KR102550235B1 - Nucleic acid amplification device using light source - Google Patents

Abstract

A nucleic acid amplification device using a light source is disclosed.
A nucleic acid amplification device using a light source according to an embodiment of the present invention includes an accommodating structure formed of a light-transmitting material that transmits light irradiated from the light source and having an accommodating space in which a sample including a target nucleic acid is accommodated; and a porous structure disposed in the accommodating space of the accommodating structure, at least a portion of which is located in a central region of the sample accommodated in the accommodating space, and having pores through which the sample is introduced or trapped, wherein the porous structure comprises: It has permeability and may be formed of a fiber in which metal nanoparticles are distributed on the surface or inside.

Description

Nucleic acid amplification device using light source}

The present invention relates to a nucleic acid amplification device using a light source, and more particularly, to a nucleic acid amplification device that amplifies a target nucleic acid by changing the temperature of the target nucleic acid using a light source.

In general, amplification of nucleic acids such as DNA is performed through PCR (Polymerase Chain Reaction) technology. This PCR technique involves the thermal denaturation of a target nucleic acid having a specific base sequence, the annealing of a primer, and the elongation of a complementary nucleic acid using a polymerase in one reaction. As a cycle, the reaction cycle is repeated about 30 to 40 times. In addition, since the thermal denaturation process in each reaction cycle proceeds at a temperature of 94 to 96 ° C, and the binding process and elongation process proceed at a temperature of 55 to 65 ° C and 72 to 75 ° C, respectively, the reaction cycle is performed at the temperature of the target nucleic acid. Respond to cycles of change.

However, as disclosed in Korean Patent Registration No. 10-1423582, the existing technology for controlling the temperature of a sample using a Peltier element during PCR performs a temperature change cycle once, from tens of seconds to several Since it takes more than a minute, there is a problem in that a long time is required for nucleic acid amplification and energy efficiency is low.

In addition, as disclosed in Korean Patent Publication No. 10-2017-0106995, a conventional technique for controlling the temperature of a sample by depositing a gold (Au) thin film on the inner surface of a well to which the sample is fixed, the surface of the sample Since only the region is in contact with the gold thin film, a relatively long time is required for heat to reach the central region of the sample through thermal convection, and when the temperature of the gold thin film is raised to shorten the sample heating time, There is a problem that causes deformation or destruction of some samples.

The technical problem to be solved by the present invention is to provide a nucleic acid amplification device that improves the heating and cooling rate of a sample including a target nucleic acid during amplification of a target nucleic acid, prevents deformation or destruction of the sample by heat, and improves energy efficiency will be.

A nucleic acid amplification device using a light source according to an embodiment of the present invention includes an accommodating structure formed of a light-transmitting material that transmits light emitted from the light source and having an accommodating space in which a sample including a target nucleic acid is accommodated; and a porous structure disposed within the accommodating space of the accommodating structure, at least a portion of which is located in a central region of the sample accommodated in the accommodating space, and having pores through which the sample is introduced or captured, wherein the porous structure comprises: It has permeability and may be formed of a fiber in which metal nanoparticles are distributed on the surface or inside.

In one embodiment, the fiber forming the porous structure, the core forming the center of the fiber; metal nanoparticles attached to the surface of the core; and a coating layer formed on the surface of the core and the metal nanoparticles.

In one embodiment, the accommodating structure is configured in the form of a tube having an opening communicating with the accommodating space at an upper end, and the porous structure is inserted into the accommodating space through the opening or in the accommodating space. It can be configured in the form of a stick, rod or block to be drawn out.

In one embodiment, the nucleic acid amplification device may include a reflector for reflecting light emitted from the light source and passing through the accommodation structure back toward the accommodation space of the accommodation structure.

In one embodiment, the accommodating structure is configured in the form of a chip having the accommodating space therein, and the porous structure is in the form of a network or block disposed in the accommodating space. can be configured.

In one embodiment, the accommodating structure may include an incident surface for incident light emitted from the light source toward the accommodating space, and a reflective surface for reflecting the light passing through the accommodating space back toward the accommodating space.

According to the present invention, a porous structure formed of fibers in which metal nanoparticles are distributed on the surface or inside and having a plurality of pores through which a sample is introduced or trapped is disposed in the central region of the sample accommodated in the receiving structure, and the porous structure By heating or cooling the corresponding sample by irradiating light or blocking the irradiated light, it is possible to prevent deformation or destruction of the sample due to heat and improve energy efficiency while increasing the heating and cooling rate of the sample.

In addition, by forming the porous structure in the form of a stick, rod, or block so that the porous structure can be inserted into or extracted from the tube-shaped accommodation structure, it is easy to separate nucleic acids from the corresponding sample after nucleic acid amplification is completed. and cost efficiency can be improved through reuse of the porous structure.

In addition, the sample heating rate and energy efficiency can be further improved by reflecting the light emitted from the light source and passing through the accommodation structure back to the accommodation space of the accommodation structure using a reflector or a reflective surface.

1 is a diagram showing a nucleic acid amplification device according to an embodiment of the present invention.
2 is a view showing an accommodation structure and a porous structure of a nucleic acid amplification device according to an embodiment of the present invention.
3 is a cross-sectional view showing an example of fibers forming a porous structure.
4 is a cross-sectional view showing another example of fibers forming a porous structure.
5 is a cross-sectional view showing an accommodation structure and a porous structure of a nucleic acid amplification device according to another embodiment of the present invention.
6 is a graph showing a change in temperature of a sample according to the operation of a light source.
7 is a graph showing comparison results of performance tests between the present invention and the existing technology.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings in order to clarify solutions to the technical problems of the present invention. However, in the description of the present invention, if the description of the related known technology makes the gist of the present invention unclear, the description thereof will be omitted. In addition, the terms used in this specification are terms defined in consideration of functions in the present invention, and they may vary according to the intention or custom of a designer or manufacturer. Therefore, the definitions of the terms to be described below will have to be made based on the content throughout this specification.

1 shows a nucleic acid amplification device 100 according to an embodiment of the present invention.

As shown in FIG. 1, the nucleic acid amplification device 100 according to an embodiment of the present invention is a device for amplifying a target nucleic acid having a specific base sequence, such as DNA, by using a light source It can be configured to change the temperature of the target nucleic acid to a temperature required for PCR (Polymerase Chain Reaction).

To this end, the nucleic acid amplification device 100 may include a light source 110, an accommodation structure 120, and a porous structure 130. In addition, according to the embodiment, the nucleic acid amplification device 100 may optionally further include a support frame 140, an optical lens 150, a reflector 160, a temperature sensor 170, a control unit 180, and the like. .

The light source 110 is configured to emit light of a specific band, such as a visible ray band or an infrared ray band, toward the accommodation structure 120 . To this end, the light source 110 may include a Light Emitting Diode (LED) or a laser diode.

The accommodating structure 120 is made of a light-transmitting material that transmits light emitted from the light source 110 and has an accommodating space 122 accommodating the sample S including the target nucleic acid. The sample S accommodated in the receiving structure 120 may include a target nucleic acid and a primer for generating a new nucleic acid using the target nucleic acid as a template, DNA polymerase, deoxynucleotide triphosphate (dNTP), etc. .

The receiving structure 120 may be made of a material having light transmittance and heat resistance. For example, the receiving structure 120 may be made of a polymer synthetic resin such as PP (Polypropylene), PE (Polyethylene), COC (Cycloolefin copolymer), PC (Polycarbonate), PET (Polyethylene terephthalate) or PDMS (Polydimethyls iloxane), or glass. It may be made of inorganic material.

In addition, the receiving structure 120 may be configured in a tube shape as shown in FIG. 1, or may be configured in a plate-shaped chip shape according to an embodiment. When the accommodating structure 120 is configured in a tube shape, a stopper 126 for preventing evaporation or leakage of the sample S may be coupled to an opening of the accommodating structure 120 . The stopper 126 may be made of a material having light transmission like the accommodation structure 120 . For example, the stopper 126 is a polymer synthetic resin such as PP (Polypropylene), PE (Polyethylene), COC (Cycloolefin copolymer), PC (Polycarbonate), PET (Polyethylene terephthalate) or PDMS (Polydimethyls iloxane), or an inorganic material such as glass It can be made of material.

In addition, the stopper 126 may be configured in various forms such as a detachable dome-type cap, a hinged dome-type cap, or a flat cap, and may be configured as a single stopper or 8 or 12 caps It may be configured in the form of a connected strip. In the case of a flat cap, labeling is possible, and in the case of a domed cap, it is possible to prevent the sample from evaporating on top.

The porous structure 130 is a three-dimensional structure having a plurality of air gaps, and is disposed in the accommodation space 122 of the accommodation structure 120 in which the sample S is accommodated, so that at least a portion of the accommodation space 122 ) Is configured to be located in the central region of the sample (S) accommodated in. When such a porous structure 130 is put into the sample (S), the sample (S) may flow into or be captured in the pores of the porous structure (130).

As will be described again below, the porous structure 130 may be formed of single or multiple fibers having light transmittance and having metal nanoparticles distributed on the surface or inside. For example, the porous structure 130 is configured by randomly folding or bending single or multiple strands of fibers to form a plurality of pores, or forming a plurality of strands of fibers into a network or scaffold. It can be configured in a way that combines in the form. That is, the fibers forming the porous structure 130 may be randomly or regularly coupled.

In addition, the porous structure 130 may be configured in various shapes that can be disposed in the accommodation space 122 of the accommodation structure 120 . In this case, the porous structure 130 is configured to be fixed in the accommodation space 122 of the accommodation structure 120, or is inserted into the accommodation structure 120 from the outside and taken out from the inside of the accommodation structure 120 to the outside. It can be configured to allow this. For example, the porous structure 130 is configured in the form of an irregular fiber bundle, or a tube, a stick, or a rod whose overall shape matches the accommodation space 122 of the accommodation structure 120 Alternatively, it may be configured in the form of a block or the like.

For reference, the samples introduced into or captured in the pores of the porous structure 130 are separated by centrifugation after completion of the nucleic acid amplification process, or the porous structure 130 is separated from the accommodation space 122 of the accommodation structure 120. It can be separated in a way that is gradually withdrawn.

On the other hand, the fibers forming the porous structure 130 may be made of a material having light transmittance and heat resistance. For example, the fiber is composed of a polymer synthetic resin such as PP (Polypropylene), PE (Polyethylene), COC (Cycloolefin copolymer), PC (Polycarbonate), PET (Polyethylene terephthalate) or PDMS (Polydimethyls iloxane), or an inorganic material such as glass It can be.

In addition, the metal nanoparticles distributed on or inside the fiber may have a size or diameter in nanometers to exhibit surface plasmon resonance characteristics by light irradiated from the light source 110. . Surface plasmon resonance refers to a phenomenon in which free electrons on the surface of metal nanoparticles oscillate collectively when light is incident between the surface of a metal nanoparticle, which is a conductor, and a dielectric such as air or water, in resonance with an electromagnetic field of a specific energy possessed by the light. As described above, the porous structure 130 can quickly and energy-efficiently heat the sample S by using a surface plasmon resonance phenomenon generated from metal nanoparticles. In this case, the frequency of the surface plasmon resonance may be changed according to the size or shape of the metal nanoparticles, the type of dielectric in contact, and the like.

In addition, the metal nanoparticles are, for example, gold (Au), silver (Ag), palladium (Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), germanium (Ge), tungsten (W) or iridium (Ir), etc., or may be composed of one or a combination of two or more of them.

On the other hand, the support frame 140 supports the accommodation structure 120 and is configured to be fixed at a predetermined position. In this case, the support frame 140 may be made of a light-transmitting material like the receiving structure 120 .

The optical lens 150 is configured to converge the light emitted from the light source 110 toward the receiving structure 120 . To this end, the optical lens 150 may be disposed between the light source 110 and the accommodation structure 120 .

The reflector 160 is configured to reflect the light emitted from the light source 110 and passing through the receiving structure 120 toward the receiving space 122 of the receiving structure 120 again. To this end, the reflector 160 may have a concave reflective surface such as a concave mirror.

The temperature sensor 170 is configured to detect the temperature of the sample S accommodated in the receiving structure 120 . In this case, the temperature sensor 170 may be configured as a contact temperature sensor using a thermocouple, thermistor, or resistance temperature detectors, or a non-contact temperature sensor using an infrared thermometer.

The controller 180 is configured to check the temperature of the sample S through the temperature sensor 170 and control the operation of the light source 110 according to the checked temperature. To this end, the control unit 180 may include one or more processors, memories, and the like.

2 shows an accommodation structure 120 and a porous structure 130 of a nucleic acid amplification device according to an embodiment of the present invention.

As shown in FIG. 2 , the accommodating structure 120 may be configured in the form of a tube having an opening 124 communicating with the accommodating space 122 at an upper end.

In addition, the porous structure 130 is inserted into the accommodating space 122 of the accommodating structure 120 through the opening 124 of the accommodating structure 120, or drawn out from the accommodating space 122, a stick, It can be configured in the form of a rod or a block. After the porous structure 130 is inserted into the accommodation space 122 of the accommodation structure 120, a stopper 126 is coupled to the opening 124 of the accommodation structure 120 as shown in FIG. ) to prevent evaporation or leakage.

In this way, since the porous structure 130 is configured to be inserted into and withdrawn from the accommodation structure 120, it is possible to facilitate the separation of nucleic acids from the sample after completion of nucleic acid amplification, and the porous structure 130 ) can improve cost efficiency.

In addition, as mentioned above, the porous structure 130 may be formed of single or multiple fibers 132 having light transmittance and having metal nanoparticles distributed thereon or on the surface thereof. For example, the porous structure 130 is configured by randomly folding or bending single-stranded fibers to form a plurality of pores G, or by forming a plurality of fiber strands into a network or scaffold ) can be configured in a way that is combined in the form.

3 shows an example of a fiber forming the porous structure 130 in cross-sectional view.

As shown in FIG. 3 , the porous structure 130 may be composed of fine fibers 132a in which metal nanoparticles 136a are distributed.

In this case, the body 134a of the fiber 132a may be made of a material having light transmittance and heat resistance. For example, the body 134a of the fiber 132a is a polymer synthetic resin such as PP (Polypropylene), PE (Polyethylene), COC (Cycloolefin copolymer), PC (Polycarbonate), PET (Polyethylene terephthalate) or PDMS (Polydimethyls iloxane) , can be made of an inorganic material such as glass.

In addition, the metal nanoparticles 136a distributed inside the fiber 132a have a size or diameter in nanometers to exhibit surface plasmon resonance characteristics by light irradiated from the light source 110. may consist of

In this case, the metal nanoparticles are, for example, gold (Au), silver (Ag), palladium (Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), germanium (Ge), It may be composed of tungsten (W) or iridium (Ir), or a combination of one or two or more of these.

4 shows another example of a fiber forming the porous structure 130 as a cross-sectional view.

As shown in FIG. 4 , the porous structure 130 may be composed of fine fibers 132b in which metal nanoparticles 136b are distributed on the surface side.

In this case, the core 134b of the fiber 132b to which the metal nanoparticles 136b are attached may be made of a material having light transmittance and heat resistance. For example, the core 134b may be made of a polymer synthetic resin such as PP (Polypropylene), PE (Polyethylene), COC (Cycloolefin copolymer), PC (Polycarbonate), PET (Polyethylene terephthalate) or PDMS (Polydimethyls Iloxane), or an inorganic material such as glass. It can be made of material.

In addition, as described above, the metal nanoparticles 136b may have a size or diameter in nanometers to exhibit surface plasmon resonance characteristics by light irradiated from the light source 110. In this case, the metal nanoparticles 136b may be, for example, gold (Au), silver (Ag), palladium (Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), germanium (Ge), tungsten (W), iridium (Ir), or the like, or may be composed of one or a combination of two or more of them.

The metal nanoparticles 136b may be attached to the surface of the core 134b by a physical method such as sputtering or evaporation or a chemical method through reduction or deposition of a precursor.

In addition, in one embodiment, the fiber 132b is placed on the surface of the core 134b and the metal nanoparticles 136b to prevent loss of the metal nanoparticles 136b attached to the surface of the core 134b. A formed coating layer 138b may be further included. In this case, the coating layer 138b may be made of a polymer synthetic resin such as PEG (Polyethylene glycol) or an inorganic material such as silica.

5 shows a vertical cross-sectional view of the accommodating structure 120a and the porous structure 130a of the nucleic acid amplification device according to another embodiment of the present invention.

As shown in FIG. 5 , the accommodating structure 120a may be configured in the form of a plate-shaped chip having a channel-shaped sample accommodating space 122a therein. In this case, the receiving structure 120a may be made of a material having permeability and heat resistance as described above. For example, the receiving structure 120a may be made of a polymer synthetic resin such as PP (Polypropylene), PE (Polyethylene), COC (Cycloolefin copolymer), PC (Polycarbonate), PET (Polyethylene terephthalate) or PDMS (Polydimethyls iloxane), or glass. It may be made of inorganic material.

In addition, as described above, the porous structure 130a may be formed of single or multiple fibers having light transmittance and having metal nanoparticles distributed thereon, and the accommodation structure 120a may contain It may be configured in the form of a network or a block disposed in the space 122a.

In one embodiment, the accommodating structure 120a has an incident surface 124a for incident light irradiated from the light source 110 toward the accommodating space 122a, and the light passing through the accommodating space 122a again. A reflective surface 126a for reflecting toward the receiving space 122a may be included. To this end, the reflective surface 126a may include a mirror plane or may be made of a material having a higher density than the material constituting the accommodation space 122a of the accommodation structure 120a so that total reflection of light occurs.

6 shows a graph of temperature change of a sample according to the operation of the light source 110 .

As shown in FIG. 6, when a sample including DNA, primer, DNA polymerase, deoxynucleotide triphosphate (dNTP), etc. is accommodated in the receiving structures 120 and 120a on which the porous structures 130 and 130a are disposed, The controller 180 of the nucleic acid amplification device 100 turns on the light source 110 to irradiate light toward the receiving structures 120 and 120a. As a result, surface plasmon resonance occurs in the metal nanoparticles 136a and 136b included in the porous structures 130 and 130a, so that the temperature of the sample rises.

When the temperature of the sample rises to about 92 ~ 96 ℃, the heat denaturation process in which the DNA contained in the sample is separated into two strands proceeds. Thereafter, when the controller 180 turns off the light source and the temperature of the sample decreases to about 50 to 65 ° C, the primer and DNA polymerase included in the sample bind to a specific site of the single-stranded DNA (annealing); DNA polymerase synthesizes DNA using dNTP as a material (elongation). These processes form one reaction cycle.

The controller 180 repeatedly turns on and off the light source 110 to repeat the reaction cycle 30 times or more.

7 shows a graph of performance test comparison results between the present invention and the conventional technology.

For the performance test, a technique of heating a sample through a Peltier element was used as a conventional technique. In addition, both the present invention and the existing technology carried out the PCR reaction by accommodating the same composition and amount of samples in tubes of the same size and structure.

First, 30 or more temperature change cycles are required to complete nucleic acid amplification, and as shown in FIG.

On the other hand, as shown in (b) of FIG. 7 , in the case of the conventional technology, it can be seen that only about 8.5 temperature change cycles are performed for 300 seconds. That is, since the existing technology raises the temperature of the sample by heating only the surface of the sample with a Peltier element and generating heat convection, it can be seen that it takes a long time to cool the sample as well as to heat the sample.

As described above, according to the present invention, the porous structures 130 and 130a formed of fibers in which metal nanoparticles are distributed on or inside the porous structures 130 and 130a having a plurality of pores through which samples are introduced or trapped are provided. (120, 120a) by placing in the central region of the sample accommodated, irradiating light to the porous structure (130, 130a) or blocking the irradiated light by heating or cooling the sample, the heating and cooling rate of the sample While improving, it is possible to prevent deformation or destruction of the sample due to heat, and to improve energy efficiency.

In addition, to allow the porous structure 130 to be inserted into or extracted from the accommodation structure 120 in the form of a tube, the porous structure 130 may be inserted into a stick or rod. Alternatively, when formed in the form of a block, it is possible to facilitate the separation of nucleic acids from the sample after completion of nucleic acid amplification, and cost efficiency can be improved through reuse of the porous structure.

In addition, by reflecting the light irradiated from the light source 110 and passing through the accommodation structures 120 and 120a back to the accommodation space of the accommodation structures 120 and 120a using the reflector 160 or the reflective surface 126a, The sample heating rate and energy efficiency can be further improved.

Furthermore, the embodiments according to the present invention can solve various technical problems other than those mentioned in this specification in the related art as well as in the related art.

So far, the present invention has been described with reference to specific examples. However, those skilled in the art will clearly understand that various modified embodiments can be implemented within the technical scope of the present invention. Therefore, the embodiments disclosed above should be considered from a descriptive point of view rather than a limiting point of view. That is, the scope of the true technical idea of the present invention is shown in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

100: nucleic acid amplification device
110: light source
120: receiving structure
130: porous structure
140: support frame
150: optical lens
160: reflector
170: temperature sensor
180: control unit

Claims (6)

A nucleic acid amplification device using a light source,
an accommodating structure made of a light-transmitting material that transmits light irradiated from the light source and having an accommodating space in which a sample including a target nucleic acid is accommodated; and
A porous structure disposed in the accommodating space of the accommodating structure, at least a portion of which is located in a central region of the sample accommodated in the accommodating space, and having pores through which the sample is introduced or trapped,
The porous structure is formed of a fiber having light transmission and having metal nanoparticles distributed on the surface or inside,
The fibers forming the porous structure,
a core constituting the center of the fiber;
metal nanoparticles attached to the surface of the core; and
A coating layer formed on the surface of the core and the metal nanoparticles,
The nucleic acid amplification device characterized in that the sample is heated by using a surface plasmon resonance phenomenon by light of the metal nanoparticles distributed on the surface of the fiber. delete According to claim 1,
The accommodating structure is configured in the form of a tube having an opening communicating with the accommodating space at an upper end,
The nucleic acid amplification device, characterized in that the porous structure is configured in the form of a stick, rod or block to be inserted into or withdrawn from the accommodation space through the opening. According to claim 1,
and a reflector for reflecting the light emitted from the light source and passing through the accommodating structure back toward the accommodating space of the accommodating structure. According to claim 1,
The accommodating structure is configured in the form of a chip having the accommodating space therein,
The nucleic acid amplification device, characterized in that the porous structure is configured in the form of a network or block disposed in the accommodation space. According to claim 5,
The accommodating structure includes an incident surface for incident light emitted from the light source toward the accommodating space, and a reflective surface for reflecting the light passing through the accommodating space back toward the accommodating space.

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