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WO2012133633A1 - Détecteur de lysine jetable - Google Patents

Détecteur de lysine jetable Download PDF

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Publication number
WO2012133633A1
WO2012133633A1 PCT/JP2012/058334 JP2012058334W WO2012133633A1 WO 2012133633 A1 WO2012133633 A1 WO 2012133633A1 JP 2012058334 W JP2012058334 W JP 2012058334W WO 2012133633 A1 WO2012133633 A1 WO 2012133633A1
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WO
WIPO (PCT)
Prior art keywords
lysine
disposable
oxidase
electrode
measurement electrode
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/JP2012/058334
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English (en)
Japanese (ja)
Inventor
武司 上村
希 稲葉
加成恵 板岡
悦夫 篠原
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Techno Medica Co Ltd
Original Assignee
Techno Medica Co Ltd
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 Techno Medica Co Ltd filed Critical Techno Medica Co Ltd
Publication of WO2012133633A1 publication Critical patent/WO2012133633A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
    • 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
    • G01N33/02—Food

Definitions

  • the present invention relates to a disposable sensor for measuring lysine.
  • Lysine which is one of the essential amino acids, is an important amino acid that repairs body tissues and participates in the growth of the body. If it is deficient, it may cause growth disorders. As described above, lysine is the most important amino acid even though it is a very important amino acid. Lysine is not so much contained in vegetable protein, but is abundant in dairy products such as milk and cheese, and soy, meat, and fish. If it is not ingested, it will be deficient in the body, resulting in modern malnutrition. As mentioned above, lysine is an important item in diagnosing modern malnutrition, but malnutrition is usually diagnosed by examining the amount of albumin. There is no lysine measurement for the diagnosis.
  • a biosensor using a metal complex such as potassium ferricyanide or an organic compound as an electron acceptor has been proposed.
  • the substrate to be measured and the enzyme The substrate concentration is obtained from the oxidation current value by oxidizing the reduced form of the electron acceptor generated by the enzymatic reaction with the electrode at the electrode (see Patent Document 1).
  • the substrate concentration is obtained from the oxidation current value by oxidizing the reduced form of the electron acceptor generated by the enzymatic reaction with the electrode at the electrode (see Patent Document 1).
  • the inventors focused on the above-mentioned present situation, and conducted intensive research on a biosensor capable of measuring lysine, and found an optimal combination of lysine oxidase and electron acceptor for measuring lysine in a disposable sensor.
  • An object of the present invention is to provide a disposable sensor capable of measuring lysine.
  • a disposable lysine sensor forms an electrode system having at least a measurement electrode and a counter electrode on an electrically insulating substrate, and reacts with an enzyme, an electron acceptor and a sample solution.
  • a sensor that electrochemically detects a change in substance concentration with the electrode system and measures the substrate concentration of the sample solution, lysine oxidase and ferricyan ion that reacts with lysine using the lysine oxidase as a catalyst are held on the measurement electrode.
  • the reaction layer is formed.
  • the lysine oxidase can be composed of, for example, a mold-derived enzyme, specifically, an enzyme derived from a Trichoderma bacterium, and more specifically, an enzyme derived from Trichoderma viridae.
  • a second measurement electrode may be provided on the electrically insulating substrate, and a reaction layer not containing lysine oxidase holding ferricyanide that reacts with lysine using lysine oxidase as a catalyst may be formed on the second measurement electrode. Good.
  • a second measurement electrode is provided on the electrically insulating substrate, and a reaction layer holding a protein having no lysine oxidase activity and a ferricyan ion that reacts with lysine using lysine oxidase as a catalyst is formed on the second measurement electrode.
  • the protein having no lysine oxidase activity may be, for example, inactivated lysine oxidase.
  • the disposable lysine sensor forms an electrode system having at least a measurement electrode and a counter electrode on an electrically insulating substrate, and electrochemically changes a substance concentration during reaction of an enzyme, an electron acceptor and a sample solution.
  • a reaction layer holding lysine oxidase and ferricyanide that reacts with lysine using the lysine oxidase as a catalyst is formed on the measurement electrode. Therefore, lysine can be easily measured. This makes it possible to measure lysine even in diagnosis in general internal medicine and the like, so that more accurate diagnosis of malnutrition can be performed.
  • the inventors use an enzyme derived from mold as the lysine oxidase, specifically an enzyme derived from Trichoderma genus, more specifically an enzyme derived from Trichoderma viridae, and electron accepting. It was confirmed that favorable results were obtained by using ferricyan ion as a body. Further, by providing a second measurement electrode on the electrically insulating substrate, and forming a reaction layer not containing lysine oxidase holding ferricyan ion that reacts with lysine using lysine oxidase as a catalyst on the second measurement electrode.
  • a second measurement electrode is provided on the electrically insulating substrate, and a protein having no lysine oxidase activity (for example, inactivated lysine oxidase) and ferricia that reacts with lysine using lysine oxidase as a catalyst.
  • a protein having no lysine oxidase activity for example, inactivated lysine oxidase
  • ferricia that reacts with lysine using lysine oxidase as a catalyst.
  • FIG.3 (b), it is the graph which showed the lysine low concentration side.
  • 1 is a schematic development view of a disposable lysine sensor according to the present invention.
  • (A)-(e) is the schematic which shows the manufacturing method of a disposable lysine sensor.
  • (A) is a schematic top view of a disposable lysine sensor and a portable analyzer
  • (b) is a schematic top view which shows the state which mounted
  • FIG. 1 is a diagram showing a measurement principle in a disposable lysine sensor according to the present invention.
  • the disposable lysine sensor according to the present invention uses lysine oxidase and ferricyan ion as an electron acceptor. Then, lysine as a substrate contained in blood is oxidized by lysine oxidase, and by transferring electrons to ferricyan ion as an electron acceptor via lysine oxidase, ferricyan ion is reduced and ferrocyan ion And the concentration of lysine is measured based on the oxidation response current that flows when ferrocyanic ions are oxidized on the working electrode.
  • curve a shows the potential (V) and current density (A / cm 2) when 5 mM ferricyanide was deposited on the working electrode and a lysine solution having a concentration of 5 ⁇ mol / dL was added thereto. Showing the relationship.
  • curve b shows the potential when 5 mM ferricyanide is deposited on the working electrode, and a 5 ⁇ mol / dL lysine solution and 1 U / ml fungal lysine oxidase are added thereto.
  • the relationship between (V) and current density (A / cm 2) is shown.
  • a lysine oxidase a lysine oxidase derived from Trichoderma viridae (EC number: 1.4.3.14 sold by Sigma-Aldrich Japan Co., Ltd.), which is an enzyme derived from mold, was used. From the curves a and b, when the reaction proceeds, ferricyan ions are consumed in the vicinity of the electrode to generate ferrocyanide ions. By adding lysine oxidase to this, ferricyan ions function as a mediator of lysine oxidase, It can be confirmed that the current value on the oxidation side is increased because ferrocyanic ions are generated.
  • the redox peak derived from ferricyan ion could be confirmed in the lysine system as well as the glucose system. From this result, it was confirmed that the ferricyan ion functions as an electron acceptor of lysine oxidase.
  • Curves c and d show cyclic voltammograms by a reaction that does not depend on ferricyan ion (by hydrogen peroxide), respectively. From the curves c and d, it can be confirmed that hydrogen peroxide generated in this reaction does not greatly affect the current value on the oxidation side.
  • FIG. 3 (a) and FIG. 3 (b) show the results of experiments conducted to confirm what kind of current response is observed in the reaction system of lysine and lysine oxidase as the lysine concentration is increased.
  • the measurement conditions are as follows.
  • FIG. 3A shows a change in current value when lysine is sequentially added
  • FIG. 3B shows a current value according to a change in concentration due to the addition of lysine.
  • FIGS. 3A and 3B it was confirmed that the current value increased stepwise as the amount of lysine added was increased.
  • FIG. 4 is an enlarged graph of 150 nmol / ml or less assumed to be a practical concentration region in the test results shown in FIG.
  • the current value increased stepwise as the amount of lysine added was increased, so that a current response due to the reaction between lysine and lysine oxidase could be confirmed. Moreover, it has confirmed that the electric current value was correlated with the addition amount of lysine.
  • FIG. 5 is a schematic development view of a disposable lysine sensor according to the present invention
  • FIGS. 6A to 6E are schematic views showing a manufacturing method of a disposable lysine sensor.
  • Reference numeral 1 in the drawing denotes an insulating substrate.
  • substrate 1 After printing gold
  • the first measurement electrode 2 and the second measurement electrode 3 are formed at positions that are symmetrical with respect to the counter electrode 4.
  • a photoresist film 8 is formed so as to cover portions of the substrate 1 other than the electrode portions 2a, 3a, 4a and the terminal portions 2b, 3b, 4b of the first measurement electrode 2, the second measurement electrode 3, and the counter electrode 4.
  • a polymer solution in which lysine oxidase, a surfactant, and ferricyan ions are dissolved is applied and dried on the electrode 2a of the first measurement electrode 2, and the first reaction layer 6 is formed on the electrode 2a of the first measurement electrode 2.
  • the same amount of the polymer solution in which the same amount of the surfactant and ferricyan ion as the first reaction layer 6 is applied is dried.
  • a second reaction layer 7 is formed on 3a (FIG. 6C).
  • a spacer 9 provided with a notch 9a for forming a specimen channel 12 is disposed on the photoresist film 8 (FIG. 6D), and a cover having an air hole 10 formed thereon.
  • the disposable lysine sensor is completed by providing 11 (FIG. 6E).
  • a notch 9 a of the spacer 9 surrounds the electrode portions 2 a, 3 a, 4 a of the first measurement electrode 2, the second measurement electrode 3 and the counter electrode 4, and the air hole 10 of the cover 11, and is arranged on the substrate 1.
  • the end portion located on the opposite side of the terminal portions 2b, 3b, and 4b is opened to form the sample introduction port 13.
  • the spacer 9 has a thickness that forms a specimen flow path 12 having a height that causes the capillary action of the specimen between the photoresist film 8 and the cover 11, specifically, for example, about 0.3 mm. It is thickness.
  • the disposable lysine sensor configured as described above is used by being mounted on a portable analyzer, for example.
  • 7A is a schematic top view of a disposable lysine sensor and a portable analyzer
  • FIG. 7B is a schematic top view showing a state in which the disposable lysine sensor is mounted on the portable analyzer
  • FIG. 8 is a portable analyzer. It is a block diagram which shows roughly the internal process of an apparatus.
  • symbol A indicates a disposable lysine sensor
  • symbol B indicates a portable analyzer
  • symbol 21 indicates a housing of the portable analyzer
  • symbol 22 indicates a display provided on the upper surface of the housing 21
  • Reference numeral 23 denotes an operation switch provided on the upper surface of the housing 21
  • reference numeral 24 denotes a sensor insertion opening provided at one end of the housing 21
  • reference numeral 25 denotes an output terminal.
  • FIG. 8 is a block diagram schematically showing the internal processing of the portable analyzer.
  • Reference numerals 26, 27, and 28 denote terminals connected to the terminal portions 2b, 3b, and 4b of the disposable lysine sensor, and reference numeral 29 denotes A control device for calculating the amount of lysine based on input signals from the terminals 26, 27 and 28, reference numeral 30 denotes a storage device, reference numeral 22 denotes the display, and reference numeral 25 denotes the output terminal.
  • the user inserts the prepared disposable lysine sensor A into the insertion port 24 of the portable analyzer B.
  • the terminal portions 2b, 3b, 4b of the disposable lysine sensor A are connected to the terminals 26, 27, 28 provided inside the portable analyzer B.
  • the user stabs his / her finger with a needle or the like to cause bleeding, and the blood is brought into contact with the sample introduction port 13, the blood is sucked into the sample flow path 12 by capillary action, and the first measurement electrode 2. And move to the electrode portions 2 a and 3 a of the second measuring electrode 3, that is, the reaction layers 6 and 7.
  • the reaction layer 7 does not contain lysine oxidase, only the contaminants in the sample solution are detected at the second measurement electrode 3.
  • the detection results detected by the first measurement electrode 2, the second measurement electrode 3 and the counter electrode 4 of the disposable lysine sensor A are transmitted through the terminal portions 2b, 3b and 4b to the terminals 26, 27,
  • the amount of lysine is calculated by calculating the difference between the detection result in the reaction layer 6 (lysine and contaminants) and the detection result in the reaction layer 7 (contamination matter). And the calculation result is stored in the storage unit 30 and the calculation result is displayed on the display 22 as necessary.
  • the control device 29 is configured to be able to output data stored in the storage unit 30 from the output terminal 25 to an external device as necessary.
  • the control device 29 can also be configured to determine whether or not malnutrition is based on the measured amount of lysine and output the determination result.
  • ferricyan ion functions as an electron acceptor of lysine oxidase, and that the current value is correlated with the amount of lysine added in the reaction system of lysine and lysine oxidase. Therefore, as in the above-described example, a lysine oxidase, a surfactant, and ferricyan ion are provided on the first measurement electrode, and the lysine concentration is determined with a simple disposable sensor configured to guide the specimen to the first measurement electrode. There is an effect that it becomes possible to measure.
  • the second reaction layer 7 containing the same amount of surfactant and ferricyan ion as the first reaction layer 6 is formed on the electrode 3a of the second measurement electrode 3, the measurement is performed. At this time, the conditions of the first measurement electrode 2 and the second measurement electrode 3 are aligned, so that an accurate measurement result can be obtained.
  • the second reaction layer 7 containing the same amount of surfactant and electron acceptor as the first reaction layer 6 is provided on the electrode 3a of the second measurement electrode 3, but the second reaction The structure of the layer 7 is not limited to this example.
  • the same amount of surfactant, electron acceptor, and deactivation as the first reaction layer 6 are used. You may form by apply
  • the second measurement electrode 3 is provided on the substrate and the second reaction layer 7 is formed on the second measurement electrode 3, but the second measurement electrode 3 is not an essential component. It is not necessary to provide it.
  • the second measurement electrode 3 it is possible to obtain a measurement result that is not affected by contaminants even without providing a filter. Examples of contaminants that have a large influence on the measurement of lysine include L-ascorbic acid.
  • ascorbate oxidase is provided on the first measurement electrode 2 as a reagent for removing L-ascorbic acid. obtain.
  • lysine oxidase derived from Trichoderma viride (EC number: 1.4.3.14 sold by Sigma-Aldrich Japan Co., Ltd.) is used as the lysine oxidase.
  • any enzyme can be used as long as the ferricyan ion functions as an electron acceptor.
  • mold-derived lysine oxidase other than Trichoderma viridae may be used, and lysine oxidase other than mold-derived, specifically, for example, extracted from immune tissue coming out of fish skin It may be lysine oxidase or human lysine oxidase.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne un détecteur jetable capable de mesurer la lysine. Le détecteur de lysine jetable selon la présente invention est un détecteur pour mesurer la concentration de matrice d'une solution d'échantillon en formant un système d'électrode comprenant au moins une électrode de mesure et une contre-électrode sur un substrat électriquement isolant et l'utilisation du système d'électrode pour détecter électrochimiquement des changements dans la concentration de substance pendant la réaction d'une enzyme, un récepteur d'électrons et une solution d'échantillon, qui se caractérise par la formation, sur l'électrode de mesure, d'une couche de réaction qui supporte une lysine oxydase et des ions ferricyanure, qui réagissent avec la lysine en présence de la lysine oxydase comme catalyseur.
PCT/JP2012/058334 2011-03-29 2012-03-29 Détecteur de lysine jetable Ceased WO2012133633A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-071654 2011-03-29
JP2011071654 2011-03-29

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WO2012133633A1 true WO2012133633A1 (fr) 2012-10-04

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017009430A (ja) * 2015-06-22 2017-01-12 株式会社村田製作所 バイオセンサ
CN114965640A (zh) * 2022-05-23 2022-08-30 中国科学院亚热带农业生态研究所 一种基于肽适体的赖氨酸生物传感器及其制备方法
JP7598188B1 (ja) * 2023-07-26 2024-12-11 株式会社ExtenD センサ、解析装置、端末装置、これらを用いた解析システムおよびコンピュータに実行させるためのプログラム
WO2025023224A1 (fr) * 2023-07-26 2025-01-30 株式会社ExtenD Capteur, dispositif d'analyse, dispositif terminal, système d'analyse les utilisant, et programme à exécuter par ordinateur

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003501627A (ja) * 1999-06-02 2003-01-14 ノヴァ バイオメディカル コーポレイション 使い捨てセンサ及び製造方法
JP2007514931A (ja) * 2003-10-31 2007-06-07 ライフスキャン・スコットランド・リミテッド 電気化学検査ストリップにおける直接干渉電流および間接干渉電流の影響を軽減する方法
JP2008209274A (ja) * 2007-02-27 2008-09-11 Citizen Holdings Co Ltd 電気化学センサ
JP2009171874A (ja) * 2008-01-23 2009-08-06 Citizen Holdings Co Ltd 糖化タンパク質濃度測定方法及びバイオセンサ

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003501627A (ja) * 1999-06-02 2003-01-14 ノヴァ バイオメディカル コーポレイション 使い捨てセンサ及び製造方法
JP2007514931A (ja) * 2003-10-31 2007-06-07 ライフスキャン・スコットランド・リミテッド 電気化学検査ストリップにおける直接干渉電流および間接干渉電流の影響を軽減する方法
JP2008209274A (ja) * 2007-02-27 2008-09-11 Citizen Holdings Co Ltd 電気化学センサ
JP2009171874A (ja) * 2008-01-23 2009-08-06 Citizen Holdings Co Ltd 糖化タンパク質濃度測定方法及びバイオセンサ

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FRANCESCO RICCI ET AL.: "Prussian Blue and enzyme bulk-modified screen-printed electrodes for hydrogen peroxide and glucose determination with improved storage and operational stability", ANALYTICA CHIMICA ACTA, vol. 485, 26 May 2003 (2003-05-26), pages 111 - 120 *
HOLGER OLSCHEWSKI ET AL.: "Screen-printed enzyme sensors for L-lysine determination", ENZYME AND MICROBIAL TECHNOLOGY, vol. 26, no. 7, April 2000 (2000-04-01), pages 537 - 543 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017009430A (ja) * 2015-06-22 2017-01-12 株式会社村田製作所 バイオセンサ
CN114965640A (zh) * 2022-05-23 2022-08-30 中国科学院亚热带农业生态研究所 一种基于肽适体的赖氨酸生物传感器及其制备方法
CN114965640B (zh) * 2022-05-23 2024-03-15 中国科学院亚热带农业生态研究所 一种基于肽适体的赖氨酸生物传感器及其制备方法
JP7598188B1 (ja) * 2023-07-26 2024-12-11 株式会社ExtenD センサ、解析装置、端末装置、これらを用いた解析システムおよびコンピュータに実行させるためのプログラム
WO2025023224A1 (fr) * 2023-07-26 2025-01-30 株式会社ExtenD Capteur, dispositif d'analyse, dispositif terminal, système d'analyse les utilisant, et programme à exécuter par ordinateur

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