WO2014069070A1 - Biocapteur - Google Patents

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Publication number
WO2014069070A1
WO2014069070A1 PCT/JP2013/071658 JP2013071658W WO2014069070A1 WO 2014069070 A1 WO2014069070 A1 WO 2014069070A1 JP 2013071658 W JP2013071658 W JP 2013071658W WO 2014069070 A1 WO2014069070 A1 WO 2014069070A1
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Prior art keywords
layer
electrode
mediator
biosensor
hydrophilic
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PCT/JP2013/071658
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English (en)
Japanese (ja)
Inventor
純 ▲高▼木
秀明 大江
淳典 平塚
典子 佐々木
憲二 横山
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株式会社村田製作所
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Publication of WO2014069070A1 publication Critical patent/WO2014069070A1/fr

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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
    • 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

Definitions

  • the present invention includes an electrode layer provided with an electrode system including a working electrode and a counter electrode, a spacer layer in which a slit for forming a cavity is formed and stacked on the electrode layer, and an air hole communicating with the cavity.
  • the present invention relates to a biosensor comprising a cover layer laminated on a spacer layer and a reaction layer provided on a working electrode and a counter electrode.
  • a biosensor 500 shown in FIG. 11 is a sensor for quantifying glucose contained in a sample, and includes an electrode layer 510 formed by providing an electrode on an insulating substrate 504 such as polyethylene terephthalate or polyimide, and a cover layer. 506 and a spacer layer 505 disposed between the electrode layer 510 and the cover layer 506 are stacked.
  • the spacer layer 505 is provided with a slit for forming a cavity 507 to which a sample is supplied, and the cover layer 506 is laminated and bonded to the electrode layer 510 via the spacer layer 505.
  • a cavity 507 to which a sample is supplied is formed by the electrode layer 510, the slit portion of the spacer layer 505, and the cover layer 506.
  • the cover layer 506 has an air hole 506a communicating with the terminal portion of the cavity 507 in order to smoothly supply the sample to the cavity 507 by capillary action.
  • the electrode layer 510 is provided with a working electrode 501 and a counter electrode 502, and an electrode system is formed by providing electrode patterns electrically connected to the electrodes 501 and 502, respectively.
  • a reaction layer 503 is provided on the working electrode 501 and the counter electrode 502, and the working electrode 501 and the counter electrode 502 are provided on the insulating substrate 504 so as to be exposed to the cavity 507 formed in the biosensor 500. It has been.
  • the electrodes 501 and 502 exposed to the cavity 507 and the reaction layer 503 come into contact with the sample, and the reaction layer 503 is dissolved in the sample.
  • a reaction layer 503 provided on the working electrode 501 and the counter electrode 502 is provided on the electrode layer 510, and is laminated on the hydrophilic layer 503a including a hydrophilic polymer carboxymethylcellulose (CMC) and the hydrophilic layer 503a.
  • the reagent layer 503b contains glucose oxidase (enzyme) that specifically reacts with glucose contained in the sample and potassium ferricyanide as a mediator (electron acceptor).
  • the hydrophilic layer 503a provided on the electrode layer 510 protects the electrodes 501 and 502 and prevents the reagent layer 503b from peeling off.
  • Ferricyanide ion ionized by dissolving potassium ferricyanide in the sample is ferrocyanide ion (ferricyanide ion) due to electrons released when glucose is oxidized to gluconolactone by reacting with glucose oxidase. Reduced form). Therefore, when a sample containing glucose is supplied to the cavity 507 formed in the biosensor 500 from the sample introduction port, ferrocyanide ions are generated in an amount corresponding to the concentration of glucose contained in the sample.
  • the oxidation current obtained by oxidizing the reduced form of the mediator resulting from the enzyme reaction on the working electrode 501 has a magnitude depending on the glucose concentration in the sample.
  • the glucose contained in the sample can be quantified by measuring the oxidation current.
  • JP 2001-281202 A paragraphs 0017, 0018, FIG. 2, etc.
  • blood samples contain blood cells such as red blood cells, and it is known that the magnitude of the oxidation current described above is influenced by the size of the hematocrit value indicating the proportion of the volume of blood cells in the blood sample. Yes.
  • This influence on the oxidation current is caused by blood cells adhering to the working electrode, the surface of the counter electrode, and the like. Therefore, in the biosensor 500 described above, the blood sample is filtered by the hydrophilic layer 503a provided on the electrode layer 510 and containing CMC which is a hydrophilic polymer, and movement of blood cells in the direction of the electrode layer is suppressed. Therefore, the influence on the measurement accuracy due to the difference in hematocrit value is reduced.
  • a hydrophilic polymer such as CMC reduces the mediator contained in the reagent layer 503b. Therefore, for example, when the mediator is reduced by the hydrophilic polymer contained in the hydrophilic layer in a state where the biosensor 500 is stored, the mediator reduced by the hydrophilic polymer is measured when the above-described oxidation current is measured. Since the oxidation current resulting from the oxidation is also measured as the background current together with the oxidation current to be measured, the measurement accuracy deteriorates.
  • the hydrophilic layer 503a containing the hydrophilic polymer and the reagent layer 503b containing the mediator are stacked in a separated state, whereby the hydrophilic polymer contained in the hydrophilic layer 503a and the reagent Reaction with the mediator contained in the layer 503b is prevented.
  • This invention is made
  • the biosensor of the present invention comprises: An insulating substrate, and an electrode layer comprising an electrode system including a working electrode and a counter electrode on one surface of the insulating substrate; A spacer layer that is laminated on the electrode system side of the electrode layer so that the slit is located on the tip side of the working electrode and the counter electrode; A cavity formed by the electrode layer and the slit and supplied with a sample; A cover layer that has an air hole communicating with the cavity and is laminated on the spacer layer so as to cover the cavity;
  • the reaction layer is A hydrophilic layer comprising a hydrophilic polymer provided on the electrode layer and having a double bond of an oxygen atom; A water repellent formed on the surface of the hydrophilic layer, including a layer containing a surfactant, a layer containing a water-soluble gelling agent, a layer containing an amphiphilic polymer,
  • the hydrophilic layer containing the hydrophilic polymer having a double bond of oxygen atoms is provided on the electrode layer provided with the electrode system including the working electrode and the counter electrode.
  • a hydrophilic polymer having a double bond of an oxygen atom is highly effective in preventing the movement of blood cells contained in a blood sample. That is, for example, when a blood sample is supplied to the cavity, the blood sample is filtered by the hydrophilic layer to prevent blood cells from moving, and components other than blood cells (for example, glucose) contact each electrode of the electrode layer.
  • the hydrophilic polymer in the hydrophilic layer prevents blood cells in the blood sample from moving toward each electrode in the electrode layer, thus reducing the effect on measurement accuracy due to the difference in hematocrit values of the blood sample. can do.
  • the hydrophilic polymer has a double bond of oxygen atom
  • the mediator is reduced by the nucleophilic attack of the functional group having the double bond of oxygen atom on the mediator. Therefore, when a reagent layer is placed on a hydrophilic layer containing a hydrophilic polymer having a double bond of an oxygen atom, the reduction reaction of the mediator by the hydrophilic polymer gradually proceeds at the contact interface between both layers in the storage state. As described above, there arises a problem that the measurement accuracy of the oxidation current in the biosensor deteriorates.
  • a layer containing a surfactant, a water-soluble gelling agent is provided between a hydrophilic layer containing a hydrophilic polymer having a double bond of oxygen atoms and a reagent layer containing a mediator or the like. Since an intermediate layer including at least one of a layer including, a layer including an amphiphilic polymer, and a water-repellent layer formed by water-repellent treatment on the surface of the hydrophilic layer is disposed, The hydrophilic polymer in the hydrophilic layer and the mediator in the reagent layer can be prevented from coming into contact with each other, whereby the mediator can be prevented from being reduced.
  • the reagent layer may further contain a hydrophilic polymer having no oxygen atom double bond.
  • the mediator is surrounded by a hydrophilic polymer that does not have an oxygen atom double bond, that is, a hydrophilic polymer that is difficult to reduce the mediator.
  • the effect of preventing the contact between the hydrophilic polymer having a double bond of oxygen atoms contained in and the mediator of the reagent layer is further improved.
  • the hydrophilic polymer functions as a thickener, so that the adhesive strength between the reagent layer and the intermediate layer is increased, so that the reagent layer can be prevented from peeling off from the intermediate layer due to external stress or the like.
  • the reagent layer comprises an enzyme layer containing a hydrophilic polymer not having a double bond of the enzyme and the oxygen atom, and a hydrophilic polymer not having a double bond of the mediator and the oxygen atom. And a mediator layer that includes the mediator layer.
  • the mediator layer is laminated on the enzyme layer. If it does in this way, since the hydrophilic polymer and mediator of a hydrophilic layer will be arrange
  • the hydrophilic polymer having no oxygen atom double bond includes at least one of hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, polyvinyl alcohol, and polyethylene glycol. It may be. With this configuration, the mediator contained in the reagent layer can be prevented from being reduced, and the reagent layer can be prevented from peeling from the intermediate layer.
  • a sensor can be provided.
  • the hydrophilic polymer having a double bond of oxygen atoms may include at least carboxymethylcellulose.
  • the hydrophilic polymer having a double bond of oxygen atoms may include at least carboxymethylcellulose.
  • the surfactant is sodium di (2-ethylhexyl) sulfosuccinate, polyethylene glycol mono-4-nonylphenyl ether (n ⁇ 7.5), sorbitan monolaurate, sorbitan monooleate, sodium cholate,
  • the structure containing at least 1 of lecithin may be sufficient.
  • the water-soluble gelling agent may include at least one of carrageenan and sodium alginate.
  • amphiphilic polymer may include at least one of polyvinyl pyrrolidone, polyethylene glycol, and polyvinyl alcohol.
  • the water repellent layer may be formed by etching the surface of the hydrophilic layer with plasma generated using a fluorine-based gas.
  • the mediator since the intermediate layer is provided between the hydrophilic layer containing a hydrophilic polymer having a double bond of oxygen atom and the reagent layer containing a mediator or the like, the mediator has a double bond of oxygen atom.
  • Contact with the hydrophilic polymer can be prevented, whereby the mediator can be prevented from being reduced by the hydrophilic polymer having a double bond of an oxygen atom in the storage state.
  • FIG. 2 is a diagram showing a method for manufacturing the biosensor of FIG. 1, wherein (a) to (c) show different processes. It is a figure which shows the relationship between the storage period and background current of the biosensor of FIG. It is a figure which shows the relationship between the storage period and background current of the biosensor concerning 2nd Embodiment of this invention.
  • FIG. 7 is a diagram showing a method for manufacturing a biosensor according to a third embodiment of the present invention, in which (a) to (d) show different processes. It is a figure which shows the relationship between the storage period of the biosensor manufactured by the manufacturing method of FIG. 6, and background current.
  • FIG. 9 is a diagram showing a method for manufacturing a biosensor according to a fourth embodiment of the present invention, in which (a) to (c) show different processes. It is a figure which shows the relationship between the storage period of the biosensor manufactured by the manufacturing method of FIG. 8, and background current. It is a cross-sectional view of the cavity part of the biosensor according to the fifth embodiment of the present invention. It is a figure which shows the conventional biosensor.
  • FIGS. 1A and 1B are diagrams showing a biosensor according to a first embodiment of the present invention, where FIG. 1A is an exploded perspective view and FIG. 1B is a perspective view.
  • FIG. 2 is a cross-sectional view of the cavity portion of the biosensor of FIG.
  • FIG. 3 is a diagram showing the biosensor manufacturing method according to the first embodiment of the present invention, wherein (a) to (c) show different steps.
  • a biosensor 100 includes an electrode system including a working electrode 101 and a counter electrode 102, and a reaction layer 106 including an enzyme that reacts with a mediator and a measurement target substance, and is attached to a measuring instrument (not shown). Used. Specifically, in a state where the biosensor 100 is mounted at a predetermined position of the measuring instrument, a sample such as blood is supplied to the cavity 103 provided on the tip side of the biosensor. The supplied sample comes into contact with the reaction layer 106 provided in the cavity 103, and the sample and the reaction layer 106 react to generate a reduced substance in which the substance contained in the reaction layer 106 is reduced.
  • glucose in the blood reacts with glucose oxidase (enzyme) contained in the reaction layer 106 to produce gluconolactone, which is a reactant obtained by oxidizing glucose.
  • glucose oxidase enzyme
  • the electrons released at this time reduce ferricyanide ions ionized from the potassium ferricyanide (mediator) contained in the reaction layer 106 to generate potassium ferrocyanide (reduced substance: reduced form of potassium ferricyanide).
  • a voltage is applied between the working electrode 101 and the counter electrode 102 to oxidize the reducing substance generated by this reaction, and the oxidation current obtained at that time is measured.
  • this oxidation current has a value that depends on the concentration of glucose contained in the blood. Therefore, by measuring the oxidation current obtained through each of the above reactions with the biosensor 100, Quantification of the measurement target substance contained in the sample (for example, glucose when the sample is blood) can be performed.
  • the biosensor 100 includes an insulating substrate 111 and an electrode layer including an electrode system including a working electrode 101 and a counter electrode 102 on one surface of the insulating substrate 111. 110, a slit 104, a spacer layer 120 stacked on the electrode system side of the electrode layer 110 so that the slit 104 is positioned on the tip side of the working electrode 101 and the counter electrode 102, and the electrode layer 110 and the spacer layer 120 A cavity 103 in which a sample is supplied, an air hole 105 communicating with the cavity 103, a cover layer 130 stacked on the spacer layer 120 so as to cover the cavity 103, and the cavity 103 A working electrode 101 exposed to the surface of the counter electrode 102 and a reaction layer 106 provided on the tip side of the counter electrode 102.
  • a spacer layer 120 disposed pinched in the cover layer 130 is formed by the sample introduction port 103a are bonded are laminated in a state where the top side is aligned provided.
  • the biosensor 100 is attached to the measuring instrument by being inserted into a predetermined insertion port of the measuring instrument from the rear end side.
  • the electrode layer 110 is formed of an insulating substrate made of an insulating material such as polyethylene terephthalate, ceramic, glass, plastic, paper, or biodegradable material. Further, a noble metal such as platinum, gold, and palladium, carbon, copper, aluminum, titanium, ITO (Indium Tin Oxide), ZnO (Zinc Oxide :) is formed on one surface of the insulating substrate 111 that forms the electrode layer 110. A conductive layer made of a conductive material such as zinc oxide) is formed by screen printing or sputtering deposition. When the conductive layer formed on one surface of the insulating substrate is subjected to patterning by laser processing or photolithography, the working electrode 101 and the counter electrode 102 and the biosensor 100 are mounted on the measuring instrument. In addition, an electrode system including electrode patterns 101a and 102a that electrically connect each of the working electrode 101 and the counter electrode 102 to the measuring instrument is formed.
  • a noble metal such as platinum, gold, and palladium, carbon, copper, aluminum, titanium
  • the working electrode 101 and the counter electrode 102 are arranged so that their respective tips are exposed to the cavity 103.
  • the electrode patterns 101a and 102a on the rear end side of the working electrode 101 and the counter electrode 102 are the edges of the electrode layer 110 on the opposite side to the sample introduction port 103a, and are the ends of the electrode layer 110 on which the spacer layer 120 is not stacked. It is stretched to the edge.
  • the spacer layer 120 is laminated on the electrode layer 110 formed as described above.
  • the spacer layer 120 is formed of an insulating substrate made of an insulating material such as polyethylene terephthalate, ceramic, glass, plastic, paper, or a biodegradable material, and is a slit for forming the cavity 103 at substantially the center of the front edge of the substrate. 104 is formed. Then, the slit 104 is disposed on the distal end side of the working electrode 101 and the counter electrode 102, and the spacer layer 120 is partially covered and laminated on one surface of the electrode layer 110, so that the electrode layer 110 and the slit 104 are stacked. A cavity 103 to which a sample is supplied is formed.
  • the reaction layer 106 is formed.
  • various plasmas used in metal activation treatment by plasma such as oxygen plasma, nitrogen plasma, and argon plasma can be used. Plasma may be used.
  • the reaction layer 106 is formed on the tip side of the working electrode 101 and the counter electrode 102 exposed to the cavity 103 before the cover layer 130 is laminated on the spacer layer 120.
  • a hydrophilizing agent such as a surfactant or phospholipid is applied to the inner wall of the cavity 103.
  • the reaction layer 106 is provided on the electrode layer 110 and is provided on the hydrophilic layer 106a including a hydrophilic polymer having a double bond of an oxygen atom, on the hydrophilic layer 106a, and included in the reagent layer 106c.
  • a predetermined amount of a reagent 201 containing CMC as a hydrophilic polymer having a double bond of an oxygen atom is dropped from the dropping device 200 into the cavity 103 and dried, thereby making the hydrophilic
  • the layer 106a is formed (hydrophilic layer forming step).
  • a predetermined amount of a reagent 202 containing sodium di (2-ethylhexyl) sulfosuccinate as a surfactant is dropped into the cavity 103 from the dropping device 200 and dried.
  • the intermediate layer 106b is formed (intermediate layer forming step).
  • the reagent 202 is obtained by dissolving a surfactant in acetone, isopropyl alcohol, or chloroform, and the intermediate layer 106b has a solution containing 0.01 to 5 wt% of the surfactant in an amount of 0.0%. It is formed by dropping 5 ⁇ L.
  • a mediator for example, potassium ferricyanide
  • an enzyme for example, glucose dehumanlogenase
  • a hydrophilic polymer for example, methylcellulose having no oxygen atom double bond.
  • Examples of the enzyme include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, lactate dehydrogenase, alcohol dehydrogenase, hydroxybutyrate dehydrogenase, and cholesterol esterase.
  • Creatininase, creatinase, DNA polymerase, etc. can be used, and these enzymes should be selected according to the substance to be measured (glucose, lactic acid, cholesterol, alcohol, sarcosine, fructosylamine, pyruvic acid, hydroxybutyric acid)
  • Various sensors can be formed.
  • glucose oxidase or glucose dehydrogenase can be used to form a glucose sensor that detects glucose in a blood sample
  • alcohol oxidase or alcohol dehydrogenase can be used to form an alcohol sensor that detects ethanol in a blood sample.
  • a lactic acid sensor for detecting lactic acid in a blood sample can be formed, and a total cholesterol sensor can be formed by using a mixture of cholesterol esterase and cholesterol oxidase.
  • ferrocene As the mediator, ferrocene, ferrocene derivative, benzoquinone, quinone derivative, osmium complex, ruthenium complex, etc. can be used in addition to the above-mentioned potassium ferricyanide.
  • hydrophilic polymers having a double bond of oxygen atom include carbonyl group, acyl group, carboxyl group, aldehyde group, sulfo group, sulfonyl group, sulfoxide group, tosyl group, nitro group, nitroso group, ester group, keto group.
  • a polymer having a group, a ketene group, or the like can be used.
  • hydrophilic polymer having no oxygen atom double bond it is possible to use hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, polyvinyl alcohol, polyethylene glycol, etc. in addition to the above-mentioned methylcellulose. it can.
  • the hydrophilic polymer which does not have the double bond of the oxygen atom mixed with the reagent 203 with an enzyme, a mediator, etc. functions as a thickener.
  • a hydrophilic polymer having a double bond of oxygen atom and a hydrophilic polymer having no double bond of oxygen atom may be used in combination of two or more.
  • the hydrophilizing agent polyethylene glycol mono-4-octylphenyl ether (n ⁇ 10) (Triton X100: manufactured by Sigma Aldrich), polyoxyethylene sorbitan monolaurate (Tween 20: manufactured by Tokyo Chemical Industry Co., Ltd.), bis ( Surfactants such as sodium 2-ethylhexyl) sulfosuccinate and phospholipids such as lecithin can be used.
  • the hydrophilizing agent may be mixed with the reagent 203 and dropped into the cavity 103, or may be dropped into the cavity 104 after the reagent layer 106c is formed.
  • a buffering agent such as phosphoric acid may be provided in order to reduce variation in the concentration of ions contained in the sample.
  • a cover layer 130 formed of an insulating substrate made of an insulating material such as polyethylene terephthalate, ceramic, glass, plastic, paper, biodegradable material is formed as a spacer layer.
  • the biosensor 100 is formed.
  • the cover layer 130 has an air hole 105 that communicates with the cavity 103 when laminated on the spacer layer 120, and the cover layer 130 has the cavity 103. And is laminated on the spacer layer 120.
  • the biosensor 100 is formed for the purpose of quantifying glucose in blood, and FAD (flavin adenine dinucleotide) is used as an enzyme that specifically reacts with glucose as a measurement target substance.
  • FAD flavin adenine dinucleotide
  • a mediator that contains GDH (glucose dehydrogenase) hereinafter referred to as FAD-GDH
  • FAD-GDH glycose dehydrogenase
  • the reaction layer 106 containing potassium ferricyanide is provided on the tip side of the working electrode 101 and the counter electrode 102 exposed to the cavity 103.
  • the sample introduction port 103a at the tip when a sample made of blood is brought into contact with the sample introduction port 103a at the tip, the sample is sucked toward the air hole 105 by capillary action and is then injected into the cavity 103. A sample is supplied. Then, when the reaction layer 106 (reagent layer 106c) is dissolved in the sample supplied to the cavity 103, electrons are released by the enzyme reaction between glucose, which is the measurement target substance in the sample, and FAD-GDH. Ferricyanide ions are reduced by electrons to produce ferrocyanide ions, which are reducing substances.
  • a voltage for example, 0.3 V
  • the glucose in the sample is quantified in the measuring instrument by measuring the oxidation current flowing between the working electrode 101 and the counter electrode 102 by oxidation.
  • a current value 3 to 5 seconds later is measured as an oxidation current.
  • FIG. 4 is a diagram illustrating the relationship between the storage period of the biosensor and the background current, where the horizontal axis indicates the storage period (h), and the vertical axis indicates the magnitude of the background current ( ⁇ A). Also, ⁇ (black rhombus) in the figure indicates the background current of the conventional biosensor in which the intermediate layer 106b is not provided between the reagent layer 106c and the hydrophilic layer 106a. A black square indicates a background current of the biosensor 100 of the present embodiment. The background current was measured by supplying a sample for measuring the background current to the cavity 103 and then measuring the oxidation current in the same manner as in a normal procedure.
  • the background current increases with time in the conventional biosensor, whereas in the biosensor 100 of the present embodiment, the background current increases. The increase is suppressed.
  • the hydrophilic layer 106a including a hydrophilic polymer having a double bond of oxygen atoms is provided on the electrode layer 110 provided with the electrode system including the working electrode 101 and the counter electrode 102.
  • the hydrophilic polymer having a double bond of oxygen atoms has an effect of preventing the movement of blood cells contained in the blood sample. For example, when the blood sample is supplied to the cavity, the hydrophilic sample 106a causes the blood sample to have a blood sample.
  • components other than blood cells for example, glucose
  • the hydrophilic polymer has an oxygen atom double bond
  • the functional group having the oxygen atom double bond is considered to reduce the mediator by nucleophilic attack on the mediator. Therefore, when the reagent layer 106c is arranged on the hydrophilic layer 106a, the reduction reaction of the mediator gradually proceeds at the contact interface between the two layers 106a and 106c in the storage state, and the measurement accuracy of the oxidation current in the biosensor 100 may deteriorate. There is.
  • a layer containing a surfactant is provided as the intermediate layer 106b between the hydrophilic layer 106a containing a hydrophilic polymer having a double bond of oxygen atoms and the reagent layer 106c containing a mediator or the like.
  • the mediator in the storage state of the biosensor 100, the mediator can be prevented from seeping out together with the moisture in the reagent layer 106c and coming into contact with the hydrophilic polymer of the hydrophilic layer 106a.
  • CMC hydrophilic polymer
  • the periphery of the mediator is a hydrophilic polymer that does not have an oxygen atom double bond, that is, a mediator. Is surrounded by a hydrophilic polymer that is difficult to reduce. Therefore, the effect of preventing contact between the hydrophilic polymer of the hydrophilic layer 106a and the mediator of the reagent layer 106c is further improved.
  • each hydrophilic polymer of the hydrophilic layer 106a and the reagent layer 106c functions as a thickener, the adhesive strength between the layers 106a, 106b, 106c constituting the reaction layer 106 is increased, and thus the layers 106a, 106b , 106c can be prevented from being peeled off by an external stress or the like on the bonding surface.
  • a highly reliable biosensor 100 can be provided.
  • FIG. 5 is a figure which shows the relationship between the storage period and background current of the biosensor concerning 2nd Embodiment.
  • This embodiment is different from the first embodiment described above in that the intermediate layer 106b of the biosensor 100 contains an amphiphilic polymer.
  • the intermediate layer 106b has a predetermined amount (for example, 0.5 ⁇ L) of a reagent in which an amphiphilic polymer is dissolved in acetone, isopropyl alcohol, chloroform, or the like at a ratio of 0.01 to 5 wt%. It is formed by being dropped and dried on 103.
  • polyvinyl pyrrolidone polyethylene glycol, polyvinyl alcohol, etc.
  • amphiphilic polymer polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, etc.
  • two or more of these amphiphilic polymers may be used in combination.
  • the increase in the background current is suppressed as in the first embodiment.
  • the background current shown in FIG. 5 is measured under the same conditions as the background current of the first embodiment described above.
  • the mediator of the reagent layer 106c contacts the hydrophilic polymer (CMC) of the hydrophilic layer 106a in the storage state. Therefore, an increase in the background current associated with the reduction of the mediator by CMC can be suppressed.
  • CMC hydrophilic polymer
  • FIG. 6 is a diagram showing a biosensor manufacturing method according to the third embodiment, and (a) to (d) show different processes.
  • FIG. 7 is a diagram showing the relationship between the storage period and the background current of the biosensor according to the third embodiment.
  • 6A is the same step as FIG. 3A showing the biosensor manufacturing method of the first embodiment
  • FIG. 6D is the same step as FIG. 3C.
  • This embodiment is different from the first embodiment described with reference to FIG. 2 in that the intermediate layer 106b contains a water-soluble gelling agent.
  • the intermediate layer 106b of the biosensor 100 is formed as follows. First, the hydrophilic layer 106a is formed in the same manner as in the first embodiment (see FIG. 6A). Next, as shown in FIG. 6B, a predetermined amount of a reagent 204 containing a water-soluble gelling agent that gels when mixed with a cation is dropped from the dropping device 200 into the cavity 103 and dried.
  • a predetermined amount of the cation-containing reagent 205 is dropped from the dropping device 200, whereby the water-soluble gelling agent is gelled to form the intermediate layer 106b.
  • the reagent layer 106c is formed in the same manner as in the first embodiment (see FIG. 6D).
  • water-soluble gelling agent a carrageenan or alginic acid compound
  • a ionic substance such as a ferricyanide compound, sodium chloride, potassium chloride, or calcium chloride can be used.
  • An aqueous solution in which either is dissolved can be used.
  • the increase in the background current is suppressed as in the first embodiment.
  • the background current shown in FIG. 7 is measured under the same conditions as the background current of the first embodiment described above.
  • the mediator of the reagent layer 106c contacts the hydrophilic polymer (CMC) of the hydrophilic layer 106a in the storage state. Therefore, an increase in the background current associated with the reduction of the mediator by CMC can be suppressed.
  • CMC hydrophilic polymer
  • FIG. 8 is a view showing a biosensor manufacturing method according to the fourth embodiment, and (a) to (c) show different processes.
  • FIG. 9 is a diagram showing the relationship between the storage period and the background current of the biosensor according to the fourth embodiment.
  • FIG. 8A is the same process as FIG. 3A showing the method of manufacturing the biosensor of the first embodiment
  • FIG. 8C is the same process as FIG. 3C.
  • This embodiment is different from the first embodiment described with reference to FIG. 2 in that the intermediate layer 106b is formed of a water repellent layer formed by subjecting the surface of the hydrophilic layer 106a to water repellent treatment. is there.
  • the intermediate layer 106b of the biosensor 100 is formed as follows. First, the hydrophilic layer 106a is formed in the same manner as in the first embodiment (see FIG. 8A). Next, as shown in FIG. 8B, the surface of the hydrophilic layer 106a is etched by plasma generated using a fluorine-based gas (for example, CHF 3 gas or CF 4 gas), so that the hydrophilic layer 106a. The surface is coated with fluorine to form a water repellent layer. Finally, the reagent layer 106c is formed in the same manner as in the first embodiment (see FIG. 8C).
  • the water repellent treatment is not limited to the above-described plasma etching of a fluorine-based gas, and various water repellent treatments such as dropping a water repellent on the hydrophilic layer 106a can be used.
  • the increase in the background current is suppressed as in the first embodiment.
  • the background current shown in FIG. 9 is measured under the same conditions as the background current of the first embodiment described above.
  • the mediator of the reagent layer 106c contacts the hydrophilic polymer (CMC) of the hydrophilic layer 106a in the storage state. Therefore, an increase in the background current associated with the reduction of the mediator by CMC can be suppressed.
  • CMC hydrophilic polymer
  • FIG. 10 is a cross-sectional view of the cavity portion of the biosensor according to the fifth embodiment.
  • the biosensor 100a differs from the biosensor 100 according to the first embodiment described with reference to FIG. 2 in that the reagent layer 106c has a double bond between a mediator and an oxygen atom as shown in FIG.
  • 106c1 is laminated. Since the other configuration is the same as that of the first embodiment, description thereof is omitted by attaching the same reference numerals.
  • hydrophilic polymer which does not have the double bond of the oxygen atom contained in each of the mediator layer 106c1 and the enzyme layer 106c2 can be the same as that in the first embodiment. Further, the order in which the mediator layer 106c1 and the enzyme layer 106c2 constituting the reagent layer 106c are stacked may be reversed.
  • the enzyme and the mediator are separated, so that the mediator can be prevented from being reduced by the enzyme in the storage state.
  • the hydrophilic polymer of the hydrophilic layer 106a and the mediator are arranged further apart from each other, so that the reduction effect of the mediator is further improved.
  • the mobility of the enzyme in the intermediate layer 106b and the hydrophilic layer 106a is smaller than the mobility of the mediator, the amount of enzyme / mediator in the vicinity of the electrodes 101 and 102 is larger than when the enzyme is disposed on the mediator.
  • the responsiveness and measurement accuracy of the sensor are improved.
  • the intermediate layer 106b is any one of a layer containing a surfactant, a layer containing an amphiphilic polymer, a layer containing a water-soluble gelling agent, and a water repellent layer.
  • the intermediate layer 106b may be formed as a multilayer structure, for example, by laminating a layer containing a water-soluble gelling agent on a layer containing a surfactant.
  • the intermediate layer 106b may be formed by dropping a reagent in which two or all of a surfactant, an amphiphilic polymer, and a water-soluble gelling agent are mixed onto the hydrophilic layer 106a. .
  • an ethanol sensor or a lactic acid sensor may be formed by changing the combination of the enzyme and the mediator included in the reaction layer 106 of the biosensor 100 described above.
  • the biosensor 100 is formed in a bipolar electrode structure having the working electrode 101 and the counter electrode 102.
  • the biosensor 100 is formed in a tripolar electrode structure by further providing a reference electrode. May be.
  • a predetermined potential based on the counter electrode 102 may be applied to the working electrode 101 in a state where the counter electrode 102 is grounded and a reference potential is applied to the reference electrode by the voltage output unit.
  • a blood sample is placed in the cavity 103 by monitoring a current flowing between the working electrode 101 and the counter electrode 102 by applying a predetermined voltage between the working electrode 101 and the counter electrode 102.
  • a detection electrode for detecting that the sample has been supplied to the cavity 103 may be further provided. In this case, by applying a predetermined voltage between the counter electrode 102 and the detection electrode, the current flowing between the counter electrode 102 and the detection electrode is monitored to detect that the sample is supplied to the cavity 103. do it.
  • the cover layer 130 is formed of a transparent member so that it can be visually recognized that the blood sample is supplied to the cavity 103. It is desirable to do.
  • the present invention can be applied to various biosensors.

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  • Chemical & Material Sciences (AREA)
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Abstract

La présente invention concerne un biocapteur, dans lequel une couche de réaction (106) dans le biocapteur (100) a une constitution telle qu'une couche intermédiaire (106b) contenant un tensioactif est disposée entre une couche hydrophile (106a) contenant un polymère hydrophile ayant une double liaison d'un atome d'oxygène et une couche de réactif (106c) contenant un médiateur ou similaire. Selon la constitution, le contact du médiateur avec le polymère hydrophile ayant une double liaison d'un atome d'oxygène peut être inhibé, et par conséquent, il est possible d'éviter la réduction du médiateur au cours du stockage du biocapteur (100).
PCT/JP2013/071658 2012-10-30 2013-08-09 Biocapteur WO2014069070A1 (fr)

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JP2012238608A JP2014089096A (ja) 2012-10-30 2012-10-30 バイオセンサ
JP2012-238608 2012-10-30

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109395244A (zh) * 2018-10-25 2019-03-01 成都碳原时代科技有限公司 基于生物电的创面修复贴

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
KR101694982B1 (ko) 2014-12-31 2017-01-10 주식회사 아이센스 전기화학적 바이오센서
KR102464005B1 (ko) * 2017-04-25 2022-11-07 한국전자기술연구원 바이오마커 검출용 전기화학센서 및 이를 이용한 바이오마커의 검출방법

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04113262A (ja) * 1990-09-04 1992-04-14 Matsushita Electric Ind Co Ltd バイオセンサおよびその製造法
JPH10232219A (ja) * 1996-12-20 1998-09-02 Matsushita Electric Ind Co Ltd コレステロールセンサおよびその製造方法
JP2000039416A (ja) * 1998-05-21 2000-02-08 Matsushita Electric Ind Co Ltd バイオセンサ
WO2002093151A1 (fr) * 2001-05-15 2002-11-21 Matsushita Electric Industrial Co., Ltd. Biocapteur
JP2004264247A (ja) * 2003-03-04 2004-09-24 Matsushita Electric Ind Co Ltd バイオセンサ
WO2006104077A1 (fr) * 2005-03-29 2006-10-05 Cci Corporation Biosonde
JP2012208101A (ja) * 2011-03-30 2012-10-25 Cci Corp 多層構造を有するバイオセンサ
WO2013137173A1 (fr) * 2012-03-15 2013-09-19 株式会社村田製作所 Procédé de fabrication d'un biocapteur
WO2013137172A1 (fr) * 2012-03-15 2013-09-19 株式会社村田製作所 Biocapteur

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04113262A (ja) * 1990-09-04 1992-04-14 Matsushita Electric Ind Co Ltd バイオセンサおよびその製造法
JPH10232219A (ja) * 1996-12-20 1998-09-02 Matsushita Electric Ind Co Ltd コレステロールセンサおよびその製造方法
JP2000039416A (ja) * 1998-05-21 2000-02-08 Matsushita Electric Ind Co Ltd バイオセンサ
WO2002093151A1 (fr) * 2001-05-15 2002-11-21 Matsushita Electric Industrial Co., Ltd. Biocapteur
JP2004264247A (ja) * 2003-03-04 2004-09-24 Matsushita Electric Ind Co Ltd バイオセンサ
WO2006104077A1 (fr) * 2005-03-29 2006-10-05 Cci Corporation Biosonde
JP2012208101A (ja) * 2011-03-30 2012-10-25 Cci Corp 多層構造を有するバイオセンサ
WO2013137173A1 (fr) * 2012-03-15 2013-09-19 株式会社村田製作所 Procédé de fabrication d'un biocapteur
WO2013137172A1 (fr) * 2012-03-15 2013-09-19 株式会社村田製作所 Biocapteur

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109395244A (zh) * 2018-10-25 2019-03-01 成都碳原时代科技有限公司 基于生物电的创面修复贴

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