JP3042370B2 - Electrophoresis device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

2. Description of the Related Art As an electrophoresis apparatus for separating and analyzing the molecular weight of a fluorescently labeled sample by electrophoresis, there is, for example, a base sequence determining apparatus for DNA using a fluorescent substance as a label. The base sequence is determined by the well-known dideoxy method of Sanger et al. The molecular weight separation is performed by electrophoresis using a polyacrylamide gel or the like. Usually, electrophoresis is performed using a polyacrylamide gel on a flat plate created between two glass plates. In recent years, a capillary gel electrophoresis method in which a gel (capillary gel) is formed in a capillary and electrophoresed is used. Is being developed. In capillary gel electrophoresis, generally, the diameter of the capillary is small, the surface area per volume of the gel is large, and the Joule heat can be easily dissipated, so a high voltage can be applied and high-speed separation can be performed. Is a useful method. As a first conventional example of capillary gel electrophoresis, "New Clay Acid Research", Vol. 18,
Pp. 1415-1419 (1990) (Nuclei
c Acid Research, 18, 1415-1
419 (1990)). As shown in FIG. 19, using a capillary having an inner diameter of 75 μm,
A high voltage of V is applied to achieve high-speed and high-separation detection of DNA fragments and the like. In the detection unit of this method, the axis of the capillary and the irradiation axis of the laser beam are inclined by about 25 degrees from the vertical direction, and the laser beam is applied to the center of the capillary by about 2 mm in diameter.
The light is condensed and radiated to 0 μm, and the generated fluorescence is detected by spectral separation using a band-pass interference filter or the like. FIG. 19 is rewritten in an easy-to-understand manner based on the device 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)), DNA was detected by capillary gel electrophoresis.
A method for determining the base sequence is described. In this method, a DNA having an inner diameter of 50 μm is used for DNA.
The fragments are separated by molecular weight. The measuring device in this example is basically a “Science” magazine, Vol. 242, Vol. 562-564.
Page (1988) (Science, 242, 562)
564 (1988)).
In order to make it easy to understand in the equipment diagram described in "Science" magazine,
FIG. 20 shows an apparatus configuration to which a laser light source, a pump for sheath flow, and the like are added based on the description. For detection of the DNA fragment, the separated solution having a molecular weight separated is introduced into a quartz flow chamber (inner size 250 μm × 250 μm) and subjected to EDT.
A tris-borate buffer solution containing A is used as a sheath liquid to flow through a liquid chromatography pump to form a sheath flow state, and a laser beam is condensed to a diameter of about 10 μm with respect to a separated liquid flowing substantially in the center of the flow chamber. And the fluorescence from the DNA fragment is detected through a spectral filter or the like. As a third conventional example, a method for determining a base sequence in one migration path is described in, for example, “Nature”, Vol. 321, pp. 674-679 (198).
6 years) (Nature, 321, 674-679 (19
86)) and the like, four types of phosphors having different maximum fluorescence wavelengths (fluorescein, 4-chloro-7-nitrobenzo-2-oxa-1-oxa) for each type of terminal base of the DNA fragment. Labeled with diazole (NBD), tetramethylrhodamine, Texas Red (Molecular Probes)), the fragments that are electrophoresed in order of molecular weight are excited with laser light, and the fluorescence of each is filtered by four kinds of bandpass interference filters. There is a method of separating and detecting fluorescence to determine a base sequence. As described above, in capillary gel electrophoresis, measurement is usually performed using only one capillary.

[0003]

In a capillary electrophoresis apparatus, it is desired to increase the detection sensitivity as the amount of sample is reduced. Further, it is desired to improve the processing capacity, for example, to increase the number of samples that can be processed simultaneously.

In general, in the measurement of fluorescence in an electrophoretic state, in addition to the fluorescence from the intended phosphor itself, as background light,
Scattered light (such as Rayleigh scattering) and fluorescence of the excitation light by the gel, scattered light on the inner and outer walls of the gel support, that is, the capillary, or fluorescence from the capillary itself are generated. For this reason, the background level increases, and the detection sensitivity decreases. In other words, how to remove such background light is an important issue to achieve highly sensitive fluorescence detection.

In order to improve the processing capacity, it is necessary to increase the migration speed and to simultaneously migrate and measure a plurality of capillaries, which are migration paths, and it is important to achieve these. Issues.

In the first conventional example, a first measure for removing excitation light (scattered light) by a band-pass interference filter or the like and separating and detecting a fluorescent component, and a method in which the axis of the capillary is aligned with the irradiation axis of the laser light. By two measures of the second measure to incline about 25 degrees from the perpendicular with respect to the plane including the fluorescence focusing axis,
Fluorescence detection with higher sensitivity is aimed at. However, with the first measure, 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, though weakly, and it is difficult to sufficiently remove background light with a band-pass interference filter or the like. The second countermeasure is an effective method that can reduce the scattered light itself incident on the fluorescence focusing lens by tilting the capillary. However, since the capillary has a circular cross section and a small diameter, the scattering is difficult. The background light is sufficient because the light intensity is high, the scattered light is not scattered, it is emitted in all directions, and in addition, the fluorescence from the gel and the glass tube itself cannot be reduced by tilting the capillary. Is difficult to remove. From the above viewpoints, it cannot be said that background light is sufficiently removed, and it is difficult to detect fluorescence with high sensitivity. In this conventional example, the number of capillaries is one, and there is no description or suggestion of a method for simultaneous processing of a plurality of samples. Although it is conceivable to simply use a plurality of the devices shown in FIG. 19, a plurality of laser light sources, detectors, optical components, and the like are required, and the devices are large and expensive, which is not practical. Further, it is conceivable to arrange a plurality of capillaries and irradiate all the capillaries with a single laser beam.However, when irradiating the capillaries with laser light or the like, the cross section of the capillaries is circular, so that the light at the interface is Refraction and scattering occur, and the light passing through the capillary diffuses widely, so it is not practical to achieve this configuration. In other words, there is no problem when one capillary is used, but when continuously irradiating a plurality of capillaries, the intensity of light applied to the next capillary becomes extremely weak each time the capillary passes, making it difficult to measure fluorescence. become. From the above, it is difficult to apply the present conventional example to the simultaneous processing of a plurality of samples to improve the processing capacity.

The second conventional example is a method in which light scattered by a capillary and a gel can be effectively removed by capillary gel electrophoresis. In other words, by using the end of the capillary gel as the sample inlet of the sheath flow chamber, the sample solution is drawn out of the capillary gel, and the fluorescence measurement is performed under the sheath flow, so that the scattered light at the capillary interface and the scattering from the gel Light and fluorescence are eliminated. In addition, in the sheath flow chamber, the sample liquid flows in a laminar flow state at substantially the center thereof, and does not come into contact with the inner wall of the sheath flow chamber. And the fluorescence intensity can be detected with high sensitivity. However, in order to form a sheath flow, it is necessary to constantly flow the sheath flow chamber at a constant flow rate using a tris-borate buffer solution as a sheath liquid using a liquid chromatography pump or the like. Therefore, there is a problem that the apparatus is expensive and complicated. Further, the sheath flow chamber has a complicated shape in which the sheath liquid injection part is thick and the fluorescence measurement part, which is the sheath flow part, is thin, and it is difficult and expensive to manufacture the sheath flow chamber. Further, similarly to the first conventional example, the present conventional example does not describe or suggest simultaneous processing of a plurality of samples. It is not practical to simply use a plurality of the devices in FIG. 20 because the devices are 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 beam does not irradiate the capillary, there is no refraction or scattering of light by the capillary. However, since there is a sheath flow chamber for each capillary, it is difficult to spatially narrow the gap between the capillaries, and as a result, the sheath flow chamber becomes large, and when using a plurality of sheath flow chambers, ,
There is a problem that the device becomes large and expensive. Furthermore, in order to irradiate all of the sample liquid bundles flowing through a plurality of sheath flow chambers under the same conditions, the laser beam cannot be narrowed down. This is because the depth of focus of the laser beam is related to the irradiation spot size, and the smaller the irradiation spot size is, the smaller the depth of focus is, while the distance between the sample liquid fluxes flowing through the plurality of sheath flow chambers is wide. . From the above, it is difficult to apply the present conventional example to the simultaneous processing of a plurality of samples to improve the processing capacity.

An object of the present invention is to solve the above-mentioned problems of the prior art, and to provide an electrophoresis apparatus capable of easily performing a fluorescence measurement or a light absorption measurement of a sample separated by electrophoresis with high sensitivity, and An object of the present invention is to provide an electrophoresis apparatus capable of easily performing simultaneous processing of a plurality of samples. Another object of the present invention is to provide a high-sensitivity, high-throughput electrophoresis apparatus which is suitable for DNA base sequence measurement, has good operability and safety, and has high sensitivity.

[0009]

In order to achieve the above object, an electrophoresis apparatus is provided, 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 supply is a migration path comprising a capillary. In (1), one end of each of one or a plurality of pairs of capillaries each connected to the cathode or anode electrode bath is substantially aligned with the other end of the capillary in the optical cell so as to maintain a constant gap. The sample is electrophoresed from the capillary end of the upstream electrophoresis path, which is the sample separation section, by forming an electrophoresis path that penetrates the optical cell by arranging them in opposition to each other, injecting and flowing the sheath liquid from the outside into the optical cell. Into a sheath flow state and guided to the opposite capillary. This gap is used as an optical detector, and the optical detector is irradiated with light from a light source to detect a sample. A plurality of capillaries immersed in the tank 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 for electrophoresis of each capillary flows through the optical cell in a sheath flow state. The electrophoresis apparatus is configured such that the sheath flow section is an optical detection section, and the optical detection section is irradiated with light from a light source to detect a sample. (3) The other ends of a plurality of capillaries, one end of which is immersed in the electrode bath, are held and fixed in the optical cell so as to be aligned in a straight line at an appropriate interval, and the sheath liquid composed of an aqueous solution is placed in the optical cell. , A sheath flow is formed near and below the end of each capillary, and a sample for migrating each capillary is caused to flow through the optical cell in a sheath flow state, and the injected sheath liquid is discharged from the optical cell and excited. The light of the light source unit is illuminated almost in parallel with the above-mentioned straight line, directly below the ends of a plurality of capillaries arranged on a straight line in the optical cell, and fluorescence is generated from the sample that migrates and flows out of each capillary, Each of the plurality of generated fluorescence images is divided, and a spectral filter is arranged at the front or the rear of the dividing means so that the divided images have light components in different wavelength ranges, respectively. It is imaged to the image, constituting the electrophoresis apparatus for detecting the formed image.

In the configuration of (1), a plurality of optical detectors formed by a plurality of capillary pairs are arranged in an optical cell. Furthermore, a plurality of capillary pairs are arranged and arranged in an optical cell so that a plurality of optical detection units by a plurality of capillary pairs are located on a straight line, and excitation light is applied so as to simultaneously irradiate all the optical detection units. Irradiation is performed along this straight line, and fluorescence measurement is simultaneously performed 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 positioned so as to be located on a straight line. Are arranged in the optical cell in an aligned manner, and the excitation light is irradiated along the straight line so as to simultaneously irradiate all the optical detection units, and the fluorescence emitted from the sample in which each capillary migrates is simultaneously detected.

In the configuration of (3), similarly to (1),
With the same number or more as the number of the capillary for electrophoretic separation, hold its end in a straight line at the same interval as the capillary for electrophoretic separation, and place it near a plane containing the end of the capillary for electrophoretic separation and the laser beam irradiation axis. The structure may be such that the aqueous solution is discharged from the optical cell via the arranged hollow capillary. The structure is such that images from a plurality of samples migrating each capillary are divided using a polyhedral prism. A structure in which an image is divided into four and each is detected simultaneously may be adopted. A fluorescence image from a sample in which a plurality of capillaries are migrated or a divided fluorescence image is simultaneously formed on a detector, and the intensity thereof is measured simultaneously. Fluorescent light emitted from the sample is collected by a lens and then split.
An image may be formed, and after collecting light in one direction with a cylindrical lens, the image may be divided, imaged, and detected.

Further, in the configurations of (1), (2) and (3), each section may be configured as follows. The sample separation section is composed of a capillary gel, the sheath liquid has the same components as the buffer solution inside the capillary, and may also contain a sample denaturant. Further, the means for holding a plurality of capillaries and the optical cell may be detachable, and an insertion guide for holding the capillary holder at a designated position may be provided inside the optical cell. 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, disposing the liquid surface at a position higher than the surface of the liquid discharged from the optical cell, or Using an optical cell whose upper part is open, means for flowing an aqueous solution into the optical cell is provided with a sheath liquid container containing the aqueous solution outside the optical cell, and the liquid level is set at a position higher than a drain port in the optical cell. It may be arranged and performed by head. A mechanism for controlling the liquid level in the optical cell to be substantially constant may be provided. This can be constituted by a mechanism that detects the liquid level in the optical cell, in which the upper part of the optical cell is open, and a mechanism that replenishes the aqueous solution in the sheath liquid container via a valve when the liquid level decreases. The optical detector measures the 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, and these laser beams may be coaxial to be one. The resulting image such as fluorescence can be detected by a two-dimensional detector. Further, the injection of the sample into each of the plurality of capillaries is performed simultaneously. The electrode on the downstream side of the electrophoresis may be not only in the container from which the sheath liquid is discharged, but also in the optical cell or the sheath liquid container. The potential of this downstream electrode is 0 V (ground), or its absolute value is smaller than the absolute value of the other upstream potential. Also, a mechanism may be provided in which the liquid surface of the aqueous solution remaining in the electrode tank on the downstream side where the sample is electrophoresed is made substantially constant, and an excessive aqueous solution is discharged to the tank. Furthermore, the ratio of the length of both ends of the fluorescence emitted from the sample migrating many 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 the electrophoresis, it is desirable that the number of electrodes on the downstream side where the sample is electrophoresed is one and the number of electrodes on the injection side of the sample matches the number of the capillaries for electrophoresis separation. Further, a mechanism is provided for measuring and displaying a current value flowing inside the capillary for electrophoresis separation for each capillary. The current values may be binarized and displayed. Further, the capillary for electrophoresis separation is subjected to a silanization treatment on at least the inner surface near the end held on the inside of the optical cell and filled with the gel, so that at least the gel near the end held on the inside of the optical cell is filled. May be chemically bonded to the inner wall of the capillary.

[0013]

In the configuration (1) or (3) of the electrophoresis apparatus,
The other end of each of one or more pairs of capillaries each having one end connected to the cathode electrode tank or the anode electrode tank, respectively, is arranged in the optical cell so that their axes are almost aligned and a certain gap is maintained therebetween. Therefore, it is possible to form a migration path that penetrates a part of the optical cell. Since the sheath liquid is injected from the outside into the optical cell and flows, the sample to be migrated from the capillary end of the upstream migration path, which is the sample separation section, can be guided to the opposite capillary in a sheath flow state, The sample can be continuously electrophoresed between the capillary at the downstream side and the capillary at the downstream side, and the sample can be reliably electrophoresed only at the axial gap between the upstream side capillary and the downstream side capillary. . The gap is used as an optical detection section, and light irradiation from the light source to the optical detection section can be performed in a liquid without a capillary, and optical measurement such as fluorescence or light absorption of the sample can be performed. Thereby, generation of scattered light and fluorescence due to the capillary or the capillary gel can be avoided, and highly sensitive fluorescence or light absorption measurement can be performed. In this way, by allowing a plurality of capillaries to face each other with their axes substantially aligned in the optical cell and keeping a constant gap, the upstream capillary serving as the sample separation unit and the gap serving as the optical detection unit can be easily configured. Can be configured. In addition, by disposing a pair of capillaries on the downstream side, the migration path of the sample can be accurately defined, light irradiation from the light source can be easily and reliably performed, and light reception such as fluorescence can be easily performed. And reliably. In addition, the sheath liquid and the sample liquid flow out of the optical cell only through the inside of the capillary on the downstream side, but since the diameter of the capillary is small, the flow rate of the sheath liquid to be injected into the optical cell can be reduced, and the sheath liquid can be reduced. The amount of liquid can be reduced. Since the flow rate of the sheath liquid is determined by the inner diameter of the capillary irrespective of the dimensions of the optical cell, the cross-sectional area inside the injection portion of the sheath liquid is increased as in a normal sheath flow chamber, and the cross-sectional area of the sheath flow section is increased. It is not necessary to adjust the optical cell to a shape that makes the optical cell thinner, and a rectangular optical cell can be used, so that the optical cell can be manufactured easily and at low cost. As described above, the plurality of optical detection units formed by the plurality of capillary pairs can be arranged close to each other in the optical cell, and the size of the device can be reduced. Since only one optical cell and one pipe for forming the sheath flow are required, the apparatus can be configured at low cost and easily. In addition, the sample migrated from each capillary enters a sheath flow state for each capillary when passing through the optical detection unit, and the flow of the sheath liquid is all 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.However, since the sheath liquid and the like flows out through each downstream capillary, the effective cross-sectional area through which the sheath liquid flows is determined by 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 may be small, the amount of the sheath liquid can be reduced, and the size of the apparatus can be reduced.

In a single optical cell, a plurality of capillary pairs are arranged and arranged so that a plurality of optical detectors are located on a straight line, and the excitation light is irradiated along this straight line.
It is possible to irradiate all the optical detection units at the same time, and it is possible to simultaneously perform the fluorescence measurement by a plurality of optical detection units. In addition, since the plurality of optical detection units are connected to each other via the liquid, the excitation light can irradiate each optical detection unit without being scattered by the capillary or the like, and the excitation light can be efficiently transmitted to each optical detection unit. The fluorescence can be detected with high precision. If a two-dimensional TV camera or the like is used as a light detector, it is possible to simultaneously receive the fluorescence images of a plurality of optical detection units. In one optical cell, a plurality of capillary pairs are arranged and arranged so that a plurality of optical detection units are located on a straight line, and the distance between the plurality of capillary pairs can be reduced, so that the apparatus can be reduced in size. Not only can the configuration be inexpensive, but also the length between the optical detection units at both ends on the straight line can be reduced. If the length between the optical detection units at both ends is reduced, all the optical detection units can be irradiated with substantially the same light beam diameter even if the excitation light is narrowed down by a lens or the like. For example, when the laser light is focused to a diameter of about 100 μm at the focal position, about 10 mm
The laser beam diameter becomes about 100 μm. 400 μm capillary with 200 μm outer diameter and 100 μm inner diameter
m, 5 mm inward about 10 mm before and after the focal point.
Approximately zero light beams can be arranged, and these can be irradiated with the same light beam diameter and light beam intensity. Than this,
Each optical detector can be irradiated with light while the excitation light is narrowed down,
The fluorescence intensity can be increased, and the sample can be detected with high sensitivity.

The sample separation section is made of a capillary gel so that the molecular weight can be easily separated. In addition, the downstream capillary is a hollow capillary, so that the sheath liquid or the like can flow efficiently. On the downstream side, a device capable of performing the same operation without using the capillary itself, for example, a device in which holes or grooves corresponding to the number of upper capillaries are formed on a flat plate can be used. By making the sheath liquid a component equivalent to the buffer solution inside the capillary, it is possible to secure the flow of electricity in the gap, that is, in the optical detection unit, and to enable electrophoresis of the sample. Furthermore, since the composition of the buffer inside the capillary is the same as that of the buffer outside, it is possible to prevent the buffer inside the capillary from flowing inside the optical cell and change the composition, and to improve the separation ability of the sample by electrophoresis. Do not spoil. When the sample is a single-stranded DNA or the like, a denaturant can be included in the sheath liquid. In this case, it is possible to prevent the sample DNA from being recombined at the time of migrating the gap, that is, the optical detection unit, and the measurement accuracy is improved. The possibility that the denaturing agent 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 arranging the liquid level of the sheath liquid in the sheath liquid container at a position higher than the liquid level in the downstream electrode tank, so that the sheath liquid can be easily reduced due to a head drop. Can be flowed, and there is no need to mechanically flow the sheath liquid, so that a simple and inexpensive device configuration is obtained. Furthermore, since a pulsating flow generated when a pump or the like is used is eliminated, a stable sheath flow is enabled, and the measurement accuracy is improved. The flow rate of the sheath liquid can be easily adjusted by changing the head of the sheath liquid in the sheath liquid container and the liquid level in the downstream electrode tank. The optical cell is
If a cell having an open top is used in addition to the shape that is sealed when the capillary is attached, the attachment of the capillary holder can be simplified. At this time, the same effect as described above can be obtained by arranging the liquid level of the sheath liquid container at a position higher than the drain port in the optical cell. In addition, 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 are provided so that the liquid level in the optical cell is 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 is not particularly limited, but is preferably about 0.1 mm to 3 mm. Generally, the shorter the gap distance of the gap, the more easily the sample migrates in the gap space. Therefore, basically, the gap length is preferably shorter. However, in assembling the apparatus, if the gap length is too short, it becomes difficult to adjust the gap length. However, it can be set to 0.1 mm or less, and the limit is determined by the width of the excitation light beam such as laser light in the gap. Conversely, when the gap distance of the gap is increased, the sample is easily diffused. 3m gap length
When it was about m, it was confirmed that the sample migrated normally. That is, by setting the gap length to be about 0.1 mm to 3 mm, the sample can be easily and efficiently electrophoresed. Also, by holding the capillary pair inside the optical cell and performing electrophoresis, the sample migrates on the line connecting the capillaries and the capillary, and does not contact the inner surface of the optical cell. Can be removed, and the detection sensitivity can be improved.

Images from a plurality of samples migrating each capillary are divided using a polyhedral prism or the like.
A plurality of pieces of information can be easily obtained from one sample by using a spectral filter so as to pass light components in different wavelength ranges. In particular, if the image is divided into four, which is the number of base types of DNA, each base type can be labeled with a different fluorescent substance, and the DNA base sequence can be determined efficiently.

A fluorescence image from a sample in which a plurality of capillaries are migrated or a divided fluorescence image is simultaneously formed on a two-dimensional detector and its intensity is measured simultaneously, so that the number of capillaries can be selected. High-speed measurement becomes possible.

An insertion guide for holding the capillary holder at a specified position is provided inside the optical cell,
By making the plurality of capillaries and the optical cell detachable, the operability of mounting and handling the capillaries is improved.

Further, the excitation light source section is a laser device, and at least two types of laser devices are used. By coaxially irradiating these laser beams, it is possible to efficiently excite DNA fragments. it can.

Electrodes equal in number to a plurality of capillaries for electrophoretic separation are provided, and the capillaries and the electrodes are immersed in a plurality of sample solutions, respectively, and a voltage is simultaneously applied to simultaneously inject a sample into each of the plurality of capillaries. By doing so, sample injection can be performed easily and with a single operation. Compared with the conventional case of performing single sample injection, time can be shortened and operability can be improved. It becomes possible to measure a current value flowing through the inside of the capillary for each capillary. The value of the current flowing inside the capillary is effective for knowing the state of the capillary, and for example, it is possible to predict in advance the separation failure due to breakage of the gel or the like. The current value is displayed by setting the binarization level arbitrarily and turning on the lamp when the current value is binarized and below the normal electrophoresis current, so that damage to the capillary gel can be monitored. It is possible to efficiently determine the necessity of exchanging and remeasurement of the gel capillary and improve the usability.

Further, the distance between adjacent capillaries held in the optical cell is set to 10 times or less of the capillary size, and the overall image magnification of the fluorescent image to be detected is set to 1/3 times or more. The solid angle of light reception can be increased as compared with that of the first embodiment, and the detection sensitivity for the same amount of phosphor can be improved.

In a part of the configuration (2) or (3) of the electrophoresis apparatus, a plurality of capillaries, one ends of which are immersed in the electrode bath, are terminated in the optical cell, and the sheath liquid is externally introduced into the optical cell. Injecting and flowing the sample, each sample to be migrated in each capillary flows through the optical cell in a sheath flow state. The sheath flow portion is used as an optical detection portion, and the configuration is the same as in the configuration (1) of the electrophoresis apparatus. As a result, the sample migrating each capillary can be measured separately, and the same operation as the configuration (1) of the electrophoresis apparatus can be obtained. Other related operations described with respect to the configuration (1) of the electrophoresis apparatus are the same as those of the configuration (2) of the electrophoresis apparatus.

[0025]

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 electrophoresis to separate the molecular weight, and detection is performed by fluorescence. A case where fluorescein isothiocyanate (FITC) is used as a fluorescent substance for labeling will be described. As a fluorescently labeled DNA fragment as a sample, a DNA fragment in which a part of the DNA fragment is labeled with a fluorescent substance can be used. For example, it is prepared by performing a DNA polymerase reaction using a fluorescently labeled primer by the well-known dideoxy method of Sanger et al. That is, a primer (labeled primer) to which FITC is bound 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, dGTP and ddATP are added and a DNA polymerase reaction is performed. Through the above operation, fluorescently labeled DNA fragments having various lengths of A at the end are obtained. This is used as a sample.

First, the configuration of the apparatus will be described. FIG.
2 shows a configuration diagram of an electrophoresis unit and a laser beam irradiation system of the electrophoresis apparatus of the present embodiment. FIG. 2 shows a configuration diagram of a fluorescence detection system of the electrophoresis apparatus. 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, 1b, 1c, 1d..., 1t are used. These have an inner diameter of 100 μm and an outer diameter of 375 μ each.
m, the same capillary made of silica having a length of 40 cm. Furthermore, 20 silica capillaries 2a, 2b, 2c, 2 having a length of 10 cm and the same inner and outer diameters are used.
d,..., 2t 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 a silane coupling treatment. Then
Degassed 3.84% acrylamide, 0.16% bisacrylamide, 7M urea, Tris containing 2mM EDTA, tetramethylethylenediamine, ammonium persulfate solution to borate buffer, and inject into capillary to polymerize. Create an acrylamide gel. Since the capillary has been subjected to the silane coupling treatment, the acrylamide gel and the capillary are chemically bonded, and the gel does not run off the capillary during electrophoresis.

The capillaries 2a to 2t are treated so that their inner surfaces have a positive charge. First, a 3- (2-aminoethylaminopropyl) trimethoxysilane solution is injected into the capillary to cause a reaction, and heat treatment is performed at 110 ° C. to convert the inner surface of the capillary to aminosilane to have a positive charge. By doing so, the direction of the electroosmotic flow inside each of the capillaries 2a to 2t changes from the negative electrode to the positive electrode, and the electrophoresis direction of the sample in the capillaries 1a to 1t filled with acrylamide gel (negative electrode → positive electrode) In addition, the moving direction of the sample in the capillary 2 (from the negative electrode to the positive electrode) coincides, and the migration of the sample can be ensured.
One end of each of the capillaries 1a to 1t and 2a to 2t is opposed to the inside of the optical cell and fixed so as to maintain a certain gap, and the sample is optically detected. Here, a fluorescent cell is used as an optical cell in order to detect a sample by fluorescence. That is, the capillaries 1a to 1
t and one end of each of 2a to 2t are square fluorescent cells 4 made of quartz (outer size: width 36 mm × depth 4.5 mm × length 3 m)
m, inner shape: width 30 mm x depth 2 mm x length 3 mm, width is the horizontal direction in the figure (the direction in which the capillaries 1a → 1t are arranged),
The depth is arranged in the direction perpendicular to the paper surface of the figure, and the length is arranged in the longitudinal direction of the figure (1 → 2 sample migration direction of each capillary). The arrangement method is such that the capillary 1a and the capillary 2a are opposed to each other so as to be coaxial with each other, and the gap 3a having a length of 1 mm is formed. Capillaries 1b and 2
Similarly, b, 1c and 2c, 1d and 2d,..., 1t and 2t are arranged so as to form the same gaps 3b, 3c, 3d,. The capillaries are fixed in the optical cell by using multi-capillary holders 5a and 5b having 20 vertical holes provided at 0.6 mm intervals in a flat block made of fluororesin, for example, ethylene tetrafluoride resin. That is, 20 of the multi-capillary holder 5a
Capillaries 1a to 1t are inserted one by one into the vertical holes at the locations, and capillaries 2a to 2t are inserted one by one into the 20 vertical holes 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 to the upper and lower portions of the fluorescent cell 4 in close contact with each other so that 1t and 2t are coaxial. Further, the gaps 3a-
Since 3t is an optical detector, here a fluorescence detector, the capillaries 1a to 1t and 2a in the quartz fluorescent cell 4 are so arranged that the gaps 3a to 3t are aligned on one straight line.
Adjust the length and so on of ~ 2t. In addition, the capillaries 1a to
1t and the other ends of the capillaries 2a to 2t, respectively.
It is immersed in the cathode-side electrode tank 6 and the 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 inlet 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 has the same components as the buffer solution in the capillary gels of the capillaries 1a to 1t. 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 the sheath liquid, and the liquid surface of the sheath liquid 11 in the sheath liquid container 10 is filled with the anode side electrode tank 7. By disposing the sheath liquid higher than the liquid surface of the buffer solution inside, the sheath liquid 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 solution, and the capillaries 1a to 1t are filled.
Is filled with a capillary gel, and a DC voltage is applied between the cathode-side electrode tank 6 and the anode-side electrode tank 7 by the DC high-voltage power supply 12 so that, for example, the capillary 1 a and the gap 3
a flows through the capillary 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 inside higher than the liquid level of the buffer solution in the anode-side electrode tank 7, the capillaries 2a to 2t
Flows through the inside of the capillaries, and flows around the upper portions of the capillaries 2a to 2t, that is, the gaps 3a to 3t. So the capillary 1a ~
The sample migrated from 1t is the capillary 2a to 2
along the flow above t, pass through each of the gaps 3a to 3t in a sheath flow state, and are guided to each capillary,
Electrophoresis is performed on the anode-side electrode tank 7 side. Capillaries 2a-2
t is processed 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, so that there is no backflow of the liquid to the gap, and the sheath liquid can flow stably toward the anode-side electrode tank 7. In particular, even when the flow rate of the sheath liquid is small, it is possible to stably flow the sheath liquid toward the anode-side electrode tank 7.

The electrophoresis is performed by applying a DC voltage from the DC high voltage power supply 12 between the cathode side electrode bath 6 and the anode side electrode bath 7. Due to the voltage application, the current flowing through the capillary 1a and the capillary 2a mainly flows around the gap 3a, and similarly, the capillaries 1b and 2b, 1c and 2c, 1
.., 1t and 2t mainly flow through the gaps 3b, 3c, 3d. Further, as described above, the sample migrated from the capillaries 1a to 1t passes through the gaps 3a to 3t in a sheath flow state along the flow above the capillaries 2a to 2t, and is guided to each capillary. . In other words, the sample migrating in each of the capillaries and gaps can migrate without contacting the sample migrating in the adjacent gap, and the sample migrating in each gap can be separated and subjected to 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. Measurement becomes possible. The fluorescent-labeled DNA fragment as a sample is introduced by temporarily immersing the end of the cathode-side capillary 1a in a sample solution and applying a voltage of 6 kV for about 20 seconds between the sample solution and the anode-side electrode tank 7. . Thereafter, 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 gels of the capillaries 1a to 1t.
The injection of the sample may be performed one by one as described above, or may be performed by immersing 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 migrates in the capillary gel of each of the capillaries 1a to 1t while being separated in molecular weight from the cathode side to the anode side, and passes through the gaps 3a to 3t, respectively. Gap 3a-
The detection of the sample passing through 3t is based on 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 light axis is adjusted so that the laser light simultaneously irradiates the gaps 3a to 3t aligned on a straight line under substantially the same conditions. It is performed by measuring the emitted fluorescence. That is, the laser beam 21 having a wavelength of 488 nm from the argon laser light source 20 is narrowed and irradiated by the lens 22 to excite the fluorescently labeled DNA fragments 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 beam at the focal point 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 this case, 0.6 mm × 19.
That is, it is about 12 mm. That is, the laser light is applied to the gap 3
20 spots a to 3t are irradiated with almost the same spot size, and the spot size is almost equal to 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 substantially equal to the thickness of the capillary gel, and all the gaps are irradiated with almost the same spot size. In addition, it is possible to uniformly and efficiently excite the fluorescence-labeled DNA fragments that migrate through the gaps 3a to 3t.

Fluorescently labeled DN for migrating gaps 3a to 3t
Fluorescence from the fragment A is detected from the direction perpendicular to the laser beam irradiation direction. The configuration diagram is shown in FIG. The fluorescence 30 emitted from the DNA fragment removes background light such as scattered light by an interference filter 32 and forms an image on a two-dimensional detector 34 such as a CCD camera by a lens 33. The two-dimensional detector 34 is controlled by the controller 35 to detect the fluorescent images of the gaps 3a to 3t, and the data processing device 36 such as a computer continuously changes the fluorescence intensity over time for each of the gaps 3a to 3t. In addition, all the gaps are measured simultaneously, the results are displayed on the monitor 37, output to the printer 38, and the memory 3
Save to 9. Thereby, it is possible to simultaneously and continuously measure the electrophoretic patterns separated by molecular weight for each of the capillaries 1a to 1t. 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 a CCD camera or a two-dimensional detector. As the interference filter 32, a band-pass interference filter passing a wavelength range of 500 nm to 540 nm is used in order to effectively detect the fluorescence from FITC. In addition, the gaps 3a to
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 capillary or the like. for that reason,
Simultaneously and homogeneously sample multiple electrophores
Light irradiation can be performed efficiently. In addition, background light such as light scattered by a capillary or a capillary gel or fluorescence can be significantly reduced, and fluorescence can be detected with high sensitivity. As the effect of reducing the background light, for example, the fluorescence measurement is performed in the gap as in the present embodiment, as compared with the case where the capillary itself from which the coating is removed is irradiated with laser light and the fluorescence is detected without forming the gap. Then, the intensity of the background light to be detected is reduced to about 1/10 or less, and a sample having a lower concentration can be detected. In addition, by aligning a plurality of gaps on one straight line, all gaps can be simultaneously and easily irradiated with laser light, and a simple device configuration can be realized. Further, since a plurality of capillary pairs can be held in one optical cell, only one optical cell is required, and only one pipe is required for injecting the sheath liquid. In addition, since the sheath flow occurs only in the vicinity of the gap, the flow velocity in each gap is substantially equal in all the gaps, and the reproducibility between the gaps is high. Therefore, the dispersion between the migration paths is small, and the optical cell having a simple structure as in this embodiment is sufficient, and it is necessary to use an optical cell having a complicated shape like a normal sheath flow chamber. And a simple device configuration as a whole. In this embodiment, the interval between adjacent capillaries was set to 0.6 mm. By thus opposing the capillary pairs while maintaining a certain gap, the position where the sample migrates can be defined, and a plurality of capillaries (and gaps) can be arranged close to each other. Can be reduced in size. This also makes it possible to irradiate the gap with a narrower laser beam. Conversely, more capillary pairs can be held in the same optical cell. According to the present embodiment, the sample can be detected with high sensitivity without impairing the molecular weight separation characteristics because the molecular weight separation section is a capillary gel similar to the conventional one.

Further, according to the present embodiment, the buffer solution can be flowed without using a mechanical means such as a liquid chromatography pump or the like.
Become cheap. In addition, since there is no pulsating flow generated when a pump or the like is used, the flow of the sheath liquid is stabilized, the fluctuation of the flow velocity is reduced, the fluctuation of the fluorescence intensity of the sample flowing in the gap is suppressed, and the measurement accuracy is improved. be able to. 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 of the buffer solution in the anode-side electrode tank downstream of the electrophoresis. It can also be adjusted by changing the inner diameter of the capillary on the downstream side of the electrophoresis. In addition, it is basically possible to flow the sheath liquid by a mechanical means such as a pump for liquid chromatography. In this case, there is an advantage that the flow rate can be set directly. However, since the fluorescence intensity tends to fluctuate due to the pulsating flow of the pump, it is necessary to sufficiently perform processing such as smoothing in data processing.

The sheath liquid inside the optical cell flows out of the cell through the downstream capillaries 2a to 2t. Because capillaries generally have small internal diameters, the flow through them is generally small. Therefore, the amount of the sheath liquid can be reduced and operability is improved. In the present embodiment, the inside of the capillaries 2a to 2t is treated 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 toward the anode-side electrode tank 7. In this way, there was no backflow of the liquid from the capillaries 2a to 2t to the gap. Accordingly, the sheath liquid can be stably flowed toward the anode-side electrode tank 7 even when the flow rate of the sheath liquid is small. 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 the capillaries 2 to 2t is large, the effect of the electroosmotic flow is reduced, so that the inner surfaces of the capillaries 2a to 2t do not necessarily need to be treated. In this example, a buffer solution containing Tris, boric acid, EDTA, and urea was used as the sheath solution and the buffer solution in the anode-side electrode bath and the cathode-side electrode bath, and had the same components as the buffer solution of the capillary gel. As a result, the constituent components of the capillary gel do not leak into the fluorescent cell 4 or the electrode tank, and the electrophoresis can be continued more stably and with good resolution including reuse of the capillary gel. A buffer containing no urea as a DNA denaturant can be used. In this case, urea in the capillary gel may leak out of the capillary gel into the electrode tank or the fluorescent cell over time, and the number of repetitive uses is slightly reduced. Is possible.

The laser device and phosphor used are as follows:
It is not limited to argon laser and FITC,
Any phosphor and suitable laser device can be used. In the present embodiment, the measurement of the DNA fragment has been described as an example. However, it can be naturally used for the analysis of proteins, sugars and the like. Further, in this embodiment, both capillaries having the same inner diameter are used, but a combination of capillaries having different inner diameters is also possible. For example, if the inner diameter of the downstream capillary is made smaller than the inner diameter of the upstream capillary, the sample migrated from the upstream capillary end is squeezed when introduced into the downstream capillary, so that the concentration of the sample liquid is high. Become
Detection can be performed with higher sensitivity. Also, if the inner diameter of the capillary on the downstream side is made larger than the inner diameter of the capillary on the upstream side, the sample migrated from the end of the upstream capillary can be more easily and reliably guided to the downstream capillary. Furthermore, according to the present embodiment, since the fluorescence from the sample can be measured without transmitting the excitation light through the capillary part, 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. In addition, it is possible to use capillaries of various materials such as capillaries made of opaque fluororesin such as ethylene tetrafluoride resin and ethylene trifluoride chloride resin. Since the fluororesin capillary does not adsorb a sample, a processing operation such as a surface treatment is unnecessary, and the operability is improved because there is no breakage. 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. However, in the case of only one capillary pair, it is also possible to migrate, detect fluorescence, and measure the sample separation pattern in the same manner. In this case, a photomultiplier tube or the like can be used as the photodetector.

[Second Embodiment] Next, a method for determining the base sequence of DNA using the apparatus of the first embodiment will be described. A fluorescently labeled DNA fragment is prepared by performing a DNA polymerase reaction using a fluorescently labeled primer according to the well-known Sanger et al. Dideoxy method. As the primer, a primer to which FITC is bound (labeled primer) is used. First, a labeled primer is added to the single-stranded DNA, and annealed (double-stranded formation) to bind the labeled primer to the single-stranded DNA. This reaction solution is
The DNA is divided and subjected to DNA polymerase reactions corresponding to A, C, G and T respectively. That is, four kinds of deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP) and ddATP serving as a terminator are added to the single-stranded DNA to which the labeled primer is bound, and a polymerase reaction is performed. By this reaction, fluorescently-labeled DNA fragments having various lengths of A are obtained. Similar reactions are performed for C, G, and T. The four A obtained as described above,
The reaction liquids of C, G, and T are injected into four of the capillaries 1a to 1t, for example, each of the capillaries 1a, 1b, 1c, and 1d. The injection method is the same as in Example 1, and the reaction solution A is 1a, the reaction solution C is 1b, the reaction solution G is 1c,
Inject the reaction into 1d. After the injection, electrophoresis is performed by applying a voltage of about 6 kV. Using the argon laser light having a wavelength of 488 nm as the excitation light, the time change of the fluorescence intensity in each of the gaps 3a to 3d is measured. Since DNA fragments are electrophoresed in ascending order of molecular weight, the nucleotide sequence can be analyzed by analyzing the gap positions where the fluorescent peaks occur in chronological order.

The apparatus 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 five types of DNA samples can be determined simultaneously. By increasing the number of capillary pairs held in the optical cell, the base sequence of more DNA samples can be determined. In this embodiment, one kind of FITC is used as the phosphor. However, it is also possible to detect the fluorescence from two or more kinds of phosphors at the same time. In this case, as is well known, for example, in FIG. 2, a set of photodetectors including the interference filter 32, the lens 33, and the detector 34 is provided in a number corresponding to the number of phosphors, and each of the photodetectors has a different wavelength range. Alternatively, a dispersing prism may be disposed after the lens 33 to separate the light, form an image on a two-dimensional detector such as a camera, receive the light, and emit fluorescent light for each migration path and each wavelength. The intensity may be measured to 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 configured to measure a plurality of phosphors simultaneously. In other words, primers labeled with different fluorophores are used for each type of terminal base, and DN
After the A polymerase reaction is performed, the reaction solutions are mixed and subjected to electrophoresis. Then, the fluorescence of the DNA fragment passing through the gap space is detected, and the type of the fluorescent substance at that time is identified, whereby the base type can be identified, and the base sequence can be determined. The type of the phosphor can be determined 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 a migration path composed of a pair of capillaries and a gap. That is, when a plurality of migration paths are provided as shown in FIG. 1, it is 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, the light absorption measurement such as the measurement of the absorbance and the measurement of the intensity of the transmitted light is described. In this case, the device can be similarly configured. FIG. 3 shows a configuration diagram of a light irradiation system and a detection system of the electrophoresis apparatus in the case of the light absorption measurement. The electrophoresis section has the same configuration as that of FIG. 1 of the first embodiment. Sample, eg DNA
Is introduced in the same manner as in Example 1, and electrophoresis for molecular weight separation is performed in the same manner as in Example 1. To DNA fragments passing through the gap 3A～3t, light from the light source 50 such as a xenon lamp or D ₂ lamp through a monochromator 51, a lens 52 is condensed to illuminate the gap 3A～3t. The irradiation light wavelength is usually set to the absorption wavelength of the sample.
The order of nm is appropriate. The light that has been absorbed and transmitted by the DNA fragments is collected 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 a one-dimensional sensor controller 55, and a migration pattern or the like for each gap is measured by a data processing device 56 such as a computer. The results are displayed on a monitor 57 and are displayed on a printer 58. It is output and stored in the memory 59. In this embodiment, the gap is provided between the capillaries, the capillary is separated from the molecular weight separation unit, and the gap space is separated from the optical detection unit that measures the absorbance.
Since the irradiation light is transmitted through the sheath liquid with extremely little light scattering, the sample can be efficiently irradiated without being scattered by a capillary, a gel, or the like, and highly accurate absorbance measurement can be performed. Specifically, assuming that the transmitted light intensity (when the sample does not pass through and the numerical aperture of the light-receiving lens is 0.19) when light is incident on the gap as in this embodiment is 1, When the light is transmitted through the 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 the molecular weight by electrophoresis, are scatterers of light, so that incident light is scattered at that part, and as a result, transmitted light is reduced. . A decrease in the transmitted light intensity means a decrease in the S / N of the photodetector or the like, and the measurement accuracy is reduced. In addition, in the conventional case, a part of the light scattered or reflected on the surface of the capillary or the like is also detected, but such light is directly applied to the detector without irradiating the sample (without being absorbed by the sample). Incident. If there is a light component 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 condensed and radiated to the gap by the lens. It is also possible to measure the light intensity by spatially separating only the light intensity.

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

Fifth Embodiment In contrast to the first embodiment, it is also possible to construct a system in which only the upstream capillary is used, and the sheath liquid is flown, without using the downstream capillary. FIG. 5 shows a configuration diagram of an electrophoresis unit of the electrophoresis apparatus. One end of each of the capillaries 1a to 1t is held inside the optical cell 4 in the same manner as in the first embodiment. 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 block made of a fluororesin, for example, an ethylene tetrafluoride resin, is used. In other words, the capillaries 1a to 1t are inserted one by one into the 20 vertical holes of the multi-capillary holder 5a, and the multi-capillary holder 5a is fixed in close contact with the upper part of the fluorescent cell 4. The lower part of the fluorescent cell 4 is connected to a seal plate 70 and a tube 71 made of ethylene tetrafluoride resin, and the end of the tube 71 is immersed in the anode-side electrode tank 7 so that the sheath liquid flows and electrophoresis is possible. To do. The fluorescent cell 4 is provided with a sheath liquid inlet 8 for filling the inside with a sheath liquid, and a structure in which the sheath liquid 11 in the sheath liquid container 10 can be injected through a tube 9 made of ethylene tetrafluoride resin. And Further, by disposing the liquid level of the sheath liquid 11 in the sheath liquid container 10 higher than the liquid level of the buffer solution in the anode-side electrode tank 7, the sample migrated from the capillaries 1a to 1t is wrapped in the sheath liquid. The inside of the fluorescent cell 4 can flow. These solutions are finally led to the anode-side 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 that has migrated through each capillary migrates in the optical cell immediately below the capillaries without contacting the sample that migrates with another capillary, and finally migrates to the anode-side electrode tank 7. The sample is detected by irradiating a portion of the fluorescent cell 4 on the downstream side of the capillary end 500 μm with laser light to receive fluorescence. The light receiving system has the same configuration as that of the first embodiment. This embodiment is basically different from the first embodiment in a part of a method of forming a sheath flow. In the present embodiment, the sheath liquid flows in a laminar state throughout 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 that the path on which the sample migrates is easily changed. However, almost the same effects as in the first embodiment can be obtained in sensitivity, simplicity of the device configuration, and the like.

[Embodiment 6] In the apparatus described in Embodiment 1, the downstream capillary may be formed as a flat plate having a plurality of grooves to form a gap. FIG. 6 shows a perspective view of a flat plate having a plurality of grooves. The figure shows a case where four grooves are provided so as to correspond to four capillaries. Four grooves 81a, 81b, 81c and 81d are formed on one side of a flat plate 80 which matches 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 a fluorescent cell portion of an electrophoresis section of an electrophoresis apparatus using the above-mentioned flat plate. The fluorescent cell 84 has a width of 20 m inside the cell.
m, depth 3 mm, length 40 mm, and a (quartz) glass plate thickness of 2 mm is used. The figure shows a state in which the glass part on the near side of the fluorescent cell is removed. The flat plate 80 is closely fitted into the inside of the fluorescent cell 84, and the grooves 81a, 81b, 81c, 81
Four flow paths 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 shape of 200 (a gel is prepared inside in the same manner as in Example 1) 8
2 a, 82 b, 82 c, and 82 d are fixed inside the fluorescent cell 84 by the multi-capillary holder 83. The distance between the capillaries is set to 1 mm so as to match the distance between the grooves, and the capillaries are adjusted so that the axes of the capillaries are at the center of each groove. These are multi-capillary holders 83
This can be handled by adjusting the position of the holes and the like provided in the holes. Also, the ends of the capillaries 82a, 82b, 82c, 82d inside the fluorescent cell are aligned, and each end of the capillaries 82a, 82b, 82c, 82d is
Are adjusted so as to be about 1 mm apart from each other and fixed inside the fluorescent cell. Electrophoresis is enabled by bringing the lower part of the fluorescent cell into contact with the buffer solution in the anode electrode tank.
Further, similarly to the fourth or fifth embodiment, it is also possible to connect to the electrode tank via a tube. Fluorescent cell 84
Is provided with a sheath liquid inlet 85 for filling the inside thereof with a sheath liquid, so that the sheath liquid 88 in the sheath liquid container 87 can be injected through a tube 86 made of ethylene tetrafluoride resin. In addition, by arranging the liquid level of the sheath liquid 88 in the sheath liquid container 87 higher than the liquid level of the buffer solution in the anode-side electrode tank, the sample migrated from the capillaries 82a to 82d is wrapped in the sheath liquid and fluorescent It can flow through the cell 84 and be guided to the anode-side electrode tank through the grooves 81a to 81d, respectively. By performing electrophoresis, laser beam irradiation, and fluorescence detection in the same manner as described in Example 1, samples separated by each of the multiple capillaries can be efficiently detected simultaneously. In this embodiment, the downstream side is formed by a flat plate having a groove, instead of a capillary.
Therefore, the end faces of the respective grooves can be easily aligned, and adjustment can be facilitated. Further, when the flat plate is made of quartz glass or the like, the laser light is irregularly reflected, and even when the flat plate strikes the flat plate, no fluorescence is generated from that portion, and the fluorescence measurement becomes easy.

Example 7 An apparatus and method for determining the base sequence of DNA using a gel-filled capillary array will be described. In the same manner as in Example 2, a sample for DNA sequencing (fluorescently labeled DNA fragment) is prepared. In order to identify 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. The fragment A was bound with a primer (abbreviated as primer A) labeled with a fluorescent substance having a maximum fluorescence wavelength of about 550 nm. Similarly, C, G,
The T fragment has a maximum fluorescence wavelength of about 520, 575, 6
Primers labeled with a phosphor of 05 nm (abbreviated as primer C, primer G, and primer T, respectively) were bound thereto. These fragment groups were finally mixed in a single container, and after ethanol precipitation, dissolved in deionized formamide (containing 1/100 volume of Tris buffer).
Before injection into the gel capillary, the fragments were heat denatured by heating at 90 ° C. for 2 minutes to make the fragments single-stranded, and then immediately cooled on ice and used for the injection operation. The sample may be prepared using a fluorescent label as a terminator instead of a primer.

Next, the measuring device will be described. FIG. 8 is a configuration diagram of a subarray for forming a capillary array, and FIG. 9 is a configuration diagram of a capillary array of the present embodiment in which a plurality of the subarrays of FIG. 8 are combined. Further, FIG. 10 is a configuration diagram of a DNA base sequencer using a capillary array, FIG. 11 is an enlarged sectional view of a sample injection part, and FIG.
FIG. 13 is a diagram showing the configuration of the display unit, FIG. 13 is an explanatory view including the cross section of the optical cell for showing the configuration near the optical cell unit, and FIG.
FIG. 15 is an image diagram of a fluorescence image obtained by a two-dimensional detector, FIG. 16 is a fluorescence characteristic diagram of the fluorescent labeling primer used and a transmission characteristic diagram of a spectral filter, FIG.
Is an example (raw data) of a time waveform of the intensity of a fluorescent signal emitted from a sample in which one gel capillary migrates, and FIG. 18 is a time waveform for each of A, C, G, and T fragments obtained by analysis.

A capillary filled with gel is filled with 96
Using this, a capillary array having one end arranged in a straight line was produced. Assembling 96 pieces into an array with one holder complicates assembly and makes it difficult to replace the capillaries. Therefore, a sub-array of eight pieces is formed, and 12 sets of these 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 capillaries 91 and 92 are arranged so that the interval between the centers of the capillaries is 9 mm at one end and 0.5 mm at the other end. Fix each and align their ends. The capillary ends 90a fixed at intervals of 9 mm are sample injection side ends, and the opposite side 90b is an optical detection side end. This subarray is shown in FIG.
And the capillary array 10
7 is configured. The twelve capillary holders 91 are fixed by an area array holder 93 so as to be arranged in 12 rows on the same plane, and the other capillary holder 92 at the other end is linearly arranged so that twelve are sequentially aligned on a straight line. Array holder 2
Fix at 14. As a result, the capillary ends 90a are arranged in a grid of eight lines and 12 rows on the same plane, and 96 capillary ends are arranged on one straight line on the 90b side. In FIG. 9, the capillaries 90 are connected only for the subarray in the first row. The capillaries in the middle of the subarrays in the second to twelfth columns are omitted. Although the sub-arrays themselves are omitted from the third to eleventh columns, they are arranged in order between the first, second, and twelfth sub-arrays. 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 acrylamide gel in the same manner as in Example 1. Gel concentration is 9
% T (ratio to the total amount of (acrylamide + bisacrylamide) in solution) and 0% C (ratio of bisacrylamide to (acrylamide + bisacrylamide)). The use of the capillary array 107 of the present embodiment enables simultaneous analysis of 96 samples.

FIG. 10 is a block diagram of a DNA base sequence determination apparatus. The ends of the capillary array 107 arranged in a grid are inserted into a buffer solution (or a sample solution) in an upper individual electrode tank 104 having a 96-well shape. In the figure, the connection state between the capillary array 107 and the upper individual electrode tank 104 is not shown, but this will be described with reference to FIG. In the figure, the number of capillaries is increased from 96 to 20 for easy understanding.
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 via a tube 122 and a valve 121, and passes through a hollow capillary array 108 disposed below a capillary array 107 to a lower electrode tank 106. Is discharged. The ends of the capillary array 107 and the hollow capillary array 108 are opposed to each other and are fixed at a distance of 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, and the other is a YAG laser device 102 having a wavelength of 532 nm, which is a mirror 109 and a dichroic mirror 11.
0, the argon laser beam 111 and the YAG laser beam 11
The optical cell 103 was focused and irradiated with a lens (not shown) so that 2 was coaxial and one laser beam 113 was formed. Fluorescence generated from the fluorescently labeled sample is imaged on a two-dimensional detector 118 by a condenser lens 114, four types of spectral filters 115a to 115d, an image splitting prism 116, and an imaging lens 117, and a DNA fragment of each sample is formed. The data processing unit 119 analyzes the time waveform of the fluorescence intensity from the data.

The injection of a sample into the capillary array 107 will be described with reference to FIG. 11 (an enlarged sectional view of the sample injection part / upper electrode tank). FIG. 11 is a cross-sectional view of a plane including a capillary held by one of the twelve capillary holders 91. Upper individual electrode tank 1 having container-like recesses 104a, 104b, ... of 8 holes x 12 rows (96 holes in total)
04 is used. For example, a 96-well microplate with V-bottom wells or 96 sampling tubes 8 x 1
Those arranged in a grid of two rows at intervals of 9 mm can be used. The distance between the capillary arrays 107 needs to match the shape of the upper individual electrode tank 104. In addition, since each of the container-like recesses 104a, 104b,.
The six platinum electrodes 120a, 120b... Were 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 recess. Further, in the electrode holding plate 120, holes 252a for passing capillaries are provided in the vicinity of each platinum electrode,
252b ... are provided. The sample 240 has a recess 104a,
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 attached to the spacer 251.
Are sandwiched between them. Individual capillaries 10
7a, 107b ... are holes 252a, 2b of the electrode holding plate 120.
52b are immersed in the sample solution 240, and similarly, the platinum electrodes 120a, 120b,. A voltage is applied almost simultaneously between these electrodes and the electrodes in the lower electrode tank 106, and D
The NA fragment is injected into the capillary. The voltage is 100
~ 200 V / cm electric field strength, for several seconds ~ 1
Apply for about 0 seconds. For example, a voltage of 5 kV is applied 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 an electric field strength of 100 to 400 V / cm, for example, 7 kV
Is applied to continue electrophoresis, thereby performing molecular weight separation. With this configuration, 96 samples can be simultaneously injected, and then 96 samples can be electrophoresed simultaneously. Further, the sample injection is only required to overlap the electrode holding plate 120 with the area array holder 93, that is, the capillary array 107, and it is not necessary to manually perform one sample at a time by pipetting as in the case of the conventional flat gel. Therefore, the operability can be greatly improved.

When an electric field is applied, as shown in FIG. 12, a mechanism for measuring and displaying the current values flowing through the individual capillaries individually and simultaneously is provided. A voltage is applied to the electrodes (120a, 120b,...) In the upper individual electrode tank 104 and the platinum electrode 209 in the lower electrode tank 106 by the migration power supply 133. A resistor 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. The current is from electrode 209 to buffer solution 245
→ Capillary array 108 → Optical cell section → Individual capillaries 107a of capillary array 107 → Buffer solution 246 in container-like recess 104a → Electrode 120a →
The resistance 131 → the terminal 260 is connected. Note that an optical cell section and the like are provided between the capillary 107a and the capillary array 108, but are omitted in FIG. The current value flowing through the capillary is converted based on the value of the voltmeter 132, and the current value is binarized by the binarizing means to convert the current value into a current display panel 140.
To be transmitted. The terminals 261 and 262 are also omitted in FIG. 12, but are similarly connected to the electrodes 120b and 120c, respectively, binarize the current flowing through the capillaries 107b and 107c, and transmit the binarized current to the current display panel 140. Hereinafter, the same applies to all capillaries of the capillary array 107. The terminal 270 is the last 96th terminal. The current display panel 140 is provided with 8 × 12 columns of display lamps 140a, 140b,..., Which respectively 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. Also, the level of binarization can be adjusted appropriately. Normally, the migration current varies depending on the applied voltage, the capillary length, and the like, but is about 0.01 mA. If the gel breaks, the current will stop flowing or will decrease. Therefore, for example, if the display lamp is turned on when the voltage becomes 0.003 mA or less, abnormality such as gel breakage can be recognized in units of capillaries. As a result, it is possible to take measures such as stopping the analysis of the measurement result for the capillary having an abnormality. Further, in this example, the capillaries are sub-arrayed in units of eight, and only the unit having the damaged capillary can be replaced, so that the capillary array can be used effectively. Thus, the operability is good and an economical device configuration is obtained.

Next, the structure near the optical cell will be described with reference to FIG. The optical cell 103 has a box shape and includes a housing 201, array guides 202 and 205, a sheath liquid inlet 203, optical windows 204 and 206, and the like. Note that a housing and an optical window are also provided on the front surface and the rear surface of the paper on which FIG. 13 is described, but they are omitted in the drawing. Also, housing 2
01 and the optical windows 204 and 206 are shown in cross section.
The upper part of the optical cell 103 is open. From this part, the capillary array 107 fixed to the linear array holder 214 is inserted into the optical cell along the array guides 202 and 205 and fixed. A 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 to the lower electrode tank 106 through the inside of the arranged hollow capillary array 108.
The optical cell 103 is open at the top, and the liquid surface 230 of the sheath liquid in the sheath liquid container and the liquid surface 231 of the sheath liquid flowing into the cell are linked, and the height and the lower electrode tank 10
The sheath liquid flows in the cell due to a difference in the liquid surface height of No. 6 and the like. Hollow capillary array 108
Has an inner diameter of 0.2 with the same inner surface treatment as in Example 1.
mm, outer diameter 0.35mm, length 3cm,
By the linear array holder 215, the capillaries 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 faced each other. The hollow capillary array 108 and the linear array holder 215 are fixed to the optical cell 103 and sealed so as not to leak water. The optical cell 103 and the lower electrode tank 106 are closely adhered to each other with good airtightness.
1. Discarded into the waste liquid container 213 via the tube 212. That is, the liquid exceeding 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, the head does not change so much, and the sheath flow is formed stably. 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 used effectively. In particular, this mechanism is required when a measurement is completed and time is left until the next measurement. In this example, the liquid levels 230 and 231 almost coincide with each other, 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 in response to a 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 is simplified.

The lower electrode tank 106 is provided with a platinum electrode 209 for applying a voltage and a terminal block 208 thereof. When using a gel capillary, a positive voltage is applied to the platinum electrode 209 (with respect to the opposite electrode). For example, platinum electrode 1
Although it is possible to apply 0 V to 20 a and 7 kV to platinum electrode 209, it is desirable to apply a voltage of −7 kV to platinum electrode 120 a and 0 V (ground) to platinum electrode 209 to ensure safety. This is because the sheath liquid as a whole is
When the electrode 209 is in a high voltage state and the electrode 209 is in a high voltage state, the sheath liquid in the sheath liquid container, the optical cell, and the waste liquid container all have a high voltage, resulting in electric shock, leakage, and dielectric breakdown due to water leakage. This is because the danger increases. When the voltage of the electrode 209 is 0 V (ground), the sample side has a negative high voltage.
Since the electric leakage can be easily prevented, the device configuration can be easily made. Further, it is easy to use a metal such as stainless steel as the housing 201 of the optical cell, the workability is improved, and the optical cell can be manufactured at low cost. Although the electrode 209 is disposed below the hollow capillary array 108 as in this example, the electrode 209 may be disposed in an optical cell or a sheath liquid container, and has the same effect.

The laser light 113 is applied to the optical window 204 or 20
The DNA fragment enters the optical cell through 6, excites a DNA fragment to be migrated in a sheath flow state as in Example 1, and generates fluorescence. The fluorescent light emitting point 220 occurs in a dot shape almost immediately below the capillary as shown in the figure. Will be. These images are detected through an optical window (not shown) in a direction perpendicular to the paper. The laser beam 113 is radiated by a lens, but it is possible to uniformly irradiate the entire width of the capillary array (about 50 mm in this example) by using a lens having a focal length of about 150 mm. it can. It is also possible to irradiate the laser beam without being coaxial, but when coaxial, the two laser beams irradiate the same location, so that D
Since the migration distance of the NA fragment can be made constant, there is no shift in the migration time, and the analysis accuracy is improved. In the case of a phosphor that absorbs at both laser light wavelengths, coaxial arrangement substantially increases the excitation light intensity and improves the detection sensitivity. Note that measurement can be performed with only one laser.

The detection of a fluorescent image will be described with reference to FIG. Fluorescence emitted from the fluorescent light emitting point 220 is condensed by the condenser lens 114, divided into four by the image dividing prism 116, and formed into an image (2) by the imaging lens 117, respectively.
21a-b). At this time, four types of spectral filters 11
By arranging 5a to 5d on the front surface (or the rear surface) of the image splitting prism, it is possible to make each split light to have a different wavelength component. This has been described for one fluorescent light emitting point 220, but the same applies to all light emitting points, and these divisions are performed simultaneously. That is, time division is not performed as in the case of laser beam scanning or mechanical scanning of the detector, and measurement can be performed at high speed without increasing the fluorescence intensity measurement interval. It is also possible to measure continuously in real time. Further, by arranging a condenser lens in front of the image splitting prism, the efficiency of condensing the fluorescence 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 is collected only in one direction, the efficiency is reduced to about half as compared with the case where a normal convex lens is used. When an image splitting prism is used, if the position of the fluorescent light is shifted, the ratio of the intensity of the split image is greatly changed. When a flat gel is used, 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 shifted, and the measurement accuracy is easily lowered. Since it passes through the inside, the laser beam irradiates a fixed position without bending, so that the measurement accuracy is improved as compared with the related art.

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 a large number of 96 migration lanes. Therefore, a light-collecting element such as a light-collecting lens or a cylindrical lens can be used before the image splitting prism, and the fluorescence can be detected with higher sensitivity. In the case of a conventional flat gel, the distance between the migration lanes at both ends is about 30 cm even in the 24 migration lanes, and when a condenser lens is placed in front of the image splitting prism, a lens having a diameter of 30 cm is required. Realistically difficult for a reason.

In the present embodiment, the width of the capillary array is about 50 mm and the width of the light receiving element of the two-dimensional detector is about 27 mm. The overall image magnification is about 1
/ 2, and in the case of the flat plate gel method, the light-collecting efficiency is better than about 1/10. Further, when the number of the capillary arrays is as small as about 20 to 30, the magnification becomes the same magnification, the light collection efficiency is improved, and the sensitivity is further improved. In order to prevent a reduction in light collection efficiency, it is desirable that the distance between the capillaries in the capillary array is about 10 times or less the capillary diameter.

FIG. 15 is an image diagram of a fluorescent image actually obtained on a two-dimensional detector, in the case of a 20-capillary array. The horizontal direction in the figure is the capillary array (arrangement) direction, and the vertical direction is the (color) division direction.
Similarly, 96 fluorescent images are obtained horizontally and 4 fluorescent images are obtained vertically in the case of 96 lines. In this way, all the samples that migrate through the capillary array can be simultaneously measured by dividing them into four (color). Therefore, by adjusting the four types of spectral filters 115a to 115d so as to transmit the fluorescence of the primers A, C, G and T, the primers A, C, G and T can be identified and measured. .

FIG. 16 is a graph showing the fluorescence characteristics of the fluorescent-labeled primers and the transmission characteristics of the spectral filter. As shown in FIG. To prevent transmission of the wavelength).

FIG. 17 shows measured values of the time waveform of the intensity of the fluorescent signal emitted from a sample that migrates through one gel capillary of the capillary array. Spectral filter 1 from above
It is a time waveform of the fluorescence intensity which transmitted 15a, b, c, and d. As described above, 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 fluorescently labeled primer is broad, and the fluorescences of the four fluorescently labeled primers overlap. Therefore, the waveform in FIG. 17 is observed not as a signal of a single fluorescently labeled primer but as a sum of fluorescence of a plurality of fluorescently labeled primers. For example, Ia is the primer T
Primer G, Primer A, Primer C
Are mixed. The same applies to Ib, Ic, and Id. When these effects are corrected based on the fluorescence characteristics of the fluorescent-labeled primer, the transmission characteristics of the spectral filter, and the sensitivity characteristics of the detector, the fluorescence intensities of the primers T, G, A, and C alone from above as shown in FIG. Is obtained. This is T,
The electrophoretic 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 conventional method.

In this embodiment, the base sequence can be determined simultaneously for 96 samples, and high throughput can be achieved. Specifically, the electrophoresis speed is about 200 bases / hour, and the overall throughput is 20 k bases / hour, which is 10 times or more higher than that of the conventional method using a plate gel.

The present embodiment has the same effect as that described in the first embodiment. Also, when the optical cell is an optical cell having the structure described in Example 1 or Examples 4 to 6, measurement for DNA base sequence determination can be performed in substantially the same manner as described above. Similar effects as described for Examples 4-6 are obtained.

In this embodiment, a four-segment prism is used, but a two-segment prism is used.
The nucleotide sequence of DNA can be determined by identifying the base species according to the method described in Japanese Patent Publication No.

In the present embodiment, the inner surface of the capillary of the capillary array was subjected to silane coupling treatment as in Example 1, and the gel was chemically bonded to the inner wall of the capillary. This process need not be performed on the entire inner wall of the capillary, but may be performed only on a part, that is, only near the end on the optical cell side. This can similarly prevent the gel from protruding into the sheath liquid inside the optical cell, simplify the processing, 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.

[0061]

According to the present invention, an electrophoresis apparatus capable of performing highly sensitive fluorescence or light absorption measurement with little influence of background light or the like by performing an optical measurement outside a sample separation section such as a capillary gel is provided. realizable. Further, a simple electrophoresis apparatus capable of simultaneously migrating a plurality of samples and measuring at the same time can be realized. In addition, an apparatus that has high operability and safety and can determine the base sequence of DNA at high throughput can be realized.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an electrophoresis unit and a laser beam irradiation system of an electrophoresis apparatus according to a first embodiment of the present invention.

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

FIG. 3 is a configuration diagram of a light irradiation system and a detection system of an electrophoresis apparatus according to a third embodiment of the present invention.

FIG. 4 is a configuration diagram near a gap of an electrophoresis apparatus according to a fourth embodiment of the present invention.

FIG. 5 is a configuration diagram of an electrophoresis unit of an electrophoresis apparatus 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 electrophoresis section of an electrophoresis apparatus according to a sixth embodiment of the present invention.

FIG. 8 is a configuration diagram of a capillary sub-array 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 electrophoresis apparatus according to a seventh embodiment of the present invention.

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

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

FIG. 13 is an explanatory view including a partial cross section showing a configuration near an optical cell unit in an electrophoresis apparatus according to a seventh embodiment of the present invention.

FIG. 14 is a view illustrating the principle configuration of a fluorescence detection / color separation unit in an electrophoresis apparatus according to a seventh embodiment of the present invention.

FIG. 15 is an image diagram of a fluorescent image obtained by a two-dimensional detector in the electrophoresis apparatus according to the seventh embodiment of the present invention.

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

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

FIG. 18 shows A, C, and C obtained by analyzing the data of FIG.
Time waveforms for G and T fragments.

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

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: Capillary, 2
a, 2b, 2c, 2d to 2t: Capillary, 4: Fluorescent cell, 3a, 3b, 3c, 3d to 3t: gap, 5
a and 5b: Multi-capillary holder, 6: Cathode-side electrode tank, 7: Anode-side electrode tank, 8: Sheath liquid inlet, 9: Tube made of ethylene tetrafluoride resin, 10: Sheath liquid container, 11: Sheath Liquid, 12 DC high voltage power supply, 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
Reference numeral 3 denotes a lens, 54 denotes a one-dimensional sensor, 55 denotes a controller for a one-dimensional sensor, 56 denotes a data processing device, 57 denotes a monitor, 58 denotes a printer, 59 denotes a memory, and 60 denotes a fluorescent cell.
61a-61t: pores, 62: flat plate, 63: 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 condenser lens, 115 a to 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 supply, 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 the sheath liquid in the sheath liquid container, 231: liquid level of the sheath liquid flowing into the cell, 240: sample, 245, 246: buffer liquid, 2
50, 251 ... spacer, 252, 253 ... hole, 26
0, 261, 262, 270... Terminals.

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 21/64 C12M 1/00 G01N 21/17 G01N 27/447 JICST file (JOIS) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A capillary for holding a plurality of capillaries including a migration medium on which a fluorescently labeled sample migrates, and arranging ends of the plurality of capillaries in the direction of migration of the sample on a straight line with an interval. Holding means, and the plurality of keys
An optical cell in which each end of the capillary is disposed therein, and the sample for migrating each of the capillaries
Flow in the optical cell in the sheath flow state, respectively.
To over, means for introducing an internal buffer solution of the optical cell, and a discharge port for discharging the buffer solution from the optical cell, the excitation light for exciting the fluorescence from the sample, the Sea
An electrophoresis apparatus comprising: means for irradiating the sample migrating in a sflow state substantially parallel to the straight line; and means for detecting the fluorescence. 2. An electrophoresis medium in which a fluorescently labeled sample migrates.
A plurality of capillaries including the plurality of capillaries
The ends of the sample in the electrophoresis direction are arranged on a straight line at intervals.
Capillary holding means for placing and holding the plurality of keys,
Each said end of the capillary is located therein
Optical cell and the sample for migrating the respective carriers
Flow in the optical cell in the sheath flow state, respectively.
To introduce a buffer solution into the optical cell.
Means for discharging the buffer solution from the optical cell
An excitation light for exciting fluorescence from the sample and the sample,
Approximately parallel to the straight line on the sample running in the sflow state
Means for irradiating the excitation light, an image dividing means for dividing the image of the fluorescence generated from the sample by the irradiation of the excitation light, a spectral filter arranged at a front or rear part of the image dividing means, and the image dividing means An electrophoresis apparatus, comprising: an image forming unit that forms an image divided by the image forming unit; and an image detecting unit that detects an image formed by the image forming unit, and detects the fluorescence. . 3. The electrophoresis apparatus according to claim 1, wherein
An electrophoresis apparatus, wherein the capillary holding means and the optical cell are detachable. 4. The electrophoretic device according to claim 1, wherein
An electrophoresis apparatus, comprising: means for displaying a current value flowing through each of the plurality of capillaries in the electrophoresis medium. 5. The electrophoretic device according to claim 1, wherein
An electrophoresis apparatus, wherein the electrophoresis medium is a gel. 6. The electrophoretic device according to claim 1, wherein
An electrophoresis apparatus, comprising: control means for maintaining the level of the buffer solution in the optical cell substantially constant.