WO2005052541A2

WO2005052541A2 - Method of separation and collection and apparatus therefor

Info

Publication number: WO2005052541A2
Application number: PCT/JP2004/017486
Authority: WO
Inventors: 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: 2003-11-28
Filing date: 2004-11-25
Publication date: 2005-06-09
Also published as: JPWO2005051524A1; AU2003284488A1; WO2005051524A1; WO2005052541A3

Abstract

A method of separation and collection, comprising feeding a liquid containing a target substance to a carrier capable of collecting the target substance to thereby separate the target substance from the liquid and cause the carrier to retain the same and thereafter collecting the target substance from the carrier, wherein the liquid is caused to flow toward the carrier by means of squirt pressure of a spray can. Preferably, this method of separation and collection further comprises the operation of after the retention of the target substance on the carrier, washing off any matter other than the target substance. Through effective contact of the target substance to be separated and collected with the carrier having the property of retaining the target substance, separation, washing and collection can be accomplished at low cost and apparatus miniaturization can be realized.

Description

Separation and collection method and device

Technical field

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.

Background art

[0002] With the technological advancement of biotechnology, in a variety of fields such as agriculture, food hygiene, clinical examination, environmental analysis, etc., 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.

When conducting analysis, testing and research, it is often necessary to collect biological substances such as sugars, proteins and nucleic acids, and chemical substances such as metal ions and organic substances. For example, for purification of deoxyribonucleic acid (DNA), the most typical method of collection is a method using a high-concentration chaotropic salt such as guaridine thiocyanate and sodium iodide (for example, Patent Reference 1). Further, a method of selectively binding DNA to an anion exchanger (for example, see Patent Document 2) is also one of the effective collection methods.

However, even though there are various principles regarding the selectivity of a substance to be trapped contained in the liquid from the liquid, even if the liquid containing the target substance is brought into contact with the carrier and separated, and then the carrier is washed, As for the method of collecting the target substance together with the washing medium, a method using centrifugal force using a centrifuge or a method using suction force using a decompression pump is generally used. However, large-scale and expensive equipment is required to actually perform them, and electrical equipment is required to use these equipment.

Furthermore, in a system in which a liquid containing a target substance is led out to a carrier using a syringe and a piston, a large amount of liquid is required to derive a large volume of liquid because the amount to be led out matches the volume of the syringe. A large amount of force is required for the pushing operation. In addition, each time the washing operation is repeated after being led out to the carrier, the carrier force syringe must be removed and the pushed-in syringe force must be pulled out of the piston, which is inefficient. It is.

Patent Document 1: Japanese Patent Publication No. 13077, 1995

Patent Document 2: Japanese Patent Publication No. 2680462

Disclosure of the invention

Problems to be solved by the invention

[0003] Therefore, it is an object of the present invention to efficiently contact a substance to be separated and trapped contained in a liquid with a carrier having a property of separating from the liquid to perform separation, washing, and trapping. It is an object of the present invention to obtain an inexpensive separation and collection method and an inexpensive and compact separation and collection device. Means for solving the problem

[0004] In the method for separating and collecting a substance according to the present invention, 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 method according to claim 1, wherein, at the time of collecting the target substance from 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. For example, examples of the target substance include biological substances such as sugars, proteins, and nucleic acids, and chemical substances such as metal ions and organic substances. Examples of 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.

In addition, 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. Also, as 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. 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.

By using the injection pressure generated from the spray can for the flow of the liquid containing the target substance, the above-mentioned problem in the method using the centrifugal force or the method using the suction force is solved. 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.

As a specific usage of the spray pressure of the spray can, 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. .

For example, 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. In order to use the spray pressure of the spray can efficiently, 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. In this case, 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. However, it is acceptable to simply pass the liquid extruded with the spray pressure of a spray can using a filter medium that selectively chemically binds the target substance as a carrier. 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.

Specifically, 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. Thereafter, an eluate for eluting the target substance is introduced into the pressure-resistant container, and the eluate is introduced into the filter medium after the target substance is collected. Releases material. More specifically, the present invention provides a method for separating and collecting a substance comprising the following three steps.

1) 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. First, the 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. In addition, 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. Further, 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.

2) If necessary, introduce a cleaning solution into the pressure-resistant container to wash the carrier (filter material) holding the target substance, and extrude the cleaning solution to the carrier (filter material) in the same manner as described in 1). By doing so, contaminants other than the target substance remaining on the carrier (filter material) may be removed by washing. The carrier (filter material) and the target substance are strongly bonded, and impurities If the size cannot be passed, the washing liquid may be flowed so as to have a flow opposite to the method described in 1).

3) 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).

That is, according to a specific preferred embodiment of the present invention, the method further comprises an operation of washing and removing components other than the target substance after holding the target substance on a carrier.

Further, according to another preferred aspect of the present invention, 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 invention's effect

[0005] 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. And a suitable carrier. This makes it possible to provide an inexpensive and compact device for performing adsorption, washing, separation and collection.

Other objects, features and advantages of the present invention will become apparent from the following description of non-limiting examples, with reference to the accompanying drawings.

Brief Description of Drawings

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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0007] In carrying out the substance collecting method of the present invention, 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.

Example 1

[0008] In this experiment, a method and an apparatus will be described using a nucleic acid as a collection target. 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. As the starting sample liquid, a liquid prepared by adding 10 μg of λ DNA to 0.5 ml of a binding buffer containing guanidine hydrochloride as a chaotropic salt was added.

As shown in FIG. 1, a syringe filter GF / B (manufactured by Whatman) 13 in which a glass fiber filter paper 11 was enclosed in a resin container 12 was used as a carrier capable of selectively binding DNA. 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. After that, 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.

Next, 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. After connecting the spray can 17 again, the remaining liquid and the washing buffer are removed from the syringe filter 13. From the surface. Finally, the syringe 15 was removed from the spray can 17, and 100 μl of the elution buffer was put into the syringe 15 and connected to the spray can 17 again. After standing at room temperature for 3 minutes, 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,

2) Wash buffer: 70% ethanol, 30% TE {10mM Tris-HCl (pH8.0), ImM ethylenediamine tetraacetate (EDTA)} solution,

3) Elution buffer: TE {10mM Tris-HCl (pH8.0), ImM ethylenediamine tetraacetate (EDTA)} solution

The collected DNA was evaluated using agarose gel electrophoresis. FIG. 2 is a schematic diagram showing the results of agarose gel electrophoresis. In the figure, lanes 21 and 24 are molecular weight markers, lane 22 is DNA added to a binding buffer (before addition), and lane 23 is collected DNA. As shown in FIG. 2, it was confirmed that the DNA to be collected was successfully collected.

Example 2

In this experiment, a method and an apparatus will be described in which a collection target is a protein. As 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. As shown in FIG. 3, 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 μl 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.

After confirming the connection, 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.

Next, 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.

(1) Equilibration buffer: 20 mM Tris-HCl (pH 8.0)

(2) Elution buffer: lOOmM Tris-HCl (pH8.0), 1M NaCl

The collected protein solution was evaluated using absorbance measurement at 280 nm and SDS-PAGE. Fig. 4 shows the measured values of absorbance at 280 nm of the solution passing through the strong ion-exchanger for each 5001. In the figure, the horizontal axis indicates the sample number, and 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. 8 to 12 are samples obtained by performing the operation of passing 500 μl of 20 mM Tris-HCl (pH 8.0) five times for washing. Sample Nos. 13 to 16 are samples obtained by performing the operation of passing 100 μl of lOOmM Tris-HCl (pH 8.0) / 500 μl 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. In the figure, 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), and 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), and 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.

By using the substance collection method of the present invention, an extremely inexpensive The target substance could be achieved only with the device. In addition, 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.

Claims

The scope of the claims

[1] After 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, and then separated to collect the target substance from the carrier. In the collection method,

A method for separating and collecting a substance, characterized in that the liquid is directed and flowed toward the carrier by a spray can pressure in which a substance which becomes a gas under normal temperature and normal pressure is filled inside the can by an injection pressure.

2. The method for separating and collecting a substance according to claim 1, further comprising an operation of washing and removing a substance other than the target substance after holding the target substance on a carrier.

3. The method for separating and collecting a substance according to claim 1, further comprising an operation of providing an eluate for eluting the target substance from the carrier holding the target substance by adsorption.

[4] An apparatus for use in the method for separating and collecting a substance according to any one of claims 13 to 13, wherein a can container in which a substance that becomes a gas at normal temperature and normal pressure is filled therein, A spray can provided with a path communicating between the injection port and the inside of the can, and opening / closing means for opening and closing the path, a blowing port connectable to the injection port of the spray can, and a discharge port communicating with the outside; A pressure-resistant container that can store liquid inside,

A substance capable of collecting the target substance in a container that is detachably connected to the discharge port and through which the liquid in the discharge rocker can pass;