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WO2020116743A1 - Méthode de mesure destinée à un biocapteur électrochimique - Google Patents

Méthode de mesure destinée à un biocapteur électrochimique Download PDF

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Publication number
WO2020116743A1
WO2020116743A1 PCT/KR2019/010455 KR2019010455W WO2020116743A1 WO 2020116743 A1 WO2020116743 A1 WO 2020116743A1 KR 2019010455 W KR2019010455 W KR 2019010455W WO 2020116743 A1 WO2020116743 A1 WO 2020116743A1
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WIPO (PCT)
Prior art keywords
time
measuring
electrode
electrochemical biosensor
voltage
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/KR2019/010455
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English (en)
Korean (ko)
Inventor
성우경
이국녕
송성아
김영주
김원효
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Korea Electronics Technology Institute
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Korea Electronics Technology Institute
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Filing date
Publication date
Application filed by Korea Electronics Technology Institute filed Critical Korea Electronics Technology Institute
Publication of WO2020116743A1 publication Critical patent/WO2020116743A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N27/416—Systems
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

  • the present invention relates to a measurement method of an electrochemical biosensor that detects biomarkers contained in body fluids such as blood, urination, saliva as an electrochemical signal measurement principle, and more specifically, time in an in vitro diagnostic device using an electrochemical biosensor. It relates to a measurement method of an electrochemical biosensor that can improve the accuracy and reproducibility of measurement in the measurement by the current method or the time charge method.
  • Consists of a method for measuring the concentration of the form the method measures the current over time after the application of the potential and before the steady state is reached, from which the diffusion coefficient and/or the diffusion coefficient of the reduced (or oxidized) form of the redox species ) It discloses a configuration for obtaining a value indicating the concentration.
  • the conventional electrochemical in vitro diagnostic device for detecting biomarkers is a complex reaction in which three reactions, such as an antigen-antibody immune reaction, an enzyme catalytic reaction, and an electrochemical reaction, occur at one electrode, despite these advantages. It is difficult to secure reproducibility and it is not a great substitute for optical in vitro diagnostic devices.
  • the immune response is an antigen-antibody immune response that immobilizes the antibody when the biosensor is produced and reacts with the antigen of the sample sample, so that reaction conditions such as reaction temperature and reaction time can be well controlled to ensure accuracy and reproducibility.
  • reaction conditions such as reaction temperature and reaction time
  • an object of the present invention is to provide a method for measuring an electrochemical biosensor capable of improving the accuracy and reproducibility of measurement in a time-current method or a time-charge method in an in vitro diagnostic device using an electrochemical biosensor.
  • Method for measuring an electrochemical biosensor for achieving the above object is a step of dipping the electrode of the electrochemical biosensor in a solution containing a substrate; And after applying the voltage to the electrode for a certain period of time, apply a voltage at a specific point in time (1) to measure the amount of charge for a certain period of time by a time-charge method, or apply a voltage to the electrode for a period of time to set a point in time (2). And measuring the flowing current value by a time-current method.
  • the specific point in time (1) is after the electrode is immersed in a solution containing a substrate and a voltage is not applied to the electrode for a certain period of time.
  • 2) is characterized in that after immersing the electrode in a solution containing a substrate and applying a voltage to the electrode for a certain period of time.
  • the electrode is characterized in that it is an ITO electrode.
  • the ITO electrode is characterized in that the antibody for binding the antigen and the marker antibody of the sample sample is immobilized.
  • the marker antibody is characterized in that it is combined with a reactive enzyme.
  • the reaction enzyme is characterized in that it is an alkaline phosphatase (alkaline phosphatase).
  • the alkaline phosphatase reacts with the substrate solution ascorbic acid 2-phosphate (AAP) to produce ascorbic acid.
  • the ascorbic acid is accumulated on the surface of the electrode.
  • the predetermined time is 2 minutes.
  • the amount of charge for 50 seconds is measured by a time charge method after a predetermined time of 2 minutes.
  • the electrode in measuring an electrochemical biosensor for measuring a biomarker by a time-current method or a time-charge method, the electrode is immersed in a measurement solution and a voltage is not applied to the electrode for a certain time before the current value or charge value is measured.
  • a voltage is not applied to the electrode for a certain time before the current value or charge value is measured.
  • 1 is a graph showing the results of measuring the change according to the enzyme concentration according to the present invention by a time-current method.
  • Figure 2 is a graph showing the change in measurement value over time immersed in a substrate solution without applying a voltage according to the present invention.
  • Example 1 Change according to the enzyme concentration measured by the time current method
  • the factors that determine the rate are kcat, the reaction rate constant, and [ALP] 0, which is the initial concentration of the enzyme.
  • kcat is mainly determined by the reaction temperature, so the enzyme catalyst reaction rate is determined by [ALP] 0 when the measurement temperature is the same.
  • [ALP] 0 is the amount of the enzyme ALP (alkaline phosphatase) attached to an antibody bound by an antigen-antibody immune response to an indium tin oxide (ITO) electrode. Since this amount is proportional to the amount of the antigen that is a biomarker, the concentration of the biomarker is detected by measuring the amount of ALP.
  • the enzyme catalytic reaction experiment was performed using ALP and AAP (ascorbic acid 2-phosphate) as the reaction enzyme and measurement substrate solution, immobilizing the antibody, and using ITO as the electrode to cause the electrochemical reaction.
  • ALP and AAP ascorbic acid 2-phosphate
  • ITO as the electrode to cause the electrochemical reaction.
  • the amount of ALP immobilized on the ITO electrode was changed by changing the concentration of ALP used during the immobilization reaction.
  • the current value measured by the time-current method stabilized after about 30 seconds, and the stabilized value obtained an experimental result that increased with increasing concentration. From this result, it can be confirmed that the reaction rate of the enzyme catalyst reaction is proportional to [ALP] 0 , shows a large current value at the beginning of the measurement, and gradually decreases, and maintains a constant value after a certain time. That is, it can be seen that if the measurement point is adopted within 30 seconds, the measurement value changes, making accurate measurement difficult.
  • the enzyme ALP will first react with the substrate AAP to continuously produce AA (ascorbic acid) to build up on the surface of the ITO electrode.
  • AA ascorbic acid
  • the electrochemical reaction does not occur, so that as much as possible is accumulated in the ITO electrode until AA is saturated.
  • AA accumulated on the surface decreases and the overall reaction stabilizes.
  • the change in the current value was measured by the time-current method using the time immersed in the AAP substrate solution without applying a voltage to the electrode.
  • the voltage applied between the working electrode and the counter electrode was 0.45V from the Ag/AgCl reference electrode.
  • Example 2 Change in measured value over time immersed in a substrate solution without applying a voltage
  • Figure 2 is a graph showing the change in measurement value over time immersed in a substrate solution without applying a voltage according to the present invention, the time current measured while changing the time to immerse without applying a voltage from 5 seconds to 7 minutes Shows the current value of the law. From the results of this experiment, it can be seen that the reaction rate increases in proportion to the time until the soaking time is 2 minutes and does not change after about 2 minutes. That is, it is determined that AA generated on the surface of the ITO electrode is saturated at a time of about 2 minutes (product saturation time required) and reaches the maximum value.
  • the results of these experiments provide important implications for the development of in vitro diagnostic devices that test biomarkers.
  • the time for immersion in the substrate solution without applying a voltage during the measurement should be constant or sufficient time for more than 2 minutes.
  • the concentration of the biomarker can be accurately and reproducibly measured by electrochemical method only after the minimum stabilization time of 50 seconds.
  • the time-current method and the time-charge method are the most used methods for measuring the electrochemical reaction in an electrochemical biosensor.
  • the measurement methods of in vitro diagnostic devices using electrochemical biosensors applied to each of these two measurement methods are as follows.
  • the measured value changes within 50 seconds.
  • the electrode is immersed in a substrate solution, and the voltage is applied for at least 50 seconds, and then a current value flowing at a specific time is measured. Since the soaking time of 50 seconds may vary depending on the enzyme, substrate solution, and electrode used, the time taken to converge after measuring the change in the measured value according to the time to soak in the substrate solution without applying voltage as shown in FIG. Determine and apply to the measurement method.
  • the amount of charge for a certain period of time must be measured after sufficient time for the product produced by the enzymatic reaction to saturate on the electrode surface.
  • the electrode is immersed in the substrate solution for 2 minutes, so that it is saturated (3 minutes, 5 minutes, and 7 minutes show the same current value as 2 minutes). After soaking for 2 minutes, measure the amount of charge during the stabilization time of 50 seconds. If it is, the enzyme concentration on the electrode surface can be measured accurately and reproducibly. Since the saturation time of 2 minutes can also vary depending on the enzyme, substrate solution, and electrode used, as shown in Figure 2, the change in the measured value over time in the substrate solution is measured and then applied to the measurement method.
  • the present invention it is possible to increase the accuracy and reproducibility of the electrochemical biosensor by controlling the measurement time during measurement by measuring the rate of the enzyme catalytic reaction and the electrochemical reaction.
  • the miniaturization and low cost of in vitro diagnostic devices are suitable for on-site diagnosis and self-diagnosis, and provide an appropriate electrochemical measurement method to simplify the measurement circuit.

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

Abstract

La présente invention concerne une méthode de mesure destinée à un biocapteur électrochimique qui détecte, au moyen d'un principe de mesure de signal électrochimique, un biomarqueur inclus dans le sang, la miction, la salive, etc., et plus précisément, une méthode de mesure d'un biocapteur électrochimique permettant d'améliorer la précision et la reproductibilité de la mesure par une mesure au moyen de la chronoampérométrie ou de la chronocoulométrie dans un dispositif de diagnostic in vitro à l'aide d'un biocapteur électrochimique. Selon la présente invention, lors de la mesure, par chronoampérométrie ou chronocoulométrie, d'un biocapteur électrochimique afin de mesurer un biomarqueur, un excellent effet d'amélioration de la précision et de la reproductibilité des résultats de mesure, par la mesure d'une valeur de charge électrique après l'immersion d'une électrode dans une solution de mesure et la non-application d'une tension à l'électrode pendant une certaine période de temps, ou par la mesure d'une valeur de courant électrique après l'application d'une tension pendant une certaine période de temps, est obtenu.
PCT/KR2019/010455 2018-12-03 2019-08-19 Méthode de mesure destinée à un biocapteur électrochimique Ceased WO2020116743A1 (fr)

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KR1020180153276A KR102178379B1 (ko) 2018-12-03 2018-12-03 전기화학식 바이오 센서의 측정방법
KR10-2018-0153276 2018-12-03

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004132954A (ja) * 2002-06-25 2004-04-30 Bayer Ag 一個または複数個の分析物を検出するための方法および装置、ならびに装置の使用
JP2007108171A (ja) * 2005-09-30 2007-04-26 Lifescan Inc 迅速な電気化学的分析のための方法および装置
KR20070089906A (ko) * 2004-07-22 2007-09-04 타겟젠, 아이엔씨. 전기화학적 검출을 위한 방법 및 장치
US20110155589A1 (en) * 2009-12-30 2011-06-30 Lifescan, Inc. Systems, Devices, and Methods for Improving Accuracy of Biosensors Using Fill Time
KR20160032974A (ko) * 2014-09-17 2016-03-25 주식회사 아이센스 생체시료 내 분석대상물질의 농도측정방법 및 측정장치

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5243516A (en) * 1989-12-15 1993-09-07 Boehringer Mannheim Corporation Biosensing instrument and method
AUPN363995A0 (en) 1995-06-19 1995-07-13 Memtec Limited Electrochemical cell
JP5696283B2 (ja) * 2010-07-28 2015-04-08 株式会社船井電機新応用技術研究所 酵素電極の製造方法
KR20120125898A (ko) * 2011-05-09 2012-11-19 부산대학교 산학협력단 아스코르브산 포스페이트를 이용한 전기화학적 바이오센서

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004132954A (ja) * 2002-06-25 2004-04-30 Bayer Ag 一個または複数個の分析物を検出するための方法および装置、ならびに装置の使用
KR20070089906A (ko) * 2004-07-22 2007-09-04 타겟젠, 아이엔씨. 전기화학적 검출을 위한 방법 및 장치
JP2007108171A (ja) * 2005-09-30 2007-04-26 Lifescan Inc 迅速な電気化学的分析のための方法および装置
US20110155589A1 (en) * 2009-12-30 2011-06-30 Lifescan, Inc. Systems, Devices, and Methods for Improving Accuracy of Biosensors Using Fill Time
KR20160032974A (ko) * 2014-09-17 2016-03-25 주식회사 아이센스 생체시료 내 분석대상물질의 농도측정방법 및 측정장치

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KR20200066772A (ko) 2020-06-11

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