WO2022005146A1 - Appareil de diagnostic moléculaire utilisant une résonance plasmonique de surface à base de graphène métallique, fabriqué par un processus rouleau à rouleau, et son procédé de fabrication - Google Patents

Appareil de diagnostic moléculaire utilisant une résonance plasmonique de surface à base de graphène métallique, fabriqué par un processus rouleau à rouleau, et son procédé de fabrication Download PDF

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Publication number
WO2022005146A1
WO2022005146A1 PCT/KR2021/008133 KR2021008133W WO2022005146A1 WO 2022005146 A1 WO2022005146 A1 WO 2022005146A1 KR 2021008133 W KR2021008133 W KR 2021008133W WO 2022005146 A1 WO2022005146 A1 WO 2022005146A1
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Prior art keywords
graphene
roll
detection device
biological
biological molecule
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PCT/KR2021/008133
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English (en)
Korean (ko)
Inventor
리루크
조규진
Original Assignee
성균관대학교산학협력단
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Priority claimed from KR1020200109710A external-priority patent/KR102539869B1/ko
Application filed by 성균관대학교산학협력단 filed Critical 성균관대학교산학협력단
Publication of WO2022005146A1 publication Critical patent/WO2022005146A1/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
    • B65H37/00—Article or web delivery apparatus incorporating devices for performing specified auxiliary operations
    • B65H37/04—Article or web delivery apparatus incorporating devices for performing specified auxiliary operations for securing together articles or webs, e.g. by adhesive, stitching or stapling
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/71—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited

Definitions

  • 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.
  • metal nanoparticles of nanometer (nm) size are subjected to collective oscillation of electrons in the conduction band of the nanoparticles by light of a specific frequency (wavelength) incident from the outside, thereby exhibiting electric dipole characteristics.
  • LSPR Localized Surface Plasmon Resonance
  • the absorbance characteristics of the metal nanoparticles for external incident light that is, the intensity or frequency (wavelength) of absorption and scattering bands, have characteristics that are determined by the type, size, and shape of the metal nanoparticles.
  • absorption and scattering wavelengths are greatly affected by the external environment of metal nanoparticles, that is, changes in the refractive index of surrounding materials on the surface of metal nanoparticles. Using these properties, biomolecules and chemical substances are detected. It is widely applied in the field of sensors, etc.
  • the present inventors have tried to develop a technology for producing a device (eg, a PCR chip, a microfluidic chip, a biosensor, etc.) for detecting biological molecules in a large quantity within a short time more efficiently by a roll-to-roll process, and also surface plasmon resonance characteristics Efforts have been made to develop a device that can perform the polymerase chain reaction (PCR) with increased sensitivity. As a result, the present invention was completed by developing a metal-graphene-based surface plasmon resonance-based molecular diagnostic device.
  • PCR polymerase chain reaction
  • an object of the present invention is to provide a device for detecting a biological molecule based on a metal-graphene-based surface plasmon resonance, manufactured by a roll-to-roll process.
  • Another object of the present invention is to provide a roll-to-roll device for manufacturing the biological molecule detection device.
  • Another object of the present invention is to provide a method for manufacturing the biological molecule detection device using a roll-to-roll process.
  • the present invention is a biological molecule detection device manufactured by a roll-to-roll process, wherein the detection device includes an upper substrate and a lower substrate, the lower end of the upper substrate or A graphene thin film is disposed on the upper end of the lower substrate, and at least one reaction space for detecting biological molecules in a sample is positioned in a space between the graphene thin film and the substrate, and the reaction space is made of gold or silver It is characterized in that it is a metal plasmonic well, manufactured by a roll-to-roll process, and provides a metal-graphene-based surface plasmon resonance-based biological molecule detection device.
  • the biological molecular detection device can be used for molecular diagnosis.
  • the graphene thin film may be disposed on the lower end of the upper substrate and the upper end of the lower substrate, respectively, and the reaction space may be positioned in a space between the graphene thin films.
  • the biological molecule detection device is a device for polymerase chain reaction (PCR), further comprising a light source for irradiating light to the graphene thin film and the reaction space,
  • the light exposed from the light source may induce plasmonic photothermal light-to-heat conversion between the graphene thin film and the reaction space to cause heating of the sample positioned in the reaction space.
  • PCR polymerase chain reaction
  • the apparatus for polymerase chain reaction may further include a temperature sensor for monitoring the temperature of biological molecules.
  • the apparatus for polymerase chain reaction further comprises a controller coupled to the light source and the temperature sensor, wherein the controller obtains one or more data from the temperature sensor and operation can be controlled.
  • the graphene thin film may be nanometer-sized graphene quantum dot particles for improving light absorption through surface plasmon resonance.
  • the graphene thin film may be a thin film including graphene quantum dots (graphene quantum dots are mixed).
  • the substrate may be translucent or transparent.
  • the apparatus for polymerase chain reaction may further include a digital camera, a photodiode, or a spectrophotometer to detect nucleic acids in real time.
  • the biological molecule detection device may be a microfluidic chip including a microfluidic channel.
  • a guide molecule capable of binding to a biological target molecule in the sample is placed in the reaction space, and a graphene thin film or metal plasmon well that is changed by the binding of the biological target molecule and the guide molecule Biological molecules can be detected by sensing the surface plasmon resonance of
  • the reaction space in a method different from the molecular diagnosis technique that uses the guide molecule to be immobilized on the surface, includes a primer having a nucleotide sequence complementary to a biological target molecule in the sample, a fluorescent die, 4 Species of dNTP molecules and polymerases are placed, and the biological molecules are detected by measuring the fluorescence intensity generated as the nucleic acid amplification reaction is initiated, or graphene that is changed by the binding of the biological target molecule and four types of dNTP molecules Biological molecules can be detected by sensing the surface plasmon resonance of a thin film or metal plasmon well.
  • the guide molecule may be a nucleic acid having a sequence complementary to the biological target molecule, or an antibody or antigen that specifically binds to the biological molecule.
  • the biological molecule may be selected from the group consisting of nucleic acids, proteins, peptides and polypeptides.
  • the present invention provides an imprinting unit for imprinting one or more reaction spaces for detecting biological molecules in a sample in the form of a metal plasmonic well made of gold or silver on a substrate; and a laminating unit for laminating an upper portion of the reaction space with a graphene-coated substrate.
  • the roll-to-roll device further comprises a coating unit for manufacturing a graphene-coated substrate by coating the substrate with graphene, wherein the imprinting unit is graphene manufactured by the coating unit.
  • - Reaction space can be imprinted on coated substrate.
  • the present invention comprises the steps of: imprinting one or more reaction spaces for detecting biological molecules in a sample in the form of a metal plasmon well made of gold or silver on a substrate; and laminating an upper portion of the reaction space with graphene.
  • the manufacturing method may further include coating the substrate with graphene before the imprinting step.
  • FIG. 1 is a diagram schematically illustrating a cross-section of a metal-graphene-based surface plasmon resonance-based biological molecule detection device manufactured by a roll-to-roll process according to an embodiment of the present invention.
  • FIG. 2 is a diagram schematically illustrating a cross-section of a metal-graphene-based surface plasmon resonance-based biological molecule detection device manufactured by a roll-to-roll process according to another embodiment of the present invention.
  • FIG. 3 is a diagram schematically showing the configuration of the biological molecule detection device of the present invention in the case of a polymerase chain reaction (PCR) device.
  • PCR polymerase chain reaction
  • FIG. 4 is a diagram schematically showing the configuration of the biological molecule detection device of the present invention in the case of a microfluidic chip in which a microfluidic channel is formed.
  • FIG 5 shows each component of a roll-to-roll device for manufacturing a biological molecule detection device according to an embodiment of the present invention.
  • FIG. 6 shows the operation of a roll-to-roll device for manufacturing a biological molecule detection device according to an embodiment of the present invention.
  • FIG. 1 is a diagram schematically showing a cross-section of a metal-graphene-based surface plasmon resonance-based biological molecule detection device manufactured by a roll-to-roll process according to an embodiment of the present invention
  • FIG. 2 is another embodiment of the present invention
  • an apparatus 100 for detecting a metal-graphene-based surface plasmon resonance based biological molecule includes an upper substrate 20 and a lower substrate 10 , and the upper substrate A graphene thin film 21 is disposed at the lower end of the 20, and in the space between the graphene thin film 21 and the lower substrate 10 there is at least one reaction space S for detecting biological molecules in the sample. Located. Unlike FIG. 1 , the graphene thin film may be disposed on top of the lower substrate 10 .
  • a graphene thin film 11 is also disposed on the upper portion of the lower substrate 10 , , the reaction space S is located in the space between the graphene thin films 21 and 11 of the upper and lower substrates 20 and 10 .
  • the material of the substrates 10 and 20 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.
  • the biological molecule detection apparatus 100 of the present invention is for polymerase chain reaction (PCR), it may be made of a substrate of a translucent or transparent material (eg, polymethyl methacrylate (PMMA)).
  • PCR polymerase chain reaction
  • PMMA polymethyl methacrylate
  • the reaction space (S) is a metal plasmonic well in the form of a well made of gold nanoparticles or silver nanoparticles, and a polymerase chain reaction for detecting a desired biological molecule present in the sample occurs, or It is a space in which a binding reaction between the biological molecule and a probe molecule binding thereto occurs.
  • the graphene thin films 11 and 21 and the metal plasmon well as the reaction space S provided in the present invention provide heat necessary for the polymerase chain reaction.
  • one cycle of temperature control (eg, 90°C-53°C-60°C) for amplification can be performed within 0.1 seconds, so it takes only 10 seconds to perform 100 amplifications, so RT using the fluorescence method -Provides a platform that can efficiently perform PCR and digital PCR.
  • the change in the surface plasmon resonance phenomenon can be detected (confirmed) a desired biological molecule by quantitatively detecting the change in proportion to the base unit of the ssDNA or ssRNA to be detected.
  • FIG. 3 is a diagram schematically illustrating the configuration of a metal-graphene-based surface plasmon resonance-based biological molecule detection device according to an embodiment of the present invention as a polymerase chain reaction (PCR) device.
  • PCR polymerase chain reaction
  • a light source 30 for irradiating light to the graphene thin films 11 and 21 is further added.
  • the light exposed from the light source 30 induces plasmonic photothermal light-to-heat conversion in the graphene thin films 11 and 21 and the reaction space S to the reaction space. (S) causes heating of the biological molecules located in it.
  • the graphene thin film may be formed by coating graphene ink prepared by mixing and homogenizing graphene flakes from which graphite is peeled with a polymer binder (eg, carboxymethyl cellulose sodium salt) on a substrate using roll-to-roll equipment.
  • a polymer binder eg, carboxymethyl cellulose sodium salt
  • the graphene thin film can also be manufactured with nanometer-sized quantum dot-shaped particles, and thus the light absorption rate through surface plasmon resonance can be controlled for each wavelength of the light source, so that plasmon heating of the graphene thin film can be designed.
  • the apparatus for polymerase chain reaction (PCR) may further include a temperature sensor for monitoring the temperature of the sample solution.
  • the temperature sensor may be coupled to or facing the platform for measuring the temperature of the sample and/or the thin film.
  • 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.
  • FIG. 4 is a diagram schematically illustrating the configuration of the metal-graphene-based surface plasmon resonance-based biological molecule detection device of the present invention in the case of a microfluidic chip 120 having a microfluidic channel formed therein.
  • a sample containing a biological molecule to be detected is injected into the sample inlet (I)
  • the injected sample moves to the reaction space (S) through the microfluidic channel.
  • a primer capable of binding to a biological target molecule in the sample four types of deoxyribonucleotides (dNTPs), a fluorescent dye, and a polymerase are placed.
  • dNTPs deoxyribonucleotides
  • the target can be quantitatively detected by the fluorescence intensity generated in the reaction space (S), and a dNTP to the base of a biological target molecule (eg, RNA or ssDNA) Protons are generated while pairing one by one, and the generated protons affect the graphene and/or metal plasmon wells, resulting in a change in the surface plasmon phenomenon of the graphene and/or metal plasmon wells.
  • a biological target molecule eg, RNA or ssDNA
  • the microfluidic chip in which the nucleic acid amplification reaction occurs may include components for nucleic acid amplification, for example, the graphene thin films 11 and 21 described with reference to FIG. 3 and a light source for irradiating light to the reaction space S. have. Additionally, the microfluidic chip may include components for sample preparation, sample reaction, and sample delivery.
  • a guide molecule capable of binding to a biological target molecule in a sample eg, DNA or RNA having a sequence complementary to that of a biological molecule
  • a biological target molecule in a sample eg, DNA or RNA having a sequence complementary to that of a biological molecule
  • four types of dNTPs and A fluorescence die is placed, and a very rapid amplification of biological molecules occurs in the reaction space (S) and fluorescence is displayed, enabling quantitative detection.
  • protons are generated, Protons affect the graphene and/or metal plasmon wells, resulting in a change in plasmonic phenomena in the graphene and/or metal plasmon wells. By detecting such a plasmon change, a desired biological molecule can be detected.
  • the surface plasmon resonance change may be detected and detected by an angle tunable surface plasmon resonance method, a wavelength tunable surface plasmon resonance method, or a surface plasmon resonance imaging method.
  • microfluidic channel a material used for manufacturing the microfluidic channel, such as PDMS, may be used without limitation.
  • FIG. 5 is a view showing each component (coating unit, imprinting unit, laminating unit) of the roll-to-roll device for manufacturing a biological molecule detection device according to an embodiment of the present invention.
  • the roll-to-roll device includes an imprinting unit for imprinting one or more reaction spaces for detecting biological molecules in a sample on a substrate in the form of a metal (gold or silver) plasmonic well, and the reaction space and a laminating unit for laminating an upper portion with a graphene-coated substrate.
  • the roll-to-roll apparatus may further include a coating unit for manufacturing a graphene-coated substrate by coating the substrate with graphene, in this case, the imprinting unit is a graphene-coated substrate manufactured in the coating unit. Imprinting the reaction space.
  • FIG. 6 is a view showing the operation of a roll-to-roll device for manufacturing a biological molecule detection device according to an embodiment of the present invention.
  • a process for manufacturing a biological molecule detection device according to an embodiment of the present invention using the roll-to-roll device of the present invention is as follows.
  • the substrate is coated with graphene in the coating unit.
  • the coating unit is a device for coating the graphene ink on the substrate.
  • the graphene ink may be prepared, for example, by the following method. Graphene flakes or graphene quantum dots from which graphite is peeled are mixed with sodium deoxycholate and carboxymethyl cellulose sodium salt in various ratios in water (for example, 10 by weight (Water):1:0.01:0.1) and homogenize for more than 12 hours using a probe sonicator. After homogenization, the surface tension and viscosity are measured, and the amount of graphene and the amount of carboxymethylcellulose sodium salt used as a binder can be adjusted to control the viscosity according to the printing method.
  • reaction spaces for detecting biological molecules in the imprinting unit are formed on the graphene-coated substrate in the form of gold (Au) plasmon wells or silver (Ag) plasmon wells (imprinting).
  • metal microwells can be printed on a substrate (eg, a plastic film) using R2R gravure or offset using a gold or silver nanoparticle-based conductive ink.
  • microwells of gold or silver can be printed from several hundred nanometers to several microns in diameter and to several microns in depth.
  • graphene or graphene quantum dots are laminated with a coated film to manufacture a biological molecule detection device.
  • the graphene-coated film may be prepared using R2R slot die or comma coating.

Abstract

La présente invention concerne : un appareil de détection de biomolécules à base de métal (or ou d'argent)-graphène, fabriqué par un processus rouleau à rouleau ; son procédé de fabrication ; et similaire. La présente invention peut fabriquer, par un processus rouleau à rouleau, un appareil de diagnostic moléculaire capable de détecter diverses biomolécules telles que l'ADN, l'ARN et les protéines. En particulier, la présente invention peut être mise en œuvre sous la forme d'une puce de PCR, et utilise à la fois un métal et du graphène pour avoir des caractéristiques de résonance de plasmon de surface plus fortes que lorsque le graphène est utilisé, et peut ainsi réaliser de manière plus sensible une amplification d'acide nucléique.
PCT/KR2021/008133 2020-06-29 2021-06-29 Appareil de diagnostic moléculaire utilisant une résonance plasmonique de surface à base de graphène métallique, fabriqué par un processus rouleau à rouleau, et son procédé de fabrication WO2022005146A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20200079358 2020-06-29
KR10-2020-0079358 2020-06-29
KR1020200109710A KR102539869B1 (ko) 2020-06-29 2020-08-28 롤투롤 공정으로 제조된 금속-그래핀 기반 표면 플라즈몬 공명 분자진단 장치 및 이의 제조방법
KR10-2020-0109710 2020-08-28

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WO2022005146A1 true WO2022005146A1 (fr) 2022-01-06

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100090480A (ko) * 2009-02-06 2010-08-16 한국과학기술연구원 단분자 검출 바이오 센서, 그 제조방법 및 바이오 센서를 이용한 단분자 검출 방법
KR20160075504A (ko) * 2013-09-04 2016-06-29 크레도 바이오메디컬 피티이 엘티디 분석 시험 장치, 키트 및 사용 방법
KR20170106995A (ko) * 2015-01-16 2017-09-22 더 리전트 오브 더 유니버시티 오브 캘리포니아 핵산 증폭용 led 구동 플라즈몬 가열 장치
US20180207920A1 (en) * 2015-07-17 2018-07-26 Illumina, Inc. Polymer sheets for sequencing applications
WO2019002678A1 (fr) * 2017-06-30 2019-01-03 Teknologian Tutkimuskeskus Vtt Oy Puce microfluidique et procédé de production d'une puce microfluidique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100090480A (ko) * 2009-02-06 2010-08-16 한국과학기술연구원 단분자 검출 바이오 센서, 그 제조방법 및 바이오 센서를 이용한 단분자 검출 방법
KR20160075504A (ko) * 2013-09-04 2016-06-29 크레도 바이오메디컬 피티이 엘티디 분석 시험 장치, 키트 및 사용 방법
KR20170106995A (ko) * 2015-01-16 2017-09-22 더 리전트 오브 더 유니버시티 오브 캘리포니아 핵산 증폭용 led 구동 플라즈몬 가열 장치
US20180207920A1 (en) * 2015-07-17 2018-07-26 Illumina, Inc. Polymer sheets for sequencing applications
WO2019002678A1 (fr) * 2017-06-30 2019-01-03 Teknologian Tutkimuskeskus Vtt Oy Puce microfluidique et procédé de production d'une puce microfluidique

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