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WO2000007003A1 - Biocapteur - Google Patents

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Publication number
WO2000007003A1
WO2000007003A1 PCT/JP1999/004065 JP9904065W WO0007003A1 WO 2000007003 A1 WO2000007003 A1 WO 2000007003A1 JP 9904065 W JP9904065 W JP 9904065W WO 0007003 A1 WO0007003 A1 WO 0007003A1
Authority
WO
WIPO (PCT)
Prior art keywords
biosensor
reagent
mediator
electrode
derivative
Prior art date
Application number
PCT/JP1999/004065
Other languages
English (en)
Japanese (ja)
Inventor
Shigeki Joko
Original Assignee
Matsushita Electric Industrial 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 Matsushita Electric Industrial Co., Ltd. filed Critical Matsushita Electric Industrial Co., Ltd.
Publication of WO2000007003A1 publication Critical patent/WO2000007003A1/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

Definitions

  • the present invention relates to a so-called electrode-type biosensor, and particularly to a reagent section to which a sample sample is administered in order to improve responsiveness and reproducibility when specifically qualifying or quantifying components in the sample sample. It relates to an improved biosensor. Background art
  • glucose was measured using a biosensor that combines glucose oxidase (hereinafter abbreviated as GOD j) as an enzyme that oxidizes glucose and an oxygen electrode or a hydrogen peroxide electrode. 8) D-Glucose reacts with GOD to produce ⁇ -Dalconolactone, and oxygen becomes an electron acceptor to generate hydrogen peroxide.
  • the sensor measures glucose by measuring oxygen consumption in an enzymatic reaction with an oxygen electrode or by measuring hydrogen peroxide production with a hydrogen peroxide electrode.
  • the glucose measurement described above does not measure the amount of oxygen consumed in the enzymatic reaction or the amount of hydrogen peroxide produced, but an enzyme reaction detection reagent (the enzyme reaction detecting reagent) that detects the enzymatic reaction itself Hereafter, "media" U. ).
  • an enzyme reaction detection reagent the enzyme reaction detecting reagent
  • media the biosensor having the above-mentioned mediator can more accurately measure glucose detection in a sample without being affected by dissolved oxygen.
  • FIG. 1 shows a configuration diagram of an electrode-type biosensor manufactured using a mediator.
  • the biosensor is composed of a measuring electrode (working electrode) 4 connected to lead 2 and a counter electrode connected to lead 3 on an insulating substrate "!
  • An electrode-based detection unit 9 having a number of 5 is formed, and an oxidoreductase and a nodator are compounded on the furnace-electrode detection unit 9 as shown in Fig. 1 (b).
  • the qualitative or quantitative determination of the specimen sample is performed by administering the specimen sample to the reagent section 10 and performing measurement by the electrode system detecting section 9.
  • GOD is used as the oxidoreductase
  • potassium ferricyanide is used as the mediator.
  • 6 designates the exposed area of the negative electrodes 4 and 5, and both electrodes 4 and 5 And rie de 2, 3 insulating layer covering the unnecessary portion of, 7 Wasupesa, 8 denotes a cover.
  • the biosensor detects the consumption of potassium ferricyanide because the ferricyanidation beam of the mediator is consumed as an electron acceptor during the enzymatic reaction.
  • Glucose is measured by measuring current values detected from leads 2 and 3 through electrodes 4 and 5 of unit 9.
  • FIG. 4 is a graph showing the sensor response performance depending on the mediator concentration in the dark-course measurement pyrosensor manufactured by the above configuration.
  • the vertical axis is the current value for detecting the amount of mediator (ca. ferricyanide) consumed as an electron receptor for each concentration
  • the horizontal axis is the concentration of glucose, which is the specimen sample.
  • FIG. 5 is a graph showing the reproducibility of the sensor response and the sensor response depending on the mediator concentration in the biosensor.
  • the line graph on the vertical axis indicates the thickness of the reagent section 10, and the bar graph on the vertical axis indicates the coefficient of variation (hereinafter abbreviated as “cv”) of the sensor response as an index of the reproducibility of the sensor response.
  • the horizontal axis is the concentration of the mediator (ca. ferricyanide).
  • the reagent section 10 is composed of 5 microliters (hereinafter referred to as “mixture”) of these mixed liquids in order to carry GOD as an oxidoreductase and potassium ferricyanide as a mediator. ] On electrodes 4 and 5 and dried.
  • Fig. 4 shows that the oxidoreductase (GOD) concentration was fixed at 200 Units Z milliliters (hereinafter abbreviated as UZml), and the mediaease (calidium ferricyanide) was used. It shows the relationship with the detected glucose concentration when ⁇ is arbitrarily increased.
  • the mediator concentration is 25 milimolnoliter (hereinafter abbreviated as mM)
  • the correlation coefficient (r) is 0.9831
  • the mediator concentration is up to 15 OmM. It can be seen that the correlation coefficient improves to 0.9931 when increasing. That is, it can be seen that when the mediator concentration is increased, the response sensitivity of the sensor is increased, and the linear correlation between the glucose concentration and the current value is also improved.
  • the poor adhesion of the reagent part 10 itself may be caused by the oxidoreductase and the mediator in the reagent part 10 and the surface of the electrodes 4 and 5 during the production, handling, and storage of the sensor. This caused a large error in the amount of reagent that had been optimized and the sensor response was significantly reduced.
  • the present invention provides an oxidoreductase and a mediator that suppress the elevation and unevenness of the reagent portion and stabilize the response and reproducibility of the biosensor, and carry the reagent on the reagent portion. It is another object of the present invention to provide a biosensor that enhances adhesion to an electrode surface. Disclosure of the invention
  • an electrode-based detection unit having a working electrode and a counter electrode is formed on an insulating substrate, and an oxidoreductase and an enzyme reaction are formed on the electrode-based detection unit.
  • a biosensor in which a reagent portion containing a detection reagent is formed and the qualitative or quantitative detection of a sample sample administered to the reagent portion is performed by the electrode-based detection portion a biomolecule is contained in the reagent portion. Characterized by having an additive containing the protein or its derivative.
  • an additive containing a biomolecular protein or a derivative thereof is blended in the reagent section. Therefore, such an additive is a polymer substance having a large cohesive force. Therefore, the redox enzyme and the enzyme reaction detection reagent (mediator) in the reagent section can be carried with good adhesion. Therefore, the media in the reagent part are caused by the coagulation action of this additive. It is possible to suppress bumps and bumps caused by the eta concentration, to enhance the adhesion to the detection section supporting the reagent section, and to prevent the oxidoreductase / mediator from peeling off and falling off from the detection section surface. As a result, there is an effect that the sensor response performance and the sensor reproduction performance are greatly improved.
  • the invention (Claim 2) is the biosensor according to Claim 1, wherein the biopolymer protein or the derivative thereof contained in the additive does not have a redox catalytic activity. It is characterized by.
  • the biosensor having such a configuration, since a biopolymer protein or a derivative thereof having no redox catalytic activity is used as the protein or the derivative thereof contained in the additive, enzymatic and chemical The oxidation-reduction reaction can prevent the oxidation-reduction reaction with an unspecified component of the sample sample, so that only the sensor response corresponding to the reaction with the specified component can be detected. it can. As a result, it has become possible to specifically measure specimen samples.
  • the invention provides the biosensor according to Claim 1, wherein the additive is gelatin, collagen, elastin, casein, peptone, or a derivative thereof, or a combination thereof. It is characterized in that arbitrarily combined mixtures are used.
  • FIG. 1 is a diagram showing the configuration of an electrode-type biosensor.
  • FIG. 1 (a) is an exploded perspective view of the electrode-type biosensor, and
  • FIG. 1 (b) is a diagram showing the configuration of the electrode-type biosensor.
  • FIG. 5 is a side view of a detecting unit that performs the detection.
  • FIG. 2 is a graph showing the reagent part thickness and the sensor response reproducibility due to the mixing of gelatin in the reagent part in the electrode type biosensor according to the embodiment of the present invention.
  • FIG. 3 is a graph showing the linear response of the electrode-type biosensor according to the embodiment of the present invention, in which the gelatin is blended in the reagent part.
  • FIG. 4 is a graph showing the sensor response performance depending on the mediator concentration in a conventional electrode type biosensor.
  • FIG. 5 is a graph showing the reproducibility of the sensor response and the thickness of the reagent depending on the mediator concentration in a conventional electrode-type biosensor.
  • the biosensor according to the embodiment of the present invention has substantially the same configuration as that described in the related art, but the biomolecule is added to the width of the reagent part 10. And an additive containing the protein or its derivative.
  • biopolymer protein or a derivative thereof contained in the above-mentioned additive one having no redox catalytic activity is preferably used, for example, gelatin, collagen, elastin, casein, peptone, or any of these. A derivative or a mixture of any of these is used.
  • Examples of the oxidoreductase contained in the reagent section include dalcosoxidase (GOD), cholesterol oxidase, lactate oxidase, pericasidase, galactose oxidase, and alcohol oxidase.
  • Examples of the enzyme include choline, cholineoxidase, ascorbate oxidase, glucose dehydrogenase, cholesterol dehydrogenase, and alcohol dehydrogenase.
  • an electron acceptor of an organic or inorganic compound is used, for example, metal cyano complex such as lithium ferricyanide, benzoquinone, etc.
  • metal cyano complex such as lithium ferricyanide, benzoquinone, etc.
  • a quinone derivative, a chlorocene derivative, or a fluorene derivative may be used.
  • the biosensor having the above configuration is manufactured, for example, as follows. First, silver paste is printed on an insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Next, an electrode system detecting section 9 including a measuring electrode (working electrode) 4 and a counter electrode 5 is formed on the leads 2 and 3 by printing a conductive carbon paste. In addition, an insulating paste is printed to define the exposed area of the electrodes 4 and 5 and to cover the unnecessary portions of the leads 2 and 3 between the electrodes 4 and 5 to form the insulating layer 6. I do.
  • the electrode type biosensor is manufactured by bonding the cover 8 and the spacer 7 in the positional relationship indicated by the dashed line in FIG. Next, the operation of the biosensor will be described.
  • a sample is directly administered to the reagent section 10 carried on the detection section 9. Then, the components in the specimen sample undergo an enzymatic reaction with the oxidoreductase contained in the reagent section 10, and the enzyme becomes a reduced form. Next, the reduced enzyme reacts with the mediator, the enzyme is oxidized again, and the mediator takes on a reduced form. By applying a voltage to this reduced mediator, the mediator emits electrons at the same time as it is oxidized again. By measuring the emitted electrons as current values from the electrodes 4 and 5, the components in the sample can be specifically qualitatively or quantitatively determined.
  • the reagent portion 10 of the biosensor according to the present embodiment contains an additive containing a biopolymer protein or a derivative thereof.
  • the oxidoreductase in the reagent part 10 can be loaded with good adhesion to the mediator. Therefore, according to the biosensor according to the present embodiment, an additive containing a biopolymer protein or a derivative thereof is blended in the reagent portion 10, and the reagent portion 10 is agglutinated by the additive. Of the oxidoreductase from the surfaces of the electrodes 4 and 5 to prevent exfoliation and falling off of the mediator. Can be. As a result, the biosensor has an effect that the sensor response performance and the sensor reproduction performance are significantly improved.
  • the biosensor of the present embodiment a protein or a derivative of a biomolecular substance contained in the above additive that does not have a redox catalytic activity is used, so that enzymatic and chemical It is possible to prevent an oxidation-reduction reaction with an unspecified component of the sample sample from occurring by a typical oxidation-reduction reaction, and it is possible to detect only a sensor response corresponding to the reaction with the specified component. As a result, the biosensor has an effect of being able to specifically measure a specimen sample.
  • the reagent part 10 supported on the electrodes 4 and 5 shown in FIG. 1 has a GOD of 200 U ml as an oxidoreductase and a ferrocyanide as a mediator 150 xn M was mixed with gelatin as an additive at various ratios of 0 to 3 W / V%, and 5 ⁇ l of this mixture was applied to the surfaces of electrodes 4 and 5 and dried. Formed. In the present embodiment, it was used to prepare a plurality of biosensor having a reagent portion 1 0 engaged gd gelatin in various ratios of 0 to 3 ⁇ 0/0.
  • FIG. 2 is a graph showing the thickness of the reagent section 10 of the glucose measuring biosensor having the reagent section 10 containing gelatin and the sensor response reproducibility.
  • the line graph on the vertical axis shows the thickness of the reagent section 10 and the bar graph on the vertical axis. Is the coefficient of variation of the sensor response (hereinafter referred to as the index of reproducibility of the sensor response).
  • the horizontal axis is the concentration of the additive (gelatin).
  • the thickness of the reagent part 10 depends on the concentration of gelatin mixed in the reagent part 10, as is evident from the line graph in the figure.
  • the characteristic is that the thickness of the reagent portion 10 decreases as the gelatin concentration increases. In other words, it can be seen that in the reagent portion 10, the higher the gelatin concentration, the more the elevation is suppressed and the unevenness is reduced.
  • the reagent part 10 formed by appropriately applying 15 O mM of potassium ferricyanide has a reagent part thickness of about 540 ⁇ m without gelatin. Although it is about ⁇ , it can be seen that when gelatin is added at about 0.25 WZV D / 0 , the reagent thickness is suppressed to about 260 ⁇ m. In other words, this result indicates that the thickness of the reagent is improved to 75 mM or less, as shown in FIG. 5, which is a conventional example, when gelatin is not blended.
  • the CV value of the reagent part 10 formed by dropping 50 mM of lithium ferricyanide without gelatin was 20 ° C. / 0 or more.
  • the bar graph in Fig. 2 when gelatin is added at about 1.5 W / V%, it is possible to improve the CV value to about 5%, and to further reduce the gelatin to 2.OW It can be seen that the CV value stabilizes to about 2% when blended at / V% or more.
  • FIG. 3 is a graph showing the response performance of a glucose measuring biosensor having a reagent part 10 containing gelatin.
  • the vertical axis indicates the current value for detecting the amount of the mediator (potassium polyfluoride) consumed as an electron acceptor, and the horizontal axis indicates the concentration of glucose in the test sample.
  • each of the polygonal lines was composed of gelatin powder (0.00 W / V%), 0.50 WZV%, 1.O owzv%, 2.O OW / V
  • the graph shows the relationship between the glucose concentration and current value (sensor response performance) detected when the mixture was formulated with% or 2.75 V%. From Fig.
  • the relationship between the glucose concentration and the current value was found to be extremely high in the relationship between the glucose concentration and the current value by blending gelatin at about 2.0 W // ⁇ % or more. It is understood that it can be done.
  • the correlation coefficient (r) was 0.9931 when gelatin was not added, but improved to 0.939 when gelatin was added at 2.75 W / V%. It can be seen that the slope of the straight line is superior to that when gelatin is not blended.
  • the biosensor was formed on the surfaces of the electrodes 4 and 5 by applying 5 ⁇ l of a reagent solution containing 1.5 to 3.0 V% of gelatin and drying the mixture.
  • a biosensor having high sensor response performance and high sensor response reproducibility can be realized.
  • the amounts of the biopolymers, enzymes, reagents, and the like, the amounts supported on the sensor, the methods, and the like described in the above examples are merely examples, and the amounts and ratios of the biosensors of the present invention are not limited. It is not limited to the method and the method, but can be changed according to the purpose. Industrial applicability
  • the biosensor according to the present invention relates to a so-called electrode-type biosensor having a wide range of applications such as clinical examination, food, environment, and industrial production processes. Enables a simple measurement system that directly administers to the sensor.In addition, the thickness of the reagent section can be suppressed to accurately measure the sample, and the adhesion between the reagent section and the electrode surface can be improved. It became. As a result, the sensor response performance and the sensor response reliability are significantly improved, and a biosensor having higher reliability can be realized.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

On peut préparer ce biocapteur, comme illustré dans la figure, par un procédé consistant à combiner par mélangeage un additif contenant une protéine, qui est un biopolymère, ou un dérivé de cette protéine, une oxydo-réductase et un médiateur, pour former une solution, à ajouter cette solution goutte-à-goutte sur une partie réactive (10) du biocapteur, et à procéder à son séchage. Un biocapteur ayant une telle constitution permet de réduire l'épaisseur de la partie réactive (10), ce qui entraîne un contact plus étroit de l'oxydoréductase et du médiateur placés les deux en support sur une partie supérieure de la partie réactive (10) avec une surface d'une électrode (4, 5), et empêche ainsi l'oxydoréductase et le médiateur de s'effriter ou de se détacher d'une surface d'une pièce de détection (9). En outre, dès lors que l'additif mentionné ci-dessus n'a pas d'activité catalytique en lui-même pour l'oxydation-réduction, une détermination spécifique d'un spécimen est possible grâce à l'utilisation d'une réaction d'oxydation-réduction enzymatique ou chimique. Il en résulte que les performances de réaction et la reproductibilité de la réaction du biocapteur sont notablement améliorées, permettant ainsi la préparation d'un biocapteur ayant une fiabilité élevée.
PCT/JP1999/004065 1998-07-30 1999-07-29 Biocapteur WO2000007003A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10216186A JP2000046782A (ja) 1998-07-30 1998-07-30 バイオセンサ
JP10/216186 1998-07-30

Publications (1)

Publication Number Publication Date
WO2000007003A1 true WO2000007003A1 (fr) 2000-02-10

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JP (1) JP2000046782A (fr)
CN (1) CN1274421A (fr)
WO (1) WO2000007003A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4913355B2 (ja) * 2005-03-29 2012-04-11 シーシーアイ株式会社 バイオセンサ
KR100829400B1 (ko) * 2006-11-30 2008-05-15 주식회사 인포피아 바이오센서

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09304329A (ja) * 1996-03-12 1997-11-28 Matsushita Electric Ind Co Ltd バイオセンサおよびそれを用いた基質の定量方法
JPH10104192A (ja) * 1996-09-30 1998-04-24 Matsushita Electric Ind Co Ltd バイオセンサ

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09304329A (ja) * 1996-03-12 1997-11-28 Matsushita Electric Ind Co Ltd バイオセンサおよびそれを用いた基質の定量方法
JPH10104192A (ja) * 1996-09-30 1998-04-24 Matsushita Electric Ind Co Ltd バイオセンサ

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JP2000046782A (ja) 2000-02-18
CN1274421A (zh) 2000-11-22

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