WO2011055873A1

WO2011055873A1 - Apparatus for extracting biochemical material from a biological sample

Info

Publication number: WO2011055873A1
Application number: PCT/KR2009/006565
Authority: WO
Inventors: 윤성준; 강상현; 박지성; 박보나; 박만순; 최윤혁
Original assignee: 주식회사 인트론바이오테크놀로지
Priority date: 2009-11-05
Filing date: 2009-11-10
Publication date: 2011-05-12
Also published as: KR20090129387A

Abstract

The present invention relates to a disposable extracting apparatus for one-time use in the extraction and recovery of biochemical material from a biological sample, wherein the extracting apparatus of the present invention comprises: a plurality of tube plates having one or more tubes formed therein; and a column plate detachably coupled to the tube plates, having one or more columns formed therethrough corresponding to the tubes of the tube plates, the column having a filter installed therein and defining a suction hole at one end thereof to form a vacuum. As a biochemical material can be extracted from a biological sample by means of elevating the vacuum pressure without a centrifugal separating process, the extraction of biochemical material from a biological sample is rendered simple and convenient and can be performed quickly. The small scale structure not only enables a target vacuum pressure to be expediently reached, but also enables the uniform application of vacuum pressure to the columns. As the extracting apparatus is not reused, unlike those of the prior art, it has the advantage of preventing the possibility of cross-contamination that can occur between extractions when one extracting apparatus is reused.

Description

Apparatus for Extracting Biochemicals from Biological Samples

The present invention relates to an extractor for extracting and recovering a biochemical material from a biological sample, and more particularly, to a single use that can be used to extract and separate biochemical materials such as DNA, RNA and proteins from a biological sample. A device for the extraction of biochemicals from depleted biological samples.

In general, biological laboratories use these biochemistry for washing of biochemical materials such as DNA, RNA and proteins, for isolation and purification of the biochemical materials from biological samples. Cylindrical columns are used that contain elements of material that can be used for adsorption and desorption of the material.

This conventional column 10 is set together with the tube 20, as shown in FIG. 1.

The column 10 includes a body 11 formed in a cylindrical tubular shape, a discharge port 12 protruding to a diameter smaller than the body 11 at the lower end of the body 11, and an upper end of the body 11. And a lid 13 to open and close the body 11. In addition, a filter 14 made of a plurality of filter membranes for adsorption and desorption of biochemical materials is installed at the inner lower end of the body 11 of the column 10. This filter 14 is not used for the concept of normal filtration but is used for the purpose of specific adsorption and desorption with the biochemical material. Accordingly, the filter 14 is only one usable embodiment, and may be implemented in the form of silica gel or in the form of fine powder or beads, which will all be included below. It is called "filter".

In addition, the tube 20 is a cylindrical tubular shape having a diameter larger than that of the column 10, and one side of the outlet 12 and the body 11 of the column 10 is inserted, and in this state, the centrifuge The liquid containing the washing solution of the biological sample loaded on the column 10 or the biochemical material itself is discharged to the tube 20 by centrifugal force provided by an apparatus such as "centrifuge". do.

That is, after the column 10 is inserted into the tube 20, biological samples containing biochemical substances such as DNA, RNA, and proteins are supplied into the body 11 of the column 10, and the biological samples are supplied. Add a wash solution to selectively separate the biochemicals or contaminants to be removed. Subsequently, the tube 20 into which the column 10 is inserted is installed in a centrifuge (not shown) and rotated at a high speed so that foreign matter other than the biochemical material to be extracted by the centrifugal force generated therefrom (biochemical substances to be removed or The cleaning liquid containing the other contaminants) is recovered into the tube 20 after passing through the filter 14, and thus the cleaning liquid containing the foreign matter recovered in the tube 20 is not necessary for subsequent operations. 20) is separated from column 10 and then discarded. After discarding the wash solution, the tube 20 is again coupled to the column 10.

This process is usually referred to as washing since it is repeated several times and is carried out for the purpose of enhancing the purity of the desired biochemical material in the biological sample while removing the unwanted material from the biological sample. In this washing step, the desired biochemical material is adsorbed to the filter 14 mounted inside the column 10 and the other material is eluted by the washing liquid. After the washing is completed, the recovery of the desired biochemical material adsorbed to the filter 14 is carried out by introducing an elution solution into the column 10, and then the eluate containing the desired biochemical material is tubed by a centrifuge. This is achieved by collecting in 20.

However, such a conventional sampling method, because the operation time according to the centrifuge operation time caused by the operator repeats the process several times during the continuous washing of the column 10 takes a long time in the work and hassle of the sample Difficulties in processing were inevitable.

In order to overcome the disadvantages of using such a centrifuge, a vacuum manifold 30 has been developed that allows the separation of desired biochemicals from biological samples using vacuum pressure. The vacuum manifold 30 is designed to replace the centrifuge in the conventional method using only the centrifuge, and the vacuum manifold 30 is equipped with a column 10 and a vacuum pump.

FIG. 2 shows a conventional vacuum manifold, which is a sealed conventional box shape, in which a mounting hole 31 into which the outlet 12 of the above-described column 10 is inserted and mounted is drilled on one side thereof. In order to suck the air inside the vacuum manifold 30 and then discharge it to the outside to form a vacuum pressure in the vacuum manifold 30, a vacuum pump (not shown) so that the vacuum pressure acts on the column 10 as a result. And a suction port 32 connected to the discharge port, and a drain hole 33 for discharging the collected solution discharged into the vacuum manifold 30 is installed at the other side, and a drain hole 33 is disposed at the drain hole 33. It is preferable that a separate valve (not shown) or a plug (not shown) or the like capable of opening and closing is installed.

On the other hand, such a vacuum manifold 30 is installed by inserting the outlet 12 of the column 10 into the mounting hole 31 of the upper surface, and then the vacuum manifold (by a vacuum pump connected through the inlet 32) ( 30) Inhale the air inside. Then, as the pressure inside the vacuum manifold 30 is lowered, a negative pressure (hereinafter referred to as "vacuum pressure") is formed, and the vacuum pressure causes the solution inside the column 10 to be vacuum manifold 30. Will be discharged to the inside of the When the washing solution is added to the column 10, the washing solution containing the foreign matter (biochemical substances or contaminants to be removed) is discharged into the vacuum manifold 30.

In the conventional method using the vacuum manifold 30, the eluent is added to the column 10 as a final step of obtaining the desired biochemical material, and then centrifuged as in the conventional method using the centrifuge described above. Done. The reason for using the centrifugation separately in this final step is that the vacuum manifold 30 used in this method is contaminated with various materials and is discharged from the individual columns 10. This is because only the solution discharged from the individual column 10 including only the desired biochemical material cannot be recovered from each other because the solutions are all combined.

Therefore, such a dependency on the centrifugation in the final step has a problem that causes inconvenience and inconvenience in the work as in the conventional method using a centrifuge.

In addition, in the conventional vacuum manifold 30, many mounting holes 31 are formed to mount a plurality of columns 10 on the upper surface thereof. In order to form such mounting holes 31, a vacuum manifold is formed. The size of the 30 must be large and in the case of such a large-scale vacuum manifold 30, it takes a long time to form the desired vacuum pressure inside the vacuum manifold 30, and as a result, each operation step such as repeated washing As the time required to wait until the vacuum pressure is formed every time, not only the overall work time is longer, but also the inconvenience is caused, and even vacuum pressure is applied to the column 10 mounted at each mounting hole 31. There was a disadvantage to be difficult.

In addition, in the extraction method based on the conventional vacuum manifold 30, although the column 10 is not reused, the vacuum manifold 30 itself is reused again, so there has always been a concern of cross contamination between extraction operations. Cross-contamination between these extraction operations leads to erroneous results in subsequent operations with the extracted biochemicals.

Accordingly, the present invention has been made to solve the problems of the prior art as described above, the biochemical material from the biological sample used to selectively extract and separate biochemical materials such as DNA, RNA and protein from the biological sample Since the extraction device is composed of small and small type for the purpose of single use that can apply vacuum extraction method, extraction of biochemical material from biological sample can be done easily, conveniently and quickly without using centrifuge. Due to the small scale, it is possible not only to quickly reach the desired vacuum pressure but also to apply a uniform vacuum pressure to the column, and since the used extraction device is not reused unlike the conventional method, one extraction device is repeated in several operations. Concerns of cross contamination between extraction operations due to use It is an object of the present invention to provide a device for the extraction of biochemicals from biological samples.

The above object is a tube plate having a tube formed therein; Removably coupled to the tube plate and a hollow column corresponding to the tube of the tube plate penetrates therein, while inside the column, an element made of a material capable of adsorption and desorption of biochemical substances in a biological sample is installed. One side is achieved by an apparatus for extracting a biochemical material from a biological sample comprising a; a column plate formed with a hollow suction port formed so as to add a vacuum pressure from the outside.

A cover slip of a flexible material that opens and closes the column is detachably coupled to the column of the column plate, and the cover slip is formed with a groove to improve adhesion to the column when the vacuum pressure is applied.

In addition, the coupling between the column plate and the tube plate is installed so that the lower end of the column is located in the tube, the tube of the tube plate is formed with an incision to be discharged out of the tube when the discharged solution is filled above a certain height, It is preferable that the cutouts are formed so as not to face each other, and the coupling plate corresponding to each other is formed on one side of the tube plate and the column plate.

In addition, a supporter for supporting the column plate coupled thereto is protruded from the outer surface of the tube plate, and the inner bottom surface of the tube plate of the tube plate is gently curved.

According to the extraction device of the biochemical material from the biological sample of the present invention, in the extraction device consisting of two tube plates and one column plate, both washing and separation of the desired biochemical material occur, so that subsequent work without the use of a centrifuge Biochemical material to be used as a sample from the biological sample to the tube has the advantage that can be easily and conveniently extracted and recovered.

In using the extraction apparatus of the present invention, the tube plate from which the primary solution (washing liquid) is recovered is discarded as it is, and the solution containing the desired biochemical material is recovered in the secondary new tube plate, and the tube plate is also repeated. By not using it, cross contamination between extraction operations can be prevented in advance.

In addition, since the tube plate and the column plate of the present invention is a small scale formed with less than 10 tubes and columns, the space in which the vacuum should be formed is small, so that the desired vacuum pressure can be quickly reached therein as well as the angle of the column plate. There is an advantage that even vacuum pressure can be applied to the column.

1 is an exploded perspective view showing columns and tubes for the extraction of biochemicals from common biological samples.

2 is a cross-sectional view showing a conventional vacuum manifold equipped with a general column.

Figure 3 is an exploded perspective view showing an apparatus for extracting biochemical material from the wasted biological sample according to the present invention.

Figure 4 is a perspective view showing the extraction device of the biochemical material from the wasted biological sample according to the present invention.

5 is an exploded view showing an apparatus for extracting biochemical material from the wasted biological sample according to the present invention.

FIG. 6 is a view illustrating a process of extracting a biochemical material using an apparatus for extracting a biochemical material from a consumable biological sample according to the present invention.

7 is a view showing an example of using a combination of the extraction device of biochemicals from the consumable biological sample according to the present invention.

8 is an exploded perspective view showing an apparatus for extracting biochemical material from the wasted biological sample according to another embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Accompanying drawings Figures 3 to 8 illustrate a device for extracting biochemical material from biological samples according to the present invention.

Extraction apparatus 100 of the present invention, as shown in Figures 3 and 5, includes two tube plate 200, column plate 300 and the cover slip 400.

The tube plate 200 is a small rectangular box shape having an upper surface opened, and a plurality of tubes 210 are integrally formed on the bottom surface of the tube plate 200, and a column 310 to be described later on the tube 210. The solution discharged from) is introduced.

The tube 210 is a hollow tube having an upper surface thereof opened and protruded to 10 or less, more preferably 5, on the tube plate 200, and the bottom surface thereof is formed of a gentle inclined surface 211. In this case, the solution discharged into the tube 210 can be easily and maximally recovered.

In addition, one side of the tube 210 is formed in the incision 212 is cut in the longitudinal direction, that is, the vertical direction of the tube 210, when the cleaning liquid containing the foreign matter discharged into the tube 210 is filled to a certain height By naturally discharged to the tube plate 200 outside the tube 210, a direct contact with the column 310 to be described later, the washing solution containing foreign matter is prevented in advance to prevent contamination of the sample.

On the other hand, the cutout 212 of the tube 210 is preferably formed so as not to face each other. That is, when the washing solution discharged into the tube 210 is discharged to the tube plate 200, the discharged solution is prevented from being reintroduced into another adjacent tube 210.

In addition, a supporter 220 for supporting the column plate 300 coupled thereto is formed on the outer surface of the tube plate 200, and the supporter 220 protrudes over the entire outer surface of the tube plate 200. It is formed and is effective to apply a vacuum pressure.

In addition, a coupling display unit 230 for inducing correct coupling of the tube plate 200 and the column plate 300 is formed at one side of the outer surface of the tube plate 200, and the coupling display unit 230 is a tube plate ( It is formed by inclining the corner of one side of the four corners of the 200.

Therefore, the tube plate 200 and the column plate 300 are always coupled only in a predetermined direction as shown in FIG. 4 by the coupling display unit 230, and the column of the column plate 300 is coupled by a constant direction. The 310 may be inserted accurately while maintaining a constant pair at all times without shifting the tube 210 of the tube plate 200.

On the other hand, the tube plate 200 configured as described above is composed of two sets (bar), one tube plate 200 is used for the recovery of the cleaning liquid containing foreign matter, the remaining tube plate 200 is the desired Used to recover the solution containing the biochemical material, the tube plates 200 used in this way are not reused for the purpose of single use, so that cross-contamination between extraction operations can be prevented in advance.

Here, the two tube plates 200 are made of the same configuration and structure, and will be described with the same reference numerals.

The column plate 300 is a small rectangular box shape corresponding to the tube plate 200, the bottom surface of which is opened, and 10 or less columns 310 corresponding to the tube 210 are formed on the ceiling surface of the column plate 300. That is, more preferably, five are formed to protrude.

The column 310 is a container for loading a biological sample including biochemical materials such as DNA, RNA, and protein therein, and an inlet 313 penetrating the column plate 300 is formed at an upper end thereof, and a lower end thereof. The outlet 311 is gradually reduced in diameter is formed, the filter 310 is composed of a plurality of filter membranes for adsorption and desorption of the biochemical material inside the column (310).

In this case, the filter 312 is not used as a conventional filtration concept as described above, but is used for the purpose of specific adsorption and desorption with a biochemical material. Therefore, the filter 312 is only one usable embodiment, and may be implemented in the form of silica gel or in the form of fine powder or beads, which will all be included below. It is called "filter".

Although not shown in the present embodiment, the filter 312 may be bent to the outlet 311 side of the column 310 by the vacuum pressure applied to the column 310 to prevent the filter 312 from deflecting. It is preferable that a lower surface is provided with a lattice-shaped support, and an upper surface of the filter 312 is provided with an O-ring fixing ring for preventing the flow and separation of the filter 312.

On the other hand, the column 310 is the outlet of the column 310 when the column plate 300 and the tube plate 200 is coupled to the bar 310 is inserted into the tube 210, the bar 310 and By the coupling method between the tubes 210, the solution discharged from the column 310 is not discharged to the outside of the tube 210, all are correctly introduced and stored inside the tube 210.

In addition, a suction port 320 protrudes from an outer surface of the column plate 300, and the vacuum pump pumps air in an internal space formed by the tube plate 200 and the column plate 300 to be coupled through the suction port 320. By suction by using a vacuum plate is formed inside the extraction device 100 formed by coupling the tube plate 200 and the column plate 300. Meanwhile, in the present embodiment, the suction port 320 is formed in the column plate 300 as a preferred example, but the suction port 320 may be formed on the outer surface of the tube plate 200.

In addition, a coupling display unit 330 corresponding to the coupling display unit 230 of the tube plate 200 is formed on the column plate 300 to have the correct orientation when the tube plate 200 and the column plate 300 are coupled to each other. Induces binding.

That is, when the tube plate 200 and the column plate 300 are coupled, the tube plate 200 and the column plate 300 are always coupled in a constant direction by both

coupling display units

230 and 330. Outlet 311 of 310 is inserted and coupled correctly inside the tube 210 to be paired with each other.

In particular, the tube plate 200 and the column plate 300 are typically injection molded, in which case the tube 210 and the column 310 formed on the tube plate 200 and the column plate 300 correspond to one-to-one. Formed so that the coupling between the tube 210 and the column 310 corresponding to each other can be made accurately with respect to the

coupling indicators

230 and 330.

In addition, a cover slip 400 is installed at the inlet 313 of the column 310 to seal the column 310 to improve the vacuum degree of the column 310.

The cover slip 400 is a thin film made of a flexible material, and is directly inserted into the inlet 313 to protrude from the cap 410 to open and close the inlet 313, and protrude outward from the outer periphery of the cap 410. And a flange portion 420 seated on an upper surface of the inlet 313.

A plurality of grooves 421 are formed in the flange part 420 to further contract the flange part 420 to the upper surface of the inlet 313 while shrinking during vacuum of the column 310 to improve the sealing property.

On the other hand, the tube plate 200 and the column plate 300 configured as described above are composed of a small number formed in the tube 210 and the column 310 corresponding to each other therein, respectively, when the action of the vacuum pressure The same vacuum pressure can be applied to column 310.

In addition, as described above, the extraction device 100 consisting of the tube plate 200 and the column plate 300 may be additionally connected as many as desired as needed.

That is, as shown in Figure 7, in order to use a plurality of extraction apparatus 100 consisting of a tube plate 200 and a column plate 300 at the same time a plurality of extraction apparatus 100 to the multi-plate plate 500 Each of the multiple plate plate 500 is to be installed, a plurality of fixing blocks (510) are formed to be fixed to install each removable device 100 detachably.

As described above, each vacuum pump is individually connected to the suction port 320 of each extraction apparatus 100 installed in the multi-plate plate 500 through the fixing block 510 to apply vacuum pressure to each. Alternatively, it may be connected to one vacuum pump by means of one tube or tube or the like to apply vacuum pressure at the same time.

The extraction process of the biochemical material from the biological sample by the extraction apparatus of the present invention configured as described above will be described.

First, the column plate 300 is coupled to the tube plate 200 in a state in which the coupling display unit 330 of the column plate 300 coincides with the coupling display unit 230 of the tube plate 200. Then, the column plate 300 is coupled to the supporter 220 of the tube plate 200 in a supported state.

In this case, before mounting the column plate 300 to the tube plate 200, a biological sample containing biochemical materials such as DNA, RNA, and protein may be loaded into each column 310 of the column plate 300, Alternatively, as described above, after coupling the column plate 300 to the tube plate 200, a biological sample containing biochemical substances such as DNA, RNA, and protein may be loaded into each column 310 of the column plate 300. It may be.

Of course, after the biological sample is contained in the column 310, the inlet 313 of the column 310 is closed with the coverslip 400 to seal the column 310.

Thereafter, by using the vacuum pump connected through the suction port 320 of the column plate 300 by sucking the air in the internal space formed by coupling the column plate 300 and the tube plate 200 to discharge to the outside, of FIG. As in (a), the inside thereof becomes a vacuum state, and its vacuum pressure acts inside the column 310 through the outlet 311 of the column 310 to filter the solution (washing liquid) inside the column 310 (312). It is discharged into the tube 210 through).

At this time, since the tube plate 200 and the column plate 300 are manufactured in a small size, the tube plate 200 and the column plate 300 may not only rapidly reach the desired vacuum pressures, but may also apply a uniform vacuum pressure to each column 310.

In addition, since the outlet 311 of the column 310 is inserted into the upper side of the tube 210, the discharged solution is not scattered to the tube plate 200 side, and each tube 210 paired with each column 310. When the solution stored in the tube 210 reaches a predetermined height, the tube 210 is discharged to the tube plate 200 outside the tube 210 through the cutout 212 of the tube 210. It is possible to prevent the solution inside the direct contact with the outlet 311 of the column 310 in advance.

Subsequently, the tube plate 200 containing the discharged solution is separated from the column plate 300, another new tube plate 200 is coupled to the column plate 300 again, and the inside thereof is vacuumed again.

At this time, the eluate for extracting the sample is supplied to the column 310, and the solution containing the desired biochemical material is inside the tube 210 as shown in FIG. 6 (b) by the vacuum pressure acting therein. The solution containing the biochemical material discharged to the tube 210 in this way is drawn out through an apparatus such as a pipette.

In addition, since the bottom surface of the tube 210 is formed of a slanted inclined surface 211, the solution in the tube 210 is collected at the bottom center of the tube 210, so that all the solutions remaining in the tube 210 are easily maintained. It is possible to recover.

Therefore, in the present invention, when extracting the desired biochemical material contained in the biological sample, it is possible to completely exclude the centrifugation step, unlike the conventional one, in addition to the extraction device can be simplified and simplified as well as the work process and operation Time can also be shortened.

Furthermore, in the present invention, the centrifugation step is completely excluded, and thus the size of the column is not limited according to the shape of the centrifuge. When the desired biochemical material is extracted using a column having a larger size than the conventionally used column, the yield of the target biochemical material which can be recovered from one column is increased. In addition, reducing the use of eluent may result in a solution containing a higher concentration of biochemicals. In other words, if a large column is used and the use of the eluent is reduced, solutions up to the concentration obtained by the conventional method (including the case where the eluate is reduced in the conventional method) can be obtained.

8 is an exploded perspective view illustrating an apparatus for extracting a biochemical material from a consumable biological sample, according to another embodiment of the present invention. Is omitted.

As shown in FIG. 8, one tube 210a and one column 310a may be formed in the tube plate 200a and the column plate 300a of the present embodiment, respectively.

As such, when the tube plate 200a and the column plate 300a having one tube 210a and the column 310a are used, the tube plate having the plurality of tubes 210 and the column 310 of the above-described embodiment ( Since the column 310a can be made larger than in the case of using the column 200 and the column plate 300, the yield of the desired biochemical substance can be increased with the same reason as described above, and a high concentration of the biochemical substance solution can be obtained. There are advantages to it.

Claims

A plurality of tube plates having one or more tubes formed therein;

A column plate detachably coupled to the tube plate and having one or more columns corresponding to the tubes of the tube plate penetrating therein while a filter is installed inside the column and a suction port formed to form a vacuum at one side thereof. Apparatus for extracting a biochemical material from a biological sample comprising a.
The method of claim 1,

Apparatus for extracting a biochemical material from a biological sample, characterized in that the cover slip of the flexible material for opening and closing the column is detachably coupled to the column of the column plate.
The method of claim 2,

The cover slip is a device for extracting a biochemical material from a biological sample, characterized in that the groove is formed to improve the adhesion to the column under the action of vacuum pressure.
The method of claim 1,

Apparatus for extracting a biochemical material from a biological sample, characterized in that the lower end of the column is installed in the tube when the column plate and the tube plate are combined.
The method according to claim 1 or 4,

The tube of the tube plate is formed with an incision to be discharged out of the tube when the solution discharged from the column is filled, wherein the incision does not face each other, apparatus for extracting biochemical material from biological samples.
The method of claim 1,

Apparatus for extracting a biochemical material from a biological sample, characterized in that the tube plate and column plate formed on one side of the coupling indicator corresponding to each other.
The method of claim 1,

Apparatus for extracting a biochemical material from a biological sample, characterized in that the outer surface of the tube plate protruding from the supporter for supporting the column plate coupled thereto.
The method of claim 1,

Apparatus for extracting a biochemical material from a biological sample, characterized in that the tube of the tube plate is formed in the inner bottom surface is gently curved.