WO2013132891A1 - 核酸増幅反応用マイクロチップの製造方法 - Google Patents

核酸増幅反応用マイクロチップの製造方法 Download PDF

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Publication number
WO2013132891A1
WO2013132891A1 PCT/JP2013/050652 JP2013050652W WO2013132891A1 WO 2013132891 A1 WO2013132891 A1 WO 2013132891A1 JP 2013050652 W JP2013050652 W JP 2013050652W WO 2013132891 A1 WO2013132891 A1 WO 2013132891A1
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Prior art keywords
nucleic acid
acid amplification
microchip
amplification reaction
reagent solution
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PCT/JP2013/050652
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English (en)
French (fr)
Japanese (ja)
Inventor
真寛 松本
佐藤　正樹
英俊 渡辺
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ソニー株式会社
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Application filed by ソニー株式会社 filed Critical ソニー株式会社
Priority to RU2014135538A priority Critical patent/RU2014135538A/ru
Priority to JP2014503511A priority patent/JP5987895B2/ja
Priority to IN1628MUN2014 priority patent/IN2014MN01628A/en
Priority to EP13758540.2A priority patent/EP2824172B1/en
Priority to CN201380012088.2A priority patent/CN104160011A/zh
Priority to KR1020147023987A priority patent/KR20140143139A/ko
Priority to US14/378,588 priority patent/US9545630B2/en
Publication of WO2013132891A1 publication Critical patent/WO2013132891A1/ja

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5088—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above confining liquids at a location by surface tension, e.g. virtual wells on plates, wires
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials

Definitions

  • This technology relates to a method for producing a microchip for nucleic acid amplification reaction. More specifically, the present invention relates to a nucleic acid amplification reaction microchip in which a solidified reagent containing one or more substances necessary for the reaction is contained in a well serving as a reaction field for the nucleic acid amplification reaction.
  • microchips having wells and channels for performing chemical and biological analysis on a silicon or glass substrate have been developed by applying microfabrication technology in the semiconductor industry. These microchips are beginning to be used in, for example, electrochemical detectors for liquid chromatography and small electrochemical sensors in medical settings.
  • ⁇ -TAS micro-Total-Analysis System
  • lab-on-chip a sample-on-chip
  • biochips a microchip that uses microchips
  • speed up and high efficiency of chemical and biological analysis As a technology that enables downsizing, integration, or downsizing of analyzers, it is attracting attention.
  • ⁇ -TAS can be analyzed with a small amount of sample and disposable use of microchips (disposable). Has been.
  • ⁇ -TAS there is an optical detection device that introduces a substance into a plurality of regions arranged on a microchip and chemically detects the substance.
  • an optical detection apparatus for example, a reaction apparatus (for example, a real-time PCR apparatus) that optically detects a substance to be generated by advancing a reaction between a plurality of substances such as a nucleic acid amplification reaction in a well on a microchip. and so on.
  • a reagent and template DNA necessary for a nucleic acid amplification reaction are all mixed in advance, and this mixed solution is introduced into a plurality of wells arranged in the microchip to perform a reaction.
  • this method requires a certain amount of time for the mixture to be introduced into the well, and during that time, the reaction proceeds in the mixture, facilitating non-specific nucleic acid amplification and improving the quantitativeness. There has been a problem of lowering.
  • Patent Document 1 discloses a microchip in which a plurality of reagents necessary for a nucleic acid amplification reaction are stacked and fixed in a predetermined order in a well.
  • This technology is mainly intended to provide a method for producing a microchip for nucleic acid amplification reaction that allows simple and highly accurate analysis.
  • the present technology provides a solidification step for drying a reagent solution containing at least a part of a substance necessary for a nucleic acid amplification reaction, and a well serving as a reaction field for the nucleic acid amplification reaction.
  • a method for producing a microchip for nucleic acid amplification reaction comprising: The solidification step preferably includes a step of freeze-drying the reagent solution. Before the solidification step, a preparation step of preparing a plurality of the reagent solutions having different compositions is included.
  • the reagent solution includes a first reagent solution that includes an oligonucleotide primer and does not include an enzyme, and an oligo that includes the enzyme.
  • the solidification step may include a step of separately lyophilizing the first reagent solution and the second reagent solution.
  • the storing step may include a step of storing the first reagent solution containing two or more kinds of solidified oligonucleotide primers in each of the plurality of wells.
  • any one of the first reagent solution and the second reagent solution is solidified by the solidification step, and the reagent not used in the solidification step before the containing step
  • a method for producing a microchip for nucleic acid amplification reaction which comprises a fixing step of dropping a liquid into the well and drying the well.
  • the fixing step preferably includes a step of vacuum drying the reagent solution.
  • This technology provides a nucleic acid amplification microchip that enables simple and accurate analysis.
  • FIG. 1 is a schematic diagram illustrating the configuration of a microchip 1a according to the first embodiment of the present technology.
  • 1A is a schematic top view
  • FIG. 1B is a schematic cross-sectional view corresponding to the PP cross section of FIG. 1A.
  • microchip for nucleic acid amplification reaction (hereinafter also referred to as “microchip”) denoted by reference numeral 1a in the figure, as an area into which a sample solution is introduced, an introduction part 2 into which a liquid such as a sample is introduced from the outside, and a nucleic acid Wells 41 to 45 serving as reaction fields for the amplification reaction, and flow paths 31 to 35 connecting the introduction part 2 and each well are provided.
  • the wells 41 to 45 contain reagents R1 and R2 containing at least a part of substances necessary for the nucleic acid amplification reaction (reagents R1 and R2, not shown in FIG. 1B).
  • the sample solution refers to a solution containing a nucleic acid such as DNA or RNA which is a template nucleic acid to be amplified in the nucleic acid amplification reaction.
  • the “nucleic acid amplification reaction” performed using the microchip according to the present technology includes a conventional PCR (Polymerase® Chain Reaction) method in which a temperature cycle is performed and various isothermal amplification methods not involving a temperature cycle.
  • PCR Polymerase® Chain Reaction
  • LAMP Loop-Mediated Isothermal Amplification
  • SMAP SMart Amplification Process
  • NASBA Nucleic Acid Sequence-Based Amplification
  • ICAN Isothermal and Chimeric primeer-initiated Amplification of Nucleic acid ⁇ method
  • TRC Transcription-Reverse-Translation-Amplification
  • SDA Stringand-Displacement-Amplification
  • TMA Transcription-Mediated Amplification
  • RCA Rolling-Circle-Amplification
  • nucleic acid amplification reaction broadly encompasses nucleic acid amplification reactions by temperature variation or isothermal for the purpose of nucleic acid amplification. These nucleic acid amplification reactions also include reactions involving quantification of amplified nucleic acids such as real-time PCR.
  • the microchip 1a is configured by bonding the substrate layer 11 to the substrate layer 12 on which the introduction part 2, the flow paths 31 to 35 and the wells 41 to 45 are formed, and further bonding the substrate layer 13 to the substrate layer 11. (See FIG. 1B).
  • the microchip 1a when the bonding of the substrate layer 11 and the substrate layer 12 is performed under a negative pressure with respect to the atmospheric pressure, the inside of the introduction unit 2, the flow paths 31 to 35, and the wells 41 to 45 is protected with respect to the atmospheric pressure. Thus, it can be hermetically sealed so as to be a negative pressure (1/100 atm).
  • the region into which the sample solution is introduced is set to a negative pressure with respect to the atmospheric pressure, whereby the sample solution is sucked by the negative pressure inside the microchip when the sample solution is introduced, and a fine channel structure is formed.
  • the sample solution can be introduced into the microchip 1a in a shorter time.
  • the material of the substrate layers 11, 12, 13 can be glass or various plastics.
  • the substrate layers 12 and 13 are made of a material having gas impermeability.
  • the sample solution introduced into the wells 41 to 45 is vaporized by heating in the nucleic acid amplification reaction. Further, it is possible to prevent disappearance (liquid loss) through the substrate layer 11. Further, when the region of the microchip 1a into which the sample solution is introduced is hermetically sealed as a negative pressure with respect to the atmospheric pressure, the negative pressure inside the microchip 1a is prevented by preventing the permeation of air from the outside of the microchip 1a. Therefore, it is preferable that the substrate layers 12 and 13 are made of a material having gas impermeability.
  • plastics polymethyl methacrylate: acrylic resin
  • PC polycarbonate
  • PS polystyrene
  • PP polypropylene
  • PE polyethylene
  • PET polyethylene terephthalate
  • SAN resin Styrene-acrylonitrile copolymer
  • MS resin MMA-styrene copolymer
  • TPX poly (4-methylpentene-1)
  • SiMA siloxanyl methacrylate monomer
  • MMA copolymer SiMA- Fluorine-containing monomer copolymers
  • silicone macromers A) -HFBuMA (heptafluorobutyl methacrylate) -MMA terpolymers, disubstituted polyacetylene polymers, and the like.
  • Examples of the metals include aluminum, copper, stainless steel (SUS), silicon, titanium, tungsten, and the like.
  • ceramics include alumina (Al 2 O 3 ), aluminum nitride (AlN), silicon carbide (SiC), titanium oxide (TiO 2 ), zirconia oxide (ZrO 2 ), and quartz.
  • the substrate layer 11 is preferably made of an elastic material.
  • the substrate layer 11 that seals the introduction part 2 is made of an elastic material, so that a part of a puncture member such as a needle can be penetrated into the introduction part 2 from the outside of the microchip 1a.
  • a puncture member such as a needle
  • the region where the sample solution is introduced is hermetically sealed as a negative pressure with respect to the atmospheric pressure, when the tip of the needle reaches the introduction part 2, the outside of the microchip 1a and the introduction part 2 The sample solution in the syringe is automatically sucked into the introduction unit 2 due to the pressure difference.
  • the puncture site is naturally sealed by the self-sealing property of the substrate layer 11 when the needle is removed from the introduction portion 2 after the sample solution is introduced. Can be.
  • self-sealing property of the substrate layer.
  • Examples of the material for the substrate layer having elasticity include acrylic elastomers, urethane elastomers, fluorine elastomers, styrene elastomers, epoxy elastomers, and natural rubbers, in addition to silicone elastomers such as polydimethylsiloxane (PDMS). .
  • PDMS polydimethylsiloxane
  • the material of each substrate layer is light transmissive, has less autofluorescence, and has less wavelength dispersion. Therefore, it is preferable to select a material with a small optical error.
  • FIG. 2 schematically shows the well 43 on behalf of each well of the microchip 1a.
  • the well 43 contains solid reagents R1 and R2.
  • Reagents R1 and R2 contain at least a part of a substance necessary for obtaining an amplified nucleic acid chain in the nucleic acid amplification reaction.
  • oligonucleotide primers hereinafter also referred to as “primers”
  • dNTPs nucleic acid monomers
  • enzymes enzymes
  • reaction buffers that are complementary to at least a part of the base sequence of DNA, RNA or the like to be amplified. Ingredients included.
  • a probe equipped with a label such as a fluorescent label for detecting the amplified nucleic acid chain
  • a detection reagent that intercalates into a double-stranded nucleic acid etc.
  • it can be a component contained in the reagents R1 and R2.
  • the reagent R1 may be a reagent solution containing a primer and not containing an enzyme (first reagent solution)
  • the reagent R2 may be a reagent solution containing an enzyme and not containing a primer (second reagent solution).
  • first reagent solution a reagent solution containing a primer and not containing an enzyme
  • second reagent solution a reagent solution containing an enzyme and not containing a primer
  • the reagent R1 containing the primer does not contain the enzyme
  • the reagent R2 containing the enzyme does not contain the primer, so that the primer and the enzyme are not mixed until the sample solution is introduced into the well. Occurrence is suppressed.
  • the reagent R1 may be a reagent solution that contains an enzyme and does not contain a primer (second reagent solution), and the reagent R2 may be a reagent solution that contains a primer and does not contain an enzyme (first reagent solution).
  • R2 can be of any composition.
  • the reagents R1 and R2 are not limited to the shape shown in FIG. 2, and may be any shape as long as the volume can be accommodated in the well 43.
  • reagents R1 and R2 having the same composition may be stored in a plurality of wells provided in the microchip 1a, and reagents R1 and R2 having different compositions may be stored in each well.
  • reference numeral S ⁇ b> 1 is a substrate layer formation step.
  • the introduction portion 2, the flow paths 31 to 35, and the wells 41 to 45 are formed on the substrate layer 12.
  • the introduction part 2 and the like can be formed on the substrate layer 12 by a known method. For example, wet etching or dry etching of a glass substrate layer, or nanoimprint, injection molding or cutting of a plastic substrate layer. Further, the introduction part 2 or the like may be formed on the substrate layer 11, or a part of the substrate layer 11 may be formed and the remaining part of the substrate layer 12 may be formed.
  • reference numeral S2 is a reagent solution preparation step.
  • a liquid or gel reagent solution is prepared according to the composition of the reagents R1 and R2 accommodated in the microchip 1a.
  • the reagent solution only needs to contain at least a part of the substances necessary for the nucleic acid amplification reaction, and the composition can be arbitrary.
  • a reagent R1 containing only a primer and a reagent R2 containing only an enzyme may be prepared.
  • the types of reagent solutions to be prepared are not limited to two, and the number of substances necessary for the nucleic acid amplification reaction contained in one reagent solution may be one or several.
  • the primer When a primer is included in the reagent solution prepared in the preparation step, the primer may be one type or plural types.
  • a primer including a different base sequence is defined as another type of primer with respect to a primer having a certain base sequence. That is, for a target nucleic acid chain to be amplified, a pair of primer sets that combine a primer designed for the base sequence of one nucleic acid chain and a primer designed for the base sequence of its complementary strand are: It is defined as including two types of primers. The definition about the kind of these primers is the same also in 2nd embodiment and 3rd embodiment which are mentioned later.
  • the sample solution introduced at the start of the nucleic acid amplification reaction is It is preferable that the primer and the enzyme are not mixed until non-specific amplification of the nucleic acid by the primer dimer is suppressed. Moreover, it is preferable that the reagent solution containing a primer contains two or more kinds of primers.
  • the reagent solution, and the primer solution and enzyme solution added thereto are preferably kept at a cold temperature.
  • Reagent solution etc. can be kept at a cold temperature by placing a container containing reagent solution on ice or placing an instrument such as an aluminum block in a freezer in advance and using it in a cooled state. is there.
  • FIG. 3 Solidification of reagent solution
  • symbol S3a is a solidification step of the reagent solution.
  • the plurality of reagent solutions prepared in the preparation step S2 are solidified. That is, the reagent solution is dried to produce solid-phase reagents R1 and R2.
  • the solidification step S3a will be described in two steps as shown in FIG. 3, followed by the step S3a-1 for “dropping reagent solution” and the step S3a-2 for “freeze drying”.
  • FIG. 3 is a flowchart when there are two types of reagent solutions prepared in the preparation step S2.
  • Step of dropping reagent solution S3a-1 the reagent solution prepared in the above-described reagent solution preparation step S2 is dropped into the solidification container used in the solidification step S3a.
  • the reagent solutions are dropped into separate solidification containers and solidified separately.
  • the reagent R1 having the same composition is accommodated in the plurality of wells 41 to 45 of the microchip 1a, the number of solidification containers corresponding to the number of wells is prepared, and the reagent solution is dropped into each solidification container.
  • the solidification container may be made of any material, but preferably has a resistance to the temperature and pressure set in the next freeze-drying step S3a-2.
  • freeze drying step S3a-2 In this step, the reagent solution dropped into the container is dried and solidified.
  • a drying method for example, freeze-drying is suitable.
  • freeze-drying includes steps of preliminary freezing, primary drying (sublimation freezing), and secondary drying (removing bound water).
  • the freezing temperature may be equal to or lower than the eutectic point (temperature at which the reagent solution freezes), but it is frozen at about -40 ° C for the purpose of preventing enzyme deactivation and freezing the reagent solution completely. It is desirable to make it.
  • the primary drying the reagent solution frozen in the preliminary freezing step is dried.
  • the degree of vacuum in the primary drying is desirably 100 Pa or less, for example. Since the boiling point of water at 100 Pa is about ⁇ 20 ° C., it is close to the eutectic point of the reagent solution described above, and dissolution of the reagent solution during drying is prevented.
  • the vacuum degree of primary drying should just select an appropriate value according to the eutectic point of the prepared reagent liquid.
  • water in a molecular state attached to components contained in the reagent solution after the primary drying is removed.
  • the dryness of the reagent solution may be increased by heating to a temperature at which the components contained in the reagent solution do not deactivate or denature.
  • the drying method in the solidifying step S3a is not limited to freeze-drying.
  • symbol S4 is an accommodating process of the reagents R1 and R2.
  • the solid reagents R1 and R2 prepared in the solidification container by the above-described reagent liquid solidification step S3a are taken out from the solidification container and formed on any of the substrate layers by the substrate layer forming step S1.
  • the number of the wells for storing the reagents R1 and R2 may be either one or one.
  • the number and type of the reagents R1 and R2 accommodated in one well may be arbitrary, and the reagents R1 and R2 having the same composition may be accommodated in a plurality of wells, and the reagents R1 and R2 having different compositions may be accommodated. R2 may be accommodated.
  • a primer is contained in reagent R1 or reagent R2
  • a reagent R1 and a reagent R2 having different primers are prepared, and the reagent R1 and the reagent R2 are accommodated in a plurality of wells provided in the microchip 1a so as to be arranged in different wells. In this case, it is possible to analyze the amplification of a plurality of nucleic acid chains having different base sequences by one nucleic acid amplification reaction, and the analysis using the microchip 1a becomes simpler.
  • symbol S5 is a bonding step of the substrate layer.
  • another substrate layer is bonded to one of the substrate layers containing the reagents R1 and R2.
  • the substrate layers 11, 12, and 13 can be bonded to each other by a known method such as heat fusion, adhesive, anodic bonding, bonding using an adhesive sheet, plasma activated bonding, ultrasonic bonding, or the like.
  • a negative pressure with respect to the atmospheric pressure
  • each region of the introduction part 2 the channels 31 to 35, and the wells 41 to 45 into which the sample solution is introduced.
  • the substrate layers 11 and 12 are bonded together and then subjected to a negative pressure (vacuum). Since the air existing in each region such as the introduction portion 2 is exhausted through the substrate layer 11, the inside of the microchip 1a can be set to a negative pressure (vacuum) with respect to the atmospheric pressure. Note that the step of making the inside of the microchip 1a negative with respect to the atmospheric pressure is not essential in the microchip manufacturing method according to the present technology.
  • the reagents R1 and R2 including a part of the substances necessary for the nucleic acid amplification reaction are stored in advance in the wells 41 to 45 serving as analysis sites. Therefore, the nucleic acid amplification reaction can be started only by supplying the sample solution containing the remaining substance and the target nucleic acid chain necessary for the nucleic acid amplification reaction into the wells 41 to 45.
  • a plurality of solid reagents R1 and R2 in the wells 41 to 45 a plurality of substances necessary for the nucleic acid amplification reaction can be held in the microchip 1a in a separated state until the start of analysis. Can do.
  • the method for producing a nucleic acid amplification reaction microchip according to the present technology makes it possible to produce a nucleic acid amplification reaction microchip capable of simple and highly accurate analysis.
  • FIG. 4 shows the well 43 for the reagent R accommodated in the well of the microchip 1a-2 according to the modified embodiment of the first embodiment. Shown schematically as a representative.
  • the configuration of the microchip 1a-2 is the same as that of the first embodiment except for the reagent R housed in each well such as the well 43.
  • symbol is attached
  • the material of the substrate layers 11, 12, and 13 constituting the microchip 1a-2 is the same as the substrate layer having the same reference numeral in the microchip 1a.
  • the well 43 of the microchip 1a-2 contains one type of reagent R.
  • the manufacturing process of the microchip 1a-2 is the same as the flowchart shown in FIG. 3 except for the type of reagent liquid to be prepared in the reagent liquid preparation process S2, and the description of the manufacturing process is omitted.
  • the reagent R accommodated in the microchip 1a-2 may be one type.
  • the reagent R containing the enzyme may be housed in the well 43, and at the start of the nucleic acid amplification reaction, other components necessary for the nucleic acid amplification reaction such as primers may be mixed with the sample solution and introduced into the microchip 1a-2.
  • the microchip 1a-2 In the microchip 1a-2 according to the present technology, some components necessary for the nucleic acid amplification reaction are stored in the wells 41 to 45 in advance until the sample solution is introduced into the wells. It is possible to separate the component contained in the reagent R from other components. For this reason, for example, the enzyme and the primer can be separated until the nucleic acid amplification reaction is started, nonspecific nucleic acid amplification by the primer dimer or the like is suppressed, and the microchip 1a-2 is used to provide high accuracy. Analysis becomes possible.
  • FIG. 5 shows the well 43 as a representative of the reagents R1 and R2 accommodated in the wells of the microchip 1b according to the second embodiment of the present technology. This is shown schematically.
  • the microchip 1b is the same as that of the first embodiment except for the shapes of the reagents R1 and R2 accommodated in each well such as the well 43.
  • symbol is attached
  • the material of the substrate layers 11, 12, 13 constituting the microchip 1b is the same as that of the substrate layer having the same reference numeral in the microchip 1a.
  • Reagents R1 and R2 shown in FIG. 5 are solid reagents, similar to the reagents housed in microchip 1a, and contain at least a part of substances necessary for obtaining an amplified nucleic acid chain in a nucleic acid amplification reaction. Yes. About the composition of reagent R1, R2, since it is the same as reagent R1, R2 accommodated in the microchip 1a, description is abbreviate
  • the reagents R1 and R2 accommodated in the microchip 1b the difference from the reagents R1 and R2 in the microchip 1a is that some of the reagents accommodated in the well 43 are fixed in the well 43. (See FIG. 5).
  • a method for producing a microchip 1b will be described with reference to a flowchart shown in FIG.
  • the substrate layer forming step S1, the reagent solution preparing step S2, and the substrate layer bonding step S5 are the same as those in the first embodiment, and thus the description thereof is omitted, and the reagent solution fixing step S3b is performed.
  • the reagent storage step S4 will be described.
  • FIG. 6 symbol S3b is a fixing step of the reagent solution.
  • this step one type of reagent solution among the plurality of types of reagent solutions prepared in the preparation step S 2 is fixed in the well 43. That is, the reagent solution is dried in the well 43, and the dried reagent solution is fixed in the well.
  • the fixing step S3b will be described in the order of “drop of reagent solution” step S3b-1 and “vacuum drying” step S3b-2.
  • other reagent liquids that are not used in the reagent liquid fixing step S3b are solidified by the reagent liquid solidifying step S3a as in the first embodiment.
  • one type of reagent solution is dropped into each well formed in the substrate layer 12 or the like in the substrate layer forming step S1. At this time, it is preferable that the substrate layer 12 on which the well is formed is cooled.
  • symbol S4 is a reagent storage step.
  • the reagent R2 is present in the well 43 in the microchip 1b.
  • the reagent R1 prepared in the reagent solution solidifying step S3a is separately accommodated in the well in which the reagent R2 is fixed in advance.
  • the solidified reagent R1 accommodated in the microchip 1b is not limited to one type, and can be arbitrary.
  • the reagents R1 and R2 including a part of a substance necessary for the nucleic acid amplification reaction are held in advance in the wells 41 to 45 serving as analysis fields. Therefore, similarly to the microchip 1a, when the nucleic acid amplification reaction is performed using the microchip 1b, only the sample solution containing the remaining substance and the target nucleic acid chain necessary for the nucleic acid amplification reaction is placed in the wells 41 to 45. What is necessary is just to introduce
  • the components contained in the plurality of solid reagents R1 and R2 having different compositions held in the wells 41 to 45 are maintained in a separated state until the start of the nucleic acid amplification reaction. For this reason, for example, by using an enzyme and a primer as components contained in the reagent R1 and the reagent R2, respectively, nonspecific amplification of nucleic acid due to generation of primer dimers or the like can be suppressed.
  • FIG. 7 schematically shows the reagent R housed in the well of the microchip 1c according to the third embodiment, with the well 43 as a representative.
  • the configuration of the microchip 1c other than the reagent R accommodated in each well such as the well 43 is the same as that of the first embodiment.
  • symbol is attached
  • the material of the substrate layers 11, 12, 13 constituting the microchip 1c is the same as the substrate layer having the same reference numeral in the microchip 1a.
  • a reagent R containing at least a part of a substance necessary for obtaining an amplified nucleic acid chain in the nucleic acid amplification reaction is fixed (FIG. 7).
  • the component required for the nucleic acid amplification reaction contained in the reagent R may be one type or a plurality of types.
  • the substrate layer forming step S1, the reagent solution preparing step S2, and the substrate layer bonding step S5 are the same as those in the first embodiment, and a description thereof will be omitted.
  • the step of fixing the reagent solution to the well 43 similar to the reagent solution fixing step S3b of the second embodiment, the reagent solution prepared in a predetermined composition is applied to each well provided on the substrate layer 12. The reagent solution is dropped and fixed in the well 43 by vacuum drying or the like.
  • the prepared reagent solution is preferably stored at a cold temperature.
  • the substrate layer 12 on which each well is formed is also preferably stored at a cold temperature.
  • a device that holds the substrate layer 12 such as an aluminum block may be cooled in a freezer in advance, the substrate layer 12 may be placed on the cooled device, and the reagent solution may be dropped.
  • the reagent R fixed to the well 43 or the like may be one kind, or may be reagents R1 and R2 having different compositions.
  • one of the reagent solutions is dropped into the well 43 and fixed by vacuum drying or the like. On the fixed reagent R1, The next reagent solution may be dropped and dried, and the dropping and drying steps may be repeated.
  • this technique can also take the following structures.
  • a method for producing a microchip for nucleic acid amplification reaction comprising: (2) The method for producing a microchip for nucleic acid amplification reaction according to (1), wherein the solidification step includes a step of freeze-drying the reagent solution.
  • (3) including a preparation step of preparing a plurality of reagent solutions having different compositions before the solidifying step, wherein the reagent solution includes an oligonucleotide primer and an enzyme-free first reagent solution;
  • the solidifying step includes a step of separately lyophilizing the first reagent solution and the second reagent solution.
  • the storing step includes a step of storing the first reagent solution containing two or more kinds of solidified oligonucleotide primers in each of the plurality of wells.
  • Either one of the first reagent solution and the second reagent solution is solidified by the solidification step, and a reagent solution not used in the solidification step is added before the containing step.
  • the method for producing a microchip for nucleic acid amplification reaction according to the above (3) comprising a fixing step of dropping into the well and drying in the well.
  • the fixing step includes a step of vacuum drying the reagent solution.
  • Example 1 Detection of Non-specific Amplification in Nucleic Acid Amplification Reaction Inhibition of non-specific amplification of nucleic acid chains in a nucleic acid amplification reaction using a microchip according to the present technology was verified.
  • microchips used in this example are four types of microchips that differ in the method of preparing reagents contained therein.
  • PDMS and glass substrates were used as materials.
  • four types of primers used for influenza A amplification, Bst DNA polymerase, dNTPs, and a reaction buffer were prepared as reagents necessary for the nucleic acid amplification reaction performed in this example. The process from the preparation process of the reagent solution to the containing process will be described below for each microchip.
  • Microchip 1 As a comparative example of the microchip for nucleic acid amplification reaction according to the present technology, a microchip 1 (hereinafter referred to as M1) was manufactured. In the production of M1, a reagent solution containing four types of primers, Bst DNA polymerase, dNTPs, and a reaction buffer was prepared. 1.2 ⁇ l of reagent solution was dropped into the well formed in the substrate layer, and the reagent solution was fixed in the well by vacuum drying (about 1000 Pa) for about 2 hours.
  • microchip 2 (hereinafter referred to as M2) is a microchip in which a solidified reagent is accommodated in a well.
  • M2 a reagent solution containing four kinds of primers, Bst DNA polymerase, dNTPs, and reaction buffer was prepared by placing a solidification container on ice and cooling.
  • the solidification container containing 1.2 ⁇ l of the reagent solution was placed at ⁇ 40 ° C. for 6 hours or more to freeze the reagent solution. After the reagent solution was frozen, the solidification container was set in a freeze dryer (FDU-2200, EYELA).
  • the reagent solution was dried for 12 hours or more under vacuum (about 6 to 8 Pa). Thereafter, the temperature of the dry chamber was set to 30 ° C., and the reagent solution was further dried for 6 hours or more.
  • the reagent solidified by freeze-drying was taken out from the solidification container and accommodated in a well formed into a substrate layer.
  • M3 is a microchip in which a plurality of solidified reagents having different contained substances are accommodated in a well.
  • a reagent solution hereinafter referred to as FluA
  • FluA a reagent solution containing primers among the four types of primers, Bst DNA polymerase, dNTPs, and components necessary for the nucleic acid amplification reaction of the reaction buffer was prepared while cooling.
  • RM A reagent solution (hereinafter referred to as RM) containing Bst DNA polymerase, dNTPs, and reaction buffer was also prepared while cooling.
  • the prepared reagent solution was added dropwise to another solidification container at 0.4 ⁇ l for FluA and 0.8 ⁇ l for RM.
  • Each reagent solution placed in the solidification container was solidified by lyophilization in the same manner as M2.
  • the solidified FluA and RM were taken out from the solidification container and accommodated in each well formed in the substrate layer so that both were accommodated in one well.
  • M4 is a microchip in which reagent solutions having different components are fixed in a well divided into a plurality of times.
  • a reagent solution FluA and a reagent solution RM were prepared in the same manner as M2.
  • 0.4 ⁇ l of FluA was dropped into the well and fixed in the well by vacuum drying in the same manner as M1.
  • the substrate layer having the well to which FluA was fixed was cooled, and 0.8 ⁇ l of RM was dropped into the well to which FluA was fixed while keeping the temperature low. Again, vacuum drying was performed in the same manner as M1, and RM was fixed in the well.
  • each substrate layer was treated by oxygen plasma irradiation (O 2 : 10 cc, RF output: 100 W, RF irradiation time: 30 seconds) and bonded under vacuum to complete the microchips M1 to M4.
  • oxygen plasma irradiation O 2 : 10 cc, RF output: 100 W, RF irradiation time: 30 seconds
  • nucleic acid amplification reaction was performed using the microchips M1 to M4 manufactured by the above steps.
  • the LAMP method was used for nucleic acid amplification.
  • a sample solution was introduced from M1 to M4, and a nucleic acid amplification reaction was performed at 63 ° C.
  • the sample solution includes an influenza A positive specimen (positive control, hereinafter referred to as PC), an influenza A negative specimen (negative control, hereinafter referred to as NC), and water (non-template control, hereinafter referred to as NTC). ) was used.
  • the amplified nucleic acid chain was detected by fluorescence detection, and SYBR Green was used as a detection reagent.
  • FIG. 8 shows the results of this example.
  • FIG. 8 shows the start of nucleic acid amplification for each sample solution in each of the M1 to M4 microchips.
  • the start time of nucleic acid amplification was defined as the time when the amplification curve obtained by plotting the fluorescence intensity obtained by SYBR Green rose and reached a predetermined threshold.
  • M1 'shown in FIG. 8 is a microchip manufactured by the same manufacturing process as M1, and was used for nucleic acid amplification reaction like M1.
  • nucleic acid amplification was detected in the wells into which PC was introduced in the M1 to 4 microchips (for M1, see M1 '). That is, it was shown that the reagent accommodated in the well was stored in a usable state for the nucleic acid amplification reaction. On the other hand, nucleic acid amplification was also observed in the wells of M1-4 microchips into which NC and NTC had been introduced. This indicates that non-specific amplification of the nucleic acid chain occurred in the wells of the microchips M1 to M4.
  • nucleic acid amplification reaction performed in this example, amplification specific to the template nucleic acid strand of the nucleic acid was detected within 30 minutes after the start of the nucleic acid amplification reaction (FIG. 8). Therefore, the occurrence of nucleic acid amplification within 30 minutes after the start of the reaction in a well into which NC and NTC, which should not cause nucleic acid amplification, is hindered in analysis performed using a microchip.
  • the start of non-specific nucleic acid amplification in M3 was 50 minutes after the start of the nucleic acid amplification reaction.
  • the start of non-specific nucleic acid amplification in M1 which is a comparative example, is detected from about 20 minutes after the start of the reaction. From this result, it was shown that non-specific nucleic acid amplification was suppressed in the nucleic acid amplification reaction using M3.
  • the start of non-specific nucleic acid amplification in M2 and M4 was around 30 minutes in some wells. Compared with the result of M3, the start time of non-specific nucleic acid amplification was earlier in the results of M2 and M4. However, no nucleic acid amplification was observed in NTC and NC within 30 minutes after the start of the nucleic acid amplification reaction. From these results, it was shown that nonspecific nucleic acid amplification was suppressed in M2 and M4 compared to M1 (comparative example). Moreover, the inhibitory effect of nonspecific nucleic acid amplification in M2 and M4 was comparable.
  • a microchip (M2) containing a solid phase reagent containing an enzyme and a primer, or a microchip prepared by dropping a reagent solution containing an enzyme into a well to which a reagent containing a primer is fixed ( In M4), it was observed that non-specific nucleic acid amplification reaction was suppressed as compared with Comparative Example (M1). This indicates that in the microchip manufacturing process, non-specific nucleic acid amplification reaction was suppressed in the nucleic acid amplification reaction performed using the reagent dried after mixing the cooled enzyme and primer. .
  • the microchip for nucleic acid amplification reaction according to the present technology can be analyzed simply by introducing a sample solution or the like, and non-specific nucleic acid amplification is suppressed, so that highly accurate analysis is possible. It was confirmed that there was.
  • the microchip for nucleic acid amplification reaction according to the present technology analysis by nucleic acid amplification can be performed easily and with high accuracy. Therefore, the microchip for nucleic acid amplification reaction according to the present technology can be used as an apparatus for performing nucleic acid amplification for clinical genotype determination, infectious pathogen determination, and the like.
  • R, R1, R2 Reagent, 1a, 1a-2, 1b, 1c: Microchip, 11, 12, 13: Substrate layer, 2: Introduction part, 31, 32, 33, 34, 35: Channel, 41, 42, 43, 44, 45: Well

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