KR101911021B1 - Film-based integrated chip and its nucleic acid detection method - Google Patents

Abstract

The present invention relates to a film-based integrated chip and a nucleic acid detection method thereof for amplifying and detecting nucleic acid in real time to rapidly detect a gene and confirm whether or not specific amplification is simultaneously performed. The constitution of the present invention comprises a film part forming a body; A sample injecting unit provided in the film unit and injecting a sample; A first flow path connected to one side of the sample injection unit to transfer the sample; An extraction unit connected to the other side of the first flow path and extracting nucleic acid from the transferred sample; A second flow path connected to one side of the extraction unit for transferring the extracted nucleic acid; A PCR solution injecting unit provided in the film unit and into which a PCR solution is injected; A third channel connected to one side of the PCR solution injecting unit and transferring the injected PCR solution; A fourth flow path connected to the second flow path and the third flow path, wherein the nucleic acid introduced from the second flow path and the PCR solution introduced from the third flow path are mixed to form a mixed material; An amplification unit connected to the other side of the fourth flow path to receive the mixed material and amplify the extracted nucleic acid; A fifth channel connected to one side of the amplification unit and transferring the mixed material discharged from the amplification unit; And a recovery unit connected to the other side of the fifth flow path for recovering the mixed material, wherein the nucleic acid is detected in real time while being amplified by the amplification unit.

Description

[0001] FILM-BASED INTEGRATED CHIP AND ITS NUCLEIC ACID DETECTION METHOD [0002]

The present invention relates to a film-based integrated chip and a nucleic acid detection method thereof, and more particularly, to a method and a device for detecting a nucleic acid in a real time by integrating simple nucleic acid extraction and nucleic acid amplification and detection using a liquid extraction method, Based integrated chip and a nucleic acid detection method thereof.

In recent years, studies on nano-biotechnology capable of directly confirming and manipulating biomolecule behavior of nano units such as genes, proteins and cells of an object have been actively conducted. Such nano-biotechnology can be used to diagnose a disease or to determine the type of a target object, and thus the recent industrial field is being expanded.

And, since general gene diagnosis technology uses a large amount of samples, expensive biochemical reagents are used in large amounts. In particular, the nucleic acid extraction method using solid phase extraction is not economical because it requires complex and repetitive operations using various chemicals.

In addition, since the general gene diagnosis technology is large, it is inconvenient to carry, and it takes a long time to identify the gene, which is not practical.

That is, the conventional gene diagnosis technology is difficult to carry, is not economical, and has a problem in that it takes a long time to discriminate the gene, resulting in poor practicality.

Particularly, in recent years, the development of a lab-on-a-chip has been actively carried out. A lab-on-a-chip is a type of biochip that allows a small chip to perform research that can be done in a laboratory. Specifically, the lab-on-a-chip is made of plastic, glass, silicon, etc. to create microchannels of nanometer (nm) or less. Through this, labs and researches that can be done in existing laboratories It is a chip made to replace. Because of this practicality, lab-on-a-chip is continuously being developed and used.

However, such a lab-on-a-chip requires an expensive photolithography process, which is complicated and inexpensive to manufacture.

Korean Patent Registration No. 1421098 (Jul. 14, 2014)

An object of the present invention to solve the above problems is to provide a film-based integrated chip and a nucleic acid detection method thereof for rapidly detecting a gene by amplifying and detecting nucleic acid in real time and confirming specific amplification .

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the precise form disclosed. There will be.

According to an aspect of the present invention, A sample injecting unit provided in the film unit and injecting a sample; A first flow path connected to one side of the sample injection unit to transfer the sample; An extraction unit connected to the other side of the first flow path and extracting nucleic acid from the transferred sample; A second flow path connected to one side of the extraction unit for transferring the extracted nucleic acid; A PCR solution injecting unit provided in the film unit and into which a PCR solution is injected; A third channel connected to one side of the PCR solution injecting unit and transferring the injected PCR solution; A fourth flow path connected to the second flow path and the third flow path, wherein the nucleic acid introduced from the second flow path and the PCR solution introduced from the third flow path are mixed to form a mixed material; An amplification unit connected to the other side of the fourth flow path to receive the mixed material and amplify the extracted nucleic acid; A fifth channel connected to one side of the amplification unit and transferring the mixed material discharged from the amplification unit; And a recovery unit connected to the other side of the fifth flow path for recovering the mixed material, wherein the nucleic acid is detected in real time while being amplified by the amplification unit.

In an embodiment of the present invention, it may further comprise a valve portion for controlling the flow of the fluid in the film portion.

In an embodiment of the present invention, the valve unit may include: a first valve provided in the first flow path to control the transfer of the sample; A second valve provided in the second flow path and controlling transfer of the extracted nucleic acid; A third valve provided in the third flow path for controlling transfer of the PCR solution; And a fourth valve provided in the fifth flow path for controlling the transfer of the mixed substance.

In an embodiment of the present invention, the PCR solution may include a signal substance.

In an embodiment of the present invention, the nucleic acid amplified by the amplification unit reacts with the signal material, and the amount of the nucleic acid is detected by receiving the fluorescence signal of the signal material.

In an embodiment of the present invention, the signal material may be any one of a fluorescent PNA probe, a hydrolysis probe, and an intercalating dye.

In an embodiment of the present invention, an extraction reagent containing an enzyme is injected into the extraction unit, and the enzyme activated at a temperature of 70 ° C to 80 ° C reacts with the sample to extract the nucleic acid .

According to another aspect of the present invention, there is provided a method for detecting nucleic acid in a film-based integrated chip, comprising the steps of: a) injecting the sample into the sample injection unit; b) transferring the injected sample to the extractor; c) extracting the nucleic acid from the sample transferred to the extraction unit; d) mixing the extracted nucleic acid with the PCR solution injected into the PCR solution injection unit to form a mixed material; And e) transferring the formed mixed material to the amplification unit to amplify and detect the nucleic acid.

In an embodiment of the present invention, the step b) comprises: b1) opening the first valve, the second valve and the third valve; And b2) transferring the sample toward the extraction unit.

In an embodiment of the present invention, the step c) includes: c1) injecting an extraction reagent containing an enzyme into the extraction unit; c2) closing the first valve and the second valve; c3) raising the temperature of the extraction unit such that the enzyme is activated; c4) extracting said nucleic acid from said sample with said activated enzyme; And c5) further raising the temperature of the extraction unit such that the enzyme is inactivated.

In the embodiment of the present invention, in the step c3), the extracting unit may be heated to a temperature of 70 ° C or more and less than 80 ° C.

In an embodiment of the present invention, the step d) comprises the steps of: d1) opening the first valve, the second valve and the fourth valve; d2) transferring the extracted nucleic acid to the fourth channel; d3) closing the second valve; And d4) transferring the PCR solution injected into the PCR solution injection unit to the fourth flow path to mix the PCR solution and the nucleic acid, wherein in the step d4), the PCR solution and the nucleic acid are mixed Thereby forming a mixed material.

In an embodiment of the present invention, the PCR solution may include a signal substance.

In an embodiment of the present invention, the step e) includes the steps of: e1) closing the valve part; e2) amplifying the nucleic acid in the amplification unit; And e3) receiving the fluorescence signal of the signal material and detecting the amount of the nucleic acid, wherein the step of amplifying the nucleic acid and the step of detecting the amount of the nucleic acid are sequentially and repeatedly performed a predetermined number of times . ≪ / RTI >

In an embodiment of the present invention, the signal material may be any one of a fluorescent PNA probe, a hydrolysis probe, and an intercalating dye.

In an embodiment of the present invention, it is preferable that the method further comprises, after the step e), f) determining whether specific amplification is performed using the PCR solution in the mixed material, wherein the step f) And the fluorescence signal of the signal substance contained therein is detected through melting curve analysis to determine whether specific amplification is possible.

In an embodiment of the present invention, after step (f), g) recovering the mixed material may be further included.

In an embodiment of the present invention, the step g) includes: g1) opening the third valve and the fourth valve; g2) injecting air into the PCR solution injection unit; And g3) collecting the mixed material discharged to the collection part by the injected air.

According to an aspect of the present invention, there is provided a gene detector using a film-based integrated chip.

According to an aspect of the present invention, there is provided a gene detector using a nucleic acid detection method of a film-based integrated chip.

The effect of the present invention according to the above configuration is that it is economical to use the expensive biochemical reagent because a small amount of sample is injected into the film-based integrated chip.

In addition, since the present invention can perform both extraction, amplification, and detection using a film-based integrated chip, it is possible to automate the extraction of nucleic acids and the detection without cross contamination between samples.

Further, since the nucleic acid is extracted using the liquid phase extraction method, the present invention can extract the nucleic acid only by adjusting the temperature, so that the extraction operation is simpler and more economical than when the chemical agent is used.

Further, since the present invention is in the form of a film, a high-cost photolithography step is unnecessary and can be manufactured at low cost.

Further, the present invention can amplify and detect the nucleic acid in the amplification unit in real time, and perform genetic analysis quickly.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

1 is a top view of a film-based integrated chip according to an embodiment of the present invention.
2 is a flowchart of a method of detecting a nucleic acid of a film-based integrated chip according to an embodiment of the present invention.
3 is a flowchart illustrating a step of transferring a sample of a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention to an extraction unit.
4 is a flowchart illustrating a step of extracting nucleic acid from a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention.
FIG. 5 is a flowchart illustrating a step of forming a mixed material in a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention.
FIG. 6 is a flowchart illustrating steps of amplifying and detecting a nucleic acid of a method of detecting a nucleic acid of a film-based integrated chip according to an exemplary embodiment of the present invention.
FIG. 7 is a flowchart illustrating a step of recovering a mixed material in a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention.
8 is a photograph showing the results of an experiment comparing a conventional example and an embodiment of the present invention.
9 is a photograph of a film-based integrated chip according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout the specification, when a part is referred to as being "connected" (connected, connected, coupled) with another part, it is not only the case where it is "directly connected" "Is included. Also, when an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

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

1 is a top view of a film-based integrated chip according to an embodiment of the present invention.

1, the film-based integrated chip 100 includes a film unit 110, a sample injection unit 120, an extraction unit 130, a PCR solution injection unit 140, an amplification unit 150, (160).

The film unit 110 forms the body of the film-based integrated chip 100. The film portion 110 is made of a film material and has a high thermal conductivity, so that efficiency in extraction and amplification of nucleic acid can be improved.

The sample injecting unit 120 is provided in the film unit 110 and the sample injecting unit 120 may be provided with a hole through which a sample is injected.

One end of the first flow path 171 is connected to the sample injection unit 120 and the sample injected through the sample injection unit 120 may be transferred to the extraction unit 130.

The extraction unit 130 is connected to the other side of the first flow path 171 and can extract nucleic acid from the transferred sample. Specifically, an extraction reagent containing an enzyme is injected into the extraction unit 130. The extraction unit 130 may be heated to a temperature at which the enzyme is activated. Preferably, the extraction section 130 may be heated to a temperature of 70 ° C or more and less than 80 ° C. When the extraction unit 130 is heated to a temperature of 70 ° C or more and less than 80 ° C, the enzyme is activated, and the activated enzyme acts as a catalyst to extract the nucleic acid from the sample. After extracting the nucleic acid for a preset time, the extracting unit 130 may reheat the enzyme to deactivate the enzyme and the temperature may be further increased. For example, the extraction unit 130 may be heated to 95 ° C to deactivate the enzyme. For this, the extraction unit 130 may be controlled to a temperature of 70 ° C or more and 95 ° C or less.

The extraction unit 130 may be heated by a heater block (not shown) that heats the amplification unit 150. That is, it is not necessary to provide additional equipment for raising the temperature of the extraction unit 130, which is economical and can reduce the volume of the entire facility.

The second flow path 172 is connected to the extraction part 130 at one side and forms a path through which the extracted nucleic acid can be transferred to a fourth flow path 174 to be described later.

The PCR solution injection unit 140 is provided in the film unit 110, and a PCR solution can be injected. Here, the PCR solution may include a fluorescent PNA (Peptide Nucleic Acid) probe.

The third flow path 173 is connected to the PCR solution injecting unit 140 at one side and forms a path for transferring the PCR solution injected through the PCR solution injecting unit 140 to the fourth flow path 174 can do.

The other end of the second flow path 172 and the other end of the third flow path 173 are connected to one side of the fourth flow path 174 to receive the nucleic acid extracted from the second flow path 172, And the PCR solution may be supplied from the third channel 173. In the fourth flow path 174, the nucleic acid and the PCR solution may be mixed to form a mixed material.

The amplification unit 150 is connected to the other side of the fourth flow path 174 to receive the mixed material and amplify the extracted nucleic acid. The nucleic acid is amplified in the amplification unit 150 and detected in real time. Specifically, the amplification unit 150 may be heated by the heater block and cooled by a cooling block (not shown). As described above, the temperature of the amplification unit 150 can be raised and cooled by the heater block and the cooling block, and the nucleic acid can be amplified by causing a polymerase chain reaction according to the temperature change.

The nucleic acid amplified by the amplification unit 150 reacts with the signal material, and the amount of the nucleic acid can be detected by receiving the fluorescence signal of the signal material. Here, the fluorescence signal of the signal material may be received through a separate fluorescence signal receiver.

The amplification unit 150 may perform the amplification and detection of the nucleic acid sequentially and repeatedly a predetermined number of times. That is, according to the present invention, the amplification unit 150 can detect the nucleic acid in real time. By detecting the nucleic acid in real time, the number of times of amplification and detection of the nucleic acid can be controlled by the number of times the gene can be analyzed.

One end of the fifth flow path 175 is connected to the amplification unit 150 and the mixed material discharged from the amplification unit 150 may be transferred to the recovery unit 160.

The recovery unit 160 is connected to the other side of the fifth flow path 175 and is capable of recovering the mixed material.

The film-based integrated chip 100 may further include a valve unit 180 for controlling the flow of the fluid in the film unit 110. The valve unit 180 may include a first valve 181, A valve 182, a third valve 183, and a fourth valve 184.

The first valve 181 is provided in the first flow path 171 to control the transport of the sample and the second valve 182 is provided in the second flow path 172 to control the transport of the extracted nucleic acid can do. The third valve 183 is provided in the third flow path 173 to control the transfer of the PCR solution and the fourth valve 184 is provided in the fifth flow path 175, Can be controlled.

Hereinafter, a nucleic acid detection method using the film-based integrated chip 1000 described above with reference to FIG. 1 and the following figures will be described in detail.

2 is a flowchart of a method of detecting a nucleic acid of a film-based integrated chip according to an embodiment of the present invention.

Referring to FIG. 2, the nuclear-ginseng detection method of the film-based integrated chip may first execute the step of injecting the sample into the sample injection unit 120 (S210). Here, the sample injecting unit 120 may inject a sample containing a gene.

3 is a flowchart illustrating a step of transferring a sample of a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention to an extraction unit.

Referring to FIG. 3, after the step S210 of injecting the sample into the sample injection unit 120, the step of transferring the injected sample to the extraction unit 130 (S220) may be performed.

The step S220 of transferring the injected sample to the extraction unit 130 may include opening the first valve 181, the second valve 182 and the third valve 183 ) Can be performed.

After the step S221 of opening the first valve 181, the second valve 182 and the third valve 183, the step of transferring the sample to the extraction unit 130 S222) may be performed. At this stage, the sample injected through the sample injecting unit 120 may be transferred to the extracting unit 130 through the first flow path 171.

4 is a flowchart illustrating a step of extracting nucleic acid from a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention.

Referring to FIG. 4, after the step S220 of transferring the injected sample to the extraction unit 130, a step S230 of extracting the nucleic acid from the sample transferred to the extraction unit 130 is performed .

In step S230 of extracting the nucleic acid from the sample transferred to the extraction unit 130, a step S231 of injecting an extraction reagent containing an enzyme into the extraction unit 130 may be performed. In this step, the enzyme refers to a specific substance that causes a biological and chemical reaction so that the nucleic acid is extracted from the sample. The extraction reagent may be injected together with the sample, or may be directly injected into the extraction unit 130.

After the step S231 of injecting the extraction reagent containing the enzyme into the extraction unit 130, the first valve 181 and the second valve 182 may be closed (S232) . When the first valve 181 and the second valve 182 are closed, the sample is suspended in the extraction part 130 and the flow is stopped.

After the first valve 181 and the second valve 182 are closed (S232), the temperature of the extraction unit 130 may be increased to activate the enzyme (S233) . At this time, the extraction unit 130 may be heated by the heater block that heats the amplification unit 150. The extraction unit 130 may be heated to a temperature of 70 ° C. or more and less than 80 ° C. at which the enzyme can be activated. However, the present invention is not limited to heating the temperature of the extraction unit 130 to 70 ° C or more and less than 80 ° C, and may be included in an embodiment as long as the temperature can activate the enzyme.

After the step S233 of raising the temperature of the extraction unit 130 to activate the enzyme, step S234 of extracting the nucleic acid from the sample by the activated enzyme may be performed. Specifically, when the temperature of the extraction unit 130 is heated to a temperature at which the enzyme is activated, the enzyme may be activated to generate a chemical and biological reaction with the sample to extract the nucleic acid from the sample. The time for extracting the nucleic acid using the enzyme may be set to a predetermined time.

After the activated nucleic acid is extracted from the sample (S234), the temperature of the extraction unit 130 may be further increased (S235) so that the enzyme is deactivated. Specifically, the enzyme is activated within a predetermined temperature range, and when the temperature exceeds the predetermined temperature range, the enzyme is lost. When extraction of the nucleic acid is completed in the extraction unit 130, the temperature of the extraction unit 130 may be raised to a temperature at which the enzyme is deactivated. For example, when the enzyme is activated at a temperature of 70 ° C or more and less than 80 ° C, the extraction unit 130 may be heated to reach 95 ° C to inactivate the enzyme. As described above, the enzyme heated to a high temperature can be maintained in an inactivated state in which the function is lost irrespective of a change in temperature at a position of the amplification unit 150 or the like. Accordingly, it is possible to prevent the nucleic acid extraction time of the enzyme from further extracting the nucleic acid after a predetermined time, so that the nucleic acid extraction time of all the samples can be made constant at all times. As a result, the reliability of the experimental results can be improved.

FIG. 5 is a flowchart illustrating a step of forming a mixed material in a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention.

5, after extracting the nucleic acid from the sample transferred to the extracting unit 130 (S230), the extracted nucleic acid and the PCR solution injected into the PCR solution injecting unit 140 And a step S240 of forming a mixed material by mixing may be performed.

The first valve 181, the second valve 182, and the second valve 182 are formed by mixing the extracted nucleic acid and the PCR solution injected into the PCR solution injecting unit 140 to form a mixed material (S240) And opening the fourth valve 184 (S241).

After the step (S241) of opening the first valve (181), the second valve (182) and the fourth valve (184), transferring the extracted nucleic acid to the fourth flow path (174) S242) may be performed. At this stage, air may be injected into the sample injecting unit 120 to transfer the nucleic acid located in the extracting unit 130 to the fourth flow path 174.

After the extracted nucleic acid is transferred to the fourth flow path 174 (S242), the step of closing the second valve 182 (S243) may be performed. In this step, the second valve 182 may be closed to prevent the nucleic acid from flowing back toward the extraction part 130 while the PCR solution is transferred to the fourth flow path 174.

After the second valve 182 is closed (S243), the PCR solution injected into the PCR solution injection unit 140 is transferred to the fourth flow path 174 to mix the PCR solution and the nucleic acid Step S244 may be performed. At this stage, the PCR solution is injected into the PCR solution injection unit 140. Here, the PCR solution contains a signal substance. Here, the signal substance may be any one of a fluorescence PNA probe, a hydrolysis probe, and an intercalating dye, but is not limited thereto, and may include a substance capable of detecting a fluorescent signal by reacting with a nucleic acid. The PCR solution is transferred to the fourth flow path 174 through the third flow path 173 and the PCR solution and the nucleic acid are mixed in the fourth flow path 174 to form a mixed material. have. That is, the nucleic acid and the PCR solution may be mixed sufficiently before being transferred to the amplification unit 150.

FIG. 6 is a flowchart illustrating steps of amplifying and detecting a nucleic acid of a method of detecting a nucleic acid of a film-based integrated chip according to an exemplary embodiment of the present invention.

6, after the extracted nucleic acid and the PCR solution injected into the PCR solution injection unit 140 are mixed to form a mixed material (S240), the mixed material is injected into the amplification unit 150, And amplifying and detecting the nucleic acid (S250).

In step S250 of transferring the mixed material to the amplification unit 150 and amplifying and detecting the nucleic acid, the first step S251 of closing the valve unit 180 may be performed. At this stage, the nucleic acid located in the fourth flow path 174 may be transferred to the amplification unit 150 while mixing with the PCR solution injected from the PCR solution injection unit 140 to form a mixed material. As the valve unit 180 is closed, the mixed material may be suspended in the amplification unit 150.

After the step S251 of closing the valve unit 180, a step S252 of amplifying the nucleic acid located in the amplification unit 150 may be performed. At this stage, the amplification unit 150 can sequentially amplify the nucleic acid by sequentially performing heating and cooling by the heater block and the cooling block.

After the step S252 of amplifying the nucleic acid in the amplification unit 150, a step S253 of receiving the fluorescence signal of the signal substance and detecting the amount of the nucleic acid may be performed. The fluorescence signal of the signal material can be received and confirmed through a separate fluorescence signal receiver or the like, thereby detecting the amount of the nucleic acid.

The step S252 of amplifying the nucleic acid located in the amplification unit 150 and the step S253 of detecting the amount of the nucleic acid by receiving the fluorescence signal of the signal substance are sequentially and repeatedly performed a predetermined number of times .

As described above, the present invention can simultaneously perform the amplification and detection of the nucleic acid in real time, so that gene analysis can be performed quickly.

Referring to FIG. 2, after the mixed material is transferred to the amplification unit 150 to amplify and detect the nucleic acid (S250), the PCR solution in the mixed material is used to amplify and detect specific amplification (S260). ≪ / RTI > Specifically, the step of determining whether specific amplification is performed using the PCR solution in the mixed material (S260) may include a melting curve analysis using the fluorescence signal received from the signal material of the amplification unit 150 analysis can be performed. The melting curve analysis may be performed to confirm whether or not the nucleic acid other than the nucleic acid to be amplified is to be detected.

More specifically, the melting curve analysis is based on the fact that when the DNA reaches the melting temperature, 50% of the dsDNA becomes ssDNA (single-stranded DNA), and the fluorescence intensity decreases sharply. In the case of other substances, they have different melting temperatures depending on the length of the DNA and the difference in the base sequence. Therefore, if an error occurs, the fluorescence signal will be drastically reduced at the melting temperature of the nucleic acid to be detected and the melting temperature of the nucleic acid to be detected as a result of the melting curve analysis. In this way, when the amplification unit 150 amplifies the fluorescence signal, the fluorescence signal of the signal material is received in real time and the melting curve analysis is performed using the fluorescence signal. . That is, it can be confirmed whether the nucleic acid to be amplified is amplified correctly.

In addition, the amplification unit 150 or the fifth flow path 175 may determine whether the specific amplification is performed using the PCR solution in the mixed material (S260). That is, the mixed material may be discharged before being discharged to the collection unit 160. The step S260 of determining whether specific amplification is performed using the PCR solution in the mixed material may be applied when the signal material is a PNA probe.

FIG. 7 is a flowchart illustrating a step of recovering a mixed material in a nucleic acid detection method of a film-based integrated chip according to an embodiment of the present invention.

Referring to FIGS. 2 and 7, after the step S260 of determining the specific amplification using the PCR solution in the mixed material, the step S270 of recovering the mixed material may be further performed.

The step of recovering the mixed material (S270) may firstly open the third valve (183) and the fourth valve (184) (S271).

After the third valve 183 and the fourth valve 184 are opened (S271), injecting air into the PCR solution injecting unit 140 (S272) may be performed. At this stage, the mixed substance, amplified and detected by the amplification unit 150, is pushed by the air injected through the PCR solution injection unit 140, passes through the fifth flow path 175, . ≪ / RTI >

After the step S272 of injecting air into the PCR solution injecting unit 140, a step S273 of collecting the mixed substance discharged to the collection unit 160 by the injected air may be performed.

8 is a photograph showing the results of an experiment comparing a conventional example and an embodiment of the present invention. More specifically, FIG. 8 is a result of extracting nucleic acid by liquid phase extraction using toxoplasma gondii, amplifying the nucleic acid, and performing gel electrophoresis. 8 (a) corresponds to a conventional example in which gel electrophoresis was carried out after tube extraction and amplification with different amounts of toxoplasmosis, and FIG. 8 (b) shows the amount of toxoplasmosis And performing gel electrophoresis after performing tube extraction and amplification.

As can be seen from FIG. 8, extraction and amplification can be smoothly performed even with a chip extraction method using a relatively small extraction reagent. Especially, when the toxoplasmosis is small, the tube extraction method shows almost no signal, while the chip extraction method shows that the signal appears.

8 shows the result of measuring the final fluorescence signal by gel electrophoresis on the recovered mixed material. However, in actual implementation, the fluorescence signal from the amplification unit 150 is transmitted through a separate fluorescence signal receiver It is not necessary to take a separate step of measuring the fluorescence signal by collecting the mixed substance as in the gel electrophoresis method.

9 is a photograph of a film-based integrated chip according to an embodiment of the present invention.

Since the film-based integrated chip 100 prepared as described above is injected with a small amount of sample, the use of expensive biochemical reagents can be reduced. Therefore, since a small amount of sample is used, The nucleic acid can be rapidly extracted and amplified.

Since the film-based integrated chip 100 is configured to perform liquid-phase nucleic acid extraction, nucleic acid amplification, nucleic acid detection, and specific amplification on a single chip, cross-contamination among the samples does not occur, There are features that can be automated.

In addition, the film-based integrated chip 100 is advantageous in that liquid nucleic acid extraction, nucleic acid amplification, detection, and specific amplification are all performed on one chip.

Further, since the nucleic acid is extracted using the liquid phase extraction method, the present invention can extract the nucleic acid only by adjusting the temperature, so that the extraction operation is simpler and more economical than when the chemical agent is used. In addition, since the film-based integrated chip 100 is formed in a film shape and is manufactured through a patterning process, a costly photolithography process is unnecessary and can be manufactured at a low cost.

The film-based integrated chip 100 prepared as described above can be applied to and used in a gene detector. The nucleic acid detection method of the film-based integrated chip may also be applied to a gene detector.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

100: Film-based integrated chip 110: Film part
120: sample injection unit 130:
140: PCR solution injection unit 150: amplification unit
160: recovery unit 171: first flow path
172: second flow path 173: third flow path
174: fourth flow path 175: fifth flow path
180: valve portion 181: first valve
182: second valve 183: third valve
184: fourth valve

Claims (20)

A film portion forming a body;
A sample injecting unit provided in the film unit and injecting a sample;
A first flow path connected to one side of the sample injection unit to transfer the sample;
An extraction unit connected to the other side of the first flow path and extracting nucleic acid from the transferred sample;
A second flow path connected to one side of the extraction unit for transferring the extracted nucleic acid;
A PCR solution injecting unit provided in the film unit and into which a PCR solution is injected;
A third channel connected to one side of the PCR solution injecting unit and transferring the injected PCR solution;
A fourth flow path connected to the second flow path and the third flow path, wherein the nucleic acid introduced from the second flow path and the PCR solution introduced from the third flow path are mixed to form a mixed material;
An amplification unit connected to the other side of the fourth flow path to receive the mixed material and amplify the extracted nucleic acid;
A fifth channel connected to one side of the amplification unit and transferring the mixed material discharged from the amplification unit; And
And a recovery unit connected to the other side of the fifth flow path for recovering the mixed substance,
Wherein the nucleic acid is detected in real time while being amplified by the amplification unit. The method according to claim 1,
Further comprising a valve portion for controlling the flow of the fluid in the film portion. 3. The method of claim 2,
The valve unit includes:
A first valve provided in the first flow path to control the transfer of the sample;
A second valve provided in the second flow path and controlling transfer of the extracted nucleic acid;
A third valve provided in the third flow path for controlling transfer of the PCR solution; And
And a fourth valve provided at the fifth flow path for controlling the transfer of the mixed material. The method according to claim 1,
Wherein the PCR solution includes a signal material. 5. The method of claim 4,
Wherein the nucleic acid amplified by the amplification unit reacts with the signal material, and the amount of the nucleic acid is detected by receiving a fluorescence signal of the signal material. The method according to claim 4 or 5,
Wherein the signal material is any one of a fluorescent PNA probe, a hydrolysis probe, and an intercalating dye. The method according to claim 1,
In the extraction unit,
Wherein an enzyme-containing extraction reagent is injected and the enzyme activated at a temperature of 70 ° C to 80 ° C reacts with the sample to extract the nucleic acid. A method for detecting nucleic acid in a film-based integrated chip according to claim 3,
a) injecting the sample into the sample injector;
b) transferring the injected sample to the extractor;
c) extracting the nucleic acid from the sample transferred to the extraction unit;
d) mixing the extracted nucleic acid with the PCR solution injected into the PCR solution injection unit to form a mixed material; And
and (e) transferring the formed mixed material to the amplification unit to amplify and detect the nucleic acid. 9. The method of claim 8,
The step b)
b1) opening said first valve, said second valve and said third valve; And
and b2) transferring the sample to the extraction unit. 9. The method of claim 8,
The step c)
c1) injecting an extraction reagent containing an enzyme into the extraction unit;
c2) closing the first valve and the second valve;
c3) raising the temperature of the extraction unit such that the enzyme is activated;
c4) extracting said nucleic acid from said sample with said activated enzyme; And
and c5) further raising the temperature of the extraction unit such that the enzyme is deactivated. 11. The method of claim 10,
In the step c3)
Wherein the extracting unit is heated to a temperature of 70 ° C or more and less than 80 ° C. 9. The method of claim 8,
The step d)
d1) opening the first valve, the second valve and the fourth valve;
d2) transferring the extracted nucleic acid to the fourth channel;
d3) closing the second valve; And
d4) transferring the PCR solution injected into the PCR solution injection unit to the fourth flow path, and mixing the PCR solution and the nucleic acid,
Wherein the PCR solution and the nucleic acid are mixed to form a mixed material in step d4). 9. The method of claim 8,
Wherein the PCR solution includes a signal material. 14. The method of claim 13,
The step e)
e1) closing the valve portion;
e2) amplifying the nucleic acid in the amplification unit; And
e3) receiving the fluorescence signal of the signal material and detecting the amount of the nucleic acid,
Wherein the step of amplifying the nucleic acid and the step of detecting the amount of the nucleic acid are sequentially and repeatedly performed a predetermined number of times. The method according to claim 13 or 14,
Wherein the signal material is any one of a fluorescent PNA probe, a hydrolysis probe, and an intercalating dye. 9. The method of claim 8,
After step e)
f) determining whether specific amplification is performed using the PCR solution in the mixed material,
Wherein the fluorescence signal of the signal substance contained in the PCR solution is detected through melting curve analysis to determine whether the amplification is specifically amplified. 17. The method of claim 16,
After the step f)
g) recovering the mixed material. < RTI ID = 0.0 > 8. < / RTI > 18. The method of claim 17,
The step g)
g1) opening the third valve and the fourth valve;
g2) injecting air into the PCR solution injection unit; And
g3) collecting the mixed material discharged to the collection unit by the injected air. A gene detector using the film-based integrated chip according to claim 1. A gene detector using the nucleic acid detection method of the film-based integrated chip according to claim 8.

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