KR20130135111A - Multi-channel device for distributing 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 distributing 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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Publication number
KR20130135111A
KR20130135111A KR1020130060993A KR20130060993A KR20130135111A KR 20130135111 A KR20130135111 A KR 20130135111A KR 1020130060993 A KR1020130060993 A KR 1020130060993A KR 20130060993 A KR20130060993 A KR 20130060993A KR 20130135111 A KR20130135111 A KR 20130135111A
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South Korea
Prior art keywords
nucleic acid
channel
liquid
microfluidic chip
inlet
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KR1020130060993A
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Korean (ko)
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김성우
김덕중
김선진
이세현
류호선
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나노바이오시스 주식회사
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Publication of KR20130135111A publication Critical patent/KR20130135111A/en

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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
    • 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
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/028—Modular arrangements
    • 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
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting

Abstract

An embodiment of the present invention relates to a multi-channel liquid dispensing apparatus, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the same. In this respect, in performing various biological reactions using a thin- It is possible to quickly dispense and dispense the same very small amount of liquid to the small one or more inflow portions and to accurately distribute a very small amount of liquid to the one or more inflow portions by only one user operation, Can be significantly shortened to allow rapid progression of a variety of subsequent biological detection or analysis reactions.

Description

[0001] The present invention relates to a multi-channel liquid distributor, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the same.

TECHNICAL FIELD The present invention relates to a liquid dispensing apparatus for accurately and simultaneously distributing a biological sample or a reagent and a liquid to a thin-film microfluidic chip having a liquid inlet, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the same.

Recently, techniques for extracting nucleic acids from biological samples such as cells, bacteria, or viruses have been widely used in connection with nucleic acid amplification reaction techniques in order to diagnose, treat, or prevent disease at a gene level. In addition to the diagnosis, treatment, or prevention of diseases, technologies for extracting nucleic acids from biological samples in various fields such as customized drug development, forensic medicine, and environmental hormone detection are required. As an example of a conventional nucleic acid extraction technique, there is a method of treating a sample containing cells with SDS or proteinase K, followed by solubilization, and denaturing the protein with phenol to purify the nucleic acid. However, since the phenol extraction method requires a lot of processing steps, it takes a lot of time and the efficiency of the nucleic acid extraction depends greatly on the experience and the skill of the researcher. In recent years, a kit using silica or glass fiber that specifically binds to nucleic acid has been used to solve this problem. Since the silica or glass fiber has a low binding ratio with proteins and cellular metabolites, a relatively high concentration of nucleic acid can be obtained. Such a method is advantageous compared with the phenol method, but since it uses a chaotropic reagent or ethanol which strongly inhibits the enzyme reaction such as PCR, it is necessary to completely remove these substances, This is a very cumbersome and time-consuming disadvantage. A method of directly purifying nucleic acids using recent filters is disclosed in WO 00/21973, which involves passing a sample through a filter to adsorb the cells to the filter, dissolving the adsorbed cells in 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 cells on the filter, there is a problem in that a filter must be selected according to the type of the cell, and there are disadvantages that the apparatuses are large and complex and can not be used easily by researchers.

In addition, it is necessary to provide a device for injecting a liquid such as a very small sample or reagent into a reaction vessel during various biological reactions. Conventional reaction vessels are mostly tube-shaped, and furthermore, multi-tubes in which a plurality of tubes having a very small volume are arranged and arranged are also used. Thus, devices commonly used to inject, mix, or withdraw liquids such as very small amounts of sample or reagent into such reaction vessels are pipettes and tips. However, since the pipette and the tip are not satisfactory in accuracy by adjusting the amount of liquid to be injected into the reaction vessel by a manual operation, particularly when injecting a liquid into one or more small-sized inflow portions of the thin- It is very troublesome that a tip having a fine outlet and a pipette designed accordingly are required. In addition, when the reaction is performed using a thin-film microfluidic chip having a plurality of reaction channels, the pipette can not accurately and quickly dispense the same sample or reagent, which is very troublesome. Therefore, in a biological reaction using a thin-film shaped microfluidic chip, it is possible to accurately and accurately distribute a very small amount of sample or reagent to one or more small-sized influent portions quickly and accurately, to improve user convenience and to perform a rapid reaction A liquid dispensing apparatus capable of discharging liquids is required.

One embodiment of the present invention is a method for rapidly and accurately dispensing a very small amount of liquid such as a biological sample or reagent to at least one inlet of a thin film microfluidic chip having at least one reaction channel, , A multi-channel liquid distributor, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the same.

A first embodiment of the present invention is a thin film transistor comprising: a thin film substrate; A single liquid inlet disposed at one end region of the substrate; And one or more even liquid outlets disposed in the other end region of the substrate and in fluid communication with the single liquid inlet through a channel, the channel having one end connected to the single liquid inlet side And the other end is divided into two halves so as to distribute the flow rate by half, and has a channel pattern connected to the liquid outlet side, wherein the multi-channel liquid distributor comprises at least one unit channel region .

In the first embodiment of the present invention,

The liquid discharge port is implemented pieces 2 N, wherein the channel and wherein the N or less the i-th unit of the channel region is 2 i-1 of start of the N units of the channel region formed by the 2 N of the liquid outlet from the single liquid inlet channel 3100 and the 2 i-1 from the start of the channel is divided into two branches each comprising a flow rate of 2 i of branch channel (3200) for dispensing a half, the start of the first unit channel domain channel side as the terminal is connected to the single liquid inlet and, connected to the N-th unit of the channel region of the branch channel ends each of the 2 N of the liquid outlet, wherein the N and i may be a natural number.

A second embodiment of the present invention is a microfluidic chip (1) in the form of a thin film having at least one to at least two reaction channels each having an inlet and an outlet at both ends thereof, Channel liquid dispensing apparatus according to the first embodiment of the present invention for down-infusing liquid into at least one reaction channel, and having a liquid outlet corresponding to the number of the at least one inlet; And fluid delivery means for fluidly connecting one or more inflow portions of the microfluidic chip to at least one liquid outlet of the multi-channel liquid distribution device.

A third embodiment of the present invention is directed to extracting 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 nucleic acid extraction in the form of a thin film, the microfluidic chip having at least one reaction channel, the microfluidic chip for nucleic acid extraction comprising: a heating unit configured to transfer externally obtained heat to the biological sample; A multi-channel liquid distributor according to the first embodiment of the present invention, having a liquid outlet coinciding with the number of the at least one inlet; And fluid delivery means for fluidly connecting at least one inlet of the microfluidic chip and at least one liquid outlet of the multi-channel liquid distributor.

In a third embodiment of the present invention,

A chip outlet region end mount adapted to fixably mount 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 of the microfluidic chip, And a liquid storage vessel having one or more liquid storage chambers fluidly connected to the upward liquid intake.

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

The microfluidic chip may include a first filter disposed in a first channel region connected to the inlet portion and disposed in a second channel region connected to the heating unit and capable of passing a substance having a size corresponding to nucleic acid, And a nucleic acid separator disposed in a third channel region connected to the first filter and having a nucleic acid binding substance capable of specifically binding to the nucleic acid.

The microfluidic chip may include a first filter disposed in a first channel region connected to the inlet portion and disposed in a second channel region connected to the heating unit and capable of passing a substance having a size corresponding to nucleic acid, And a nucleic acid separator disposed in a third channel region connected to the first filter and having a nucleic acid binding substance capable of specifically binding with the nucleic acid, the nucleic acid separator being disposed in a fourth channel region connected to the nucleic acid separator, And a second filter capable of passing a substance having a size corresponding to the nucleic acid.

The microfluidic chip may further include a nucleic acid separator disposed in a channel region connected to the heating unit and having a heating portion disposed in a channel region connected to the inflow portion and having a nucleic acid binding material capable of specifically binding to the nucleic acid, .

The microfluidic chip may further include a nucleic acid separator disposed in a channel region connected to the heating unit and having a heating unit disposed in a channel region connected to the inlet unit and having a nucleic acid binding material capable of specifically binding with the nucleic acid, And a second filter disposed in a channel region connected to the nucleic acid separation unit and capable of passing a substance having a size corresponding to the nucleic acid.

The fourth embodiment of the present invention includes the steps of providing a nucleic acid extracting apparatus according to the third embodiment of the present invention; Injecting a biological sample or reagent into said microfluidic chip for nucleic acid extraction through said multi-channel liquid distributor and fluid delivery means; And driving the microfluidic chip for nucleic acid extraction to extract nucleic acid from the biological sample.

The fifth embodiment of the present invention provides a nucleic acid extracting apparatus according to the third embodiment of the present invention; Injecting a biological sample or reagent into said microfluidic chip for nucleic acid extraction through said multi-channel liquid distributor and fluid delivery means; Driving the microfluidic chip for nucleic acid extraction to extract nucleic acid from the biological sample; And storing the nucleic acid extracted product in a liquid storage chamber of the liquid storage container.

An embodiment of the present invention relates to a multi-channel liquid dispensing apparatus, a nucleic acid extracting apparatus including the same, and a nucleic acid extracting method using the same. In this respect, in performing various biological reactions using a thin- It is possible to quickly dispense and dispense the same very small amount of liquid to the small one or more inflow portions and to accurately distribute a very small amount of liquid to the one or more inflow portions by only one user operation, Can be significantly shortened to allow rapid progression of a variety of subsequent biological detection or analysis reactions.

1 illustrates a multi-channel liquid dispensing apparatus in accordance with an embodiment of the present invention.
Figures 2 to 3 show the unit channel area of the channels of the multi-channel liquid distributor according to Figure 1.
Figures 4 to 5 schematically illustrate a microfluidic chip according to an embodiment of the present invention.
Figure 6 illustrates a multi-channel liquid dispensing device in accordance with an embodiment of the present invention.
FIGS. 7 to 10 illustrate a microfluidic chip according to an embodiment of the present invention and illustrate a nucleic acid extraction method using the same.
11 to 13 show a liquid storage container according to an embodiment of the present invention.
FIG. 14 shows a flow path of a liquid such as a biological sample or a reagent in a state where the microfluid chip and the liquid storage container are combined according to an embodiment of the present invention.
Figure 15 illustrates the path of movement of a liquid, such as a biological sample or reagent, in conjunction with a multi-channel liquid distributor, microfluidic chip, and liquid reservoir according to one embodiment of the present invention.
16 to 17 show results of nucleic acid extraction experiments using a third-party nucleic acid extracting apparatus and a nucleic acid extracting apparatus according to an embodiment of the present invention, respectively.

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

1 shows a multi-channel liquid dispensing apparatus 2 according to an embodiment of the present invention.

1, the multi-channel liquid distributor 2 comprises a substrate 1000 in the form of a thin film; A single liquid inlet (2000) disposed at one end region of the substrate; And one or more even liquid outlets (4000) disposed in the other end region of the substrate and in fluid communication with the single liquid inlet through a channel (3000), wherein the channel (3000) And one or more unit channel regions, each having one end connected to the single liquid injection port side and the other end divided into two to distribute the flow rate to one half, and having a channel pattern connected to the liquid discharge port side.

The substrate 1000 has a microspace, or channel, that is adapted to receive a very small amount of liquid and to allow the received liquid to migrate. The substrate 1000 is implemented in the form of a thin film like a thin plate, so that the amount of the sample or the reagent can be saved in a biological or biochemical reaction requiring a very small amount of liquid, and a minute amount of the substrate 1000 can be adjusted. The substrate 1000 supports the modules 2000, 3000, and 4000 because it functions as a base of a single liquid inlet 2000, a channel 3000, and a plurality of liquid outlets 4000 For example, plastic, silicon, metal, ceramics or the like, but the present invention is not limited thereto.

The multi-channel liquid distributor (2) is for simultaneously distributing the same amount of sample or reagent in a single operation to a reaction vessel having at least one inlet. Thus, the multi-channel liquid distributor 2 may have one, i.e., a single liquid inlet 2000 and a plurality of, e.g., one or more, even liquid outlet ports 4000 in fluid communication therewith . More specifically, the single liquid inlet 2000 is disposed at one end region of the substrate 1000, the at least one even liquid outlet 4000 is disposed at the other end region of the substrate, (3000) to be movable in fluid communication from the single liquid inlet (2000) to the at least one even liquid outlet (4000). The single liquid injection port 2000 and the at least one even liquid discharge port 4000 may be disposed on one surface of the thin film substrate 1000 and may be disposed on both surfaces of the thin film substrate 1000 May be disposed upward and downward, respectively. 1, a single liquid inlet 2000 of the multi-channel liquid distributor 2 and at least one even liquid outlet 4000 are formed on one surface of the thin film substrate 1000 As shown in Fig.

Figures 2-3 illustrate the unit channel region 3500 in the channel 3000 of the multi-channel liquid distributor 2 according to Figure 1.

2, the channel 3000 of the multi-channel liquid distributor 2 has one end connected to the single liquid inlet 2000 side and the other end divided into two to divide the flow rate into 1/2 And has at least one unit channel region 3500 having a channel pattern connected to the liquid outlets 4000a and 4000b. The unit channel region 3500 may be a region of the channel 3000 that is fluidly communicatively and continuously implemented from the single liquid inlet 2000 to the at least one even liquid outlet 4000. Accordingly, the unit channel region 3500 may be implemented in the multi-channel liquid distributor 2 to form a continuous flow path between the single liquid inlet 2000 and the liquid outlet 4000 . It is assumed that one unit channel region 3500 shown in FIG. One end of the one unit channel region 3500 is directly connected to the single liquid inlet 2000 and the other end is directly connected to the at least one even numbered liquid outlet 4000a and 4000b. In this case, when a user injects a certain amount of liquid F into the single liquid injection opening 2000, the injected liquid moves along the channel connected to one end of the unit channel region 3500 (F) The flow rate is divided by 1/2 (1/2 F) along the bifurcated channels to move to the two liquid outlets 4000a and 4000b. In this case, a general electric circuit formula can be used to distribute the dispensed flow rate exactly 1/2. That is, since the liquid introduced into the single liquid inlet 2000 has the same physical properties, the cross-sectional area of two channels divided into two is compared with the cross-sectional area of one channel before the division, taking into consideration the electric circuit formula and the resistance value thereof . ≪ / RTI >

Furthermore, the multi-channel liquid distributor 2 can determine the number of liquid distributions easily by predetermining the number of the one or more even liquid discharge ports 4000. Specifically, the liquid discharge port is implemented pieces 2 N, wherein the channel comprises a channel region formed by the N units of the 2 N of the liquid outlet from the single liquid inlet, the N or less the i-th unit of the channel region 2 i- is divided into two streams respectively from one start channel and the 2 i-1 of the start channel, including, but 2 i of branch channel for distributing a flow rate of 1/2, the first start of the second unit of the channel region is the channel-side ends as being associated with a single liquid injection port and, coupled to the N-th unit of the channel region of the branch channel ends each of the 2 N of the liquid outlet, wherein the N and i is a natural number of multi-channel liquid dispensing device can be implemented. For example, according to FIG. 3, when four liquid outlets 4000 are determined (4000a, 4000b, 4000c, and 4000d), the channel 3000 is separated from the single liquid inlet 2000 by four liquid outlets 4000a, And a first unit channel region 3500 of the unit channel regions includes one start channel 3100 and one start channel 3100. The first unit channel region 3500 includes two unit channel regions 3500 and 3500 ' 3100, and divides the flow rate by half, and the second unit channel region 3500 'of the unit channels includes two branch channels 3200, 1 / 2F, And four branch channels 3200 ', 1 / 4F for dividing the flow rate into two by two branches from the two start channels 3100' and the two start channels 3100 ', respectively. In this case, The end of the unit channel region 3500 corresponding to the start channel 3100 is connected to the single liquid inlet 2000 and the second unit channel region 35 00 ', the ends of the branch channels 3200' are connected to the four liquid outlets 4000a, 4000b, 4000c and 4000d.

Figures 4 to 5 schematically illustrate a microfluidic chip 1 according to one embodiment of the present invention.

The microfluidic chip 1 has one or more reaction channels 70 which are used for various reactions, for example biological or biochemical reactions, in which such reactions take place. 4 to 6, the microfluidic chip 1 has eight reaction channels 70, but 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 reagent is introduced into the reaction channel 70 through the inlet 10, And a liquid such as the biological reaction product or waste is discharged through the outlet 60. The microfluidic chip 1 may be formed in the form of a thin film such as a thin plate and may include a space capable of accommodating a small amount of liquid. The microfluidic chip 1 may be useful for biological reactions using very small quantities 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.

Figure 6 illustrates a multi-channel liquid dispensing device in accordance with an embodiment of the present invention.

6, a multi-channel liquid distribution injection apparatus according to an embodiment of the present invention includes a microfluidic chip having a thin film-shaped microfluidic chip having at least one and at most two reaction channels each having an inlet and an outlet, (1, 2, 3, 4) for injecting liquid downward into the at least one reaction channel through the at least one inlet (10), wherein the liquid outlets (4000, Channel liquid dispensing device (2) comprising: a) a multi-channel liquid dispensing device (2); And fluid delivery means (4) for fluidly connecting at least one inlet (10) of the microfluidic chip (1) and at least one liquid outlet (4000) of the multi-channel liquid distributor (2) do. In this case, the microfluidic chip 1 can be used for a nucleic acid extraction reaction, a polymerase chain reaction (PCR), and the like. In the multi-channel liquid distribution device 2 and the fluid delivery means 4 ) May be a sample or a reagent necessary for each reaction. The fluid delivery means 4 is associated with the orientation of the one or more even liquid outlets 4000 of the multi-channel liquid dispensing device 2 according to an embodiment of the present invention. 6, since the liquid discharge direction of the liquid discharge port 4000 is implemented upward and the inlet 10 of the thin-film shaped microfluidic chip 1 is also upward, In order to transfer the liquid from the liquid distributing device 2 to the microfluidic chip 1, there is a case where a separate fluid transmitting means 4 for connecting them in fluid communication is required. However, although not shown, if the liquid outlet 4000 of the multi-channel liquid dispensing apparatus 2 according to the embodiment of the present invention is downwardly implemented, and the liquid outlet 4000 of the odd- If the inflow portion of the microfluidic chip 1 is in close contact with the downflow liquid outlet 4000 and the inflow portion of the microfluidic chip 1 is in close contact with the inflow portion of the microfluidic chip 1, 4) may not be required.

FIGS. 7 to 10 illustrate a microfluidic chip according to an embodiment of the present invention and illustrate a nucleic acid extraction method using the same. 7 to 10, the microfluidic chip according to an embodiment of the present invention can be used for nucleic acid extraction. Hereinafter, in Figs. 7 to 10, the microfluidic chip is referred to as "microfluidic chip for nucleic acid extraction ".

The microfluidic chip for nucleic acid extraction is a structure for extracting nucleic acid, that is, an inlet, an outlet, a channel connecting the inlet and the outlet, a first filter, 2 filter or the like is implemented in units of millimeters (mm) or micrometers (탆).

7A, a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention includes an inlet 10, a channel region 70 connected to the inlet 10, Wherein the channel region includes a heating portion configured to transfer externally obtained heat to a biological sample introduced through the inlet portion, Various modules may be provided for efficiently extracting nucleic acid from a biological sample, such as 7g.

The microfluid chip for nucleic acid extraction according to an embodiment of the present invention shown in FIG. 7B has a heating unit 20 disposed in a first channel region connected to the inlet 10, and is connected to the heating unit 20 And a first filter (30) disposed in the second channel region and capable of passing a substance having a size corresponding to the nucleic acid. The microfluid chip for nucleic acid extraction according to an embodiment of the present invention shown in FIG. A heating unit 20 is disposed in a first channel region connected to the inlet unit 10 and a first channel region disposed in a second channel region connected to the heating unit 20 and capable of passing a substance having a size corresponding to a 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 with the nucleic acid, , And may be used for nucleic acid extraction according to an embodiment of the present invention shown in FIG. The three-flow chip has a heating portion 20 disposed in a first channel region connected to the inlet portion 10 and disposed in a second channel region connected to the heating portion 20, 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 with the nucleic acid is provided And a second filter 50 disposed in a fourth channel region connected to the nucleic acid separation unit 40 and capable of passing a substance having a size corresponding to the nucleic acid, The microfluid chip for nucleic acid extraction according to an embodiment of the present invention shown in FIG. 7E has a heating unit 20 disposed in a first channel region connected to the inlet 10, A material having a size corresponding to that of the nucleic acid can be passed through the second channel region And a nucleic acid-binding substance (45, membrane) provided in a third channel region connected to the first filter (10) and capable of specifically binding with the nucleic acid, And a second filter 50 disposed in a fourth channel region connected to the nucleic acid separation unit 40 and capable of passing a substance having a size corresponding to the nucleic acid, The microfluidic chip for nucleic acid extraction according to an embodiment of the present invention shown in FIG. 7f has a heating portion 20 disposed in a channel region connected to the inlet portion 10, and a channel region connected to the heating portion 20 And a nucleic acid separating unit 40 provided with a nucleic acid binding material 45 (membrane) capable of specifically binding with the nucleic acid. In addition, The microfluid chip for extraction has a channel region (10) connected to the inlet (10) A nucleic acid separation unit 40 having a heating unit 20 disposed in a channel region connected to the heating unit 20 and having a nucleic acid binding material 45 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 substance having a size corresponding to the nucleic acid.

The biological sample may be a biological sample containing 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.

The inlet (10) is a portion where the biological sample or a solution for nucleic acid extraction is introduced into the microfluidic chip, and the outlet (60) is a portion for extracting the nucleic acid, the solution for nucleic acid extraction, And other waste is discharged to the outside of the microfluidic chip. In this case, the inflow section 10 and the outflow section 60 may function as an outlet and an inflow section, respectively, if necessary. The solution for nucleic acid extraction includes all the solutions required for nucleic acid extraction, for example, distilled water, a nucleic acid binding buffer, an elution buffer, and the like. The inlet 10 and the outlet 60 are connected in fluid communication through a channel 70 and are connected to a heating unit 20, a first filter 30, The first filter 40, and the second filter 50 may be connected to the channel 70 to perform respective functions. The channel 70 may be implemented in various standards, but the width and depth of the channel are preferably within a range of 0.001 to 10 millimeters (mm), but the present invention is not limited thereto. The first, second, third, and fourth channel regions, which will be described below, mean sequential placement from the inlet 10 to the outlet 60, But is not limited to.

The heating unit 20 is disposed in a first channel region connected to the inlet 10, and is a part of the solution (including biological sample) introduced through the inlet unit 10, which is externally heated. For example, when a sample containing cells, bacteria, or viruses is introduced through the inlet 10, when the cell, bacteria, or virus reaches the heating unit 20, (° C) so that the outer wall of the cell, bacteria, or virus is destroyed and the substance in the cell is released to the outside (cell lysis). The heating unit 20 can receive heat from a heating module 600 of a nucleic acid extracting apparatus, which will be described later, in a contact or non-contact manner.

The first filter 30 serves to distinguish a passing material from a non-passing material by a size in a fluid flow direction. For example, the first filter 30 may be a structure having a pore of a predetermined size. In one embodiment of the present invention, the first filter 30 is disposed in a second channel region connected to the heating unit 20, and is configured to pass a substance having a size corresponding to the nucleic acid. The first filter (30) collects a substance of a size larger than a nucleic acid in a soluble product generated by heating in the heating unit (20) in the heating unit (20), and a nucleic acid and a substance having a size corresponding thereto pass And transfers it to the nucleic acid separator 40, which will be described below. The first filter 30 may be implemented in a variety of sizes but may include pores having a diameter in the range of 0.1 to 0.4 micrometers and may have a thickness in the range of 0.01 to 10 millimeters . More preferably, the first filter 30 has a pore having a diameter of 0.2 micrometers (mu m), and 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 to the nucleic acid. 7, the nucleic acid separating unit 40 is a space between the first filter 30 and the second filter 50 to be described below. The nucleic acid separating unit 40 includes a nucleic acid binding material 45 (specifically, . The nucleic acid binding material 45 may be any substance capable of specifically binding to the nucleic acid. The nucleic acid binding material 45 may be a silica (SiO 2) bead, a biotin, a strptavidin-attached bead, or a membrane, to which a nucleic acid binding functional group is attached, for example. The beads or membranes to which the nucleic acid-binding functional groups are attached may be implemented in various standards, but preferably have diameters in the range of 0.001 to 20 millimeters (mm). In addition, the nucleic acid separation unit 40 may include beads or membranes having the nucleic acid-binding functional groups in various amounts and sizes, but it is preferably within the range of 1 microgram (μg) to 200 milligram (mg). After the nucleic acid is specifically bound to the nucleic acid binding material 45 and then the inside of the nucleic acid separating part 40 is washed to remove the foreign substance, the nucleic acid binding material 40 is immersed in the target nucleic acid- Only the complex remains. Thereafter, when the elution buffer is provided to the nucleic acid separation unit 40, the target nucleic acid can be separated from the complex.

The second filter 50 serves to distinguish between a passing material and a non-passing material through the pores in the fluid flow direction. For example, the second filter 50 may be a structure having a pore of a predetermined size have. In an embodiment of the present invention, the second filter 50 is disposed in a fourth channel region connected to the nucleic acid separator 40, and is capable of passing a substance having a size corresponding to the nucleic acid. The second filter 50 collects the nucleic acid binding material 45 in the nucleic acid separator 40 and passes the nucleic acid separated from the nucleic acid binding material 45 to the outlet 60 . The second filter 50 may be implemented in a variety of sizes, but it is preferred to have a pore having a diameter in the range of 0.1 to 100 micrometers (mu m), with 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 micrometers (mu m), preferably a thickness of 0.3 millimeters (mm).

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

8 is a cross-sectional view of a microfluidic chip for nucleic acid extraction according to an embodiment of the present invention. The microfluidic chip for nucleic acid extraction according to an embodiment of the present invention comprises a first silver plate 100; A second plate (200) disposed on the first plate and having a channel (70) including the first to fourth channel regions disposed therein; And a third plate 300 disposed on the second plate 200 on which the inlet 10 and the outlet 60 are disposed. The microfluidic chip for nucleic acid extraction according to an exemplary embodiment of the present invention may be formed of various materials, but it may be formed of a plastic material. As described above, when a plastic material is used, the heat transfer efficiency can be increased only by adjusting the thickness of the plastic, and the manufacturing process can be simplified, thereby greatly reducing the manufacturing cost. The first plate 100 and the third plate 300 may be formed of a material selected from the group consisting of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate (PMMA) A material selected from the group consisting of polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the second plate 200 may be formed of a material selected from the group consisting of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA) (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyether Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT) A thermoplastic resin or a thermosetting resin material selected from the group consisting of fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof. Wherein the inlet of the third plate is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, the outlet is implemented within a range of 0.1 to 5.0 millimeters (mm) in diameter, and the thickness of the first and third plates Is realized within a range of 0.01 to 20 millimeters (mm), and the thickness of the second plate can be realized within a range of 30 micrometers (占 퐉) to 10 millimeters (mm). In addition, the microfluidic chip for nucleic acid extraction according to an embodiment of the present invention may be embodied as one or more inlet units, outlet units, and channels for connecting the microfluidic chips to one or more biological samples The nucleic acid can be extracted and the nucleic acid can be extracted quickly and efficiently.

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

Referring to FIG. 9, a nucleic acid extracting apparatus according to an embodiment of the present invention includes: a microfluidic chip 1 for nucleic acid extraction; A chip mounting module 500 configured to mount the microfluidic chip 1; A heating module 600 configured to apply heat to the heating unit 20 of the microfluidic chip 1 mounted on the chip mounting module 500; And a solution for nucleic acid extraction into the microfluidic chip 1 by being connected to the inflow part 10 and / or the outflow part 60 of the microfluidic chip 1 mounted on the chip mounting module 500, And / or a fluid control module 700 implemented to be able to discharge the solution present inside the microfluidic chip 1 to the outside.

The nucleic acid extracting apparatus is implemented to perform all steps for nucleic acid extraction in a state where the microfluidic chip 1 according to an embodiment of the present invention is mounted. The above-described chip mounting module 500, The heating module 600, and the fluid control module 700, as well as various modules required to extract other nucleic acids. In addition, the nucleic acid extracting apparatus according to an embodiment of the present invention can be implemented so that all the steps can be implemented in an automated manner, and the nucleic acid amplification reaction can be performed immediately after the nucleic acid extraction in conjunction with the PCR apparatus have.

The microfluidic chip 1 for nucleic acid extraction has already been described.

The chip mounting module 500 is a portion on which the microfluidic chip 1 is mounted. The chip mounting module 500 may be variously formed in correspondence with 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 variously implemented, but a contact heating block is preferred.

The fluid control module 700 is connected to the inlet 10 and / or the outlet 60 of the microfluidic chip 1 mounted on the chip mounting module 500, To introduce a solution for nucleic acid extraction into the microfluidic chip 1 and / or to discharge the solution present inside the microfluidic chip 1 to the outside. The fluid control module 700 may include various components including, for example, microchannels that are fluid passages, pneumatic pumps that provide the driving force of fluid movement, valves that can control the opening and closing of fluid movement, A storage chamber containing various solutions required for nucleic acid extraction such as binding buffer, elution buffer, silica gel, distilled water (DW), and the like.

Meanwhile, the nucleic acid extracting apparatus according to an embodiment of the present invention includes an electronic control module (not shown) for automatically controlling the microfluid chip 1, the heating module 600, and the fluid control module 700 ). ≪ / RTI > The electronic control module can precisely control the modules so that a predetermined amount of nucleic acid can be extracted in the microfluidic chip 1 according to a program stored in advance. The pre-stored program includes, for example, a program for a series of steps relating to a nucleic acid extraction method, which will be described in detail below.

10 is a flowchart of a nucleic acid extraction method according to an embodiment of the present invention. Specifically, FIGS. 10A to 10D show various nucleic acid extraction methods based on the microfluidic chip 1 for nucleic acid extraction according to an embodiment of the present invention.

Referring to FIG. 10A, a method of extracting a nucleic acid from a biological sample according to an embodiment of the present invention includes: providing a microfluidic chip for nucleic acid extraction according to FIG. 7F (providing a microfluidic chip); Introducing a biological sample selected from the group consisting of cells, bacteria, and virus (biological sample introduction step) through the inlet of the microfluidic chip; Moving the introduced biological sample to a heating section of the microfluidic chip and then applying heat to the heating section of the microfluidic chip to lyse the biological sample (biological sample dissolution step); Separating the nucleic acid from the dissolving material through a nucleic acid binding material (membrane) (nucleic acid separation step); As a selectable step, there is a step of removing foreign substances generated in the nucleic acid separation process (a foreign substance removing step); And a step (nucleic acid extraction step) of transferring the nucleic acid to the outflow portion and then extracting the nucleic acid through the outflow portion.

Referring to FIG. 10B, a method of extracting nucleic acid from a biological sample according to an embodiment of the present invention includes the steps of providing a microfluidic chip for nucleic acid extraction according to FIG. 7B or 7C (providing a microfluidic chip); Introducing a biological sample selected from the group consisting of cells, bacteria, and virus (biological sample introduction step) through the inlet of the microfluidic chip; Moving the introduced biological sample to a heating section of the microfluidic chip and then applying heat to the heating section of the microfluidic chip to lyse the biological sample (biological sample dissolution step); Moving the material obtained from the dissolving step to a first filter of the microfluidic chip, passing through the first filter, and removing material that has not passed through the first filter (filtration through a first filter ); Separating the nucleic acid from the substance that has passed through the first filter (nucleic acid separation step); As a selectable step, there is a step of removing foreign substances generated in the nucleic acid separation process (a foreign substance removing step); And a step (nucleic acid extraction step) of transferring the nucleic acid to the outflow portion and then extracting the nucleic acid through the outflow portion.

Referring to FIG. 10C, a method of extracting nucleic acid from a biological sample according to an embodiment of the present invention includes the steps of providing a microfluidic chip for nucleic acid extraction according to FIG. 7G (providing a microfluidic chip); Introducing a biological sample selected from the group consisting of cells, bacteria, and virus (biological sample introduction step) through the inlet of the microfluidic chip; Moving the introduced biological sample to a heating section of the microfluidic chip and then applying heat to the heating section of the microfluidic chip to lyse the biological sample (biological sample dissolution step); Separating the nucleic acid from the dissolution material through a nucleic acid binding material (bead) (nucleic acid separation step); As a selectable step, there is a step of removing foreign substances generated in the nucleic acid separation process (a foreign substance removing step); Separating the nucleic acid from the nucleic acid binding material, transferring the separated nucleic acid to the second filter, and passing the separated nucleic acid through a second filter (filtering step through a second filter); And a step (nucleic acid extraction step) of transferring the nucleic acid to the outflow portion and then extracting the nucleic acid through the outflow portion.

Referring to FIG. 10d, a method of extracting nucleic acid from a biological sample according to an embodiment of the present invention includes the steps of providing a microfluidic chip for nucleic acid extraction according to FIG. 7d or 7e (providing a microfluidic chip); Introducing a biological sample selected from the group consisting of cells, bacteria, and virus (biological sample introduction step) through the inlet of the microfluidic chip; Moving the introduced biological sample to a heating section of the microfluidic chip and then applying heat to the heating section of the microfluidic chip to lyse the biological sample (biological sample dissolution step); Moving the material obtained from the dissolving step to a first filter of the microfluidic chip, passing through the first filter, and removing material that has not passed through the first filter (filtration through a first filter ); Separating the nucleic acid from the dissolution material through a nucleic acid binding material (bead or membrane) (nucleic acid separation step); As a selectable step, there is a step of removing foreign substances generated in the nucleic acid separation process (a foreign substance removing step); Separating the nucleic acid from the nucleic acid binding material, transferring the separated nucleic acid to the second filter, and passing the separated nucleic acid through a second filter (filtering step through a second filter); And a step (nucleic acid extraction step) of transferring the nucleic acid to the outflow portion and then extracting the nucleic acid through the outflow portion.

11 to 13 show a liquid storage container according to an embodiment of the present invention.

11, the liquid storage container 5000 is used for storing and storing the reaction product after the reaction by the microfluidic chip 1 is completed. The liquid storage container 5000 may include one or more outflow ports (not shown) of the microfluidic chip 1 A chip outlet region end mounting portion 5100 implemented to be fixedly mounted at an end of the at least one outflow portion 60 of the microfluidic chip 1, at least one upward liquid inlet 5200 corresponding to an upper end of at least one outflow portion 60 of the microfluidic chip 1, And at least one liquid storage chamber 5300 fluidly connected to the at least one upward liquid inlet 5200. [ FIGS. 12 to 13 illustrate a process in which the liquid discharged through one or more outlet portions 60 of the microfluidic chip 1 moves in the liquid storage container 5000. For example, after the nucleic acid extraction reaction is completed in the microfluidic chip 1, the liquid storage container 5000 is fixedly mounted on the chip outlet region end mounting portion 5100, and a solution E1 and E2 are discharged through the at least one outlet 60, the nucleic acid-containing solutions E1 and E2 are introduced through one or more upward liquid inlets 5200 of the liquid storage container 5000, (F1, F2) through the channels in the liquid storage vessel 5000 to reach the one or more liquid storage chambers 5300 (S1, S2). Thereafter, the nucleic acid-containing solution can be separately stored and stored by separating the liquid storage container 5000 from the microfluidic chip 1, and the beneficial effect of further utilizing the nucleic acid-containing solution in the subsequent nucleic acid detection and analysis process . FIG. 14 shows a flow path of a liquid such as a biological sample or a reagent in a state where the microfluidic chip 1 and the liquid storage container 5000 are combined according to an embodiment of the present invention. In this case, a driving force enabling the continuous movement of the liquid in the microfluidic chip 1 and the liquid storage container 5000 is transmitted to the microfluidic chip 1 through the one or more inflow parts 10 of the microfluidic chip 1, A multi-channel liquid distributor 2 according to one embodiment, or any pump or syringe. Fig. 15 is a schematic view showing a flow path of a liquid such as a biological sample or reagent in a state where the multi-channel liquid distributing apparatus 2, the microfluidic chip 1, and the liquid storage container 5000 according to the embodiment of the present invention are combined. / RTI >

Based on the above-described nucleic acid extracting apparatus, an embodiment of the present invention can provide a convenient, quick and efficient ultra-fast nucleic acid extracting method. For example, the first nucleic acid extraction method comprises the steps of: providing the nucleic acid extraction apparatus described above; Injecting a biological sample or reagent into said microfluidic chip for nucleic acid extraction through said multi-channel liquid distributor and fluid delivery means; And driving the microfluidic chip for nucleic acid extraction to extract nucleic acid from the biological sample. The second nucleic acid extraction method may further include the steps of: providing the nucleic acid extraction device; Injecting a biological sample or reagent into said microfluidic chip for nucleic acid extraction through said multi-channel liquid distributor and fluid delivery means; Driving the microfluidic chip for nucleic acid extraction to extract nucleic acid from the biological sample; And storing the nucleic acid extracted product in a liquid storage chamber of the liquid storage container.

Hereinafter, in Examples 1 and 2, the amount and time of the nucleic acid extract were determined while extracting the nucleic acid from the biological sample in comparison with the third-party nucleic acid extracting apparatus (Qiagen), and further, The reliability of the result of the extract was confirmed again.

Example  1. Identification of nucleic acid extraction yield and run time

First, DNA is extracted from a tubercle main cell using a general tube included in a third-party product and a microfluidic chip 1 for nucleic acid extraction according to an embodiment of the present invention, Respectively.

The steps of nucleic acid extraction using a third-party nucleic acid separator are as follows. The Mycobacterium tuberculosis main cell was prepared and mixed with 6% NaOH and 4% NaLC at a ratio of 1: 1: 1 to prepare a sample solution. The sample solution was then centrifuged to remove supernatant (10 min, 7500 rpm, 4 캜). Then, 20 占 퐇 of Proteinase K was added to the sample solution and left at 56 占 폚 until the sample solution became transparent. Then, 200 μl of 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 (8000 rpm) for 1 minute. Then, 500 μl of AW 1 buffer was added, followed by centrifugation (8000 rpm) for 1 minute. Then, 500 μl of AW 2 buffer was added, followed by centrifugation for 1 minute (14,000 rpm). Thereafter, centrifugation was again carried out for 1 minute (14,000 rpm). The column was then placed in a new tube and 200 A of AE buffer was added and left 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, nucleic acids were extracted from the same M. tuberculosis cell using the multi-channel liquid distributor 2, the fluid delivery means 4, and the microfluidic chip 1 for nucleic acid extraction according to an embodiment of the present invention, The detailed procedure is as follows.

The Mycobacterium tuberculosis main cell was prepared and mixed with 6% NaOH and 4% NaLC at a ratio of 1: 1: 1 to prepare a sample solution. Thereafter, it was introduced into at least one inlet of a microfluidic chip for nucleic acid extraction (25 x 72 x 2 mm, silica bead (OPS Diagnostics, LLC), filter (Whatman)) according to Fig. 300 [micro] l of silica gel and 1X DNA binding buffer were introduced into the microfluidic chip inflow section according to the instant embodiment of the present invention, and then the heating section of the microfluid chip according to the instant embodiment of the present invention was heated to 95 [ . Thereafter, wastes in the sample solution were removed through an inlet of the microfluidic chip according to a temporary example of the present invention, and 100 μl of an elution buffer was introduced. In this case, the reagent was introduced into the microfluidic chip for nucleic acid extraction using the multi-channel liquid distributor 2 and the fluid delivery means 4 according to an embodiment of the present invention. Thereafter, the final product was obtained through the outflow of the microfluidic chip according to the instant 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 obtain the final DNA product.

As a result of the above experiment, when the multi-channel liquid distributor 2, the fluid delivery means 4 and the microfluidic chip 1 for nucleic acid extraction according to the embodiment of the present invention are used, The total amount of time required can be significantly reduced, unlike the conventional nucleic acid extraction method.

Example  2. Third party products and work of the invention In the embodiment  Obtained by the nucleic acid extraction method according to DNA  Polymerase chain reaction of products PCR ) result

In order to ensure the reliability of the DNA product obtained in Example 1, PCR was carried out based on the DNA product.

The polymerase chain reaction (PCR) was performed using a third-party product (Bio-Rad: CFX connect equipment). PCR samples and reagents for PCR were prepared by mixing 10 μl of real-time PCR mixed solution (TOYOBO SYBR qPCR mix), 2 μl of forward primer (μl), reverse primer (Reverse) A total of 20 microliters (占 퐇) including 2 microliters (쨉 l) of primer, 10 占), 1 microliter (쨉 l) of template DNA (1ng) and 5 占 퐇 l of distilled water . Thereafter, pre-denaturation step (95 ° C, 30 sec) was performed (1 cycle), denaturation step at 95 ° C for 5 sec and anealing & extension step at 72-65 ° C for 30 sec cycle.

FIG. 16 is a graph showing the results of real-time PCR on the nucleic acid-extracted products obtained using the nucleic acid extraction method and fluorescence of each PCR cycle. FIG. Figure 17 is a photograph of gel electrophoresis of the final PCR product. The graph curve of FIG. 16 is a PCR result curve (X axis: cycle, Y axis: fluorescence) of the DNA product by each nucleic acid extraction method.

Classification (gDNA copies / rxn) Ct value Non template (1) - Plasmid DNA (1 x 10 5 copies) (2) 18.32 1 x 10 6 (3) 16.77 1 x 10 3 (4) 26.00

(1) is a negative control group, and (2) is a positive control group.

The nucleic acid extracting method using the nucleic acid extracting apparatus according to an embodiment of the present invention can maintain or improve the result reliability of the nucleic acid extract product and significantly reduce the nucleic acid extracting step, I can confirm that I can do it.

Claims (12)

A thin film substrate;
A single liquid inlet disposed at one end region of the substrate; And
At least one liquid outlet disposed in the other end region of the substrate and fluidly connected through the channel with the single liquid inlet;
, ≪ / RTI &
The channel includes at least one unit channel region having one end connected to the single liquid injection port side and the other end divided into two to distribute the flow rate to one half and having a channel pattern connected to the liquid discharge port side However,
Multi-channel liquid distributor. The method according to claim 1,
The liquid discharge port is implemented pieces 2 N, wherein the channel and wherein the N or less the i-th unit of the channel region is 2 i-1 of start of the N units of the channel region formed by the 2 N of the liquid outlet from the single liquid inlet channel and the 2 i-1 from the start of the channel is divided into two branches each comprising a 2 i of branch channels for distributing the flow rate of 1/2, the first unit of the start of the channel section a channel-side terminal is the single liquid inlet is connected to the N-th unit as a channel region of the branch channel ends are respectively connected to the 2 N of the liquid outlet, wherein the N and i is the one which, the natural-channel liquid dispensing device. A microfluidic chip in the form of a thin film having one or more even-numbered reaction channels having inlet and outlet sections at both ends, the microfluidic chip comprising: at least one inlet for injecting liquid downward into the at least one inlet channel; As such,
The multi-channel liquid distributor according to any of the preceding claims, comprising a liquid outlet coinciding with the at least one inlet number; And
Fluid delivery means for fluidly communicating at least one inlet of the microfluidic chip and at least one liquid outlet of the multi-channel liquid distributor;
Channel liquid dispensing device. A channel region connected to the inlet, and an outlet connected to the channel region, wherein the channel region comprises a heat source for externally obtaining a biological sample introduced into the biological sample through the inlet, A microfluidic chip for nucleic acid extraction in the form of a thin film having at least one reaction channel;
The multi-channel liquid distributor according to any of the preceding claims, comprising a liquid outlet coinciding with the at least one inlet number; And
Fluid delivery means for fluidly communicating at least one inlet of the microfluidic chip and at least one liquid outlet of the multi-channel liquid distributor;
And a nucleic acid extracting device. 5. The method of claim 4,
A chip outlet region end mount adapted to fixably mount 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 of the microfluidic chip, Further comprising a liquid reservoir having at least one liquid storage chamber fluidly connected to the upstream liquid inlet. 5. The method of claim 4,
The microfluidic chip may include a first filter disposed in a first channel region connected to the inlet portion and disposed in a second channel region connected to the heating unit and capable of passing a substance having a size corresponding to the nucleic acid, Wherein the nucleic acid extracting device is a nucleic acid extracting device. 5. The method of claim 4,
The microfluidic chip may include a first filter disposed in a first channel region connected to the inlet portion and disposed in a second channel region connected to the heating unit and capable of passing a substance having a size corresponding to nucleic acid, And a nucleic acid separator disposed in a third channel region connected to the first filter and having a nucleic acid binding substance capable of specifically binding to the nucleic acid. 5. The method of claim 4,
The microfluidic chip may include a first filter disposed in a first channel region connected to the inlet portion and disposed in a second channel region connected to the heating unit and capable of passing a substance having a size corresponding to nucleic acid, And a nucleic acid separator disposed in a third channel region connected to the first filter and having a nucleic acid binding substance capable of specifically binding with the nucleic acid, the nucleic acid separator being disposed in a fourth channel region connected to the nucleic acid separator, And a second filter capable of passing a substance of a size corresponding to the nucleic acid. 5. The method of claim 4,
Wherein the microfluid chip includes a nucleic acid separator disposed in a channel region connected to the heating unit and having a heating portion disposed in a channel region connected to the inflow portion and having a nucleic acid binding material capable of specifically binding with the nucleic acid, Wherein the nucleic acid extracting device is a nucleic acid extracting device. 5. The method of claim 4,
The microfluidic chip may further include a nucleic acid separator disposed in a channel region connected to the heating unit and having a heating unit disposed in a channel region connected to the inlet unit and having a nucleic acid binding material capable of specifically binding with the nucleic acid, And a second filter disposed in a channel region connected to the nucleic acid separation unit and capable of passing a substance having a size corresponding to the nucleic acid. Providing a nucleic acid extraction device according to claim 4;
Injecting a biological sample or reagent into said microfluidic chip for nucleic acid extraction through said multi-channel liquid distributor and fluid delivery means; And
Driving the microfluidic chip for nucleic acid extraction to extract nucleic acid from the biological sample;
/ RTI > Providing a nucleic acid extraction device according to claim 5;
Injecting a biological sample or reagent into said microfluidic chip for nucleic acid extraction through said multi-channel liquid distributor and fluid delivery means;
Driving the microfluidic chip for nucleic acid extraction to extract nucleic acid from the biological sample; And
Storing the nucleic acid extracted product in a liquid storage chamber of the liquid storage container;
/ RTI >
KR1020130060993A 2012-05-30 2013-05-29 Multi-channel device for distributing liquid sample, device for extracting nucleic acid comprising the same, and method for extracting nucleic acid using the same KR20130135111A (en)

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