JP6350349B2 - Capillary electrophoresis apparatus and sample analysis method using capillary electrophoresis - Google Patents

Description

  The present invention relates to a capillary electrophoresis apparatus for detecting a component by introducing a sample into a capillary channel, and then applying a voltage between both ends of the capillary channel to cause electrophoresis of the component contained in the sample, and the sample by capillary electrophoresis It relates to the analysis method.

  An analysis method by electrophoresis is known as a method for analyzing a very small amount of protein or nucleic acid. A typical example is capillary electrophoresis. As a method for introducing a sample into a capillary channel for analysis by electrophoresis, an electrokinetic method and a cross injection method are known (see, for example, Patent Document 1).

  In the electrokinetic method, a sample is directly introduced into one end of a capillary channel for analysis. In the electrokinetic method, a marker is set at a position different from a component to be measured whose concentration is to be known, for example, a fragment to be measured.

  In the cross-injection method, a sample is introduced into a loading channel for introducing a sample that intersects a capillary channel for analysis, and is introduced from the loading channel into a capillary channel for analysis.

In the analysis method by capillary electrophoresis, the concentration of the fragment contained in the sample is reflected in the detection signal intensity.
As shown in FIG. 9, in the cross injection method (see solid line), the concentration ratio of each fragment contained in the sample is reflected in the signal intensity. In addition, in the electrokinetic method (see dotted line), the concentration of each fragment introduced into the capillary channel varies depending on the injection conditions, and the concentration ratio of each fragment is not uniform, resulting in a decrease in quantitative accuracy. Therefore, when the concentration is quantified by capillary electrophoresis, a cross injection method in which the concentration ratio is uniform is generally used.

JP 2002-310858 A

  The cross injection method has a problem that the filling of the separation medium into the capillary channel becomes complicated. In addition, there is a problem that throughput is deteriorated because the loading channel needs to be filled with the sample. Further, the cross-injection method is not suitable for a multi-channel in which analysis is performed simultaneously using a plurality of capillary channels because the channel configuration is complicated.

  For example, when the electrokinetic method is used in order to simplify the system, there is a problem in that quantitativeness is sacrificed because the injection amount of each fragment varies depending on the electrophoresis conditions even if the fragment has the same concentration.

  An object of the present invention is to improve the quantitativeness of a measurement target component even when a sample is introduced into a capillary channel by an electrokinetic method.

  In one aspect of the capillary electrophoresis apparatus of the embodiment of the present invention, after a sample is introduced into a capillary channel, a voltage is applied between both ends of the capillary channel to detect components contained in the sample by electrophoresis. A capillary electrophoresis apparatus, comprising: a measurement target component labeled with a first dye as a sample; a measurement target reference labeled with a second dye different from the first dye and having a known concentration; A marker component labeled with the first dye and having an electrophoretic mobility different from the component to be measured; a marker reference labeled with the second dye and having a known concentration ratio with the marker component; When the component including is used, the measurement target component, the measurement target reference, the marker component, and the marker reference are detected, respectively. A concentration ratio between the marker component and the marker reference, a detection signal of the marker component, and a detection signal of the marker reference, and a detection signal intensity ratio of the first dye and the second dye, A calculation unit that calculates a concentration of the measurement target component based on a detection signal intensity ratio between the detection signal of the measurement target component and the detection signal of the measurement target reference, and a known concentration of the measurement target reference; It is.

  In another aspect of the capillary electrophoresis apparatus according to the embodiment of the present invention, after a sample is introduced into the capillary channel, a voltage is applied between both ends of the capillary channel to detect components contained in the sample by electrophoresis. A capillary electrophoresis apparatus for detecting the intensity of a detection signal between a measurement target component labeled with a first dye and a dye different from the first dye and having the same concentration as the first dye as the sample. A detection unit for detecting the measurement target component and the measurement target reference, respectively, when a measurement target reference that is labeled with a known second dye and has a known concentration is used. A known detection signal intensity ratio between one dye and the second dye, a detection signal intensity ratio between the detection signal of the measurement target component and the detection signal of the measurement target reference, and the measurement target reference And known concentration of the Reference, in which and a calculation unit for calculating a concentration of the measurement target component based on.

  One aspect of the sample analysis method by capillary electrophoresis according to an embodiment of the present invention is that, after a sample is introduced into a capillary channel, a voltage is applied across the capillary channel to cause electrophoresis of components contained in the sample. Sample analysis method using capillary electrophoresis to detect the component, wherein the sample is a measurement target component labeled with a first dye and a component labeled with a second dye different from the first dye and having a known concentration The concentration ratio of the measurement target reference, the marker component labeled with the first dye and having an electrophoretic mobility different from that of the measurement target component, and the marker component labeled with the second dye. Using a known marker reference, the measurement target component, the measurement target reference, the marker component and the marker reference A detection signal intensity ratio between the first dye and the second dye obtained by the detection step, the concentration ratio between the marker component and the marker reference, the detection signal of the marker component, and the detection signal of the marker reference; And calculating the concentration of the measurement target component based on the detection signal intensity ratio of the detection signal of the measurement target component and the detection signal of the measurement target reference, and the known concentration of the measurement target reference. .

  Another aspect of the sample analysis method by capillary electrophoresis according to the embodiment of the present invention is that, after a sample is introduced into a capillary channel, a voltage is applied between both ends of the capillary channel to cause electrophoresis of components contained in the sample. A sample analysis method by capillary electrophoresis for detection, wherein the measurement target component labeled with the first dye and the first dye having a concentration different from that of the first dye and the same concentration are used as the sample. Detecting the measurement target component and the measurement target reference, respectively, using a measurement target reference that is labeled with a second dye whose detection signal intensity ratio is known and has a known concentration. The known detection signal intensity ratio of the first dye and the second dye, the detection signal intensity ratio of the detection signal of the measurement target component and the detection signal of the measurement target reference, Comprising a known concentration of the measurement object reference, and a step of calculating a concentration of the measurement target component based on.

  The sample analysis method using the capillary electrophoresis apparatus and the capillary electrophoresis according to the embodiment of the present invention can improve the quantitativeness of the measurement target component even when the sample is introduced into the capillary channel by the electrokinetic method. it can.

It is a flowchart for demonstrating one Embodiment of the sample analysis method by capillary electrophoresis. It is a schematic block diagram for demonstrating the structural example of one Embodiment of a capillary electrophoresis apparatus. It is a schematic block diagram for demonstrating one structural example of the optical system of the detector of the same capillary electrophoresis apparatus. It is a schematic block diagram for demonstrating one structural example of the density | concentration calculation function of the control part of the same capillary electrophoresis apparatus. It is a figure showing an example of a detection signal in one embodiment of a sample analysis method. It is a flowchart for demonstrating other embodiment of the sample analysis method by capillary electrophoresis. It is the figure which showed an example of the detection signal in embodiment of the sample analysis method. It is a schematic block diagram for demonstrating the structural example of other embodiment of a capillary electrophoresis apparatus. It is a figure for demonstrating the difference of a detection signal when a sample is introduce | transduced by the cross injection method and the electrokinetic method.

In the capillary electrophoresis apparatus according to the embodiment of the present invention, for example, the calculation unit uses a peak area and / or a peak height as the intensity of the detection signal.
In the sample analysis method by capillary electrophoresis according to the embodiment of the present invention, for example, the intensity of the detection signal is a peak area, a peak height, or both.
However, the intensity of the detection signal is not limited to the peak area or peak height.

In the capillary electrophoresis apparatus and the sample analysis method using capillary electrophoresis according to the embodiment of the present invention, a detector capable of detecting at least two types of dyes is used.
FIG. 2 is a schematic configuration diagram for explaining a configuration example of an embodiment of a capillary electrophoresis apparatus.

  For example, a capillary channel 2 made of a fused silica capillary is arranged. At the time of electrophoresis, one end of the capillary channel 2 is immersed in the electrophoresis buffer solution 4a, and the other end is immersed in the electrophoresis buffer solution 6a (see solid line). The electrophoresis buffer solution 4 a is accommodated in the buffer solution container 4. The electrophoresis buffer solution 6 a is accommodated in the buffer solution container 6. An electrophoresis voltage is applied across the capillary channel 2 from the power supply device 8 via the electrophoresis buffer solutions 4a and 6a. The power supply device 8 includes an ammeter that detects a current flowing through the capillary channel 2.

  An auto sampler 10 is provided to inject a sample into one end of the capillary channel 2 prior to electrophoresis. The autosampler 10 holds a plurality of sample containers 12 on a sample plate. At the time of sample analysis, the auto sampler 10 moves the sample container 12 containing the sample to be analyzed to the sample injection position. At the time of sample injection, one end of the capillary channel 2 and the electrode are immersed in the sample in the sample container 12 (see the wavy line). A power supply device 8 applies a voltage for sample injection between the sample container 12 and the migration buffer solution 6a. In this way, the sample is injected into one end of the capillary channel 2.

The capillary channel 2 is accommodated in the thermostatic chamber 14 and maintained at a constant temperature.
A detector 16 (detection unit) is arranged at a detection position on the other end side of the capillary channel 2. The detector 16 can detect at least two types of dyes. The detector 16 is, for example, a laser excitation fluorescence detector.

  A control unit 18 is provided. The control unit 18 controls the operation of the autosampler 10, application of sample injection voltage by the power supply device 8, application of migration voltage by the power supply device 8, and the like. Further, the control unit 18 takes in the detection signal of the detector 16 and processes the data, and performs the operation of identifying and quantifying the sample component separated by electrophoresis.

An input unit 20 for inputting analysis information and the like to the control unit 18 is provided. The input unit 20 is an input device such as a keyboard or a touch panel connected to the CPU, for example.
For example, a display unit 22 is provided for displaying analysis information and the concentration of the measurement target fragment (measurement target component) calculated by the control unit 18. The display unit 22 is a display device such as a monitor or a digital display.

FIG. 3 is a schematic configuration diagram for explaining a configuration example of the optical system of the detector of the capillary electrophoresis apparatus.
The light source of excitation light is a laser, for example. A short pass filter 16a and a dichroic mirror 16b are arranged in this order on the optical path of the laser beam of the excitation light. A lens 16c is disposed on the reflected light path of the dichroic mirror 16b. The light transmitted through the lens 16 c is collected on the capillary channel 2.

  A high frequency cut filter 16d and a dichroic mirror 16e are disposed on the reflected light path from the capillary channel 2 via a lens 16c and a dichroic mirror 16b. A photodetector 16f is arranged at a position for receiving light transmitted through the dichroic mirror 16e.

  A dichroic mirror 16g is disposed on the reflected light path of the dichroic mirror 16e. A photodetector 16h is arranged at a position for receiving light transmitted through the dichroic mirror 16g.

  A dichroic mirror 16i is disposed on the reflected light path of the dichroic mirror 16g. A photodetector 16j is disposed at a position for receiving light transmitted through the dichroic mirror 16i. A photodetector 16k is disposed at a position where the reflected light of the dichroic mirror 16i is received.

An example of the excitation light source, the labeled dye of the sample to be electrophoresed, and the optical characteristics of each optical element will be described.
An argon laser is used as an excitation light source, and the 488 nm laser light is used as excitation light. As labeling dyes, dR110 (generated fluorescence wavelength is 530-535 nm), dR6G (generated fluorescence wavelength is 560-565 nm), dTAMURA (generated fluorescence wavelength is 590-595 nm) and dROX (generated fluorescence wavelength is 615-620 nm). ) At least two kinds of fluorescent dyes are used.

  A filter that transmits light having a wavelength shorter than 750 nm is used as the short pass filter 16a.

  A dichroic mirror 16b that is adjusted to transmit light having a wavelength longer than 525 nm and reflect light having a wavelength shorter than 525 nm is used. The dichroic mirror 16b reflects the excitation light of 488 nm and transmits the fluorescence from the labeling dye used.

  As the high frequency cut filter 16d, a filter that shields light having a wavelength shorter than 525 nm and transmits light having a wavelength longer than 525 nm is used. The high frequency cut filter 16d shields the excitation light component and transmits the fluorescence.

  A dichroic mirror 16e that is adjusted to transmit light having a wavelength longer than 600 nm and reflect light having a wavelength shorter than 600 nm is used. The fluorescence that passes through the dichroic mirror 16e and is detected by the photodetector 16f is only the fluorescence (615-620 nm) generated from dROX.

  A dichroic mirror 16g that is adjusted so as to transmit light having a wavelength longer than 575 nm and reflect light having a wavelength shorter than 575 nm is used. Since the fluorescence transmitted through the dichroic mirror 16g and detected by the photodetector 16h is in the range of 575-600 nm, only the fluorescence (590-595 nm) generated from dTAMURA is obtained.

  A dichroic mirror 16i that is adjusted so as to transmit light having a wavelength longer than 545 nm and reflect light having a wavelength shorter than 545 nm is used. Since the fluorescence transmitted through the dichroic mirror 16i and detected by the photodetector 16j is in the range of 545-575 nm, only the fluorescence (560-565 nm) generated from dR6G is present.

  Since the fluorescence reflected by the dichroic mirror 16i and detected by the photodetector 16k has a wavelength shorter than 545 nm, only the fluorescence (530-535 nm) generated from the dR110 is obtained.

  Excitation light from the excitation light source passes through the short pass filter 16a, is reflected by the dichroic mirror 16b, and is irradiated onto the capillary channel 2 through the lens 16c. The fluorescence generated in the capillary channel 2 is returned to the dichroic mirror 16b through the lens 16c, passes through the dichroic mirror 16b, and the excitation light component is removed by the high frequency cut filter 16d.

  The fluorescence that has passed through the high-frequency cut filter 16d is separated into fluorescence from four types of labeling dyes by the dichroic mirrors 16e, 16g, and 16i, and detected by the photodetectors 16f, 16h, 16j, and 16k. The photodetectors 16f, 16h, 16j, and 16k are, for example, photomultiplier tubes.

  FIG. 4 is a schematic block diagram for explaining a configuration example of the concentration calculation function of the control unit of the capillary electrophoresis apparatus.

  The control unit 18 includes, for example, a micro processing unit including a CPU (Central Processing Unit), a RAM (Random Access Memory), a ROM (Read Only Memory), an EPROM (Erasable Programmable ROM), and the like dedicated to the capillary electrophoresis apparatus provided in the apparatus main body. It is structured around a computer.

  The control unit 18 can be realized by, for example, a workstation outside the capillary electrophoresis apparatus or a personal computer other than the CPU dedicated to the capillary electrophoresis apparatus. The control unit 18 may be configured by a plurality of CPUs, a plurality of workstations, a plurality of personal computers, or a combination thereof.

  The configuration of the control unit 18 includes, for example, functions executed by a program installed in a microcomputer and data held in the EPROM. Instead of EPROM, electrically erasable EEPROM (Electrically Erasable Programmable ROM) or flash memory can be used.

The control unit 18 includes a data holding unit 24 and a calculation unit 26.
The data holding unit 24 holds sample information. The sample information includes concentration information of a measurement target reference that is a fragment having a known concentration. In addition, the sample information includes, for example, the concentration ratio between the marker fragment (marker component) and the marker reference, and the measurement target fragment, the measurement target reference, the marker fragment, and the dye information labeled on the marker reference. The data holding unit 24 also holds necessary analysis information other than sample information.

  The data holding unit 24 is composed of, for example, an EPROM. The data holding unit 24 may be stored in a storage medium outside the microcomputer. The storage medium may be any storage medium that can hold data indicating the relationship between an appropriate column flow rate and carrier gas flow rate. , Magneto-optical disk, CD-ROM, DVD-ROM, DVD-RAM, nonvolatile memory card, and the like.

  The calculation unit 26 calculates the concentration of the measurement target fragment based on the detection signal from the detector 16 and the concentration information of the measurement target reference held in the data holding unit 24. The calculation of the concentration of the measurement target fragment by the calculation unit 26 is realized by, for example, a CPU program. Specific calculation processing will be described later.

  The input unit 20 is for inputting concentration information of the measurement target reference and analysis information such as measurement target fragment, measurement target reference, marker fragment, and dye information labeled on the marker reference. For example, the analysis information input from the input unit 20 is displayed on the display unit 22 by the calculation unit 26. The input unit 20 can also input information other than the analysis information.

  FIG. 1 is a flowchart for explaining an embodiment of a sample analysis method by capillary electrophoresis.

  A sample including a measurement target fragment, a measurement target reference, two marker fragments, and a marker reference is prepared as a sample (step S1).

  The fragment to be measured is a target for concentration measurement and is labeled with a first dye. The measurement target reference is a fragment that is labeled with a second dye different from the first dye and has a known concentration. For example, the measurement target fragment and the measurement target reference are the same fragment except for the label.

  Note that the measurement target fragment and the measurement target reference may be different from each other. Since the measurement target fragment and the measurement target reference are compared in the calculation of the concentration of the measurement target fragment, it is preferable that the detected migration time positions be close to each other. Furthermore, it is preferable that the electrophoretic mobility of the measurement target fragment and the electrophoretic mobility of the measurement target reference are approximately the same.

  The two marker fragments are both labeled with the first dye, have different electrophoretic mobilities, and have different electrophoretic mobilities from the fragments to be measured. The marker reference is labeled with the second dye and has a known concentration ratio with one marker fragment. For example, one marker fragment and the marker reference are the same fragment except for the label, and have the same concentration.

  One marker fragment and the marker reference may be different from each other. Here, since the detection signal of one marker fragment and the marker reference are compared when calculating the concentration of the fragment to be measured, it is preferable that the detected migration time positions are close to each other. Furthermore, it is preferable that the electrophoretic mobility of one marker fragment and the electrophoretic mobility of the marker reference are approximately the same.

  Further, one marker fragment and the marker reference may have different concentrations as long as the concentration ratio is known.

  The first dye and the second dye are different from each other. A 1st pigment | dye and a 2nd pigment | dye are any one of said dR110, dR6G, dTAMURA, and dROX, for example.

  Sample information is input from the input unit 20 and held in the data holding unit 24. Further, the sample is accommodated in the sample container 12 and placed on the autosampler 10 (step S2).

  The power supply device 8 and the autosampler 10 are operated under the control of the control unit 18, and the sample is introduced into one end of the capillary channel 2 by the electrokinetic method (step S3).

  After one end of the capillary channel 2 is immersed in the electrophoresis buffer solution 4 a, the power supply device 8 is operated under the control of the control unit 18 to cause electrophoresis of components contained in the sample in the capillary channel 2. The sample component migrated to the detection position is detected by the detector 16 (step S4). The detection signal of the detector 16 is sent to the calculation unit 26 of the control unit 18.

  FIG. 5 is a diagram showing an example of a detection signal in the embodiment of the sample analysis method. The vertical axis represents the detection signal intensity. The horizontal axis indicates the migration time.

  In the first dye series (solid line), a detection signal MA of one marker, a detection signal S of a measurement target fragment, and a detection signal MB of the other marker appear. In the second dye series (broken line), the detection signal SR of the measurement target reference and the detection signal MAR of the marker reference appear.

  For example, since the measurement object fragment and the measurement object reference are the same fragment except for the label, their electrophoretic mobilities are comparable. In this case, the measurement target fragment detection signal S and the measurement target reference detection signal SR appear at substantially the same migration time position.

  Further, for example, one marker fragment and the marker reference are the same fragment except for the label, and therefore, their electrophoretic mobility is approximately the same. In this case, the marker fragment detection signal MA and the marker reference detection signal MAR appear at substantially the same migration time position. The detection signals MA and MB of the marker fragments at both ends are used for mobility correction.

  The calculation unit 26 detects the concentration ratio between the marker fragment and the marker reference, the detection signal of the marker fragment and the detection signal of the marker reference, the detection signal intensity ratio of the first dye and the second dye, and the detection signal of the measurement target fragment. The concentration of the measurement target fragment is calculated based on the detection signal intensity ratio of the detection signal of the measurement target reference and the known concentration of the measurement target reference (step S5).

For example, the concentration C _S of the fragment to be measured can be calculated by the following equation (1).
C _S = {(S _S × S _MAR ) / (S _SR × S _MA )} × C _SR (1)
In the above equation (1), S _S is the area of the detection signal S of the measurement target fragment. _SSR is the area of the detection signal SR of the measurement target reference. S _MA is the area of the marker fragment detection signal MA. S _MAR is the area of the marker reference detection signal MAR. _CSR is the known concentration of the reference to be measured.

Here, the marker fragment detected by the detection signal MA and the marker reference detected by the detection signal MAR have the same concentration, for example. Accordingly, in the above formula (1), (S _MAR / S _MA ) represents the detection signal intensity ratio between the first dye and the second dye. Note that the marker fragments and the marker reference concentration when a different but the concentration ratio is known to each other, a first dye by integrating or dividing the concentration ratio in the detection signal intensity ratio (S _MAR / S _MA) A detection signal intensity ratio of the second dye is obtained.

  Thus, this embodiment of the sample analysis method by capillary electrophoresis can improve the quantitativeness of the measurement target fragment even when the sample is introduced into the capillary channel 2 by the electrokinetic method.

  In addition, when the detection signal intensity ratio when the first dye and the second dye having the same concentration are detected is known in advance, the concentration of the measurement target fragment can be calculated without mixing the marker reference into the sample. This embodiment will be described.

  FIG. 6 is a flowchart for explaining another embodiment of the sample analysis method by capillary electrophoresis.

  A sample including a measurement target fragment, a measurement target reference, and two marker fragments is prepared as a sample (step S11). These fragments are the same as the fragments in the embodiment described with reference to FIG. 1, for example. However, for the first dye or the second dye labeled with each fragment, the detection signal intensity ratio between the detection signal of the first dye and the detection signal of the second dye when the first dye and the second dye have the same concentration is Known.

  Sample information is input from the input unit 20 and held in the data holding unit 24. At this time, the detection signal intensity ratio between the detection signal of the first dye and the detection signal of the second dye is also input and held as part of the sample information. Further, the sample is accommodated in the sample container 12 and placed on the autosampler 10 (step S12).

  Similar to step S3 described with reference to FIG. 1, the sample is introduced into one end of the capillary channel 2 by the electrokinetic method (step S13). Similar to step S4 described with reference to FIG. 1, components contained in the sample are electrophoresed in the capillary channel 2 and detected by the detector 16 (step S14). The detection signal of the detector 16 is sent to the calculation unit 26 of the control unit 18.

  FIG. 7 is a diagram showing an example of a detection signal in the embodiment of the sample analysis method. The vertical axis represents the detection signal intensity. The horizontal axis indicates the migration time.

  In FIG. 7, as in the description of FIG. 5, the detection signal MA of one marker, the detection signal S of the measurement target fragment, and the detection signal MB of the other marker appear in the first dye series (solid line). In addition, the detection signal SR of the measurement target reference appears in the second dye series (dashed line). However, a marker reference detection signal that is not mixed in the sample in this embodiment does not appear.

  The calculation unit 26 is based on the known detection signal intensity ratio of the first dye and the second dye, the detection signal intensity ratio of the detection signal of the measurement target fragment and the detection signal of the measurement target reference, and the known concentration of the measurement target reference. Thus, the concentration of the measurement target fragment is calculated (step S15).

For example, the concentration C _S of the measurement target fragment can be calculated by the following equation (2).
C _S = k (S _S / S _SR ) × C _SR (2)
In the above equation (2), S _S is the area of the detection signal S of the measurement target fragment. _SSR is the area of the detection signal SR of the measurement target reference. k is a known detection signal intensity ratio between the first dye and the second dye. _CSR is the known concentration of the reference to be measured.

  Thus, this embodiment of the sample analysis method by capillary electrophoresis can improve the quantitativeness of the measurement target fragment even when the sample is introduced into the capillary channel 2 by the electrokinetic method.

  The capillary electrophoresis apparatus used in the embodiment of the sample analysis method by capillary electrophoresis is not limited to the apparatus shown in FIG. A configuration example of another capillary electrophoresis apparatus will be described.

  FIG. 8 is a schematic configuration diagram for explaining a configuration example of another embodiment of the capillary electrophoresis apparatus. In FIG. 8, parts having the same functions as those in FIG. 2 are denoted by the same reference numerals. In this capillary electrophoresis apparatus, a plate in which a plurality of capillary channels are formed is used.

  For example, a plurality of capillary channels 2 are arranged in a substrate 28 made of glass so as not to cross each other. For example, the capillary channel 2 has a width of 90 μm and a depth of 40 μm. The capillary channel 2 extends in the longitudinal direction of the substrate 28, and a plurality of, for example, 384 capillaries are arranged so as not to cross each other in a radial region extending from the anode side toward the cathode side.

  A buffer solution container 4 is attached to the surface of the substrate 28 corresponding to the position of the end of the capillary channel 2 on the cathode side. A buffer solution container 6 is attached corresponding to the position of the end of the capillary channel 2 on the anode side.

  The buffer liquid containers 4 and 6 constitute a container having an open top. Each of the buffer liquid containers 4 and 6 accommodates an electrophoresis buffer solution, and is electrically connected to the end of the capillary channel 2 through the electrophoresis buffer solution at the bottom.

  A plurality of openings 2 a connected to one end of the capillary channel 2 are disposed at the bottom of the buffer solution container 4 on the cathode side (see FIGS. 8B and 8C). In this example, the number of openings 2a is 384. Each of the openings 2a constitutes a sample dispensing position. The buffer liquid container 4 is a single container that surrounds a region where all the openings 2a are disposed. The buffer solution container 6 on the anode side is also connected to the other end of the capillary channel 2 at the bottom.

  The material of the substrate 28 is, for example, quartz glass or borosilicate glass. Further, as the material of the substrate 28, another material such as a resin can be used instead of glass. Here, since the components separated by electrophoresis are optically detected, a material having optical transparency is selected as the material of the substrate 28. However, when using a detection means other than the optical detector, the material of the substrate 28 is not limited to one having optical transparency.

  For example, the substrate 28 may be a laminate of two glass plates. The capillary channel 2 can be formed on the joining surface of one glass plate by photolithography and etching techniques (wet etching or dry etching). The openings 2a and the like are formed as through holes in the same glass plate or the other glass plate at positions corresponding to the positions of both ends of the capillary channel 2 by a method such as etching, sandblasting, or laser drilling. Can do.

  A detector 16 such as a laser excitation fluorescence detector is disposed at a detection position on the buffer liquid container 6 side on the anode side of the capillary channel 2. The detector 16 can detect a labeled fragment for each capillary channel 2. Moreover, the power supply device 8, the control part 18, the input part 20, and the display part 22 are provided similarly to the capillary electrophoresis apparatus demonstrated with reference to FIG.

The operation of this capillary electrophoresis apparatus will be briefly described.
In order to inject a sample into one end of the capillary channel 2 prior to electrophoresis, the sample is injected into the sample dispensing opening 2a by, for example, a pipetter mechanism. Thereafter, the tip of each electrode is immersed in the sample injected into the opening 2a, and a predetermined voltage is applied between the electrode immersed in the migration buffer solution of the buffer solution container 6 on the anode side, and the electrokinetic method is applied. Thus, the sample is introduced into the capillary channel 2.

  The sample remaining in the sample dispensing opening 2a is removed by suction with a suction mechanism such as an aspirator. Thereafter, the electrophoresis buffer solution is supplied into the buffer solution container 4 on the cathode side. A voltage is applied to both ends of the capillary channel 2 by the power supply device 8 via the electrophoresis buffer solution, and the components contained in the sample are electrophoresed in the capillary channel 2. The sample component migrated to the detection position is detected for each capillary channel 2 by the detector 16. The calculation unit 26 of the control unit 18 calculates the concentration of the measurement target fragment for each capillary channel 2 in the same manner as described in the embodiment of the sample analysis method described with reference to FIG. 1 or FIG.

  As mentioned above, although embodiment of this invention was described, the structure, arrangement | positioning, a numerical value, material, etc. in embodiment are examples, This invention is not limited to this, This invention described in the claim Various changes can be made within the range.

  For example, in the above embodiment, the concentration of the measurement target fragment is calculated based on the peak area. However, the concentration of the measurement target fragment may be calculated based on the peak height or the peak area and the peak height.

  Note that the capillary electrophoresis apparatus and the sample analysis method using capillary electrophoresis according to the embodiment of the present invention are also effective when the sample is introduced by the cross injection method.

  Further, the capillary electrophoresis apparatus according to the embodiment of the present invention is not limited to a dedicated apparatus for analyzing the sample. The capillary electrophoresis apparatus according to the embodiment of the present invention may have a function of analyzing a sample different from the sample by electrophoresis in addition to the function of analyzing the sample.

2 Capillary flow path 16 Detector 28 Calculation unit

Claims (6)

A capillary electrophoresis apparatus for detecting a component contained in a sample by applying a voltage between both ends of the capillary channel after introducing the sample into the capillary channel,
As the sample,
A measurement target component labeled with a first dye;
A measurement target reference labeled with a second dye different from the first dye and having a known concentration;
A marker component labeled with the first dye and having an electrophoretic mobility different from that of the measurement target component;
When a marker label that is labeled with the second dye and has a known concentration ratio with the marker component is used,
A detector for detecting the measurement target component, the measurement target reference, the marker component, and the marker reference;
The concentration ratio between the marker component and the marker reference, the detection signal intensity ratio of the first dye and the second dye obtained by the detection signal of the marker component and the detection signal of the marker reference, and the measurement target component A capillary electrophoresis apparatus comprising: a detection signal intensity ratio between a detection signal and a detection signal of the measurement target reference; and a calculation unit that calculates the concentration of the measurement target component based on a known concentration of the measurement target reference. A capillary electrophoresis apparatus for detecting a component contained in a sample by applying a voltage between both ends of the capillary channel after introducing the sample into the capillary channel,
As the sample,
A measurement target component labeled with a first dye;
A measurement target reference that is a dye different from the first dye, labeled with a second dye having a known detection signal intensity ratio with the first dye having the same concentration, and a component having a known concentration Is used when
A detection unit for detecting the measurement target component and the measurement target reference, and
Based on the known detection signal intensity ratio of the first dye and the second dye, the detection signal intensity ratio of the detection signal of the measurement target component and the detection signal of the measurement target reference, and the known concentration of the measurement target reference A capillary electrophoresis apparatus comprising: a calculation unit that calculates the concentration of the measurement target component.   The capillary electrophoresis apparatus according to claim 1, wherein the calculation unit uses a peak area and / or a peak height as the intensity of the detection signal. A sample analysis method by capillary electrophoresis in which a sample is introduced into a capillary channel, and then a voltage is applied between both ends of the capillary channel to detect components contained in the sample by electrophoresis.
As the sample,
A measurement target component labeled with a first dye;
A measurement target reference labeled with a second dye different from the first dye and having a known concentration;
A marker component labeled with the first dye and having an electrophoretic mobility different from that of the measurement target component;
Using a marker reference that is labeled with the second dye and has a known concentration ratio with the marker component,
Detecting each of the measurement target component, the measurement target reference, the marker component, and the marker reference;
The concentration ratio between the marker component and the marker reference, the detection signal intensity ratio of the first dye and the second dye obtained by the detection signal of the marker component and the detection signal of the marker reference, and the measurement target component A sample analysis method by capillary electrophoresis, comprising: a detection signal intensity ratio between a detection signal and a detection signal of the measurement target reference; and a concentration of the measurement target component based on a known concentration of the measurement target reference . A sample analysis method by capillary electrophoresis in which a sample is introduced into a capillary channel, and then a voltage is applied between both ends of the capillary channel to detect components contained in the sample by electrophoresis.
As the sample,
A measurement target component labeled with a first dye;
A measurement target reference that is a dye different from the first dye, labeled with a second dye having a known detection signal intensity ratio with the first dye having the same concentration, and a component having a known concentration Use
Detecting each of the measurement target component and the measurement target reference;
Based on the known detection signal intensity ratio of the first dye and the second dye, the detection signal intensity ratio of the detection signal of the measurement target component and the detection signal of the measurement target reference, and the known concentration of the measurement target reference And a step of calculating the concentration of the component to be measured, and a sample analysis method by capillary electrophoresis.   The sample analysis method by capillary electrophoresis according to claim 4 or 5, wherein the intensity of the detection signal is a peak area and / or a peak height.

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