Classifications

• • 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
  • B01L3/502746—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 means for controlling flow resistance, e.g. flow controllers, baffles
• • 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
  • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
• • 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
• • 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
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
• • 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
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • 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
  • B01J2219/00781—Aspects relating to microreactors
  • B01J2219/00783—Laminate assemblies, i.e. the reactor comprising a stack of plates
• • 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
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0668—Trapping microscopic beads
• • 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
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/08—Regulating or influencing the flow resistance
  • B01L2400/084—Passive control of flow resistance
  • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

• the present invention relates to a microchip, a kit for extracting nucleic acids, and a method for extracting nucleic acids.
• the present application relates to a method for extracting nucleic acids in Japan in 2003.
• This application claims priority based on Japanese Patent Application No. 2000-2016 filed on May 24, which is incorporated herein by reference. . 2.
• a groove having a depth of about 100 ⁇ irn and a width of about 500iLim is formed on a surface of a substrate such as glass by a fine processing technique such as lithography.
• a microchip technology that uses a gas microchannel to enable chemical reaction, biochemical reaction, solvent extraction, gas-liquid separation, and chemical analysis or non-contact optical analysis of trace components based on these.
• the inventors of the present application have also proposed a method of inserting microbeads as a reaction carrier into a fine channel and providing a dam-shaped damming portion in the microchannel. It has been proposed (Reference 1).
• the present invention The purpose of this method is to produce and prepare a low-cost microchip technology using a microphone mouth bead that is not only effective for extracting nucleic acids but also useful as various reaction carriers.
• New technology microchips that can suppress the generation of stagnation, facilitate smooth and easy injection, filling, and transportation of microbeads, and also reduce the generation of dust. Is to provide a way.
• a fine channel is formed by a groove provided on a joint surface between upper and lower substrates, and the fine channel has upper, lower, right and left sides of its cross section.
• a gap with a reduced flow channel cross section is provided Have been.
• the gap may be formed by a protrusion in the groove. Further, the gap may be formed by opposing the protrusions in the grooves provided in each of the upper and lower substrates. Further, the gap may be formed by inserting a protrusion of the other substrate into a groove of one substrate. Further, the size of the cross section of the gap may be variable by at least one of the movable protrusions of the upper and lower substrates.
• the size of the cross section of the gap portion is a size for blocking the micro peas inserted into the fine channel.
• the inner wall surface of the fine channel may be modified with a surface treatment agent.
• a kit for nucleic acid extraction according to the present invention includes the above-described microchip and microbeads having a surface hydroxyl group.
• the microbeads having surface hydroxyl groups may be at least one of silica microbeads having a diameter of 10 m or less, hollow silica microbeads, and resin microbeads.
• the surface hydroxyl group may be coated with a surface treating agent.
• This surface treatment agent is a silane coupling agent containing a trialkylhalogenosilane as a main component.
• FIG. 1 is an exploded perspective view schematically illustrating a microchip.
• FIG. 2A and 2B are cross-sectional views of the gap 31 from the directions A and B in FIG. 3A to 3C are cross-sectional views showing other examples of the gap 31.
• FIG. 4A and 4B are cross-sectional views showing still another example of the gap portion 31.
• FIG. 4A and 4B are cross-sectional views showing still another example of the gap portion 31.
• FIG. 5 is a schematic diagram of an optical microscope photograph illustrating DNA adsorption as an example.
• BEST MODE FOR CARRYING OUT THE INVENTION FIGS. 1 and 2 schematically show a partial structure of a microchip according to the present invention, and FIG. 1 shows a state in which a lower substrate 1 and an upper substrate 2 are separated.
• FIG. 2 shows a partial cross section of the gap 3 1 provided in the fine flow path 3 in a state where the upper and lower substrates 1 and 2 are joined, as viewed from the directions of arrows A and B in FIG. .
• the fine channels 3 are formed by the grooves 11 and 21 provided on the joint surface between the upper and lower substrates 1 and 2.
• the fine flow path 3 is provided with a gap portion 31 having a reduced flow path cross section at the upper and lower central portions of the cross section.
• the fine channels 3 are formed by opposing the grooves 11, 21 of the upper and lower substrates 1, 2.
• the gaps 31 are formed by the projections 12 and 22 provided in the grooves 11 and 21 facing each other.
• the gap 31 may be a central portion of the cross section of the fine flow channel 3, and is not limited to the upper and lower central portions as in the slit-shaped opening illustrated in FIG. It may be the left and right central part as shown, or the upper, lower, left and right central part as shown in FIG. 3B.
• the cross-sectional shape of the gap 31 may be various, for example, as shown in FIG. 3C, may be various such as a circular cross-section.
• the formation of the gap 31 may not be based on the protrusions 12 and 22 provided in the grooves 11 and 21 of the upper and lower substrates 1 and 2.
• the fine channel 3 itself is formed, for example, by a groove 11 provided in the lower substrate 1, and the upper substrate 2 is made to exist as a cover thereof, and the fine channel 3 is formed.
• the protrusions 23 provided on the upper substrate 2 may be inserted into the grooves 11 and the gaps 31 may be formed by facing the protrusions 12 in the grooves 11. It is possible.
• the gap 31 may be various other than the configuration of the protrusions 12, 22, and 23.
• these projections 12, 22, 23 may be formed not only by microfabrication of the substrates 1 and 2 by lithography etching or the like, but also by means such as formation by curing of a polymer. Good.
• the gap 31 may be formed by an action of a minute member from the outside on the fine flow path 3, or by a means such as deformation of the fine flow path 3 itself.
• the production and preparation of the microchannel 3 and the gap 31 can be performed easily and at low cost.
• the gap 31 at the center of the cross-section, when micro-beads are injected into the micro-channel 3, the generation of stagnation in the flow of fluids such as liquids and gases is suppressed, and micro-beads are injected.
• microbeads can be blocked.
• the size of the cross section of the microchannel using the microbeads can block the microphone mouthpiece injected into the microchannel 3. Shall be.
• the above-described protrusion or a member having the same function as the above-described protrusion may be made movable, and the size of the cross section may be made variable.
• the cross-section is greatly enlarged, and the microbeads can be transferred to the downstream region. .
• the microbeads must be discharged in a direction opposite to the injection.
• the microbeads can be discharged in the same forward direction as the injection.
• the sample substance, impurities, dust, etc. are prevented from adhering to the inside of the fine channel 3.
• the inner wall surface can be modified with a surface treatment agent.
• surface treatment agents it is effective to cover and inactivate the surface hydroxyl groups with the above.
• the micromouth chip according to the present invention becomes extremely useful as a kit for characteristic chemical synthesis or analysis by being combined with micropease.
• the present invention provides, as such a kit, a kit for nucleic acid extraction comprising the microchip as described above, and micropore beads having a surface hydroxyl group.
• microbeads having a surface hydroxyl group in this case, silica microbeads having a diameter of lO / m or less, hollow silica microbeads, and resin beads such as polystyrene having a hydroxyl group on the surface are preferable. As shown. In the case of silica mic mouth beads having a diameter of 10 m or more, it is difficult to transport the beads in the microchannel 3 due to the specific gravity.
• the kit for nucleic acid extraction of the present invention for those having a surface hydroxyl group on the inner wall surface of the fine channel, it is preferable to coat the surface hydroxyl group with a silane coupling agent.
• a trialkylhalogenosilane having one halogen atom that is, the general formula is
• X represents a halogen atom such as a chlorine atom.
• nucleic acid extraction for example, in the example of the microchip shown in FIGS. 1 and 2, a microbead-containing liquid is injected from the liquid inlet 4 and the gap is formed in the fine channel 3. A dam is interposed at the gap 31 and then a sample is introduced from the liquid inlet 5 to adsorb the nucleic acid to the microbeads. The remaining liquid is discharged from the discharge port 6. The nucleic acid is extracted by this adsorption. The adsorbed nucleic acid can be desorbed from the microbead by introducing a desorbing solution. It is also preferable that the nucleic acid extraction is performed in the presence of chaotropic ions.
• a glass microchip having a fine channel with a diameter of 50 ⁇ and a length of 60 mm having a dam structure in the center was prepared (FIG. 1). Gaps in a dam-shaped damming structure with protrusions were formed on both the upper and lower sides of the microchannel.
• the gap at the center of the microchannel had a slit shape of 2 mm at the top and bottom and 50 ⁇ um at the left and right.
• One inlet of the microchannel was used as an inlet, and the other was used as an outlet.
• a liquid reservoir was provided at the inlet, and a microtube was connected to the outlet.
• a syringe pump was connected to the microtube at the outlet, and an experimental system was constructed in which the liquid injected into the liquid reservoir was sucked by the operation of the syringe pump and could be injected into the microchannel.
• the microbeads suspended in pure water were injected into a liquid reservoir, and the transport of the microbeads in the microchannel was observed using an optical microscope.
• microbeads silica microbeads (10 m or less in diameter) were used.
• Silica microphone mouth beads (diameter 10; m or more), hollow silica microphone mouth beads (diameter 2 to 20 m), and polystyrene microbeads with a hydroxyl group added to the surface (diameter 20 m) were prepared. Suction was performed at a flow rate of 101 per minute. All microbeads other than silica microbeads (with a diameter of 10 ⁇ m or more) were successfully transported in the microchannel, and a bead column could be formed immediately before the gap in the dam structure. On the other hand, when a similar experiment was performed using silica mic mouth beads larger than 10 microns in diameter, transport stopped halfway through the microchannel and column formation was insufficient.
• hollow silica microbeads and polystyrene microbeads with hydroxyl groups added to the surface were as good in transport as silica nanobeads (beads less than 1 m in diameter).
• Pure water is injected into the syringe pump and injected into the microchannel.
• Microbeads other than silica micropipes are transported through the microchannel and discharged into the liquid reservoir.
• Example 2 Surface modification treatment of the inner wall of a microchannel
• a surface modification treatment using a silane coupling agent having an alkyl group and a reactive group is widely used. Therefore, the inner surface of the microchannel may be subjected to surface modification using a silane coupling agent.
• oc decyltrichlorosilane which is commonly used, has three chlorine groups, which are reactive groups for surface modification, and not only reacts with the hydroxyl groups on the inner wall of the microchannel, but also polymerizes due to the polymerization of molecules.
• the molecules of octadecyltrichlorosilane react with each other and become polymerized, generating garbage.
• the dust generated in the surface modification treatment of a flat substrate such as a slide glass instead of a fine channel can be washed away with a solvent, and there is no problem.
• dust generated in the treatment of the inner wall of the fine channel cannot be easily washed away. It is particularly difficult to remove when a damming structure is provided in the fine channel.
• the dust remaining in the microchannel not only hinders the transport of liquids and beads in the microchannel, but also hinders the nucleic acid adsorption process.
• the chlorine group which is the reactive group is 2 ⁇ (10!
• trietylchlorosilane has only one chlorine group, which is the reactive site, and does not in principle polymerize.
• it since it has a boiling point of 145 ° C and a melting point of 150 ° C, it has low volatility at room temperature and is liquid, so it can be easily diluted in a solvent such as toluene and handled.
• tdethylclilorosi lane or octadecyltrichlorosilane for the inner wall of the microchannel was subjected to a surface modification treatment.
• dehydrated toluene as a solvent
• 5% solutions of each were prepared. The two were compared using the experimental system described in Example 1.
• the prepared solution was injected into the liquid reservoir, and the solution was injected into the fine channel by sucking with a syringe pump.
• the flow rate was set at 101 per minute, and a 50 X 1 solution was injected to perform surface modification.
• 50 ⁇ l or more of dehydrated toluene was injected, and the fine channel after the surface treatment was washed.
• Example 1 Adsorption of nucleic acids was tested using the experimental system described in 1.
• the inner wall surface of the microchannel was formed as described in Example 2 using 10% triethylchlorosilane.
• Co1E1 DNA was used as the nucleic acid.
• An aqueous saturated sodium iodide solution was used as the chaotropic ion solution.
• 0.2 g of the microbeads described in Example 1 was suspended in a sodium iodide solution at a ratio of 11 and 25 1 was injected into the microchannel from the liquid reservoir to form a column.
• sodium iodide as a solvent, a 1% DNA solution was prepared and injected into the microchannel from the solution reservoir.
• a SYBRGreen I solution which is a fluorescent dye for DNA, was injected into the microchannel from the reservoir. Observation of the microchannel with an optical microscope confirmed that only the column 42 of the silica microbeads 41 emitted fluorescence as shown in FIG. It was confirmed that DNA was adsorbed to the silica microbeads 41 used in Example 1. No fluorescence was observed from the microchannels other than the column section 42, and it was confirmed that DNA was not adsorbed to the inner wall surface of the microchannel.
• Example 2 Using the experimental system described in Example 1, adsorption and desorption of nucleic acids were tested. The experimental system was the same as above. The microbeads described in Example 1 were injected into the microchannel to form a column, and then the nucleic acid was injected. Lambda DNA was used as the nucleic acid. The adsorption of lambda DNA to the microbeads was confirmed using an optical microscope. Hot pure water at 60 ° C or higher was prepared and injected into the fine channel from the liquid reservoir. Desorption of lambda DNA from microbeads was confirmed. From the above, it was confirmed that the adsorption and desorption of nucleic acids to and from the microbeads was confirmed, and that the extraction of nucleic acids could be realized in the microchannel.
• the present invention enables low-cost production as a microchip technology using microbeads that is not only effective for nucleic acid extraction but also useful as various reaction carriers.
• New technology means that can suppress the formation of stagnation in the flow of fluid samples, facilitate the injection, filling, and transportation of microbeads, and reduce the generation of dust. And a new method for extracting nucleic acid using the same.

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• 2004
  • 2004-03-24 EP EP04722945.5A patent/EP1607748B1/en not_active Expired - Fee Related
  • 2004-03-24 US US10/550,302 patent/US8012680B2/en not_active Expired - Fee Related
  • 2004-03-24 JP JP2005504072A patent/JP5162095B2/ja not_active Expired - Fee Related
  • 2004-03-24 CN CN2004800082843A patent/CN1764842B/zh not_active Expired - Fee Related
  • 2004-03-24 WO PCT/JP2004/003998 patent/WO2004086055A1/ja active Application Filing
  • 2004-03-24 KR KR1020057017815A patent/KR20050111785A/ko not_active Application Discontinuation

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