KR20200099491A - Extracting apparatus, extracting method and fluid flow chip for extracting target substance - Google Patents

Abstract

The present invention relates to an apparatus, a method and a fluid flow chip for extracting a target material. In the apparatus according to the present invention, when an eluent solution and a magnetic solution pass through an extraction kit sequentially, while a target material is bound to the porous membrane in the extraction kit, the magnetic solution pushes out the eluent solution remaining in the porous membrane due to a difference in polarity, and then the eluent solution is collected in a collection chamber. In addition, the eluent solution and the magnetic solution are maintained in a layered state also in the collection chamber due to different polarities. While the magnetic solution in the collection chamber is physically separated from the eluent solution by the magnetic force applied from a magnetic force-applying unit, the eluent solution in the collection chamber can be recovered. Therefore, the eluent solution is collected in the collection chamber together with the magnetic solution, and then the eluent solution can be recovered, while the magnetic solution is physically separated from the eluent solution in the collection chamber by the magnetic force.

Description

Extraction device, extraction method and fluid flow chip for extracting target material {EXTRACTING APPARATUS, EXTRACTING METHOD AND FLUID FLOW CHIP FOR EXTRACTING TARGET SUBSTANCE}

The present invention relates to an extraction device, an extraction method, and a fluid flow chip for extracting a target material, and more particularly, to extract a target material such as nucleic acid without using a centrifugation method or applying high-speed centrifugation. It relates to a possible extraction device, extraction method and fluid flow chip.

In the medical field, efficient diagnosis and treatment methods are being actively developed to implement customized medicine, and the extraction and purification process of target substances such as nucleic acids is key in tests such as biotechnology, molecular biology, biochemistry, and diagnostic test medicine. It is a process of becoming.

In recent years, non-invasive liquid biopsy is attracting attention as a practical alternative to existing tissue biopsies as its high detection accuracy and potential as an early detection marker have been proven.CfDNA (cell free DNA), exosomes, and CTCs from blood or other body fluids The demand for separation and purification technologies such as (Circulating Tumor Cell) is increasing.

As an example of a typical target material pretreatment method for rapid and accurate diagnosis, the spin column method of nucleic acid extraction is a process of sequentially flowing a sample and buffers for nucleic acid extraction into a column including a porous membrane made of silica. Includes. As a result, the spin column method is a method in which nucleic acids are bound to a porous membrane, followed by washing, and then concentrated to a desired concentration and extracted. 1 is a diagram showing an example of a column disclosed in Korean Patent Registration No. 10-1495631, and FIG. 2 is a diagram illustrating a process of extracting a nucleic acid through a spin column method.

Referring to FIG. 2, when a sample containing nucleic acid is introduced into a column and then subjected to centrifugation in a centrifuge, the nucleic acid in the sample is bound to the porous membrane. Then, when the washing solution is injected into the column and centrifuged again through a centrifuge, impurities remaining in the porous membrane are removed. Here, the washing process may be performed multiple times or using various washing solutions depending on the type of nucleic acid or the body fluid from which the nucleic acid is extracted, or a subsequent nucleic acid processing process.

When the washing process is completed, a drying process is performed so that the washing liquid remaining in the porous membrane is completely removed, and even in this case, a centrifugal separation process is generally performed through a centrifuge.

When the drying process is completed, an elution buffer is injected into the column, and then centrifuged again through a centrifuge, so that the nucleic acid bound to the porous membrane can be extracted together with the elution solution.

As described above, in the case of nucleic acid extraction using the spin column method, centrifugation has to be performed in most of the processes. Recently, a method of using negative pressure in the binding process or washing process has been proposed, but the drying process or the elution process, especially the elution process Since 12,000G or more must be applied to the column during the process, it is difficult to replace this with negative pressure.

Such a centrifugal separation process makes it difficult to separate nucleic acids through a series of processes in a single chip because, in each process, a column must be passed through a centrifuge and then removed again in each process to extract nucleic acids.

In addition, in the field diagnosis in which a centrifuge cannot be disposed, it is difficult to apply the spin column method, and there is a problem of cross contamination in the process of moving a sample extracted from the field to a place where the centrifuge is provided.

In addition, since a series of processes must be manually performed by the user, the amount of nucleic acid extraction or the repetition accuracy of the purity is affected according to the user's skill level.

Accordingly, the present invention has been devised to solve the above problems, an extraction device capable of extracting a target material such as nucleic acid without using a centrifugation method or applying high-speed centrifugation, an extraction method, and a fluid Its purpose is to provide a floating chip.

The object is, according to the present invention, in an extraction device for extracting a target material, an extraction kit, a porous membrane installed inside the extraction kit to which the target material is bound, and passing through the porous membrane in the extraction kit And an elution solution that elutes the target material from the porous membrane, a magnetic solution that has a magnetic property and is not mixed with the elution solution because of a different polarity from the elution solution, and the elution solution and the magnetic that sequentially pass through the porous membrane A collection chamber in which services are collected in a stacked state, and a magnetic force application unit for applying a magnetic force to the collection chamber; When the magnetic solution passes through the porous membrane, the elution solution remaining in the porous membrane is pushed out by a different polarity from the elution solution; In a state in which the magnetic solution in the collection chamber is physically separated from the elution solution by the magnetic force of the magnetic force application unit, the elution solution in the collection chamber is recoverable by an extraction device for extracting a target material, characterized in that Is achieved.

Here, the magnetic force applying unit may apply a magnetic force to the side of the collection chamber so that the magnetic solution is aggregated on the wall surface inside the collection chamber to expose the eluted solution at the bottom.

In addition, the elution solution may be a polar solution, and the magnetic solution may be a non-polar solution.

In addition, when the target material is a nucleic acid, the elution solution may include polar distilled water or an elution buffer for nucleic acid extraction, and the magnetic solution may include non-polar mineral oil or silicone oil in which magnetic particles are dispersed.

In addition, a flow force applying unit for applying a flow force for passing the elution solution and the magnetic solution through the porous membrane to the extraction kit may be further included.

In addition, the extraction kit includes an inlet through which the elution solution and the magnetic solution are injected into the extraction kit, and an outlet through which the elution solution passing through the porous membrane is discharged; The flow force application unit may include a pressure pump that applies a positive pressure into the extraction kit through the inlet or provides a negative pressure into the extraction kit through the outlet.

On the other hand, the object is, in accordance with another embodiment of the present invention, in an extraction method for extracting a target material by an extraction device, providing an extraction kit having a porous membrane installed therein, and the target material in the porous membrane Binding, and eluting the target material while an elution solution for eluting the target material passes through the porous membrane, and a magnetic solution having a magnetic property and a polarity different from that of the elution solution passes through the porous membrane, The elution solution remaining in the sclera is pushed out, and the elution solution and the magnetic solution sequentially passing through the porous membrane are collected in a collection chamber, and in the collection chamber, the elution solution and the magnetic solution have different polarities. Stacking accordingly, and recovering the elution solution from the collection chamber; When recovering the elution solution, a target material characterized in that the elution solution in the collection chamber is recovered in a state where the magnetic solution is physically separated from the elution solution by applying a magnetic force from the outside of the collection chamber. It is also achieved by an extraction method for extraction.

Here, when the elution solution is recovered, a magnetic force is applied to a side surface of the collection chamber so that the magnetic solution aggregates on a wall surface inside the collection chamber to expose the elution solution upward; The elution solution exposed to the upper portion of the collection chamber may be recovered.

In addition, the elution solution may be a polar solution, and the magnetic solution may be a non-polar solution.

In addition, when the target material is a nucleic acid, the elution solution may include polar distilled water or an elution buffer for nucleic acid extraction, and the magnetic solution may include non-polar mineral oil or silicone oil in which magnetic particles are dispersed.

In addition, the extraction kit includes an inlet through which the elution solution and the magnetic solution are injected into the extraction kit, and an outlet through which the elution solution passing through the porous membrane is discharged; The flow force for flowing the elution solution and the magnetic solution may be applied as a positive pressure into the extraction kit through the inlet or may be applied as a negative pressure into the extraction kit through the outlet.

On the other hand, the object is, according to another embodiment of the present invention, in a fluid flow chip for extracting a target material, a chip body, and a sample formed in the chip body to store a sample solution containing the target material A chamber, an elution chamber formed inside the chip body to store an elution solution for elution of the target material, and an elution chamber formed inside the chip body to have magnetic properties and have a different polarity from the elution solution so that the elution solution does not mix. A magnetic chamber in which a magnetic solution is stored, an extraction chamber formed inside the chip body, a porous membrane installed inside the extraction chamber to which the target material is bound, and a porous film installed in the chip body, the sample chamber, and the elution A chamber selection valve selectively connecting one of the chamber and the magnetic chamber to an inlet of the extraction chamber, and a collection chamber formed in the chip body and connected to an outlet of the extraction chamber; The chamber selection valve sequentially connects the extraction chamber to the elution container and the magnetic container so that the elution solution and the magnetic solution in the elution chamber and the magnetic chamber flow by a flow force from the outside, The porous membrane is introduced through the inlet and sequentially passes through the porous membrane, and when the magnetic solution passes through the porous membrane, the elution solution remaining in the porous membrane is pushed out and collected into the collection chamber through the outlet, The elution solution and the magnetic solution are stacked according to different polarities in the collection chamber; When a magnetic force is applied to the collection chamber from the outside so that the magnetic solution is physically separated from the elution solution, the elution solution in the collection chamber may be recovered.

Here, the magnetic force applied from the outside to the collection chamber may be applied to the side of the collection chamber so that the magnetic solution aggregates on the wall surface inside the collection chamber to expose the elution solution at the bottom.

And, the chip body includes a lower body in which the sample chamber, the elution chamber, the magnetic chamber, the extraction chamber, and the collection chamber are formed, and an upper cover coupled to an upper portion of the lower body; A flow channel for connecting the sample chamber, the elution chamber, and the magnetic chamber to an inlet of the extraction chamber is formed in the lower body; The end of the flow channel and the inlet of the extraction chamber are opened to the upper part of the chip body, and the chamber selection valve coupled to the lower body through the upper cover is respectively provided in the sample chamber, the elution chamber, and the magnetic chamber. Any one of the ends of the connected flow channels may be selectively connected to the inlet of the extraction chamber.

And, at least one washing chamber formed in the lower body to receive a washing solution, a waste solution chamber formed in the lower body to store the sample solution and the washing solution passed through the extraction chamber, and the upper cover. And a discharge selection valve coupled to the lower body through the outlet and selectively connecting the outlet of the extraction chamber to one of the collection chamber and the waste solution chamber; The cleaning solution in the cleaning chamber may clean the porous membrane after the target material in the sample solution is bound to the porous membrane while passing through the extraction chamber of the sample solution.

In addition, the chip body further includes a negative pressure inlet connected to the collection chamber and the waste solution chamber, through which negative pressure from the outside for fluid flow is introduced; The negative pressure inlet may apply negative pressure for flow to the outlet of the collection chamber through the collection chamber or the waste solution chamber according to the selection of the discharge selection valve.

In addition, the elution solution may be a polar solution, and the magnetic solution may be a non-polar solution.

In addition, when the target material is a nucleic acid, the elution solution may include polar distilled water or an elution buffer for nucleic acid extraction, and the magnetic solution may include non-polar mineral oil or silicone oil in which magnetic particles are dispersed.

And, when the target material is a nucleic acid, the porous membrane is any one of a silica membrane, an ion exchange resin, a silica mesh, a packing tube packed with silica beads, and a membrane having a functional group capable of specific binding to the target material formed on the surface. Includes; When the target material includes any one of cells including CTC (Circulating Tumor Cell), extracellular vesicles including exosomes, and proteins, the porous membrane is an ion exchange resin, the target A functional group capable of specific binding to a substance may include any one of the films formed on the surface.

In addition, the target material may include any one of a nucleic acid including DNA and RNA, a cell including a circulating tumor cell (CTC), an extracellular vesicles including an exosome, and a protein. .

1 is a diagram showing an example of a column disclosed in Korean Patent Registration No. 10-1495631,
2 is a diagram showing a process of extracting a nucleic acid through a spin column method,
3 is a view showing the configuration of an extraction device for extracting a target material according to the present invention,
4 is a view for explaining the principle of operation of the extraction device for extracting a target material according to the present invention,
5 is a view for explaining an extraction method for extracting a target material according to the present invention,
6 is a view for explaining a process of recovering the elution solution from the elution solution and the magnetic solution collected in the collection chamber in the extraction device for extracting the target material according to the present invention,
7 is a perspective view of a fluid flow chip for extracting a target material according to the present invention,
8 is an exploded perspective view of the fluid flow chip shown in FIG. 7,
Figure 9 (a) is a plan view of the fluid flow chip shown in Figure 7,
FIG. 9B is a plan view of the lower body of the fluid flow chip shown in FIG. 8,
10 is a cross-sectional view taken along line XX in FIG. 8,
11 to 14 are views for explaining a process of extracting a target material using a fluid flow chip according to the present invention,
15 is a diagram for explaining an experiment result using a fluid flow chip according to the present invention,
16 is a diagram showing an experiment result of monitoring a mutant gene through blood analysis of a patient in an actual clinical trial using the fluid flow chip according to the present invention.

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

3 is a diagram showing the configuration of an extraction device 100 for extracting a target material according to the present invention. Referring to FIG. 3, the extraction apparatus 100 according to the present invention includes an extraction kit 110, a porous membrane 120, an elution solution 130, and a magnetic solution 140. In addition, the extraction apparatus 100 according to the present invention may include a collection chamber 200 (refer to FIG. 6) and a flow force application unit 150.

The extraction kit 110 has a cylindrical shape in which the inlet 111 and the outlet 112 are formed, and in the present invention, it is assumed that it has a cylindrical shape. Then, the elution solution 130 and the magnetic solution 140 are introduced through the inlet 111, and the eluted solution 130 and the magnetic solution 140 passed through the porous membrane 120 through the outlet 112 are discharged. do. In the present invention, it is assumed that the inner diameter of the outlet 112 is narrowed.

The porous membrane 120 is installed inside the extraction kit 110, and a target material (TS, see FIG. 4) is bound to the porous membrane 120. Here, the binding process of the target material TS may include a known method, for example, a binding step, a washing step, and a drying step, and binding through the extraction method according to the present invention or the fluid flow chip 300 to be described later. As possible, a detailed description thereof will be provided later.

Here, the target material (TS) to which the extraction device 100 according to the present invention is applied is a nucleic acid including DNA and RNA, a cell including a circulating tumor cell (CTC), and an extracellular vesicle including an exosome. (Extracellular vesicles), may contain any of the proteins.

In addition, when the target material (TS) is a nucleic acid, the porous membrane 120 includes a silica membrane, an ion exchange resin, a silica mesh, a packing tube packed with silica beads, and a functional group capable of specific binding to the target material (TS). It may include any one of the films formed on. In addition, when the target substance (TS) contains any one of cells including CTC (Circulating Tumor Cell), extracellular vesicles including exosomes, and proteins, the porous membrane 120 It may include any one of an ion exchange resin or a membrane in which a functional group capable of specific binding to the target material TS is formed on the surface. This can be applied to various types of membrane structures in which the target material TS is attached or specifically bound to the porous membrane 120.

The elution solution 130 is stacked on the porous membrane 120 in the extraction kit 110. And, when the elution solution 130 passes through the porous membrane 120, the target material (TS) bound to the porous membrane 120 is dissolved in the elution solution 130 or dispersed in the elution solution 130 to elute. It is possible to be separated from the porous membrane 120 together with the solution 130.

The magnetic solution 140 has a polarity different from that of the elution solution 130 and is maintained in a state not mixed with the elution solution 130 when injected into the extraction kit 110. In addition, the magnetic solution 140 is provided to have magnetism. For example, in the magnetic solution, magnetic particles are dispersed in a solution having a polarity different from that of the elution solution 130 so that the entire solution has a magnetic shape.

Here, the magnetic solution 140 is stacked on the elution solution 130 in the extraction kit 110, as shown in FIG. 3, that is, the porous membrane 120, the elution solution 130, and the magnetic solution Maintaining the form in which 140 is stacked, the elution solution 130 passes through the porous membrane 120 and then the magnetic solution 140 passes through the porous membrane 120.

In the present invention, it is assumed that the elution solution 130 is a polar solution such as distilled water, and the magnetic solution 140 is a non-polar solution such as oil. For example, when the target material TS is a nucleic acid, the elution solution 130 may include polar distilled water or an elution buffer for nucleic acid extraction. Here, the elution buffer for nucleic acid extraction is a commercial buffer solution applied to extract nucleic acids, for example, 10 mM Tris-Cl, 0.1 M EDTA, 20 μg/ml pancreatic RNase A, pH 8.0 mixed solution of 0.5% SDS is applied. I can. In this case, the magnetic solution 140 may be non-polar mineral oil or silicone oil in which magnetic particles are dispersed.

According to the configuration as described above, when the flow force for passing the elution solution 130 and the magnetic solution 140 through the porous membrane 120 is applied to the extraction kit 110 through the flow force application unit 150, Fig. As shown in (a) of 4, while the elution solution 130 first passes through the porous membrane 120, it flows to the opposite side of the porous membrane 120 together with the target material TS in the porous membrane 120.

However, in order to separate most of the target material TS from the porous membrane 120 while the elution solution 130 passes through the porous membrane 120, 12,000G was applied, but according to the present invention In the extraction device 100, while the magnetic solution 140 passes through the porous membrane 120 after the elution solution 130 passes, the elution remaining in the porous membrane 120 as shown in FIG. 4(b). By pushing the solution 130, the elution solution 130 and the target material TS are completely detachable from the porous membrane 120. Accordingly, even if a centrifugal separator is not used or a centrifugal force is used, extraction of the target material TS using the elution solution 130 is possible even at a pressure of 12,000 G or less.

In the present invention, the flow force applied by the flow force applying unit 150 may include pressure such as positive or negative pressure, centrifugal force, gravity, or inertial force. For example, the flow force application unit 150 applies a positive pressure into the extraction kit 110 through the inlet 111 of the extraction kit 110 or provides a negative pressure into the extraction kit 110 through the outlet 112 A pressure pump may be included, and a compressor, a syringe pump, a tube peristaltic pump, and the like may be applied as a configuration of the pressure pump.

In addition, in the case of centrifugal force, when compared with the prior art using centrifugal force, it is possible to easily separate even at a smaller rotational speed, that is, a pressure of 12,000 G or less, as described above.

When gravity is used as the flow force, the flow resistance of the porous membrane 120 to the eluted solution 103 is small, so that the remaining eluted solution can pass through the porous membrane 120 even with the force of gravity, but not all Even if 130 remains in the porous membrane 120, a target using the elution solution 130 by pushing the remaining elution solution 130 using a magnetic solution 140 that can penetrate into the porous membrane 120 only by gravity The extraction of the substance TS becomes possible.

In addition, when using the inertial force as the flow force, the inertial force generated by repeatedly moving and stopping in the direction of gravity in a state in which the elution solution 130 and the magnetic solution 140 are stacked on the porous membrane 120 is used. Extraction of the target material TS using the elution solution 130 is possible.

On the other hand, the elution solution 130 and the magnetic solution 140 sequentially passing through the porous membrane 120 in the extraction kit 110 are collected in the collection chamber 200 connected to the outlet 112 of the extraction kit 110. , As shown in FIG. 6, the elution solution 130 and the magnetic solution 140 are collected in a stacked state according to different polarities. Then, the process of recovering the elution solution 130 from the collection chamber by using the magnetism of the magnetic solution 140 is performed, and a detailed description thereof is provided with reference to FIGS. 5 and 6 as the target material TS according to the present invention. It will be described through the extraction method for extracting.

First, as shown in FIG. 3, an extraction kit 110 with a porous membrane 120 installed therein is prepared. Then, the target material TS is bound to the porous membrane 120 of the extraction kit 110. In more detail with reference to FIG. 5, a sample solution containing the target material TS is injected into the extraction kit 110 in which the porous membrane 120 is installed (S40).

Then, when a flow force for the flow of the sample solution is applied to the extraction kit 110, the target material TS in the sample solution is bound to the porous membrane 120 while the sample solution passes through the porous membrane 120 ( S41). Then, the washing solution is injected into the extraction kit 110 (S42), and when a flow force is applied, the washing solution passes through the porous membrane 120 to clean the interior of the porous membrane 120 (S43). In addition, when the drying process is performed after completion of the washing step, binding of only the target material TS is completed in the porous membrane 120.

Then, the elution solution 130 and the magnetic solution 140 are injected into the extraction kit 110 (S45, S46). In FIG. 5, the injection of the magnetic solution 140 after injection of the elution solution 130 is illustrated as an example, but when the elution solution 130 is distilled water of polarity and the magnetic solution 140 is non-polar oil, the injection sequence Regardless, as shown in FIG. 3, an elution solution 130 and a magnetic solution 140 are sequentially stacked on the porous membrane 120.

After the injection of the elution solution 130 and the magnetic solution 140 is completed, when a flow force is applied to the extraction kit 110 (S47), as described above, the elution solution 130 first becomes the porous membrane 120 After passing through, the remaining target material (TS) together with the elution solution 130 is pushed out of the porous membrane 120 so that the remaining target material TS is completely removed with the elution solution 130. The extraction becomes possible. Here, the method of applying the flow force to the extraction kit 110 is as described above, and a detailed description thereof will be omitted.

And, when part or all of the magnetic solution 140 passes through the porous membrane 120, the elution solution 130 and the magnetic solution 140 are, as shown in Fig. 6A, the collection chamber 200 It is collected in (S48). In this case, in the collection chamber 200, the elution solution 130 and the magnetic solution 140 are collected in a stacked state, as shown in FIG. 6A according to different polarities.

In the state as shown in (a) of FIG. 6, in the case of recovering the lower elution solution 130 using the pipette tip 170, a part of the magnetic solution 140 is recovered together in the recovery process. Purity may be reduced, and if the recovery is stopped before the magnetic solution 140 is recovered to improve the purity, there is a problem in that the recovery efficiency of the target material TS is reduced.

Thus, in the present invention, as shown in (b) of FIG. 6, the magnetic force application unit 160 applies a magnetic force from the outside of the collection chamber 200 (S49) to dissolve the magnetic solution 140 into the elution solution 130 The eluted solution 130 is recovered in a state physically separated from (S50). In the present invention, the magnetic force applying unit 160 has a permanent magnet shape as an example, but it is also possible to use an electromagnet.

In addition, in (b) of FIG. 6, it is an example that the magnetic force application unit 160 applies a magnetic force to the side of the collection chamber 200 so that the magnetic solution 140 is aggregated to the side of the collection chamber 200. As shown in (c) of FIG. 6, the pipette tip 170 is immediately immersed in the elution solution 130 without passing through the magnetic solution 140, as shown in (d) of FIG. 6, The elution solution 130 can be recovered. Then, after the elution solution 130 is recovered, the target material TS is extracted from the elution solution 130.

In the above-described embodiment, the elution solution 130 and the magnetic solution 140 are injected into the extraction kit 110 and the elution solution 130 and the magnetic solution 140 are not mixed and are stacked on the porous membrane 120 It is an example that the flow force is applied to the furnace. In addition, without the lamination process of the elution solution 130 and the magnetic solution 140 in the extraction kit 110, the elution solution 130 first flows into the extraction kit 110 to pass through the porous membrane 120 and elute. It goes without saying that the solution 130 may be first collected in the collection chamber 200 and then collected in the collection chamber 200 by passing the magnetic solution 140 through the porous membrane 120 in the same manner.

Hereinafter, a fluid flow chip 300 for extracting a target material according to the present invention will be described in detail with reference to FIGS. 7 to 14. The fluid flow chip 300 according to the present invention is an example in which the above-described extraction device is implemented in a chip form, and the extraction kit 110 is implemented as an extraction chamber 350 in the fluid flow chip 300. In addition, the flow force applying unit 150 in the above-described embodiment will be described as an example consisting of the negative pressure pump 151 and the pressure gauge 152 shown in Figs. 11 to 14, the porous membrane 120 Reference numbers are used identically.

7 to 10, the fluid flow chip 300 according to the present invention includes a chip body 310, a sample chamber 321, an elution chamber 323, a magnetic chamber 324, and an extraction chamber 350. ), a porous membrane 120, a chamber selection valve 330, and a collection chamber 325.

The chip body 310 is made of a transparent or translucent plastic material, for example. In the present invention, as shown in FIG. 8, it is assumed to include a lower body 311 and an upper cover 312. A sample chamber 321, an elution chamber 323, a magnetic chamber 324, an extraction chamber 350, and a collection chamber 325 are formed in the lower body 311, and a flow channel 371 for connecting these chambers Are formed.

The upper cover 312 is coupled to the upper portion of the lower body 311, blocks all or part of the above-described chambers, has a structure in which a stopper for opening and closing each chamber is coupled, and a chamber selection valve 330 and will be described later. The discharge selection valve 340 is coupled to the lower body 311 through the upper cover 312.

The sample chamber 321 is formed inside the lower body 311 of the chip body 310, and a sample solution containing a target material is stored. In addition, the elution chamber 323 and the magnetic chamber 324 are similarly formed in the lower body 311 of the chip body 310 to store the elution solution and the magnetic solution, respectively. Here, the elution solution and the magnetic solution correspond to the above-described extraction device 100, and detailed descriptions thereof will be omitted.

The extraction chamber 350 is formed inside the lower chamber of the chip body 310 and corresponds to the extraction kit 110 of the extraction apparatus 100 described above. That is, inside the extraction chamber 350, as shown in FIGS. 7 and 10, a porous membrane 120 to which a target material is bound is installed. In addition, the collection chamber 325 is formed inside the lower body 311 of the chip body 310 and is connected to the outlet 352 (not shown) of the extraction chamber 350.

On the other hand, the chamber selection valve 330 is installed in the chip body 310, and one of the sample chamber 321, the elution chamber 323, and the magnetic chamber 324 is connected to the inlet 351 of the extraction chamber 350. Connect selectively. In the present invention, it is assumed that the chamber selection valve 330 is coupled to the lower body 311 through the upper cover 312, as shown in FIG. 8.

Referring to FIGS. 8 and 9B, the sample chamber 321, the elution chamber 323, and the magnetic chamber 324 are respectively provided in the lower body 311 in the inlet 351 of the extraction chamber 350. A flow channel 371 for connecting with is formed. At this time, the end of the flow channel 371 has an open state to the upper surface of the lower body 311, as shown in (b) of FIG. 9, similarly, the inlet 351 of the extraction chamber 350 ) It is also in a state that is open to the upper portion of the lower body 311. In this case, the chamber selection valve 330 is coupled to the upper surface of the lower body 311 through the upper cover 312 and is connected to the sample chamber 321, the elution chamber 323, and the magnetic chamber 324, respectively. Any one of the ends of 371 is selectively connected to the inlet 351 of the extraction chamber 350.

In the present invention, as shown in FIG. 8, the chamber selection valve 330 includes a gasket 311 made of silicon, a chamber switching valve 332, and a first fixing jig 333 for fixing them. do. Here, in the gasket 311, communication holes communicating with the end of each flow channel 371 and the inlet 351 of the extraction chamber 350 are formed, and the extraction chamber 350 according to the rotation of the chamber switching valve 332 Any one of the inlet 351 of the flow channel 371 is in communication with each other.

Meanwhile, the fluid flow chip 300 according to the present invention may include at least one washing chamber 322a, 322b, and 322c, a waste solution chamber 326, and a discharge selection valve 340. The washing chambers 322a, 322b, and 322c are formed in the lower body 311 to accommodate the washing solution. In the present invention, it is assumed that three washing chambers 322a, 322b, and 322c are formed. And, each washing chamber (322a, 322b, 322c) is accommodated in each washing solution having a different concentration of ethanol, the three washing chambers (322a, 322b, 322c) sequentially in the order of the washing solution having a low concentration of ethanol. For example, it is provided to clean the porous membrane 120.

The waste solution chamber 326 is formed in the lower body 311 and passed through the extraction chamber 350, and waste solutions such as a sample solution and a washing solution are stored. Here, the discharge selection valve 340 is coupled to the lower body 311 through the upper cover 312, and the discharge port 352 of the extraction chamber 350 is provided with any one of the collection chamber 325 and the waste solution chamber 326. Selectively connect with one. As shown in FIG. 8, it is assumed that the discharge selection valve 340 includes a discharge switching valve 342 and a second fixing jig 343.

In addition, a negative pressure inlet 360 connected to the collection chamber 325 and the waste solution chamber 326 is formed in the chip body 310. A negative pressure pump 151 is connected to the negative pressure inlet to apply negative pressure for fluid flow. That is, the negative pressure inlet 360 has a negative pressure for the flow of the fluid to the outlet 352 of the collection chamber 325 through the collection chamber 325 or the waste solution chamber 326 according to the selection of the discharge selection valve 340. It is approved, a detailed description thereof will be described later.

According to the above configuration, a process of extracting the target material using the fluid flow chip 300 according to the present invention will be described in detail with reference to FIGS. 11 to 14.

First, as shown in FIG. 11, in a state in which the chamber selection valve 330 connects the extraction chamber 350 to the sample chamber 321, the sample solution accommodated in the sample chamber 321 is transferred to the extraction chamber 350. The binding step that is injected is performed.

In the binding step, the discharge selection valve 340 is switched so that the negative pressure pump 151 is connected through the discharge port 352 side of the extraction chamber 350 and the waste solution chamber 326 as shown in FIG. 11. . Through this, the sample solution in the sample chamber 321 is bound into the extraction chamber 350 through the chamber selection valve 330 by the negative pressure applied to the outlet 352 of the extraction chamber 350. Here, the lines indicated in bold color in FIGS. 11 to 14 indicate lines connected by the discharge selection valve 340 and the chamber selection valve 330.

Then, as shown in FIG. 12, in a state in which the chamber selection valve 330 connects the extraction chamber 350 to the washing chambers 322a, 322b, and 322c, it is accommodated in the washing chambers 322a, 322b, and 322c. A washing step in which the washing solution is injected into the extraction chamber 350 is performed.

In the washing step, the discharge selection valve 340 is switched so that the negative pressure pump 151 is connected through the discharge port 352 side of the extraction chamber 350 and the waste solution chamber 326 as shown in FIG. 12. . Through this, after the cleaning solution in the cleaning chambers 322a, 322b, 322c is injected into the extraction chamber 350 through the chamber selection valve 330 by the negative pressure applied to the outlet 352 of the extraction chamber 350 , While passing through the porous membrane 120, a washing process is performed, and the cleaning solution that has passed through the porous membrane 120 is discharged to the waste solution chamber 326 through the discharge port 352 and the discharge selection valve 340.

11 to 14 show examples in which one washing chamber 322a, 322b, and 322c is applied, but as described above, three washing solutions sequentially flow from the three washing chambers 322a, 322b, and 322c. As a matter of course, it may be provided to clean the porous membrane 120.

And, as described above, after the washing step is performed, the drying step may be performed, and after the drying step is completed, the chamber selection valve 330 is switched to connect the extraction chamber 350 and the elution chamber 323 to the elution chamber. The eluted solution in the 323 flows into the extraction chamber 350.

At this time, as shown in FIG. 13, the discharge selection valve 340 is switched so that the negative pressure pump 151 is connected through the outlet 352 side of the extraction chamber 350 and the collection chamber 325. Through this, the elution solution in the elution chamber 323 is injected into the extraction chamber 350 through the chamber selection valve 330 by the negative pressure applied to the outlet 352 of the extraction chamber 350, and the porous membrane 120 After passing through, it is collected into the collection chamber 325.

Then, the chamber selection valve 330 is switched so that the extraction chamber 350 and the magnetic chamber 324 are connected, as shown in FIG. 14, so that the magnetic solution in the magnetic chamber 324 is inside the extraction chamber 350. It is injected into the porous membrane 120 and the remaining elution solution is pushed out and collected into the collection chamber 325 together with the elution solution.

In this case, as shown in FIG. 14, the discharge selection valve 340 is switched so that the negative pressure pump 151 is connected through the outlet 352 side of the extraction chamber 350 and the collection chamber 325. Through this, the magnetic solution in the magnetic chamber 324 flows into the extraction chamber 350 through the chamber selection valve 330 due to the negative pressure applied to the outlet 352 of the extraction chamber 350, and the porous membrane 120 After passing through, it is collected in the collection chamber 325.

Then, as shown in Figure 6, by applying a magnetic force from the outside of the collection chamber 325 to physically separate the magnetic solution from the elution solution, and then through a process of recovering the elution solution, the target material from the elution solution The extraction of is possible.

15 is a diagram for explaining an experiment result using a fluid flow chip according to the present invention. Referring to FIG. 15, in the experiment, a cfDNA standard sample was used to test whether a very small proportion of mutant genes could be extracted. For the comparative experiment, a commercially available centrifugation method, QIA amp, was used. And, the cfDNA standard sample used in the experiment was a Horizon product. The sample volume was 500 μl, and an experiment was conducted to extract ctDNA of PIK3CA E545K, EGFR L858R, KRAS G12D, and NRAS Q61K from a total of 4 sample sets (0%, 0.1%, 1%, 5%). In FIG. 15, PIBEX is an experimental result of the fluid flow chip according to the present invention.

As shown in Figure 15, it can be confirmed that the amount of ctDNA extraction is almost the same as the QIA amp to which the conventional centrifugation method is applied, and it has been confirmed that ctDNA extraction is possible despite the very small ratio of 0.1% to 6% mutation. .

16 is a diagram showing an experiment result of monitoring a mutant gene through blood analysis of a patient in an actual clinical trial using the fluid flow chip according to the present invention. PIK3CA H1047R mutation was monitored using 500 μl of plasma from round bottoms of HER-2 breast cancer.

As cancer metastasis to the liver progresses, as shown in FIG. 16B, it can be seen that the mutant gene in the blood increases. The tumor metastasized to the liver can be confirmed through an MRI image as shown in FIG. 16A.

Although some embodiments of the present invention have been shown and described, those skilled in the art of ordinary skill in the art to which the present invention pertains will appreciate that the present embodiments can be modified without departing from the principles or spirit of the present invention. . The scope of the invention will be determined by the appended claims and their equivalents.

100: extraction device 110: extraction kit
111: inlet 112: outlet
120: porous membrane 130: elution solution
140: magnetic solution 150: flow force application unit
151: negative pressure pump 152: pressure gauge
300: fluid flow chip 310: chip body
311: lower body 312: upper cover
321: sample chamber 322a, 322b, 322c: washing chamber
323: elution chamber 324: magnetic chamber
325: collection chamber 326: waste solution chamber
330: chamber selection valve 331: gasket
332: chamber switching valve 333: first fixing jig
340: discharge selection valve 342: discharge switching valve
343: second fixing jig 350: extraction chamber
351: inlet 352: outlet
360: negative pressure inlet 371: flow channel

Claims (18)

In the extraction device for extracting the target material,
With an extraction kit,
A porous membrane installed inside the extraction kit to which the target material is bound,
An elution solution for eluting the target material from the porous membrane while passing through the porous membrane in the extraction kit,
A bipolar solution that has a different polarity from the elution solution and is not mixed with the elution solution,
A collection chamber in which the elution solution and the bipolar solution sequentially passing through the porous membrane are collected in a stacked state;
An extraction device for extracting a target material, characterized in that when the dipolar solution passes through the porous membrane, the elution solution remaining in the porous membrane is pushed out by a different polarity from the elution solution. The method of claim 1,
The extraction device for extracting the target material, characterized in that the elution solution is a polar solution, and the bipolar solution is a non-polar solution. The method of claim 2,
When the target material is a nucleic acid, the elution solution includes polar distilled water or an elution buffer for nucleic acid extraction, and the bipolar solution includes non-polar mineral oil or silicone oil. Extraction device. The method of claim 1,
An extraction apparatus for extracting a target material, further comprising a flow force application unit for applying a flow force for passing the elution solution and the bipolar solution through the porous membrane to the extraction kit. The method of claim 4,
The extraction kit includes an inlet through which the elution solution and the bipolar solution are injected into the extraction kit,
And an outlet through which the elution solution that has passed through the porous membrane is discharged;
The flow force application unit includes a pressure pump for applying a positive pressure into the extraction kit through the inlet or providing a negative pressure into the extraction kit through the outlet. In the extraction method for the extraction device to extract the target material,
(a) preparing an extraction kit with a porous membrane installed therein,
(b) binding the target material to the porous membrane,
(c) eluting the target material while an elution solution for eluting the target material passes through the porous membrane,
(d) pushing the elution solution remaining in the porous membrane while a dipolar solution having a polarity different from that of the elution solution passes through the porous membrane,
(e) an extraction method for extracting a target material comprising the step of collecting the elution solution and the bipolar solution sequentially passing through the porous membrane in a collection chamber. The method of claim 6,
Step (b)
(b1) passing the sample containing the target material through the porous membrane to bind the target material to the porous membrane;
(b2) injecting a cleaning solution into the sample kit;
(b3) an extraction method for extracting a target material comprising the step of passing the washing solution through the porous membrane to wash the porous membrane. The method of claim 7,
The extraction device for extracting the target material, characterized in that the elution solution is a polar solution, and the bipolar solution is a non-polar solution. The method of claim 8,
When the target material is a nucleic acid, the elution solution includes polar distilled water or an elution buffer for nucleic acid extraction, and the bipolar solution includes non-polar mineral oil or silicone oil. Extraction method. The method of claim 6,
The extraction kit includes an inlet through which the elution solution and the bipolar solution are injected into the extraction kit, and an outlet through which the elution solution passing through the porous membrane is discharged;
The flow force for flowing the elution solution and the bipolar solution is applied at a positive pressure into the extraction kit through the inlet, or a negative pressure is applied into the extraction kit through the outlet. Extraction method for doing. In the fluid flow chip for extracting the target material,
The chip body,
A sample chamber formed inside the chip body to store a sample solution containing the target material,
An elution chamber formed inside the chip body to store an elution solution for elution of the target material,
A bipolar chamber in which a bipolar solution that is formed inside the chip body and has a different polarity from the elution solution and is not mixed with the elution solution is stored,
An extraction chamber formed inside the chip body,
A porous membrane installed inside the extraction chamber to which the target material is bound,
A chamber selection valve installed in the chip body and selectively connecting any one of the sample chamber, the elution chamber, and the bipolar chamber to an inlet of the extraction chamber,
A collection chamber formed inside the chip body and connected to an outlet of the extraction chamber;
The chamber selection valve sequentially connects the extraction chamber to the elution container and the bipolar container, so that the elution solution and the bipolar solution in the elution chamber and the bipolar chamber flow by a flow force from the outside. The porous membrane is introduced through the inlet of the extraction chamber to sequentially pass through the porous membrane, and when the bipolar solution passes through the porous membrane, the elution solution remaining in the porous membrane is pushed out and the collection chamber through the outlet. Fluid flow chip for extracting the target material, characterized in that collected as. The method of claim 11,
The chip body is
A lower body in which the sample chamber, the elution chamber, the bipolar chamber, the extraction chamber, and the collection chamber are formed,
And an upper cover coupled to an upper portion of the lower body;
A flow channel for connecting the sample chamber, the elution chamber, and the bipolar chamber to an inlet of the extraction chamber is formed in the lower body;
The end of the flow channel and the inlet of the extraction chamber are opened to an upper portion of the chip body, and the chamber selection valve coupled to the lower body through the upper cover is provided in the sample chamber, the elution chamber, and the bipolar chamber. A fluid flow chip for extracting a target material, characterized in that one of the ends of each of the connected flow channels is selectively connected to the inlet of the extraction chamber. The method of claim 12,
At least one cleaning chamber formed in the lower body to accommodate a cleaning solution,
A waste solution chamber formed in the lower body to store the sample solution and the washing solution that have passed through the extraction chamber,
A discharge selection valve coupled to the lower body through the upper cover and selectively connecting the outlet of the extraction chamber to one of the collection chamber and the waste solution chamber;
Fluid flow for extracting a target material, characterized in that the cleaning solution in the washing chamber cleans the porous membrane after the target material in the sample solution is bound to the porous membrane while passing through the extraction chamber of the sample solution chip. The method of claim 13,
The chip body further includes a negative pressure inlet connected to the collection chamber and the waste solution chamber, through which negative pressure from the outside for fluid flow is introduced;
The negative pressure inlet is a fluid flow chip for extracting a target material, characterized in that for applying a negative pressure for flow to the outlet of the collection chamber through the collection chamber or the waste solution chamber according to the selection of the discharge selection valve. The method of claim 11,
The elution solution is a polar solution, the bipolar solution is a fluid flow chip for extracting a target material, characterized in that the non-polar solution. The method of claim 15,
When the target material is a nucleic acid, the elution solution includes polar distilled water or an elution buffer for nucleic acid extraction, and the bipolar solution includes non-polar mineral oil or silicone oil. Fluid flow chip. The method of claim 11,
When the target material is a nucleic acid, the porous membrane includes any one of a silica membrane, an ion exchange resin, a silica mesh, a packing tube packed with silica beads, and a membrane having a functional group capable of specific binding to the target material formed on the surface. And;
When the target material contains any one of cells including CTC (Circulating Tumor Cell), extracellular vesicles including exosome, and protein, the porous membrane is an ion exchange resin, the target A fluid flow chip for extracting a target material, comprising any one of a film formed on a surface of a functional group capable of specific binding to a material. The method of claim 11,
The target material is characterized in that it contains any one of nucleic acids including DNA and RNA, cells including circulating tumor cells (CTC), extracellular vesicles including exosomes, and proteins. A fluid flow chip for extracting the target material.

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