WO2004008132A1 - Bio-molecule separation cell, manufacturing method thereof, and dna fragmentation apparatus - Google Patents

Abstract

An electrophoresis cell has a substrate (2), and a groove (3) is formed on an upper surface of this substrate (2). The substrate (2) is formed of a transparent material enabling a user seeing through the substrate (2). The groove (3) includes a separation matrix (5) comprising a large number of stays (4). The upper surface of the substrate (2) and the groove (3) are covered by a transparent cover (6). In addition, the electrophoresis cell (1) has two sample inlets (7) and two electrodes (8). The electrode (8) are inserted into the groove (3) through corresponding sample inlets (7). Both of the electrodes (8) are connected to a DC power source (10) via a lead wire (9). This electrophoresis cell (1) can solve disadvantages in agarose gel and polyacrylic amide gel used in electrophoresis analysis, or in a conventional artificial separation matrix.

Description

 Description Biomolecule separation cell, method for producing the same, and DNA separation device

 TECHNICAL FIELD The present invention relates to a biomolecule separation cell such as an electrophoresis cell and a method for producing the same, and a DNA separation apparatus using the electrophoresis cell. Background art

 Biomolecules such as DNA, proteins, and fragments thereof extracted from biological samples are essential elements for medical examination, medical diagnosis, and biological examination. By biochemically analyzing these biomolecules and examining their molecular structures, diseases are diagnosed, microorganisms are detected, and genes are determined. These biomolecules are characterized by high molecular weight and high molecules. The most common of these methods is the electrophoresis method. In electrophoresis, biomolecules are separated in a voltage-separated matrix by the difference in molecular size (molecular weight) ゃ charge. As the electrophoresis method, a method using agarose gel (agar gel) or polyacrylamide gel as the separation matrix has been established. In this method, DNA and proteins are separated while passing through the molecular chain network of agarose gel or polyacrylamide. It is estimated that the mesh holes of these gels will be between 5 and 200 nm. However, these gels are heterogeneous in microstructure and morphologically unstable. For this reason, it is difficult to handle, and there are the following problems.

 (Problems of gel)

 (1) Since the form is unstable, storage stability is poor and handling is poor.

 ② Moisture must always be included.

 (3) The separation speed is low.

 A relatively large amount of sample, on the order of ④ μg, is required.

 で き な い Cannot be reused.

⑥High sensitivity optical detection cannot be performed. ⑦ It is difficult to reduce the size.

 In order to solve these problems, hard gels for electric swimming using semiconductor lithography technology have been proposed in recent years. Specifically, for example, an artificial microstructure is formed as an obstacle on a silicon substrate / glass substrate, and this is used as a substitute for a gel network, or optically transparent. Artificial separation matrices formed by fine molding of a suitable plastic have been proposed.

 Examples of the former include the report by Volkmuth et al. Published in Nature, Vol. 358, pp. 60-62, published in 1992. Bakajin et al., Published in Analytical Chemistry, Vol. 73, pp. 603-656, published in 2001. Hard gel. In these reports, the authors described a microartificial obstacle made of silicon dioxide or glass targeted at the analysis of DNA with a length of 50 to 200 kilobase pairs (kilobase pairs). We propose a separation matrix with. This separation matrix consists of regularly arranged struts of a few m / m in diameter. The DNA of the sample is separated according to its size in the separation matrix by applying an electric field in which the direction and angle are alternately changed, and the DNA is not observed or observed with a fluorescence microscope installed at the sample outlet. Is detected.

 On the other hand, as an example of the latter, there is an artificial separation matrix using plastics disclosed in Japanese Patent Application Laid-Open No. Hei 11-508082 by Yager et al. This artificial separation matrix differs from a separation matrix based on a silicon substrate or a glass substrate in the following points.

 (Difference)

 ① Use organic polymer as separation matrix material.

 (2) The distance between the arrangements of the artificial mouth structure is 100 nm or less, and preferably 10 to 30 nm.

③ The polymer is polymerized in the micro-processed mold ② to produce a separation matrix. However, these conventional technologies have the following problems or problems. That is, the following problems occur when silicon or glass is used for separation matrices for electrophoresis. (problem)

 (1) Silicon and glass are poor in workability, expensive and unsuitable for mass production.

 (2) The diameter and gap of the support are several μm or more, but this value is about 100 times larger than that of agarose gel or polyacrylamide gel, and is not suitable for analysis of small DNA.

 On the other hand, artificial separation matrices using plastic have the following problems.

 (problem)

 (1) At the nanometer (nm) to micrometer (/ m) level in the production of separation matrices, plastic molding technology has not been established.

 (2) The gap between the struts is as narrow as 100 nm or less, and the hydrated DNA molecules are easily clogged and smooth separation is difficult.

 (3) In the manufacturing technology using the microfabricated mold (2), the plastic peeling process and the fine pattern transfer process have not reached the level of theoretical discussion at this time and have not been put to practical use. Disclosure of the invention

 The present invention has been made to solve the above-mentioned conventional problems, and overcomes the above-mentioned drawbacks in, for example, agarose gel or polyacrylamide gel used for electrophoresis analysis, or a conventional artificial separation matrix. An object of the present invention is to provide a separation matrix which can be used or a biomolecule separation cell such as an electrophoresis cell, and a method for producing the same. Furthermore, it is another object to provide an application technique of such a biomolecule separation cell.

The biomolecule separation cell according to the present invention separates or detects biomolecules in a liquid sample, for example, by electrophoresis. The biomolecule separation cell includes a substrate having a groove (flow path) formed on the surface thereof, a separation matrix, a cover, a buffer filling the groove, and at least two electrodes. The separation matrix consists of a plurality of struts arranged in a partial area of the groove, with the narrowest part of the gap between the struts being 0.1-5 μπι. The cover covers the surface of the substrate and the groove, and is in close contact with the upper surface of the support. The electrodes are in direct contact with the buffer and apply a voltage to the buffer. Substrate and separation matrix It is formed of a child material.

 In this biomolecule separation cell, a separation matrix using artificial micro obstacles is used as an alternative to a gel for electrophoresis, for example. For this reason, the separation matrix can be reduced in size and weight, and can be stored in a dry state. In other words, biomolecules such as DNA can be separated or detected quickly and with high sensitivity even if the sample is small.

 In this biomolecule separation cell, an optimal structure can be selected according to the size and properties of the biomolecule to be separated. In other words, the degree of freedom in design is large. For this reason, the cross section of the support can be of various shapes, for example, a circle, an ellipse, a polygon, or a polygon with rounded or cut corners.

 Part or all of the substrate and the separation matrix are preferably formed of, for example, an elastomer, plastic, or the like, which is a polymer material that is inexpensive and easy to mass-produce. As the elastomer, for example, it is preferable to use a silicone rubber which is a general material and which is inexpensive and has good moldability. Further, as the plastic, for example, it is preferable to use an acrylic resin, a polystyrene resin, a polycarbonate resin, or the like, which is a general material and which is inexpensive and has good moldability.

 The width of the groove is set to 20 to 100 microns, the depth of the groove is set to 1 to 50 μπι, and the length of the groove is set to 0.1 to 5 O mm. preferable. In this way, biomolecules such as DNA can be electrophoresed uniformly and smoothly. For this reason, an electrophoresis cell capable of high-sensitivity detection can be realized.

The arrangement density of the pillars is preferably 100 or more Zmm ² or more. In the present invention, for example, the basic principle is that when DNA passes through sub-micron-order gaps in the column, it affects the migration speed of DNA, so the number of gaps greatly affects the resolution. . Then, if the arrangement density of the columns is 1000 or more / mm ² or more, it is possible to separate DNAs of 1000 base pairs or less.

If the cover is provided with a sample inlet for injecting biomolecule sample into the groove, the separation matrix has a region or column near the sample inlet where columns are less densely arranged than other regions. It is preferable to have a region that does not. For example, it is difficult for the DNA to enter the groove from the gap at the top of the column. Injection becomes easy.

 In one manufacturing method of the biomolecule separation cell according to the present invention, first, a silicon substrate, a glass substrate, or a polymer substrate is subjected to fine processing to form a shape having a negative pattern corresponding to a groove and a support. . Next, a liquid elastomer or plastic, or a monomer or prepolymer as a raw material of the elastomer or plastic is poured into the mold and solidified. After that, the solidified product is separated from the 铸 type to obtain a biomolecule separation cell including a substrate and a separation matrix. According to this manufacturing method, a 铸 -shaped fine structure can be faithfully transferred, and mass production of electrophoresis cells becomes possible.

 In another method for manufacturing a biomolecule separation cell according to the present invention, first, a silicon substrate, a glass substrate, or a polymer substrate is subjected to fine processing to provide a press mold having a negative pattern corresponding to grooves and columns. To form Next, after the plastic heated above the glass transition temperature is pressed against the press mold, the temperature is reduced and the plastic is cured. Thereafter, the cured product is separated from the press mold to obtain a biomolecule separation cell including a substrate and a separation matrix. According to this manufacturing method, since it is possible to transfer a 微細 -shaped fine structure faithfully and at high speed, mass production of electrophoresis cells becomes possible.

 The DNA sorting apparatus according to the present invention uses a biomolecule cell according to the present invention that separates biomolecules in a liquid sample by electrophoresis, from a liquid sample containing DNA supplied in a groove, and The DNA is separated (separated) or fractionated based on the properties and structure of the DNA. As described above, by using the electrophoresis cell according to the present invention for DNA analysis and medical inspection, a genetic test that had conventionally been difficult to perform and required a long time for analysis can be quickly and highly sensitively performed. Can be done with BRIEF DESCRIPTION OF THE FIGURES

 The present invention will become more fully understood from the detailed description and the accompanying drawings, wherein: FIG. In the attached drawings, common constituent elements are denoted by the same reference numerals.

FIG. 1 is an overall perspective view (perspective view) of an electrophoresis cell according to the present invention provided with a separation matrix formed of a polymer material. FIG. 2 is an enlarged perspective view showing a part of the electrophoresis cell or the separation matrix shown in FIG.

 FIG. 3 is an elevational sectional view of the electrophoresis cell shown in FIG.

 4A to 4E are three-dimensional schematic diagrams showing the shapes and arrangements of various columns.

 5A to 5E are top views of the separation matrix, showing various strut arrangements.

 FIGS. 6A to 6C are one perspective view and two cross-sectional elevation views showing the structures of the sample inlet and the separation matrix, respectively.

 FIGS. 7A to 7G are diagrams showing a method for producing a type III for producing a separation matrix, and a method for producing an electrophoresis cell using the type III.

 FIG. 8 is a schematic diagram showing a configuration or layout of a fluorescence microscope for an electrophoresis cell and its attached equipment.

 FIG. 9 is a graph showing the relationship between the number of DNA bases in the electrophoresis cell and the electrophoresis speed.

 FIG. 10 is a diagram showing the movement of DNA in the gap between the columns.

 FIG. 11 is a schematic diagram showing a schematic configuration of one DNA sorting apparatus using the electrophoresis cell according to the present invention.

 FIGS. 12A and 12B are schematic diagrams showing the schematic configuration of another DNA sorting apparatus using the electrophoresis cell according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, in each of the drawings, common members, that is, members having substantially the same configuration and function are denoted by common reference numerals, and redundant description will be omitted in principle.

 (Outline of electrophoresis cell)

First, the outline of the electrophoresis cell according to the present invention will be described. FIG. 1 shows the overall structure of the basic form of the electrophoresis cell according to the present invention. FIG. 2 shows an enlarged part of the electrophoresis cell, and FIG. 3 shows an elevational cross section of the electrophoresis cell. As shown in FIGS. 1 to 3, the electrophoresis cell 1 has a substrate 2, and a groove 3 is formed on the upper surface of the substrate 2. The substrate 2 is not necessarily required to be transparent, but is preferably transparent because the inside of the substrate 2 can be seen through. The groove 3 contains a separation matrix 5 consisting of a number of struts 4 (pillars). The upper surface of the substrate 2 and the groove 3 are covered with a transparent cover 6. Further, the electrophoresis cell 1 is provided with two sample inlets 7 and two electrodes 8 (positive electrode and negative electrode). Each electrode 8 is inserted into the groove 3 through the corresponding sample inlet 7. That is, the sample injection port 7 also serves as an electrode insertion hole. Further, both electrodes 8 are connected to DC power supply 10 via lead wire 9.

 In the electrophoresis cell 1, each column 4 constituting the separation matrix 5 has a geometrical cross section. The upper surface of each column is in close contact with the lower surface of the cover 6. The groove 3 containing the separation matrix 5 is filled with a buffer solution for electrophoresis, and therefore, a buffer solution is present in the gap between the columns 4. Each electrode 8 inserted in the groove 3 comes into direct contact with the buffer solution in the groove 3 and lays. When a biomolecule is present in the buffer, when a voltage is applied between the two electrodes 8, the biomolecule is charged and moves toward one of the electrodes 8 according to the charging mode. I do. For example, if the biomolecule is DNA (deoxyribonucleic acid), it is negatively charged in the buffer solution, so that the electrode which is the positive electrode along the direction in which the groove 3 extends (the direction connecting both electrodes 8) 8 (in Fig. 1, the left electrode).

The shape and thickness of the substrate 2 are not particularly limited to m, but can be any desired shape according to the application and purpose. However, since the molding is easy and the strength is high, it is preferable that the substrate 2 has a plate shape. When the material of the substrate 2 is an elastomer, the thickness of the substrate 2 is preferably 3 mm or more in order to secure adhesion to the cover 6. The groove 3 including the separation matrix 5 may be provided in plural or many on the surface of the substrate 2. Grooves 3 serving as flow paths for biomolecules such as DNA may cross each other in order to facilitate sample injection and mixing with other biomolecules or reagents such as DNA. Also, a circular or polygonal part is formed in the groove 3 (flow path) to control the resistance to the liquid in the groove 3 and the moving speed of the liquid, and to perform fluid adjustment at the micro level. May be performed. (Groove configuration)

 Hereinafter, a specific configuration or function of the groove 3 will be described. In the example shown in FIGS. 1 to 3, the groove 3 has a linear shape extending in the longitudinal direction of the substrate 2. However, the shape of the groove 3 is not limited to this, and may be, for example, U-shaped, S-shaped, or circular. However, in this case, the distribution of the electric field is not parallel to the groove 3 in the curved portion of the groove 3, so that it is necessary to perform the electric field correction and the detection correction by devising the shape of the groove 3.

 Although the electrophoresis cell 1 according to the present invention can be easily miniaturized, the width of the groove 3 is preferably 1 μm or more, and more preferably 2 μππι or more. If the width of the groove 3 is smaller than 1/2 m, the detection sensitivity is reduced, and a high-magnification optical device is required for detecting biomolecules, which increases the detection cost. Further, the width of the groove 3 is preferably not more than 1000 / xm, more preferably not more than 300 zm. If the width of the groove 3 exceeds 1 OO O / im, the uniformity of electrophoresis is impaired, and the effect of the present invention is reduced. However, when the present invention is applied to a separation matrix 5 having a two-dimensional spread provided with a plurality of sets of electrodes 8, for example, a separation matrix 5 of pulse field electrophoresis, the width of the groove 3 is 10. It may exceed 0 0 / zm.

 It is preferable that the depth of the groove 3 is 1 μπι or more. If the depth of the groove 3 is smaller than this, the effect of the present invention is reduced and the detection sensitivity is reduced. The depth of the groove 3 is preferably 500 μππ or less, more preferably 50 / zm or less. When the depth of the groove 3 exceeds 500 im, biomolecules such as DNA are diffused in the vertical direction, thereby reducing the effect of the present invention and making it difficult to focus optically on the detection of the biomolecules. This is because the sensitivity may be reduced.

 The length of the groove 3 and the length of the separation matrix 5 can be set arbitrarily. Specifically, for example, it can be determined in the range of 0.1 to 5 Omm depending on the target of the biomolecule to be separated and the resolving power required for each target. However, the upper limit of these lengths may be limited by the size of a silicon wafer to be described later, which will be described later.

 (Configuration of electrode)

Hereinafter, a specific configuration or function of the electrode 8 will be described. In this electrophoresis cell 1, Electrode 8 is a platinum wire having a diameter of 0.2 mm, and is connected to lead wire 9 of power supply 10 by soldering. As long as the electrode 8 can contact the buffer solution in the groove 3, the electrode 8 may have any shape of a line, a plate, and a film (a vapor-deposited film), and may have a size and a thickness. , Can be set arbitrarily. The material of the electrode 8 can be any material as long as a voltage of 500 V can be applied, a current of several tens of mA can flow, and the elution of the electrolysis material does not occur. You may. Examples of strong materials include platinum, gold, carbon, and conductive polymers.

 In the electrophoresis cell 1, the electrode 8 composed of a platinum wire is mostly disposed on the upper surface of the cover 6, and the vicinity of the tip is inserted into the groove 3 through the sample inlet 7 provided in the cover 6. Has been inserted. However, the electrode 8 may be directly adhered or deposited on the cover 6. Alternatively, wiring may be performed from an external support through the sample inlet 7 or through the substrate 2. Note that the vicinity of the tip of the electrode 8 may be inserted into the groove 3 through an electrode insertion hole separately formed in the cover 6 instead of passing through the sample injection port 7.

 In this electrophoresis cell 1, a DC voltage in the range of 0.1 to 500 V / cm is applied to the electrode 8. A pulse voltage may be applied. When a plurality of sets of electrodes 8 are provided, the direction of the current may be changed periodically.

 In this electrophoresis cell 1, as a buffer for electrophoresis filling the flow path in the groove 3 and the gap between the columns 4 constituting the separation matrix 5, the biomolecules to be separated are DNA. In the case of, use TBE buffer (89 mM Tris-borate, 2 mM E

DTA) or TAE buffer (4 OmM Tris-acetate 1 mM EDTA). The pH of the buffer is around 8. When the biomolecule to be separated is a protein, an optimal buffer for electrophoresis is used depending on the type of the protein.

 (Structure of pillar)

 Hereinafter, the specific configuration or function of the support 4 will be described. In this electrophoresis cell 1, as can be seen from FIG. 2, the shape of the top surface or the cross section (cross section cut along a plane perpendicular to the center axis of the column) of each column 4 constituting the separation matrix 5 (hereinafter, referred to as simply

It is called “cross-sectional shape”. ) Is circular. However, the cross-sectional shape of the support 4 does not need to be circular, and can be any shape. For example, as shown in FIGS. 4A to 4E, the cross-section of the column 4 is a rectangle with rounded corners (FIG. 4A), an oval (FIG. 4B), a square or a diamond (FIG. 4C). , Hexagon

(Fig. 4D) or rectangular (Fig. 4E). Alternatively, other polygons or polygons with corners cut off may be used. Furthermore, as long as the flow of biomolecules such as DNA is smooth, the cross-sectional shape may be a star shape, a geometric shape having protrusions, or may have no fixed shape. The cross-sectional shapes of the columns 4 need not be all the same, and the above-mentioned shapes may be mixed. For example, the cross-sectional shape of the column 4 may be elliptical near the sample inlet 7 and hexagonal at other portions.

 The cross-sectional area of each support 4 can be set arbitrarily as long as the dimension of the support 4 in the groove width direction is smaller than the width of the groove 3. However, when it is necessary to equalize the vertical flow of biomolecules such as DNA, it is preferable that the cross-sectional area of the column 4 be uniform in the vertical direction (the column axis direction). In the case where the biomolecules are separated in the depth direction of the groove 3, the support 4 may have a tapered shape that becomes thinner in any of the upper and lower directions. As long as molding is possible, the support 4 may have a three-dimensional geometric shape, for example, a shape in which a plurality of spheres continuously form a support.

 (Arrangement of pillars)

 Hereinafter, a specific arrangement form of the columns 4 will be described. In this electrophoresis cell 1, as is clear from FIGS. 1 and 2, the struts 4 are arranged in such a manner that a plurality of struts 4 spaced apart at equal intervals in the groove width direction are arranged at equal intervals in the longitudinal direction of the groove. Are arranged. However, the arrangement of the columns 4 does not need to be as described above, and can be arbitrary. In order to exert the function as the separation matrix 5, the struts 4 are arranged in predetermined regions of the grooves 3, but their arrangement or arrangement is regular as long as biological molecules such as DNA flow smoothly. It may or may not exist. However, if the arrangement of the pillars 4 is not regular, it is necessary to arrange the pillars 4 at random in order to generate a uniform sample flow.

FIGS. 5A to 5E show five specific examples of the arrangement of the columns 4 in the grooves 3 (top view of the substrate 2). The arrangement shown in FIG. 5A is a two-dimensional array pattern that can move biomolecules two-dimensionally in plan view. In this configuration, the rectangular Around the groove 3, four sample inlets 7 and two sets (four) of electrodes 8 are arranged. The arrangement form shown in FIG. 5B is an arrangement pattern in which the arrangement density of the columns 4 or the gap between the columns 4 changes stepwise in the longitudinal direction of the groove 3. The arrangement shown in FIG. 5C is an arrangement pattern in which the columns 4 are arranged in a row in the groove 3. The arrangement form shown in FIG. 5D is an arrangement pattern in which the columns 4 are arranged in a row in the groove 3 and the wall surface of the groove 3 functions as a column. That is, the separation matrix 5 is composed of an independent support 4 and a support 4 ′ which is a part of the wall surface of the groove 3. The arrangement shown in FIG. 4E is an arrangement pattern in which a plurality of columns 4 are arranged in the groove 3, and two sample injection ports 0.7 are connected to one longitudinal end of the groove 3.

 As described above, when the present invention is applied to a separation matrix for pulse field electrophoresis, for example, as shown in FIG. 5A, a two-dimensional filter having a plurality of sample inlets 7 and a plurality of sets of electrodes 8 is used. The separation matrix 5 of the self-pattern is required. In order to facilitate the injection of the sample into the groove 3, the arrangement density of the columns 4 may be reduced near the sample inlet 7 (see FIG. 5B). Further, a concentration region for biomolecules such as DNA may be provided, or a gate for selectively separating biomolecules may be provided. Further, a one-dimensional or two-dimensional gradient may be provided in the gap between the columns 4.

 The columns 4 may be arranged so as to be in contact with other plural columns 4 or may be arranged so as to be continuously contacted in the arrangement direction. Note that the column 4 may be in contact with the side surface of the groove 3. Further, the wall surface of the groove 3 may be repeatedly protruded into the groove in a shape corresponding to the column 4, and this protruding portion may be used as a column. In the simplest case, the separation matrix 5 is composed of only the protrusions on one wall surface of the groove 3 or only the protrusions on both wall surfaces. In this case, the electrophoresis cell 1 is a simple channel device without an independent support structure.

 (Sample injection □)

Hereinafter, the specific configuration and function of the sample inlet 7 will be described. The sample inlet 7 for injecting a sample containing a biomolecule into the groove 3 may be provided only one, or may be provided in plural or in number. When a plurality of sample inlets 7 are provided, the electrophoresis cell 1 exhibits a function of mixing a plurality of biomolecules in addition to a function of separating biomolecules (see FIG. 5E). In addition, as shown in FIG.6A, in order to make it easier to inject a sample containing a biomolecule such as DNA into the groove 3, a column 4 is arranged in the groove 3 where the sample inlet 7 is located. It is preferable to provide a region where no support is provided or a region where the arrangement density of the columns 4 is reduced. As shown in Fig. 6B, when the column 4 is provided at the same location as the other site at the site where the sample inlet 7 is located, the sample is introduced onto the separation matrix 5, It flows curvilinearly into the separation matrix 5 as shown by the arrow P1. In this case, it is difficult to smoothly inject the sample into the groove 3 because the resistance to the flow of the sample is large.

 On the other hand, as shown in FIG. 6C, when the column 4 is not provided at the position where the sample inlet 7 is arranged, the sample is introduced into the space where the column 4 does not exist, and then the arrow P 2 As shown, it flows straight and enters the separation matrix 5 from the side of the column 4. In this case, the sample can be smoothly injected into the groove 3 because the resistance to the flow of the sample is small.

 (Biomolecular separation principle)

 Hereinafter, the principle of separation of biomolecules in the electrophoresis cell 1 will be described. The spacing of struts 4 is the most important factor for the separation of biomolecules. In the following, for convenience, the narrowest part of the gap between the columns will be referred to as “column gap”, and the size or distance of the column gap will be referred to as “gap distance”. The gap distance must be large enough to allow the biomolecules to be separated through. In addition, the gap distance acts as an obstacle to the passage of biomolecules, and further affects the speed of passage of biomolecules, depending on the size of the molecule, for example, a parameter defined by the penetration radius of the molecule. Must be able to The separation principle based on the size (molecular weight) of a biomolecule such as DNA in the separation matrix 5 according to the present invention is the same as that in agarose genole or polyacrylamide genole. Therefore, it can be said that the electrophoresis cell 1 according to the present invention replaces the role of the gel network in electrophoresis with the separation matrix 5 including the plurality of columns 4.

Generally, the DNA in the buffer is a linear polymer in a random coil state, and the length of these displacements can be expressed as the mean squared end-to-end distance. For example, according to Paul J. Flory, who is famous for polymer solution theory, the change in DNA The order length R is defined by the following equation 1.

R = 0.3 X (number of bases) ^1/2 [nm] Equation 1 According to Equation 1, the size (displacement length or radius of gyration) of DNA increases in proportion to the half power of the number of bases. I do. The role of agarose or polyacrylamide gel networks in electrophoresis is to provide an obstacle to passing DNA. Therefore, if the size of the DNA and the gap distance of the separation matrix 5 are of the same order, the motion of the DNA molecular chain passing through the support gap is affected by the gap distance, and the support gap is closed. It is thought to play a role as a barrier. That is, when DNA passes through a pillar gap that is smaller than its size, the DNA must take a thermodynamically low entropy (deformed form) conformation. This is the principle that a difference occurs in the electrophoresis speed of DNA depending on the molecular weight of DNA.

 (Prop gap)

 Hereinafter, the support gap or gap distance will be described in more detail. As inferred from the above principle, if the gap distance is about the same as the size of DNA, the column gap becomes an obstacle to DNA movement, and separation depending on the molecular weight of DNA can be achieved. . Usually, agarose or polyacrylamide gels used for separation of oligonucleotides by electrophoresis and sequencing of 10 to 1000 bases of DNA have a network size of 100 to 100 nm (for example, published in 2000). Also, see the report by Yoshinobu Baba described in “Protein / Nucleic Acid 'Enzyme Journal,” Vol. 45, No. 1, pp. 76-84. Therefore, when separating a DNA fragment of 1000 bases or less, it can be said that the gap distance must be 100 nm or less (for example, see Japanese Patent Application Laid-Open No. 11-508042).

On the other hand, in the separation matrix 5 according to the present invention, the optimum gap distance is 0.1 to 5 / m. Hereinafter, the reason will be described. Table 1 shows the results of examining the relationship between gap distance and DNA mobility for three DNAs with different numbers of bases. According to Table 1, it can be seen that T4DNA (166 kbp) and LDNA (48.5 kbp) do not easily pass through the strut gap when the gap distance is 0.1 / im or less. This is due to the influence of water molecules hydrated on DNA, which causes This is probably because the force is larger than the actual size of the DNA. Table 1 Relationship between gap distance and DNA mobility

 X: DNA cannot pass. Δ: Partial pass. 〇: Smooth pass. That is, the physical size of a mesh such as agarose gel is 10 to 100 nm. In fact, the effects of sugar molecules and polyacrylamide molecular chains, or the effects of hydrated water molecules on DNA are added. In the case of agarose or polyacrylamide, these water molecules interact with the DNA and the separation matrix, which facilitates the separation of the DNA. Since these molecular chains are resilient, it is presumed that they use the effect of hydration water such as DNA or absorb the effects of water molecules. The size of these waters of hydration at the molecular level is hardly known at this time, but it is thought that DNA is unlikely to penetrate into the gaps at the 100 nm level due to the effects of water of hydration. Water can be permeated by artificially applying a physical force (such as pressure) to the gap by means of reverse osmosis or high pressure liquid chromatography. However, in this case, biomolecules such as DNA to be separated may be destroyed.

On the other hand, in the separation matrix 5 according to the present invention, the upper limit of the gap distance is 5 μm. Hereinafter, the reason will be described. Table 2 shows the results of examining the relationship between the gap distance and the migration speed of DNA for T4 DNA and LDNA. According to Table 2, when the gap distance exceeds 5 μπι, the difference in moving speed cannot be obtained reliably. Therefore, the upper limit of the gap distance is set to 5 μπι. Table 2 Relationship between gap distance and travel speed of DNA

 X: No speed difference △: Depending on conditions 〇: Speed difference

(Structure arrangement density)

Hereinafter, the arrangement density of the columns 4 will be described. The arrangement density of the columns 4 in the separation matrix 5 can be basically set arbitrarily. However, since the separation matrix 5 according to the present invention is based on the principle that the passage speed of DNA is affected by the column gap, the DNA separation ability is proportional to the number of column gaps through which DNA passes. And improve. According to experiments by the present inventor, the electrophoretic migration speed of DNA of 50 to 200 kbp (kilobase pairs) under the condition of 50 to 100 V / cm was 50 m / sec on average. The number of struts 4 (cylinders with a diameter of 15 μπι) that passed in one second was about three, and the number of strut gaps was six. At this time, a difference in the movement distance of about 0.1 / m per 1000 base pairs (move faster as the base pairs are shorter) was observed. Assuming that separation matrix 5 in groove 3 is 5 mm long, this DNA will pass through separation matrix 5 in about 100 seconds and travel about 10 / im per 1000 base pairs at the detection point 5 mm ahead. This will result in a difference in distance. If the optical resolution of the detection system is 5 μπι, this difference in travel distance can be detected sufficiently. In this case, the DNA passes through 60 pillars 4 and about 120 pillar gaps per lmm of its travel distance. Assuming that the DNA does not swim in the column gap perpendicular to the direction of the electric field, the number of column gaps is almost twice the number of columns 4. Therefore, the arrangement density of the pillars 4 is about 4000 pieces / mm ² , and the arrangement density of the pillar gaps is about 14000 pieces / mm ² . Since DNA shorter than this DNA migrates faster, the arrangement density of the support 4 or the arrangement gap of the support gap should be higher than 4000 pieces Zmm ² or 14000 pieces Zmm ² , respectively. Table 3 shows that the above experiment was repeated using T4 DNA and LDNA at various arrangement densities of the columns 4, and the relationship between the arrangement density of the columns 4 and the difference in the migration speed of DNA was determined. The results are shown below. According to Table 3, it is understood that the arrangement density of the columns 4 of the separation matrix 5 needs to be at least 1000 / mm ² . Table 3 Relationship between the arrangement density of the pillars and the difference in the migration speed of DNA

 X: No speed difference △: Depending on conditions 〇: Speed difference

(Material of substrate and separation matrix)

 Hereinafter, the materials of the substrate 2 and the separation matrix 5 will be described. The material of the substrate 2 and the separation matrix 5 is not particularly limited as long as it is an inexpensive and mass-produced polymer material. However, these materials are desirably transparent because of their ease of optical detection. Therefore, for example, elastomer can be used as the soft material, and plastic can be used as the hard material. The most common transparent materials that are elastomers include transparent synthetic rubbers such as silicone rubber, butadiene rubber, isoprene rubber, fluorine-based rubber, and styrene-based transparent rubber, and blended rubbers thereof. The performance should satisfy the following conditions.

 Density: ~ 1 g / m1

 Water supply rate: 0.1% or less

 Total light transmittance: 85% or more

 Mold shrinkage: 1 ° /. Less than

 Flexural modulus: 30 O Mpa or more

Any curing method may be used, but from the viewpoint of moldability, chemical initiators and crosslinking A method of initiating curing with an agent or a method of curing with energy rays such as heat curing or light is preferred.

 On the other hand, the plastic material preferably transmits the excitation light and the fluorescence. Examples of such a material include a polystyrene resin, a polysulfone resin, an acrylic resin, a polycarbonate resin, a polyolefin resin, a chlorine-containing polymer, a polyether resin, and a polyester resin. These resins can be in any form of homopolymer, copolymer, blend, and prepolymer. Although a thermoplastic resin is preferable in terms of molding, a thermosetting or energy ray-curable crosslinked polymer may be used. Further, it is desirable that the substrate 2, the groove 3, and the separation matrix 5 are simultaneously formed of the same material. However, if the bonding is sufficient, different materials may be laminated or fitted. In this case, as the material of the substrate 2, for example, ceramitter, glass, silicon, polymer alloy, or the like can be used.

 (Cover material)

 Hereinafter, the material of the cover 6 will be described. The material of the cover 6 can sufficiently transmit the excitation light and the fluorescent light, and the lower surface of the cover 6 is sufficiently adhered to the upper surface of the substrate 2, the groove 3 and the separation matrix 5 (post 4) to cause liquid leakage. There is no particular limitation as long as it does not cause the biomolecules such as DNA to be adsorbed. For example, glass, plastic, elastomer, and the like can be used. The surface of the cover 6 is preferably hydrophilic, but may be hydrophobic. The thickness of the cover 6 can be set arbitrarily. However, considering the optical focus of the high-magnification detection system, the thickness of the cover 6 is preferably 2 mm or less.

 (Surface treatment of material)

Hereinafter, the surface treatment of the materials of the substrate 2, the separation matrix 5, and the cover 6 will be described. The surface of the substrate 2 is preferably hydrophobic and has a contact angle with water of 45 degrees or more. On the other hand, the surface of the groove 3, the surface of the separation matrix 5 (post 4), and the surface of the portion of the force bar 6 covering the groove 3 make it easy for the buffer solution to come into contact with the buffer solution, and the buffer solution becomes It is preferably hydrophilic so that it is easy to spontaneously fill the gap of No. 4. Therefore, it is preferable to apply a hydrophilic treatment to these surfaces. The hydrophilization may be performed by any method. Specifically, for example, plasma processing, Plasma polymerization, corona discharge, coating of hydrophilic chemicals such as coating polymers, hydrophilic treatment by physical microstructure, and the like can be used.

 Examples of the coating polymer include polyhydroxymethyl methacrylate, polyacrylamide, and cellulosic polymer. It should be noted that these coating polymers must be cross-linked so that they do not dissolve during electrophoresis. Further, since the electroosmotic flow and electrophoresis can be controlled by the type and concentration of the ionic substance, the same effect can be obtained by adding an ionic substance to the buffer solution.

 The surface of the groove 3, the surface of the separation matrix 5 (post 4), and the surface of the cover 6 covering the groove 3 are the same as those of the substrate 2 even if they are hydrophobic. It is possible to use. In general, typical elastomers and plastics are hydrophobic without a surface treatment. If these are used as they are, the buffer cannot be filled in the groove 3 or the separation matrix 5 (the gap between the columns 4) by capillary action. Therefore, in this case, it is necessary to immerse the electrophoresis cell 1 in a buffer solution to perform a degassing treatment. However, once the buffer solution is filled in the groove 3 or the separation matrix 5, the effect of the present invention can be sufficiently exerted thereafter. If the electrophoresis cell 1 is entirely hydrophobic, for example, when an elastomer and a plastic are used, the adhesion between them is increased. Furthermore, the effect of reducing nonspecific adsorption of biomolecules and making the electrophoresis cell 1 less susceptible to contamination can be obtained.

 (Method of manufacturing electrophoresis cell)

Hereinafter, a specific method for manufacturing the electrophoresis cell 1 will be described. The electrophoresis cell 1 is manufactured by the following procedure. The materials of the electrophoresis cell 1 are all polymeric materials, and these polymeric materials are processed by molding. As the molding method of polymer materials, compression molding method, injection molding method, extrusion molding method, blow molding method, molding method, thin molding method, etc. are known. Any method can be used as long as the structure can be formed. In this embodiment, the following two methods are used. That is, one method is to use a microfabricated silicon (S i) as a 銬 type and cast an elastomer to transfer a microstructure. The other is to add a glass roll to a micromachined silicon mold. This is a method in which a fine structure is transferred by pressing a thermoplastic polymer heated above the transfer temperature.

 Examples of the 铸 -type material include glass, silicon, ceramic, metal, and plastic. However, any material can be used as long as it can finely process a negative pattern on a nanometer to submicron level. As a method for fabricating a mold, it is possible to use a method in which a mask is formed and then dry-etched, or a method in which the resist is directly finely processed by lithography. However, in this embodiment, a silicon etching technique is used to improve the peelability. According to this method, the surface of the fine structure of the negative pattern can be smoothed, whereby the releasability and transferability of the fine structure can be further improved.

 (Production of type 铸)

 Hereinafter, with reference to FIGS. 7A to 7G, a method for manufacturing a mold, and a substrate 2 provided with a groove 3 including a separation matrix 5 using the mold (hereinafter, referred to as a “device”). The method for manufacturing the will be described more specifically.

 First, as shown in FIG. 7A, a silicon wafer 11 is prepared. Subsequently, as shown in FIG. 7B, a photosensitive organic film 12 is applied to the upper surface of the silicon wafer 11. Next, as shown in FIG. 7C, on the silicon wafer 11 with the photosensitive organic film 12, a region 14 a transmitting light and a region 14 b not transmitting light are formed in a predetermined pattern. Place the formed mask 14. Then, a predetermined area of the photosensitive organic film 12 is irradiated with light 13 via the mask 14 and developed using a developing solution. For example, when a so-called positive photosensitive organic film in which the exposed portion is dissolved is used as the photosensitive organic film 12, the photosensitive organic film 12 is removed at a portion corresponding to the light-transmitting region 14 a, A hole 15 is formed.

Next, as shown in FIG. 7D, the silicon wafer # 1 is etched. As an etching method, an inductive electromagnetic coupling plasma reactive plasma etching, which is an existing technology, is used. As the reaction gas, a gas mainly containing fluorocarbon is used. By this etching, the silicon wafer 11 is etched at a portion corresponding to the hole 15 formed in the photosensitive organic film 12, that is, a portion not covered with the photosensitive organic film 12, and the concave portion 16 is formed. It is formed. After that, the photosensitive organic film 1 2 Using an organic solvent. As a result, as shown in FIG. 7E, a type 17 force S made of silicon is obtained.

 Then, a device is manufactured using this type 17. Specifically, first, as shown in FIG. 7F, a silicone rubber melt 18 is applied to a mold 17 made of silicon. The surface of the mold 17 is preliminarily subjected to a treatment for maintaining hydrophobicity, for example, by applying a silane coupling agent. By the treatment for maintaining the hydrophobicity, it is possible to improve the releasability between the silicone rubber and the mold 17 in the peeling step described later.

 After solidifying the silicone rubber solution 18 at room temperature, the solidified product is peeled off from the mold 17. As a result, a device made of silicone rubber (silicone rubber structure), that is, a substrate 2 having a groove 3 containing a separation matrix 5 is obtained. Further, as shown in FIG. 7G, a cover 16 having a sample injection port (through hole) is arranged at a predetermined position on the device. As the cover 6, a glass substrate or a plastic substrate can be used. Then, by pressing the cover 6 to the device, the electrophoresis cell 1 in which the cover 6 and the device made of silicone rubber are in close contact with each other is completed. Since the silicone rubber has adhesiveness, the cover 6 made of a glass substrate or a plastic substrate and the device can be sealed simply by pressing the cover 6 on the device, and there is no problem of liquid leakage.

 Thus, the type 17 or electrophoresis cell 1 can be manufactured by a very simple method. Further, since the type 17 made of silicon can be used repeatedly, the productivity and production cost of the electrophoresis cell 1 can be improved. The device, that is, the substrate 2 provided with the groove 3 including the separation matrix 5 may be manufactured by a method different from the above-described manufacturing method. For example, a plastic device can be obtained by pressing a plastic plate against a press die while applying heat to a silicon die. After that, if the cover 6 is attached to the device, the electrophoresis cell 1 is completed. As described above, in the device or the electrophoresis cell 1 using plastic, the strength is improved as compared with the case where silicone rubber is used.

Also, instead of the type 17 made of silicon, the type 17 made of a thick photosensitive organic film is used. May be used. In this case, for example, a thick photosensitive organic film may be patterned by light irradiation, and the patterned organic film may be used as it is as a 錄 type. According to this method, the etching step of the silicon wafer 11 shown in FIG. 7D can be omitted, so that the productivity can be further improved.

 (Method of manufacturing PDMS chip)

 Hereinafter, the process or results of actually manufacturing the electrophoresis cell 1 using PDMS (polydimethylsiloxane), which is a typical silicone rubber, as a device material will be described. In this manufacturing method, the negative pattern of the silicon wafer was formed by the fine processing technology. This mold has a mold surface corresponding to the groove containing the separation matrix. This mold was placed in a plastic petri dish having a diameter of 90 cm, and PDMS was injected from above. The PDMS used was purchased from Toray Industries, Inc. (trade name “SYLGARD”).

 The silicon wafer (silicon substrate) was pre-treated with a silane coupling agent. After applying a solution of 4% dimethyldichlorosilane in acetonitrile on the surface of the silicon wafer, the silicon wafer was dried at 100 ° C. for 30 minutes. About 4 Om 1 of PDMS was placed in a 10 Om 1 beaker, and further 4 mL of a catalyst was added and mixed well. The beaker was placed in a desiccator, and deaerated with a vacuum pump for 30 minutes.

 Thereafter, 2 Oml of degassed PDMS was put into a plastic petri dish containing a silicon wafer, and this plastic petri dish was put in a desiccator, and degassed by a vacuum pump for 30 minutes. After sufficiently removing bubbles from the groove containing the separation matrix, air was introduced into the desiccator, and the plastic petri dish was taken out. Then, the degassed PDMS was laminated (overlaid) to a thickness of about 5 to 7 mm on the silicon wafer. The plastic petri dish on which the PDMS was laminated was placed in a constant temperature box at 40 ° C to polymerize the PDMS. After more than 48 hours, the plastic dish was removed. Then, a notch was cut with a cutter around the required part of the separation matrix, and the polymerized PDMS was slowly peeled off from the silicon wafer type III to obtain a PDMS chip.

Same as PDMS chip (width 5mm, length 20mm, thickness 5mm) as cover A polystyrene board of one dimension (5mm in width, 20mm in length, lmm in thickness) was covered. If the adhesion is poor, apply oxygen plasma treatment (oxygen pressure 1.0 kPa, excitation power 100 w, treatment time 5 min) or ultraviolet treatment (20 w ultraviolet lamp, treatment) on the groove side surface of the PDMS chip. A time of 20 minutes and a distance of 10 cm) should be applied to improve the adhesion. On the cover, two sample injection holes (holes) with a diameter of lmm were formed along the groove. An electrode consisting of a platinum wire (diameter: 0.2 mm) was wired so that the vicinity of the tip directly touched the buffer solution in the groove through the sample inlet (see Fig. 1). It should be noted that when the electrodes are formed by platinum vapor deposition, the same effect as when a platinum wire is used is obviously obtained.

 As a buffer for electrophoresis of DNA, TBE buffer (89 mM Tris-borate, 2 mM EDTA) was used. When the oxygen plasma treatment is applied to the PDMS chip, the surface of the PDMS chip becomes hydrophilic, so that the buffer solution enters the gap between the columns of the separation matrix by capillary action. When such a hydrophilic treatment is not performed, buffer solution is supplied into the groove from one sample inlet, and the other sample inlet is sucked by a syringe to fill the buffer into the separation matrix. Just fine. Alternatively, the PDMS chip may be immersed in a buffer solution, the pressure may be reduced in a vacuum desiccator, and the buffer solution may be filled in the separation matrix. In the latter case, the cover must be carefully placed on the PDMS chip after degassing.

 (Perform electrophoresis)

Hereinafter, the process and results of actual electrophoresis using the electrophoresis cell according to the present invention will be described. A DNA sample was used as a biomolecule sample. As DNA samples, LDNA, T4 phage DNA, and 1/2 DNA obtained by cutting DNA into about half with restriction enzyme Xbo-I were used. The base numbers of these DNAs were 48500, 166000 and 24000, respectively. Each of these DNAs contains 0.05 / X g of TBE buffer per 11 and contains YOPRO-I (manufactured by Molecular Probes), Cyber Green (manufactured by Takara Shuzo) or Cyber Gold (manufactured by Takara Shuzo) as a fluorescent staining sample. A DNA mixture containing μΜ was used as a sample. This sample was used in an amount of 1 μL per electrophoresis. Then, the sample was injected into one of the two sample inlets formed on the cover, and electrophoresis was performed. The power supply for electrophoresis is 0 to: A 1000 V, 1 A rated ATTO cross power 1000 was used. With this power supply, a voltage of 50 to 10 OV / cm was applied to both ends of an electrode made of a platinum wire. The current was less than 1 OmA. Since DNA is negatively charged in the TBE buffer, it migrates toward the positive electrode. When the groove length was 1 cm, the electrophoresis time was 2 to 5 minutes.

 (Electrophoresis observation)

 Hereinafter, an example of an electrophoresis observation method will be described. FIG. 8 shows a schematic configuration of an electrophoresis evaluation apparatus for observing or recording DNA electrophoresis.

 As shown in FIG. 8, the electrophoresis evaluation device includes a fluorescence microscope 20. The fluorescence microscope 20 includes a sample holder 22 on which a sample 21 (electrophoresis cell) is placed, a light source 23 having an excitation-side filter (not shown), a dichroic mirror 24, and an observation device for observing DNA. A window 25, a switching mirror 26 for switching an optical path, a photomultiplier tube 27 for converting light into an electric signal, and a video camera 28 (high-sensitivity CCD camera) are provided. The electrophoresis evaluation apparatus also includes a VTR 29 (video 'tape' recorder) for recording images, a personal computer 30, and a display 31.

 In the fluorescence microscope 20, the light L1 emitted from the light source 23 and having its wavelength adjusted to 420 to 490 nm by the excitation side filter is reflected by the dichroic mirror 24, and then irradiated to the sample 21. As a result, the DNA in the sample emits fluorescence. This fluorescence is adjusted to light L2 having a wavelength of 520 nm by an absorption filter (not shown). The light L 2 is split by the dichroic mirror 24 into light L 3 traveling toward the observation window 25 and light L 4 traveling toward the photomultiplier tube 27 or video camera 28.

Thus, by observing the observation window 25, it is possible to observe a state in which the fluorescent DNA molecule passes through the inside of the separation matrix (the gap between the columns) by electrophoresis. The light L4 is input to the photomultiplier tube 27 or the video camera 28 by switching the optical path by the switching mirror 26. The photomultiplier 27 converts the light L4 into an electric signal and outputs it to an external device. Video camera 28, a high-sensitivity CCD camera, allows DNA molecules to pass through the separation matrix by electrophoresis. Take a picture of the situation and send the resulting image to VTR 29. VTR 29 records this image. The image recorded on the VTR 29 can be analyzed using the personal computer 30 and can be displayed on the display 31.

 (Relationship between base number and moving speed)

 Hereinafter, the relationship between the number of bases of DNA and the migration speed of DNA by electrophoresis (electrophoresis speed) will be described.

 FIG. 9 shows the relationship between the number of bases of DNA and the migration speed of DNA under the following conditions.

 Groove width: 200 μm

 Groove depth: 10 ju m

 Separating Matritus Length: 1 Omm

 Prop shape: cylinder,

 Prop diameter: 15 μπι

 Support gap: approx. 1 μm

Support density: 4000 pieces Zmm ²

 Voltage: 50 V / cm

 Buffer: T BE buffer

 DNA concentration: 50 ηg / μ1

 Stain: Cyber Green 1 mM

 As is evident from FIG. 9, the migration speed of DNA by electrophoresis changes significantly depending on the number of bases in DNA. Therefore, it can be seen that the separation matrix according to the present invention, which includes artificial columns and gaps, has the same effect as the mesh of agarose gel or polyacrylamide gel, and can be substituted with these phenols.

 (Dna's mobile model)

FIG. 10 shows a columnar column 4 and a motion model of the DNA passing through a gap between the column 4. As shown in FIG. 10, it is considered that the gap between the pillars 4 serving as an obstacle plays a role of an entropy barrier against the movement of the DNA passing through the gap. In other words, when the globular DNA molecule passes through the gap of the strut 4, it temporarily takes on a thermodynamically low entropy conformation, deforms, and passes through the gap. You. This is thought to affect the speed of DNA migration by electrophoresis. (Application Example of Electrophoresis Cell According to the Present Invention)

 The basic electrophoresis cell according to the present invention has been described in detail in terms of its shape, material, production method, principle of DNA separation, kinetic model of DNA, and the like. A DNA sampler (biological sample sampler) using a typical electrophoresis cell is described.

 (DNA sampler 1: Application example 1)

 FIG. 11 shows one DNA sorting apparatus to which the electrophoresis cell according to the present invention is applied. As shown in Fig. 11, in this DNA sorting apparatus, the separation matrix is divided into five pillar areas A to E in which the arrangement density of pillars or the gaps between pillars (pillar gaps) are different. Here, in pillar areas A, B, C, D, and E, the arrangement density of the pillars increases in this order (the gap between the pillars decreases). The DNA separation apparatus has one sample inlet 33 with a cathode and five sample recovery ports 34 a to 34 e provided in the pillar areas A to E with anodes, respectively. .

 Thus, in this DNA sorting apparatus, for example, if samples containing DNAs having different molecular weights are supplied to the sample injection port 33, the DNA having a large molecular weight can be placed in the pillar area (the pillar area E side) where the arrangement density of the columns is large. ), It is concentrated in the pillar area (pillar area A side) where the arrangement density of the columns is low, and collected from the sample collection port of this pillar area. Therefore, the DNA present in the pillar areas A, B, C, D, and E, or the DNA recovered at the sample recovery ports 34a to 34e has a smaller molecular weight as the latter. Therefore, the DNA in the sample can be fractionated (fractionated) into five types according to the molecular weight.

 (DN A fractionation device 2: Application example 2)

 12A and 12B show another example of the application of the electrophoresis cell according to the present invention.

1 shows a DNA sorting apparatus. As shown in FIGS. 12A and 12B, in this DNA sorting apparatus, the separation matrix 5 is two-dimensional in a vertical direction (XI-X2 direction) and a horizontal direction (Yl-Y2 direction) in plan view. It is composed of a large number of prism-shaped pillars 4 that are spaced apart from each other. In addition, this DNA sorter has a cathode It has one sample inlet 35 and six sample recovery ports 36a to 36f, each with an anode.

 In this separation matrix 5, in a certain column 4 and a gap formed between the column 4 and two columns 4 adjacent in the X1-X2 direction on the X2 side and extending in the Y1-Y2 direction, The gap 38 located on the Y1 side is wide, and the gap 39 located on the Y2 side is narrow. On the other hand, the gaps 40 extending in the X1-X2 direction between the columns 4 adjacent in the Yl-Y2 direction are all wide. A substantially rectangular space 4 is provided at the connection between the end on the X2 side of the gap 40 extending in the X1-X2 direction and the gaps 38, 39 extending in the Y1-Y2 direction. 1 is formed.

 Thus, in this DNA sorting apparatus, for example, if a sample containing DNAs having different molecular weights is supplied to the sample inlet 35, the DNA having a large molecular weight cannot pass through the narrow gap 39, so that, for example, R As shown by 1, in the 丫 1- ¥ 2 direction (lateral direction), it does not move in the Y1 direction, but moves only in the Y2 direction. In contrast, DNA with a small molecular weight can move in both the Y1 and Y2 directions. However, as described above, since the DNA with a large molecular weight almost moves in the Y2 direction, the influence of the influence increases the tendency of the DNA with a small molecular weight to move in the Y1 direction as shown by, for example, R2. Therefore, in the separation matrix 5, the DNA recovered at the sample recovery ports 36a to 36f has a smaller molecular weight as the latter. Therefore, the DNA in the sump can be fractionated (fractionated) into six types according to the molecular weight.

 (Application to medical diagnosis)

The electrophoresis cell or the DNA sorting apparatus (biological sample sorting apparatus) according to the present invention can be applied to medical diagnosis or gene polymorphism detection. For example, in human genes, there are slight differences (genetic polymorphisms) in the nucleotide sequence of each individual. It is said that the difference lies in the gene regulatory region, the intron region, or the region encoding a protein. The difference between the gene regulatory region and the synchrotron region indicates that the expression level of the gene varies among individuals. In addition, regions encoding proteins and the like indicate that the activity of the enzyme varies from individual to individual. It is said that individual differences are determined by combinations of genetic polymorphisms. For example, the metabolism of drugs There are differences, which are closely related to the efficacy and side effects of the drug. In other words, these drug effects and side effects are greatly affected by genetic polymorphisms in the drug metabolism gene group possessed by the individual. Although there are various types of genetic polymorphisms, single nucleotide polymorphisms (SNPs: single nucleotide polymorphisms) are currently the largest in number and are said to be easy for mass analysis. SNP s is the part where one gene code (one base) is replaced by another base between individuals, and it is estimated that there are 300-1000 thousand human genomes. Therefore, it is expected that genetic polymorphism information of drug metabolism genes will be useful for drug development, effective use of drugs, and prevention of side effects.

 Currently, SNPs analysis methods include restriction enzyme method, SCSP (single-strand conformation polymorphisms) angle? Analysis methods, direct sequencing methods, and electrochemical detection methods are known. Among these, the ones that are analyzed by electrophoresis are the restriction enzyme method, SSCP angle analysis, and the direct sequencing method. An electrophoresis cell using the separation matrix according to the present invention can be applied to such rapid separation and detection. By using the electrophoresis cell according to the present invention, SNPs can be rapidly detected with a small amount of sample. Among these, the SSCP method is suitable for the present invention because it is amplified using the PCR technique and a change in the three-dimensional structure is detected by electrophoresis. In this specification, DNA and biomolecules are targeted, but the targets are not limited to biomolecules. It can also be applied to separation and detection of environmental chemicals and high molecular compounds. In addition, the separation matrix and its manufacturing method are expected to be useful for separation and detection in microchannels such as chemical microdevices and lab-on-a-chip, which have recently been talked about.

 While the present invention has been described with reference to specific embodiments thereof, it will be obvious to those skilled in the art that many more variations and modifications are possible. Therefore, the present invention should not be limited by such embodiments, but should be limited by the appended claims. Industrial potential

As described above, the biomolecule separation cell such as the electrophoresis cell according to the present invention and the method for producing the same, and the DNA sampler or the biological sample sorter are particularly suitable for DNA and the like. It is useful for detecting or sorting biomolecules, and is suitable for use as a medical testing device or a diagnostic device.

Claims

The scope of the claims 1. A biomolecule separation cell for separating or detecting biomolecules in a liquid sample, the substrate having a groove formed on a surface thereof;  A separation matrix comprising a plurality of struts arranged in a partial area of the groove, wherein the narrowest part of the gap between the struts is 0.1 to 5 // m;  A force bar that covers the surface of the substrate and the groove, and a close contact with the upper surface of the support, and a buffer solution that fills the groove,  At least two electrodes that directly contact the buffer and apply a voltage to the buffer;  A biomolecule separation cell formed of the above substrate, the above separation matrix, and a polymer material.  2. The biomolecule separation cell according to claim 1, wherein the biomolecule is separated by electrophoresis. 3. The biomolecule separation cell according to claim 2, wherein the cross section of the support is a circle, an ellipse, a polygon, or a polygon in which a corner is rounded or cut off.  4. The biomolecule separation cell according to claim 2, wherein at least a part of the substrate and the separation matrix is formed of an elastomer or a plastic. 5. The biomolecule separation catalyst according to claim 4, wherein the elastomer is silicone rubber.  6. The biomolecule separation cell according to claim 4, wherein the plastic is an acrylic resin, a polystyrene resin, or a polycarbonate resin.  7. The groove, wherein the groove has a width of 20 to: L000 microns, a depth of l to 50 / xm, and a length of 0.1 to 50 mm. 3. The biomolecule separation cell according to 2. 8. The biomolecule separation cell according to claim 2, wherein the arrangement density of the columns is 1000 mm ² or more. 9. A sample inlet for injecting a biomolecule sample into the groove is provided on the cover, and a region or a column having a lower arrangement density of the columns than the other regions is provided near the sample inlet of the separation matrix. The living body according to claim 2, which has a non-existent region. Molecular separation cell.  10. A biomolecule for separating or detecting a biomolecule in a liquid sample, comprising: a substrate having a groove formed on its surface; and a separation matrix consisting of a plurality of columns arranged in a part of the groove. A method for manufacturing a separation cell, comprising:  A step of subjecting a silicon substrate, a glass substrate, or a polymer substrate to microfabrication to form a mold having a negative pattern corresponding to the groove and the support;  A step of pouring a liquid elastomer or plastic, or a monomer or prepolymer as a raw material of the elastomer or plastic into the mold 铸, and solidifying;  Separating the solidified product from the mold to obtain a biomolecule separation cell including the substrate and the separation matrix.  1 1. Biomolecules for separating or detecting biomolecules in a liquid sample, comprising: a substrate having a groove formed on the surface thereof; and a separation matrix including a plurality of columns arranged in a part of the groove. A method for manufacturing a separation cell, comprising:  A step of subjecting a silicon substrate, a glass substrate, or a polymer substrate to fine processing to form a mold having a negative pattern corresponding to the groove and the support,  After pressing the plastic heated above the glass transition temperature against the mold, lowering the temperature and curing the plastic;  Separating the cured product from the mold to obtain a biomolecule separation cell including the substrate and the separation matrix.  1 2. A substrate with a groove formed on the surface,  A separation matrix formed of a plurality of struts arranged in a partial area of the groove and having a narrowest gap between the struts of 0.1 to 5 μm;  A force bar that covers the surface of the substrate and the groove, and a close contact with the upper surface of the support, and a buffer solution that fills the groove,  At least two electrodes that directly contact the buffer and apply a voltage to the buffer;  The substrate and the separation matrix are formed of a polymer material, From the liquid sample containing DNA supplied into the groove, the characteristics and structure of the DNA A DNA sorting device adapted to sort or fractionate the DNA based on the DNA.