WO2017043893A1

WO2017043893A1 - Pcr-based point-of-care testing device having microfluidic chip for electrophoresis coupled thereto

Info

Publication number: WO2017043893A1
Application number: PCT/KR2016/010111
Authority: WO
Inventors: 이재훈
Original assignee: 주식회사 하임바이오텍
Priority date: 2015-09-08
Filing date: 2016-09-08
Publication date: 2017-03-16
Also published as: KR20170030154A; KR101751913B1

Abstract

The present invention relates to a point-of-care testing device, and provides a PCR-based point-of-care testing device comprising: i) a microtube comprising a cap and a container fastened to the cap, the upper portion of the container comprising a corrugated elastic wall, the lower portion thereof having a conical shape such that the same becomes narrower downwardly, the bottom surface thereof being made of a thin film, the container comprising a junction portion protruding downwards from the bottom surface in a hollow pillar shape; and ii) a microfluidic chip having a hollow protrusion formed on the upper portion thereof, the protrusion having a sharp pointed portion fastened to the junction portion, a microfluidic channel, which is filled with an electrophoresis matrix, being connected to the lower portion of the protrusion, and electrodes being provided on the upper and lower ends of the microfluidic channel, respectively.

Description

PCR based field analysis device combined with microfluidic chip for electrophoresis

The present invention relates to a field analysis device (Point of Care Testing, hereinafter 'POCT'), and more particularly to a PCR-based field analysis device combined with a microfluidic chip for electrophoresis.

The field of microfluidics offers the possibility to reduce the size of complete chemistry laboratories to the size of credit cards. Microfluidics is achieved through a network of micrometer-sized channels connecting many microsized devices such as valves, mixers, heating elements, and sensors. It is implemented through a microfluidic device that drives it.

Microfluidics is also widely used in the fields of biochemistry and molecular biology. Examples of the microfluidics include blood glucose measurement devices, capillary electrophoresis, and PCR chips (Toriello et al., Anal. Chem. 78 (23): 79978003, 2006). exist.

In particular, the PCR chip has the advantage of being able to amplify and analyze DNA samples in a short time, but has the disadvantages of PCR inhibition and contamination due to the large surface-to-volume ratio that increases the surface-agent interaction.

Meanwhile, polymerase chain reaction (PCR) has become an essential technique in the field of molecular biology. However, traditional PCR has a relatively long reaction time, and the analysis requires labor-intensive experiments such as electrophoresis, which is not suitable for high capacity analysis. Of course, there is a real-time PCR method that can perform the analysis at the same time, but this real-time PCR method is not only unsuitable for the analysis of PCR reactions, such as multiflex PCR (multiflex PCR), but also can quickly amplify and confirm the sample in the field, such as a hospital There is a disadvantage that cannot be used as a field analysis device.

Therefore, there is an urgent need for the development of methods and devices that can perform more efficient and precise PCR reactions and subsequent analysis of amplified DNA fragments at once for rapid and accurate in situ analysis.

The present invention is to solve a number of problems including the above problems, it is an object of the present invention to provide a PCR-based field analysis device (POCT) that can perform PCR and electrophoresis at once. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to one aspect of the invention, i) a microtube consisting of a lid and a container fastened to the lid, wherein the container is a pleated elastic wall at the top and a narrower conical shape at the bottom, the bottom surface is composed of a thin film and the A microtube consisting of a hollow cylindrical joint surrounding the bottom surface; And ii) a hollow protrusion having a sharp attachment fastened to the junction portion is formed at an upper portion thereof, and a microfluidic channel filled with an electrophoretic medium is connected to the lower portion of the protrusion, and an electrode is provided at the top and bottom of the microfluidic channel, respectively. Provided is a PCR based field analysis device comprising the microfluidic chip provided.

According to another aspect of the invention, the PCR microtube insertion hole is formed on the upper surface, a hollow protrusion having a sharp attachment to the bottom of the PCR microtube insertion hole is formed, the electrophoretic medium (matrix) below the protrusion There is provided a PCR-based on-site analysis device including a microfluidic channel filled with), and a microfluidic chip having electrodes at the top and bottom of the channel, respectively.

According to another aspect of the invention, i) a PCR microtube drilling step of drilling a lower surface of the PCR microtube by applying a first pressure to the PCR microtube of the PCR-based field analysis device is complete; ii) a PCR reaction solution loading step of opening the lid of the PCR microtube and applying a second pressure to the inside of the PCR microtube to load the PCR reaction solution into the microfluidic channel of the microfluidic chip; iii) an electrophoresis step of performing electrophoresis by applying an alternating current to electrodes connected to both ends of the microfluidic channel; And iv) a nucleic acid molecule detecting step of detecting a nucleic acid molecule electrophoresed using fluorescence or ultraviolet light.

In addition, according to another aspect of the present invention, i) PCR tube loading step of inserting the PCR microtube completion of the PCR reaction in the PCR microtube insertion hole of the PCR-based field analysis apparatus of claim 7; ii) perforating the PCR microtube by applying a first pressure to the inserted PCR microtube to perforate the lower surface of the PCR microtube; iii) a PCR reaction solution loading step of opening the lid of the PCR microtube and applying a second pressure to the inside of the PCR microtube to load the PCR reaction solution into the microfluidic channel of the microfluidic chip; iv) an electrophoresis step of performing electrophoresis by applying an alternating current to electrodes connected to both ends of the microfluidic channel; And v) a nucleic acid molecule detection step of detecting a nucleic acid molecule electrophoresed using fluorescence or ultraviolet light.

According to an embodiment of the present invention, the PCR reaction is performed in the micro tube of the PCR-based field analysis apparatus, and when the PCR reaction is completed, the attachment of the protrusion formed on the microfluidic chip of the PCR-based field analysis apparatus is the micro The PCR reaction solution was made by drilling a thin film-like bottom surface of the tube and moved to the microfluidic channel filled with the electrophoretic medium through the hole in the protrusion, and the channel was inversely proportional to the size of the DNA fragment amplified by the application of an electric current. As it moves, it is detected through a detection means in a reaction apparatus that simultaneously performs PCR and electrophoresis, thereby having the advantage of automating identification of amplification products through PCR reaction and electrophoresis. Of course, the scope of the present invention is not limited by these effects.

1 is a schematic diagram schematically showing a PCR-based field analysis apparatus according to an embodiment of the present invention.

Figure 2 is a schematic diagram schematically showing a reaction apparatus equipped with a PCR-based field analysis apparatus according to an embodiment of the present invention is carried out PCR and electrophoresis.

Figure 3 is a schematic diagram showing the operating principle of the PCR-based field analysis apparatus according to an embodiment of the present invention.

4 is a schematic diagram schematically showing a PCR-based field analysis apparatus according to another embodiment of the present invention.

5 is a schematic diagram schematically showing the operating principle of the PCR-based field analysis apparatus shown in FIG.

용어의 정의:Definition of Terms:

The terms used in this document are defined below:

As used herein, the term "nucleotide" refers to a monomer molecule constituting a nucleic acid and is composed of a base, an pentose sugar, and a phosphoric acid. In the case of DNA, the base is adenine, guanine, and cytosine. Four kinds of thymine exist, and in the case of RNA, uracil is used instead of the thymine. The pentose sugar is 2'-deoxyribose in the case of DNA, ribose is used in the case of RNA.

As used herein, the term "oligonucleotide" refers to a short single-stranded nucleic acid chain consisting of 13 to 25 nucleotides as a polymer of the nucleotide monomer molecule, in some cases 6-mer, 7 It may refer to a nucleic acid chain consisting of less than 13 nucleotides or more than 25 nucleotides, such as -mer, 8-mer, 9-mer, 10-mer, 11-mer, and 12-mer.

As used herein, the term "polynucleotide" generally refers to a polymer chain of nucleotide units longer than the oligonucleotide, but is also used interchangeably with the oligonucleotide. Polynucleotides include both single-stranded or double-stranded nucleic acid chains.

As used herein, the term "sense strand" refers to a single-stranded nucleic acid molecule in the same direction as the direction of the code of the gene of the double-stranded DNA molecule, "antisense strand" is another single strand complementary to the sense strand. Although it refers to a nucleic acid molecule, it is also possible to define a strand in which the nucleic acid sequence is first identified as a "sense strand" and its complementary strand as an "antisense strand" irrespective of the direction of the gene's code progression.

As used herein, the term "polymerase chain reaction" or "nucleic acid amplification reaction" refers to a reaction that amplifies a particular target nucleic acid molecule using a thermostable DNA polymerase. PCR, the DNA in addition to the polymerase target nucleic acid oligonucleotide that can specifically hybridize to a nucleotide of the primer (forward primer, reverse primer), deoxy-nucleotide mixture (dNTP mixture), Mg ² ^+, such as divalent reaction buffer containing ions such as This is used. The DNA molecules produced by the PCR reactions are referred to herein as "amplification products".

As used herein, the term "primer" refers to an oligonucleotide or polynucleotide that complementarily hybridizes to template DNA, which is used for initiation of a PCR reaction or a primer extension reaction. A primer for a PCR reaction is a pair of forward primers (or sense primers) selected from the same sense strand as the direction of genetic code of the nucleic acid molecule to be amplified and reverse primers (or antisense primers) selected from antisense strands complementary to the sense strands. In the case of primer extension, a single extension primer is usually used.

The term "point of care testing" used in this document is a container and device used for small, portable analysis that can be analyzed immediately at the site where the sample is taken, rather than being taken to the laboratory for analysis. Are referred to generically.

발명의 상세한 설명Detailed description of the invention

According to one aspect of the invention, i) a microtube consisting of a lid and a container fastened to the lid, the container is a concave shape of the upper portion is a corrugated elastic wall and the lower narrower, the bottom surface is composed of a thin film and the A micro tube consisting of a hollow columnar junction projecting downward from the bottom surface; And ii) a hollow projection having a sharp attachment fastened to the junction portion is formed at an upper portion thereof, and a microfluidic channel filled with an electrophoretic matrix is connected to the lower portion of the projection, and upper and lower ends of the microfluidic channel are connected. PCR-based field analysis apparatus comprising a microfluidic chip each provided with an electrode is provided.

In the device, the microtube may be made of polyethylene, polystyrene or polypropylene material.

In the device, the bottom surface may have a thickness of 50 to 200 μm.

In the device, the electrophoretic medium may be an agarose gel, starch gel, or polyacrylamide gel.

In the apparatus, the microfluidic channel may be formed in a “1” shape or may have a shape in which a “d” shaped pipe portion and a “l” shaped pipe portion are mixed.

According to another aspect of the invention, the PCR microtube insertion hole is formed on the upper surface, a hollow protrusion having a sharp attachment to the bottom of the PCR microtube insertion hole is formed, the electrophoretic medium (matrix) below the protrusion There is provided a PCR-based on-site analysis device including a microfluidic chip filled with) and a microfluidic chip having electrodes at the top and bottom of the microfluidic channel, respectively.

In the apparatus, the channel may be formed in the shape of "I" or in the shape of a mixture of "D" tube and "I" tube.

In the method, the first pressure may be a pressure exceeding the maximum shear stress of the bottom surface of the PCR microtube, the second pressure is applied to the extent that the volume of the pleated elastic wall can be reduced or the PCR It may be applied by a piston inserted into the microtube.

Hereinafter, a kit and a method according to an embodiment of the present invention will be described in more detail with reference to the accompanying drawings.

1 is a schematic diagram schematically showing a microfluidic device according to an embodiment of the present invention.

As shown in FIG. 1, the PCR-based on-site analysis device 1 according to an embodiment of the present invention includes a microtube 10 in which a PCR reaction occurs and a microfluidic chip 20 in which electrophoresis is performed up and down. As a form, the microtube 10 has a hollow pipe-like coupling portion formed at the bottom of the bottom, unlike the general PCR reaction microtube, and the bottom is formed of a very thin film. It can be easily pierced by an attachment (not shown) at the end of the protrusion 21 protruding into the corrugated wall. When pressed by the first pressure, wrinkles do not fold, and after punching, when pressed by the second pressure and the height is reduced, the amount of the PCR reaction solution is reduced by the volume of electricity in the channel 22 through the passage formed in the protrusion 21. Yeongdong Hawk The load is to be able to have. At this time, the channel 22 for electrophoresis formed in the microfluidic chip 20 was vertically formed as shown in FIG. 1, and DC current was applied through the

electrodes

24 and 24 ′ formed at the upper and lower ends, respectively. When the amplified DNA is moved to the positive (+) in the channel. The detection of the amplified DNA can be detected by various detection means. In general, it is possible to monitor in real time by irradiating a laser to a channel using a fluorescent material and then detecting excitation light of a specific wavelength.

Optionally, when the microfluidic chip is elongated in the horizontal direction without being elongated in the vertical direction, the channel may also be formed horizontally in the longitudinal direction (not shown).

PCR-based field analysis apparatus according to an embodiment of the present invention can be usefully used for automation of the analysis in that the PCR reaction and electrophoresis may occur automatically except for transferring the PCR reaction solution to the microtube as described above.

2 is a view schematically showing a PCR-based field analysis apparatus according to another embodiment of the present invention. As shown in FIG. 2, the other components are the same as those of FIG. 1, but may have an effect of extending the electrophoretic development length by forming the channel 22 for electrophoresis in a 'd' shape (22 '). have. In this case, there is an advantage in that the resolution can be improved when the difference between the lengths of the amplified DNA fragments is not large. However, in this case as well, detection of the amplified DNA can be performed through the portion 22 "

A template polynucleotide, the oligonucleotide primers, thermostable DNA polymerase in the PCR reaction solution, toothed containing the ions and dNTP, such as Mg ² ⁺ made up in micro-tube 10, as shown in the left side of Figure 3 added The microtube 10 and the microfluidic chip 20 are coupled to each other during the PCR reaction which repeats cycles of thermal denaturation, hybridization, and polymerization several times. The bottom surface 15 of the lower end of the microfluidic chip 20 is not in communication with the state. However, when the PCR reaction is terminated, a first pressure is applied from a cover (not shown) of the PCR reaction apparatus so that the microtube 10 is lowered downward and the upper end of the protrusion 21 formed on the microfluidic chip 20. The attachment 23 formed in the penetrates through the bottom surface 15 of the lower end of the thin film of the microtube 10. Then, when the second pressure is applied to the cover of the PCR reactor, the corrugated wall 12 on the upper portion of the microtube 10 is pressed to reduce the volume inside the microtube 10, and the microtube is caused by the pressure generated at this time. The reaction solution therein is loaded on the electrophoretic medium of the microfluidic chip 20. Subsequently, when a DC current is applied to both electrodes of the electrophoretic microfluidic chip, the amplified DNA fragment moved into the channel is electrophoresed according to its size. The microfluidic chip 200 may further include a waste solution reservoir 205 for supporting excess solution.

As shown in FIG. 4, the PCR-based on-site analysis device 1 ′ according to an embodiment of the present invention is coupled to a micro tube 100 in which a PCR reaction occurs and a microfluidic chip 200 in which electrophoresis is performed. This possible but separate form microtube 100 is a special PCR reaction microtube made of a very thin thickness (several to several tens of micrometers) so that it is easily punched by a general PCR reaction microtube or a pointed protrusion at the bottom. As a hole easily attached by the end of the protrusion 201 protruding upward on the bottom of the bottom of the PCR reaction microtube receiving hole 206 formed on the top of the PCR-based field analysis apparatus 1 'according to an embodiment of the present invention as This may fly, and after being punched by the pressure applied to the PCR reaction microtube 100, the PCR reaction liquid is formed in the protrusion 201 by the second pressure applied in the PCR reaction microtube 100. Through such a passage, it may be possible to load the electrophoretic medium in the channel 202. At this time, the channel 202 for the electrophoresis formed in the microfluidic chip 200 is vertically formed as shown in FIG. 4, and DC current is applied through the

electrodes

204 and 204 ′ formed at the upper and lower ends, respectively. When the amplified DNA is moved to the anode (+) in the channel. The detection of the amplified DNA can be detected by various detection means. In general, it is possible to monitor in real time by irradiating a laser to a channel using a fluorescent material and then detecting excitation light of a specific wavelength.

5 is a piston 207 provided in the PCR based field analysis apparatus 1 'into the PCR microtube inserted into the PCR microtube insertion hole 206 of the PCR based field analysis apparatus 1' shown in FIG. It is a schematic diagram schematically showing a process of loading the PCR reaction solution into the microfluidic channel 202 by descending downward and pushing the PCR reaction solution in the PCR microtube downward. The outer shape of the piston 207 is manufactured according to the shape of the PCR microtube 100, the outer diameter is preferably slightly smaller than the inner diameter of the PCR microtube 100, the rubber or silicone material on the outer peripheral surface to properly apply pressure Gasket 208 may be provided.

Although the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

The apparatus and method of the present invention can be very useful in the environmental and medical fields because of the rapid detection of nucleic acid molecules, in particular the rapid detection of nucleic acids in situ.

Claims

i) a microtube consisting of a lid and a container fastened to the lid, wherein the container is a conical shape with a corrugated elastic wall at the top and a narrower taper at the bottom, the bottom being made of a thin film and hollowed out from the bottom Consisting of a columnar junction of micro-tubes; And

ii) a hollow projection having a sharp attachment fastened to the junction portion is formed at the top, and a microfluidic channel filled with an electrophoretic matrix is connected to the lower portion of the projection, and at the top and bottom of the microfluidic channel. PCR-based field analysis device comprising a microfluidic chip each equipped with an electrode.
The method of claim 1,

The electrophoretic medium is agarose gel, starch gel, or polyacrylamide gel, PCR based field analysis device.
The method of claim 1,

The micro tube is made of polyethylene, polystyrene or polypropylene material, PCR-based field analysis device.
The method of claim 1,

The bottom surface is 20 to 200 μm thick, PCR-based field analysis device.
The method of claim 1,

The electrophoretic medium is agarose gel, starch gel, or polyacrylamide gel, PCR based field analysis device.
The method of claim 1,

The microfluidic channel is formed in a "一" shape or "r" shaped pipe portion and the "一" shaped pipe portion, formed in the shape of a PCR-based field analysis device.
A PCR microtube insertion hole is formed on the upper surface, and a hollow protrusion having a sharp attachment is formed at the bottom of the PCR microtube insertion hole, and a microfluidic channel filled with an electrophoretic matrix is connected to the lower portion of the protrusion. And a PCR-based on-site analysis device comprising a microfluidic chip having electrodes on top and bottom of the microfluidic channel, respectively.
The method according to claim 7,

The electrophoretic medium is agarose gel, starch gel, or polyacrylamide gel, PCR based field analysis device.
The method of claim 7, wherein

Wherein the microfluidic channel is formed in a "一" shape or "r" shaped pipe portion and "一" shaped pipe portion is formed in a shape, PCR-based field analysis device.
i) a PCR microtube drilling step of drilling a lower surface of the PCR microtube by applying a first pressure to the PCR microtube of the PCR-based field analysis apparatus of claim 1, wherein the PCR reaction is completed;

ii) a PCR reaction solution loading step of opening the lid of the PCR microtube and applying a second pressure to the inside of the PCR microtube to load the PCR reaction solution into the microfluidic channel of the microfluidic chip;

iii) an electrophoresis step of performing electrophoresis by applying an alternating current to electrodes connected to both ends of the microfluidic channel; And

iv) PCR-based in situ nucleic acid analysis method comprising a nucleic acid molecule detection step of detecting the electrophoretic nucleic acid molecule using fluorescence or ultraviolet light.
i) PCR tube loading step of inserting the PCR microtube completion of the PCR reaction in the PCR microtube insertion hole of the PCR-based field analysis apparatus of claim 7;

ii) perforating the PCR microtube by applying a first pressure to the inserted PCR microtube to perforate the lower surface of the PCR microtube;

iii) a PCR reaction solution loading step of opening the lid of the PCR microtube and applying a second pressure to the inside of the PCR microtube to load the PCR reaction solution into the microfluidic channel of the microfluidic chip;

iv) an electrophoresis step of performing electrophoresis by applying an alternating current to electrodes connected to both ends of the microfluidic channel; And

v) PCR-based in situ nucleic acid analysis method comprising a nucleic acid molecule detection step for detecting the electrophoretic nucleic acid molecule using fluorescence or ultraviolet light.
The method according to claim 10 or 11, wherein

Wherein the first pressure is a pressure that exceeds a maximum shear stress of the bottom surface of the PCR microtube.
The method of claim 10,

And said second pressure is applied by a piston inserted into said PCR microtube.