WO2015002184A1

WO2015002184A1 - Biosensor

Info

Publication number: WO2015002184A1
Application number: PCT/JP2014/067510
Authority: WO
Inventors: 純 ▲高▼木; 勝重小谷; 功二田中; 淳典平塚; 典子佐々木; 一道小澤; 憲二横山
Original assignee: 株式会社村田製作所
Priority date: 2013-07-05
Filing date: 2014-07-01
Publication date: 2015-01-08

Abstract

A biosensor which is provided with: an insulating substrate; an electrode layer that contains a working electrode and a counter electrode; a spacer layer that has a slit; a cover layer that is laminated on a surface of the spacer layer, said surface being on the reverse side of the electrode layer-side surface; a cavity that is formed by the electrode layer, the slit and the cover layer; an enzyme layer that contains an enzyme, which reacts with a substance to be measured, but does not contain a mediator; and a mediator layer that contains a mediator but does not contain an enzyme, while containing no hydrophilic polymer that has a double-bonded oxygen atom. One of the enzyme layer and the mediator layer is provided on at least a part of respective cavity-side surfaces of the working electrode and the counter electrode, which are exposed to the cavity, and the other one is provided on at least a part of the surface of the cover layer which is exposed to the cavity. The enzyme layer and the mediator layer are separated from each other by having a space therebetween.

Description

Biosensor

The present invention relates to a biosensor using an electrochemical method.

An electrochemical biosensor (for example, a glucose sensor such as a blood glucose level sensor) has a slit 41 for forming a cavity on an electrode layer including a working electrode 21 and a counter electrode 22 formed on an insulating substrate 1. After the spacer 4 is bonded, a reagent (enzyme, mediator, etc.) is dropped on the bottom surface (surface of the electrode layer exposed in the cavity) 3 to form a reagent layer, and the cover layer 5 is formed on the spacer 4. It has a bonded structure (see FIG. 1).

The operating principle of the biosensor is as follows. First, a sample (blood or the like) is introduced into a groove (slit 41) formed in the spacer 4. A component (such as glucose) contained in the specimen reduces the mediator through the enzyme. Here, when a predetermined voltage is applied between the working electrode 21 and the counter electrode 22, the reduced mediator is reversely oxidized by an electrochemical reaction. By measuring the oxidation current generated at this time, the amount of the component of interest can be detected.

Representative of conventional biosensors disclosed in Patent Document 1 (JP-A-1-134242), Patent Document 2 (JP-A 2002-207022), and Patent Document 3 (JP-A 2009-244012) Sectional schematic diagrams of the reagent structure are shown in FIGS. 15 (a) to 15 (c), respectively.

In the biosensor disclosed in Patent Document 1 (FIG. 15A), a layer of carboxymethyl cellulose (CMC), which is a hydrophilic polymer, is formed on an electrode layer. The CMC layer has a function of filtering blood cells and preventing adsorption of proteins to the electrode surface. Further, a layer containing an enzyme and an electron acceptor (mediator) is formed on the CMC layer via a porous film or the like (not shown).

The biosensor disclosed in Patent Document 2 (FIG. 15B) has a one-layer structure in which a reagent layer containing an enzyme, an electron carrier (mediator) and a hydrophilic polymer (such as CMC) is formed on an electrode layer. is there.

In the biosensor of Patent Document 3 (FIG. 15C), a first reaction layer containing an electron carrier (mediator) and a hydrophilic polymer (such as CMC) is formed on an electrode layer, A second reaction layer containing an enzyme and a hydrophilic polymer (such as CMC) is formed on the upper side (electrode layer side) separated from the first reaction layer. In the first reaction layer, the hydrophilic polymer functions to filter blood cells and prevent adsorption of proteins to the electrode surface. In the second reaction layer, the hydrophilic polymer immobilizes mediators. It functions as an agent.

JP-A-1-134242 JP 2002-207022 A JP 2009-244012 A

In the biosensors of

Patent Documents

1 and 2, the enzyme and the mediator are in contact with each other. However, since the enzyme has an action of reducing the mediator, if the enzyme is in contact with the mediator, the background current is increased.

Moreover, in the biosensor of Patent Document 3, the enzyme and the mediator are separated by providing a space between them, so that it is considered that the above reduction is suppressed. However, according to the study by the present inventors, the hydrophilic polymer such as CMC also has an action of reducing the mediator, and the CMC reduces the mediator in the first reaction layer on the electrode layer side. Turned out to increase. Note that this phenomenon is thought to be due to the nucleophilic attack of the functional group containing oxygen with a double bond on the mediator and electron donation.

Therefore, in any of the above prior art sensors, there is a problem that the current value detected by the blood glucose meter during actual measurement is larger than the actual value (only for the enzyme reaction). In particular, when these blood glucose level sensors are stored in a high-temperature environment or a high-humidity environment exceeding room temperature (30 ° C.), the reduction of the mediator proceeds greatly and the increase in background current becomes significant. It should be noted that, since the background current is corrected, the fluctuation of the background current increases due to the increase of the background current, so that the measurement accuracy is lowered.
The present invention has been made in view of the above problems, and provides a biosensor capable of suppressing an increase in background current due to reduction of a mediator before use in a storage state or the like and suppressing a decrease in measurement accuracy. The purpose is to do.

(1) an insulating substrate;
An electrode layer including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A spacer layer that has a slit, and is laminated on the surface of the electrode layer opposite to the insulating substrate so that the slit is located on at least a part of the surface of the working electrode and the counter electrode;
A cover layer laminated on the surface of the spacer layer opposite to the electrode layer;
A cavity for supplying a sample formed by the electrode layer, the slit and the cover layer;
An enzyme layer that contains an enzyme that reacts with the substance to be measured and does not contain a mediator;
A biosensor comprising a mediator layer that includes a mediator, does not include an enzyme, and does not include a hydrophilic polymer having a double-bonded oxygen atom,
One of the enzyme layer and the mediator layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed to the cavity, and the other of the cover layer exposed to the cavity. Provided on at least part of the surface,
A biosensor characterized in that a space is provided between the enzyme layer and the mediator layer to separate them.

(2) The enzyme layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed to the cavity,
The biosensor according to (1), wherein the mediator layer is provided on at least a part of the surface of the cover layer exposed to the cavity.

(3) The biosensor according to (1) or (2), wherein the mediator layer includes a hydrophilic polymer that does not have a double-bonded oxygen atom.

(4) The hydrophilic polymer having no double-bonded oxygen atom is a carboxyl group, carbonyl group, acyl group, aldehyde group, sulfo group, sulfonyl group, sulfoxide group, tosyl group, nitro group, nitroso group, ester. The biosensor according to (3), which does not have a group, a keto group, or a ketene group.

(5) The hydrophilic polymer having no double-bonded oxygen atom contains at least one of hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, polyvinyl alcohol, and polyethylene glycol. The biosensor as described in (3) or (4) above, wherein

(6) The biosensor according to any one of (1) to (5), wherein the enzyme layer includes a hydrophilic polymer.

(7) The enzyme layer includes a hydrophilic polymer,
The biosensor according to (2), further comprising a polymer layer containing a hydrophilic polymer between the enzyme layer and the electrode layer so as to be in contact with both.

(8) The biosensor according to (7), wherein the polymer layer includes a hydrophilic polymer having a double-bonded oxygen atom.

(9) The hydrophilic polymer having a double-bonded oxygen atom is a carboxyl group, a carbonyl group, an acyl group, an aldehyde group, a sulfo group, a sulfonyl group, a sulfoxide group, a tosyl group, a nitro group, a nitroso group, or an ester group. The biosensor according to (8), which has at least one selected from the group consisting of a keto group and a ketene group.

(10) The biosensor according to (9), wherein the hydrophilic polymer having a double-bonded oxygen atom contains at least carboxymethylcellulose.

(11) The biosensor according to (7), wherein the polymer layer includes a hydrophilic polymer that does not have a double-bonded oxygen atom.

(12) The biosensor according to any one of (1) to (11), wherein the mediator is a substance that itself undergoes a redox reaction.

In the present invention, an enzyme layer that contains an enzyme that reacts with the substance to be measured and does not contain a mediator, a mediator layer that contains a mediator, does not contain an enzyme, and does not contain a hydrophilic polymer having a double-bonded oxygen atom; By providing a space between the two and completely separating them, it is possible to suppress an increase in the background current due to the reduction of the mediator before use such as storage state, etc. Can provide a highly accurate biosensor.

It is a perspective view which shows an example of the basic composition aspect of a biosensor. 2 is a schematic cross-sectional view showing a reagent configuration of Embodiment 1. FIG. 6 is a schematic cross-sectional view showing a reagent configuration of Embodiment 2. FIG. 6 is a schematic cross-sectional view showing a reagent configuration of Embodiment 3. FIG. 6 is a schematic cross-sectional view showing a reagent configuration of Embodiment 4. FIG. 6 is a schematic cross-sectional view showing a reagent configuration of Embodiment 5. FIG. 10 is a schematic cross-sectional view showing a reagent configuration of Embodiment 6. FIG. It is a perspective view for demonstrating an example of the manufacturing process of a biosensor. It is another perspective view for demonstrating an example of the manufacturing process of a biosensor. It is another perspective view for demonstrating an example of the manufacturing process of a biosensor. It is another perspective view for demonstrating an example of the manufacturing process of a biosensor. It is another perspective view for demonstrating an example of the manufacturing process of a biosensor. 3 is a graph showing the results of a heat resistance test of Example 1. 3 is a graph showing the results of a moisture resistance test of Example 1. It is the cross-sectional schematic which shows the reagent structure in the conventional biosensor.

The biosensor of the present invention is
An insulating substrate;
An electrode layer including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A spacer having a slit and laminated on the surface of the electrode layer opposite to the insulating substrate so that the slit is positioned on at least a part of the surface of the working electrode and the counter electrode;
And a cover layer laminated on a surface opposite to the electrode layer of the spacer.

In the biosensor of the present invention, a cavity for supplying a sample is formed by such an electrode layer, a spacer slit, and a cover layer. One of the enzyme layer and the mediator layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed to the cavity, and the other is exposed to the cavity. It is provided on at least a part of the surface of the layer, and both are separated by having a space between the enzyme layer and the mediator layer.

Here, the enzyme layer contains an enzyme that reacts with the substance to be measured and does not contain a mediator. The mediator layer contains a mediator, does not contain an enzyme, and does not contain a hydrophilic polymer having a double-bonded oxygen atom (hereinafter abbreviated as “double-bonded oxygen”).

In this way, by providing a space between the enzyme layer and the mediator layer and separating the two, the mediator is reduced by the enzyme before use in a storage state or the like, and the hydrophilic polymer having double bond oxygen Can reduce the reduction of mediators. It is preferable that the enzyme layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed in the cavity, and the mediator layer is provided on at least a part of the surface of the cover layer exposed in the cavity.

The mediator is a compound (electron carrier) that mediates electron transfer between the working electrode and the counter electrode, and is preferably a substance that itself undergoes a redox reaction. As the mediator, for example, potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, ruthenium complexes and the like can be used.

The mediator layer preferably contains a hydrophilic polymer having no double-bonded oxygen atom. It is possible to easily fix the mediator layer to the surface of the cover layer or the electrode layer while suppressing reduction of the mediator by the hydrophilic polymer having double bond oxygen before use in a storage state or the like.

Examples of the hydrophilic polymer having no double-bonded oxygen atom (double-bonded oxygen) include a carboxyl group, a carbonyl group, an acyl group, an aldehyde group, a sulfo group, a sulfonyl group, a sulfoxide group, a tosyl group, and a nitro group. And hydrophilic polymers having no group, nitroso group, ester group, keto group and ketene group. Specific examples include hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, polyvinyl alcohol, and polyethylene glycol.

Examples of the enzyme include glucose oxidase, glucose dehydrogenase, alcohol oxidase, alcohol dehydrogenase, lactate oxidase, lactate dehydrogenase, cholesterol esterase, cholesterol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, hydroxybutyrate dehydrogenase, creatininase , Creatinase and DNA polymerase. Various biosensors can be produced by selecting these enzymes according to the substance to be detected (glucose, alcohol, lactic acid, cholesterol, sarcosine, fructosylamine, pyruvic acid, hydroxybutyric acid, etc.).

For example, glucose oxidase or glucose dehydrogenase can be used to produce a glucose sensor that detects glucose in a blood sample, and alcohol oxidase or alcohol dehydrogenase can be used to produce 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 produced, and a total cholesterol sensor can be produced by using a mixture of cholesterol esterase and cholesterol oxidase.

The enzyme layer preferably contains a hydrophilic polymer. In this case, the enzyme layer can be easily immobilized on the surface of the cover layer or the electrode layer.

It is preferable that a polymer layer containing a hydrophilic polymer is further provided between the enzyme layer and the electrode layer so as to be in contact with both. Thereby, it can suppress more effectively that the obstruction substance in a sample (the blood cell etc. in a blood sample) contacts an electrode, and a measurement precision improves.

Here, the hydrophilic polymer constituting the polymer layer formed on the electrode preferably includes a hydrophilic polymer having a double-bonded oxygen atom (double-bonded oxygen). This is because a hydrophilic polymer having a double bond oxygen generally has a higher effect of preventing the movement of blood cells and the like than a hydrophilic polymer having no double bond oxygen.

Examples of the hydrophilic polymer having a double-bonded oxygen atom (double-bonded oxygen) include, for example, carboxyl group, carbonyl group, acyl group, aldehyde group, sulfo group, sulfonyl group, sulfoxide group, tosyl group, nitro group, Examples include hydrophilic polymers having a nitroso group, an ester group, a keto group, and a ketene group, and a hydrophilic polymer having a carboxyl group is preferred. Examples of the hydrophilic polymer having a carboxyl group include carboxymethyl cellulose and carboxymethyl ethyl cellulose, and carboxymethyl cellulose (CMC) is preferable.

The hydrophilic polymer having double bond oxygen and the hydrophilic polymer having no double bond oxygen may be used in combination of two or more. In addition, the hydrophilic polymer in the present invention includes an amphiphilic polymer.

Hereinafter, an embodiment of a reagent configuration in the biosensor of the present invention will be described with reference to the drawings.

(Embodiment 1)
FIG. 2 shows a schematic cross-sectional view of the reagent configuration in the biosensor of this embodiment. As shown in FIG. 2, the cover layer side includes a “mediator layer” that includes a mediator, includes a hydrophilic polymer that does not have double bond oxygen, and does not include an enzyme. On the electrode layer side, an “enzyme layer” containing an enzyme, a hydrophilic polymer having no double bond oxygen, and no mediator is provided.

According to this embodiment, since the enzyme and the mediator are completely separated and the mediator is immobilized with a polymer having a small reducing action, the mediator can be reduced even when stored in a high temperature environment and a high humidity environment. While suppressing, peeling of the mediator can be suppressed and the characteristic value is stabilized. As a result, it is possible to provide a biosensor having high heat resistance and moisture resistance, accurate and reliable.

Since the mobility of the enzyme is smaller than that of the mediator, forming the enzyme layer on the electrode layer side and the mediator layer on the cover layer side increases the amount of enzyme and mediator in the vicinity of the electrode, and the sensor response. And measurement accuracy are improved.

(Embodiment 2)
FIG. 3 shows a schematic cross-sectional view of the reagent configuration in the biosensor of this embodiment. As shown in FIG. 3, the mediator layer on the cover layer side is the same as that in the first embodiment, except that a hydrophilic polymer having double bond oxygen is used in the enzyme layer on the electrode layer side. Different from Form 1.

Generally, a hydrophilic polymer having double bond oxygen has a higher effect of blocking the movement of blood cells and the like than a hydrophilic polymer having no double bond oxygen. For this reason, in the present embodiment, when the enzyme layer provided on the electrode layer contains a hydrophilic polymer having double-bonded oxygen, for example, when a blood sample is supplied to the cavity of the biosensor In addition, since blood cells are efficiently filtered by the hydrophilic polymer and movement of blood cells contained in the blood sample is prevented, the influence on measurement accuracy due to the difference in hematocrit values of the blood sample can be reduced.

(Embodiment 3)
FIG. 4 shows a schematic cross-sectional view of the reagent configuration in the biosensor of this embodiment. As shown in FIG. 4, the mediator layer and the enzyme layer are the same as in Embodiment 1, but further include a hydrophilic polymer having double bond oxygen between the enzyme layer and the electrode layer, It differs from Embodiment 1 in having a “polymer layer” that does not include a mediator.

In this embodiment, since a hydrophilic polymer layer having double bond oxygen is separately provided, movement of blood cells contained in the blood sample is more effectively prevented, so that the hematocrit value of the blood sample is reduced. The influence on the measurement accuracy due to the difference can be further reduced.

(Embodiment 4)
FIG. 5 shows a schematic cross-sectional view of the reagent configuration in the biosensor of this embodiment. As shown in FIG. 5, the mediator layer and the enzyme layer are the same as in Embodiment 2, but further include a hydrophilic polymer having double-bonded oxygen between the enzyme layer and the electrode layer, It differs from Embodiment 2 in having a “polymer layer” that does not include a mediator.

Also in the present embodiment, as in the third embodiment, the movement of blood cells contained in the blood sample is more effectively prevented, and the influence on the measurement accuracy due to the difference in the hematocrit value of the blood sample can be further reduced. .

In Embodiments 3 and 4 (FIGS. 4 and 5), the arrangement of the enzyme layer and the polymer layer may be reversed. In addition, as the hydrophilic polymer having double bond oxygen added to each layer or the hydrophilic polymer having no double bond oxygen, the same type or different types may be used.

(Embodiment 5)
FIG. 6 shows a schematic cross-sectional view of the reagent configuration in the biosensor of this embodiment. As shown in FIG. 6, the present embodiment is different from the third embodiment in that the hydrophilic polymer constituting the “polymer layer” does not have double bond oxygen, but other points are implemented. The same as in the third mode.

(Embodiment 6)
FIG. 7 shows a schematic cross-sectional view of the reagent configuration in the biosensor of this embodiment. As shown in FIG. 7, the present embodiment is different from the fourth embodiment in that the hydrophilic polymer constituting the “polymer layer” does not have double bond oxygen, but other points are implemented. This is the same as in the fourth mode.

In Embodiments 5 and 6 (FIGS. 6 and 7), the arrangement of the enzyme layer and the polymer layer may be reversed. In addition, as the hydrophilic polymer having double bond oxygen added to each layer or the hydrophilic polymer having no double bond oxygen, the same type or different types may be used.

<Configuration and manufacturing method of biosensor>
Hereinafter, an example of a basic configuration aspect of the entire biosensor and an example of a method for manufacturing the biosensor will be described with reference to FIGS. 1 and 8 to 12. FIG. 1 is a perspective view illustrating an example of a basic configuration aspect of a biosensor. FIGS. 8 to 12 are diagrams for explaining an example of the manufacturing process of the biosensor, and show different processes.

In the biosensor shown in FIG. 1, a spacer 4 having a slit 41 for forming a cavity is bonded to an electrode layer including a working electrode 21 and a counter electrode 22 formed on an insulating substrate 1. A reagent layer is formed in a cavity surrounded by the bottom surface 3 (surface of the electrode layer), the inner wall surface of the slit 41, and the surface (lower surface) of the cover layer 5, and the cover layer 5 is bonded onto the spacer 4 (See FIG. 1). The reagent layer includes the above-described enzyme layer, mediator layer, polymer layer, and the like.

In the biosensor of the present invention, the enzyme layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed in the cavity, and the mediator layer is at least on the surface of the cover layer exposed in the cavity. It is provided in a part.

This biosensor is used by being mounted on a measuring instrument. That is, a sample (blood or the like) is supplied to the cavity of the biosensor mounted on the measuring instrument, and a reducing substance is generated by the reaction of the substance to be measured (such as glucose) in the sample with the enzyme and mediator. A voltage is applied between the working electrode 21 and the counter electrode 22 by a measuring device electrically connected to the working electrode 21 and the counter electrode 22 of the biosensor, and an oxidation current obtained by oxidizing this reducing substance is obtained. By measuring, the measurement target substance contained in the sample is quantified.

The material of the insulating substrate 1 is not particularly limited as long as it has insulating properties, and examples thereof include plastic such as PET film, paper, glass, ceramic, and biodegradable material.

In addition to the working electrode and the counter electrode, the electrode layer may include a reference electrode serving as a potential reference when measuring the electrode potential, and a detection electrode for detecting that the sample has been supplied to the cavity.

As materials for these electrodes (working electrode, counter electrode, reference electrode, detection electrode, etc.), noble metals such as platinum, gold, palladium, carbon, copper, aluminum, titanium, ITO (Indium Tin Oxide), ZnO (zinc oxide) etc. are mentioned. The electrode layer is formed, for example, by forming a conductive layer made of the above material on one surface of the insulating substrate using screen printing or sputtering vapor deposition, and further patterning using laser processing, photolithography, etc. Can be produced. In addition, the working electrode 21 and the counter electrode 22 are arranged so that their respective distal end sides are exposed to cavities formed by the slits 41 and the like.

Next, the electrode surface is cleaned with plasma. Note that as the plasma used in the plasma cleaning step, various plasmas used in metal activation treatment by plasma can be used, and examples thereof include oxygen plasma, nitrogen plasma, and argon plasma. Further, the plasma may be a low pressure plasma or an atmospheric pressure plasma.
The spacer 4 is laminated on the electrode layer formed as described above. The spacer 4 is formed of a substrate made of an insulating material, and a slit 41 for forming a cavity is formed at substantially the center of the front edge portion of the substrate. Then, the spacer 4 is laminated so as to partially cover one surface of the electrode layer so that the slit 41 is disposed on the tip side of the working electrode 21 and the counter electrode 22. Examples of the insulating material constituting the spacer 4 include plastic (such as PET film), paper, glass, ceramic, and biodegradable material.

Next, the above-described enzyme layer or polymer layer is formed on the surface of the electrode layer in the cavity formed as described above. Specifically, a predetermined amount of a hydrophilic polymer solution such as CMC is dropped from a dropping device so as to cover the electrode layer, and dried to form a polymer layer (polymer layer forming step).

Next, a predetermined amount of a reagent containing an enzyme and a hydrophilic polymer having double-bonded oxygen is dropped into the cavity from the dropping device and dried to form an enzyme layer (enzyme layer forming step).

The material of the cover layer 5 is preferably an insulating material, and for example, plastic such as PET film, paper, glass, ceramic, and biodegradable material can be used. The cover layer 5 is preferably formed with an air hole 51 that communicates with the cavity when stacked on the spacer 4. In the biosensor configured as described above, by bringing a sample such as blood into contact with the opening of the cavity, the sample is sucked toward the air hole 51 by capillary action, so that the sample can be easily introduced into the cavity. Because it becomes.

The above-mentioned mediator layer is formed on at least a part of the surface exposed to the cavity of the cover layer 5 (the surface on the spacer 4 side).

Next, the biosensor is formed by laminating the cover layer 5 having the mediator layer on the spacer 4 so that the mediator layer is located in the cavity (in the slit 41).

In addition, as a material constituting the reagent layer (enzyme layer, mediator layer, polymer layer, etc.), a reagent such as a hydrophilizing agent for promoting the introduction of the specimen (sample) may be blended as necessary. As the hydrophilizing agent, a surfactant such as Triton X100 (manufactured by Sigma Aldrich), Tween 20 (manufactured by Tokyo Chemical Industry Co., Ltd.), sodium bis (2-ethylhexyl) sulfosuccinate, or a phospholipid such as lecithin can be used. Further, a buffering agent such as phosphoric acid may be blended in order to reduce the variation in ion concentration contained in the sample. These hydrophilizing agents and buffering agents may be applied in advance to the inner wall surface of the cavity before forming the enzyme layer, mediator layer, polymer layer and the like.

<How to use the biosensor>
Hereinafter, an example of a method for using the biosensor of the present invention will be described.

First, blood is brought into contact with the tip of the cavity, and the blood is introduced into the cavity using capillary action.

Next, a voltage is applied between the working electrode and the counter electrode, and the current value is measured at a constant timing. The applied voltage is, for example, 0.3V.

Next, measure the current value after a certain period of time has elapsed since voltage application. For example, the current value after 3 to 5 seconds is measured. Using this current value, the analyte concentration can be determined from a calibration curve obtained in advance.

When blood is introduced into the cavity, the analyte in the blood reduces the mediator via the enzyme. The current that flows when a voltage is applied between the working electrode and the counter electrode correlates with the reductant concentration of the mediator, that is, the analyte concentration.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
First, an electrode layer was formed on an insulating substrate made of an insulating material such as polyethylene terephthalate. Specifically, a conductive layer made of a noble metal such as platinum, gold, or palladium, or a conductive material such as carbon, copper, aluminum, titanium, ITO, or ZnO is formed on one surface of the insulating substrate that forms the electrode layer. It was formed by screen printing or sputtering deposition. The conductive layer formed on one surface of the insulating substrate was subjected to pattern formation by laser processing or photolithography (FIG. 8).

Next, spacers 4 (made of polyethylene terephthalate or the like) having slits (cuts) 41 were bonded together (FIG. 9). Next, a polymer layer was formed by dropping a solution of CMC (blood cell filter agent) on an electrode (working electrode, counter electrode) for measuring the analyte concentration and drying it (FIG. 10).

Next, a mixed solution of CMC and enzyme was dropped on the polymer layer and dried to form an enzyme layer.

Separately, a liquid mixture of mediator and hydroxypropylmethylcellulose as a hydrophilic polymer is dropped into a portion exposed in the cavity on one surface of the cover layer 5 made of an insulating material such as polyethylene terephthalate and dried. Thus, a mediator layer was formed.

Next, the cover layer 5 having a mediator layer was bonded onto the spacer 4 so that the mediator layer was positioned in the slit 41 (in the cavity) (FIG. 11). The cover layer 5 is provided with an air hole 51 for letting air escape.

Next, the biosensor was obtained by cutting the laminate so that a part of the cavity formed by the spacer 4 and the like was exposed (FIG. 12).

[Heat resistance test]
A heat resistance test was performed on the biosensor obtained in Example 1 and the conventional biosensor (configuration described in Patent Document 3: FIG. 15C). Specifically, it is stored in a constant temperature bath at a temperature of 50 ° C., and a voltage of 0.3 V is applied between the working electrode and the counter electrode with respect to the biosensor after a predetermined time (200, 400, 700, 900 hours). Applied and measured the value of the background current. The measurement results are shown in FIG.

As shown in FIG. 13, when stored in a high temperature environment of 50 ° C., the increase in background current is significant in the conventional biosensor, whereas in the biosensor of Example 1, the measurement current changes from the initial value. Without increasing the background current.

[Moisture resistance test]
The moisture resistance test was performed on the biosensor obtained in Example 1 and the conventional biosensor (configuration described in Patent Document 3: FIG. 15 (c)). Specifically, it is stored in a thermo-hygrostat set at 90% relative humidity (RH) and a temperature of 30 ° C., and 0.3 V between the working electrode and the counter electrode with respect to the biosensor after a predetermined time has elapsed. Then, the background current value was measured. The measurement results are shown in FIG.

As shown in FIG. 14, when stored in a high humidity environment of 90%, the conventional biosensor has a significant increase in background current, whereas the biosensor of Example 1 has a measured current from the initial value. It can be seen that the increase in the background current is suppressed without changing.

This is because the enzyme and CMC are formed on the electrode side and the mediator is formed on the cover side, and the enzyme, CMC and mediator are completely separated, and the thickener added to the mediator layer does not have double bond oxygen. This is considered to be because, by immobilizing with a polymer having a small reducing action, it was possible to achieve both suppression of reduction of the mediator and suppression of property deterioration due to peeling of the mediator. As a result, it can be seen that according to the present invention, it is possible to provide a sensor for detecting a blood glucose level that has high heat resistance and high humidity resistance, is accurate, and has high reliability.

1 Insulating substrate, 21 working electrode, 22 counter electrode, 3 bottom of cavity, 4 spacer, 41 slit, 5 cover layer, 51 air hole.

Claims

An insulating substrate;
An electrode layer including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A spacer layer that has a slit, and is laminated on the surface of the electrode layer opposite to the insulating substrate so that the slit is located on at least a part of the surface of the working electrode and the counter electrode;
A cover layer laminated on the surface of the spacer layer opposite to the electrode layer;
A cavity for supplying a sample formed by the electrode layer, the slit and the cover layer;
An enzyme layer that contains an enzyme that reacts with the substance to be measured and does not contain a mediator;
A biosensor comprising a mediator layer that includes a mediator, does not include an enzyme, and does not include a hydrophilic polymer having a double-bonded oxygen atom,
One of the enzyme layer and the mediator layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed to the cavity, and the other of the cover layer exposed to the cavity. Provided on at least part of the surface,
A biosensor characterized in that a space is provided between the enzyme layer and the mediator layer to separate them.
The enzyme layer is provided on at least a part of the cavity-side surface of the working electrode and the counter electrode exposed in the cavity;
The biosensor according to claim 1, wherein the mediator layer is provided on at least a part of the surface of the cover layer exposed to the cavity.
The biosensor according to claim 1, wherein the mediator layer includes a hydrophilic polymer that does not have a double-bonded oxygen atom.
The hydrophilic polymer having no double-bonded oxygen atom includes carboxyl group, carbonyl group, acyl group, aldehyde group, sulfo group, sulfonyl group, sulfoxide group, tosyl group, nitro group, nitroso group, ester group, keto group. The biosensor according to claim 3, which has no group and no ketene group.
The hydrophilic polymer having no double-bonded oxygen atom includes at least one of hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, polyvinyl alcohol, and polyethylene glycol. The biosensor according to claim 3 or 4.
The biosensor according to any one of claims 1 to 5, wherein the enzyme layer contains a hydrophilic polymer.
The enzyme layer includes a hydrophilic polymer,
The biosensor according to claim 2, further comprising a polymer layer containing a hydrophilic polymer between the enzyme layer and the electrode layer so as to be in contact with both.
The biosensor according to claim 7, wherein the polymer layer includes a hydrophilic polymer having a double-bonded oxygen atom.
The hydrophilic polymer having a double-bonded oxygen atom includes a carboxyl group, a carbonyl group, an acyl group, an aldehyde group, a sulfo group, a sulfonyl group, a sulfoxide group, a tosyl group, a nitro group, a nitroso group, an ester group, and a keto group. And at least one selected from the group consisting of ketene groups.
The biosensor according to claim 9, comprising at least carboxymethylcellulose as the hydrophilic polymer having a double-bonded oxygen atom.
The biosensor according to claim 7, wherein the polymer layer includes a hydrophilic polymer having no double-bonded oxygen atom.
The biosensor according to any one of claims 1 to 11, wherein the mediator is a substance that itself undergoes an oxidation-reduction reaction.