WO2014069070A1

WO2014069070A1 - Biosensor

Info

Publication number: WO2014069070A1
Application number: PCT/JP2013/071658
Authority: WO
Inventors: 純 ▲高▼木; 秀明大江; 淳典平塚; 典子佐々木; 憲二横山
Original assignee: 株式会社村田製作所
Priority date: 2012-10-30
Filing date: 2013-08-09
Publication date: 2014-05-08
Also published as: JP2014089096A

Abstract

In a biosensor according to the present invention, a reaction layer (106) in the biosensor (100) has such a constitution that an intermediate layer (106b) containing a surfactant is provided between a hydrophilic layer (106a) containing a hydrophilic polymer having a double bond of an oxygen atom and a reagent layer (106c) containing a mediator or the like. According to the constitution, the contact of the mediator with the hydrophilic polymer having a double bond of an oxygen atom can be prevented, and therefore the mediator can be prevented from being reduced during the storage of the biosensor (100).

Description

Biosensor

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.

As shown in the conventional biosensor of FIG. 11, using a biosensor 500 having an electrode system including a working electrode 501 and a counter electrode 502 and a reaction layer 503 including an enzyme that specifically reacts with a measurement target substance, By measuring the oxidation current obtained by applying a voltage between the working electrode 501 and the counter electrode 502 to oxidize the reducing substance produced by the reaction between the measurement target substance contained in the reaction layer 503 and the reaction layer 503. A method for measuring a substance for quantifying a substance to be measured is known (for example, see Patent Document 1: Japanese Patent Laid-Open No. 2001-281202).

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. Then, a sample is supplied to the cavity 507 from the sample inlet formed on the side surface of the biosensor 500 by the opening portion of the slit. 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.

Further, 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.

Therefore, when a liquid sample is supplied to the cavity 507 from the sample inlet, 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.

In the biosensor 500 configured as described above, 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.)

By the way, 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.

However, it is known that 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. Therefore, in the biosensor 500, 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.

However, as described above, even when the hydrophilic layer 503a and the reagent layer 503b are separated from each other, the reaction between the hydrophilic polymer and the mediator is gradually performed from the interface where the hydrophilic layer 503a and the reagent layer 503b are in contact with each other. Since the measurement accuracy of the oxidation current is deteriorated by proceeding to, improvement measures for preventing the deterioration have been demanded.

This invention is made | formed in view of the said subject, and it aims at providing the technique which can prevent that a mediator reduces in a storage state.

In order to achieve the above object, 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;
In a biosensor comprising the working electrode exposed in the cavity and a reaction layer provided on the tip side of the counter electrode,
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, and a surface of the hydrophilic layer. An intermediate layer comprising at least one of the layers;
A reagent layer provided on the intermediate layer and containing an enzyme and a mediator that reacts with the substance to be measured.

According to such a configuration, 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. In general, 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. In this way, 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.

Further, when the hydrophilic polymer has a double bond of oxygen atom, it is considered that 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.

However, according to the configuration described above, 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.

Therefore, it becomes possible to achieve both the prevention of reduction of the mediator and the reduction of the influence on the measurement accuracy due to the difference in hematocrit value, and an accurate and highly reliable biosensor can be provided.

The reagent layer may further contain a hydrophilic polymer having no oxygen atom double bond. With this configuration, 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.

Also, since the hydrophilic polymer functions as a thickener, 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. By comprising in this way, since it will be in the state from which the enzyme and the mediator were isolate | separated, it can prevent that a mediator reduces by an enzyme in a storage state.

Further, it is preferable that 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 | positioned further apart, the reduction prevention effect of a mediator will further improve. In addition, since the mobility of the enzyme in the intermediate layer and the hydrophilic layer is smaller than the mobility of the mediator, the amount of enzyme / mediator near the electrode is larger than when the enzyme is placed on the mediator, and the sensor response And measurement accuracy are improved.

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.

Further, the hydrophilic polymer having a double bond of oxygen atoms may include at least carboxymethylcellulose. By comprising in this way, it can prevent that the intermediate | middle layer provided on a hydrophilic layer peels. In addition, for example, when a blood sample is supplied to the cavity of the biosensor, the blood sample is filtered by the hydrophilic polymer of the hydrophilic layer and the movement of the blood cell to each electrode is prevented, so the hematocrit value of the blood sample Therefore, the influence on the measurement accuracy of the oxidation current due to the difference in the above can be reduced.

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. By comprising in this way, it can prevent that the mediator of the reagent layer provided on the intermediate | middle layer contacts the hydrophilic polymer of a hydrophilic layer, and, thereby, a mediator becomes hydrophilic polymer of a hydrophilic layer. Can be prevented from being reduced.

In addition, the water-soluble gelling agent may include at least one of carrageenan and sodium alginate. By comprising in this way, it can prevent that the mediator of the reagent layer provided on the intermediate | middle layer contacts the hydrophilic polymer of a hydrophilic layer, and, thereby, a mediator becomes hydrophilic polymer of a hydrophilic layer. Can be prevented from being reduced.

Further, the amphiphilic polymer may include at least one of polyvinyl pyrrolidone, polyethylene glycol, and polyvinyl alcohol. By comprising in this way, it can prevent that the mediator of the reagent layer provided on the intermediate | middle layer contacts the hydrophilic polymer of a hydrophilic layer, and, thereby, a mediator becomes hydrophilic polymer of a hydrophilic layer. Can be prevented from being reduced.

In addition, the water repellent layer may be formed by etching the surface of the hydrophilic layer with plasma generated using a fluorine-based gas. By comprising in this way, it can prevent that the mediator of the reagent layer provided on the intermediate | middle layer contacts the hydrophilic polymer of a hydrophilic layer, and, thereby, a mediator becomes hydrophilic polymer of a hydrophilic layer. Can be prevented from being reduced.

According to the present invention, 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.

It is a figure which shows the biosensor concerning 1st Embodiment of this invention, Comprising: (a) is a disassembled perspective view, (b) is a perspective view. It is a cross-sectional view of the cavity part of the biosensor of FIG. 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.

<First Embodiment>
A biosensor according to a first embodiment of the present invention and a method for manufacturing the biosensor will be described with reference to 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.

(Configuration and manufacturing method of biosensor)
A biosensor 100 according to this embodiment 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.

For example, when the sample is blood, 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. 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).

In the biosensor 100, 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. For example, when the sample is blood, 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.

More specifically, as shown in FIGS. 1 and 2, 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. And 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.

In addition, the working electrode 101 and the counter electrode 102 are arranged so that their respective tips are exposed to the cavity 103. Further, 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.

Next, 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.

Subsequently, after the cavity 103 is cleaned with plasma, the reaction layer 106 is formed. As the plasma used in the plasma cleaning process, 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.

As shown in FIGS. 3A to 3C, 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 reagent 201 containing a hydrophilic polymer having a double bond of an oxygen atom such as methylcellulose (CMC), a reagent 202 containing a surfactant such as sodium di (2-ethylhexyl) sulfosuccinate, a mediator, an enzyme And a reagent 203 containing a hydrophilic polymer or the like that does not have a double bond of an oxygen atom, and is formed by dropping them in order. Further, in order to smoothly supply a sample such as blood to the cavity 104, a hydrophilizing agent such as a surfactant or phospholipid is applied to the inner wall of the cavity 103.

Specifically, 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. An intermediate layer 106b containing a surfactant for preventing the mediator from contacting the hydrophilic polymer of the hydrophilic layer 106a, and an enzyme and a mediator (electron acceptor) that are provided on the intermediate layer 106b and react with the substance to be measured. And a reagent layer 106c containing a hydrophilic polymer that does not have a double bond of an oxygen atom, and is formed as follows.

That is, as shown in FIG. 3A, 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). Next, as shown in FIG. 3B, 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). In this embodiment, 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 μL.

Subsequently, as shown in FIG. 3 (c), a mediator (for example, potassium ferricyanide), an enzyme (for example, glucose dehumanlogenase), and a hydrophilic polymer (for example, methylcellulose) having no oxygen atom double bond. ) Is dropped into the cavity 103 by a predetermined amount from the dropping device 200 and dried to form the reagent layer 106c (reagent layer forming step), and the reaction layer 106 is formed by these steps. In this case, there may be no hydrophilic polymer that does not have an oxygen atom double bond contained in the reagent 203. In addition, the reagent 203 may include an amphiphilic polymer in place of the hydrophilic polymer having no oxygen atom double bond or together with the hydrophilic polymer.

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.

For example, glucose oxidase or glucose dehydrogenase can be used to form a glucose sensor that detects glucose in a blood sample, and alcohol oxidase or alcohol dehydrogenase can be used to form an alcohol sensor that detects ethanol in a blood sample. For example, 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.

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.

In addition, 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.

Further, as the 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. In addition, 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. In addition, 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.

In addition to the above-mentioned sodium di (2-ethylhexyl) sulfosuccinate (Aerosol OT: Wako Pure Chemical Industries, Ltd.), polyethylene glycol mono-4-nonylphenyl ether (n≈7.5) (Igepal CO-210: Rhone-Poulenc), sorbitan monolaurate (Span20: Tokyo Chemical Industry Co., Ltd.), sorbitan monooleate (Span80: Tokyo Chemical Industry Co., Ltd.), sodium cholate, lecithin, etc. it can.

Further, as 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. In addition to applying to the cavity 103 as described above, 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. Moreover, you may apply | coat to the surface at the side of the spacer layer of the cover layer 130. FIG. Further, a buffering agent such as phosphoric acid may be provided in order to reduce variation in the concentration of ions contained in the sample.

Next, after the reaction layer 106 is formed in the cavity 103, 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. By being laminated on 120, the biosensor 100 is formed. As shown in FIGS. 1A and 1B, 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.

In this embodiment, 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. A mediator that contains GDH (glucose dehydrogenase) (hereinafter referred to as FAD-GDH), which is a coenzyme, and is reduced by electrons generated by the reaction of glucose as a measurement object with FAD-GDH to become a reducing substance 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.

In the biosensor 100 configured as described above, as described above, 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. Then, a voltage (for example, 0.3 V) is applied between the working electrode 101 and the counter electrode 102 of the biosensor 100 with respect to the reducing substance generated by the oxidation-reduction reaction caused by the reaction layer 106 dissolving in the sample. 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. In addition, after a voltage is applied between the working electrode 101 and the counter electrode 102 of the biosensor 100, a current value 3 to 5 seconds later is measured as an oxidation current.

(Comparison example of background 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 (μA). 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.

As shown in FIG. 4, as the storage period of the biosensor becomes longer, 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.

Therefore, in this embodiment, 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. In addition, 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. Is filtered to block the movement of blood cells, and components other than blood cells (for example, glucose) come into contact with the

electrodes

101 and 102 of the electrode layer 110, so that the influence on the measurement accuracy due to the difference in the hematocrit value of the blood sample Can be reduced.

By the way, when 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.

Therefore, in this embodiment, 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. Thus, 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. By configuring in this way, it is possible to prevent the mediator from being reduced by the hydrophilic polymer (CMC) having a double bond of oxygen atoms contained in the hydrophilic layer 106a in the storage state of the biosensor 100. In addition, it is possible to suppress an increase in background current accompanying the reduction of the mediator by CMC.

In addition, since the reagent layer 106c includes a hydrophilic polymer that does not have an oxygen atom double bond, 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.

Further, since 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.

As described above, according to the configuration of the present embodiment, it is possible to achieve both the suppression of the increase in the background current accompanying the reduction of the mediator by CMC and the reduction in the influence on the measurement accuracy due to the difference in the hematocrit value. A highly reliable biosensor 100 can be provided.

Second Embodiment
A biosensor according to a second embodiment of the present invention will be described with reference to FIGS. In addition, 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.

In this case, the intermediate layer 106b has a predetermined amount (for example, 0.5 μL) 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.

In addition, polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, etc. can be used as the amphiphilic polymer. Further, two or more of these amphiphilic polymers may be used in combination.

In addition, regarding the relationship between the storage period of the biosensor 100 and the background current in this embodiment, as shown in FIG. 5, 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.

Therefore, according to the present embodiment, even when the intermediate layer 106b is composed of an amphiphilic polymer, 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.

<Third Embodiment>
A biosensor according to a third embodiment of the present invention will be described with reference to FIG. 2, FIG. 6, and FIG. 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, and 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.

In this embodiment, 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.

Subsequently, as shown in FIG. 6C, 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. Finally, the reagent layer 106c is formed in the same manner as in the first embodiment (see FIG. 6D).

As the water-soluble gelling agent, a carrageenan or alginic acid compound can be used, and as the reagent 205 containing a cation, an 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. Moreover, you may use these water-soluble gelling agents in combination of 2 or more types, respectively.

Further, regarding the relationship between the storage period of the biosensor 100 and the background current in this embodiment, as shown in FIG. 7, the increase in the background current is suppressed as in the first embodiment. Note that the background current shown in FIG. 7 is measured under the same conditions as the background current of the first embodiment described above.

Therefore, according to the present embodiment, even when the intermediate layer 106b is composed of a water-soluble gelling agent, 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.

<Fourth embodiment>
A biosensor according to a fourth embodiment of the present invention will be described with reference to FIGS. 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, and 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.

In this embodiment, 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.

Further, regarding the relationship between the storage period of the biosensor 100 and the background current in this embodiment, as shown in FIG. 9, the increase in the background current is suppressed as in the first embodiment. Note that the background current shown in FIG. 9 is measured under the same conditions as the background current of the first embodiment described above.

Therefore, according to the present embodiment, even when the intermediate layer 106b is formed of a water repellent layer, 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.

<Fifth Embodiment>
A biosensor according to a fifth embodiment of the present invention will be described with reference to FIG. FIG. 10 is a cross-sectional view of the cavity portion of the biosensor according to the fifth embodiment.

The biosensor 100a according to this embodiment 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. A mediator layer 106c1 containing a hydrophilic polymer not containing oxygen and an enzyme layer 106c2 containing a hydrophilic polymer not containing a double bond between an enzyme and an oxygen atom, and a mediator layer on the enzyme layer 106c2. 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. In addition, the 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.

With such a configuration, the enzyme and the mediator are separated, so that the mediator can be prevented from being reduced by the enzyme in the storage state. In addition, by laminating the mediator layer 106c1 on the enzyme layer 106c2, 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. . In addition, since 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. Thus, the responsiveness and measurement accuracy of the sensor are improved.

The present invention is not limited to the above-described embodiments, and various modifications other than those described above can be made without departing from the spirit of the invention.

For example, in each of the above-described embodiments, 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. Although formed, 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. Alternatively, 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. .

In addition, by combining a plurality of hydrophilic polymers having no oxygen atom double bond and appropriately mixing them in the reagent layer 106c, it is possible to effectively prevent the movement of blood cells in the blood sample, In addition, diffusion and mixing of the mediator layer are reduced, and the prevention effect of preventing reduction of the mediator can be improved.

Further, 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.

In each of the embodiments described above, the biosensor 100 is formed in a bipolar electrode structure having the working electrode 101 and the counter electrode 102. However, the biosensor 100 is formed in a tripolar electrode structure by further providing a reference electrode. May be. In this case, 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.

In the above-described embodiment, 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. Although it is detected that the sample has been supplied, 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. *

Of the electrode layer 110, the spacer layer 120, and the cover layer 130 that form the

biosensors

100 and 100a, at least 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.

Moreover, the present invention can be applied to various biosensors.

100, 100a, 500

Biosensor

101, 501

Working electrode

102, 502

Counter electrode

101a, 102a

Electrode pattern

103, 104, 507 Cavity 103a Sample inlet 104

Slit

105,

506a Air hole

106, 503

Reaction layer

106a, 503a Hydrophilic layer

106b Middle Layer

106c, 503b Reagent layer 106c1 Mediator layer

106c2 Enzyme layer

110, 510

Electrode layer

111, 504 Insulating

substrate

120, 505

Spacer layer

130, 506 Cover layer 200

Dropping device

201, 202, 203, 204, 205 Reagent

Claims

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;
In a biosensor comprising the working electrode exposed in the cavity and a reaction layer provided on the tip side of the counter electrode,
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, and a surface of the hydrophilic layer. An intermediate layer comprising at least one of the layers;
A biosensor comprising: a reagent layer provided on the intermediate layer and containing an enzyme and a mediator that reacts with a substance to be measured.
The biosensor according to claim 1, wherein the reagent layer further includes a hydrophilic polymer having no oxygen atom double bond.
The reagent layer is
An enzyme layer comprising a hydrophilic polymer having no double bond of the enzyme and the oxygen atom;
The biosensor according to claim 2, further comprising: a mediator layer including a hydrophilic polymer not having a double bond of the mediator and the oxygen atom.
The biosensor according to claim 3, wherein the mediator layer is laminated on the enzyme layer.
The hydrophilic polymer having no oxygen atom double bond includes at least one of hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, polyvinyl alcohol, and polyethylene glycol. The biosensor according to any one of 2 to 4.
The biosensor according to any one of claims 1 to 5, wherein the hydrophilic polymer having a double bond of an oxygen atom contains 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, lecithin. The biosensor according to any one of claims 1 to 6, comprising at least one of them.
The biosensor according to any one of claims 1 to 7, wherein the water-soluble gelling agent contains at least one of carrageenan and sodium alginate.
The biosensor according to any one of claims 1 to 8, wherein the amphiphilic polymer contains at least one of polyvinyl pyrrolidone, polyethylene glycol, and polyvinyl alcohol.
10. The biosensor according to claim 1, wherein the water repellent layer is formed by etching the surface of the hydrophilic layer with plasma generated using a fluorine-based gas.