WO2015147594A1 - Electrochemical biosensor using dual electrode pair - Google Patents

Electrochemical biosensor using dual electrode pair Download PDF

Info

Publication number
WO2015147594A1
WO2015147594A1 PCT/KR2015/003046 KR2015003046W WO2015147594A1 WO 2015147594 A1 WO2015147594 A1 WO 2015147594A1 KR 2015003046 W KR2015003046 W KR 2015003046W WO 2015147594 A1 WO2015147594 A1 WO 2015147594A1
Authority
WO
WIPO (PCT)
Prior art keywords
electrode
electrochemical sensor
clause
glucose
enzyme
Prior art date
Application number
PCT/KR2015/003046
Other languages
French (fr)
Korean (ko)
Inventor
신흥주
임영진
이윤정
샤마 딥티
Original Assignee
에스케이이노베이션 주식회사
국립대학법인 울산과학기술대학교 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
Priority claimed from KR1020150042538A external-priority patent/KR102347669B1/en
Application filed by 에스케이이노베이션 주식회사, 국립대학법인 울산과학기술대학교 산학협력단 filed Critical 에스케이이노베이션 주식회사
Priority to US15/300,192 priority Critical patent/US20170166946A1/en
Publication of WO2015147594A1 publication Critical patent/WO2015147594A1/en
Priority to US16/258,326 priority patent/US11060125B2/en

Links

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
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • 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

Definitions

  • the present invention relates to an electrochemical sensor, and more particularly, to an electrochemical biosensor using a sensor system comprising an active electrode including an active surface whose surface is modified through a linker, and an auxiliary electrode.
  • Glucose is a very common fuel in biology and is used as an energy source in most living things, from microorganisms to humans.
  • the concentration of glucose in human blood is comprised between 4.4 and 6.6 mM. Therefore, it is necessary to develop a method for continuous quantitative detection of glucose in blood, urine and other body fluids. Since abnormal improvement in glucose levels leads to diabetes, detection of glucose is a critical factor.
  • the research can be applied for sensing on all kinds of carbon surfaces (free carbon, flax, surface printed electrode carbon nanotube diamonds) as well as metals, silicon and indium tin oxide.
  • Electrochemical reduction of diazonium salts has been used successfully to form various functional groups on carbon electrodes for biomolecule immobilization.
  • adsorption of diazonium salts to nanoscale electrodes remains a challenge.
  • Most recent studies on glucose sensors have been published in the Republic of Korea Publication 2000-
  • Glucose dehydrogenase such as glucose oxidase or Korean Laid-Open Publication No. 2010-0131495 and Korean Laid-Open Publication No. 2005-0019139, such as 0008880, which promotes oxidation of glucose to gluconolactone. It is based on fixing the same enzymes. In most cases, enzyme-based sensors require charge carriers to improve the sensitivity and selectivity of the sensor. However, the current measurement electrode has a relatively low output current and sensitivity, and there is a problem that the reaction time is delayed.
  • An object of the present invention is to provide a glucose sensor having a high output current density, extremely smooth charge transfer, high specific surface area, and excellent nanoelectrode.
  • the present invention relates to an electrochemical sensor formed on the substrate and including a working electrode to which an enzyme is attached through a linker.
  • the electrochemical sensor may lead to a matrix in which P working electrodes (natural numbers of P ⁇ l or more) are arranged longitudinally and / or laterally.
  • FIG. 1 shows a schematic describing glucose sensitivity using a single electrode coated with glucose oxidase.
  • glucose and oxygen labeled '0' in the sample solution react in the presence of glucose oxidase to produce gluconolactone and hydrogen peroxide labeled 'Red'.
  • the concentration of lucose can be measured.
  • the current level can be increased.
  • the current level obtained using this strategy is very low due to a decrease in surface response due to the area of the enzyme coating and the limited electrode area. Therefore, it is necessary to study the geometry of the electrode to obtain high current levels.
  • the electrode may be manufactured using various kinds of conductive materials such as carbon, gold, platinum, palladium, conductive polymers, and the like.
  • the IDA nanoelectrode consists of two comb-shaped electrodes. As shown in FIG. 2, any electrochemically reversible redox species recycles redox reactions between the electrodes, thereby increasing the current signal according to the number of recycling. Current signal amplification by redox eye relies on mass transfer of redox species between electrodes. In particular, mass transfer through diff fusion in micro / nano electrodes can be improved by reducing the gap between the electrodes and increasing the aspect ratio of the electrodes.
  • Redox mediators play an important role in the detection of various molecules using electrochemical sensors.
  • the mediator deposits electrons between the enzyme and the electrode through an electrochemical-enzyme redox cycle.
  • oxygen reacts very quickly with the reduced form of GOx to produce hydrogen peroxide as a byproduct of the reaction.
  • oxygen is ruthenium, hexamine com lex, ferr i cyanide, ferrocenemethanol and ferrocenemono ⁇ 1 "carboxylic acid (6 ] "0: 611 ⁇ 21110110031 * 130 1 ⁇ ; acid), etc. It can be replaced with various types of redox mediators.
  • the electron mediators have a rapid electron-transfer reaction on the upper surface of the electrode and a rapid electron transfer reaction with redox enzymes. For example, redox reactions using ferricyanide can occur as follows.
  • the linker is not limited, but may be attached on the nanoparticles formed directly on the electrode.
  • the nanoparticles are not limited but may be selected from gold, platinum or palladium.
  • Another aspect of the present invention relates to an electrochemical sensor, wherein the sensor further comprises an auxiliary electrode formed to be spaced apart in the horizontal direction of the working electrode and the substrate.
  • the electrochemical sensor of the present invention using one pair of the working electrode and one auxiliary electrode as a unit may include M (M> 1 natural number) XN (N> 1 natural number) units.
  • M units may be spaced apart in the longitudinal direction, and N units may be spaced apart in the lateral direction to form an M ⁇ N matrix.
  • glucose oxidase is one of the IDA electrodes.
  • the enzyme can be selectively immobilized on a specific electrode by reducing the aryl diazonium salt.
  • oxygen in the glucose sensor can be recycled between the enzyme and electrode, ruthenium, hexamine com lex, ferr i cyanide and ferrocenemethanol. It can be replaced with various forms of redox mediators such as ferrocenemethanol and ferrocenemonocarboxyl ic acid.
  • the redox species have the advantage of being able to react electrochemically at the electrode surface without hydrolysis, which limits the stable glucose sensing when oxygen is used as the redox species.
  • the analyte-enzyme is not limited to:
  • diazonium salt binding can provide various functional groups on the electrode surface such as NH 2 and -C00H, which enable the binding of various biomolecules.
  • selective combination of biomolecules on diazonium-based IDA nanoelectrodes may contribute to the detection of various types of molecules as follows.
  • the working electrode is formed spaced apart in the vertical direction from the substrate, and relates to an electrochemical sensor having a mesh (mesh) shape.
  • the sensor is not limited, but may further include an auxiliary electrode formed spaced apart from the working electrode under the working electrode.
  • the electrochemical sensor manufactured in the form of the mesh-shaped electrode compared to the case of using the IDA nano-electrode, it can exhibit a good efficiency in the redox recycling (redox recycling).
  • the linker is formed by converting a functional group of a surface modification base material containing diazonium, a diazonium salt or a derivative thereof into an amine group, and then mixing a dialdehyde crosslinking reagent, and forming a biomaterial on the electrode surface. Can be attached.
  • the present invention is formed by converting a functional group of a surface modification base material containing diazonium, a diazonium salt or a derivative thereof into an amine group, and then mixing a dialdehyde crosslinking reagent, and forming a biomaterial on the electrode surface. Can be attached.
  • the present invention is formed by converting a functional group of a surface modification base material containing diazonium, a diazonium salt or a derivative thereof into an amine group, and then mixing a dialdehyde crosslinking reagent, and forming a biomaterial on the electrode surface. Can be attached.
  • the present invention is formed by converting a functional group of a surface modification
  • the present invention relates to a method for manufacturing an electrochemical sensor, including bonding.
  • the surface modification base material is not limited to diazonium.
  • diazonium diazonium salts or derivatives thereof may be used, and more preferably, 4-nitrophenyl diazonium tetrafluoroborate (4-NP) may be used as the surface modification base material.
  • 4-NP 4-nitrophenyl diazonium tetrafluoroborate
  • the cross-linking reagent is not limited to a dialdehyde-based compound, but examples thereof include glyoxal, makmdi aklehyde, succinic aldehyde, and glue.
  • Tar aldehyde (glutaraldehyde) and phthalaldehyde (phthalaldehyde) and the like can be selected and used, preferably glutaraldehyde (glutaraldehyde) can be used.
  • Another aspect of the invention further includes forming nanoparticles on the electrode, wherein the surface modification base material relates to a method of manufacturing an electrochemical sensor formed directly on the nanoparticles.
  • the nanoparticles are not limited, and preferably, gold, platinum, or palladium may be used.
  • electrochemically improved characteristics may be obtained by applying various nanoparticles to the electrode surface. This improves the surface area and reaction properties of the electrode, resulting in an improvement in the electrochemical signal.
  • the electrode is not limited, but an IDA interdigitated array (nano) electrode may be used.
  • the distance between the working electrode and the auxiliary electrode is not limited but may be in the range of lOnm to ⁇ .
  • the redox reaction can occur more smoothly, thereby providing a high level of current.
  • the sensor of the present invention has a higher current value than the conventional sensor, and is expected to be easily used for the sensing of various kinds of biomaterials due to its excellent stability and sensitivity.
  • FIG. 1 is a schematic diagram of glucose sensing using a single electrode functionalized by glucose oxidase.
  • FIG. 2 is a schematic diagram of an oxidation / reduction cycle in (a) a thin band IDA electrode and (b) a high aspect ratio IDA electrode.
  • FIG. 3 is a schematic of glucose sensing using IDA electrodes selectively functionalized by glucose oxidase.
  • FIG. 5 is a schematic diagram for selective fixation of biomolecules using diazonium salts.
  • FIG. 6 is a schematic process chart of selective electrode functionalization.
  • Figure 7 shows 1 mM 4-nitrophenyl diazonium tetrafluoroborate (4-NP) and 0.1 M tetra butylammonium tetrafluor at a scan rate of 200 mV / sec. Robo rate, I is the circulating current voltage curve of acetonitrile (tetrabutyl ammonium tetraf luoroborate I acetoni tr i le) a carbon nano-IDA electrode (comb 1) in the solution.
  • acetonitrile tetrabutyl ammonium tetraf luoroborate I acetoni tr i le
  • FIG. 9 is a graph illustrating oxidation current values collected from IDA electrodes according to glucose concentration using carbon IDA nanoelectrodes.
  • FIG. 10 is a schematic diagram of immobilizing biomolecules on IDA nanoelectrodes modified with metal nanoparticles.
  • A Both electrodes are coated with nanoparticles,
  • B Only one electrode (comb) is coated with these nanoparticles.
  • FIG. 11 is an SEM image of (a, b) a polymer set laminated before pyrolysis and (c, d) an SEM image of the laminated carbon electrode set after pyrolysis.
  • FIG. 12 is a schematic diagram of glucose sensitivity using a stacked electrode set selectively modified with an enzyme.
  • Carbon IDA nanoelectrodes were fabricated on 6 inch (100) silicon wafers.
  • a 700 nm thick silicon dioxide (SiC> 2) layer was deposited on a silicon wafer by thermal oxidation.
  • Photolithography was used to pattern the SU-8 negative photoresist IDA structure.
  • a predefined photoresist IDA pattern was pyrolyzed at 900 ° C. under vacuum. During pyrolysis, the size of the IDA structure decreased by 60% in width and 90% in height.
  • the carbon electrode was passivated except for the interdigitated electrode region.
  • the immobilization of the enzyme may be composed of three steps. 4—Nitrophenyl diazonium tetrafluoroborate (4-NP) was used as the base. To link the enzyme to diazonium, the functional group of the diazonium was converted to an amine group and glutaraldehyde was used between the amine group and the enzyme of the diazonium.
  • 4—Nitrophenyl diazonium tetrafluoroborate (4-NP) was used as the base.
  • the functional group of the diazonium was converted to an amine group and glutaraldehyde was used between the amine group and the enzyme of the diazonium.
  • the electrochemical modification of the carbon electrode was performed at 0.5 to -0.7 V in acetonitrile containing 1 mM 4-NP in 0.1 M NBu 4 BF 4 at a scanning rate of 200 mV / s for the reference electrode Ag / AgCl. It measured by scanning the electron potential of an electrode until. Prior to modification, the solvent was removed impurities for 30 minutes using argon gas. After the modifications were made, the electrodes were washed with DI water for 30 minutes. Reduction with nitro groupol amine groups
  • a proton solvent containing 0.1 M potassium chloride and water / ethanol (90: 10 v / v) was used to generate the amine group through reduction of the nitro group.
  • the potential of the electrode was measured by scanning the electron potential of the electrode from 0 to 0.8 V at a scanning speed of 100 mV / s relative to the reference electrode Ag / AgCl.
  • Glutaraldehyde is a bifunctional crosslinking reagent for enzyme immobilization via a linker.
  • the electrode was then washed with deionized water and dried using nitrogen gas.
  • the electrode was combined with 0.1% digested cyanoborohydride in 50 mM PBS buffer (pH 7.4). Incubated overnight in 4 ° C buffer solution containing mg / mL glucose oxidase.
  • Electrodes range from 0 to 0.6 V at a scanning rate of 50 mV / s for the reference electrode Ag / AgCl at 0.5 M potassium chloride and 10 mM [Fe (CN) 6 ] 4_ in deionized water . Scanning electron potentials were characterized using cyclic voltammetry. Glucose Sensing is 05 ⁇
  • Glucose solution was prepared at a concentration of 0.1 M in 100 mM PBS and anomeric
  • Carbon IDA nano-electrode was prepared by the procedure of Preparation Example 1. Electrode surface using linker and enzyme for carbon IDA nanoelectrode as shown in FIG. 3 was functionalized. One comb of the carbon IDA nanoelectrode was functionalized with glucose oxidase (GOx), while the other adjacent comb was used to collect the oxidation current of the ferrocyanide redox species.
  • GOx glucose oxidase
  • FIG. 7 shows a cyclic voltagram in a comb of 1 mM 4-NP and 0.1 M NBu 4 BF 4 / acetonitrile solution.
  • the irreversible reduction curve at the first cycle -0.05 V contributed to the formation of the 4-nitrophenyl radical from the diazonium salt derivative. Since the first irreversible curve disappeared in the second scan, it meant that the nitrophenyl group bonded to the carbon surface interfered with the electron transfer. From the last reversible curve, it shows that the radical anions formed by the cleavage were reduced to aryl anions at the surface of the carbon electrode.
  • the amine group that activated the carbon electrode was incubated with glucose oxidase using 2.5% glutaraldehyde. Finally, one side of the carbon IDA nanoelectrode was functionalized with glucose oxidase using the Preparation Example 2 method, and adjacent electrodes were used to collect the oxidation current of the ferrocyanide.
  • Glucose sensing was performed in the presence of ferri cyanide redox mediators with fast electron kinetics and stable oxidation / reduction forms.
  • the glucose molecule was oxidized by the flavin-adenine dinucleotide (FAD) redox core element of the glucose oxidase enzyme.
  • FAD flavin-adenine dinucleotide
  • FAD3 ⁇ 4 G0x red
  • oxygen in the reaction can be replaced with ferricyanide as a substitute electron acceptor.
  • FADH 2 was oxidized back to FAD while being reduced to [Fe (CN) 6 ] 3 [Fe (CN) 6 ;
  • the reaction occurred at a carbon electrode (comb 1) functionalized with glucose oxidase
  • [Fe (CN) 6 ] is the electrode comb 1 and the adjacent carbon electrode as shown in FIG.
  • nanoparticles such as (pal ladium)
  • a biosensor comprising a glucose oxidase connected to a linker is connected to an electrode through the nanoparticles.
  • a stacked electrode set including a suspended carbon mesh and a substrate-bound planar electrode is similar to the method described in Preparation Examples 1 and 2. It was made in a configuration that replaced the IDA electrode for use as a biosensor platform. The redox mechanism and manufacturing process for biosensing used the method described in Preparation Examples 1 to 3.
  • the electrode was modified by attaching and modifying a substrate-boundary planar electrode in the same manner as in Preparation Example 2 for the enzyme immobilization. Produced.
  • the redox couple was recycled between the enzyme and the electrode surface, and glucose concentration was measured by measuring the oxidation current at the electrode surface. Measured.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Emergency Medicine (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Electrochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to an electrochemical sensor and, more specifically, to an electrochemical biosensor using a sensing system comprising a working electrode including an active surface modified through a linker; and an auxiliary electrode. The sensor of the present invention has a high current value compared with an existing sensor and retains excellent stability and sensitivity, and thus can be expected to be easily used for sensing various kinds of biomaterials.

Description

【명세서】  【Specification】
[발명의 명칭】  [Name of invention]
이중 전극쌍을 이용한 전기화학 바이오 센서  Electrochemical Biosensor Using Double Electrode Pair
【기술분야】  Technical Field
<1> 본 발명은 전기화학 센서에 관한 것으로， 상세하게는 링커를 통하여 표면이 개질된 활성 표면을 포함하는 작용 전극과， 보조 전극으로 이루어진 센성 시스템을 이용한 전기화학 바이오 센서에 관한 것이다.  The present invention relates to an electrochemical sensor, and more particularly, to an electrochemical biosensor using a sensor system comprising an active electrode including an active surface whose surface is modified through a linker, and an auxiliary electrode.
[배경기술]  [Background]
<2> 생명과학에서 나노의학에 대한 목표 중 하나는 다양한 생물학적 공정을 실험 실 내에서 연구할 수 있으며， 나노물질들이 호환가능하다는 점이다. 대부분의 생물 화학 공정들이 생체 내에서의 전자의 이동을 수반하기 때문에 전기화학 분야의 관 심의 대상이 되고 있다. 상기 분야에서 나노물질과 함께 효소 또는 다른 생체 물질 을 결합시킨 새로운 형태의 전기화학 바이오센서의 개발의 중요성이 부각되고 있 다. 글루코스는 생물학에서 아주 흔한 연료로 미생물로부터 인간까지 대부분의 생 물체에서 에너지 원료로서 사용되고 있다. 인간의 혈액에서 글루코스의 농도는 4.4 내지 6.6 mM로 구성된다. 따라서， 혈액, 소변 및 다른 체액에서 글루코스의 연속적 인 정량 검출 방법을 개발하는 것이 필요하다. 비정상적인 글루코스 레밸의 향상이 당뇨병을 초래하기 때문에， 글루코스의 검출은 증요한 요소이다. 현재까지， 글루코 스 센싱을 위하여 폴리아닐린 (polyani l ine) , 폴리파이를 (polypyrrole) 또는 폴리인 돌 (polyindole)과 같은 다양한 전도성 고분자가 사용되었다. 그러나， 이와 같은 방 법은 많은.시간을 필요로 하고 제조 단가를 상승시키는 복잡한 화학 합성을 수반한 다. 이와 함께， 자가조립단층 (sel f -assembled monolayer , SAM)은 글루코스 옥시다 제 (glucose oxidase)의 공유결합을 위해 널리 사용된다. 그러나， 글루코스 센서의 민감도는 일반적으로 SAMs의 낮은 안정성과 제한된 전위창 (potent i al window)의 절 층에 의한 것이다. 따라서， 이러한 SAMs의 제약을 극복하기 위해서， 탄소 전극에서 아릴 디아조니움 (aryl di azonium) 염의 환원적 흡착법 연구가 이루어졌다. 상기 연 ' 구는 모든 종류의 탄소 표면 (유리 탄소, 혹연， 표면 인쇄 전극 탄소 나노튜브 다이 아몬드) 뿐만 아니라 금속， 실리콘 및 인디움 틴 옥사이드 ( indiom t in oxi de)에서 센싱하기 위해 적용될 수 있다. 디아조니움 염의 전기화학적 환원은 생체분자 고정 화를 위해 탄소 전극에서 다양한 기능성 그룹을 형성하기 위해 성공적으로 사용되 어 왔다. 그러나， 나노크기의 전극에 디아조니움 염을 흡착시키는 것은 여전히 과 제로 남아 있다. <3> 글루코스 센서에 대한 대부분의 최근 연구들은 대한민국 공개공보 제 2000-<2> One of the goals for nanomedicine in life science is that various biological processes can be studied in the laboratory, and the nanomaterials are compatible. Most biochemical processes are of interest in the field of electrochemistry because they involve the movement of electrons in vivo. In this field, the importance of the development of new types of electrochemical biosensors combining enzymes or other biological materials with nanomaterials is highlighted. Glucose is a very common fuel in biology and is used as an energy source in most living things, from microorganisms to humans. The concentration of glucose in human blood is comprised between 4.4 and 6.6 mM. Therefore, it is necessary to develop a method for continuous quantitative detection of glucose in blood, urine and other body fluids. Since abnormal improvement in glucose levels leads to diabetes, detection of glucose is a critical factor. To date, various conductive polymers have been used for glucose sensing, such as polyaniline, polypyrrole or polyindole. However, this method involves complex chemical synthesis that requires a lot of time and increases manufacturing costs. In addition, self-assembled monolayers (SAMs) are widely used for covalent binding of glucose oxidase. However, the sensitivity of the glucose sensor is generally due to the low stability of the SAMs and the lamination of the limited potential window. Therefore, in order to overcome these limitations of SAMs, research on the reductive adsorption of aryl di azonium salts on carbon electrodes has been conducted. The research can be applied for sensing on all kinds of carbon surfaces (free carbon, flax, surface printed electrode carbon nanotube diamonds) as well as metals, silicon and indium tin oxide. Electrochemical reduction of diazonium salts has been used successfully to form various functional groups on carbon electrodes for biomolecule immobilization. However, adsorption of diazonium salts to nanoscale electrodes remains a challenge. <3> Most recent studies on glucose sensors have been published in the Republic of Korea Publication 2000-
0008880호와 같이， 글루코스의 글루코노락톤 (gluconolactone)으로의 산화를 촉진 하는 글루코스 산화효소 (glucose oxidase) 또는 대한민국 공개공보 제 2010-0131495 호 및 대한민국 공개공보 제 2005-0019139호와 같이 글루코스 탈수소효소와 같은 효 소의 고정에 기반을 두고 있다. 대부분의 경우 효소를 기반으로 한 센서는 센서의 감도와 선택도를 향상시키기 위해 전하전달자를 필요로 한다. 그러나, 전류측정 효 소 전극은 상대적으로 낮은 출력 전류 및 감웅도를 가지며， 반응시간이 지연되는 문제점이 있다. Glucose dehydrogenase, such as glucose oxidase or Korean Laid-Open Publication No. 2010-0131495 and Korean Laid-Open Publication No. 2005-0019139, such as 0008880, which promotes oxidation of glucose to gluconolactone. It is based on fixing the same enzymes. In most cases, enzyme-based sensors require charge carriers to improve the sensitivity and selectivity of the sensor. However, the current measurement electrode has a relatively low output current and sensitivity, and there is a problem that the reaction time is delayed.
<4> 특히, 당뇨환자의 혈액속의 혈당물질인 글루코스 측정 시에는 혈액속의 여러 가지 간섭물질인 이온 끌림 현상을 극 저전압 (0.055V이하)의 인가에 의해 저지시키 면서도 빠른 효소산화반웅을 유도할 수 있어야 함에 따라， 저전압 인가시의 높은 전류 밀도 및 우수한 감웅도를 갖는 글루코스 센서의 개발이 절실히 요구되고 있 다.  In particular, when measuring glucose, a blood glucose substance in diabetic patients, it is possible to induce rapid reaction of enzyme oxidation while preventing ion attraction phenomenon, which is various interference substances in blood, by applying a very low voltage (below 0.055V). As such, there is an urgent need for the development of a glucose sensor having a high current density and excellent sensitivity when low voltage is applied.
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
<5> 본 발명의 목적은 높은 출력 전류밀도를 가지며， 전하의 이동이 극히 원활하 고， 높은 비표면적을 가지며， 감웅도가 우수한 나노전극이 구비된 글루코스 센서를 제공하는 것이다.  An object of the present invention is to provide a glucose sensor having a high output current density, extremely smooth charge transfer, high specific surface area, and excellent nanoelectrode.
【기술적 해결방법】  Technical Solution
<6> 본 발명은  <6> The present invention
<7> 유체 중의 분석물의 존재 또는 농도를 결정하기 위한 전기화학 센서로서， <7> An electrochemical sensor for determining the presence or concentration of an analyte in a fluid,
<8> 기판; 및 <8> substrate; And
<9> 상기 기판 상에 형성되며， 링커 ( l inker )를 통하여 효소가 부착된 작용 전극 을 포함하는 전기화학 센서에 관한 것이다.  The present invention relates to an electrochemical sensor formed on the substrate and including a working electrode to which an enzyme is attached through a linker.
<ιο> 상기 전기화학 센서는 P개 (P≥l 이상인 자연수)의 작용 전극이 종 또는 /및 횡으로 배열된 매트릭스를 이를 수 있다.  The electrochemical sensor may lead to a matrix in which P working electrodes (natural numbers of P ≧ l or more) are arranged longitudinally and / or laterally.
<π> 단일 전극 표면에의 생체분자 고정화는 전극과 효소 간의 직접적인 전자 전 달을 가능하게 해 준다. 도 1은 글루코스 옥시다제 (glucose oxidase)로 코팅된 단 일 전극을 사용하여 글루코스 센성을 기술하는 개략도를 나타내고 있다. 도 1에서 표본 용액의 글루코스와 ' 0 '로 표기된 산소가 글루코스 옥시다제 존재하에 반웅하 여 글루코노락톤 (gluconolactone)과 ' Red '로 표기된 과산화수소 (hydrogen peroxide)를 생산한다. 상기 과산화수소의 산화 전류를 수집함으로써， 전극에서 글 루코스의 농도를 측정할 수 있다. 수소가 전극에서 산화된 후， 생성물인 산소는 효 소 사이트로 이동되어 환원 반웅을 거친다 . 이와 같은 산화환원 반응의 반복을 통 해 전류 레벨 (current level )이 증가될 수 있다. 그러나, 일반적으로 이와 같은 전 략을 사용하여 얻은 전류 레벨은， 효소 코팅과 한정된 전극의 면적으로 인해 표면 반웅성이 감소함으로써 매우 낮아진다. 따라서， 높은 전류 레벨을 얻기 위해 전극 의 기하학적 구조 연구를 필요로 한다. Immobilization of biomolecules on the surface of a single electrode enables direct electron transfer between the electrode and the enzyme. 1 shows a schematic describing glucose sensitivity using a single electrode coated with glucose oxidase. In FIG. 1, glucose and oxygen labeled '0' in the sample solution react in the presence of glucose oxidase to produce gluconolactone and hydrogen peroxide labeled 'Red'. By collecting the oxidation current of the hydrogen peroxide, The concentration of lucose can be measured. After hydrogen is oxidized at the electrode, the product, oxygen, is transported to the site and undergoes a reduction reaction. By repeating such a redox reaction, the current level can be increased. In general, however, the current level obtained using this strategy is very low due to a decrease in surface response due to the area of the enzyme coating and the limited electrode area. Therefore, it is necessary to study the geometry of the electrode to obtain high current levels.
<12> 산화환원 전류의 레벨을 향상시키기 위하여 IDA interdigi tated array) 나노 전극에 대한 연구가 활발히 이루어지고 있다. 전극은 탄소， 금， 백금, 팔라듐， 전 도성 고분자 등의 다양한 종류의 전도성 물질을 사용하여 제조될 수 있다. IDA 나 노전극은 두 가지의 빗 -형태 (comb— shaped)의 전극으로 구성된다. 도 2에서와 같이 임의의 전기화학적 가역 산화환원 종 (redox species )은 전극 사이에서의 산화환원 반웅을 재활용하게 되고， 이를 통해 재활용 횟수에 따라 전류 신호가 증가하게 된 다. 산화환원 순환 (redox eye l ing)에 의한 전류 신호 증폭은 전극간 산화환원 종의 질량 전달에 의존한다. 특히 마이크로 /나노 전극에 있어서의 확산 (di f fusion)을 통 한 질량 전달은 전극 사이의 간극을 감소시키고 전극의 종횡비 (aspect rat io)를 증 가시킴에 따라 향상될 수 있다.  In order to improve the level of redox current, researches on IDA interdigiated array nano electrodes have been actively conducted. The electrode may be manufactured using various kinds of conductive materials such as carbon, gold, platinum, palladium, conductive polymers, and the like. The IDA nanoelectrode consists of two comb-shaped electrodes. As shown in FIG. 2, any electrochemically reversible redox species recycles redox reactions between the electrodes, thereby increasing the current signal according to the number of recycling. Current signal amplification by redox eye relies on mass transfer of redox species between electrodes. In particular, mass transfer through diff fusion in micro / nano electrodes can be improved by reducing the gap between the electrodes and increasing the aspect ratio of the electrodes.
<13> 산화환원 매개체는 전기화학 센서를 이용하여 다양한 분자들을 검출하는 동 한 중요한 역할을 수행한다. 상기 매개체는 전기화학 -효소 산화환원 순환을 통해 효소 및 전극 사이에서 전자들을 증재한다. 글루코스 옥시다제 (glucose oxidase , GOx) 반웅 기작에 있어， 산소는 환원된 형태의 GOx와 매우 빠르게 반응하여 반응의 부산물로 과산화수소를 생산한다. 여기서 산소는 루테늄 (ruthenium) , 핵사민 복합 체 (hexamine com lex) , 페리시아니드 ( ferr i cyanide)， 페로세네메탄올 ( f er r ocenemethano 1 ) 및 페로세네모노^1"르복실산( (6]"0 ：61½1110110031*130 1 ^； acid) 등 다양한 형태의 산화환원 매개체로 대체될 수 있다. 상기 전자 매개체는 전극 위 부 표면에서의 빠른 전자 -전달 반응과 산화환원 효소와의 빠른 전자 전달 반응을 거친다. 예를 들어， 페리시아니드를 이용한 산화환원 반웅은 다음과 같이 일어날 수 있다. Redox mediators play an important role in the detection of various molecules using electrochemical sensors. The mediator deposits electrons between the enzyme and the electrode through an electrochemical-enzyme redox cycle. In the reaction of glucose oxidase (GOx) reactions, oxygen reacts very quickly with the reduced form of GOx to produce hydrogen peroxide as a byproduct of the reaction. Where oxygen is ruthenium, hexamine com lex, ferr i cyanide, ferrocenemethanol and ferrocenemono ^ 1 "carboxylic acid (6 ] "0: 61½1110110031 * 130 1 ^; acid), etc. It can be replaced with various types of redox mediators. The electron mediators have a rapid electron-transfer reaction on the upper surface of the electrode and a rapid electron transfer reaction with redox enzymes. For example, redox reactions using ferricyanide can occur as follows.
<14>  <14>
<15> [화학식 1]  <15> [Formula 1]
<i6> Glucose + G0x(FAD)-> Gluconi c acid + G0x(FADH2)+2H+ <i6> Glucose + G0x (FAD)-> Gluconi c acid + G0x (FADH 2 ) + 2H +
<17> [화학식 2] <i8> G0x(FADH2) + [Fe(CN)6] -> GOx(FADH) + [Fe(CN)6] + H <17> [Formula 2] <i8> G0x (FADH 2 ) + [Fe (CN) 6 ]-> GOx (FADH) + [Fe (CN) 6 ] + H
<19> [화학식 3] <19> [Formula 3]
<20> G0x(FADH2) + [Fe(CN)6]3" -> GOx(FADH) + [Fe(CN)6] 4" <20> G0x (FADH 2 ) + [Fe (CN) 6 ] 3 " -> GOx (FADH) + [Fe (CN) 6 ] 4"
<21> <21>
<22> 본 발명에서 상기 링커는 제한되지는 않으나 상기 전극 상에 직접 형성된 나 노입자 상에 부착될 수 있다.  In the present invention, the linker is not limited, but may be attached on the nanoparticles formed directly on the electrode.
<23> 본 발명에서 상기 나노입자는 제한되지는 않으나 금, 백금 또는 팔라듐으로 부터 선택할 수 있다. In the present invention, the nanoparticles are not limited but may be selected from gold, platinum or palladium.
<24>  <24>
<25> 본 발명의 다른 양태는， 상기 센서는 상기 작용 전극과 상기 기판의 수평 방 향으로 이격되어 형성된 보조 전극을 더 포함하는 전기화학 센서에 관한 것이다. Another aspect of the present invention relates to an electrochemical sensor, wherein the sensor further comprises an auxiliary electrode formed to be spaced apart in the horizontal direction of the working electrode and the substrate.
<26> 본 발명에서， 상기 작용 전극 1개와 보조 전극 1개의 쌍을 단위체로 하여 본 발명의 전기화학 센서는 M(M>1인 자연수) X N(N>1인 자연수)개의 단위체를 포함할 수 있으며， 종방향으로 M개의 단위체가 이격 배열되고， 횡방향으로 N개의 단위체가 이격 배열되어, MxN 매트릭스를 이를 수 있다. In the present invention, the electrochemical sensor of the present invention using one pair of the working electrode and one auxiliary electrode as a unit may include M (M> 1 natural number) XN (N> 1 natural number) units. M units may be spaced apart in the longitudinal direction, and N units may be spaced apart in the lateral direction to form an M × N matrix.
<27> 도 3에 나타낸 것과 같이， 글루코스 옥시다제는 IDA 전극들 중 어느 하나  As shown in Fig. 3, glucose oxidase is one of the IDA electrodes.
(comb 1)에만 선택적으로 고정화될 수 있으며， 이를 통해 환원된 종은 어떤 물질로 도 코팅되지 않아 표면이 보다 전기화학적으로 반웅성이 높은 나머지 전극 (comb 2) 에서 보다 효율적으로 산화될 수 있다. 이를 통해 단일 전극 기반의 센성에 비하여 고 신호 증폭이 가능해진다. 두 전극을 보다 가까이 위치시킬수록 전류 레벨이 향 상될 수 있다. 효소는 아릴 디아조니움 염을 환원시킴으로써 특정 전극에 선택적으 로 고정화시킬 수 있다.  It can be selectively immobilized only in (comb 1), so that the reduced species can not be coated with any material so that the surface can be more efficiently oxidized in the remaining electrode (comb 2) having a more electrochemical reaction. This enables higher signal amplification compared to single electrode-based sensors. The closer the two electrodes are to be positioned, the higher the current level can be. The enzyme can be selectively immobilized on a specific electrode by reducing the aryl diazonium salt.
<28> 상기 언급한 바와 같이 본 글루코스 센서에서 산소는 효소와 전극 사이에서 재활용될 수 있는 루테늄 ( ruthenium)， 핵사민 복합체 (hexamine com lex) , 페리시아 니드 ( ferr i cyanide) , 페로세네메탄올 ( ferrocenemethanol ) 및 페로세네모노카르복실 산 ( ferrocenemonocarboxyl i c acid) 등 다양한 형태의 산화환원 매개체로 대체될 수 있다. 상기 산화환원 종은 산소를 산화환원 종으로써 사용하였을 때 안정된 글루코 스 센싱을 제한하는 가수분해 없이 전극 표면에서 전기화학적으로 반웅할 수 있는 장점이 있다.  As mentioned above, oxygen in the glucose sensor can be recycled between the enzyme and electrode, ruthenium, hexamine com lex, ferr i cyanide and ferrocenemethanol. It can be replaced with various forms of redox mediators such as ferrocenemethanol and ferrocenemonocarboxyl ic acid. The redox species have the advantage of being able to react electrochemically at the electrode surface without hydrolysis, which limits the stable glucose sensing when oxygen is used as the redox species.
<29> 본 발명에서， 상기 분석물-효소는 제한되지는 않으나:  In the present invention, the analyte-enzyme is not limited to:
<30> 글루코스 (glucose)-글루코스 옥시다제 (glucose oxidase) ; 글루코스 (glucose)ᅳ글루코스 디하이드로게나제 (glucose dehydrogenase)； 아데노신 트리포스 페이트 (adenosine tr iphosphate)-글루코스 옥시다제 (glucose oxidase) 및 핵소키나 제 (hexoki nase)； 콜레스테를 ( cho 1 est er o 1 ) _콜레스테를 옥시다제 ( cho 1 est er o 1 oxidase , ChOx) 및 콜레스테를 에스터라제 ( cholesterol esterase) ; 및 락테이트 ( lactate)-락테이트 디하이드로게나제 ( lactate dehydrogenase)；으로 이루어진 군으 로부터 선택될 수 있다. <30> glucose (gl ucose) - glucose oxidase (glucose oxidase); Glucose glucose dehydrogenase; adenosine tr iphosphate-glucose oxidase and hexoki nase; cho 1 est er o 1 ) _ Cholesterol oxidase (cho 1 est er o 1 oxidase, ChOx) and cholesterol esterase (cholesterol esterase); And lactate-lactate dehydrogenase; and the group consisting of lactate dehydrogenase.
<3 i> 도 4에서와 같이 디아조니움 염 결합은 다양한 생체분자들의 결합을 가능하 게 하는 NH2와 -C00H와 같은 전극 표면에 다양한 작용 그룹을 제공할 수 있다. 또 한 도 5에서와 같이 디아조니움에 기반한 IDA 나노전극 상의 생체분자의 선택적 결 합은 하기와 같이 다양한 타입의 분자 검출에 기여할 수 있다. As shown in FIG. 4, diazonium salt binding can provide various functional groups on the electrode surface such as NH 2 and -C00H, which enable the binding of various biomolecules. In addition, as shown in FIG. 5, selective combination of biomolecules on diazonium-based IDA nanoelectrodes may contribute to the detection of various types of molecules as follows.
<32> (a) 글루코스 옥시다제 (GOx)를 이용한 글루코스,
Figure imgf000007_0001
(A) glucose using glucose oxidase (GOx),
Figure imgf000007_0001
(b) 글루코스 옥시다제 및 핵소키나아제를 이용한 아데노신 트리포스페이트 (adenos ine tr iphosphate , ATP) , (b) adenosine triphosphate using glucose oxidase and nucleokinase (adenos ine tr iphosphate, ATP),
Figure imgf000007_0002
Figure imgf000007_0003
Figure imgf000007_0002
Figure imgf000007_0003
(c) 콜레스테를 옥시다제 (cholesterol oxidase , ChOx) 및 콜레스테롤 에스터 라제 (cholesterol esterase)를 이용한 콜레스테를 (cholesterol )，
Figure imgf000008_0001
i fatty adds
Figure imgf000008_0002
(c) cholesterol using cholesterol oxidase (ChOx) and cholesterol esterase;
Figure imgf000008_0001
i fatty adds
Figure imgf000008_0002
(d) 락테이트 디하이드로게나제 ( lactate dehydrogenase)를 이용한 락테이트 ( lactate) , (d) lactate using lactate dehydrogenase,
+腳 ^ 隱: 丽 + 腳 ^ 隱: 丽
본 발명의 또 다른 양태는， 상기 작용 전극은 상기 기판으로부터 수직 방향 으로 이격되어 형성되며， 메시 (mesh) 형상을 가지는 전기화학 센서에 관한 것이다. 본 발명에서 상기 센서는 제한되지는 않으나 상기 작용 전극 하부에 상기 작 용 전극과 이격되어 형성된 보조 전극을 더 포함할 수 있다. Another aspect of the invention, the working electrode is formed spaced apart in the vertical direction from the substrate, and relates to an electrochemical sensor having a mesh (mesh) shape. In the present invention, the sensor is not limited, but may further include an auxiliary electrode formed spaced apart from the working electrode under the working electrode.
상기 메시 형상의 전극 형태로 제조된 전기화학 센서의 경우， IDA 나노전극 을 사용한 경우에 비해， 산화환원의 순환 (redox recycl ing)에 있어서 좋은 효율을 나타낼 수 있다.  In the case of the electrochemical sensor manufactured in the form of the mesh-shaped electrode, compared to the case of using the IDA nano-electrode, it can exhibit a good efficiency in the redox recycling (redox recycling).
본 발명에서 상기 링커는 디아조니움, 디아조니움 염 또는 그 유도체를 포함 하는 표면 개질 베이스 물질의 작용 그룹을 아민 그룹으로 변환한 후 디알데히르 가교 시약을 흔합하여 형성되며 생체물질을 전극 표면에 부착시킬 수 있다. 또한 본 발명은  In the present invention, the linker is formed by converting a functional group of a surface modification base material containing diazonium, a diazonium salt or a derivative thereof into an amine group, and then mixing a dialdehyde crosslinking reagent, and forming a biomaterial on the electrode surface. Can be attached. In addition, the present invention
기판 상에 전극을 형성하고，  Forming an electrode on the substrate,
상기 전극 상에 표면 개질 베이스 물질올 형성하고，  Forming a surface-modified base material on the electrode;
상기 표면 개질 베이스 물질과 가교 시약을 반응시키고，  Reacting the surface modification base material with a crosslinking reagent,
상기 반웅된 가교 시약과 효소를 반웅시켜, 상기 효소를 상기 전극 상에 부 착하는 것을 포함하는 전기 화학 센서의 제조 방법에 관한 것이다. Reaction of the reacted crosslinking reagent and enzyme is carried out so that the enzyme is loaded on the electrode. The present invention relates to a method for manufacturing an electrochemical sensor, including bonding.
<52> 본 발명에서 상기 표면 개질 베이스 물질은 제한되지는 않으나 디아조니움 In the present invention, the surface modification base material is not limited to diazonium.
(di azonium) , 디아조니움 염 또는 그 유도체를 사용할 수 았으며, 보다 바람직하게 는 상기 표면 개질 베이스 물질은 4-니트로페닐 디아조니움 테트라플루오로보레이 트 (4-NP)를 사용할 수 있다.  (di azonium), diazonium salts or derivatives thereof may be used, and more preferably, 4-nitrophenyl diazonium tetrafluoroborate (4-NP) may be used as the surface modification base material.
<53> 본 발명에서 상기 가교 시약은 디알데히드계의 화합물인 경우 제한되지는 않 으나, 일예로 글리옥살 (glyoxal ) , 말론디알데히드 (makmdi aklehyde)， 숙신디알데히 드 (succ indi aldehyde) , 글루타르알데히드 (glutaraldehyde) 및 프탈알데히드 (phthalaldehyde) 등으로부터 선택하여 사용할 수 있으며， 바람직하게는 글루타르 알데히드 (glutaraldehyde)를 사용할 수 있다. In the present invention, the cross-linking reagent is not limited to a dialdehyde-based compound, but examples thereof include glyoxal, makmdi aklehyde, succinic aldehyde, and glue. Tar aldehyde (glutaraldehyde) and phthalaldehyde (phthalaldehyde) and the like can be selected and used, preferably glutaraldehyde (glutaraldehyde) can be used.
<54>  <54>
<55> 본 발명의 다른 양태는， 상기 전극 상에 나노입자를 형성하는 것을 더 포함 하며， 상기 표면 개질 베이스 물질은 상기 나노입자 상에 직접 형성된 전기 화학 센서의 제조 방법에 관한 것이다. - Another aspect of the invention further includes forming nanoparticles on the electrode, wherein the surface modification base material relates to a method of manufacturing an electrochemical sensor formed directly on the nanoparticles. -
<56> 본 발명에서 상기 나노입자는 제한되지는 않으나， 바람직하게 금 (gold) , 백 금 (pl at inum) 또는 팔라듐 (pal ladi )을 사용할 수 있다. In the present invention, the nanoparticles are not limited, and preferably, gold, platinum, or palladium may be used.
<57> 상기와 같은 나노입자의 코팅을 통해 IDA 나노 전극을 이용한 바이오 센싱을 수행함에 있어 전극 표면에 다양한 나노 입자를 도포함으로써 전기화학적으로 향상 된 특성을 얻을 수 있다. 이를 통해 전극의 표면적과 반웅성이 향상되어 전기화학 적 신호의 향상을 가져올 수 있다. In the biosensing using the IDA nanoelectrode through the coating of the nanoparticles as described above, electrochemically improved characteristics may be obtained by applying various nanoparticles to the electrode surface. This improves the surface area and reaction properties of the electrode, resulting in an improvement in the electrochemical signal.
<58> 본 발명에서， 상기 전극은 제한되지는 않으나 IDA interdigi tated array) 나 노 전극을 사용할 수 있다. In the present invention, the electrode is not limited, but an IDA interdigitated array (nano) electrode may be used.
<59> 본 발명에서， 상기 작용 전극과 보조 전극 간의 거리는 제한되지는 않으나 lOnm 내지 ΙΟ μ ιη의 범위일 수 있다.  In the present invention, the distance between the working electrode and the auxiliary electrode is not limited but may be in the range of lOnm to ΙΟμιη.
<60> 본 발명의 일 실시예에서 알 수 있는 바와 같이 상기와 같은 범위로 작용 전 극과 보조 전극의 거리를 비치함에 따라， 산화환원 반웅이 보다 원활하게 일어날 수 있게 되어， 높은 수준의 전류를 얻을 수 있었다. As can be seen in an embodiment of the present invention, by having the distance between the working electrode and the auxiliary electrode in the above-described range, the redox reaction can occur more smoothly, thereby providing a high level of current. Could get
【유리한 효과】  Advantageous Effects
<61> 본 발명의 센서는 종래의 센서 대비 높은 전류값을 가지며， 안정성 및 감웅 도가 우수하여 다양한 종류의 생체물질의 센성을 위해 용이하게 사용할 수 있을 것 으로 기대된다.  The sensor of the present invention has a higher current value than the conventional sensor, and is expected to be easily used for the sensing of various kinds of biomaterials due to its excellent stability and sensitivity.
【도면의 간단한 설명】 <62> 도 1은 글루코스 옥시다제에 의해 기능화된 단일 전극을 이용한 글루코스 센 싱의 모식도이다. [Brief Description of Drawings] 1 is a schematic diagram of glucose sensing using a single electrode functionalized by glucose oxidase.
<63> 도 2는 (a) 얇은 밴드의 IDA 전극 및 (b) 높은 종횡비의 IDA 전극에서의 산 화환원 순환의 모식도이다.  FIG. 2 is a schematic diagram of an oxidation / reduction cycle in (a) a thin band IDA electrode and (b) a high aspect ratio IDA electrode.
<64> 도 3은 글루코스 옥시다제에 의해 선택적으로 기능화된 IDA 전극을 이용한 글루코스 센싱의 모식도이다. FIG. 3 is a schematic of glucose sensing using IDA electrodes selectively functionalized by glucose oxidase. FIG.
<65> 도 4는 디아조니움-변형된 전극을 이용한 효소 고정화이다.  4 is enzyme immobilization using diazonium-modified electrodes.
<66> 도 5는 디아조니움 염을 이용한 생체분자의 선택적 고정을 위한 모식도이다. FIG. 5 is a schematic diagram for selective fixation of biomolecules using diazonium salts. FIG.
<67> 도 6은 선택적 전극 기능화의 모식 공정도이다. 6 is a schematic process chart of selective electrode functionalization.
<68> 도 7은 스캔 속도 200 mV/sec에서 1 mM 4-니트로페닐 디아조니움 테트라플루 오로보레이트 (4— ni trophenyl di azonium tetraf luoroborate , 4-NP)와 0. 1 M 테트라 부틸암모늄 테트라플루오로보레이트 , I 아세토니트릴 ( tetrabutyl ammonium tetraf luoroborate I acetoni tr i le) 용액에서 탄소 IDA 나노전극 (comb 1)의 순환전 압전류곡선이다. Figure 7 shows 1 mM 4-nitrophenyl diazonium tetrafluoroborate (4-NP) and 0.1 M tetra butylammonium tetrafluor at a scan rate of 200 mV / sec. Robo rate, I is the circulating current voltage curve of acetonitrile (tetrabutyl ammonium tetraf luoroborate I acetoni tr i le) a carbon nano-IDA electrode (comb 1) in the solution.
<69> 도 8은 스캔 속도 100 mV/sec , 0. 1 M KC1 + 탈이온화수 /에탄을 (9 : 1)에서 4一  8 shows a scanning speed of 100 mV / sec, 0.1 M KC1 + deionized water / ethane at (9: 1).
NP의 니트로 그룹 (ni tro group)을 아민 그룹 (amine group)으로 환원시키기 위한 순 환전압전류곡선이다.  The cyclic voltammetry curve for reducing the nitro group of NP to amine group.
<70> 도 9는 탄소 IDA 나노전극을 이용한 글루코스 농도에 따른 IDA 전극에서 수 집된 산화전류 값을 나타낸 도면이다.  FIG. 9 is a graph illustrating oxidation current values collected from IDA electrodes according to glucose concentration using carbon IDA nanoelectrodes.
<7i> 도 10은 금속 나노입자로 개질된 IDA 나노전극상에 생체분자를 고정화한 모 식도이다. (A) 양쪽 전극이 모두 나노입자로 코팅된 경우, (B) 한쪽 전극 (comb)만 이 나노입자로 코팅된 경우. FIG. 10 is a schematic diagram of immobilizing biomolecules on IDA nanoelectrodes modified with metal nanoparticles. (A) Both electrodes are coated with nanoparticles, (B) Only one electrode (comb) is coated with these nanoparticles.
<72> 도 11은 ( a , b) 열분해 전에 적층된 고분자 세트의 SEM 이미지와 ( c , d) 열 분해 후의 상기 적층된 탄소 전극 세트의 SEM 이미지이다. FIG. 11 is an SEM image of (a, b) a polymer set laminated before pyrolysis and (c, d) an SEM image of the laminated carbon electrode set after pyrolysis.
<73> 도 12는 효소로 선택적으로 개질된 적층된 전극 세트를 사용한 글루코스 센 성의 모식도이다.  12 is a schematic diagram of glucose sensitivity using a stacked electrode set selectively modified with an enzyme.
【발명의 실시를 위한 최선의 형태】  [Best form for implementation of the invention]
<74> 이하 본 발명을 실시예와 첨부된 도면을 참조하여 상세히 설명한다 . 그러나 이들은 본 발명을 보다 상세하게 설명하기 위한 것으로 본 발명의 권리범위가 하 기의 실시예에 의해 한정되는 것은 아니다.  Hereinafter, the present invention will be described in detail with reference to the embodiments and the accompanying drawings. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited by the following examples.
<75>  <75>
<76> (화학물질) <77> 아세토니트릴 (acetonitrile; Fisher Scientific), 테트라부틸암모늄 테트라 플루오로보레이트 (tetrabutylammonium tetraf luoroborate, NBu4BF4; Fluka), 4-니트 로페닐 디아조니움 테트라플루오로보레이트 (4-nitrophenyl diazonium tetraf luoroborate, 4-NP) , 포타슘 페리入 1아니드 (pottassium ferri cyanide) , 포타 슘 페로시 01"니드 (pottassium ferrocyanide) , 글루타르알데히드 (glutaraldehyde)， 포타슘 클로라이드 (pottasium chloride), 소듐 시아노보로하이드라이드 (sodium cyanoborohydride), 아스퍼질러스 니거
Figure imgf000011_0001
niger) 유래 글루코스 옥시다 제 (glucose oxidase; Type X-S, 100,000-250,000 units/g solid, Sigma Aldrich), 포스페이트 버퍼 (phosphate buffer, PBS, pH 7.4； Life Technologies). <76> (chemicals) Acetonitrile (fisher scientific), tetrabutylammonium tetraf luoroborate (NBu 4 BF 4 ; Fluka), 4-nitrophenyl diazonium tetrafluoroborate (4-nitrophenyl diazonium tetraf) luoroborate, a 4-NP), potassium Perry入1 No de (pottassium ferri cyanide), Porta syum Perot 0:00 1 "Need (pottassium ferrocyanide), glutaraldehyde (glutaraldehyde), potassium chloride (pottasium chloride), sodium cyanoborohydride Hydride (sodium cyanoborohydride), Aspergillus niger
Figure imgf000011_0001
niger) glucose oxidase (Type XS, 100,000-250,000 units / g solid, Sigma Aldrich), phosphate buffer (PBS, pH 7.4; Life Technologies).
<78>  <78>
<79> (제조예 1) 전극 제조 (electrode fabrication)  Preparation Example 1 Electrode Fabrication
<80> 탄소 IDA 나노전극은 6 인치 (100) 규소 웨이퍼 (Si wafer) 상에 제조되었다. Carbon IDA nanoelectrodes were fabricated on 6 inch (100) silicon wafers.
<8i> 첫째로, 700 nm 두께의 이산화규소 (SiC>2)층이 열산화 (thermal oxidation)에 의해 규소 웨이퍼 상에 용착되었다. 포토리소그래피 (photolithography)를 이용하여 SU-8 음성 감광제 (negative photoresist) IDA 구조를 패턴화하였다. 감광제 구조를 탄소 전극으로 변환하기 위해， 사전정의된 감광제 IDA 패턴을 진공상태， 900 °C에 서 열분해시켰다. 열분해 동안, IDA 구조의 크기가 폭이 60%， 높이가 90%만큼 감소 하였다. 최종적으로， 탄소 전극은 맞물린 (interdigitated) 전극 영역을 제외하고 부동태화 (passivate)되었다. First, a 700 nm thick silicon dioxide (SiC> 2) layer was deposited on a silicon wafer by thermal oxidation. Photolithography was used to pattern the SU-8 negative photoresist IDA structure. In order to convert the photoresist structure into a carbon electrode, a predefined photoresist IDA pattern was pyrolyzed at 900 ° C. under vacuum. During pyrolysis, the size of the IDA structure decreased by 60% in width and 90% in height. Finally, the carbon electrode was passivated except for the interdigitated electrode region.
<82>  <82>
<83> (제조예 2) 선택적 표면 기능화 (selective surface functional izat ion) Preparation Example 2 Selective surface functional izat ion
<84> 도 6에서와 같이 효소의 고정화는 3 단계로 구성될 수 있다. 4—니트로페닐 디아조니움 테트라플루오로보레이트 (4-NP)를 베이스로 사용하였다. 효소를 디아조 니움에 연결하기 위해， 디아조니움의 작용 그룹을 아민그룹으로 변환시키고 디아조 니움의 아민 그룹과 효소 사이에서 글루타르알데히드가 사용되었다. As shown in FIG. 6, the immobilization of the enzyme may be composed of three steps. 4—Nitrophenyl diazonium tetrafluoroborate (4-NP) was used as the base. To link the enzyme to diazonium, the functional group of the diazonium was converted to an amine group and glutaraldehyde was used between the amine group and the enzyme of the diazonium.
<85> 4-NP의 전기화학적 흡착 ᅳ <85> Electrochemical Adsorption of 4-NP ᅳ
<86> 탄소 전극의 전기화학적 개질은 0.1 M NBu4BF4에 1 mM 4-NP를 포함하는 아세 토니트릴에서， 기준전극 Ag/AgCl에 대해 200 mV/s의 스캐닝 속도로 0.5에서 -0.7 V 까지 전극의 전자전위를 스캔함으로써 측정하였다. 개질하기 전， 용매는 아르곤 가 스를 이용하여 30분간 불순물을 제거하였다. 개질이 이루어진 후， 전극은 30분간 탈이온화수 (DI water)로 세척하였다. <87> 니트로 그룹올 아민 그룹으로 환원 The electrochemical modification of the carbon electrode was performed at 0.5 to -0.7 V in acetonitrile containing 1 mM 4-NP in 0.1 M NBu 4 BF 4 at a scanning rate of 200 mV / s for the reference electrode Ag / AgCl. It measured by scanning the electron potential of an electrode until. Prior to modification, the solvent was removed impurities for 30 minutes using argon gas. After the modifications were made, the electrodes were washed with DI water for 30 minutes. Reduction with nitro groupol amine groups
<88> 니트로 그룹의 환원을 통한 아민 그룹의 생성을 위해 0. 1 M 염화칼륨과 물 / 에탄올 (90 : 10 v/v)를 함유한 프로톤 용매 (prot i c solvent )를 사용하였다. 기준전극 Ag/AgCl에 대해 100 mV/s의 스캐닝 속도로 0에서 0.8 V까지 전극의 전자전위를 스 캔함으로써 전극의 전위를 측정하였다.  A proton solvent containing 0.1 M potassium chloride and water / ethanol (90: 10 v / v) was used to generate the amine group through reduction of the nitro group. The potential of the electrode was measured by scanning the electron potential of the electrode from 0 to 0.8 V at a scanning speed of 100 mV / s relative to the reference electrode Ag / AgCl.
<89> 효소 고정화  <89> Enzyme Immobilization
<90> 링커를 통한 효소 고정화를 위해 글루타르알데히드를 이작용기 가교 시약  Glutaraldehyde is a bifunctional crosslinking reagent for enzyme immobilization via a linker.
(bi funct ional cross-l inking agent )으로 사용하였다. 아민 그룹 전환 이후， 탄소 IDA 전극을 0. 1% 소듬 시아노보로하이드라이드 (sodium cyan?borohydr ide)와 2.5 중 량% 글루타르알데히드 용액 200 에 실온에서 2 시간 동안 담갔다가 꺼냈다. (bi funct ional cross-l inking agent). After amine group conversion, the carbon IDA electrode was immersed in 0.1% sodium cyan? Borohydride and 200% of a 2.5% by weight glutaraldehyde solution at room temperature for 2 hours.
<91> 이후 전극은 탈이온화수로 세정하고 질소 가스를 이용하여 건조하였다. 글루 코스 옥시다제 효소를 탄소 전극상에 생성된 알데히드 그룹과 결합 (coupl ing)시키 기 위해, 상기 전극을 50 mM PBS 버퍼 (pH 7.4)에 0. 1% 소듬 시아노보로하이드라이 드와 함께 10 mg/mL 글루코스 옥시다제를 포함하는 4 °C 버퍼 용액에서 하룻밤 배 양하였다. The electrode was then washed with deionized water and dried using nitrogen gas. In order to couple the glucos oxidase enzyme to the aldehyde groups produced on the carbon electrode, the electrode was combined with 0.1% digested cyanoborohydride in 50 mM PBS buffer (pH 7.4). Incubated overnight in 4 ° C buffer solution containing mg / mL glucose oxidase.
<92>- <92>-
<93> (제조예 3) 전극의 전기화학적 특성화 Preparation Example 3 Electrochemical Characterization of Electrodes
<94> 모든 전극은 탈이온화수에 0.5 M의 염화칼륨과 10 mM의 [Fe(CN)6]4_에서 기준 전극 Ag/AgCl에 대해 50 mV/s의 스캐닝 속도로 0에서 0.6 V까지ᅵ전극의 전자전위를 스캔함으로써 순환전압전류법을 사용하여 특성화하였다. 글루코스 센싱은 05 ιηΜ All of the electrodes range from 0 to 0.6 V at a scanning rate of 50 mV / s for the reference electrode Ag / AgCl at 0.5 M potassium chloride and 10 mM [Fe (CN) 6 ] 4_ in deionized water . Scanning electron potentials were characterized using cyclic voltammetry. Glucose Sensing is 05 ιηΜ
PBS(pH 7.4)에서 산화환원 매개체로 10 mM [Fe(CN)6]3—을 이용하여 수행하였다. 10 mM [Fe (CN) 6 ] 3 — was used as the redox mediator in PBS pH 7.4.
<95> 글루코스 용액은 100 mM PBS에 0. 1 M의 농도로 준비되었고 아노머릭 Glucose solution was prepared at a concentration of 0.1 M in 100 mM PBS and anomeric
(anomer i c) 평형에 도달하도록 24 시간 동안 실온에서 변선광화 (mutarotate)를 허 용하였다. 글루코스 센싱을 위한 모든 용액은 전기화학적 테스트를 수행하기 전에 최소 30분간 용액에 아르곤 가스를 배출하여 정화하였다. 대조전극으로는 백금선을 사용하였다. 전기화학적 검출은 다증 전위 측정기 (CHI 1020； CH Instrument Inc . , USA)를 이용하여 수행하였다.  (anomer i c) allowed mutarotation at room temperature for 24 hours to reach equilibrium. All solutions for glucose sensing were clarified by releasing argon gas into the solution for at least 30 minutes prior to performing the electrochemical test. Platinum wire was used as a control electrode. Electrochemical detection was performed using a multipotential potentiometer (CHI 1020; CH Instrument Inc., USA).
<96>  <96>
<97> (실시예 1) 탄소 IDA 나노 전극의 표면 개질  Example 1 Surface Modification of a Carbon IDA Nano Electrode
<98> 상기 제조예 1의 절차에 의하여 탄소 IDA 나노 전극을 제작하였다. 도 3에 나타난 것과 같이 탄소 IDA 나노 전극에 대해 링커 및 효소를 이용하여 전극 표면 을 기능화하였다. 탄소 IDA 나노 전극 중 한 콤 (comb)은 글루코스 옥시다제 (GOx)로 기능화한 반면， 다른 인접한 콤 (comb)은 페로시아니드 산화환원 종의 산화 전류를 수집하기 위해 사용되었다. Carbon IDA nano-electrode was prepared by the procedure of Preparation Example 1. Electrode surface using linker and enzyme for carbon IDA nanoelectrode as shown in FIG. 3 Was functionalized. One comb of the carbon IDA nanoelectrode was functionalized with glucose oxidase (GOx), while the other adjacent comb was used to collect the oxidation current of the ferrocyanide redox species.
<99> 도 7에는 한 쪽 콤에 1 mM 4-NP 및 0.1 M NBu4BF4/아세토니트릴 용액상에서 의 순환전압전류 곡선 (cyclic volta瞧 ogram)을 나타내었다. 첫 번째 사이클 -0.05 V에서 비가역적 환원 곡선은 디아조니움 염 유도체로부터 4-니트로페닐 라디칼 (radical)의 형성에 기여하였다. 상기 첫 번째 비가역적 곡선이 두 번째 스캔에서 사라진 것으로부터， 탄소 표면에 접합된 니트로페닐 그룹이 전자 전달을 방해한다 는 것을 의미하였다. 마지막 가역 곡선으로부터， 분열로 형성된 라디칼 음이온 (radical anion)이 탄소 전극 표면에서 아릴 음이온 (aryl anion)으로 환원되었음을 보여준다. FIG. 7 shows a cyclic voltagram in a comb of 1 mM 4-NP and 0.1 M NBu 4 BF 4 / acetonitrile solution. The irreversible reduction curve at the first cycle -0.05 V contributed to the formation of the 4-nitrophenyl radical from the diazonium salt derivative. Since the first irreversible curve disappeared in the second scan, it meant that the nitrophenyl group bonded to the carbon surface interfered with the electron transfer. From the last reversible curve, it shows that the radical anions formed by the cleavage were reduced to aryl anions at the surface of the carbon electrode.
<ιοο> 제조예 2에서 니트로 그룹을 아민 그룹으로 환원시킨 결과를 도 8에 도시하 였다. 환원 전류의 증가는 니트로 그룹이 아민 그룹으로 전환되도록 하였다. ； <ιοο> The result of reducing the nitro group to the amine group in Preparation Example 2 is shown in FIG. 8. An increase in the reduction current caused the nitro group to be converted to an amine group. ;
<10!> <10!>
<102> *탄소 전극을 활성화시킨 아민 그룹은 2.5% 글루타르알데히드를 사용하여 글 루코스 옥시다제와 함께 배양되었다. 마지.막으로 탄소 IDA 나노전극 한쪽은 제조예 2 방법을 사용하여 글루코스 옥시다제로 기능화시키고， 인접한 전극은 페로시아니 드의 산화 전류를 수집하기 위해 사용되었다.  The amine group that activated the carbon electrode was incubated with glucose oxidase using 2.5% glutaraldehyde. Finally, one side of the carbon IDA nanoelectrode was functionalized with glucose oxidase using the Preparation Example 2 method, and adjacent electrodes were used to collect the oxidation current of the ferrocyanide.
<103>  <103>
<104> (실시예 2) 글루코스 센싱  Example 2 Glucose Sensing
<105> 빠른 전자 전달 동특성 (transfer kinetics)과 안정된 산화 /환원 형태를 갖는 페리시아니드 산화환원 매개체 (ferri cyanide redox mediator) 존재하에 글루코스 센싱을 수행하였다.  Glucose sensing was performed in the presence of ferri cyanide redox mediators with fast electron kinetics and stable oxidation / reduction forms.
<106> 효소 반웅이 이루어지는 동안， 글루코스 분자는 글루코스 옥시다제 효소의 플라빈-아데닌 디뉴클레이티드 (f lavin-adenine dinucleot ide, FAD) 산화환원 핵심 요소에 의해 산화되었다. 상기 산화 공정에서， FAD는 하기 [화학식 4]에 기술한 바 와 같이 FAD¾(G0xred)로 환원되었다. 이 시스템에서 [화학식 5] 반웅에서 산소는 대 체 전자 수용체로 페리시아니드 (ferricyanide)로 대치될 수 있다. 이후, FADH2는 [ 화학식 6~기에서와 같이 [Fe(CN)6]3 [Fe(CN)6;广로 환원되면서 FAD로 다시 산화되 었다. 상기 반웅은 글루코스 옥시다제로 기능화된 탄소 전극 (comb 1)에서 일어난 반면, [Fe(CN)6] 는 도 3에서 나타난 바와 같이 전극 (comb 1) 및 인접한 탄소 전극 During the enzyme reaction, the glucose molecule was oxidized by the flavin-adenine dinucleotide (FAD) redox core element of the glucose oxidase enzyme. In the oxidation process, FAD was reduced to FAD¾ (G0x red ) as described in [Formula 4]. In this system, oxygen in the reaction can be replaced with ferricyanide as a substitute electron acceptor. Thereafter, FADH 2 was oxidized back to FAD while being reduced to [Fe (CN) 6 ] 3 [Fe (CN) 6 ; The reaction occurred at a carbon electrode (comb 1) functionalized with glucose oxidase On the other hand, [Fe (CN) 6 ] is the electrode comb 1 and the adjacent carbon electrode as shown in FIG.
(comb 2)에서 [Fe(CN)6]3_으로 다시 산화되었다. 상기 최종 단계의 반웅은 매개체의 산화형태가 재생됨에 따라 글루코스 농도에 직접적으로 비례하여 측정가능한 전류 를 생산하였다. It was oxidized again to [Fe (CN) 6 ] 3_ in (comb 2). The reaction of this final step produced a measurable current in direct proportion to the glucose concentration as the oxidized form of the medium was regenerated.
<107>  <107>
<108> [화학식 4]  <108> [Formula 4]
<i09> Glucose + FAD -> Gluconic acid + FADH2 + 2H+ <i09> Glucose + FAD-> Gluconic acid + FADH 2 + 2H +
<ιιο> [화학식 5] <ιιο> [Formula 5]
<ιπ> FADH2 + 02 -> FAD + ¾02 <ιπ> FADH 2 + 0 2- > FAD + ¾0 2
<112> ' [화학식 6] <112>' [Formula 6]
<ii3> FADH2 + [Fe(CN)6]3" -> FADH + [Fe(CN)6]4" + H+ <ii3> FADH 2 + [Fe (CN) 6 ] 3 " -> FADH + [Fe (CN) 6 ] 4" + H +
<114> [화학식 7] <114> [Formula 7]
<ii5> FADH2 + [Fe(CN)6]3" -〉 FADH + [Fe(CN)6]4" <ii5> FADH 2 + [Fe (CN) 6 ] 3 " -> FADH + [Fe (CN) 6 ] 4"
<116> <116>
<ιΐ7> 상기 결과로부터 효소와 두 탄소 IDA 나노전극 combs 사이에 산화환원 순환  <ιΐ7> From these results redox cycle between enzyme and two carbon IDA nanoelectrode combs
(redox cycling)이 발생하였다. 글루코스 옥시다제로 개질된 탄소 전극 (comb 1)에 서는 글루코스의 산화와 [Fe(CN)6]3—의 환원을 포함하는 효소 반웅이 일어났다. 반 면， 개질된 탄소 전극 (comb 1)과 개질되지 않은 인접한 탄소 전극 (comb 2)에서는 [Fe(CN)6]4ᅭ의 산화반웅이 일어났다. 따라서， IDA 나노 전극의 두 전극 모두 페리시 아니드의 산화환원 순환에 관여하고 있으며， 이는 글루코스 검출에 있어 높은 민감 도를 부여하는 데 기여함을 알 수 있었다. (redox cycling) occurred. At the carbon electrode (comb 1) modified with glucose oxidase, an enzyme reaction involving oxidation of glucose and reduction of [Fe (CN) 6 ] 3 — occurred. On the other hand, an oxide reaction of [Fe (CN) 6 ] 4 일어 났 occurred in the modified carbon electrode comb 1 and the unmodified adjacent carbon electrode comb 2. Therefore, both electrodes of the IDA nano-electrode are involved in the redox circulation of ferric anide, and it can be seen that this contributes to high sensitivity in the detection of glucose.
<Π8> 도 9에 IDA 나노 전극의 두 전극에서 글루코스 농도의 증가에 따라 전류가 선형적으로 증가함을 나타내고 있다. Comb 2에서의 전류는 Comb 1에서의 전류보다 큰데, 이는 Comb 2의 효소 사이트 사이의 거리와 전극 표면이 Comb 1에 비해서 큼 에도 불구하고， Comb 2의 개질되지 않은표면이 다수의 분자로 개질된 Comb 1에 비 해 표면 반응성이 더 좋기 때문이다. 따라서， 추가로 개질되지 않은 탄속 전극을 사용한 IDA 나노 전극을 이용하여 보다 많은 전류의 수집이 가능하였다. <1 I9> 9 shows that the current increases linearly with increasing glucose concentration in the two electrodes of the IDA nanoelectrode. The current at Comb 2 is greater than the current at Comb 1, which means that the unmodified surface of Comb 2 is modified by a number of molecules, despite the distance between the enzyme sites of Comb 2 and the electrode surface being larger than Comb 1. This is because the surface reactivity is better than that of Comb 1. Thus, more current collection was possible using IDA nanoelectrodes using unmodified carbon velocity electrodes. <1 I9>
<i20> (제조예 4) 금속 나노입자로 개질된 IDA intergidi tated array) 나노 전극을 사용한 바이오센서  <i20> (Production Example 4) Biosensor using IDA intergidated array nano electrode modified with metal nanoparticles
<i2i> 제조예 1 및 제조예 2의 전극 제조 방법과 유사하게, 도 10과 같이 디아조니 움 염을 이용한 링커를 탄소 전극에 부착시키기 전에 금 (gold) , 백금 (pl at inum) 또 는 팔라듐 (pal ladium) 등의 나노입자를 코팅하여， 링커에 연결된 글루코스 옥시다 제를 포함하는 생체분자가 나노입자를 통하여 전극과 연결되는 바이오 센서를 제작 하였다.  <i2i> Similar to the electrode preparation methods of Preparation Examples 1 and 2, before attaching the linker using the diazonium salt to the carbon electrode as shown in FIG. 10, gold, platinum or palladium By coating nanoparticles such as (pal ladium), a biosensor comprising a glucose oxidase connected to a linker is connected to an electrode through the nanoparticles.
<\22>  <\ 22>
<123> (제조예 5) 적층된 탄소 전극 세트를 사용한 글루코스 센싱  (Production Example 5) Glucose Sensing Using a Stacked Carbon Electrode Set
<124> 도 11에서와 같이 서스펜디드 카본 메쉬 ( suspended carbon mesh)와 기질-경 계 평면 전극을 포함하는 적층된 전극 세트 ( stacked electrode set )를 상기 제조예 1 및 제조예 2에서 기술한 방법과 유사한 바이오센서 플랫품으로서 사용하기 위한 IDA 전극을 대체하는 구성으로 제조하였다. 바이오센싱을 위한 산화환원 기작 및 제조 공정은 상기 제조예 1 내지 제조예 3에 기술한 방법을 사용하였다. As shown in FIG. 11, a stacked electrode set including a suspended carbon mesh and a substrate-bound planar electrode is similar to the method described in Preparation Examples 1 and 2. It was made in a configuration that replaced the IDA electrode for use as a biosensor platform. The redox mechanism and manufacturing process for biosensing used the method described in Preparation Examples 1 to 3.
<125> 이와 같이 제조한 적층된 전극 세트의 경우 IDA 나노전극을 사용한 경우에 비해 산화환원 순환에 있어 좋은 효율을 보이는 것을 확인하였다. It was confirmed that the stacked electrode sets manufactured as described above showed better efficiency in the redox circulation than when the IDA nanoelectrode was used.
<126> 글루코스 센성을 위하여 서스펜디드 나노메쉬 ( suspended nanomesh)의 선택적 디아조니움 개질은 상기 제조예 2에서 기술한 4-NP의 전기화학적 환원 흡착에 의해 이루어졌다. 이후 글루타르알데히드를 사용하여 글루코스 옥시다제를 고정화하였 다. 도 12(a)에서와 같이 평면 전극은 페리시아니드 /페로시아니드 산화환원 커플의 산화환원 순환을 위해 아무런 개질 없이 남겨두었다.  Selective diazonium modification of suspended nanomesh for glucose sensitivity was performed by electrochemical reduction adsorption of 4-NP as described in Preparation Example 2 above. Glutaraldehyde was then used to immobilize glucose oxidase. As shown in FIG. 12 (a), the planar electrode was left without modification for redox circulation of the ferricyanide / ferrocyanide redox couple.
<127> 상기 제작한 전극과 다른 방법으로， 효소 고정화를 위하여 도 12(b)에 도시 한 바와 같이 상기 제조예 2의 방법과 마찬가지로 기질 -경계 평면 전극을 부착하여 개질하는 방법을 사용하여 전극을 제작하였다. 12. As shown in FIG. 12 (b), the electrode was modified by attaching and modifying a substrate-boundary planar electrode in the same manner as in Preparation Example 2 for the enzyme immobilization. Produced.
<128> 도 12 (a) , (b)에서 도시한 각각에 대하여， 효소와 전극 표면 사이에서 산화 환원 커플 (redox couple)이 재순환되었으며， 전극 표면에서의 산화 전류를 측정하 여 글루코스의 농도를 측정하였다.  12 (a) and 12 (b), the redox couple was recycled between the enzyme and the electrode surface, and glucose concentration was measured by measuring the oxidation current at the electrode surface. Measured.

Claims

【청구의 범위】 【Scope of Claim】
【청구항 1】 【Claim 1】
유체 중의 분석물의 존재 또는 농도를 결정하기 위한 전기화학 센서로서, 기판; 및 An electrochemical sensor for determining the presence or concentration of an analyte in a fluid, comprising: a substrate; and
상기 기판 상에 형성되며， 링커 ( l inker )를 통하여 효소가 부착된 작용 전극 을 포함하는 전기화학 센서 . An electrochemical sensor formed on the substrate and including a working electrode to which an enzyme is attached through a linker.
【청구항 2】 【Claim 2】
제 1항에 있어서， In clause 1,
상기 링커는 상기 전극 상에 직접 형성된 나노입자 상에 부착된 전기 화학 센서 - The linker is an electrochemical sensor attached to nanoparticles formed directly on the electrode -
【청구항 3] [Claim 3]
제 2항에 있어서， In paragraph 2,
상기 나노입자는 금, 백금 또는 팔라듐으로부터 선택되는 전기 화학 센서. An electrochemical sensor wherein the nanoparticles are selected from gold, platinum or palladium.
【청구항 4】 【Claim 4】
제 1항에 있어서， In clause 1,
상기 센서는 상기 작용 전극과 상기 기판의 수평 방향으로 이격되어 형성된 보조 전극을 더 포함하는 전기 화학 센서 . The sensor is an electrochemical sensor further comprising an auxiliary electrode formed to be spaced apart from the working electrode in a horizontal direction of the substrate.
【청구항 5】 【Claim 5】
제 1항에 있어서, According to clause 1,
상기 작용 전극은 상기 기판으로부터 수직 방향으로 이격되어 형성되며， 메 시 (mesh) 형상을 가지는 전기 화학 센서 . The working electrode is formed to be vertically spaced from the substrate, and is an electrochemical sensor having a mesh shape.
【청구항 6】 【Claim 6】
제 5항에 있어서, According to clause 5,
상기 센서는 상기 작용 전극 하부에 상기 작용 전극과 이격되어 형성된 보조 전극을 더 포함하는 전기 화학 센서 . The sensor is an electrochemical sensor further comprising an auxiliary electrode formed below the working electrode and spaced apart from the working electrode.
【청구항 71 【Claim 71
제 1항에 있어서， In clause 1,
상기 링커는 디아조니움, 디아조니움 염 또는 그 유도체를 포함하는 표면 개 질 베이스 물질의 작용 그룹을 아민 그룹으로 변환한 후 디알데히르 가교 시약을 흔합하여 형성되는 전기 화학 센서 . The linker is an electrochemical sensor formed by converting the functional group of a surface-modifying base material containing diazonium, diazonium salt, or its derivative into an amine group and then mixing it with a dialdehyde cross-linking reagent.
【청구항 8】 【Claim 8】
기판 상에 전극을 형성하고， 상기 전극 상에 표면 개질 베이스 물질을 형성하고 , Form an electrode on the substrate, Forming a surface modification base material on the electrode,
상기 표면 개질 베이스 물질과 가교 시약을 반응시키고， React the surface modification base material and the crosslinking reagent,
상기 반웅된 가교 시약과 효소를 반웅시켜， 상기 효소를 상기 전극 상에 부 착하는 것을 포함하는 전기 화학 센서의 제조 방법 . A method of producing an electrochemical sensor comprising reacting the crosslinking reagent and the enzyme and attaching the enzyme to the electrode.
【청구항 9】 【Claim 9】
제 8항에 있어서， In clause 8,
상기 표면 개질 베이스 물질은 디아조니움 (di azonium) , 디아조니움 염 또는 그 유도체인 전기 화학 센서의 제조 방법 . The surface modification base material is diazonium, a diazonium salt, or a derivative thereof. Method for producing an electrochemical sensor.
【청구항 10】 【Claim 10】
제 9항에 있어서, According to clause 9,
상기 표면 개질 베이스 물질은 4-니트로페닐 디아조니움 테트라플루오로보레 이트 (4-NP)인 전기 화학 센서의 제조 방법. A method of manufacturing an electrochemical sensor wherein the surface modification base material is 4-nitrophenyl diazonium tetrafluoroborate (4-NP).
【청구항 11】 【Claim 11】
제 8항에 있어서， In clause 8,
상기 가교 시약은 *디알데히드인 전기 화학 센서의 제조 방법 . The cross-linking reagent is * dialdehyde. Method for producing an electrochemical sensor.
【청구항 12] [Claim 12]
제 11항에 있어서， In clause 11,
상기 가교 시약은 글루타르알데히드 (glutaraldehyde)인 전기 화학 센서의 제 조 방법 . Method for producing an electrochemical sensor wherein the crosslinking reagent is glutaraldehyde.
【청구항 13] [Claim 13]
제 8항에 있어서， In clause 8,
상기 전극 상에 나노입자를 형성하는 것을 더 포함하며， It further includes forming nanoparticles on the electrode,
상기 표면 개질 베이스 물질은 상기 나노입자 상에 직접 형성된 전기 화학 센서의 제조 방법 . The surface modification base material is a method of manufacturing an electrochemical sensor formed directly on the nanoparticles.
PCT/KR2015/003046 2014-03-28 2015-03-27 Electrochemical biosensor using dual electrode pair WO2015147594A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US15/300,192 US20170166946A1 (en) 2014-03-28 2015-03-27 Electrochemical biosensor using dual electrode pair
US16/258,326 US11060125B2 (en) 2014-03-28 2019-01-25 Electrochemical biosensor using dual electrode pair

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20140036876 2014-03-28
KR10-2014-0036876 2014-03-28
KR1020150042538A KR102347669B1 (en) 2014-03-28 2015-03-26 Biosensor using dual electrode array
KR10-2015-0042538 2015-03-26

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/300,192 A-371-Of-International US20170166946A1 (en) 2014-03-28 2015-03-27 Electrochemical biosensor using dual electrode pair
US16/258,326 Continuation US11060125B2 (en) 2014-03-28 2019-01-25 Electrochemical biosensor using dual electrode pair

Publications (1)

Publication Number Publication Date
WO2015147594A1 true WO2015147594A1 (en) 2015-10-01

Family

ID=54196005

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/003046 WO2015147594A1 (en) 2014-03-28 2015-03-27 Electrochemical biosensor using dual electrode pair

Country Status (1)

Country Link
WO (1) WO2015147594A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101947639B1 (en) 2017-08-08 2019-02-14 한국화학연구원 Glucose sensor for ex-vivo diagnosis comprising graphene-based electrode

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040102534A (en) * 2003-05-28 2004-12-08 삼성엔지니어링 주식회사 Electrode for measuring nitrogenic nutrient salts and biosensor comprising the same
KR20070116608A (en) * 2005-03-29 2007-12-10 씨씨아이 가부시키가이샤 Biosensor
JP2009158458A (en) * 2007-12-06 2009-07-16 Sony Corp Fuel cell, method of manufacturing fuel cell, electronic apparatus, enzyme immobilization electrode, biosensor, bioreactor, energy conversion element, and enzyme reaction utilization device
WO2013029115A1 (en) * 2011-09-01 2013-03-07 Newsouth Innovations Pty Limited Electrochemical competition sensor
KR20130106044A (en) * 2012-03-19 2013-09-27 한국과학기술원 Biosensor using the graphene oxide by the polymer of enzyme substrate and method of detecting using it

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040102534A (en) * 2003-05-28 2004-12-08 삼성엔지니어링 주식회사 Electrode for measuring nitrogenic nutrient salts and biosensor comprising the same
KR20070116608A (en) * 2005-03-29 2007-12-10 씨씨아이 가부시키가이샤 Biosensor
JP2009158458A (en) * 2007-12-06 2009-07-16 Sony Corp Fuel cell, method of manufacturing fuel cell, electronic apparatus, enzyme immobilization electrode, biosensor, bioreactor, energy conversion element, and enzyme reaction utilization device
WO2013029115A1 (en) * 2011-09-01 2013-03-07 Newsouth Innovations Pty Limited Electrochemical competition sensor
KR20130106044A (en) * 2012-03-19 2013-09-27 한국과학기술원 Biosensor using the graphene oxide by the polymer of enzyme substrate and method of detecting using it

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101947639B1 (en) 2017-08-08 2019-02-14 한국화학연구원 Glucose sensor for ex-vivo diagnosis comprising graphene-based electrode

Similar Documents

Publication Publication Date Title
Sun et al. In situ growth of TiO2 nanowires on Ti3C2 MXenes nanosheets as highly sensitive luminol electrochemiluminescent nanoplatform for glucose detection in fruits, sweat and serum samples
Chen et al. Recent advances in electrochemical glucose biosensors: a review
Rahman et al. A lactate biosensor based on lactate dehydrogenase/nictotinamide adenine dinucleotide (oxidized form) immobilized on a conducting polymer/multiwall carbon nanotube composite film
Zhu et al. Electrochemical sensors and biosensors based on nanomaterials and nanostructures
Privett et al. Electrochemical sensors
Lawal Synthesis and utilisation of graphene for fabrication of electrochemical sensors
Imran et al. Platinum and zinc oxide modified carbon nitride electrode as non-enzymatic highly selective and reusable electrochemical diabetic sensor in human blood
Kaushik et al. Carbon nanotubes—chitosan nanobiocomposite for immunosensor
Sharma et al. Glucose sensor based on redox-cycling between selectively modified and unmodified combs of carbon interdigitated array nanoelectrodes
EP3634907B1 (en) Modified nanostructured carbon matrix, containing such a matrix system and including such system biosensor and a method for the determination of an analyte in a sample using such biosensor system as well as the application of a pyrrole benzoic acid derivative for the modification of the nanostructured carbon matrix
Pannell et al. Versatile sarcosine and creatinine biosensing schemes utilizing layer-by-layer construction of carbon nanotube-chitosan composite films
Isoaho et al. Carbon nanostructure based platform for enzymatic glutamate biosensors
Kuralay Nanomaterials-based enzyme biosensors for electrochemical applications: recent trends and future prospects
Dutta et al. Fabrication of electrochemical biosensor using zinc oxide nanoflowers for the detection of uric acid
Khatri et al. Electrochemical studies of biofunctionalized MoS 2 matrix for highly stable immobilization of antibodies and detection of lung cancer protein biomarker
Huseinov et al. Chemically Bonded Carbon Nanotube Film to a Nanostructured Gold Electrode for Electrochemical Sensing of Hydrogen Peroxide
Demir et al. An overview of stability and lifetime of electrochemical biosensors
WO2015147594A1 (en) Electrochemical biosensor using dual electrode pair
Manesh et al. Fabrication of a gold nanoparticles decorated carbon nanotubes based novel modified electrode for the electrochemical detection of glucose
US11060125B2 (en) Electrochemical biosensor using dual electrode pair
Xiao et al. Fabrication of ultrafine PtPd alloy/ionic liquid-carbon dots nanocomposites modified flexible carbon fiber as enzyme-free amperometric sensing for H2O2 in living cells
Aziz et al. Nanomaterials in electrochemical biosensor
Horozova et al. Electrooxidation of phenol by catalase immobilized on graphite electrodes
Ngo et al. Electrochemical biosensors based on nanocomposites of carbon-based dots
Razzino et al. Graphene-based sensors: applications in electrochemical (Bio) sensing

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: 15767923

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 15300192

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 15767923

Country of ref document: EP

Kind code of ref document: A1