KR102549976B1 - Apparatus for continuously detecting nucelic acid - Google Patents

Abstract

A PCR comprising a substrate provided with one or more sample preprocessing wells and one or more PCR wells, and a nanoantenna attached to a lower surface of the substrate, wherein the nanoantenna includes carbon black and metal nanowires. plate; and a near-infrared light source, wherein the PCR plate is designed to move.

Description

Continuous nucleic acid detection device {APPARATUS FOR CONTINUOUSLY DETECTING NUCELIC ACID}

The present invention relates to a continuous nucleic acid detection device.

As daily life activities are restricted due to the corona epidemic around the world, ultra-rapid nucleic acid tests for coronavirus in one hour are performed on more than 1,000 people at shopping centers, schools, churches, bus terminals, train stations, and airports where many people gather. It is necessary to secure a detection system. The ultra-rapid nucleic acid detection system not only protects the life and health of the people, but is also a very important technology for the continued growth and development of the national economy. However, a system that can test 1,000 samples worldwide within 1 hour has not yet been developed, and Roche's COBAS system can automatically test 1,000 samples within 8 hours worldwide.

Existing nucleic acid diagnostic techniques take a sample, perform cell lysis, purify RNA, perform reverse transcription to clone cDNA, mix the DNA sample with a polymerase, and then heat and cool cycles (95 ℃-72 ℃-60 ℃) is performed more than 30 times to amplify and examine DNA.

Since a general nucleic acid test system requires at least one hour or more to be performed, it is not possible to quickly test a large number of samples. Therefore, it has been an obstacle in the development of nucleic acid diagnostic test technology that can quickly test more than 1000 samples. In particular, the heating and cooling cycle (95℃-72℃-60℃), which is the core of PCR (Polymerase Chain Reaction), is performed. Due to the temperature control problem of the Joule heater for heating and the cooling system for cooling, There were many problems in developing a technology that significantly reduced the cooling time to less than 30 minutes.

In order to overcome these disadvantages, UV rays are irradiated on nano metal particles and metal films with nanostructures to generate Surface Plasmon Resonance to perform rapid heating while applying an existing cooling system to perform heating and cooling cycles within 10 minutes. Rapid nucleic acid diagnostic technologies are being developed. However, PCR technology using nano-metal particles and nano-structured metal films has not yet been applied to the development of a nucleic acid diagnostic test technology capable of continuously testing many samples due to cost and reliability problems. In addition, since most of the time required to perform Lysis, RNA purification, and reverse transcription required before PCR testing is between 1 hour and 30 minutes, RNA purification, reverse transcription, and PCR from samples are performed using 96 mini samples in the form of automatic pipettes and cartridges. Even if a nucleic acid test equipment is manufactured using tubes to continuously perform tests, it is impossible to test 1000 samples in 1 hour with conventionally known automated nucleic acid test technologies.

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

An object of the present invention is to provide a continuous nucleic acid detection device capable of remarkably increasing the speed of nucleic acid detection of a plurality of samples.

One aspect of the present invention is a continuous nucleic acid detection device.

1. A continuous nucleic acid detection device includes a substrate having one or more sample pretreatment wells and one or more PCR wells, and a nanoantenna attached to a lower surface of the substrate, wherein the nanoantenna includes carbon black and metal A PCR plate comprising nanowires; and a near-infrared irradiation light source, wherein the PCR plate is designed to move.

In 2.1, the carbon black may be included in an amount of 1% to 10% by weight of the nanoantenna.

In 3.1-2, the metal nanowires may include silver nanowires.

In 4.1-3, the sample preparation well may contain a material for cell lysis and reverse transcription.

In 5.1-4, the PCR plate may move at a speed of 1 cm to 10 cm per minute.

In 6.1-5, the PCR well includes a core well, a satellite first well, and a satellite second well, and the satellite first well and the satellite second well are connected to the core well through a microfluidic channel. can

The present invention provides a continuous nucleic acid detection device capable of remarkably increasing the speed of detecting nucleic acids in a plurality of samples.

1 is an overall drawing of the R2R continuous nucleic acid detection device of the present invention.
2 is a conceptual diagram of the continuous nucleic acid detection device of the present invention.
3 is a cross-sectional view of the continuous nucleic acid detection device of the present invention.
4 is a top plan view of a sample preprocessing well and a PCR well.
5 is a schematic diagram of a method for manufacturing a PCR plate using an R2R imprinter.
6 is a graph showing a temperature increase and decrease according to irradiation time when the near-infrared irradiation light source is repeatedly turned on and off.
7 shows electrophoresis results of 55 bp and 100 bp DNA amplified by performing 30 cycles of heating and cooling while irradiating a near-infrared light source.
8 is a graph of a temperature increase according to an input voltage according to a combination of metal nanowires and carbon black.

With reference to the accompanying drawings, the embodiments will be described in detail so that those skilled in the art can easily carry out the embodiments. This invention may be embodied in many different forms and is not limited to the embodiments set forth herein. In order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted.

The inventors of the present invention have provided a continuous nucleic acid detection device capable of remarkably increasing the speed of detecting nucleic acids in a plurality of samples. The continuous nucleic acid detection device of the present invention can detect nucleic acids in 1000 or more samples within 1 hour.

The present invention includes a substrate provided with one or more sample preprocessing wells and one or more PCR wells, and a nanoantenna attached to a lower surface of the substrate, wherein the nanoantenna includes carbon black and metal nanowires. Provided is a continuous nucleic acid detection device comprising a PCR plate and a near-infrared irradiation light source, wherein the PCR plate is designed to move.

Referring to Figures 1 and 2, the concept of the continuous nucleic acid detection device of the present invention will be described.

Referring to FIGS. 1 and 2 , a nanoantenna (not shown in FIG. 1 ) and a near-infrared light source (not shown in FIG. 1 ) are disposed under a substrate having sample preprocessing wells and PCR wells. Light emitted from the near-infrared irradiation light source is absorbed by the nanoantenna and converted into heat by plasmonic resonance. The generated heat may increase the temperature of the sample pretreatment well and the buffer in the PCR well to perform sample pretreatment (eg, lysis) and PCR.

The wells for sample pretreatment contain substances for sample dissolution and reverse transcription, and the PCR wells contain substances for PCR (PCR Mix). Through this, the speed of nucleic acid detection can be increased by rapidly performing sample lysis, reverse transcription, and PCR.

By continuously moving the substrate provided with the sample preprocessing well and the PCR well in a roll-to-roll manner, the rate of sample dissolution, reverse transcription, and PCR nucleic acid detection can be increased.

Referring to Figure 3, the continuous nucleic acid detection device of the present invention will be described. 2 is a partial cross-sectional view of the continuous nucleic acid detection device of the present invention.

Referring to FIG. 3 , the continuous nucleic acid detection device includes a support layer 100, a nanoantenna 200, a substrate 300, a protective layer 400, a first pipette 500, a second pipette 600, and a sensor 700. ) and a light source 800 .

The support layer 100 may be disposed between the nanoantenna 200 and the substrate 300 to support the nanoantenna 200 and the substrate 300 . The support layer 100 may be formed of one or more of polymers, paper, and metal thin films. The polymer is PET (Polyethylene terephthalate), PEN (Polyethylene naphthalate), PMMA (Poly (methyl methacrylate)), PE (Polyethylene), PP (Polypropylene), PC (Polycarbonate), PI (Polyimide), PES (Polyethersulfone), poly At least one of ester (Polyester), PS (Polystyrene), and PDMS (Polydimethylsiloxane) may be included, but is not limited thereto.

The light source 800 may include a light source that emits light in the near-infrared region. For example, the light source 800 may emit light having a wavelength of 700 nm or more, specifically within a range of 700 nm to 1000 nm.

Light irradiated from the light source 800 is absorbed by the nanoantenna described in detail below and generates surface plasmonic resonance, so that the temperature of the sample pretreatment well and the PCR well formed in the substrate 300 can be rapidly increased. there is. On the other hand, if irradiation of light from the light source 800 is stopped, the temperature can be rapidly decreased.

The sensor 700 may detect the amplified nucleic acid in real time by using a fluorescence detection camera to detect the amplified nucleic acid.

The substrate 300 is provided with one or more sample preprocessing wells 310 and 320 and one or more PCR wells 330, so that sample preprocessing and PCR can be performed.

The sample preprocessing wells 310 and 320 include a well 310 for storing and storing a sample and a well 320 for lysis, purification, and reverse transcription of the sample. The well 310 for storing and storing a sample is a well for temporarily storing and dissolving cells having nucleic acids to be amplified. The well 320 for lysis, purification, and reverse transcription of the sample allows cells entering from the well 310 for storage and storage of the sample to be lysed, purified, and then reverse transcribed. Materials for cell lysis and reverse transcription are supported in the well 320 for sample lysis, purification, and reverse transcription.

Cells contained in the well 310 for preservation and storage of the sample may be dispensed through the first pipette 500 and put into the well 320 for lysis, purification, and reverse transcription of the sample. The nucleic acid reverse transcripts contained in the well 320 for dissolution, purification, and reverse transcription of the sample may be introduced into the well 330 for PCR through the second pipette 600 .

A nanoantenna 200 is attached to the bottom of the well 320 for dissolution, purification, and reverse transcription of the sample, and a light source 800 is disposed. Therefore, the temperature increase required for dissolution and reverse transcription of the sample can be performed by the nanoantenna 200 and the light source 800 . That is, light irradiated from the light source 800 is absorbed by the nanoantenna 200 and the absorbed light generates surface plasmonic resonance, thereby increasing the temperature of the buffer in the well for dissolution, purification, and reverse transcription of the sample. Dissolution of the sample may be performed at 90° C. for 2 seconds, and after 2 seconds, reverse transcription may be performed at 50° C. for 2 minutes. The temperature at the time of dissolving the sample and performing the reverse transcription may be adjusted by controlling the presence or absence of light irradiation from the light source 800 and the intensity thereof.

The well 310 for sample dissolution, purification, and reverse transcription may have a radius of 1 mm to 5 mm and a depth of 0.1 mm to 2 mm. Within the above range, the effect of the present invention can be obtained well.

The reverse transcript placed into the PCR well 330 may undergo a PCR reaction by the PCR mixer in the PCR well 330 . A PCR mixer may be supported in a well for PCR in a conventional manner. The PCR mixer may include, but is not limited to, DNA polymerase, reverse transcriptase, buffer, dNTPs, gene-specific primers, RNA template, and the like.

A nanoantenna 200 is attached to the bottom of the PCR well 330, and a light source 800 is disposed. Therefore, the temperature rise necessary for PCR can be performed by the nanoantenna 200 and the light source 800 . That is, light irradiated from the light source 800 is absorbed by the nanoantenna 200 and the absorbed light generates surface plasmonic resonance, thereby increasing the temperature of the buffer in the well for PCR. When PCR is performed, the temperature may be adjusted to 62° C. to 94° C. corresponding to three stages of denaturation, annealing, and extension. The temperature at the time of performing PCR can be adjusted by controlling the presence or absence of light irradiation from the light source 800 and the intensity.

The well 330 for PCR may have a radius of 1 to 5 mm and a depth of 0.1 to 2 mm. Within the above range, the effect of the present invention can be obtained well.

The well 330 for PCR may be formed in the shape shown in FIG. 3 for effective PCR. 3 is a top plan view of a sample preprocessing well and a PCR well.

Referring to FIG. 4 , sample preprocessing wells 310 and 320 and PCR wells 340 , 350 and 370 are provided on the substrate.

Sample preprocessing wells 310 and 320 are the same as described above.

The PCR wells 340 , 350 , and 370 include a core well 340 , a first satellite well 350 and a second satellite well 370 . The first satellite well 350 and the second satellite well 370 are connected to the core well 340 through the microfluidic channel 360 . When the reverse transcript is dispensed from the sample preprocessing well 320 to the core well 340, the reverse transcript moves to the first satellite well 350 and the second satellite well 370 through the microfluidic channel 360. . The satellite first well 350 may amplify nucleic acids in the sample. In the second satellite well 370, injection of the target nucleic acid is limited for pitch control, and thus always gives a negative result.

After the sample is injected into the satellite first well 350 and the satellite second well 370 within 1-2 seconds, PCR is performed for 70 seconds to 300 seconds by automatically turning on and off the near-infrared light source 30 times. . At this time, while the near-infrared light source is turned off, a laser with a wavelength of 530 nm is irradiated through a filter, CYBER Green is activated, and fluorescence of a wavelength of 460 nm is monitored through the filter to quantitatively monitor the amplification of nucleic acids. At this time, after completing 30 cycles of PCR, it can be confirmed whether amplification has occurred through electrophoresis gel.

The nanoantenna 200 includes a mixture of metal nanowires and carbon black. In the present invention, by mixing carbon black with metal nanowires to form the nanoantenna 100, temperature control can be more easily performed in cell lysis, reverse transcription, and PCR. A nanoantenna containing only metal nanowires has a disadvantage in that it is difficult to perform high-speed PCR due to a problem in that the efficiency of heat rise and fall is lower than that of nanoantennas containing metal nanowires and carbon black.

Metal nanowires may include metal nanowires such as silver, gold, copper, nickel, etc., but are not limited thereto. By controlling the length and thickness of the metal nanowires, near-infrared absorption and light-to-heat conversion efficiency can be increased.

The length of the metal nanowire may be 1 μm to 100 μm, specifically 10 μm to 40 μm, preferably 20 μm, and the thickness of the metal nanowire may be 1 nm to 100 nm, specifically 15 nm to 25 nm, preferably may be 20 nm. Within this range, absorption of near-infrared rays and light-heat conversion efficiency may be increased.

The metal nanowires may be included in the amount of 1 wt% to 20 wt%, specifically 1 wt% to 10 wt%, and specifically 2 wt% to 5 wt%, of the nanoantenna 100 . Within this range, absorption of near-infrared rays and light-heat conversion efficiency may be increased.

Carbon black may be included in an amount of 80 wt% to 99 wt%, specifically 95 wt% to 99 wt%, of the nanoantenna 100 . Within this range, absorption of near-infrared rays and light-heat conversion efficiency may be increased.

The PCR plate may be designed to move at a rate of 1 cm to 10 cm per minute. Through this, it is possible to remarkably increase the speed of detecting nucleic acids in a plurality of samples.

A roll-to-roll method can be applied to move the PCR plate. This refers to a method of printing while a flexible substrate moves between rolls in contact with each other. As shown in Figure 1, the PCR solution is sprayed at the start of the roll with a dispenser in such a way that the flexible substrate moves while the rolls come into contact with each other, and then the PCR solution is moved at a rate of 1 cm to 10 cm per minute, PCR amplification product with fluorescence at the end of the roll Check the

A PCR plate including a substrate 300 having sample preprocessing wells and PCR wells, nanoantennas 200, and support layer 100 may be manufactured by a roll-to-roll imprinting method. 5 is a schematic diagram of a method of manufacturing a PCR plate by a roll-to-roll imprinting method. First, a mixture containing carbon black and metal nanowires is printed using a flexible substrate (PET, polyethylene terephthalate) through the roll-to-roll gravure equipment 401, and then polydimethylsiloxane is introduced and roll-to-roll imprinting 402 A well is formed by giving an intaglio pattern through. At this time, in order to maintain the shape of the intaglio pattern of rolloutol imprinting, drying is performed in real time through a UV-lamp 403 during the process. The solution is added through the dispenser 404, and plasma treatment 405 is performed to seal the well to prevent evaporation and contamination of the PCR solution. Through this printing method, the continuous nucleic acid detection device proposed in this patent can be manufactured.

Referring to FIG. 5, a mixture containing carbon black and metal nanowires is applied to a support layer by a dispenser and dried to form a nanoantenna on the upper surface of the support layer, and a resin for forming a substrate on the nanoantenna, for example, polydimethylsiloxane ( PDMS) is applied and then imprinted in the shape of a well, so that a PCR plate can be manufactured.

The continuous nucleic acid detection device may further include two or more temperature sensors for monitoring the temperature of the well. A temperature sensor may be coupled to or directed to the PCR plate to measure the temperature of the sample. The temperature sensor may include, but is not limited to, a thermocouple or a camera (eg, an IR camera).

The continuous nucleic acid detection device may further employ a digital camera, photodiode, spectrophotometer, or similar device to detect nucleic acids and fluorescence signals in the sample solution in real time.

Referring to FIG. 6, FIG. 6 shows that PCR can be performed by repeating continuous heat rise and fall by irradiating near-infrared rays on a mixed substrate of carbon black and metal nanowires, and when 30 cycles are repeated for nucleic acid amplification, this patent proposes It is shown that the method is performed at high speed within about 5 minutes. This starts at an initial temperature of 55°C and reaches up to 90°C, whereby 30 cycles can be repeated as cooling is performed quickly. Nucleic acid denaturation occurs in the 90°C section, and nucleic acid amplification proceeds by binding the nucleic acid in the 55°C section. If these steps are repeated about 30 times, about 100,000 nucleic acids are amplified from the original number of DNAs, and thus the presence or absence of the nucleic acids can be confirmed.

Referring to FIG. 7, FIG. 7 shows electrophoresis graph results of amplification of nucleic acids (55 bp and 100 bp) obtained through the method proposed in this patent. Referring to FIG. 7, it can be confirmed that nucleic acids with a length of 55 bp (base pair) and nucleic acids with a length of 100 bp can be amplified within 5 minutes according to the method proposed in this patent. M on the left side of the graph in FIG. 7 means a marker, meaning 50 bp per band, and indicates 50 bp, 100 bp, 150 bp, 200 bp, etc. sequentially from the bottom. Electrophoresis is performed using the amplified nucleic acid, and it is possible to confirm that the amplification is appropriate by comparing with the marker. In this patent, it can be confirmed that nucleic acids of 55 bp and 100 bp are amplified well without additional reaction when performing R2R PCR as target amplification materials.

Referring to FIG. 8 , FIG. 8 shows the rate of heat rise compared to the input power when only metal nanowires or carbon black are present. According to FIG. 8, since high-speed PCR cannot be performed due to the low thermal rise rate when only a single material is used, a mixture of metal nanoantenna and carbon black can be suitable for performing high-speed PCR proposed in this patent. When the nanoantenna was formed of only the metal nanowire or only the carbon black, the effect was significantly lowered compared to the case where the nanoantenna was formed of the metal nanowire and carbon black of the present application.

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

Claims (6)

A substrate having one or more sample preprocessing wells and one or more PCR wells, and a nanoantenna attached to a lower surface of the substrate, wherein the nanoantenna comprises 80 to 99% by weight of carbon black and A PCR plate containing 1 to 20% by weight of metal nanowires; and
Including a near-infrared irradiation light source,
The PCR plate is designed to move, continuous nucleic acid detection device.
delete The continuous nucleic acid detection device according to claim 1, wherein the metal nanowires include silver nanowires.
The continuous nucleic acid detection device according to claim 1, wherein the well for sample pretreatment is loaded with a material for cell lysis and reverse transcription.
The continuous nucleic acid detection device according to claim 1, wherein the PCR plate moves at a speed of 1 cm to 10 cm per minute.
The method of claim 1, wherein the PCR well includes a core well, a first satellite well, and a second satellite well, and the first satellite well and the second satellite well are connected to the core well through a microfluidic channel. What is, continuous nucleic acid detection device.

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