JP4078324B2 - Electrophoresis apparatus and capillary array - 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 detection of DNA (nucleic acid) and the like, analysis of DNA base sequence, and the like.

An example of an electrophoresis apparatus for separating and analyzing a fluorescently labeled sample by molecular weight by electrophoresis is a DNA base sequence determination apparatus using a fluorescent substance as a label. The nucleotide sequence is determined by the well-known dideoxy method of Sanger et al. Molecular weight separation is performed by electrophoresis using a polyacrylamide gel or the like. Usually, electrophoresis is performed using a polyacrylamide gel formed on a flat plate formed between two glass plates. Recently, however, a capillary gel electrophoresis method in which a gel (capillary gel) is prepared in a capillary for electrophoresis is used. Has been developed. In capillary gel electrophoresis, since the capillary diameter is generally small, the surface area per volume of the gel is large, and Joule heat can be easily dissipated, so that a high voltage can be applied and high-speed separation can be achieved. Is a useful method. As a first conventional example of capillary gel electrophoresis, “Nuclideic Acid Research”, Vol. 18, pages 1415-1419 (1990) (Nucleic Acid Research, 18, 1415-1419 (1990)) There is a method described. As shown in FIG. 19, a capillary with an inner diameter of 75 μm is used, and 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 capillary axis and the laser beam irradiation axis are tilted by about 25 degrees from the vertical direction, and the laser beam is condensed and irradiated to the central part of the capillary at a diameter of about 20 μm. Spectral detection is performed using a bandpass interference filter or the like. Note that FIG. 19 has been rewritten in an easy-to-understand manner based on the device diagram described in the prior art. Furthermore, as a second conventional example, “Journal of Chromatography”, Vol. 516, pages 61-67 (1990) (Journal of Chromatography, 516, 61-67 (1990)), 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 described in “Science”, Vol. 242, pages 562-564 (1988) (Science, 242, 562-564 (1988)). FIG. 20 shows a device configuration in which a laser light source, a sheath flow pump, and the like are added to the device diagram described in “Science” for easy understanding. For detection of DNA fragments, the molecular weight-separated separation liquid is introduced into a quartz flow chamber (inner shape 250 μm × 250 μm), and tris-borate buffer containing EDTA is used as a sheath liquid to flow through a liquid chromatography pump. In this state, the laser beam is condensed and irradiated to the separation liquid flowing in the substantially central part of the flow chamber to a diameter of about 10 μm, and the fluorescence from the DNA fragment is detected through a spectral filter or the like. In addition, as a method for determining a base sequence in one migration path, which is a third conventional example, for example, “Nature”, Vol. 321, pages 674-679 (1986) (Nature, 321, 674-679). (1986)) and the like, for each type of terminal base of the DNA fragment, phosphors having four different maximum fluorescence wavelengths (fluorescein, 4-chloro-7-nitrobenzo-2-oxa-1). -Labeled with diazole (NBD), tetramethylrhodamine, Texas red (product of Molecular Probes)), the fragments migrated in order of molecular weight are excited with laser light, and each fluorescence is detected by four types of bandpass interference filters. There is a method in which the nucleotide sequence is determined by separating and detecting fluorescence. As described above, in capillary gel electrophoresis, measurement is usually performed using only one capillary.
“Nuclideic Acid Research”, Vol. 18, pp. 1415-1419 (1990) (Nucleic Acid Research, 18, 1415-1419 (1990))

  Capillary electrophoresis apparatuses are desired to have high detection sensitivity as the amount of sample is reduced. Furthermore, it is desired to improve the processing capability, for example, increase the number of samples that can be processed simultaneously.

  Generally, in fluorescence measurement in an electrophoretic state, in addition to fluorescence from the target phosphor itself, as background light, scattering light (ex. Rayleigh scattering) of excitation light from the gel and fluorescence, gel support, that is, the inner wall of the capillary And scattered light on the outer wall or fluorescence from the capillary itself. For this reason, the background level becomes high and the detection sensitivity is lowered. That is, in order to achieve highly sensitive fluorescence detection, how to remove such background light is an important issue.

  In order to improve throughput, it is necessary to increase the migration speed, and to simultaneously migrate and measure multiple capillaries as 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 the fluorescence component, and the capillary axis as the laser beam irradiation axis and the fluorescence condensing. Higher sensitivity fluorescence detection is achieved by two measures of the second measure that is inclined by about 25 degrees from the perpendicular to the plane including the axis. However, with the first countermeasure, 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 although it is weak, and it is difficult to sufficiently remove the background light with a band-pass interference filter or the like. The second countermeasure is an effective method that can reduce the scattered light incident on the fluorescent light collecting lens itself by tilting the capillary. However, since the capillary has a circular cross section and a small diameter, it is scattered. Because the light intensity is high, the scattered light is radiated in all directions, and the fluorescence from the gel and the glass tube itself cannot be reduced by tilting the capillary. It is difficult to remove. From the above viewpoint, it is difficult to detect fluorescence with high sensitivity because the background light is not sufficiently removed. In this conventional example, there is one capillary, and there is no description or suggestion of a method for simultaneously processing a plurality of samples. It is conceivable to simply use a plurality of the devices shown in FIG. 19, but a plurality of laser light sources, detectors, optical components and the like are required, which makes the device 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 the capillaries are irradiated with light such as a laser beam, the capillary has a circular cross section, so that light is transmitted at the interface. Refraction / scattering occurs, and light passing through the capillary diffuses widely, so this configuration cannot be practically achieved. In other words, there is no problem with a single capillary, but when multiple capillaries are continuously irradiated, the intensity of light irradiated to the next capillary is extremely weak every time it passes through the capillary, making it difficult to measure fluorescence. become. From the above, it is difficult to apply this conventional example to simultaneous processing of a plurality of samples and improve the processing capability.

  The second conventional example is a method capable of effectively removing light scattered by the capillary and the 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 liquid is led out of the capillary gel and fluorescence measurement is performed under the sheath flow. Generation of light and fluorescence is eliminated. In the sheath flow chamber, the sample liquid flows almost in the center in a laminar flow state and does not come into contact with the inner wall of the sheath flow chamber, so that the scattered light from the sheath flow chamber and the fluorescence from the sample are separated spatially. And the fluorescence intensity can be detected with high sensitivity. However, in order to form a sheath flow, it is necessary to always flow the tris-borate buffer solution in the sheath flow chamber at a constant flow rate using a liquid chromatography pump or the like as a sheath solution. Therefore, there is a problem that the apparatus is expensive and complicated. Further, the sheath flow chamber has a complicated shape in which the sheath liquid injection part is thick and the fluorescence measurement part as the sheath flow part is thin, and it is difficult to manufacture the sheath flow chamber and it is expensive. Further, as in the first conventional example, there is no description or suggestion about simultaneous processing of a plurality of samples in this conventional example. It is not practical to simply use a plurality of devices shown 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 light refraction / scattering by the capillary. However, since there is a sheath flow chamber for each capillary, it is difficult to spatially narrow the gap between the capillaries, and as a result, the sheath flow chamber becomes large, and when multiple sheath flow chambers are used There is a problem that the apparatus becomes large and expensive. Furthermore, in order to irradiate all the sample liquid bundles flowing in the plurality of sheath flow chambers under the same conditions, the laser beam cannot be narrowed down. This is because the focal depth of the laser beam is related to the irradiation spot size, and when the irradiation spot size becomes smaller, the focal depth becomes shallower, whereas the interval between the sample liquid bundles flowing in the plurality of sheath flow chambers is wider. . From the above, it is difficult to apply this conventional example to simultaneous processing of a plurality of samples and improve the processing capability.

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

  In order to achieve the above object, in an electrophoresis apparatus in which a sample separation part provided between a cathode electrode tank and an anode electrode tank to which a voltage is applied by a power source is an electrophoresis path consisting of capillaries, (1) An optical cell in which the other end of each of a pair of capillaries or a plurality of capillaries connected to the cathode electrode tank or the anode electrode tank is arranged in the optical cell so as to face each other while maintaining a constant gap in the axis. Forming a migration path that passes through the capillary tube, injecting and flowing sheath liquid into the optical cell from the outside, and setting the sample to be migrated from the capillary end of the upstream migration path, which is the sample separation section, in a sheath flow state and facing the capillary In order to detect the sample by irradiating the optical detection unit with light from a light source, or (2) a plurality of cartridges whose one end is immersed in an electrode tank. The rally is terminated in the optical cell, and the sheath liquid is injected into the optical cell from the outside to flow, so that the sample migrating each capillary flows in the optical cell in the sheath flow state, and the sheath flow part is the optical detection part. The electrophoresis apparatus is configured to detect the sample by irradiating the optical detection unit with light from the light source. (3) The other end of a plurality of capillaries whose one ends are immersed in an electrode tank are held and fixed in an optical cell so as to be aligned in a straight line, and a sheath liquid made of an aqueous solution is placed in the optical cell. The sheath flow is formed in the vicinity of and under the end of each capillary, and the sample migrating through each capillary flows through the optical cell in the sheath flow state, and the injected sheath liquid is discharged from the optical cell and excited. The light from the light source part is irradiated almost directly in parallel with the straight line directly below the ends of the plurality of capillaries arranged in a straight line in the optical cell, and fluorescence is generated from the sample that flows out of each capillary, A plurality of generated fluorescent images are divided, and spectral filters are respectively arranged in front or rear of the dividing means so that the divided images have light components in different wavelength ranges. It is imaged to the image, constituting the electrophoresis apparatus for detecting the formed image.

  In the configuration (1), a plurality of optical detection units formed by a plurality of capillary pairs are arranged in an optical cell. In addition, a plurality of capillary pairs are arranged in an optical cell so that a plurality of optical detection units by a plurality of capillary pairs are positioned on a straight line, and excitation light is irradiated so as to irradiate all the optical detection units simultaneously. Irradiation is performed along this straight line, and fluorescence measurement is simultaneously performed by the plurality of optical detection units. In the configuration (2), the plurality of optical detection units formed by the plurality of capillaries are positioned on a straight line. The capillaries are arranged in an optical cell, and excitation light is irradiated along the straight line so as to irradiate all the optical detectors simultaneously, so that the fluorescence emitted from the sample migrating each capillary is simultaneously detected.

  In the configuration of (3), as in (1), the ends are held in a straight line at the same number or more as the electrophoresis separation capillaries and at the same intervals as the electrophoresis separation capillaries. A structure in which the aqueous solution is discharged from the optical cell through a hollow capillary disposed in the vicinity of a plane including the capillary end and the laser beam irradiation axis may be employed. A structure is used in which images from a plurality of samples that migrate through each capillary are divided using a polyhedral prism. The image may be divided into four and each may be detected simultaneously. The fluorescence images from the samples respectively migrating through the plurality of capillaries or the divided fluorescence images are formed on the detector at the same time, and their intensities are measured simultaneously. Fluorescence emitted from a sample may be divided and imaged after being collected by a lens, or may be detected after being condensed and imaged in one direction by a cylindrical lens.

  In the configurations of (1), (2), and (3), each unit may be configured as follows. The sample separation part is composed of a capillary gel, and the sheath liquid is made of the same component as the buffer solution inside the capillary, and can also contain a sample denaturing agent. Further, the means for holding a plurality of capillaries and the optical cell can be detachable, and an insertion guide for holding the capillary holder at a specified position can be provided inside the optical cell. The injection of the aqueous solution into the optical cell is performed by providing a sheath liquid container containing the aqueous solution outside the optical cell and placing the liquid surface at a position higher than the surface of the liquid drained from the optical cell, or Using an optical cell with an open top, the 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 the drainage port in the optical cell. It may be arranged and performed by a drop. 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 for detecting the liquid level in the optical cell, and a mechanism for supplying the aqueous solution in the sheath liquid container via a valve when the liquid level is lowered. The optical detection unit measures fluorescence or light absorption of the sample. Further, the excitation light source unit may be a laser device, or at least two types of laser devices, and these laser beams may be coaxially combined into one. The resulting image such as fluorescence can be detected by a two-dimensional detector. In addition, the sample is injected into each of the plurality of capillaries simultaneously. The electrode on the downstream side of electrophoresis can be not only inside the container from which the sheath liquid is discharged, but also inside the optical cell or inside the sheath liquid container. The electrode on the downstream side of the migration has a potential of 0 V (ground), or the absolute value thereof is smaller than the absolute value of the potential on the other migration upstream side. Also, a mechanism may be provided in which the level of the aqueous solution accumulated in the electrode tank on the downstream side where the sample is migrated is made substantially constant, and the excess aqueous solution is drained into the tank. Further, the ratio of the length of both ends of the fluorescence emitted from the sample running through a plurality 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. In addition, it is desirable that the distance between adjacent capillaries is 10 times the capillary size or less. In electrophoresis, it is desirable to have a structure in which the number of electrodes on the downstream side on which the sample is migrated is one and the number of electrodes on the sample injection side matches the number of capillaries for electrophoresis separation. Furthermore, a mechanism is provided for measuring and displaying the value of the current flowing in the electrophoresis separation capillary for each capillary. The current values may be binarized and displayed. The electrophoresis separation capillary is silanized at least on the inner surface in the vicinity of the end held inside the optical cell and filled with a gel so that at least the gel in the vicinity of the end held in the optical cell. May be chemically bonded to the inner wall of the capillary.

  In the configuration (1) or (3) of the electrophoresis apparatus, the other end of one or more pairs of capillaries each having one end connected to the cathode electrode tank or the anode electrode tank is connected to the axis of the optical cell in the optical cell. Since they are arranged so as to be substantially coincident with each other while maintaining a certain gap, an electrophoresis path penetrating a part of the optical cell can be formed. Since the sheath liquid is injected into the optical cell from the outside and flowed, the sample migrated from the capillary end of the upstream migration path, which is the sample separation unit, can be guided to the opposing capillary in a sheath flow state. The sample can be continuously electrophoresed between the capillary and the downstream capillary, and the sample can be reliably electrophoresed only in the axial gap between the upstream capillary and the downstream capillary. . Using this gap as an optical detection unit, light irradiation from the light source to the optical detection unit 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 by the capillary or capillary gel can be avoided, and highly sensitive fluorescence or light absorption measurement can be performed. In this way, by aligning the plurality of capillaries in the optical cell so that the axes substantially coincide with each other while maintaining a certain gap, the capillary on the upstream side that is the sample separation part and the gap part that is the optical detection part can be easily provided. Can be configured. Moreover, by arranging a pair of capillaries on the downstream side, the sample migration path can be accurately defined, light irradiation from the light source can be made easily and reliably, and light such as fluorescence can be easily received. And can be sure. In addition, the sheath liquid and the sample liquid flow out of the optical cell only through the inside of the downstream capillary. However, since the capillary has a small diameter, the flow rate of the sheath liquid injected into the optical cell 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, regardless of the dimensions of the optical cell, the cross-sectional area inside the sheath liquid injection part is made thicker as in a normal sheath flow chamber, and the cross-sectional area of the sheath flow part is increased. Therefore, it is not necessary to adjust the optical cell so as to make it thinner, and a rectangular optical cell can be used. Therefore, the optical cell can be manufactured easily and inexpensively. In this way, a plurality of optical detection units formed by a plurality of capillary pairs can be arranged close to each other in the optical cell, and the apparatus can be reduced in size. Since only one optical cell and one pipe for forming the sheath flow are required, the apparatus can be configured easily and inexpensively. In addition, the sample migrated from each capillary is in a sheath flow state for each capillary when passing through the optical detection unit, and the flow of the sheath liquid is all 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, the cross-sectional area thereof becomes large. However, since the sheath liquid flows out through each capillary on the downstream side, the effective cross-sectional area through which the sheath liquid flows is The total inner diameter area 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 apparatus can be downsized.

  In one optical cell, a plurality of pairs of capillaries are arranged in a line so that a plurality of optical detectors are positioned on a straight line, and excitation light is irradiated along this straight line. Irradiation can be performed simultaneously, and fluorescence measurement using a plurality of optical detection units can be performed simultaneously. In addition, since a plurality of optical detection units are connected to each other via a liquid, the excitation light can irradiate each optical detection unit without being scattered by a capillary or the like, and the excitation light can be efficiently applied to each optical detection unit. The fluorescence can be detected with high accuracy. If a two-dimensional TV camera or the like is used as the light detector, fluorescence images of a plurality of optical detection units can be received simultaneously. In a single optical cell, a plurality of capillary pairs are aligned and arranged so that a plurality of optical detectors are positioned in a straight line, and the intervals between the plurality of capillary pairs can be made close to each other. In addition to being able to be configured at low cost, the length between the optical detection units at both ends on the straight line can be shortened. If the length between the optical detection units at both ends is shortened, all the optical detection units can be irradiated with substantially the same light beam diameter even if the excitation light is narrowed down with a lens or the like. For example, when the laser beam is focused to about 100 μm in the focal position, the laser beam diameter is about 100 μm over about 10 mm before and after that. When capillaries having an outer diameter of 200 μm and an inner diameter of 100 μm are arranged every 400 μm, about 50 capillaries 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 with the excitation light narrowed down, the fluorescence intensity can be increased, and the sample can be detected with high sensitivity.

In addition, the sample separation part is composed of a capillary gel, so that molecular weight separation can be easily performed.
In addition, the downstream capillary can be a hollow capillary, allowing sheath fluid or the like to flow efficiently. On the downstream side, not the capillaries themselves but those capable of performing the same operation, for example, a plate formed with holes and grooves corresponding to the number of upper capillaries can be used. By making the sheath liquid the same component as the buffer solution inside the capillary, it is possible to ensure the flow of electricity in the gap, that is, the optical detection section, and to enable electrophoresis of the sample. Furthermore, since the composition of the buffer solution inside the capillary is the same as that of the external buffer, it can be prevented that the buffer solution inside the capillary flows into the optical cell and the composition fluctuates. No damage. When the sample is single-stranded DNA or the like, a denaturant can be included in the sheath liquid. In this case, the sample DNA can be prevented from recombining when migrating through the gap portion, that is, the optical detection portion, and the measurement accuracy is improved. The possibility of the denaturant trapped inside the capillary leaking into the optical cell is reduced, and the sample separation ability in electrophoresis is not impaired.

  In addition, the sheath liquid is injected into the optical cell by allowing the sheath liquid to flow easily by a drop by disposing the liquid level of the sheath liquid in the sheath liquid container at a position higher than the liquid level in the downstream electrode tank. Therefore, it is not necessary to mechanically flow the sheath liquid, and the apparatus configuration is simple and inexpensive. Furthermore, since the pulsating flow generated when using a pump or the like is eliminated, a stable sheath flow is possible and the measurement accuracy is increased. The flow rate of the sheath liquid can be easily adjusted by changing the drop between the liquid level of the sheath liquid in the sheath liquid container and the liquid level in the downstream electrode tank. The optical cell can be easily attached by using a cell having a shape that is sealed when a capillary is attached and a shape having an open top. At this time, the same effect as described above can be obtained by disposing the liquid level of the sheath liquid container at a position higher than the drain outlet in the optical cell. In addition, a mechanism that detects the liquid level in the optical cell and a mechanism that replenishes the aqueous solution in the sheath liquid container via a valve when the liquid level drops to control the liquid level in the optical cell to be almost constant. In addition, a stable sheath flow with little pulsating flow can be achieved, and the apparatus can be configured simply and inexpensively.

The optical detection unit can measure either fluorescence or light absorption of the sample. The gap distance of the gap is not particularly specified, but is preferably about 0.1 mm to 3 mm. In general, the shorter the gap distance of the gap portion, the easier it is for the sample to migrate in the gap space, so basically a shorter gap length is preferred. However, when assembling the apparatus, if the gap length is too short, it becomes difficult to adjust the gap length. However, it can also be set to 0.1 mm or less, and the limit is determined by the width of the excitation light beam such as laser light in the gap. Conversely, if the gap distance of the gap is increased, the sample is likely to diffuse.
If the gap length was about 3 mm, it was confirmed that the sample migrated normally. That is, by setting the gap length to about 0.1 mm to 3 mm, the sample can be simply and efficiently electrophoresed. Also, by holding the capillary pair inside the optical cell and performing electrophoresis, the sample migrates on the line connecting the capillaries and does not come into contact with the inner surface of the optical cell. The influence of the scattered light can be removed, and the detection sensitivity can be improved.

  In addition, images from multiple samples that migrate through each capillary are divided using a polyhedral prism, etc., and a spectral filter is used so that each passes light components in different wavelength ranges. Can get to. In particular, if the image is divided into four, which is the number of DNA base species, each base species can be labeled with a separate phosphor to efficiently determine the DNA base sequence.

  Fluorescent images from samples that migrate through multiple capillaries or divided fluorescent images are simultaneously imaged on a two-dimensional detector, and the intensity is measured at the same time, allowing 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 in the specified position, and multiple capillaries and the optical cell can be attached and detached, improving the operability of attaching and handling capillaries. To do.

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

  By providing electrodes equal to the number of capillaries for electrophoretic separation, immersing the capillaries and electrodes in a plurality of sample solutions, and simultaneously applying a voltage to inject samples into each of the plurality of capillaries. The sample can be injected easily and in one operation, and the time can be shortened and the operability can be improved as compared with the case where it is performed in a single unit as in the prior art. The value of the current flowing through the inside can be measured for each capillary. The value of the current flowing inside the capillary is effective to know the state of the capillary, and for example, it is possible to predict in advance a separation failure due to gel breakage or the like. The current value can be displayed by arbitrarily setting the binarization level and binarizing the current value so that the lamp lights up when the current is below the normal electrophoresis current. In addition, it is possible to efficiently determine the necessity of exchanging and re-measuring gel capillaries, thereby improving usability.

  Furthermore, the distance between adjacent capillaries held inside the optical cell is set to 10 times the capillary size or less, and the overall image magnification of the fluorescent image to be detected is set to 1/3 times or more, thereby receiving light compared to the plate gel method. 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 whose one ends are immersed in an electrode tank are terminated in the optical cell, and sheath liquid is injected into the optical cell from the outside. The sample to be migrated through each capillary flows through the optical cell in a sheath flow state, and the sheath flow part is used as an optical detection part, and in the same manner as in the configuration (1) of the electrophoresis apparatus, Samples that migrate through the capillaries can be separately measured, and the same effect as in the configuration (1) of the electrophoresis apparatus can be obtained. Other related operations described regarding the configuration (1) of the electrophoresis apparatus are the same as those of the configuration (2) of the electrophoresis apparatus.

  According to the present invention, an electrophoretic apparatus capable of 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. In addition, a simple electrophoresis apparatus that can simultaneously migrate and measure a plurality of samples can be realized. In addition, it is possible to realize an apparatus that has high operability and safety and can determine the base sequence of DNA with high throughput.

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

[Example 1]
In this example, a fluorescently labeled DNA fragment is separated by molecular weight by electrophoresis and detected by fluorescence. The case where fluorescein isothiocyanate (FITC) is used as the fluorescent substance for labeling will be described. As a fluorescently labeled DNA fragment as a sample, a part of the DNA fragment 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 template single-stranded DNA 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. By the above operation, fluorescent-labeled DNA fragments having various lengths whose ends are A are obtained. This is used as a sample.

  First, the apparatus configuration will be described. FIG. 1 shows a configuration diagram of an electrophoresis unit and a laser beam irradiation system of the electrophoresis apparatus of this 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 sample separation units are arranged to measure a plurality of samples simultaneously. Twenty silica capillaries 1a, 1b, 1c, 1d,..., 1t are used as the sample separation unit. These are the same capillaries made of silica each having an inner diameter of 100 μm, an outer diameter of 375 μm, and a length of 40 cm. Further, 20 silica capillaries 2a, 2b, 2c, 2d,..., 2t having the same inner and outer diameters and having a length of 10 cm are used. For the capillaries 1a to 1t, capillary gels filled with polyacrylamide gel containing urea as a denaturant are prepared. First, the inside of the capillary is washed and subjected to silane coupling treatment. Next, degassed 3.84% acrylamide, 0.16% bisacrylamide, 7M urea, Tris containing 2mM EDTA, tetramethylethylenediamine and ammonium persulfate solution were added to borate buffer and injected into the capillary. Polymerize to make an acrylamide gel. Since the capillary is silane-coupled, the acrylamide gel and the capillary are chemically bonded, and the gel does not protrude from the capillary 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. so that the capillary inner surface is aminosilanized to have a positive charge. In this way, the direction of electroosmotic flow in each of the capillaries 2a to 2t is changed from the negative electrode to the positive electrode, and the sample migration direction (negative electrode → positive electrode) in the capillaries 1a to 1t filled with acrylamide gel The moving direction of the sample in the capillary 2 (negative electrode → positive electrode) coincides, so that sample migration 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, since the sample is detected by fluorescence, a fluorescence cell is used as the optical cell. That is, one end of each of the capillaries 1a to 1t and 2a to 2t is connected to a rectangular quartz fluorescent cell 4 (outer shape: width 36 mm × depth 4.5 mm × length 3 mm, inner shape: width 30 mm × depth 2 mm × length 3 mm). ,: The width is the horizontal direction in the figure (capillary 1a → 1t arrangement direction), the depth is the vertical direction with respect to the paper surface of the figure, and the length is the vertical direction in the figure (the direction of sample migration 1 → 2 of each capillary)) To place. The arrangement method is such that the capillary 1a and the capillary 2a are coaxial with each other, and a gap portion 3a having a length of 1 mm is formed to face each other. Similarly, the capillaries 1b and 2b, 1c and 2c, 1d and 2d,..., 1t and 2t are arranged so as to form the same gap portions 3b, 3c, 3d. For fixing the capillaries in the optical cell, multicapillary holders 5a and 5b having 20 vertical holes at 0.6 mm intervals in a flat block made of a fluororesin, for example, ethylene tetrafluoride resin, are used. That is, one capillary 1a to 1t is inserted into 20 vertical holes of the multi-capillary holder 5a, and one capillary 2a to 2t is inserted into 20 vertical holes of the multi-capillary holder 5b. 2a, 1b and 2b, 1c and 2c, 1d and 2d..., 1t and 2t are fixed in close contact with the upper and lower portions of the fluorescent cell 4 so that the multicapillary holders 5a and 5b are coaxial. Further, since the gaps 3a to 3t are optical detection units, here, fluorescence detection units, the capillaries 1a to 1t in the quartz fluorescent cell 4 and the gaps 3a to 3t and the gaps 3a to 3t are aligned on a straight line. The lengths 2a to 2t are adjusted. The other ends of the capillaries 1a to 1t and capillaries 2a to 2t are immersed in a cathode side electrode tank 6 and an anode side electrode tank 7 containing a buffer solution (buffer solution containing Tris, boric acid, EDTA, and urea). The fluorescent cell 4 is provided with a sheath liquid injection port 8 for filling the inside thereof with a sheath liquid, and the sheath liquid 11 in the sheath liquid container 10 can be injected through a tube 9 made of ethylene tetrafluoride resin. It has a structure. As the sheath liquid, a buffer solution containing tris, boric acid, EDTA, and urea is used, and the same components as the buffer solution in the capillary gel of the capillaries 1a to 1t are used, and the constituent 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 1 a to 1 t and the capillaries 2 a to 2 t 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 side electrode tank 7. By disposing the liquid higher than the liquid level, 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, and the cathode side electrode tank 6 and the anode side electrode tank 7 By applying a direct current voltage between the direct current high voltage power supply 12 between them, for example, a current flows so as to penetrate the capillary 1a, the gap 3a and the capillary 2a, and the sample can be electrophoresed. In addition, by disposing the liquid level of the sheath liquid 11 in the sheath liquid container 10 higher than the liquid level of the buffer liquid in the anode-side electrode tank 7, the sheath liquid flows inside the capillaries 2a to 2t. A flow is generated around each of the capillaries 2a to 2t, that is, the gaps 3a to 3t. Therefore, the sample migrated from the capillaries 1a to 1t passes through the gaps 3a to 3t in a sheath flow state along the flow of the upper portions of the capillaries 2a to 2t, and is guided to the capillaries. 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 is directed from the capillaries 1a to 1t to the anode side electrode tank 7, and the liquid flowing into the gaps Therefore, even when the flow rate of the sheath liquid is small, the sheath liquid can be stably flowed in the direction of the anode side electrode tank 7.

Electrophoresis is performed by applying a DC voltage from a DC high voltage power source 12 between the cathode side electrode tank 6 and the anode side electrode tank 7. When a voltage is applied, the currents flowing through the capillaries 1a and 2a mainly flow around the gap 3a. Similarly, the currents flowing through the capillaries 1b and 2b, 1c and 2c, 1d and 2d,. The gaps 3b, 3c, 3d, ... flow around 3t. Further, as described above, the sample migrated from the capillaries 1a to 1t passes through the gaps 3a to 3t in a sheath flow state along the flow above the capillaries 2a to 2t, and is guided to the capillaries. . That is, the sample that migrates in each capillary and the gap can be migrated without contacting the sample that migrates in the adjacent gap, and the sample that migrates in each gap can be separated to measure fluorescence. In addition, since the sample does not come into contact with the inner surface of the fluorescent cell 4, there is no influence of the sample adsorbing to the fluorescent cell, and the scattered light on the fluorescent cell surface can be spatially removed by a slit, etc. Fluorescence measurement becomes possible.
The fluorescently labeled DNA fragment as the sample is introduced by temporarily immersing the end of the cathode side capillary 1a in the sample solution and applying a voltage of 6 kV between the sample solution and the anode side electrode tank 7 for about 20 seconds. . Thereafter, the end of the capillary is returned to the original cathode side electrode tank 6. This operation is performed for each of the capillaries 1b to 1t, and a sample is injected into the capillary gel of the capillaries 1a to 1t. Note that the sample may be injected one by one as described above, or by immersing the capillaries 1a to 1t in the sample solution and simultaneously applying a voltage. The molecular weight separation of the sample by electrophoresis is performed by applying a DC voltage of 6 kV between the cathode side electrode tank 6 and the anode side electrode tank 7. The sample injected into each of the capillaries 1a to 1t is migrated in the capillary gels 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 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 gap 3a in which the laser beams are aligned on a straight line. The laser light axis is adjusted so as to irradiate .about.3t at the same time and under substantially the same conditions, and the emitted fluorescence is measured. That is, 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, a lens 22 having a focal length of 100 mm is used, and focusing is performed in 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 in this example is 0.6 mm × 19, that is, about 12 mm. That is, the laser light irradiates 20 portions of the gaps 3a to 3t with substantially the same spot size, and the spot size is almost equal to the thickness (100 μm) of the capillary gel. As described above, by selecting the light source and the lens system so that the spot size of the laser beam can be made approximately the same as the thickness of the capillary gel, and all the gaps are irradiated with almost the same spot size. The fluorescently labeled DNA fragments that migrate through the gaps 3a to 3t can be excited uniformly and efficiently.

  The fluorescence from the fluorescence-labeled DNA fragments that migrate through the gaps 3a to 3t is detected from the direction perpendicular to the laser beam irradiation direction. The configuration diagram is shown in FIG. The fluorescence 30 emitted from the DNA fragment removes background light such as scattered light with an interference filter 32 and forms an image with a lens 33 on a two-dimensional detector 34 such as a CCD camera. The two-dimensional detector 34 is controlled by the controller 35 to detect the fluorescence images of the gaps 3a to 3t, and the data processor 36 such as a computer continuously changes the fluorescence intensity over time for each of the gaps 3a to 3t. In addition, all the gaps are measured simultaneously, and the results are displayed on the monitor 37, output to the printer 38, and stored in the memory 39. Thereby, it is possible to simultaneously and continuously measure the migration pattern separated by molecular weight for each capillary of 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. The interference filter 32 is a band-pass interference filter that passes through a wavelength range of 500 nm to 540 nm in order to effectively detect the fluorescence from FITC. Further, the image magnification of the lens 33 is set so that all the images of the gaps 3a to 3t are 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 a capillary or the like. Therefore, it is possible to irradiate the sample migrating a plurality of capillaries simultaneously, uniformly and efficiently. In addition, background light such as scattered light and fluorescence due to capillaries or capillary gel can be greatly reduced, and fluorescence can be detected with high sensitivity. As an effect of reducing the background light, for example, the fluorescence measurement is performed at the gap portion as in this embodiment, compared with the case where fluorescence is detected by irradiating the capillary itself with the coating removed without forming the gap portion. If it does, the detected background light intensity will be about 1/10 or less, and a sample with a lower concentration can be detected. Further, by aligning the plurality of gaps on a straight line, all the gaps can be simultaneously irradiated with laser light, and a simple apparatus configuration can be obtained. Further, since a plurality of capillary pairs can be held in one optical cell, only one optical cell is required, and only one system of piping is required for injecting sheath liquid. Further, since the sheath flow occurs only in the vicinity of the gaps, the flow velocity in each gap is almost the same in all the gaps, and the reproducibility between the gaps is high. Therefore, there is little variation between the migration paths, and an optical cell having a simple structure as in this embodiment is sufficient, and it is necessary to use an optical cell with a complicated shape like a normal sheath flow chamber. As a whole, the apparatus configuration is simple. In this example, the interval between adjacent capillaries was set to 0.6 mm. In this way, by facing the capillary pair 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 miniaturized. This also makes it possible to irradiate the gap portion by narrowing the laser beam more narrowly. Conversely, more capillary pairs can be held in the same optical cell. According to this embodiment, since the molecular weight separation unit is a capillary gel similar to the conventional one, the sample can be detected with high sensitivity without impairing the molecular weight separation characteristics.

  Further, according to the present embodiment, the buffer solution can be flowed without using mechanical means such as a liquid chromatography pump, so that the apparatus configuration becomes simple and inexpensive. In addition, since there is no pulsating flow that occurs when using a pump, etc., the flow of the sheath liquid becomes stable, fluctuations in the flow velocity are reduced, fluctuations in the fluorescence intensity of the sample flowing through the gap are suppressed, and measurement accuracy is increased. 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 liquid in the anode side electrode tank on the downstream side of migration. 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 mechanical means such as a liquid chromatography pump. 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 the data processing.

  The sheath liquid inside the optical cell flows out of the cell through the downstream capillaries 2a to 2t. Capillaries generally have a narrow inner diameter, so the flow rate through them is generally less. Therefore, the amount of sheath liquid can be reduced and operability is improved. Further, in this embodiment, the capillaries 2a to 2t are 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 to the anode side electrode tank 7. In this way, there was no back flow of the liquid from the capillaries 2a to 2t to the gap. Thus, even when the flow rate of the sheath liquid is small, the sheath liquid can flow stably toward the anode-side electrode tank 7. In addition, when the flow velocity in the gap portion of the sheath liquid is large, such as when the inner diameters of the capillaries 2a to 2t are large, the effect of the electroosmotic flow is reduced, so that the inner surfaces of the capillaries 2a to 2t are necessarily processed. There is 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 tank and the cathode side electrode tank, and the same components as the buffer solution of the capillary gel were used. As a result, the constituent components of the capillary gel do not leak into the fluorescent cell 4 or the electrode tank, and electrophoresis that is more stable and has good resolution can be continued including the reuse of the capillary gel. It is also possible to use a buffer solution that does not contain urea, which is a DNA denaturing agent. In this case, urea in the capillary gel may leak from the capillary gel into the electrode tank or fluorescent cell over time, and the number of repeated uses will be somewhat reduced, but the same migration as in the case of a buffer solution containing urea. Is possible.

  Note that the laser device and phosphor used are not limited to argon laser and FITC, and any phosphor and appropriate laser device can be used. In this example, measurement of DNA fragments has been described as an example, but it can also be used for analysis of proteins, sugars and the like. In the present embodiment, both capillaries having the same inner diameter are used, but capillaries having different inner diameters can be combined. For example, if the inner diameter of the downstream capillary is made thinner than the inner diameter of the upstream capillary, the sample migrated from the upstream capillary end is throttled when introduced into the downstream capillary, so the concentration of the sample liquid is high. Therefore, it can be detected with higher sensitivity. Further, if the inner diameter of the downstream capillary is made larger than the inner diameter of the upstream capillary, the sample migrated from the upstream capillary end can be more easily and reliably guided to the downstream capillary. Furthermore, according to this embodiment, since the fluorescence from the sample can be measured without passing the excitation light through the capillary portion, the capillary need not be transparent. That is, since it is not necessary to remove the capillary coating, handling becomes easy. Furthermore, it is possible to use capillaries of various materials such as capillaries made of opaque fluororesin such as tetrafluoroethylene resin or trifluoride ethylene chloride resin. Since the fluororesin capillary has little sample adsorption, it does not require a processing operation such as a surface treatment and is not damaged, so that the operability is improved. Moreover, since it is excellent in chemical resistance, it becomes possible to use a solvent in a wide pH range. In the present embodiment, the case of a plurality of capillary pairs has been described. Similarly, when only one capillary pair is used, it is possible to migrate, detect fluorescence, and measure the separation pattern of the sample. 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 dideoxy method of Sanger et al. As a primer, a FITC-bound primer (labeled primer) is used. First, a labeled primer is added to the single-stranded DNA, annealed (double-stranded formation), and the labeled primer is bound to the single-stranded DNA. This reaction solution is divided into four, and DNA polymerase reactions corresponding to A, C, G, and T are performed on each. That is, four kinds of deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP) and ddATP serving as a terminator are added to a single-stranded DNA to which a labeled primer is bound to cause a polymerase reaction. By this reaction, fluorescently labeled DNA fragments having various lengths A are obtained. The same reaction is performed for C, G, and T. The four A, C, G, and T reaction liquids obtained as described above are injected into four of the capillaries 1a to 1t, for example, the capillaries 1a, 1b, 1c, and 1d. The injection method is the same as in Example 1. The reaction solution A is injected into 1a, the reaction solution C into 1b, the reaction solution G into 1c, and the reaction solution T into 1d. After the injection, electrophoresis is performed by applying a voltage of about 6 kV. Using an argon laser beam having a wavelength of 488 nm as excitation light, the temporal change in fluorescence intensity in each of the gaps 3a to 3d is measured. Since DNA fragments are migrated in ascending order of molecular weight, the base sequence can be analyzed by analyzing the gap positions where the fluorescence peaks occur in time order.

  The apparatus of Example 1 has 20 capillaries into which a sample can be injected. According to the above DNA base sequence determination method, the base sequences of five types of DNA samples can be determined simultaneously. If the number of capillary pairs held in the optical cell is increased, the base sequences of more DNA samples can be determined. In this embodiment, one type of FITC is used as the phosphor. However, it is also possible to detect fluorescence from two or more phosphors at the same time. In that case, for example, as is well known, a set of photodetecting devices including the interference filter 32, the lens 33, and the detector 34 in FIG. Alternatively, a dispersive prism may be arranged after the lens 33 for spectroscopic analysis, imaged on a two-dimensional detector such as a camera, and received, and fluorescent for each migration path and each wavelength. The intensity may be measured, and the fluorescence intensity for each migration path and each phosphor may be calculated.

  As described above, the DNA base sequence can be determined even when the apparatus configuration is such that a plurality of phosphors can be measured simultaneously. That is, for each type of terminal base, a primer labeled with a different phosphor is used, and after each DNA polymerase reaction is performed, the reaction solution is mixed and electrophoresed. By detecting the fluorescence of the DNA fragment passing through the gap space and identifying the type of the phosphor at that time, the base type can be identified and the base sequence can be determined. The type of phosphor can be identified by comparing the fluorescence intensities of the four phosphors at the maximum fluorescence wavelength. In this case, the base sequence of the DNA sample can be determined by an electrophoresis 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, the base sequences of a plurality of DNA samples can be determined simultaneously.

Example 3
In the first embodiment and the second embodiment, the apparatus for detecting a sample by fluorescence measurement has been described. However, the apparatus is similarly configured in the case of light absorption measurement such as measurement of absorbance and measurement of transmitted light intensity. be able to. FIG. 3 shows a configuration diagram of the light irradiation system / detection system portion of the electrophoresis apparatus in the case of light absorption measurement. The electrophoresis part has the same configuration as that of FIG. A sample, for example, a restriction enzyme-cleavage fragment of DNA is introduced in the same manner as in Example 1, and electrophoresis for molecular weight separation is also carried out in the same manner as in Example 1. Light from a light source 50 such as a xenon lamp or a D2 lamp passes through the monochromator 51 and is condensed by a lens 52 to irradiate the gaps 3a to 3t with respect to the DNA fragments passing through the gaps 3a to 3t. The light wavelength for irradiation is usually set to the absorption wavelength of the sample. For example, about 260 nm is appropriate for DNA fragments. The light that has been absorbed and transmitted by the DNA fragment is again collected by the lens 53 and detected by a 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. Is output and stored in the memory 59. In this embodiment, the gap is provided between the capillaries, the capillary is separated from the molecular weight separator, and the gap space is separated from the optical detector for measuring the absorbance, so that the irradiation light is transmitted through the sheath liquid with very little light scattering. Therefore, the sample can be efficiently irradiated without being scattered by a capillary, a gel, or the like, and highly accurate absorbance measurement can be performed. Specifically, if the transmitted light intensity (when the sample does not pass through and the numerical aperture of the light receiving lens is 0.19) when light is incident on the gap as in the present embodiment is 1, When the light is transmitted through the capillary as in the prior art, 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 molecular weight separation by electrophoresis, are light scatterers, so that incident light is scattered at that portion, resulting in a decrease in transmitted light. . Decreasing the transmitted light intensity means that the S / N of the photodetector or the like is reduced accordingly, 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 detected, but these lights are not directly irradiated to the sample (without being absorbed by the sample). Incident. If there is a light component that is detected regardless of the presence of such a sample, it is difficult to measure a minute change in light intensity, that is, it is difficult to measure the absorbance with high accuracy. In this embodiment, the light that has passed through the monochromator 51 is condensed and irradiated on the gap portion by the lens. However, the light that has passed through the gap portion is irradiated as almost parallel light without being condensed. It is possible to measure in the same way by spatially separating only the light and measuring the light intensity.

Example 4
In the first to third embodiments, the gap portion is formed by the capillary pair, but the gap portion is not formed only by the capillary pair. For example, the molecular weight separation part may be a capillary as in the above embodiment, and the gap part may be formed by this capillary end and a flat plate having a pore. FIG. 4 shows a configuration diagram of the gap. As in Example 1, the 20 capillaries 1a to 1t, which are molecular weight separators, are held by 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 facing each of the capillaries 1a to 1t, gaps are formed, and by flowing the sheath liquid, the sample migrated from each capillary can be guided to the corresponding pores. it can. A liquid reservoir 63 is disposed below the flat plate 62, and the solution flowing from the pores is temporarily stored and guided to the electrode tank 65 through 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 instead of the downstream capillary and the sheath liquid is allowed to flow for migration. FIG. 5 shows a configuration diagram of the electrophoresis unit of the electrophoresis apparatus. Similarly to the first embodiment, one end of each of the capillaries 1a to 1t is held in the optical cell 4 with the respective tips aligned. The capillary is fixed in the optical cell by using a multi-capillary holder 5a in which 20 vertical holes are provided at intervals of 0.6 mm in a flat block made of a fluorine resin, for example, ethylene tetrafluoride resin. That is, the capillaries 1 a to 1 t are inserted one by one into the 20 vertical holes of the multi-capillary holder 5 a, and the multi-capillary holder 5 a is brought into close contact with the upper portion of the fluorescent cell 4 and fixed. The lower part of the fluorescent cell 4 is connected to a seal plate 70 and a tube 71 made of ethylene tetrafluoride resin, so that 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. Like that. The fluorescent cell 4 is provided with a sheath liquid injection port 8 for filling the inside thereof with a sheath liquid, and the sheath liquid 11 in the sheath liquid container 10 can be injected through a tube 9 made of ethylene tetrafluoride resin. And In addition, by placing the liquid level of the sheath liquid 11 in the sheath liquid container 10 higher than the liquid level of the buffer liquid in the anode electrode tank 7, the sample migrated from the capillaries 1a to 1t is wrapped in the sheath liquid. The inside of the fluorescent cell 4 can be flowed. These solutions are finally led to the anode electrode tank 7 side. Electrophoresis is performed by applying a DC voltage from a DC high voltage power source 12 between the cathode side electrode tank 6 and the anode side electrode tank 7. Samples migrated through the capillaries migrate in the optical cell immediately below the capillaries without coming into contact with the samples migrating other capillaries, and finally migrate to the anode electrode tank 7. The sample is detected by irradiating a portion 500 μm downstream from the capillary end in the fluorescent cell 4 with laser light to receive the fluorescence. The light receiving system has the same configuration as in the first embodiment. The present embodiment is basically different from the first embodiment in the part of the sheath flow forming method. In this embodiment, the sheath liquid flows in a laminar flow state inside the optical cell. For this reason, the flow rate of the sheath liquid is somewhat different depending on the position inside the optical cell, and there is instability that the path along which the sample migrates easily changes. However, effects similar to those of the first embodiment can be obtained in terms of sensitivity, simplicity of the apparatus configuration, and the like.

Example 6
In the apparatus described in the first embodiment, the downstream capillary can be formed as a flat plate having a plurality of grooves to form the gap. FIG. 6 shows a perspective view of a flat plate having a plurality of grooves. In the figure, a case where four grooves are provided so as to correspond to four capillaries is illustrated. Four grooves 81a, 81b, 81c, 81d are formed on one side of the flat plate 80 that 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 wide and 600 μm deep. The interval between the grooves is 1 mm. Note that these intervals and groove shapes can be freely changed. FIG. 7 is a perspective view of a cross-sectional view of the fluorescent cell portion of the electrophoresis part of the electrophoresis apparatus using the flat plate. The fluorescent cell 84 uses a flow cell type in which the inside of the cell is 20 mm wide, 3 mm deep, and 40 mm long, and the thickness of (quartz) glass is 2 mm and the upper and lower ends are open. The figure shows a state in which the glass portion on the front side of the fluorescent cell is removed. The flat plate 80 is fitted inside the fluorescent cell 84 so as to form four flow paths formed by the grooves 81a, 81b, 81c, 81d and the glass of the fluorescent cell. Also, capillaries 82a, 82b, 82c, and 82d having an inner diameter of 100 μm and an outer shape of 200 (a gel is produced in the same manner as in Example 1) are fixed inside the fluorescent cell 84 by the multicapillary holder 83. The interval between the capillaries is set to 1 mm so as to coincide with the interval between the grooves, and the capillary axis is adjusted to be at the center of each groove. These can be dealt with by adjusting the positions of holes or the like provided in the multi-capillary holder 83. Further, the ends of the capillaries 82a, 82b, 82c, and 82d inside the fluorescent cell are aligned, and the ends of the capillaries and the flat plate 80 are adjusted so as to be separated by about 1 mm and fixed inside the fluorescent cell. In addition, electrophoresis is enabled by making the lower part of a fluorescent cell contact the buffer solution in an anode electrode tank. Moreover, it is also possible to connect with an electrode tank through a tube similarly to Example 4 or Example 5. The fluorescent cell 84 is provided with a sheath liquid injection port 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 ethylene tetrafluoride resin. And Further, by placing the liquid surface of the sheath liquid 88 in the sheath liquid container 87 higher than the liquid surface of the buffer solution in the anode electrode tank, the sample migrated from the capillaries 82a to 82d is wrapped in the sheath liquid and fluorescent. It can flow in the cell 84 and can be led to the anode side electrode tank through the grooves 81a to 81d.
Electrophoresis, laser light irradiation, and fluorescence detection are performed in the same manner as described in Example 1, so that a sample separated by each of a plurality of capillaries can be detected efficiently at the same time. In this embodiment, the downstream side is not a capillary but a flat plate having grooves. Therefore, the end surfaces of the respective grooves can be easily aligned and adjustment is facilitated. Further, if the flat plate is made of quartz glass or the like, the laser light is irregularly reflected, and no fluorescence is generated from the portion even on the flat plate, thereby facilitating fluorescence measurement.

Example 7
An apparatus and method for determining the base sequence of DNA using a gel-filled capillary array will be described. A sample for DNA sequencing (fluorescence-labeled DNA fragment) is prepared in the same manner as in Example 2. In order to distinguish four base species in one migration path (gel capillary), a primer labeled with a different phosphor 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 fluorescence maximum wavelength of about 550 nm was bound to the A fragment. Similarly, primers (abbreviated as “primer C”, “primer G”, and “primer T”, respectively) labeled with phosphors having fluorescence maximum wavelengths of about 520, 575, and 605 nm were bound to the C, G, and T fragments. These fragments were finally put in a single container, mixed, and after ethanol precipitation, dissolved in deionized formamide (containing 1/100 amount of Tris buffer). Prior to injection into the gel capillary, the fragments were heated to 90 ° C. for 2 minutes to be thermally denatured to make the fragments single-stranded, and then immediately cooled on ice and used for the injection operation. Note that the sample may be prepared using a fluorescent label as a terminator instead of a primer.

  Next, the measuring apparatus will be described. FIG. 8 is a block diagram of a subarray for constituting the capillary array, and FIG. 9 is a block diagram of the capillary array of this embodiment in which a plurality of subarrays of FIG. 8 are combined. 10 is a block diagram of a DNA base sequence determination apparatus using a capillary array, FIG. 11 is an enlarged cross-sectional view of a sample injection unit, and FIG. 12 is a block diagram of an electrophoretic current measurement / display unit of the DNA base sequence determination apparatus. 13 is an explanatory diagram including a cross section of the optical cell to show the configuration in the vicinity of the optical cell unit, FIG. 14 is a diagram illustrating the principle configuration of the fluorescence detection / color separation unit, and FIG. 16 shows the fluorescence characteristics of the fluorescently labeled primer used and the transmission characteristics of the spectral filter. FIG. 17 shows an example (raw data) of the time waveform of the fluorescence signal intensity emitted from the sample that migrates through one gel capillary. FIG. 18 shows the analysis. It is the time waveform for every A, C, G, and T fragment obtained in this way.

Using 96 capillaries filled with gel inside, 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 the assembly and makes it difficult to replace the capillaries. Therefore, a sub-array of 8 capillaries is formed, and 12 sets of these sub-arrays are arranged to form a 96-capillary array. Configured. As shown in FIG. 8, eight capillaries 90 having slightly different lengths are arranged on the same plane, and the capillaries are held so that the distance between the centers of the capillaries is 9 mm at one end and 0.5 mm at the other end. They are fixed with tools 91 and 92, respectively, and their ends are aligned. The capillary end 90a fixed at an interval of 9 mm is the sample injection side end, and the opposite 90b is the optical detection side end.
As shown in FIG. 9, twelve sets of subarrays are combined to form a capillary array 107. The twelve capillary holders 91 are fixed by an area array holder 93 so as to be arranged in 12 rows on the same plane, and the capillary holders 92 on the other end are linearly arranged so that twelve capillary holders 92 are arranged in a straight line in order. Fix with the array holder 214. As a result, the capillary ends 90a are arranged in a grid of 8 rows and 12 rows on the same plane, and 96 capillary ends are arranged on a straight line on the 90b side. FIG. 9 shows the capillaries 90 connected only to the subarray in the first row. The capillaries in the middle of the subarrays in the 2nd to 12th columns are omitted. Although the subarrays themselves are omitted for the 3rd to 11th columns, they are arranged in order between the 1st, 2nd, and 12th subarrays. The capillaries used had an inner diameter of 0.1 mm, an outer diameter of 0.2 mm, and a length of about 35 cm, and were filled with an acrylamide gel in the same manner as in Example 1. The gel concentration was 9% T (ratio of (acrylamide + bisacrylamide) to the total solution amount) and 0% C (ratio of bisacrylamide to (acrylamide + bisacrylamide)). By using the capillary array 107 of this embodiment, 96 samples can be analyzed simultaneously.

  FIG. 10 is a configuration diagram of a DNA base sequence determination apparatus. The capillary array 107 is inserted into the buffer solution (or sample solution) of the upper individual electrode tank 104 like a microplate having 96 wells at the ends arranged in a grid pattern. In the drawing, 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 capillary array 107 arranged in a straight line is fixed inside the optical cell 103. A sheath liquid (buffer solution) is injected into the optical cell 103 from the sheath liquid container 105 through the tube 122 and the valve 121, passes through the inside of the hollow capillary array 108 disposed under the capillary array 107, and enters the lower electrode tank 106. Discharged. The capillary array 107 and the hollow capillary array 108 are fixed with their ends facing each other and separated by 2 mm. Two types of laser devices were used as light sources for exciting the sample. One is an argon laser apparatus 101 having a wavelength of 488 nm, and the other is a YAG laser apparatus 102 having a wavelength of 532 nm. These are arranged such that an argon laser beam 111 and a YAG laser beam 112 are coaxially formed by a mirror 109 and a dichroic mirror 110. The optical cell 103 was irradiated with being squeezed with a lens (not shown) so as to become one laser beam 113. The fluorescence generated from the fluorescently labeled sample is imaged on the two-dimensional detector 118 by the condensing lens 114, the four types of spectral filters 115a to 115d, the image dividing prism 116, and the imaging lens 117, and DNA fragments of each sample. The data processing unit 119 analyzes the time waveform of the fluorescence intensity from.

  Sample injection into the capillary array 107 will be described with reference to FIG. 11 (sample injection portion / enlarged cross-sectional view of the 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. The upper individual electrode tank 104 having the container-like recesses 104a, 104b... In 8 holes × 12 rows (96 holes in total) is used. For example, a 96-well V-bottom well microplate or 96 sampling tubes arranged in a grid of 8 × 12 rows at 9 mm intervals can be used. The interval 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,... Serve as upper electrode tanks, the 96 platinum electrodes 120a, 120b,... Are formed by the electrode holding plate 120 in the same manner as the ends of the capillary array 107 corresponding to the container-like recesses. They were fixed in a grid of × 12 rows. Further, the electrode holding plate 120 is provided with holes 252a, 252b,... For passing capillaries in the vicinity of each platinum electrode. The sample 240 is placed inside the recesses 104a, 104b. The electrode holding plate 120 is stacked on the upper individual electrode tank 104 with the spacer 250 interposed therebetween, and the area array holder 93 is further stacked with the spacer 251 interposed therebetween. The individual capillaries 107a, 107b,... Penetrate the holes 252a, 252b,... Of the electrode holding plate 120 and are immersed in the sample liquid 240, and the platinum electrodes 120a, 120b,. A voltage is applied almost simultaneously between these electrodes and the electrode in the lower electrode chamber 106 to inject the DNA fragment in the sample 240 into the capillary. The voltage is set to an electric field strength of 100 to 200 V / cm, and is applied for several seconds to 10 seconds. For example, it is applied at 5 kV for 10 seconds. After sample injection, remove the upper individual electrode tank 104, set another upper individual electrode tank 104 containing a buffer solution, and continue electrophoresis by applying an electric field strength of 100 to 400 V / cm, for example, 7 kV. Thus, molecular weight separation is performed. With this configuration, 96 samples can be injected simultaneously, and then the 96 samples can be electrophoresed simultaneously. In addition, the injection of the sample only needs to overlap the electrode holding plate 120 and the area array holder 93, that is, the capillary array 107, and there is no need to manually perform each sample by pipetting as in the case of the conventional flat gel. The operability can be greatly improved.

  In addition, when an electric field was applied, as shown in FIG. 12, a mechanism for measuring and displaying the current values flowing through the individual capillaries individually and simultaneously was provided. 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 source 133. A resistor 131 and a voltmeter 132 of about 10 kΩ are connected between the electrode 120a and the electrophoresis power supply 133 as shown in FIG. The current flows in the order of electrode 209 → buffer solution 245 → capillary array 108 → optical cell section → individual capillaries 107a of the capillary array 107 → buffer solution 246 in the container-like recess 104a → electrode 120a → resistance 131 → terminal 260. Although there is an optical cell portion between the capillary 107a and the capillary array 108, it is omitted in FIG. The current value flowing through the capillary is converted by the value of the voltmeter 132, and the current value is binarized by the binarization means and transmitted to the current display panel 140. Although not shown in FIG. 12, the terminals 261 and 262 are also connected to the electrodes 120b and 120c, respectively, and the current values flowing through the capillaries 107b and 107c are binarized and transmitted to the current display panel 140. The same applies to all the capillaries of the capillary array 107 below. Terminal 270 is the last 96th terminal. The current display panel 140 has 8 × 12 rows of display lamps 140a, 140b,..., And displays the current values of the capillaries 107a, 107b,. The resistor 131 has a resistance value that is sufficiently smaller than the resistance value of the capillary gel. Usually, there is no problem if it is 100 kΩ or less. The binarization level can be adjusted appropriately. The normal electrophoretic current is about 0.01 mA, although it varies depending on the applied voltage, capillary length, and the like. If the gel breaks, the current will stop flowing or become smaller. Therefore, for example, if the display lamp is turned on when the current becomes 0.003 mA or less, abnormalities such as gel breakage can be recognized in units of capillaries. As a result, it is possible to take measures such as stopping the analysis of the measurement result for the capillary having an abnormality. Further, in this example, the capillaries are sub-arrayed in units of eight, so that only a unit with a broken capillary can be replaced, and the capillary array can be used effectively. Thus, the operability is good and the apparatus configuration is economical.

Next, the structure in the vicinity of the optical cell portion 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 injection port 203, optical windows 204 and 206, and the like. In addition, although the housing | casing and the optical window are also in the front surface and back surface of the paper surface in which FIG. The casing 201 and the optical windows 204 and 206 are shown in cross section. The upper portion 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 from this portion and fixed. A sheath liquid (buffer solution) is injected into the optical cell 103 from the sheath liquid container 105 through the tube 122 and the valve 121, and a sheath flow is formed in the cell as in the first embodiment. It is discharged to the lower electrode tank 106 through the inside of the arranged hollow capillary array 108. The optical cell 103 is open to the top, 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. The sheath liquid flows in the cell due to a difference in height or the like.
A hollow capillary array 108 having an inner diameter of 0.2 mm, an outer diameter of 0.35 mm, and a length of 3 cm subjected to the same inner surface treatment as in Example 1 is used, and the linear array holder 215 is arranged at intervals of 0.5 mm. The capillary arrays 107 and 108 were fixed so that the ends of the capillaries of the capillary arrays 107 and 108 face each other. The hollow capillary array 108 and the linear array holder 215 are fixed to the optical cell 103 and sealed so as not to leak water. Further, the optical cell 103 and the lower electrode tank 106 are brought into close contact with each other with good airtightness, and the liquid accumulated in the optical cell 103 is discarded from the discharge port 210 to the waste liquid container 213 through the valve 211 and the tube 212. That is, the liquid exceeding the height of the discharge port 210 is drained, and the height of the liquid level in the lower electrode tank 106 is substantially constant. Therefore, even if the sheath liquid flows in, the drop does not change so much and the sheath flow is stably formed. Further, when the valve 211 is closed, the inside of the lower electrode tank 106 is filled with the sheath liquid, so that the flow of the sheath liquid can be stopped and the sheath liquid can be used effectively. In particular, this mechanism is necessary when a measurement is finished and time is available until the next measurement. In this example, the liquid levels 230 and 231 substantially coincide with each other. For example, a liquid level sensor for the liquid level 231 may be provided, and a mechanism for opening and closing the valve 121 according to the decrease in the liquid level may be provided. In this case, the sheath liquid container 105 can be disposed at a position higher than the optical cell, and the apparatus configuration is facilitated.

  The lower electrode tank 106 is provided with a platinum electrode 209 for voltage application and its terminal block 208. When a gel capillary is used, a positive voltage is applied to the platinum electrode 209 (relative to the opposite electrode). For example, it is possible to apply 0V to the platinum electrode 120a and 7kV to the platinum electrode 209, but it is desirable to apply a voltage of -7kV to the platinum electrode 120a and 0V (ground) to the platinum electrode 209 to ensure safety. This is because the sheath liquid as a whole has almost 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 are all at a high voltage, This is because there is an increased risk of electric shock, electric leakage and dielectric breakdown due to water leakage. When the electrode 209 is 0 V (grounded), the sample side becomes a negative high voltage, but since there is no flow of buffer solution, there is no water leakage, etc., and it is possible to easily prevent electric shocks and leakages, thus simplifying the device configuration. To be able to. In addition, it becomes easy to use a metal such as stainless steel as the casing 201 of the optical cell, the workability is improved, and the optical cell can be manufactured at low cost. Although the electrode 209 is disposed below the hollow capillary array 108 as in this example, it may be disposed in the optical cell or in the sheath liquid container, and has the same effect.

The laser beam 113 enters the optical cell through the optical window 204 or 206.
In the same manner as above, DNA fragments that migrate in a sheath flow state are excited to generate fluorescence. As shown in the figure, the fluorescent light emission points 220 are generated in the form of dots almost directly under the capillaries . These images are detected through an optical window (not shown) from the direction perpendicular to the paper surface. The laser beam 113 is radiated while being focused by a lens. However, by using a lens having a focal length of about 150 mm, the laser beam 113 can be irradiated uniformly over the entire width of the capillary array (about 50 mm in this example). it can. Although it is possible to irradiate the laser beam without being coaxial, in the case where the laser beam is coaxial, two laser beams irradiate the same part, so that the DN excited by each laser is used.
Since the migration distance of the A fragment can be made constant, the migration time is eliminated, and the analysis accuracy is improved.
Further, in the case of a phosphor having absorption at both laser light wavelengths, when it is made coaxial, the excitation light intensity substantially increases, so that the detection sensitivity is improved. Note that measurement is possible with only one laser.

  The detection of the fluorescence image will be described with reference to FIG. The fluorescence emitted from the fluorescent light emitting point 220 is condensed by the condensing lens 114, divided into four by the image dividing prism 116, and imaged (221a-b) by the imaging lens 117, respectively. At this time, by arranging the four types of spectral filters 115a to 115d on the front surface (or rear surface) of the image dividing prism, it is possible to make the divided light components have different wavelength components. This has been described for one fluorescent light emission point 220, but the same applies to all light emission points, and these divisions are performed simultaneously. In other words, time division is not performed unlike in the case of laser beam scanning or mechanical scanning of the detector, and the measurement interval of fluorescence intensity is not lengthened and can be measured at high speed. It is also possible to measure continuously in real time. Further, by arranging a condensing lens in front of the image dividing prism, the fluorescence condensing efficiency is improved, and the detection sensitivity can be improved. It is also possible to use a cylindrical lens instead of the condenser lens. In this case, since the light in only one direction is condensed, the efficiency is about half that in the case where a normal convex lens is used, but the detection sensitivity can be greatly improved as compared with the case where it is not used. In addition, when the image dividing prism is used, the intensity ratio of the divided image greatly fluctuates when the fluorescence emission position is shifted. When using a slab gel, there is a problem that the laser light incident on the gel is bent due to the heat generation of the gel and the fluorescence emission position is shifted, and the measurement accuracy is liable to decrease. In this example, the laser light is an aqueous solution. Since the laser beam irradiates a certain position without bending because it passes through, the measurement accuracy is improved as compared with the conventional case.

  In addition, since the capillaries are densely arranged in the optical cell, the distance between both ends of the arrangement is about 5 cm or less even in a large number of 96 electrophoresis lanes. Therefore, a condensing element such as a condensing lens or a cylindrical lens can be used in front of the image dividing prism, and fluorescence can be detected with higher sensitivity. In the case of a conventional slab gel, even in the case of 24 lanes, the distance between the lanes at both ends is about 30 cm. When a condenser lens is placed in front of the image dividing prism, a lens with a diameter of 30 cm is required. Really difficult for a reason.

In this embodiment, a capillary array having a width of about 50 mm and a light receiving element of the two-dimensional detector having a width of about 27 mm was used. The image magnification as a whole is about 1/2, and in the case of the slab gel system, the light collection efficiency is better than about 1/10.
Furthermore, when the number of capillary arrays is as small as about 20 to 30, the enlargement ratio is the same, the light collection efficiency is improved, and the sensitivity is further increased. In order to prevent a reduction in the light collection efficiency, it is desirable that the distance between the capillaries in the capillary array be about 10 times the capillary diameter or less.

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

  FIG. 16 is a graph showing the fluorescence characteristics of the fluorescently labeled primers and the transmission characteristics of the spectral filters. As described above, the spectral filters having the maximum transmittance near the fluorescence maximum wavelength of each fluorescently labeled primer (however, the wavelength of the excitation laser light is transmitted). To measure) and measure.

  FIG. 17 is an actual measurement value of the time waveform of the fluorescence signal intensity emitted from the sample that migrates through one gel capillary in the capillary array. It is a time waveform of the fluorescence intensity which permeate | transmitted the spectral filters 115a, b, c, and d from the top. Thus, four types of fluorescence information can be obtained simultaneously from one sample / one capillary. However, as can be seen from FIG. 16, the fluorescence of the fluorescently labeled primer is broad, and the fluorescence of the four fluorescently labeled primers overlaps. Therefore, the waveform in FIG. 17 is observed as the sum of the fluorescence of a plurality of fluorescently labeled primers, not the signal of a single fluorescently labeled primer. For example, Ia is a mixture of the fluorescence of primer T and the fluorescence components of primer G, primer A, and primer C. The same applies to Ib, Ic, and Id. When these influences are corrected based on the fluorescence characteristics of the fluorescently labeled primer, the transmission characteristics of the spectral filter, and the sensitivity characteristics of the detector, the fluorescence intensities of primer T, primer G, primer A, and primer C alone from the top as shown in FIG. The time waveform is obtained. This becomes the migration spectrum of each of the T, G, A, and C fragments, and the base sequence of the DNA can be determined from this waveform by an ordinary method.

  In this example, 96 samples can be determined simultaneously, and high throughput can be achieved. Specifically, the migration speed is about 200 bases / hour, and the overall throughput is 20 k bases / hour, which can achieve a high throughput of 10 times or more compared to the conventional method using a slab gel.

  In addition, this embodiment has the same effect as described in connection with the first embodiment. In addition, the optical cell having the structure described in Example 1 or Examples 4 to 6 can be used for measuring the DNA base sequence in substantially the same manner as described above. There are similar effects as described for Examples 4-6.

  In this embodiment, a quadrant prism is used. However, the base sequence of DNA can be determined by discriminating the base species according to the method described in Japanese Patent Application Laid-Open No. 5-118991 using a bipartite prism.

  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 capillary inner wall. This process does not need to be performed on the entire inner wall of the capillary, but may be performed only partially, that is, near the end on the optical cell side. As a result, the gel can be prevented from protruding into the sheath liquid inside the optical cell, the processing can be simplified, and the gel can be stably prepared. 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.

The block diagram of the electrophoresis part and laser beam irradiation system of the electrophoresis apparatus of the 1st Example of this invention. The block diagram of the fluorescence detection system of the electrophoresis apparatus of the 1st Example of this invention. The block diagram of the part of the light irradiation system and the detection system of the electrophoresis apparatus of the 3rd Example of this invention. The block diagram of the gap | interval vicinity of the electrophoresis apparatus of the 4th Example of this invention. The block diagram of the electrophoresis part of the electrophoresis apparatus of the 5th Example of this invention. The perspective view of the flat plate which has the some groove | channel of the 6th Example of this invention. The figure which shows the fluorescence cell part of the electrophoresis part of the electrophoresis apparatus of the 6th Example of this invention. The block diagram of the subarray of the capillary used in the 7th Example of this invention. The block diagram of the capillary array used in the 7th Example of this invention. The block diagram of the electrophoresis apparatus of the 7th Example of this invention. The expanded sectional view of the sample injection | pouring part / upper electrode tank in the 7th Example of this invention. The block diagram of the electrophoretic current measurement and the display part in the electrophoresis apparatus of the 7th Example of this invention. Explanatory drawing including the partial cross section which shows the structure of the vicinity of the optical cell part in the electrophoresis apparatus of the 7th Example of this invention. The principle block diagram of the fluorescence detection and color separation part in the electrophoresis apparatus of the 7th Example of this invention. The image figure of the fluorescence image obtained with the two-dimensional detector in the electrophoresis apparatus of the 7th Example of this invention. The fluorescence characteristic of the fluorescent labeling primer used in the 7th Example of this invention, and the transmission characteristic figure of a spectral filter. The figure which shows an example of the measurement data of the time waveform of the fluorescence signal intensity | strength emitted from the sample which migrates the one gel capillary by the 7th Example of this invention. The time waveform with respect to A, C, G, and T fragment obtained by analyzing the data of FIG. The block diagram of the fluorescence measurement part of the capillary electrophoresis apparatus of a prior art example. The block diagram of the fluorescence measurement part using the sheath flow cuvette of the capillary electrophoresis apparatus of a prior art example.

Explanation of symbols

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 ... direct current High voltage power source, 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 ... Controller for one-dimensional sensor, 5 Data processing device 57 ... Monitor 58 ... Printer 59 ... Memory 60 ... Fluorescent cell 61a-61t ... Fine pore 62 ... Flat 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. Tanks, 104a, 104b, ..., container-like recesses, 105, sheath liquid container, 106, lower electrode tank, 107, capillary array, 107a, 107b, ... capillary, 108, hollow capillary array, 109, mirror, 110, dichroic mirror 111 ... Argon laser light, 112 ... YAG laser light, 113 ... Laser light, 114 ... Condensing lens, 115a-d ... Spectral filter, 116 ... Image division prism, 117 ... Imaging lens, 118 ... Two-dimensional detector, 119 ... Data processing unit, 120 ... Electrode holding plate, 120a, 120b ... Platinum electrode, 121 ... Valve, 122 ... Tube, 131 ... Resistance, 132 ... Voltmeter, 133 ... Electrophoresis power supply, 140 ... Current display panel, 140a, 140b ... Display lamp, 201 ... Case, 202,205 ... Array guide DESCRIPTION OF SYMBOLS 203 ... Sheath liquid inlet, 204, 206 ... Optical window, 208 ... Terminal block, 209 ... Platinum electrode, 210 ... Discharge port, 211 ... Valve, 212 ... Tube, 213 ... Waste liquid container, 214 ... Linear array holder DESCRIPTION OF SYMBOLS 215 ... Linear array holder, 220 ... Fluorescence emission point, 221a-221d ... Imaging, 230 ... Sheath liquid level of sheath liquid container, 231 ... Sheath liquid level flowing into cell, 240 ... Sample 245, 246, buffer solution, 250, 251, spacer, 252, 253, hole, 260, 261, 262, 270, terminal.

Claims (15)

A plurality of capillaries for electrophoretic separation of the sample;
A plurality of electrodes provided near a sample injection end of the plurality of capillaries and applying a voltage to the sample to be migrated;
Having a first member,
One electrode is provided for each of the sample injection ends of the plurality of capillaries,
The number of pairs of one capillary and one electrode, the number of capillaries, and the number of electrodes are equal, and the first member holds a plurality of the pairs. Electrophoresis device. Said first member electrophoretic device according to claim 1, characterized in that for holding a plurality of said pairs at regular intervals.   The electrophoresis apparatus according to claim 1, wherein the pair is immersed in different samples. Electrophoretic device according to any one of claims 1 to 3, wherein the pair are those each are simultaneously immersed in the sample. The electrodes each electrophoretic device according to any one of claims 1 to 4, characterized in that to be simultaneously applying a voltage. A second member having a plurality of container part Furthermore, the different samples are electrically according to any one of claims 3 to 5, characterized in that it is contained in each of the plurality of container portions Electrophoresis device.   The electrophoretic device according to claim 6, wherein an interval between the centers of the plurality of container portions coincides with an interval between the centers of the plurality of capillaries. Electrophoretic device according to any one of claims 1 to 7, characterized in that it further comprises means for measuring the current flowing through the electrode. Applying a voltage to each of a plurality of electrodes provided in the vicinity of a sample injection end of a plurality of capillaries, and injecting a fluorescently labeled sample into the capillaries;
Applying a voltage to the electrode to electrophoresis the sample;
Detecting fluorescence generated from the fluorescent label,
The electrodes are provided one by one corresponding to each of the sample injection ends, and one capillary and one electrode form a pair, and are held by a first member that holds a plurality of the pairs. that is intended, equal to the number of the number of the capillaries and the number of the counter electrode, an electrophoretic method characterized by electrophoresing each different said samples in said plurality of capillaries.   10. The electrophoresis method according to claim 9, wherein, in the injecting step, a voltage is applied to the electrode almost simultaneously and the sample is injected into the capillary almost simultaneously. Electrophoresis method according to any one of claims 9 to 10, characterized in that immersing the sample injection end after the step of said injection in a buffer. In the process of the electrophoresis according to substantially any one of claims 9 to 11 and performing electrophoresis by applying a voltage and substantially simultaneously in each of said plurality of capillaries simultaneously to the electrode Electrophoresis method. Electrophoresis method according to any one of claims 9 to 12, characterized in that to measure the current flowing through the electrodes when the voltage to the electrode is applied. A plurality of capillaries for electrophoretic separation of the sample;
A plurality of electrodes provided near a sample injection end of the plurality of capillaries;
One capillary and one electrode form a pair, and have a first member holding a plurality of the pairs ,
One capillary electrode is provided for each of the sample injection ends of the plurality of capillaries, and the number of pairs, the number of capillaries, and the number of electrodes are equal.   The capillary array according to claim 14, wherein the first member holds a plurality of the pairs at a constant interval.

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