WO2005037983A1 - 核酸分離精製カートリッジおよびその製造方法 - Google Patents
核酸分離精製カートリッジおよびその製造方法 Download PDFInfo
- Publication number
- WO2005037983A1 WO2005037983A1 PCT/JP2004/013735 JP2004013735W WO2005037983A1 WO 2005037983 A1 WO2005037983 A1 WO 2005037983A1 JP 2004013735 W JP2004013735 W JP 2004013735W WO 2005037983 A1 WO2005037983 A1 WO 2005037983A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- porous membrane
- adsorbing porous
- cartridge
- opening
- Prior art date
Links
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 287
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 287
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 287
- 238000000926 separation method Methods 0.000 title claims abstract description 169
- 238000000746 purification Methods 0.000 title claims abstract description 168
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000012528 membrane Substances 0.000 claims abstract description 234
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- 238000001746 injection moulding Methods 0.000 claims abstract description 15
- 230000002093 peripheral effect Effects 0.000 claims description 63
- 238000002347 injection Methods 0.000 claims description 54
- 239000007924 injection Substances 0.000 claims description 54
- 239000012488 sample solution Substances 0.000 claims description 46
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- 239000011347 resin Substances 0.000 claims description 39
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- NNLVGZFZQQXQNW-ADJNRHBOSA-N [(2r,3r,4s,5r,6s)-4,5-diacetyloxy-3-[(2s,3r,4s,5r,6r)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6s)-4,5,6-triacetyloxy-2-(acetyloxymethyl)oxan-3-yl]oxyoxan-2-yl]methyl acetate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](OC(C)=O)[C@H]1OC(C)=O)O[C@H]1[C@@H]([C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](COC(C)=O)O1)OC(C)=O)COC(=O)C)[C@@H]1[C@@H](COC(C)=O)O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O NNLVGZFZQQXQNW-ADJNRHBOSA-N 0.000 description 15
- 239000011148 porous material Substances 0.000 description 13
- 238000011084 recovery Methods 0.000 description 13
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- SMEGJBVQLJJKKX-HOTMZDKISA-N [(2R,3S,4S,5R,6R)-5-acetyloxy-3,4,6-trihydroxyoxan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@@H](O1)O)OC(=O)C)O)O SMEGJBVQLJJKKX-HOTMZDKISA-N 0.000 description 9
- 229940081735 acetylcellulose Drugs 0.000 description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
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- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 6
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- 238000002474 experimental method Methods 0.000 description 5
- 230000002209 hydrophobic effect Effects 0.000 description 5
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- 238000007789 sealing Methods 0.000 description 5
- 229920003002 synthetic resin Polymers 0.000 description 5
- 239000000057 synthetic resin Substances 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- 229920002477 rna polymer Polymers 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 210000000633 nuclear envelope Anatomy 0.000 description 3
- 229920000620 organic polymer Polymers 0.000 description 3
- -1 polypropylene Polymers 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
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- 230000000274 adsorptive effect Effects 0.000 description 2
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- 238000009832 plasma treatment Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
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- 230000037303 wrinkles Effects 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
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- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 1
- OKBPCTLSPGDQBO-UHFFFAOYSA-L disodium;dichloride Chemical group [Na+].[Na+].[Cl-].[Cl-] OKBPCTLSPGDQBO-UHFFFAOYSA-L 0.000 description 1
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- 230000005484 gravity Effects 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
- B01D63/08—Flat membrane modules
- B01D63/081—Manufacturing thereof
-
- 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
- B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
- B01L3/50255—Multi-well filtration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
- B29C45/14—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles
- B29C45/14336—Coating a portion of the article, e.g. the edge of the article
- B29C45/14377—Coating a portion of the article, e.g. the edge of the article using an additional insert, e.g. a fastening element
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2626—Absorption or adsorption
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2313/00—Details relating to membrane modules or apparatus
- B01D2313/04—Specific sealing means
- B01D2313/042—Adhesives or glues
-
- 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
- B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
-
- 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
- B01L2200/12—Specific details about manufacturing devices
-
- 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
- B01L2300/0829—Multi-well plates; Microtitration plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
- B29C45/14—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles
- B29C45/14311—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles using means for bonding the coating to the articles
- B29C2045/14319—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles using means for bonding the coating to the articles bonding by a fusion bond
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
- B29C45/14—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles
- B29C45/14311—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles using means for bonding the coating to the articles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
- B29C45/14—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles
- B29C45/14467—Joining articles or parts of a single article
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29L—INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
- B29L2031/00—Other particular articles
- B29L2031/14—Filters
Definitions
- the present invention relates to a cartridge for separating and purifying nucleic acids for separating nucleic acids. More specifically, a nucleic acid-adsorbing porous membrane is provided inside a cylindrical body having a first opening and a second opening, and a sample solution containing nucleic acids is supplied from a first opening side to a second opening side with a pressurized gas.
- the present invention relates to a nucleic acid separation / purification cartridge for adsorbing nucleic acid to a nucleic acid-adsorbing porous membrane by allowing the nucleic acid to adsorb and separating and purifying the nucleic acid, a nucleic acid separation / purification cartridge manufactured by insert injection molding, and a method for manufacturing the same.
- nucleic acids are used in various forms in various fields. In many cases, nucleic acids are extremely small and cannot be obtained, and isolation and purification operations are complicated and time-consuming.
- a nucleic acid separation / purification unit in which a solid phase composed of an organic polymer having a hydroxyl group is accommodated in a container having at least two openings is used.
- the method used is described in Japanese Patent Application Laid-Open No. 2003-128691 (hereinafter sometimes referred to as “Patent Document 1”) (see FIG. 19).
- Patent Document 1 Japanese Patent Application Laid-Open No. 2003-128691
- one opening Z of the nucleic acid separation / purification unit is immersed in a sample solution containing nucleic acids.
- the inside of the nucleic acid separation / purification unit is depressurized, and the sample solution is sucked into the container.
- the sample solution comes into contact with the solid phase, and the nucleic acid present in the sample solution is adsorbed on the solid phase.
- the inside of the container of the unit is pressurized using a pressure difference generator, and the sucked sample solution is discharged.
- the container is washed out by discharging it from the container.
- This washing solution has a function of washing away the sample solution remaining in the container and washing away impurities in the sample solution adsorbed on the solid phase together with the nucleic acid.
- the recovered liquid for releasing the nucleic acid adsorbed on the solid phase is sucked into the container by the same depressurization and pressure operation as described above, and the container is discharged. Since the discharged recovery liquid contains the target nucleic acid, the separation and purification are completed by recovering the recovered nucleic acid.
- a nucleic acid-adsorbing porous membrane is generally used as a solid phase for adsorbing nucleic acids.
- nucleic acid separation / purification unit generally has a structure in which a nucleic acid-adsorbing porous membrane is sandwiched and held between two cylindrical parts constituting a container of the unit. It is.
- a method for fixing the two cylindrical components a method of fixing by ultrasonic welding, heat welding by laser, an adhesive, a screw, or the like is used.
- the nucleic acid separation / purification unit is capable of pressing a sample solution through a nucleic acid-adsorbing porous membrane by pressurizing the sample solution.
- it is necessary to hold down the periphery of the nucleic acid-adsorbing porous membrane tightly by the two parts constituting the container.
- the washing solution is not left as much as possible in the container when washing and flowing with the washing solution.
- the cleaning solution is mixed with the discharged recovery solution, and depending on the concentration of the mixed cleaning solution, it may be used in the next step, for example, PCR (Polymerase Chain Reaction). May have an adverse effect.
- the attached foam is collected together with the nucleic acid purified in the subsequent collection step using a collection solution. Since the foam thus recovered is an unwashed solution, it becomes an impurity for the nucleic acid recovered by the recovery liquid. That is, there is a problem that the purification rate of the nucleic acid is deteriorated, and an enzymatic reaction may be inhibited when conducting an experiment or research using the nucleic acid recovered in the subsequent step.
- the present invention has been made to solve these problems, and does not require a dedicated facility for fixing a container, and a nucleic acid separation and purification cartridge capable of simultaneously producing a large number of containers. It is a first object to provide a manufacturing method thereof.
- the present invention provides a sample solution comprising a nucleic acid-adsorbing porous membrane inside a cylindrical body having a first opening and a second opening, and comprising a nucleic acid from the first opening side to the second opening side.
- a sample solution comprising a nucleic acid-adsorbing porous membrane inside a cylindrical body having a first opening and a second opening, and comprising a nucleic acid from the first opening side to the second opening side.
- a third object of the present invention is to provide a cartridge for purifying and isolating nucleic acid having a structure in which a solution or the like does not adhere to the outer wall surface of the discharge unit.
- a nucleic acid separation and purification cartridge has a nucleus having an opening at the bottom of a cylindrical body having a bottom and a nucleic acid-adsorbing porous membrane supported at the bottom.
- the nucleic acid-adsorbing porous membrane By disposing the nucleic acid-adsorbing porous membrane on a bottom member forming the bottom portion, inserting the same into a cavity of an injection mold, and further injecting a molding material into the cavity, the nucleic acid is removed.
- the other part of the tubular main body which is the other part that sandwiches the adsorptive porous membrane, is formed integrally with the bottom member and molded, and simultaneously sandwiches the nucleic acid-adsorptive porous membrane. It is characterized by.
- the nucleic acid-adsorbing porous membrane is placed on the bottom member forming the bottom, which is one of the parts holding the nucleic acid-adsorbing porous membrane.
- the molding material By inserting the molding material into the cavity of the injection mold and injecting the molding material into the cavity, the other part of the tubular main body that holds the nucleic acid-adsorbing porous membrane is formed. Is molded integrally with the bottom member, and at the same time, sandwiches the nucleic acid-adsorptive porous membrane, so that special equipment for fixing such as an ultrasonic welding machine, which was conventionally required, becomes unnecessary, and injection is performed.
- nucleic acid separation and purification cartridge it is possible to manufacture a nucleic acid separation and purification cartridge using only a molding device. Also, there is no possibility that the nucleic acid-adsorbing porous membrane is broken or the sealing is insufficient due to a difference in the pressing force of the nucleic acid-adsorbing porous membrane due to a manufacturing error of the component.
- the bottom member further includes a cylindrical discharge portion (nozzle) communicating with an opening of the bottom portion.
- nozzle communicating with an opening of the bottom portion.
- a peripheral portion of the nucleic acid-adsorbing porous membrane is crushed and sandwiched by an injection pressure of a molding material forming a cylindrical portion of the cylindrical main body.
- the voids (pores) innumerably existing inside the peripheral portion of the nucleic acid-adsorbing porous membrane are crushed, so that the sample solution and the washing solution do not remain on the peripheral portion, and the nucleic acid-adsorbing porous film is not damaged.
- the sample solution does not flow around the side of the membrane.
- the peripheral portion of the nucleic acid-adsorbing porous membrane is crushed until there is no internal void. As a result, it is possible to reliably prevent the sample solution or the like from remaining or wrapping around. Further, it is preferable that the peripheral portion of the nucleic acid-adsorbing porous membrane is crushed to a thickness of 10% to 70% of the original thickness. As a result, countless voids (holes) inside the peripheral portion of the nucleic acid-adsorbing porous membrane are not crushed, so that the sample solution or the like can be reliably prevented from remaining or wrapping around.
- the injection mold includes a plurality of the cavities, and the plurality of cavities are provided. It is preferable that the nucleic acid-adsorptive porous membrane is arranged on the bottom member and inserted therein, and the plurality of cavities are connected to each other. .
- the injection mold has a plurality of the cavities, and the nucleic acid-adsorbing porous membrane is disposed on the bottom member in the plurality of cavities. Each of these is inserted, and the plurality of cavities are in communication with each other, so that the resin injected at the same injection pressure is filled in each of the cavities.
- a large number of nucleic acid separation / purification cartridges or a group of nucleic acid separation / purification cartridges connected with a large number of nucleic acid separation / purification cartridges can be simultaneously formed without causing insufficient sealing or breakage of the porous membrane. , Can be manufactured.
- the “connected state” may be a state in which the cartridges are directly connected, or a state in which the cartridges are connected by a runner.
- the method for producing a cartridge for nucleic acid separation and purification provides a nucleic acid separation and purification cartridge having an opening at the bottom of a cylindrical body having a bottom and supporting a nucleic acid-adsorbing porous membrane at the bottom.
- the nucleic acid-adsorbing porous membrane is disposed on the bottom of the bottom member having the bottom of the tubular body and forming a part of the tubular body.
- the cylindrical parts are fixed to each other using an ultrasonic welding machine or the like. Process is not required, and the core can be It is possible to manufacture the cartridge for separation and purification. Also, the difference in the pressing force of the nucleic acid-adsorbing porous membrane due to the manufacturing error (dimension error) of the parts does not cause the nucleic acid-adsorbing porous membrane to be broken or the seal to be insufficient.
- the core pin may have a thickness of 10% to 70% of the original thickness of the nucleic acid-adsorbing porous membrane. It is preferable to hold it by crushing it. Thus, the nucleic acid-adsorbing porous membrane does not shift or wrinkle due to the injection pressure of the molding material.
- a tip portion of the core pin is formed in a conical shape from a peripheral portion toward a center portion. Thereby, the core pin is self-centered.
- the peripheral portion of the nucleic acid-adsorbing porous membrane protruding around the core pin may be internally pressurized by an injection pressure of a molding material injected into the cavity. Is preferably crushed until the voids disappear. As a result, it is possible to reliably prevent the sample solution or the like from remaining or wrapping around.
- the periphery of the core pin may be provided.
- the rim of the nucleic acid-adsorbing porous membrane that has protruded may be crushed by the injection pressure of the molding material injected into the cavity until the thickness becomes 10% to 70% of the original thickness. preferable.
- countless voids (holes) inside the peripheral edge portion of the nucleic acid-adsorbing porous membrane are not crushed, so that it is possible to reliably prevent the sample solution or the like from remaining or wrapping around.
- the injection mold includes a plurality of the cavities, and the plurality of cavities are provided.
- the nucleic acid-adsorbing porous membrane is placed on the bottom member in the cavity, and these are inserted respectively, and the plurality of cavities are connected to each other.
- the injection mold has a plurality of cavities, and the plurality of cavities have a nucleic acid-adsorbing porous material on the bottom member.
- the membranes After the membranes are placed, they are inserted, and a plurality of cavities are in communication with each other, so that the resin injected at the same injection pressure is filled in each cavity.
- the force for pressing the nucleic acid-adsorbing porous membrane becomes equal, and a large number of nucleic acid separation / purification cartridges or a large number of nucleic acid separation / purification cartridges are connected at the same time without insufficient sealing or tearing.
- a group of refined cartridges can be manufactured.
- the cartridge for nucleic acid separation and purification includes a nucleic acid-adsorbing porous membrane inside a cylindrical body having a first opening and a second opening, and extends from the first opening side to the second opening side.
- a plurality of protrusions support the nucleic acid-adsorbing porous membrane at least at a part of the top thereof, and the nucleic acid-adsorbing porous membrane is used during use. So that it is displaced toward the discharge section as it approaches the bottom opening It is characterized by being formed.
- the plurality of projections provided on the bottom surface support the nucleic acid-adsorbing porous membrane on at least a part of its top, and at the time of use, Since the nucleic acid-adsorbing porous membrane is formed so as to be displaced toward the discharge section as the nucleic acid-adsorbing porous membrane approaches the bottom opening, the nucleic acid-adsorbing porous membrane is made to flow when the cleaning solution is caused to flow by the pressurized gas in the washing step. The film is deformed in a convex shape along the protrusion toward the discharge portion. As a result, the cleaning liquid is quickly discharged from the discharge part without remaining at the bottom of the tubular main body.
- the plurality of protrusions are preferably a plurality of ribs extending radially from the bottom opening.
- the number of the ribs is preferably three or more.
- the inclination angle of each of the ribs with respect to the radial direction of the cylindrical body is preferably 3 ° or more, more preferably 5 ° or more.
- the bottom surface has a slope that is displaced toward the discharge portion as approaching the bottom opening. Thereby, the cleaning liquid is discharged more quickly.
- the inclination angle of the bottom surface with respect to the radial direction of the cylindrical body is preferably 10 ° or more, more preferably 15 ° or more, and most preferably 20 ° or more.
- the rib has a top formed in an arc shape. This makes it difficult for the cleaning liquid to stay between the top of the rib and the nucleic acid-adsorbing porous membrane, so that the cleaning liquid is discharged more quickly.
- the nucleic acid separation / purification cartridge has the corners and the corners present on the inner surface formed in an arc shape. This makes it difficult for the cleaning liquid to stay at the corners and corners present on the inner surface, so that the cleaning liquid is discharged more quickly.
- the radius of curvature at the top of the rib is preferably 1Z4 or more with respect to the rib width, more preferably 1Z3 or more, and most preferably 1Z2 or more.
- the radius of curvature of the corner is preferably 0.1 mm or more, more preferably 0.2 mm or more, and most preferably 0.3 mm or more.
- the radius of curvature at the corner is preferably 0.1 mm or more, more preferably 0.15 mm or more, and most preferably 0.2 mm or more.
- the angle formed by the axis of the tubular body and the inner peripheral surface of the tubular body is preferably 10 ° or less, more preferably 5 ° or less. This makes it easier for the cleaning liquid to flow along the inner peripheral surface of the cylindrical body, so that the cleaning liquid is more quickly discharged.
- the inner wall surface of the cartridge for nucleic acid separation and purification is made of a material having a contact angle force of ⁇ 0 ° or less or 90 ° or more, or a surface treatment so as to have such a contact angle. It is preferable to use a material subjected to the above.
- the contact angle is 80 ° or less, the cleaning liquid is more quickly discharged because the cleaning liquid wettability to the inner wall surface of the nucleic acid separation / purification cartridge is improved, and the cleaning liquid is less likely to remain as droplets.
- the contact angle is more preferably 60 ° or less, most preferably 50 ° or less.
- the contact angle is 90 ° or more, even if the washing liquid remains as droplets on the inner wall surface of the nucleic acid separation and purification cartridge, the droplets become substantially spherical due to surface tension. It will be easier. Thereby, the cleaning liquid is discharged more quickly.
- the nucleic acid-adsorbing porous membrane is held in a state where its peripheral edge is crushed. Since the nucleic acid-adsorbing porous membrane has pores, if the peripheral edge is held in a crushed state, the pores are crushed so as to close the pores, so that the liquid does not flow from that part. Thereby, it is possible to prevent a problem that a liquid (a sample solution or the like) that should pass through the nucleic acid-adsorbing porous membrane goes around the side of the nucleic acid-adsorbing porous membrane.
- the nucleic acid separation and purification cartridge includes a nucleic acid-adsorbing porous membrane inside a cylindrical body having a first opening and a second opening, and extends from the first opening side to the second opening side.
- the thickness of the portion forming the second opening be 0.5 mm or more.
- the opening diameter of the second opening is not less than 1.0 mm, and the thickness of the portion forming the second opening is not less than 0.2 mm.
- the outer diameter of the portion forming the second opening is 1.4 mm or more. It is more preferable that the outer diameter of the portion forming the second opening is not less than 2. Omm.
- the angle formed between the end face of the discharge section and the outer wall surface of the discharge section is 105 ° or less. It is more preferable that the angle between the end face of the discharge portion and the outer wall surface is 100 ° or less, and it is more preferable that the force S is 95 ° or less. Further, as the shape of the end face of the discharge portion approaches the second opening, the opening diameter may be made to be a wide-angle funnel shape. In this case, it is preferable that the angle between the end face of the discharge section and the outer wall surface is 30 ° or more.
- nucleic acid separation / purification cartridge in which bubbles of the sample solution hardly flow around the outer wall surface of the discharge section can be realized. Further, even if bubbles adhere to the outer wall surface, a nucleic acid separation / purification cartridge that can easily remove the bubbles with a washing liquid that easily returns to the vicinity of the second opening of the discharge portion can be realized. As a result, a nucleic acid separation / purification cartridge that can prevent the untreated sample solution from being mixed into the recovery liquid can be realized.
- the shape of the end face of the discharge part is a funnel shape in which the opening diameter increases as approaching the second opening, it is difficult for the generated foam to reach the outer wall surface. Further, even if the angle between the end face of the discharge portion and the outer wall surface is formed to be 30 ° or more, the wraparound of bubbles can be prevented.
- the material constituting the cartridge for separating and purifying nucleic acid be made easily wettable.
- “wetability” refers to the ease with which a solution and an object (an end face or an outer wall surface) fit together, and is defined by the contact angle between the solution and the object. From the experiments, bubbles can be retained on the end surface in the dimensions described above, and the contact angle between the solution (washing solution) and the object for washing off impurities in the washing process is preferably 100 ° or less. Preferably it is 95 ° or less, more preferably 90 ° or less.
- a claw member for inducing bubbles is provided on an end face of the discharge section. It is desirable to provide a plurality of these claw members. It is particularly desirable that the position inside the claw member be provided so as to coincide with the inner wall surface of the discharge portion. It is more preferable that the claw member is formed in a rod shape.
- the foam that has reached the second opening propagates along the claw member extending on the inner wall surface and aggregates at the tip portion, that is, at a position below the second opening.
- the coagulated foam easily falls into the waste liquid container, so that the foam hardly adheres to the outer wall surface. Also, even if bubbles are blown up by air, they will not reach the outer wall!
- the washing solution is quickly discharged without remaining inside the cartridge for nucleic acid separation and purification in the washing step. Mixing of the cleaning liquid into the collected liquid can be suppressed. As a result, in the next step, problems caused by the cleaning solution can be prevented.
- the nucleic acid separation and purification cartridge of the present invention it is possible to prevent bubbles of the sample solution from adhering to the outer wall surface of the discharge portion of the nucleic acid separation and purification cartridge. Further, since it is possible to prevent bubbles from being mixed in the untreated sample solution, it is possible to improve the nucleic acid purification rate. In experiments and research using the recovered nucleic acid, it is possible to prevent problems such as inhibition of the reaction by the enzyme.
- FIG. 1 is an exploded perspective view of a nucleic acid separation and purification cartridge according to a first embodiment.
- FIG. 2 is an enlarged sectional perspective view of an insert used in the first embodiment.
- FIG. 3 is a cross-sectional view of a cartridge for nucleic acid separation and purification and an injection mold, wherein (a) shows a state when an insert material is installed, and (b) shows a state when the mold is closed.
- FIG. 4 is a cross-sectional view of a nucleic acid separation / purification cartridge and an injection mold, wherein (a) shows a state at the time of resin injection, and (b) shows a state at the time of injection completion.
- FIG. 5 is an enlarged cross-sectional view of a portion A in FIG. 4 (b), wherein (a) shows the state when the mold is closed, (b) shows the state when the mold is completely closed, (c) shows the state when the resin is injected, (D) shows the state when the injection is completed.
- FIG. 6 is a cross-sectional view of the cartridge for separating and purifying nucleic acid according to the first embodiment.
- FIG. 7 is a perspective view of a nucleic acid separation / purification cartridge group according to a second embodiment.
- FIG. 8 is an exploded perspective view of a nucleic acid separation and purification cartridge according to a third embodiment of the present invention.
- FIG. 9 is a cross-sectional view of a cartridge for separating and purifying nucleic acid according to a third embodiment of the present invention.
- FIG. 10 is an enlarged sectional perspective view of a cap used in a third embodiment of the present invention.
- FIG. 11 is a sectional view taken along line XX of FIG. 10.
- FIG. 12 is an enlarged view of a portion B in FIG. 9.
- FIG. 13 is a cross-sectional view showing a washing step of the nucleic acid separation / purification cartridge according to the third embodiment of the present invention.
- FIG. 14 is an exploded perspective view of a cartridge for separating and purifying nucleic acid according to a fourth embodiment of the present invention.
- FIG. 15 is a cross-sectional view of a cartridge for separating and purifying nucleic acid according to a fourth embodiment of the present invention.
- FIG. 16 is an enlarged cross-sectional view of a cap of the cartridge for separating and purifying nucleic acid according to the fourth embodiment of the present invention.
- FIG. 17 is an enlarged cross-sectional view of a cap of the cartridge for separating and purifying nucleic acid according to the fifth embodiment of the present invention.
- FIG. 18 is an enlarged cross section of a cap of a nucleic acid separation and purification cartridge according to a sixth embodiment of the present invention.
- FIG. 19 is a longitudinal sectional view of a nucleic acid separation and purification unit.
- FIG. 20 is a view showing a nucleic acid separation / purification cartridge for discharging a solution by pressurization.
- FIG. 1 to be referred to is an exploded perspective view of a nucleic acid separation and purification cartridge according to the first embodiment
- FIG. 2 is an enlarged sectional perspective view of an insert material used in the first embodiment.
- a nucleic acid separation / purification cartridge 100 includes an insert material 110 including a bottom member 120 and a nucleic acid-adsorbing porous membrane F, and an insert for the insert material 110.
- the barrel 140 is formed by injection molding.
- the barrel 140 of the cartridge 100 for nucleic acid separation and purification according to the first embodiment is formed integrally with the bottom member 120 and the nucleic acid-adsorbing porous membrane F by insert injection molding. , The barrel 140 is shown separately for convenience of explanation.
- the barrel 140 is a “portion forming the tubular portion of the tubular main body”.
- the insert material 110 is composed of a bottom member 120 constituting the bottom side of the cartridge 100 for nucleic acid separation and purification, and a nucleic acid-adsorbing porous membrane F for adsorbing and collecting nucleic acid.
- the insert material 110 is previously set in an injection mold (bottom mold 150 and barrel mold 160) for molding the nucleic acid separation / purification cartridge 100 (see FIG. 3), and the cavity 151 is an example of a molding material. When a certain melted resin J is injected, it is fused with the barrel 140 formed by the resin J.
- the bottom member 120 includes a bottom 121 having an opening 121a formed at the center, a lower surface of the bottom 121, a nozzle 122 (discharge portion) extending therefrom, and And a bottom-side fused portion 123 extending in a cylindrical shape along the circumference.
- a second opening 122a is formed at the tip of the nozzle 122, and communicates with the opening 121a of the bottom 121.
- the bottom-side fused portion 123 is a portion to be fused with the barrel-side fused portion 142 of the barrel 140 described later, and has an inner diameter substantially equal to the diameter of the nucleic acid-adsorbing porous membrane F.
- the bottom 121 of the bottom member 120 has an annular holding surface 125 that is one step higher than the bottom 121b, along the outer periphery of the bottom 121b.
- the sandwiching surface 125 is a surface that comes into contact with a peripheral edge Fa of the nucleic acid-adsorbing porous membrane F described later, and is formed flat.
- the bottom surface 121b is inclined so as to become lower toward the opening 121a side from the holding surface 125 side (closer to the second opening 122a side), so that the sample solution is easily discharged.
- six ribs 126 (only three ribs are shown in FIG. 2) are radially formed on the bottom surface 121b. The rib 126 protrudes from the bottom surface 121b, and is inclined at an angle smaller than the inclination angle of the bottom surface 121b so as to become lower toward the opening 121a side from the holding surface 125 side.
- the nucleic acid-adsorbing porous membrane F is a circular membrane member having a diameter substantially the same as the inner diameter of the above-mentioned bottom-side fused portion 123.
- the nucleic acid-adsorbing porous membrane F has a myriad of fine pores so that a nucleic acid can be extracted by filtering a sample solution. Further, the nucleic acid-adsorbing porous membrane F is placed on the holding surface 125 of the bottom member 120 to form the insert 110 (see FIG. 2).
- the peripheral portion Fa of the nucleic acid-adsorbing porous membrane F is a portion that comes into contact with the holding surface 125, and is pressed and held against the holding surface 125 by injection pressure during injection molding of the barrel 140 described later.
- the barrel 140 also has a cylindrical barrel body 141 and a cylindrical barrel-side fused portion 142 connected to the barrel body 141.
- the barrel 140 is formed by injecting resin J into the cavity 151 after placing the insert material 110 in the bottom mold 150 (see FIG. 3).
- the hollow portion 143 of the barrel 140 is a portion for temporarily storing a sample solution and the like, and is formed by a core pin 161 provided in a barrel-side mold 160 described later (see FIG. 4).
- the upper end of the hollow portion 143 is open (first opening 143a), The lower end of the part 143 is closed by the nucleic acid-adsorbing porous membrane F.
- the barrel-side fused portion 142 is formed by the resin J that has flowed into a gap (cavity 151a in FIG. 5B) formed between the core pin 161 and the bottom-side fused portion 123 of the bottom member 120. Therefore, in practice, the heat of the resin J flowing into the gap melts the inner peripheral surface 123a (see FIG. 2) of the bottom-side fused portion 123, and the barrel 140 and the insert material 110 are deformed. The Rukoto.
- FIG. 3 is a cross-sectional view of a nucleic acid separation and purification cartridge and an injection mold, and (a) shows a state when an insert material is installed, and (b) shows a state when the mold is closed.
- FIGS. 4A and 4B are cross-sectional views of the cartridge for nucleic acid separation and purification and the injection mold, respectively.
- FIG. 4A shows a state at the time of resin injection
- FIG. 4B shows a state at the time of resin injection
- a known injection molding machine can be used for manufacturing the cartridge 100 for separating and purifying nucleic acid.
- the injection molding machine is preferably a rigid injection molding machine because it is necessary to install the insert material 110 in the injection mold, but the insert material 110 (nucleic acid-adsorbing porous membrane F) is held at a predetermined position. If possible, it may be horizontal! / ⁇ .
- an “injection mold” is configured by the bottom mold 150 and the barrel mold 160.
- the nucleic acid-adsorbing porous membrane F is set so as to be supported by the clamping surface 125 and the rib 126 of the bottom 121 of the bottom member 120 manufactured in advance. Then, an insert material 110 is manufactured. Then, the insert material 110 is set in a cavity 151 formed in the bottom mold 150.
- the insert material 110 may be prepared in advance. It is preferable that the production of the insert material 110 and the installation of the insert material 110 be performed using a known assembling robot or the like.
- the barrel is placed in the bottom mold 150 where the insert material 110 is installed.
- the mold is closed by combining the metal mold 160 on the side.
- the barrel-side mold 160 has a columnar core pin 161 at a position corresponding to the hollow portion 143 of the nucleic acid separation / purification cartridge 100.
- the tip 162 of the core pin 161 contacts the upper surface of the nucleic acid-adsorbing porous membrane F, and the core pin 161 adsorbs nucleic acid between the holding surface 125 of the bottom member 120 and the core pin 161.
- the porous porous membrane F is sandwiched therebetween. At this time, the nucleic acid-adsorbing porous membrane F is compressed to a predetermined thickness so that the resin J injected in the next step does not leak.
- the length of the core pin 161 is adjusted so as to compress the nucleic acid-adsorbing porous membrane F to a certain thickness such that the resin J injected in the next step does not leak.
- the fixing (pressing) of the nucleic acid-adsorbing porous membrane F will be described in detail later.
- the barrel-side mold 160 has a gate 163 for injecting the resin J, and the resin J is injected into the cavity 151. Injectable! /
- the molten resin J is injected from the gate 163 into the cavity 151 formed by the bottom mold 150, the barrel mold 160, and the insert 110.
- the peripheral portion Fa of the nucleic acid-adsorbing porous membrane F is crushed by the injection pressure of the resin J filled in the cavity 151.
- the molten resin J is filled into the cavity 151 by applying an injection pressure to the extent that the peripheral edge Fa of the nucleic acid-adsorbing porous membrane F is suitably crushed. This will be described later in detail.
- Fig. 4 (b) when filling of resin J is completed and resin J is cooled and hardened, the mold is opened by operating an injection molding machine (not shown) to separate and purify nucleic acid. Remove cartridge 100.
- FIG. 5 is an enlarged cross-sectional view of a portion A in FIG. 4 (b), (a) when the mold is closed, (b) when the mold is completely closed, and (c) At the time of resin injection, (d) shows the state at the time of injection completion.
- a bottom member 120 and a nucleic acid-adsorptive porous membrane F are provided in an insert 151 as an insert material 110.
- the nucleic acid-adsorbing porous membrane F is placed on a sandwiching surface 125 formed on the bottom 121 of the bottom member 120 so that the peripheral edge Fa contacts.
- the barrel-side mold 160 is lowered by operating an injection molding machine (not shown).
- the diameter of the core pin 161 of the barrel-side mold 160 is formed to be smaller than the inner diameter of the bottom-side fused portion 123 of the bottom member 120 and larger than the inner diameter of the holding surface 125. Accordingly, when the mold closing is completed, the peripheral edge 162a of the tip 162 of the core pin 161 is located between the inner peripheral edge of the holding surface 125 and the nucleic acid, as shown in FIG. 5 (b). A part of the peripheral portion Fa of the adsorptive porous membrane F is sandwiched.
- the tip 162 of the core pin 161 is formed so as to incline toward the center from the peripheral edge 162a in accordance with the inclination of the upper end surface of the rib 126. Therefore, when the mold closing is completed, the nucleic acid-adsorbing porous membrane F is placed between the upper end face of the rib 126 and the tip 162 of the core pin 161 as well as inside the holding face 125 as shown in FIG. 5 (b). It is sandwiched and held between the peripheral edge and the peripheral edge 162a of the tip 162 of the core pin 161.
- the tip 162 of the core pin 161 is formed in a mountain shape (cone-shaped body) in accordance with the shape of the bottom 121, the core pin 161 is autonomously centered on the center of the bottom member 120. You. Therefore, a predetermined member thickness (width of the cavity of the cavity 151a) can be ensured so that the core pin 161 is not eccentric.
- the degree of compression of the nucleic acid-adsorbing porous membrane F by the core pin 161 was such that the nucleic acid-adsorbing porous membrane F was displaced by the injection pressure of the resin J without breaking the nucleic acid-adsorbing porous membrane F. It is necessary to compress it so that it does not wrinkle or leak resin J. Specifically, it is preferable to compress to a thickness of about 10% to 70% of the film thickness.
- the width W of the cavity 151a is preferably set to 0.2 mm or more, preferably 0.5 mm or more, in consideration of the occurrence of an error in the thickness of the member. Is more preferred
- nucleic acid-adsorbing porous membrane F (80 ⁇ m in thickness) made of triacetyl cellulose that has been subjected to an oxidation treatment
- the sample solution is compressed to 30 ⁇ m. It has been confirmed that sneaking around can be prevented.
- a part of the peripheral portion Fa of the nucleic acid-adsorbing porous membrane F is sandwiched between the inner peripheral edge portion of the clamping surface 125 and the peripheral portion 162a of the core pin 161 in an annular shape, so that the cavity is removed. Resin J injected into 151a does not flow into the center of nucleic acid-adsorbing porous membrane F.
- the sample solution wrapped around the side surface of the nucleic acid-adsorbing porous membrane F.
- the injection pressure of resin J should be set within the range of greater than 14.7 MPa and less than 147 MPa.
- the core pin 161 is pulled out from the hollow portion 143 of the barrel 140.
- the peripheral portion Fa of the nucleic acid-adsorbing porous membrane F is sandwiched between the injection-molded barrel side fusion portion 142 and the sandwiching surface 125. It is sandwiched and held at the bottom 121 of the cartridge 100 for separating and purifying nucleic acid.
- the inner peripheral surface 123a of the bottom-side fused portion 123 is melted by the heat of the resin J at the time of injection, and is integrated with the outer peripheral surface 142a of the barrel-side fused portion 142.
- the nucleic acid-adsorbing porous membrane F is held at the same time that the barrel 140 is molded, and a dedicated device for fixing the two components constituting the cartridge as in the related art is unnecessary. Become. Further, since the nucleic acid-adsorbing porous membrane F is compressed and held by the injection pressure of the resin J, there is no need to worry about insufficient sealing or breakage of the nucleic acid-adsorbing porous membrane F due to dimensional errors of parts.
- FIG. 6 to be referred to is a sectional view of the cartridge for separating and purifying nucleic acid according to the first embodiment.
- the step of separating and purifying nucleic acid from a sample containing nucleic acid using the nucleic acid separation / purification cartridge 100 is preferably performed using an automatic device that automatically performs this step. This makes it possible to obtain a certain level of nucleic acid irrespective of the skill of the operator who not only simplifies and speeds up the operation.
- the above-described automatic device uses the first opening 143a of the barrel 140 of the nucleic acid separation / purification cartridge 100 toward the second opening 122a of the nozzle 122 to transfer a sample solution containing nucleic acid,
- pressurized air which is an example of a pressurized gas
- the washing solution S is similarly passed through to remove impurities, and subsequently,
- a nucleic acid separation / purification device that automatically performs a separation / purification operation in which a nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane F is released by flowing the recovery liquid and recovered together with the recovered liquid.
- a mounting mechanism for holding a container and a recovery container for storing a recovery solution containing nucleic acids, a pressurized air supply mechanism for introducing pressurized air into the nucleic acid separation and purification cartridge 100, and a washing solution S and A dispensing mechanism for dispensing the collected liquid is used.
- the mounting mechanism includes a stand mounted on the main body of the apparatus, a cartridge holder that is vertically movably supported by the stand and holds the nucleic acid separation / purification cartridge 100, It is preferable to provide a container holder for holding the waste liquid container and the recovery container so that the position with respect to the nucleic acid separation / purification cartridge 100 can be exchanged below the cartridge holder.
- the pressurized air supply mechanism raises and lowers the air nozzle with respect to a nucleic acid separation / purification cartridge 100 held by the cartridge holder while supporting the air nozzle for ejecting pressurized air from the lower end.
- the apparatus includes a pressure head to be moved and positioning means for positioning the nucleic acid separation / purification cartridge 100 in the rack of the mounting mechanism, which is mounted on the pressure head.
- the dispensing mechanism includes a cleaning liquid dispensing nozzle for dispensing the cleaning liquid S, and dispensing the recovered liquid.
- a nozzle moving table that holds the cleaning liquid dispensing nozzle, the cleaning liquid dispensing nozzle, and the recovered liquid dispensing nozzle, and that can sequentially move on the nucleic acid separation and purification cartridge 100 held by the mounting mechanism;
- the cleaning solution S is sucked from the cleaning solution bottle containing
- a cleaning liquid supply pump for supplying a purified liquid dispensing nozzle and a recovered liquid supply pump for sucking the recovered liquid from a recovered liquid bottle containing the recovered liquid and supplying the recovered liquid to the recovered liquid dispensing nozzle are preferable. It is.
- the sample that can be used in the present invention is not limited.
- a body fluid such as whole blood, plasma, serum, urine, stool, semen, saliva, or a plant (or a sample thereof) collected as a sample is used. Solutions prepared from biological materials such as part), animals (or part thereof), etc., or their lysates and homogenates are covered.
- these specimens are treated with an aqueous solution containing a reagent for dissolving the cell membrane and the nuclear membrane to dissolve the nucleic acid.
- a reagent for dissolving the cell membrane and the nuclear membrane to dissolve the nucleic acid.
- the cell membrane and the nuclear membrane are dissolved, the nucleic acid is dispersed in the aqueous solution, and a sample solution containing the nucleic acid is obtained.
- the sample is whole blood
- add guanidine hydrochloride, Tris, Triton-X100, and protease K manufactured by SIGMA
- SIGMA protease K
- the sample solution thus obtained is put into the hollow portion 143 of the barrel 140 (see FIG. 6), and is caused to flow toward the nozzle 122 by applying pressure. In this way, the nucleic acid in the sample solution is adsorbed on the nucleic acid-adsorbing porous membrane F. [0087] Next, as shown in FIG.
- the cleaning solution S when pressurized, the nucleic acid-adsorbing porous membrane F is deformed in a convex shape along the inclined shape of the rib 126 toward the nozzle 122 as shown in FIG. As a result, the cleaning solution S
- the washing liquid S is used for both the water-soluble organic solvent and the salt, or the water-soluble organic solvent or the salt.
- the solution contains at least one of them.
- a water-soluble organic solvent such as alcohol is suitable for releasing components other than nucleic acids while retaining the nucleic acids, since the nucleic acids are hardly soluble.
- the addition of a salt enhances the nucleic acid adsorption effect.
- Examples of the water-soluble organic solvent contained in the washing solution s include methanol, ethanol, and isopropanol.
- the water-soluble organic solvent contained in the washing solution S is preferably 20 to 100% by volume.
- the salt contained in the washing liquid S is preferably a salt of a halogenated product.
- the salt has a monovalent or divalent cation and the salt thereof is contained in an amount of lOmM or more. More preferably, the salt power is sodium salt sodium chloride, and more preferably, the sodium salt sodium salt contains 20 mM or more! /.
- a purified liquid such as purified distilled water or a TE buffer is passed from the first opening 143a of the barrel 140 toward the nozzle 122 while applying pressure, and the nucleic acid is released from the nucleic acid-adsorbing porous membrane F. And collects the recovered liquid (recovered liquid containing nucleic acids) discharged from the nozzle 122.
- the recovered solution can be desorbed by adjusting the volume of the recovered solution with respect to the volume of the sample solution containing the nucleic acid prepared according to the sample size.
- the amount of the recovery solution containing the separated and purified nucleic acid depends on the amount of the sample used at that time.
- the amount of recovered solution that is commonly used is several tens to several hundred hundreds.However, when the sample volume is extremely small, or when a large amount of nucleic acid is to be separated and purified, the recovered solution volume is 1 ⁇ m. It can vary from one to several tens of ml.
- the pH of the recovered solution is preferably pH 2-11. Furthermore, it may be pH5-9 preferable.
- ionic strength and salt concentration have an effect on elution of the adsorbed nucleic acid.
- the recovered solution preferably has an ionic strength of 290 mmol Zl or less, and more preferably a salt concentration of 90 mmol Zl or less. By doing so, the recovery rate of nucleic acids is improved, and more nucleic acids can be recovered.
- the nucleic acid to be recovered may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and may be single-stranded or double-stranded.
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the nucleic acid to be collected is RNA
- the water used to prepare the washing solution S and the recovery solution is DEPC (d
- the nucleic acid contained in the recovered solution obtained as described above is measured with an ultraviolet-visible spectrophotometer (260 nm / 280 nm).
- an ultraviolet-visible spectrophotometer 260 nm / 280 nm
- DNA 1.6-2.0, 1 ⁇ ⁇
- it has a purity of 1.8-2.2. That is, a high-purity nucleic acid with a small amount of impurities can be constantly obtained.
- nucleic acids having a purity of about 1.8 when the measurement value (260 nm Z280 nm) with an ultraviolet-visible spectrophotometer is DNA and about 2.0 for RNA can be recovered.
- the nucleic acid separation / purification cartridge 100 can be suitably used by an automatic apparatus as described above, but can be used even when operated manually.
- a syringe and a pitter can be used as a means for pressurizing the sample solution or the like.
- the syringe pipitter can be detachably connected to one opening (the first opening 143a side) of the nucleic acid separation / purification cartridge 100.
- plastics such as polypropylene, polystyrene, polycarbonate, and polyvinyl chloride can be used. Further, biodegradable materials can also be suitably used. Further, the barrel 140 and the bottom member 120 may be transparent or colored.
- the nucleic acid-adsorbing porous membrane F a porous membrane in which ionic bonds are not involved and nucleic acids are adsorbed by interaction is preferable. More preferably, the nucleic acid-adsorbing porous membrane F is a porous membrane having a hydrophilic group, and the material forming the porous membrane itself is a porous membrane having a hydrophilic group. It is a porous membrane into which a hydrophilic group has been introduced by treating or coating a film or a material forming the porous membrane. The material forming the porous film may be any of an organic substance and an inorganic substance.
- the porous film itself is an organic material having a hydrophilic group
- the porous film itself is an organic material having a hydrophilic group
- a porous film obtained by treating a porous film of an organic material to introduce a hydrophilic group Does not have a hydrophilic group!
- a porous film made of a porous film made of an organic material coated with a material having a hydrophilic group to introduce a hydrophilic group, or a material that forms a porous film itself has a hydrophilic group.
- Examples of the porous film having a hydrophilic group include a porous film of an organic material having a hydroxyl group.
- Examples of the organic material having a hydroxyl group include a surface acetate of acetyl cellulose described in Patent Document 1 described above.
- the acetyl cellulose may be any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose, but is particularly preferably triacetyl cellulose.
- the amount (density) of hydroxyl groups on the surface of the solid phase can be controlled by the degree of the zirid treatment (the zirid degree).
- the amount (density) of hydroxyl groups is large.
- the oxidation ratio is preferably 5% or more, more preferably 10% or more.
- the amount (density) of hydroxyl groups in the solid phase can be controlled by a combination of the degree of the oxidizing treatment (the oxidizing degree) and the pore size of the porous film.
- the porous membrane may be a front-back symmetric porous membrane, but a front-back asymmetric porous membrane can be suitably used.
- a porous film of an organic material having a hydroxyl group a porous film of an organic polymer having a mixed power of acetyl-cellulose having different acetyl values can also be suitably used.
- a mixture of acetyl cellulose having different acetyl values a mixture of triacetyl cellulose and diacetyl cellulose can be suitably used.
- the mixing ratio of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99. Yo More preferably, the mixing ratio of triacetyl cellulose and diacetyl cellulose is 90: 10-50: 50.
- Examples of a mixture of acetyl cellulose having different acetyl values include a mixture of triacetyl cellulose and monoacetyl cellulose, a mixture of triacetyl cellulose, diacetyl cellulose and monoacetyl cellulose, and a mixture of diacetyl cellulose and monoacetyl cellulose. Can also be used.
- Examples of the porous film of an organic material having a hydroxyl group include a porous film having a strong organic material obtained by oxidizing a mixture of acetyl cellulose having different acetyl values.
- a peroxide of a mixture of triacetyl cellulose and diacetyl cellulose can be suitably used.
- the mixing ratio of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99. Further, the mixing ratio of triacetyl cellulose and diacetyl cellulose is preferably 90:10 to 50:50.
- an organic material obtained by subjecting a mixture of acetyl cellulose having different acetyl values to oxidation treatment an oxidation product of a mixture of triacetyl cellulose and monoacetyl cellulose, and a mixture of a mixture of triacetyl cellulose and diacetyl cellulose and monoacetyl cellulose are used.
- Oxide, an oxide of a mixture of diacetyl cellulose and monoacetyl cellulose can also be used.
- a porous membrane having a thickness of 10 to 500 ⁇ m can be suitably used. More preferably, a porous membrane having a thickness of 50 to 250 m is used.
- a porous membrane having a minimum pore size of 0.22 ⁇ m or more can be suitably used. More preferably, a porous membrane having a minimum pore size of 0.5 m or more can be used.
- a porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 2 or more can be suitably used. More preferably, a porous membrane having a ratio of the maximum pore size to the minimum pore size of 5 or more can be used.
- a porous membrane having a porosity of 50 to 95% can be suitably used. More preferably, a porous membrane having a porosity of 65-80% is used. Can. Further, as the nucleic acid-adsorbing porous membrane F, a porous membrane having a bubble point of 9.8 to 980 kPa (0.1 lOkgfZcm 2 ) can be suitably used. More preferably, a porous membrane having a bubble point of 19.6 to 392 kPa (0.2 to 4 kgf / cm 2 ) can be used.
- a porous membrane having a pressure loss of 0.1 lOOkPa can be suitably used. More preferably, a porous membrane having a pressure loss of 0.5 to 50 kPa can be used.
- the pressure loss is the minimum pressure required to pass water per 100 m of membrane thickness.
- nucleic acid-adsorbing porous membrane F a porous membrane in which the amount of nucleic acid adsorbed per 1 mg of the porous membrane is 0.1 ⁇ g or more can be suitably used. More preferably, a porous membrane having a nucleic acid adsorption amount of 0.9 g or more per 1 mg of the porous membrane can be used.
- FIG. 7 is a perspective view of a cartridge group for nucleic acid separation and purification according to the second embodiment.
- the nucleic acid separation / purification cartridge group 190 has a large number of nucleic acid separation / purification cartridges 192 formed continuously.
- the nucleic acid separation / purification cartridge 192 constituting the nucleic acid separation / purification cartridge group 190 is similar to the nucleic acid separation / purification cartridge 100 described in the first embodiment in that its structure is almost the same. Part of the cartridge 192 (connection part 193) is continuous with each other.
- the nucleic acid separation / purification cartridge group 190 is manufactured using an injection mold (not shown) in which a number of cavities 151 (see FIG. 3) communicating with each other are formed. Therefore, the pressure of the resin J pressing the peripheral portion Fa of the large number of nucleic acid-adsorbing porous membranes F set in the large number of cavities 151 becomes uniform. Therefore, a large number of nucleic acid separation / purification cartridges 192 (nucleic acid) can be used at a time without the nucleic acid-adsorbing porous membrane F being torn or being insufficiently sealed due to the variation in the force pressing the nucleic acid-adsorbing porous membrane F. Separation and purification cartridges 190) can be manufactured efficiently.
- the cartridges for nucleic acid separation and purification according to the first and second embodiments of the present invention are described.
- the present invention is not limited to the above embodiment.
- the nucleic acid separation / purification cartridge group 190 according to the second embodiment is in a state where the nucleic acid separation / purification cartridges 192 are directly connected to each other, but the injection molding type in which the cavities communicate with each other by a runner is used.
- the nucleic acid separation / purification cartridge 192 can be configured not to be directly connected.
- the nucleic acid separation / purification cartridge 100 of the first embodiment may be manufactured by communicating with the runner and injection molding, and then cutting off the runner to produce a large number of nucleic acid separation / purification cartridges 100.
- FIG. 8 to be referred to is an exploded perspective view of the cartridge for nucleic acid separation and purification according to the third embodiment of the present invention
- FIG. 9 is a cross-sectional view of the cartridge for nucleic acid separation and purification according to the third embodiment of the present invention
- 10 is an enlarged sectional perspective view of a cap used in the third embodiment of the present invention
- FIG. 11 is an XX sectional view of FIG. 10
- FIG. 12 is an enlarged view of a portion B in FIG.
- FIG. 13 is a cross-sectional view showing a washing step of the cartridge for separating and purifying nucleic acid according to the third embodiment of the present invention.
- the terms “up” and “down” are based on FIG.
- the nucleic acid separation / purification cartridge 200 holds the nucleic acid-adsorbing porous membrane F, the nucleic acid-adsorbing porous membrane F, and allows liquid to pass through.
- the barrel 210 and the cap 220 which are cylindrical bodies forming a flowing passage, also have a force.
- the barrel 210 is formed by a cylindrical barrel body 212 and a cylindrical barrel-side fitting part 213 connected to the barrel body 212, and a first opening 211a is provided at the upper part of the barrel body 212.
- An opening 21 lb is formed in the lower part of the fitting part 213. Therefore, the liquid can flow downward from above the barrel 210.
- the outer diameter of the barrel-side fitting portion 213 is slightly smaller than the outer diameter of the barrel main body 212.
- the cap 220 includes a cylindrical cap-side fitting portion 222 and a discharge portion 224 connected to an opening 223 provided at the bottom 222a of the cap-side fitting portion 222.
- the opening 223, which is an example of the bottom opening has a smaller diameter than the first opening 211a provided above the barrel main body 212.
- an opening 221a is formed at an upper portion of the cap-side fitting portion 222, and a second opening 221b is formed at a lower portion of the discharge portion 224. Therefore, the liquid can flow downward from above the cap 220.
- the inner diameter of the cap-side fitting portion 222 is formed to a diameter that can be fitted to the outer diameter of the barrel-side fitting portion 213 of the barrel 210.
- the barrel-side fitting portion 213 of the barrel 210 is attached to the bottom side 222a of the cap-side fitting portion 222 of the cap 220 with the nucleic acid-adsorbing porous membrane F arranged.
- the nucleic acid-adsorbing porous membrane F can be sandwiched between the barrel 210 and the cap 220 by being fitted into the cap-side fitting portion 222 of the cap 220.
- a “cylindrical main body” is constituted by the barrel 210 and the cap-side fitting portion 222.
- the cap 220 has six (only three are shown in the figure) radial ribs 226 formed on the bottom surface 222b of the cap-side fitting portion 222.
- the rib 226 has a top 226c of the outer peripheral end 226a abutting on the nucleic acid-adsorbing porous membrane F and supporting the nucleic acid-adsorbing porous membrane F. (See Figure 9).
- the rib 226 is moved from the outer end 226a to the inner end so that, when the nucleic acid separation / purification cartridge 200 is used, the nucleic acid-adsorbing porous membrane F is displaced toward the outlet 224 as the nucleic acid-adsorbing porous membrane F approaches the opening 223. It is inclined toward the discharge section 224 toward the section 226b. Further, a clamping surface 225 that is one step higher than the bottom surface 222b is formed on the outer periphery of the bottom surface 222b so as to be continuous with the outer peripheral end 226a of the rib 226.
- the sandwiching surface 225 is a surface that sandwiches the nucleic acid-adsorbing porous membrane F between the barrel 210 and an opening edge 214 (see FIG. 8) corresponding to the edge of the opening 21 lb. It is preferable that the width of the holding surface 225 is formed in accordance with the width of the opening edge portion 214.
- the rib 226 is an example of a “projection”, and the “projection” may be formed by forming a plurality of mountain-shaped projections that are not necessarily in the form of a rib.
- the ribs 226 are formed radially, when the liquid flows from above to below, the liquid smoothly flows into the discharge portion 224. Further, since the top 226c of the rib 226 is inclined toward the discharge part 224 from the outer end 226a to the inner end 226b, the cleaning liquid S (see FIG.
- the inclination angle ⁇ 1 of the rib 226 with respect to the radial direction of the cap-side fitting portion 222 is preferably 3 ° or more, more preferably 5 ° or more.
- the rib 226 has a top 226c formed in an arc shape when viewed in a cross section. This makes it difficult for the cleaning solution S to stay between the top portion 226c of the rib 226 and the nucleic acid-adsorbing porous membrane F, so that the cleaning solution S is discharged more quickly.
- the rib 22
- the radius of curvature of the top 226c of 6 is preferably 1Z4 or more, more preferably 1Z3 or more, most preferably 1Z2 or more with respect to the width of the rib 226.
- the bottom surface 222b of the cap-side fitting portion 222 has a slope that is displaced toward the discharge portion 224 as approaching the opening 223.
- the inclination angle 02 of the bottom surface 222b with respect to the radial direction of the cap-side fitting portion 222 is preferably 10 ° or more, more preferably 15 ° or more, and most preferably 20 ° or more.
- the corners (eg, the edge 223a of the opening 223) and the corners (eg, the outer edge 222c of the bottom surface 222b) of the inner surface are circular. It is formed in an arc shape. This makes it difficult for the cleaning liquid S to stay at the corners and corners present on the inner surface, so that the cleaning liquid S is discharged more quickly.
- the radius is preferably at least 0.1 mm, more preferably at least 0.2 mm, most preferably at least 0.3 mm.
- the radius of curvature of the corner is preferably 0.1 mm or more, more preferably 0.15 mm or more, and most preferably 0.2 mm or more.
- the angle 3 formed by the axis of the barrel 210 and the inner peripheral surface 210a of the barrel 210 is preferably 10 ° or less, more preferably 5 ° or less.
- the nucleic acid-adsorbing porous membrane F is held between the opening edge 214 of the barrel 210 and the sandwiching surface 225 of the cap 220 with the peripheral edge Fa crushed.
- a liquid a sample solution or the like
- a nucleic acid-containing sample solution is caused to flow by pressurized air from the first opening 211a of the barrel 210 (see FIG. 9) to the second opening 221b of the discharge unit 224. Thereby, the nucleic acid in the sample solution is adsorbed on the nucleic acid-adsorbing porous membrane F.
- the cleaning liquid S is discharged from the first opening 21 la of the barrel 210 to the discharge section 224.
- the nucleic acid-adsorbing porous membrane F is deformed in a convex shape toward the discharge portion 224 along the inclined shape of the rib 226.
- the washing solution S power bottom 222a
- the liquid is quickly discharged from the discharge portion 224 without remaining at the corner 227 formed by the porous film F and the inner peripheral surface 210a of the barrel 210.
- the contact angle of the droplet Wd of the washing liquid S to the inner wall surface (the inner peripheral surface 210a of the barrel 210, etc.) of the nucleic acid separation / purification cartridge 200 is 90 ° or more, for example, as shown in FIG.
- the cleaning solution S As described above, even if the droplet Wd remains on the inner peripheral surface 210a of the barrel 210, the droplet Wd Since it becomes substantially spherical by force, it is easy to remove by pressurized air. As a result, the cleaning solution S
- the droplet Wd of the cleaning solution S contacts the inner wall surface of the nucleic acid separation and purification cartridge 200.
- the angle is not more than 80 °, the wettability of the cleaning solution S to the inner wall surface of the nucleic acid separation / purification cartridge 200 is improved, and the cleaning solution S is less likely to remain as droplets Wd.
- the washing solution S is quickly discharged without remaining inside the nucleic acid separation / purification cartridge 200.
- the cylindrical body is formed by fitting the barrel into the cap, but the present invention is not limited to this.
- a barrel and a cap that are integrally formed may be used as a cylindrical body.
- FIG. 14 is an exploded perspective view of the cartridge for nucleic acid separation and purification according to the fourth embodiment of the present invention
- FIG. 15 is a cross-sectional view of the cartridge for nucleic acid separation and purification according to the fourth embodiment of the present invention. is there.
- FIG. 16 is an enlarged sectional view of the discharge part of the cartridge for separating and purifying nucleic acid according to the fourth embodiment of the present invention.
- FIG. 17 is an enlarged cross-sectional view of the discharge part of the cartridge for separating and purifying nucleic acid according to the fifth embodiment of the present invention.
- FIG. 18 is an enlarged cross-sectional view of the discharge unit of the cartridge for separating and purifying nucleic acid according to the sixth embodiment of the present invention.
- the expressions “up” and “down” are based on the state of use of the cartridge for separating and purifying nucleic acids, specifically, the state as shown in FIG. (Configuration of Cartridge 300 for Separation and Purification of Nucleic Acid)
- the nucleic acid separation / purification cartridge 300 includes a nucleic acid-adsorbing porous membrane F, a barrel 310 and a cap 320 that hold the nucleic acid-adsorbing porous membrane F and form a passage through which a solution flows. It is composed of
- the barrel 310 also has a cylindrical barrel body 312 and a cylindrical barrel-side fitting portion 313 connected to the barrel body 312, and the first opening 311 and the barrel-side fitting are formed at the top of the barrel body 312.
- An opening 314 is formed at a lower portion of the joint 313. Therefore, a solution S (see FIG. 15) can flow downward from above the barrel 310.
- the outer diameter of the barrel side fitting portion 313 is slightly smaller than the outer diameter of the barrel main body 312.
- the cap 320 includes a cylindrical cap-side fitting portion 325 and a discharge portion 302 connected to a bottom opening 323 provided in the bottom portion 322 of the cap-side fitting portion 325.
- the bottom opening 323 provided in the bottom 322 has a smaller diameter than the first opening 311 provided above the barrel main body 312.
- an opening 327 is formed at an upper portion of the cap side fitting portion 325, and a second opening 321 is formed at a lower portion of the discharge portion 302. Therefore, the liquid can flow downward from above the cap 320.
- the inner diameter of the cap-side fitting portion 325 is formed to be a diameter that can be fitted to the outer diameter of the barrel-side fitting portion 313 of the barrel 310.
- the barrel-side fitting portion 313 of the barrel 310 is attached with the nucleic acid-adsorbing porous membrane F disposed on the bottom 322 of the cap-side fitting portion 325 of the cap 320.
- the nucleic acid-adsorbing porous membrane F can be sandwiched between the barrel 310 and the cap 320 by being fitted into the cap-side fitting portion 325 of the cap 320.
- at least three, preferably six, radially formed ribs 326 are formed on the bottom 322 of the cap 320.
- the corner of the rib 326 is formed at the center of the bottom 322 of the cap-side fitting portion 325.
- the opening 323 is formed so as to have a downwardly inclined shape toward the opening 323.
- a discharge portion 302 configured as a tube communicating the bottom opening 323 and the second opening 321 of the cap 320 corresponds to a “discharge portion”, and the cylindrical body and the discharge portion are formed in a cylindrical shape. Corresponds to a “cylindrical body”.
- FIGS. 16 and 17 will be described with reference to FIGS. 14 and 15 for preferred configurations of the discharge section 302 and the end face 304 of the discharge section 302 of the cartridge 300 for nucleic acid separation and purification according to the fourth embodiment of the present invention. This will be described in detail with reference to FIG.
- the discharge section 302 of the cartridge 300 for nucleic acid separation and purification constitutes the lower half of the cap 320, and one end is formed at the bottom opening 323 of the bottom 322 of the cylindrical main body. The other end is configured as a cylinder forming a second opening 321 for discharging the waste liquid.
- the waste liquid can be accurately guided toward the waste liquid container 400 (see FIG. 20) disposed below the nucleic acid separation / purification cartridge 300. This is effective, for example, in preventing contamination of another sample solution when the nucleic acid separation / purification cartridge 300 is used in multiple units by an automatic device described later.
- the thickness T of the end face 324 of the portion of the cap 320 where the second opening 321 is formed is preferably set to 0.2 mm or more. If the thickness T of the portion forming the second opening 321 is formed to be 0.2 mm or more, the bubbles of the sample solution S (see FIG.
- the thickness T is set to 0.5 mm or more.
- the opening diameter r of the second opening 321 of the cap 320 in the nucleic acid separation / purification cartridge 300 is preferably formed to be equal to or greater than 1.0 mm.
- the opening diameter r is set to 1. Omm or more, the waste liquid can be discharged well.
- the outer diameter R of the end face 324 of the cap 320 is preferably formed to be 1.4 mm or more. If the outer diameter R of the end face 324 of the discharge portion 302 is set to 1.4 mm or more, the above-described thickness T can be secured to 0.2 mm or more, and the strength of the portion forming the second opening 321 is kept high. thing Can do. For the above reason, it is more preferable that the outer diameter R is equal to or greater than 2. Omm.
- the output 302 of the cap 320 preferably has an angle ⁇ 4 between the end surface 324 and the outer wall surface 302a of 105 ° or less.
- the angle ⁇ 4 between the discharge portion 302 and the outer wall surface 302a is more preferably 100 ° or less, more preferably 95 ° or less.
- the opening diameter r becomes larger. It may be shaped. In this case, since the outer edge of the second opening 321 is formed at an acute angle with the outer wall surface 302a, even when the downward force and the air roll up, the foam passes over the outer edge portion to the outer wall surface 302a. It becomes difficult to adhere.
- the angle ⁇ 5 between the end surface 324 of the discharge portion 302 and the outer wall surface 302a is preferably set to 30 ° or more (see FIG. 17). If the angle is less than 30 °, the sample solution S
- the resin hydrophilicity of the end surface 324 and the outer wall surface 302a it is preferable that the material constituting the cartridge 300 for separating and purifying nucleic acid be easily wetted. By making the end surface 324 and the outer wall surface 302a hydrophilic, the water repellency of the cleaning solution S is suppressed.
- bubbles can be drawn in by the cleaning liquid S.
- the inner wall surface 302b is preferably made of a hydrophobic synthetic resin which is preferably water-repellent. It is preferable to make 300.
- the following treatment is performed. It can be done by doing.
- a cap 320 in which the second opening 321 side is sealed is manufactured by injection molding using polystyrene which is a hydrophobic synthetic resin.
- the shape of the second opening 321 is preferably adjusted to the above-mentioned arbitrary shape.
- the hydrophilicity of the end surface 324 and the outer wall surface 302a is improved by subjecting the end surface 324 of the cap 320 and the outer wall surface 302a (preferably the outer wall surface 302a near the second opening 321) to plasma treatment.
- the inner wall surface 302b has high water repellency, and the outer wall surface 302a and the end surface 324 of the discharge portion 302 have high hydrophilicity, so that the cap 320 can be obtained.
- the hydrophilicity of the end surface 324 and the outer wall surface 302a can be increased by performing the same treatment.
- the cartridge for separating and purifying nucleic acid of the present invention it is preferable to form a claw member 328 for inducing bubbles on the end face 324 of the cap 320. Further, it is more preferable that the claw member 328 is formed in a rod shape. Further, it is preferable that the inner surface of the claw member 328 is formed to coincide with the inner wall surface 302b. With this configuration, the foam that has reached the second opening 321 travels along the rod-shaped claw member 328 extending on the inner wall surface 302b, and at the tip portion, that is, at a position further below the second opening 321. As a result, the foam easily aggregates and falls into the waste liquid container 400, so that the adhesion of the foam to the outer wall surface 302a can be prevented.
- the number of the bar-shaped claw members 328 can be any number from one to a plurality as long as the above-described object can be achieved.
- the rib 326 is formed so as to have a downwardly inclined shape on the side of the bottom opening 323, when the solution or the like S is pressurized from the first opening 311 side of the barrel 310, the nucleic acid-adsorbing porous membrane F Along the inclined shape of the S rib 326, the S rib 326 is deformed in a convex shape toward the bottom opening 323 side. As a result, the solution S or the like is quickly discharged from the bottom opening 323 without remaining in the bottom 322.
- nucleic acid separation / purification cartridge containing one nucleic acid-adsorbing porous membrane is used.
- a nucleic acid separation / purification cartridge containing a plurality of nucleic acid-adsorbing porous membranes may be used.
- the plurality of nucleic acid-adsorptive porous membranes to be accommodated may be the same or different.
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04787918A EP1676906A4 (en) | 2003-10-21 | 2004-09-21 | NUCLEIC ACID CLEANING CARTRIDGE AND METHOD FOR THE PRODUCTION THEREOF |
US10/576,756 US20070148649A1 (en) | 2003-10-21 | 2004-09-21 | Cartridge for nucleic acid separation and purification and method for producing the same |
JP2005514724A JP4478110B2 (ja) | 2003-10-21 | 2004-09-21 | 核酸分離精製カートリッジの製造方法 |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003-360550 | 2003-10-21 | ||
JP2003360550 | 2003-10-21 | ||
JP2003361183 | 2003-10-21 | ||
JP2003-361019 | 2003-10-21 | ||
JP2003361019 | 2003-10-21 | ||
JP2003-361183 | 2003-10-21 | ||
JP2004051811 | 2004-02-26 | ||
JP2004-051811 | 2004-02-26 |
Publications (1)
Publication Number | Publication Date |
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WO2005037983A1 true WO2005037983A1 (ja) | 2005-04-28 |
Family
ID=34468469
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/JP2004/013735 WO2005037983A1 (ja) | 2003-10-21 | 2004-09-21 | 核酸分離精製カートリッジおよびその製造方法 |
Country Status (4)
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US (1) | US20070148649A1 (ja) |
EP (1) | EP1676906A4 (ja) |
JP (2) | JP4478110B2 (ja) |
WO (1) | WO2005037983A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100015694A1 (en) * | 2008-07-18 | 2010-01-21 | Carlo Acosta | Filtered petri dish |
JP2010228206A (ja) * | 2009-03-26 | 2010-10-14 | Seiko Epson Corp | 液体流路ユニットの製造方法、液体流路ユニット、液体噴射ヘッドユニット及び液体噴射装置 |
Families Citing this family (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4878977B2 (ja) * | 2006-09-29 | 2012-02-15 | 富士フイルム株式会社 | インサート部材、及びこれを備えた多孔質フィルターカートリッジの製造方法 |
US8298763B2 (en) * | 2007-03-02 | 2012-10-30 | Lawrence Livermore National Security, Llc | Automated high-throughput flow-through real-time diagnostic system |
DE102007043614B3 (de) * | 2007-09-13 | 2008-11-20 | Biocrates Life Sciences Gmbh | Halterung für ein Trägermittel zum Einsetzen in eine zylinderförmige Öffnung |
FR2921490B1 (fr) * | 2007-09-21 | 2010-09-10 | Metagenex | Procede et dispositif pour recueillir du materiel cellulaire de cellules isolees sur filtre |
US8664377B2 (en) * | 2009-09-30 | 2014-03-04 | Samsung Electronics Co., Ltd. | Method and apparatus for isolating nucleic acids |
US8685749B2 (en) * | 2009-12-02 | 2014-04-01 | Whatman International Limited | Methods and systems for processing samples on porous substrates |
DE102010062064A1 (de) * | 2010-11-26 | 2012-05-31 | Hamilton Bonaduz Ag | Probenbehälter zur Aufbewahrung und Verarbeitung von mit einem Probenentnahmewerkzeug entnommenen Proben |
RU2602897C2 (ru) * | 2011-10-17 | 2016-11-20 | Зульцер Микспэк Аг | Картридж, способ его изготовления, а также многокомпонентный картридж |
US20150252356A1 (en) | 2012-08-28 | 2015-09-10 | Bio Cube System Co., Ltd. | Porous solid phase for rapidly isolating biological molecules for nucleic acid amplification reaction from biological sample, and use thereof |
CN103753775B (zh) * | 2013-12-30 | 2016-04-20 | 苏州恒辉科技有限公司 | U形尼龙油滤网制件模具 |
JP7092787B2 (ja) * | 2017-03-10 | 2022-06-28 | 昭和電工マテリアルズ株式会社 | ろ過デバイス、捕捉デバイス、及びそれらの使用 |
AU2018239436B2 (en) * | 2017-03-22 | 2022-12-15 | Unchained Labs | Sample plates for buffer exchange and methods of manufacture |
CN109207340B (zh) * | 2017-06-30 | 2022-08-12 | 开启基因股份有限公司 | 核酸萃取组件 |
US20210302289A1 (en) * | 2018-08-31 | 2021-09-30 | Horiba Advanced Techno, Co., Ltd. | Sample preprocessor and analysis system |
JP7172745B2 (ja) * | 2019-03-06 | 2022-11-16 | 株式会社Jvcケンウッド | 分析用ユニット、洗浄装置、及び洗浄方法 |
CN111778241A (zh) * | 2020-07-23 | 2020-10-16 | 广州捷瑞生物工程有限公司 | 一种核酸回收柱的吸附膜安装装置 |
CN217526452U (zh) * | 2022-02-25 | 2022-10-04 | 武汉医蒂生物科技有限公司 | 一种纯化柱 |
KR102473201B1 (ko) * | 2022-05-10 | 2022-12-01 | 곽태문 | 감염병 진단 키트의 필터결합장치 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0275109U (ja) * | 1988-11-30 | 1990-06-08 | ||
JPH03230919A (ja) * | 1990-02-03 | 1991-10-14 | Japan Steel Works Ltd:The | オイルストレーナの製造方法 |
JP2003128691A (ja) * | 2001-08-01 | 2003-05-08 | Fuji Photo Film Co Ltd | 核酸の分離精製方法 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5108704A (en) * | 1988-09-16 | 1992-04-28 | W. R. Grace & Co.-Conn. | Microfiltration apparatus with radially spaced nozzles |
SE9400436D0 (sv) * | 1994-02-10 | 1994-02-10 | Pharmacia Lkb Biotech | Sätt att tillverka filterbrunnar |
US7014049B2 (en) * | 1996-12-23 | 2006-03-21 | Glycorex Transplantation Ab | Device for bio-affinity material |
US6391241B1 (en) * | 1997-06-06 | 2002-05-21 | Corning Incorporated | Method of manufacture for a multiwell plate and/or filter plate |
US6451260B1 (en) * | 1997-08-26 | 2002-09-17 | Dyax Corp. | Method for producing microporous elements, the microporous elements thus produced and uses thereof |
US6419827B1 (en) * | 1998-10-29 | 2002-07-16 | Applera Corporation | Purification apparatus and method |
US6159368A (en) * | 1998-10-29 | 2000-12-12 | The Perkin-Elmer Corporation | Multi-well microfiltration apparatus |
US7135117B2 (en) * | 2001-05-31 | 2006-11-14 | Pall Corporation | Well for processing a fluid |
US6896144B2 (en) * | 2001-06-25 | 2005-05-24 | Innovative Microplate | Filtration and separation apparatus and method of assembly |
CN100543126C (zh) * | 2003-08-19 | 2009-09-23 | 富士胶片株式会社 | 提取装置 |
-
2004
- 2004-09-21 US US10/576,756 patent/US20070148649A1/en not_active Abandoned
- 2004-09-21 EP EP04787918A patent/EP1676906A4/en not_active Withdrawn
- 2004-09-21 WO PCT/JP2004/013735 patent/WO2005037983A1/ja active Application Filing
- 2004-09-21 JP JP2005514724A patent/JP4478110B2/ja not_active Expired - Fee Related
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2010
- 2010-02-01 JP JP2010020578A patent/JP5087096B2/ja active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0275109U (ja) * | 1988-11-30 | 1990-06-08 | ||
JPH03230919A (ja) * | 1990-02-03 | 1991-10-14 | Japan Steel Works Ltd:The | オイルストレーナの製造方法 |
JP2003128691A (ja) * | 2001-08-01 | 2003-05-08 | Fuji Photo Film Co Ltd | 核酸の分離精製方法 |
Non-Patent Citations (1)
Title |
---|
See also references of EP1676906A4 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100015694A1 (en) * | 2008-07-18 | 2010-01-21 | Carlo Acosta | Filtered petri dish |
US8163540B2 (en) * | 2008-07-18 | 2012-04-24 | Carlo Acosta | Filtered petri dish |
JP2010228206A (ja) * | 2009-03-26 | 2010-10-14 | Seiko Epson Corp | 液体流路ユニットの製造方法、液体流路ユニット、液体噴射ヘッドユニット及び液体噴射装置 |
Also Published As
Publication number | Publication date |
---|---|
JP2010094136A (ja) | 2010-04-30 |
JPWO2005037983A1 (ja) | 2007-11-22 |
EP1676906A4 (en) | 2011-03-09 |
JP5087096B2 (ja) | 2012-11-28 |
EP1676906A1 (en) | 2006-07-05 |
US20070148649A1 (en) | 2007-06-28 |
JP4478110B2 (ja) | 2010-06-09 |
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