JP3481831B2 - Capillary electrophoresis apparatus and capillary state determination method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an electrophoresis apparatus for separating and analyzing nucleic acids, proteins, sugars and the like and a state of a capillary.
In particular, the present invention relates to a capillary electrophoresis apparatus and a capillary state determination method suitable for detection of DNA (nucleic acid) or the like, determination of base sequence, etc. I'm concerned.

[0002]

2. Description of the Related Art Conventionally, many analyzes using electrophoresis have been used, but an important analysis among them is DNA sequencing. For the base sequence determination, conventionally, an electrophoretic medium formed by sandwiching a polyacrylamide gel between two glass plates was used. On the other hand, recently, as described in, for example, Japanese Patent Laid-Open No. 6-138037, an electrophoretic medium in which a gel is filled in a capillary that facilitates handling and enables analysis in a short time is used. The method is being developed.

[0003]

However, in the conventional method, even when the capillary is used as the electrophoretic medium, the filled gel is consumed every measurement and is replaced every time. Replace the gel with an inner diameter of 100μ
A method of pressurizing and pressing the gel into a capillary of m or less using a cylinder or the like is used, but the operation of filling the gel of a large number of capillaries takes time. Further, when the capillaries are replaced every time, it is difficult to automatically analyze a plurality of samples because the capillaries are replaced manually.

In order to reduce the exchange work and to promote automation, it is sufficient to make it possible to use the same gel repeatedly for a plurality of times. However, simple repeated use causes deterioration in performance, so that the reliability of data is improved. There was a problem that could not be secured.

An object of the present invention is to provide a capillary electrophoresis apparatus capable of ensuring the reliability of data even when it is repeatedly used for a plurality of analyzes without exchanging the gel.
Another object of the present invention is to provide a method for determining the state of a capillary .

[0006]

(1) To achieve the above object, the present invention provides a plurality of capillaries filled with an electrophoretic medium and a plurality of capillaries introduced into these capillaries and electrophoretically separated. Having a detector for optically detecting sample components, applying a voltage to both ends of the capillary, separating and developing while moving the sample introduced to one end of the capillary toward the other end, sample a capillary electrophoresis apparatus for separating and analyzing the components in the above
The signal corresponding to each component in the sample detected by the detector
Find the half-width of a given single peak in the No. peak,
If the calculated half width is narrower than the predetermined reference level
First, a capillary determining means for determining that the state of the capillary is good is provided. With such a configuration, the reliability of the data can be secured even when the gel is repeatedly used for a plurality of analyzes without exchanging it.

[0007]

[0008] (2) A (1), preferably,
The detector optically detects a plurality of sample components electrophoretically separated by laser-excited multi-wavelength fluorescence detection, and a predetermined single peak used by the capillary determination means for determining the quality of the capillary state is detected. Among the plurality of sample component peaks, the fluorescence spectrum has a maximum peak on the short wavelength side.
With such a configuration, it is possible to reduce the influence of the peaks of other sample components and obtain the full width at half maximum.

[0009] (3) A (2), preferably,
Furthermore, the peaks appearing on both sides of a predetermined single peak used by the capillary determining means for determining the quality of the capillary state, among the peaks of a plurality of sample components to be detected, the fluorescence spectrum has a maximum peak on the long wavelength side. It is supposed to have. With this configuration, the full width at half maximum can be obtained without being affected by peaks of other sample components.

[0010] (4) The above (1), preferably,
The detector optically detects a plurality of sample components electrophoretically separated by laser-excited multi-wavelength fluorescence detection, and a predetermined single peak used by the capillary determination means for determining the quality of the capillary state is detected. Among the multiple sample component peaks, the fluorescence spectrum has the second maximum peak from the short wavelength side, and the peaks appearing on both sides of this peak are among the multiple sample component peaks to be detected. The fluorescence spectrum has a maximum peak on the long wavelength side. With such a configuration, even if there is no one having a maximum peak on the short wavelength side, it is possible to determine the quality of the capillary state using a predetermined peak.

[0011] (5) The above (1), preferably,
The predetermined single peak used by the capillary judging means for judging the quality of the capillary is the predetermined peak in the standard sample introduced into the capillary. With such a configuration, the already known peak can be used, so that the quality of the capillary state can be easily determined.

[0012] (7) To achieve the above object, the present invention includes a Capilla Larry capable of holding an electrophoretic medium, this key
A power supply that applies voltage across the capillary and the above cap
Introduced from one end of the rally and separated by electrophoresis
The detector that detects the sample and the detector detected by this detector
Obtain the half-value width of a single peak from the signal and compare it with the reference value
Then, the capillary that determines the state of the above-mentioned capillary
And a determination device.

(8) In the above (7), preferably,
A table displaying the determination result by the above capillary determination device.
The display part is provided.

(9) In the above (8), preferably,
The display section displays the full width at half maximum of the single peak.
It is a scam.

(10) The above (8), preferably
Indicates that the display unit is in a good capillary state and / or
Is for displaying defects. (11) In the above (7), preferably, the cap
The capillary is judged to be defective by the rally judgment device.
Input section for instructing to continue or stop the analysis when specified.
It was prepared. (12) In the above (7), preferably the cap
The rally decision device uses the detected signal to identify a single
The peak is selected and its half-width is calculated.
is there. (13) In order to achieve the above-mentioned object, the present invention provides an electric swimming.
It is a method of determining the state of a capillary that can hold a moving medium.
The sample into one end of the capillary and
Apply voltage to both ends of the pillary and separate by electrophoresis.
Detected sample with a detector, and with a judgment device
The half value width of the single peak is calculated from the measured signal and the reference value
Compare with the above to determine the status of the above capillaries
It was done. (14) In the above (13), preferably, the above syn
If the full width at half maximum of the glupeak is wider than the reference value, the capillary
The Lee is determined to be defective. (15) In the above (13), preferably, the above syn
If the full width at half maximum of the glupeak is narrower than the reference value, the capillary
Lee is judged to be normal. (16) In the above (13), preferably the above detection
The container is equipped with multiple fluorescent dyes corresponding to the base type.
It detects the components of the sample optically and uses the above single
Among the peaks of multiple sample components detected,
The fluorescence spectrum has a maximum peak on the short wavelength side.
Of. (17) In the above (16), preferably the syn
Multiple peaks appearing on either side of the glu peak are detected.
Among the peaks of the sample components, the fluorescence spectrum has a long wavelength
It has a maximum peak on the side. (18) In the above (13), preferably the syn
Glue peak is the peak of multiple sample components detected.
Then, the fluorescence spectrum has the second maximum peak from the short wavelength side.
Peaks on either side of this single peak.
In the peaks of the multiple sample components detected.
The fluorescence spectrum has a maximum peak on the long wavelength side.
is there. (19) In the above (13), preferably the above sample
Is the standard sample. (20) In the above (13), preferably the above sample
Is an unknown sample.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The configuration and operation of a capillary electrophoresis apparatus according to an embodiment of the present invention will be described below with reference to FIGS. First, using FIG.
The overall configuration of the capillary electrophoresis device according to the present embodiment will be described. FIG. 1 is a block diagram showing the overall configuration of a capillary electrophoresis device according to an embodiment of the present invention.

First, the overall configuration will be described. An electrophoretic buffer tank 12 containing a buffer (electrolytic solution) is arranged on the moving mechanism 10. Migration buffer tank 12
A platinum electrode 13 is stretched inside the platinum electrode 1
3 is in contact with the buffer. Further, three sample trays 100A, 100B, 100C are arranged on the moving mechanism 10 via the sample tray holder 14. The sample tray 100A includes 48 sample containers as described later with reference to FIG. The sample tray 100A is fixed to the sample tray holder 14 with set screws S1 and S2, and is removable from the sample tray holder 14. Sample tray 1
The bottom of 00A is made of a conductive metal such as SUS, and is made of a conductive metal such as SUS and is electrically connected to the sample tray holder 14. Similarly, the sample trays 100B and 100C are also provided with 48 sample containers.
Is electrically connected to the sample tray holder 14.

The moving mechanism 10 includes a vertical moving motor 16Z.
Can be moved up and down along the vertical slide guide 18Z in the Z-axis direction. Further, the moving mechanism 10 uses the front-rear movement motor 16X to move the front-rear slide guide 18
It is possible to move back and forth along the X in the X-axis direction. The vertical movement motor 16Z and the longitudinal movement motor 16X are controlled by the control device 80. A transparent cover 19 made of vinyl chloride, acrylic resin, or the like is arranged so as to cover the three sample trays 100A, 100B, and 100C, so that evaporation of the sample held in the sample container can be suppressed and externally applied. Prevents the entry of dust.

Forty-eight capillaries 20 are arranged in parallel, and a cross-linked polyacrylamide gel for separation is filled therein as an electrophoretic medium. The lower end side of the capillary 20 is fixed by a capillary retainer 22, and the lower end thereof is inserted into a buffer in the migration buffer tank 12. The upper end side of the capillary 20 is fixed by a capillary retainer 24, and the upper end thereof is connected to the coupler 2
6 is connected and fixed.

The length of the capillary 20 is 30 cm. The cross-linked polyacrylamide gel packed in the capillary 20 consists of all-acrylamide and bis-acrylamide. Total acrylamide is 5% by weight of total gel and bis-acrylamide is 10% by weight of total acrylamide.

The coupler 26 is connected to the electrode plug 32 on the ground side, and the electrode plug 32 is connected to the ground electrode of the high voltage power supply 30. In addition, the sample tray holder 14
Is connected to the electrode plug 34 on the high voltage side, and the buffer tank 1
The second platinum electrode 13 is connected to the electrode plug 36 on the high voltage side, and the electrode plugs 34, 36 are connected to the high voltage (−) pole of the high voltage power supply 30.

The coupler 26 is connected to the sheath flow cell 40. The sheath liquid 44 held in the sheath liquid tank 42 is introduced into the sheath flow cell 40 by gravity. Electrophoretically separated in the capillary 20,
The separated sample flowing out from the migration end portion of the capillary 20 is carried to the upper side while the sample components of the capillaries are separated from each other by the sheath liquid.

Side direction of the sheath flow cell 40 (Y in the drawing)
From the direction), the laser light emitted from the laser 50 is collimated into a parallel light flux by the lens 42 and is then irradiated to excite the separated sample in the sheath flow cell 40. A shutter 5 is provided between the laser 50 and the lens 52.
4 is provided so that the excitation of the sample can be selectively performed.

Fluorescence generated by laser light irradiation is Y
It is taken out from the X-axis direction orthogonal to the axial direction and detected by the detector unit 60. The detector unit 60 is
As will be described later with reference to FIG. 2, it includes a condenser lens 62, a filter 64, an imaging lens 66, and an optical sensor 68. The fluorescence generated by the laser light irradiation is condensed by the condensing lens 62, the light of the wavelength to be detected is selected by the filter 64, and further, the imaging lens 66 is used by the optical sensor 6 such as a two-dimensional CCD sensor.
Image on 8. The signal detected by the optical sensor 68 is sent to the signal processing device 70, signal processing is performed, the type of terminal base is identified by the wavelength of fluorescence, and the base sequence of the nucleic acid sample is analyzed based on the measurement signal.

In determining the base sequence of DNA (deoxyribonucleic acid), generally four wavelengths are measured. A fluorescent dye is previously bound by a reaction operation so that each maximum wavelength corresponds to the type of base at the end of the DNA fragment.

The signal detected by the optical sensor 68 is input to the capillary determination device 200. The capillary determination device 200 obtains the half width of a specific peak in the signal detected by the optical sensor 68, and when the obtained half width is narrower than a reference level, the capillary 20 is in a good state. If it is determined that the capillary 20 is wider than the reference level, the capillary 20 is determined to be in a bad state. Capillary determination device 2
Details of the determination method in 00 will be described later using FIG. The determination result by the capillary determination device 200 is displayed on the display unit 210. The operator determines whether to continue the analysis or replace the capillary based on the determination result of the capillary 20 displayed on the display unit 210, and inputs an instruction based on this determination to the capillary determination device 200 from the input unit 220.

Further, as will be described later, the capillary determination device 200 determines whether the state of the capillary 20 is good or bad based on the current value detected by the current detection circuit 90 connected between the high voltage power supply 30 and the electrode plug 32. A decision can be made. If the detected current value is higher than the reference level, it is determined that the state of the capillary 20 is good, and if it is lower than the reference level, the state of the capillary 20 is bad. Judge that there is. A method for determining the quality based on this current value will also be described later.

A drain adapter 46 is attached to the upper end of the sheath flow cell 40, and the sample flowing into the sheath flow cell 40 from the capillary 20 is discharged as a waste liquid through a drain tube 47 into a drain bottle 49. . A flow controller including an orifice and a plurality of capillaries is provided in the middle of the drain tube 47.
The flow rate is controlled by keeping the flow path resistance of 1 constant.

Next, the overall operation of the electrophoretic analysis device according to the present embodiment will be explained. First, pre-migration before measurement is performed. In the pre-migration, the standard sample and the analytical sample are not introduced into the capillary 20. The lower end of the capillary 20 is inserted into the buffer tank 12, and a voltage is applied from the high voltage power supply 30 to both ends of the capillary 20 to equilibrate the capillary 20. In the pre-electrophoresis, the voltage applied from the power supply 30 is 120 V / cm. If the length of the capillary 20 is 30 cm, the power source 3
From 0 to 3600 V is applied to both ends of the capillary 20. The pre-migration time is 20 minutes.

On the other hand, sample trays 100A, 100
A predetermined amount of the standard sample is dispensed in advance into one of the 48 sample containers of B and 100C.
Here, for example, M13mp18 is used as the standard sample. Other than this, for example, pUC18 or p
GEM can be used as a standard sample. Further, a predetermined amount of analysis sample is dispensed into the remaining 47 sample containers. A sample tray 100A containing a sample,
100B and 100C are fixed to the sample tray holder 14 with set screws S1 and S2. The control device 80 is
The vertical movement motor 16Z is driven to move the movement mechanism 10 to Z1.
Descend in the direction. The lower end of the capillary 20 stops the lowering of the moving mechanism 10 at a position sufficiently separated from the buffer tank 12. Next, the control device 80 controls the front-back movement motor 16X.
To move the moving mechanism 10 in the X1 direction. When the sample tray 100A comes directly under the capillary 20, the movement of the moving mechanism 10 is stopped. Further, the control device 80 drives the vertical movement motor 16Z to move the movement mechanism 1
0 is increased in the Z2 direction. Then, at the position where the tip of the capillary 20 is inserted into the sample in the sample container in the sample tray 100A, the raising of the moving mechanism 10 is stopped.
Positioning of the moving mechanism 10 in the vertical movement operation and the front-back movement operation is performed using a position detection mechanism such as a switch provided in the movement mechanism 10.

With the lower end of the capillary 20 inserted into the sample, the sample tray 100A and the coupler 26 are
The sample in the sample container is introduced into the capillary 20 by applying a high voltage from the high voltage power supply 30 during the period. When the sample is introduced, the voltage applied from the power supply 30 is 50 V / cm for 10 seconds. As a result, the standard sample and the analytical sample contained in the sample container 100A are introduced into the capillary 20.

Next, the controller 80 controls the vertical movement motor 1
6Z is driven to lower the moving mechanism 10 in the Z1 direction.
The lower end of the capillary 20 stops the lowering of the moving mechanism 10 at a position sufficiently separated from the sample tray 100A.
Next, the control device 80 drives the forward / backward moving motor 16X to move the moving mechanism 10 in the X2 direction. Buffer tank 1
When 2 is directly below the capillary 20, the moving mechanism 10
Stop moving. Further, the control device 80 drives the vertical movement motor 16Z to raise the moving mechanism 10 in the Z2 direction. Then, at the position where the tip of the capillary 20 is inserted into the buffer in the buffer tank 12, the moving mechanism 10
Stop rising. Positioning of the moving mechanism 10 in the vertical movement operation and the front-back movement operation is performed using a position detection mechanism such as a switch provided in the movement mechanism 10.

The sample introduced into the capillary 20 by applying a high voltage from the high-voltage power supply 30 between the platinum electrode 13 and the coupler 26 with the lower end of the capillary 20 inserted in the buffer, Separated by electrophoresis. The electrophoretic voltage is 120 V / cm.
The detector unit 60 detects each separated DNA fragment component. Laser-excited fluorescence detection is used for the detection. The signal processing unit 70 identifies the type of terminal base based on the wavelength of the fluorescence detected by the detector unit 60, and determines the base sequence of the sample DNA based on the DNA fragment component.

The state of the capillary 20 is also monitored during the electrophoretic analysis of the sample. The capillary determination device 200 calculates the half width of a specific peak in each DNA fragment component detected by the detector unit 60, and the capillary 2 is calculated based on the calculated half width.
The quality of the state of 0 is judged.

When the analysis of the sample in the sample tray 100A is completed, the samples in the sample trays 100B and 100C are introduced into the capillary 20 and electrophoretically separated by the same procedure as described above.

Since it takes about 2 hours to analyze one sample, as shown in FIG.
3 sample trays 100A, 100B, 1 on 4
By installing 00C, it is possible to perform automatic analysis for about 6 hours. The number of sample trays is not limited to three and may be larger.

Further, the optical detection method of the sample electrophoretically separated is not limited to fluorescence detection, and absorbance detection or the like can be used.

Next, the detailed structure of the detector unit 60 according to the present embodiment and the image on the light receiving surface will be described with reference to FIGS. First, the detailed configuration of the detector unit 60 according to the present embodiment will be described with reference to FIG. FIG. 2 is a block diagram of an optical system showing a configuration of a detector unit used in the capillary electrophoresis analyzer according to the embodiment of the present invention.

In determining the base sequence of DNA (deoxyribonucleic acid), generally four wavelengths are measured. The fluorescent dye is bound by a reaction operation in advance so that each maximum wavelength corresponds to the type of base at the end of the DNA fragment.

The fluorescence emitted from the light emitting point EP is reflected by the lens 62.
Is collected at. The emission point EP is the fluorescence emitted by irradiating the sample separated by the capillary 20 shown in FIG. 1 with the light from the laser 50. Lens 6
Although 2 is shown as one lens in the illustrated example, it can be composed of a plurality of lenses. By arranging the emission point EP and the lens 62 so that the emission point EP is located at the focal position of the lens 62, the light condensed by the lens 62 becomes a parallel light flux and is guided to the detector unit 60.

Here, for the purpose of making a correlation with FIG. 1, the direction in which the capillary 20 extends is the Z-axis direction, and the lens 6
The optical axis that has become a parallel light flux by 2 is the X axis. Also,
As shown in FIG. 1, it is assumed that the plurality of capillaries 20 are arranged parallel to the Y-axis direction perpendicular to the paper surface.

The detector unit 60 includes a condenser lens 62.
, Filter 64, right-angle prisms 65A to 65D
Imaging lenses 66A and 66B, spatial filters (slits) 67A and 67B, and four one-dimensional optical sensors 68A
˜68D.

The filter 64 is a bandpass filter, and is a filter 64 for selecting light of four kinds of wavelengths.
It is composed of A, 64B, 64C and 64D. The filter 64A selects light of the first wavelength λ1, and the wavelength λ1 is, for example, 520 nm, which is a fluorescence peak wavelength of guanine (G) which is one of purine bases which is a nucleic acid constituent. . The filter 64B selects light of the third wavelength λ3, and the wavelength λ3 is, for example, 580 nm, which is the fluorescence peak wavelength of thymine (T), which is one of the purine bases constituting the nucleic acid. . The filter 64C selects light of the second wavelength λ2, and the wavelength λ2 is, for example, 540 nm, and adenine (A) which is one of purine bases which is a nucleic acid constituent component.
It is the fluorescence peak wavelength of. The filter 64D is for selecting the light of the fourth wavelength λ4, and the wavelength λ4 is, for example, 605 nm, which is the fluorescence peak wavelength of cytosine (C) which is one of the purine bases constituting the nucleic acid. .
The bandpass of the filters 64A, 64B, 64C and 64D is 20 nm.

The light flux passing through the filter 64 is bent at a right angle by the four right-angle prisms 65A, 65B, 65C and 65D and is split into four light fluxes. That is, in the optical system of the present embodiment, one light beam is split into four light beams.

The four light beams are converged by the lenses 66A and 66B, and the one-dimensional photosensors 68A, 68B, 68C,
An image is formed on the light receiving surface of 68D. That is, the light emitted from one light emitting point EP is the light of four kinds of wavelengths filtered by the filter 64.
After being selected by A to 64D, the four one-dimensional photosensors 68A to 68D detect the fluorescence intensity of each of the four wavelengths. Here, for example, when 48 capillaries 20 are arranged in parallel, the one-dimensional optical sensor 68 has 48 light receiving surfaces. 48
The individual light receiving surfaces are arranged in the Y-axis direction (direction perpendicular to the paper surface). Therefore, 4 out of 48 capillaries 20
The light emitted from the eight light emitting points is selected by the filters 64A to 64D for the light of four kinds of wavelengths, and then the light receiving surfaces on the four one-dimensional photosensors 68A to 68D respectively The light intensity of fluorescence for four types of wavelengths is detected.

Spatial filters (slits) 67A and 67B are arranged between the lenses 66A and 66B and the one-dimensional photosensors 68A, 68B, 68C and 68D to prevent stray light.

Next, the relationship between the light emitting point and the image formed on the light receiving surface of the one-dimensional photosensor in this embodiment will be described with reference to FIG. FIG. 3 is an explanatory diagram of a relationship between a light emitting point and an image formed on the light receiving surface of the one-dimensional photosensor in the multicolor fluorescence detection electrophoretic analysis device according to the embodiment of the present invention.

FIG. 3A shows the state of the light emitting point. Here, for convenience of description, the case where the number of capillaries 20 is 5 is shown as an example.
In the above, fluorescence is generated by irradiating the separated sample with the light from the laser, and the emission point EP is formed.

FIG. 3B shows one-dimensional photosensors 68A-6A.
The image of the light emission point EP formed on the light receiving surface of 8D is shown. The images formed on the light receiving surfaces of the one-dimensional photosensors 68A to 68D are images for lights of different wavelengths.

In the present embodiment, only the necessary information is imaged on the light receiving surfaces of the one-dimensional photosensors 68A to 68D and can be used as it is, so that the processing can be simplified.

As described above, in the present embodiment, the light flux is split using the four rectangular prisms 65A to 65D. The distance L1 between the one-dimensional photosensor 68A and the one-dimensional photosensor 68B is preferably large, and the size of the detector in which the one-dimensional photosensor is arranged on the substrate is 50 mm.
If so, the distance L1 needs to be about 50 mm.
Here, in the present embodiment, since the light beam is divided by using the right-angle prisms arranged in the parallel light beam, the distance L2 between the right-angle prism 65A and the right-angle prism 65B can be freely changed. It is easy to expand L2 to about 50 mm.

Further, in the present embodiment, since the light of four kinds of wavelengths is simultaneously measured by one detector unit 60, the distance L3 between the lens 62 and the light emitting point EP is the lens 62. Since it can be easily shortened by shortening the focal length of the
Since the solid angle at which the above light is taken into the detector unit 60 can be increased, the detection sensitivity can be improved.

Further, the right-angle prism 65 is used as a means for splitting the light beam, and the optical axis can be easily matched by disposing one side of the right-angle prism 65 in parallel with the optical base. Easy to manufacture.

The positions of the filters 64A to 64D may be arranged between the right-angle prism 65 and the lens 66 in the parallel light beam, instead of between the lens 62 and the right-angle prism 65. Is. Further, between the lens 66 and the spatial filter 67, there is a convergent light beam,
The filter 64 may be arranged at this position.

Further, although the lens 62 makes the light emitted from the light emitting point EP a parallel light flux, it may be a light flux which is a substantially parallel light flux and converges to some extent.

Instead of the rectangular prism 65, a plane mirror may be used.

Further, the spatial filter (slit) is provided in front of or behind the lens 66 or at the right-angle prism 65 as required.
Inserted before and after.

Next, the method of determining the state of the capillaries in the capillary determining device 200 according to the present embodiment will be described with reference to FIGS. First, a chromatogram obtained by electrophoretic separation analysis will be described with reference to FIG.

FIG. 4 is a schematic diagram of a chromatogram obtained by the capillary electrophoresis apparatus according to one embodiment of the present invention.

In FIG. 4, the horizontal axis represents the base length (bas).
e) is shown, and the vertical axis shows the signal intensity of the detected fluorescence. The optical sensor 68 of the detector unit 60
The horizontal axis represents time (time) and the vertical axis represents signal intensity. The horizontal axis represents time (t).
It is shown by converting from (ime) to a base length (base).

In FIG. 4, FIG. 4A shows a chromatogram when the state of the used capillary is good. That is, the single peak appearing at the base length bn shows a sharp peak with a narrow half width w. Also, the peaks appearing at the base length bi and the base length bi + 1 are multi-peaks that overlap each other near their bases, but the two peaks are still separated at the top thereof,
It is clear that it is composed of two peaks.

On the other hand, FIG. 4 (B) shows a chromatogram when the used capillary is in a bad state. That is, the half width w of the single peak appearing at the base length bn is widened. The peaks appearing in the base length bi and the base length bi + 1 are multi-peaks that overlap each other.
It is uncertain whether it is composed of two peaks or a single peak.

Therefore, in the present embodiment, the detected full width at half maximum w is based on the full width at half maximum w of the single peak.
If it is narrower than the reference half-value width w0, it is determined that the capillary is good, and if the half-value width w is wider than the reference half-value width w0, the capillary is determined to be defective. ing.

Next, the fluorescence spectrum detected when the terminal base is excited by laser light will be described with reference to FIG. FIG. 5 is a fluorescence spectrum obtained from each terminal base using the capillary electrophoresis apparatus according to one embodiment of the present invention. In FIG. 5, the horizontal axis represents the wavelength (n
m), and the vertical axis represents fluorescence intensity (arbitrary unit).

The fluorescence peak wavelength of guanine (G), which is one of the purine bases constituting the nucleic acid, is 527 nm. The fluorescence peak wavelength of adenine (A), which is one of the purine bases constituting the nucleic acid, is 555 nm. In addition, the fluorescence peak wavelength of thymine (T), which is one of the purine bases as a nucleic acid constituent, is 577 nm. Furthermore, the fluorescence peak wavelength of cytosine (C), which is one of the purine bases constituting the nucleic acid, is 602 nm.

For example, in the fluorescence spectrum of guanine (G), when the short wavelength side and the long wavelength side are compared centering on the maximum wavelength of the peak, the signal intensity changes relatively sharply on the short wavelength side and the long wavelength side changes. The characteristic shows that the signal strength changes gently on the side. The same applies to other adenine (A), thymine (T), and cytosine (C). on the other hand,
The bandpass of the bandpass filter 64 used in the detector unit 60 is 20 nm, and the center wavelengths of the four types of bandpass filters 64A, 64B, 64C, 64D are
They are 520 nm, 550 nm, 580 nm and 605 nm. In FIG. 5, the shaded areas in the figure are the light transmission areas of the bandpass filters 64A, 64B, 64C and 64D. With respect to guanine (G), adenine (A), thymine (T), and cytosine (C), the maximum peak wavelength of each fluorescence spectrum and the center wavelength of the bandpass filter are substantially matched.

Therefore, if cytosine (C) is used as the specific peak used for determining the quality of the capillary,
When a bandpass filter having a center wavelength of 605 nm and a bandpass of 20 nm is used, as shown in FIG.
The fluorescence components of guanine (G), adenine (A), and thymine (T) are mixed in the fluorescence having a wavelength of 5 nm to 615 nm, and the half-width of only cytosine (C) is accurately measured. Is difficult.

On the other hand, when guanine (G) is used as the specific peak used for determining whether the capillary is good or bad, when a bandpass filter having a center wavelength of 520 nm and a bandpass of 20 nm is used, a wavelength of 510
Since there is almost no influence of fluorescence of adenine (A) and other bases in the fluorescence of wavelength of nm to 530 nm, the half width of guanine (G) can be accurately measured by using guanine (G). It is possible to Therefore, the use of a single peak of guanine (G) is the most preferable for judging the quality of the capillary state.

However, there is a case where the single peak of guanine (G) does not have an appropriate base length. In such a case, a single peak of adenine (A) can be used. However, the fluorescence spectrum of adenine (A) is
Since the fluorescence spectra of guanine (G) are superposed, the condition is that there is no peak of guanine (G) on the short base length side and the long base length side of the base length of adenine (A) used for determination. Further, since the fluorescence spectrum of thymine (T) is also superimposed on adenine (A), there is no peak of thymine (T) on the short base length side and the long base length side of the base length of adenine (A) used for determination. Is a condition. In other words, in the base lengths on both sides of the single peak of adenine (A) used for the determination,
When there is a cytosine (C) peak, it is not affected by the fluorescence, so this single peak of adenine (A) can be used for judging the quality of the capillary state.

In summary, the specific peak used for judging the quality of the capillary needs to be a single peak. Then, the specific peak needs to have a maximum peak on the short wavelength side in the fluorescence spectrum among the peaks of a plurality of bases to be detected. When there is no specific peak on the shortest wavelength side, it is assumed that the fluorescence spectrum has the next maximum peak on the shortest wavelength side. However, in this case, the base lengths on both sides of this specific peak are:
It is necessary that the maximum peak of the fluorescence spectrum does not have peaks of adjacent bases on the short wavelength side and the long wavelength side.

Next, with reference to FIGS. 6 and 7, a specific example of selecting a specific peak used for determining the quality of the state of the capillary will be described using a chromatogram actually obtained. 6 and 7 are chromatograms obtained by electrophoretic separation analysis of a standard sample measured by the capillary electrophoresis apparatus according to one embodiment of the present invention.

Here, in this embodiment, the analyzed base length is from 0 base length (base) to 300 base length (bas).
e). In such an analyzed base length, the specific peak used for the quality judgment of the capillary state is the maximum base length side in the range of the analyzed base length, that is, in the above example, a single peak near 300 base length is To use.

FIG. 6 shows that M13m is used as a standard sample.
FIG. 7 shows a chromatogram in the vicinity of 236 base length to 300 base length in the case of using p18, and FIG. 7 shows a chromatogram in the vicinity of 301 base length to 365 base length. That is, the range of ± 65 base length is shown centering on the 300 base length. 6 and 7, the horizontal axis represents the base length (base) and the vertical axis represents the fluorescence intensity (arbitrary unit). Further, in FIGS. 6 and 7, G represents guanine, A represents adenine, T represents thymine, and C represents cytosine.

First, in FIG. 6, 300 bases long (b
When a single peak of guanine (G) is searched for in the vicinity of (ase), 288G or 345G is a single peak of guanine (G). Also, 300 base length (base)
Although a little away from 258G or 260G, 258G or 260G is also a single peak of guanine (G). Moreover, 288G,
The bases on the short base length side and the long base length side of 345G, 258G, or 260G are cytosines (C). As described in FIG. 5, since the fluorescence spectrum of cytosine (C) is different in wavelength region from the fluorescence spectrum of guanine (G), a single peak of guanine (G) having cytosine (C) on both sides is most preferable. It is a thing.

Comparing the single peaks of guanine (G) of 288G and 345G, 288G has one cytosine (C) on the short base length side and 2 on the long base length side.
3 cytosines (C) exist, while 345G
Has two cytosines (C) on both the short base length side and the long base length side. Therefore, from the viewpoint of being less likely to be affected by other bases, the single peak of 345 G guanine (G) is better than the single peak of 288 G guanine (G) for determining the quality of the capillary. Is preferred.

Further, 277G, 279G, 303G, 3
The guanine (G) single peaks such as 05G and 307G have thymine (T) and adenine (A) on both sides thereof, and therefore, compared to the above-mentioned 345G, in order to use it for judging the quality of the capillary state. It is not preferable. However, it can also be used in the absence of a suitable single peak such as 345 G guanine (G).

Incidentally, for example, 292G, 293G and 29
4G is a multi-peak with a length of 3 bases, which is 296G or 2
Since 97G is a multi-peak having a length of 2 bases, 97G is not used for judging the quality of the capillary state.

Furthermore, when there is no suitable guanine (G) single peak, it is necessary to select an appropriate peak from among the adenine (A) single peaks. For example, a single peak of adenine (A) of 309A has cytosine (C) on both the short base length side and the long base length side, and therefore, from the viewpoint of being less likely to be affected by other bases, The quality of the state can be used for the determination. The single peaks of adenine (A) of 312A and 291A have guanine (G) on the long base length side, which is not preferable for use in determining the quality of the capillary state.

As described above, as a standard sample, M
When using 13mp18 and setting the analysis base length to 300 bases, 345 G or 288 G guanine (G)
It is preferable to use the single peak of.

Here, as a standard sample, M13mp18
When the analysis base length is set to 600 bases, a single peak of guanine (G) that satisfies the above requirements is used. As a standard sample, pUC18
When using pGEM or pGEM, an appropriate single peak of guanine (G) or adenine (A) can be used depending on the analyzed base length.

Next, with reference to FIG. 8, description will be made on the half-value width level which is a reference for judging the quality of the capillary using the half-value width of the specific single peak detected by the capillary electrophoresis apparatus according to the present embodiment. To do.
FIG. 8 is an explanatory diagram of a half-value width of a single peak in the capillary electrophoresis device according to the embodiment of the present invention.

FIG. 8 shows a chromatogram in which two adjacent peaks are completely separated. Here, the width of the bottom of peak 1 is W1, and the width of the bottom of peak 2 is W2.
Let tR1 be the time at which the maximum value of peak 1 appears, tR2 be the time at which the maximum value of peak 2 appears, and let Δt be the time between the maximum values of peak 1 and peak 2, and the resolution Rs of the two peaks is , Is given by the following formula.

Rs = (2 (tR2-tR1)) / (W1 + W2) (1) The state where the resolution Rs is greater than 1 is the state where the two peaks are completely separated. Therefore, in the present embodiment, when Rs is 0.5 or more, it is determined that the capillary state is good, and when it is smaller than this, it is determined that the capillary state is bad.

Here, W1, W2, tR1, t in the above equation
It takes a long time to obtain R2 and Δt from the chromatogram obtained by the capillary electrophoresis apparatus according to the present embodiment, and it is unsuitable as it is for use in the determination of the capillary state. Therefore, the concept of the degree of separation is converted into the full width at half maximum of the peak of the electrophoretically separated base. That is, in the above equation, tR2-tR1,
That is, Δt is 1 in the electrophoretic separation analysis of nucleic acid.
Corresponds to the base length. Assuming that the widths of the bases of the adjacent peaks 1 and 2 are equal, W1 = W2 = W. Then, the above equation becomes as follows.

Rs = Δt / W = 1 base length / W (2) Here, since it is easier to detect the half-value width than to automatically detect W itself, the half-value width W / 2 Is W / 2 = 1 base length / 2.Rs = 1 base length (3) (assuming Rs = 0.5) FIG. 8 (B) shows the peaks of the bases electrophoretically separated, The horizontal axis is time. A peak value of a single peak of the electrophoretically separated base is H, and a peak width ΔT that gives H / 2, which is one half of the peak value, is a half-value width of the single peak. Based on the above concept, the half-width ΔT
When the half-width ΔT is 8.5 s or more, it can be determined that the state of the capillary is good.

Further, it is possible to convert the above-mentioned idea into the half-value width of the base in the nucleic acid which is actually electrophoretically separated. That is, FIG. 8C schematically shows the state of the bases electrophoretically separated by the capillary 20.
When the analytical sample is introduced from the left side of the capillary 20 in the figure and subjected to electrophoretic separation, the bases separated at the end of the capillary 20 have respective half widths ΔL. FIG. 8D shows the fluorescence spectrum of the bases separated by electrophoresis, and the horizontal axis shows the length L of the separated bases. The half width ΔL is the half width Δ described above.
It can be obtained from T as follows.

That is, assuming that the total length of the capillary is l and the time when a single peak used for the determination appears at the end of the capillary is T, the moving speed v of 345 G guanine (G) can be expressed by the following equation.

V = l / T (4) Further, the half-value width ΔL of the single peak is ΔL = v · ΔT (5) = (ΔT / T) · l (6) For example, for 345G In the case of guanine (G), the time T is
Since the total length 1 of the capillary is 30 cm, ΔL is 0.45 m when ΔT is 8.5 s.
m. That is, when the half-value width ΔL of the single peak of 345 G guanine (G) is 0.45 mm or more, it can be determined that the capillary state is good.

In the above description, as a prerequisite, the length of the capillary is 30 cm, the inner diameter is 75 μm, and the cross-linked polyacrylamide gel packed in the capillary is composed of all acrylamide and bis-acrylamide. Acrylamide was 5% by weight based on the total gel, and bis-acrylamide was 10% by weight based on the total acrylamide. The electrophoretic voltage applied to the capillary during the electrophoretic separation was 120 V / cm, and the analyzed base length was 300 base length. (Base) and 3
A single peak near the 00 base length is used for the determination. If these conditions are different, the half widths ΔL and ΔT, which are the criteria for determining the quality of the capillary, are also different. For example, under the conditions described above, if the analyzed base length is 600 base lengths (base), the half width ΔL is 0.35 mm.

However, in a general capillary electrophoresis apparatus, the length, inner diameter, and
Since the type of gel to be used, the electrophoretic voltage, the analysis base length, etc. are determined in advance, if these conditions are determined, the half widths ΔL, ΔT, which are the criteria for determining the quality of the capillary, are also determined in advance. Can be set.

Next, with reference to FIG. 9, a specific method will be described in which the above-described determination method according to the present embodiment is used by the capillary determination device 200 according to the present embodiment to determine whether the capillary state is good or bad. FIG. 9 is a flowchart showing the determination process of the capillary determination device in the capillary electrophoresis device according to the embodiment of the present invention.

In step 910, the controller 80
Starts pre-migration. That is, in order to equilibrate the capillary 20 before measurement,
The voltage is only applied from the high-voltage power supply 30 to both ends of the capillary 20 without injecting into the. Pre-electrophoresis conditions are 120V
/ Cm for 20 minutes.

At step 920, the controller 80
Introduces an analytical sample and a standard sample into the capillary.
The analysis sample and the standard sample are accommodated in the sample tray 100, the capillary 20 is replaced with the buffer tank 12 and the sample tray 100 is replaced, and the high-voltage power supply 30 is controlled by the controller 80 to apply a voltage to the sample. It is introduced into the capillary 20. The introduction condition is 50 V / cm for 10 seconds.

At step 930, the controller 80
Again, the capillary 20 is again connected to the buffer tank 12 from the sample tray 100, the high voltage power source 30 is controlled, and a voltage is applied to cause each DNA fragment component in the sample to electrophorese into the capillary 20. Electrophoresis voltage of 120
V / cm. The detector unit 60 detects the separation of each DNA fragment component. The signal processing unit 70 determines the base sequence of the sample DNA.

In step 940, the capillary determining device 200 calculates the half width of the specific peak. As described above, the specific peak is 345 G guanine (G).
The single peak of is selected and the half width ΔL is calculated.

Next, in step 950, the capillary determination device 200 determines whether the capillary state is good or bad based on the obtained half width ΔL. here,
For example, if the half width ΔL is 0.45 mm or less, it is determined to be good. If the determination is good, the process returns to step 920 and shifts to the next measurement using the same capillary 20. Also, if the half-value width exceeds 0.45 mm, it is judged as defective,
Go to step 960.

In step 960, the capillary determination device 200 displays an abnormal message on the display unit 210.

Here, an example of the abnormal message displayed on the display unit 210 in this embodiment will be described with reference to FIG. FIG. 10 is an explanatory diagram of a display example of an abnormal message in the capillary electrophoresis apparatus according to the embodiment of the present invention.

As shown in FIG. 10, the measurement result display section 210A of the display section 210 displays the half-value width (0.55 mm in the illustrated example) as the measurement result, and the reference value display section 2
10B, the half value width (0.
45 mm) and the chromatogram of the detected peak is displayed on the chromatogram display unit 210C.

Further, a message for the judgment result is displayed on the judgment display section 210D, and in the example shown in the figure, "Half width is too wide. Please check" is displayed. A judgment input dialog 210E is displayed below the judgment display portion 210D.

In step 970, the operator
Based on the determination result shown in FIG. 10, it is determined whether to continue or end the use of the capillary. Selection of continuation or termination is performed by using the input unit 220 and clicking a “continue” or “end” button of the judgment input dialog 210E with a mouse or the like. If it is to continue, the procedure returns to step 920 to analyze the next sample, and if it is to be finished, it is finished at that point and the capillary is replaced.

Next, referring to FIG. 11, an example of the result of capillary determination performed in this embodiment will be described. FIG. 11 is an explanatory diagram of an example of the result of capillary determination in the capillary electrophoresis apparatus according to the embodiment of the present invention.

As shown in FIG. 11, in the first electrophoretic analysis, the half-value width is 0.26 mm, and the determination result is “good”. After that, until the 5th time,
The full width at half maximum is 0.45 mm or less, and the judgment result is “good”.
Has become. However, in the sixth time, the detected half-width was 0.47 mm, so the determination result is
It is “No”. Based on this result, the analysis up to the sixth time is automatically repeated, and further repeated measurement is stopped at the operator's discretion.

In the above description, the quality of the capillary state is determined by using the specific peak of the standard sample, for example, the single peak of 345 G of M13mp18. However, in the method using the standard sample, as shown in FIG.
When one sample can be stored, one of them needs to store the standard sample, and therefore, the number of samples that can be simultaneously analyzed is 47.

On the other hand, as described below, the quality of the capillary can be determined without using the standard sample, that is, without using the single peak in the analytical sample. The capillary determination device 200 determines a chromatogram as shown in FIG. 6 or 7 based on the signal obtained from the detector unit 60. Then, the capillary determination device 200 is a single peak in the vicinity of the analyzed base length, and the peak is the peak of a plurality of bases to be detected, whose fluorescence spectrum has a maximum peak on the short wavelength side, or the most. If there is no specific peak on the short wavelength side, the fluorescence spectrum shall have a maximum peak on the short wavelength side next, and the base length on both sides of this peak, the maximum peak of the fluorescence spectrum is on the short wavelength side and the long wavelength side. A peak having no peaks of adjacent bases is designated as a specific peak used for judging the quality of the capillary. If the base length of the specific peak is around 300 base length, the reference half-value width ΔL is 0.45 mm, and if it is near 600 base length, the half-value width ΔL is 0.35 mm. By setting the reference value of the half-width ΔL, the quality of the capillary state can be determined in the same manner as when the specific peak of the standard sample is used. When the number of samples that can be stored in the sample tray is 48, , The number of analysis samples is 4
The number of samples that can be analyzed simultaneously can be increased to eight.

When the number of samples is 48, the quality of the above-mentioned capillaries is judged for all of the 48 capillaries. Since it is time-consuming to replace capillaries one by one, it is preferable to carry out 48 capillaries at the same time. Therefore, as a result of the determination of 48 capillaries, 80% of all capillaries (that is, 48 capillaries) are replaced. If it is determined that 39 of the capillaries are good, the capillaries are not replaced, and if the number of defective capillaries is further increased, the entire capillaries are replaced.

Further, since it is considered that the capillaries change in the same manner as a whole, the capillaries are not judged at the same time for all the capillaries, and for example, as shown in FIG. In this case, the quality of a typical capillary state may be determined as in the case of a total of three capillaries, two at both ends and one at the center. By doing so, it becomes possible to reduce the load of the determination process of the capillary determination device 200.

As described above, according to the present embodiment, it is possible to judge the quality of the state of the capillary by using the half width of a predetermined single peak. Therefore, the reliability of the data can be ensured even when the gel filled in the capillary is repeatedly used for a plurality of analyzes without replacement.

Next, a capillary electrophoresis device according to another embodiment of the present invention will be described with reference to FIGS. The overall system configuration of the capillary electrophoresis apparatus according to this embodiment is the same as that shown in FIG. In the first embodiment, the capillary determination device 200 determines whether the capillary state is good or bad based on the signal detected by the detector unit 60, whereas in the present embodiment, As shown in FIG. 1, the current detection unit 90 detects the migration current, and the capillary determination device 200 determines the quality of the capillary based on the detected migration current.

As described above, when the capillary is in a bad state, the peak half-width increases. There are various possible causes for the capillary failure. For example, voids may be generated in the gel filled in the capillary, an analytical sample may be attached to the gel, or the gel network may be broken. Among these, the largest factor is the generation of voids in the gel. When a void is generated in the gel filled in the capillary, the base separated at the position before the void continues to migrate so as to avoid the void, and the separated state deteriorates when avoiding the void. This result is detected as a half-width spread.

On the other hand, when voids are generated in the gel in the capillaries, it is considered that it becomes difficult for electricity to flow because the electrical conductivity of the voids is different. Then, the inventors of the present application conducted research on the correlation between the increase and decrease in the half width and the electrophoretic current, and found that there was a correlation between them. It was found that the migration current was low in the initial state of the capillary and increased as the analysis was repeated. Then, under the same conditions such as the above-mentioned inner diameter and length of the capillary and the kind of gel to be filled, the standard sample M13m
The full width at half maximum ΔL of the peak at 345 G of p18 is 0.45 mm
Then, the migration current was calculated to be 2.8μ.
It was A. That is, when the electrophoretic current is larger than 2.8 μA, it can be judged that the capillary state is good,
When it is smaller than 2.8 μA, it can be judged that the capillary is in a bad state.

Further, in electrophoresis, there is pre-electrophoresis for equilibrating the capillaries in addition to the main electrophoresis in which the analysis sample is actually electrophoresed. A decision can be made. When the half width ΔL of the peak of 345G of M13mp18 of the standard sample is 0.45 mm under the same conditions such as the inner diameter and length of the capillary and the kind of gel to be filled as described above, the migration current of the pre-electrophoresis is determined. When found, it was 3.5 μA. That is, when the migration current of the pre-electrophoresis is smaller than 3.5 μA, it can be judged that the capillary state is good,
When it becomes higher than 3.5 μA, it can be judged that the capillary is in a bad state.

Now, with reference to FIG. 12, a specific method will be described in which the capillary determination device 200 determines the quality of the capillary state by using the determination method based on the migration current according to the present embodiment. FIG. 12 is a flowchart showing the determination process of the capillary determination device in the capillary electrophoresis device according to another embodiment of the present invention.
Note that the same reference numerals as those in FIG. 9 indicate the same processing, and in the present embodiment, in addition to the processing shown in FIG. 9, the determination processing based on the migration current value in steps 1210 to 1250 is added, In particular, these processes will be described.

At step 910, the controller 80
Starts pre-migration. That is, in order to equilibrate the capillary 20 before measurement,
The voltage is only applied from the high-voltage power supply 30 to both ends of the capillary 20 without injecting into the. Pre-electrophoresis conditions are 120V
/ Cm for 20 minutes.

In step 1210, the capillary determining device 200 measures the current value detected by the current detecting means 90.

Next, in step 1220, the capillary determination device 200 determines whether or not the capillary state is good based on the measured current width of the preliminary migration. Here, for example, if the current value is 3.5 μA or less, it is determined to be good. If the determination is good, the process proceeds to step 920 to shift to the measurement of the main migration. Also, the current value is 3.5μ
If A is exceeded, it is determined to be defective, and the process proceeds to step 1230.

In step 1230, the capillary determination device 200 displays an abnormality message on the display section 210.

Here, an example of the abnormal message displayed on the display unit 210 in this embodiment will be described with reference to FIG. FIG. 13 is an explanatory diagram of a display example of an abnormality message in the capillary electrophoresis apparatus according to another embodiment of the present invention.

As shown in FIG. 13, the measurement result display section 210A of the display section 210 displays the current value (4.5 μA in the illustrated example) as the measurement result, and the reference value display section 21
In 0B, the range of the current value that is the reference value (in the illustrated example,
2.8 μA to 3.5 μA) is displayed.

Further, a message for the judgment result is displayed on the judgment display section 210D, and in the example shown in the figure, "current value is too high. Please check" is displayed. A judgment input dialog 210E is displayed below the judgment display portion 210D.

At step 1240, the operator determines whether to continue or end the use of the capillary based on the determination result shown in FIG. Selection of continuation or termination is performed by using the input unit 220 and clicking a “continue” or “end” button of the judgment input dialog 210E with a mouse or the like. If it is to continue, the procedure proceeds to step 920, where the main migration is performed, and if it is to be terminated, it is terminated at that point and the capillary is replaced.

At step 920, the controller 80
Introduces an analytical sample and a standard sample into the capillary.
The analysis sample and the standard sample are accommodated in the sample tray 100, the capillary 20 is changed from the buffer tank 12 to the sample tray 100, and the high voltage power source 30 is controlled by the controller 80 to apply a voltage to the sample. It is introduced into the capillary 20. The introduction condition is 50 V / cm for 10 seconds.

At step 930, the controller 80
Again, the capillary 20 is again connected to the buffer tank 12 from the sample tray 100, the high voltage power source 30 is controlled, and a voltage is applied to cause each DNA fragment component in the sample to electrophorese into the capillary 20. Electrophoresis voltage of 120
V / cm. The detector unit 60 detects the separation of each DNA fragment component. The signal processing unit 70 determines the base sequence of the sample DNA.

Further, in step 1250, the current value during the main migration is determined. If the determination value is larger than the predetermined value, the process proceeds to steps 1230 and 1240, and the above-described message display and processing continuation determination are performed.

After step 940, the capillary determining device 200 determines whether the capillary state is good or bad depending on the half-value width of the specific peak.

Next, with reference to FIG. 14, an example of the result of capillary determination performed in this embodiment will be described. FIG. 14 is an explanatory diagram of an example of a capillary determination result in a capillary electrophoresis apparatus according to another embodiment of the present invention.

As shown in FIG. 14, in the electrophoretic analysis of the pre-electrophoresis, the current value of the pre-electrophoresis was 3.2 μA, which was less than the reference level of 3.5 μA, and the judgment result was “good”. Has become. Next, in the first time of the main migration, the current value of the main migration was 3.0 μA, and thereafter, until the fifth time, the current value was 2.8 μA or more, and the determination result was “good”. It has become. However, at the sixth time, the detected current value was 2.7 μA, so the determination result is “no”. Based on this result, the analysis up to the sixth time is automatically repeated, and further repeated measurement is stopped at the operator's discretion.

In the above description, the current detecting means 90
The current value detected by is the sum of the currents flowing through the 48 capillaries 20, and therefore the capillary determination device 200
Converts the current value per one to determine the state of the capillary. When the current detection means 90 can detect the current value of each capillary, the quality of the capillary state is determined based on the current value of each capillary.

As described above, according to this embodiment, it is possible to easily determine the quality of the capillary state by using the migration current. Therefore, the reliability of the data can be ensured even when the gel filled in the capillary is repeatedly used for a plurality of analyzes without replacement.

[0130]

According to the present invention, the reliability of data can be ensured even when the gel is repeatedly used for a plurality of analyzes without exchanging the gel in the capillary electrophoresis apparatus.

[Brief description of drawings]

FIG. 1 is a block diagram showing an overall configuration of a capillary electrophoresis device according to an embodiment of the present invention.

FIG. 2 is a block diagram of an optical system showing a configuration of a detector unit used in the capillary electrophoresis analyzer according to the embodiment of the present invention.

FIG. 3 is an explanatory diagram of a relationship between a light emitting point and an image formed on a light receiving surface of the one-dimensional photosensor in the multicolor fluorescence detection electrophoretic analysis device according to the embodiment of the present invention.

FIG. 4 is a schematic diagram of a chromatogram obtained by the capillary electrophoresis apparatus according to the embodiment of the present invention.

FIG. 5 is a fluorescence spectrum obtained from each terminal base using the capillary electrophoresis apparatus according to one embodiment of the present invention.

FIG. 6 is a chromatogram obtained by electrophoretic separation analysis of a standard sample measured in a capillary electrophoresis apparatus according to an embodiment of the present invention.

FIG. 7 is a chromatogram obtained by electrophoretic separation analysis of a standard sample measured in a capillary electrophoresis apparatus according to an embodiment of the present invention.

FIG. 8 is an explanatory diagram of a half-value width of a single peak in the capillary electrophoresis device according to the embodiment of the present invention.

FIG. 9 is a flowchart showing a determination process of the capillary determination device in the capillary electrophoresis apparatus according to the embodiment of the present invention.

FIG. 10 is an explanatory diagram of a display example of an abnormality message in the capillary electrophoresis device according to the embodiment of the present invention.

FIG. 11 is an explanatory diagram showing an example of a capillary determination result in the capillary electrophoresis apparatus according to the embodiment of the present invention.

FIG. 12 is a flowchart showing a determination process of a capillary determination device in a capillary electrophoresis device according to another embodiment of the present invention. Note that it is the same as in FIG.

FIG. 13 is an explanatory diagram of a display example of an abnormality message in the capillary electrophoresis device according to another embodiment of the present invention.

FIG. 14 is an explanatory diagram showing an example of a capillary determination result in a capillary electrophoresis apparatus according to another embodiment of the present invention.

[Explanation of symbols]

10 ... Moving mechanism 12 ... Migration buffer tank 13 ... Platinum electrode 14 ... Sample tray holder 16 ... Motor 18 ... Slide guide 19 ... Cover 20 ... Capillary 22, 24 ... Capillary holder 26 ... Coupler 30 ... High-voltage power supply 40 ... Sheath flow cell 44 ... Sheath liquid 50 ... Laser 60 ... Detector unit 64 ... Filter 68 ... Optical sensor 70 ... Signal processing device 80 ... Control device 90 ... Current detection means 100 ... Sample tray 200 ... Capillary determination device

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Susumu Watanabe 832 Nagakubo, Horiguchi, Hitachinaka City, Ibaraki Prefecture 2 Hitachi Measurement Engineering Co., Ltd. In-house (56) Reference JP-A-6-138037 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/447 G01N 21/64

Claims (20)

(57) [Claims] 1. A capillary having a plurality of capillaries filled with an electrophoretic medium and a detector for optically detecting a plurality of electrophoretically separated sample components introduced into these capillaries. A capillary electrophoresis apparatus for separating and developing each component in a sample by applying a voltage to the sample, moving the sample introduced to one end of the capillary toward the other end, and separating and analyzing each component in the sample . Corresponding to each component in the sample detected by
The half-value width of a given single peak among the signal peaks
Therefore, the obtained half width is narrower than the specified reference level.
When the capillaries are in good condition,
Ruki Yapirari electrophoresis apparatus comprising a capillary determining means for. 2. The capillary electrophoresis apparatus according to claim 1.
In the above, the detector optically detects a plurality of sample components electrophoretically separated by laser-excited multi-wavelength fluorescence detection, and a predetermined single peak used by the capillary determination means for determining the quality of the capillary state. is among the peaks of the plurality of sample components to be detected, Ruki Yapirari electrophoresis apparatus fluorescence spectrum having a maximum peak on the short wavelength side. 3. The capillary electrophoresis apparatus according to claim 2.
In addition, the peaks appearing on both sides of a predetermined single peak used by the capillary determining means for determining the quality of the capillary are the peaks of a plurality of sample components to be detected, and their fluorescence spectra are on the long wavelength side. Ruki catcher pin <br/> Larry electrophoresis apparatus having a maximum peak at. 4. The capillary electrophoresis apparatus according to claim 1.
In the above, the detector optically detects a plurality of sample components electrophoretically separated by laser-excited multi-wavelength fluorescence detection, and a predetermined single peak used by the capillary determination means for determining the quality of the capillary state. Of the plurality of sample components to be detected, the fluorescence spectrum has the second maximum peak from the short wavelength side, and the peaks appearing on both sides of this peak are the peaks of the plurality of sample components to be detected. among the peaks, Ruki Yapirari electrophoresis apparatus fluorescence spectrum having a maximum peak to the long wavelength side. 5. The capillary electrophoresis apparatus according to claim 1.
A is a predetermined single peak which the capillary determination means used in quality determination of the state of the capillary predetermined peak der Ruki catcher <br/> Pirari electrophoretic device in the standard sample introduced into the capillary . 6. The capillary electrophoresis apparatus according to claim 1.
The predetermined single peak used by the capillary determination means for determining the quality of the capillary is a predetermined peak among unknown samples introduced into the plurality of capillaries, and a predetermined number of unknown samples. Ruki Yapirari electrophoresis apparatus to determine the quality of the capillary state using the peak. 7. A capillary capable of holding an electrophoretic medium.
And a power supply for applying a voltage to both ends of this capillary, and one that is introduced from one end of the above-mentioned capillary,
A detector that detects the separated sample and a single peak from the signal detected by this detector
Obtain the full width at half maximum, compare it with the reference value, and
A capillary electrophoresis device comprising a capillary determination device for determining a state . 8. The capillary electrophoresis apparatus according to claim 7.
And a table displaying the determination result by the above capillary determination device.
A capillary electrophoresis apparatus including a display section . 9. A capillary electrophoresis apparatus according to claim 8.
A capillary electrophoresis device in which the display unit displays the half-value width of the single peak . 10. The capillary electrophoresis apparatus according to claim 8.
A location, the display section of the capillary Good Condition, and / or unsaturated
Capillary electrophoresis device that displays goodness . 11. The capillary electrophoresis apparatus according to claim 7.
The capillary determination device determines that the capillary is
Instruct to continue or stop the analysis when it is judged to be in good condition
Capillary electrophoresis apparatus having an input section for 12. A capillary electrophoresis device according to claim 7.
And the capillary determination device identifies the detected signal.
Select the single peak of and select the full width at half maximum.
Rally electrophoresis device. 13. A capillary capable of holding an electrophoretic medium.
In the state determination method, the sample is introduced into one end of this capillary , a voltage is applied to both ends of the capillary, the sample separated by electrophoresis is detected by a detector, and the signal detected by the determination device is used. From single peak
Obtain the full width at half maximum, compare it with the reference value, and
Capillary state determination method for determining the state. 14. The state determination of a capillary according to claim 13.
If the half width of the single peak is wider than the reference value,
Capillary status is determined to be defective Capillary status determination
Method. 15. The state determination of the capillary according to claim 13.
If the half width of the single peak is narrower than the reference value,
Capillary status is judged normal Capillary status judgment
Method. 16. The state determination of a capillary according to claim 13.
This is a standard method, in which the detector is linked with a fluorescent dye corresponding to the base type.
In addition , the above single peak is the peak of the plurality of sample components to be detected.
Peak in the peak of the fluorescence spectrum on the short wavelength side.
A method for determining the state of a capillary having a gap. 17. The state determination of a capillary according to claim 16.
Method, the peaks appearing on both sides of the above single peak are detected.
The fluorescence spectra of the peaks of multiple sample components
Has a maximum peak on the long wavelength side
How to judge the condition of the user. 18. A state determination of a capillary according to claim 13.
The method wherein the single peak is the peak of multiple sample components detected.
Of the second fluorescence spectrum from the short wavelength side
Exists on both sides of this single peak.
Of the sample components detected
And its fluorescence spectrum has a maximum peak on the long wavelength side.
Capillary state determination method. 19. A status determination of a capillary according to claim 13.
A method for determining the state of a capillary in which the above sample is a standard sample
Law . 20. The state determination of the capillary according to claim 13.
Method for determining the state of capillaries where the above sample is an unknown sample
Law.