KR102041217B1 - Multi-channel device for downwardly injecting liquid sample, device for extracting nucleic acid comprising the same, and method for extracting nucleic acid using the same - Google Patents

Multi-channel device for downwardly injecting liquid sample, device for extracting nucleic acid comprising the same, and method for extracting nucleic acid using the same Download PDF

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KR102041217B1
KR102041217B1 KR1020130060996A KR20130060996A KR102041217B1 KR 102041217 B1 KR102041217 B1 KR 102041217B1 KR 1020130060996 A KR1020130060996 A KR 1020130060996A KR 20130060996 A KR20130060996 A KR 20130060996A KR 102041217 B1 KR102041217 B1 KR 102041217B1
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South Korea
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nucleic acid
channel
microfluidic chip
inlet
liquid
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KR1020130060996A
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Korean (ko)
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KR20130135112A (en
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김성우
김덕중
김선진
이세현
류호선
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주식회사 미코바이오메드
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons

Abstract

An embodiment of the present invention relates to a multi-channel downward liquid injection device, a nucleic acid extraction device comprising the same, and a nucleic acid extraction method using the same, and accordingly, in performing various biological reactions using a microfluidic chip having a thin film shape, It is possible to rapidly inject a very small amount of the same or different liquid into one or more inlets, and to accurately dispense the amount of liquid injected into the one or more inlets with only one user operation, and the one or more inlets It is expected that the user's convenience may be considerably improved by implementing a relatively larger liquid inlet, and the nucleic acid extraction reaction time may be considerably shortened, so that various biological analysis reactions may be rapidly progressed.

Description

Multi-channel device for downwardly injecting liquid sample, device for extracting nucleic acid comprising the same, and method for extracting nucleic acid using the same }

The present invention relates to a liquid injection device for injecting a biological sample or a reagent and a liquid downward to the thin-film microfluidic chip having a liquid inlet, a nucleic acid extracting apparatus comprising the same, and a nucleic acid extraction method using the same.

Recently, a technique for extracting nucleic acids from biological samples such as cells, bacteria, or viruses to diagnose, treat, or prevent diseases at the genetic level has been widely used in connection with nucleic acid amplification reaction technology. In addition to the diagnosis, treatment, or prevention of diseases, there is a need for a technology for extracting nucleic acids from biological samples in various fields such as development of customized new drugs, forensic medicine, and detection of environmental hormones. As an example of the conventional nucleic acid extraction technology, there is a method of purifying nucleic acid by denatured protein with phenol after solubilizing a sample including cells by treatment with SDS or proteinase K. However, the phenol extraction method is not only time-consuming because many processing steps have to be performed, but also has a problem in that the nucleic acid extraction efficiency is highly dependent on the researcher's experience and experience, and thus the reliability is greatly reduced. Recently, in order to solve this problem, kits using silica or glass fibers that specifically bind to nucleic acids have been used. The silica or glass fiber has a low binding ratio with proteins and cellular metabolites, so that nucleic acids having a relatively high concentration can be obtained. This method has the advantage of being simpler than the phenol method. However, the use of chaotropic reagents or ethanol, which strongly inhibit enzymatic reactions such as polymerase chain reaction (PCR), requires the complete removal of these substances. This is very cumbersome and time consuming. Recently, a method of directly purifying nucleic acid using a filter has been disclosed in International Patent Publication No. 00/21973, which passes a sample through a filter to adsorb the cells to the filter, and then dissolves the cells adsorbed on the filter. After filtration, the nucleic acid adsorbed on the filter is washed and eluted. However, in order to elute the nucleic acid after adsorbing the cell to the filter, there is a problem in that the filter must be selected according to the type of the cell, and the devices used are large and complex, so that the researcher cannot easily use the filter.

In addition, a device for injecting a very small amount of liquid such as a sample or a reagent into the reaction vessel is essential when performing various biological reactions. Conventional reaction vessels are mostly tube-shaped, and moreover, multi-tubes and the like, which are equipped with a plurality of tubes having a very small volume, are also used. Thus, devices commonly used for injecting, mixing, or dispensing liquids, such as trace amounts of samples or reagents, into such reaction vessels are pipettes and tips. However, the pipette and tip are unsatisfactory in controlling the amount of liquid injected into the reaction vessel by the user's hand operation, and are particularly useful when injecting liquid into one or more inlets of a small size of a thin microfluidic chip. It is very cumbersome, requiring a tip with a fine outlet and a pipette designed accordingly. Therefore, in a biological reaction using a thin-film microfluidic chip, it is possible to rapidly and accurately inject a small amount of sample or reagent into one or more inlets of a small size, and improve the user convenience and perform the reaction quickly. There is a need for a liquid injection device.

One embodiment of the present invention is a multi-channel downward liquid injection, capable of quickly and accurately injecting a small amount of liquid, such as a biological sample or a reagent, into one or more inlets of a thin-film microfluidic chip, and improving user convenience. An apparatus, a nucleic acid extracting apparatus comprising the same, and a nucleic acid extracting method using the same are provided.

A first embodiment of the present invention is a thin-film microfluidic chip having at least one reaction channel having an inlet and an outlet at both ends thereof, wherein the at least one inlet is connected to the at least one reaction channel. One or more downward liquid outlets for respectively injecting a liquid such as a biological sample or a reagent downwardly, one or more downward through holes corresponding to the upper end of one or more inlet portions of the microfluidic chip, and one or more vertical through holes penetrating upward and downward from the one or more downward liquid outlets. A body having a channel and at least one liquid inlet connected to the interior of the at least one vertical through channel; And at least one vertical pressurizing module, each inserted into the at least one vertical through-hole channel to move the inside thereof in a vertical direction, the lower distal end being in close contact with the inner surface of the at least one vertical through-hole channel, and the at least one vertical. And a cover having a press plate integrally connected with the upper end of the pressurizing module to provide simultaneous up and down movement of the at least one vertical pressurizing module by user manipulation, wherein the liquid such as the biological sample or reagent is pressurized with the at least one vertical pressurization. After the lower end of the module is introduced through the at least one liquid inlet that is open upon upward movement, the lower end of the at least one vertical pressurizing module moves through the at least one vertical through channel when the downward movement is directed to the at least one downward liquid outlet. Being injected into at least one inlet of the microfluidic chip Provides a channel down the liquid injection device, and that the gong.

In the multi-channel downward liquid injection device according to the first embodiment of the present invention,

Implemented at the bottom of one or more downward liquid outlet of the main body, it may include a chip inlet region end mounting portion implemented to be fixedly mounted at least one inlet region end of the microfluidic chip.

In addition, the main body and the cover may include a guide means for supporting the vertical movement of the cover in the engaged state.

In addition, the main body and the cover may include a detachable means implemented to be separated from each other.

In addition, the one or more liquid inlets may be connected to a portion of each inner surface of the one or more vertically through channels.

In addition, the one or more vertical through-channels may be disposed in a horizontal cross-sectional center region of the body, and the one or more liquid inlets may be disposed in an edge region adjacent to the one or more vertical through-channels.

In addition, the vertical through channel may be implemented in three or more, but may be implemented in a zigzag form based on the horizontal cross-section of the main body.

A second embodiment of the present invention is for extracting a nucleic acid from a biological sample, comprising an inlet, a channel region connected to the inlet, and an outlet connected to the channel region, wherein the channel region is introduced through the inlet. A microfluidic chip for extracting nucleic acids having a thin film shape having one or more reaction channels, including a heating unit configured to transfer heat obtained from the outside to the biological sample; A multi-channel liquid dispensing apparatus according to a first embodiment of the present invention, having a liquid outlet corresponding to the number of said at least one inlet; And fluid delivery means for fluidly connecting the one or more inlets of the microfluidic chip and the one or more liquid outlets of the multi-channel liquid dispensing device.

In the nucleic acid extracting apparatus according to the second embodiment of the present invention,

A chip outlet region end mounting portion configured to be fixedly mounted to at least one outlet region end of the microfluidic chip, at least one upward liquid inlet corresponding to an upper end of at least one outlet portion of the microfluidic chip, and at least one It may further comprise a liquid storage container having at least one liquid storage chamber in fluid communication with the upward liquid inlet.

The microfluidic chip may include a first filter disposed in a first channel region connected to the inlet, and disposed in a second channel region connected to the heating unit and capable of passing a material having a size corresponding to a nucleic acid. Can be.

The microfluidic chip has a heating unit disposed in a first channel region connected to the inlet, and disposed in a second channel region connected to the heating unit, and having a first filter through which a material having a size corresponding to a nucleic acid can pass therethrough. And a nucleic acid separation unit disposed in a third channel region connected to the first filter and having a nucleic acid binding material capable of specifically binding to the nucleic acid.

The microfluidic chip has a heating unit disposed in a first channel region connected to the inlet, and disposed in a second channel region connected to the heating unit, and having a first filter through which a material having a size corresponding to a nucleic acid can pass therethrough. And a nucleic acid separation unit disposed in a third channel region connected to the first filter and having a nucleic acid binding material capable of specifically binding to the nucleic acid, and disposed in a fourth channel region connected to the nucleic acid separation unit. It may be provided with a second filter capable of passing a substance of a size corresponding to the nucleic acid.

The microfluidic chip may include a nucleic acid separation unit including a heating unit disposed in a channel region connected to the inlet unit and disposed in a channel region connected to the heating unit and provided with a nucleic acid binding material capable of specifically binding to the nucleic acid. Can be.

The microfluidic chip is provided with a nucleic acid separation unit which is disposed in the channel region connected to the inlet, the nucleic acid binding material is disposed in the channel region connected to the heating portion and is provided with a nucleic acid binding material that can specifically bind to the nucleic acid. And a second filter disposed in a channel region connected to the nucleic acid separation unit and capable of passing a material having a size corresponding to the nucleic acid.

According to a third embodiment of the present invention, there is provided a nucleic acid extracting apparatus according to a second embodiment of the present invention; Injecting a biological sample or reagent into the microfluidic chip for nucleic acid extraction through the multi-channel downward liquid injection device; And extracting nucleic acids from the biological sample by driving the nucleic acid extracting microfluidic chip.

A fourth embodiment of the present invention includes the steps of providing a nucleic acid extracting apparatus according to a second embodiment of the present invention; Injecting a biological sample or reagent into the microfluidic chip for nucleic acid extraction through the multi-channel downward liquid injection device; Extracting nucleic acids from the biological sample by driving the nucleic acid extracting microfluidic chip; And storing the nucleic acid extraction product in a liquid storage chamber of the liquid storage container.

An embodiment of the present invention relates to a multi-channel downward liquid injection device, a nucleic acid extraction device comprising the same, and a nucleic acid extraction method using the same, and accordingly, in performing various biological reactions using a microfluidic chip having a thin film shape, It is possible to rapidly inject the same or different trace amounts of liquid into a significantly smaller one or more inlets, and to accurately dispense a very small amount of liquid into the one or more inlets with only one user operation, the one or more inlets It is expected that a relatively large liquid inlet can be implemented, which greatly improves user convenience during liquid injection, and further, can significantly shorten the nucleic acid extraction reaction time to rapidly proceed with a variety of biological detection or analytical reactions.

1 to 2 schematically show a microfluidic chip according to one embodiment of the invention.
3 through 6 illustrate a multi-channel downward liquid injection device according to one embodiment of the invention.
Figure 7 illustrates a guide means for supporting the vertical movement of the cover of the multi-channel downward liquid injection device according to an embodiment of the present invention.
FIG. 8 shows detachable means allowing the body and cover of the multi-channel downward liquid injection device according to one embodiment of the invention to be detachable.
Figure 9 illustrates a vertically channeled and liquid inlet of a multi-channel downward liquid injection device according to one embodiment of the invention.
10-11 illustrate the paths of movement of liquids, such as biological samples or reagents, in a multi-channel downward liquid injection device according to one embodiment of the invention.
12 to 13 show the arrangement of the vertically penetrating channel and the liquid inlet in the cross section of the body of the multi-channel downward liquid injection device according to one embodiment of the invention.
14 to 17 illustrate a microfluidic chip according to an embodiment of the present invention in detail and illustrate a nucleic acid extraction method using the same.
18-20 illustrate a liquid storage container in accordance with one embodiment of the present invention.
FIG. 21 illustrates a flow path of a liquid such as a biological sample or a reagent in a state in which a microfluidic chip and a liquid storage container are combined according to an embodiment of the present invention.
FIG. 22 illustrates a flow path of a liquid, such as a biological sample or reagent, in combination with a multi-channel downward liquid injection device, microfluidic chip, and liquid storage container in accordance with one embodiment of the present invention.
23 to 26 show the results of nucleic acid extraction experiments using the nucleic acid extracting apparatus and the nucleic acid extracting apparatus according to an embodiment of the present invention, respectively.

Hereinafter, with reference to the accompanying drawings will be described in detail an embodiment of the present invention. The description set forth below is only for easily understanding the embodiments of the present invention, and is not intended to limit the protection scope of the present invention from such description.

1 to 2 schematically show a microfluidic chip according to one embodiment of the invention.

1 to 2, the microfluidic chip 1 according to one embodiment of the present invention has one or more reaction channels 70 in which such reactions occur as used in various reactions, for example biological reactions. 1 to 2, the microfluidic chip 1 includes eight reaction channels 70, but the number of reaction channels is not limited thereto. The reaction channel 70 has an inlet 10 and an outlet 60 at both ends, and a liquid such as a biological sample or a reagent is introduced into the reaction channel 70 through the inlet 10. Liquid, such as a product or waste product of the biological reaction, is discharged through the outlet 60. The microfluidic chip 1 is embodied in a thin film shape such as a thin plate and includes a space capable of accommodating a small amount of liquid. The microfluidic chip 1 can be usefully used for biological reactions using very small amounts of liquids, for example biological samples, and reagents for extracting nucleic acids therefrom. The detailed structure and use of the microfluidic chip 1 will be described later.

3 through 6 illustrate a multi-channel downward liquid injection device according to one embodiment of the invention.

The multi-channel downward liquid injection device 2 according to one embodiment of the invention is introduced into the one or more reaction channels 70 through one or more inlets 10 of the microfluidic chip 1 according to FIGS. 1 to 2. For injecting a liquid, such as a biological sample or reagent, downward. As described above, the microfluidic chip 1 is implemented in a thin film shape, and the inlet 10 and the outlet 60 of the reaction channel 70 are based on the microfluidic chip 1 of the thin film shape. Implemented upwards. Therefore, in order for a liquid such as a biological sample or a reagent to be introduced into the microfluidic chip 1, the liquid must be injected downward into the at least one inlet 10. In order to achieve this object, the liquid injection device must have a liquid discharge port implemented downward.

According to FIGS. 3 to 6, the multi-channel downward liquid injection device 2 according to an embodiment of the present invention includes a body 1000 and a cover 2000.

The body 1000 includes one or more downward liquid outlets 1100, one or more vertical through channels 1200, and one or more liquid inlets 1300, each of which is unitary, integrally, within the body 1000. It is composed. The downward liquid outlet 1100 is disposed to correspond to the upper end of one or more inlet portions 10 of the microfluidic chip 1, respectively, and serves to inject liquid downwardly into the one or more inlet portions 10. In this case, the downward liquid discharge port 1100 is preferably implemented to be in close contact with the one or more inlet portion 10 to prevent leakage of the liquid injected downward. The vertical through channel 1200 is implemented to penetrate in the vertical direction from the at least one downward liquid outlet 1100 to serve to allow the liquid introduced through the liquid inlet 1300 to move downward, It also serves to provide a path of vertical movement of the vertical pressing module 2100 to be described in detail below. The liquid injection hole 1300 is implemented to be connected to the inside of the one or more vertical through-hole channel 1200 at any position of the main body 1000 serves to introduce the liquid from the outside. In this case, the area of the outer exposed portion of the liquid inlet 1300 is the area connected to the vertical through-channel 1200 of the liquid inlet 1300 and the area of the bottommost portion of the liquid outlet 1100 to be described in detail below. A wider implementation can improve the convenience and accuracy of the user's liquid injection (see FIGS. 5 and 6). Furthermore, even if the amount of liquid injected by the user into the liquid inlet 1300 is slightly different, the amount of liquid moving downward through the vertical through channel 1200 may be constantly adjusted.

The cover 2000 includes one or more vertical pressing modules 2100 and a pressing plate 2200, which are integrally configured within the cover 2000. The vertical pressurizing module 2100 is inserted into the one or more vertical through channels 1200, respectively, and moves up and down inside thereof, and the lower distal end 2110 is connected to the inner surface of the one or more vertical through channels 1200. It is implemented to maintain close contact to push down the liquid introduced into the one or more vertical through-channel 1200 through the one or more liquid inlet 1300. In this case, the lower end portion 2110 may be made of a material, for example, rubber or silicone, which may be kept in close contact without physically damaging the inner surface of the vertically channeled channel 1200, such as a close contact pressure module of a general syringe. Can be implemented. The pressing plate 2200 is integrally connected with the upper end of the one or more vertical pressing modules 2110 to perform simultaneous vertical movement of the one or more vertical pressing modules 2110 by a user operation. 3 and 5 illustrate that the cover 2000 is disposed above the main body 1000, and FIGS. 4 and 6 illustrate that the cover 2000 is disposed below the main body 1000. When downward pressure (see dotted arrow in FIGS. 4 and 6) is applied by a user operation while the cover 2000 is disposed at an upper position, the cover 2000 moves to a lower position, in which case the at least one vertical pressurization is applied. The module 2110 causes the interior of the one or more vertical through channels 1200 to move downward.

Meanwhile, according to FIGS. 5 to 6, the multi-channel downward liquid injection device 2 according to an embodiment of the present invention is implemented at the bottom of one or more downward liquid discharge ports 1100 of the main body 1000 and the microfluidic chip. And a chip inlet region end mounting portion 3000 implemented such that one or more inlet 10 region ends of (1) can be fixedly mounted. 3 to 4 show that the inlet portion 10 region of the thin-film microfluidic chip 1 is mounted to the chip inlet region end mounting portion 3000 to be driven with the multi-channel downward liquid injection device 2. Shows a coupled state. In this case, it is assumed that one or more inlets 10 of the microfluidic chip 1 are closely connected in fluid communication with the one or more downward liquid outlets 1100 of the multi-channel downward liquid injection device 2. . On the other hand, a liquid such as a biological sample or reagent is introduced through the one or more liquid inlet 1300, which is opened when the lower distal end 2110 of the one or more vertical pressurization module 2100 is moved upward (see FIG. 3), the user When downward pressure is applied to the pressing plate 2200 by the operation and the lower end portion 2110 of the one or more vertical pressurizing modules 2100 moves downward, it moves through the one or more vertical through channels 1200 and moves to the one. It is injected into the one or more inlet portion 10 of the microfluidic chip 1 through the above downward liquid outlet 1100 (see FIG. 4).

FIG. 7 shows guide means for supporting a vertical movement of a cover of a multi-channel downward liquid injection device according to an embodiment of the present invention, and FIG. 8 shows a multi-channel downward liquid injection device according to an embodiment of the present invention. It shows a removable means for allowing the body and the cover of the to be separated.

The guide means 4000 serves to support the vertical movement of the cover 2000. For example, according to FIG. 7, the guide means 4000 is embodied in the cover 2000 to be inserted and moved up and down and the main body 1000 so that the insertion means 4000a is not separated. It may include a support means (4000b) surrounding the one direction to maintain the vertical movement, if the one that can serve to support the vertical movement of the cover 2000 can be implemented in a variety of shapes and structures, of course. . On the other hand, the removal means 5000 serves to allow the main body 1000 and the cover 2000 to be separated. For example, according to FIG. 8, a recess means 5000a having a groove (not shown) implemented in the main body 1000 and disposed at an arbitrary position of the main body 1000 may be implemented in the cover 2000. And a protruding means 5000b having a locking jaw (not shown) disposed at an arbitrary position of the cover 2000, but the main body 1000 and the cover 2000 may be separated from each other. Of course, if the role can be implemented in a variety of shapes and structures. As the detaching means 5000 is present, the cover 2000 is firmly maintained so that the cover 2000 does not detach from the main body 1000 due to the vertical movement of the cover 2000 when the liquid is injected. The cover 2000 may be separated from the main body 1000 when the downward liquid outlet 1100, the vertical through channel 1200, and the liquid inlet 1300 are washed, thereby facilitating washing. have.

9 illustrates a vertically penetrating channel and a liquid inlet of a multi-channel downward liquid injection device according to an embodiment of the present invention, and FIGS. 10 to 11 illustrate a multi-channel downward liquid injection device according to an embodiment of the present invention. It shows the path of movement of a liquid, such as a biological sample or reagent, within. 9-11, the right figure is the figure which expanded the dotted line part of the upper left.

According to FIG. 9, the one or more liquid inlets 1300 may be connected to a portion of each inner surface of the one or more vertical through channels 1200. In this case, the liquid injection hole 1300 may penetrate from the outside to the inner surface of the vertical through channel 1200 to inject liquid from the outside into the vertical through channel 1200. As described above, the liquid inlet 1300 is connected to the inner surface of the vertical through-channel 1200, but is exposed to the outside of the main body, the user is in contact with the liquid at one or more inlet of the microfluidic chip (1) It is easier to inject. According to FIG. 10, the vertical pressurizing module 2100 is moved upward by a user manipulation (empty arrow) such that the lower distal end 2110 is positioned at any position of the inner surface of the vertical through channel 1200 (X-X). '), The liquid inlet 1300 penetratingly connected to the inner surface of the vertically channeled channel 1200 is opened. In this case, the liquid (arrow filled) may be introduced into the vertical through channel 1200 through the liquid inlet 1300. Subsequently, according to FIG. 11, the vertical pressurizing module 2100 is moved downward by a user operation (empty arrow) such that the lower distal end 2110 is positioned at any position Y of the inner surface of the vertical through channel 1200. -Y '), the liquid inlet 1300 penetratingly connected to the inner surface of the vertically channeled channel 1200 is closed and the liquid (full arrow) introduced into the vertically channeled channel 1200 is closed. The lower end portion 2110 is pushed downward to reach the inner surface of the vertically penetrating channel 1200 and reaches the downward liquid outlet 1100.

12 to 13 show the arrangement of the vertically penetrating channel and the liquid inlet in the cross section of the body of the multi-channel downward liquid injection device according to one embodiment of the invention.

According to FIG. 12, the one or more vertical through channels 1200 are disposed in the horizontal cross-sectional center region “C” of the main body 1000, and the one or more liquid inlets 1300 are disposed through the one or more vertical through channels. It is disposed in the border region "P" adjacent to the type channel 1200. In this case, the user may easily inject liquid into the liquid inlet 1300 which is disposed at the edge area “P” and is exposed to the outside, and the user subsequently applies downward pressure to the pressing plate 2200 to provide a central area ( The liquid moved to the vertical through channel 1200 disposed in the "C" can be moved downward to easily discharge the liquid through the downward liquid outlet 1100. According to FIG. 13, the vertical through channel 1200 may be implemented as three or more, but may be implemented in a zigzag form based on a horizontal cross section of the main body 1000 (see the dotted line). In this case, in manufacturing the multi-channel downward liquid injection device 2 according to an embodiment of the present invention, the size and volume of the main body 1000 may be considerably reduced than that of the straight form. The zigzag shape may be the same as that of the arrangement of the three or more inlet portions 10 of the microfluidic chip 1. On the other hand, according to Figures 12 to 13, although the vertical through-hole channel 1200 is implemented in eight, for example, the number is not limited to three or more.

14 to 17 illustrate a microfluidic chip according to an embodiment of the present invention in detail and illustrate a nucleic acid extraction method using the same. According to Figures 14 to 17, the microfluidic chip (microfluidic chip) according to an embodiment of the present invention can be used for nucleic acid extraction. 14 to 17, the microfluidic chip is referred to as "nucleic acid extraction microfluidic chip".

The microfluidic chip for nucleic acid extraction is a component for nucleic acid extraction, that is, an inlet, an outlet, a channel connecting the inlet and the outlet, and a first filter. , And the second filter, etc., refers to a microchip implemented in millimeter (mm) or micrometer (μm) units.

According to FIG. 14A, a nucleic acid extracting microfluidic chip according to an embodiment of the present invention has an inlet 10, a channel region 70 connected to the inlet 10, and an outflow connected to the channel region 70. Including a portion 60, the channel region 70 includes a heating portion 20 implemented to transfer heat obtained from the outside to the biological sample introduced through the inlet portion 10, 14b to Various modules may be provided for efficiently extracting nucleic acids from biological samples such as 14 g.

In the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention illustrated in FIG. 14B, a heating unit 20 is disposed in a first channel region connected to the inlet unit 10, and connected to the heating unit 20. The microfluidic chip for nucleic acid extraction according to an embodiment of the present invention shown in FIG. 14C includes a first filter 30 disposed in the second channel region and capable of passing a material having a size corresponding to the nucleic acid. The heating unit 20 is disposed in the first channel region connected to the inlet unit 10, and the first channel region is disposed in the second channel region connected to the heating unit 20 to pass a material having a size corresponding to the nucleic acid. A nucleic acid separation unit 40 having a filter 30 and disposed in a third channel region connected to the first filter 30 and having a nucleic acid binding material 45 capable of specifically binding to the nucleic acid. Can be provided, nucleic acid extraction according to an embodiment of the present invention shown in Figure 14d In the microfluidic chip, a heating unit 20 is disposed in a first channel region connected to the inlet 10, and is disposed in a second channel region connected to the heating unit 20, and passes through a material having a size corresponding to a nucleic acid. And a nucleic acid binding material 45 (bead) disposed in a third channel region connected to the first filter 10 and capable of specifically binding to the nucleic acid. And a second filter 50 disposed in a fourth channel region connected to the nucleic acid separator 40 to pass a material having a size corresponding to the nucleic acid. In the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention illustrated in FIG. 14E, a heating unit 20 is disposed in a first channel region connected to the inlet unit 10, the heating unit 20. Disposed in a second channel region associated with the nucleic acid and capable of passing a substance of a size corresponding to the nucleic acid. And a nucleic acid having a nucleic acid binding material 45 (membrane) disposed in a third channel region connected to the first filter 10 and capable of specifically binding to the nucleic acid. And a second filter 50 disposed in a fourth channel region connected to the nucleic acid separator 40 and capable of passing a material having a size corresponding to the nucleic acid. In the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention illustrated in FIG. 14F, a heating unit 20 is disposed in a channel region connected to the inlet 10, and a channel region connected to the heating unit 20. It is provided with a nucleic acid separation unit 40 which is disposed in the but is provided with a nucleic acid binding material (45, membrane) that can specifically bind to the nucleic acid, according to an embodiment of the present invention shown in Figure 14g Microfluidic chip for nucleic acid extraction is a channel connected to the inlet (10) A nucleic acid separation unit 40 having a heating unit 20 disposed in a region, and having a nucleic acid binding material 45 disposed in a channel region connected to the heating unit 20 and capable of specifically binding to the nucleic acid. And a second filter 50 disposed in a channel region connected to the nucleic acid separation unit 40 and capable of passing a material having a size corresponding to the nucleic acid.

The biological sample is a biological material including a nucleic acid such as DNA or RNA, and may be, for example, a liquid sample including animal cells, plant cells, pathogens, fungi, bacteria, viruses, and the like, but is not limited thereto.

The inlet 10 is a portion into which the biological sample or the solution for nucleic acid extraction is introduced into the microfluidic chip, and the outlet 60 is a nucleic acid obtained from the biological sample, a solution for nucleic acid extraction, Other waste (waste) and the like is discharged to the outside of the microfluidic chip. In this case, if necessary, the inlet 10 and the outlet 60 may serve as outlets and inlets, respectively. The solution for nucleic acid extraction includes all the solutions required for nucleic acid extraction, and may be, for example, distilled water, a nucleic acid binding buffer, an elution buffer, or the like. On the other hand, the inlet 10 and the outlet 60 is connected in fluid communication by the channel 70, the heating unit 20, the first filter 30, the nucleic acid separation unit will be described in detail below Components 40, the second filter 50, and the like are arranged to be driven in the channel 70 to perform each function. The channel 70 may be implemented in various standards, but the width and depth of the channel are preferably implemented in a range of 0.001 to 10 millimeters (mm), respectively. The first, second, third, and fourth channel regions to be described below mean a sequential arrangement from the inlet portion 10 to the outlet portion 60 and are limited to a specific position in the channel 70. It is not.

The heating part 20 is a portion in which heat obtained from the outside is applied to a solution (including a biological sample) introduced through the inlet part 10, and is disposed in a first channel region connected to the inlet part 10. For example, when a sample containing cells, bacteria, or viruses is introduced through the inlet 10, when the cells, bacteria, or viruses reach the heating unit 20, about 80 to 100 degrees are instantaneously. Heating to (° C.) causes the outer wall of the cell, bacteria, or virus to break, causing the cell material to be released to the outside (cell lysis). The heating unit 20 may be supplied with heat in a contact or non-contact manner from the heating module 600 of the nucleic acid extraction apparatus to be described below.

The first filter 30 is a structure having a pore of a predetermined size, and serves to distinguish the passing material and the non-passing material by size through the pore in the fluid flow direction. In one embodiment of the present invention, the first filter 30 is disposed in the second channel region connected to the heating unit 20, it is implemented to pass a material of a size corresponding to the nucleic acid. The first filter 30 collects a material having a size larger than that of the nucleic acid in the dissolution product generated by the heating in the heating unit 20 in the heating unit 20, the nucleic acid and the material having a corresponding size is filtered It is moved to the nucleic acid separation unit 40 to be described below. The first filter 30 may be implemented in various standards, but having a pore having a diameter in the range of 0.1 to 0.4 micrometers (μm), but having a thickness in the range of 0.01 to 10 millimeters (mm). desirable. More preferably, the first filter 30 has a pore having a diameter of 0.2 micrometer (μm), but preferably has a thickness of 0.01 to 0.5 millimeters (mm).

The nucleic acid separation unit 40 is for selectively separating the nucleic acid from a nucleic acid or a substance having a size corresponding thereto. According to FIG. 1, the nucleic acid separation unit 40 is a space between the first filter 30 and the second filter 50 to be described below, and the nucleic acid binding material 45 capable of specifically binding to the nucleic acid. ) Is provided. The nucleic acid binding material 45 includes all materials capable of specifically binding to nucleic acids. The nucleic acid binding material 45 has a nucleic acid binding functional group attached thereto, and may be, for example, silica (SiO 2) beads, biotin, strptavidin attachment beads, or a membrane. The bead or membrane to which the nucleic acid binding functional group is attached may be implemented in various standards, but preferably has a diameter within the range of 0.001 to 20 millimeters (mm). In addition, the nucleic acid separation unit 40 may include the bead or membrane to which the nucleic acid binding functional group is attached in various contents and specifications, but preferably includes within a range of 1 microgram (μg) to 200 mg (mg). Do. After the nucleic acid is specifically bound to the nucleic acid binding material 45, the foreign matter is removed by washing the inside of the nucleic acid separation part 40, and the nucleic acid binding part 40 of the target nucleic acid-nucleic acid binding material 45 is removed. Only the complex remains. Thereafter, when an elution buffer is provided to the nucleic acid separation unit 40, the target nucleic acid is separated from the complex.

The second filter 50, like the previously described first filter 30, is a structure having a pore of a constant size, and passes through the non-pass material by size through the pore in the direction of fluid flow. It plays a role of distinguishing. In one embodiment of the present invention, the second filter 50 is disposed in the fourth channel region connected to the nucleic acid separation unit 40, it is implemented to pass a material of a size corresponding to the nucleic acid. The second filter 50 collects the nucleic acid binding material 45 in the nucleic acid separation unit 40, and filters the nucleic acid separated from the nucleic acid binding material 45 to the outlet 60. . The second filter 50 may be implemented in various standards, but having a pore having a diameter in the range of 0.1 to 100 micrometers (μm), but having a thickness in the range of 0.01 to 0.5 millimeters (mm). desirable. More preferably, the second filter 50 has a pore having a diameter of 0.2 micrometer (μm), but preferably has a thickness of 0.3 millimeter (mm).

15 is a cross-sectional view of the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.

According to Figure 15, it is possible to check the cross-sectional view of the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention. Microfluidic chip for nucleic acid extraction according to an embodiment of the present invention is a silver first plate (100); A second plate (200) disposed on the first plate and having a channel (70) including the first to fourth channel regions; And a third plate 300 disposed on the second plate 200 and having the inlet 10 and the outlet 60 disposed thereon. Nucleic acid extraction microfluidic chip according to an embodiment of the present invention may be implemented in a variety of materials, preferably may be implemented in a plastic material. As such, when the plastic material is used, heat transfer efficiency may be increased only by adjusting the thickness of the plastic, and the manufacturing process may be simplified, thereby greatly reducing the manufacturing cost. Meanwhile, the first plate 100 and the third plate 300 may include polydimethylsiloxane (PDMS), cyclo olefin copolymer (COC), polymethyl methacrylate (PMMA), Material selected from the group consisting of polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof The second plate 200 includes polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA), Polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyether Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT) It may include a thermoplastic resin or a thermosetting resin material selected from the group consisting of, fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), and combinations thereof. In addition, the inlet portion of the third plate is implemented in the range of 0.1 to 5.0 millimeters (mm) in diameter, the outlet portion is implemented in the range of 0.1 to 5.0 millimeters (mm) in diameter, the thickness of the first plate and the third plate Is implemented within the range of 0.01 to 20 millimeters (mm), the thickness of the second plate may be implemented within the range of 30 micrometers (μm) to 10 millimeters (mm). In addition, the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention may be implemented as one or more inlets, outlets, and channels connecting them, if necessary, in this case from one or more biological samples on one chip Nucleic acid can be extracted, and nucleic acid can be extracted quickly and efficiently.

16 is a schematic diagram of a nucleic acid extraction apparatus equipped with a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention.

According to Figure 16, the nucleic acid extracting apparatus according to an embodiment of the present invention is a nucleic acid extracting microfluidic chip (1) already described; A chip mounting module 500 implemented to mount the microfluidic chip 1; A heating module 600 implemented to apply heat to the heating unit 20 of the microfluidic chip 1 mounted on the chip mounting module 500; And a solution for extracting nucleic acids into the microfluidic chip 1 by being connected to the inlet 10 and / or the outlet 60 of the microfluidic chip 1 mounted on the chip mounting module 500. It may include a fluid control module 700 implemented to introduce and / or to discharge the solution present in the microfluidic chip (1) to the outside.

The nucleic acid extraction apparatus is a device implemented to perform all the steps for nucleic acid extraction in the state in which the microfluidic chip 1 according to an embodiment of the present invention, the chip mounting module 500, In addition to the heating module 600 and the fluid control module 700, it may further include various modules required for extracting other nucleic acids. In addition, the nucleic acid extracting apparatus according to an embodiment of the present invention can be implemented so that all steps can be implemented in an automated manner, the nucleic acid amplification reaction can proceed immediately after nucleic acid extraction in conjunction with the polymerase chain reaction (PCR) apparatus have.

The microfluidic chip 1 for nucleic acid extraction is as described above.

The chip mounting module 500 is a portion on which the microfluidic chip 1 is mounted. The chip mounting module 500 may be implemented in various ways corresponding to the shape of the contact surface of the microfluidic chip 1.

The heating module 600 is a module for supplying heat to the heating unit 20 of the microfluidic chip 1 when the microfluidic chip 1 is mounted on the chip mounting module 500. The heating module 600 may be implemented in various ways, but a contact heating block is preferable.

The fluid control module 700 is connected to the inlet part 10 and / or the outlet part 60 of the microfluidic chip 1 mounted on the chip mounting module 500 to be inside the microfluidic chip 1. It is a module implemented to introduce a solution for nucleic acid extraction and / or to discharge the solution existing in the microfluidic chip (1) to the outside. The fluid control module 700 may include various components, for example, a microchannel that is a fluid movement passage, a pneumatic pump providing a driving force for fluid movement, a valve for controlling opening and closing of fluid movement, and a nucleic acid. It may further include a storage chamber containing a variety of solutions required for nucleic acid extraction, such as binding buffer, elution buffer, silica gel (silica gel), distilled water (DW).

Meanwhile, the nucleic acid extracting apparatus according to an embodiment of the present invention is an electronic control module (not shown) for automatically controlling the microfluidic chip 1, the heating module 600, and the fluid control module 700. ) May be further included. The electronic control module can precisely control the respective modules so that the quantitative nucleic acid can be extracted from the microfluidic chip 1 according to a pre-stored program. The prestored program includes, for example, a program relating to a series of steps relating to a nucleic acid extraction method which will be described in detail below.

17 is a flowchart of a nucleic acid extraction method according to an embodiment of the present invention. Specifically, FIGS. 17A to 17D show various nucleic acid extraction methods based on the microfluidic chip 1 for nucleic acid extraction according to an embodiment of the present invention.

According to FIG. 17A, a method of extracting nucleic acids from a biological sample according to an embodiment of the present invention includes providing a microfluidic chip for nucleic acid extraction according to FIG. 14F (providing a microfluidic chip); Introducing a biological sample selected from the group consisting of cells, bacteria, and viruses through an inlet of the microfluidic chip (biological sample introduction step); Moving the introduced biological sample to a heating part of the microfluidic chip and then heating the heating part of the microfluidic chip to dissolve the biological sample (biological sample dissolution step); Separating the nucleic acid from the soluble material through a nucleic acid binding material (membrane) (nucleic acid separation step); As an optional step, the step of removing foreign matters generated in the nucleic acid separation process (foreign matter removing step); And extracting the nucleic acid through the outlet after moving the nucleic acid to the outlet (nucleic acid extraction step).

According to Figure 17b, a method for extracting a nucleic acid from a biological sample according to an embodiment of the present invention comprises the steps of providing a microfluidic chip for nucleic acid extraction according to Figure 14b or 14c (microfluidic chip providing step); Introducing a biological sample selected from the group consisting of cells, bacteria, and viruses through an inlet of the microfluidic chip (biological sample introduction step); Moving the introduced biological sample to a heating part of the microfluidic chip and then heating the heating part of the microfluidic chip to dissolve the biological sample (biological sample dissolution step); The material obtained from the dissolution step is transferred to the first filter of the microfluidic chip and then passed through the first filter, and removing the material not passed through the first filter (filtration step through the first filter) ); Separating the nucleic acid from the material passing through the first filter (nucleic acid separation step); As an optional step, the step of removing foreign matters generated in the nucleic acid separation process (foreign matter removing step); And extracting the nucleic acid through the outlet part after moving the nucleic acid to the outlet part (nucleic acid extraction step).

According to Figure 17c, a method for extracting a nucleic acid from a biological sample according to an embodiment of the present invention comprises the steps of providing a microfluidic chip for nucleic acid extraction according to Figure 14g (microfluidic chip providing step); Introducing a biological sample selected from the group consisting of cells, bacteria, and viruses through an inlet of the microfluidic chip (biological sample introduction step); Moving the introduced biological sample to a heating part of the microfluidic chip and then heating the heating part of the microfluidic chip to dissolve the biological sample (biological sample dissolution step); Separating the nucleic acid from the soluble material through a nucleic acid binding material (bead) (nucleic acid separation step); As an optional step, the step of removing foreign matters generated in the nucleic acid separation process (foreign matter removing step); Separating the nucleic acid from the nucleic acid binding material, passing the separated nucleic acid to the second filter and passing it through a second filter (filtration through a second filter); And extracting the nucleic acid through the outlet after moving the nucleic acid to the outlet (nucleic acid extraction step).

According to FIG. 17D, a method of extracting nucleic acids from a biological sample according to an embodiment of the present invention includes providing a microfluidic chip for nucleic acid extraction according to FIG. 14D or 14E (providing a microfluidic chip); Introducing a biological sample selected from the group consisting of cells, bacteria, and viruses through an inlet of the microfluidic chip (biological sample introduction step); Moving the introduced biological sample to a heating part of the microfluidic chip and then heating the heating part of the microfluidic chip to dissolve the biological sample (biological sample dissolution step); The material obtained from the dissolution step is transferred to the first filter of the microfluidic chip and then passed through the first filter, and removing the material not passed through the first filter (filtration step through the first filter) ); Separating the nucleic acid from the soluble material through a nucleic acid binding material (bead or membrane) (nucleic acid separation step); As an optional step, the step of removing foreign matters generated in the nucleic acid separation process (foreign matter removing step); Separating the nucleic acid from the nucleic acid binding material, passing the separated nucleic acid to the second filter and passing it through a second filter (filtration through a second filter); And extracting the nucleic acid through the outlet part after moving the nucleic acid to the outlet part (nucleic acid extraction step).

18-20 illustrate a liquid storage container in accordance with one embodiment of the present invention.

According to FIG. 18, the liquid storage container 5000 is for storing and storing the reaction product after the reaction by the microfluidic chip 1 is completed, and at least one outlet portion of the microfluidic chip 1 ( 60) a chip outlet region end mounting portion 5100 implemented such that the region end is fixedly mounted, and one or more upward liquid suction ports 5200 respectively corresponding to upper ends of the one or more outlet portions 60 of the microfluidic chip 1. And one or more liquid storage chambers 5300 in fluid communication with the one or more upward liquid inlets 5200. 19 to 20 illustrate a process in which liquid discharged through at least one outlet 60 of the microfluidic chip 1 moves in the liquid storage container 5000. For example, after the nucleic acid extraction reaction in the microfluidic chip 1 is completed, the liquid storage container 5000 is fixedly mounted to the chip outlet region end mounting portion 5100, and contains a solution containing a desired nucleic acid ( When E1, E2 is discharged through the one or more outlets 60, the nucleic acid-containing solution E1, E2 is introduced through one or more upward liquid inlet 5200 of the liquid storage container 5000, The liquid storage container 5000 moves through the channel (F1, F2) to reach the one or more liquid storage chambers 5300 (S1, S2). Thereafter, the liquid storage container 5000 may be separated from the microfluidic chip 1 to separately store and store the nucleic acid-containing solution, and may further utilize the nucleic acid-containing solution in subsequent nucleic acid detection and analysis. Will be. FIG. 21 illustrates a flow path of a liquid such as a biological sample or a reagent in a state in which the microfluidic chip 1 and the liquid storage container 5000 are combined according to an embodiment of the present invention. In this case, the driving force for the continuous movement of the liquid in the microfluidic chip 1 and the liquid storage container 5000 is connected to one or more inlets 10 of the microfluidic chip 1. It may be provided from a multi-channel downward liquid injection device 2 according to one embodiment, or any pump or syringe. 22 illustrates the movement of a liquid, such as a biological sample or a reagent, in a state in which the multi-channel downward liquid injection device 2, the microfluidic chip 1, and the liquid storage container 5000 are combined according to an embodiment of the present invention. Show the route.

Under the premise of the nucleic acid extracting apparatus as described above, an embodiment of the present invention can provide a fast and efficient ultra-fast nucleic acid extracting method. For example, the first nucleic acid extracting method may include providing the nucleic acid extracting apparatus described above; Injecting a biological sample or reagent into the microfluidic chip for nucleic acid extraction through the multi-channel downward liquid injection device; And extracting nucleic acids from the biological sample by driving the nucleic acid extracting microfluidic chip, wherein the second nucleic acid extracting method comprises the steps of providing the nucleic acid extracting apparatus described above; Injecting a biological sample or reagent into the microfluidic chip for nucleic acid extraction through the multi-channel downward liquid injection device; Extracting nucleic acids from the biological sample by driving the nucleic acid extracting microfluidic chip; And storing the nucleic acid extraction product in a liquid storage chamber of the liquid storage container.

Hereinafter, in Examples 1 to 2, compared to other nucleic acid extracting apparatuses (Qiagen), while extracting the nucleic acid from the biological sample to determine the yield and the running time of the nucleic acid extract, and further through the polymerase chain reaction (PCR) The result reliability of the extract was again confirmed.

Example  1. Confirmation of yield and running time of nucleic acid extraction

First, DNA is extracted using a general tube included in a third-party product and a nucleic acid extracting microfluidic chip 1 according to an embodiment of the present invention, and then the yield and duration of the tuberculosis strain cells. Confirmed.

Nucleic acid extraction step using a third-party nucleic acid separation device is as follows. Tuberculosis strain cells were prepared, and the tuberculosis strain cells were mixed with 6% NaOH and 4% NaLC in a 1: 1: 1 ratio to prepare a sample solution. The sample solution was then centrifuged to remove supernatant (10 min, 7500 rpm, 4 ° C.). Thereafter, 20 μl Proteinase K was added to the sample solution, and the sample solution was left at 56 ° C. until it became clear. Then, 200 μl AL buffer was added to the sample solution, mixed for 15 seconds, and left at 56 ° C. for 10 minutes. The sample solution was then transferred to a column and centrifuged for 1 minute (8000 rpm). Then, 500 μl AW 1 buffer was added and centrifuged for 1 minute (8000 rpm). Thereafter, 500 µl AW 2 buffer was added and centrifuged for 1 minute (14,000 rpm). Then centrifuged again for 1 minute (14,000 rpm). Thereafter, the column was placed in a new tube, and 200 µl of AE buffer was added, followed by standing for 3 minutes. Thereafter, DNA was eluted after centrifugation for 1 minute. As a result, about 100 μl of the final DNA product was obtained and it took about 30 minutes to obtain the final DNA product.

Subsequently, the nucleic acid was extracted from the same tuberculosis strain cells using the multi-channel downward liquid injection device 2 and the nucleic acid extracting microfluidic chip 1 according to an embodiment of the present invention.

Tuberculosis strain cells were prepared, and the tuberculosis strain cells were mixed with 6% NaOH and 4% NaLC in a 1: 1: 1 ratio to prepare a sample solution. Thereafter, at least one inlet portion of the nucleic acid extraction microfluidic chip {25 × 72 × 2 mm, silica beads (OPS Diagnostics, LLC), filter (Whatman) according to FIG. The sample solution was introduced using a channel down liquid injection device. Then, 300 μl of silica gel and 1X DNA binding buffer were introduced into the inlet of the microfluidic chip according to the present invention, followed by heating of the microfluidic chip according to the present invention. The part was heated rapidly to 95 ° C. Thereafter, waste in the sample solution was removed through the inlet of the microfluidic chip according to the embodiment of the present invention, and 100 µl of an elution buffer was introduced. Then, the final product was obtained through the outlet of the microfluidic chip according to one embodiment of the present invention (using a liquid storage container according to one embodiment of the present invention), and as a result, about 100 μl of the final DNA product was obtained. It took about 5 minutes to get the final DNA product.

As a result of the experiment, using the multi-channel downward liquid injection device and the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention, the amount of the nucleic acid extraction product can be maintained as it is, unlike the conventional nucleic acid extraction method It can be seen that the time required can be significantly shortened.

Example  2. Third party products and work of the present invention Example  Each obtained by the nucleic acid extraction method according to DNA  Polymerase chain reaction of product ( PCR ) result

In order to secure the reliability of the DNA product obtained in Example 1, a polymerase chain reaction (PCR) was performed based on the DNA product.

The polymerase chain reaction (PCR) used a PCR device comprising two heat blocks described in the applicant's Korean Patent Application No. 2011-0037352. Applicant's PCR apparatus is a real time PCR apparatus, comprising: a first row block disposed on a substrate; A second thermal block spaced apart from the first thermal block on the substrate; And a chip holder which is movable left and right and / or up and down by a driving means over the first row block and the second row block, and is equipped with a PCR chip made of a transparent plastic material. In addition, the driving means includes a rail extending in the left and right direction, and a sliding member disposed to be slidably movable in the left and right direction through the rail, the connecting member slidable in the vertical direction, one end of the connection member is the chip holder Characterized in that arranged. In addition, a light source is further disposed between the first column block and the second column block, and a light detector for detecting light emitted from the light source is further disposed on the chip holder, or the first column block and the second column block. A light detector for detecting light emitted from the light source is further disposed between the heat blocks, and the light source is further disposed on the chip holder. By using the PCR chip and the PCR device, it is possible to significantly shorten the PCR execution time within about 5 to 15 minutes, the PCR chip and the PCR device for nucleic acid extraction microfluidic chip and nucleic acid extraction according to an embodiment of the present invention In conjunction with the device, the nucleic acid extraction time can be shortened to within about 5-7 minutes and at least about 20 minutes before obtaining the final nucleic acid amplification product. On the other hand, PCR samples and reagents for carrying out the polymerase chain reaction (PCR) are 10 microliters (μl), real-time PCR mixed solution (TOYOBO SYBR qPCR mix), 2 microliters (μl), Forward Primer (10μM), A total of 20 microliters (μl) containing 2 microliters (μl) of reverse primer, 1 microliter (μl) of template DNA (1 ng), 5 microliters (μl) of distilled water (DW), and the like Ready. Then, the pre-denaturation step was performed at 95 ° C. and 30 sec conditions (1 cycle), the denaturation step was performed at 95 ° C. and 5 sec conditions, and the anealing & extension step was performed at 72 ° C. and 30 sec conditions (40 cycles). .

Table 1 below shows real-time PCR results (Ct values) of nucleic acid extraction products obtained using a nucleic acid extraction method using a third-party product, and FIG. 23 shows nucleic acid extraction products obtained using a nucleic acid extraction method using a third-party product. Real-time PCR results for the measurement is a graph measured by the fluorescence by PCR cycle, Figure 24 is a photograph of the gel (electrophoresis) gel electrophoresis of the final PCR product. 23 is a PCR result curve (X-axis: period, Y-axis: fluorescence) of the DNA product by each nucleic acid extraction method.

Classification (gDNA copies / rxn) Ct value Non template - 1 × 10 6 16.36 1 × 10 5 19.79 1 × 10 4 23.22 1 × 10 3 26.72 1 × 10 2 29.94 1 × 10 1 33.27 1 × 10 0 > 35

In addition, Table 2 below shows the real-time PCR results (Ct value) for the nucleic acid extraction product obtained by using the nucleic acid extraction method according to an embodiment of the present invention, Figure 25 is a nucleic acid according to an embodiment of the present invention Real-time PCR results of the nucleic acid extraction product obtained by using the extraction method is a graph measured by the fluorescence for each PCR cycle, Figure 26 is a photograph of gel electrophoresis of the final PCR product. 25 is a PCR result curve (X-axis: period, Y-axis: fluorescence) of the DNA product by each nucleic acid extraction method.

Classification (gDNA copies / rxn) Ct value Non template (7) - 5 × 10 5 (1) 18.89 5 × 10 4 (2) 21.84 5 × 10 3 (3) 23.84 5 × 10 2 (4) 26.32 5 × 10 1 (5) 30.14 1 × 10 7 (Positive Control) (6) 13.49

Through the PCR results, the nucleic acid extraction method using the nucleic acid extraction apparatus according to an embodiment of the present invention significantly reduces the nucleic acid extraction step while maintaining or improving the result reliability of the nucleic acid extraction product, compared to using a third-party product. It was confirmed that the reaction time can be much shorter.

Claims (16)

In a thin microfluidic chip having at least one reaction channel having inlets and outlets at both ends, a liquid such as a biological sample or a reagent is introduced into the at least one reaction channel through the at least one inlet. For injecting downwards,
At least one downward liquid outlet corresponding to an upper end of at least one inlet of the microfluidic chip, at least one vertical through channel penetrating upward and downward from the at least one downward liquid outlet, and at least one inside of the at least one vertical through channel A main body having at least one liquid inlet; And
At least one vertical pressurizing module, each inserted into the at least one vertical through-hole channel to move its interior in a vertical direction, the lower distal end being in close contact with the inner surface of the at least one vertical through-channel, and the at least one vertical pressurization A cover having a pressing plate integrally connected with the upper end of the module to implement simultaneous vertical movement of the at least one vertical pressing module by user operation;
Including,
The liquid, such as the biological sample or reagent, is introduced through the one or more liquid inlets, wherein the lower end of the one or more vertical press modules is opened upon upward movement, and then the one or more vertical when the lower end of the one or more vertical press modules moves downward. Moved through the through channel and injected into at least one inlet of the microfluidic chip through the at least one downward liquid outlet,
A multi-channel downward, characterized in that it comprises a chip inlet region end mounting portion which is implemented at the bottom of the at least one downward liquid outlet of the body, the one or more inlet region end of the microfluidic chip is fixedly mounted. Liquid injection device. delete The method of claim 1,
The body and the cover, characterized in that it comprises a guide means for supporting the vertical movement of the cover in the engaged state, multi-channel downward liquid injection device. The method of claim 1,
Wherein said body and cover comprise detachable means embodied separably from each other. The method of claim 1,
And the at least one liquid inlet is connected to a portion of each inner surface of the at least one vertically through channel. The method of claim 1,
Wherein said at least one vertically through channel is disposed in a central cross-sectional reference central region of said body and said at least one liquid inlet is disposed at an edge region adjacent to said at least one vertically through channel. Injection device. The method of claim 1,
The vertical through-channel is implemented in three or more, characterized in that the zigzag (zigzag) on the basis of the horizontal cross-section of the main body, multi-channel downward liquid injection device. A nucleic acid for extracting a nucleic acid from a biological sample, comprising an inlet, a channel region connected to the inlet, and an outlet connected to the channel region, wherein the channel region is heat obtained from the outside of the biological sample introduced through the inlet. A microfluidic chip for extracting nucleic acids having a thin film-like shape having one or more reaction channels, including a heating unit implemented to deliver the; And
A multi-channel downward liquid injection device according to any one of the preceding claims;
Comprising, nucleic acid extraction apparatus. The method of claim 8,
A chip outlet region end mounting portion configured to be fixedly mounted to at least one outlet region end of the microfluidic chip, at least one upward liquid inlet corresponding to an upper end of at least one outlet portion of the microfluidic chip, and at least one And a liquid storage container having at least one liquid storage chamber in fluid communication with an upward liquid inlet. The method of claim 8,
The microfluidic chip may include a heating unit disposed in a first channel region connected to the inlet, and disposed in a second channel region connected to the heating unit, and including a first filter configured to pass a material having a size corresponding to a nucleic acid. Characterized in that, the nucleic acid extracting device. The method of claim 8,
The microfluidic chip has a heating unit disposed in a first channel region connected to the inlet, and disposed in a second channel region connected to the heating unit, and having a first filter through which a material having a size corresponding to a nucleic acid can pass therethrough. And a nucleic acid separation unit disposed in a third channel region connected to the first filter and having a nucleic acid binding material capable of specifically binding to the nucleic acid. The method of claim 8,
The microfluidic chip has a heating unit disposed in a first channel region connected to the inlet, and disposed in a second channel region connected to the heating unit, and having a first filter through which a material having a size corresponding to a nucleic acid can pass therethrough. And a nucleic acid separation unit disposed in a third channel region connected to the first filter and having a nucleic acid binding material capable of specifically binding to the nucleic acid, and disposed in a fourth channel region connected to the nucleic acid separation unit. And a second filter capable of passing a substance of a size corresponding to the nucleic acid. The method of claim 8,
The microfluidic chip has a heating unit disposed in a channel region connected to the inlet unit, and a nucleic acid separation unit including a nucleic acid binding material disposed in the channel region connected to the heating unit and specifically capable of specifically binding to the nucleic acid. Characterized in that, the nucleic acid extracting device. The method of claim 8,
The microfluidic chip is provided with a nucleic acid separation unit which is disposed in the channel region connected to the inlet, the nucleic acid binding material is disposed in the channel region connected to the heating portion and is provided with a nucleic acid binding material that can specifically bind to the nucleic acid. And a second filter disposed in a channel region connected to the nucleic acid separation unit and capable of passing a material having a size corresponding to the nucleic acid. Providing a nucleic acid extracting apparatus according to claim 8;
Injecting a biological sample or reagent into the microfluidic chip for nucleic acid extraction through the multi-channel downward liquid injection device; And
Extracting nucleic acids from the biological sample by driving the nucleic acid extracting microfluidic chip;
Including, nucleic acid extraction method. Providing a nucleic acid extracting apparatus according to claim 9;
Injecting a biological sample or reagent into the microfluidic chip for nucleic acid extraction through the multi-channel downward liquid injection device;
Extracting nucleic acids from the biological sample by driving the nucleic acid extracting microfluidic chip; And
Storing the nucleic acid extract product in a liquid storage chamber of the liquid storage container;
Including, nucleic acid extraction method.
KR1020130060996A 2012-05-30 2013-05-29 Multi-channel device for downwardly injecting liquid sample, device for extracting nucleic acid comprising the same, and method for extracting nucleic acid using the same KR102041217B1 (en)

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