WO2002018924A1 - Procede de determination d'un substrat et biodetecteur - Google Patents

Procede de determination d'un substrat et biodetecteur Download PDF

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Publication number
WO2002018924A1
WO2002018924A1 PCT/JP2000/005789 JP0005789W WO0218924A1 WO 2002018924 A1 WO2002018924 A1 WO 2002018924A1 JP 0005789 W JP0005789 W JP 0005789W WO 0218924 A1 WO0218924 A1 WO 0218924A1
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WO
WIPO (PCT)
Prior art keywords
sample
acid
electrode
substrate
biosensor
Prior art date
Application number
PCT/JP2000/005789
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English (en)
Japanese (ja)
Inventor
Naoki Shinozuka
Toru Yokoyama
Kenji Nakamura
Original Assignee
Sapporo Immuno Diagnostic Laboratory
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sapporo Immuno Diagnostic Laboratory filed Critical Sapporo Immuno Diagnostic Laboratory
Priority to PCT/JP2000/005789 priority Critical patent/WO2002018924A1/fr
Priority to AU2000267324A priority patent/AU2000267324A1/en
Publication of WO2002018924A1 publication Critical patent/WO2002018924A1/fr

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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
    • 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
    • C12Q1/004—Enzyme electrodes mediator-assisted

Definitions

  • the present invention relates to a method and a biosensor for specifically and simply quantifying a substrate in a sample without requiring complicated pretreatment of the sample.
  • a reaction reagent comprising at least a dehydrogenase, a coenzyme, an electron mediator, and a tetrazolium salt was immobilized on an absorbent carrier and placed on an electrode formed on the same plane using a conductive material.
  • a method for quantifying a substrate and a biosensor are provided. Background of the Invention
  • a biosensor is a technology that enables quantification by combining a molecular recognition function that specifically reacts with a substrate contained in a sample, a transducer that can electrically convert information, and electronics technology.
  • enzyme sensors immunosensors, DNA sensors, etc.
  • enzyme sensors have been the most studied since ancient times and widely used.
  • Enzyme sensors are also classified into reaction principles using oxidases or dehydrogenases. Enzyme sensors using oxidases have been applied to many blood glucose measurement sensors. However, an enzyme sensor using an oxidase is greatly affected by dissolved oxygen, which poses a major problem when measuring low-concentration regions. For this reason, enzymatic sensors using dehydrogenase that are not affected by dissolved oxygen are being actively studied.
  • biosensor that integrates oxidized nicotinamide adenine dinucleotide (NAD +) of various dehydrogenases and coenzymes and 1-methoxy PMS of electron mediator with an electrode as a reaction reagent.
  • NAD + oxidized nicotinamide adenine dinucleotide
  • 1-methoxy PMS 1-methoxy PMS of electron mediator with an electrode as a reaction reagent.
  • a biosensor using a reagent consisting of a tetrazolium salt has been developed (PC TZ JP99 / 0 1 3 9 2). Compared to biosensors using reduced coenzyme direct oxidation or various electronic media, this biosensor ultimately produces chemically stable formazan, resulting in less responsive response. A current is obtained. In addition, a large increase in response current and an improvement in detection sensitivity were observed, enabling highly sensitive quantification of the substrate in a low concentration range.
  • At least a dehydrogenase, a coenzyme, an electron mediator and a tetrazolium salt are formed on at least an electrode composed of a working electrode and a counter electrode formed on the same plane using a conductive material. It is intended to provide a method for quantifying a substrate and a biosensor on which an absorptive carrier in which a reaction reagent consisting of:
  • the biosensor of the present invention is on the same plane
  • the electrode system is formed in a simple manner.Since it has a simple structure in which an absorptive carrier on which all the reaction reagents are immobilized is simply laminated on the electrodes, the biosensor can be easily manufactured, Production has become possible. Description of the invention
  • an electrode system comprising at least a working electrode and a counter electrode formed on the same plane using a conductive material, and absorbing a reaction reagent comprising at least a dehydrogenase, a coenzyme, an electron mediator, and a tetrazolium salt.
  • the reducing coenzyme is generated by a specific enzymatic reaction of a substrate in a sample with a dehydrogenase and a coenzyme as reaction reagents, and further, a redox reaction with an electron mediator and a tetrazolium salt. Progress and eventually produce chemically stable formazan. Subsequently, when a potential is applied to the electrode, formazan is oxidized, and the concentration of the substrate in the sample can be determined from the obtained oxidation current.
  • the feature of the present invention is that by disposing the absorbent carrier on both electrodes formed on the same plane on the insulating substrate, an effect of the filter by the absorbent carrier can be obtained, and the measurement contained in the sample can be performed. Separation of the influential solid components etc. becomes possible, and only the separated sample reaches both electrodes. Further, the separation effect can be further improved by using several kinds of absorbent carriers and gradually reducing the pore size. In addition, since it has a simple structure in which an absorbent carrier is simply laminated on the electrode, the biosensor can be easily manufactured and mass production is possible. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a method for manufacturing an electrode according to one embodiment of the present invention
  • FIG. 2 is a configuration diagram of a method (1) for manufacturing a sensor chip in Example 2
  • FIG. 3 is a configuration diagram of a method (2) of manufacturing a sensor chip in Example 3.
  • FIG. It is a block diagram of the manufacturing method (3).
  • FIG. 5 is a configuration diagram of a method (4) for manufacturing a sensor chip in Example 5
  • FIG. 6 is a rough drawing showing a result of an addition test of glucose in saliva in Example 6;
  • FIG. 7 is a graph showing the results of a dilution test of glucose in saliva in Example 7.
  • the structures of the substrate to be measured, the reaction reagent, and the sensor chip will be described in order.
  • the measurable substrate of the present invention is not particularly limited as long as it can generate a reduced coenzyme using dehydrogenase as a catalyst, and any substrate can be quantified.
  • alanine, alcohol, aldehyde, isochenoic acid, peridine-5, -diphosphoglucose, galactose, formic acid, dariceraldehyde-13-monophosphate, glycerol, glycerol-13-phosphate, glucose, glucose-16 —Phosphoric acid, glutamic acid, cholesterol, sarcosine, sorbitol, carbonic acid, lactic acid, 3-hydroxybutyric acid, pyruvic acid, phenylalanine, flux 1, 6-phosphogluconic acid, formaldehyde, mannitol, malic acid, mouth isocyanate Etc. are available.
  • the dehydrogenase used in the present invention is not particularly limited as long as it is an enzyme that produces a reduced coenzyme, and the origin is not particularly limited.
  • alanine dehydrogenase alcohol dehydrogenase, aldehyde dehydrogenase, isocenoic acid dehydrogenase, peridine-1,5-diphosphoglucose dehydrogenase, galactose dehydrogenase, formate dehydrogenase, darisel Aldehyde-13-phosphate dehydrogenase, glycerol dehydrogenase, glycerol-13-phosphate dehydrogenase, glucose dehydrogenase, glucose-16-phosphate dehydrogenase, glutamate dehydrogenase, kore Sterol dehydrogenase, sarcosine dehydrogenase, sorbitol dehydrogenase, carbonic acid dehydrogenase, lactate
  • the electron mediator is a substance that promptly undergoes an oxidative reduction reaction with a reduced coenzyme and a tetrazolium salt.
  • the electron mediator for example, quinones, diaphorases, cytochromes, porogens, phenazines, 'phenoxazines, phenothiazines, ferricyanides, ferredoxins, phenocens and derivatives thereof can be used.
  • phenazines show stable response here, and in particular, 1-methoxy PMS has good storage stability and excellent reactivity with reduced coenzyme and tetrazolium salt. —I found it to be a good evening.
  • the tetrazolium salt is not particularly limited as long as it forms formazan, and among them, 2- (4-1-dophenyl) -1,3- (412-trophenyl) 1,5-1 (2,4-disulfophenyl) 1-1 2H-tetrazolium, monosodium salt (WST-1) is a water-soluble and chemically stable formazane produced upon reduction, and the produced formazan shows good response in electrochemical detection methods From this, it has been found that the tetrazolium salt in the present invention is preferable.
  • the electrode used in the present invention is a conductive substance and is not particularly limited as long as it is electrochemically stable.
  • the material is carbon, gold, silver, silver / silver chloride, nickel, platinum, platinum black, and platinum black. Palladium, etc., and their alloys can be used. As a result of examining various materials among them, they found that carbon materials are inexpensive and chemically stable, and are preferable as working electrodes of the electrodes in the present invention.
  • the carbon material means all materials containing carbon.
  • the carbon materials that can be used are not particularly limited, and are used in conventional carbon electrodes. Any material can be used, for example, pon fiber, carbon black, bon paste, glass, bonbon, graphite, and the like.
  • Such a carbon material is formed as an electrode portion on an insulating support by a conventional method. Usually, it can be formed by printing a paste made from a carbon fiber material using a resin binder or the like and then heating and drying the paste.
  • the printing method is not particularly limited to screen printing, and gravure printing, offset printing, inkjet printing, and the like can be applied.
  • the counter electrode also serves as the reference electrode, silver / silver silver is selected here.
  • the lead that is the connection between the electrode reaction part and the electrochemical detection circuit is the most conductive and inexpensive silver. Was selected and formed by printing.
  • a two-electrode system including only the working electrode and the counter electrode is used.
  • a three-electrode system including a reference electrode is used, more accurate quantification can be performed.
  • the insulating support examples include glass, glass epoxy, ceramics, and plastic.
  • plastic films such as polyester, polyethylene, polyethylene terephthalate (PET), polystyrene, and polypropylene are inexpensive, and PET films are preferred here because of their good adhesion to conductive inks and good workability. I found it.
  • the cover, spacer, and the material that adheres to it can be shaped as long as the sample can be retained, the absorbent carrier can be retained, there is no hindrance to the series of reactions, and external contact with the reaction area can be protected. There are no particular restrictions on the material, etc.
  • the absorbent carrier used in the present invention is not particularly limited as long as it is a material that can absorb the reaction reagent solution and does not affect the reaction reagent.
  • the material include glass fiber, silica fiber, and cellulose fiber. Fibers and carboxymethylcellulose, getylaminoethylcellulose, cellulose acetate, cellulose mixed ester, nylon woven fabric, nitrocellulose, polypropylene, polyether sulfone, polyester nonwoven fabric, polytetrafluoroethylene (PTFE), Polypropylene or the like can be used.
  • the three types of absorbent carriers of the absorption layer, the spreading layer, and the reaction layer are respectively It uses polypropylene, cellulose fiber, and nitrocellulose, and forms a reaction reagent layer by layering them.
  • a single-layer structure is also possible, in which case the constituent materials can be reduced.
  • Example 1 Method for producing electrode
  • FIG. 1 shows the electrode manufactured in this example. It should be noted that this electrode can be manufactured more easily by screen printing two types of electrodes on the same plane.
  • FIG. 2 shows the sensor chip manufactured in this example.
  • This sensor chip is composed of the electrode prepared in Example 1 and three kinds of absorbent carriers.
  • the sample is supplied into the sensor chip by bringing the sample into contact with the supply port 7 of the force par 6. It is a structure that can be used.
  • a polypropylene membrane (manufactured by Nippon Millipore Co., Ltd.) is used as the absorbent carrier 8 of the absorption layer, and a buffer component for adjusting the optimum pH of the enzyme reaction is absorbed in a solution and dried (at 40 ° C). , 15 minutes).
  • a nitrocellulose membrane (manufactured by Nippon Millipore Co., Ltd.) was used as the absorbent carrier 10 in the reaction layer, and a tetrazolium salt WST-1 (manufactured by Dojin Chemical Lab.) And a dehydrogenase (Toyobo) Co., Ltd.) and oxidized nicotinamide adenine dinucleotide (NAD '+) (manufactured by Oriental Co., Ltd.) as a coenzyme were dissolved in a phosphate buffer solution (pH 7.4, 2 O mM). After absorption, drying (40. C, 10 minutes), the cells were immobilized.
  • a phosphate buffer solution pH 7.4, 2 O mM
  • FIG. 3 shows the sensor chip manufactured in this example.
  • the sensor chip is composed of the electrode prepared in Example '1 and one kind of absorbent carrier', and the sample is supplied into the sensor chip by bringing the sample into contact with the supply port 7 on the side of the sensor chip. It is a structure that can do it.
  • Buffer solution, 1-methoxy PMS, WST-1 and enzyme are absorbed into each part of the nitrocellulose membrane of absorbent carrier 10 and dried (40 ° C, 10 minutes) and fixed. It has become.
  • Example 4 Manufacturing method of sensor chip (3)
  • FIG. 4 shows the sensor chip manufactured in this example.
  • This sensor chip is composed of the electrode prepared in Example 1 and three types of absorbent carriers.
  • the sample is immediately supplied into the sensor chip. Do Can be.
  • Example 5 Sensor chip fabrication method (4)
  • FIG. 5 shows the sensor chip manufactured in this example.
  • This sensor chip is composed of the electrode prepared in Example 1 and three types of absorbent carriers. By bringing the sample into contact with the supply port 7 on the side of the sensor chip, the sample can be supplied into the sensor chip. It has a structure that can be used.
  • Example 6 Addition test of gafflecose in saliva
  • FIG. 6 shows the results of measurement using a biosensor produced using glucose dehydrogenase in Example 2 and saliva samples adjusted to various concentrations by adding glucose to saliva.
  • a saliva sample was added 70, and after 60 seconds, a potential of +50 OmV was applied with reference to the counter electrode, and the response current value was measured 7 seconds after the potential was applied. This result shows the concentration obtained by calculating the obtained response current value by the conversion formula.
  • FIG. 7 shows the results of measurement using the biosensor prepared in Example 6 and a sample obtained by diluting saliva having a high glucose concentration with physiological saline.
  • Example 6 In the same manner as in the measurement of Example 6, a sample containing glucose was immersed in 70 L, and after 60 seconds, a potential of +50 OmV was applied with reference to the counter electrode. The response current value was measured. This result shows the concentration obtained by calculating the obtained response current value by the conversion formula.

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  • Chemical & Material Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Immunology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détermination d'un substrat dans lequel un substrat dans un échantillon est déterminé par arrangement d'un biodétecteur ayant une structure dans laquelle des électrodes sont formées sur le même plan par l'utilisation de matériaux électroconducteurs, et des réactifs de réaction comprenant au moins une déshydrogénase, une coenzyme ainsi qu'un médiateur électronique et un sel de tétrazolium sont fixés sur des supports absorbants et arrangés sur ou au-dessus des électrodes, et par détection électrochimique d'un formazan produit par l'alimentation en un échantillon; ainsi qu'un biodétecteur utilisé dans ce procédé. Le procédé et le biodétecteur peuvent être utilisés pour déterminer un substrat dans un échantillon de manière spécifique, rapide et avec facilité, sans prétraitement compliqué de l'échantillon.
PCT/JP2000/005789 2000-08-28 2000-08-28 Procede de determination d'un substrat et biodetecteur WO2002018924A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2000/005789 WO2002018924A1 (fr) 2000-08-28 2000-08-28 Procede de determination d'un substrat et biodetecteur
AU2000267324A AU2000267324A1 (en) 2000-08-28 2000-08-28 Method for determination of substrate and biosensor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2000/005789 WO2002018924A1 (fr) 2000-08-28 2000-08-28 Procede de determination d'un substrat et biodetecteur

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086453A1 (fr) 2001-04-20 2002-10-31 Sapporo Immuno Diagnostic Laboratory Instrument servant a prelever et a recuperer une secretion liquide d'une cavite orale
EP3561507A4 (fr) * 2016-12-21 2020-09-30 Dongwoon Anatech Co., Ltd. Dispositif de diagnostic utilisant la salive et procédé de diagnostic correspondant
CN114527266A (zh) * 2021-12-31 2022-05-24 攸太科技(台州)有限公司 尿液电化学检测方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5298414A (en) * 1989-05-12 1994-03-29 British Technology Group Limited Detection of morphine using morphine dehydrogenase
JPH09286784A (ja) * 1996-04-18 1997-11-04 Doujin Kagaku Kenkyusho:Kk 新規水溶性テトラゾリウム塩化合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5298414A (en) * 1989-05-12 1994-03-29 British Technology Group Limited Detection of morphine using morphine dehydrogenase
JPH09286784A (ja) * 1996-04-18 1997-11-04 Doujin Kagaku Kenkyusho:Kk 新規水溶性テトラゾリウム塩化合物

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086453A1 (fr) 2001-04-20 2002-10-31 Sapporo Immuno Diagnostic Laboratory Instrument servant a prelever et a recuperer une secretion liquide d'une cavite orale
EP3561507A4 (fr) * 2016-12-21 2020-09-30 Dongwoon Anatech Co., Ltd. Dispositif de diagnostic utilisant la salive et procédé de diagnostic correspondant
EP4227678A1 (fr) * 2016-12-21 2023-08-16 Dongwoon Anatech Co., Ltd. Appareil de diagnostic et procédé de diagnostic pour des mesures sur la salive
EP4227679A1 (fr) * 2016-12-21 2023-08-16 Dongwoon Anatech Co., Ltd. Appareil de diagnostic et procédé de diagnostic pour des mesures sur la salive
CN114527266A (zh) * 2021-12-31 2022-05-24 攸太科技(台州)有限公司 尿液电化学检测方法

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