WO2005052541A2 - Procede et dispositif de separation et de collecte - Google Patents

Procede et dispositif de separation et de collecte Download PDF

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Publication number
WO2005052541A2
WO2005052541A2 PCT/JP2004/017486 JP2004017486W WO2005052541A2 WO 2005052541 A2 WO2005052541 A2 WO 2005052541A2 JP 2004017486 W JP2004017486 W JP 2004017486W WO 2005052541 A2 WO2005052541 A2 WO 2005052541A2
Authority
WO
WIPO (PCT)
Prior art keywords
target substance
carrier
substance
liquid
spray
Prior art date
Application number
PCT/JP2004/017486
Other languages
English (en)
Japanese (ja)
Other versions
WO2005052541A1 (fr
WO2005052541A3 (fr
Inventor
Akihiro Hino
Hideo Kuribara
Takashi Kodama
Satoshi Furui
Yasutaka Minegishi
Shinji Kanayama
Original Assignee
Nat Food Res
Nippon Gene Co Ltd
Akihiro Hino
Hideo Kuribara
Takashi Kodama
Satoshi Furui
Yasutaka Minegishi
Shinji Kanayama
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nat Food Res, Nippon Gene Co Ltd, Akihiro Hino, Hideo Kuribara, Takashi Kodama, Satoshi Furui, Yasutaka Minegishi, Shinji Kanayama filed Critical Nat Food Res
Priority to JP2005515787A priority Critical patent/JP5061288B2/ja
Publication of WO2005052541A1 publication Critical patent/WO2005052541A1/fr
Publication of WO2005052541A2 publication Critical patent/WO2005052541A2/fr
Publication of WO2005052541A3 publication Critical patent/WO2005052541A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N2001/317—Apparatus therefor spraying liquids onto surfaces

Definitions

  • the present invention relates to a method and an apparatus for separating and collecting a substance contained in a liquid such as a suspension or a solution.
  • a target substance can be selectively selected from a liquid in which various substances are suspended or dissolved.
  • Technology has been developed for separation and collection! Puru.
  • Patent Document 1 Japanese Patent Publication No. 13077, 1995
  • Patent Document 2 Japanese Patent Publication No. 2680462
  • a liquid containing the target substance is supplied to a carrier capable of collecting the target substance, the target substance is separated from the liquid, and held on the carrier.
  • the spray pressure of the spray can is used to flow the liquid toward the carrier. That is, in the present invention, the liquid is caused to flow toward the carrier by using the injection pressure of a spray can that is available at a low cost.
  • the target substance to which the present invention is applied and the liquid containing the target substance include the target substance which can be retained on the carrier when it comes into contact with the carrier, and the target substance in any form such as dissolution or suspension.
  • Any liquid may be used.
  • the target substance include biological substances such as sugars, proteins, and nucleic acids, and chemical substances such as metal ions and organic substances.
  • the liquid include solutions and suspensions containing one or more of these target substances.
  • the carrier capable of collecting the target substance referred to in the present invention is a physical substance that can adsorb, bind or hold and separate the target substance when contacted with a liquid containing the target substance. Includes those that select target substances with a sieving effect. Preferably, those having a chemical property of binding to the target substance, particularly those having a chemical property of selectively binding to the target substance are also suitable.
  • the shape of the carrier may be any shape as long as the liquid containing the target substance flows and comes into contact with the carrier, so that the target substance is collected without escaping.
  • examples thereof include those used as a so-called "filtering material” such as a sponge having porous holes and an aggregate of porous particles, and a membrane or sponge is particularly preferable.
  • the material of the carrier various materials such as paper and ceramic can be used. Particularly, those made of glass fiber or resin fiber are preferable.
  • the spray can used in the present invention is a pressure can filled with a substance which becomes a gas at normal temperature and normal pressure, such as air, oxygen, dioxygen carbon or chlorofluorocarbon, and a mixture thereof into a pressure-resistant can. And a switching means for opening and closing the path.
  • a substance which becomes a gas at normal temperature and normal pressure such as air, oxygen, dioxygen carbon or chlorofluorocarbon, and a mixture thereof into a pressure-resistant can.
  • a switching means for opening and closing the path Any substance that does not adversely affect the target substance is acceptable as a substance to be filled into the pipe under pressure.For example, oxygen, carbon dioxide, nitrogen, or alternative fluorocarbons are preferred. What is used to blow off dust and the like adhering to the contacts of the product is commercially available at low cost! Puru.
  • the trapping device can be used anywhere.
  • the injection pressure is sufficient to the extent permitted for commercially available spray cans, and is generally 0.8 MPa or less.
  • any method can be used as long as the liquid containing the target substance is directed toward the carrier for collecting the target substance by applying pressure by the injection pressure. .
  • the carrier is formed into a tube, and the liquid is circulated through the tubular carrier to remove unnecessary liquid or non-target substances contained in the liquid while concentrating and separating and collecting the target substance inside the liquid.
  • spray the gas into the pressure-resistant container in order to make efficient use of the spray pressure of the spray can. It is preferable that the gas in the can is introduced, and the liquid in the pressure-resistant container is extruded into the flow path by the spray pressure of the spray can and discharged toward the carrier.
  • the carrier that separates the target substance from the liquid forms a pipe path using a carrier that has a chemical property that selectively binds to the target substance, and converts the liquid extruded with the spray pressure of the spray can.
  • the target substance is selectively chemically bonded while passing through the pipe route.
  • the method in which the liquid is passed through the filter medium is preferred in the present invention using a spray can in which a limited volume of gas is sealed.
  • the present invention provides the following separation and collection method. That is, after supplying the liquid containing the target substance to a carrier capable of collecting the target substance, separating the target substance from the liquid and holding the target substance on the carrier, when collecting the target substance from the carrier, The liquid containing the target substance is introduced into a pressure-resistant container, connected to a filter material made of a carrier capable of collecting the target substance, and then connected to the gas injection port of the spray can to convert the liquid to the filter material by the injection pressure of the spray can. At the same time, the target substance is collected by the filter medium by extrusion, and the liquid from which the target substance has been removed is discharged from the filter medium from which the target substance has been collected.
  • the present invention provides a method for separating and collecting a substance comprising the following three steps.
  • a spray can Removably connect a spray can, a pressure-resistant container such as a syringe, and a filter material made of a carrier capable of collecting the target substance.
  • a pressure vessel and the filter medium are connected, and a liquid containing the target substance is added into the pressure vessel.
  • This is connected to the gas injection port of the spray can and the liquid is extruded to the filter medium by the spray pressure of the spray can, so that the target substance is collected on the filter medium and at the same time, the liquid from which the target substance has been removed is subjected to the target substance. Is discharged from the collected filter media.
  • the operation of pushing out the liquid containing the target substance to the filter medium may be repeated not only once but also several times as necessary to hold almost all of the target substance in the liquid on the filter medium.
  • the liquid containing the target substance may contain a reagent composition for promoting the binding of the target substance to a carrier (filter material), if necessary.
  • the eluate for eluting the target substance is introduced into the pressure vessel, and the eluate is introduced into the carrier (filter material) to collect the liquid containing the target substance released from the carrier. If the target substance has a size that cannot be eluted and pass through the carrier (filter material), the eluate may be flowed in the reverse flow to the method described in 1).
  • the method further comprises an operation of washing and removing components other than the target substance after holding the target substance on a carrier.
  • the carrier force holding the target substance by adsorption is provided with an eluate for eluting the target substance, thereby releasing the carrier force target substance. Is further provided.
  • the substance separation and collection device of the present invention is an apparatus used for the above-described substance separation and collection method, which includes a can container in which a substance that becomes a gas under normal temperature and normal pressure is filled.
  • a spray can having an injection port, a path communicating between the injection port and the inside of the can, and opening / closing means for opening and closing the path; an injection port connectable to the injection port of the spray can; and a discharge port communicating with the outside.
  • a pressure-resistant container having an outlet and capable of storing a liquid therein, and a target container which is detachably connected to the discharge port and held in a container through which the liquid from the discharge port can pass to collect the target substance.
  • a suitable carrier is provided. This makes it possible to provide an inexpensive and compact device for performing adsorption, washing, separation and collection.
  • FIG. 1 is an explanatory view including a configuration and a partial cross section of a DNA collecting apparatus according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the results of agarose gel electrophoresis.
  • FIG. 3 is an explanatory diagram showing a configuration of a protein collecting device according to another embodiment of the present invention.
  • FIG. 4 is a graph showing absorbance at 280 nm.
  • FIG. 5 is a schematic diagram showing the results of SDS-PAGE.
  • the target substance and some principle of capturing the target substance are not important. Therefore, the target substance is not limited.
  • the gist of the present invention is that the implementation of the above principle is portable and can be realized by the injection pressure of a disposable spray can.
  • FIG. 1 is an explanatory diagram including a configuration and a partial cross section of a DNA collecting device according to an embodiment of the present invention.
  • a liquid prepared by adding 10 ⁇ g of ⁇ DNA to 0.5 ml of a binding buffer containing guanidine hydrochloride as a chaotropic salt was added.
  • the inlet 14 of the syringe filter 13 was connected to the outlet 16 of a 1 ml syringe (made of thermone earth) 15 as a pressure-resistant container, and the liquid was added to the 1 ml syringe 15.
  • a spray can (trade name: OA dust cleaner, manufactured by RIX Co., Ltd.) 17 is connected to the injection port 19 of the head part 18 of the syringe 19 and the injection port 20 at the rear end of the syringe 15 to collect the target substance.
  • the device was assembled. After confirming the connection, the gas injection button 10 of the spray can 17 was pressed to spray the gas inside, and the liquid in the syringe 15 was discharged from the syringe filter 13.
  • the 1 ml syringe 15 connected to the syringe filter 13 is removed from the spray can 17, and 1 ml of the washing buffer is put into the syringe 15.
  • the remaining liquid and the washing buffer are removed from the syringe filter 13. From the surface.
  • the syringe 15 was removed from the spray can 17, and 100 ⁇ l of the elution buffer was put into the syringe 15 and connected to the spray can 17 again.
  • the gas injection button 19 of the spray can 17 was pressed to inject the gas inside, the elution buffer in the syringe 15 was discharged from the syringe filter 13, and the discharged liquid was collected.
  • the buffer was of the following composition: 1) Binding buffer: 8M guanidine hydrochloride,
  • FIG. 2 is a schematic diagram showing the results of agarose gel electrophoresis.
  • lanes 21 and 24 are molecular weight markers
  • lane 22 is DNA added to a binding buffer (before addition)
  • lane 23 is collected DNA. As shown in FIG. 2, it was confirmed that the DNA to be collected was successfully collected.
  • a method and an apparatus will be described in which a collection target is a protein.
  • a starting sample solution a solution prepared by adding 100 g of BSA (silicone albumin) to 1 ml of an equilibration buffer was used.
  • SAX manufactured by Whatman, which is a strong ion exchanger, was used as a carrier capable of binding BSA.
  • FIG. 3 is an explanatory diagram showing a configuration of a protein collecting device according to another embodiment of the present invention.
  • a porous membrane 34 made of polyethylene was incorporated into an outlet 32 of a 1 ml syringe 31 and 0.5 ml of a strong ion exchanger 35 swollen with an equilibration buffer was filled. Thereafter, 500 ⁇ l of the equilibration buffer is put into the syringe 31, and the rear end 33 of the syringe 31 is connected to the injection port 36 at the head of the spray can, thereby forming a device for collecting the target substance. I stood up.
  • the gas injection button on the spray can was pressed to inject the gas inside, and the equilibration buffer in the syringe was discharged from the syringe outlet. Since it is necessary to wash with 5-6 times the equilibration buffer of the strong ion exchanger, this operation is performed 5 times, and a total of 2.5 ml is added to the strong ion exchanger. Of equilibration buffer.
  • the 1 ml syringe was removed from the spray can, and 0.5 ml of the starting sample solution was put into the syringe. After connecting the spray can again, the solution was discharged. This operation was performed twice in total to add 1 ml. Then, put the equilibration buffer 5001 into the syringe for washing, and After connection with the play can, the strong ion exchanger was washed. This operation was repeated five times, and a total of 2.5 ml of the washing buffer was passed. Finally, the syringe was removed from the spray can, and the syringe was filled with Elution Buffer 5001 and connected to the spray can again. The gas in the spray can was pressed to inject the gas inside, the elution buffer in the syringe was discharged, and the discharged solution was collected. This operation was repeated four times, and a total of 2 ml was collected.
  • the buffer used had the following composition.
  • Fig. 4 shows the measured values of absorbance at 280 nm of the solution passing through the strong ion-exchanger for each 5001.
  • the horizontal axis indicates the sample number
  • the vertical axis indicates the absorption at a wavelength of 280 nm of the collected permeate.
  • the sample numbers of 1 to 5 are the samples obtained when the operation of passing 500 mM 1 of 20 mM Tris-HCl (pH 8.0) 5 times was performed 5 times to equilibrate the column.
  • the sample numbers 6 to 7 are the samples obtained by passing 500 g of 100 g / ml BSA at a time. Sample Nos.
  • Sample Nos. 13 to 16 are samples obtained by performing the operation of passing 100 ⁇ l of lOOmM Tris-HCl (pH 8.0) / 500 ⁇ l of 1M NaCl four times to elute the protein. In sample number 13, which is the beginning of protein elution, the absorbance measurement at 280 nm is the highest.
  • FIG. 5 is a schematic diagram showing the results of acrylamide gel electrophoresis.
  • lane A is the elution buffer lOOmM Tris-HCl (pH 8.0) / lM NaCl
  • lane M is the molecular weight marker
  • lane 51 is the flow-through of the equilibration buffer (sample numbers 15)
  • lane 57 is the starting material.
  • the flow-through of the solution (sample numbers 6-7)
  • lanes 58, 59, and 60 are the flow-through of the washing step with the equilibration buffer (sample numbers 8, 9, and 10)
  • lanes 64 and 65 are the collected BSA (sample numbers). 14, 15).
  • Lane B is 100 g / ml BSA before addition. As shown in FIG. 5, as shown in lanes 64, 65 and B, it was confirmed that the protein to be collected was collected well.
  • the present invention does not limit the location in the process of collecting the target substance to the vicinity of the place where the apparatus is installed, so that the target substance can be collected even in a place where there is no experimental facility.

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  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Extraction Or Liquid Replacement (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

Cette invention concerne un procédé de séparation et de collecte, consistant à amener un liquide qui contient une substance cible à un support capable de séparer ladite substance cible du liquide et de conserver cette substance, puis à extraire la substance cible du support, le liquide atteignant le support sous l'effet d'une pression appliquée sur un récipient de pulvérisation. Ce procédé de séparation et de collecte s'accompagne, après rétention de la substance cible sur le support, d'une opération d'élimination par lavage de toute substance autre que la substance cible. Un bon contact entre la substance à séparer et à collecter d'une part, le support d'autre part, a pour effet de retenir la substance cible, la séparation, le lavage et la collecte pouvant se faire à coût réduit, avec possibilité de miniaturiser le dispositif.
PCT/JP2004/017486 2003-11-28 2004-11-25 Procede et dispositif de separation et de collecte WO2005052541A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005515787A JP5061288B2 (ja) 2003-11-28 2004-11-25 分離捕集方法及び装置

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/JP2003/015264 WO2005051524A1 (fr) 2003-11-28 2003-11-28 Procede et dispositif de separation et de recueil
JPPCT/JP03/15264 2003-11-28

Publications (3)

Publication Number Publication Date
WO2005052541A1 WO2005052541A1 (fr) 2005-06-09
WO2005052541A2 true WO2005052541A2 (fr) 2005-06-09
WO2005052541A3 WO2005052541A3 (fr) 2005-08-04

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PCT/JP2003/015264 WO2005051524A1 (fr) 2003-11-28 2003-11-28 Procede et dispositif de separation et de recueil
PCT/JP2004/017486 WO2005052541A2 (fr) 2003-11-28 2004-11-25 Procede et dispositif de separation et de collecte

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AU (1) AU2003284488A1 (fr)
WO (2) WO2005051524A1 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02182260A (ja) * 1989-01-06 1990-07-16 Koken Co Ltd 骨形成活性を有する粒状骨補てつ材
JPH0376585A (ja) * 1989-08-18 1991-04-02 Ngk Insulators Ltd 微生物による生成物の製造方法
JP2717315B2 (ja) * 1989-10-30 1998-02-18 トヨタ自動車株式会社 溶接粉塵洗浄廃液の浄化再生方法
JPH06238265A (ja) * 1993-02-16 1994-08-30 Netsushii Kogyo Kk 汚水処理装置

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Publication number Publication date
JPWO2005051524A1 (ja) 2007-08-09
AU2003284488A1 (en) 2005-06-17
WO2005052541A3 (fr) 2005-08-04
WO2005051524A1 (fr) 2005-06-09

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