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WO2004008132A1 - Cellule de separation biomoleculaire, procede de fabrication de celle-ci et appareil de fragmentation de l'adn - Google Patents

Cellule de separation biomoleculaire, procede de fabrication de celle-ci et appareil de fragmentation de l'adn Download PDF

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Publication number
WO2004008132A1
WO2004008132A1 PCT/JP2002/007044 JP0207044W WO2004008132A1 WO 2004008132 A1 WO2004008132 A1 WO 2004008132A1 JP 0207044 W JP0207044 W JP 0207044W WO 2004008132 A1 WO2004008132 A1 WO 2004008132A1
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WIPO (PCT)
Prior art keywords
groove
substrate
dna
separation
biomolecule
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2002/007044
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English (en)
Japanese (ja)
Inventor
Kenichi Inatomi
Shinichi Izuo
Hiroshi Ohji
Satoru Shiono
Kazuhiko Tsutsumi
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Mitsubishi Electric Corp
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Mitsubishi Electric Corp
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Priority to JP2004521103A priority Critical patent/JPWO2004008132A1/ja
Priority to PCT/JP2002/007044 priority patent/WO2004008132A1/fr
Publication of WO2004008132A1 publication Critical patent/WO2004008132A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • 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.
  • 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) ⁇ charge. As the electrophoresis method, a method using agarose gel (agar gel) or polyacrylamide gel as the separation matrix has been established.
  • 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.
  • the distance between the arrangements of the artificial mouth structure is 100 nm or less, and preferably 10 to 30 nm.
  • the diameter and gap of the support are several ⁇ m 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.
  • 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.
  • 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 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 ⁇ .
  • 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.
  • a separation matrix using artificial micro obstacles is used as an alternative to a gel for electrophoresis, for example.
  • the separation matrix can be reduced in size and weight, and can be stored in a dry state.
  • biomolecules such as DNA can be separated or detected quickly and with high sensitivity even if the sample is small.
  • 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.
  • an elastomer for example, it is preferable to use a silicone rubber which is a general material and which is inexpensive and has good moldability.
  • 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 ⁇ , 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solidified product is separated from the ⁇ type to obtain a biomolecule separation cell including a substrate and a separation matrix.
  • a ⁇ -shaped fine structure can be faithfully transferred, and mass production of electrophoresis cells becomes possible.
  • 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.
  • a press mold having a negative pattern corresponding to grooves and columns.
  • the plastic heated above the glass transition temperature is pressed against the press mold, the temperature is reduced and the plastic is cured.
  • the cured product is separated from the press mold to obtain a biomolecule separation cell including a substrate and a separation matrix.
  • the DNA sorting apparatus 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • the groove 3 has a linear shape extending in the longitudinal direction of the substrate 2.
  • the shape of the groove 3 is not limited to this, and may be, for example, U-shaped, S-shaped, or circular.
  • 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.
  • the width of the groove 3 is preferably 1 ⁇ m or more, and more preferably 2 ⁇ 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.
  • the width of the groove 3 is 10. It may exceed 0 0 / zm.
  • the depth of the groove 3 is 1 ⁇ 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 ⁇ 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.
  • 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.
  • 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.
  • the electrode 8 may be directly adhered or deposited on the cover 6.
  • wiring may be performed from an external support through the sample inlet 7 or through the substrate 2.
  • 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.
  • 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.
  • the direction of the current may be changed periodically.
  • the biomolecules to be separated are DNA.
  • TBE buffer 89 mM Tris-borate, 2 mM E
  • TAE buffer OmM Tris-acetate 1 mM EDTA.
  • the pH of the buffer is around 8.
  • an optimal buffer for electrophoresis is used depending on the type of the protein.
  • cross-sectional shape Is circular.
  • the cross-sectional shape of the support 4 does not need to be circular, and can be any shape.
  • 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).
  • 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 Hexagon
  • 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.
  • the cross-sectional shape of the column 4 may be elliptical near the sample inlet 7 and hexagonal at other portions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the arrangement density of the columns 4 may be reduced near the sample inlet 7 (see FIG. 5B).
  • a concentration region for biomolecules such as DNA may be provided, or a gate for selectively separating biomolecules may be provided.
  • 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.
  • 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.
  • the electrophoresis cell 1 is a simple channel device without an independent support structure.
  • 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.
  • the electrophoresis cell 1 exhibits a function of mixing a plurality of biomolecules in addition to a function of separating biomolecules (see FIG. 5E).
  • a column 4 is arranged in the groove 3 where the sample inlet 7 is located.
  • the sample 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.
  • the spacing of struts 4 is the most important factor for the separation of biomolecules.
  • 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.
  • 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.
  • 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.
  • 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.
  • the change in DNA is defined by the following equation 1.
  • 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.
  • the support gap or gap distance 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. .
  • 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).
  • the optimum gap distance is 0.1 to 5 / m.
  • 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.
  • X DNA cannot pass.
  • ⁇ Partial pass.
  • ⁇ Smooth pass. That is, the physical size of a mesh such as agarose gel is 10 to 100 nm.
  • the effects of sugar molecules and polyacrylamide molecular chains, or the effects of hydrated water molecules on DNA are added.
  • 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 upper limit of the gap distance is 5 ⁇ m.
  • 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 ⁇ , the difference in moving speed cannot be obtained reliably. Therefore, the upper limit of the gap distance is set to 5 ⁇ . Table 2 Relationship between gap distance and travel speed of DNA
  • the arrangement density of the columns 4 in the separation matrix 5 can be basically set arbitrarily.
  • 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.
  • 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 ⁇ ) that passed in one second was about three, and the number of strut gaps was six.
  • the arrangement density of the pillars 4 is about 4000 pieces / mm 2
  • 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
  • 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.
  • elastomer can be used as the soft material
  • plastic can be used as the hard material.
  • 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.
  • 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.
  • the plastic material preferably transmits the excitation light and the fluorescence.
  • 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.
  • a thermoplastic resin is preferable in terms of molding, a thermosetting or energy ray-curable crosslinked polymer may be used.
  • 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.
  • 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.
  • the surface of the substrate 2 is preferably hydrophobic and has a contact angle with water of 45 degrees or more.
  • 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.
  • coating polymer examples 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.
  • 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.
  • 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.
  • the following two methods are used. That is, one method is to use a microfabricated silicon (S i) as a ⁇ 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.
  • the ⁇ -type material examples include glass, silicon, ceramic, metal, and plastic.
  • any material can be used as long as it can finely process a negative pattern on a nanometer to submicron level.
  • 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.
  • 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.
  • 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.
  • a silicon wafer 11 is prepared.
  • a photosensitive organic film 12 is applied to the upper surface of the silicon wafer 11.
  • 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.
  • 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.
  • the silicon wafer # 1 is etched.
  • an etching method an inductive electromagnetic coupling plasma reactive plasma etching, which is an existing technology, is used.
  • the reaction gas a gas mainly containing fluorocarbon is used.
  • 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.
  • the photosensitive organic film 1 2 Using an organic solvent.
  • a type 17 force S made of silicon is obtained.
  • 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.
  • a 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.
  • a device made of silicone rubber that is, a substrate 2 having a groove 3 containing a separation matrix 5 is obtained.
  • a cover 16 having a sample injection port (through hole) is arranged at a predetermined position on the device.
  • a glass substrate or a plastic substrate can be used as the cover 6, a glass substrate or a plastic substrate can be used.
  • 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.
  • 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.
  • the type 17 made of silicon instead of the type 17 made of silicon, the type 17 made of a thick photosensitive organic film is used. May be used.
  • a thick photosensitive organic film may be patterned by light irradiation, and the patterned organic film may be used as it is as a ⁇ 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.
  • the process or results of actually manufacturing the electrophoresis cell 1 using PDMS polydimethylsiloxane
  • PDMS polydimethylsiloxane
  • 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.
  • PDMS chip width 5mm, length 20mm, thickness 5mm
  • 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.
  • oxygen plasma treatment oxygen pressure 1.0 kPa, excitation power 100 w, treatment time 5 min
  • ultraviolet treatment (20 w ultraviolet lamp, treatment
  • TBE buffer 89 mM Tris-borate, 2 mM EDTA
  • 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.
  • 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.
  • 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.
  • a DNA sample was used as a biomolecule sample.
  • 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 ⁇ was used as a sample. This sample was used in an amount of 1 ⁇ L 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.
  • FIG. 8 shows a schematic configuration of an electrophoresis evaluation apparatus for observing or recording DNA electrophoresis.
  • 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.
  • VTR 29 video 'tape' recorder
  • 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.
  • 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.
  • 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.
  • FIG. 9 shows the relationship between the number of bases of DNA and the migration speed of DNA under the following conditions.
  • Support density 4000 pieces Zmm 2
  • 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.
  • FIG. 10 shows a columnar column 4 and a motion model of the DNA passing through a gap between the column 4.
  • 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.
  • 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.
  • FIG. 11 shows one DNA sorting apparatus to which the electrophoresis cell according to the present invention is applied.
  • 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.
  • 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. .
  • 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.
  • 12A and 12B show another example of the application of the electrophoresis cell according to the present invention.
  • 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.
  • this DNA sorter has a cathode It has one sample inlet 35 and six sample recovery ports 36a to 36f, each with an anode.
  • 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 ⁇ 1- ⁇ 2 direction (lateral direction), it does not move in the Y1 direction, but moves only in the Y2 direction.
  • DNA with a small molecular weight can move in both the Y1 and Y2 directions.
  • 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.
  • 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.
  • 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.
  • 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.
  • SNPs single nucleotide polymorphisms
  • SNPs analysis methods include restriction enzyme method, SCSP (single-strand conformation polymorphisms) angle? Analysis methods, direct sequencing methods, and electrochemical detection methods are known.
  • 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.
  • SNPs can be rapidly detected with a small amount of sample.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne une cellule d'électrophorèse comprenant un substrat (2), une rainure (3) étant formée sur une surface supérieure de ce substrat (2). Celui-ci (2) est constitué d'un matériau transparent permettant à un utilisateur de voir à travers le substrat (2). La rainure (3) comprend une matrice de séparation (5) présentant un nombre important de renforts (4). La surface supérieure du substrat (2) et la rainure (3) sont recouvertes d'un élément couvrant transparent (6). De plus, la cellule d'électrophorèse (1) comprend deux orifices d'admission (7) d'échantillons et deux électrodes (8). Celles-ci sont introduites dans la rainure (3) par l'intermédiaire des orifices d'admission (7) d'échantillons correspondants. Les deux électrodes (8) sont connectées à une source de courant CC (10), par l'intermédiaire d'un fil conducteur (9). Cette cellule d'électrophorèse (1) permet de palier aux désavantages du gel d'agarose et du gel d'amide polyacrylique utilisés dans l'analyse par électrophorèse ou dans une matrice de séparation artificielle classique.
PCT/JP2002/007044 2002-07-11 2002-07-11 Cellule de separation biomoleculaire, procede de fabrication de celle-ci et appareil de fragmentation de l'adn Ceased WO2004008132A1 (fr)

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PCT/JP2002/007044 WO2004008132A1 (fr) 2002-07-11 2002-07-11 Cellule de separation biomoleculaire, procede de fabrication de celle-ci et appareil de fragmentation de l'adn

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JP2007130836A (ja) * 2005-11-09 2007-05-31 Ushio Inc 接合方法
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JP2009541737A (ja) * 2006-06-20 2009-11-26 オーミック・アクチボラゲット アッセイ装置と方法
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JP4763690B2 (ja) * 2004-06-04 2011-08-31 ユニベールシテ・デ・スジャンス・エ・テクノロジー・ドゥ・リル 生化学的分析のために液滴を扱う装置、前記装置を製造する方法及びマイクロ流体分析
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US8012430B2 (en) 2004-03-04 2011-09-06 National Institute Of Advanced Industrial Science And Technology Methods for producing microchannel chips, microchannel chips, methods for separating biomolecules using the microchannel chips, and electrophoretic apparatus having the microchannel chips
JP4763690B2 (ja) * 2004-06-04 2011-08-31 ユニベールシテ・デ・スジャンス・エ・テクノロジー・ドゥ・リル 生化学的分析のために液滴を扱う装置、前記装置を製造する方法及びマイクロ流体分析
JP2006187730A (ja) * 2005-01-06 2006-07-20 Nippon Filcon Co Ltd 樹脂製微小流路化学デバイスの製造方法並びに該製法により製造された樹脂製微小流路化学デバイス構造体
JP2008533485A (ja) * 2005-03-18 2008-08-21 キヤノン株式会社 構造体、分離素子、分離装置、捕捉素子、検出装置、及びその製造方法、ならびに標的物質の分離方法及び検出方法
JP2007130836A (ja) * 2005-11-09 2007-05-31 Ushio Inc 接合方法
JP2009541737A (ja) * 2006-06-20 2009-11-26 オーミック・アクチボラゲット アッセイ装置と方法
US8085578B2 (en) 2009-03-13 2011-12-27 Paul Scherrer Institut Method and system for coding and read out of information in a microscopic cluster comprising coupled functional islands
WO2010122720A1 (fr) * 2009-04-20 2010-10-28 パナソニック株式会社 Dispositif de trajet d'écoulement
JP2011252776A (ja) * 2010-06-01 2011-12-15 Hiroshima Univ 解析装置及び解析装置の製造方法
WO2013069122A1 (fr) * 2011-11-09 2013-05-16 株式会社日立製作所 Dispositif et procédé de séparation de particules
JPWO2013069122A1 (ja) * 2011-11-09 2015-04-02 株式会社日立製作所 微粒子分離装置及び方法

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