JPH06138037A - Cataphoresis device - Google Patents

Abstract

PURPOSE:To provide a cataphoresis device for measuring fluorescence and light absorption of a sample which has been molecular weight-isolated, with ease and high sensitivity and then a device for measuring the DNA base arrangement with a high throughput. CONSTITUTION:Capillaries 1a-1t and 2a-2t where gel is filled are fixed into a fluorescent cell 4 coaxially so that space parts 3a-3t with constant length are retained. Sheath liquid is poured into the fluorescent cell by a fall, thus causing a sample migrating at the space parts 3a-3t to be in sheath flow state. Fluorescence or light absorption measurement is performed at the space parts 3a-3t at a part without the capillaries and gel. Four bases are identified simultaneously for a plurality of samples simultaneously by the multiplexed capillary and an image-splitting prism, thus achieving the cataphoresis device and the DNA base arrangement measuring device which allow a plurality of samples to migrate and can measure them simultaneously.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoretic device for separating and analyzing nucleic acids, proteins, sugars and the like, and more particularly to an electrophoretic device suitable for detecting DNA (nucleic acid) and analyzing the base sequence of DNA. Is.

[0002]

2. Description of the Related Art As an electrophoretic device for separating a molecular weight of a fluorescently labeled sample by electrophoresis and analyzing it, there is, for example, a DNA base sequencer for labeling a fluorescent substance. The nucleotide sequence is determined by the well-known dideoxy method of Sanger et al. Molecular weight separation is performed by electrophoresis using polyacrylamide gel or the like. Usually, electrophoresis is performed using a polyacrylamide gel on a flat plate created between two glass plates, but in recent years, a capillary gel electrophoresis method in which a gel (capillary gel) is created in a capillary and electrophoresis is performed. Being developed. In the capillary gel electrophoresis method, the surface area per volume of the gel is generally large due to the small diameter of the capillaries, and it is easy to dissipate the Joule heat. Is a useful method. As a first conventional example of the capillary gel electrophoresis method, “Newclaic Acid Research”, Vol. 18,
1415-1419 (1990) (Nucleei
c Acid Research, 18, 1415-1
419 (1990)). As shown in FIG. 19, using a capillary with an inner diameter of 75 μm,
By applying a high voltage of V, high speed and high separation detection of DNA fragments etc. is achieved. In the detection part of this method, the axis of the capillary and the irradiation axis of the laser light are tilted by about 25 degrees from the vertical direction, and the diameter of the laser light is about 2 at the center of the capillary.
The fluorescence is condensed and emitted to 0 μm, and the generated fluorescence is spectrally detected by a bandpass interference filter or the like and detected. Note that FIG. 19 has been rewritten for easier understanding based on the apparatus diagram described in the above-mentioned conventional example. Further, as a second conventional example, "Journal of Chromatography", Vol. 516, Nos. 61-67.
Page (1990) (Journal of Chrom
atomography, 516, 61-67 (199
0)) to DNA by capillary gel electrophoresis.
Has been described. In this method, a capillary with an inner diameter of 50 μm is used
The fragments are separated by molecular weight. The measuring device in this example is basically "Science" magazine, Volume 242, 562-564.
Page (1988) (Science, 242, 562-)
564 (1988)).
In order to make it easier to understand in the device diagram described in "Science" magazine,
FIG. 20 shows an apparatus configuration in which a laser light source, a pump for sheath flow, and the like are added based on the described contents. For detection of DNA fragments, the separated solution whose molecular weight has been separated is introduced into a quartz flow chamber (internal shape 250 μm × 250 μm), and EDT is performed.
A tris-borate buffer solution containing A is used as a sheath liquid to flow by a pump for liquid chromatography to make a sheath flow state, and a laser beam is condensed to a diameter of about 10 μm with respect to the separation liquid flowing in almost the center of the flow chamber. The fluorescence from the DNA fragment is detected through a spectral filter or the like. In addition, as a method of determining the base sequence by one migration path, which is the third conventional example, for example, "Nature", Vol. 321, pp. 674-679 (198).
6 years) (Nature, 321, 674-679 (19)
86)) and the like, four types of fluorophores having different maximum fluorescence wavelengths (fluorescein, 4-chloro-7-nitrobenzo-2-oxa-1- Labeled with diazole (NBD), tetramethylrhodamine, Texas red (product of Molecular Probes)), the fragments that migrate in order of molecular weight are excited by laser light, and each fluorescence is detected by four kinds of bandpass interference filters. There is a method of determining the base sequence by separating and detecting fluorescence. Thus, in capillary gel electrophoresis, measurement is usually performed using only one capillary.

[0003]

In the capillary electrophoresis apparatus, there is a demand for higher detection sensitivity as the amount of sample is reduced. Further, it is desired to improve the processing capacity, for example, increase the number of samples that can be processed simultaneously. Generally, in fluorescence measurement in an electrophoresis state, in addition to fluorescence from the target phosphor itself, scattered light of excitation light by the gel (Rayleigh scattering, etc.) and fluorescence as the background light, the inner wall of the gel support, that is, the capillary. And, scattered light on the outer wall or fluorescence from the capillary itself is generated. Therefore, the background level becomes high and the detection sensitivity is lowered. In other words, how to remove such background light is an important issue in achieving highly sensitive fluorescence detection. In addition, in order to improve the processing capacity, it is necessary to make the migration speed faster, and to simultaneously migrate and measure multiple capillaries, which are migration paths, and how to achieve these is an important issue. Become. In the first conventional example, the first measure for removing the excitation light (scattered light) by a bandpass interference filter or the like and separating and detecting the fluorescent component, and the capillary axis and the laser light irradiation axis and the fluorescent light condensing With the two measures of the second measure in which the plane including the axis is inclined by about 25 degrees from the perpendicular, the fluorescence detection with higher sensitivity is achieved. However, with the first countermeasure, it is difficult to completely remove the scattered light due to the characteristics of the interference filter. In addition, fluorescence may be generated from the gel and the glass tube itself although they are weak, and it is difficult to sufficiently remove the background light with a bandpass interference filter or the like. The second measure is an effective method in which the scattered light itself incident on the fluorescence condensing lens can be reduced by tilting the capillary. However, since the capillary has a circular cross section and its diameter is small, it is scattered. Since the light intensity is high, scattered light is emitted in all directions without being scooped, and in addition, it is not possible to tilt the capillaries to reduce the fluorescence from the gel and glass tube itself. Difficult to remove. From the above viewpoint, the background light is not sufficiently removed, and it is difficult to detect fluorescence with high sensitivity. It should be noted that this conventional example has only one capillary, and the method for simultaneous treatment of a plurality of samples is neither described nor suggested. Although it is conceivable to simply use a plurality of devices of FIG. 19, a plurality of laser light sources, detectors, optical parts, etc. are also required, and the device becomes large and expensive, which is not realistic. Further, it is conceivable to arrange a plurality of capillaries and irradiate all capillaries with a single laser light, but when irradiating light such as laser light to the capillaries,
Since the capillary has a circular cross section, light is refracted / scattered at the interface, and the light passing through the capillary is widely diffused. Therefore, it is practically impossible to achieve this configuration. In other words, there is no problem if there is only one capillary,
When continuously irradiating a plurality of capillaries, the intensity of light irradiated to the next capillary becomes extremely weak every time it passes through the capillary, and fluorescence measurement becomes difficult. From the above, applying this conventional example to the simultaneous processing of a plurality of samples,
It is difficult to improve the processing capacity.

The second conventional example is a method capable of effectively removing scattered light by the capillary and gel by capillary gel electrophoresis. In other words, the capillary gel end is used as the sample injection port of the sheath flow chamber to draw the sample solution out of the capillary gel and perform fluorescence measurement under the sheath flow. Therefore, scattered light at the capillary interface and scattering from the gel Elimination of light and fluorescence. Further, in the sheath flow chamber, the sample liquid flows in a laminar flow state almost at the center thereof and does not come into contact with the inner wall of the sheath flow chamber. Therefore, the scattered light from the sheath flow chamber and the fluorescence from the sample should be spatially separated. The fluorescence intensity can be detected with high sensitivity. However, in order to form the sheath flow, it is necessary to constantly flow the Tris-borate buffer solution as a sheath liquid into the sheath flow chamber at a constant flow rate by a liquid chromatography pump or the like. Therefore, there is a problem that the device is expensive and complicated. Further, the sheath flow chamber has a complicated shape such that the sheath liquid injection portion is thick and the fluorescence measurement portion which is the sheath flow portion is thin, and it is difficult and expensive to manufacture the sheath flow chamber. Further, like the first conventional example, this conventional example does not describe or suggest the simultaneous treatment of a plurality of samples. It is not realistic to simply use a plurality of devices shown in FIG. 20 because the device becomes large and expensive. It is also conceivable to use a plurality of sheath flow chambers, arrange a capillary in each sheath flow chamber, and irradiate a single laser beam. In this case, since the laser light does not irradiate the capillary, there is no refraction / scattering of light by the capillary. However, since there is a sheath flow chamber for each capillary, it is difficult to narrow the space between the capillaries spatially, and as a result, the sheath flow chamber part becomes large, and if multiple sheath flow chambers are used. ,
There is a problem that the device becomes large and expensive. Further, in order to irradiate all the sample liquid bundles flowing in the plurality of sheath flow chambers under the same condition, the laser light cannot be narrowed down. This is because the focal depth of the laser light is related to the irradiation spot size, and as the irradiation spot size becomes smaller, the focal depth becomes shallower, whereas the interval between the sample liquid bundles flowing in the plurality of sheath flow chambers is wide. . From the above, it is difficult to improve the processing capacity by applying this conventional example to the simultaneous processing of a plurality of samples. An object of the present invention is to solve the above-mentioned problems of the prior art, and an electrophoresis apparatus capable of easily performing fluorescence measurement or light absorption measurement of a sample separated by electrophoresis with high sensitivity, and a plurality of samples. It is an object of the present invention to provide an electrophoretic device capable of easily performing the simultaneous treatment of. Another object of the present invention is to provide an electrophoretic apparatus suitable for measuring DNA base sequences, having good operability and safety, high sensitivity, and high throughput.

[0005]

In order to achieve the above object, an electrophoretic device in which a sample separating section provided between a cathode electrode tank and an anode electrode tank to which a voltage is applied by a power source is a migration path composed of capillaries. In (1), one end of each of the one or more pairs of capillaries whose one end is connected to the cathode electrode tank or the anode electrode tank respectively has the other ends in the optical cell so that their axes are substantially aligned and a constant gap is maintained. The sample to be migrated from the capillary end of the upstream migration path, which is the sample separation section, is formed by injecting a sheath liquid into the optical cell from the outside In a sheath flow state and guide them to the opposite capillaries, and use this gap as an optical detection section so that the optical detection section is irradiated with light from a light source to detect a sample, or (2) one end is electrically charged. A plurality of capillaries immersed in the bath are terminated in the optical cell, and a sheath liquid is injected into the optical cell from the outside and allowed to flow, so that the sample migrating in each capillary flows in the optical cell in the sheath flow state. The electrophoretic device is configured so that the sheath flow section serves as an optical detection section and the optical detection section is irradiated with light from a light source to detect a sample. (3) The other end of a plurality of capillaries, one end of which is immersed in the electrode tank, is held and fixed in the optical cell so as to be aligned in a straight line at an appropriate interval, and the sheath liquid is formed of an aqueous solution in the optical cell. To form a sheath flow in the vicinity of the end of each capillary and its lower part, and the sample that migrates each capillary flows in the optical cell in the sheath flow state, and the injected sheath liquid is discharged from the optical cell and excited. The light of the light source unit, immediately below the ends of the plurality of capillaries aligned on a straight line in the optical cell, is irradiated substantially parallel to the above straight line, and fluorescence is generated from the sample flowing out from each capillary, Each of the plurality of generated fluorescence images is divided, and a spectral filter is arranged in front of or behind the dividing means so that the divided images have light components in different wavelength regions, and It is imaged to the image, constituting the electrophoresis apparatus for detecting the formed image.

In the structure of (1), a plurality of optical detecting portions formed by a plurality of capillary pairs are arranged in the optical cell. Furthermore, a plurality of pairs of capillaries are arranged in an optical cell so that the plurality of pairs of capillary detectors are located on a straight line, and the excitation light is irradiated so as to irradiate all the pairs of detectors at the same time. Irradiation is performed along this straight line, and fluorescence measurement is performed simultaneously by the plurality of optical detection units. In the configuration of (2), the plurality of optical detection units formed by the plurality of capillaries are arranged so as to be aligned. The capillaries are arranged side by side in the optical cell, and excitation light is irradiated along the straight line so that all the optical detection parts are irradiated at the same time, and fluorescence emitted from the sample running through each capillary is detected at the same time. Further, in the configuration of (3), as in the case of (1), the number of capillaries for electrophoretic separation is equal to or more than the number of capillaries for electrophoretic separation. A structure may be adopted in which the aqueous solution is discharged from the optical cell via a hollow capillary arranged in the vicinity of the plane including the capillary end and the laser light irradiation axis. An image from a plurality of samples migrating each capillary is divided by using a polyhedral prism. The image may be divided into four and each may be detected simultaneously. Fluorescent images from a sample on which each of a plurality of capillaries migrates or fluorescent images divided into each are simultaneously formed on a detector, and their intensities are measured at the same time. Fluorescence or the like emitted from the sample may be focused by a lens and then split to form an image. Alternatively, the light may be focused in one direction by a cylindrical lens and then split and imaged to be detected.

Further, in the configurations of (1), (2) and (3), each part may be configured as follows. The sample separation unit is composed of a capillary gel, the sheath liquid has a component equivalent to the buffer solution inside the capillary, and a denaturant for the sample can be included. Further, the means for holding a plurality of capillaries and the optical cell can be made detachable, and an insertion guide for holding the capillary holder at a designated position can be provided inside the optical cell. The injection of the aqueous solution into the optical cell is performed by providing a sheath liquid container containing the aqueous solution outside the optical cell, arranging the liquid surface at a position higher than the surface of the liquid discharged from the optical cell, and dropping it, or Using an optical cell with an open upper part, the means for pouring the aqueous solution inside the optical cell is to install a sheath liquid container containing the aqueous solution outside the optical cell, and place the liquid surface at a position higher than the drain port in the optical cell. You may arrange and carry out by a head. It is also possible to provide a mechanism for controlling the liquid level in the optical cell to be substantially constant. This can be configured by a mechanism in which the upper part of the optical cell is open and a mechanism for detecting the liquid level in the optical cell and a mechanism for replenishing the aqueous solution in the sheath liquid container via a valve when the liquid level is lowered. The optical detector measures fluorescence or light absorption of the sample. Further, the excitation light source unit may be a laser device, or at least two types of laser devices may be used, and these laser lights may be coaxially formed into one. The resulting image, such as fluorescence, can be detected by a two-dimensional detector. In addition, the sample is injected into each of the plurality of capillaries at the same time. The electrode on the downstream side of the migration can be not only inside the container from which the sheath liquid is discharged, but also inside the optical cell or inside the sheath liquid container. The potential of the electrode on the downstream side of migration is set to 0 V (ground) or its absolute value is smaller than the absolute value of the potential on the upstream side of the other migration. Further, a mechanism may be provided in which the liquid surface of the aqueous solution accumulated in the electrode bath on the downstream side where the sample is migrated is kept substantially constant and the excess aqueous solution is discharged into the bath. Further, the ratio of the length of both ends of the fluorescence emitted from the sample which migrates a large number of capillaries to the light receiving size of the two-dimensional detector is 3 or less, that is, the overall image magnification is 1/3 or more. Further, it is desirable that the distance between adjacent capillaries is 10 times or less the capillary size. In electrophoresis, it is preferable that the number of electrodes on the downstream side on which the sample is migrated is one and the number of electrodes on the injection side of the sample is equal to the number of capillaries for electrophoretic separation. Further, a mechanism for measuring and displaying the value of the current flowing through the capillary for electrophoretic separation is provided. The current values may be binarized and displayed. Further, in the capillary for electrophoresis separation, at least the inner surface in the vicinity of the end on the side held inside the optical cell is silanized and filled with gel, so that at least the gel near the end on the side held inside the optical cell is filled. May be chemically bonded to the inner wall of the capillary.

[0008]

In the configuration (1) or (3) of the electrophoretic device,
One end of each pair of capillaries whose one end is connected to the cathode electrode tank or the anode electrode tank, respectively, and the other ends of the capillaries are arranged to face each other in the optical cell with their axes being substantially aligned with each other while maintaining a constant gap. Therefore, a migration path that penetrates a part of the optical cell can be formed. Since the sheath liquid is injected into the optical cell from the outside and allowed to flow, the sample migrated from the capillary end of the migration path on the upstream side, which is the sample separation unit, can be guided to the opposite capillary in a sheath flow state. The sample can be continuously electrophoresed in the capillary and the downstream capillary, and the sample can be surely electrophoresed only in the axial gap between the upstream capillary and the downstream capillary. . By using this gap portion as an optical detection portion, light irradiation from the light source to the optical detection portion can be performed in a liquid without a capillary, and optical measurement such as fluorescence or light absorption of the sample can be performed. As a result, it is possible to avoid the generation of scattered light and fluorescence due to the capillary or the capillary gel, and it is possible to perform highly sensitive fluorescence or light absorption measurement. In this way, by making a plurality of capillaries face each other in the optical cell so that their axes are substantially aligned with each other while maintaining a constant gap, the capillary on the upstream side that is the sample separation unit and the gap that is the optical detection unit can be easily Can be configured to. Moreover, by arranging a pair of capillaries on the downstream side, the migration path of the sample can be accurately defined, the light irradiation from the light source can be made easy and reliable, and the reception of fluorescence etc. is also easy. And can be assured. Further, the sheath liquid and the sample liquid flow out to the outside of the optical cell only through the inside of the capillary on the downstream side, but since the capillary has a small diameter, it is possible to reduce the flow rate of the sheath liquid to be injected into the optical cell. The amount of liquid can be reduced. Since the flow rate of the sheath liquid is regulated by the inner diameter of the capillary, not by the size of the optical cell, the cross-sectional area inside the sheath liquid injection part must be thickened like the normal sheath flow chamber, Since it is not necessary to adjust the optical cell into a shape that makes the shape narrower and a prismatic optical cell can be used, the optical cell can be easily manufactured at low cost. In this way, it is possible to arrange a plurality of optical detection units formed by a plurality of capillary pairs in the optical cell in close proximity, and it is possible to achieve miniaturization of the device. Since only one optical cell and one pipe for forming the sheath flow are required, the device can be constructed inexpensively and easily. When passing through the optical detection unit, the sample migrated from each capillary is in a sheath flow state for each capillary, and the sheath liquid flows under the same conditions. It becomes uniform for each capillary, and the accuracy of optical detection can be improved. Since the inside of the optical cell holds a plurality of capillary pairs, its cross-sectional area becomes large, but since the sheath liquid and the like flow out through each capillary on the downstream side, the effective cross-sectional area through which the sheath liquid flows is that of the capillary. This is the sum of the inner diameter areas, which is smaller than the cross-sectional area of the optical cell itself. Therefore, the flow rate of the sheath liquid is small, the amount of sheath liquid can be reduced, and the device can be downsized.

In a single optical cell, a plurality of pairs of capillaries are aligned so that a plurality of optical detectors are located on a straight line, and excitation light is irradiated along this straight line.
It is possible to irradiate all the optical detection sections at the same time, and it is possible to simultaneously perform fluorescence measurement in a plurality of optical detection sections. Further, since the plurality of optical detection units are connected to each other through the liquid, each optical detection unit can be irradiated without the excitation light being scattered by the capillary or the like, and the excitation light can be efficiently transmitted to each optical detection unit. Therefore, the fluorescence can be detected with high accuracy. If a two-dimensional TV camera or the like is used as the light detector, it is possible to simultaneously receive the fluorescent images of the plurality of optical detection units. In a single optical cell, a plurality of pairs of capillaries can be aligned and arranged so that a plurality of optical detectors are located on a straight line, and the intervals of the plurality of pairs of capillaries can be close to each other. Not only can it be constructed at a low cost, but the length between the optical detection portions at both ends on the above straight line can be shortened. If the length between the optical detection portions at both ends is shortened, even if the excitation light is narrowed down by a lens or the like, it is possible to irradiate all the optical detection portions with a substantially equal light beam diameter. For example, when the laser light is focused to a diameter of about 100 μm at the focal position, about 10 mm before and after that
The laser beam diameter becomes about 100 μm. 400μ with a capillary with an outer diameter of 200μm and an inner diameter of 100μm
If you arrange every m, 5 will be inside about 10mm before and after the focal point.
About 0 lines can be arranged and they can be irradiated with the same light beam diameter and light beam intensity. Than this,
You can irradiate each optical detection part with the excitation light narrowed down,
The fluorescence intensity can be increased, and the sample can be detected with high sensitivity.

The sample separation section is composed of a capillary gel so that the molecular weight can be easily separated. In addition, the capillary on the downstream side is a hollow capillary, so that the sheath liquid or the like can be efficiently flown. On the downstream side, it is possible to use one that can perform the same operation without being the capillary itself, for example, a flat plate on which holes or grooves corresponding to the number of upper capillaries are formed. By using the sheath liquid as a component equivalent to the buffer liquid inside the capillary, the flow of electricity in the gap portion, that is, the optical detection portion can be secured, and the sample can be electrophoresed. Furthermore, since the composition of the buffer solution inside the capillary is the same as the composition of the buffer solution outside the capillary, it is possible to prevent the buffer solution inside the capillary from flowing into the inside of the optical cell and to fluctuate the composition, and to improve the resolution of the sample by electrophoresis. Does not spoil. Further, when the sample is single-stranded DNA or the like, the denaturing agent can be included in the sheath liquid. In this case, it is possible to prevent the sample DNA from recombining during migration in the gap, that is, the optical detection section, and the measurement accuracy is improved. The possibility that the denaturant confined inside the capillary leaks into the optical cell is reduced, and the resolution of the sample in electrophoresis is not impaired. Further, the sheath liquid is injected into the optical cell by placing the sheath liquid level in the sheath liquid container at a position higher than the liquid level in the downstream electrode tank, so that the sheath liquid easily flows due to the drop. Therefore, it is not necessary to mechanically flow the sheath liquid, and the device configuration is simple and inexpensive. Furthermore, since the pulsating flow that occurs when using a pump or the like is eliminated, a stable sheath flow is possible and the measurement accuracy is improved. The flow rate of the sheath liquid can be easily adjusted by changing the drop between the liquid level of the sheath liquid in the sheath liquid container and the liquid level in the downstream electrode tank. The optical cell can be easily attached to the capillary holder by using a cell having a shape in which it is sealed when the capillary is attached and the shape of the optical cell is open. At this time, by arranging the liquid surface of the sheath liquid container at a position higher than the drain port in the optical cell,
The same effect as described above can be obtained. In addition, a mechanism that detects the liquid level in the optical cell and a mechanism that replenishes the aqueous solution in the sheath liquid container via a valve when the liquid level drops is provided, and the liquid level in the optical cell is controlled to be almost constant. However, a stable sheath flow with little pulsation can be achieved, and the device can be configured simply and inexpensively. The optical detector can measure either fluorescence or light absorption of the sample. The gap distance of the gap portion is not particularly limited, but is preferably about 0.1 mm to 3 mm. Generally, the shorter the gap distance of the gap portion, the easier the sample migrates in the gap space. Therefore, basically, the shorter gap length is preferable. However, when assembling the apparatus, if the gap length is too short, it is difficult to adjust the gap length. However, it is also possible to set it to 0.1 mm or less, and its limit is determined by the width of the excitation light beam such as laser light in the gap. On the contrary, if the gap distance of the gap portion is increased, the sample is likely to diffuse. Gap length is 3m
It was confirmed that the sample migrated normally when it was about m. That is, by setting the gap length to about 0.1 mm to 3 mm, the sample can be easily and efficiently electrophoresed. In addition, by holding the pair of capillaries inside the optical cell and performing electrophoresis, the sample migrates on the line connecting the capillaries and does not come into contact with the inner surface of the optical cell. The influence of scattered light can be eliminated, and the detection sensitivity can be improved.

Further, images from a plurality of samples migrating each capillary are divided by using a polyhedral prism or the like,
It is possible to easily obtain a plurality of pieces of information from one sample by using a spectral filter so that light components of different wavelength ranges pass through. In particular, if the image is divided into four, which is the number of DNA base species, each base species can be labeled with a different fluorophore to efficiently determine the DNA base sequence. A fluorescence image from a sample that migrates multiple capillaries or divided fluorescence images is simultaneously imaged on a two-dimensional detector and its intensity is measured at the same time, enabling high-speed measurement regardless of the number of capillaries. It becomes possible to do. In addition, an insertion guide for holding the capillary holder at the specified position is provided inside the optical cell, and multiple capillaries and optical cells can be attached and detached, improving the operability of mounting and handling the capillaries. To do. Further, the excitation light source unit is a laser device, and at least two types of laser devices are used, and by irradiating these laser lights coaxially into one, the DNA fragments can be efficiently excited. By providing electrodes equal in number to multiple capillaries for electrophoretic separation, immersing each capillary and electrode in multiple sample solutions, and applying voltage simultaneously to inject sample into each of multiple capillaries simultaneously. In addition, sample injection can be performed easily and in a single operation, which saves time and improves operability as compared to the conventional case where a single unit is used. Furthermore, a capillary for electrophoresis separation is used. It is possible to measure the value of the current flowing inside each capillary. The value of the current flowing inside the capillary is effective for knowing the state of the capillary, and for example, a separation failure due to gel breakage or the like can be predicted in advance. The current level can be displayed by setting the binarization level to any value and turning on the lamp when the current level is below the normal electrophoretic current so that damage to the capillary gel can be monitored. Therefore, it is possible to efficiently judge the necessity of exchanging the gel capillary and re-measurement, and improve the usability.

Further, the distance between adjacent capillaries held inside the optical cell is 10 times or less the capillary size, and the overall image magnification of the fluorescent image to be detected is 1/3 or more, whereby the flat gel system is adopted. It is possible to increase the light receiving solid angle as compared with, and it is possible to improve the detection sensitivity for the same amount of phosphor. In a part of the configuration (2) or (3) of the electrophoretic device, a plurality of capillaries whose one end is immersed in the electrode tank are terminated in the optical cell, and the sheath liquid is injected into the optical cell from the outside. Flow through the optical cell in a sheath flow state, and the sheath flow section serves as an optical detection section, and each sample is run in the same manner as in the configuration (1) of the electrophoresis apparatus. It is possible to separately measure the samples to be migrated through the capillaries, and obtain the same effect as the configuration (1) of the electrophoresis apparatus. Other related operations described with respect to the configuration (1) of the electrophoretic device are the same as for the configuration (2) of the electrophoretic device.

[0013]

The present invention will be described in more detail with reference to the following examples. [Example 1] In this example, a fluorescently labeled DNA fragment is subjected to molecular weight separation by electrophoresis and detected by fluorescence. A case where fluorescein isothiocyanate (FITC) is used as a fluorescent substance for labeling will be described. The sample of the fluorescently labeled DNA fragment is DN
A part of the A fragment labeled with a fluorescent substance can be used.
For example, it is prepared by carrying out a DNA polymerase reaction using a fluorescently labeled primer by the well-known Sanger et al. Dideoxy method. That is, a FITC-bound primer (labeled primer) is used as a primer, the labeled primer is added to the single-stranded DNA of the template and annealed, and the labeled primer is bound to the single-stranded DNA. Next, dATP, dTTP, dCTP, d
GTP and ddATP are added and the DNA polymerase reaction is carried out. By the above operation, fluorescent labeled DNA fragments having various lengths with A at the ends are obtained. This is used as a sample. First, the device configuration will be described. FIG. 1 shows a configuration diagram of an electrophoretic unit and a laser light irradiation system of the electrophoretic device of this embodiment.
FIG. 2 shows a configuration diagram of the fluorescence detection system of the electrophoretic device. The electrophoresis apparatus has a configuration in which 20 capillaries serving as a sample separation unit are arranged and a plurality of samples are simultaneously measured. As the sample separation unit, 20 silica capillaries 1a and 1 were used.
b, 1c, 1d ... 1t are used. These are the same capillaries made of silica, each having an inner diameter of 100 μm, an outer diameter of 375 μm, and a length of 40 cm. Furthermore, 20 silica capillaries 2a, 2b, 2c, 2d, ..., 2t having the same inner and outer diameters and 10 cm in length are used.
For the capillaries 1a to 1t, a capillary gel filled with a polyacrylamide gel containing urea as a denaturant is prepared. First, the inside of the capillary is washed and subjected to silane coupling treatment. Then, degassed 3.84% acrylamide, 0.16% bisacrylamide, 7 M urea, Tris containing 2 mM EDTA, tetramethylethylenediamine and ammonium persulfate solution were added to the borate buffer solution and the mixture was injected into the capillary. Polymerize to make an acrylamide gel. Since the capillaries have been subjected to silane coupling treatment, the acrylamide gel and the capillaries are chemically bonded, and the gel does not stick out from the capillaries during the migration.

The capillaries 2a to 2t are processed so that their inner surfaces have a positive charge. First, a 3- (2-aminoethylaminopropyl) trimethoxysilane solution is injected into the capillaries to cause a reaction, and heat treatment is performed at 110 ° C. to aminosilanize the inner surfaces of the capillaries so that they have a positive charge. By doing so, the direction of the electroosmotic flow inside each of the capillaries 2a to 2t is changed from the negative electrode to the positive electrode, and the migration direction of the sample in the capillaries 1a to 1t filled with acrylamide gel (negative electrode → positive electrode). , The moving direction of the sample in the capillary 2 (negative electrode → positive electrode) is the same, and the migration of the sample can be ensured.
One end of each of the above-mentioned capillaries 1a to 1t and 2a to 2t is opposed to the inside of the optical cell and fixed so as to maintain a constant gap, and the sample is optically detected. Since a sample is detected by fluorescence here, a fluorescence cell is used as an optical cell. That is, the capillaries 1a-1
t and 2a to 2t each have one end having a prismatic quartz fluorescent cell 4 (outer shape: width 36 mm x depth 4.5 mm x length 3 m).
m, inner shape: width 30 mm x depth 2 mm x length 3 mm ,: width is the lateral direction of the figure (capillary 1a → 1t arrangement direction),
The depth is arranged in the vertical direction with respect to the paper surface of the drawing, and the length is arranged in the vertical direction of the drawing (the 1 → 2 sample migration direction of each capillary). The arrangement method is such that the capillaries 1a and the capillaries 2a are coaxial with each other, and a gap portion 3a having a length of 1 mm is formed so as to face each other. Capillaries 1b and 2
Similarly, b, 1c and 2c, 1d and 2d, ..., 1t and 2t are arranged so as to form the same gap portions 3b, 3c, 3d ,. The capillaries are fixed in the optical cell by using multi-capillary holders 5a and 5b in which a flat plate block made of a fluororesin such as ethylene tetrafluoride resin is provided with 20 vertical holes at 0.6 mm intervals. That is, 20 of the multi-capillary holder 5a
Insert the capillaries 1a to 1t into the vertical holes at each position, and insert the capillaries 2a to 2t into the vertical holes at 20 positions of the multi-capillary holder 5b.
Capillaries 1a and 2a, 1b and 2b, 1c and 2c, 1
d and 2d ... The multi-capillary holders 5a and 5b are fixed in close contact with the upper and lower portions of the fluorescent cell 4 so that 1t and 2t are coaxial with each other. Furthermore, the gap 3a-
Since 3t is an optical detection unit, here a fluorescence detection unit, the capillaries 1a to 1t and 2a in the quartz fluorescence cell 4 are arranged so that the gaps 3a to 3t are aligned on a straight line.
Adjust the length of ~ 2t, etc. The capillaries 1a-
The other ends of the 1t and the capillaries 2a to 2t are
It is dipped in a cathode side electrode tank 6 and an anode side electrode tank 7 containing a buffer solution (a buffer solution containing Tris, boric acid, EDTA and urea). The fluorescent cell 4 is provided with a sheath liquid injection port 8 for filling the inside thereof with a sheath liquid, and the sheath liquid 11 in the sheath liquid container 10 can be injected through a tube 9 made of ethylene tetrafluoride resin. It has a structure. A buffer solution containing Tris, boric acid, EDTA, and urea is used as the sheath liquid, and the same component as the buffer solution in the capillary gel of the capillaries 1a to 1t is used to leak the constituent components of the capillary gel into the fluorescent cell 4. To prevent.

Further, the inside of the fluorescent cell 4 in which the capillaries 1a to 1t and the capillaries 2a to 2t are arranged is filled with a sheath liquid, and the surface of the sheath liquid 11 in the sheath liquid container 10 is filled with the anode side electrode tank 7 By arranging it higher than the liquid level of the buffer solution inside, the sheath solution flows into the anode-side electrode tank 7 through the capillaries 2a to 2t. In this state, the inside of the fluorescent cell 4 and the inside of the capillaries 2a to 2t are filled with the sheath liquid, that is, the buffer liquid, and the capillaries 1a to 1t.
Is also filled with the capillary gel, and by applying a DC voltage between the cathode-side electrode tank 6 and the anode-side electrode tank 7 by the DC high-voltage power supply 12, for example, the capillary 1a and the gap 3
An electric current flows so as to penetrate a and the capillary 2a, and the sample can be electrophoresed. In addition, the sheath liquid container 10
By arranging the liquid level of the sheath liquid 11 in the inner side higher than the liquid level of the buffer liquid in the anode-side electrode tank 7, the capillaries 2a to 2t are provided.
The sheath liquid flows inside the inside of the tube, and a flow is generated around the upper portions of the capillaries 2a to 2t, that is, the gaps 3a to 3t. Therefore, capillary 1a ~
Samples migrated from 1t are capillaries 2a to 2
Along the flow of the upper part of t, they pass through the gaps 3a to 3t in the sheath flow state and are guided to the respective capillaries,
It migrates to the side of the anode-side electrode tank 7. Capillaries 2a-2
t is treated so that the inside has a positive charge,
The electroosmotic flow in the capillaries 2a to 2t is directed from the capillaries 1a to 1t toward the anode side electrode tank 7, and there is no backflow of the liquid into the gap portion, and the sheath liquid can be stably flowed toward the anode side electrode tank 7. In particular, even when the flow rate of the sheath liquid is small, the sheath liquid can stably flow in the direction of the anode-side electrode tank 7.

The electrophoresis is performed by applying a DC voltage between the cathode side electrode tank 6 and the anode side electrode tank 7 by a DC high voltage power supply 12. When a voltage is applied, the current flowing through the capillaries 1a and 2a mainly flows through the gap 3a, and similarly the capillaries 1b and 2b, 1c and 2c, 1
The currents flowing through d and 2d ... 1t and 2t mainly flow through the gaps 3b, 3c, 3d. Further, as described above, the samples migrated from the capillaries 1a to 1t pass through the gaps 3a to 3t in the sheath flow state along the upper flow of the capillaries 2a to 2t and are guided to the respective capillaries. . That is, the sample migrating in each capillary and the gap can be migrated in the adjacent gap without contacting the migrating sample, and the sample migrating in each gap can be separated for fluorescence measurement. Also,
Since the sample does not come into contact with the inner surface of the fluorescent cell 4, there is no influence of the adsorption of the sample on the fluorescent cell, and the scattered light on the fluorescent cell surface can be spatially removed by a slit or the like, thus providing highly sensitive fluorescent light. Measurement becomes possible. The fluorescence-labeled DNA fragment as a sample is introduced by temporarily immersing the end of the capillary 1a on the cathode side in the sample solution and applying a voltage of 6 kV for about 20 seconds between the sample solution and the anode-side electrode tank 7. . After that, the end of the capillary is returned to the original cathode-side electrode tank 6. This operation is performed for each of the capillaries 1b to 1t, and the sample is injected into the capillary gel of the capillaries 1a to 1t.
The samples may be injected one by one as described above, or by immersing each of the capillaries 1a to 1t in the sample solution and applying a voltage at the same time. The molecular weight separation of the sample by electrophoresis is performed by applying a DC voltage of 6 kV between the cathode side electrode tank 6 and the anode side electrode tank 7. The sample injected into each of the capillaries 1a to 1t is electrophoresed in the capillary gel of each of the capillaries 1a to 1t while being separated in molecular weight from the cathode side toward the anode side, and passes through the gaps 3a to 3t, respectively. Gap 3a ~
The detection of the sample passing 3t is carried out by using F which is a fluorescent substance for labeling.
Argon laser light having a wavelength of 488 nm is used to excite the ITC, and the laser optical axis is adjusted so that the laser light irradiates the gap portions 3a to 3t aligned in a straight line at the same time and under substantially the same conditions. It is performed by measuring the amount of fluorescence emitted. That is, the laser light 21 of the wavelength of 488 nm of the argon laser light source 20 is narrowed down by the lens 22 and irradiated to excite the fluorescence labeled DNA fragment passing through the gaps 3a to 3t. A laser beam having a beam diameter of about 0.7 mm is used, and a lens 22 having a focal length of 100 mm is used to focus on the middle of the gaps 3a to 3t. In this case, the spot size of the laser light at the focus is about 150 μm, and the depth of focus is about 20 mm. The distance from the gap 3a to the gap 3t is equal to the distance from the capillary 1a to the capillary 1t, and in the case of this example, 0.6 mm × 19.
That is, it is about 12 mm. That is, the laser light is generated in the gap 3
The 20 spots a to 3t are irradiated with almost the same spot size, and the spot size is almost the same as the thickness (100 μm) of the capillary gel. As described above, by selecting the light source and the lens system so that the spot size of the laser beam can be made almost the same as the thickness of the capillary gel and all gaps can be irradiated with almost the same spot size. The fluorescently labeled DNA fragments that migrate in the gaps 3a to 3t can be excited uniformly and efficiently.

Fluorescently labeled DN that migrates in the gaps 3a to 3t
The fluorescence from the A fragment is detected in the direction perpendicular to the laser light irradiation direction. The block diagram is shown in FIG. The fluorescence 30 emitted from the DNA fragment is filtered by an interference filter 32 to remove background light such as scattered light, and is imaged by a lens 33 on a two-dimensional detector 34 such as a CCD camera. The two-dimensional detector 34 is controlled by the controller 35 to detect the fluorescence image of the gaps 3a to 3t, and the data processing device 36 such as a computer continuously changes the fluorescence intensity of each gap 3a to 3t with time. Moreover, all the gaps are measured at the same time, the results are displayed on the monitor 37, output to the printer 38, and the memory 3
Save in 9. As a result, the migration patterns whose molecular weights have been separated for each of the capillaries 1a to 1t can be measured simultaneously and continuously. In the case of this example, since the fluorescent images in the gap are arranged one-dimensionally, a one-dimensional detector such as a photodiode array can be used instead of the CCD camera or the two-dimensional detector. Further, as the interference filter 32, a bandpass interference filter that passes a wavelength range of 500 nm to 540 nm is used in order to effectively detect the fluorescence from the FITC. In addition, the gap 3a-
The image magnification of the lens 33 is set so that the 3t image is entirely formed on the light receiving surface of the two-dimensional detector. In this embodiment, since the gap is basically a buffer solution, the laser light propagates through the gap without being scattered by the capillaries and the like. for that reason,
Simultaneously and homogenize samples running multiple capillaries
Light can be efficiently irradiated. Further, background light such as scattered light or fluorescence due to the capillaries or the capillary gel can be significantly reduced, and fluorescence can be detected with high sensitivity. As the effect of reducing the background light, for example, as compared with the case where the capillary itself without the coating is irradiated with laser light and fluorescence is detected without forming a gap, fluorescence measurement is performed in the gap as in the present embodiment. When this is done, the background light intensity to be detected becomes about 1/10 or less, and it becomes possible to detect a sample having a lower concentration. Further, by arranging the plurality of gaps on a straight line, it is possible to irradiate all the gaps with the laser light simultaneously and easily, and it is possible to provide a simple device configuration. Further, since a plurality of pairs of capillaries can be held in one optical cell, only one optical cell is required, and only one pipe is required for injecting the sheath liquid. Moreover, since the sheath flow occurs only in the vicinity of the gaps, the flow velocity in each gap is almost the same in all the gaps, and the reproducibility between the gaps is high. Therefore, there is little variation between migration paths, and an optical cell with a simple structure as in the present embodiment is sufficient, and it is necessary to use an optical cell with a complicated shape like a normal sheath flow chamber. There is no problem, and the device configuration is simple as a whole. In this embodiment, the distance between adjacent capillaries is set to 0.6 mm. In this way, by allowing the pair of capillaries to face each other while holding a certain gap, the position where the sample migrates can be defined, a plurality of capillaries (and gaps) can be arranged in close proximity, and the optical cell Can be miniaturized. Further, this also makes it possible to squeeze the laser beam more finely and irradiate the gap. On the contrary, it becomes possible to hold more capillary pairs in the same optical cell. According to this example, since the molecular weight separation unit is a capillary gel similar to the conventional one, it is possible to detect a sample with high sensitivity without impairing the molecular weight separation characteristics.

Further, according to the present embodiment, since the buffer solution can be flowed without using a mechanical means such as a liquid chromatography pump, the apparatus structure is simplified,
It will be cheaper. In addition, since there is no pulsating flow that occurs when using a pump or the like, the flow of the sheath liquid is stable, fluctuations in the flow velocity are reduced, fluctuations in the fluorescence intensity of the sample flowing through the gap are suppressed, and measurement accuracy is improved. be able to. The flow rate of the sheath liquid can be easily adjusted by changing the difference between the liquid level of the sheath liquid in the sheath liquid container and the liquid level of the buffer liquid in the anode-side electrode tank downstream of the migration. It can also be adjusted by changing the inner diameter of the capillary on the downstream side of the migration. It is basically possible to flow the sheath liquid by a mechanical means such as a liquid chromatography pump. In this case, there is an advantage that the flow rate can be set directly. However, the fluorescence intensity is likely to change due to the pulsating flow of the pump, and therefore it is necessary to sufficiently perform smoothing processing in data processing.

The sheath liquid inside the optical cell flows out of the cell through the capillaries 2a to 2t on the downstream side. Since the inner diameter of a capillary is generally small, the flow rate therethrough is generally low. Therefore, the amount of sheath liquid can be reduced and the operability is improved. In addition, in the present embodiment, the capillaries 2a to 2t are processed so as to have a positive charge, and the direction of the electroosmotic flow in the capillaries 2a to 2t is changed from the capillaries 1a to 1t to the anode side electrode tank 7. As a result, there was no backflow of liquid from the capillaries 2a to 2t into the gap. As a result, even when the flow rate of the sheath liquid is low, the sheath liquid can be stably flown toward the anode electrode chamber 7. In addition, the capillary 2a
When the flow velocity of the sheath liquid in the gap is large, such as when the inner diameter of ~ 2t is large, the effect of the electroosmotic flow becomes small, so that it is not always necessary to treat the inner surfaces of the capillaries 2a to 2t. In this example, a buffer solution containing Tris, boric acid, EDTA, and urea was used as the buffer solution in the sheath solution, the anode-side electrode tank, and the cathode-side electrode tank, and the same component as the buffer solution of the capillary gel was used. This prevents the constituents of the capillary gel from leaking into the fluorescent cell 4 or into the electrode tank, and electrophoresis, which is more stable and has good resolution, can be continued including reuse of the capillary gel. It is also possible to use a buffer solution containing no urea that is a denaturant for DNA. In this case, there is a possibility that urea in the capillary gel will leak from the capillary gel into the electrode tank or fluorescent cell over time, and the number of times of repeated use will decrease somewhat, but the same migration as in the case of a buffer solution containing urea will occur. Is possible.

The laser device and phosphor used are:
Not limited to argon laser and FITC,
Any phosphor and suitable laser device can be used. In the present embodiment, the measurement of DNA fragments has been described as an example, but it can be naturally used for analysis of proteins, sugars and the like. Further, in this embodiment, both capillaries having the same inner diameter are used, but capillaries having different inner diameters can be combined. For example, if the inner diameter of the capillary on the downstream side is made smaller than the inner diameter of the capillary on the upstream side, the sample migrated from the end of the capillary on the upstream side is narrowed down when it is introduced into the capillary on the downstream side, so that the concentration of the sample solution is high. Becomes
It is also possible to detect with higher sensitivity. Further, by making the inner diameter of the downstream capillary larger than the inner diameter of the upstream capillary, the sample migrated from the upstream capillary end can be more easily and reliably guided to the downstream capillary. Further, according to the present embodiment, the fluorescence from the sample can be measured without transmitting the excitation light through the capillary portion, and therefore the capillary does not need to be transparent. That is, since it is not necessary to remove the coating of the capillary, the handling becomes easy. Furthermore, capillaries made of various materials such as opaque fluororesin capillaries such as tetrafluoroethylene resin and trifluoroethylene chloride resin can be used. Since the capillary made of fluororesin absorbs a small amount of a sample, it does not require a treatment operation such as surface treatment, and since it does not break, the operability is improved. Further, since it has excellent chemical resistance, it is possible to use a solvent having a wide pH range. In the present embodiment, the case of a plurality of capillary pairs has been described, but in the case of only one capillary pair, migration, fluorescence detection, and sample separation pattern can be similarly measured. In this case, a photomultiplier tube or the like can be used as the photodetector.

[Example 2] Next, a method for determining the nucleotide sequence of DNA using the apparatus of Example 1 will be described. By the well-known Sanger et al. Dideoxy method, a fluorescently labeled primer is used to carry out a DNA polymerase reaction to prepare a fluorescently labeled DNA fragment. A FITC-bound primer (labeled primer) is used as the primer. First, a labeled primer is added to the single-stranded DNA and annealed (double-strand formation) to bind the labeled primer to the single-stranded DNA. Add this reaction mixture to 4
It is divided, and each is subjected to a DNA polymerase reaction corresponding to A, C, G, and T. That is, four kinds of deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP) and ddATP serving as a terminator are added to the single-stranded DNA bound with the labeled primer to carry out a polymerase reaction. By this reaction, fluorescently labeled DNA fragments having various lengths with A at the ends are obtained. The same reaction is performed for C, G and T. Four A's obtained as described above,
The reaction liquids of C, G, and T are injected into four of the capillaries 1a to 1t, for example, capillaries 1a, 1b, 1c, and 1d. The injection method was the same as in Example 1, except that A reaction liquid was 1a, C reaction liquid was 1b, G reaction liquid was 1c, and T reaction liquid was 1T.
The reaction solution is poured into 1d. Electrophoresis is performed by applying a voltage of about 6 kV after the injection. The time change of the fluorescence intensity in each of the gaps 3a to 3d is measured by using the argon laser light having the wavelength of 488 nm as the excitation light. Since the DNA fragments are electrophoresed in ascending order of molecular weight, the base sequence can be analyzed by analyzing the gap positions where the fluorescence peaks occur in chronological order. The device of Example 1 has 20 capillaries into which a sample can be injected. According to the above DNA base sequence determination method, the base sequences of 5 types of DNA samples can be determined at the same time. It is also possible to determine the base sequence of more DNA samples by increasing the number of capillary pairs retained in the optical cell. In this embodiment, one type of FITC is used as the phosphor. However, it is also possible to detect fluorescence from two or more kinds of phosphors at the same time. In that case, for example, as is well known, as shown in FIG. 2, a set of photodetecting devices each including an interference filter 32, a lens 33, and a detector 34 is provided in a number corresponding to the number of phosphors, and each set has a different wavelength range. May be detected, or a dispersive prism may be arranged after the lens 33 to disperse light,
It is also possible to form an image on a two-dimensional detector such as a camera to receive the light, measure the fluorescence intensity for each migration path and each wavelength, and calculate the fluorescence intensity for each migration path and each phosphor.

As described above, the base sequence of DNA can be determined even when the apparatus is constructed so that a plurality of fluorescent substances can be simultaneously measured. In other words, primers labeled with different fluorophores are used for each type of terminal base, and
After carrying out the A polymerase reaction, the reaction solutions are mixed and electrophoresed. The base species can be identified and the base sequence can be determined by detecting the fluorescence of the DNA fragment passing through the gap space and identifying the type of the fluorophore at that time. The type of the phosphor can be identified by comparing the fluorescence intensities of the four types of phosphors at the maximum fluorescence wavelength. In this case, the base sequence of the DNA sample can be determined by the migration path composed of a pair of capillaries and a gap. That is, in the case of having a plurality of migration paths as shown in FIG. 1, it becomes possible to simultaneously determine the base sequences of a plurality of DNA samples.

[Embodiment 3] In the above-mentioned first and second embodiments, the apparatus for detecting a sample by fluorescence measurement has been described. However, it can be used for light absorption measurement such as absorbance measurement and transmitted light intensity measurement. In such a case, the device can be similarly configured. FIG. 3 shows a configuration diagram of the light irradiation system / detection system portion of the electrophoretic apparatus in the case of light absorption measurement. The electrophoretic portion has the same configuration as that of FIG. 1 of the first embodiment. Sample, eg DNA
The introduction of the restriction enzyme-cleavage fragment is carried out in the same manner as in Example 1, and the electrophoresis for molecular weight separation is carried out in the same manner as in Example 1. Light from a light source 50 such as a xenon lamp or a D ₂ lamp is passed through a monochromator 51 to a DNA fragment passing through the gaps 3a to 3t, and is condensed by a lens 52 to irradiate the gaps 3a to 3t. The light wavelength for irradiation is usually set to the absorption wavelength of the sample.
About nm is suitable. The light absorbed and transmitted by the DNA fragment is condensed again by the lens 53 and detected by the one-dimensional sensor 54 such as a photodiode array. The operation of the one-dimensional sensor 54 is controlled by the controller 55 for the one-dimensional sensor, the migration pattern and the like for each gap is measured by the data processing device 56 such as a computer, and the results are displayed on the monitor 57 and displayed on the printer 58. It is output and stored in the memory 59. In the present embodiment, the gap portion is provided between the capillaries, the capillary portion is separated from the molecular weight separation portion, and the gap portion space is separated from the optical detection portion for measuring the absorbance,
Since the irradiation light passes through the sheath liquid with extremely little light scattering, the sample can be efficiently irradiated without being scattered by the capillaries, gels, etc., and highly accurate absorbance measurement can be performed. Specifically, when the transmitted light intensity (when the sample does not pass and the numerical aperture of the light-receiving lens is 0.19) when light is incident on the gap portion as in this embodiment, is 1. When transmitting through a capillary as in the conventional case, the transmitted light intensity is reduced to about 0.6. This is because the capillary itself and a gel such as polyacrylamide gel, which is a medium for separating molecular weights by electrophoresis, are light scatterers, so that incident light is scattered at that portion, and as a result, transmitted light is reduced. . A decrease in transmitted light intensity means a corresponding decrease in S / N ratio of the photodetector and the like, which results in a decrease in measurement accuracy. Moreover, in the conventional case, a part of the light scattered or reflected by the surface of the capillary is also detected, but the light is directly detected by the detector without irradiating the sample (without being absorbed by the sample). Incident. If there is a light component that is detected regardless of the presence of such a sample,
It becomes difficult to measure a minute change in light intensity, that is, it becomes difficult to measure the absorbance with high accuracy. In the present embodiment, the light passing through the monochromator 51 is focused on the gap portion by the lens for irradiation, but it is irradiated as substantially parallel light without being focused and the light passing through the gap portion. The same measurement can be performed by spatially separating only the light and measuring the light intensity.

[Embodiment 4] In the first to third embodiments, the gap portion is formed by the capillary pair, but the gap portion is not formed by only the capillary pair. For example, the molecular weight separation part may be a capillary as in the above-mentioned embodiment, and the gap part may be formed by the end of the capillary and a flat plate having pores. FIG. 4 shows a configuration diagram of the gap portion. As in Example 1, the molecular weight separating unit 20
The book capillaries 1a to 1t are held by the multi-capillary holder 5a and fixed to the fluorescent cell 60. Fluorescent cell 6
On the opposite side of 0, a flat plate 62 having pores 61a to 61t having an inner diameter of 200 μm is fixed. By providing the pores 61a to 61t at positions facing each of the capillaries 1a to 1t, a gap is formed, and by flowing the sheath liquid, the sample migrated from each capillary can be guided to the corresponding pore. it can. In addition, below the flat plate 62, a liquid reservoir 63 is arranged to temporarily store the solution flowing in through the pores, and a tube 64
Through the electrode tank 65. Electrophoresis is performed by applying a voltage to the electrode tank 65 and the other electrode tank (not shown),
It can be performed in the same manner as in the first embodiment. Irradiation of laser light,
The same applies to fluorescence detection and the like.

[Embodiment 5] In contrast to Embodiment 1, it is also possible to construct a system in which only the capillary on the upstream side is used without using the capillary on the downstream side and a sheath liquid is caused to flow to migrate. FIG. 5 shows a configuration diagram of the electrophoretic section of the electrophoretic device. Similar to the first embodiment, one end of each of the capillaries 1a to 1t is held inside the optical cell 4 with its tip aligned. For fixing the capillaries in the optical cell, a multi-capillary holder 5a having 20 vertical holes provided at 0.6 mm intervals in a flat plate block made of fluororesin, for example, ethylene tetrafluoride resin, is used. That is, the capillaries 1a to 1t are inserted into the 20 vertical holes of the multi-capillary holder 5a one by one, and the multi-capillary holder 5a is fixed in close contact with the upper portion of the fluorescent cell 4. A sealing plate 70 and a tube 71 made of ethylene tetrafluoride resin are connected to the lower part of the fluorescent cell 4, and the end of the tube 71 is immersed in the anode-side electrode tank 7 so that the sheath liquid can flow and electrophoresis can be performed. To do so. The fluorescent cell 4 is provided with a sheath liquid injection port 8 for filling the inside thereof with a sheath liquid so that the sheath liquid 11 in the sheath liquid container 10 can be injected through a tetrafluoroethylene resin tube 9. And In addition, by arranging the liquid level of the sheath liquid 11 in the sheath liquid container 10 higher than the liquid level of the buffer liquid in the anode electrode tank 7, the sample migrated from the capillaries 1a to 1t is wrapped in the sheath liquid. It can flow in the fluorescent cell 4. These solutions are finally led to the anode electrode tank 7 side. The electrophoresis is performed by applying a DC voltage between the cathode-side electrode tank 6 and the anode-side electrode tank 7 by the DC high-voltage power supply 12. The sample migrated out of each capillary migrates in the optical cell immediately below the capillary without contacting with the sample migrated in other capillaries, and finally migrates into the anode-side electrode tank 7. The sample is detected by irradiating the portion of the fluorescent cell 4 on the downstream side of 500 μm from the capillary end with laser light to receive the fluorescent light. The light receiving system has the same configuration as that of the first embodiment. This embodiment is basically different from the first embodiment in the method of forming the sheath flow. In this embodiment, the sheath liquid flows in a laminar flow state inside the optical cell. Therefore, there is an instability that the flow velocity of the sheath liquid is slightly different depending on the position inside the optical cell, and the migration path of the sample is easily changed. However, substantially the same effects as in Example 1 can be obtained in terms of sensitivity, simplicity of device configuration, and the like.

[Embodiment 6] In the apparatus described in Embodiment 1, the gap portion can be formed by using the downstream capillary as a flat plate having a plurality of grooves. FIG. 6 shows a perspective view of a flat plate having a plurality of grooves. In the figure, the case where four grooves are provided so as to correspond to four capillaries is illustrated. Four grooves 81a, 81b, 81c, 81d are formed on one surface of the flat plate 80 that conforms to the internal shape of the fluorescent cell. The shape of the groove corresponds to the size of the capillary for molecular weight separation,
For example, the width is 300 μm and the depth is 600 μm. Also,
The distance between the grooves is 1 mm. In addition, these intervals,
The shape of the groove can be freely changed. FIG. 7 shows a perspective view of a cross-sectional view of the fluorescent cell portion of the electrophoretic unit of the electrophoretic device using the flat plate. The width of the fluorescent cell 84 is 20 m inside the cell.
m, depth 3 mm, length 40 mm, and a flow cell type of (quartz) glass having a plate thickness of 2 mm with open upper and lower ends is used. Further, the figure shows a state in which the glass portion on the front side of the fluorescent cell is removed. The flat plate 80 is closely fitted into the inside of the fluorescent cell 84 and fitted into the groove 81a, 81b, 81c, 81.
Four channels formed by d and the glass of the fluorescent cell are formed. Further, a capillary having an inner diameter of 100 μm and an outer diameter of 200 (a gel is prepared inside in the same manner as in Example 1) 8
2a, 82b, 82c and 82d are fixed inside the fluorescent cell 84 by the multi-capillary holder 83. The distance between the capillaries is set to 1 mm to match the distance between the grooves, and the capillaries are adjusted so that the axes of the capillaries are located at the center of each groove. These are multi-capillary holders 83
This can be dealt with by adjusting the positions of holes and the like provided in the. In addition, the ends of the capillaries 82a, 82b, 82c, and 82d inside the fluorescent cell are aligned, and the ends of the capillaries and the flat plate 80 are aligned.
And are adjusted to be separated by about 1 mm and fixed inside the fluorescent cell. In addition, electrophoresis can be performed by bringing the lower part of the fluorescent cell into contact with the buffer solution in the anode electrode tank.
It is also possible to connect to the electrode tank via a tube as in the case of the fourth or fifth embodiment. Fluorescent cell 84
Is provided with a sheath liquid injection port 85 for filling the inside thereof with a sheath liquid, and the sheath liquid 88 in the sheath liquid container 87 can be injected through a tube 86 made of a tetrafluoroethylene resin. Further, by arranging the liquid level of the sheath liquid 88 in the sheath liquid container 87 to be higher than the liquid level of the buffer liquid in the anode-side electrode tank, the sample migrated from the capillaries 82a to 82d is wrapped in the sheath liquid to cause fluorescence. It can flow in the cell 84 and can be guided to the anode-side electrode tank through the grooves 81a to 81d. By performing the electrophoresis, laser light irradiation, and fluorescence detection in the same manner as described in the first embodiment, the samples separated by each of the plurality of capillaries can be simultaneously and efficiently detected. In this embodiment, the downstream side is not a capillary but a flat plate having grooves.
Therefore, the end faces of the respective grooves can be easily aligned, and the adjustment becomes easy. Further, if the flat plate is made of quartz glass or the like, the laser light is not diffusely reflected and even if the flat plate hits the flat plate, fluorescence is not generated from that portion, and the fluorescence measurement becomes easy.

[Embodiment 7] An apparatus and method for determining the base sequence of DNA using a gel-filled capillary array will be described. A sample for DNA sequencing (fluorescently labeled DNA fragment) is prepared in the same manner as in Example 2. In order to identify the four base species in one migration path (gel capillary), a primer labeled with a different fluorescent substance for each of the A, C, G, and T fragments is used. A primer (abbreviated as primer A) labeled with a fluorescent substance having a maximum fluorescence wavelength of about 550 nm was bound to the A fragment. Similarly C, G,
The T-fragments have maximum fluorescence wavelengths of about 520, 575, and 6 respectively.
Primers labeled with a 05 nm fluorescent substance (abbreviated as primer C, primer G, and primer T, respectively) were bound. These fragments were finally mixed in a single container, mixed with ethanol, and then dissolved in deionized formamide (containing 1/100 amount of Tris buffer).
Prior to injection into the gel capillaries, the fragments were heat-denatured by heating at 90 ° C. for 2 minutes to make the fragments single-stranded, and immediately thereafter, ice-cooled and used for the injection operation. The sample may be prepared by using the fluorescent label as a terminator instead of the primer. Next, the measuring device will be described. FIG. 8 is a block diagram of a sub-array for forming a capillary array, and FIG. 9 is a block diagram of the capillary array of the present embodiment in which a plurality of sub-arrays of FIG. 8 are combined. further,
10 is a configuration diagram of a DNA base sequence determination device using a capillary array, FIG. 11 is an enlarged cross-sectional view of a sample injection part, FIG. 12 is a configuration diagram of a migration current measurement / display part of the DNA base sequence determination device, and FIG. FIG. 14 is an explanatory diagram including an optical cell cross section for showing the configuration in the vicinity of the optical cell portion, FIG. 14 is a principle configuration diagram of a fluorescence detection / color separation portion, FIG. 15 is an image diagram of a fluorescence image obtained by a two-dimensional detector, and FIG. 16 is FIG. 17 shows an example (raw data) of a time waveform of the fluorescence signal intensity emitted from a sample which migrates one gel capillary, and FIG. 18 is analyzed. It is a time waveform for each of the obtained A, C, G, and T fragments.

96 capillaries filled with gel inside
Using this, a capillary array was prepared in which one end was arranged in a straight line. Assembling 96 pieces in an array with one holder complicates the assembly and makes it difficult to replace the capillaries. Therefore, a sub-array of 8 units is formed, and 12 sub-arrays are arranged to form a 96-capillary array. Configured. As shown in FIG. 8, eight capillaries 90 are arranged on the same plane, and the capillary holders 91 and 92 are arranged so that the centers of the capillaries are spaced at 9 mm at one end and 0.5 mm at the other end. Fix each and align their ends. Capillary ends 90a fixed at 9 mm intervals are the sample injection side ends, and 90b on the opposite side is the optical detection side end. This sub-array is shown in Figure 9.
12 sets are combined as shown in FIG.
7 are configured. The twelve capillary holders 91 are fixed by the area array holder 93 so that they are arranged in 12 rows on the same plane, and the capillary holders 92 at the other end are linear so that 12 pieces are arranged in a straight line in order. Array holder 2
Fix at 14. As a result, the capillary ends 90a are arranged in a grid pattern of eight 12 columns on the same plane, and on the 90b side, 96 capillary ends are arranged in a straight line. In FIG. 9, the capillaries 90 are connected and shown only for the first row sub-array. The capillaries in the middle of the subarrays in the 2nd to 12th columns are omitted. Although the sub-arrays themselves are omitted for the 3rd to 11th columns, they are arranged in order between the subarrays of the 1st, 2nd, and 12th columns. The capillary used had an inner diameter of 0.1 mm, an outer diameter of 0.2 mm, and a length of about 35 cm, and was filled with an acrylamide gel in the same manner as in Example 1. Gel concentration is 9
% T (ratio of (acrylamide + bisacrylamide) to the total amount of solution) and 0% C (ratio of bisacrylamide to (acrylamide + bisacrylamide)). The use of the capillary array 107 of this embodiment enables simultaneous analysis of 96 samples.

FIG. 10 is a block diagram of a DNA base sequence determination device. The ends of the capillary array 107 arranged in a grid pattern are inserted into the buffer solution (or sample solution) of the upper individual electrode tank 104 like a microplate having 96 wells. Although the connection state between the capillary array 107 and the upper individual electrode tank 104 is not shown in the figure, this will be described with reference to FIG. 11. In the figure, the number of capillaries is changed from 96 to 20 for the sake of clarity.
Shown less in the book. The other end of the linearly arranged capillary array 107 is fixed inside the optical cell 103. A sheath liquid (buffer solution) is injected into the optical cell 103 from a sheath liquid container 105 through a tube 122 and a valve 121, passes through the inside of a hollow capillary array 108 arranged in the lower part of the capillary array 107, and enters a lower electrode tank 106. Is discharged. The capillary array 107 and the hollow capillary array 108 are fixed with their ends facing each other and separated by 2 mm. Two types of laser devices were used as light sources for exciting the sample. One is an argon laser device 101 having a wavelength of 488 nm, the other is a YAG laser device 102 having a wavelength of 532 nm, and these are a mirror 109 and a dichroic mirror 11.
0 for argon laser light 111 and YAG laser light 11
The optical cell 103 was irradiated with the laser beam 113 so that the laser beam 113 becomes a single laser beam 113 with the lens 2 (not shown). Fluorescence generated from the fluorescently labeled sample is imaged on the two-dimensional detector 118 by the condensing lens 114, the four kinds of spectral filters 115a to 115d, the image division prism 116, and the imaging lens 117, and the DNA fragment of each sample. The data processing unit 119 analyzes the time waveform of the fluorescence intensity from Eq.

Injection of the sample into the capillary array 107 will be described with reference to FIG. 11 (enlarged sectional view of the sample injection part / upper electrode tank). FIG. 11 shows a cross-sectional view of a surface including a capillary held by one of the 12 capillary holders 91. Upper individual electrode tank 1 having 8 holes x 12 rows (96 holes in total) of container-like depressions 104a, 104b ...
Use 04. For example, a 96-well V-bottom well microplate or 96 sampling tubes 8 x 1
It is possible to use two rows of grids arranged at 9 mm intervals. The space between the capillary arrays 107 needs to match the shape of the upper individual electrode tank 104. Further, since the container-like depressions 104a, 104b ...
Six platinum electrodes 120a, 120b ... Are arranged and fixed in a grid of 8 × 12 rows by the electrode holding plate 120, similarly to the end of the capillary array 107 corresponding to the container-like depression. Further, in the electrode holding plate 120, holes 252a for passing capillaries are provided in the vicinity of the respective platinum electrodes,
252b ... Are provided. The sample 240 has a recess 104a,
Put it inside 104b. The electrode holding plate 120 is overlapped with the spacer 250 on the upper part of the upper individual electrode tank 104, and the area array holder 93 is further attached with the spacer 251.
The structure is such that they are sandwiched between. Individual capillaries 10
7a, 107b ... Are holes 252a, 2 of the electrode holding plate 120.
52b ... Are soaked in the sample liquid 240, and similarly, the platinum electrodes 120a, 120b. A voltage is applied between these electrodes and the electrodes in the lower electrode tank 106 almost at the same time, and
The NA fragment is injected into the capillary. The voltage is 100
To 200 V / cm for several seconds to 1
Apply for about 0 seconds. For example, it is applied at 5 kV for 10 seconds.
After the sample injection, the upper individual electrode tank 104 is removed, and another upper individual electrode tank 104 containing a buffer solution is set instead, and the electric field strength of 100 to 400 V / cm, for example, 7 kV.
The molecular weight separation is performed by applying and continuing the migration. With this configuration, 96 samples can be simultaneously injected, and then 96 samples can be simultaneously electrophoresed. Further, the sample injection need only be performed by stacking the electrode holding plate 120 and the area array holder 93, that is, the capillary array 107, and it is not necessary to manually perform each sample by pipetting as in the case of the conventional flat gel. , The operability can be greatly improved.

When an electric field is applied, as shown in FIG. 12, a mechanism is provided for continuously and simultaneously measuring and displaying the current value flowing in each capillary. A voltage is applied from the migration power supply 133 to the electrodes (120a, 120b ...) In the upper individual electrode tank 104 and the platinum electrode 209 in the lower electrode tank 106. A resistance 131 of about 10 kΩ and a voltmeter 132 are connected between the electrode 120a and the migration power supply 133 as shown in FIG. Current is electrode 209 → buffer solution 245
→ capillary array 108 → optical cell portion → individual capillaries 107a of the capillary array 107 → buffer solution 246 in the container-like recess 104a → electrode 120a →
The resistance 131 can be connected to the terminal 260. Although there is an optical cell section or the like between the capillary 107a and the capillary array 108, it is omitted in FIG. The current value flowing through the capillary is converted by the value of the voltmeter 132, and the current value is binarized by the binarizing means to display the current display panel 140.
To transmit. Although not shown in FIG. 12, the terminals 261 and 262 are similarly connected to the electrodes 120b and 120c, respectively, and the current values flowing through the capillaries 107b and 107c are binarized and transmitted to the current display panel 140. The same applies to all capillaries of the capillary array 107 below. The terminal 270 is the last 96th terminal. .. are arranged on the current display panel 140. The display lamps 140a, 140b ... Are arranged in 8 rows and 12 columns to display the current values of the capillaries 107a, 107b. In addition,
The resistor 131 has a resistance value sufficiently smaller than the resistance value of the capillary gel. Usually, there is no problem if it is 100 kΩ or less. The binarization level should be adjusted appropriately. The electrophoretic current is usually about 0.01 mA, though it depends on the applied voltage, the capillary length, and the like. If the gel is broken, the current will stop flowing or it will become smaller. Therefore, for example, when the display lamp is turned on when the current becomes 0.003 mA or less, an abnormality such as breakage of gel can be recognized on a capillary unit basis. As a result, it is possible to take a measure such as stopping the analysis of the measurement result for the abnormal capillary. Further, in this example, the capillaries are made into sub-arrays in units of eight, and only the unit having a broken capillary can be replaced, and the capillary array can be used effectively. In this way, the operability is good and the device configuration is economical.

Next, the structure near the optical cell portion will be described with reference to FIG. The optical cell 103 has a box-like shape and includes a housing 201, array guides 202 and 205, a sheath liquid injection port 203, optical windows 204 and 206, and the like. It should be noted that although a housing and an optical window are provided on the front surface and the rear surface of the paper in which FIG. 13 is described, they are omitted in the drawing. Also, the housing 2
01, the optical windows 204 and 206 are shown in cross section.
The upper part of the optical cell 103 is open, and the capillary array 107 fixed to the linear array holder 214 is inserted from this portion along the array guides 202 and 205 into the optical cell and fixed. The sheath liquid (buffer solution) is injected into the optical cell 103 from the sheath liquid container 105 via the tube 122 and the valve 121, and a sheath flow is formed in the cell as in the first embodiment. It is discharged into the lower electrode tank 106 through the inside of the arranged hollow capillary array 108.
The optical cell 103 is opened to the upper side, and the liquid level 230 of the sheath liquid in the sheath liquid container and the liquid level 231 of the sheath liquid flowing into the cell are interlocked, and this height and the lower electrode tank 10 are connected.
The sheath liquid flows in the cell due to the difference in the height of the liquid surface of No. 6 and the like. Hollow capillary array 108
Has an inner diameter of 0.2 after being subjected to the same inner surface treatment as in Example 1.
mm, outer diameter 0.35 mm, length 3 cm,
The linear array holders 215 were evenly arranged at intervals of 0.5 mm, separated by 2 mm from the end of the capillary array 107 inside the optical cell 103, and fixed so that the ends of the capillaries of the capillary arrays 107 and 108 face each other. The hollow capillary array 108 and the linear array holder 215 are fixed to the optical cell 103 and sealed to prevent water leakage. Further, the optical cell 103 and the lower electrode tank 106 are brought into close contact with each other with good airtightness, and the liquid accumulated therein is discharged from the discharge port 210 to the valve 21.
1, and is discarded into the waste liquid container 213 via the tube 212. That is, the liquid that exceeds the height of the discharge port 210 is discharged, and the height of the liquid surface in the lower electrode tank 106 becomes substantially constant. Therefore, even if the sheath liquid flows in, the head does not change much, and the sheath flow is stably formed. Further, when the valve 211 is closed, the inside of the lower electrode tank 106 is filled with the sheath liquid, so that the flow of the sheath liquid can be stopped and the sheath liquid can be effectively used. In particular, this mechanism is necessary when the measurement is finished and there is time until the next measurement. In this example, the liquid levels 230 and 231 are substantially the same, but for example, a liquid level sensor for the liquid level 231 may be provided and a mechanism for opening and closing the valve 121 according to the decrease in the liquid level may be provided. In this case, the sheath liquid container 10
5 can be arranged at a position higher than the optical cell, and the device configuration becomes easy.

The lower electrode tank 106 is provided with a platinum electrode 209 for voltage application and its terminal block 208. When using a gel capillary, a positive voltage is applied to the platinum electrode 209 (relative to the opposite electrode). For example, platinum electrode 1
It is possible to apply 0V to 20a and 7kV to the platinum electrode 209, but it is desirable to apply a voltage of -7kV to the platinum electrode 120a and 0V (ground) to the platinum electrode 209 to ensure safety. This is because the whole sheath liquid is the platinum electrode 20.
When the potential of the electrode 209 becomes a high voltage and the potential of the electrode 209 becomes a high voltage, the sheath liquid in the sheath liquid container, the optical cell, and the waste liquid container all become a high voltage, resulting in electric shock, leakage, insulation breakdown due to water leakage, etc. This increases the risk of. When the electrode 209 is at 0 V (grounded), the sample side has a negative high voltage, but since there is no buffer solution flow, there is no water leakage, and electric shock,
Since the electric leakage can be easily prevented, the device can be easily constructed. Further, it becomes easy to use metal such as stainless steel as the casing 201 of the optical cell, the workability is improved, and the optical cell can be manufactured at low cost. Although the electrode 209 is arranged in the lower part of the hollow capillary array 108 as in this example, it may be arranged in the optical cell or the sheath liquid container, and the same effect is obtained.

The laser light 113 is transmitted through the optical window 204 or 20.
The light enters through 6 to enter the optical cell, and excites the DNA fragment that migrates in the sheath flow state in the same manner as in Example 1 to generate fluorescence. The fluorescent light emission points 220 occur in a dot shape just below the capillary as shown in the figure. It will be. These images are detected from the direction perpendicular to the paper surface through an optical window (not shown). Note that the laser light 113 is squeezed and irradiated by a lens, but by using a lens having a focal length of about 150 mm, the laser light 113 can be uniformly irradiated over the entire width of the capillary array (about 50 mm in this example). it can. It is also possible to irradiate the laser light without making it coaxial, but in the case of making it coaxial, two laser lights irradiate the same location, so that each laser excites D
Since the migration distance of NA fragments can be made constant, there is no shift in migration time, and analysis accuracy is improved. Further, in the case of a phosphor that absorbs both laser light wavelengths, if it is coaxial, the excitation light intensity will increase substantially, and the detection sensitivity will improve. It should be noted that measurement is possible with only one laser.

The detection of the fluorescent image will be described with reference to FIG. The fluorescence emitted from the fluorescence emission point 220 is condensed by the condensing lens 114, divided into four by the image division prism 116, and imaged by the imaging lens 117 (2
21a-b). At this time, the four types of spectral filters 11
By arranging 5a to 5d on the front surface (or the rear surface) of the image division prism, it is possible to make the respective divided lights have different wavelength components. This has been described for one fluorescent emission point 220, but the same applies to all the emission points, and these divisions are performed simultaneously. That is, it is not time-divided as in the case of laser beam scanning or mechanical scanning of the detector, and the fluorescence intensity measurement interval does not become long and high-speed measurement is possible. It is also possible to measure continuously in real time. Further, by disposing the condenser lens in front of the image division prism, the fluorescence condensing efficiency is improved and the detection sensitivity can be improved. It is also possible to use a cylindrical lens instead of the condenser lens. In this case, since the light in only one direction is condensed, the efficiency is about half that in the case where a normal convex lens is used, but the detection sensitivity can be greatly improved compared to the case where it is not used. Further, in the case of using the image division prism, if the fluorescence emission position shifts, the ratio of the intensity of the divided image changes greatly. In the case of using a flat gel, there is a problem that the laser light incident on the gel is bent due to heat generation of the gel and the fluorescence emission position is displaced, and the measurement accuracy is easily deteriorated. Since the laser light passes through the inside, the laser light irradiates a fixed position without bending, so that the measurement accuracy is improved as compared with the conventional case.

Further, since the capillaries are densely arranged in the optical cell, the distance between both ends of the arrangement is about 5 cm or less even in the case of a large number of 96 migration lanes. Therefore, a condenser element such as a condenser lens or a cylindrical lens can be used in front of the image division prism, and fluorescence can be detected with higher sensitivity. In the case of a conventional slab gel, even with 24 migration lanes, the distance between the migration lanes at both ends is about 30 cm, and if a condenser lens is placed in front of the image division prism, a lens with a diameter of 30 cm is required. Really difficult for a reason. In this example, the width of the capillary array was about 50 mm, and the width of the light receiving element of the two-dimensional detector was about 27 mm. The image magnification as a whole is about 1/2, which is about 1/10 in the case of the flat gel method, which is better in light collection efficiency. Further, when the number of the capillary arrays is as small as about 20 to 30, the enlargement ratio becomes the same size, the light collection efficiency is improved, and the sensitivity is further enhanced.
In order to prevent a decrease in the light collection efficiency, it is desirable that the distance between the capillaries in the capillary array be about 10 times the capillary diameter or less. FIG. 15 is an image diagram of a fluorescence image on a two-dimensional detector that is actually obtained, and is a case of 20 capillary arrays. The horizontal direction in the figure is the capillary array (arrangement) direction, and the vertical direction is the (color) division direction. Similarly, in the case of 96 pieces, 96 pieces horizontally and 4 pieces vertically
Individual fluorescent images are obtained. In this way, all the samples that migrate on the capillary array can be divided into 4 (color) and measured simultaneously. Therefore, four types of spectral filters 115a-
By adjusting d so that the fluorescence of the primer A, the primer C, the primer G, and the primer T is transmitted,
The primer A, the primer C, the primer G, and the primer T can be identified and measured. FIG. 16 is a diagram showing the fluorescence characteristics of the fluorescent-labeled primer and the transmission characteristic of the spectral filter. Do not use) to make measurements.

FIG. 17 is an actual measurement value of the time waveform of the fluorescence signal intensity emitted from the sample in which one gel capillary of the capillary array is electrophoresed. Spectral filter 1 from above
15A, 15B, 15C, and 15D are time waveforms of fluorescence intensity transmitted through 15a, b, c, and d. In this way, four types of fluorescence information can be obtained simultaneously from one sample / one capillary. However, as can be seen from FIG. 16, the fluorescence of the fluorescence-labeled primer is broad, and the fluorescences of the four types of fluorescence-labeled primers overlap. Therefore, the waveform of FIG. 17 is observed as the sum of the fluorescence of a plurality of fluorescent-labeled primers, not the signal of a single fluorescent-labeled primer. For example, Ia is the primer T
To the fluorescence of primer G, primer A, primer C
It is a mixture of fluorescent components. The same applies to Ib, Ic, and Id. When these influences are corrected based on the fluorescence characteristics of the fluorescence-labeled primer, the transmission characteristics of the spectral filter, and the sensitivity characteristics of the detector, the fluorescence intensities of the primer T, the primer G, the primer A, and the primer C alone as shown in FIG. The time waveform of is obtained. This is T,
The migration spectra of the G, A, and C fragments are obtained, and the base sequence of the DNA can be determined from this waveform by a usual method. In this example, the nucleotide sequence determination can be performed simultaneously on 96 samples, and high throughput can be achieved. Specifically, the migration rate is about 200 bases / hour, and the overall throughput is 20 k bases / hour, which is 10 times higher than the conventional method using a slab gel. Further, this embodiment also has the same effect as described in the first embodiment. Further, even when the above optical cell is used as the optical cell having the structure described in Example 1 or Examples 4 to 6, the measurement for determining the nucleotide sequence of DNA can be carried out in substantially the same manner as described above. There are similar effects as described for Examples 4-6. Further, in this embodiment, the four-division prism is used, but the two-division prism is used.
The base sequence of DNA can be determined by identifying the base species according to the method described in 18991. In addition,
In the present embodiment, the capillaries of the capillary array are subjected to silane coupling treatment on the inner surface in the same manner as in the first embodiment,
The gel was chemically bound to the inner wall of the capillary. It is not necessary to perform this treatment on the entire inner wall of the capillary,
That is, it may be only near the end on the optical cell side. This makes it possible to prevent the gel from squeezing out into the sheath liquid inside the optical cell, simplify the process, and stably prepare the gel. Further, a surfactant or the like may be added to the gel. These can be similarly applied to the first to sixth embodiments described above.

[0038]

According to the present invention, an electrophoretic device capable of highly sensitive fluorescence or optical absorption measurement with little influence of background light or the like is obtained by performing optical measurement outside a sample separation unit such as a capillary gel. realizable. Further, it is possible to realize a simple electrophoretic device capable of simultaneously migrating a plurality of samples and simultaneously measuring them. Further, it is possible to realize an apparatus which has high operability and safety and can determine the base sequence of DNA with high throughput.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an electrophoretic unit and a laser light irradiation system of an electrophoretic device according to a first embodiment of the present invention.

FIG. 2 is a configuration diagram of a fluorescence detection system of the electrophoretic device according to the first embodiment of the present invention.

FIG. 3 is a configuration diagram of a light irradiation system / detection system portion of an electrophoretic device according to a third embodiment of the present invention.

FIG. 4 is a configuration diagram in the vicinity of a gap of an electrophoretic device according to a fourth embodiment of the present invention.

FIG. 5 is a configuration diagram of an electrophoretic unit of an electrophoretic device according to a fifth embodiment of the present invention.

FIG. 6 is a perspective view of a flat plate having a plurality of grooves according to a sixth embodiment of the present invention.

FIG. 7 is a diagram showing a fluorescent cell portion of an electrophoretic unit of an electrophoretic device according to a sixth embodiment of the present invention.

FIG. 8 is a configuration diagram of a sub-array of capillaries used in a seventh embodiment of the present invention.

FIG. 9 is a configuration diagram of a capillary array used in a seventh embodiment of the present invention.

FIG. 10 is a configuration diagram of an electrophoretic device according to a seventh embodiment of the present invention.

FIG. 11 shows a sample injection part / in a seventh embodiment of the present invention.
The expanded sectional view of an upper electrode tank.

FIG. 12 is a configuration diagram of an electrophoretic current measurement / display unit in an electrophoretic device according to a seventh embodiment of the present invention.

FIG. 13 is an explanatory diagram including a partial cross section showing the configuration in the vicinity of the optical cell portion in the electrophoretic device according to the seventh embodiment of the present invention.

FIG. 14 is a principle configuration diagram of a fluorescence detection / color separation unit in an electrophoretic device according to a seventh embodiment of the present invention.

FIG. 15 is an image diagram of a fluorescence image obtained by a two-dimensional detector in the electrophoretic device according to the seventh embodiment of the present invention.

FIG. 16 is a diagram showing the fluorescence characteristics of the fluorescence-labeled primer used in the seventh embodiment of the present invention and the transmission characteristics of the spectral filter.

FIG. 17 is a diagram showing an example of measurement data of time waveform of fluorescence signal intensity emitted from a sample on which one gel capillary is migrated according to the seventh embodiment of the present invention.

FIG. 18 is a graph obtained by analyzing the data in FIG.
Time waveforms for G and T fragments.

FIG. 19 is a configuration diagram of a fluorescence measurement unit of a conventional capillary electrophoresis device.

FIG. 20 is a configuration diagram of a fluorescence measurement unit using a sheath flow cuvette of a conventional capillary electrophoresis apparatus.

[Explanation of symbols]

1a, 1b, 1c, 1d to 1t ... Capillaries, 2
a, 2b, 2c, 2d to 2t ... Capillary, 4 ... Fluorescent cell, 3a, 3b, 3c, 3d to 3t ... Gap portion, 5
a and 5b ... Multi-capillary holder, 6 ... Cathode side electrode tank, 7 ... Anode side electrode tank, 8 ... Sheath liquid injection port, 9 ... Tetrafluoroethylene resin tube, 10 ... Sheath liquid container, 11 ... Sheath Liquid, 12 ... DC high voltage power source, 20 ... Argon laser light source, 21 ... Laser light, 22 ... Lens, 3
0 ... Fluorescence, 32 ... Interference filter, 33 ... Lens, 34 ...
Two-dimensional detector, 35 ... Controller, 36 ... Data processing device, 37 ... Monitor, 38 ... Printer, 39 ... Memory,
50 ... Light source, 51 ... Monochromator, 52 ... Lens, 5
3 ... Lens, 54 ... One-dimensional sensor, 55 ... One-dimensional sensor controller, 56 ... Data processing device, 57 ... Monitor, 58 ... Printer, 59 ... Memory, 60 ... Fluorescent cell,
61a to 61t ... Pores, 62 ... Flat plate, 63 ... Liquid reservoir, 6
4 ... Tube, 65 ... Electrode tank, 70 ... Seal plate, 71 ...
Tube, 80 ... Flat plate, 81a, 81b, 81c, 81
d ... Groove, 84 ... Fluorescent cell, 82a, 82b, 82c, 8
2d ... Capillary, 83 ... Multi-capillary holder, 85 ... Sheath liquid inlet, 86 ... Tube, 87 ... Sheath liquid container, 88 ... Sheath liquid, 90 ... Capillary, 9
0a, 90b ... Capillary end, 91, 92 ... Capillary holder, 93 ... Area array holder, 107 ... Capillary array, 214 ... Linear array holder, 1
01 ... Argon laser device, 102 ... YAG laser device, 103 ... Optical cell, 104 ... Upper individual electrode tank, 10
4a, 104b -... Container-like recess, 105 ... Sheath liquid container, 106 ... Lower electrode tank, 107 ... Capillary array, 107a, 107b -... Capillary, 108 ... Hollow capillary array, 109 ... Mirror, 110 ... Dichroic mirror, 111 … Argon laser light, 11
2 ... YAG laser light, 113 ... Laser light, 114 ... Condensing lens, 115a-d ... Spectral filter, 116 ... Image splitting prism, 117 ... Imaging lens, 118 ... Two-dimensional detector, 119 ... Data processing unit, 120 ... Electrode holding plate, 120a, 120b ... Platinum electrode, 121 ... Valve,
122 ... Tube, 131 ... Resistance, 132 ... Voltmeter, 1
33 ... Migration power source, 140 ... Current display panel, 140a,
140b -... Indicator lamp, 201 ... Housing, 202, 20
5 ... Array guide, 203 ... Sheath liquid inlet, 204,
206 ... Optical window, 208 ... Terminal block, 209 ... Platinum electrode,
210 ... outlet, 211 ... valve, 212 ... tube,
213 ... Waste liquid container, 214 ... Linear array holder, 2
15 ... Linear array holder, 220 ... Fluorescent emission point, 2
21a to 221d ... Imaging, 230 ... Liquid level of sheath liquid in sheath liquid container, 231 ... Liquid level of sheath liquid flowing into cell, 240 ... Sample, 245, 246 ... Buffer liquid, 2
50, 251 ... Spacer, 252, 253 ... Hole, 26
0, 261, 262, 270 ... Terminals.

Claims (42)

[Claims] 1. An electrophoretic device in which a sample separating portion provided between a cathode electrode tank and an anode electrode tank to which a voltage is applied by a power source is a migration path composed of a capillary, and one end of which is the cathode electrode tank or The other end of the pair of capillaries connected to the anode electrode tank are arranged in the optical cell so that their axes are substantially aligned with each other and are opposed to each other with a certain gap therebetween, and a migration path that penetrates the optical cell, and the optical cell. And a sample to be electrophoresed from the capillary end for electrophoretic separation, which is an electrophoretic passage on the upstream side that is a sample separation unit, by injecting and flowing the sheath liquid into the optical cell from the sheath liquid container. Sheath flow forming means for introducing the sheath flow state to the opposite capillary, and light detecting means for detecting the sample by irradiating the optical detecting portion with light from a light source. Electrophoresis apparatus according to claim Rukoto. 2. A plurality of optical detectors formed by a plurality of capillary pairs are arranged in the optical cell, and the plurality of optical detectors are connected to each other through an electrolytic solution. Item 1. The electrophoresis apparatus according to Item 1. 3. The plurality of pairs of capillaries are aligned and arranged so that the plurality of optical detectors are located on a straight line.
3. The electrophoresis according to claim 2, wherein the excitation light is irradiated along the straight line so as to simultaneously irradiate all the optical detection units, and fluorescence emitted from the sample flowing out of the respective capillaries is detected at the same time. apparatus. 4. An electrophoretic device in which a sample separating portion provided between a cathode electrode tank and an anode electrode tank to which a voltage is applied by a power source is a migration path composed of capillaries, and one end of which is immersed in the electrode tank. An optical cell in which a plurality of electrophoretic separation capillaries serving as sample separation units terminate, a sheath liquid container provided outside the optical cell, and a sheath liquid injected into the optical cell from the sheath liquid container. Sheath flow forming means for causing a sample to flow out from each of the plurality of phoretic separation capillaries by flowing in the optical cell in a sheath flow state, and the sheath flow part as an optical detection part. An electrophoretic device comprising: a light detection unit that irradiates light from a light source to detect a sample. 5. The plurality of electrophoretic separation capillaries are arranged in line so that the plurality of optical detection sections are positioned on a straight line, and the excitation light is linearly irradiated so that all the optical detection sections are simultaneously irradiated. 5. The electrophoresis apparatus according to claim 4, wherein the fluorescence emitted from the sample migrating from each of the plurality of capillaries for electrophoretic separation is simultaneously detected by irradiating the sample along the path. 6. The electrophoretic device according to claim 1, wherein the insides of the plurality of electrophoresis separation capillaries are filled with a gel. 7. The electrophoresis apparatus according to claim 1, wherein the sheath liquid is a component equivalent to a buffer liquid filled in the capillary. 8. The electrophoretic device according to claim 1, wherein the sheath liquid contains a denaturant for the sample. 9. The sheath flow forming means is configured to inject the sheath liquid into the optical cell, and to move a surface of the sheath liquid in the sheath liquid container to a liquid surface in an electrode tank on a downstream side where the sample is migrated. The electrophoretic device according to claim 1 or 4, wherein the electrophoretic device is arranged at a higher position than the above, and the sheath liquid is caused to flow by a drop. 10. The electrophoretic device according to claim 1, wherein the photodetection means detects the fluorescence emitted from the sample in the optical detection section. 11. The electrophoretic device according to claim 1, wherein the light detecting means measures the light absorption by the sample in the optical detecting section. 12. An electrophoretic device in which a sample separation section provided between a cathode electrode tank and an anode electrode tank to which a voltage is applied by a power source is a migration path composed of capillaries, and a plurality of samples are electrophoretically separated. A capillary for electrophoretic separation, a capillary holding means for holding the ends of the plurality of capillaries in the migration direction of the sample so as to be aligned on a straight line at a predetermined interval, and the plurality of capillaries in the migration direction of the sample. An optical cell for fixing the ends of the capillaries for electrophoretic separation therein, and a sheath flow forming means for forming a sheath flow at least in the vicinity of the ends of the plurality of capillaries for electrophoretic separation and in the lower part thereof by flowing an aqueous solution into the optical cells. And discharge means for discharging the aqueous solution from the optical cell, and for generating fluorescence from the sample migrating in the sheath flow. An excitation light source unit that emits light, and irradiates the light from the excitation light source unit substantially parallel to the straight line directly below the ends of the plurality of electrophoresis separation capillaries arranged on the straight line in the optical cell, Excitation light irradiation means for generating fluorescence from the sample flowing out from each of the plurality of capillaries for electrophoretic separation, and image division means for respectively dividing a plurality of fluorescence images generated from the sample,
A spectral filter arranged in front of or behind the image dividing means so that the divided images have light components in different wavelength ranges, an image forming means for forming the divided image, An electrophoretic device comprising: an image detection unit that detects an image, and simultaneously detecting fluorescence emitted from a sample migrated from the inside of each of the plurality of capillaries for electrophoretic separation. 13. The electrophoresis apparatus according to claim 12, wherein a gel-like substance is filled in the plurality of capillaries for electrophoretic separation on which the measurement sample is electrophoretically separated. 14. The electrophoretic device according to claim 1, wherein the capillary holding means and the optical cell are detachable. 15. An insertion guide for holding a predetermined portion of the capillary holding means at a predetermined position inside the optical cell, wherein the insertion guide is provided. 15. The electrophoretic device according to claim 14. 16. The sheath flow forming means arranges a sheath liquid container outside the optical cell, and sets a liquid level in the sheath liquid container at a position higher than a liquid surface discharged from the optical cell and held therein. 13. The electrophoresis apparatus according to claim 12, wherein the electrophoresis apparatus is arranged, and the sheath liquid is caused to flow by utilizing the drop. 17. An upper part of the optical cell is open, the sheath flow forming means arranges a sheath liquid container outside the optical cell, and a liquid level in the sheath liquid container is inside the optical cell. It arrange | positions in a position higher than the position of a drainage port, and a sheath liquid is made to flow and a sheath flow is formed using a fall, The Claim 1, Claim 4, Claim 12, Claim 1 characterized by the above-mentioned.
The electrophoretic device according to any one of 3 above. 18. A means for controlling the liquid level in the optical cell to be substantially constant is provided. The electrophoretic device according to any one of claims. 19. An upper part of the optical cell is opened, and means for detecting the liquid level in the optical cell and a mechanism for replenishing the aqueous solution from the sheath liquid container through a valve when the liquid level is lowered are provided. The electrophoretic device according to any one of claims 1, 4, 12, 17, and 18, which is provided. 20. The discharge means is a hollow capillary having the same number as or more than the capillaries for electrophoretic separation, one end of which is held in a straight line at the same interval as the capillaries for electrophoretic separation, and the end of the capillary for electrophoretic separation. The electrophoretic device according to claim 12, wherein the electrophoretic device is a hollow capillary disposed in the vicinity of a plane including an axis on which light is emitted to generate fluorescence from the sample. 21. The electrophoretic device according to claim 1, wherein the excitation light source unit is a laser device. 22. The excitation light source section has at least two kinds of laser devices, and the excitation light irradiating means coaxially combines these laser lights into one. The electrophoretic device according to claim 4, claim 12, or claim 21. 23. The electrophoretic device according to claim 12, wherein the image dividing means is a polyhedral prism. 24. The electrophoretic device according to claim 12, wherein the image dividing means divides the image into four. 25. The electrophoretic device according to claim 3, wherein the image detecting means is a two-dimensional detector. 26. The electrophoresis apparatus according to claim 12 or 25, wherein the intensities of the fluorescent images from the samples migrated from a plurality of the electrophoresis separation capillaries are simultaneously measured. 27. The electrophoretic device according to claim 12, 25, or 26, wherein the intensities of the images formed by the image detection means are measured at the same time. 28. The electrophoretic device according to claim 1, wherein a sample is simultaneously injected into each of the plurality of capillaries for electrophoretic separation. 29. The electrode contacting an aqueous solution inside the optical cell or inside the sheath liquid container, claim 1, claim 4, claim 12, claim 16 or claim 1.
7. The electrophoresis apparatus according to any one of 7. 30. An electrode in contact with the aqueous solution inside the optical cell, an electrode in an electrode tank on the side where the sheath liquid is discharged from the optical cell, an electrode in contact with the aqueous solution inside the sheath liquid container,
Alternatively, the potential applied to the electrode of the electrode bath on the downstream side where the sample is migrated is 0 V (ground), or the potential whose absolute value is applied to the electrode of the electrode bath on the upstream side where the sample starts to migrate. The electrophoretic device according to any one of claims 1, 4, 12, 16, 17, and 29, which is a value smaller than the absolute value of. 31. The method according to claim 1, wherein the height of the liquid surface of the aqueous solution accumulated in the electrode tank on the downstream side where the sample is migrated is made substantially constant. 31. The electrophoretic device according to claim 30. 32. A means for discharging an excessive aqueous solution is provided in an electrode tank on the downstream side where the sample is electrophoresed, wherein the method is provided in claim 1, claim 4, claim 12, claim 30, and claim 30. Item 32. The electrophoretic device according to any one of Items 31. 33. The electrophoresis according to claim 12, 23, or 24, wherein the fluorescence emitted by the laser light is condensed by a lens and then divided to form an image. apparatus. 34. The light for generating fluorescence from the sample is laser light, and the fluorescence emitted is condensed in one direction by a cylindrical lens and then divided and imaged. The electrophoretic device according to any one of claims 23 and 24. 35. An image by fluorescence emitted from a sample electrophoresed from the plurality of electrophoretic separation capillaries arranged in the optical cell is detected at an image magnification of 1/3 or more. The electrophoretic device according to any one of claim 3, claim 5, claim 12 to claim 15, claim 25 to claim 27, claim 33, and claim 34. 36. The method according to claim 3, wherein the distance between the plurality of capillaries for electrophoretic separation and the adjacent capillaries is 10 times or less the diameter size of the capillaries. 15, claim 20, claim 3
The electrophoretic device according to any one of 5 above. 37. Any one of claim 1, claim 4, claim 12, claim 29, and claim 30, wherein the number of electrodes provided on the downstream side on which the sample is electrophoresed is one. An electrophoretic device according to item 1. 38. The number of electrodes provided on the injection side of the electrophoresis separation capillaries of the sample matches the number of the electrophoresis separation capillaries.
The electrophoretic device according to any one of claims 4, 12, and 28. 39. The method according to claim 1, wherein the current value flowing through the inside of the capillary for electrophoresis separation is measured and / or displayed for each capillary.
The electrophoretic device according to claim 38. 40. The method according to claim 1, wherein the current value flowing inside the capillary for electrophoretic separation is binarized and displayed. Electrophoresis device. 41. The method according to any one of claims 1 to 6, wherein at least the inner surface of the capillary for electrophoresis separation on the side held inside the optical cell is silanized.
The electrophoretic device according to claim 2 or claim 13. 42. The method according to any one of claims 1 to 12, wherein at least the inner wall of the capillary for electrophoresis separation on the side held inside the optical cell and the gel-like substance are chemically bonded. 42. The electrophoretic device according to any one of claims 13 and 41.