JP3599060B2 - Electrophoresis device - Google Patents

Description

  The present invention relates to an electrophoresis apparatus for separating and analyzing nucleic acids, proteins, sugars, and the like, and more particularly to an electrophoresis apparatus suitable for detecting DNA (nucleic acid) and the like, analyzing the base sequence of DNA, and the like.

As an electrophoresis apparatus for separating and analyzing the molecular weight of a fluorescently labeled sample by electrophoresis, for example, there is an apparatus for determining the base sequence of DNA using a fluorescent substance as a label. The nucleotide 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. Capillary gel electrophoresis generally has a large surface area per gel volume due to the small diameter of the capillary and easy dissipation of Joule heat, so high voltage can be applied and high-speed separation can be performed. Is a useful way. A first conventional example of capillary gel electrophoresis is described in "Nucleic Acid Research", Vol. 18, pp. 1415-1419 (1990) (Nucleic Acid Research, 18, 1415-1419 (1990)). There is a method described. As shown in FIG. 19, using a capillary having an inner diameter of 75 μm, a high voltage of 9 kV 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 light are inclined by about 25 degrees from the vertical direction, and the laser light is condensed and radiated to the center of the capillary to a diameter of about 20 μm, and the generated fluorescence is emitted. The spectrum is detected by 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, pp. 61-67 (1990) (Journal of Chromatography, 516, 61-67 (1990)), describes a capillary gel electrophoresis. A method for determining the base sequence of DNA by the method is described. In this method, DNA fragments are separated by molecular weight using a capillary having an inner diameter of 50 μm. The measuring apparatus in this example is basically the one described in "Science", Vol. 242, pp. 562-564 (1988) (Science, 242, 562-564 (1988)). FIG. 20 shows an apparatus configuration in which a laser light source, a pump for sheath flow, and the like are added based on the contents of the description for easy understanding in the apparatus diagram described in “Science” magazine. The DNA fragment is detected by introducing the separated solution from which the molecular weight has been separated into a quartz flow chamber (inner size 250 μm × 250 μm), and using a Tris-borate buffer solution containing EDTA as a sheath solution to flow by a liquid chromatography pump. In this state, the laser light is condensed to a diameter of about 10 μm to irradiate the separated liquid flowing substantially in the center of the flow chamber, and the fluorescence from the DNA fragments 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 to 679 (1986) (Nature, 321, 674 to 679). (1986)) and the like, a fluorophore (fluorescein, 4-chloro-7-nitrobenzo-2-oxa-1) having four different maximum fluorescence wavelengths for each type of terminal base of the DNA fragment. -Labeled with diazole (NBD), tetramethylrhodamine, Texas Red (Molecular Probe Co.), excite the fragments migrated in order of molecular weight with laser light, and fluoresce each with four kinds of bandpass interference filters. And then detecting the fluorescence to determine the base sequence. As described above, in capillary gel electrophoresis, measurement is usually performed using only one capillary.
"Nucleic Acid Research", Vol. 18, pp. 1415-1419 (1990) (Nucleic Acid Research, 18, 1415-1419 (1990))

  In a capillary electrophoresis apparatus, with the miniaturization of samples and the like, higher sensitivity of detection is desired. 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 fluorescence measurement in an electrophoretic state, in addition to the fluorescence from the intended phosphor itself, background light includes scattered light (eg, Rayleigh scattering) of excitation light by the gel, fluorescence, and the gel support, that is, the inner wall of the capillary. And scattered light on the outer wall or fluorescence from the capillary itself is 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 in achieving 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 multiple capillaries, which are migration paths, and how to achieve these is an important issue. Become.

  In the first conventional example, a first countermeasure for removing excitation light (scattered light) by a band-pass interference filter or the like and separating and detecting a fluorescent component, and setting the axis of the capillary to the laser light irradiation axis and the fluorescence condensing With the two measures of the second measure of inclining about 25 degrees from the perpendicular to the plane including the axis, more highly sensitive fluorescence detection is achieved. However, with the first measure, it is difficult to completely remove 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 measure is an effective method that can reduce the scattered light itself incident on the fluorescence focusing lens by tilting the capillary. However, the capillary has a circular cross section and its diameter is small, so it is an effective method. Sufficient background light is required because the light intensity is high, scattered light is emitted in all directions without being reduced, and in addition, it is not possible to reduce the fluorescence from the gel and the glass tube itself 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 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. Furthermore, 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 Since the refraction and scattering occur and the light passing through the capillary is widely diffused, it is practically impossible to achieve this configuration. In other words, there is no problem in the case of one capillary, but when continuously irradiating a plurality of capillaries, the intensity of light irradiated to the next capillary becomes extremely weak each time it passes through the capillary, making it difficult to measure fluorescence. become. From the above, it is difficult to apply the present conventional example to simultaneous processing of a plurality of samples to improve the processing capability.

  The second conventional example is a method capable of effectively removing light scattered by a capillary and a gel 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 led out of the capillary gel, and the fluorescence is measured under the sheath flow, so that the scattered light at the interface of the capillary and the scattering from the gel Light and fluorescence are eliminated. In the sheath flow chamber, the sample liquid flows in a laminar flow at almost the center thereof and does not contact the inner wall of the sheath flow chamber. Therefore, the scattered light from the sheath flow chamber and the fluorescence from the sample are spatially separated. And the fluorescence intensity can be detected with high sensitivity. However, in order to form a sheath flow, it is necessary to constantly flow at a constant flow rate into a sheath flow chamber by a liquid chromatography pump or the like using a Tris-borate buffer solution as a sheath solution. Therefore, there are problems 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 capillaries 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 / scattering of light by the capillary. However, since there is a sheath flow chamber for each capillary, it is difficult to spatially narrow the space between the capillaries and the capillaries.As a result, the sheath flow chamber becomes large, and when using a plurality of sheath flow chambers, However, there is a problem that the apparatus 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 condition, 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 simultaneous processing of a plurality of samples to improve the processing capability.

  An object of the present invention is to solve the above-mentioned problems of the prior art, and to easily and easily perform a fluorescence measurement or a light absorption measurement of a sample separated by electrophoresis with high sensitivity, and a plurality of samples. An object of the present invention is to provide an electrophoresis apparatus which can easily perform the simultaneous processing of the electrophoresis. 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.

  In order to achieve the above object, in an electrophoresis apparatus in which a sample separation unit 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 composed of a capillary, (1) one end of each is provided. The other end of each of one or a plurality of pairs of capillaries connected to the cathode electrode tank or the anode electrode tank is arranged in the optical cell so that their axes are substantially aligned and opposed to each other with a certain gap therebetween. A sheath solution is injected into the optical cell from the outside, flows through the capillary, and the sample to be migrated from the capillary end of the upstream migration path, which is the sample separation section, is placed in a sheath flow state and the opposite capillary is used. The gap is used as an optical detector, and the optical detector is irradiated with light from a light source to detect a sample, or (2) a plurality of caps having one end immersed in an electrode bath. By terminating the rally in the optical cell and injecting and flowing a sheath liquid from the outside into the optical cell, the sample for migrating each capillary is caused to flow through the optical cell in a sheath flow state, and the sheath flow section is detected by the optical detection section. Then, the electrophoresis apparatus is configured to detect the sample by irradiating the optical detection unit with light from a light source. (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 radiated almost in parallel to the straight line just below the ends of a plurality of capillaries arranged on a straight line in the optical cell, and fluorescence is generated from a 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 (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 performed simultaneously by the plurality of optical detection units. In the configuration of (2), the plurality of optical detection units formed by the plurality of capillaries are positioned so as to be located on a straight line. Are arranged in an optical cell, and excitation light is irradiated along the above-mentioned straight line so as to simultaneously irradiate all the optical detection units, and simultaneously, fluorescence emitted from a sample which migrates each capillary is detected.

  In the configuration of (3), similarly to (1), the ends are held in a straight line with the same number or more than the number of capillaries for electrophoretic separation and at the same interval as the capillaries for electrophoretic separation. The structure may be such that the aqueous solution is discharged from the optical cell via a hollow capillary arranged near a plane including the end of the capillary and the laser beam irradiation axis. 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 is simultaneously measured. Fluorescence or the like emitted from the sample may be collected by a lens, divided, and imaged. Alternatively, the light may be collected in one direction by a cylindrical lens, divided, imaged, and detected.

  In the configurations of (1), (2), and (3), each unit 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 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. For the injection of the aqueous solution into the optical cell, a sheath liquid container containing the aqueous solution is provided outside the optical cell, the liquid surface is arranged at a position higher than the surface of the liquid discharged from the optical cell, or by dropping, or Using an optical cell whose upper part is open, means for pouring 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 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 configured by a mechanism in which the upper part of the optical cell is open and a liquid level in the optical cell is detected, and a mechanism for replenishing the aqueous solution in the sheath liquid container via a valve when the liquid level is lowered. The optical detector measures 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 one. The resulting image such as fluorescence can be detected by a two-dimensional detector. 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. Further, a mechanism may be provided in which the liquid surface of the aqueous solution remaining in the electrode tank on the downstream side on which the sample is electrophoresed is made substantially constant, and an excessive aqueous solution is discharged to the tank. Further, the ratio of the length of both ends of the fluorescence emitted from the sample that migrates a large number of capillaries to the light receiving size of the two-dimensional detector is 3 or less, that is, the overall image magnification is 1/3 or more. Further, it is desirable that the distance between adjacent capillaries is 10 times or less the capillary size. In the electrophoresis, it is preferable 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 for measuring and displaying a current value flowing through the inside of the capillary for electrophoretic separation for each capillary is provided. The current value may be binarized and displayed. Also, the capillary for electrophoresis separation is subjected to a silanization treatment on the inner surface at least near the end held inside the optical cell and filled with gel, so that at least the gel near the end held inside the optical cell is filled. May be chemically bonded to the inner wall of the capillary.

  In the configuration (1) or (3) of the electrophoresis apparatus, one end of each of one or more pairs of capillaries connected to the cathode or anode electrode tank is connected to the other end of the capillary in the optical cell. Since they are arranged so as to face each other while maintaining a substantially constant gap and a certain gap, it is possible to form an electrophoresis path penetrating a part of the optical cell. Since the sheath liquid is injected into the optical cell from the outside and flows, the sample to be migrated from the capillary end of the upstream migration path, which is the sample separation section, can be put into the sheath flow state and guided to the opposing capillary. The sample can be electrophoresed continuously 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 maintaining a constant gap, the upstream capillary serving as the sample separation section and the gap section serving as the optical detection section can be easily formed. 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.However, 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, a plurality of optical detectors formed by a plurality of capillary pairs can be arranged close to each other in an 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, but 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 can be reduced, the amount of the sheath liquid can be reduced, and the size of the apparatus can be reduced.

  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 excitation light is irradiated along this straight line. Irradiation can be performed at the same time, and fluorescence measurement by a plurality of optical detection units can be performed simultaneously. 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 subjected to light scattering by a 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 a 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 short, even if the excitation light is narrowed down by a lens or the like, all the optical detection units can be irradiated with almost the same light beam diameter. For example, when the laser light is focused to a diameter of about 100 μm at the focal position, the diameter of the laser light becomes about 100 μm over about 10 mm before and after the focus. When capillaries having an outer diameter of 200 μm and an inner diameter of 100 μm are arranged every 400 μm, about 50 can be arranged inside about 10 mm before and after the focal point, and these can be irradiated with the same light beam diameter and light beam intensity. . Accordingly, each optical detection unit 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.

It should be noted that the molecular weight can be easily separated by forming the sample separation section with a capillary gel.
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 fluctuate the composition, and to improve the separation ability of the sample by electrophoresis. Do not spoil. When the sample is single-stranded DNA or the like, a denaturing agent can be included in the sheath liquid. In this case, it is possible to prevent the sample DNA from being recombined when migrating through the gap, that is, the optical detection unit, and the measurement accuracy is improved. The possibility that the denaturant confined inside the capillary leaks into the optical cell is reduced, and the resolution of the sample in electrophoresis is not impaired.

  In addition, when the sheath liquid is injected into the optical cell, the liquid level of the sheath liquid in the sheath liquid container is arranged at a position higher than the liquid level in the downstream electrode tank, so that the sheath liquid flows easily due to a head drop. Therefore, there is no need to mechanically flow the sheath liquid, and a simple and inexpensive device configuration is obtained. Further, since a pulsating flow generated when a pump or the like is used is eliminated, a stable sheath flow is enabled, and 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 can be easily attached to the capillary holder by using a cell having an open top in addition to a shape that is sealed when the capillary is attached. 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. In addition, 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. In general, 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, when 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 its 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.
When the gap length was about 3 mm, 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 capillary and the capillary, and does not contact the inner surface of the optical cell, so that the sample is adsorbed on the inner surface of the cell and from the inner surface of the cell. Can be removed and the detection sensitivity can be improved.

  In addition, images from multiple samples migrating each capillary are divided using a polyhedral prism or the like, and a spectral filter is used to pass light components in different wavelength ranges. Can be obtained. In particular, if the image is divided into four, which is the number of base types of DNA, each base type is labeled with a different fluorescent substance, and the DNA base sequence can be determined efficiently.

  Fluorescence images from a sample migrating multiple capillaries or split fluorescence images are simultaneously formed on a two-dimensional detector and their intensities are measured at the same time, enabling high-speed measurement regardless of the number of capillaries. It becomes possible to do.

  In addition, an insertion guide is provided inside the optical cell to hold the capillary holder at the specified position, and multiple capillaries and the optical cell can be attached and detached, improving operability in mounting and handling the capillary. I do.

  In addition, a laser device is used as the excitation light source unit, and at least two types of laser devices are used, and these laser beams are coaxially irradiated with one laser beam, so that the DNA fragments can be efficiently excited.

  By providing electrodes equal in number to a plurality of capillaries for electrophoretic separation, immersing the capillaries and electrodes in a plurality of sample solutions, simultaneously applying a voltage and simultaneously injecting a sample into each of the plurality of capillaries. In addition, the sample injection can be performed easily and by one operation, which can shorten the time and improve the operability as compared with the conventional case where the sample is injected one by one, and further, the capillary for electrophoretic separation. The value of the current flowing inside can be measured for each capillary. The value of the current flowing through the inside of the capillary is effective for knowing the state of the capillary, and for example, it is possible to predict in advance a failure in separation due to breakage of the gel or the like. The current value can be displayed by setting the binarization level arbitrarily and displaying the lamp so that the lamp is turned on when the current value is binarized and the current is lower than the normal electrophoresis current, so that damage to the capillary gel can be monitored. It is possible to efficiently judge the necessity of exchanging and remeasurement of the gel capillary, thereby improving the usability.

  Furthermore, 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 can be increased, 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 tank, are terminated in the optical cell, and a sheath liquid is injected into the optical cell from outside. As a result, the sample that migrates through each capillary flows through the optical cell in a sheath flow state, and the sheath flow portion is used as an optical detection portion. Can be separately measured, and the same operation as that of 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 similar to the configuration (2) of the electrophoresis apparatus.

  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 can be realized by performing optical measurement outside a sample separation unit such as a capillary gel. 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.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1]
In the present embodiment, a fluorescently labeled DNA fragment is subjected to molecular weight separation by electrophoresis, and detection is performed by fluorescence. The case where fluorescein isothiocyanate (FITC) is used as the fluorescent substance for labeling will be described. As the 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 device configuration will be described. FIG. 1 shows a configuration diagram of an electrophoresis section 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 are the same capillary made of silica having an inner diameter of 100 μm, an outer diameter of 375 μm, and a length of 40 cm. Further, 20 silica capillaries 2a, 2b, 2c, 2d,..., 2t having a length of 10 cm and the same inner and outer diameters 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. Next, tetramethylethylenediamine and ammonium persulfate solution were added to the degassed 3.84% acrylamide, 0.16% bisacrylamide, 7 M urea, Tris containing 2 mM EDTA, and borate buffer, and the mixture was injected into the capillary. Polymerize to make acrylamide gel. Since the capillaries have been subjected to silane coupling treatment, the acrylamide gel and the capillaries are chemically bonded, and the gel does not protrude from the capillaries during electrophoresis.

  The capillaries 2a to 2t are processed so that their inner surfaces have a positive charge. First, a 3- (2-aminoethylaminopropyl) trimethoxysilane solution is injected into the 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) The moving direction (negative electrode → positive electrode) of the sample in the capillary 2 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 to detect a sample by fluorescence. That is, one end of each of the capillaries 1a to 1t and 2a to 2t is connected to a square fluorescent cell 4 made of quartz (outer size: width 36 mm × depth 4.5 mm × length 3 mm, inner shape: width 30 mm × depth 2 mm × length 3 mm). , Inside: width is the horizontal direction of the figure (the direction of the capillary 1a → 1t arrangement), depth is the direction perpendicular to the plane of the figure, and length is the vertical direction of the figure (the sample migration direction of 1 → 2 of each capillary). To place. 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. Similarly, the capillaries 1b and 2b, 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 using multi-capillary holders 5a and 5b having 20 vertical holes at 0.6 mm intervals in a flat block made of a fluororesin, for example, an ethylene tetrafluoride resin. That is, the capillaries 1a to 1t are inserted one by one into the 20 vertical holes of the multi-capillary holder 5a, and the capillaries 2a to 2t are inserted one by one into the 20 vertical holes of the multi-capillary holder 5b. The multi-capillary holders 5a and 5b are fixed in close contact with the upper and lower portions of the fluorescent cell 4 so that 2a, 1b and 2b, 1c and 2c, 1d and 2d are coaxial with each other. Further, since the gaps 3a to 3t are used as an optical detection unit, here, a fluorescence detection unit, the capillaries 1a to 1t and the capillaries 1a to 1t in the quartz fluorescent cell 4 are arranged so that the gaps 3a to 3t are aligned on one straight line. Adjust the length and the like of 2a to 2t. The other end of each of the capillaries 1a to 1t and the capillaries 2a to 2t is immersed in the cathode-side electrode bath 6 and the anode-side electrode bath 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 tetrafluoroethylene resin. It has a structure. A buffer solution containing Tris, boric acid, EDTA, and urea is used as the sheath solution, and has the same components as the buffer solution in the capillary gels of the capillaries 1a to 1t, and the components of the capillary gel leak into the fluorescent cell 4. To prevent

  Further, the inside of the fluorescent cell 4 in which the capillaries 1a to 1t and the capillaries 2a to 2t are arranged is filled with the sheath liquid, and the liquid surface of the sheath liquid 11 in the sheath liquid container 10 is buffered in the anode electrode tank 7. By disposing the sheath liquid higher than the liquid surface, 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 also filled with the capillary gel. By applying a DC voltage from the DC high-voltage power supply 12 to the gap, a current flows through the capillary 1a, the gap 3a, and the capillary 2a, for example, and the sample can be electrophoresed. Further, by arranging 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 sheath liquid flows inside the capillaries 2a to 2t, A flow occurs at the upper part of each of the capillaries 2a to 2t, that is, each of the gaps 3a to 3t. Then, the sample electrophoresed 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, is led to each capillary, and is supplied to the anode side electrode tank. Electrophoresed on the 7 side. The capillaries 2a to 2t are treated so that the inside thereof has a positive charge, and the electroosmotic flow in the capillaries 2a to 2t flows from the capillaries 1a to 1t toward the anode-side electrode tank 7, and the liquid flows into the gap. And the sheath liquid can flow stably in the direction of the anode-side electrode tank 7, and in particular, even when the flow rate of the sheath liquid is small, the sheath liquid can flow stably in the direction of the anode-side electrode tank 7.

The electrophoresis is performed by applying a DC voltage between the cathode-side electrode tank 6 and the anode-side electrode tank 7 by the DC high-voltage power supply 12. Due to the voltage application, the current flowing through the capillary 1a and the capillary 2a mainly flows through the gap 3a, and similarly, the current flowing through the capillaries 1b and 2b, 1c and 2c, 1d and 2d,. Flow around the gaps 3b, 3c, 3d,..., 3t. Furthermore, 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. Further, 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. Fluorescence measurement becomes possible.
The introduction of the fluorescent-labeled DNA fragment as a sample is performed by temporarily immersing the end of the capillary 1a on the cathode side in the sample solution and applying a voltage of 6 kV for about 20 seconds between the sample solution and the anode-side electrode tank 7. . Then, the end of the capillary is returned to the original cathode-side electrode tank 6. This operation is performed for each of the capillaries 1b to 1t, and the sample is injected into the capillary gel of the capillaries 1a to 1t. The 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. For the detection of the sample passing through the gaps 3a to 3t, an argon laser beam having a wavelength of 488 nm is used to excite FITC, which is a fluorescent substance for labeling, and the gaps 3a in which the laser beams are aligned in a straight line. The irradiation is performed by adjusting the laser optical axis so as to irradiate 〜3t simultaneously and under substantially the same conditions, and measuring the emitted fluorescence. In other words, the laser beam 21 having a wavelength of 488 nm from the argon laser light source 20 is squeezed 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 focal depth 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 is 0.6 mm × 19, that is, about 12 mm in this example. In other words, the laser light irradiates 20 places in the gaps 3a to 3t with substantially the same spot size, and the spot size becomes substantially 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 approximately equal to the thickness of the capillary gel, and all the gaps can be irradiated with almost the same spot size. In addition, it is possible to uniformly and efficiently excite the fluorescently labeled DNA fragments that migrate through the gaps 3a to 3t.

  Fluorescence from the fluorescence-labeled DNA fragment migrating in the gaps 3a to 3t is detected in a direction perpendicular to the laser light irradiation direction. The configuration diagram is shown in FIG. The fluorescence 30 emitted from the DNA fragment removes background light such as scattered light with an interference filter 32 and forms an image on a two-dimensional detector 34 such as a CCD camera with a lens 33. The two-dimensional detector 34 is controlled by the controller 35 to detect the fluorescence images of the gaps 3a to 3t, and the data processing device 36 such as a computer continuously changes the fluorescence intensity of each of the gaps 3a to 3t with time. In addition, all the gaps are measured simultaneously, and the results are displayed on the monitor 37, output to the printer 38, and stored in the memory 39. As a result, it is possible to simultaneously and continuously measure the migration patterns of which the molecular weight has been separated 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 that passes a wavelength range of 500 nm to 540 nm is used to effectively detect the fluorescence from FITC. Further, the image magnification of the lens 33 is set so that the images of the gaps 3a to 3t are all 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. Therefore, it is possible to uniformly and efficiently irradiate a sample, which migrates a plurality of capillaries, with light at the same time. 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 to detect the fluorescence 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. In addition, 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 almost equal in all the gaps, and the reproducibility between the gaps is high. Therefore, there is little variation between electrophoresis paths, 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 an ordinary 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 example, the sample can be detected with high sensitivity without impairing the molecular weight separation characteristics, because the molecular weight separation section is the same capillary gel as the conventional one.

  Further, according to the present embodiment, the buffer solution can be caused to flow without using a mechanical means such as a liquid chromatography pump or the like, so that the device configuration is simplified and the cost is reduced. 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. 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 the operability is improved. In this 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 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. Note that when the flow velocity of the sheath liquid in the gap is large, such as when the inner diameter of the capillaries 2a to 2t is large, the effect of the electroosmotic flow is reduced, so that the inner surfaces of the capillaries 2a to 2t are necessarily treated. No need. 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. This prevents the constituent components of the capillary gel from leaking into the fluorescent cell 4 or into the electrode tank, so that 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, the 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 times of repeated use is slightly reduced. Is possible.

  The laser device and the phosphor used are not limited to the argon laser and FITC, but any phosphor and an appropriate laser device can be used. In the present embodiment, the measurement of DNA fragments has been described as an example, but the present invention can naturally be used for analysis of proteins, sugars, and the like. 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 capillary on the downstream side is made smaller than the inner diameter of the capillary on the upstream side, the sample migrated from the end of the upstream capillary is squeezed when introduced into the downstream capillary, so that the concentration of the sample liquid is high. That is, it is possible to detect with higher sensitivity. 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 capillary on the upstream side can be more easily and reliably guided to the capillary on the downstream side. Furthermore, according to the present embodiment, since the fluorescence from the sample can be measured without transmitting the excitation light through the capillary portion, the capillary does not need to be transparent. That is, since it is not necessary to remove the coating of the capillary, the handling becomes easy. Furthermore, capillaries of various materials such as capillaries made of opaque fluororesin such as ethylene tetrafluoride resin and ethylene trifluoride chloride resin can be used. Since the fluororesin capillary does not adsorb any sample, a treatment operation such as a surface treatment is unnecessary, and the operability is improved because there is no breakage. In addition, 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, also in the case of only one capillary pair, it is 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.

[Example 2]
Next, a method for determining the base sequence of DNA using the apparatus of Example 1 will be described. A fluorescently labeled DNA fragment is prepared by performing a DNA polymerase reaction using a fluorescently labeled primer by 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 divided into four parts, and DNA polymerase reactions corresponding to A, C, G, and T are respectively performed. 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 at the end are obtained. Similar reactions are performed for C, G, and T. The four reaction solutions of A, C, G, and T obtained as described above are injected into four of the capillaries 1a to 1t, for example, the capillaries 1a, 1b, 1c, and 1d, respectively. The injection method is the same as in Example 1, and the A reaction liquid is injected into 1a, the C reaction liquid into 1b, the G reaction liquid into 1c, and the T reaction liquid into 1d. Electrophoresis is performed by applying a voltage of about 6 kV after the injection. 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 base sequence can be analyzed by analyzing the gap positions where fluorescent peaks occur in chronological order.

  The apparatus of the first embodiment 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 that case, as is well known, for example, as shown 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 for 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 fluorescent substances simultaneously. That is, for each type of terminal base, a primer labeled with a different fluorescent substance is used, and after each DNA polymerase reaction is performed, the reaction solution is 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 identified by comparing the fluorescence intensities of the four types of phosphors at the maximum fluorescence wavelength. In this case, the base sequence of the DNA sample can be determined by 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.

[Example 3]
In the above-described first and second embodiments, the apparatus for detecting a sample by fluorescence measurement has been described. However, the apparatus is similarly configured for light absorption measurement such as measurement of absorbance and measurement of transmitted light intensity. be able to. FIG. 3 shows a configuration diagram of a light irradiation system and a detection system of the electrophoresis apparatus in the case of light absorption measurement. The electrophoresis part has the same configuration as that of FIG. The introduction of a sample, for example, a restriction fragment of DNA is performed in the same manner as in Example 1, and the electrophoresis for separating the molecular weight is performed in the same manner as in Example 1. Light from a light source 50 such as a xenon lamp or a D2 lamp is passed through a monochromator 51 to a DNA fragment passing through the gaps 3a to 3t and condensed by a lens 52 to irradiate the gaps 3a to 3t. The irradiation light wavelength is usually set to the absorption wavelength of the sample. For example, about 260 nm is appropriate for a DNA fragment. 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 controller 55 for the one-dimensional sensor, 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 displayed on a printer 58. It is output and stored in the memory 59. In the present embodiment, the gap is provided between the capillaries, the capillary is divided into the molecular weight separation section, and the gap space is separated from the optical detection section for measuring the absorbance, so that the irradiation light passes through the sheath liquid with extremely little light scattering. Therefore, it is possible to efficiently irradiate the sample without being scattered by the capillary, the gel, or the like, and it becomes possible to measure the absorbance with high accuracy. Specifically, assuming that the transmitted light intensity when the light is made to enter the gap as in the present embodiment (when the sample does not pass and the numerical aperture of the light receiving lens is 0.19) 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. . Decreasing the transmitted light intensity means that the S / N of the photodetector and the like is correspondingly reduced, 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 irrespective 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 this embodiment, the light that has passed 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.

[Example 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, a capillary may be used as the molecular weight separating portion as in the above embodiment, and a gap may be formed between the capillary end and a flat plate having pores. FIG. 4 shows a configuration diagram of the gap. As in the first embodiment, the 20 capillaries 1a to 1t, which are the molecular weight separating sections, are held in the multi-capillary holder 5a and fixed to the fluorescent cell 60. On the opposite side of the fluorescent cell 60, a flat plate 62 having pores 61a to 61t having an inner diameter of 200 μm is fixed. By providing the pores 61a to 61t at positions opposed to 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 guide the solution to the electrode tank 65 via the tube 64. Electrophoresis can be performed in the same manner as in Example 1 by applying a voltage to the electrode tank 65 and the other electrode tank (not shown). The same applies to laser light irradiation, fluorescence detection, and the like.

[Example 5]
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 flowed and electrophoresed, without using the downstream capillary. FIG. 5 shows a configuration diagram of an electrophoresis unit of the electrophoresis apparatus. As in the first embodiment, one end of each of the capillaries 1a to 1t is held inside the optical cell 4 with their respective tips aligned. For fixing the capillaries in the optical cell, a multi-capillary holder 5a having 20 vertical holes provided at 0.6 mm intervals in a flat block made of a fluororesin, for example, an ethylene tetrafluoride resin, is used. That is, 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 to the upper part of the fluorescent cell 4 in close contact. 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 thereof 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 tetrafluoroethylene resin. And Further, by disposing the liquid surface of the sheath liquid 11 in the sheath liquid container 10 higher than the liquid surface 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 capillary 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 downstream of the capillary end with a laser beam of 500 μm 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.

[Example 6]
In the apparatus described in the first embodiment, the gap may be formed by using the downstream capillary as a flat plate having a plurality of grooves. FIG. 6 shows a perspective view of a flat plate having a plurality of grooves. 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, and is, for example, 300 μm in width and 600 μm in depth. The distance between the grooves is 1 mm. In addition, these intervals and the shapes of the grooves 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 is of a flow cell type in which the inside of the cell is 20 mm wide, 3 mm deep, and 40 mm long, and the (quartz) glass plate thickness is 2 mm and the upper and lower ends are open. 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 to form four flow paths formed by the grooves 81a, 81b, 81c, 81d and the glass of the fluorescent cell. In addition, capillaries 82 a, 82 b, 82 c, and 82 d having an inner diameter of 100 μm and an outer shape 200 (in which gel is prepared in the same manner as in Example 1) 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 can be dealt with by adjusting the positions of holes and the like provided in the multi-capillary holder 83. The ends of the capillaries 82a, 82b, 82c, and 82d inside the fluorescent cell are aligned, and each capillary end is adjusted to be separated from the flat plate 80 by about 1 mm 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. The fluorescent cell 84 is provided with a sheath liquid inlet 85 for filling the inside thereof with a sheath liquid, and a structure in which the sheath liquid 88 in the sheath liquid container 87 can be injected through a tube 86 made of tetrafluoroethylene resin. And 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 the fluorescent It can flow in 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, if the flat plate is made of quartz glass or the like, the laser light is irregularly reflected, and even when the flat plate hits the flat plate, no fluorescence is generated from the portion, and the fluorescence measurement becomes easy.

[Example 7]
An apparatus and a method for determining a DNA base sequence 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 distinguish four base species in one migration path (gel capillary), a primer labeled with a different fluorescent substance for each of the A, C, G, and T fragments is used. A primer (abbreviated as primer A) labeled with a fluorescent substance having a maximum fluorescence wavelength of about 550 nm was bound to the fragment A. Similarly, to the C, G, and T fragments, primers (abbreviated as primer C, primer G, and primer T, respectively) labeled with fluorescent substances having fluorescence maximum wavelengths of about 520, 575, and 605 nm, respectively, were bound. 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 fragment was heat-denatured by heating at 90 ° C. for 2 minutes to form a single strand, 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 sub-array 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 sub-arrays of FIG. 8 are combined. 10 is a configuration diagram of a DNA base sequence determination device using a capillary array, FIG. 11 is an enlarged cross-sectional view of a sample injection unit, and FIG. 12 is a configuration diagram of a migration current measurement / display unit of the DNA base sequence determination device. 13 is an explanatory diagram including an optical cell cross section for showing a configuration in the vicinity of the optical cell unit, FIG. 14 is a principle configuration diagram of a fluorescence detection / color separation unit, and FIG. 15 is an image diagram of a fluorescence image obtained by a two-dimensional detector. 16 is a diagram showing the fluorescence characteristics of the fluorescently labeled primers used and the transmission characteristics of the spectral filter, FIG. 17 is an example of a time waveform of the fluorescence signal intensity emitted from a sample in which one gel capillary is migrated (raw data), and FIG. 5 is a time waveform for each of the A, C, G, and T fragments obtained.

Using 96 capillaries filled with gel, a capillary array was prepared in which one end was arranged in a straight line. 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 capillaries is formed, and 12 sets of these sub-arrays are arranged to form a 96-capillary array. Was configured. As shown in FIG. 8, eight capillaries 90 having slightly different lengths are arranged on the same plane, and the capillaries are held 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 them with tools 91 and 92, respectively, 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.
As shown in FIG. 9, 12 sub-arrays are combined to form a capillary array 107. The twelve capillary holders 91 are fixed by the area array holder 93 so as to be arranged in 12 rows on the same plane, and the other capillary holders 92 at the other end are linearly arranged so that the twelve capillary holders are sequentially arranged on one straight line. It is fixed with the array holder 214. 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. Capillaries in the middle of the subarrays in the second to twelfth columns are omitted. Although the subarrays themselves are omitted from the third to eleventh columns, they are arranged in order between the first, second, and twelfth subarrays. 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 as in Example 1 and used. The gel concentration was 9% T (ratio to the total amount of (acrylamide + bisacrylamide) solution) and 0% C (ratio of bisacrylamide to (acrylamide + bisacrylamide)). The use of the capillary array 107 of this embodiment enables simultaneous analysis of 96 samples.

  FIG. 10 is a block diagram of the DNA base sequencer. The ends of the capillary array 107 arranged in a lattice are inserted into the buffer solution (or sample solution) of the upper individual electrode tank 104 having a 96-well shape and having the shape of a microplate. 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 reduced from 96 to 20 for easy understanding. 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. These components are made coaxial with an argon laser beam 111 and a YAG laser beam 112 by a mirror 109 and a dichroic mirror 110. The optical cell 103 was squeezed and irradiated with a lens (not shown) so as to become one laser beam 113. Fluorescence generated from the fluorescently labeled sample is imaged on a two-dimensional detector 118 by a condenser lens 114, four kinds 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 the sample into the capillary array 107 will be described with reference to FIG. 11 (an enlarged cross-sectional view of the sample injection part / upper electrode tank). FIG. 11 is a cross-sectional view of a surface including a capillary held by one of the twelve capillary holders 91. An upper individual electrode tank 104 having container-like recesses 104a, 104b,... Of 8 holes × 12 rows (96 holes in total) is used. For example, a 96-well microplate with V-bottom wells or 96 sampling tubes arranged in a grid of 8 × 12 rows at 9 mm intervals 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 the container-like recesses 104a, 104b,... Each serve as an upper electrode tank, 96 platinum electrodes 120a, 120b... Are formed by the electrode holding plate 120 in the same manner as the end of the capillary array 107 corresponding to the container-like recesses. It was arranged and fixed in a grid of × 12 rows. The electrode holding plate 120 is provided with holes 252a, 252b,... For passing capillaries near each platinum electrode. The sample 240 is put in the recesses 104a, 104b,. The electrode holding plate 120 is stacked on the upper part of the upper individual electrode tank 104 with the spacer 250 interposed therebetween, and the area / array holder 93 is stacked with the spacer 251 interposed therebetween. Each of the capillaries 107a, 107b,... Penetrates through the holes 252a, 252b,... Of the electrode holding plate 120 and is immersed in the sample liquid 240. Similarly, the platinum electrodes 120a, 120b,. A voltage is applied almost simultaneously between these electrodes and the electrodes in the lower electrode tank 106 to inject the DNA fragments in the sample 240 into the capillary. The voltage is set to an electric field intensity of 100 to 200 V / cm and applied for several seconds to 10 seconds. For example, a voltage of 5 kV is applied for 10 seconds. After the sample is injected, the upper individual electrode tank 104 is removed, and another upper individual electrode tank 104 containing a buffer solution is set instead, and electrophoresis is continued by applying an electric field strength of 100 to 400 V / cm, for example, 7 kV. Then, the molecular weight separation is performed. 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 there is no need 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.

  At the time of electric field application, as shown in FIG. 12, a mechanism was provided for simultaneously and continuously measuring current values flowing through individual capillaries individually and continuously. 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 flows from the electrode 209 → the buffer solution 245 → the capillary array 108 → the optical cell section → the individual capillaries 107a of the capillary array 107 → the buffer solution 246 in the container-like recess 104a → the electrode 120a → the resistor 131 → the terminal 260. Note that an optical cell portion 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 binarization means and transmitted to the current display panel 140. 12, the terminals 261 and 262 are also connected to the electrodes 120b and 120c, respectively, and 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. .. Are arranged on the current display panel 140 to display the current values of the capillaries 107a, 107b,. The resistor 131 has a resistance value sufficiently smaller than the resistance value of the capillary gel. Usually, there is no problem if it is 100 kΩ or less. The level of binarization can be adjusted appropriately. Normally, the electrophoresis 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. In this example, the capillaries are sub-arrayed in units of eight, so that only the unit having the damaged capillary can be replaced, and 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 unit 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. Although a housing and an optical window are also provided on the front surface and the rear surface of the paper in which FIG. 13 is described, they are omitted in the drawing. The sections of the housing 201 and the optical windows 204 and 206 are shown in cross section. The upper part of the optical cell 103 is open, and the capillary array 107 fixed to the linear array holder 214 is inserted into the optical cell along the array guides 202 and 205 and fixed from this part. 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, and the sheath liquid is formed below the capillary array 107. 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 to the upper part, and the liquid level 230 of the sheath liquid in the sheath liquid container and the liquid level 231 of the sheath liquid flowing into the cell are linked, and the height and the liquid level of the lower electrode tank 106 are adjusted. The sheath liquid flows in the cell due to a difference in height or the like.
The hollow capillary array 108 having an inner surface of 0.2 mm, an outer diameter of 0.35 mm, and a length of 3 cm which has been subjected to the same inner surface treatment as that of the first embodiment is used. The capillary array 107 inside the optical cell 103 was fixed at a distance of 2 mm from the end of the capillary array 107 so that the ends of the capillaries of the capillary arrays 107 and 108 face each other. The hollow capillary array 108 and the linear array holder 215 are fixed to the optical cell 103 and sealed so as not to leak water. Further, the optical cell 103 and the lower electrode tank 106 are closely adhered to each other with good airtightness, and the liquid remaining in the optical cell 103 is discarded from a discharge port 210 to a waste liquid container 213 via a valve 211 and a 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 is 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. However, 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 105 can be arranged at a position higher than the optical cell, and the configuration of the apparatus is simplified.

  The lower electrode tank 106 is provided with a platinum electrode 209 for voltage application and a terminal block 208 thereof. When using a gel capillary, a positive voltage is applied to the platinum electrode 209 (relative to the opposite electrode). For example, it is possible to apply 0 V to the platinum electrode 120 a and 7 kV to the platinum electrode 209. However, it is desirable to apply a voltage of −7 kV to the platinum electrode 120 a and 0 V (ground) to the platinum electrode 209 to ensure safety. This is because the entire sheath liquid has substantially the same potential as the voltage of the platinum electrode 209, and when 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, This is because the risk of electric shock, electric leakage, and insulation breakdown due to water leakage or the like increases. When the voltage of the electrode 209 is 0 V (ground), the sample side has a negative high voltage. However, since there is no flow of a buffer solution, there is no water leakage, and electric shock and electric leakage can be easily prevented. Will be able to In addition, 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, it may be disposed in an optical cell or a sheath liquid container, and has the same effect.

The laser beam 113 enters the optical cell through the optical window 204 or 206, and excites a DNA fragment that migrates in a sheath flow state as in the first embodiment to generate 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) from a direction perpendicular to the paper surface. The laser beam 113 is focused and radiated by a lens. However, by using a lens having a focal length of about 150 mm, the laser beam 113 can be uniformly radiated over the entire width of the capillary array (about 50 mm in this example). it can. It is also possible to irradiate the laser beam without being coaxial, but when coaxial, the two laser beams irradiate the same spot, so that the migration distance of the DNA fragment excited by each laser is constant. As a result, 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, thereby improving detection sensitivity. The measurement can be performed with only one laser.

  The detection of the fluorescent image will be described with reference to FIG. Fluorescence emitted from the fluorescent light emission point 220 is condensed by the condenser lens 114, divided into four by the image dividing prism 116, and formed into respective images (221a and 221b) by the imaging lens 117. At this time, by arranging the four types of spectral filters 115a to 115d on the front surface (or the rear surface) of the image splitting prism, it is possible to make the split light beams have different wavelength components. Although this has been described for one fluorescent light emitting point 220, 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 light 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 condensed in only 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 fluorescence emission position is shifted, the ratio of the intensity of the split image greatly changes. In the case of using a flat gel, there is a problem that the laser light incident on the gel is bent due to heat generation of the gel and the fluorescence emission position is 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, even in a large number of 96 migration lanes, the distance between both ends of the arrangement is about 5 cm or less. 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 spacing 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 with a diameter of 30 cm is required. Realistically difficult for a reason.

In the present embodiment, a two-dimensional detector having a width of about 27 mm was used for a capillary array having a width of about 50 mm. The image magnification as a whole is about 、, 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-collecting efficiency, it is desirable that the distance between the capillaries in the capillary array be 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, in the case of 96 fluorescent images, 96 fluorescent images in the horizontal direction and 4 fluorescent images in the vertical direction are obtained. In this way, all the samples that migrate through the capillary array can be simultaneously measured in four (color) divisions. 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, it is possible to identify and measure the primers A, C, G, and T. .

  FIG. 16 is a graph showing the fluorescence characteristics of the fluorescent-labeled primers and the transmission characteristics of the spectral filters. Thus, the spectral filters having the maximum transmittance near the maximum fluorescence wavelength of each fluorescent-labeled primer (however, the wavelength of the excitation laser light is transmitted) Measurement).

  FIG. 17 shows measured values of the time waveform of the intensity of the fluorescent signal emitted from a sample in which one gel capillary in the capillary array is migrated. It is a time waveform of the fluorescence intensity transmitted through the spectral filters 115a, b, c, and d from above. In this way, four types of fluorescence information can be obtained simultaneously from one sample / one capillary. However, as can be seen from FIG. 16, the fluorescence of the fluorescent-labeled primer is broad, and the fluorescence of the four fluorescent-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 a mixture of the fluorescence of the primer T and the fluorescent components of the primer G, the primer A, and the primer C. 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 the top as shown in FIG. Is obtained. These become the electrophoretic spectra of the T, G, A, and C fragments, respectively, from which the base sequence of the DNA can be determined by a usual method.

  In this example, 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.

  This embodiment also 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-piece prism is used. However, a two-piece prism is used, and the base type of the DNA can be determined by identifying the base type according to the method described in Japanese Patent Application Laid-Open No. 5-118991.

  In this example, as in Example 1, the capillaries of the capillary array were subjected to silane coupling treatment on the inner surface, 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.

FIG. 2 is a configuration diagram of an electrophoresis unit and a laser beam irradiation system of the electrophoresis apparatus according to the 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. 9 is a configuration diagram of a light irradiation system / detection system of an electrophoresis apparatus according to a third embodiment of the present invention. FIG. 9 is a configuration diagram near a gap of an electrophoresis apparatus according to a fourth embodiment of the present invention. FIG. 13 is a configuration diagram of an electrophoresis unit of an electrophoresis apparatus according to a fifth embodiment of the present invention. FIG. 14 is a perspective view of a flat plate having a plurality of grooves according to a sixth embodiment of the present invention. FIG. 14 is a diagram illustrating a fluorescent cell part of an electrophoresis unit of an electrophoresis apparatus according to a sixth embodiment of the present invention. FIG. 14 is a configuration diagram of a subarray of a capillary used in a seventh embodiment of the present invention. FIG. 17 is a configuration diagram of a capillary array used in a seventh embodiment of the present invention. FIG. 13 is a configuration diagram of an electrophoresis apparatus according to a seventh embodiment of the present invention. FIG. 14 is an enlarged sectional view of a sample injection part / upper electrode tank according to a seventh embodiment of the present invention. FIG. 17 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 diagram 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 diagram illustrating the principle of a configuration of a fluorescence detection / color separation unit in an electrophoresis apparatus according to a seventh embodiment of the present invention. FIG. 19 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. 19 is a diagram showing the fluorescence characteristics of the fluorescent-labeled primer used in the seventh embodiment of the present invention and the transmission characteristics of the spectral filter. FIG. 14 is a diagram showing an example of measured data of a time waveform of the intensity of a fluorescence signal emitted from a sample in which one gel capillary migrates according to a seventh embodiment of the present invention. 18 is a time waveform for A, C, G, and T fragments obtained by analyzing the data in FIG. FIG. 3 is a configuration diagram of a fluorescence measurement unit of a conventional capillary electrophoresis apparatus. FIG. 4 is a configuration diagram of a fluorescence measurement unit using a sheath flow cuvette of a conventional capillary electrophoresis apparatus.

Explanation of reference numerals

1a, 1b, 1c, 1d to 1t: Capillary, 2a, 2b, 2c, 2d to 2t: Capillary, 4: Fluorescent cell, 3a, 3b, 3c, 3d to 3t: Gap, 5a 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, 30 ... 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, 53 ... Lens, 54 ... One-dimensional sensor, 55 ... One-dimensional sensor controller, 5 ... Data processing device, 57 ... Monitor, 58 ... Printer, 59 ... Memory, 60 ... Fluorescent cell, 61a-61t ... Pore, 62 ... Plate, 63 ... Liquid reservoir, 64 ... Tube, 65 ... Electrode tank, 70 ... Seal Plate, 71: Tube, 80: Flat plate, 81a, 81b, 81c, 81d: Groove, 84: Fluorescent cell, 82a, 82b, 82c, 82d: Capillary, 83: Multi-capillary holder, 85: Sheath liquid inlet, 86 ... tube, 87 ... sheath liquid container, 88 ... sheath liquid, 90 ... capillary, 90a, 90b ... capillary end, 91, 92 ... capillary holder, 93 ... area array holder, 107 ... capillary array, 214 ... linear Array holder, 101: argon laser device, 102: YAG laser device, 103: optical cell, 104: upper individual electrode Vessels, 104a, 104b ~ ... container-like recess, 105 ... sheath liquid container, 106 ... lower electrode vessel, 107 ... capillary array, 107a, 107b ~ ... capillary, 108 ... hollow capillary array, 109 ... mirror, 110 ... dichroic mirror 111, argon laser light, 112, YAG laser light, 113, laser light, 114, condenser lens, 115a-d, spectral filter, 116, image splitting prism, 117, imaging lens, 118, two-dimensional detector, 119: data processing unit, 120: electrode holding plate, 120a, 120b: platinum electrode, 121: valve, 122: tube, 131: resistor, 132: voltmeter, 133: electrophoresis power supply, 140: current display panel, 140a, 140b ~ ... Indication lamp, 201 ... Housing, 202, 205 ... 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 215: Linear array holder, 220: Fluorescence emission point, 221a to 221d: Image formation, 230: Liquid surface of sheath liquid in sheath liquid container, 231: Liquid surface of sheath liquid flowing into cell, 240: Sample 245, 246: buffer solution, 250, 251: spacer, 252, 253: hole, 260, 261, 262, 270: terminal.

Claims (2)

A plurality of capillaries for electrophoretically separating the sample,
Near the end of the plurality of capillaries on the migration termination side, a flat plate having a plurality of pores,
The electrophoresis apparatus according to claim 1, wherein the plurality of pores are provided at positions facing ends of the plurality of capillaries on a migration end side.   2. The electrophoresis apparatus according to claim 1, wherein a sheath liquid for guiding the sample to the pores flows in a gap between the end on the migration termination side and the flat plate. .

Images