A point that takes the average value of one specific point and a second specific point is determined as an average value point PA. The time point of this average value point PA is determined as the time point of reaching the interface in this example.

In the waveform shown in FIG. 21, the most distant point PL located on the direction y2 side as the point most distant from the reference value Ls in the most distant point determination step S322 with reference to the reference value Ls determined in the reference value determination step S321. Has been decided. Next, in the first to fourth feature point determination steps S323, the point located closest to the direction y1 within the predetermined search time with reference to the farthest point PL (in other words, with reference to the reference value Ls) is the first. It is determined as a feature point P1. In this example, as the first feature point P1, a point located on the direction x2 side with respect to the farthest distance point PL is selected. Next, a point located on the direction x1 side with respect to the first feature point P1 and on the direction y2 side is determined as the second feature point P2. In this example, the farthest distance point PL is determined as the second feature point P2. Next, the point located on the direction x2 side with respect to the first feature point P1 and on the most direction y2 side is determined as the third feature point P3. In this example, the third feature point P3 is a point that takes substantially the same value as the reference value Ls, and in the illustrated example, the bending point of the differential waveform is selected for convenience. Next, the point located on the direction x2 side with respect to the third feature point P3 and on the most direction y1 side is determined as the fourth feature point P4. In this example, for convenience, the same point as the third feature point P3 is determined as the fourth feature point P4.

Next, as shown in FIG. 22, in the first and second specific point selection steps S324, the first specific point and the second specific point are selected. As described above, in the comparison of the values of the second feature point P2 and the third feature point P3, the value of the second feature point P2 is larger than the value of the third feature point P3. Therefore, the second feature point P2 is selected as the first specific point. Here, since the second feature point P2 is the point of the first to fourth feature points that is most in the x1 direction on the time axis x, there is only the first feature point P1 that is adjacent to the second feature point, and the first feature point is inevitably the first. The feature point P1 is selected as the second specific point. Then, in the mean value point specifying step S325, the differential waveform is traced starting from the first feature point P1 located on the direction x2 side of the first feature point P1 and the second feature point P2 on the time axis x, and first the first. A point that takes the average value of one specific point and a second specific point is determined as an average value point PA. The time point of this average value point PA is determined as the time point of reaching the interface in this example.

In the waveform shown in FIG. 23, the most distant point PL located on the direction y2 side as the point most distant from the reference value Ls in the most distant point determination step S322 with reference to the reference value Ls determined in the reference value determination step S321. Has been decided. Next, in the first to fourth feature point determination steps S323, the point located closest to the direction y1 within the predetermined search time with reference to the farthest point PL (in other words, with reference to the reference value Ls) is the first. It is determined as a feature point P1. In this example, the first feature point P1 is a point that takes substantially the same value as the reference value Ls, and in the illustrated example, the bending point of the differential waveform is selected for convenience. Next, a point located on the direction x1 side with respect to the first feature point P1 and on the direction y2 side is determined as the second feature point P2. In this example, for convenience, the same point as the first feature point P1 is determined as the second feature point P2. Next, the point located on the direction x2 side with respect to the first feature point P1 and on the most direction y2 side is determined as the third feature point P3. In this example, the same point as the farthest point PL is determined as the third feature point P3. Next, the point located on the direction x2 side with respect to the third feature point P3 and on the most direction y1 side is determined as the fourth feature point P4. In this example, the fourth feature point P4 is a point that takes substantially the same value as the reference value Ls, and in the illustrated example, the bending point of the differential waveform is selected for convenience.

Next, as shown in FIG. 24, in the first and second specific point selection steps S324, the first specific point and the second specific point are selected. As described above, in the comparison of the values of the second feature point P2 and the third feature point P3, the value of the third feature point P3 is larger than the value of the second feature point P2. Therefore, the third feature point P3 is selected as the first specific point. Next, it is determined whether or not the value of the first feature point P1 is sufficiently smaller than the reference value Ls. This judgment is made, for example, between the difference between the reference value Ls and the value of the first feature point P1 which is lower than the reference value Ls and the value of 5% of the difference between the value of the third feature point P3 and the reference value Ls. Perform by comparison. In this example, since the first feature point P1 is almost the same value as the reference value Ls, the difference between the reference value Ls and the first feature point P1 is the difference between the value of the third feature point P3 and the reference value Ls. It is less than the value of 5% of. Therefore, it is not determined that the first feature point P1 is sufficiently smaller than the reference value Ls, and it is determined that the graph does not form a clear valley at the first feature point P1. As a result, the fourth feature point P4 is selected as the second specific point. Then, in the mean value point specifying step S325, the differential waveform is traced starting from the fourth feature point P4 located on the direction x2 side of the third feature point P3 and the fourth feature point P4 on the time axis x, and first the first. A point that takes the average value of one specific point and a second specific point is determined as an average value point PA. The time point of this average value point PA is determined as the time point of reaching the interface in this example.

<Component identification step S33>
In this step, the components contained in the sample solution are identified by using the relationship between the elapsed time from the start of voltage application and the optical measured values such as absorbance and the interface arrival time point obtained in the interface arrival time determination step S32. .. Alternatively, the components contained in the sample solution may be identified by using an optical measurement value such as an absorbance at an elapsed time elapsed from the time when the interface is reached. Since the relationship between the elapsed time from the start of voltage application and the absorbance is represented by the measured waveform obtained in the waveform forming step S31, the measured waveform can also be used. For example, with respect to the measured waveform obtained in the waveform forming step S31, the elapsed time from the time when the interface is reached is assumed to be the time axis. Then, by comparing the configuration of the plurality of peak waveform portions appearing in this waveform with the basic waveform data for each component prepared in advance for the components that can be contained in the mixed sample Sm (sample Sa), the sample Sa is obtained. Identify the components involved. Such basic waveform data is stored in, for example, the memory of the control unit 8. The identification using the basic waveform data is an example of a specific identification method in the component identification step S33, and the specific method is not limited as long as it is an identification method based on the time when the interface is reached.

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

According to the present embodiment, the time when the interface between the mixed sample Sm as the sample solution and the running solution Lm reaches the measurement unit is determined as the interface arrival time, and the component identification step is performed using this interface arrival time. Therefore, for example, due to differences in the specific configuration and analysis conditions of the analysis chip 2, even if the arrival of the interface is before or after the time axis, the time point that becomes the reference of the component identification process is appropriate for the actual timing of arrival at the interface. Can be adapted to. The event of the arrival of the interface has a significant and certain association with the subsequent arrival and arrival of the various specific components on the time axis of the waveform. Therefore, it is possible to suppress the identification error caused by the inaccuracy of the reference time in the component identification step, and it is possible to perform more accurate analysis.

FIG. 25 shows an example of the component identification step S33 in the present embodiment. In this example, the peaks of HbF and HbA1c are identified as having an accurate range at an accurate time axis position by using the interface arrival time point determined by the interface arrival time point determination step S32. On the other hand, FIG. 26 shows the identification result of the component identification step S33 in the comparative example. In this example, the time point of the farthest separation point PL is adopted as the interface arrival time point itself, and the time point shifted by 0.5 seconds from the accurate interface arrival time point is adopted as the interface arrival time point in the component identification step S33. It ends up. Therefore, due to the error at the time of reaching the interface, a peak at an irregular position different from that in FIG. 25 has been identified as the peak of HbF. Further, as the peak of HbA1c, a peak at substantially the same position as the peak of FIG. 25 has been identified. However, regarding the HbA1c measured value, in the comparative example (FIG. 26) in which the time point of the farthest distance point PL was adopted as the interface arrival time point itself, the HbA1c measured value was 4.89%, and in this embodiment (FIG. 25), the HbA1c measured value was 4.89%. The HbA1c value is 5.36%, which includes an error of 0.47%. The cause of this error is considered as follows. First, the identification of the HbA1c peak is determined based on the ratio of the integrated area value of the HbA1c peak to the area value of the total HbA. Here, the area value of HbF is not included in the area value of total HbA. Therefore, it is considered that the area value of the total HbA changed due to the erroneous recognition of HbF, and the measured value of HbA1c contained an error. As described above, if the interface arrival time point is determined by the interface arrival time point determination step S32 of the present embodiment, the component identification step S33 can be executed more accurately.

Further, by executing the differential waveform forming step S311 in the waveform forming step S31, the interface reaching time point is determined using the differential waveform of the absorbance in the interface reaching time point determination step S32. The differential waveform can express the change in absorbance due to the arrival of the interface more steeply. Therefore, the time when the interface is reached can be determined more accurately.

Differentiation waveform by selecting the first specific point and the second specific point in the first and second specific point selection steps S324 and determining the point to take the average value of these in the average value point identification step S325. The time point of arrival at the interface can be determined more accurately based on the peak caused by the interface appearing in.

Further, in the reference value determination step S321, the reference value Ls is determined, and in the maximum distance point determination step S322, the maximum distance point PL is determined with reference to the reference value Ls.

In the first to fourth feature point determination steps S323, the first feature point P1 to the fourth feature point P4 are determined with reference to the farthest point PL (in other words, with reference to the reference value Ls). It is a rational logic that the predetermined search time with respect to the farthest point PL includes a time point suitable for assuming that the time point when the interface actually arrives or the time point when the interface reaches. Then, by determining the first feature point P1 to the fourth feature point P4 with reference to the farthest point PL (in other words, with reference to the reference value Ls), FIGS. 11 (17), 19 and 21, It is possible to objectively and automatically determine the characteristic points of the various typical differential waveforms shown in FIG. 23 regardless of the subjectivity. This is suitable for performing more accurate analysis with good reproducibility regardless of the skill and experience of the user or the like when performing automatic analysis using the analysis system A1 on various samples.

Selecting the first specific point and the second specific point from the first feature points P1 to the fourth feature points P4 is suitable for performing more accurate analysis automatically and with good reproducibility. Further, in the mean value point specifying step S325, when determining the mean value point PA that takes the average value of the first specific point and the second specific point, either one side of the time axis x (in the above example, the direction). By searching the mean value point PA starting from the point located on the x2 side), the mean value point PA is determined with good reproducibility even when a fine partial peak is included within the predetermined search time. be able to.

In the above, the first and second specific points are the two points most distant from the first feature point, the second feature point, the third feature point, and the fourth feature point along the differential value axis. Although it was selected as a point, if two of these feature points are separated by a predetermined interval or more, they are selected as the first and second specific points, even if they are not necessarily the two most separated points. It may be that.

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

A1 Analytical system 1 Analytical device 2 Analytical chip 2A Upper part 2B Lower part 5 Detector 6 Dispenser 8 Control unit 21 Main body 22 Mixing tank 23 Introduction tank 24 Filter 25 Discharge tank 26 Electrode tank 27 Capillary pipe 28 Communication flow path 31 , 32 Electrode 41 Light source 42 Optical filter 43 Lens 44 Slit 61 Pump 71 Diluting solution tank 72 Running solution tank Bn1, Bn2 Bin Ld Diluting liquid Lm Running solution Ls Reference value P1 First feature point P2 Second feature point P3 Third feature point P4 4th feature point PA average value point PL maximum distance point S1 preparation step S11 sampling step S12 mixing step S13 running solution filling step S14 introduction step S2 electrophoresis step S3 analysis step S31 waveform forming step S311 differential waveform forming step S32 reaching the interface Time point determination step S321 Reference value determination step S322 Maximum distance point determination step S323 First to fourth feature point determination steps S324 First and second specific point selection steps S325 Average value point identification step S33 Component identification step Sa Sample Sm Mixed sample x Time axis x1, x2 Direction along the time axis y Differential value axis y1, y2 Direction along the differential value axis

Claims (13)

Analysis of the sample by capillary electrophoresis, in which a voltage is applied to the sample solution introduced into the microchannel, the components contained in the sample solution are separated and analyzed, and the optical measurement value corresponding to the elapsed time after the start of measurement is measured. It's a method
A step of determining the interface arrival time point based on the optical measurement value when the interface between the sample solution and the running solution reaches a predetermined measurement position in the microchannel.
A step of identifying a component contained in the sample solution using the optical measurement value in the elapsed time elapsed from the time of reaching the interface, and a step of identifying the component contained in the sample solution.
Analytical methods, including. The analysis method according to claim 1, wherein the sample solution is a solution containing blood as a sample, and the component is hemoglobin. The analysis according to claim 1 or 2, wherein in the step of determining the interface arrival time point, the interface arrival time point is determined based on the change of the optical measurement value when the interface reaches the predetermined measurement position. Method. The optical measurement value is the absorbance of the sample solution.
Prior to the step of determining the time point of reaching the interface, there is a step of forming a waveform related to the absorbance corresponding to the elapsed time after the start of measurement.
The analysis method according to claim 3, wherein the change in the optical measurement value in the step of determining the interface arrival time point is a change in the waveform. The step of forming the waveform includes a step of forming a differential waveform in which the differential value obtained by time-differentiating the waveform related to the absorbance is represented as a waveform corresponding to the elapsed time.
The analysis method according to claim 4, wherein the differential waveform is used in the step of determining the time point of reaching the interface. The step of determining the interface arrival time point includes a step of determining a reference value determined based on the differential waveform within a predetermined search time.
A step of determining a first specific point and a second specific point based on the degree of separation from the reference value within the predetermined search time, and
Specify an average value point located between the first specific point and the second specific point on the time axis and having the average value of the differential values of the first specific point and the second specific point as differential values. Steps and
The analysis method according to claim 5, further comprising a step of determining the time point of the average value point as the time point of reaching the interface. The step of determining the first specific point and the second specific point is
A step in which the point having the value most distant from the reference value in the negative direction along the differential value axis within the predetermined search time is set as the first feature point.
A step in which the second feature point is a point that takes the value farthest from the first feature point along the differential value axis on the negative direction side along the time axis within the predetermined search time.
A step in which a point having a value farthest from the first feature point along the differential value axis on the positive direction side along the time axis within the predetermined search time is set as the third feature point.
A step in which the fourth feature point is a point that takes a value farthest from the third feature point on the positive direction side along the time axis within the predetermined search time.
The two points most distant from the first feature point, the second feature point, the third feature point, and the fourth feature point along the differential value axis are designated as the first specific point and the second specific point. The analysis method according to claim 6, which comprises a step of selection. The analysis method according to any one of claims 1 to 7, which is carried out using a disposable type analysis chip in which the fine flow path is formed. A measuring unit that measures the optical measurement value of the liquid in the microchannel,
A sample analysis system based on a separation analysis method, comprising a control unit that performs analysis processing using the measurement results of the measurement unit.
In the separation analysis method, a voltage is applied to the sample solution introduced into the microchannel, the components contained in the sample solution are separated and analyzed, and the optical measurement value corresponding to the elapsed time after the start of measurement is measured. The sample is analyzed by capillary electrophoresis.
The control unit
A means for determining the interface arrival time based on the optical measurement value when the interface between the sample solution and the running solution reaches a predetermined measurement position in the microchannel.
A means for identifying a component contained in the sample solution using the optical measurement value in the elapsed time elapsed from the time when the interface is reached, and
Analytical system, including. The analysis system according to claim 9, wherein the sample solution is a solution containing blood as a sample, and the component is hemoglobin. The analysis system according to claim 9 or 10, wherein the means for determining the interface arrival time determination determines the interface arrival time point based on a change in the optical measurement value when the interface reaches the predetermined measurement position. .. The optical measurement value is the absorbance of the sample solution.
It has a means for forming a waveform related to the absorbance corresponding to the elapsed time after the start of measurement.
The analysis system according to claim 11, wherein the means for determining the interface arrival time is determined as the change in the optical measurement value based on the change generated in the waveform. The analysis system according to any one of claims 9 to 12, which uses a disposable type analysis chip in which the fine flow path is formed.

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