WO2008038507A1 - Substratum and method of reactive liquid reaction - Google Patents

Substratum and method of reactive liquid reaction Download PDF

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Publication number
WO2008038507A1
WO2008038507A1 PCT/JP2007/067523 JP2007067523W WO2008038507A1 WO 2008038507 A1 WO2008038507 A1 WO 2008038507A1 JP 2007067523 W JP2007067523 W JP 2007067523W WO 2008038507 A1 WO2008038507 A1 WO 2008038507A1
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Prior art keywords
region
hydrophilic
water
reaction
liquid
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PCT/JP2007/067523
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French (fr)
Japanese (ja)
Inventor
Yoshinobu Akimoto
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Olympus Corporation
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Publication of WO2008038507A1 publication Critical patent/WO2008038507A1/en

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B01J2219/00662—Two-dimensional arrays within two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides

Definitions

  • the present invention performs a chemical reaction of a substrate such as a slide glass used in a biochemical experiment, and a water-soluble reaction solution such as a PCR (Polymerase Chain Reaction), an isothermal amplification reaction, or a ligation reaction.
  • a substrate such as a slide glass used in a biochemical experiment
  • a water-soluble reaction solution such as a PCR (Polymerase Chain Reaction), an isothermal amplification reaction, or a ligation reaction.
  • the present invention relates to a reaction solution reaction method.
  • PCR has been widely used in the field of biotechnology. PCR can amplify the desired DNA fragment (hundred-type DNA) several hundred thousand times by binding the primer across the target DNA region and repeating the DNA synthesis reaction in the DNA polymerase reaction. Is the method.
  • PCR-dedicated tube or microplate is used as a reaction vessel.
  • a PCR-dedicated tube or microplate is used as a reaction vessel.
  • approximately 30 microliters 1) of a water-soluble reaction solution dedicated to PCR is used.
  • Patent Document 2 discloses a technique for reducing the amount of water-soluble reaction liquid used.
  • Patent Document 2 discloses a sample handling tool in which a minute hydrophilic region is arranged on a hydrophobic surface. Thereby, operations such as holding or mixing a trace amount of the water-soluble reaction liquid can be performed.
  • Patent Document 2 shows that the entire sample nodding tool is placed in a humidity-controlled environment in order to prevent evaporation of the water-soluble reaction solution.
  • Patent Document 3 is disclosed as a technique for preventing evaporation of the water-soluble reaction liquid on the slide glass.
  • Patent Document 3 discloses a technique for covering the entire slide glass with mineral oil. ing. If this technique is applied to the technique of Patent Document 2, evaporation of the water-soluble reaction solution can be prevented even in PCR performed at a high temperature of 95 ° C.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 5-244950
  • Patent Document 2 Japanese Patent Laid-Open No. 11 304666
  • Patent Document 3 Japanese Patent Publication No. 8-73
  • Patent Document 3 since the entire slide glass is covered with mineral oil, there is a possibility that the mineral oil is dispersed non-uniformly on the slide glass. Therefore, the technique of Patent Document 3 is used in Patent Document 2. When applied to the technology, there was a problem that the water-soluble reaction solution could not be sufficiently covered and its evaporation could not be prevented reliably.
  • Patent Document 3 since the entire slide glass is covered with mineral oil, it is possible that an excessive amount of mineral oil is used to reliably prevent evaporation of the water-soluble reaction liquid. Therefore, when the technique of Patent Document 3 is applied to the technique of Patent Document 2, there is a problem that there is a risk that the cost will be increased more than necessary.
  • the present invention has been made in view of the above-mentioned problems, and performs a chemical reaction of a water-soluble reaction liquid! /, While reliably preventing evaporation of the water-soluble reaction liquid during water reaction.
  • a substrate that can effectively reduce the cost by optimizing the amount of oily liquid used for the purpose of preventing evaporation of the reactive reaction liquid, and a reaction liquid that can reliably carry out the chemical reaction of the water-soluble reaction liquid
  • the purpose is to provide a response method.
  • the substrate according to claim 1 which has the power of the present invention, has a water repellent surface as a whole, and has a circular shape.
  • a hydrophilic circular region that is partially provided on the surface of the substrate, wherein the hydrophilic circular region is a water-repellent region that forms a ring shape and surrounds the hydrophilic circular region.
  • the width of the ring in the water-repellent ring region is not less than 0.6 mm and not more than 0.8 mm.
  • the width of the ring in the hydrophilic ring region is 0.15 mm or more and 0.35 mm or less.
  • the substrate according to claim 3 according to the present invention is the substrate according to claim 2, wherein the water-repellent annular region and the hydrophilic annular region are held in the hydrophilic circular region. so as to cover the liquid being lifting, 2 ⁇ (r + O. 6 ) 3 / 3- ⁇ ⁇ 3 microliters Honoré or more and 2 ⁇ (r + O. 8) 3 / 3- ⁇ ⁇ 3/3 micro It is characterized in that an oily liquid in an amount of 1 liter or less is retained (wherein r is a radius (unit: millimeter) of the hydrophilic circular region, and ⁇ is a circumference).
  • the substrate according to claim 4 according to the present invention is the substrate according to any one of claims 1 to 3, wherein the hydrophilic circular region is prevented from adsorbing enzymes and / or nucleic acids. Processed! /, Characterized in that
  • the substrate according to claim 5 which is the power of the present invention, is the substrate according to any one of claims 1 to 4, wherein the hydrophilic circular region, the water-repellent annular region, and the A plurality of sets of hydrophilic annular regions are provided.
  • the substrate according to claim 6 according to the present invention is the substrate according to claim 5, wherein an outer peripheral portion of the hydrophilic annular region and an outer peripheral portion of another hydrophilic annular region are present. Is characterized by a distance of 1 mm or more.
  • the present invention relates to a reaction liquid reaction method, and the reaction liquid reaction method according to claim 7 according to the present invention includes the substrate according to any one of claims 1 to 6. It is used to perform chemical reaction of water-soluble reaction liquid.
  • the reaction liquid reaction method according to claim 8 according to the present invention is the reaction liquid reaction method according to claim 7, which is provided on the substrate.
  • ⁇ r 3/3 of the water-soluble reaction following amounts microliters or more and [pi r 3 microliters be held in the hydrophilic circular region ( R is before The radius of the hydrophilic circular region (unit: millimeter), and ⁇ is the circumference. ).
  • the reaction liquid reaction method according to claim 9 is the reaction liquid reaction method according to claim 8, which is provided on the substrate.
  • the reaction liquid reaction method according to claim 10 according to the present invention is the reaction liquid reaction method according to any one of claims 7 to 9, wherein the water-soluble reaction liquid is a polymerase chain reaction. It is for conducting one of the reaction, isothermal amplification reaction and ligation reaction.
  • the surface has a water repellency as a whole, and a hydrophilic circular region that is a hydrophilic region forming a circle is partially provided on the surface.
  • a water-repellent region in the form of an annulus, surrounding the hydrophilic circular region and adjacent to the hydrophilic circular region, and an annular shape.
  • a hydrophilic ring region surrounding the water-repellent ring region and adjacent to the water-repellent ring region, and the width of the ring in the water-repellent ring region is 0.6.
  • the width of the ring in the hydrophilic annular region is not less than 0.15 millimeters and not more than 0.35 millimeters.
  • FIG. 1 is a diagram showing an example of a substrate 100 that exerts a force on the present embodiment.
  • FIG. 2 is a diagram showing an example of a liquid holding region 102A that exerts a force on the present embodiment.
  • FIG. 3 is a diagram showing an example of a liquid holding region 102B that exerts a force on the present embodiment.
  • FIG. 4 is a diagram showing a liquid holding region 102A in a state where water is dispensed.
  • FIG. 5 is a diagram showing a liquid holding region 102B in a state where water is dispensed.
  • Fig. 6 is a view showing a liquid holding region 102A in a state where sealing oil is dispensed.
  • FIG. 7 is a view showing a liquid holding region 102B in a state where sealing oil is dispensed.
  • FIG. 1 is a diagram showing an example of a substrate 100 that is effective in the present embodiment.
  • the substrate 100 is a commercially available microscope slide glass or the like.
  • the surface of the substrate 100 has water repellency as a whole, and a liquid holding region that is a region for holding a liquid such as a water-soluble reaction liquid or an oily liquid partially (spot-like) on the surface.
  • the substrate 100 includes a total of 32 liquid holding regions 102 as shown in the drawing and a water repellent region.
  • An aqueous region 104 is provided.
  • a total of 32 liquid holding regions 102 are arranged on the surface of the substrate 100 in two groups (group A and group as shown in the figure. Each group has 4 rows and 4 columns as shown in the figure. There are 16 liquid holding areas 102 forces, spaced apart.
  • FIG. 2 is a diagram showing an example of the liquid holding region 102A according to the present embodiment.
  • the liquid holding region 102A is a characteristic part of the present invention, and includes a hydrophilic circular region 102a, a water-repellent annular region 102b, and a hydrophilic annular region 102c.
  • the hydrophilic circular region 102a is a hydrophilic region having a circular shape with a radius of 0.8 millimeter (mm).
  • the hydrophilic circular region 102a preferably holds liquid (specifically, water-soluble) in an amount (volume) of ⁇ / 3 microliters 1) or more and ⁇ r 3 1 or less (volume). Is the radius (in millimeters) of the hydrophilic circular region 102a, and ⁇ is the circumference.)
  • the hydrophilic circular region 102a may be subjected to an enzyme and / or nucleic acid adsorption prevention treatment.
  • the water repellent ring region 102b is a water repellent region having a ring shape with a width of 0.7 mm.
  • the water-repellent annular region 102b surrounds the hydrophilic circular region 102a as shown and is adjacent to the hydrophilic circular region 102a.
  • the hydrophilic ring region 102c is a hydrophilic region having a circular shape with a width of 0.25 mm.
  • the hydrophilic annular region 102c surrounds the water repellent annular region 102b as shown and is adjacent to the water repellent annular region 102b.
  • the water-repellent toric region 102b and the hydrophilic toric region 102c have 2 ⁇ (r + ⁇ ⁇ 6) 3 / 3— ⁇ ⁇ 1 or more and 2 ⁇ (r + ⁇ ⁇ 8) 3 / 3— ⁇ ⁇ It is preferable that a liquid (specifically, oily) in an amount of 3 / 3 ⁇ 1 or less is retained (r is the radius of the hydrophilic circular region 102a (unit: millimeter), and ⁇ is the ratio of the circumference. is there.).
  • FIG. 3 is a diagram showing an example of the liquid holding region 102B that applies force to the present embodiment.
  • the liquid holding region 102B is an example of the prior art, and is a hydrophilic region in the shape of a circle having a radius of 0.8 mm, similar to the hydrophilic circular region 102a.
  • Present The outer peripheral portion of the liquid holding region 102B and the outer peripheral portion of the other liquid holding region 102B are separated by 2.9 mm! /.
  • the configuration of the substrate that is applicable to the present invention is not limited to that shown in FIG. 1, FIG. 2, and FIG.
  • the arrangement state and the number of the liquid holding regions 102 are not limited to those shown in FIG. 1, FIG. 2, and FIG.
  • the shape of the hydrophilic circular region 102a is not limited to a circular shape, and may be, for example, a substantially circular shape (eg, an ellipse).
  • the radius of the hydrophilic circular region 102a is preferably not less than 0.8 mm and preferably not less than 0.6 mm and not more than 1. Omm.
  • the width of the water-repellent annular region 102b is not limited to 0.7 mm, but is preferably 0.6 mm or more and 0.8 mm or less.
  • the width of the hydrophilic annular region 102c is not limited to 0.25 mm, and is preferably 0.15 mm or more and 0.35 mm or less.
  • the water-repellent region 104 may be provided on the entire surface of the substrate 100 as shown in FIGS. 1, 2, and 3 as long as it surrounds the liquid holding region 102 and is adjacent to the liquid holding region 102. In addition, it may be partially provided on the surface of the substrate 100.
  • a method for manufacturing the substrate 100 in which the hydrophilic circular region 102a is subjected to an enzyme and nucleic acid adsorption prevention treatment will be described.
  • a commercially available microscope slide glass for example, a microscope slide glass manufactured by Matsunami Glass Industrial Co., Ltd.
  • Silicone coat the minutes.
  • the surface half of the slide glass was silanized, and the surface half of the slide glass could be subjected to enzyme and nucleic acid adsorption prevention treatment.
  • a polyfluorocarbon (Teflon (registered trademark)) thin film is formed on the entire surface of the slide glass in the pattern shown in FIGS. 1, 2, and 3 by screen printing.
  • the water-repellent annular region 102b and the water-repellent region 104 are printed on the surface of the slide glass with the pattern shown in FIG. 1 and FIG.
  • a water-repellent region 104 is printed in a pattern shown in FIGS. 1 and 3 on a portion not coated with silicon.
  • FIG. 4 shows the liquid holding region 102A in a state where water Wt is dispensed
  • FIG. 5 shows the liquid holding region 102B in a state where water Wt is dispensed! /.
  • both the liquid holding region 102A and the liquid holding region 102B were able to hold 1 ⁇ l of water Wt.
  • FIG. 6 shows the liquid holding region 102 A in a state where the sealing oil Oi is dispensed! /
  • FIG. 7 shows the liquid holding region 102 B in a state where the sealing oil Oi is dispensed!
  • the sealing oil Oi dispensed in the liquid holding region 102A does not spread outside the hydrophilic annular region 102c and stably covers the water Wt.
  • the sealing oil Oi dispensed into the liquid holding area 102B becomes unstable on the slide glass, and as shown in FIG. 7, the sealing oil Oi may be combined to sufficiently cover the water Wt. could not.
  • water Wt and sealing oil Oi are dispensed into all the liquid holding regions 102A and all the liquid holding regions 102B so that the water Wt is covered with the sealing oil Oi and heated to 95 ° C.
  • the water-repellent annular region 102b and the hydrophilic annular region 102c are further provided to provide the force.
  • the circular width of the water-repellent annular region 102b is 0.6 mm or more and 0.8 mm or less (0.7 mm in FIG. 2), and the circular width of the hydrophilic annular region 102c is 0.15 mm or more. In addition, since it is 0.35 mm or less (0.25 mm in FIG.
  • the substrate 100 which is effective in this embodiment, by optimizing the sizes of the water-repellent annular region 102b and the hydrophilic annular region 102c, a small amount of water-soluble reaction liquid can be reliably retained. In addition, evaporation of the water-soluble reaction liquid can be reliably prevented.
  • the volume of the oily liquid is determined by the size of the water-repellent annular region 102b and the hydrophilic annular region 102c.
  • the volume of the oily liquid is also small, and when the water-repellent ring region 102b and the hydrophilic ring region 102c is large, the volume of the oily liquid is small. Also grows. If the volume of the oily liquid is too small, the water-soluble reaction liquid held in the hydrophilic circular region 102a cannot be sufficiently covered with the oily liquid, and the water-soluble reaction liquid evaporates. Also, the hydrophilic ring region 102c is too small Then, since the oily liquid becomes unstable on the substrate, there is a problem that the oily liquid cannot be properly held on the substrate.
  • the water-repellent annular region 102b and the hydrophilic annular region 102c are too large, there is a problem that the number of liquid holding regions 102A that can be formed on a substrate having a limited area is reduced. Therefore, according to the substrate 100 which is effective in the present embodiment, in addition to the hydrophilic circular region 102a and the water repellent region 104, the water repellent annular region 102b and the hydrophilic annular region 102c are further provided, and the water repellent property is provided. Since the sizes of the annular region 102b and the hydrophilic annular region 102c are optimized! /, The above-mentioned problems can be solved reliably.
  • the force to this embodiment according to mow the substrate 100, the hydrophilic circular area 102a, ⁇ ⁇ 3/3 1 or more and [pi r 3 1
  • the following amounts of the liquid specifically, water Therefore, it is possible to optimize the amount of the water-soluble reaction solution used for the chemical reaction of the water-soluble reaction solution.
  • the amount of the water-soluble reaction liquid held in the hydrophilic circular region 102a is too small, the amount of the sample for performing the chemical reaction of the water-soluble reaction liquid also decreases. There is a problem that it is inconvenient to do.
  • the amount of the water-soluble reaction liquid held in the hydrophilic circular area 102a is too large, there is a problem that the water-soluble reaction liquid cannot be held in the hydrophilic circular area 102a. Therefore, according to this embodiment, according to the substrate 100, the amount of liquid retained in the hydrophilic circular region 102a is optimized! /, So that the above-mentioned numerous problems can be reliably solved. it can.
  • the water repellent annular region 102b and the hydrophilic hydrophilic region 102c are held in the hydrophilic circular region 102a! Specifically, 2 ⁇ (r + ⁇ ⁇ 6) 3 / 3— ⁇ ⁇ 3 ⁇ 1 or more and 2 ⁇ (r + 0.8) 3 /3- ⁇ r 3 / 3, ⁇ 1 or less liquid (specifically oily) may be retained! /, So it is used to prevent evaporation of the water-soluble reaction liquid in the chemical reaction of the water-soluble reaction liquid.
  • the amount of oily liquid used can be optimized.
  • the amount of the oily liquid retained in the water-repellent annular region 102b and the hydrophilic annular region 102c is too small, the water-soluble reaction liquid cannot be sufficiently covered and evaporation thereof can be prevented. There is a problem that it can not be done.
  • the amount of oily liquid retained in the water-repellent ring region 102b and the hydrophilic ring region 102c If there is too much, there is a problem that the oily liquid cannot be retained in the hydrophilic ring region 102c. Therefore, according to the substrate 100 which is effective in this embodiment, since the amount of liquid retained in the water-repellent annular region 102b and the hydrophilic annular region 102c is optimized, the above-mentioned problems are surely confirmed. Can be solved.
  • the hydrophilic circular region 102a may be subjected to a nucleic acid and / or enzyme adsorption prevention treatment.
  • nucleic acids and enzymes contained in a small amount of the water-soluble reaction solution can be reliably and easily recovered.
  • a plurality of liquid holding regions 102 (specifically, the liquid holding regions 102A) are provided!
  • the power of S can be measured simultaneously with S.
  • the outer peripheral portion of the existing liquid holding region 102A (specifically, the existing hydrophilic ring region 102c) and the other liquid holding region 102A ( Specifically, since the outer peripheral portion of the other hydrophilic ring region 102c) is separated by 1 mm or more (1 mm in FIG. 2), the oily liquids held in the individual liquid holding regions 102A are not bonded to each other. Thus, the oily liquid can be reliably held in the individual liquid holding regions 102A.
  • the oily liquid held in the existing liquid holding area 102A and the oily liquid held in the other liquid holding area 102A do not bind to each other, and the water-soluble reaction liquid does not enter the liquid holding area 102A. Since it is held stably, the water-soluble reaction liquid can be easily recovered from each liquid holding area 102A.
  • the distance between the outer peripheral portions of the hydrophilic ring region 102c is less than 1 mm, the possibility that oily liquids are bonded to each other increases. When the oily liquids are combined, the oily liquid is in the state shown in FIG. 7, so that the water-soluble reaction liquid cannot be sufficiently covered, and as a result, the water-soluble reaction liquid evaporates. There is a problem.
  • the outer peripheral portions of the hydrophilic annular region 102c are separated from each other by 1 mm or more, so that the above problem can be solved reliably.
  • a water-soluble reaction liquid or the like is obtained using a substrate 100 in which the hydrophilic circular region 102a has been subjected to an enzyme and nucleic acid adsorption prevention treatment. A warm amplification reaction was performed.
  • Example 1 the isothermal amplification reaction sample corresponding to the water-soluble reaction liquid in the embodiment described above is prepared by adjusting the following composition ratios.
  • the sealing oil corresponding to the oily liquid in the above-described embodiment is mineral oil “M5904” (product name) manufactured by Sigma-Aldrich Japan.
  • gDNA 1 nanogram (ng) / ⁇ 1
  • Isothermal amplification reagent “Loopamp DNA amplification reagent kit master mix” (product name)
  • the experimenter is provided on the substrate 100! /, The hydrophilic circular region 102a and the liquid holding region.
  • an isothermal amplification reaction sample was pipetted for 1 to 1 minutes, and sealing oil was pipetted for 51 minutes to cover the dispensed isothermal amplification reaction sample. Then, the experimenter sets the substrate 100 on which the isothermal amplification reaction sample and the sealing oil are dispensed to the prepared thermal cycler (Eppendorf Master Cycler), and performs the thermal cycle necessary for the isothermal amplification reaction. When applied, an isothermal amplification reaction was performed on the isothermal amplification reaction sample. This thermal cycle is performed in the following order (11) and (12).
  • the experimenter collects only the isothermal amplification reaction sample from each of the liquid holding region 102A and the liquid holding region 102B with a pipette, and the recovered isothermal amplification reaction sample is collected from Agilent Technologies. “Agilent bioanalyze rj (product name)”
  • Example 2 As an example of the reaction liquid reaction method according to the present invention, PCR of the water-soluble reaction liquid is performed using the substrate 100 in which the hydrophilic circular region 102a is subjected to the enzyme and nucleic acid adsorption prevention treatment. It was.
  • Example 2 the PCR sample corresponding to the water-soluble reaction solution in the above-described embodiment is prepared by adjusting the following composition ratio.
  • the sealing oil corresponding to the oily liquid in the above-described embodiment is Minera Leo Inore “M5904” (product name) manufactured by Sigma-Aldrich Japan.
  • the experimenter is provided on the substrate 100! /, The hydrophilic circular region 102a and the liquid holding region.
  • a PCR sample was pipetted into each of 102B by 1 ⁇ l, and sealing oil was pipetted 51 times so as to cover the dispensed PCR sample. Then, the experimenter placed the substrate 100 on which the CR sample and sealing oil had been dispensed into a pre-prepared Thermorecycler (Eppendorf Master Cycler), applied the thermal cycle necessary for PCR, and applied the PCR. Samples were PCRed. This thermal cycle is performed in the following order (21) to (24).
  • the experimenter pipettes only the PCR sample from each of the liquid holding area 102A and the liquid holding area 102B, and collects the collected PCR samples using Agilent Technologies' “Agilent Analysis was performed using bioanalyzerj (product name).
  • reaction liquid reaction method a ligation reaction of a water-soluble reaction liquid is performed using the substrate 100 in which the hydrophilic circular region 102a is subjected to an enzyme and nucleic acid adsorption prevention treatment. went.
  • Example 3 the ligation reaction sample corresponding to the water-soluble reaction liquid in the above-described embodiment was obtained by mixing the following in the tube at the following composition ratio: Furthermore, it is supplemented with ultrapure water, the amount of which is 100 ⁇ 1.
  • the sealing oil corresponding to the oily liquid in the above-described embodiment is a mineral oil “M5904” (product name) manufactured by Sigma-Aldrich Japan.
  • Ligase enzyme “TaqDNA ligase kit M0208S” (product name) (New England
  • Kit included 10x notfer: 5 ⁇ 1
  • the experimenter pipettes the ligation reaction sample from the tube, and dispenses the extracted ligation reaction sample into the hydrophilic circular region 102a and the liquid holding region 102B provided on the substrate 100, respectively. Further, 51 ml of sealing oil was pipetted to cover the dispensed ligation reaction sample. Then, the experimenter sets the substrate 100 on which the ligation reaction sample and the sealing oil are dispensed on a hot plate prepared in advance, and leaves it at 50 ° C. for 30 minutes to perform the ligation reaction of the ligation reaction sample. went.
  • the experimenter can determine that each liquid holding region 102A and Collect only the ligation reaction sample from the liquid holding area 102B with a pipette and perform denaturation gel electrophoresis of the collected ligation reaction sample with a DNA sequencer (product name “ABI PRISM 8100”) together with a marker DNA modified with 5'TAMRA. Fragment analysis.
  • the substrate and the reaction liquid reaction method according to the present invention can reliably perform a chemical reaction of an aqueous reaction liquid such as an isothermal amplification reaction or a PCR or a ligation reaction. It can be suitably used in various industrial fields such as medical care.
  • an aqueous reaction liquid such as an isothermal amplification reaction or a PCR or a ligation reaction. It can be suitably used in various industrial fields such as medical care.

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Abstract

A substratum that while securely preventing the evaporation of a water-soluble reactive liquid during the execution of chemical reaction thereof, optimizes the amount of oily liquid used for the purpose of preventing the evaporation of the water-soluble reactive liquid, thereby attaining effective reduction of cost; and a method of reactive liquid reaction in which the chemical reaction of the water-soluble reactive liquid can be securely performed. The substratum is one on its surface wholly exhibiting water repellency, provided partially on the surface thereof with a hydrophilic circular region, which substratum is further provided with awater-repellent annular region surrounding the hydrophilic circular region in relationship abutting on the hydrophilic circular region and provided with a hydrophilic annular region surrounding the water-repellent annular region in relationship abutting on the water-repellent annular region.

Description

明 細 書  Specification
基板および反応液反応方法  Substrate and reaction liquid reaction method
技術分野  Technical field
[0001] 本発明は、生化学実験で用いるスライドガラスのような基板、および PCR (Polymer ase Chain Reaction：ポリメラーゼ連鎖反応）や等温増幅反応やライゲーシヨン反 応などの水溶性反応液の化学反応を行う反応液反応方法に関するものである。 背景技術  [0001] The present invention performs a chemical reaction of a substrate such as a slide glass used in a biochemical experiment, and a water-soluble reaction solution such as a PCR (Polymerase Chain Reaction), an isothermal amplification reaction, or a ligation reaction. The present invention relates to a reaction solution reaction method. Background art
[0002] 近年、バイオテクノロジーの分野では、 PCRが広く利用されている。 PCRは、 目的と する DNA領域を挟んでプライマーを結合し、 DNAポリメラーゼ反応で DNA合成反 応を繰り返すことによって、 目的の DNA (铸型 DNA)断片を数十万倍にも増幅する ことができる方法である。  In recent years, PCR has been widely used in the field of biotechnology. PCR can amplify the desired DNA fragment (hundred-type DNA) several hundred thousand times by binding the primer across the target DNA region and repeating the DNA synthesis reaction in the DNA polymerase reaction. Is the method.
[0003] 一般的に PCRを行うときは反応容器として PCR専用のチューブやマイクロプレート を用いる。そして、例えば特許文献 1では、約 30マイクロリットル 1)の PCR専用の 水溶性反応液を使用してレ、る。  [0003] Generally, when PCR is performed, a PCR-dedicated tube or microplate is used as a reaction vessel. For example, in Patent Document 1, approximately 30 microliters 1) of a water-soluble reaction solution dedicated to PCR is used.
[0004] ここで、水溶性反応液に力、かる費用は決して安価ではない。そのため、コストの削 減や微量検体の検出という観点から、水溶性反応液の使用量を微量化することが重 要視されている。水溶性反応液の使用量を微量化する技術として、特許文献 2が開 示されている。特許文献 2には、疎水性の面上に微小な親水性領域を配置した試料 ハンドリングツールが示されている。これにより、微量な水溶性反応液を保持したり混 合したりする等の操作を行うことができる。  [0004] Here, the cost for the water-soluble reaction liquid is not low. For this reason, it is important to reduce the amount of water-soluble reaction solution used from the viewpoint of cost reduction and detection of a small amount of sample. Patent Document 2 discloses a technique for reducing the amount of water-soluble reaction liquid used. Patent Document 2 discloses a sample handling tool in which a minute hydrophilic region is arranged on a hydrophobic surface. Thereby, operations such as holding or mixing a trace amount of the water-soluble reaction liquid can be performed.
[0005] 一方で、 PCRを行うときには、微量な水溶性反応液の蒸発を防止する必要がある。  On the other hand, when performing PCR, it is necessary to prevent evaporation of a small amount of water-soluble reaction solution.
特許文献 2には、水溶性反応液の蒸発を防止するために、湿度がコントロールされた 環境に試料ノヽンドリングツール全体を置くことが示されている。  Patent Document 2 shows that the entire sample nodding tool is placed in a humidity-controlled environment in order to prevent evaporation of the water-soluble reaction solution.
[0006] しかし、そのような環境に試料ノ、ンドリングツール全体を置いても、 95°Cという高温 で行う PCRでは、水溶性反応液は蒸発してしまう。  [0006] However, even if the entire sample and the ring tool are placed in such an environment, the water-soluble reaction solution evaporates in PCR performed at a high temperature of 95 ° C.
[0007] そこで、スライドガラス上の水溶性反応液の蒸発を防止する技術として、特許文献 3 が開示されている。特許文献 3には、スライドガラス全体を鉱物油で覆う技術が示され ている。この技術を特許文献 2の技術に応用すれば、 95°Cという高温で行う PCRに おいても水溶性反応液の蒸発を防止することができる。 [0007] Patent Document 3 is disclosed as a technique for preventing evaporation of the water-soluble reaction liquid on the slide glass. Patent Document 3 discloses a technique for covering the entire slide glass with mineral oil. ing. If this technique is applied to the technique of Patent Document 2, evaporation of the water-soluble reaction solution can be prevented even in PCR performed at a high temperature of 95 ° C.
[0008] 特許文献 1 :特開平 5— 244950号公報 Patent Document 1: Japanese Patent Application Laid-Open No. 5-244950
特許文献 2：特開平 11 304666号公報  Patent Document 2: Japanese Patent Laid-Open No. 11 304666
特許文献 3：特公平 8— 73号公報  Patent Document 3: Japanese Patent Publication No. 8-73
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] しかしながら、特許文献 3ではスライドガラス全体を鉱物油で覆うので、鉱物油がス ライドガラス上で不均一に分散する可能性があり、それゆえに、特許文献 3の技術を 特許文献 2の技術に応用した際に、水溶性反応液を十分に覆うことができずその蒸 発を確実に防止することができない虞があるという問題点があった。  However, in Patent Document 3, since the entire slide glass is covered with mineral oil, there is a possibility that the mineral oil is dispersed non-uniformly on the slide glass. Therefore, the technique of Patent Document 3 is used in Patent Document 2. When applied to the technology, there was a problem that the water-soluble reaction solution could not be sufficiently covered and its evaporation could not be prevented reliably.
[0010] また、特許文献 3ではスライドガラス全体を鉱物油で覆うので、水溶性反応液の蒸 発を確実に防止するのに必要以上の量の鉱物油を使用している可能性があり、それ ゆえに、特許文献 3の技術を特許文献 2の技術に応用した際に、必要以上にコストを 掛けてレ、る虞があるとレ、う問題点があった。  [0010] In Patent Document 3, since the entire slide glass is covered with mineral oil, it is possible that an excessive amount of mineral oil is used to reliably prevent evaporation of the water-soluble reaction liquid. Therefore, when the technique of Patent Document 3 is applied to the technique of Patent Document 2, there is a problem that there is a risk that the cost will be increased more than necessary.
[0011] 本発明は、上記問題点に鑑みてなされたものであって、水溶性反応液の化学反応 を行って!/、る際の水溶性反応液の蒸発を確実に防止しつつ、水溶性反応液の蒸発 を防止する目的で使用する油性液体の量を最適化してコストを効果的に削減するこ とができる基板、および水溶性反応液の化学反応を確実に行うことができる反応液反 応方法を提供することを目的とする。  [0011] The present invention has been made in view of the above-mentioned problems, and performs a chemical reaction of a water-soluble reaction liquid! /, While reliably preventing evaporation of the water-soluble reaction liquid during water reaction. A substrate that can effectively reduce the cost by optimizing the amount of oily liquid used for the purpose of preventing evaporation of the reactive reaction liquid, and a reaction liquid that can reliably carry out the chemical reaction of the water-soluble reaction liquid The purpose is to provide a response method.
課題を解決するための手段  Means for solving the problem
[0012] 上述した課題を解決し、 目的を達成するために、本発明に力、かる請求項 1に記載の 基板は、その面が全体的に撥水性を有し、円の形を成す親水性の領域である親水 性円形領域をその面上に部分的に設けている基板において、円環の形を成す撥水 性の領域であって前記親水性円形領域を囲み且つ前記親水性円形領域に隣接す る撥水性円環領域と、円環の形を成す親水性の領域であつて前記撥水性円環領域 を囲み且つ前記撥水性円環領域に隣接する親水性円環領域と、をさらに設け、前記 撥水性円環領域における前記円環の幅は 0. 6ミリメートル以上且つ 0. 8ミリメートノレ 以下であり、前記親水性円環領域における前記円環の幅は 0. 15ミリメートル以上且 つ 0. 35ミリメートル以下であることを特徴とする。 [0012] In order to solve the above-described problems and achieve the object, the substrate according to claim 1, which has the power of the present invention, has a water repellent surface as a whole, and has a circular shape. A hydrophilic circular region that is partially provided on the surface of the substrate, wherein the hydrophilic circular region is a water-repellent region that forms a ring shape and surrounds the hydrophilic circular region. A water-repellent annular region adjacent to a hydrophilic region in the shape of an annulus that surrounds the water-repellent annular region and is adjacent to the water-repellent annular region. Further, the width of the ring in the water-repellent ring region is not less than 0.6 mm and not more than 0.8 mm. The width of the ring in the hydrophilic ring region is 0.15 mm or more and 0.35 mm or less.
[0013] また、本発明にかかる請求項 2に記載の基板は、請求項 1に記載の基板において、 前記親水性円形領域には、 π r3/3マイクロリットル以上且つ π r3マイクロリットル以下 の量の液体が保持されること（前記 rは前記親水性円形領域の半径（単位はミリメート ル)であり、前記 πは円周率である。）を特徴とする。 [0013] The substrate of claim 2 according to the present invention, in the substrate according to claim 1, wherein the hydrophilic circular area, π r 3/3 or more microliters and [pi r 3 microliters or less (R is the radius of the hydrophilic circular region (unit is millimeter), and π is the circumference).
[0014] また、本発明にかかる請求項 3に記載の基板は、請求項 2に記載の基板において、 前記撥水性円環領域および前記親水性円環領域には、前記親水性円形領域に保 持されている液体を覆うように、 2 π (r + O. 6) 3/3— π Γ3マイクロリットノレ以上且つ 2 π (r + O. 8) 3/3— π Γ3/3マイクロリットル以下の量の油性液体が保持されること（ 前記 rは前記親水性円形領域の半径（単位はミリメートル)であり、前記 πは円周率で ある。）を特徴とする。 [0014] Further, the substrate according to claim 3 according to the present invention is the substrate according to claim 2, wherein the water-repellent annular region and the hydrophilic annular region are held in the hydrophilic circular region. so as to cover the liquid being lifting, 2 π (r + O. 6 ) 3 / 3- π Γ 3 microliters Honoré or more and 2 π (r + O. 8) 3 / 3- π Γ 3/3 micro It is characterized in that an oily liquid in an amount of 1 liter or less is retained (wherein r is a radius (unit: millimeter) of the hydrophilic circular region, and π is a circumference).
[0015] また、本発明にかかる請求項 4に記載の基板は、請求項 1から 3のいずれか 1つに 記載の基板において、前記親水性円形領域には、酵素および/または核酸の吸着 防止処理が施されて!/、ることを特徴とする。  [0015] Further, the substrate according to claim 4 according to the present invention is the substrate according to any one of claims 1 to 3, wherein the hydrophilic circular region is prevented from adsorbing enzymes and / or nucleic acids. Processed! /, Characterized in that
[0016] また、本発明に力、かる請求項 5に記載の基板は、請求項 1から 4のいずれか 1つに 記載の基板において、前記親水性円形領域と前記撥水性円環領域と前記親水性円 環領域とを複数組設けたことを特徴とする。 [0016] In addition, the substrate according to claim 5, which is the power of the present invention, is the substrate according to any one of claims 1 to 4, wherein the hydrophilic circular region, the water-repellent annular region, and the A plurality of sets of hydrophilic annular regions are provided.
[0017] また、本発明にかかる請求項 6に記載の基板は、請求項 5に記載の基板において、 在る前記親水性円環領域の外周部分と他の前記親水性円環領域の外周部分とは 1 ミリメートル以上離れてレ、ることを特徴とする。 [0017] Further, the substrate according to claim 6 according to the present invention is the substrate according to claim 5, wherein an outer peripheral portion of the hydrophilic annular region and an outer peripheral portion of another hydrophilic annular region are present. Is characterized by a distance of 1 mm or more.
[0018] また、本発明は反応液反応方法に関するものであり、本発明にかかる請求項 7に記 載の反応液反応方法は、前記請求項 1から 6のいずれか 1つに記載の基板を用いて 水溶性反応液の化学反応を行うことを特徴とする。 [0018] Further, the present invention relates to a reaction liquid reaction method, and the reaction liquid reaction method according to claim 7 according to the present invention includes the substrate according to any one of claims 1 to 6. It is used to perform chemical reaction of water-soluble reaction liquid.
[0019] また、本発明にかかる請求項 8に記載の反応液反応方法は、請求項 7に記載の反 応液反応方法にお!/、て、前記基板に設けられて!/、る前記親水性円形領域に前記水 溶性反応液を滴下することで、 π r3/3マイクロリットル以上且つ π r3マイクロリットル 以下の量の前記水溶性反応液を前記親水性円形領域に保持させること（前記 rは前 記親水性円形領域の半径（単位はミリメートル)であり、前記 πは円周率である。）を 特徴とする。 [0019] The reaction liquid reaction method according to claim 8 according to the present invention is the reaction liquid reaction method according to claim 7, which is provided on the substrate. by dropping the water-soluble reaction liquid hydrophilic circular region, π r 3/3 of the water-soluble reaction following amounts microliters or more and [pi r 3 microliters be held in the hydrophilic circular region ( R is before The radius of the hydrophilic circular region (unit: millimeter), and π is the circumference. ).
[0020] また、本発明にかかる請求項 9に記載の反応液反応方法は、請求項 8に記載の反 応液反応方法にお!/、て、前記基板に設けられて!/、る前記撥水性円環領域および前 記親水性円環領域に、前記水溶性反応液を覆うように油性液体を滴下することで、 2 π (r + Ο· 6) 3/3— π Γ3マイクロリットル以上且つ 2 π (r + 0. 8) 3/3— π r3/3マイク 口リットル以下の量の前記油性液体を前記撥水性円環領域および前記親水性円環 領域に保持させること（前記 rは前記親水性円形領域の半径（単位はミリメートル)で あり、前記 πは円周率である。）を特徴とする。 [0020] Further, the reaction liquid reaction method according to claim 9 according to the present invention is the reaction liquid reaction method according to claim 8, which is provided on the substrate. 2 π (r + Ο 6) 3 / 3— π Γ 3 microliters by dropping an oily liquid over the water-repellent annular region and the hydrophilic annular region so as to cover the water-soluble reaction liquid. or more and 2 π (r + 0. 8) 3 / 3- π r 3/3 thereby holding the oil liquid microphone port liter quantities in the water-repellent annular region and the hydrophilic annular region (the r is a radius of the hydrophilic circular region (unit is millimeter), and π is a circumference ratio).
[0021] また、本発明にかかる請求項 10に記載の反応液反応方法は、請求項 7から 9のい ずれか 1つに記載の反応液反応方法において、前記水溶性反応液は、ポリメラーゼ 連鎖反応、等温増幅反応およびライゲーシヨン反応のいずれ力、 1つを行うためのもの であることを特徴とする。  [0021] The reaction liquid reaction method according to claim 10 according to the present invention is the reaction liquid reaction method according to any one of claims 7 to 9, wherein the water-soluble reaction liquid is a polymerase chain reaction. It is for conducting one of the reaction, isothermal amplification reaction and ligation reaction.
発明の効果  The invention's effect
[0022] 本発明に力、かる基板によれば、その面が全体的に撥水性を有し、円の形を成す親 水性の領域である親水性円形領域をその面上に部分的に設けてレ、る基板にぉレ、て 、円環の形を成す撥水性の領域であって親水性円形領域を囲み且つ親水性円形領 域に隣接する撥水性円環領域と、円環の形を成す親水性の領域であって撥水性円 環領域を囲み且つ撥水性円環領域に隣接する親水性円環領域と、をさらに設け、撥 水性円環領域における円環の幅は 0. 6ミリメートル以上且つ 0. 8ミリメートル以下で あり、親水性円環領域における円環の幅は 0. 15ミリメートル以上且つ 0. 35ミリメート ル以下であるので、水溶性反応液の化学反応を行って!/、る際の水溶性反応液の蒸 発を確実に防止しつつ、水溶性反応液の蒸発を防止する目的で使用する油性液体 の量を最適化してコストを効果的に削減することができるという効果を奏する。  [0022] According to the substrate of the present invention, the surface has a water repellency as a whole, and a hydrophilic circular region that is a hydrophilic region forming a circle is partially provided on the surface. A water-repellent region in the form of an annulus, surrounding the hydrophilic circular region and adjacent to the hydrophilic circular region, and an annular shape. A hydrophilic ring region surrounding the water-repellent ring region and adjacent to the water-repellent ring region, and the width of the ring in the water-repellent ring region is 0.6. It is not less than millimeters and not more than 0.8 millimeters, and the width of the ring in the hydrophilic annular region is not less than 0.15 millimeters and not more than 0.35 millimeters. Used to prevent evaporation of water-soluble reaction liquid while reliably preventing evaporation of water-soluble reaction liquid An effect that it is possible to reduce the cost effectively by optimizing the amount of sex liquid.
[0023] 本発明にかかる反応液反応方法によれば、本発明にかかる基板を用いて水溶性 反応液の化学反応を行うので、本発明にかかる基板で得られる上記の効果を享受す ることで水溶性反応液の化学反応を確実に行うことができるという効果を奏する。 図面の簡単な説明 [0024] [図 1]図 1は、本実施の形態に力、かる基板 100の一例を示す図である。 [0023] According to the reaction liquid reaction method according to the present invention, a chemical reaction of a water-soluble reaction liquid is performed using the substrate according to the present invention, so that the above-described effects obtained with the substrate according to the present invention can be enjoyed. Thus, the chemical reaction of the water-soluble reaction solution can be reliably performed. Brief Description of Drawings [0024] FIG. 1 is a diagram showing an example of a substrate 100 that exerts a force on the present embodiment.
[図 2]図 2は、本実施の形態に力、かる液体保持領域 102Aの一例を示す図である。  FIG. 2 is a diagram showing an example of a liquid holding region 102A that exerts a force on the present embodiment.
[図 3]図 3は、本実施の形態に力、かる液体保持領域 102Bの一例を示す図である。  FIG. 3 is a diagram showing an example of a liquid holding region 102B that exerts a force on the present embodiment.
[図 4]図 4は、水が分注されている状態の液体保持領域 102Aを示す図である。  FIG. 4 is a diagram showing a liquid holding region 102A in a state where water is dispensed.
[図 5]図 5は、水が分注されている状態の液体保持領域 102Bを示す図である。  FIG. 5 is a diagram showing a liquid holding region 102B in a state where water is dispensed.
[図 6]図 6は、シーリングオイルが分注されている状態の液体保持領域 102Aを示す 図である。  [Fig. 6] Fig. 6 is a view showing a liquid holding region 102A in a state where sealing oil is dispensed.
[図 7]図 7は、シーリングオイルが分注されている状態の液体保持領域 102Bを示す 図である。  FIG. 7 is a view showing a liquid holding region 102B in a state where sealing oil is dispensed.
符号の説明  Explanation of symbols
[0025] 100 基板 [0025] 100 substrates
102 液体保持領域  102 Liquid holding area
102A 液体保持領域 (本発明）  102A Liquid holding area (Invention)
102a 親水性円形領域  102a hydrophilic circular region
102b 撥水性円環領域  102b Water repellent ring region
102c 親水性円環領域  102c hydrophilic ring region
102B 液体保持領域 (従来例）  102B Liquid holding area (conventional example)
104 撥水性領域  104 Water repellent area
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0026] 以下に、本発明にかかる基板および反応液反応方法の実施の形態を、図面に基 づいて詳細に説明する。なお、本実施の形態により本発明が限定されるものではな い。 Hereinafter, embodiments of a substrate and a reaction liquid reaction method according to the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.
[0027] まず、本実施の形態に力、かる基板 100の構成について、図 1や図 2、図 3を参照し て説明する。図 1は、本実施の形態に力、かる基板 100の一例を示す図である。基板 1 00は、市販の顕微鏡用スライドガラスなどである。基板 100は、その面が全体的に撥 水性を有し、その面上に部分的 (スポット状）に、水溶性反応液や油性液体などの液 体を保持するための領域である液体保持領域 102を複数設けて!/、る。具体的には、 基板 100は、図示の如ぐ合計 32個の液体保持領域 102と、撥水性の領域である撥 水性領域 104とを設けている。合計 32個の液体保持領域 102は、図示の如ぐ 2つ のグループ（グループ A、グループ に分けて基板 100の表面に配置されている。 各々のグループは、図示の如く 4行 4列で等間隔に配置された 16個の液体保持領域 102力、らなる。 [0027] First, the configuration of the substrate 100 that is effective in the present embodiment will be described with reference to FIG. 1, FIG. 2, and FIG. FIG. 1 is a diagram showing an example of a substrate 100 that is effective in the present embodiment. The substrate 100 is a commercially available microscope slide glass or the like. The surface of the substrate 100 has water repellency as a whole, and a liquid holding region that is a region for holding a liquid such as a water-soluble reaction liquid or an oily liquid partially (spot-like) on the surface. Set up multiple 102! Specifically, the substrate 100 includes a total of 32 liquid holding regions 102 as shown in the drawing and a water repellent region. An aqueous region 104 is provided. A total of 32 liquid holding regions 102 are arranged on the surface of the substrate 100 in two groups (group A and group as shown in the figure. Each group has 4 rows and 4 columns as shown in the figure. There are 16 liquid holding areas 102 forces, spaced apart.
[0028] ここで、グループ Aを構成する液体保持領域 102である液体保持領域 102Aの構 成について、図 2を参照して説明する。図 2は、本実施の形態にかかる液体保持領域 102Aの一例を示す図である。液体保持領域 102Aは、本発明の特徴部分であり、 親水性円形領域 102aと、撥水性円環領域 102bと、親水性円環領域 102cとからな る。親水性円形領域 102aは、半径が 0. 8ミリメートル (mm)の円の形を成す親水性 の領域である。親水性円形領域 102aには、兀 /3マイクロリットル 1)以上且つ π r3 1以下の量 (体積）の液体（具体的には水溶性のもの）が保持されることが好まし い（rは親水性円形領域 102aの半径（単位はミリメートル）であり、 πは円周率である 。）。親水性円形領域 102aには、酵素および/または核酸の吸着防止処理が施され てもよい。撥水性円環領域 102bは、幅が 0. 7mmの円環（リング）の形を成す撥水性 の領域である。撥水性円環領域 102bは、図示の如ぐ親水性円形領域 102aを囲み 且つ親水性円形領域 102aに隣接する。親水性円環領域 102cは、幅が 0. 25mm の円環の形を成す親水性の領域である。親水性円環領域 102cは、図示の如ぐ撥 水性円環領域 102bを囲み且つ撥水性円環領域 102bに隣接する。撥水性円環領 域 102bおよび親水性円環領域 102cには、 2 π (r + Ο· 6) 3/3— π Γ 1以上且つ 2 π (r + Ο· 8) 3/3— π Γ3/3〃1以下の量の液体（具体的には油性のもの）が保持さ れることが好ましい（rは親水性円形領域 102aの半径（単位はミリメートル）であり、 π は円周率である。）。在る液体保持領域 102A (具体的には在る親水性円環領域 102 c)の外周部分と他の液体保持領域 102Α (具体的には他の親水性円環領域 102c) の外周部分とは、 1mm離れている。 [0028] Here, the configuration of the liquid holding region 102A, which is the liquid holding region 102 constituting the group A, will be described with reference to FIG. FIG. 2 is a diagram showing an example of the liquid holding region 102A according to the present embodiment. The liquid holding region 102A is a characteristic part of the present invention, and includes a hydrophilic circular region 102a, a water-repellent annular region 102b, and a hydrophilic annular region 102c. The hydrophilic circular region 102a is a hydrophilic region having a circular shape with a radius of 0.8 millimeter (mm). The hydrophilic circular region 102a preferably holds liquid (specifically, water-soluble) in an amount (volume) of 兀 / 3 microliters 1) or more and π r 3 1 or less (volume). Is the radius (in millimeters) of the hydrophilic circular region 102a, and π is the circumference.) The hydrophilic circular region 102a may be subjected to an enzyme and / or nucleic acid adsorption prevention treatment. The water repellent ring region 102b is a water repellent region having a ring shape with a width of 0.7 mm. The water-repellent annular region 102b surrounds the hydrophilic circular region 102a as shown and is adjacent to the hydrophilic circular region 102a. The hydrophilic ring region 102c is a hydrophilic region having a circular shape with a width of 0.25 mm. The hydrophilic annular region 102c surrounds the water repellent annular region 102b as shown and is adjacent to the water repellent annular region 102b. The water-repellent toric region 102b and the hydrophilic toric region 102c have 2 π (r + Ο · 6) 3 / 3—π Γ 1 or more and 2 π (r + Ο · 8) 3 / 3— π Γ It is preferable that a liquid (specifically, oily) in an amount of 3 / 3〃1 or less is retained (r is the radius of the hydrophilic circular region 102a (unit: millimeter), and π is the ratio of the circumference. is there.). What is the outer peripheral part of the existing liquid holding area 102A (specifically, the hydrophilic ring area 102c) and the outer peripheral part of the other liquid holding area 102Α (specifically, the other hydrophilic ring area 102c)? 1mm away.
[0029] また、グループ Bを構成する液体保持領域 102である液体保持領域 102Bの構成 について、図 3を参照して説明する。図 3は、本実施の形態に力、かる液体保持領域 1 02Bの一例を示す図である。液体保持領域 102Bは、従来技術の一例であり、親水 性円形領域 102aと同様、半径が 0. 8mmの円の形を成す親水性の領域である。在 る液体保持領域 102Bの外周部分と他の液体保持領域 102Bの外周部分とは、 2. 9 mm離れて!/、る。 [0029] The configuration of the liquid holding region 102B, which is the liquid holding region 102 constituting the group B, will be described with reference to FIG. FIG. 3 is a diagram showing an example of the liquid holding region 102B that applies force to the present embodiment. The liquid holding region 102B is an example of the prior art, and is a hydrophilic region in the shape of a circle having a radius of 0.8 mm, similar to the hydrophilic circular region 102a. Present The outer peripheral portion of the liquid holding region 102B and the outer peripheral portion of the other liquid holding region 102B are separated by 2.9 mm! /.
[0030] なお、本発明に力、かる基板の構成は、図 1や図 2、図 3に示したものに限定されない 。例えば、液体保持領域 102の配列状態およびその数は、図 1や図 2、図 3に示した ものに限定されない。親水性円形領域 102aの形状は、円形に限定されることなぐ 例えば略円形（例えば楕円など）でもよい。親水性円形領域 102aの半径は、 0. 8m mに限定されることなぐ好ましくは 0. 6mm以上且つ 1. Omm以下である。撥水性円 環領域 102bの幅は、 0. 7mmに限定されることなぐ好ましくは 0. 6mm以上且つ 0 . 8mm以下である。親水性円環領域 102cの幅は、 0. 25mmに限定されることなぐ 好ましくは 0. 15mm以上且つ 0. 35mm以下である。在る液体保持領域 102A (具 体的には在る親水性円環領域 102c)の外周部分と他の液体保持領域 102A (具体 的には他の親水性円環領域 102c)の外周部分との間の距離は、 1mmに限定される ことなく、好ましくは lmm以上である。撥水性領域 104は、液体保持領域 102を囲み 且つ液体保持領域 102に隣接するように設ければよぐ図 1や図 2、図 3に示すように 基板 100の表面に全体的に設けたりする以外にも、基板 100の表面に部分的に設け てもよい。  [0030] It should be noted that the configuration of the substrate that is applicable to the present invention is not limited to that shown in FIG. 1, FIG. 2, and FIG. For example, the arrangement state and the number of the liquid holding regions 102 are not limited to those shown in FIG. 1, FIG. 2, and FIG. The shape of the hydrophilic circular region 102a is not limited to a circular shape, and may be, for example, a substantially circular shape (eg, an ellipse). The radius of the hydrophilic circular region 102a is preferably not less than 0.8 mm and preferably not less than 0.6 mm and not more than 1. Omm. The width of the water-repellent annular region 102b is not limited to 0.7 mm, but is preferably 0.6 mm or more and 0.8 mm or less. The width of the hydrophilic annular region 102c is not limited to 0.25 mm, and is preferably 0.15 mm or more and 0.35 mm or less. Between the outer peripheral portion of the existing liquid holding region 102A (specifically, the hydrophilic annular region 102c) and the outer peripheral portion of the other liquid holding region 102A (specifically, another hydrophilic annular region 102c). The distance between them is not limited to 1 mm, but is preferably 1 mm or more. The water-repellent region 104 may be provided on the entire surface of the substrate 100 as shown in FIGS. 1, 2, and 3 as long as it surrounds the liquid holding region 102 and is adjacent to the liquid holding region 102. In addition, it may be partially provided on the surface of the substrate 100.
[0031] つぎに、図 1や図 2、図 3に示す基板 100の作製方法を説明する。その面が全体的 に親水性を有する市販の顕微鏡用スライドガラス (例えば松浪硝子工業社製の顕微 鏡用スライドガラスなど）の表面に、スクリーン印刷法を用いて、撥水性であるポリフル ォロカーボン (テフロン (登録商標））薄膜を、図 1や図 2、図 3に示すパターンで形成 する。具体的には、撥水性円環領域 102bおよび撥水性領域 104を図 1や図 2に示 すパターンで親水性のスライドガラスの表面の半分に印刷すると共に、撥水性領域 1 04を図 1や図 3に示すパターンで親水性のスライドガラスの表面の残り半分に印刷す る。これにより、スライドガラスの表面に、 16個の液体保持領域 102Aおよび 16個の 液体保持領域 102Bが作製された。  Next, a method for manufacturing the substrate 100 shown in FIGS. 1, 2, and 3 will be described. On the surface of a commercially available microscope slide glass (for example, a microscope slide glass manufactured by Matsunami Glass Industrial Co., Ltd.) whose surface is entirely hydrophilic, water-repellent polyfluorocarbon (Teflon) is used by screen printing. (Registered trademark)) A thin film is formed in the pattern shown in Fig. 1, Fig. 2 or Fig. 3. Specifically, the water-repellent annular region 102b and the water-repellent region 104 are printed on the half of the surface of the hydrophilic glass slide in the pattern shown in FIG. 1 and FIG. Print on the other half of the surface of the hydrophilic glass slide in the pattern shown in Fig. 3. As a result, 16 liquid holding regions 102A and 16 liquid holding regions 102B were formed on the surface of the slide glass.
[0032] また、親水性円形領域 102aに酵素および核酸の吸着防止処理が施された基板 1 00の作製方法を説明する。まず、その面が全体的に親水性を有する市販の顕微鏡 用スライドガラス（例えば松浪硝子工業社製の顕微鏡用スライドガラスなど）の表面半 分をシリコンコートする。これにより、スライドガラスの表面半分はシラン化され、スライ ドガラスの表面半分に酵素および核酸の吸着防止処理を施すことができた。つぎに、 このスライドガラスの表面全体に、スクリーン印刷法を用いて、ポリフルォロカーボン（ テフロン (登録商標））薄膜を図 1や図 2、図 3に示すパターンで形成する。具体的に は、スライドガラスの表面においてシリコンコートされた部分に、図 1や図 2に示すパタ 一ンで撥水性円環領域 102bおよび撥水性領域 104を印刷すると共に、スライドガラ スの表面においてシリコンコートされてない部分に、図 1や図 3に示すパターンで撥 水性領域 104を印刷する。これにより、スライドガラスの表面において、シリコンコート された部分には 16個の液体保持領域 102Aが作製され、シリコンコートされてない部 分には 16個の液体保持領域 102Bが作製された。つぎに、作製された 16個の液体 保持領域 102Aの個々の親水性円形領域 102aに、 5 %ゥシ血清アルブミン水溶液 をピペットで; 1分注して自然乾燥させる。これにより、液体保持領域 102Aにおい てシリコンコートのみされている領域が親水化された。以上の手順により、スライドガラ スの表面に、酵素および核酸の吸着防止処理が施された 16個の液体保持領域 102 Aおよび酵素および核酸の吸着防止処理が施されてな!/、 16個の液体保持領域 102 Bが作製された。 [0032] A method for manufacturing the substrate 100 in which the hydrophilic circular region 102a is subjected to an enzyme and nucleic acid adsorption prevention treatment will be described. First, the surface half of a commercially available microscope slide glass (for example, a microscope slide glass manufactured by Matsunami Glass Industrial Co., Ltd.) whose surface is entirely hydrophilic. Silicone coat the minutes. As a result, the surface half of the slide glass was silanized, and the surface half of the slide glass could be subjected to enzyme and nucleic acid adsorption prevention treatment. Next, a polyfluorocarbon (Teflon (registered trademark)) thin film is formed on the entire surface of the slide glass in the pattern shown in FIGS. 1, 2, and 3 by screen printing. Specifically, the water-repellent annular region 102b and the water-repellent region 104 are printed on the surface of the slide glass with the pattern shown in FIG. 1 and FIG. A water-repellent region 104 is printed in a pattern shown in FIGS. 1 and 3 on a portion not coated with silicon. As a result, on the surface of the slide glass, 16 liquid holding regions 102A were produced in the silicon-coated portion, and 16 liquid holding regions 102B were produced in the non-silicon-coated portion. Next, pipette 5% sushi serum albumin aqueous solution into each hydrophilic circular region 102a of the prepared 16 liquid holding regions 102A; As a result, the region where only the silicon coating is applied in the liquid holding region 102A is hydrophilized. According to the above procedure, the surface of the slide glass has not been subjected to the 16 liquid holding regions 102 A subjected to the enzyme and nucleic acid adsorption prevention treatment and the enzyme and nucleic acid adsorption prevention treatment! /, 16 pieces. A liquid holding region 102 B was created.
[0033] つぎに、本実施の形態に力、かる基板 100の性能について、図 4や図 5、図 6、図 7を 参照して説明する。まず、液体の保持に関する性能を検証した。図 1や図 2、図 3に 示す基板 100において、個々の液体保持領域 102Aおよび個々の液体保持領域 10 2Bに、ピペットを用いて水 Wtを 1 1ずつ分注した。水 Wtが分注されている状態の 液体保持領域 102Aを図 4に示し、水 Wtが分注されて!/、る状態の液体保持領域 10 2Bを図 5に示す。図 4および図 5に示すように、液体保持領域 102Aおよび液体保持 領域 102B共に、 1 μ 1の水 Wtを保持することができた。  Next, the power of the present embodiment and the performance of the substrate 100 will be described with reference to FIG. 4, FIG. 5, FIG. 6, and FIG. First, the performance related to liquid retention was verified. In the substrate 100 shown in FIG. 1, FIG. 2, and FIG. 3, water Wt was dispensed 11 by 1 into each liquid holding area 102A and each liquid holding area 102B using a pipette. FIG. 4 shows the liquid holding region 102A in a state where water Wt is dispensed, and FIG. 5 shows the liquid holding region 102B in a state where water Wt is dispensed! /. As shown in FIGS. 4 and 5, both the liquid holding region 102A and the liquid holding region 102B were able to hold 1 μl of water Wt.
[0034] また、液体の蒸発防止に関する性能を検証した。図 4および図 5に示すように、水の みが分注されて!/、る場合、常温'常湿の状態にぉレ、ては数分でその水は蒸発してし ま!/、、 PCRで印可する 95°Cと!/、う温度の状態にお!/、ては数秒でその水は蒸発してし まう。そこで、水の蒸発を防止するために、ピペットを用いて 5 1のシーリングオイル Oi (具体的にはシグマアルドリッチジャパン社製のミネラルオイル「M5904」）を、水 Wtを覆うように分注した。シーリングオイル Oiが分注されて!/、る状態の液体保持領域 102 Aを図 6に示し、シーリングオイル Oiが分注されて!/、る状態の液体保持領域 102 Bを図 7に示す。液体保持領域 102Aに分注されたシーリングオイル Oiは、図 6に示 すように、親水性円環領域 102cの外側には広がらず安定に水 Wtを覆っている。一 方、液体保持領域 102Bに分注されたシーリングオイル Oiは、スライドガラス上で不 安定になり、図 7に示すように、シーリングオイル Oi同士が結合して十分に水 Wtを覆 うことができなかった。そして、全ての液体保持領域 102Aおよび全ての液体保持領 域 102Bに水 Wtとシーリングオイル Oiとを分注して水 Wtがシーリングオイル Oiに覆 われている状態にし、 95°Cに加熱されているホットプレートの上にスライドガラスを 10 分間置いて、水 Wtの状態を観察した。 16個の液体保持領域 102A全てにおいて水 Wtは蒸発しなかった。一方、 16個の液体保持領域 102Bのうち 6個の液体保持領域 102Bにお!/、て水 Wtが蒸発して!/、た。 [0034] Further, the performance with respect to prevention of liquid evaporation was verified. As shown in Figs. 4 and 5, when only water is dispensed! /, The water will evaporate at room temperature and normal humidity, and the water will evaporate within a few minutes! /, The temperature of 95 ° C applied by PCR! /, And the temperature of the water! / Will evaporate in a few seconds. Therefore, in order to prevent evaporation of the water, use a pipette to remove 51 of the sealing oil Oi (specifically, mineral oil “M5904” manufactured by Sigma-Aldrich Japan) Dispensing to cover Wt. FIG. 6 shows the liquid holding region 102 A in a state where the sealing oil Oi is dispensed! /, And FIG. 7 shows the liquid holding region 102 B in a state where the sealing oil Oi is dispensed! As shown in FIG. 6, the sealing oil Oi dispensed in the liquid holding region 102A does not spread outside the hydrophilic annular region 102c and stably covers the water Wt. On the other hand, the sealing oil Oi dispensed into the liquid holding area 102B becomes unstable on the slide glass, and as shown in FIG. 7, the sealing oil Oi may be combined to sufficiently cover the water Wt. could not. Then, water Wt and sealing oil Oi are dispensed into all the liquid holding regions 102A and all the liquid holding regions 102B so that the water Wt is covered with the sealing oil Oi and heated to 95 ° C. A slide glass was placed on the hot plate for 10 minutes, and the state of water Wt was observed. Water Wt did not evaporate in all 16 liquid holding regions 102A. On the other hand, out of the 16 liquid holding regions 102B, the water Wt evaporated in the 6 liquid holding regions 102B.
以上、本実施の形態に力、かる基板 100によれば、親水性円形領域 102aおよび撥 水性領域 104に加えて、撥水性円環領域 102bおよび親水性円環領域 102cをさら に設け、し力、も撥水性円環領域 102bの円環の幅は 0. 6mm以上且つ 0. 8mm以下 (図 2では 0. 7mm)であり、親水性円環領域 102cの円環の幅は 0. 15mm以上且つ 0. 35mm以下（図 2では 0. 25mm)であるので、水溶性反応液の化学反応を行って いる際の水溶性反応液の蒸発を確実に防止しつつ、水溶性反応液の蒸発を防止す る目的で使用する油性液体の量を最適化してコストを効果的に削減することができる 。また、本実施の形態に力、かる基板 100によれば、撥水性円環領域 102bと親水性 円環領域 102cの大きさを最適化することで、微量の水溶性反応液を確実に保持で き且つ水溶性反応液の蒸発を確実に防止することができる。ここで、油性液体の体 積は撥水性円環領域 102bおよび親水性円環領域 102cの大きさで決まる。具体的 には、撥水性円環領域 102bおよび親水性円環領域 102cが小さいと油性液体の体 積も小さくなり、撥水性円環領域 102bおよび親水性円環領域 102cが大きいと油性 液体の体積も大きくなる。そして、油性液体の体積が小さすぎると、親水性円形領域 102aに保持される水溶性反応液を十分に油性液体で覆うことができなくなり、水溶 性反応液が蒸発してしまうという問題がある。また、親水性円環領域 102cが小さすぎ ると、油性液体は基板上で不安定になるので、油性液体を基板上で適切に保持する ことができなくなるという問題がある。一方、油性液体の体積が大きすぎると、必要以 上に油性液体を使用することとなり、コストが掛かってしまうという問題がある。また、 撥水性円環領域 102bおよび親水性円環領域 102cが大きすぎると、限られた面積 の基板上に形成できる液体保持領域 102Aの数が少なくなつてしまうという問題があ る。そこで、本実施の形態に力、かる基板 100によれば、親水性円形領域 102aおよび 撥水性領域 104に加えて撥水性円環領域 102bおよび親水性円環領域 102cをさら に設けつつ、撥水性円環領域 102bおよび親水性円環領域 102cの大きさを最適化 して!/、るので、上記の数々の問題を確実に解決することができる。 As described above, according to the substrate 100 according to the present embodiment, in addition to the hydrophilic circular region 102a and the water-repellent region 104, the water-repellent annular region 102b and the hydrophilic annular region 102c are further provided to provide the force. The circular width of the water-repellent annular region 102b is 0.6 mm or more and 0.8 mm or less (0.7 mm in FIG. 2), and the circular width of the hydrophilic annular region 102c is 0.15 mm or more. In addition, since it is 0.35 mm or less (0.25 mm in FIG. 2), it is possible to prevent evaporation of the water-soluble reaction liquid while reliably preventing evaporation of the water-soluble reaction liquid during the chemical reaction of the water-soluble reaction liquid. Costs can be effectively reduced by optimizing the amount of oily liquid used to prevent it. In addition, according to the substrate 100 which is effective in this embodiment, by optimizing the sizes of the water-repellent annular region 102b and the hydrophilic annular region 102c, a small amount of water-soluble reaction liquid can be reliably retained. In addition, evaporation of the water-soluble reaction liquid can be reliably prevented. Here, the volume of the oily liquid is determined by the size of the water-repellent annular region 102b and the hydrophilic annular region 102c. Specifically, when the water-repellent ring region 102b and the hydrophilic ring region 102c are small, the volume of the oily liquid is also small, and when the water-repellent ring region 102b and the hydrophilic ring region 102c is large, the volume of the oily liquid is small. Also grows. If the volume of the oily liquid is too small, the water-soluble reaction liquid held in the hydrophilic circular region 102a cannot be sufficiently covered with the oily liquid, and the water-soluble reaction liquid evaporates. Also, the hydrophilic ring region 102c is too small Then, since the oily liquid becomes unstable on the substrate, there is a problem that the oily liquid cannot be properly held on the substrate. On the other hand, if the volume of the oily liquid is too large, the oily liquid will be used more than necessary, and there is a problem that costs increase. Further, if the water-repellent annular region 102b and the hydrophilic annular region 102c are too large, there is a problem that the number of liquid holding regions 102A that can be formed on a substrate having a limited area is reduced. Therefore, according to the substrate 100 which is effective in the present embodiment, in addition to the hydrophilic circular region 102a and the water repellent region 104, the water repellent annular region 102b and the hydrophilic annular region 102c are further provided, and the water repellent property is provided. Since the sizes of the annular region 102b and the hydrophilic annular region 102c are optimized! /, The above-mentioned problems can be solved reliably.
[0036] また、本実施の形態に力、かる基板 100によれば、親水性円形領域 102aには、 π Γ3 /3 1以上且つ π r3 1以下の量の液体（具体的には水溶性のもの）が保持されても よ!/、ので、水溶性反応液の化学反応にお!/、て使用する当該水溶性反応液の量を最 適化すること力 Sできる。ここで、親水性円形領域 102aに保持される水溶性反応液の 量が少なすぎると、水溶性反応液の化学反応を行うための試料の量も少なくなるの で、水溶性反応液の化学反応を行うには不都合であるという問題がある。一方、親水 性円形領域 102aに保持される水溶性反応液の量が多すぎると、親水性円形領域 1 02aで水溶性反応液を保持することができなくなるという問題がある。そこで、本実施 の形態に力、かる基板 100によれば、親水性円形領域 102aで保持する液体の量を最 適化して!/、るので、上記の数々の問題を確実に解決することができる。 [0036] In addition, the force to this embodiment, according to mow the substrate 100, the hydrophilic circular area 102a, π Γ 3/3 1 or more and [pi r 3 1 The following amounts of the liquid (specifically, water Therefore, it is possible to optimize the amount of the water-soluble reaction solution used for the chemical reaction of the water-soluble reaction solution. Here, if the amount of the water-soluble reaction liquid held in the hydrophilic circular region 102a is too small, the amount of the sample for performing the chemical reaction of the water-soluble reaction liquid also decreases. There is a problem that it is inconvenient to do. On the other hand, if the amount of the water-soluble reaction liquid held in the hydrophilic circular area 102a is too large, there is a problem that the water-soluble reaction liquid cannot be held in the hydrophilic circular area 102a. Therefore, according to this embodiment, according to the substrate 100, the amount of liquid retained in the hydrophilic circular region 102a is optimized! /, So that the above-mentioned numerous problems can be reliably solved. it can.
[0037] また、本実施の形態に力、かる基板 100によれば、撥水性円環領域 102bおよび親 水性円環領域 102cには、親水性円形領域 102aに保持されて!/、る液体（具体的に は水溶性のもの）を覆うように、 2 π (r + Ο · 6) 3/3— π Γ3〃1以上且つ 2 π (r+ 0. 8 ) 3 /3 - π r3/3 ,ι 1以下の量の液体（具体的には油性のもの）が保持されてもよ!/、ので 、水溶性反応液の化学反応において当該水溶性反応液の蒸発を防止するために使 用する油性液体の量を最適化することができる。ここで、撥水性円環領域 102bおよ び親水性円環領域 102cに保持される油性液体の量が少なすぎると、水溶性反応液 を十分に覆うことができずその蒸発を防止することができなくなるという問題がる。一 方、撥水性円環領域 102bおよび親水性円環領域 102cに保持される油性液体の量 が多すぎると、親水性円環領域 102cで油性液体を保持することができなくなるという 問題がある。そこで、本実施の形態に力、かる基板 100によれば、撥水性円環領域 10 2bおよび親水性円環領域 102cで保持する液体の量を最適化しているので、上記の 数々の問題を確実に解決することができる。 [0037] Further, according to the substrate 100 which is effective in the present embodiment, the water repellent annular region 102b and the hydrophilic hydrophilic region 102c are held in the hydrophilic circular region 102a! Specifically, 2 π (r + Ο · 6) 3 / 3— π Γ 3 〃1 or more and 2 π (r + 0.8) 3 /3-π r 3 / 3, ι 1 or less liquid (specifically oily) may be retained! /, So it is used to prevent evaporation of the water-soluble reaction liquid in the chemical reaction of the water-soluble reaction liquid. The amount of oily liquid used can be optimized. Here, if the amount of the oily liquid retained in the water-repellent annular region 102b and the hydrophilic annular region 102c is too small, the water-soluble reaction liquid cannot be sufficiently covered and evaporation thereof can be prevented. There is a problem that it can not be done. On the other hand, the amount of oily liquid retained in the water-repellent ring region 102b and the hydrophilic ring region 102c. If there is too much, there is a problem that the oily liquid cannot be retained in the hydrophilic ring region 102c. Therefore, according to the substrate 100 which is effective in this embodiment, since the amount of liquid retained in the water-repellent annular region 102b and the hydrophilic annular region 102c is optimized, the above-mentioned problems are surely confirmed. Can be solved.
[0038] また、本実施の形態に力、かる基板 100によれば、親水性円形領域 102aには核酸 および/または酵素の吸着防止処理が施されてもよいので、当該吸着防止処理を親 水性円形領域 102aに施した場合には微量の水溶性反応液に含まれる核酸や酵素 を確実且つ容易に回収することができる。  [0038] Further, according to the substrate 100 which is effective in the present embodiment, the hydrophilic circular region 102a may be subjected to a nucleic acid and / or enzyme adsorption prevention treatment. When applied to the circular region 102a, nucleic acids and enzymes contained in a small amount of the water-soluble reaction solution can be reliably and easily recovered.
[0039] また、本実施の形態に力、かる基板 100によれば、液体保持領域 102 (具体的には 液体保持領域 102A)を複数設けて!/、るで、水溶性反応液の化学反応を複数同時に fiうこと力 Sでさる。  [0039] Further, according to the present embodiment, according to the substrate 100, a plurality of liquid holding regions 102 (specifically, the liquid holding regions 102A) are provided! The power of S can be measured simultaneously with S.
[0040] また、本実施の形態に力、かる基板 100によれば、在る液体保持領域 102A (具体的 には在る親水性円環領域 102c)の外周部分と他の液体保持領域 102A (具体的に は他の親水性円環領域 102c)の外周部分とは lmm以上（図 2では lmm)離れてい るので、個々の液体保持領域 102Aで保持している油性液体同士が結合することな ぐ個々の液体保持領域 102Aで油性液体を確実に保持することができる。また、在 る液体保持領域 102Aに保持されている油性液体と他の液体保持領域 102Aに保 持されている油性液体とが結合することは無ぐまた水溶性反応液は液体保持領域 1 02Aに安定に保持されるので、水溶性反応液を個々の液体保持領域 102Aから容 易に回収することができる。ここで、親水性円環領域 102cの外周部分同士の距離が lmm未満であると、油性液体同士が結合する可能性が高まる。そして、油性液体同 士が結合すると、油性液体が図 7で示したような状態になるので、水溶性反応液を十 分に覆うことができなくなり、結果として水溶性反応液が蒸発してしまうという問題があ る。そこで、本実施の形態に力、かる基板 100によれば、親水性円環領域 102cの外周 部分同士を lmm以上離しているので、上記の問題を確実に解決することができる。 実施例 1  [0040] Further, according to this embodiment, according to the substrate 100, the outer peripheral portion of the existing liquid holding region 102A (specifically, the existing hydrophilic ring region 102c) and the other liquid holding region 102A ( Specifically, since the outer peripheral portion of the other hydrophilic ring region 102c) is separated by 1 mm or more (1 mm in FIG. 2), the oily liquids held in the individual liquid holding regions 102A are not bonded to each other. Thus, the oily liquid can be reliably held in the individual liquid holding regions 102A. In addition, the oily liquid held in the existing liquid holding area 102A and the oily liquid held in the other liquid holding area 102A do not bind to each other, and the water-soluble reaction liquid does not enter the liquid holding area 102A. Since it is held stably, the water-soluble reaction liquid can be easily recovered from each liquid holding area 102A. Here, if the distance between the outer peripheral portions of the hydrophilic ring region 102c is less than 1 mm, the possibility that oily liquids are bonded to each other increases. When the oily liquids are combined, the oily liquid is in the state shown in FIG. 7, so that the water-soluble reaction liquid cannot be sufficiently covered, and as a result, the water-soluble reaction liquid evaporates. There is a problem. In view of this, according to the substrate 100 which is effective in the present embodiment, the outer peripheral portions of the hydrophilic annular region 102c are separated from each other by 1 mm or more, so that the above problem can be solved reliably. Example 1
[0041] ここでは、本発明にかかる反応液反応方法の実施例として、親水性円形領域 102a に酵素および核酸の吸着防止処理が施された基板 100を用いて水溶性反応液の等 温増幅反応を行った。 [0041] Here, as an example of the reaction liquid reaction method according to the present invention, a water-soluble reaction liquid or the like is obtained using a substrate 100 in which the hydrophilic circular region 102a has been subjected to an enzyme and nucleic acid adsorption prevention treatment. A warm amplification reaction was performed.
[0042] 実施例 1にお!/、て、上述した実施の形態における水溶性反応液に相当する等温増 幅反応サンプルは、以下のものが以下の組成比で調整されたものである。上述した 実施の形態における油性液体に相当するシーリングオイルは、シグマアルドリッチジ ャパン社製のミネラルオイル「M5904」（製品名）である。  [0042] In Example 1, the isothermal amplification reaction sample corresponding to the water-soluble reaction liquid in the embodiment described above is prepared by adjusting the following composition ratios. The sealing oil corresponding to the oily liquid in the above-described embodiment is mineral oil “M5904” (product name) manufactured by Sigma-Aldrich Japan.
gDNA： 1ナノグラム（ng) / μ 1  gDNA: 1 nanogram (ng) / μ 1
等温増幅反応試薬：「Loopamp DNA増幅試薬キット マスターミックス」（製品名）  Isothermal amplification reagent: “Loopamp DNA amplification reagent kit master mix” (product name)
[0043] 実験者は、基板 100に設けられて!/、る親水性円形領域 102aおよび液体保持領域 [0043] The experimenter is provided on the substrate 100! /, The hydrophilic circular region 102a and the liquid holding region.
102Bのそれぞれに、等温増幅反応サンプルをピペットで 1〃1分注し、さらに、分注し た等温増幅反応サンプルを覆う様にシーリングオイルをピペットで 5 1分注した。そし て、実験者は、等温増幅反応サンプルおよびシーリングオイルが分注されている基 板 100を、予め用意したサーマルサイクラ一（Eppendorf Master Cycler)にセッ トし、等温増幅反応に必要なサーマルサイクルを印可して等温増幅反応サンプルの 等温増幅反応を行った。なお、このサーマルサイクルは、以下の（11)および（12)の 順で行われるものである。  To each of 102B, an isothermal amplification reaction sample was pipetted for 1 to 1 minutes, and sealing oil was pipetted for 51 minutes to cover the dispensed isothermal amplification reaction sample. Then, the experimenter sets the substrate 100 on which the isothermal amplification reaction sample and the sealing oil are dispensed to the prepared thermal cycler (Eppendorf Master Cycler), and performs the thermal cycle necessary for the isothermal amplification reaction. When applied, an isothermal amplification reaction was performed on the isothermal amplification reaction sample. This thermal cycle is performed in the following order (11) and (12).
(11 ) 63°Cを 30分間維持する。  (11) Maintain at 63 ° C for 30 minutes.
(12) 80°Cを 2分維持する。  (12) Maintain 80 ° C for 2 minutes.
[0044] サーマルサイクルが終了した後、実験者は、それぞれの液体保持領域 102Aおよ び液体保持領域 102Bから等温増幅反応サンプルのみをピペットで回収し、回収し た等温増幅反応サンプルを Agilent Technologies社製の「Agilent bioanalyze rj (製品名）で解析した。  [0044] After the thermal cycle is completed, the experimenter collects only the isothermal amplification reaction sample from each of the liquid holding region 102A and the liquid holding region 102B with a pipette, and the recovered isothermal amplification reaction sample is collected from Agilent Technologies. “Agilent bioanalyze rj (product name)”
[0045] 解析の結果、液体保持領域 102Aから回収した全ての等温増幅反応サンプルから は、バンドが検出され、等温増幅反応による核酸の増幅が確認された。一方、液体保 持領域 102Bから回収したすべての等温増幅反応サンプルからは、バンドが検出さ れず、等温増幅反応による核酸の増幅が確認されなかった。なお、等温増幅反応サ ンプルは液体保持領域 102Aおよび液体保持領域 102Bのどちらにおいても安定に 保持されていたので、ピペットのみで容易に回収することができた。  [0045] As a result of the analysis, bands were detected from all isothermal amplification reaction samples collected from the liquid holding region 102A, and nucleic acid amplification was confirmed by the isothermal amplification reaction. On the other hand, no band was detected from all the isothermal amplification reaction samples collected from the liquid holding region 102B, and nucleic acid amplification by the isothermal amplification reaction was not confirmed. Since the isothermal amplification reaction sample was stably held in both the liquid holding region 102A and the liquid holding region 102B, it could be easily recovered with a pipette alone.
実施例 2 [0046] ここでは、本発明にかかる反応液反応方法の実施例として、親水性円形領域 102a に酵素および核酸の吸着防止処理が施された基板 100を用いて水溶性反応液の P CRを行った。 Example 2 [0046] Here, as an example of the reaction liquid reaction method according to the present invention, PCR of the water-soluble reaction liquid is performed using the substrate 100 in which the hydrophilic circular region 102a is subjected to the enzyme and nucleic acid adsorption prevention treatment. It was.
[0047] 実施例 2において、上述した実施の形態における水溶性反応液に相当する PCRサ ンプルは、以下のものが以下の組成比で調整されたものである。上述した実施の形 態における油性液体に相当するシーリングオイルは、シグマアルドリッチジャパン社 製のミネラノレオイノレ「M5904」（製品名）である。 [0047] In Example 2, the PCR sample corresponding to the water-soluble reaction solution in the above-described embodiment is prepared by adjusting the following composition ratio. The sealing oil corresponding to the oily liquid in the above-described embodiment is Minera Leo Inore “M5904” (product name) manufactured by Sigma-Aldrich Japan.
Figure imgf000015_0001
Figure imgf000015_0001
Forward Primer： 0. 5 μ Μ  Forward Primer: 0.5 μ Μ
Reverse Primer： 0. 5 μ Μ  Reverse Primer: 0.5 μΜ
PCR試薬：「Ampli Taq Gold Master Mix」（製品名）（ABI社製）  PCR reagent: “Ampli Taq Gold Master Mix” (product name) (ABI)
[0048] 実験者は、基板 100に設けられて!/、る親水性円形領域 102aおよび液体保持領域 [0048] The experimenter is provided on the substrate 100! /, The hydrophilic circular region 102a and the liquid holding region.
102Bのそれぞれに、 PCRサンプルをピペットで 1 μ 1分注し、さらに、分注した PCR サンプルを覆う様にシーリングオイルをピペットで 5 1分注した。そして、実験者は、 Ρ CRサンプルおよびシーリングオイルが分注されている基板 100を、予め用意したサ 一マノレサイクラ一（Eppendorf Master Cycler)にセットし、 PCRに必要なサーマ ルサイクルを印可して PCRサンプルの PCRを行った。なお、このサーマルサイクルは 、以下の（21 )から（24)の順で行われるものである。  A PCR sample was pipetted into each of 102B by 1 μl, and sealing oil was pipetted 51 times so as to cover the dispensed PCR sample. Then, the experimenter placed the substrate 100 on which the CR sample and sealing oil had been dispensed into a pre-prepared Thermorecycler (Eppendorf Master Cycler), applied the thermal cycle necessary for PCR, and applied the PCR. Samples were PCRed. This thermal cycle is performed in the following order (21) to (24).
(21 ) 95°Cを 10分間維持する。  (21) Maintain 95 ° C for 10 minutes.
(22) 95°Cを 30秒維持し、引き続いて 60°Cを 30秒維持し、さらに引き続いて 72°Cを 30秒維持する。  (22) Maintain 95 ° C for 30 seconds, continue to maintain 60 ° C for 30 seconds, and continue to maintain 72 ° C for 30 seconds.
(23) (22)を 35回繰り返す。  (23) Repeat (22) 35 times.
(24) 72°Cを 10分間維持する。  (24) Maintain 72 ° C for 10 minutes.
[0049] サーマルサイクルが終了した後、実験者は、それぞれの液体保持領域 102Aおよ び液体保持領域 102Bから PCRサンプルのみをピペットで回収し、回収した PCRサ ンプルを Agilent Technologies社製の「Agilent bioanalyzerj (製品名）で解析 した。  [0049] After the thermal cycle is completed, the experimenter pipettes only the PCR sample from each of the liquid holding area 102A and the liquid holding area 102B, and collects the collected PCR samples using Agilent Technologies' “Agilent Analysis was performed using bioanalyzerj (product name).
[0050] 解析の結果、液体保持領域 102Aから回収した全ての PCRサンプルからは、铸型 DNAに特異的なバンドが検出され、 PCRによる核酸の増幅が確認された。一方、液 体保持領域 102Bから回収したすべての PCRサンプルからは、バンドが検出されず 、 PCRによる核酸の増幅が確認されな力、つた。なお、 PCRサンプルは液体保持領域 102Aおよび液体保持領域 102Bのどちらにおいても安定に保持されていたので、ピ ペットのみで容易に回収することができた。 [0050] As a result of analysis, all PCR samples collected from the liquid holding region 102A were A band specific to DNA was detected, confirming amplification of the nucleic acid by PCR. On the other hand, no band was detected from all the PCR samples collected from the liquid holding region 102B, and the amplification of nucleic acid by PCR was confirmed. Since the PCR sample was stably held in both the liquid holding region 102A and the liquid holding region 102B, it could be easily recovered with only a pipette.
実施例 3  Example 3
[0051] ここでは、本発明にかかる反応液反応方法の実施例として、親水性円形領域 102a に酵素および核酸の吸着防止処理が施された基板 100を用いて水溶性反応液のラ ィゲーシヨン反応を行った。  [0051] Here, as an example of the reaction liquid reaction method according to the present invention, a ligation reaction of a water-soluble reaction liquid is performed using the substrate 100 in which the hydrophilic circular region 102a is subjected to an enzyme and nucleic acid adsorption prevention treatment. went.
[0052] 実施例 3にお!/ヽて、上述した実施の形態における水溶性反応液に相当するライゲ ーシヨン反応サンプルは、チューブ内で以下のものが以下の組成比で混合されたも のに更に超純水が補われたもので、その量は 100 ^ 1である。上述した実施の形態に おける油性液体に相当するシーリングオイルは、シグマアルドリッチジャパン社製のミ ネラルオイル「M5904」（製品名）である。  [0052] In Example 3, the ligation reaction sample corresponding to the water-soluble reaction liquid in the above-described embodiment was obtained by mixing the following in the tube at the following composition ratio: Furthermore, it is supplemented with ultrapure water, the amount of which is 100 ^ 1. The sealing oil corresponding to the oily liquid in the above-described embodiment is a mineral oil “M5904” (product name) manufactured by Sigma-Aldrich Japan.
プローブ l (20mer, 5'TAMRA修飾）： 10ピコモル（pmol)  Probe l (20mer, 5'TAMRA modification): 10 pmol
プローブ 2 (20mer, 5'リン酸修飾） : 10pmol  Probe 2 (20mer, 5 'phosphate modification): 10pmol
Taqライゲース： 5ユニット  Taq Ligate: 5 units
ライゲース酵素：「TaqDNAライゲースキット M0208S」（製品名）（New England Ligase enzyme: “TaqDNA ligase kit M0208S” (product name) (New England
Biolab社製） Biolab)
キット添付 10xノ ッファー : 5 ^ 1  Kit included 10x notfer: 5 ^ 1
[0053] 実験者は、ライゲーシヨン反応サンプルをピペットでチューブから取り出し、取り出し たライゲーシヨン反応サンプルを、基板 100に設けられている親水性円形領域 102a および液体保持領域 102Bのそれぞれに 1 1分注し、さらに、分注したライグーショ ン反応サンプルを覆う様にシーリングオイルをピペットで 5 1分注した。そして、実験 者は、ライゲーシヨン反応サンプルおよびシーリングオイルが分注されている基板 10 0を、予め用意したホットプレート上にセットし、 50°Cで 30分間静置してライゲーシヨン 反応サンプルのライゲーシヨン反応を行った。  [0053] The experimenter pipettes the ligation reaction sample from the tube, and dispenses the extracted ligation reaction sample into the hydrophilic circular region 102a and the liquid holding region 102B provided on the substrate 100, respectively. Further, 51 ml of sealing oil was pipetted to cover the dispensed ligation reaction sample. Then, the experimenter sets the substrate 100 on which the ligation reaction sample and the sealing oil are dispensed on a hot plate prepared in advance, and leaves it at 50 ° C. for 30 minutes to perform the ligation reaction of the ligation reaction sample. went.
[0054] 30分間の静置が終了した後、実験者は、それぞれの液体保持領域 102Aおよび 液体保持領域 102Bからライゲーシヨン反応サンプルのみをピペットで回収し、回収 したライゲーシヨン反応サンプルを 5'TAMRA修飾のマーカー DNAと共に DNAシ 一ケンサ一（製品名「ABI PRISM 8100」）で変性ゲル電気泳動を行い、断片解 析した。 [0054] After the 30-minute standing is complete, the experimenter can determine that each liquid holding region 102A and Collect only the ligation reaction sample from the liquid holding area 102B with a pipette and perform denaturation gel electrophoresis of the collected ligation reaction sample with a DNA sequencer (product name “ABI PRISM 8100”) together with a marker DNA modified with 5'TAMRA. Fragment analysis.
[0055] 解析の結果、液体保持領域 102Aから回収した全てのライゲーシヨン反応サンプル からは、 20mer、 40mer相当のピークとマーカーのピークが検出され、プローブ 1と プローブ 2とのライゲーシヨン反応が確認された。一方、液体保持領域 102Bから回 収したすベてのライゲーシヨン反応サンプルからは、 20mer、 40mer相当のピークと マーカーのピークが検出されず、プローブ 1とプローブ 2とのライゲーシヨン反応が確 認されなかった。なお、ライゲーシヨン反応サンプルは液体保持領域 102Aおよび液 体保持領域 102Bのどちらにおいても安定に保持されていたので、ピペットのみで容 易に回収することができた。  As a result of the analysis, 20 mer and 40 mer peaks and marker peaks were detected from all the ligation reaction samples collected from the liquid holding region 102A, and the ligation reaction between probe 1 and probe 2 was confirmed. On the other hand, from all ligation reaction samples collected from the liquid holding region 102B, peaks corresponding to 20 mer and 40 mer and a marker peak were not detected, and ligation reaction between probe 1 and probe 2 was not confirmed. . Since the ligation reaction sample was stably held in both the liquid holding region 102A and the liquid holding region 102B, it could be easily recovered with only a pipette.
産業上の利用可能性  Industrial applicability
[0056] 以上のように、本発明にかかる基板および反応液反応方法は、等温増幅反応や P CRやライゲーシヨン反応などの水溶性反応液の化学反応を確実に行うことができ、 バイオや製薬、医療など様々な産業上の分野において好適に用いることができる。 [0056] As described above, the substrate and the reaction liquid reaction method according to the present invention can reliably perform a chemical reaction of an aqueous reaction liquid such as an isothermal amplification reaction or a PCR or a ligation reaction. It can be suitably used in various industrial fields such as medical care.

Claims

請求の範囲 The scope of the claims
[1] その面が全体的に撥水性を有し、円の形を成す親水性の領域である親水性円形 領域をその面上に部分的に設けてレ、る基板にお!/、て、  [1] The substrate has a water-repellent surface as a whole, and a hydrophilic circular region, which is a circular region, is partially provided on the surface. ,
円環の形を成す撥水性の領域であって前記親水性円形領域を囲み且つ前記親水 性円形領域に隣接する撥水性円環領域と、円環の形を成す親水性の領域であって 前記撥水性円環領域を囲み且つ前記撥水性円環領域に隣接する親水性円環領域 と、をさらに設け、  A water-repellent region in the shape of a ring, surrounding the hydrophilic circular region and adjacent to the hydrophilic circular region, and a hydrophilic region in the shape of a ring, A hydrophilic ring region that surrounds the water-repellent ring region and is adjacent to the water-repellent ring region; and
前記撥水性円環領域における前記円環の幅は 0. 6ミリメートル以上且つ 0. 8ミリメ 一トル以下であり、前記親水性円環領域における前記円環の幅は 0. 15ミリメートノレ 以上且つ 0. 35ミリメートル以下であること  The width of the ring in the water-repellent ring region is 0.6 mm or more and 0.8 mm or less, and the width of the ring in the hydrophilic ring region is 0.15 mm or more and 0. Be 35mm or less
を特徴とする基板。  A substrate characterized by.
[2] 前記親水性円形領域には、 π r3/3マイクロリットル以上且つ π r3マイクロリットル以 下の量の液体が保持されること [2] wherein the hydrophilic circular area, π r 3/3 to microliters or more and [pi r 3 of the amount of under microliters or less liquid is held
(前記 rは前記親水性円形領域の半径（単位はミリメートル)であり、前記 πは円周率 である。 )  (Where r is the radius (in millimeters) of the hydrophilic circular region, and π is the circumference)
を特徴とする請求項 1に記載の基板。  The substrate according to claim 1, wherein:
[3] 前記撥水性円環領域および前記親水性円環領域には、前記親水性円形領域に 保持されている液体を覆うように、 2 π (r + Ο· 6) 3/3— π Γ3マイクロリットノレ以上且つ 2 π (r + 0. 8) 3/3— π Γ3/3マイクロリットル以下の量の油性液体が保持されること (前記 rは前記親水性円形領域の半径（単位はミリメートル)であり、前記 πは円周率 である。 ) [3] In the water-repellent annular region and the hydrophilic annular region, 2 π (r + Ο · 6) 3 / 3- π Γ is provided so as to cover the liquid retained in the hydrophilic circular region. 3 microliters Honoré or more and 2 π (r + 0. 8) 3 / 3- π Γ 3/3 to microliters or less of the amount of oil liquid is held (the r is the radius (unit of the hydrophilic circular area Is millimeter), and π is the circumference.
を特徴とする請求項 2に記載の基板。  The substrate according to claim 2.
[4] 前記親水性円形領域には、酵素および/または核酸の吸着防止処理が施されて レ、ること [4] The hydrophilic circular region is subjected to an enzyme and / or nucleic acid adsorption prevention treatment.
を特徴とする請求項 1から 3のいずれか 1つに記載の基板。  The substrate according to any one of claims 1 to 3, wherein:
[5] 前記親水性円形領域と前記撥水性円環領域と前記親水性円環領域とを複数組設 けたこと [5] A plurality of sets of the hydrophilic circular region, the water-repellent annular region, and the hydrophilic annular region are provided.
を特徴とする請求項 1から 4のいずれか 1つに記載の基板。 The substrate according to any one of claims 1 to 4, wherein:
[6] 在る前記親水性円環領域の外周部分と他の前記親水性円環領域の外周部分とは 1ミリメートル以上離れて!/、ること [6] The outer peripheral portion of the hydrophilic annular region and the outer peripheral portion of the other hydrophilic annular region are separated from each other by 1 mm or more! /
を特徴とする請求項 5に記載の基板。  The substrate according to claim 5.
[7] 前記請求項 1から 6のいずれか 1つに記載の基板を用いて水溶性反応液の化学反 応を fiうこと [7] Using the substrate according to any one of claims 1 to 6 to perform chemical reaction of the water-soluble reaction liquid.
を特徴とする反応液反応方法。  The reaction liquid reaction method characterized by these.
[8] 前記基板に設けられて!/、る前記親水性円形領域に前記水溶性反応液を滴下する ことで、 π r3/3マイクロリットル以上且つ π r3マイクロリットル以下の量の前記水溶性 反応液を前記親水性円形領域に保持させること [8] provided on the substrate! /, Ru said by dropwise addition of hydrophilic the circular region aqueous reaction solution, the water of [pi r 3/3 or more microliters and [pi r 3 microliters or less of the amount Holding the reaction solution in the hydrophilic circular region
(前記 rは前記親水性円形領域の半径（単位はミリメートル)であり、前記 πは円周率 である。 )  (Where r is the radius (in millimeters) of the hydrophilic circular region, and π is the circumference)
を特徴とする請求項 7に記載の反応液反応方法。  The reaction solution reaction method according to claim 7, wherein:
[9] 前記基板に設けられて!/、る前記撥水性円環領域および前記親水性円環領域に、 前記水溶性反応液を覆うように油性液体を滴下することで、 2 π (r + O. 6) 3/3— 3マイクロリットル以上且つ 2 π (r+ O. 8) 3/3— π Γ3/3マイクロリットル以下の量の前 記油性液体を前記撥水性円環領域および前記親水性円環領域に保持させること (前記 rは前記親水性円形領域の半径（単位はミリメートル)であり、前記 πは円周率 である。 ) [9] An oily liquid is dropped onto the water-repellent annular region and the hydrophilic annular region provided on the substrate so as to cover the water-soluble reaction liquid, thereby obtaining 2 π (r + O. 6) 3 / 3- 3 microliters or more and 2 π (r + O. 8) 3 / 3- π Γ 3/3 the previous SL oil liquid following amounts microliters water repellent annular region and the hydrophilic (Where r is the radius (in millimeters) of the hydrophilic circular region, and π is the circumference).
を特徴とする請求項 8に記載の反応液反応方法。  The reaction liquid reaction method according to claim 8, wherein:
[10] 前記水溶性反応液は、ポリメラーゼ連鎖反応、等温増幅反応およびライゲーシヨン 反応のいずれ力、 1つを行うためのものであること [10] The water-soluble reaction solution is for performing one of a polymerase chain reaction, an isothermal amplification reaction, and a ligation reaction.
を特徴とする請求項 7から 9のいずれか 1つに記載の反応液反応方法。  10. The reaction solution reaction method according to any one of claims 7 to 9, wherein:
PCT/JP2007/067523 2006-09-26 2007-09-07 Substratum and method of reactive liquid reaction WO2008038507A1 (en)

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JP2013526700A (en) * 2010-05-07 2013-06-24 ユーティ―バテル エルエルシー System and method for extracting a sample from a surface

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JPH11304666A (en) * 1998-04-24 1999-11-05 Hitachi Ltd Sample handling tool and method for using same
JP2006349558A (en) * 2005-06-17 2006-12-28 Toppan Printing Co Ltd Container

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JPH05336971A (en) * 1991-07-23 1993-12-21 F Hoffmann La Roche Ag Improvement of pcr
JPH11304666A (en) * 1998-04-24 1999-11-05 Hitachi Ltd Sample handling tool and method for using same
JP2006349558A (en) * 2005-06-17 2006-12-28 Toppan Printing Co Ltd Container

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Publication number Priority date Publication date Assignee Title
JP2013526700A (en) * 2010-05-07 2013-06-24 ユーティ―バテル エルエルシー System and method for extracting a sample from a surface
US9063047B2 (en) 2010-05-07 2015-06-23 Ut-Battelle, Llc System and method for extracting a sample from a surface

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