WO2023027491A1 - Enzyme-based electrode, manufacturing method therefor and application thereof - Google Patents

Enzyme-based electrode, manufacturing method therefor and application thereof Download PDF

Info

Publication number
WO2023027491A1
WO2023027491A1 PCT/KR2022/012615 KR2022012615W WO2023027491A1 WO 2023027491 A1 WO2023027491 A1 WO 2023027491A1 KR 2022012615 W KR2022012615 W KR 2022012615W WO 2023027491 A1 WO2023027491 A1 WO 2023027491A1
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
layer
conductive particle
electrode
support
Prior art date
Application number
PCT/KR2022/012615
Other languages
French (fr)
Korean (ko)
Inventor
조진한
권정훈
Original Assignee
고려대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 고려대학교 산학협력단 filed Critical 고려대학교 산학협력단
Publication of WO2023027491A1 publication Critical patent/WO2023027491A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • 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
    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the present invention relates to an enzyme-based electrode, a method for manufacturing the same, and an application thereof, and more particularly, an enzyme electrode having excellent electron transfer efficiency and high output by having a thin thickness while maintaining a high packing density of the enzyme, and a method for manufacturing the same and its applications.
  • Enzyme electrodes have long been widely used in various fields including biosensing systems and electrochemical devices.
  • biofuel cells BFCs
  • BFCs which can convert biochemical energy into electricity under non-extreme biological conditions, are emerging as promising candidates for powering various bioelectronic devices.
  • NPs metal nanoparticles
  • NPs charged metal nanoparticles
  • various conductive carbon-based nanomaterials e.g., carbon nanotubes, graphene, reduced graphene oxide, nitrogen-doped graphite carbon nitride nanosheets, etc. are used to increase the active area of the electrode as well as the electrode and active site (oxidation). It is possible to reduce the gap between the buried areas in the reduced protein tissue).
  • the thickness of the GOx composite film is It is widely distributed from tens of nanometers (nm) (manufactured by Au NP-recombinant method) to several micrometers (micrometers), and it is too thick to be implemented in ordinary thin films with high density GOx (glucose oxidase) in the factory. There are limitations, and there are unreasonable electrodes suitable for efficient electron transfer, high output, and long-term use.
  • the problem to be solved by the present invention is to provide an enzyme electrode having excellent electron transfer efficiency and high output by having a thin thickness while maintaining a high packing density of the enzyme, a manufacturing method thereof, and an application thereof.
  • the present invention supports; Enzyme layer bound to the support; It provides an enzyme electrode comprising a conductive particle layer chemically bonded to the enzyme layer, wherein the enzyme layer and the conductive particle layer are directly bonded to the conductive particle through a functional group of the enzyme.
  • the functional group is an amine group
  • the binding between the conductive particle layer and the enzyme layer is achieved by removing a ligand from the surface of the conductive particle in an organic solvent.
  • the enzyme electrode has a laminated structure in which the enzyme layer and the conductive particle layer alternate and repeat.
  • the support is a fibrous support.
  • the present invention is an enzyme electrode manufacturing method comprising the steps of contacting a support with a first solution containing an enzyme to form an enzyme layer on the support; and bonding the conductive particle layer to the enzyme layer by bringing the support on which the enzyme layer is formed into contact with a second solution in which conductive particles are dispersed, wherein the solvent of the first solution is a polar solvent, and the second solution is organic. is a solvent
  • the enzyme is at least one selected from the group consisting of glucose oxidase (Gox), catalase (CAT, hemoglomin and feratin, and the conductive particles are gold, copper, platinum and It is at least one selected from the group consisting of indium tin oxide (ITO).
  • a hydrophilic ligand is bound to the surface of the conductive particle, and a covalent bond between the conductive particle and the enzyme is formed by a substitution reaction between the hydrophilic ligand of the conductive particle and the functional period of the enzyme in the organic solvent. do.
  • the hydrophilic ligand of the conductive particles is removed from the organic solvent, thereby reducing the electrostatic repulsive force between the conductive particles.
  • the first solution includes water
  • the second solution includes toluene
  • the support is in the form of a fiber.
  • the present invention also provides an enzyme electrode manufactured by the above-described method, and a biofuel cell and a biosensor including the same as an application example.
  • an amphiphilic assembly method in which an enzyme in a polar solvent such as water and conductive nanoparticles are alternately deposited in an organic solvent is introduced to a conductive support to obtain a cotton fabric-type/fiber-type enzyme electrode with improved electron transfer capability.
  • the amphiphilic assembly method developed through the present invention maintains the stability of the enzyme used in organic solvents as well as in polar solvents such as water, and thus applications such as biosensors or fuel cells using the enzyme electrode according to the present invention
  • the product can maintain high stability and efficiency, and the amount of enzyme adsorbed to the electrode can be absolutely increased through assembly with the introduced conductive nanoparticles. Due to this, accuracy and high efficiency of biosensors and fuel cells can be implemented.
  • FIG. 1 is a schematic diagram of a method for manufacturing an enzyme electrode according to an embodiment of the present invention.
  • FIG. 3 is a diagram explaining LbL stacking between TOA-AuNP/GOx in the solvent conditions of FIGS. 1 and 2.
  • FIG. 5 is a result of manufacturing an enzyme electrode by an amphiphilic LbL assembly method according to the method shown in FIG. 1 by using different types of conductive metal particles and enzymes.
  • FIG. 6 is a result of depositing various biomaterials and gold nanoparticles by LbL according to the method shown in FIG. 1 .
  • the present invention comprises the steps of contacting the support with a first solution containing an enzyme to form an enzyme layer on the support;
  • a method for manufacturing an amphiphilic enzyme electrode in which the support on which the enzyme layer is formed is brought into contact with a second solution in which conductive particles are dispersed to bind the conductive particle layer to the enzyme layer.
  • the solvent of the first solution is a polar solvent
  • the second solution is an organic solvent, for example, an enzyme layer in water and a non-polar solvent such as toluene to bind conductive particles to the enzyme layer can make it
  • the conductive particle is a particle that is conductive and to which a hydrophilic ligand can be bound, and any material to which a ligand capable of binding at least through a substitution reaction with a functional group such as an amine of an enzyme is a conductive particle of the present specification. belong to the range
  • FIG. 1 is a schematic diagram of a method for manufacturing an enzyme electrode according to an embodiment of the present invention.
  • an enzyme electrode is manufactured by crossing an enzyme material (GOx) and conductive nanoparticles in an organic solvent (toluene) and an aqueous solvent (water) and depositing them on a substrate in an LbL method. That is, an enzyme material is deposited in an aqueous solvent and conductive nanoparticles are deposited in an organic solvent, and through this, a support; Enzyme layer bound to the support; An enzyme electrode having a structure including a conductive particle layer chemically bonded to the enzyme layer, wherein the enzyme layer and the conductive particle layer are directly bonded to the conductive particle through a functional group of the enzyme.
  • conductivity is imparted to the electrode layer through a ligand bond between the conductive particle and the enzyme layer in an organic solvent, and in particular, a covalent bond between the conductive particle and the enzyme layer is induced due to the hydrophilic ligand removed from the organic solvent.
  • PEI poly(ethylene imine)
  • the resulting PEI-coated fabric prepared through hydrogen bonding interactions between the NH2 groups of PEI and the OH groups of the cotton surface was immersed in TOA-AuNP suspension (10 mg mL-1) for 40 min and then washed with toluene to obtain weak adsorption.
  • TOA-AuNPs were removed. Again, the bulky TOA ligand bound to the surface of the AuNPs was exchanged with the NH2 groups of the PEI-coated cotton fabric due to the high affinity between the Au surface and the NH2 groups.
  • the formed TOA-AuNP-coated cotton fabric was immersed in a solution of diethylenetriamine (DETA) dissolved in ethanol (1 mg mL-1) for 40 min.
  • DETA diethylenetriamine
  • the adsorption mechanism between TOA-AuNPs and NH2-functionalized DETA is the same as that between TOA-AuNPs and PEI, and these deposition and washing steps for the preparation of MCT were successively repeated, (TOA-AuNP/DETA) 20
  • the fiber support was used as the MCT electrode support according to the present invention.
  • the conductive nanoparticle layer/enzyme was sequentially stacked in the amphiphilic LbL method, which will be described in detail as follows.
  • the prepared MCT electrode was first immersed in a GOx solution containing water as a polar solvent (concentration of 5 mg mL-1 in a PBS solution containing 0.5 M NaCl) for 20 minutes and then washed with distilled water (pH 5.8) to weakly adsorb GOx. has been removed.
  • a GOx solution containing water as a polar solvent concentration of 5 mg mL-1 in a PBS solution containing 0.5 M NaCl
  • distilled water pH 5.8
  • the amine group which is the representative two functional groups of GOx capable of bonding with metal nanoparticles, and the carboxylic acid functional group among the carboxylic acid functional groups were rarely bonded. Bonding between nanoparticles takes place.
  • the support was immersed in a toluene solution, and the dispersed TOA-AuNPs were deposited on the GOx layer for 20 minutes and washed with toluene to remove weakly adsorbed TOA-AuNPs.
  • the enzyme material in an aqueous solvent, when the conductive particles are adsorbed in an organic solvent (non-polar solvent) such as toluene, the bulky ligand of TOA-AuNPs is removed and GOx is directly adsorbed.
  • FIG. 3 is a diagram explaining LbL stacking between TOA-AuNP/GOx in the solvent conditions of FIGS. 1 and 2.
  • the total loading amount of GOx and TOA-AuNPs per layer was measured to be about 2.3 ⁇ 0.3 ⁇ g cm -2 , and (GOx/TOA-AuNP)p measured by field emission scanning electron microscopy (FE-SEM)
  • the total film thickness of the multilayer increased from about 18 to 193 nm as p increased from 2 to 20.
  • the thickness of each adsorbed TOA-AuNP layer and each GOx layer was measured to be about 5.5 ⁇ 1.3 nm and 3.3 ⁇ 0.8 nm, respectively.
  • TOA-AuNPs in toluene are LbL assembled into amine-functionalized PEI and DETA and carboxylic acid (COOH)-functionalized poly(acrylic acid) (PAA) in water at pH 7.4.
  • the pKa (pH value at which 50% of the functional groups are ionized) of the amine group in aqueous solution is about 9,39, and the ratio of the uncharged amine moiety in PEI and DETA is about 15-25%.
  • TOA-AuNPs can be LbL-assembled through relatively high ligand exchange with PEI or DETA dissolved in water, but the affinity for the carboxylate ion (COO-) group of PAA formed at pH 7,4 is There was no
  • the water contact angle was measured to be about 48 ⁇ 2°.
  • TOA-AuNPs were continuously deposited on the outermost GOx layer, the water contact angle increased to 62 ⁇ 1°, and it was found that this water contact angle changed periodically according to the stagnation of the outermost layer.
  • FIG. 5 is a result of manufacturing an enzyme electrode by an amphiphilic LbL assembly method according to the method shown in FIG. 1 by using different types of conductive metal particles and enzymes.
  • (a) is copper nanoparticles (TOA-Cu NP), (b) is platinum nanoparticles (Pt NP) and oleic acid, and (c) is indium (ITO) and oleamine
  • (OAm) indium
  • the amphiphilic enzyme electrode according to the present invention can be applied to various enzymes (biomaterials) and conductive nanoparticles, and at least as long as the enzyme layer is deposited in a polar solvent and the conductive nanoparticles are deposited in a non-polar solvent, all of these are in accordance with the present invention. belong
  • FIG. 6 is a result of depositing various biomaterials and gold nanoparticles by LbL according to the method shown in FIG. 1 .
  • the LbL enzyme electrode obtained through three iterations obtains a concentration-dependent electrical detection result for Ag as a counter electrode.
  • the detected current value becomes stronger as the concentration increases, and the impedance clearly shows a concentration-dependent tendency.
  • the liquid metal according to the present invention includes particles surrounded by a polymer film, and in this case, the thickness can be controlled by adjusting the solution processing conditions, and a large-area process is possible while maintaining high resolution.
  • the liquid metal ink prepared for the solution process can form thin films on various types of substrates and transfer patterns to various materials.
  • An electrode combining GOx using a cross linker which is a traditional method of stacking GOx in FIG. 9 (Conventional GOx layer); an electrode ((GOX/DETA)3/MCT) obtained by dissolving DETA, a unimolecular linker, in water and layering GOx;
  • the present invention introduces an amphiphilic assembly method in which an enzyme in a polar solvent such as water and conductive nanoparticles are alternately deposited in an organic solvent to a conductive support, so that the cotton fabric/fiber enzyme has improved electron transfer ability. electrodes can be made.
  • the amphiphilic assembly method developed through the present invention maintains the stability of the enzyme used in organic solvents as well as in polar solvents such as water, and thus applications such as biosensors or fuel cells using the enzyme electrode according to the present invention
  • the product can maintain high stability and efficiency, and can absolutely increase the amount of enzyme adsorbed to the electrode through the conductive nanoparticles and assembly introduced. , accuracy and high efficiency of biosensors and fuel cells can be realized.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Emergency Medicine (AREA)
  • Hematology (AREA)
  • Inert Electrodes (AREA)

Abstract

Provided is an enzyme electrode comprising: a support; an enzyme layer bonded to the support; and a conductive particle layer chemically bonded to the enzyme layer, wherein, in the enzyme layer and the conductive particle layer, conductive particles are directly bonded through functional groups of the enzyme.

Description

효소기반 전극, 그 제조방법 및 그 응용Enzyme-based electrode, its manufacturing method and its applications
본 발명은 효소기반 전극, 그 제조방법 및 그 응용에 관한 것으로, 보다 상세하게는 효소의 높은 충전밀도를 유지하면서 얇은 두께를 가짐으로써 우수한 전자전달 효율, 높은 출력량 등을 갖는 효소 전극, 그 제조방법 및 그 응용에 관한 것이다. The present invention relates to an enzyme-based electrode, a method for manufacturing the same, and an application thereof, and more particularly, an enzyme electrode having excellent electron transfer efficiency and high output by having a thin thickness while maintaining a high packing density of the enzyme, and a method for manufacturing the same and its applications.
효소 전극은 바이오 센싱 시스템 및 전기화학 장치를 포함한 다양한 분야에서 오랫동안 널리 사용되어 왔다. 특히 극심하지 않은 생물학적 조건에서 생화학 에너지를 전기로 변환할 수 있는 바이오 연료전지(BFC)는 다양한 생체 전자 장치에 전력을 공급하기 위한 유망한 후보로 각광을 받고 있다. Enzyme electrodes have long been widely used in various fields including biosensing systems and electrochemical devices. In particular, biofuel cells (BFCs), which can convert biochemical energy into electricity under non-extreme biological conditions, are emerging as promising candidates for powering various bioelectronic devices.
하지만 BFC의 유망한 특성에도 불구하고 저전력 출력은 지난 수십 년 동안 치명적인 단점으로 남아 있으며, 이 문제를 해결하기 위해 많은 연구 노력은 효소/호스트 전극 및 효소/효소 인터페이스 및 보다 효과적인 효소 고정화 방법 개발에 집중되고 있다.However, despite the promising properties of BFCs, their low power output has remained a fatal drawback in the past decades. To address this issue, much research effort has been focused on the development of enzyme/host electrodes and enzyme/enzyme interfaces and more effective enzyme immobilization methods. there is.
이러한 시도 중 하나는 산화환원 매개체를 BFC 전극에 결합시켜 "매개된 전자 전달 기반 BFC(MET-BFC)"를 개발하는 것으로, 전극 내 전자 전달을 개선하는 데 가장 널리 사용되고 있다. 하지만, 생물학적 독성, 불안정성 및 많은 산화환원 매개체의 복잡한 합성방법의 문제로 인하여, 최근 연구에서는 산화환원 매개체가 없는 "직접 전자 이동 기반 BFC(이하 DET-BFC)" 개발을 통하여 전자 이동 및 전력 출력을 높이는 데 집중하고 있다. 대부분의 DET-BFC(수~수십 μW cm-2) 출력은 MET-BFC(수십~수백 μW cm-2)보다 훨씬 열등하지만,1 전자의 전력 출력은 효소와 전극 사이의 전자 전달을 개선하기 위한 다양한 도전적인 접근법을 통해 점진적으로 향상되고 있다. One of these attempts is to develop “mediated electron transport-based BFC (MET-BFC)” by incorporating redox mediators into the BFC electrode, which is most widely used to improve electron transport within the electrode. However, due to problems of biological toxicity, instability, and complex synthesis method of many redox mediators, recent studies have improved electron transfer and power output through the development of "direct electron transfer-based BFC (hereinafter referred to as DET-BFC)" without redox mediators. I'm concentrating on getting high. Although the power output of most DET-BFCs (tens to tens of μW cm −2 ) is far inferior to that of MET-BFCs (tens to hundreds of μW cm −2 ), the power output of 1 electron is used to improve electron transfer between the enzyme and the electrode. It is progressively improved through various challenging approaches.
효과적인 전자 전달을 실현하기 위한 주목할만한 접근 방식으로, 효소 사이에 금속 나노입자(NP)를 삽입하여 전자 릴레이 효과를 활용하는 것이다. A noteworthy approach to realizing effective electron transport is to utilize the electron relay effect by intercalating metal nanoparticles (NPs) between enzymes.
이 경우 대부분, 물에서 전하를 띤 금속 나노입자(NP)를 물리적 흡착과 간단한 혼합방식으로 효소와 결합시키는 방법으로 수행된다. 더 나아가, 다양하나 전도성 탄소 기반 나노물질(예를 들어 탄소나노튜브, 그래핀, 환원 그래핀 산화물, 질소 도핑 흑연 탄소 질화물 나노시트 등)이 사용되어 전극의 활성면적 뿐만 아니라 전극과 활성사이트(산화환원 단백직에서 묻힌 부위) 사이의 간격을 줄여줄 수 있다. In most cases, this is performed by combining charged metal nanoparticles (NPs) with enzymes in water through physical adsorption and a simple mixing method. Furthermore, various conductive carbon-based nanomaterials (e.g., carbon nanotubes, graphene, reduced graphene oxide, nitrogen-doped graphite carbon nitride nanosheets, etc.) are used to increase the active area of the electrode as well as the electrode and active site (oxidation). It is possible to reduce the gap between the buried areas in the reduced protein tissue).
하지만, 그러나 이러한 접근 방식은 비활성 유기물(가교성 고분자 포함)의 사용, 금속 NP의 낮은 충전 밀도(물에서 정전기적 반발에 의한) 및/또는 탄소기반 전도성 물질이 가지는 본질적으로 낮은 전기 전도도로 인하여 전자 전달을 더욱 개선하는 데 한계가 있다. However, this approach is not suitable for electrons due to the use of inert organics (including crosslinkable polymers), the low charge density of metal NPs (due to electrostatic repulsion from water) and/or the inherently low electrical conductivity of carbon-based conductive materials. Further improvement of delivery is limited.
또한, 두께를 증가시키면 결국 효소층의 전자 전달 효율이 억제되므로 최적의 두께에 대한 연구가 더 필요한 상황이며, 구체적으로, GOx(glucose oxidase)를 전극에 고정하는 기존 방법의 경우 GOx 복합막의 두께는 수십 나노미터(nm)(Au NP-재조합 방법으로 제작)에서 수 마이크로미터(마이크로미터)까지 광범위하게 분포되며, 공장상 높은 밀도의 GOx(glucose oxidase)를 갖는 보통의 박막에서 구현되기에는 두께에 그 한계가 있으며, 효율적인 전자전달, 높은 출력, 및 장시간 사용 등에 적합한 전극에는 무리가 있다. In addition, since increasing the thickness eventually suppresses the electron transfer efficiency of the enzyme layer, further research on the optimal thickness is needed. Specifically, in the case of the existing method of fixing GOx (glucose oxidase) to the electrode, the thickness of the GOx composite film is It is widely distributed from tens of nanometers (nm) (manufactured by Au NP-recombinant method) to several micrometers (micrometers), and it is too thick to be implemented in ordinary thin films with high density GOx (glucose oxidase) in the factory. There are limitations, and there are unreasonable electrodes suitable for efficient electron transfer, high output, and long-term use.
따라서, 본 발명이 해결하고자 하는 과제는 효소의 높은 충전밀도를 유지하면서 얇은 두께를 가짐으로써 우수한 전자전달 효율, 높은 출력량 등을 갖는 효소 전극, 그 제조방법, 그 응용을 제공하는 것이다. Therefore, the problem to be solved by the present invention is to provide an enzyme electrode having excellent electron transfer efficiency and high output by having a thin thickness while maintaining a high packing density of the enzyme, a manufacturing method thereof, and an application thereof.
상기 과제를 해결하기 위하여, 본 발명은 지지체; 상기 지지체에 결합된 효소층; 상기 효소층과 화학결합된 전도성 입자층을 포함하며, 상기 효소층과 상기 전도성 입자층은 상기 효소의 기능기를 통하여 상기 전도성 입자와 직접 결합한 것을 특징으로 하는 효소전극을 제공한다. In order to solve the above problems, the present invention supports; Enzyme layer bound to the support; It provides an enzyme electrode comprising a conductive particle layer chemically bonded to the enzyme layer, wherein the enzyme layer and the conductive particle layer are directly bonded to the conductive particle through a functional group of the enzyme.
본 발명의 일 실시예에서, 상기 기능기는 아민기이며, 상기 전도성 입자층과 상기 효소층간 결합은 상기 전도성 입자 표면의 리간드를 유기용매에서 제거함으로써 이루어진다. In one embodiment of the present invention, the functional group is an amine group, and the binding between the conductive particle layer and the enzyme layer is achieved by removing a ligand from the surface of the conductive particle in an organic solvent.
본 발명의 일 실시예에서, 상기 효소전극은, 상기 효소층과 상기 전도성 입자층이 교차하여 반복하는 적층구조이다. In one embodiment of the present invention, the enzyme electrode has a laminated structure in which the enzyme layer and the conductive particle layer alternate and repeat.
본 발명의 일 실시예에서, 상기 지지체는 섬유형 지지체이다. In one embodiment of the present invention, the support is a fibrous support.
본 발명은 효소전극 제조방법으로, 지지체를 효소를 포함하는 제 1 용액과 접촉시켜 지지체에 효소층을 형성하는 단계; 상기 효소층이 형성된 지지체를 전도성 입자가 분산된 제 2 용액과 접촉시켜 상기 전도성 입자층을 상기 효소층에 결합시키는 단계를 포함하며, 상기 제 1 용액의 용매는 극성 용매이며, 상기 제 2 용액은 유기 용매이다. The present invention is an enzyme electrode manufacturing method comprising the steps of contacting a support with a first solution containing an enzyme to form an enzyme layer on the support; and bonding the conductive particle layer to the enzyme layer by bringing the support on which the enzyme layer is formed into contact with a second solution in which conductive particles are dispersed, wherein the solvent of the first solution is a polar solvent, and the second solution is organic. is a solvent
본 발명의 일 실시예에서, 상기 효소는 글로코스 옥시데이즈(Gox), 카탈라아제(CAT, 헤모글로민 및 페라틴으로 이루어진 군으로부터 선택된 적어도 어느 하나이며, 상기 전도성 입자는 금, 구리, 백금 및 인듐주석산화물(ITO)로 이루어진 군으로부터 선택된 적어도 어느 하나이다. In one embodiment of the present invention, the enzyme is at least one selected from the group consisting of glucose oxidase (Gox), catalase (CAT, hemoglomin and feratin, and the conductive particles are gold, copper, platinum and It is at least one selected from the group consisting of indium tin oxide (ITO).
본 발명의 일 실시예에서, 상기 전도성 입자 표면에는 친수성 리간드가 결합되며, 상기 유기용매에서 상기 전도성 입자의 친수성 리간드와 상기 효소의 기능기간 치환 반응에 의하여 상기 전도성 입자와 상기 효소간 공유결합이 형성된다. In one embodiment of the present invention, a hydrophilic ligand is bound to the surface of the conductive particle, and a covalent bond between the conductive particle and the enzyme is formed by a substitution reaction between the hydrophilic ligand of the conductive particle and the functional period of the enzyme in the organic solvent. do.
본 발명의 일 실시예에서, 상기 전도성 입자의 친수성 리간드는 상기 유기용매에서 제거되며, 이로써 상기 전도성 입자간 정전전기적 반발력은 감소된다. In one embodiment of the present invention, the hydrophilic ligand of the conductive particles is removed from the organic solvent, thereby reducing the electrostatic repulsive force between the conductive particles.
본 발명의 일 실시예에서, 상기 제 1 용액은 물, 상기 제 2 용액은 톨루엔을 포함하며, 상기 지지체는 섬유형태이다. In one embodiment of the present invention, the first solution includes water, the second solution includes toluene, and the support is in the form of a fiber.
본 발명은 또한 상술한 방법에 의하여 제조된 효소전극과, 그 응용예로 이를 포함하는 바이오 연료전지 및 바이오 센서를 제공한다. The present invention also provides an enzyme electrode manufactured by the above-described method, and a biofuel cell and a biosensor including the same as an application example.
본 발명에 따르면, 물과 같은 극성 용매에서는 효소, 유기용매에서는 전도성 나노입자를 교차하여 증착시키는 양친화성 어셈블리리 방법을 전도성 지지체에 도입하여, 전자전달 능력이 향상된 면 직물형/섬유형 효소 전극을 제조할 수 있다. 또한, 본 발명을 통해 개발된 양친화성 어셈블리 방법은 물과 같은 극성용매 뿐만 아니라 유기용매 내에서도 사용된 효소의 안정성이 유지되며, 이로써 본 발명에 따른 효소 전극을 활용하는 바이오 센서나 연료전지와 같은 응용제품은 안정성과 효율을 높게 유지할 수 있고, 도입되는 전도성 나노입자와의 어셈블리를 통해 전극에 흡착되는 효소의 양을 절대적으로 증가시킬 수 있었으며, 이를 통해 효과적으로 전자전달을 일으킬 수 있는 효소층을 디자인함으로 인해, 바이오 센서 및 연료전지의 정확도 및 고효율을 구현할 수 있다. According to the present invention, an amphiphilic assembly method in which an enzyme in a polar solvent such as water and conductive nanoparticles are alternately deposited in an organic solvent is introduced to a conductive support to obtain a cotton fabric-type/fiber-type enzyme electrode with improved electron transfer capability. can be manufactured In addition, the amphiphilic assembly method developed through the present invention maintains the stability of the enzyme used in organic solvents as well as in polar solvents such as water, and thus applications such as biosensors or fuel cells using the enzyme electrode according to the present invention The product can maintain high stability and efficiency, and the amount of enzyme adsorbed to the electrode can be absolutely increased through assembly with the introduced conductive nanoparticles. Due to this, accuracy and high efficiency of biosensors and fuel cells can be implemented.
도 1은 본 발명의 일 실시예에 따른 효소전극 제조방법의 모식도이다. 1 is a schematic diagram of a method for manufacturing an enzyme electrode according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 전극 제조방법과 이에 따라 얻어진 사진이다. 2 is an electrode manufacturing method according to an embodiment of the present invention and a photograph obtained thereby.
도 3은 도 1 및 2의 용매 조건에서 TOA-AuNP/GOx간 LbL 적층을 설명하는 도면이다. FIG. 3 is a diagram explaining LbL stacking between TOA-AuNP/GOx in the solvent conditions of FIGS. 1 and 2.
도 4는 본 발명의 일 실시예에 따른 효소전극의 접촉각 분석결과이다. 4 is a contact angle analysis result of an enzyme electrode according to an embodiment of the present invention.
도 5는 전도성 금속임자와 효소 종류를 달리하여, 도 1에서 도시된 방법에 따라 양친매성 LbL 어셈블리 방식으로 효소전극을 제조한 결과이다. 5 is a result of manufacturing an enzyme electrode by an amphiphilic LbL assembly method according to the method shown in FIG. 1 by using different types of conductive metal particles and enzymes.
도 6은 다양한 바이오 물질과 금 나노입자를 도 1에서 도시된 방식에 따라 LbL로 증착시킨 결과이다. FIG. 6 is a result of depositing various biomaterials and gold nanoparticles by LbL according to the method shown in FIG. 1 .
도 7은 본 발명에 따른 효소전극을 활용한 글루코스 농도 및 임피던스 측정 결과이다. 7 is a result of glucose concentration and impedance measurement using the enzyme electrode according to the present invention.
도 8은 산화수소 농도에 따른 카탈라아제 (CAT)의 촉매활성 정도 및 임피던스 측정 결과이다. 8 shows the degree of catalytic activity of catalase (CAT) and the impedance measurement results according to the concentration of hydrogen oxide.
도 9는 전도성 입자의 용매 종류에 따른 전극 효율 특성을 비교한 결과이다. 9 is a result of comparing electrode efficiency characteristics according to the type of solvent of conductive particles.
상술한 과제를 해결하기 위하여, 본 발명은 지지체를 효소를 포함하는 제 1 용액과 접촉시켜 지지체에 효소층을 형성하는 단계; 상기 효소층이 형성된 지지체를 전도성 입자가 분산된 제 2 용액과 접촉시켜 상기 전도성 입자층을 상기 효소층에 결합시키는 양친매성 효소전극 제조방법을 제공한다. In order to solve the above problems, the present invention comprises the steps of contacting the support with a first solution containing an enzyme to form an enzyme layer on the support; Provided is a method for manufacturing an amphiphilic enzyme electrode in which the support on which the enzyme layer is formed is brought into contact with a second solution in which conductive particles are dispersed to bind the conductive particle layer to the enzyme layer.
본 발명의 일 실시예에서, 상기 제 1 용액의 용매는 극성 용매이며, 상기 제 2 용액은 유기 용매로서, 예를 들어 물에서 효소층을, 톨루엔과 같은 비극성 용매에서는 전도성 입자를 효소층에 결합시킬 수 있다. In one embodiment of the present invention, the solvent of the first solution is a polar solvent, and the second solution is an organic solvent, for example, an enzyme layer in water and a non-polar solvent such as toluene to bind conductive particles to the enzyme layer can make it
본 명세서에서 전도성 입자는 전도성을 띠며 친수성 리간드가 결합될 수 있는 입자로서 적어도 효소의 아민과 같은 기능기와 치환 반응을 통하여 결합할 수 있는 리간드가 결합될 수 있는 임의의 모든 물질이 본 명세서의 전도성 입자 범위에 속한다. In this specification, the conductive particle is a particle that is conductive and to which a hydrophilic ligand can be bound, and any material to which a ligand capable of binding at least through a substitution reaction with a functional group such as an amine of an enzyme is a conductive particle of the present specification. belong to the range
도 1은 본 발명의 일 실시예에 따른 효소전극 제조방법의 모식도이다. 1 is a schematic diagram of a method for manufacturing an enzyme electrode according to an embodiment of the present invention.
도 1을 참조하면, 유기용매(톨루엔)과 수성용매(물)에서 각각 효소물질(GOx)와 전도성 나노입자를 교차하여 LbL 방식으로 기판상에 증착하여 효소전극을 제조한다. 즉, 수성용매에서는 효소물질을 증착하고, 유기용매에서는 전도성 나노입자를 증착하게 되며, 이를 통하여 지지체; 상기 지지체에 결합된 효소층; 상기 효소층과 화학결합된 전도성 입자층을 포함하며, 상기 효소층과 상기 전도성 입자층은 상기 효소의 기능기를 통하여 상기 전도성 입자와 직접 결합한 구조의 효소전극을 제조된다. Referring to FIG. 1, an enzyme electrode is manufactured by crossing an enzyme material (GOx) and conductive nanoparticles in an organic solvent (toluene) and an aqueous solvent (water) and depositing them on a substrate in an LbL method. That is, an enzyme material is deposited in an aqueous solvent and conductive nanoparticles are deposited in an organic solvent, and through this, a support; Enzyme layer bound to the support; An enzyme electrode having a structure including a conductive particle layer chemically bonded to the enzyme layer, wherein the enzyme layer and the conductive particle layer are directly bonded to the conductive particle through a functional group of the enzyme.
본 발명에서는 특히 유기용매에서 전도성 입자와 효소층간 리간드 결합을 통하여 전극층에 전도성을 부여하며, 특히 유기용매에서 제거되는 친수성 리간드로 인하여 전도성 입자와 효소층간 공유결합이 유도되며, 동일 전하를 띠는 벌키한 친수성 리간드가 제거됨으로써 입자간 반발을 최소화하여 높은 충전밀도로 전극층을 제조할 수 있다. In the present invention, in particular, conductivity is imparted to the electrode layer through a ligand bond between the conductive particle and the enzyme layer in an organic solvent, and in particular, a covalent bond between the conductive particle and the enzyme layer is induced due to the hydrophilic ligand removed from the organic solvent. By removing one hydrophilic ligand, it is possible to manufacture an electrode layer with high packing density by minimizing repulsion between particles.
이하 바람직한 실시예를 통하여 본 발명은 보다 상세히 설명한다. The present invention will be described in more detail through the following preferred examples.
실시예Example
지지체 제조support fabrication
먼저 면직물을 폴리(에틸렌 이민)의 에탄올 용액(PEI, Mw = 800 g mol-1, 1 mg mL-1)에 40분 동안 침지시켰다. First, the cotton fabric was soaked in an ethanol solution of poly(ethylene imine) (PEI, Mw = 800 g mol-1, 1 mg mL-1) for 40 minutes.
PEI의 NH2 그룹과 면 표면의 OH 그룹 사이의 수소 결합 상호작용을 통해 제조된 생성된 PEI 코팅 직물을 TOA-AuNP 현탁액(10 mg mL-1)에 40분 동안 담근 다음 톨루엔으로 세척하여 약하게 흡착된 TOA-AuNP를 제거하였다. 다시 AuNPs의 표면에 결합된 부피가 큰 TOA 리간드는 Au 표면과 NH2 그룹 사이의 높은 친화도로 인해 PEI 코팅된 면직물의 NH2 그룹으로 교환되었다. 다음으로, 형성된 TOA-AuNP 코팅된 면직물을 에탄올(1 mg mL-1)에 용해된 디에틸렌트리아민(DETA) 용액에 40분 동안 담그었다. 이 과정을 반복하고 면직물을 에탄올로 세척하여 과량의 DETA 분자를 제거하여 하나의 이중층(즉, (TOA-AuNP/DETA)1-면직물)으로 코팅된 면직물 기반 지지체(MCT)를 얻었다. The resulting PEI-coated fabric prepared through hydrogen bonding interactions between the NH2 groups of PEI and the OH groups of the cotton surface was immersed in TOA-AuNP suspension (10 mg mL-1) for 40 min and then washed with toluene to obtain weak adsorption. TOA-AuNPs were removed. Again, the bulky TOA ligand bound to the surface of the AuNPs was exchanged with the NH2 groups of the PEI-coated cotton fabric due to the high affinity between the Au surface and the NH2 groups. Next, the formed TOA-AuNP-coated cotton fabric was immersed in a solution of diethylenetriamine (DETA) dissolved in ethanol (1 mg mL-1) for 40 min. This process was repeated and the cotton fabric was washed with ethanol to remove excess DETA molecules, resulting in a cotton fabric-based scaffold (MCT) coated with one bilayer (i.e., (TOA-AuNP/DETA) 1 -cotton fabric).
TOA-AuNPs와 NH2 기능화된 DETA 사이의 흡착 메커니즘은 TOA-AuNPs와 PEI 사이의 흡착 메커니즘과 동일하며, MCT의 제조를 위한 이러한 증착 및 세척 단계는 연속적으로 반복되었으며, (TOA-AuNP/DETA)20 섬유지지체를 본 발명에 따른 MCT 전극지지체로 사용하였다.The adsorption mechanism between TOA-AuNPs and NH2-functionalized DETA is the same as that between TOA-AuNPs and PEI, and these deposition and washing steps for the preparation of MCT were successively repeated, (TOA-AuNP/DETA) 20 The fiber support was used as the MCT electrode support according to the present invention.
효소전극 제조Enzyme electrode manufacturing
상기 제조된 MCT 지지체 상에 도 1에 설명한 바와 같이 양친매 LbL 방식으로 전도성 나노입자층/효소를 순차적으로 적층시켰으며, 이를 보다 상세히 설명하면 다음과 같다. On the prepared MCT support, as described in FIG. 1, the conductive nanoparticle layer/enzyme was sequentially stacked in the amphiphilic LbL method, which will be described in detail as follows.
상기 제조된 MCT 전극을 먼저 극성용매인 물을 포함하는 GOx 용액(0.5 M NaCl을 포함하는 PBS 용액에 5 mg mL-1 농도)에 20분 동안 담그고 증류수(pH 5.8)로 세척하여 약하게 흡착된 GOx를 제거하였다. 이로써 물에서 전하를 띤 금속 나노입자(NP)를 물리적 흡착과 간단한 혼합방식으로 효소와 금속나노입자는 결합을 하게 된다The prepared MCT electrode was first immersed in a GOx solution containing water as a polar solvent (concentration of 5 mg mL-1 in a PBS solution containing 0.5 M NaCl) for 20 minutes and then washed with distilled water (pH 5.8) to weakly adsorb GOx. has been removed. As a result, the enzyme and metal nanoparticles are bonded by physical adsorption of charged metal nanoparticles (NP) in water and a simple mixing method.
즉, 본 발명은 금속나노입자와 함께 결합할 수 있는 GOx의 대표적인 두개의 기능기인 아민기와 카르복실산 작용기 중 카르복실산 작용기의 결합은 거의 나타나지 않았고, Gox와 아민기간의 결합에 의하여 효소와 금속나노입자간 결합이 이루어진다. That is, in the present invention, the amine group, which is the representative two functional groups of GOx capable of bonding with metal nanoparticles, and the carboxylic acid functional group among the carboxylic acid functional groups were rarely bonded. Bonding between nanoparticles takes place.
그 다음, 톨루엔 용액에 상기 지지체를 침지하여 분산된 TOA-AuNPs를 GOx 층 위에 20분 동안 증착하고 톨루엔으로 세척하여 약하게 흡착된 TOA-AuNP를 제거하였다. 수성 용매에서 효소물질을 증착한 다음, 톨루엔과 같은 유기용매(비극성 용매)에서 전도성 입자를 흡착시키게 되면 TOA-AuNPs의 벌키한 리간드가 제거되면서 직접적으로 GOx와 흡착하게 된다. 이때, 아민과 같은 친수성 리간드 제거를 통하여 얻어지는 낮은 정전기적 반발력으로 인해 더 높은 GOx의 충전밀도를 확보할 수 있고, 더 나아가 GOx 층 주변에 만들어진 수화층의 효과로 인해 비극성 용매 내에서도 GOx의 활성도를 잃지 않는 상태에서 안정적으로 보호될 수 있다. Then, the support was immersed in a toluene solution, and the dispersed TOA-AuNPs were deposited on the GOx layer for 20 minutes and washed with toluene to remove weakly adsorbed TOA-AuNPs. After depositing the enzyme material in an aqueous solvent, when the conductive particles are adsorbed in an organic solvent (non-polar solvent) such as toluene, the bulky ligand of TOA-AuNPs is removed and GOx is directly adsorbed. At this time, a higher packing density of GOx can be secured due to the low electrostatic repulsive force obtained through the removal of hydrophilic ligands such as amine, and furthermore, the activity of GOx is not lost even in non-polar solvents due to the effect of the hydration layer formed around the GOx layer. can be reliably protected in the absence of
이와 같이 도 1에서 도시된 바와 같이 극성 용매에서 효소인 GOx를, 비극성 용매에서 전도성 나노입자를 반복하여 증착하여 수용액의 친수성 GOx 분자및 전도성 나노입자를 MC 지지체상에 안정적으로 흡착시켜 LbL 어셈블리 기반 효소 전극을 얻었다. As shown in FIG. 1, by repeatedly depositing GOx as an enzyme in a polar solvent and conductive nanoparticles in a non-polar solvent, the hydrophilic GOx molecules and conductive nanoparticles in an aqueous solution are stably adsorbed on the MC support, resulting in LbL assembly-based enzymes. electrode was obtained.
도 2는 본 발명의 일 실시예에 따른 전극 제조방법과 이에 따라 얻어진 사진이다. 2 is an electrode manufacturing method according to an embodiment of the present invention and a photograph obtained thereby.
도 2를 참조하면, GOx 및 TOA-AuNP가 순차적으로 적층된 효소전극을 확인할 수 있다. Referring to FIG. 2, it can be seen that the enzyme electrode in which GOx and TOA-AuNP are sequentially stacked.
실험예 Experimental example
먼저 소수성의 TOA-AuNP가 톨루엔 용매에서, 음이온인 GOx와 수성 용매에서 LbL을 형성하는 현상을 UV-vis 분광법 및 QCM 측정을 사용, 분석하였다. First, the phenomenon in which hydrophobic TOA-AuNPs form LbL in toluene solvent, anion GOx and aqueous solvent was analyzed using UV-vis spectroscopy and QCM measurement.
도 3은 도 1 및 2의 용매 조건에서 TOA-AuNP/GOx간 LbL 적층을 설명하는 도면이다. FIG. 3 is a diagram explaining LbL stacking between TOA-AuNP/GOx in the solvent conditions of FIGS. 1 and 2.
도 3을 참조하면, 반복횟수(p)가 1에서 10으로 증가함에 따라, (GOx/TOA-AuNP)p 다층의 UV-vis 흡광도가 선형으로 증가하는 것을 알 수 있으며, GOx에서 관찰된 일반적인 흡광도 피크(277 nm)를 보여주는 것을 알 수 있다.Referring to FIG. 3, as the repetition number (p) increases from 1 to 10, it can be seen that the UV-vis absorbance of the (GOx/TOA-AuNP)p multilayer increases linearly, and the general absorbance observed in GOx It can be seen that it shows a peak (277 nm).
또한 반복횟수에서 증가되는 층당 GOx 및 TOA-AuNP의 총 로딩량은 약 2.3 ± 0.3 μg cm-2로 측정되었으며, 전계 방출 주사 전자 현미경(FE-SEM)으로 측정한 (GOx/TOA-AuNP)p 다층의 총 막 두께는 p가 2에서 20으로 증가함에 따라 약 18에서 193 nm로 증가하였다. 또한 AFM(Atomic Force Microscopy) 측정의 경우 흡착된 각 TOA-AuNP 층과 각 GOx 층의 두께는 각각 약 5.5 ± 1.3 nm 및 3.3 ± 0.8 nm로 측정되었다. In addition, the total loading amount of GOx and TOA-AuNPs per layer, which increases with the number of iterations, was measured to be about 2.3 ± 0.3 μg cm -2 , and (GOx/TOA-AuNP)p measured by field emission scanning electron microscopy (FE-SEM) The total film thickness of the multilayer increased from about 18 to 193 nm as p increased from 2 to 20. In addition, in the case of AFM (Atomic Force Microscopy) measurement, the thickness of each adsorbed TOA-AuNP layer and each GOx layer was measured to be about 5.5 ± 1.3 nm and 3.3 ± 0.8 nm, respectively.
TOA-AuNPs의 직경이 약 6 nm임을 감안할 때, 이러한 결과는 TOA-AuNPs와 GOx가 기판에 조밀하게 패킹되었음을 의미하며, 더 나아가 친수성(음이온성) GOx가 양친매성 리간드 교환 반응을 통해 소수성 전도성 NP와 거의 완벽하게 나노 블렌딩되었음을 의미한다. Given that the diameter of the TOA-AuNPs is about 6 nm, these results imply that the TOA-AuNPs and GOx are densely packed on the substrate, and furthermore, the hydrophilic (anionic) GOx is converted into hydrophobic conductive NPs through an amphiphilic ligand exchange reaction. and almost perfectly nano-blended.
극도로 상이한 용매 극성을 갖는 비극성 및 극성 용매에서 수행되는 이 독특한 양친매성 어셈블리 공정은, AuNP에 결합된 TOA 리간드와 GOx의 아미노산 내의 아민(-NH2) 부분의 연속적인 리간드 교환 반응에 의해 이루어지는 것으로 예상된다. This unique amphiphilic assembly process, performed in non-polar and polar solvents with extremely different solvent polarities, is expected to be achieved by successive ligand exchange reactions between the TOA ligand bound to AuNPs and the amine (-NH2) moiety in the amino acid of GOx. do.
이를 위하여, GOx와 TOA-AuNP 사이의 흡착 거동을, GOx 대신 amine-functionalized organic linker와 carboxylic acid (COOH)-functionalized linker를 사용하여 분석하였다. 이때 톨루엔의 TOA-AuNP는 pH 7.4인 물에서 아민 작용기화된 PEI 및 DETA 및 카르복실산(COOH) 작용기화된 폴리(아크릴산)(PAA)으로 LbL 조립되는 점을 고려하였다. To this end, the adsorption behavior between GOx and TOA-AuNP was analyzed using an amine-functionalized organic linker and a carboxylic acid (COOH)-functionalized linker instead of GOx. At this time, it was considered that TOA-AuNPs in toluene are LbL assembled into amine-functionalized PEI and DETA and carboxylic acid (COOH)-functionalized poly(acrylic acid) (PAA) in water at pH 7.4.
보통 수용액에서 아민기의 pKa(관능기의 50%가 이온화되는 pH 값)가 약 9,39이며, 이때 PEI 및 DETA 내에서 전하를 띠지 않는 아민 부분의 비율은 약 15~25%이었다. 이때 TOA-AuNPs는 물에 용해된 PEI 또는 DETA와 상대적으로 높은 리간드 교환을 통하여 LbL-조립될 수 있지만, pH 7,4에서 형성되는 PAA의 카르복실레이트 이온(COO-) 그룹에 대한 친화도는 없었다. The pKa (pH value at which 50% of the functional groups are ionized) of the amine group in aqueous solution is about 9,39, and the ratio of the uncharged amine moiety in PEI and DETA is about 15-25%. At this time, TOA-AuNPs can be LbL-assembled through relatively high ligand exchange with PEI or DETA dissolved in water, but the affinity for the carboxylate ion (COO-) group of PAA formed at pH 7,4 is There was no
이러한 결과는 GOx/TOA-AuNP 다층의 축적이 GOx의 아민 부분과 AuNP의 표면 사이의 안정적인 공유 결합을 통한 연속 조립에 기반함을 시사한다. 이때, 금 나노입자의 벌키한 리간드가 제거되면서 AuNP의 표면과 GOx 사이에서 직접적인 조립이 이루어 지게 된다. 푸리에 변환 적외선 분광법 (FTIR) 분석에 따르면, 비극성 용매에서 TOA-AuNP에 존재하는 리간드 중 68%가 치환반응에 의해 GOx와의 연속 조립에 참여하였음을 확인하였다.These results suggest that the accumulation of GOx/TOA-AuNP multilayers is based on continuous assembly through stable covalent bonds between the amine moiety of GOx and the surface of AuNPs. At this time, as the bulky ligands of the gold nanoparticles are removed, direct assembly between the AuNP surface and GOx is achieved. According to Fourier Transform Infrared Spectroscopy (FTIR) analysis, it was confirmed that 68% of the ligands present in TOA-AuNPs in non-polar solvents participated in continuous assembly with GOx by substitution reaction.
도 4는 본 발명의 일 실시에에 따른 효소전극의 접촉각 분석결과이다. 4 is a contact angle analysis result of an enzyme electrode according to an embodiment of the present invention.
도 4를 참조하면, 친수성(또는 음이온성) GOx가 최외각층일 때 물 접촉각은 약 48±2°로 측정되었다. 그러나 TOA-AuNPs가 최외곽 GOx 층에 연속적으로 증착되면 물 접촉각이 62±1°로 증가하였으며, 이러한 물 접촉각은 최외곽층의 정체에 따라 주기적으로 변하는 것을 알 수 있다. Referring to FIG. 4 , when the hydrophilic (or anionic) GOx is the outermost layer, the water contact angle was measured to be about 48±2°. However, when TOA-AuNPs were continuously deposited on the outermost GOx layer, the water contact angle increased to 62±1°, and it was found that this water contact angle changed periodically according to the stagnation of the outermost layer.
도 5는 전도성 금속임자와 효소 종류를 달리하여, 도 1에서 도시된 방법에 따라 양친매성 LbL 어셈블리 방식으로 효소전극을 제조한 결과이다. 5 is a result of manufacturing an enzyme electrode by an amphiphilic LbL assembly method according to the method shown in FIG. 1 by using different types of conductive metal particles and enzymes.
도 5를 참조하면, (a)는 구리 나노입자(TOA-Cu NP), (b)는 백금 나노입자(Pt NP) 및 올레산(oleic acid), 그리고 (c)는 인듐(ITO) 및 올레아민(OAm)을 사용하는 경우에 대한 결과이며, 이를 참조하면, 반복횟수(p)가 증가함에 따라 흡광도는 도 3과 같이 증가함을 알 수 있다. 이것은 결국 본 발명에 따른 양친매성 효소전극은 다양한 효소(바이오 물질)과 전도성 나노입자에 적용될 수 있으며, 적어도 효소층은 극성용매에서, 그리고 전도성 나노입자는 비극성 용매에서 증착되는 한 이것은 모두 본 발명에 속한다Referring to FIG. 5, (a) is copper nanoparticles (TOA-Cu NP), (b) is platinum nanoparticles (Pt NP) and oleic acid, and (c) is indium (ITO) and oleamine This is the result for the case of using (OAm), and referring to this, it can be seen that the absorbance increases as the number of repetitions (p) increases, as shown in FIG. 3. After all, the amphiphilic enzyme electrode according to the present invention can be applied to various enzymes (biomaterials) and conductive nanoparticles, and at least as long as the enzyme layer is deposited in a polar solvent and the conductive nanoparticles are deposited in a non-polar solvent, all of these are in accordance with the present invention. belong
도 6은 다양한 바이오 물질과 금 나노입자를 도 1에서 도시된 방식에 따라 LbL로 증착시킨 결과이다. FIG. 6 is a result of depositing various biomaterials and gold nanoparticles by LbL according to the method shown in FIG. 1 .
도 6을 참조하면, (a)는 카탈라아제(CAT), (b)는 헤모글로민, (c)는 페라틴을 사용하였으며, 반복횟수(p)가 증가함에 따라 LbL 어셈블리 층수가 올라감에 따라 흡광도가 증가하는 것을 알 수 있다.Referring to FIG. 6, (a) catalase (CAT), (b) hemoglomin, and (c) feratin were used, and as the number of repetitions (p) increased, the number of LbL assembly layers increased. It can be seen that the absorbance increases.
도 7은 본 발명에 따른 효소전극을 활용한 글루코스 농도 및 임피던스 측정 결과이다. 7 is a result of glucose concentration and impedance measurement using the enzyme electrode according to the present invention.
도 7을 참조하면, 3번의 반복을 통하여 얻어진 LbL 효소전극은 상대전극인 Ag에 대하여 농도의존적 전기적 검출 결과를 얻는 것을 알 수 있다.Referring to FIG. 7 , it can be seen that the LbL enzyme electrode obtained through three iterations obtains a concentration-dependent electrical detection result for Ag as a counter electrode.
도 8은 산화수소 농도에 따른 카탈라아제 (CAT)의 촉매활성 정도 및 임피던스 측정 결과이다. 8 shows the degree of catalytic activity of catalase (CAT) and the impedance measurement results according to the concentration of hydrogen oxide.
도 8을 참조하면, GOx가 아닌 CAT를 사용하는 경우, 농도가 높아짐에 따라 검출되는 전류값이 강해지며, 임피던스도 농도의존적인 경향을 뚜렷이 보여준다. Referring to FIG. 8 , when using CAT instead of GOx, the detected current value becomes stronger as the concentration increases, and the impedance clearly shows a concentration-dependent tendency.
이상 설명한 바와 같이 본 발명에 따른 액체금속은 고분자막으로 둘러싸인 입자를 포함하며, 이 경우 용액 공정 조건의 조절을 통해 두께 조절이 가능하며, 높은 해상도를 유지함과 동시에 대면적 공정이 가능하다. 또한 고분자막의 안정성으로 인하여 외부 조건에 대한 안정성이 있으며, 용액 공정을 위해 제조한 액체 금속 잉크는 여러 종류의 기판에 박막 형성이 가능하며 다양한 물질에 패턴을 옮길 수 있다.As described above, the liquid metal according to the present invention includes particles surrounded by a polymer film, and in this case, the thickness can be controlled by adjusting the solution processing conditions, and a large-area process is possible while maintaining high resolution. In addition, there is stability against external conditions due to the stability of the polymer film, and the liquid metal ink prepared for the solution process can form thin films on various types of substrates and transfer patterns to various materials.
도 9는 전도성 입자의 용매 종류에 따른 전극 효율 특성을 비교한 결과이다. 9 is a result of comparing electrode efficiency characteristics according to the type of solvent of conductive particles.
도 9에서 GOx를 적층하는 전통적인 방법인 크로스 링커를 이용해 GOx를 결합한 전극 (Conventional GOx layer); 단분자 링커인 DETA를 물에 녹여 GOx를 적층한 후 얻은 전극((GOX/DETA)3/MCT); Citrate 이온에 의해 안정화된 금 나노입자 (C-AuNP) 및 DETA와 함께 정전기적 인력으로 적층한 전극 ((GOx/DETA/C-AuNP/MCT)4/MCT; 그리고 본 발명에 따른 전극((GOx/TOA-AuNPs)3)의 성능을 직접 비교하였다. 이 경우 본 발명에 따른 전극을 제외하고는 모두 물에서 금속 나노입자를 분산하여 적층하였다. An electrode combining GOx using a cross linker, which is a traditional method of stacking GOx in FIG. 9 (Conventional GOx layer); an electrode ((GOX/DETA)3/MCT) obtained by dissolving DETA, a unimolecular linker, in water and layering GOx; An electrode ((GOx/DETA/C-AuNP/MCT)4/MCT stacked by electrostatic attraction with gold nanoparticles (C-AuNP) stabilized by citrate ions and DETA; and an electrode according to the present invention ((GOx The performances of /TOA-AuNPs)3) were directly compared, except for the electrode according to the present invention, in which metal nanoparticles were dispersed in water and stacked.
도 9를 참조하면, 물 기반의 GOx와 비극성용매 기반의 TOA-AuNP가 적층 되었을 때 (즉, GOX/TOA-AuNP)3/MCT), 낮은 정전기적 반발력으로 인해 더 높은 GOx의 충전밀도를 확보할 수 있었고, 더 높은 산화전극의 전류밀도를 얻을 수 있음을 확인할 수 있다. Referring to FIG. 9, when water-based GOx and non-polar solvent-based TOA-AuNPs are laminated (i.e., GOX/TOA-AuNP)3/MCT), a higher packing density of GOx is secured due to low electrostatic repulsive force It can be confirmed that a higher current density of the anode can be obtained.
이상 설명한 바와 같이 본 발명은 물과 같은 극성 용매에서는 효소, 유기용매에서는 전도성 나노입자를 교차하여 증착시키는 양친화성 어셈블리리 방법을 전도성 지지체에 도입하여, 전자전달 능력이 향상된 면 직물형/섬유형 효소 전극을 제조할 수 있다. 또한, 본 발명을 통해 개발된 양친화성 어셈블리 방법은 물과 같은 극성용매 뿐만 아니라 유기용매 내에서도 사용된 효소의 안정성이 유지되며, 이로써 본 발명에 따른 효소 전극을 활용하는 바이오센서나 연료전지와 같은 응용제품은 안정성과 효율을 높게 유지할 수 있고, 도입되는 전도성 나노입자와 어셈블리를 통해 전극에 흡착되는 효소의 양을 절대적으로 증가시킬 수 있었으며, 이를 통해 효과적으로 전자전달을 일으킬 수 있는 효소층을 디자인함으로 인해, 바이오 센서 및 연료전지의 정확도 및 고효율을 구현할 수 있다.As described above, the present invention introduces an amphiphilic assembly method in which an enzyme in a polar solvent such as water and conductive nanoparticles are alternately deposited in an organic solvent to a conductive support, so that the cotton fabric/fiber enzyme has improved electron transfer ability. electrodes can be made. In addition, the amphiphilic assembly method developed through the present invention maintains the stability of the enzyme used in organic solvents as well as in polar solvents such as water, and thus applications such as biosensors or fuel cells using the enzyme electrode according to the present invention The product can maintain high stability and efficiency, and can absolutely increase the amount of enzyme adsorbed to the electrode through the conductive nanoparticles and assembly introduced. , accuracy and high efficiency of biosensors and fuel cells can be realized.

Claims (13)

  1. 지지체; support;
    상기 지지체에 결합된 효소층; Enzyme layer bound to the support;
    상기 효소층과 화학결합된 전도성 입자층을 포함하며, It includes a conductive particle layer chemically bonded with the enzyme layer,
    상기 효소층과 상기 전도성 입자층은 상기 효소의 기능기를 통하여 상기 전도성 입자와 직접 결합한 것을 특징으로 하는 효소전극.The enzyme electrode, characterized in that the enzyme layer and the conductive particle layer are directly bonded to the conductive particle through the functional group of the enzyme.
  2. 제 1항에 있어서, According to claim 1,
    상기 기능기는 아민기이며, 상기 전도성 입자층과 상기 효소층간 결합은 상기 전도성 입자 표면의 리간드를 유기용매에서 제거함으로써 이루어지는 것을 특징으로 하는 효소전극.The enzyme electrode, characterized in that the functional group is an amine group, and the binding between the conductive particle layer and the enzyme layer is achieved by removing a ligand on the surface of the conductive particle in an organic solvent.
  3. 제 1항에 있어서, 상기 효소전극은, The method of claim 1, wherein the enzyme electrode,
    상기 효소층과 상기 전도성 입자층이 교차하여 반복하는 적층구조인 것을 특징으로 하는 효소전극.Enzyme electrode, characterized in that the laminated structure in which the enzyme layer and the conductive particle layer are alternately repeated.
  4. 제 1항에 있어서, According to claim 1,
    상기 지지체는 섬유형 지지체인 것을 특징으로 하는 효소전극. The support is an enzyme electrode, characterized in that the fibrous support.
  5. 효소전극 제조방법으로, As an enzyme electrode manufacturing method,
    지지체를 효소를 포함하는 제 1 용액과 접촉시켜 지지체에 효소층을 형성하는 단계;contacting the support with a first solution containing an enzyme to form an enzyme layer on the support;
    상기 효소층이 형성된 지지체를 전도성 입자가 분산된 제 2 용액과 접촉시켜 상기 전도성 입자층을 상기 효소층에 결합시키는 단계를 포함하며, Contacting the support on which the enzyme layer is formed with a second solution in which conductive particles are dispersed to bind the conductive particle layer to the enzyme layer,
    상기 제 1 용액의 용매는 극성 용매이며, 상기 제 2 용액은 유기 용매인 것을 특징으로 하는 효소전극 제조방법Enzyme electrode manufacturing method, characterized in that the solvent of the first solution is a polar solvent, and the second solution is an organic solvent
  6. 제 5항에 있어서, According to claim 5,
    상기 효소는 글로코스 옥시데이즈(Gox), 카탈라아제(CAT, 헤모글로민 및 페라틴으로 이루어진 군으로부터 선택된 적어도 어느 하나를 포함하며, 상기 전도성 입자는 금, 구리, 백금 및 인듐주석산화물(ITO)로 이루어진 군으로부터 선택된 적어도 어느 하나를 포함하는 것을 특징으로 하는 효소전극 제조방법.The enzyme includes at least one selected from the group consisting of glucose oxidase (Gox), catalase (CAT, hemoglomin, and feratin, and the conductive particles are gold, copper, platinum, and indium tin oxide (ITO) Enzyme electrode manufacturing method comprising at least one selected from the group consisting of.
  7. 제 5항에 있어서, According to claim 5,
    상기 전도성 입자 표면에는 친수성 리간드가 결합되며, 상기 유기용매에서 상기 전도성 입자의 친수성 리간드와 상기 효소의 기능기간 치환 반응에 의하여 상기 전도성 입자와 상기 효소간 공유결합이 형성되는 것을 특징으로 하는 효소전극 제조방법.A hydrophilic ligand is bound to the surface of the conductive particle, and a covalent bond between the conductive particle and the enzyme is formed by a substitution reaction between the hydrophilic ligand of the conductive particle and the functional period of the enzyme in the organic solvent. method.
  8. 제 7항에 있어서, According to claim 7,
    상기 전도성 입자의 친수성 리간드는 상기 유기용매에서 제거되며, 이로써 상기 전도성 입자간 정전전기적 반발력은 감소되는 것을 특징으로 하는 효소전극 제조방법.The method of manufacturing an enzyme electrode, characterized in that the hydrophilic ligand of the conductive particles is removed from the organic solvent, thereby reducing the electrostatic repulsive force between the conductive particles.
  9. 제 5항에 있어서, According to claim 5,
    상기 제 1 용액은 물, 상기 제 2 용액은 톨루엔을 포함하는 것을 특징으로 하는 효소전극 제조방법. The enzyme electrode manufacturing method, characterized in that the first solution contains water, the second solution contains toluene.
  10. 제 5항에 있어서, According to claim 5,
    상기 지지체는 섬유형태인 것을 특징으로 하는 효소전극 제조방법.The enzyme electrode manufacturing method, characterized in that the support is in the form of a fiber.
  11. 제 5항 내지 제 10항 중 어느 한 항에 따른 방법에 의하여 제조된 효소전극. An enzyme electrode manufactured by the method according to any one of claims 5 to 10.
  12. 제 11항에 따른 효소전극을 포함하는 바이오 연료전지. A biofuel cell comprising the enzyme electrode according to claim 11.
  13. 제 11항에 따른 효소전극을 포함하는 바이오 센서. A biosensor comprising the enzyme electrode according to claim 11.
PCT/KR2022/012615 2021-08-27 2022-08-24 Enzyme-based electrode, manufacturing method therefor and application thereof WO2023027491A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2021-0113844 2021-08-27
KR1020210113844A KR102661260B1 (en) 2021-08-27 2021-08-27 Enzyme based electrode with facile electron transfer, manufacturing, and the application thereof

Publications (1)

Publication Number Publication Date
WO2023027491A1 true WO2023027491A1 (en) 2023-03-02

Family

ID=85321902

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/012615 WO2023027491A1 (en) 2021-08-27 2022-08-24 Enzyme-based electrode, manufacturing method therefor and application thereof

Country Status (2)

Country Link
KR (1) KR102661260B1 (en)
WO (1) WO2023027491A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR0128159B1 (en) * 1988-07-28 1998-04-01 캠브리지 라이프 사이언시즈 피엘시 Enzyne electrondes and improvements in the manufacturing thereof
KR20140097896A (en) * 2013-01-30 2014-08-07 고려대학교 산학협력단 Multifunctional material with reversible phase transfer via layer-by-layer assembly and method for preparing the same
KR20170105929A (en) * 2016-03-11 2017-09-20 고려대학교 산학협력단 Method of preparing nanocomposite film using amphiphilic layer-by-layer assembly and nanocomposite film prepared thereby

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR0123078B1 (en) * 1993-07-21 1997-11-17 배순훈 Circuit for decreasing noise signal when a television weak signal
US9416390B2 (en) * 2012-07-27 2016-08-16 Ohmx Corporation Electric measurement of monolayers following pro-cleave detection of presence and activity of enzymes and other target analytes
KR101670581B1 (en) * 2015-03-09 2016-10-28 한양대학교 산학협력단 A fiber shaped mediatorless enzymatic biofuel cell
KR101877681B1 (en) * 2017-07-14 2018-07-13 고려대학교 산학협력단 Flexible electrode, biofuel cell and its preparing method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR0128159B1 (en) * 1988-07-28 1998-04-01 캠브리지 라이프 사이언시즈 피엘시 Enzyne electrondes and improvements in the manufacturing thereof
KR20140097896A (en) * 2013-01-30 2014-08-07 고려대학교 산학협력단 Multifunctional material with reversible phase transfer via layer-by-layer assembly and method for preparing the same
KR20170105929A (en) * 2016-03-11 2017-09-20 고려대학교 산학협력단 Method of preparing nanocomposite film using amphiphilic layer-by-layer assembly and nanocomposite film prepared thereby

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAVIES, BETHAN: "Amphiphilic Assembly-Based Electrode for High-Performance Hybrid Biofuel Cells", AZO MATERIALS, AZONETWORK, UK, UK, pages 1 - 4, XP009544002, Retrieved from the Internet <URL:https://www.azom.com/news.aspx?newsID=59161> *
KWON CHEONG HOON, KO YONGMIN, SHIN DONGYEEB, KWON MINSEONG, PARK JINHO, BAE WAN KI, LEE SEUNG WOO, CHO JINHAN: "High-power hybrid biofuel cells using layer-by-layer assembled glucose oxidase-coated metallic cotton fibers", NATURE COMMUNICATIONS, vol. 9, no. 1, XP093039962, DOI: 10.1038/s41467-018-06994-5 *
PARK MINKYUNG, KIM YOUNGHOON, KO YONGMIN, CHEONG SANGHYUK, RYU SOOK WON, CHO JINHAN: "Amphiphilic Layer-by-Layer Assembly Overcoming Solvent Polarity between Aqueous and Nonpolar Media", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 136, no. 49, 10 December 2014 (2014-12-10), pages 17213 - 17223, XP093039965, ISSN: 0002-7863, DOI: 10.1021/ja509168g *

Also Published As

Publication number Publication date
KR102661260B1 (en) 2024-04-29
KR20230031516A (en) 2023-03-07

Similar Documents

Publication Publication Date Title
Zamani et al. Current trends in the development of conducting polymers-based biosensors
Gan et al. Electrochemical sensors based on graphene materials
Zhang et al. Design and synthesis of multifunctional materials based on an ionic‐liquid backbone
Zuo et al. Graphene oxide-facilitated electron transfer of metalloproteins at electrode surfaces
Mao et al. Fabrication of electrochemical sensor for paracetamol based on multi-walled carbon nanotubes and chitosan–copper complex by self-assembly technique
Xi et al. Non-enzymatic detection of hydrogen peroxide using a functionalized three-dimensional graphene electrode
US5208154A (en) Reversibly immobilized biological materials in monolayer films on electrodes
Zhang et al. Layer-by-layer assembly for immobilizing enzymes in enzymatic biofuel cells
Xu et al. Biopolymer and carbon nanotubes interface prepared by self-assembly for studying the electrochemistry of microperoxidase-11
Ratlam et al. Development of dopamine biosensor based on polyaniline/carbon quantum dots composite
Li et al. Controlled electrophoretic patterning of polyaniline from a colloidal suspension
CN108585544B (en) Method for surface modification of substrate material based on carbon nanotube assembly composite material
Zhang et al. Electrochemical preparation of surface molecularly imprinted poly (3-aminophenylboronic acid)/MWCNTs nanocomposite for sensitive sensing of epinephrine
WO2013165101A1 (en) Hybrid electrode using silver nanowires and graphene, and preparation method thereof
Kwon et al. Highly conductive electrocatalytic gold nanoparticle-assembled carbon fiber electrode for high-performance glucose-based biofuel cells
Sayre et al. Electrooxidative deposition of polypyrrole and polyaniline on self-assembled monolayer modified electrodes
Mittal Surface modification of nanotube fillers
Gao et al. Electro-oxidative polymerization of phenothiazine dyes into a multilayer-containing carbon nanotube on a glassy carbon electrode for the sensitive and low-potential detection of NADH
Luo et al. Layer-by-layer assembled ionic-liquid functionalized graphene–polyaniline nanocomposite with enhanced electrochemical sensing properties
Wang et al. Direct electrochemistry and electrocatalysis of hemoglobin incorporated in composite film based on diblock weak polyelectrolyte PHAEMA-b-PDMAEMA and multi-walled carbon nanotubes
Li et al. Amperometric phenol biosensor based on a new immobilization matrix: Polypyrrole nanotubes derived from methyl orange as dopant
Weliwatte et al. Unbranched hybrid conducting redox polymers for intact chloroplast-based photobioelectrocatalysis
WO2023027491A1 (en) Enzyme-based electrode, manufacturing method therefor and application thereof
Garrett et al. Robust forests of vertically aligned carbon nanotubes chemically assembled on carbon substrates
Deng et al. Molecular “wiring” glucose oxidase in supramolecular architecture

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22861700

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE