KR102090863B1 - Thermal cycler including microchamber integrated metal mesh film, polymerase chain reaction system including the same, and method for manufacturing the same - Google Patents

Abstract

According to an embodiment of the present invention, a flexible substrate (flexible substrate); A reaction chamber formed on an upper surface of the flexible substrate; And a metal mesh film formed on a lower surface of the flexible substrate.

Description

THERMAL CYCLER INCLUDING MICROCHAMBER INTEGRATED METAL MESH FILM, POLYMERASE CHAIN REACTION SYSTEM INCLUDING THE SAME, AND METHOD FOR MANUFACTURING THE SAME }

The present invention relates to a heat circulation device comprising a microchamber-integrated metal mesh film, a gene amplification system comprising the same, and a method for manufacturing the heat cycle device.

Polymerase Chain Reaction (PCR) is a technology that repeatedly heats and cools a specific part of the template nucleic acid, replicates it in a chain, and amplifies the nucleic acid having the specific part exponentially, life science, genetic engineering , It is widely used for analysis and diagnostic purposes in the medical field. With the application field of PCR technology expanding, a number of devices have been recently developed to efficiently perform PCR.

Conventional PCR devices are equipped with a tube-shaped reaction vessel in which a plurality of sample solutions containing template nucleic acids are introduced into a single heater, and the reaction vessel is repeatedly heated and cooled to perform gene amplification. Perform. Other types of conventional PCR devices are equipped with a plurality of heaters, and gene amplification is performed by perfusion of a sample solution containing nucleic acids in a single flow path through them.

However, as shown in FIG. 1, the conventional PCR device has a metal block-based thermal cycler and a reaction chamber independently using a Peltier effect, so high-power application is required for rapid heating. Since it is required, power consumption is not only high, but overshoot may occur.

In addition, such a PCR device uses a large-capacity tube, which results in unnecessary loss of expensive PCR reagents. Moreover, the existing PCR device has a weak heat transfer efficiency between the heat cycler and the reaction chamber, and there is a limitation that requires expensive equipment for uniform temperature gradient.

The present invention is to solve the above-mentioned problems of the prior art, the object of the present invention is a rapid heat transfer between the thermal cycler (termal cycler) and the reaction chamber (reaction chamber), fast heating / cooling rate (ramping rate), low power operation And to provide a thermal circulation device capable of simultaneously implementing a uniform temperature distribution, a gene amplification system comprising the same, and a method of manufacturing the thermal cycle device.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, a flexible substrate (flexible substrate); A reaction chamber provided on an upper surface of the flexible substrate; And a metal mesh film provided on a lower surface of the flexible substrate.

According to one side, the flexible substrate is polyethylene terephthalate (PET, Polyethylene terephthalate), polyimide (PI, Polyimide), polyethylene naphthalate (PEN, Polyethylene naphthalate), polyether sulfone (PES, Polyether sulfone), polycarbonate ( PC, PolyCarbonate) or a mixture of two or more of them.

According to one side, the reaction chamber may include a plurality of reaction grooves formed in a vertical direction from the top surface.

According to one side, the height of the reaction groove may be 100 nm to 500 nm, and the cross-sectional diameter may be 0.1 μm to 3 μm.

According to one side, the thermal circulation device further includes a hydrophobic film provided on the upper surface of the reaction chamber, the hydrophobic film may be formed in a hole corresponding to the reaction groove.

According to one side, the thickness of the hydrophobic film may be 0.1 nm to 10 nm.

According to one side, the reaction chamber may be made of a hydrophilic material.

According to one side, the metal mesh film is silver (Ag), copper (Cu), aluminum (Al), a magnesium (Mg), gold (Au), nickel (Ni), titanium (Ti), molybdenum (Mo) , Tungsten (W), chromium (Cr), platinum (Pt), or two or more of these.

According to one side, the thickness of the metal mesh film may be 15 nm to 350 nm.

According to one side, the line width of the metal mesh may be 0.25㎛ to 3.5㎛, the distance between the lines may be 15㎛ to 300㎛.

According to another embodiment of the present invention, a gene amplification system is provided that includes the thermal cycling device.

According to another embodiment of the present invention, depositing a metal on the first stamp is formed a mesh pattern; Forming a metal mesh film by bringing the first stamp into contact with a lower surface of a flexible substrate; Depositing a hydrophilic material on a second stamp having a plurality of grooves; And forming a reaction chamber having a plurality of reaction grooves by bringing the second stamp into contact with the upper surface of the flexible substrate.

According to one side, after the step of forming the reaction chamber, forming a hydrophobic film on the upper surface of the reaction chamber; further comprising, the hydrophobic film may be formed in a hole corresponding to the reaction groove.

By using the micro-sized metal mesh film as a heat transfer medium, the thermal cycle device of the present invention can form a fast heating / cooling rate and a uniform temperature distribution in the device even at low power.

In addition, the thermal circulation device may improve the heat transfer efficiency between the two by integrating the reaction chamber and the metal mesh film.

Therefore, the gene amplification system including the thermal cycle device can rapidly and effectively amplify multi-target genes simultaneously.

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

1 shows a conventional gene amplification device.
2 is a side view of a thermal cycle device according to an embodiment of the present invention.
3 is an enlarged view of a reaction chamber according to an embodiment of the present invention.
Figure 4 is a side view of a thermal cycle device according to an aspect of the present invention.
5 is an enlarged view of a metal mesh film according to an embodiment of the present invention.
6 is a schematic diagram of a method of manufacturing a thermal cycle device according to an embodiment of the present invention.
7 shows a scanning electron microscope (SEM) image of a reaction chamber according to an embodiment of the present invention.
8 shows a scanning electron microscope (SEM) image of a metal mesh film according to an embodiment of the present invention.
9 is a result of measuring the temperature increase rate and the temperature distribution of the thermal circulation device according to an embodiment of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. The same reference numerals in each drawing denote the same members.

Various changes can be made to the embodiments described below. The examples described below are not intended to be limiting with respect to the embodiments, and should be understood to include all modifications, equivalents, or substitutes thereof.

The terms used in the examples are only used to describe specific examples, and are not intended to limit the examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts or combinations thereof described in the specification, one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, actions, components, parts or combinations thereof are not excluded in advance.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related known technologies may unnecessarily obscure the subject matter of the embodiments, detailed descriptions thereof will be omitted.

2 to 5 show a thermal circulation device according to an embodiment of the present invention.

Referring to Figure 2, an embodiment of the present invention is a flexible substrate (flexible substrate) (10); A reaction chamber 20 provided on an upper surface of the flexible substrate; And a metal mesh film 30 provided on a lower surface of the flexible substrate.

The flexible substrate 10 may be formed of an organic material-based reaction chamber and a material that facilitates injection of reagents in the chamber. In addition, by using a flexible substrate capable of relatively free modification, the application of a gene amplification system (device) including the same can be expanded in many ways.

The flexible substrate 10 is polyethylene terephthalate (PET), polyimide (PI), polyethylene naphthalate (PEN), polyether sulfone (PES), polyether sulfone (PC), PolyCarbonate) or a mixture of two or more of them, preferably polyimide (PI), polyethersulfone (PES) or polycarbonate (PC), and more preferably a transparent polyimide material. , But is not limited thereto.

The reaction chamber 20 is a space for performing an amplification reaction by introducing a gene, a primer, a buffer, and the like to be amplified. 2 and 3, the reaction chamber may include a reaction groove 21 formed in a vertical direction from the top surface. The reaction groove is formed to extend into the chamber to accommodate samples, reagents, and the like.

At this time, the height h of the reaction groove 21 may be 100 nm to 500 nm, and the cross-sectional area diameter d may be 0.1 μm to 3 μm. By adjusting the internal space of the reaction groove as described above, the thermal cycling device can be manufactured in a compact size to improve portability, and the thermal cycling device can be implemented in a form suitable for digital PCR.

Referring to FIG. 4, the thermal circulation device further includes a hydrophobic film 40 provided on the upper surface 20 of the reaction chamber, and the hydrophobic film has holes formed at positions corresponding to the reaction grooves 21. Can be. That is, the shape of the plurality of reaction grooves formed in the reaction chamber may extend to the hydrophobic film. Since samples for gene amplification usually exhibit hydrophilicity, by forming the hydrophobic film, the sample remaining on the upper surface of the reaction chamber can be introduced into the reaction groove to improve reaction efficiency.

The hydrophobic film may be a film in which perfluorooctyltrichlorosilane (FOTS) or octadecyltrichlorosilane (OTS) is surface-treated or dispersed in a nano pillar form, but is not limited thereto.

At this time, the thickness t of the hydrophobic film 40 may be 0.1 nm to 10 nm. If the thickness of the hydrophobic film is less than 0.1 nm, the sample remaining on the upper surface of the reaction chamber cannot be easily introduced into the reaction groove, and if it is more than 10 nm, excessive hydrophobic material may be used to deteriorate economic efficiency.

Furthermore, the reaction chamber 20 may be made of a hydrophilic material in order to further improve the effect of removing the residue of the hydrophobic film 40. As described above, due to the hydrophilic properties of the sample for gene amplification, when the reaction chamber is made of a hydrophilic material, it is possible to more smoothly introduce the residual sample from the hydrophobic film formed thereon to the reaction groove.

The hydrophilic material may be polyvinyl alcohol (PVA), polyethyleneimine (PEI), polycarbonate (PC), or a mixture of two or more of them, but is not limited thereto.

Referring to FIG. 5, the metal mesh film 30 may be formed on the lower surface of the flexible substrate 10, that is, on the opposite surface of the surface on which the reaction chamber 20 is formed. Specifically, the mesh film may have an overall film form by crossing a metal mesh 31, that is, a plurality of metal lines in a mesh shape.

The metal mesh film 30 may be heated / cooled according to the supply / stop of DC voltage from the outside to control the temperature of the flexible substrate and reaction chamber. At this time, the film is formed in the form of a mesh, so that a more uniform temperature distribution in the device can be formed compared to a Peltier element including a heat sink.

The metal mesh film is silver (Ag), copper (Cu), aluminum (Al), magnesium (Mg), gold (Au), nickel (Ni), titanium (Ti), molybdenum (Mo), tungsten (W), It may be made of chromium (Cr), platinum (Pt) or an alloy of two or more of them, and preferably may be formed of an alloy based on copper or aluminum, silver nanowire (AgNW), or an alloy ink containing copper, It is not limited to this.

The metal mesh film 30 may have a thickness t of 15 nm to 350 nm. When the thickness of the metal mesh film is out of the above range, the heat transfer efficiency of the metal mesh may be reduced.

Further, the line width w of the metal mesh 31 may be 0.25 μm to 3.5 μm, and the line distance d may be 15 μm to 300 μm, preferably 50 μm to 150 μm. When the line width of the metal mesh is less than 0.25 μm or more than 3.5 μm, heat transfer efficiency of the metal mesh may be deteriorated. If the distance between the lines of the metal mesh is less than 15 μm, temperature control is not easy, and if it exceeds 300 μm, the temperature control efficiency of the reaction chamber may decrease.

According to another embodiment of the present invention, a gene amplification system is provided that includes the thermal cycling device. As described above, when gene amplification is performed using the thermal cycle device, rapid heat transfer to the reaction chamber and uniform temperature gradient can be realized, thereby improving the overall gene amplification rate and efficiency.

6 is a schematic diagram of a method of manufacturing a thermal cycle device according to an embodiment of the present invention.

Referring to FIG. 6, another embodiment of the present invention comprises depositing a metal 32 on a first stamp 50 on which a mesh pattern is formed; Forming a metal mesh film 30 by bringing the first stamp into contact with a lower surface of a flexible substrate 10; Depositing a hydrophilic material (22) on a plurality of grooved second stamps (60); And forming a reaction chamber 20 provided with a plurality of reaction grooves by contacting the second stamp with the upper surface of the flexible substrate 10. do.

The first stamp 50 is for forming a mesh pattern on the flexible substrate 10, and may be manufactured using a master in which the mesh pattern is engraved. At this time, the master may be made of silicon (Si) material, but is not limited thereto. The first stamp may be made of a photocurable polymer such as polyurethane acrylate (PUA), but is not limited thereto. The material of the flexible substrate is the same as described above.

A metal (32) is deposited on the first stamp (50) using bar coating, spin coating or spray coating, and an adhesive (not shown) is applied to one surface (lower surface) of the flexible substrate (10), and then contacted therebetween. The metal can be transferred to the flexible substrate by pressing and pressing. At this time, the pressing may be -0.05㎜ to -0.3㎜ interval rolling (rolling) pressing. In addition, in order to improve the transfer efficiency of the metal, the photocurable polymer may be cured by irradiating ultraviolet rays. Specifically, the ultraviolet light may be irradiated for 45 seconds to 1 minute at a rate of 1 m / min to 5 m / min. The types of metals are as described above.

After UV curing is completed, when the first stamp 50 is removed from the flexible substrate 10, the metal deposited on the first stamp is transferred onto the flexible substrate in the same mesh pattern as the first stamp to form a metal mesh film ( 30). The line width, line distance and film thickness of the metal mesh are the same as described above.

The second stamp 60 having a plurality of grooves may be manufactured from a master such as silicon (Si) using the same method as the first stamp. The reaction chamber 20 having a plurality of reaction grooves may be formed by depositing a hydrophilic material 22 on the second stamp and transferring it to the upper surface of the flexible substrate. The specific deposition, transfer conditions, and the like are the same as the method of forming the metal mesh film using the first stamp.

On the other hand, after the step of forming the reaction chamber, forming a hydrophobic film 40 on the upper surface of the reaction chamber; further comprising, the hydrophobic film may be formed in a hole corresponding to the reaction groove. The method for forming the hydrophobic film may be a known method such as bar coating, spin coating, spray coating. Other functions and thicknesses of the hydrophobic film are the same as described above.

Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  One

A first stamp of polyurethane acrylate (PUA) material was produced on a PET substrate by an imprint method using a silicon (Si) master on which an intaglio mesh pattern was formed. Separately, an adhesive layer was formed on a flexible substrate made of polyether sulfone (PES) using spin coating (5,000 rpm), and then partially cured by irradiating ultraviolet rays. After depositing 140 µm of Cu-based alloy (CuMg) metal on the first stamp, contacting the adhesive layer formed and partially cured on the flexible substrate, pressurized using a roll of -0.3 mm intervals. 100% ultraviolet light was irradiated at a rate of 3 m / min for 1 minute. After completion of ultraviolet irradiation, the first stamp was separated from the flexible substrate to form a film (200 nm thick) made of a metal mesh having a line width of 1.5 µm and a line-to-line distance of 50 µm on one side (lower surface) of the flexible substrate.

After producing a second stamp having a plurality of cylindrical grooves in the same way as the first stamp production, by depositing a hydrophilic material on the second stamp on the other side (top) of the flexible substrate is not formed of the metal mesh film Contact. Subsequently, after pressurizing using a roll of -0.3 mm intervals and irradiating 100% ultraviolet rays at a rate of 3 m / min for 1 minute, the second stamp was separated to form a reaction chamber in which a plurality of reaction grooves were formed. At this time, the cross-sectional diameter of the reaction groove was 300 nm and the height was 200 nm.

Next, after depositing a hydrophobic material on the second stamp and contacting the upper surface of the reaction chamber, pressurized using a roll of -0.3 mm intervals and 100% UV light at a rate of 3 m / min for 1 minute. Was investigated. By separating the second stamp from the upper surface of the reaction chamber to form a hydrophobic film to correspond to the upper surface of the reaction chamber, a metal cycle film, a flexible substrate, a reaction chamber and a thermal cycler in which the hydrophobic film is integrated were manufactured.

Example  2

In the same manner as in Example 1, a thermal circulation device was prepared, but the distance between the lines of the metal mesh film was changed to 100 μm.

Example  3

In the same manner as in Example 1, a thermal circulation device was prepared, but the distance between the lines of the metal mesh film was changed to 150 μm.

The thermal circulation devices of Examples 1 to 3 were observed using a scanning electron microscope (SEM), and the observation results of the reaction chambers formed on their upper surfaces are shown in FIG. 7, and the observation results of metal mesh films formed on their respective lower surfaces. 8 is shown.

Referring to FIG. 7, it can be seen that a plurality of reaction grooves were formed at regular intervals, diameters, and heights in the reaction chamber, and referring to FIG. 8, it was confirmed that films made of metal meshes at regular intervals were formed.

Experimental Example : metal Mesh  Measurement of temperature increase rate and temperature distribution according to the distance between lines of film

In order to confirm the temperature characteristics according to the distance between the lines of the metal mesh film in the thermal cycling device of Examples 1 to 3, electrodes were prepared by depositing Al on both left and right ends of each film through a deposition process using a shadow mask, DC voltage of 3V was applied to each electrode to stop supply at 200 seconds. The temperature change with time was observed for the device of each example, and the results are shown in FIG. 9.

9A, it can be seen that as the distance between the lines decreases, that is, as the inner space of the mesh becomes narrower, the temperature increase rate of the film increases, which is most dramatic when the distance between the lines is 50 µm (Example 1), It appeared most gently at 150 μm (Example 3).

Through these results, it can be seen that the gene amplification system can be driven according to each application because the gene amplification rate or efficiency can be controlled by adjusting the line-to-line distance of the thermal cycle device.

In addition, referring to FIG. 9B, it can be seen that the temperature distribution of the metal mesh film is uniformly formed while applying the voltage, thereby improving the efficiency of gene amplification.

As described above, although the embodiments have been described by a limited embodiment and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order than the described method, and / or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

10: flexible substrate
20: reaction chamber
21: reaction home
22: hydrophilic material
30: Metal mesh film
31: Metal mesh
32: Metal
40: hydrophobic film
50: first stamp
60: second stamp

Claims (13)

Flexible substrates;
A reaction chamber provided on an upper surface of the flexible substrate; And
Includes a metal mesh film (metal mesh film) of 15nm to 350nm thickness provided on the lower surface of the flexible substrate;
The reaction chamber is made of a hydrophilic material,
A hydrophobic film is provided on the upper surface of the reaction chamber, and a thermal circulation device for a gene amplification system.
According to claim 1,
The flexible substrate is polyethylene terephthalate (PET), polyimide (PI), polyethylene naphthalate (PEN), polyether sulfone (PES), polycarbonate (PC, PolyCarbonate) or A thermocycle device for a gene amplification system, consisting of a mixture of two or more of these.
According to claim 1,
The reaction chamber includes a plurality of reaction grooves formed in the vertical direction from the top surface,
The hydrophobic film has a hole formed at a position corresponding to the reaction groove, a thermal cycle device for a gene amplification system.
According to claim 3,
The height of the reaction groove is 100 ㎚ to 500 ㎚, the cross-sectional diameter of 0.1㎛ to 3㎛, thermal circulation device for a gene amplification system.
delete According to claim 1,
The hydrophobic film has a thickness of 0.1 to 10 nm, a thermal cycle device for a gene amplification system.
delete According to claim 1,
The metal mesh film is silver (Ag), copper (Cu), aluminum (Al), magnesium (Mg), gold (Au), nickel (Ni), titanium (Ti), molybdenum (Mo), tungsten (W), Chromium (Cr), platinum (Pt) or a thermocycler for a gene amplification system, consisting of two or more of these.
delete According to claim 1,
The metal mesh has a line width of 0.25 µm to 3.5 µm and a line-to-line distance of 15 µm to 300 µm, a thermal cycle device for a gene amplification system.
A gene amplification system comprising the thermocycling device of any one of claims 1 to 4, 6, 8 or 10.
Depositing metal on the first stamp on which the mesh pattern is formed;
Forming a metal mesh film by bringing the first stamp into contact with a lower surface of a flexible substrate;
Depositing a hydrophilic material on a second stamp having a plurality of grooves; And
And forming a reaction chamber having a plurality of reaction grooves by bringing the second stamp into contact with an upper surface of the flexible substrate.
The method of claim 12,
After the reaction chamber forming step,
Further comprising; forming a hydrophobic film on the upper surface of the reaction chamber,
The hydrophobic film has a hole formed in a position corresponding to the reaction groove, a method of manufacturing a thermal cycle device.

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