WO2022235058A1

WO2022235058A1 - Pcr device

Info

Publication number: WO2022235058A1
Application number: PCT/KR2022/006350
Authority: WO
Inventors: 조규진; 정연수; 이루크
Original assignee: 성균관대학교산학협력단
Priority date: 2021-05-03
Filing date: 2022-05-03
Publication date: 2022-11-10
Also published as: KR20220150502A; KR102597565B1

Abstract

Provided is a PCR device comprising: a PCR plate; a plurality of LED light sources disposed on a lower surface of the PCR plate; and a display disposed above the PCR plate, wherein the PCR plate includes: a PCR chamber having a plurality of wells; and an alumina substrate disposed on a lower surface of the PCR chamber, and containing nano-structured gold nanoparticles and gold nanoclusters.

Description

PRC device

The present invention relates to a PCR (polymerase chain reaction) apparatus.

Polymerase chain reaction (PCR) is a chain reaction of a polymerase to obtain a large amount of copies of a specific helix of a nucleic acid. Polymerase chain reaction is an essential technology that is currently widely applied in medical and biological research, and it is also introduced as a general method for testing coronavirus-19 (SARS-CoV-2), which has recently caused a global pandemic. In the COVID-19 test, PCR test is used for the purpose of sufficiently obtaining a sample of a specific DNA sequence through nucleic acid (DNA or RNA) amplification technology. This is because it is a technology that replicates a very small amount of DNA and amplifies it as much as you want. In particular, the patient's saliva or sputum does not contain enough virus to make a confirmed diagnosis. Therefore, by amplifying the DNA, it is possible to confirm the presence or absence of the coronavirus in the sample by reading it using a nucleotide sequence that can distinguish the coronavirus.

However, as it takes about 1 to 2 days for a conventional PCR test, it is difficult to quickly and accurately diagnose the corona virus in the field. This can be explained as a three-step process of denaturation, annealing, and extension, which are the basic operating principles of PCR. It is important to shorten the reaction time.

However, the existing PCR apparatus takes a long time of 1 hour or more to complete the nucleic acid amplification reaction by increasing or decreasing the temperature through the conversion of electrical energy to thermal energy. In addition, since additional accessory devices such as a heating device and a cooling device are required, as the volume of the device increases, it is difficult to apply it to a field requiring rapid disease diagnosis. Due to the inconvenience of portability, expensive equipment price, long detection time, and difficulty in handling, research on portable PCR equipment that is easy to carry and inexpensive is steadily progressing.

Background art of the present invention is disclosed in Korean Patent No. 10-1768146 and the like.

It is an object of the present invention to provide a PCR apparatus capable of performing PCR at high speed by increasing the rate of rise and fall of the temperature required in PCR.

Another object of the present invention is to provide a PCR apparatus that is portable and can be used quickly in the field by minimizing the volume of the PCR apparatus.

Another object of the present invention is to control the structure and formation of gold nanoparticles and gold nanoclusters using alumina nanostructures to generate surface plasmonic resonance to convert light into heat, thereby reducing the temperature rise-fall cycle required in PCR. It is to provide an apparatus in which amplification of nucleic acids occurs by controlling the

One aspect of the present invention is a PCR device.

1. The PCR apparatus includes a PCR plate, a plurality of LED light sources disposed on a lower surface of the PCR plate, and a display disposed on an upper portion of the PCR plate, wherein the PCR plate includes a PCR chamber having a plurality of wells and the PCR and an alumina (Al ₂ O ₃ ) substrate disposed on the lower surface of the chamber and containing nano-aligned, gold nanoparticles and gold nanoclusters.

In 2.1, the gold nanoparticles may have different sizes compared to the gold nanoclusters.

In 3.1-2, the LED light source may include a light source irradiating light having a wavelength of 500 nm or more and 600 nm or less and a light source irradiating light having a wavelength of more than 600 nm and 700 nm or less.

In 4.1-3, the alumina substrate is an alumina substrate; a barrier rib structure formed integrally with the alumina substrate on an upper surface of the alumina substrate and formed by crossing a plurality of barrier ribs; the gold nanoparticles integrally formed with the alumina substrate in an empty space within the barrier rib structure; Among the barrier rib structures, the gold nanoclusters formed on the upper surface of the barrier rib may be included.

In 5.4, the upper surface of the partition wall may be curved.

In 6.1-5, the alumina may be a porous anodic aluminum oxide (AAO).

In 7.1-6, the alumina substrate is formed by anodizing and wet etching the aluminum substrate to form a plurality of barrier ribs on the alumina substrate; and forming a barrier rib structure including an empty space formed by crossing the barrier ribs (first step), depositing gold on the barrier rib structure (second step); and heat-treating the barrier rib structure on which gold is deposited (third step).

In 8.7, the alumina substrate may be manufactured by performing the first step, the second step, and the third step by a roll-to-roll hybrid manufacturing process.

The present invention provides a PCR apparatus capable of performing PCR at high speed by increasing the rate of rise and fall of the temperature required in PCR.

The present invention provides a PCR device that is portable and can be used quickly in the field by minimizing the volume of the PCR device.

The present invention controls the structure and formation of gold nanoparticles and gold nanoclusters using alumina nanostructures to generate surface plasmonic resonance to convert light into heat, thereby controlling the temperature rise-fall cycle required in PCR with light to control nucleic acid A PCR test apparatus was provided by amplification of the.

1 is an internal schematic diagram of a portable PCR device according to an embodiment of the present invention.

2 is a schematic diagram of an alumina substrate comprising nano-ordered, gold nanoparticles and gold nanoclusters.

3 is a schematic diagram of a manufacturing process of an alumina substrate comprising nano-ordered, gold nanoparticles and gold nanoclusters.

4 is a schematic diagram of a full roll to roll (R2R) hybrid vacuum process for preparing nano-aligned, alumina substrates comprising gold nanoparticles and gold nanogluster.

5 is a scanning electron microscopy (SEM) image of an object manufactured in each step of FIG. 3 and an upper surface of the object.

6 is a conceptual diagram illustrating an action when light of different wavelengths is irradiated to an alumina substrate including nano-aligned, gold nanoparticles and gold nanoclusters.

7 shows a state in which the light is irradiated to the alumina substrate by installing the LED light source on the upper part.

8 shows the temperature rise (Y-axis, unit: °C) according to the LED wavelength and irradiation time (X-axis, unit: minutes).

9 is a graph showing the rise and fall of temperature according to time when the LED light source is turned on and off when an LED light source (simultaneous irradiation of light with a wavelength of 530 nm and 660 nm) is irradiated to an alumina substrate including gold nanoparticles and gold nanocluster; to be.

10 is a graph showing temperature rise and fall according to DNA amplification using a PCR device.

11 is an electrophoretic gel result after amplification of DNA (55 bp and 100 bp) using a PCR device.

With reference to the accompanying drawings, the embodiments will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily implement them. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts not related to the description are omitted.

The present inventors, as a means for raising and lowering the sample temperature required in the DNA amplification process in the PCR device, include a plurality of LED light sources irradiating light of different wavelengths, arranged on top of the LED light source, and arranged nano-aligned, Alumina substrates containing gold nanoparticles and gold nanoclusters were used.

Through this, the PCR device rapidly converts light into heat due to the plasmonic resonance effect caused by the nano-aligned, gold nanoparticles and gold nanoclusters in the substrate by the light of a plurality of wavelengths emitted from the LED light source, thereby increasing the sample temperature and As the descent is repeated rapidly, the PCR reaction can be performed at high speed by increasing the rate of rise and fall of the temperature required for PCR. In addition, the plurality of LED light sources have low power consumption and are very inexpensive compared to the existing light sources, so that the PCR device is portable and can be used quickly in the field.

In one embodiment, the PCR apparatus increases the temperature from room temperature to 90°C to 95°C (denaturation), lowers it from 90°C to 95°C to 60°C to 65°C (annealing), and again from 60°C to 65°C to 70°C to The 75° C. rise (extension) cycle can be repeated.

The PCR apparatus of the present invention includes a PCR plate, a plurality of LED light sources disposed on the lower surface of the PCR plate, and a display disposed on the PCR plate, wherein the PCR plate includes one or more sample pretreatment wells and one or more A PCR chamber having a PCR performing well, and an alumina substrate containing nano-aligned, gold nanoparticles and gold nanoclusters disposed on a lower surface of the PCR chamber.

Hereinafter, a PCR apparatus according to an embodiment of the present invention will be described with reference to FIG. 1 .

Referring to Figure 1, the PCR apparatus includes a PCR plate (S150), LED light sources (S130, S140), a display (S160) and a power supply (S110).

The PCR plate (S150), the LED light sources (S130, S140), the display (S160), and the power supply source (S110) are included in the housing (S170). The PCR apparatus of the present invention can be used as a portable device. The PCR device may further include a cooling fan (S120) to further increase the cooling rate when the PCR device is driven. Alternatively, the PCR apparatus may circulate low-temperature air in the PCR apparatus through a chiller (not shown in FIG. 1) to further increase the cooling rate.

Although not shown in Figure 1, the PCR plate (S150) is a PCR chamber disposed toward the display (S160); and an alumina substrate disposed on the lower surface of the PCR chamber and disposed toward the LED light sources S130 and S140 and containing nano-aligned, gold nanoparticles and gold nanoclusters.

The PCR chamber includes one or more sample preparation wells and one or more PCR performing wells. The sample pretreatment well and the PCR performing well may be included by adopting or modifying the wells that are normally included in the PCR apparatus, respectively. The PCR chamber is not particularly limited, but polydimethylsiloxane (PDMS) wells may be used.

A mix of enzymes, biomolecules, and chemicals necessary for cell lysis (Lysis), RNA purification, reverse transcription and PCR is applied in the well. Purification, reverse transcription and PCR can be performed. The enzymes, biomolecules, and chemical agents may employ conventional types known to those skilled in the art that are necessary when performing PCR.

The display S160 may confirm the amplification of the nucleic acid by confirming the rise and fall of the temperature of the sample. The display S160 may confirm whether or not the actual nucleic acid is actually amplified by additionally including a fluorescence detector as well as a cycle of increasing and decreasing the temperature.

The LED light sources S130 and S140 may emit light of different wavelengths, respectively. When irradiating light of the same wavelength, the effect of increasing the temperature may be insignificant. In one embodiment, the LED light source may employ a light source that irradiates light of a wavelength of 500 nm or more and 600 nm or less, and light of a wavelength of 600 nm or more and 700 nm or less, respectively. In the above range, the conversion efficiency from light energy to thermal energy can be maximized. Preferably, light having a wavelength of 530 nm to 550 nm, more preferably 530 nm and 640 nm to 660 nm, and more preferably 660 nm may be used. This is also explained in detail below with reference to FIG. 8 .

As the LED light sources S130 and S140 irradiate light of different wavelengths, the focal length of the two light sources is important. Accordingly, it may further include an adjustment means capable of adjusting the distance and position of the LED light source.

LED light sources (S130, S140) are arranged in the lower part of the PCR device. However, as shown in FIG. 7 , the conversion efficiency can be further increased by installing the LED light source ( S130 ) on the top of the PCR plate and irradiating light onto the nano-aligned, gold nanoparticles and gold nanoclusters-containing alumina substrate ( S240 ). may be

With reference to FIG. 2 , an alumina substrate containing nano-aligned, gold nanoparticles and gold nanoclusters is described.

2 and 3 , the nano-aligned alumina substrate containing gold nanoparticles and gold nanoclusters includes an alumina substrate S240; a barrier rib structure (S221) formed integrally with the alumina substrate (S240) on the upper surface of the alumina substrate (S240) and formed by crossing a plurality of barrier ribs; gold nanoparticles S320 integrally formed with the alumina substrate S240 in an empty space within the barrier rib structure S221; Among the barrier rib structures S221 , the gold nano-clusters S330 formed in contact with the upper surface of the barrier ribs are included.

The gold nanoparticles S320 have a size different from that of the gold nanocluster S330 or include gold particles of a different size. Accordingly, by using the LED light sources S130 and S140 irradiating two different wavelengths of light, the plasmon effect by each light source can be increased, which can further increase the rate of temperature rise.

Referring to FIG. 5 , when the gold nanoparticles S320 formed on the alumina substrate S240 are irradiated with light S400 from the LED light source, light energy can be converted into thermal energy, and the emitted thermal energy is the sample. can increase the rate of temperature rise. When light is irradiated, light is absorbed by the gold nanoparticles, and at this time, electrons move rapidly and generate heat. Then, when the light irradiation is stopped, the temperature rapidly drops as the plasmon effect disappears.

On the other hand, when the gold nanoclusters S330 formed on the alumina substrate S240 are irradiated with light S410 of a wavelength different from that of the light S400 from the LED light source, light energy can be converted into thermal energy, The released thermal energy may further increase the rate of temperature increase of the sample.

The present invention converts light emitted from different wavelength bands into thermal energy by the gold nanoparticles S320 and the gold nanocluster S330 to maximize conversion efficiency, thereby increasing the rate of temperature increase of the sample. When the light irradiation from the light source is stopped, the alumina substrate S240 cools quickly, so that the temperature of the sample can be rapidly decreased. In this way, by increasing the rate of rise and fall of the temperature required for PCR by the nano-aligned, gold nanoparticles, gold nanoclusters, and alumina substrates, it is possible to perform a PCR reaction at high speed and quickly.

Preferably, as shown in FIG. 5 , the gold nanoparticles S320 and the gold nanoclusters S330 are disposed to face the LED light sources S130 and S140, so that the gold nanoparticles S320 and the gold nanoclusters (S320) and the gold nanoclusters ( S330) may directly receive the light emitted from the LED light sources (S130, S140).

Referring back to FIG. 3 , the gold nanoparticles S320 are formed integrally with the alumina substrate in an empty space where the barrier ribs cross each other and are spaced apart from the barrier rib.

The gold nanoparticles S320 may have a particle diameter of 50 nm to 70 nm, specifically 60 nm to 70 nm. Within the above range, it may be easy to prepare gold nanoparticles, and it may be easy to implement the effects of the present invention. The gold nanoparticles S320 are spaced apart from each other at the same distance from each other on the alumina substrate S240 and are formed in nano-alignment. Through this, it may be easier to implement the effects of the present invention.

In one embodiment, the gold nanoparticles S320 are nano-aligned, and the separation distance between the gold nanoparticles S320 may be 0.001 nm to 1000 nm. In the above range, the rate of rise and fall of the temperature may be faster when the LED light source of the present invention is irradiated.

The gold nanocluster S330 is formed in direct contact with the upper surface of the barrier rib, and fine gold particles form a cluster. The gold nanoclusters S330 may have an average particle diameter of 5 nm or less, specifically 5 nm to 10 nm. Within the above range, it may be easy to implement the effects of the present invention.

The gold nanoclusters S330 are also nano-aligned, and as shown in FIG. 2 , they are combined in a regular hexagonal shape with a side length of 0.001 nm to 1000 nm.

The barrier rib structure S221 includes a plurality of barrier ribs; and an empty space in which the partition walls intersect each other. The barrier rib structure S221 is formed by aligning the barrier rib with the empty space, thereby increasing the rate of temperature increase due to the plasmon effect. The barrier rib structure S221 has an outermost barrier rib, so that it can be easily mounted on a PCR plate.

Since the barrier rib structure S221 and the barrier rib are formed of alumina, the manufacturing processability of the nano-aligned alumina substrate containing gold nanoparticles and gold nanoclusters may be increased, and the temperature drop rate of the sample may be increased. In one embodiment, alumina can be made porous as anodic aluminum oxide (AAO) by anodization. The porosity of alumina can facilitate the formation of gold nanoparticles and gold nanoclusters of the present invention.

The upper surface of the partition wall ( S222 in FIG. 3 ) may be curved. The curved surface allows the gold nanoclusters to be stably formed compared to the plane, the effect of the present invention can be further enhanced, and an additional plasmonic effect can be provided.

The height of the barrier rib may be adjusted according to the size of the PCR apparatus, the PCR plate and/or the type of light source included in the PCR apparatus. For example, the height of the barrier rib may be 1 mm to 3 mm. Within the above range, it may be easy to implement the effects of the present invention. The barrier rib may form a barrier rib structure while regular hexagons are joined to each other to have a honeycomb structure.

The empty space formed by crossing the barrier ribs may have a predetermined cross-sectional shape. For example, it may have a regular hexagonal cross section as shown in FIG. 2 , but is not limited thereto.

The manufacturing process of the nano-aligned alumina substrate containing gold nanoparticles and gold nanoclusters will be described with reference to FIGS. 3 and 4 .

An alumina substrate containing nano-aligned, gold nanoparticles and gold nanoclusters was formed by anodizing and wet etching an aluminum substrate to form a plurality of barrier ribs on the alumina substrate; and forming a barrier rib structure including an empty space formed by crossing the barrier ribs (step 1), depositing gold on the barrier rib structure (step 2); It may be manufactured by a method comprising the step of heat-treating the barrier structure on which gold is deposited (step 3).

Referring to FIG. 3 , an aluminum substrate S210 is prepared.

The aluminum substrate S210 may be an aluminum substrate having a purity of 99.99% or more, for example, 99.99% to 100%. Within the purity range, the arrangement of empty spaces among the finally formed barrier rib structures may be aligned.

The aluminum substrate S210 may be used without additional processing. However, for the rapid increase and decrease of the temperature and the manufacturing yield of the nano-aligned, gold nanoparticles and gold nanoclusters-containing alumina substrate of the present invention, the aluminum substrate S210 is surface treated to remove impurities on the surface of the aluminum substrate. may be subjected to a pretreatment process.

The pretreatment process may include electrolytic polishing of the surface of the aluminum substrate. Electropolishing is performed by impregnating an aluminum substrate in a mixed solution of HClO ₄ and ethanol in a volume ratio of 1:1 to 10:1, preferably 3:1 to 5:1, more preferably 4:1, and then 10 It can be carried out by applying a direct current voltage of 10V to 30V, preferably 20V under the condition of ℃ to 20℃, preferably 15℃.

Next, referring to FIG. 3 , a plurality of barrier ribs are formed on the alumina substrate S240 from the aluminum substrate S210; and a barrier rib structure S221 including an empty space formed by crossing the barrier ribs with each other. The upper surface of the barrier rib of the barrier rib structure S221 may be a curved surface S222 .

The barrier rib structure may be performed by anodization and wet etching. Although the anodization and wet etching may each be performed once, in order to facilitate the formation of the barrier rib structure of the present invention and to form a nano-ordered structure well, the first anodization process, the first wet etching process, and the second An anodization process and a second wet etching process may be performed in the order of the process. The second wet etching process may be omitted.

In the first anodization process, the aluminum substrate (S210) is impregnated in 0.1 to 0.5M, preferably 0.3M of oxalic acid, and under a DC voltage of 10V to 100V, preferably 60V, 1 hour to 20 hours, preferably 12 hours. It can be done by proceeding. Through this, an aluminum oxide (alumina) film may be formed on the aluminum substrate S210 .

The first wet etching process is a process of removing the formed aluminum oxide. The first wet etching process includes impregnating an aluminum substrate in a mixed solution of phosphoric acid and chromic acid. Phosphoric acid: chromic acid may be included in a weight ratio of 1:1 to 10, preferably 4:1. In the mixed solution, 1 to 10% by weight of phosphoric acid and 1 to 5% by weight of chromic acid may be included. In order to efficiently remove aluminum oxide, the first wet etching process is preferably performed at a high temperature of 50°C to 100°C, preferably 60°C. In order to efficiently remove aluminum oxide, the first wet etching process may be performed at a high temperature for 1 hour to 10 hours, preferably for 6 hours.

The second anodization process and the second wet etching process include an aligned, plurality of barrier ribs; and a barrier rib structure including an empty space formed by crossing the barrier ribs with each other.

The second anodization process is substantially the same as the first anodization process described above, but may be performed for a shorter time than the first anodization process in order to control the height of the barrier rib.

The second wet etching process may be performed at 20° C. to 30° C., preferably at 28° C. in 0.01 to 1 M, preferably 0.1 M phosphoric acid.

Next, referring to FIG. 3 , gold is deposited on the barrier rib structure S221 to obtain a structure in which gold S310 is deposited on the empty space of the barrier rib structure S221 and the upper surface of the barrier rib.

A method of depositing gold may be performed by a conventional method known to those skilled in the art. For example, it may be performed using a typical deposition apparatus (E-BEAM, THERMAL EVAPORATOR). The thickness of the deposited gold may be between 5 nm and 10 nm, preferably 7 nm. Within the above range, gold nanoparticles are efficiently formed, and as shown in FIGS. 2 and 3 , gold nanoparticles and gold nanoclusters can be formed to have a 3D flower shape.

Next, referring to FIG. 3 , the barrier rib structure S221 and nano-aligned, gold nanoparticles S320 and gold nanocluster S330 are formed on the alumina substrate S240 by heat-treating the barrier rib structure on which gold is deposited. do.

The heat treatment causes the gold deposited in the empty space of the barrier rib structure to form gold nanoparticles, and the gold deposited on the upper surface of the barrier rib to form gold nanoclusters. Gold deposited in the empty space has a larger deposition area compared to gold deposited on the upper surface of the barrier rib, so that gold nanoparticles can be formed with a relatively large particle diameter compared to gold nanoclusters.

The heat treatment may be performed at 300° C. to 600° C., preferably at 500° C. for 1 hour to 5 hours, preferably 3 hours. The heat treatment may be performed by a conventional method.

If heat treatment is not performed when forming an alumina substrate, it is not suitable because it cannot quickly reach a temperature of 90°C or higher suitable for PCR even if an LED of the same wavelength band or an LED of a different wavelength band is used. Therefore, in the present invention, a rapid temperature rise suitable for PCR was induced by dewetting into a nanostructure through heat treatment.

The nano-aligned, gold nanoparticles and gold nanoclusters-containing alumina substrates can be manufactured through the above process in a roll-to-roll (R2R) manner to increase the manufacturing processability.

In this regard, it will be described with reference to FIG. 4 .

Figure 4 shows the overall R2R hybrid vacuum process for preparing nano-ordered, gold nanoparticles and gold nanoclusters containing alumina substrates. In the case of PCR amplification, since it should be carried out for a large number of people within a short time, in the present invention, an alumina substrate containing nano-aligned gold nanoparticles and gold nanoclusters is prepared through an R2R hybrid vacuum process.

For the production of porous alumina, the existing known manufacturing process has to go through one step at a time, so it is possible to obtain highly ordered porous pores, but it has the disadvantages of high cost and long time. Accordingly, the present invention proposes a roll-to-roll hybrid vacuum process along with the existing process. In a general roll-to-roll process, it is difficult to deposit high-purity gold, and since heat treatment at a high temperature is impossible, a vacuum deposition apparatus and a dryer capable of drying at a high temperature may be used. Preferably, as the aluminum substrate (S210), high-purity aluminum foil (purity: 99.99% or more, thickness 0.1-0.5 mm) is preferred, which directly affects the arrangement of the porous holes to be finally formed.

First, electrolytic polishing is performed to remove impurities on the surface of the aluminum substrate. For electropolishing, a mixed solution of HClO ₄ and ethanol 4:1 may be used, and the process may be performed by immersing it in a chamber. Impurities on the aluminum surface can be removed by applying a DC voltage of 20 V under the temperature of 15-25°C.

In order to make a porous hole, it undergoes a primary anodization process (S220), and 0.3 M of oxalic acid can be used for 12 hours (1 minute in the roll-to-roll process) under a direct current voltage of 60V. In the roll-to-roll process proposed above, 0.1-0.5 mm/s of web moving speed and 5-7 kg of tension are preferred for sufficient polishing and anodization. For a fast manufacturing process it can be 10-50 mm/s, which can be different depending on the given process and solution conditions.

The alumina oxide film is removed through the wet etching (S230) process, and a mixed solution of 6 wt% phosphoric acid and 1.5 wt% chromic acid is used. In order to efficiently remove the alumina oxide film, a temperature higher than room temperature is required, and in some cases, the process is performed at a high temperature of 60° C. for 1 minute.

Thereafter, porous alumina is formed through the secondary anodization process (S240). At this time, the same as the primary anodization conditions, a DC voltage is applied for a short time (several seconds to several minutes) to control the height of the porous pores.

Thereafter, an alumina plate including aligned porous alumina can be obtained through a secondary wet etching process. 0.1 M phosphoric acid is used for the etching process, and depending on the given temperature (optionally 28°C is required), alumina with various types of porous pores can be prepared. It may optionally be introduced as an additional process.

The prepared alumina substrate S240 with porous pores is determined according to the applied DC voltage value, the applied time, the moving speed of the web, the concentration of the acid used, and the purity of the aluminum plate, and preferably the A size of 50-100 nm is preferred.

Gold is the preferred metallic material for light-to-heat conversion efficiency. In the present invention, representative deposition equipment (e-beam, thermal evaporator), etc. can be used to coat gold with a thickness of several nano nm. In the roll-to-roll process, a hybrid type vacuum deposition equipment (S250) is preferred and can be performed under the same conditions as the existing deposition equipment. Ideally, gold deposition with a thickness of 5-7 nm is required to form efficient gold nanoparticles and have a 3D flower structure.

Then, gold nanoparticles and gold nanoclusters may be formed through a heat treatment process ( S260 ).

5 is an SEM photograph of the upper surface of the alumina substrate S240 on which the barrier rib structure is formed, the alumina substrate S240 on which gold is deposited on the barrier rib structure, and the alumina substrate S240 in which gold nanoparticles and gold nanoclusters are aligned. will be.

Referring to FIG. 5 , it can be seen that gold is deposited in the empty space and the upper surface of the barrier rib, and gold nanoparticles and gold nanoclusters are formed.

When the LED wavelength was irradiated to the nano-aligned alumina substrate containing the nano-aligned, gold nanoparticles and gold nanoclusters of the present invention, the degree of temperature increase according to the irradiation time according to the LED wavelength is shown in FIG. 8 .

Referring to FIG. 8 , the nano-aligned, alumina substrate including gold nanoparticles and gold nanoclusters of the present invention exhibits different efficiencies according to the wavelength band of the LED used.

It is advantageous to use a light source of preferably a wavelength band of 530 nm to 550 nm, more preferably a wavelength band of 530 nm and 640 nm to 660 nm, more preferably 660 nm.

The 530nm LED causes a rapid temperature rise (from room temperature to 90℃) within a given time, and the 520nm LED with the same 500nm wavelength shows low thermal conversion efficiency. However, in the present invention, LEDs having two different wavelength bands are preferred because a desired temperature cannot be obtained within a short time by using only a short wavelength band.

In the case of nano-aligned, gold nanoparticles proposed in the present invention, since gold nanoparticles having two sizes exist, heat conversion efficiency can be maximized by using LEDs of two or more wavelength bands. In the present invention, it is possible to obtain maximized heat conversion efficiency by preferably using the LED of the wavelength band of 530 nm to 550 nm and the LED of the 640 nm to 660 nm wavelength band.

9 shows graphs of temperature rise and fall over time when an LED light source (simultaneous irradiation of wavelengths 530 nm and 660 nm) is irradiated with an alumina substrate including nano-aligned, gold nanoparticles and gold nanoclusters of the present invention It was.

Referring to FIG. 9 , in the case of an alumina substrate containing nano-aligned gold nanoparticles and gold nanoclusters, denaturation, annealing, and extension are used for PCR amplification. If 30 cycles (a cycle capable of sufficiently amplifying DNA) are repeated in the temperature range of 94°C, it can be reached within 3 minutes. This is an essential factor for implementing high-speed PCR, allowing PCR to be performed in a short time.

10 shows graphs of actual temperature rise and fall according to DNA amplification using a PCR apparatus having an alumina substrate including nano-aligned, gold nanoparticles and gold nanoclusters of the present invention.

Referring to FIG. 10 , a PCR device is configured to prepare a mix of biomolecules and chemicals for cell lysis (Lysis), RNA purification, and trans reverse transcription, and nano-arranged, gold nanoparticles including a PDMS well chamber Put about 1 μl of an alumina substrate containing particles and gold nanoclusters, add about 2 μl of oil to prevent evaporation, and perform PCR. In this case, the execution time ranges from 300 seconds to 500 seconds, which may vary depending on the size of the target DNA and the design of the primer. When amplification is performed using DNA having a length of 55 bp (or 100 bp), about 300 seconds are consumed, and it can be confirmed that the time is significantly reduced compared to the existing PCR apparatus.

Amplification of DNA (55 bp and 100 bp) was performed using a PCR apparatus having an alumina substrate including nano-aligned, gold nanoparticles and gold nano-clusters of the present invention, followed by electrophoresis gel. The results are shown in FIG. 11 . .

Referring to FIG. 11 , it can be confirmed that DNA (S510) having a length of 55 bp and DNA having a length of 100 bp (S520) are also amplified in the PCR apparatus of the present invention.

Simple modifications or changes of the present invention can be easily carried out by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims

A PCR plate, a plurality of LED light sources disposed on the lower surface of the PCR plate, and a display disposed on the top of the PCR plate,

The PCR plate includes a PCR chamber having a plurality of wells; and an alumina (Al 2 O 3 ) substrate disposed on the lower surface of the PCR chamber and nano-aligned, containing gold nanoparticles and gold nanoclusters.
The PCR apparatus according to claim 1, wherein the gold nanoparticles have a size different from that of the gold nanoclusters.
The PCR apparatus according to claim 1, wherein the LED light source comprises a light source irradiating light with a wavelength of 500 nm or more and 600 nm or less and a light source irradiating light with a wavelength of more than 600 nm and 700 nm or less.
According to claim 1, wherein the alumina substrate is an alumina substrate; a barrier rib structure formed integrally with the alumina substrate on an upper surface of the alumina substrate and formed by crossing a plurality of barrier ribs; the gold nanoparticles integrally formed with the alumina substrate in an empty space within the barrier rib structure; The PCR apparatus comprising the gold nanoclusters formed on the upper surface of the barrier ribs among the barrier rib structures.
The PCR apparatus according to claim 4, wherein the upper surface of the partition wall is curved.
The PCR apparatus according to claim 1, wherein the alumina is aluminum oxide (AAO, anodic aluminum oxide) by anodic oxidation of porosity.
According to claim 1, wherein the alumina substrate is

A plurality of barrier ribs on the alumina substrate by anodizing and wet etching the aluminum substrate; and forming a barrier rib structure including an empty space formed by intersecting barrier ribs (first step);

depositing gold on the barrier rib structure (second step); and

The PCR apparatus, which is manufactured by a method comprising the step (third step) of heat-treating the barrier rib structure on which gold is deposited.
The PCR apparatus according to claim 7, wherein the alumina substrate is manufactured by performing the first step, the second step, and the third step by a roll-to-roll hybrid manufacturing process.