WO2021235797A1 - Appareil de détection d'acide nucléique à base de nanostructure de tio2 et son procédé de fabrication à l'aide d'un procédé rouleau à rouleau - Google Patents

Appareil de détection d'acide nucléique à base de nanostructure de tio2 et son procédé de fabrication à l'aide d'un procédé rouleau à rouleau Download PDF

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Publication number
WO2021235797A1
WO2021235797A1 PCT/KR2021/006150 KR2021006150W WO2021235797A1 WO 2021235797 A1 WO2021235797 A1 WO 2021235797A1 KR 2021006150 W KR2021006150 W KR 2021006150W WO 2021235797 A1 WO2021235797 A1 WO 2021235797A1
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Prior art keywords
nucleic acid
acid detection
roll
detection device
sample
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PCT/KR2021/006150
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English (en)
Korean (ko)
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리루크
조규진
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성균관대학교산학협력단
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Priority claimed from KR1020200109719A external-priority patent/KR102449073B1/ko
Application filed by 성균관대학교산학협력단 filed Critical 성균관대학교산학협력단
Publication of WO2021235797A1 publication Critical patent/WO2021235797A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65H—HANDLING THIN OR FILAMENTARY MATERIAL, e.g. SHEETS, WEBS, CABLES
    • B65H18/00—Winding webs
    • B65H18/08—Web-winding mechanisms
    • B65H18/10—Mechanisms in which power is applied to web-roll spindle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Definitions

  • the present invention relates to a nucleic acid detection device having a TiO 2 nanostructure-based photocatalytic 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.
  • 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.
  • 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 are 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 a small number of pathogens, once invaded into the human body, rapidly grow 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.
  • 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).
  • 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.
  • a roll-to-roll (R2R) process apparatus refers to an apparatus for performing various types of processes on a roll-type film or web.
  • 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.
  • 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 used.
  • roll-to-roll printing apparatuses are being used in various ways to manufacture various electronic components such as electronic circuits, sensors, and flexible displays.
  • 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.
  • R2R roll-to-roll
  • nucleic acid detection device 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.
  • 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 light is irradiated to the TiO 2 nanostructure-based photocatalytic unit.
  • TiO 2 titanium dioxide
  • the photocatalytic unit includes a titanium dioxide nanomaterial doped with metal nanoparticles, and light is irradiated to the TiO 2 nanostructure-based photocatalytic unit.
  • 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
  • the nucleic acid detection device characterized in that the nucleic acid amplification reaction occurs through photonic polymerase chain reaction (PCR) by light.
  • 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 it provides a method of manufacturing the nucleic acid detection device using a roll-to-roll device, comprising the step of roll-to-roll printing the metal nanoparticles on the titanium dioxide nanostructure unit.
  • the biological molecular detection device can be used for molecular diagnosis.
  • the nucleic acid detection device may be manufactured by a roll-to-roll process.
  • 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.
  • the titanium dioxide nanomaterial may be a titanium dioxide nanostructure having a hexagonal surface (hexagonal TiO 2 nanostructure).
  • the metal nanoparticles of the TiO 2 nanostructure-based photocatalytic unit may be selected from the group consisting of silver nanoparticles, gold nanoparticles, and copper nanoparticles.
  • the polymer membrane may be a PVDF membrane (polyvinylidene difluoride membrane).
  • the nucleic acid detection device may further include a light source for irradiating light to the photocatalyst layer.
  • the nucleic acid detection device may further include a temperature sensor for monitoring the temperature of the nucleic acid molecule.
  • 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.
  • 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 are bound
  • a target nucleic acid molecule can be detected by detecting a change in a photonic bandgap of a photonic crystal of a photocatalytic layer that is changed by the .
  • the change in the bandgap can be detected by absorption spectroscopy or fluorescence analysis.
  • 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.
  • a target nucleic acid DNA, RNA
  • 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 utilized for the detection and diagnosis (point-of-care testing) in the field of pathogenic bacteria, viruses, etc. .
  • 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.
  • FIG. 1 is a diagram schematically showing a cross-section of a nucleic acid detection apparatus according to an embodiment of the present invention.
  • FIG. 2 is a diagram showing the structure of a nucleic acid detection apparatus according to an embodiment of the present invention.
  • FIG. 3 is a flowchart of a method for manufacturing a nucleic acid detection apparatus using a roll-to-roll apparatus according to an embodiment of the present invention.
  • FIG. 1 is a diagram schematically showing a cross-section of a nucleic acid detection apparatus according to an embodiment of the present invention
  • FIG. 2 is an exemplary view showing the structure of the nucleic acid detection apparatus.
  • the nucleic acid detecting apparatus 100 includes a sample inlet 20 into which a sample provided on a substrate 10 is injected, and a sample inlet 20 through which the sample is moved. It includes a microfluidic channel 30, and a nucleic acid detection unit 40 communicating with the microfluidic channel 30 to receive a sample and separate and detect nucleic acids in the sample.
  • the material of the substrate 10 is not particularly limited, and materials commonly used for manufacturing devices for detecting biomolecules such as biosensors, biochips, and microfluidic chips (eg, plastics, glass, etc.) can be used without limitation. .
  • 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 so that there is 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.).
  • the titanium dioxide nano-material may be of hexagonal titanium dioxide having a surface nano-structure (hexagonal TiO 2 nanostructure), for example, titanium dioxide nanotubes (hexagonal nanotube TiO 2) 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.
  • 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 (by-products) excluding nucleic acids (DNA, RNA) adsorbed to the photocatalytic unit (M) in the cell or virus lysate. Silver is discharged through the polymer film 42 to be removed.
  • a polyvinylidene difluoride membrane (PVDF) may be used as the polymer membrane 42 .
  • the nucleic acid detection device may include a microfluidic channel 60 for accommodating impurities that have passed 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 detection 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) with 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.
  • photonic crystals have a characteristic in that different materials having a period of several hundred nanometers to several micrometers are arranged in one-dimensional, two-dimensional or three-dimensional order, and when 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 arrives at 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 are the photocatalyst layer 41 through the micropores of the photocatalyst layer 41 (eg, micropores in the photocatalyst unit M).
  • the nucleic acid adsorbed to the photocatalyst layer 41 is amplified by photonic PCR.
  • the pH of the adsorbed nucleic acid may be appropriately adjusted for the nucleic acid amplification reaction.
  • a sample solution for PCR such as a primer having a nucleotide 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 occurs rapidly.
  • temperature control for nucleic acid amplification may be performed by controlling light irradiation.
  • dNTPs are paired one by one with the bases of the target nucleic acid molecule (eg, RNA or ssDNA), and protons are generated, and the generated protons change the photonic crystal band gap 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.
  • target nucleic acid molecule eg, RNA or ssDNA
  • a technique known in the art for detecting the change in the photo-bandgap may be used without limitation.
  • it can be detected by absorption spectroscopy or fluorescence analysis.
  • the nucleic acid detection apparatus 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, diode laser array, quantum well (vertical)-cavity laser, or the like.
  • the emission wavelength of the light source may be ultraviolet (UV) light, visible light or infrared (IR) light.
  • the nucleic acid detection apparatus 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.
  • 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.
  • 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.
  • the camera may be a smartphone camera, and the smartphone includes application software for analyzing a sample solution.
  • the sensor and light source may be coupled to a computing unit for obtaining sensor data and controlling the light source.
  • a computing device obtains data from a sensor, including a processor and memory stored in application software executable by the processor for driving the light source (eg, for controlling 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 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, a fluid flow can be induced without a separate microfluidic pumping, which is advantageous in reducing the weight and size of the detection device.
  • a vacuum battery 50 for example, reference may be made to US Patent No. 9,970,423.
  • FIG. 3 is a flowchart of a method for manufacturing a nucleic acid detection apparatus using a roll-to-roll apparatus according to an embodiment of the present invention.
  • the method of manufacturing a nucleic acid detection device 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 moves 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.
  • a roll-to-roll device 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
  • FIGS. 1 to 3 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 .
  • 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.
  • a 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, in this case, the coating method is continuous in roll-to-roll using a gravure coater, comma coater, or slot die. can be coated.
  • a target nucleic acid can be easily detected, and thus it is expected to be usefully utilized for on-site detection and diagnosis of pathogenic bacteria and viruses (Point-of-care testing).

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Abstract

La présente invention concerne un appareil de diagnostic moléculaire apte à détecter un acide nucléique cible (ADN, ARN) présent dans un échantillon et une technologie de fabrication de celui-ci par un procédé de rouleau à rouleau (R2R). Lorsque l'échantillon est injecté dans un dispositif de détection, la séparation et l'amplification de l'acide nucléique provenant de l'échantillon se produisent en continu, et aucune étape supplémentaire n'est nécessaire pour isoler l'acide nucléique de l'échantillon. En conséquence, à l'aide de l'appareil de détection d'acide nucléique, l'acide nucléique cible peut être facilement détecté, et l'appareil de détection d'acide nucléique est conçu pour être utilisé de manière utile pour un test sur lieu d'intervention portant sur des bactéries et des virus pathogènes.
PCT/KR2021/006150 2020-05-18 2021-05-17 Appareil de détection d'acide nucléique à base de nanostructure de tio2 et son procédé de fabrication à l'aide d'un procédé rouleau à rouleau WO2021235797A1 (fr)

Applications Claiming Priority (4)

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KR10-2020-0058996 2020-05-18
KR20200058996 2020-05-18
KR10-2020-0109719 2020-08-28
KR1020200109719A KR102449073B1 (ko) 2020-05-18 2020-08-28 TiO2 나노구조 기반 핵산 검출장치 및 롤투롤 공정을 이용한 이의 제조방법

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008526255A (ja) * 2005-01-13 2008-07-24 マイクロニクス， インコーポレイテッド 微量流体希薄細胞検出デバイス
KR20110000091A (ko) * 2009-06-26 2011-01-03 강원대학교산학협력단 체외진단 시약용 금／ＴｉＯ₂ 복합나노입자 및 이의 제조방법
KR20160143795A (ko) * 2014-04-14 2016-12-14 에스알아이 인터내셔널 휴대용 핵산 분석 시스템 및 고성능 미세유체 전기활성 중합체 작동기
KR20170083229A (ko) * 2016-01-08 2017-07-18 고려대학교 산학협력단 표면 측정 센싱 기반의 실시간 핵산 증폭 측정 장치
KR101766283B1 (ko) * 2016-03-02 2017-08-24 경북대학교 산학협력단 수질분석용 마이크로 유체칩 및 이의 제조 방법
KR20180098744A (ko) * 2017-02-27 2018-09-05 한국과학기술원 필름기반 통합칩 및 이의 핵산 검출 방법

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008526255A (ja) * 2005-01-13 2008-07-24 マイクロニクス， インコーポレイテッド 微量流体希薄細胞検出デバイス
KR20110000091A (ko) * 2009-06-26 2011-01-03 강원대학교산학협력단 체외진단 시약용 금／ＴｉＯ₂ 복합나노입자 및 이의 제조방법
KR20160143795A (ko) * 2014-04-14 2016-12-14 에스알아이 인터내셔널 휴대용 핵산 분석 시스템 및 고성능 미세유체 전기활성 중합체 작동기
KR20170083229A (ko) * 2016-01-08 2017-07-18 고려대학교 산학협력단 표면 측정 센싱 기반의 실시간 핵산 증폭 측정 장치
KR101766283B1 (ko) * 2016-03-02 2017-08-24 경북대학교 산학협력단 수질분석용 마이크로 유체칩 및 이의 제조 방법
KR20180098744A (ko) * 2017-02-27 2018-09-05 한국과학기술원 필름기반 통합칩 및 이의 핵산 검출 방법

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