KR20140071213A

KR20140071213A - Reagent container for amplification nucleic acid, method for manufacturing the same, method for storing the reagent, and micro-fluidic system for analysis of nucleic acid

Info

Publication number: KR20140071213A
Application number: KR1020130029921A
Authority: KR
Inventors: 정성욱; 지성민; 이수관; 권성홍; 김준호; 남궁각; 박진성; 황규연
Original assignee: 삼성전자주식회사
Priority date: 2012-12-03
Filing date: 2013-03-20
Publication date: 2014-06-11
Also published as: KR102046101B1

Abstract

The present invention provides a reagent container capable of maintaining the stability of a reagent, a manufacturing method of the reagent container, a method for storing the reagent, and a micro-fluid system which performs cell capture, destruction, and nucleic acid extraction and amplification. According to the method for storing the reagent, the stability of the reagent is maintained and the activity is maintained in a reaction using the reagent.

Description

TECHNICAL FIELD [0001] The present invention relates to a reagent container for nucleic acid analysis, a reagent container manufacturing method, a reagent storing method, and a microfluidic system for nucleic acid analysis. for analysis of nucleic acid}

A reagent vessel for amplifying a nucleic acid, a method for producing the reagent vessel, a method for storing the reagent, and a microfluid system for performing cell capture, destruction, nucleic acid extraction and amplification.

Polymerase chain reaction (PCR) is a method of amplification of a specific target genetic material that is desired to be detected, which is used in almost all processes of manipulating genetic material. Polymerase chain reaction can amplify large amounts of the same genetic material from a small amount of genetic material, so it is used to amplify human genetic material and diagnose various genetic diseases. In addition, it can be applied to the genetic material of bacteria, viruses and fungi to be used for diagnosis of infectious diseases.

In addition, diagnostic devices are becoming smaller and more automated due to the user's safety and convenience and the need for rapid on-site testing (POCT). Such miniaturized and automated diagnostic devices are preferred to liquid reagents rather than solid reagents, i.e., lyophilized reagents. This is because liquid reagents are difficult to store and less stable, while solid reagents increase shelf life and reduce the size of the product itself due to the small storage container volume. Studies are being conducted to prepare reagents and PCR premixes used in PCR as solid reagents.

On the other hand, in order to accurately determine the presence of a specific DNA or the amount of DNA in the sample, it is required to sufficiently amplify the actual sample so that it can be measured after purification / extraction. For the PCR process, it is necessary to perform a process of capturing cells from a biological sample, a process of extracting nucleic acid through cell disruption, and a process of mixing nucleic acid with a PCR reagent.

One aspect provides a reagent vessel for nucleic acid amplification.

Another aspect provides a microfluidic system for nucleic acid analysis capable of performing a series of steps of capturing cells from a sample, disrupting the captured cells to extract nucleic acids, and performing a nucleic acid amplification reaction in one device.

Another aspect provides a method of producing the reagent vessel for amplifying the nucleic acid.

Another aspect provides a method of storing the nucleic acid amplification reagent using the reagent vessel.

One aspect is a reagent vessel comprising a first well in which a first reagent is received, a first well in which the first reagent comprises a nucleotide or a nucleic acid component, and a second well in which the second reagent is received, The two wells comprise a reaction buffer, and the first reagent and the second reagent are nucleic acid amplification reagents.

In the reagent vessel, the nucleotide or nucleic acid component may be a nucleotide, a deoxynucleotide, or a ribonucleotide triphosphate, a primer, and a probe nucleic acid. The first reagent may further comprise an enzyme.

The shape of the first well and / or the second well on the plan view may vary. The shape may be circular, paired circular, elliptical or polygonal. For example, it may be a square, or a pentagon. The size of the first well and / or the second well may be a well in the microliter range. For example, the size of the well can be from 1 to 10 ul, such as 1 to 9 ul, 1 to 8 ul, 1 to 7 ul, 1 to 6 ul, 1 to 5 ul, 1 to 4 ul, mu] l, or a volume of 1-2 [mu] l.

The vessel may further include a first aperture connected to the first well, and a second aperture connected to the second well. The first opening may be disposed substantially spaced apart from the first well, or may be fluidly connectable. The second opening may be disposed substantially spaced apart from the first well, or may be fluidly connectable. The first and second openings may operate with an inlet and / or an outlet of the reagent vessel. The first and second openings may be located on top of the reagent vessel. The first and / or second opening may be one in which the top is open.

In the reagent vessel, the vessel may be configured to be mounted in the rehydration chamber. When the container is mounted in the rehydration chamber, the container can act as a cover, i.e. the container can be a rehydration cover.

The first and second openings may be configured to be fluidly connected to the rehydration chamber. The reagent vessel may be turned upside down and mounted to the rehydration chamber so that the open top opening may contact the outer surface of the rehydration chamber to form one channel. The rehydration chamber may be one that can be used in a device for analyzing nucleic acid and / or a microfluidic system. The nucleic acid analysis may include polymerase chain reaction (PCR).

The container may further include a connection portion connecting the first well and the second well to each other. The connecting portion may be a groove, a channel, a partition wall, or a membrane. The groove may be open at the top. The channel may further include a valve therein. The valve may be opened or closed. The valve that can be opened and closed can be appropriately selected by those skilled in the art. The barrier may be defined by a common sidewall in which the first and second wells are in contact with each other, and the height of the barrier may be smaller than the height of the first and second wells. The film may be a friable film. The film can be a fragile film that can be easily broken by the pressure of the fluid being injected. The membrane may be a porous membrane.

The reagent vessel is provided with a plurality of protrusions, and the first well and the second well may be two sub grooves separated from each other, which are formed in the grooves drawn in a predetermined shape in the protrusions. The side surfaces of the first well and the second well may be curved, and the width of the center portion may be the narrowest. The outer edges of both side edges of the positions forming the narrowest widths of the first well and the second well may be in the range of 30 degrees to 90 degrees.

In the reagent vessel, the nucleic acid amplification reagent may be a polymerase chain reaction premix.

As used herein, the term "primer" is intended to encompass all types of nucleotides, such as four different nucleoside nucleotides, deoxynucleotides, or ribonucleotide triphosphates and DNA, RNA polymerase or reverse transcriptase Polymerases and single-stranded oligonucleotides which can act as a starting point for template-directed DNA synthesis under appropriate temperatures. The appropriate length of the primer may vary depending on the purpose of use, but may be 15 to 30 nucleotides. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. This primer is used in pairs with a second primer that hybridizes to the opposite side.

As used herein, the term "probe" refers to a polynucleotide capable of specifically binding to a specific target nucleic acid and confirming the presence of the target nucleic acid. The probe may be a single stranded nucleic acid. "Target nucleic acid" refers to a nucleic acid to be analyzed. The nucleic acid may contain a sequence complementary to the probe nucleic acid. The target nucleic acid may comprise a sequence complementary to the probe nucleic acid, and when hybridized with the probe nucleic acid, a nucleic acid composed of a sequence having an internal mismatch of 0 to 5 bp. The probe nucleic acid may be labeled with a detectable label. The detectable label is known. For example, the label may be selected from a label for generating an optical signal, a radioactive label, and a label for generating an electrical signal. For example, the label may be a fluorescent material that generates a fluorescent signal. The fluorescent material may include Cal610, fluorescein, rhodamine, cyanines including Cy3 and Cy5, and metal porphyrin complex. Examples of fluororesin dyes include 6-carboxyl fluorosine (6-FAM) 1,2 ', 4', 1,4, -tetrachlorofluorescein (TET) and 2 ', 4' , 7 ', 1,4'-hexachlorofluorescein (HEX), 2', 7'-dimethoxy-4 ', 5'-dichloro-6- carboxydodamine (JOE) Fluoro-7 ', 8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein, 2'-chloro-7'- . The detectable signal material may be attached to an atom in the nucleobase of the probe nucleic acid.

The enzyme may be an enzyme commonly used in the art. The enzyme may be selected from the group consisting of DNA polymerase, reverse transcriptase, RNA polymerase, RNAase H, and combinations thereof. The DNA polymerase may be a DNA polymerase that can be used for polymerase chain reaction (PCR). The DNA polymerase may be thermally stable. The DNA polymerase may be a DNA polymerase isolated from thermophiles. The DNA polymerase may be a DNA polymerase isolated from Thermus aquaticus or Thermococcus litoralis.

The nucleotide, deoxynucleotide, or ribonucleotide triphosphate may be NTP, dNTP, or rNTP. NTP refers to the nucleotide triphosphates of ATP, CTP, GTP and TTP. dNTP refers to deoxynucleotide triphosphates of dATP, dCTP, dGTP and dTTP. rNTP refers to ribonucleotide triphosphates of rATP, rCTP, rGTP and rTTP. The nucleotide, deoxynucleotide, or ribonucleotide triphosphate may or may not be labeled with a detectable label. The above-described detectable label is as described above.

The first reagent may further comprise a stabilizer. The safener may be an enzyme stabilizer. The enzyme stabilizing agent may be a substance that assists in maintaining the activity of the enzyme. The enzyme stabilizing agent may be an enzyme stabilizing agent selected from the group consisting of glycerol, glucose, sucrose, fructose, sorbitol, trehalose, raffinose, meleletose or a combination thereof. The stabilizing agent can stabilize the activity of the enzyme. The stabilizer may be a lyophilized stabilizer in a concentrated state above the concentration used in the reaction.

The buffer is to provide buffer conditions that allow the polymerase to have activity. The buffer can be suitably selected by those skilled in the art according to the polymerase chosen. For example, the buffer may be a polymerase buffer that is commercially available for the selected polymerase. For example, it may be a polymerase buffer provided for a Taq polymerase. The buffer may be one that provides a buffering condition that allows the polymerase to be active and a buffering condition that allows the ligase to have activity. In general, the buffering conditions for the polymerase are considered to be compatiable with the buffering conditions for the ligase. The buffer may be, for example, MgCl2, Na2HPO4, NaH2PO4, MOPS-KOH, HEPES-NaOH, tris (hydroxymethyl) aminomethane-HCl, borate or glycine-NaOH. The buffer may be a lyophilized buffer in a concentrated state above the concentration used in the reaction.

In the reagent vessel, the first solid reagent or the second solid reagent may further include an additive. The additive may be an antifoamer or a surfactant. The antifoaming agent or the surfactant can be appropriately selected by those skilled in the art. The first reagent or the second reagent may further comprise other components required for the reaction, for example, water, a substrate, a cofactor, or a coenzyme. The water may be sterilized distilled water.

In the reagent vessel, the first reagent may be a solid reagent. The second reagent may be a solidified reagent. The first reagent and / or the second reagent may be a dried reagent. The drying may include natural drying, freeze drying, or vacuum drying. The drying can be appropriately selected by those skilled in the art. The second reagent may be a liquid reagent. The reagent vessel may be a reagent vessel for storing the reagent. The enzyme; Nucleotides, deoxynucleotides, or ribonucleotide triphosphates; primer; Probe; buffer; Stabilizers; Or the additive may be lyophilized in a concentrated state above the concentration used in the reaction. The buffer has a concentration of from about 1.5 to about 2.5. 1.6 to 2.4, 1.7 to 2.3, 1.8 to 2.2, and 1.9 to 2.1 times in concentration.

The reagent vessel may be made of a material which is easy to mold and whose surface is biologically inert. The container may be made of a material having chemical or biological stability. The container may be made of a material having mechanical workability. The container may be made of an optically transparent material. The container may be formed from a polymeric material. The polymer may be selected from polypropylene, polyethylene, polystyrene, polymethyl methacrylate, polyolefins, and combinations thereof. The polymer may be an oxygen containing polymer. The oxygen may be oxygen of siloxane, carbonyl, ester, or ether. The polymer may comprise a polysiloxane. The polymer may include PDMS (polydimethylsiloxane), PMPS (polymethylphenylsiloxane), polydimethyldiphenylsiloxane, or PVS (polyvinylsiloxane). The polymer may be a silicone polymer comprising an alkylsiloxane or an organosiloxane, generally described as a polysiloxane.

The reagent vessel may further include a plurality of wells. The plurality of wells may be aligned with each other in the X-axis direction, the Y-axis direction, or the other direction. The plurality of wells may separately contain the reagents in the first reagent, which are mixed when they are mixed together and dried.

Another aspect relates to a nucleic acid analysis microfluidic device comprising a rehydration chamber, the reagent vessel mounted in the rehydration chamber, an amplification chamber, and a flow system forming an integrated fluid flow between the rehydration chamber and the amplification chamber. The system of claim 1, wherein the rehydration chamber comprises a cell lysate, a nucleic acid amplification reagent in the reagent vessel to form an amplification reaction mixture, and the amplification chamber comprises a nucleic acid amplification And the reaction proceeds.

The plurality of rehydration chambers may each include two separate sub-chambers. The nucleic acid amplification reagent may be divided into two sub-chambers. The two sub-chambers correspond to the first and second wells of the reagent vessel, respectively, and may accommodate the first well and the second well, respectively.

A nucleic acid amplification reagent may be disposed in each of the plurality of rehydration chambers.

The side surface of the sub chamber may have a curved shape, and the width of the flow path of the inflowed nucleic acid dissolved product may be the narrowest at the center.

A plurality of second through holes are formed to form a space of the plurality of rehydration chambers. The reagent container, that is, the rehydration cover covers the plurality of second through-holes, a plurality of protrusions are formed at positions corresponding to the plurality of second through-holes, a plurality of protrusions Grooves < / RTI >

The diameter of the protrusion may be greater than the diameter of the second through-hole, and the protrusion may be fitted in the second through-hole to seal the groove.

A plurality of grooves drawn in a predetermined shape at positions corresponding to the plurality of second through holes are formed so as to cover the plurality of second through holes and the nucleic acid amplification reagents are arranged in a lyophilized state in the grooves A rehydration cover may be provided.

Each of the plurality of grooves includes two sub-grooves separated from each other, and the nucleic acid amplification reagent may be divided into the two sub-grooves. The two sub-grooves may be the first well and the second well of the reagent vessel.

The nucleic acid amplification reagent can be disposed in the first well and the second well in each of the plurality of grooves and is the same as the nucleic acid amplification reagent disposed in the first well and the second well.

The side surface of the sub-groove may have a curved shape and the width of the center portion may be the narrowest.

The outer angle of both side edges of the position forming the narrowest width of the sub-groove may be in the range of 30 DEG to 90 DEG.

The microfluidic system for nucleic acid analysis includes a reagent supply device having a sample chamber into which a sample to be examined is injected, a plurality of reagent chambers into which a reagent for extracting nucleic acid from the sample is injected, and a waste chamber in which used reagent is discarded; A binding-lysis chamber in which a plurality of particles for capturing a cell are placed, and a binding-lysis chamber in which a plurality of particles for capturing a cell are placed, wherein a cell is captured from the sample, and the captured cells are disrupted to form a cell lysate containing nucleic acid ; A plurality of rehydration chambers for mixing the cell lysate and the nucleic acid amplification reagent in the reagent vessel to form an amplification reaction mixture; The reagent vessel mounted in the rehydration chamber; A plurality of nucleic acid amplification chambers for performing a nucleic acid amplification reaction on the amplification reaction mixture introduced from the plurality of rehydration chambers; A flow path system for forming an integrated fluid flow between the binding-lysis chamber, the rehydration chamber, and the nucleic acid amplification chamber, the flow path system including an outlet connected to the reagent supply device and a plurality of inlets, System.

The cell lysate may be formed in the binding-lysis chamber. The cell lysate may be dispensed into the plurality of rehydration chambers.

The plurality of reagent chambers may include a lysis buffer chamber into which a lysis buffer is injected and a washing buffer chamber into which a washing buffer is injected.

Wherein the sample chamber, the lysis buffer chamber, and the bottom surface of the washing buffer chamber are each provided with a crushing pattern which is broken by an external impact and discharges the injected solution to the outside, A needle may be used.

The bottom surface of the waste chamber is formed with a crushing pattern that is broken by an external impact, and the outlet may have a needle shape that impacts the crushing pattern.

One or more metering chambers may be further provided for quantifying the amount of lysis buffer supplied from the lysis buffer chamber of the reagent supply apparatus.

In the binding-lysis chamber, one or more bubble trap chambers may be further provided to remove bubbles that may occur upon cell disruption.

The particles provided in the binding-lysis chamber may have a diameter of from 1 to 1000 μm, and the amount of the particles may be from 1 to 100 mg.

A plurality of metering chambers for quantifying the amount of cell lysate formed in the binding-lysis chamber and distributing the lysate to the plurality of rehydration chambers may be further provided.

The microfluidic system for nucleic acid analysis has a first through hole formed in the upper surface of the binding-lysis chamber space and an inlet and an outlet connected to the reagent supply device. The first through hole forms the binding- A fluid part in which a plurality of second through holes are formed and a groove pattern is formed on the bottom surface to form the plurality of nucleic acid amplification chamber spaces; A membrane part joined to a lower surface of the fluid part to form a bottom surface of the binding-lysis chamber and the plurality of ridation chambers, the membrane part being made of an elastic material; And a pneumatic part joined to a lower surface of the membrane and having a plurality of ports for applying a pneumatic pressure to a predetermined position of the membrane.

A microvalve that can block the flow of the fluid passing through the microchannel by the pneumatic pressure applied by the pneumatic part and the microchannel that implements the flowpath system may be formed on the lower surface of the fluidic part.

A plurality of particles for cell trapping may be disposed in the first through-hole, and a cover covering the first through-hole may be provided.

A plurality of protrusions are formed at positions corresponding to the plurality of second through-holes by covering the plurality of second through-holes, a plurality of grooves drawn in a predetermined shape are formed in the plurality of protrusions, And a rehydration cover in which the nucleic acid amplification reagent is lyophilized.

A PCR film may be provided to cover the groove pattern formed on the bottom surface of the fluid part by forming the bottom surface of the nucleic acid amplification chamber.

The upper surface of the fluid part may form a path through which the amplification reaction mixture formed in the rehydration chamber moves to the nucleic acid amplification chamber, and a bridging pattern of a shape drawn from the upper surface of the fluid part may be formed.

Wherein the bridge pattern comprises a plurality of subpatterns, each of the plurality of subpatterns having a hole facing the membrane through the fluid part, a hole penetrating the fluid part to face the PCR film, And connecting the two holes in the upper surface and including the inserted bridge grooves.

A bridge cover covering the plurality of sub patterns entirely may be provided on the upper surface of the fluid part.

The bottom surface of the fluid part may be further provided with a drawing pattern for forming one or more metering chambers to quantify the amount of lysis buffer supplied from the lysis buffer chamber of the reagent supply device.

A bottom surface of the fluid part may be further provided with a drawing pattern for forming at least one bubble trap chamber for removing bubbles which may occur during cell disruption in the binding-lysis chamber.

A bottom surface of the fluid part may be provided with a drawing pattern to form a plurality of metering chambers for quantifying the amount of lysate formed in the binding-lycechamber and distributing it to the plurality of rehydration chambers .

A guide part for mounting the reagent supply device may further be disposed on the upper part of the fluid part.

The fluid part may be formed of a transparent polymer material such as polycarbonate, polymethyl methacrylate (PMMA), polystyrene (PS), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS) As shown in FIG.

The membrane may be formed of PDMS (polydimethylsiloxane) or silicone.

The pneumatic part may be formed of a transparent polymer material.

Another aspect relates to a method of preparing a first reagent comprising mixing an enzyme, a nucleotide, a deoxynucleotide, or a ribonucleotide triphosphate, a primer and a probe to prepare a first reagent, placing the first reagent in the first well of the reagent vessel, Placing the reagent in a second well of the reagent vessel, and drying and solidifying the first reagent, wherein the second reagent comprises a buffer.

Another aspect relates to a method of preparing a reagent, comprising the steps of preparing a first reagent by mixing an enzyme, a nucleotide, a deoxynucleotide, a ribonucleotide triphosphate, or a primer, placing the first reagent in a first well of the reagent vessel, Placing the reagent in a second well of the vessel, the second reagent comprising a buffer, and drying and solidifying the first reagent, wherein the reagent vessel is a first reagent Wherein the first well is a first well containing a nucleotide or nucleic acid component and a second well in which a second reagent is received, wherein the second well comprises a reaction buffer Wherein the first reagent and the second reagent are reagents for nucleic acid amplification. The nucleic acid amplification reagent may be a PCR primer. The first reagent may further comprise a probe.

The enzyme; Nucleotides, deoxynucleotides, or ribonucleotide triphosphates; primer; Probe; Stabilizers; And additives are as described above. In the step of preparing the first reagent by mixing an enzyme, a nucleotide, a deoxynucleotide, or a ribonucleotide triphosphate, or a primer, the first reagent may further comprise a probe or a stabilizer. The enzyme; Nucleotides, deoxynucleotides, or ribonucleotide triphosphates; primer; Probe; Stabilizers; Or the additive may be liquid. The buffer can be dried and solidified. The first reagent and / or the second reagent may further comprise an additive. The drying may be at least one drying selected from the group consisting of lyophilization and natural drying. The buffer can be lyophilized in a concentrated state above the concentration used in the reaction. The reagent vessel is as described above.

According to one aspect of the reagent vessel, it is possible to provide a vessel containing a reagent capable of maintaining stability for a long time, and the reagent can maintain its activity in the reaction in which it is used.

According to the method of storing a reagent according to one aspect, the reagent can be stored for a long period of time to maintain stability, and the reagent can maintain its activity in the reaction in which it is used.

According to one aspect of the microfluidic system for nucleic acid analysis, when a sample to be examined is injected, the cells present in the sample are captured, the nucleic acid is extracted from the captured cells, and then mixed with the nucleic acid amplification reagent, Since a series of steps are performed sequentially in the apparatus, a simple and accurate inspection is possible. In addition, it is possible to prevent contamination from the outside, which may occur during the process from nucleic acid extraction to nucleic acid amplification reaction, from the sample, so that it is possible to perform stable inspection in comparison with the case where each step proceeds in a separate system. In addition, since multiplex PCR can be performed by dividing one sample into the same plurality of chambers and performing PCR, it can be usefully used for various clinical diagnosis purposes.

1 is a front view of a reagent container according to one embodiment.
2 is a plan view of a reagent vessel according to one embodiment.
3 to 6 are plan views showing a reagent vessel including an example of a connection portion according to one embodiment.
7 is a front view showing a reagent vessel according to an embodiment mounted on a rehydration chamber;
8 and 9 are graphs showing the stability of the control group 1 and the experimental group 1.
10 and 11 are graphs showing the stability of the target nucleic acid with respect to the control group 2 and the test group 2 by concentration.
12 is a block diagram showing a schematic structure of a microfluidic system according to an embodiment.
13 is a flowchart illustrating a series of steps performed in a microfluidic system according to an embodiment.
14 is a perspective view showing a schematic outer shape of a microfluidic system according to an embodiment.
FIG. 15 is an exploded perspective view of the components constituting the microfluid system shown in FIG. 14 in a disassembled state.
16 is a plan view of the microfluidic system of Fig.
17A shows a groove pattern to form a PCR chamber space formed on the bottom surface of the fluid part of the microfluidic system of FIG.
17B is a sectional view taken along the line AA in Fig.
Fig. 18 shows the needle-shaped inlet and outlet shapes formed on the upper surface of the fluid part of the microfluidic system of Fig.
19A is a plan view showing the structure of the rehydration cover.
FIG. 19B is a sectional view taken along the line AA in FIG. 19A. FIG.
Fig. 19C is a sectional view taken along the line BB of Fig. 19A.
20A is a plan view showing a state in which the rehydration cover and the fluid part are engaged.
20B is a sectional view taken along the line AA in Fig. 20A.
20C is an enlarged view showing a part of FIG. 20B in detail.
21A is a plan view showing a state in which a bridge cover and a fluid part are engaged.
Fig. 21B is a sectional view taken along the line AA in Fig.
FIG. 21C is an enlarged view showing a part of FIG. 21B in detail.
22A to 22C show the detailed structure of the guide part on which the reagent supply device is mounted.
23A to 23C show the external structure of the reagent supply device.
FIGS. 24A to 24T are plan views showing a process in which a step according to the flowchart shown in FIG. 13 is executed in a microfluidic system according to an embodiment together with a valve operation required for fluid movement.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

1 is a front view of a reagent container according to one embodiment. Referring to FIG. 1, a reagent vessel 1000 includes a first well 100 in which a first reagent 600 is accommodated, and a second well 200 in which a second reagent 700 is accommodated. The first reagent may comprise an enzyme, a nucleotide, a deoxynucleotide, or a ribonucleotide triphosphate, a primer, and a probe. The first reagent may optionally comprise a stabilizer or additive of the enzyme. The second reagent may comprise a buffer. The second reagent may optionally comprise an additive. The first reagent and the second reagent may be reagents for use in the PCR premix.

After the first reagent 600 is prepared, it may be placed in the first well 100 of the reagent vessel 1000. The second reagent 700 can be placed in the second well 200 of the reagent vessel 1000. The first reagent 600 can be dried and solidified. The second reagent 700 can be selectively dried and solidified. The reagent vessel 1000 can be manufactured by the above-described method. Also, the first reagent 700 and / or the second reagent 800 may be stored in the reagent vessel 1000.

2 is a plan view of a reagent vessel according to one embodiment. Referring to FIG. 2, the reagent vessel 1000 may include a connection part 400 connecting the first well 100 and the second well 200 to each other. The reagent vessel 100 may include a first opening 310 connected to the first well 100 and a second opening 320 connected to the second well. The first and second openings 310 and 320 may operate as an inlet and / or an outlet of the reagent vessel 1000. The first and second openings may be disposed in the interior of the container or may be located at the top of the container. The opening disposed in the interior of the vessel may form a channel in fluid communication with the well. The opening located at the top of the vessel may define a groove in fluid communication with the well. A sample containing the target nucleic acid can be injected through the first and / or the second opening, and a sample containing the target nucleic acid, the first reagent, and the second reagent through the first and / The mixture may be drained.

3 to 6 are plan views showing a reagent vessel including an example of a connection portion according to one embodiment.

Referring to FIG. 3, the reagent vessel 1000 may include a groove 410 connecting the first well 100 and the second well 200 to each other. The groove 410 may be open at the top.

Referring to FIG. 4, the reagent vessel 1000 may include a channel 420 connecting the first well 100 and the second well 200 to each other. The channel 420 may further include a valve in the channel (not shown). The valve may be opened or closed. The valve can be suitably selected by those skilled in the art.

Referring to FIG. 5, the reagent vessel 1000 may include a partition 430 connecting the first well 100 and the second well 200 to each other. The partition 430 may be defined as a common sidewall in which the first well 100 and the second well 200 are in contact with each other. The height h3 of the barrier ribs 430 may be less than or equal to the heights h1 and h2 of the first and second wells. The small height h3 of the partition wall allows the reagents contained in the first well 100 and the second well 200 to be contacted, for example, mixed.

Referring to FIG. 6, the reagent vessel 1000 may include a membrane 440 connecting the first well 100 and the second well 200 to each other. The membrane 440 can be a film that can be easily broken into common sidewalls where the first and second wells are in contact with each other. The membrane 440 may be a friable membrane. The membrane 440 can be easily broken by the pressure of the fluid being injected. When the membrane 440 is broken, the reagents contained in the first well 100 and the second well 200 may be contacted, for example, mixed. The membrane 440 may be a porous membrane. The dissolved reagent can pass through the pores of the membrane. The first reagent 600 and the second reagent 700 are dissolved in the fluid to be injected into the container 1000 so that the porous membrane can be permeated.

7 is a front view showing a reagent vessel 1000 according to one embodiment mounted in the rehydration chamber 2000. As shown in FIG. When the reagent vessel 1000 is mounted on one side of the rehydration chamber 2000, the first and second openings 310 and 320 are fluidly connected to a channel (not shown) of the rehydration chamber And a sample containing the target nucleic acid can be injected into the reagent vessel through the rehydration chamber. The reagent vessel 1000 is inverted and mounted in the rehydration chamber 2000 so that the upper openings 310 and 320 are in contact with the outer surface of the rehydration chamber 2000, The channel 2500 can be formed. The channel may be a channel surrounded by the first well 100, the second well 200, the connection 400 and the outer surface of the rehydration chamber 2000 of the reagent vessel 1000. The inlet and / or outlet of the channel may be formed by a first opening and / or a second opening and an outer surface of the rehydration chamber 2000. The rehydration chamber may be a rehydration chamber used in a polymerase chain reaction apparatus. The polymerase chain reaction device may comprise a chamber (not shown) where the polymerase chain reaction takes place. A mixture of the sample containing the target nucleic acid, the first reagent, and the second reagent may be discharged from the reagent container and introduced into the chamber.

FIG. 12 is a block diagram showing a schematic structure of a microfluidic system 1 according to an embodiment, and FIG. 13 is a flowchart showing a series of steps performed in a microfluidic system 1 according to an embodiment.

The microfluidic system 1 includes a reagent supply device 50, a binding-lysing chamber 117, rehydration chambers R1 to R7, nucleic acid amplification chambers P1 to P7 and a reagent supply device 50, And a flow path system (not shown) for forming an integrated fluid flow between the lysis chamber 117, the rehydration chambers R1 to R7, and the nucleic acid amplification chambers P1 to P7.

The reagent supply device 50 is a device capable of storing, moving, and supplying a sample to be inspected, a reaction reagent used for sample inspection, a sample chamber into which a sample is injected, a plurality of reagent chambers and a waste chamber Respectively. The plurality of reagent chambers may be, for example, a lysis buffer chamber into which a lysis buffer is injected for cell lysis, and a washing buffer chamber into which a washing buffer is injected.

The binding-lysis chamber 117 is where a series of processes for cell binding and DNA elution are performed. The binding-lysis chamber 117 has a plurality of particles for cell capture. The particles may have a diameter of about 1 to 1000 um, and the amount of the particles may be about 1 to 100 mg. The particles may have any shape. The particles may be in the form of a bead, a sphere, a flat plate, a column, a sieve or a combination of a filter, a gel, a membrane, a fiber, or an aide. Further, the particles may be ones having magnetism. The particles can be made, for example, from glass, silica, latex or polymeric materials.

When a sample is injected from the sample chamber into the binding-lysis chamber 117, the cells are bound to a plurality of particles provided in the binding-lysis chamber 117. The surface of the particle may contain a substance that binds to the cell, and the substance may specifically or nonspecifically bind to the cell. The material may comprise a substance that specifically binds to a substance on the cell surface, for example, an antibody or a ligand. The material may be a material having a water contact angle of 70-90 degrees of hydrophobicity, or a material having one or more amino groups. Examples of the hydrophobic substance include those having a surface such as octadecyltrichlorosilane (OTS), tridecafluorotetrahydrooctyltrimethoxysilane (DTS), octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (OTC) Polyethyleneimine trimethoxysilane (PEIM), and the like.

The captured cells are then washed from the wash buffer chamber in a manner that the wash buffer is injected into the binding-lysis chamber 117 to wash out various debris or buffers used in cell trapping, for example, The particles can be dried by injection of a gas such as air.

Next, the lysis buffer is injected into the binding-lysis chamber 117 from the lysis buffer chamber, and vibrations are applied to the binding-lysis chamber 117 from the outside to vibrate the particles, thereby causing the cells to be disrupted and nucleic acids to flow out .

In the rehydration chambers R1 to R6, a cell lysate formed in the binding-lysis chamber 117 is mixed with a nucleic acid amplification reagent, for example, a PCR reagent. A plurality of rehydration chambers R1 to R6 are provided for multiplex PCR, but the present invention is not limited thereto. A cell lysate formed in the binding-lysis chamber 117 is distributed and introduced into each of a plurality of rehydration chambers R1 to R6. The nucleic acid amplification reagent may comprise, for example, a probe, a primer, an enzyme, or a combination thereof, and may also be in lyophilized form in a rehydration chamber (R1 to R6 As shown in FIG. The enzyme may comprise a polymerase. The rehydration chambers R1 to R6 are thus capable of exiting the rehydration chambers R1 to R6 after a lyophilized nucleic acid amplification reagent and a cell lysate are well mixed Chamber shape, and the detailed shape will be described later.

The nucleic acid amplification chamber may be, for example, a plurality of PCR chambers P1 to P6, and may be provided in a plurality corresponding to the plurality of rehydration chambers R1 to R6. In each of the plurality of PCR chambers P1 to P6, a nucleic acid amplification reaction is performed on an amplification reaction mixture, for example, a PCR mixture, which is formed and introduced into the plurality of rehydration chambers R1 to R6 .

Hereinafter, PCR will be exemplified by nucleic acid amplification reactions performed in the microfluidic system 1, using expressions such as PCR chambers, PCR reagents, and PCR mixtures, each of which can be used as an amplification chamber, a nucleic acid amplification reagent, Is described as an example of a reaction mixture. That is, in the microfluidic system 1, various types of nucleic acid amplification reactions can be performed in addition to PCR.

The microfluidic system 1 may also include one or more metering chambers for quantifying the amount of buffer supplied from the reagent supply device 50 to the binding-lysis chamber 117, And one or more bubble trap chambers for removing bubbles that may occur.

The metering chamber can be located in the flow path from the reagent supply device 50 to the binding-dissipation chamber 117 and also from the binding-dissipation chamber 117 to the rehydration chambers R1- As shown in FIG.

The bubble trap chambers are arranged in the flow path from the binding-lysis chamber 117 to the rehydration chambers R1 to R6 and / or from the rehydration chambers R1 to R6 toward the PCR chambers P1 to P6 .

Hereinafter, a detailed configuration of the microfluidic system 1 implementing the channel system integrated between the reagent supply device, the plurality of chambers, and the plurality of chambers will be described.

FIG. 14 is a perspective view showing a schematic outer shape of the microfluidic system 1 according to the embodiment, and FIG. 15 is an exploded perspective view showing the components constituting the microfluidic system 1 of FIG. 14 in an exploded view.

The microfluidic system 1 mainly comprises a fluid part 10, a pneumatic part 20 and a membrane part 30 and further comprises a guide part 40 for mounting a reagent supply device (not shown) .

The fluid part 10 has patterns such as various through holes and inlet parts for constituting various channels, valves, chambers, etc. for controlling the flow of the fluid to be inspected, It is made of plastic material. For example, the transparent polymer may be any one of polycarbonate (PC), poly methyl methacrylate (PMMA), polystyrene (PS), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), and silicone. The fluid part 10 includes an inlet 110 (111) 112 connected to a reagent supply device and an outlet 113. Inlets 110, 111 and 112 and an outlet 113 are connected to a reagent supply device The bottom surface of the reagent may be crushed to have a needle shape so that the reagent can flow out. Figure 18 shows the needle-shaped inlet 110, 111, 112, and outlet 113 shapes in detail. The fluid part 10 further includes a first through hole H1 forming a binding-lyce chamber space and a plurality of second through holes H2 forming a plurality of re-hydration chamber spaces. In addition, on the bottom surface, a groove pattern (not shown) is formed and drawn in order to form a plurality of PCR chamber spaces. Further, on the lower surface of the fluid part 10, a plurality of patterns (not shown) drawn in for forming a microchannel that implements a flow path system and a flow of fluid passing through the microchannel by pneumatic pressure applied from the pneumatic part 20 A valve seat (not shown), which is a protruding pattern, is formed to form a microvalve capable of closing.

The membrane part 30 is bonded to the bottom surface of the fluid part 10 to form the bottom surface of the binding-lycechamber, the plurality of rehydration chambers, the metering chamber, the bubble trap chamber, and various other channels. The membrane part 20 is made of an elastic material, for example, PDMS or silicone.

The pneumatic part 20 is for applying a pneumatic pressure to the fluid part 10 and is joined to the lower surface of the membrane part 30 and has a plurality of ports for applying a pneumatic pressure to a predetermined position of the membrane part 30 have. The pneumatic pressure applied at the pneumatic part 20 may result in, for example, particle beating, for example bead beating, for the cell disruption process in the binding-lysis chamber, And it plays a role of mixing cell lysate with PCR reagent. That is, the membrane part 30 vibrates in accordance with the pneumatic pressure applied by the pneumatic part 20 and transmits vibration energy within the binding-lysis chamber or rehydration chamber. The pneumatic pressure applied by the pneumatic part 20 serves to open / close a plurality of microvalves formed by the fluid part 10, that is, , The valve portion is brought into close contact with the valve seat formed on the lower surface of the fluid part 10 to close the valve or open the valve away from the valve seat.

A large number of particles (not shown) for capturing cells are arranged in the first through hole H1 formed in the fluid part 10 and the particle cover 15 covers the first through hole H1.

Further, a rehydration cover 14 covering the plurality of second through holes H2 formed in the fluid part 10 is provided. A protrusion 145 is formed in the rehydration cover 14 at a position corresponding to the second through hole H2 and a groove 140 drawn in a predetermined shape is formed on the protrusion 145. A PCR reagent (Not shown) are placed in a lyophilized state.

Cell lysate requires various reagents necessary for PCR reaction in order to perform PCR reaction, and may include a probe, a primer, an enzyme, or a combination thereof. These reagents, when present in a liquid phase, have problems such as evaporation or loss of enzyme activity, and therefore they are placed in the reagent vessel, i.e., the rehydration cover 14, in a lyophilized form. The grooves 140 formed in the rehydration cover 14 include a first well and a second well separated from each other, that is, two sub grooves 141 and 142, and the PCR reagent includes two sub grooves 141, (142). For example, in each of the plurality of grooves, the first well, i.e., one sub-groove 141, contains a nucleic acid-containing sample, for example, a sample containing at least one of a probe and a primer, And a buffer may be disposed in the second well, that is, the remaining one sub-groove 142. [ An enzyme or a stabilizer including a polymerase may be disposed in the first well. Also, the additives may be disposed in the first well and / or the second well.

The protrusion 145 of the rehydration cover 14 may be formed to have a size slightly larger than the size of the second through hole H2 and about 10 .mu.m. . Adhesives, etc., are likely to cause problems with freeze-dried reagents. Further, the rehydration cover 14 may be formed of an elastic material, for example, silicon or rubber, for more reliable sealing.

The PCR film 11 forms the bottom surface of the PCR chamber. That is, the PCR film 11 is provided at a position covering the recessed groove pattern (not shown) to form a PCR chamber space on the lower surface of the fluid part 1.

The upper part of the fluid part 10 is for forming a path for moving the PCR mixture formed in the rehydration chamber to the PCR chamber and a bridge pattern BP of the shape drawn from the upper surface of the fluid part 10 is formed . The bridge pattern BP is for constituting a necessary channel since the fluid must flow over the upper surface of the fluid part 10 when the PCR mixture formed in the rehydration chamber moves to the PCR chamber. The bridge pattern BP includes a plurality of sub patterns SP and each of the plurality of sub patterns SP includes a hole h1 passing through the fluid part 10 and facing the membrane part 30, Hole 126 facing the PCR film 11 and a bridge groove bg extending from the upper surface of the fluid part 10 and connecting the two holes h1 and 126. The hole h2 forms an inflow hole toward the PCR chamber or an outflow hole from the PCR chamber, which will be described later. Further, on the upper surface of the fluid part 10, a bridge cover 12 covering the plurality of sub patterns SP as a whole is provided. The bridge cover 12 may be formed with an ultrasonic welding acid for ultrasonic bonding with the fluid part 10. Alternatively, the ultrasonic welding acid may be formed on the fluid part 10, but not on the bridge cover 12. For example, ultrasonic welding may be performed around the two holes h1 and h2 and the bridging group bridge bg, An acid may be formed.

A vent cover 122 covering the vent channel 122 and the vent channel 122 is provided on the upper surface of the fluid part 10. The vent channel 122 is prepared for errors that may occur in the system. For example, if the flow of fluid is not accurately detected and the fluid continues to flow without stopping while filling the chamber, the vent channel 122 flows through the vent channel 122 I will be confined to the space designated by me. The vent channel 122 may be composed of a region drawn in a predetermined shape and a plurality of vent holes formed therein, as shown in FIG.

Further, a plurality of inflow patterns (not shown) may be formed on the lower surface of the fluid part 10 to form a metering chamber, a bubble trap chamber, and the like. For example, an infeed pattern may be formed to form one or more metering chambers that quantify the amount of lysis buffer supplied from the lysis buffer chamber of the reagent supply device, and also, in cell lysis in the binding- An inflow pattern can be formed to form one or more bubble trap chambers for removing bubbles that may occur and the amount of cell lysate formed in the binding-lysis chamber can be quantified, A drawing pattern may be formed to form a plurality of metering chambers for dispensing into the hydration chamber.

An assembling process for forming the assembly of the form as shown in FIG. 14 will be described below. First, the fluid part 10 is manufactured, and the PCR film 11 is ultrasonically welded to the lower surface of the fluid part 10. However, the present invention is not limited to this, and it is also possible to use an adhesive or a tape. Further, the bridge cover 12 and the vent cover 13 are ultrasonically welded to the upper surface of the fluid part 10. Adhesion using adhesive or tape is also possible.

Is coated on the lower surface of the fluid part 10, that is, the surface bonded to the membrane part 30, with about 2,000 angstroms of SiO ₂ .

Next, the pneumatic part 20 is manufactured, each surface on which the pneumatic part 20 and the membrane part 30 are joined is subjected to plasma treatment, and the pneumatic part 20 and the membrane part 30 are bonded.

SiO ₂ The plasma processing and, SiO ₂ coating of the fluid part 10 and the membrane 30 is joined to the joint surface of the coated fluid part 10, the joint surfaces and the membrane parts pneumatic part 20 is 30, the junction of the pneumatic The part 20 is bonded.

After the particles are injected into the first through-hole H1 forming the binding-lyocyte chamber, the particle cover 15 is bonded by ultrasonic welding or using an adhesive or tape.

An O ring 16 is inserted into the inlet 110, 111 and 112 and the outlet 113 and the guide part 40 is aligned on the upper part of the fluid part 10 and ultrasonic welding or adhesive or tape is used .

The reagent supply device is inserted into the guide part 40, and the O-ring 16 inserted between the reagent supply device and the fluid part 10 serves to prevent leakage of the solution.

Assemble the rehydration cover (14) in which the lyophilized PCR reagent is disposed to the fluid part (10).

16 is a plan view of the microfluidic system 1 of Fig.

The inlet through which the reagent is injected from the reagent supply device includes an inlet 110 (111) 112 and an outlet 113 through which a lysing buffer, a washing buffer, and a sample are respectively introduced. 15, the inlet ports 110, 111 and 112 and the outlet port 113 are inclined toward the insertion direction when the reagent supply device is inserted from the open direction of the guide part 40 , And can be inserted while slipping over the needle-shaped portion. In addition, the inlet ports 110, 111 and 112 and the outlet port 113 serve to form a passage through which a reagent, which penetrates the membrane forming the bottom surface of the reagent supply apparatus, It is in the form of a needle with a pointed tip.

The metering chambers 114, 115 and 116 are for quantifying the lysis buffer entering through the inlet 110. As a lysis buffer for cell disruption, for example, NaOH can be used, and the lysis buffer is used as little as possible, and the lysis buffer is moved to the PCR chamber without loss to increase the concentration effect. The metering chambers 114, 115 and 116 may have different volumes. For example, they may have a volume of 8 [mu] l, 8 [mu] l and 12 [mu] l, respectively. If only one metering chamber 114 is used, only 12 μl of the lysis buffer is used. For example, if only two PCR chambers among the six PCR chambers P1 to P6 having a volume of 4 μl are to be used . Using two metering chambers 115 and 116 simultaneously, 20 l of lysis buffer can be used, so four PCR chambers can be used and three metering chambers 114, 115 and 116 can be used simultaneously 28 μl of the lysis buffer was used so that all six PCR chambers could be used. There is no problem in filling the PCR chambers even if some combinations are used, even if some samples are lost due to the presence of dead volume of about 4 mu l. The number or the respective volumes of the metering chambers 114, 115, 116 are illustrative and may be varied variously.

A channel connected to the binding-lysis chamber 117 is provided with a weir having a gap of about 20 탆 from the bottom of the channel to the ceiling so as to prevent particles injected for cell binding from flowing into the binding-lysis chamber 117 .

The bubble trap chambers 118 (119) and 120 (b) are chambers each having a volume of about 28 μl. The bubble trap chambers 118, 119 and 120 function to remove air bubbles which may occur after the cell rupture through the movement of the membrane part 30, . (The bubble trap chamber 119 → the binding-raisis chamber 117 → the bubble trap chamber 119) and the backward direction (the bubble trap chamber 118 → the bubble trap chamber 118) with respect to the binding- And a function of reciprocating the elution buffer in the direction of the binding-lysis chamber 117 → bubble trap chamber 119). The elution buffer may be the same as the lysing buffer, and may be used as an elution buffer after additional lysing. It can be a volume capable of accommodating a volume of up to 28 [mu] l when the buffer is reciprocated. The bubble trap chamber 120 removes the cell lysate bubbles that have undergone the DNA elution process and prevents various errors due to bubbles in the process. The number or volume of the bubble trap chambers 118, 119, 120 is exemplary and can be varied variously.

The two chambers 121 on both sides of the vent channel 122 serve to trap fluid such as reagents and samples through the vent channel 122 due to system errors that may occur . That is, even if the PCR chambers P1 to P6 are correctly filled and the flow of the solution is sensed by the system and stopped correctly, it does not flow into the vent channel 122 but is not detected even after filling the PCR chambers P1 to P6 And flows along the vent channel 122 to be collected in the chambers 121 on both sides.

The region 124 is an optical window for observing the change of the amount of fluorescence upon the progress of the PCR as the upper part of the PCR chambers P1 to P6. This area is made thinner than the surrounding area so that very little fluorescence is transmitted as much as possible. Each of the metering chambers M1 to M6 can store 4 쨉 l of lysate passed through the bubble trap chamber 120 as a chamber having a volume of 4 쨉 l each. Cell lysates stored in the metering chambers M1 to M6 are respectively injected into the rehydration chambers R1 to R6 and stored in the rehydration chambers R1 to R6 freeze- A PCR mixture is prepared by mixing the probe with a probe, a primer, an enzyme, or a combination thereof by the motion of the membrane part 30.

The PCR, which is the last step of the analysis using the microfluidic system 1, proceeds in the PCR chambers P1 to P6. The PCR mixture passed through the rehydration chambers R1 to R6 is finally injected into the PCR chambers P1 to P6 after passing through the bubble trap chambers B1 to B6. 4 μl of the PCR mixture is used to fill all of the channel regions connected to the PCR chambers (P1 to P6) as well as the PCR chambers (P1 to P6) without bubbles, so that the volume involved in the actual PCR can be about 2.5 μl have.

Fig. 17A shows a groove pattern for forming the PCR chamber spaces P1 to P6 formed on the lower surface of the fluid part 1 of Fig. 15, and Fig. 17B is a sectional view taken along line A-A of Fig. 17A.

17A and 17B illustrate only three PCR chambers P1 to P3 by way of illustration and the remaining three PCR chambers P4 to P6 have the same structure. An inlet hole 126, an inlet channel 128, and an outlet hole 127 are connected to each of the PCR chambers P1 to P6. The PCR mixture flowing through the inlet hole 126 flows along the inlet channel 128 to fill the respective PCR chambers P1 to P6 and then escape to the outside through the outlet hole 127. [ The inlet holes 126 and the outlet holes 127 are positioned on the same side without being positioned on both sides of the PCR chambers P1 to P6 for miniaturization and maximization of fluorescence signals. By doing so, it is possible to obtain a higher fluorescence signal by securing a predetermined depth of the chamber, and to arrange the six PCR chambers P1 to P6 as close as possible to minimize the temperature deviation between the PCR chambers P1 to P6 . The bottom surface of the PCR chambers P1 to P6 is formed and the PCR film 11 is attached to the lower surface of the fluid part 10 for efficient heat transfer. At this time, ultrasonic welding can be used. For ultrasonic welding, a deposition acid 129 of about 100 [mu] m height may be formed. As shown in FIG. 6B, the deposition source 129 may be formed at regular intervals in the corners of the inlet channel 128 and the PCR chambers P1 to P6.

19A is a plan view showing the structure of the reagent container, i.e., the rehydration cover 14, FIG. 19B is a cross-sectional view taken along line A-A of FIG. 19A, and FIG. 19C is a cross-

The rehydration cover 14 is for forming six rehydration chambers and includes six protrusions 145 corresponding to the six through-holes forming the rehydration chamber space. Each protrusion 145 is formed with a drawn groove 140, and each groove includes a first well and a second well, that is, two sub grooves 141 and 142. The arrows indicate the direction of movement of the fluid. The two sub-grooves 141 and 142 are connected through the micro-channel 125. [ The cell lysate flows from the upper side of the sub groove 141 to fill the sub groove 141 and then the micro groove 125 to fill the sub groove 142. The shape of the sub-grooves 141 and 142 is such that the cell lysate fills the sub-grooves 141 and 142 without bubbles and is mixed with the PCR reagents through the movement of the membrane part, ) 142 without leaving a residue. The shape of the sub-grooves 141 and 142 can be derived from the hydrodynamic analysis taking into consideration the surface properties of the inner surfaces of the sub-grooves 141 and 142 and the solution properties of the nucleic acid solution. As shown, the side surfaces of the sub grooves 141 and 142 may have a curved shape, and the width of the center portion may be the narrowest. The outer angle [theta] formed by the narrow central corners in the sub-grooves 141 and 142 may be in the range of about 30 [deg.] To 90 [deg.].

20A is a plan view showing a state in which the rehydration cover 14 and the fluid part 10 are engaged, FIG. 20B is a cross-sectional view taken along line A-A of FIG. 20A, and FIG. 20C is an enlarged view showing a part of FIG. 20B in detail.

When the rehydration cover 14 and the fluid part 10 are joined together, sealing is formed using the properties of the respective materials without using a separate adhesive. Adhesives, etc., are likely to cause problems with lyophilized PCR reagents. The diameter of the protrusion 145 of the rehydration cover 14 is formed to be slightly larger than the diameter of the second through hole H2 in which the protrusion 145 is fitted in the fluid part 10 to form a seal . Figure 20c shows the position of the microchannel 125 connecting the two sub-grooves 141 and 142 after being fully engaged and when this is properly engaged the interface between the fluid part 10 and the rehydration cover 14 The solution moves only along the microchannel 125 without leakage.

21A is a plan view showing a state in which the bridge cover 12 and the fluid part 10 are engaged, FIG. 21B is a cross-sectional view taken along the line A-A in FIG. 21A, and FIG. 21C is an enlarged view showing a part of FIG.

The bridge cover 12 is shown with a bridge pattern formed on the upper surface of the fluid part 10 to enable vertical movement of the fluid. The bridging pattern comprises a plurality of subpatterns SP each having a hole h1 facing the membrane part 30 through the fluid part 10 and a hole h2 passing through the fluid part 10 A hole h2 facing the PCR film 11 and a bridge groove bg connected to the two holes h1 and h2. 21B, the hole h2 becomes an inflow hole (126 in Fig. 17A) connecting with the inflow channel 128 toward the PCR chamber. Since the membrane part 30 is attached to the lower surface of the fluid part 10 and the lower surface of the PCR chamber is formed of the PCR film 11, a channel through which the membrane part 30 and the PCR film 11 are connected It is difficult. The fluid that has passed through the bubble trap chambers B1 to B6 of the fluid part 10 moves through the hole h1 to the top of the fluid part 10 and flows into the bridge groove bg for fluid flow to the PCR chamber. And flows into the inlet channel 128 through the hole h2 toward the PCR chamber. The bridge cover 12 may be welded onto the fluid part 10 forming the bridge pattern through ultrasonic welding.

Further, the bridge groove (bg) serves as a channel for sensing the flow of the solution filling the entire PCR chamber. That is, once the flow of the solution is detected, the PCR mixture is no longer pushed into the PCR chamber and stopped.

Fig. 21C shows the detail of part B of Fig. 21B. As the membrane part 30 attaches to or falls off the valve seat 130, the flow of the fluid is opened and closed. The valve seat 130 is separated from the membrane part 30 in the absence of external pressure, that is, the microvalve is opened. This form implements a normally open type valve. This structure is distinguished from a normally closed type in which the membrane part 30 and the valve seat 130 are brought into contact with each other without external pressure. In the case of the normally closed type, when the microvalve is not operated for a long time , The membrane part 30 may naturally be adhered to the valve seat 130 by chemical or physical reaction and therefore the membrane part 30 may be removed from the valve seat 130 when the microvalve is not used for a long time Initialization is required to drop. In this embodiment, a micro valve is implemented more easily by using a normally open type valve.

When the PCR chamber is filled, the valve at the B site is opened, that is, the fluid part 30 is not closed to the valve seat 130 to form an exhaust path toward the vent channel 122, When the flow of the solution is detected in the bridge groove (bg) and the flow of the PCR mixture is stopped, the valve at the B site is closed.

FIGS. 22A to 22C show the detailed structure of the guide part 40 on which the reagent supply device 50 is mounted, and FIGS. 23A to 23C show the outline structure of the reagent supply device 50. FIG.

The horizontal axis of the upper side of the structures 401 and 404 serves as a pedestal for preventing the structures 501 and 502 from being slid while the reagent feeder 50 is inserted and damaging the membrane part such as being pushed downward . When the reagent supply device 50 is inserted to the correct position, a hook located on the longitudinal axis of the structure 404 is fastened to the structure 504 and can not be pushed back in the inserted direction. 23C, a crushing pattern P is formed on the lower surface of the reagent supply device 50. This portion is a needle-like inlet 110 (shown in FIG. 15) 112, and the outlet 113, so that the reagent and the sample are flowed in or out. The structures 501, 502 and 503 are fastened with the structures 402, 403 and 405 so that the reagent supply device 50 can not be lifted upwards.

FIGS. 24A to 24T are plan views for explaining a process in which the steps according to the flowchart shown in FIG. 13 are executed in the microfluidic system 1 according to the embodiment together with the valve operation required for fluid movement.

The microfluidic system 1 is used to capture cells from a sample containing the cells to be examined and lyses to form a cell lysate containing the nucleic acid (DNA), followed by PCR to determine the amount of DNA May be performed through the following exemplary processes.

As shown in FIG. 24A, in a microfluidic system, a valve in a portion indicated by a black circle (?) Is opened to apply about 1 ml of a sample S containing a sample through an inlet 112 using external pressure, (117). In this process, the cells are bound to a plurality of particles disposed in the binding-lyocis chamber 117, and the remaining solution flows toward the outlet 113 of the reagent supply device to the waste chamber. At this time, when the solution flows from the solution sensing area indicated by the double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in FIG. 24B, the valve in the portion indicated by the black circle (?) Is opened, and 0.5 mL of the washing buffer (WB) is injected using the external pressure, and the cells and the buffer are flowed toward the outlet 113 to the waste chamber. At this time, when the solution flows from the solution sensing area indicated by the double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in FIG. 24C, the valve is opened at a portion indicated by a black circle (?) And air is injected through the inlet 111 to dry the particles.

The valve in the portion indicated by the black circle (?) Is opened to flow the lysis buffer LB through the inlet 110 to fill the binding-lysis chamber 117 and the solution sensing area marked with double circle (Air to liquefied) is detected when the liquid flows into the solution sensing area and the fluid passes through the solution sensing area, the process proceeds to the next step.

As shown in Figure 24 (e), the valve in the portion indicated by the black circle (●) is opened to create a path that can be vented, and then the membrane portion of the position portion corresponding to the bottom surface of the binding-lysis chamber 117 is vibrated. The membrane part is vibrated at a vibration frequency of about 5 Hz to cause particle beating in the binding-lysis chamber 117 to last for about 5 minutes to break up the cells.

As shown in Figure 24f, the valve marked with a black circle (●) is opened to fill 4 metric chambers (M1 to M6) in six metering chambers and the solution flows into the solution detection area marked with double circle (⊚) When the passing fluid changes from air to liquid (air to liquid), it stops and proceeds to the next step.

The valve in the portion indicated by the black circle (?) Is opened to push the cell lysate in the metering chamber M1 into the rehydration chamber R1 as shown in Fig. 24G. At this time, when the solution flows into the solution sensing area indicated by double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in FIG. 24H, the valve in the portion indicated by the black circle (?) Is opened to push the cell lysate in the metering chamber M2 into the rehydration chamber R2. At this time, when the solution flows into the solution sensing area indicated by double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in FIG. 24i, the valve in the portion indicated by the black circle (?) Is opened to push the cell lysate in the metering chamber M3 into the rehydration chamber R3. At this time, when the solution flows into the solution sensing area indicated by double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in FIG. 24j, the valve in the portion indicated by the black circle (?) Is opened to push the cell lysate in the metering chamber M4 into the rehydration chamber R4. At this time, when the solution flows into the solution sensing area indicated by double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in FIG. 24K, the valve in the portion indicated by the black circle (?) Is opened to push the cell lysate in the metering chamber M5 into the rehydration chamber R5. At this time, when the solution flows into the solution sensing area indicated by double circle (⊚) and liquid to air is detected, the flow stops and the process proceeds to the next step.

As shown in Figure 241, the valve in the portion indicated by the black circle (●) is opened to push the cell lysate in the metering chamber M6 into the rehydration chamber R6. At this time, when the solution flows into the sensing area indicated by the double circle (⊚) and the moment that the liquid is changed into air (air) is detected, the process is stopped.

As shown in FIG. 24M, the valve in the portion indicated by the black circle (?) Is opened to vibrate the membrane part of the region forming the bottom surface of the rehydration chamber (R1 to R6). At this time, the membrane part can be vibrated at a vibration cycle of about 0.2 Hz. In this process, the PCR reagent in the rehydration chambers (R1 to R6) is dissolved in a cell lysate and mixed to form a PCR mixture. .

As shown in FIG. 24N, air is injected into the inlet 111 and the PCR mixture (PCR mixture) is pushed into the PCR chamber P1 by opening a valve indicated by a black circle (?). At this time, when the fluid passing through the solution sensing area indicated by the double circle (⊚) is detected as an air to liquid, the flow stops and the process proceeds to the next step.

Open the valve marked with a black circle (●) as in Figure 24o and push the PCR mixture into the PCR chamber (P2). At this time, when the fluid passing through the solution sensing area indicated by the double circle (⊚) is detected as an air to liquid, the flow stops and the process proceeds to the next step.

Open the valve indicated by the black circle (●) as shown in FIG. 24P and push the PCR mixture into the PCR chamber (P3). At this time, when the fluid passing through the solution sensing area indicated by the double circle (⊚) is detected as an air to liquid, the flow stops and the process proceeds to the next step.

Open the valve shown in Figure 24q and the black circles (●) and push the PCR mixture into the PCR chamber (P4). At this time, when the fluid passing through the solution sensing area indicated by the double circle (⊚) is detected as an air to liquid, the flow stops and the process proceeds to the next step.

Open the valve marked with a black circle (●) as in Figure 24r and push the PCR mixture into the PCR chamber (P5). At this time, when the fluid passing through the solution sensing part indicated by the double circle (⊚) is detected as an air to liquid, the solution stops and proceeds to the next step.

Open the valve marked with a black circle (●) as in Figure 24s and push the PCR mixture into the PCR chamber (P6). At this time, when the fluid passing through the solution sensing part indicated by the double circle (⊚) is detected as an air to liquid, the solution stops and proceeds to the next step.

As shown in FIG. 24 (t), the valve in the portion indicated by the black circle (?) Is opened and the PCR is carried out with only the valves located at the front ends of the PCR chambers (P1 to P6) closed.

Using the microfluidic system 1 described above, a series of processes such as a process of distributing a sample to be tested from a reagent supply device to a plurality of PCR chambers to progress a PCR, that is, mixing with a cell capture, a lysing and a PCR reagent Can be performed precisely and reproducibly in an integrated system.

Example One: PCR Premix Freeze-dried body Manufacturing and stability comparison

Primers and probes used in this embodiment a PCR reaction is Staphylococcus aureus (Staphylococcus mure < / RTI > sequence of Table 1 was used. The Tm of the following forward primer was 55 캜, the Tm of the reverse primer was 51 캜, and the Tm of the probe was 46 캜.

Oligo ID order Label / End Location [3a] mecA-99
Forward primer SEQ ID NO: 1 none [3a] mecA-99
Reverse primer SEQ ID NO: 2 none [3a] mecA-99 probe SEQ ID NO: 3 Cal610

In order to compare the stability of the PCR primers prepared with the compositions shown in Table 1 of Control 1 and the stability of the PCR primers prepared with the compositions shown in Table 3 and Experimental Group 1,

The first reagent and the second reagent shown in Table 2 were prepared. The first reagent and the second reagent were injected into the first well and the second well of the PDMS reagent vessel, respectively, in an amount of 2 ul. Thereafter, the PDMS reagent vessel was placed in a freeze dryer (FDUT-6002, Operon) and lyophilized to prepare a control 1.

The first reagent and the second reagent shown in Table 3 were prepared in the same manner as in the above-mentioned method, and the first reagent and the second reagent were injected into the first and second wells of the PDMS reagent vessel, And dried.

The control group 1 and the test group 1 were placed in an oven at 40 ° C, and the reagent containers of the control group 1 and the test group 1 were taken out on days 7, 14, 21, and 28, respectively. 10 into the respective ³ GeneBank EF190335.1 target template of the reagent vessel proceeds for PCR we compared the change in the initial value compared to the PCR value. Comparison of the PCR values was performed by measuring mecA signal changes. PCR was carried out with a total of 45 cycles of denaturation at 95 ° C for 1 second and extension at 60 ° C for 5 seconds.

8 and 9 are graphs showing the stability of the control group 1 and the experimental group 1. As shown in FIG. 8, in the case of the control group 1, the mecA signal, that is, the PCR signal, decreased from 7 days after the temperature at 40 ° C. Also, it was confirmed that the threshold cycle (Ct) was more than 5% higher than the initial value after 14 days. In addition, as shown in Fig. 9, it was confirmed that the experimental group 1 retained initial values up to 4th week at 40 ° C. Comparing FIG. 8 and FIG. 9, it was confirmed that the experimental group 1 had a stable PCR signal at 4th week, unlike the control group 1. Therefore, in Experiment 1, it can be seen that the PCR primer composition is more stable than the control group. In the case of the control 1, the stability of the PCR primer is low because the primer and / or the probe in the second reagent are mixed to solidify the gel state and the primer and / or the probe are adsorbed to the PDMS constituting the reagent container.

First reagent Second reagent ingredient Volume (ul) ingredient Volume (ul) dNTP 16 mecA
Primer / probe 12 z-taq 2 Trehalose stabilizer 10 10X buffer
(Takara PCR buffer) 20 Defoamer (SHA646, Saehan Chemical) 646 (1%) 10 Triton-100 (1%) 5 water 68 water 57

First reagent Second reagent ingredient Volume (ul) ingredient Volume (ul) dNTP 16 10X buffer
(Takara PCR buffer) 20 z-taq 2 Defoamer (SHA646, Saehan Chemical) 646 (1%) 10 Trehalose stabilizer 10 Triton-100 (1%) 5 mecA primer / probe 12 water 65 water 60

Example 2: PDMS Dried in a reagent vessel PCR Premix Reagent performance evaluation

In order to confirm that the PCR pre-mix according to one embodiment of the present invention has stability similar to the liquid PCR pre-mix, the following procedure was performed. Specifically, the PCR reaction was carried out in the same manner as in Example 1. Control 2 is a liquid PCR pre-mix, and the components of the PCR pre-mix of the liquid are shown in Table 4. Experimental group 2 is the PCR premix in the PDMS reagent vessel, and the components of the premix are the same as those in Table 3.

ingredient Volume (ul) dNTP 16 z-taq 2 mecA primer / probe 12 10X buffer
(Takara PCR buffer) 20 water 68

For control 2, the PCR primer mix of the liquid was mixed with the PCR tube and 4 μl of each were pipetted into different PCR tubes. 4 μl of a solution containing 10 ⁴ , 10 ³ , 10 ² , and 10 ¹ template nucleic acids was added to the PCR tube, followed by PCR reaction.

For group 2, and Table 3 for the first reagent and the second reagent a 2 ul by freezing by 10 ^4, 10 ^3, 10 ^2, 10 ¹ a mold the target solution in the four dry samples taken indicated in the PDMS reagent vessel by 4 ul The dried product was melted to carry out the PCR reaction.

10 and 11 are graphs showing the stability of the target nucleic acid with respect to the control group 2 and the test group 2 by concentration. _Ct for each concentration of the target nucleic acid shown in FIGS. 10 and 11 are shown in Table 5. [ The Y-axis in FIGS. 10 and 11 represents fluorescence intensity. As shown in FIGS. 10 and 11, a comparison of FIGS. 10 and 11 shows no difference in fluorescence intensity between the two PCR premixes due to PCR. Indicating no difference in stability between the two PCR premixes. Thus, it was confirmed that the PCR primmix in the reagent vessel according to one embodiment of the present invention exhibits stability similar to that of the liquid PCR primmix.

The concentration of the target nucleic acid Control group 2 Experiment 2 10 ⁴ 22.9 22.7 10 ³ 26.3 26.1 10 ² 29.9 29.6 10 ¹ 32.0 32.9

1: microfluidic system 10: fluid part
20: Pneumatic part 30: Membrane part
40: Guide part 50: Reagent supply device
11: PCR film 12: bridge cover
13: Vent cover 14: Reagent container, rehydration cover
15: particle cover 16: O-ring
110, 111, 112: inlet portion 113: outlet portion
114, 115, 116: metering chamber 117: binding-lysis chamber
118, 119, 120: bubble trap chamber 121: chamber
122: vent channel 125: microchannel
126: inlet hole 127: outlet hole
128: Inlet channel 129:
130: valve seat 140: groove
141, 142: Sub-groove 145:
H1: first through hole H2: second through hole
h1, h2: hole bg: bridge groove
BP: Bridge pattern SP: Sub pattern
R1 to R6: Rehydration chambers P1 to P6: PCR chambers
M1 to M6: Metering chamber
100: first well 200: second well
310: first opening 320: second opening
400: connection part 410: home
420: channel 430: bulkhead
440: membrane 600: first reagent
700: Second reagent 1000: Reagent container, rehydration cover
2000: Cartridge 2500: Channel

<110> Samsung Electronics Co. Ltd <120> Reagent container for amplification nucleic acid, method for manufacturing the same, and method for storing the reagent, and micro-fluidic system for analysis of nucleic acid <130> PN100087 <150> KR 12/139266 <151> 2012-12-03 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> [3a] mecA-99 forward primer <400> 1 attaacccag tacagatcct ttcaatc 27 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> [3a] mecA-99 reverse primer <400> 2 ccaaactttg tttttcgtgt ctttt 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <3a> mecA-99 probe <400> 3 tattaacgca cctcacttat 20

Claims

A first well in which a first reagent is received, wherein the first reagent comprises a nucleotide or a nucleic acid component, and a second well in which a second reagent is received, Reaction buffer, wherein the first reagent and the second reagent are nucleic acid amplification reagents.

The reagent vessel of claim 1, wherein the nucleotide or nucleic acid component is a nucleotide, a deoxynucleotide, or a ribonucleotide triphosphate, a primer, and a probe nucleic acid.

The reagent container according to claim 1, wherein the first reagent further comprises an enzyme or a stabilizer.

The reagent vessel of claim 1, wherein the first reagent is a solid reagent.

The reagent vessel of claim 1, wherein the second reagent is a solidified reagent.

The reagent container according to claim 1, wherein the first reagent or the second reagent further comprises an additive.

The reagent vessel according to claim 1, wherein the first reagent or the second reagent is lyophilized in a concentrated state above a concentration used in the reaction.

The reagent vessel of claim 1, wherein the buffer is a lyophilized buffer in a concentrated state at a concentration above the concentration used in the reaction.

The reagent container according to claim 1, wherein the reagent container further comprises a connection portion connecting the first well and the second well to each other.

The reagent container according to claim 9, wherein the connecting portion is a groove, a channel, a partition wall, or a membrane.

The reagent vessel according to claim 1, wherein the reagent vessel is formed with a plurality of protrusions, and the first well and the second well are two sub grooves separated from each other, which are formed in grooves drawn in a predetermined shape in the protrusions.

12. The reagent vessel according to claim 11, wherein the side surfaces of the first well and the second well are curved and the width of the center portion is the narrowest.

12. The reagent vessel according to claim 11, wherein outer edge angles of both side edges of the first and second wells forming the narrowest width are in the range of 30 DEG to 90 DEG.

The reagent vessel of claim 1, wherein the reagent vessel further comprises a first aperture connected to the first well, a second aperture connected to the second well, and the first or second aperture is open at the top.

The reagent vessel of claim 1, wherein the reagent vessel is adapted to be mounted in a rehydration chamber.

16. The reagent vessel of claim 15, wherein the reagent vessel is flipped over and mounted in the rehydration chamber such that the open top opens into contact with the exterior surface of the rehydration chamber to form a channel.

A microfluidic system for nucleic acid analysis comprising a rehydration chamber, a reagent vessel of claim 1 attached to the rehydration chamber, an amplification chamber, and a flow system forming an integrated fluid flow between the rehydration chamber and the amplification chamber. As a result,
The rehydration chamber mixes a cell lysate and a nucleic acid amplification reagent in the reagent vessel to form an amplification reaction mixture. The amplification chamber performs a nucleic acid amplification reaction on the amplification reaction mixture introduced from the rehydration chamber Wherein the microfluidic system for nucleic acid analysis is a microfluidic system.

18. The microfluidic system for nucleic acid analysis according to claim 17, wherein each of the rehydration chambers comprises two separate sub-chambers.

19. The microfluidic system for nucleic acid analysis according to claim 18, wherein the side surface of the subchamber is curved and the width of the flow path of the inflowed nucleic acid solute is the narrowest at the center.

18. The microfluidic system for nucleic acid analysis according to claim 17, further comprising a reagent supply device and a binding-lyse chamber,
The reagent supply apparatus is provided with a sample chamber into which a sample to be inspected is injected, at least one reagent chamber into which a reagent for extracting nucleic acid from the sample is injected, and a waste chamber from which used reagent is discarded,
Wherein the binding-lysis chamber captures cells from the sample and breaks up the captured cells to form a cell lysate containing the nucleic acid, wherein a plurality of particles for cell trapping are arranged,
Wherein the flow path system has an outlet connected to the reagent supply device and a plurality of inlets to form an integrated fluid flow between the binding-lysing chamber, the rehydration chamber, and the amplification chamber. .

Preparing a first reagent by mixing an enzyme, a nucleotide, a deoxynucleotide, a ribonucleotide triphosphate, or a primer;
Placing the first reagent in a first well of the reagent vessel,
Placing a second reagent in a second well of the reagent vessel, wherein the second reagent comprises a buffer; and
A method of storing a reagent comprising drying and solidifying a first reagent,
The reagent vessel comprising a first well in which a first reagent is received, wherein the first reagent comprises a nucleotide or a nucleic acid component, and a second well in which a second reagent is received, The second well comprises a reaction buffer, and the first reagent and the second reagent are reagents for nucleic acid amplification.