JPH06294771A - Electrophoretic medium and analyzing device using the same - Google Patents

Abstract

PURPOSE:To obtain an electrophoretic device having a capillary array which holds the features of a capillary system and is easy to handle and inexpensive. CONSTITUTION:This device has a structure wherein a spacer sheet 3 having a number of grooves is held between two macromolecular sheets 1 and 2. Gel is dilled up in the grooves of the spacer sheet 3 and thereby gel capillaries 6 are constructed. The density of the gel capillaries 6 is large on the detecting part side 8 and small on the sample injection part side 7. On the sample injection part side 7, each capillary 5 is separated from others by cutting in order to the device easy to handle.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for separating and detecting DNA, RNA or protein, and more particularly to a novel gel electrophoresis medium and an analyzer using the same.

[0002]

2. Description of the Related Art A technique for separating and analyzing DNA, RNA and the like is important as a basic technique in the field of life science including gene analysis and gene diagnosis. Particularly in recent years, it has been desired to develop an apparatus capable of analyzing DNA and the like at high speed and high throughput in connection with human genome analysis.

Conventionally, a gel electrophoresis apparatus has been used for analysis of these substances such as DNA nucleotide sequence determination. For example, in a DNA sequencer using fluorescence detection, a polyacrylamide gel having a thickness of 0.2 to 0.3 mm was prepared between two glass plates (30 cm to 40 cm) and used as an electrophoretic separation medium. Recently, gel capillary electrophoresis, in particular, a method of arranging a large number of capillaries and performing electrophoresis at the same time has been developed in response to a need for a faster and higher throughput analysis method. This method has an inner diameter of 0.05 to 0.1 mm and an outer diameter of 0.2 to
Gel is packed in a 0.3 mm fused silica capillary [Analytical Chemistry, No. 62]
Vol. 900 (1990) (Analytical Chemistr
y 62,900 (1990))]. This method has an advantage that a large electrophoretic electric field can be applied because it generates less heat than a flat gel, and is suitable for high-speed electrophoresis. Actually, it has been noted that the electrophoresis separation, which took 4 to 5 hours in the past, can be performed in less than 1 hour, but the results are remarkable, but the capillaries are expensive, and many samples cannot be simultaneously migrated. There was a drawback. Particularly for the latter, it has been desired to realize a multi-capillary type detection device in which a plurality of capillaries are arranged.

Recently, Mathies et al. Have reported a capillary array electrophoresis in which a large number of capillaries are arranged [Analytical Chemistry, Vol. 64, 96].
Page 7 (1992) (Analytical Chemistry 64, 9
67 (1992))].

[0005]

However, this multi-capillary system uses a large number of expensive capillaries arranged side by side, and even if they are used several times, the gel deteriorates and becomes unusable, so the running cost is very high. There was an expensive difficulty. In addition, the time and effort required for the measurement are large, such as the need to align and arrange the capillaries for each measurement and to remove the coating of the measurement part.

Therefore, the advantages of the capillary system are as follows: High-speed, high-throughput migration is possible 1. It has been desired to develop a method that eliminates the above-mentioned drawbacks while maintaining advantages such as easy sample injection because electric field injection is possible. It is an object of the present invention to provide a novel gel electrophoretic medium that meets such requirements and an analyzer using the same.

[0007]

In the present invention, the capillaries are composed of a polymer material, or a large number of capillaries are formed in a polymer sheet and filled with a gel, which is used as an electrophoretic medium. To achieve. The plurality of capillary migration paths can be constituted by grooves provided between at least two sheet-like polymer films, and as an example, a spacer sheet sandwiched between the two sheet-like polymer films can be used. It is formed.

The arrangement density of the migration paths at one end of the capillary array sheet is different from the arrangement density of the migration paths at the other end, and the end of which the arrangement density is rough is such that each capillary has a predetermined length from the tip. The end portions that are separated from each other to form the sample injection portion and the arrangement density is dense are connected to the detection portion. The electrophoretic medium of the present invention constitutes an analysis device in combination with an optical detection device. The detection is preferably performed after the sample is eluted from the electrophoretic medium.

[0009]

The capillaries made of a polymeric substance can be manufactured at low cost by using a technique of pressure molding or etching using a mold in a polymeric sheet, or by a hollow fiber manufacturing method. The sample injection side and the detection side are manufactured by changing the density distribution of the capillaries, and on the sample injection side, the capillary density is made coarse and divided into each other, so that each sample can be injected independently into the capillary. It can be done efficiently and efficiently. Further, since the detection side is densely arranged, the detection unit becomes compact and highly efficient detection can be performed. In the analyzer, since the detection is performed in the state where the sample is eluted from the capillary made of the organic polymer substance, strong fluorescence due to the organic polymer substance is not generated, and therefore high-sensitivity measurement can be performed.

[0010]

EXAMPLES Examples of the present invention will be described below. FIG. 1 is a partially cutaway perspective view of a gel capillary array sheet 9 according to an embodiment of the present invention. This gel capillary array sheet 9 consists of two PET sheets with a thickness of about 0.2 mm.
It has a structure in which a spacer sheet 3 having a thickness of about 0.1 mm is sandwiched between holding sheets 1 and 2 made of a (polyethylene terephthalate) film. The spacer sheet 3 has
The fine groove 6 as shown in the drawing is formed by photoetching or the like. The width of the groove 6 provided in the spacer sheet 3 is 0.1 to 0.2 mm, and the pitch of the groove is about 0.5 mm at the lower part of the sheet and about 2 mm at the upper part of the sheet. Although ten grooves 6 are provided in the illustrated example, the number of grooves is not particularly limited. Each groove 6 is filled with gel to form a migration path, and the migration path length is about 30 cm.

The upper portion of the gel capillary array sheet 9 is divided into individual migration paths 5 by cutting a length of about 5 cm from the tip. The side 7 where the groove is roughly arranged and the tip is divided serves as a sample injection section, and the densely arranged side 8 is connected to the detection section during measurement. In this way, by arranging the capillaries 6 roughly on the sample injection part side 7 of the gel capillary array sheet 9 and dividing the individual capillaries 5, handling of the gel capillary array sheet and to the sample injection part 4 of each migration path 6 are performed. The sample injection operation can be easily performed. Further, by making the capillaries on the detection unit side 8 dense, the detection device becomes compact and the reliability of analysis can be improved.

The gel capillary array sheet 9 is
It is produced as follows. First PE as a holding sheet
A photosensitive resin film such as an acrylic sheet containing a photosensitive group to be the spacer sheet 3 is bonded to the T film 1 with an adhesive. It is exposed using a narrow groove mask pattern, and the exposed or unexposed portion is melted to form a groove pattern serving as a migration path. The groove pattern has a divergent arrangement from one side 8 of the sheet 9 to the other side 7.
After forming a groove pattern serving as a migration path on the spacer sheet 3, the PET film 2 as a second holding sheet is formed thereon.
Are bonded with an adhesive. Then, a cut is made from the tip to a predetermined length on the side 7 in which the groove is roughly cut by cutting into a desired shape, and the tip portion of each capillary tube 5 is separated to produce a capillary sheet.

Next, one end of the thus-prepared capillary sheet is pushed into a syringe and hermetically fixed, and 3% by weight [3% C (Cross Cross
5% by weight including linking material concentration)]
A polyacrylamide gel solution (5% T, 3% C) of [5% T (Total concentration)] is pressed into the narrow groove to form the gel capillary 6. As the material of the holding sheets 1 and 2, besides PET, a polymer such as polyacrylic resin or polyimide resin, which is well compatible with polyacrylamide gel, can be used. Instead of using a shaped polymer sheet, the spacer sheet 3 may be obtained by applying a liquid material to a certain thickness to obtain a sheet.

In this embodiment, three polymer sheets are stacked and the spacer sheet in the middle between them is etched to form a capillary array sheet.
The sheets may be stacked to form a capillary array. That is, a capillary array sheet may be prepared by forming a narrow groove on one holding sheet 1 by pressure molding or etching using a mold, and adhering the other holding sheet 2 thereon.

The capillary array sheet 9 shown in FIG.
After standing for a day to form a gel, the gel is attached to the device of FIG. 2 for use. When the gel formation at the lower end portion or the upper end portion of the sheet is disturbed, a part of the sheet end is cut and removed, and the gel end face is used in a clean state. In order to keep the end of the gel clean, as shown in FIG. 3, cover the end of the capillary array sheet 9 with a guide cover 18,
From the other end of the sheet, the gel is filled into the space between the guide covers 18 using the syringe as described above.
Auxiliary part gel 19 is prepared between 8 parts. Then, the guide cover 18 is removed at the time of use, and a sharp blade is used in FIG.
Advantageously, the auxiliary gel 19 is cut off at the position indicated by 0.

The gel capillary array sheet 9 is set in the flow cell 11 with the lower portion 8 sandwiched by the holder guides 21. The holder guide 21 is provided with a plurality of fluid flow passages that communicate with each other in the vertical direction on the side surface in contact with the capillary array sheet 9. In the cell 11, a buffer solution (1 × TBE; 90 mM Tris borate, pH 8.3,
0.2 mM EDTA), and this buffer solution flows around the capillary array from top to bottom through the channel provided in the holder guide 21, and after passing through the detection part 12 of the cell 11, It flows into the lower buffer solution 14 through the sheath flow solution outflow passage 10.

The sample injection into the capillaries is performed by inserting the separated capillaries 5 above the capillary array sheet 9 into a sample reservoir (not shown) one by one and applying an electric field for a short time to inject the electric field from the sample injection unit 4 into each capillary. To be done. The end of the capillary 5 into which the sample is injected is immersed in a common buffer solution tank 13, and an electrophoretic voltage is applied to electrodes (not shown) inserted in the upper and lower buffer solution tanks 13 and 14 to generate the sample electricity. Electrophoretic separation is performed.

The sample separated in the gel capillary part flows out from the end part of the capillary and migrates downward in the flow cell, but is irradiated by a laser beam (not shown) incident in the direction perpendicular to the plane of the drawing at the detection part 12 on the way. To be done. Then, the fluorescence emitted from the sample is filtered by the filter 1 arranged toward the surface of the capillary array sheet 9.
5 are simultaneously detected by the high-sensitivity CCD camera 16. The detection signal from the CCD camera 16 is sent to the data processing device 17 and subjected to necessary arithmetic processing. Since fluorescence is detected by irradiating excitation light on the migration path without the organic capillary array, the fluorescence emitted from the capillary array does not interfere with the detection.

The sheath flow liquid outflow passage 10 does not necessarily have to be provided separately for each capillary, but if a hollow capillary array corresponding to each capillary is used to form a large number of sheath flows around the migration passage. This is advantageous because it is possible to reliably prevent the eluate of the adjacent capillaries from mixing in the detection unit 12. Of course, the cell 11 can be omitted by connecting the lower end portion of the capillary array sheet 9 to a capillary array made of quartz with less fluorescence.

Further, the sample injection part of the capillary is apt to generate heat by application of an electrophoretic voltage to cause gel breakage, and therefore it is difficult to repeatedly use it. When the sample injection side 7 is used by connecting it to a short capillary, there is an advantage that the gel sheet can be used many times only by exchanging the connection capillary in which the gel has deteriorated. As an example of the detection method, FIG.
A system was shown in which the migration paths arranged in a row were simultaneously irradiated with light from the side, and a linear fluorescence image was taken by a CCD camera. Figure 2
Is an example of detecting a fluorescent image from one type of phosphor,
It is also possible to measure the wavelengths of a plurality of phosphors by using a prism or the like. Alternatively, the fluorescence from each sample may be sequentially detected by moving and scanning the moving table on which the light source and the detector are fixed along the capillary array sheet.

Further, in the above-mentioned embodiment, a plurality of capillaries are formed by polymer sheets, but a hollow tube of organic polymer, that is, a capillary is made by the hollow fiber manufacturing technique currently used in various fields, and the capillaries are formed. A large number can be bundled and used. In that case, it is convenient to arrange one end of the bundled capillaries in a straight line when measuring by a fluorescence detection method or the like. PET, polyacrylic resin, polyimide resin or the like can be used as the polymer. The gel can be injected into the capillaries by a press-in method using a syringe as in the above-mentioned embodiment.

[0022]

As described above, according to the present invention, it is possible to obtain an inexpensive capillary array which is flexible and easy to handle and has the advantages of the capillaries. Further, since the thickness and width of the capillaries can be controlled with high accuracy, there is an advantage that the migration speed difference between migration paths can be reduced.

[Brief description of drawings]

FIG. 1 is a conceptual diagram of a gel capillary array sheet according to an embodiment of the present invention.

FIG. 2 is a conceptual diagram of an electrophoretic analysis device using the gel capillary array sheet of FIG.

FIG. 3 is an explanatory view of a capillary array sheet with an auxiliary gel.

[Explanation of symbols]

1, 2 ... Organic polymer holding sheet, 3 ... Spacer sheet, 4 ... Gel capillary sample injection section, 5 ... Separated capillary tube, 6 ... Gel capillary, 7 ... Separated capillary section, 8 ... Measuring section Packed capillary tube section , 9 ...
Gel capillary array sheet, 10 ... Capillary for outflow of sheath flow solution, 11 ... Flow cell, 12 ... Detection part, 13 ... Upper buffer solution, 14 ... Lower buffer solution, 15 ... Filter, 16 ... High sensitivity CCD camera, 1
7 ... Data processing device, 18 ... Guide cover, 19 ... Auxiliary part gel, 20 ... Cutting site, 21 ... Holder guide

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Takashi Anazawa, 1-280, Higashi Koikeku, Kokubunji, Tokyo Metropolitan Research Center, Hitachi, Ltd. (72) Keiichi Nagai, 1-280, Higashi Koikeku, Kokubunji, Tokyo Hitachi, Ltd. Central Research Center

Claims (14)

[Claims] 1. An electrophoretic medium which is made of a polymer material and has a plurality of capillary migration paths therein. 2. The capillary migration path is at least two.
The electrophoretic medium according to claim 1, wherein the electrophoretic medium is composed of a groove provided between a sheet of polymer film. 3. The electrophoretic medium according to claim 2, wherein the capillary migration path is formed by a spacer sheet sandwiched between two sheet-shaped polymer films. 4. The electrophoretic medium according to claim 2, wherein the arrangement density of the capillary migration paths at one end is different from the arrangement density of the capillary migration paths at the other end. 5. The electrophoresis according to claim 2, 3 or 4, wherein the capillaries are separated from each other by a predetermined length by cutting a sheet-like polymer film at one end. Medium. 6. The capillaries are separated from each other by a predetermined length by making a cut in the sheet-like polymer film at the end portion where the arrangement density is rough, and the capillaries are separated from each other by a predetermined length. The described electrophoretic medium. 7. The electrophoretic medium according to claim 4, wherein the end portion on the side where the arrangement density of the capillary migration paths is rough is the sample injection side. 8. The electrophoretic medium according to claim 5, further comprising a sample injection section at an end of the capillary migration path on the separated side. 9. The migration path comprises a plurality of polymer capillary tubes,
An electrophoretic medium characterized in that at least one end of the plurality of polymer capillaries is linearly bundled. 10. The electrophoretic medium according to claim 1, wherein a gel is filled in the migration path. 11. An analysis device comprising the electrophoretic medium according to claim 1 and an optical detection means. 12. The analyzer according to claim 11, wherein the optical detection means includes a cell connected to the electrophoretic medium, a light source, and a photodetector. 13. The analyzer according to claim 12, wherein the detection of the sample is performed by irradiating the sample eluted from the electrophoretic medium with excitation light and detecting fluorescence. 14. The analyzer according to claim 11, wherein the photodetector is an imaging device.