KR102449073B1 - Apparatus for detecting nucleic acid based on TiO2 Nano-structure and method for fabricating the same using Roll-to-Roll processing - Google Patents

Abstract

The present invention relates to a molecular diagnostic device capable of detecting a target nucleic acid (DNA, RNA) present in a sample and a technology for manufacturing it using the R2R process. Occurs continuously, no additional steps are required to isolate the nucleic acid from the sample. Accordingly, by using the nucleic acid detection apparatus of the present invention, it is possible to easily detect a target nucleic acid, and it is expected that it will be usefully used for detection and diagnosis (point-of-care testing) in the field of pathogenic bacteria, viruses, etc. .

Description

Apparatus for detecting nucleic acid based on TiO2 Nano-structure and method for fabricating the same using Roll-to-Roll processing

The present invention relates to a nucleic acid detection device having a TiO ₂ nanostructure-based photocatalyst layer required for separation and amplification of nucleic acids in a sample, and a method for manufacturing the nucleic acid detection device using a roll-to-roll process.

In the intestines of all warm-blooded animals including humans, a very favorable environment is provided for various pathogens such as bacteria and viruses to inhabit. Pathogens are widely distributed in the surrounding environment, and specifically, bacterial pathogens are found in soil, animal organs, and water contaminated by animal feces. On average, the human body also has more than 150 types of bacteria inside and outside the human body, and although many microorganisms are harmless to the human body, some types of bacteria can cause botulism, cholera, diarrhea, emesis, It causes various infectious diseases, including pneumonia and typhoid. In particular, more than 200 diseases can be transmitted through food and drinking water alone.

Pathogens can easily infect humans through contaminated soil and water environments. Because the reproduction rate of pathogens is very fast under normal conditions, even if a small number of pathogens once invade the human body, they grow rapidly in the intestine suitable for their growth environment and reach a level that can threaten human health. Accordingly, there is a need for a diagnostic technology capable of quickly and easily detecting the presence or absence of a pathogen (particularly, a virus) from a contaminated environment.

As such, clinical diagnostic techniques based on genetic analysis typically recover nucleic acids from bacteria, fungi, or viruses, perform an amplification reaction, and then perform an amplification reaction using various detection means (eg, optical, electrochemical, or mechanical biotechnology). A method of analyzing an amplicon using a sensor device) is used. This is because, in general, trace amounts of bacteria or viruses to be detected are present in biological samples such as blood or environmental samples such as drinking water and food.

Meanwhile, in general, a roll-to-roll (R2R) process apparatus refers to an apparatus for performing various types of processes on a roll-type film or web. Such a roll-to-roll process device includes an unwinder that unwinds a film wound in a roll form, process units that perform various processes such as a printing process on a film, and a rewinder that winds the film back into a roll form. And, it may be provided with various transport units for transporting the film between them.

As an example of the roll-to-roll process apparatus, a roll-to-roll printing apparatus for forming various patterns on the surface of a film, which is an object to be processed, may be mentioned. A recent roll-to-roll printing apparatus is used in various ways to manufacture various electronic components such as electronic circuits, sensors, and flexible displays.

US registered patent US 9,970,423B2

The present inventors strive to develop a technology for mass-producing devices (eg, PCR chips, microfluidic chips, biosensors, etc.) for detecting biological molecules (nucleic acids) more efficiently and at low cost in a short time. , a nucleic acid detection device having a TiO ₂ nanostructure-based photocatalyst layer for isolating and amplifying nucleic acids from cells or viruses present in a sample in a short time by a roll-to-roll (R2R) process, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a nucleic acid detection apparatus capable of sensitively detecting nucleic acids by making TiO ₂ nanostructures act as photocatalysts and at the same time as photonic crystals.

Another object of the present invention is to provide a method for manufacturing the nucleic acid detection device using a roll-to-roll process.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention, the present invention provides a sample injection hole provided on a substrate is injected; a microfluidic channel through which the sample injected into the sample inlet moves; and a nucleic acid detection unit communicating with the microfluidic channel to receive a sample and isolating and detecting nucleic acid in the sample, wherein the nucleic acid detection unit includes a plurality of titanium dioxide (TiO ₂ ) nanostructure-based a photocatalyst layer formed by a photocatalyst unit; and a polymer membrane provided at the bottom of the photocatalyst layer, wherein the photocatalytic unit includes a titanium dioxide nanomaterial doped with metal nanoparticles, and the TiO ₂ nanostructure-based photocatalytic unit is irradiated with light When the cell (eg, red blood cells, white blood cells, bacteria, etc.) or virus in the sample is lysed, the nucleic acid present in the cell or virus lysate is adsorbed to the photocatalytic unit, and the adsorbed nucleic acid is irradiated to the photocatalyst layer It provides a nucleic acid detection apparatus, characterized in that the nucleic acid amplification reaction occurs through photonic polymerase chain reaction (PCR) by light.

In addition, the present invention comprises the steps of: imprinting a sample injection hole into which a sample is injected and a microfluidic channel through which the injected sample moves on one end face of a substrate; laminating a polymer film from one end of the microfluidic channel; laminating a titanium foil on the polymer film and performing roll-to-roll anodization to form a titanium dioxide nanostructured unit; and roll-to-roll printing of metal nanoparticles on the titanium dioxide nanostructured unit.

In one embodiment of the present invention, the biological molecular detection device can be used for molecular diagnosis.

In another embodiment of the present invention, the nucleic acid detection device may be manufactured by a roll-to-roll process.

In another embodiment of the present invention, the titanium dioxide nanomaterial of the TiO ₂ nanostructure-based photocatalytic unit is a titanium dioxide nanostructure having a polygonal surface selected from the group consisting of a triangle, a square, a pentagon, a hexagon, a heptagon, and an octagon. can

In another embodiment of the present invention, the titanium dioxide nanomaterial may be a titanium dioxide nanostructure (hexagonal TiO ₂ nanostructure) having a hexagonal surface.

In another embodiment of the present invention, the metal nanoparticles of the TiO ₂ nanostructure-based photocatalytic unit may be selected from silver nanoparticles, gold nanoparticles, and copper nanoparticles.

In another embodiment of the present invention, the polymer membrane may be a PVDF membrane (polyvinylidene difluoride membrane).

In another embodiment of the present invention, the nucleic acid detection device may further include a light source for irradiating light to the photocatalyst layer.

In another embodiment of the present invention, the nucleic acid detection device may further include a temperature sensor for monitoring the temperature of the nucleic acid molecule.

In another embodiment of the present invention, the nucleic acid detection device further comprises a controller coupled to the light source and the temperature sensor, wherein the controller controls the acquisition of one or more data from the temperature sensor and the operation of the light source can do.

In another embodiment of the present invention, a primer having a base sequence complementary to a target nucleic acid molecule, four kinds of dNTP molecules, and a polymerase are placed on the photocatalyst layer, and the target nucleic acid molecule and the four kinds of dNTP molecules bind A target nucleic acid molecule can be detected by detecting a change in a photonic bandgap of a photonic crystal of a photocatalyst layer that is changed by the .

In another embodiment of the present invention, the change in the bandgap can be detected by absorption spectroscopy or fluorescence analysis.

In another embodiment of the present invention, by-products other than the nucleic acid of the cell or virus lysate may be discharged and removed through the polymer membrane through the micropores of the photocatalyst layer.

The present invention relates to a molecular diagnostic device capable of detecting a target nucleic acid (DNA, RNA) present in a sample. When the sample is injected into the detection device, separation and amplification reactions of the nucleic acid from the sample occur continuously, thereby extracting the nucleic acid from the sample. No additional steps are required for separation. Accordingly, by using the nucleic acid detection apparatus of the present invention, it is possible to easily detect a target nucleic acid, and it is expected that it will be usefully used for detection and diagnosis (point-of-care testing) in the field of pathogenic bacteria, viruses, etc. .

In addition, the nucleic acid detection apparatus of the present invention can be mass-produced at low cost within a short time through a roll-to-roll process.

1 is a diagram schematically showing a cross-section of a nucleic acid detection apparatus according to an embodiment of the present invention.
2 is a diagram showing the structure of a nucleic acid detection apparatus according to an embodiment of the present invention.
3 is a flowchart of a method for manufacturing a nucleic acid detection device according to an embodiment of the present invention using a roll-to-roll device.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. This example is provided to more completely explain the present invention to those of ordinary skill in the art. Accordingly, the shapes of elements in the drawings are exaggerated in some cases to emphasize a clearer description. In addition, the terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor appropriately defines the concept of the term in order to best describe his invention. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that it can be done.

When describing with reference to the drawings, the same or corresponding components are given the same reference numerals, and overlapping descriptions thereof will be omitted.

1 is a diagram schematically showing a cross-section of a nucleic acid detection apparatus according to an embodiment of the present invention, and FIG. 2 is an exemplary view showing the structure of the nucleic acid detection apparatus.

Referring to FIG. 1 , the nucleic acid detecting apparatus 100 according to an embodiment of the present invention includes a sample inlet 20 into which a sample provided on a substrate 10 is injected, and a sample in which the sample injected into the sample inlet 20 moves. It includes a microfluidic channel 30, and a nucleic acid detection unit 40 communicating with the microfluidic channel 30 to receive a sample to separate and detect nucleic acids in the sample.

The material of the substrate 10 is not particularly limited, and materials (eg, plastic, glass, etc.) commonly used for manufacturing devices for detecting biomolecules such as biosensors, biochips, and microfluidic chips can be used without limitation. . As described below, since the nucleic acid detection device 100 of the present invention includes the photocatalyst layer 41, when irradiating light to the photocatalyst layer 41 from the outside of the detection device, a translucent or transparent material (eg, polymethyl It can be prepared as a substrate of methacrylate (PMMA)).

The sample introduced into the detection device 100 through the sample inlet 20 passes through the microfluidic channel 30 and moves to the nucleic acid detection unit 40 . The microfluidic channel 30 may be designed for pretreatment of a sample (eg, blood). For example, by designing each channel of the microfluidic channel 30 to be higher in one direction to have a step difference, impurities (eg, red blood cells, etc.) included in the sample can be gradually removed by gravity.

The nucleic acid detection unit 40 is a space for separating the nucleic acid in the sample received from the microfluidic channel 30 and performing a nucleic acid amplification reaction. and a polymer film 42 .

The photocatalyst layer 41 is composed of a plurality of TiO ₂ nanostructure-based photocatalytic units M, and the photocatalytic unit M may be a titanium dioxide nanomaterial doped with metal nanoparticles.

The titanium dioxide nanomaterial of the photocatalytic unit M may be a titanium dioxide nanostructure having a surface of a polygon (eg, a triangle, a square, a pentagon, a hexagon, a heptagon, an octagon, etc.). For example, the titanium dioxide nanomaterial may be a titanium dioxide nanostructure having a hexagonal surface, for example, _{a hexagonal TiO 2 nanotube} having a hexagonal surface.

The hexagonal titanium dioxide may be formed in the photocatalyst layer 41 by performing roll-to-roll anodization on a titanium foil. Thereafter, the metal nanoparticles are roll-to-roll printed (doped) on the titanium dioxide nanostructure to prepare a titanium dioxide pattern doped with the metal nanoparticles.

As the metal nanoparticles of the photocatalytic unit (M), silver nanoparticles, gold nanoparticles, copper nanoparticles, or a combination thereof may be used. Titanium dioxide (TiO ₂ ) semiconductor material has a bandgap energy of 3.0 eV or more, so there is a limit in the absorption of visible light in a wide wavelength band except for light in the ultraviolet (UV) region. The photocatalytic function can be improved by increasing the electron-hole pair recombination rate and decreasing the electron-hole pair recombination rate. If the titanium dioxide nanomaterial surface is doped with Ag, which has its own antibacterial properties, in a nano size, the photofunctionality and lysis effect can be enhanced.

The polymer membrane 42 is disposed at the bottom of the photocatalyst layer 41 to serve as a support layer and a separation membrane, and impurities (byproducts) except for nucleic acids (DNA, RNA) adsorbed to the photocatalytic unit (M) in cell or virus lysate. Silver is discharged through the polymer film 42 and removed. For example, a polyvinylidene difluoride membrane (PVDF) may be used as the polymer membrane 42 .

The nucleic acid detection apparatus according to an embodiment of the present invention may include a microfluidic channel 60 for receiving impurities passing through the polymer film 42 at the lower end of the polymer film 42 . One end of the microfluidic channel 60 is in communication with a waste reservoir 70 , so that impurities passing through the polymer film 42 can be stored in the impurity reservoir 70 through the microfluidic channel 60 . have.

A process of detecting a target nucleic acid using the nucleic acid detecting apparatus according to an embodiment of the present invention is as follows.

Titanium dioxide and metal (eg, silver, gold) nanoparticles of the photocatalyst layer 41 act as photonic crystals to match the photonic bandgap (PBG) to light (eg, UV, visible light) When irradiated, electron transition occurs and photocatalytic activity appears. The photonic crystal is a structure having an optical band gap and means a material having a periodic repeating structure of dielectric constant. In general, photonic crystals have a characteristic in that different materials having a period of several hundred nanometers to several micrometers are arranged in one, two, or three dimensions, and light having a certain wavelength region meets the photonic crystal structure. In this case, part is transmitted and part is reflected.

The sample injected into the sample inlet 20 reaches the nucleic acid detection unit 40 through the microfluidic channel 30, and the virus or bacteria contained in the reached sample is deposited on the TiO ₂ nanostructure-based photocatalyst layer 41. concentrated When the photocatalyst layer 41 is irradiated with light after the virus or bacteria is concentrated in the photocatalyst layer 41, destruction (dissolution) of the virus or bacteria occurs due to the photocatalytic activity induced by the light irradiation, and the lysate The contained nucleic acid is adsorbed to the photocatalyst layer (mainly titanium dioxide), and the remaining impurities (by-products) that are not adsorbed pass through the micropores of the photocatalyst layer 41 (eg, micropores in the photocatalytic unit M) through the photocatalyst layer 41 ) separated and discharged to the polymer membrane 42 provided at the bottom. For example, when a washing solution is injected through the sample inlet 20 after dissolution of viruses or bacteria in the sample occurs as described above, by-products not adsorbed to the photocatalyst layer 41 are washed out through the polymer film 42 . can be removed.

Thereafter, the nucleic acid adsorbed to the photocatalyst layer 41 is amplified by photonic PCR. In this case, before the nucleic acid amplification occurs, the pH of the adsorbed nucleic acid may be appropriately adjusted for the nucleic acid amplification reaction. Specifically, a sample solution for PCR, such as a primer having a base sequence complementary to a target nucleic acid molecule, four dNTP molecules, and a polymerase, is placed on the photocatalyst layer 41, and the pH is adjusted (eg, pH 6- 7), when light is irradiated to the photocatalyst layer 41, heat is generated by light-to-heat conversion, so that heating and cooling of the nucleic acid and PCR sample solution located in the photocatalyst layer 41 is fast The cycle takes place so that the nucleic acid amplification reaction takes place rapidly. In this case, temperature control for nucleic acid amplification may be performed by controlling light irradiation.

As the nucleic acid amplification reaction starts, dNTPs are paired one by one to the bases of the target nucleic acid molecule (eg, RNA or ssDNA), and protons are generated, and the generated protons change the optical band gap of the photonic crystal of the photocatalytic layer 41. is brought about By detecting such a change in the optical bandgap, a desired target nucleic acid can be detected.

As the change in the photo-bandgap of the photocatalyst layer 41, any technique known in the art capable of detecting the change in the photo-bandgap may be used without limitation. For example, it can be detected by absorption spectroscopy or fluorescence analysis.

For such photocatalytic activity and photoPCR, the nucleic acid detection apparatus according to an embodiment of the present invention may further include a light source for irradiating light to the photocatalytic layer 41 . The light source may be located inside the detection device 100 or outside the device.

The light source may be implemented as an LED, diode lasers, a diode laser array, a quantum well (vertical)-cavity laser, or the like. In addition, the emission wavelength of the light source may be ultraviolet (UV) light, visible light or infrared (IR) light.

The nucleic acid detection apparatus according to an embodiment of the present invention may further include a temperature sensor for monitoring the temperature of the PCR sample solution for nucleic acid amplification. The temperature sensor may be coupled to or facing the platform for measuring the temperature of the sample. Such temperature sensors may include multiple sensor types, such as thermocouples or cameras (eg, IR cameras) facing the platform.

In addition, the PCR system may be integrated or compatible with a diagnostic device such as a digital camera, photodiode, spectrophotometer or similar imaging device that detects nucleic acid and/or fluorescence signals in a sample solution in real time. It can be understood that For example, the camera may be a smartphone camera, and the smartphone includes application software for analyzing a sample solution.

In one embodiment, the sensor and light source may be coupled to a computing unit for obtaining sensor data and controlling the light source. In general, a computing device obtains data from a sensor, including a processor and memory stored in application software executable by the processor to drive the light source (eg, to control LED timing, intensity/injection current, etc.) and/or process data from the diagnostic device, such as digital camera real-time detection of fluorescent signals of nucleic acids and/or sample solutions. The computing device may include a separate computer or device, or may be integrated into a microcontroller module having the remaining components.

The nucleic acid detection apparatus according to an embodiment of the present invention may include a vacuum battery 50 for moving the injected sample to the nucleic acid detection unit 40 through the microfluidic channel 30 . If the vacuum battery 50 is provided, it is possible to induce a fluid flow without a separate microfluidic pumping, which is advantageous in reducing the weight and size of the detection device. For details on the vacuum battery 50, for example, reference may be made to US Patent No. 9,970,423.

3 is a flowchart of a method for manufacturing a nucleic acid detection device according to an embodiment of the present invention using a roll-to-roll device.

Referring to FIG. 3 , the method for manufacturing a nucleic acid detection device according to an embodiment of the present invention is performed using a roll-to-roll device, and a sample inlet through which a sample is injected and a microscopic hole through which the injected sample is moved are performed on one end surface of a substrate. Imprinting the fluid channel (S100), laminating a polymer film from one end of the microfluidic channel (S200), laminating a titanium foil on the polymer film, and performing roll-to-roll anodization Forming a titanium nanostructured unit (S300) and a step (S400) of roll-to-roll printing of metal nanoparticles on the titanium dioxide nanostructured unit.

Hereinafter, a method of manufacturing a nucleic acid detecting apparatus according to an embodiment of the present invention will be described with reference to FIGS. 1 to 3 .

In S100, the sample injection hole 20 and the microfluidic channel 30 are imprinted by a roll-to-roll process and formed on the substrate. The microfluidic channel 30 may use any material used for manufacturing the microfluidic channel, such as PDMS, without limitation. Imprinting is performed by manufacturing an embossed roll according to the required depth and width of the microfluidic channel 30, re-coating the graphene with UV acrylic resin or epoxy resin, and curing with UV while imprinting through the embossed roll. The microfluidic channel 30 can be printed.

Next, laminating the polymer film 42 from one end of the microfluidic channel 30 (S200), laminating a titanium foil directly on the polymer film 42, and performing roll-to-roll anodization to form a titanium dioxide nanostructure A unit (M) is formed (S300).

In S400, the photocatalyst layer 41 is prepared by roll-to-roll printing of metal nanoparticles on the titanium dioxide nanostructure unit (M) to form a titanium dioxide nanomaterial doped with metal nanoparticles.

The coating speed at the time of roll-to-roll coating can be appropriately controlled, for example, it can be coated by controlling from 1 m to 6 m per minute, and the coating method is continuous in roll-to-roll using a gravure coater, comma coater, or slot die can be coated.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

10: substrate
20: sample inlet
30: microfluidic channel
40: nucleic acid detection unit
41: photocatalyst layer
42: polymer film
50: vacuum battery
60: microfluidic channel
70: impurity reservoir

Claims (12)

a sample inlet through which a sample provided on the substrate is injected;
a microfluidic channel through which the sample injected into the sample inlet moves; and
A nucleic acid detection device communicating with the microfluidic channel to receive a sample and comprising a nucleic acid detection unit for separating and detecting nucleic acids in the sample,
The nucleic acid detection unit may include: a photocatalyst layer formed by a plurality of titanium dioxide (TiO ₂ ) nanostructure-based photocatalytic units; and a polymer membrane provided at the lower end of the photocatalyst layer,
The photocatalytic unit includes a titanium dioxide nanomaterial doped with metal nanoparticles,
When light is irradiated to the TiO ₂ nanostructure-based photocatalytic unit, cells or viruses in the sample are lysed, and nucleic acids present in the cell or virus lysate are adsorbed to the photocatalytic unit,
The nucleic acid detection device, characterized in that the adsorbed nucleic acid undergoes a nucleic acid amplification reaction through photonic polymerase chain reaction (PCR) by light irradiated to the photocatalyst layer. According to claim 1,
The nucleic acid detection device, characterized in that manufactured by a roll-to-roll process, a nucleic acid detection device. According to claim 1,
The titanium dioxide nanomaterial of the TiO ₂ nanostructure-based photocatalytic unit is a titanium dioxide nanostructure having a surface of a polygon selected from the group consisting of a triangle, a square, a pentagon, a hexagon, a heptagon, and an octagon, characterized in that it is a nucleic acid detection device. 4. The method of claim 3,
The titanium dioxide nanomaterial is a nucleic acid detection device, characterized in that the titanium dioxide nanostructure (hexagonal TiO ₂ nanostructure) having a hexagonal surface. According to claim 1,
The TiO ₂ Nanostructure-based metal nanoparticles of the photocatalytic unit is a nucleic acid detection device, characterized in that selected from silver nanoparticles, gold nanoparticles and copper nanoparticles. According to claim 1,
The polymer membrane is a PVDF membrane (polyvinylidene difluoride membrane), characterized in that the nucleic acid detection device. According to claim 1,
The nucleic acid detection apparatus, characterized in that it further comprises a light source (light source) for irradiating light to the photocatalyst layer, nucleic acid detection apparatus. 8. The method of claim 7,
The nucleic acid detection device, characterized in that it further comprises a temperature sensor for monitoring the temperature of the nucleic acid molecule, nucleic acid detection device. 9. The method of claim 8,
The nucleic acid detecting device further comprises a controller coupled to the light source and the temperature sensor, wherein the controller controls the acquisition of one or more data from the temperature sensor and operation of the light source, the nucleic acid detecting device. According to claim 1,
A primer having a nucleotide sequence complementary to a target nucleic acid molecule and a polymerase are placed on the photocatalytic layer, and a photonic crystal of the photocatalyst layer that is changed by binding of the target nucleic acid molecule and four dNTP molecules ( A nucleic acid detection device, characterized in that for detecting a target nucleic acid molecule by detecting a change in a bandgap of a photonic crystal. According to claim 1,
A nucleic acid detection device, characterized in that the by-products other than the nucleic acid of the cell or virus lysate are discharged and removed through the polymer membrane through the micropores of the photocatalyst layer. Imprinting a sample injection hole through which a sample is injected and a microfluidic channel through which the injected sample moves on one end surface of the substrate;
laminating a polymer film from one end of the microfluidic channel;
laminating a titanium foil on the polymer film and performing roll-to-roll anodization to form a titanium dioxide nanostructured unit; and
A method for manufacturing a nucleic acid detection device according to any one of claims 1 to 11 using a roll-to-roll device, comprising the step of roll-to-roll printing of metal nanoparticles on the titanium dioxide nanostructured unit.

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