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WO2008038507A1 - Substrat et procédé de réaction d'un liquide réactif - Google Patents

Substrat et procédé de réaction d'un liquide réactif 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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English (en)
Japanese (ja)
Inventor
Yoshinobu Akimoto
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Olympus Corp
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Olympus Corp
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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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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Structural Engineering (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un substrat qui, tout en empêchant de manière sûre l'évaporation d'un liquide réactif hydrosoluble pendant la réalisation de sa réaction chimique, optimise la quantité de liquide huileux utilisée dans le but d'empêcher l'évaporation du liquide réactif hydrosoluble, ce qui permet d'obtenir une réduction effective des coûts ; et un procédé de réaction du liquide réactif dans lequel la réaction chimique du liquide réactif hydrosoluble peut être réalisée de manière sûre. La surface du substrat présente une caractéristique hydrophobe et est partiellement dotée d'une zone circulaire hydrophile. Ledit substrat est en outre munid'une zone annulaire hydrophobe entourant la zone circulaire hydrophile et venant en butée contre celle-ci, et muni d'une zone annulaire hydrophile entourant la zone annulaire hydrophobe et venant en butée contre celle-ci.
PCT/JP2007/067523 2006-09-26 2007-09-07 Substrat et procédé de réaction d'un liquide réactif Ceased WO2008038507A1 (fr)

Applications Claiming Priority (2)

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JP2006260547 2006-09-26
JP2006-260547 2006-09-26

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WO2008038507A1 true WO2008038507A1 (fr) 2008-04-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013526700A (ja) * 2010-05-07 2013-06-24 ユーティ―バテル エルエルシー 表面からサンプルを抽出するためのシステムおよび方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05336971A (ja) * 1991-07-23 1993-12-21 F Hoffmann La Roche Ag Ｐｃｒの改良方法
JPH11304666A (ja) * 1998-04-24 1999-11-05 Hitachi Ltd 試料ハンドリングツールおよびその使用方法
JP2006349558A (ja) * 2005-06-17 2006-12-28 Toppan Printing Co Ltd 容器

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05336971A (ja) * 1991-07-23 1993-12-21 F Hoffmann La Roche Ag Ｐｃｒの改良方法
JPH11304666A (ja) * 1998-04-24 1999-11-05 Hitachi Ltd 試料ハンドリングツールおよびその使用方法
JP2006349558A (ja) * 2005-06-17 2006-12-28 Toppan Printing Co Ltd 容器

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013526700A (ja) * 2010-05-07 2013-06-24 ユーティ―バテル エルエルシー 表面からサンプルを抽出するためのシステムおよび方法
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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