JP2013190212A - Biosensor and manufacturing method of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor including a carbon electrode layer for suppressing corrosion of a metal layer, and a manufacturing method of the biosensor.SOLUTION: A biosensor includes: a first substrate; a metal layer arranged on a first surface of the first substrate; a first electrode layer including carbon provided on one end of the metal layer and a first resin; a second electrode layer including carbon provided so as to cover the first electrode layer; and a reaction part positioned on the second electrode layer. The first electrode layer includes the first resin with lower water absorption coefficient than the second electrode layer.

Description

The present invention relates to a biosensor, and more particularly to a biosensor that electrochemically measures the concentration of a substance in a solution.

Biosensors using electrochemical detection means have been put into practical use as a method for quickly and easily measuring the concentration and the like of a specific component in a biological sample such as blood. A biosensor generally includes an electrode system including a working electrode and a counter electrode, an enzyme, and an electron acceptor as a basic configuration. An example of such a biosensor is a glucose sensor that electrochemically quantifies glucose in blood.

In the glucose sensor, the enzyme selectively oxidizes glucose in blood to produce gluconic acid, and simultaneously reduces the electron acceptor to produce a reduced form. By applying a constant voltage to the reductant with an electrode system, the reductant is oxidized again, and current is generated at that time. Since this current depends on the glucose concentration in the blood, glucose in the blood can be quantified.

Conventionally, a lead wiring is formed by screen printing of a silver paste, and a conductive carbon paste is printed on the lead wiring to form an electrode system, thereby producing a biosensor (see Patent Documents 1 and 2). ). In an electrode system formed of conductive carbon in a conventional biosensor, moisture and electron acceptors (mediators) in the sample enter the electrode from the voids existing in the carbon electrode layer, and are oxidized under the carbon electrode layer by an oxidation-reduction reaction. Corrosion of the metal layer (or wiring) disposed on the biosensor reduces the electrical characteristics of the biosensor.

JP-A-8-15220 JP-A-8-5600

This invention solves the above-mentioned problem, Comprising: It aims at providing the biosensor provided with the carbon electrode layer which suppresses corrosion of a metal layer, and its manufacturing method.

According to an embodiment of the present invention, a first base, a metal layer disposed on a first surface of the first base, carbon disposed on one end of the metal layer, and a first A first electrode layer containing a resin, a second electrode layer containing carbon disposed to cover the first electrode layer, and a second electrode layer disposed on the second electrode layer. A biosensor comprising a first resin having a lower water absorption rate than the second electrode layer.

The biosensor according to this embodiment includes a first electrode layer including carbon disposed on one end of a metal layer, and a second electrode layer including carbon disposed so as to cover the first electrode layer. And the first electrode layer includes an electrode layer containing carbon having a two-layer structure including a first resin having a lower water absorption rate than the second electrode layer. Suppressing the contained electron acceptor from entering the second electrode layer and inhibiting the metal layer disposed under the second electrode layer from being corroded by the redox reaction of the electron acceptor. Can do.

In the biosensor, the second electrode layer may include a second resin having higher hydrophilicity than the first electrode layer.

Since the second electrode layer includes an electrode layer including carbon having a two-layer structure including the second resin having higher hydrophilicity than the first electrode layer, the second electrode layer is caused by a reaction between an object to be detected and an enzyme contained in the sample. The generated electric signal can be easily transmitted to the first electrode layer through the electron acceptor.

According to one embodiment of the present invention, a metal layer having a predetermined pattern is formed on the first surface of the first base material, and carbon and the first resin are formed on one end of the metal layer. And a first coating solution containing a first solvent to form a first electrode layer, carbon and a second resin so as to be positioned on the first electrode layer And applying a second coating solution containing a second solvent to form a second electrode layer, forming a reaction portion so as to be positioned on the second electrode layer, and A method for producing a biosensor is provided in which the resin is a resin having a lower water absorption rate than the second resin.

The biosensor manufacturing method according to the present embodiment forms a first electrode layer by applying a first coating liquid containing carbon and a first resin on one end of a metal layer, A second coating layer containing carbon and a second resin is applied to form the second electrode layer so as to be positioned on the electrode layer, and the first resin is more than the second resin. By being a resin having a low water absorption rate, it is possible to suppress the moisture in the sample and the electron acceptor contained in the reaction part from entering the second electrode layer, and the second electrode by the redox reaction of the electron acceptor. Corrosion of the metal layer disposed under the layer can be suppressed.

In the biosensor manufacturing method, the second resin may be more hydrophilic than the first resin.

Since the second electrode layer includes an electrode layer including carbon having a two-layer structure including a second resin having higher wettability than the first electrode layer, the second electrode layer is caused by a reaction between an object to be detected and an enzyme included in the sample. The generated electric signal can be easily transmitted to the first electrode layer through the electron acceptor.

ADVANTAGE OF THE INVENTION According to this invention, the biosensor provided with the carbon electrode layer which suppresses corrosion of a metal layer, and its manufacturing method can be provided.

It is a schematic diagram of the biosensor 1000 which concerns on one Embodiment of this invention, (a) shows the whole biosensor 1000, (b) is an exploded view of the biosensor 1000. 1A and 1B are schematic views of an electrode system 100 according to an embodiment of the present invention, in which FIG. 1A is a perspective view of an upper surface of the electrode system 100, and FIG. It is a schematic diagram which shows the manufacturing method of the biosensor 1000 which concerns on one Embodiment of this invention. It is a schematic diagram which shows the manufacturing method of the biosensor 1000 which concerns on one Embodiment of this invention. It is a schematic diagram which shows a mode that it connected to the biosensor 1000 and measuring device 10000 which concern on one Embodiment of this invention. It is a schematic diagram which shows the electrode system 900 of the conventional biosensor.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a first electrode layer containing carbon on one end of the metal layer, carbon disposed so as to cover the first electrode layer, and the first An electrode layer containing carbon having a two-layer structure with a second electrode layer containing one resin is disposed, and the first electrode layer has a lower water absorption (or higher hydrophobicity) than the second electrode layer. By containing the first resin, the moisture in the sample and the electron acceptor contained in the reaction part are prevented from entering the second electrode layer, and the second electrode layer is formed by the redox reaction of the electron acceptor. It has been found that it is possible to suppress corrosion of the metal layer disposed underneath.

Hereinafter, a biosensor and a manufacturing method thereof according to the present invention will be described with reference to the drawings. However, the biosensor of the present invention can be implemented in many different modes and should not be construed as being limited to the description of the embodiments and examples shown below. Note that in the drawings referred to in this embodiment mode and examples, the same portions or portions having similar functions are denoted by the same reference numerals, and repetitive description thereof is omitted.

(1. Biosensor structure)
The electrode structure of the biosensor 1000 according to the embodiment of the present invention will be described. FIG. 1 is a schematic diagram showing an example of a biosensor 1000 according to an embodiment of the present invention. FIG. 1A is an overall view of the biosensor 1000, and is a perspective view through which the second base material 1100 and the third base material 1200 are transmitted for convenience of explanation. FIG. 1B is an exploded view of the biosensor 1000. 2A and 2B are diagrams for explaining the electrode system 100 of the biosensor 1000 according to the embodiment of the present invention. FIG. 2A is a perspective view of the upper surface of the electrode system 100, and FIG. AA 'sectional drawing in a) is shown.

The biosensor 1000 according to the present embodiment includes a first base 170, a metal layer 101 disposed on the first surface (upper surface) of the first base 170, and one end of the metal layer 101. A first electrode layer 103 containing carbon formed, a second electrode layer 105 containing carbon disposed so as to cover the first electrode layer 103, and a second electrode layer 105. The reaction unit 140 is provided. In the present embodiment, the first electrode layer 103 includes a first resin having a lower water absorption rate (higher hydrophobicity) than the second electrode layer 105. By including the first resin, the water resistance of the first electrode layer can be improved. An electrode system including a working electrode 110, a counter electrode 120, and a reference electrode 130 formed by the electrode layer (first electrode layer 103 and second electrode layer 105) containing carbon having such a two-layer structure and the metal layer 101. 100 is configured.

On the other hand, in the electrode system 900 in the conventional biosensor shown in FIG. 6, only one electrode layer 907 containing carbon is disposed on the metal layer 101. In the conventional biosensor having such a structure, since the electrode layer 907 containing carbon does not have sufficient corrosion resistance, corrosion of the metal layer 101 is suppressed during the period from manufacture to use. It was difficult. At the time of measurement, moisture and electron acceptors (mediators) in the sample enter the inside of the electrode from the voids present in the electrode layer 907 containing carbon, and are disposed under the electrode layer 907 containing carbon by an oxidation-reduction reaction. In addition, the metal layer 101 (or the wiring) is corroded to deteriorate the electrical characteristics of the biosensor.

The biosensor 1000 according to this embodiment includes a second base material 1100 that is a spacer for forming the sample supply path 1510 above the electrode system 100 and the wiring part 150, and a first cover that is an upper cover of the sample supply path 1510. The three base materials 1200 are sequentially laminated and fixed so as to cover the second base material. The working electrode 110 is further formed with a reaction portion 140 on a part of the upper surface of the second electrode layer 105.

The second base material 1100 formed on the electrode system 100 and the wiring part 150 is formed on the reaction electrode 140 of the working electrode 110, the counter electrode 120, and the second electrode layer 105 of the reference electrode 130, for example, on the second electrode layer 105. It arrange | positions so that the T-shaped flow path leading to the outer edge of the base material 1100 may be formed. The T-shaped channel is perpendicular to the air vent channel 1530 and the air vent channel 1530 that is linearly disposed on the reaction electrode 140 of the working electrode 110, the counter electrode 120, and the second electrode layer 105 of the reference electrode 130. The sample supply path 1510 passes through the upper part of the reaction part 140 of the working electrode 110. The biosensor 1000 includes the sample supply path 1510 and the air vent channel 1530, and thus the sample to be measured passes through the upper part of the working electrode 110, the counter electrode 120, and the reference electrode 130 using the capillary phenomenon from the sample supply path 1510. The target component of the sample can be measured.

In the electrode system 100, the metal layer 101 is disposed on the first surface (upper surface) of the first base material 170. In the electrode system 100 of the biosensor according to the present embodiment, the metal layer 101 and the wiring portion 150 are integrally formed, and the first electrode layer 103 and the second electrode layer 105 are formed on at least a part of the metal layer 101. It is characterized by being stacked. By disposing the first electrode layer 103 containing the first resin having a low water absorption rate (having hydrophobicity), corrosion of the metal layer 101 can be suppressed. In the present embodiment, the first electrode layer 103 and the second electrode layer 105 also cover the side surface of the metal layer 101, but the first electrode layer 103 and the second electrode layer 105 are metal layers. It may be formed only on the upper surface of 101.

(Base material)
The first base material 170 is a base material that supports the electrode system 100, and at least the surface on which the electrode system 100 is disposed has an insulating property. The first base material 170 is preferably a highly rigid base material. For example, a resin base material, a ceramic base material, a glass base material, a semiconductor base material or a metal base material having at least a surface insulation is used. it can. The first base material 170 may be set to have a sufficient thickness that can realize strength sufficient for handling performance at the time of measurement according to these materials. As the first base material 170, a PET base material is preferably used. This is because a biosensor can be manufactured at low cost by using PET. The thickness of the first base material 10 may be appropriately set within a range of 6 μm to 1 mm, for example.

(Electrode system)
The electrode system 100 includes a working electrode 110, a counter electrode 120, and a reference electrode 130, and the working electrode 110 is one electrode for applying a voltage to the reductant electron acceptor. The counter electrode 120 is one electrode for measuring a current flowing by electrons emitted from the electron acceptor to the working electrode 110. The reference electrode 130 is an electrode that serves as a reference when determining the potential of the working electrode 110. Each electrode includes a metal layer 101 disposed on the first substrate 170, a first electrode layer 103 disposed on the surface of the metal layer 101, and carbon disposed so as to cover the first electrode layer 103. The 2nd electrode layer 105 containing is provided. In the present embodiment, the reaction unit 140 is described as being directly disposed on the upper surface of the working electrode 110. However, the reaction unit 140 may be disposed so as to face the working electrode 110 through a space. .

(Metal layer)
The metal layer 101 according to this embodiment is a layer containing a highly conductive metal. For the metal layer 101, for example, aluminum, iron, nickel, chromium, titanium, tantalum, silver, or the like can be used. These metals include metals that are susceptible to corrosion due to the redox reaction of the electron acceptor described later. By providing an electrode layer containing carbon having a two-layer structure according to the present invention, a sufficient anticorrosive effect is obtained. be able to. Considering that such an anticorrosion effect can be obtained, and conductivity and manufacturing cost, aluminum can be suitably used in the present embodiment.

The thickness of the metal layer 101 is preferably in the range of 0.02 μm to 40 μm. When the thickness is less than 0.02 μm, the resistance value of the metal layer 101 is sufficiently high and the intended electrode cannot be obtained. When the thickness is greater than 40 μm, the first electrode layer 103 and the second electrode layer 105 are laminated. The formation of the sample supply path 1510 and the air vent path 1530 requires high three-dimensional processing accuracy, and the number of processing dies used, processing steps, and time are dramatically increased. The metal layer 101 may be a single metal layer made of the above metal material, or may be an embodiment in which a plurality of metal layers are stacked. The metal layer 101 according to the present embodiment may be formed by any method of printing a metal paste, etching a metal foil, vapor deposition of metal, or sputtering film formation. The metal layer 101 according to the present embodiment is preferably formed by metal foil etching or metal vapor deposition from the viewpoint of reducing the amount of metal used and suppressing the manufacturing cost.

(First electrode layer)
The first electrode layer 103 according to the present embodiment is an electrode layer that exhibits resistance to water, chemicals, and the like, and includes a first resin having a lower water absorption rate than the second electrode layer 105. The water absorption rate of the first electrode layer 103 is preferably 0.1% or less. The water absorption rate may be measured according to JIS K7209 (plastic water absorption rate test method). The first electrode layer 103 is a coating layer formed of a conductive material, and serves to protect the surface of the metal layer 101 and function as part of the electrode. For example, the first electrode layer 103 can be formed of a mixture of a carbon pigment and a hydrophobic first resin. Examples of the carbon pigment used for the first electrode layer 103 include graphite, amorphous carbon, diamond-like carbon, carbon fiber, carbon black, acetylene black, ketjen black (registered trademark), carbon nanotube, carbon nanohorn, and carbon nanofiber. Etc. can be used.

Examples of the first hydrophobic resin having a low water absorption rate as described above include vinyl chloride / vinyl acetate copolymer, vinyl chloride resin, ethylene vinyl acetate copolymer, ethylene vinyl acetate resin, ethylene vinyl acetate chloride. Any one or more selected from vinyl graft copolymers can be used. Since these resins have a low water absorption rate and high moisture resistance, chemical resistance and water resistance can be imparted to the first electrode layer 103, and the anticorrosive effect of the metal layer 101 can be obtained. “Chemical resistance” in the first electrode layer 103 means resistance to mediators applied to the reaction unit 140 described later, components in the sample, or chemical substances that oxidize or corrode the metal layer 101. Means.

In the first electrode layer 103 according to this embodiment, the carbon pigment and the first resin are preferably 5% by weight to 60% by weight and 10% by weight to 40% by weight, respectively. If the carbon pigment is less than 5% by weight, the probability that the carbon pigments are electrically connected to each other rapidly decreases, and the conductivity as an electrode layer is lost. On the other hand, when the amount of carbon pigment is more than 60% by weight, voids between the pigments increase, and physical shaping strength, adhesive strength, and friction resistance strength are lost, and the required structure and function cannot be maintained. Furthermore, the redox state of the carbon surface is not stable, and enzymes and electron acceptors added in the post-process are also adsorbed unevenly in the voids. Further, if the first resin is less than 10% by weight, the coating characteristics of the first electrode layer 103 are lowered and the coating film strength is also lowered. The coating characteristics of the electrode layer 103 are lowered, and the conductivity is also lowered. Note that the first electrode layer 103 may contain other conductive pigments, reaction reagents such as curing agents and cross-linking agents, auxiliary agents and additives for improving processability, and the like as needed. It may be mixed with the resin.

The thickness of the first electrode layer 103 is preferably in the range of 0.1 μm to 10 μm, more preferably 0.3 μm to 2.0 μm. The first electrode layer 103 has a high conductive carbon pigment ratio, and if it is less than 0.1 μm, the effects of water resistance and chemical resistance are reduced. Moreover, when the conductive carbon pigment ratio is low and the thickness is larger than 10 μm, the resistance of the carbon layer increases. The first electrode layer 103 according to this embodiment contributes to the rust prevention of the metal layer 101 by coating the metal layer 101 with a carbon pigment, and the moisture and metal in the enzyme, electron acceptor, sample or air Direct contact with the layer 101 can be prevented.

(Second electrode layer)
The second electrode layer 105 is a coating layer formed so as to cover the first electrode layer 103 with a conductive material, and functions as a part of the electrode. The second electrode layer 105 is preferably an electrode that increases the affinity for an aqueous solution in which an enzyme or mediator is dissolved and protects the surface. For example, the second electrode layer 105 can be formed of a mixture of a carbon pigment and a second resin. As the carbon pigment used for the second electrode layer 105, the same pigment as that used for the first electrode layer can be used.

As the second resin, a water-insoluble resin having lower hydrophobicity (or higher hydrophilicity) than the first resin can be preferably used. For example, acrylic resin, styrene resin, acetal resin, polyvinyl acetal resin Any one or more selected from can be used. Acrylic resins and styrene resins have hydrophilic side chains, and the degree of hydrophilicity can be changed as appropriate. Polyvinyl acetal resins can have the same hydrophilicity by changing the degree of acetalization. In addition, polyvinyl acetal resins, especially butyral resins, are resistant to scratches and scratches on the printed matter because the coating film strength is increased by adding a phenol resin, an epoxy resin, a melamine resin or the like to cause a crosslinking reaction. This is particularly suitable when the electrode layer is positioned on the outermost surface. For the purpose of assisting this crosslinking reaction, a curing agent such as isocyanate or dialdehyde can be used in combination. By using the above resin having higher hydrophilicity than the first resin, it is possible to obtain the second electrode layer having a lower contact angle with respect to the aqueous liquid and higher wettability than the first electrode layer. The water absorption rate of the second electrode layer 105 is preferably 0.2% or more.

Since the wettability of the second electrode layer is high, a uniform coating surface can be obtained when an aqueous solution in which an enzyme or mediator is dissolved is applied to the reaction part by a dispenser, ink jet, mask transfer, photocopying or the like. The degree of hydrophilicity is appropriately set according to the coating solution used and the coating process.

In the second electrode layer 105 according to this embodiment, the carbon pigment and the second resin are preferably 5% by weight to 60% by weight and 10% by weight to 40% by weight, respectively. If the carbon pigment is less than 5% by weight, the probability that the carbon pigments are electrically connected to each other rapidly decreases, and the conductivity as an electrode layer is lost. On the other hand, when the amount of carbon pigment is more than 60% by weight, voids between the pigments increase, and physical shaping strength, adhesive strength, and friction resistance strength are lost, and the required structure and function cannot be maintained. Furthermore, the redox state of the carbon surface is not stable, and enzymes and electron acceptors added in the post-process are also adsorbed unevenly in the voids. Further, if the second resin is less than 10% by weight, the coating characteristics of the second electrode layer 105 are lowered and the coating film strength is also lowered. The coating characteristics of the electrode layer 105 are lowered, and the conductivity is also lowered. The second electrode layer 105 may contain other conductive pigments, reaction reagents such as curing agents and crosslinking agents, auxiliary agents and additives for improving processability, as necessary, with carbon pigments and organic binders. And may be mixed. Alternatively, the surface of the second electrode layer 105 may be mechanically polished or physically etched by a discharge method such as corona plasma to improve the surface activation.

The thickness of the second electrode layer 105 is preferably in the range of 0.1 μm to 10 μm, more preferably 0.3 μm to 2.0 μm. If the second electrode layer 105 has a high conductive carbon pigment ratio and is less than 0.1 μm, it is difficult to isolate it from the reaction part 140 in order to guarantee the manufacturing, storage, enzyme reaction conditions, and stability over time. Moreover, when the conductive carbon pigment ratio is low and the thickness is larger than 10 μm, the resistance of the carbon layer increases. The second electrode layer 105 according to this embodiment provides the affinity for the electron acceptor contained in the reaction unit 140 by covering the first electrode layer 103, and the detection target contained in the sample An electric signal generated by the reaction between the enzyme and the enzyme can be easily transmitted to the first electrode layer 103 through the electron acceptor. In order to obtain an electrode system with low resistance, it is more preferable that the thickness of the second electrode layer 105 be larger than the thickness of the first electrode layer 103. More preferably, the thickness of the second electrode layer 105 is 1.5 times or more the thickness of the first electrode layer 103.

The reaction unit 140 of the biosensor 1000 to be described later contains chemical substances that are easily degraded by oxidoreductases such as potassium ferricyanide, benzoquinone compounds, osmume complexes, ferrocene derivatives, phenazine methosulfate, and the like. There are concerns about side reactions when the voltage is applied by corrosion of the metal or a potentiostat. For this reason, the conventional metal layer was water-resistant and insulated with respect to the reaction part. In the present invention, a chemical substance contributing to the reaction penetrates and transmits electrons up to the second electrode layer containing the second resin, but the first electrode layer containing the first resin having water resistance is an electron. It contributes only to transmission and does not cause corrosion or side reactions to the metal layer.

Each of the working electrode 110, the counter electrode 120, and the reference electrode 130 is electrically connected to a wiring portion 150 formed of the metal layer 101, and the electrode system 100 and the wiring portion 150 are integrally formed. The wiring part 150 can apply a voltage to the electrode system 100 and take out an electric signal. By making the working electrode 110 and the counter electrode 120 have a laminated structure of a metal material having a lower resistance than carbon and a carbon material, the resistance value of the electrode system 100 can be greatly reduced as compared with the conventional one, and a highly sensitive biosensor. Can be provided. In addition, by configuring the wiring part 150 and the electrode system 100 integrally, a more sensitive biosensor can be provided. Further, since it is not necessary to use a noble metal for the reference electrode 130, the manufacturing cost can be reduced.

(Reaction part)
In the present embodiment, the reaction unit 140 includes a biological substance, and converts a chemical potential, heat, or an optical change accompanying the change movement of the substrate-specific substance into an electrical signal. The reaction unit 140 includes, for example, an enzyme and an electron acceptor as a biological substance. When measuring the glucose concentration, glucose oxidase (GOD) or glucose dehydrogenase (GDH) can be used as an enzyme. As the electron acceptor, potassium ferricyanide, a ferrocene derivative, a quinone derivative, an osmume derivative, or the like can be used. The enzyme and electron acceptor are used after appropriately diluted with a solvent. Examples of the solvent according to this embodiment include water, alcohol, and a water-alcohol mixed solvent. Further, it may be uniformly dispersed in a linear or cyclic hydrocarbon poor solvent. It is preferable that the enzyme and the electron acceptor are 0.3 unit or more and 10 units or less and 0.5 μg or more and 200 μg or less, respectively, per test specimen. Glucose oxidase and glucose dehydrogenase preferably have high purity, and the species of origin is not particularly limited as long as it has an activity in the above range. For example, glucose oxidase includes GLO-201 manufactured by Toyobo Co., Ltd. Can be used. The enzyme and electron acceptor of the reaction unit 140 can obtain a reaction amount in accordance with the enzyme amount (titer / unit), but may be a small excess of the optimum weight part that ensures the performance of the reaction unit 140.

Moreover, since the detection part 140 proportional to the area is obtained, it is preferable to set the reaction part 140 as wide as possible. The reaction unit 140 may be configured by mixing with a hydrophilic polymer or by mixing with a hydrophilic polymer and a surfactant. As the hydrophilic polymer, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetic acid, polyvinyl butyral, or a mixture thereof can be used. When mixed with a hydrophilic polymer, the blood becomes a gel and the response current value is slightly reduced, but the influence on the sensor response of red blood cells and other proteins can be reduced. The inclusion of a surfactant is preferable because even a highly viscous sample solution can be easily introduced into the sensor. Examples of the surfactant used in the reaction unit 140 include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and polyethylene glycols. When the reaction part 140 is formed, the enzyme loses its activity when left at a temperature of 40 ° C. or higher for a long time. Therefore, it is preferable to dry the solvent at 40 ° C. or lower and quickly return to room temperature after drying.

In the present embodiment, an example in which the reaction unit 140 is disposed on the upper surface of the second electrode layer 105 has been described. However, the biosensor 1000 according to the present embodiment is not limited thereto, and the reaction unit 140 is not limited thereto. As long as it is an upper layer of the second electrode layer 105, it may be at another position, and may be disposed between the second base material 1100 and the third base material 1200.

(Second base material)
The second base material 1100 is a base material for providing a gap between the first base material 170 and the third base material 1200 and providing a channel for supplying a sample to the biosensor 1000 from the outside. is there. At least one sample supply path 1510 is formed in the second base material 1100. The sample supply path 1510 is a flow path provided through the second base material 1100 in the horizontal direction, and guides a sample supplied from the outside to the working electrode 110. The second substrate 1100 may be a rigid body or an elastic body. Among them, it is preferable to use an elastic body having electrical insulation, and for example, a film of polyethylene terephthalate (PET) resin, vinyl chloride resin, polystyrene (PS) resin, polypropylene (PP) resin, or the like can be suitably used.

The width of the sample supply path 1510 is preferably in the range of 0.5 mm to 5 mm. If the width of the sample supply path 1510 is less than 0.5 mm, stable sample supply is not possible due to capillary action to the flow path. Moreover, since the electrode area of the reaction part 140 cannot be made large, sensitivity becomes low. When the thickness exceeds 5 mm, when the biosensor 1000 is cut perpendicularly to the electrode system 100 formation surface of the first base material 170, the first base material and the first base material toward the center line of the width of the sample supply path 1510. 3 is crushed into an arch shape, and the volume in the sample supply path 1510 is easily changed. The width of the sample supply path 1510 may be uniform throughout the entire shape, or the width of the sample supply path 1510 increases toward the outer edge that is the introduction portion of the sample supply path 1510 of the second base material 1100. May be.

The second base material 1100 may further include an air vent channel 1530 different from the sample supply channel 1510. In the biosensor 1000 according to the present embodiment, the air vent channel 1530 is connected to the sample supply channel 1510, and the sample supply channel 1510 and the air vent channel 1530 are combined to form a T-shaped channel. With such a configuration, when a sample is supplied from the outside, the air vent channel 1530 through which air in the sample supply channel 1510 escapes functions. The width of the air vent channel 1530 is preferably in the range of 0.3 mm to 10 mm. If the width of the air vent channel 1530 is less than 0.3 mm, the burr of the adhesive or the base material obstructs air venting, and if the width is larger than 10 mm, when the base material is deformed, the adjacent supply path is also deformed and the supply path. It becomes a fluctuation factor of the volume. The thickness of the second base material 1100 is the height of the sample supply channel 1510 and the air vent channel 1530, and is preferably in the range of 15 μm to 500 μm. If the thickness of the second base material 1100 is less than 15 μm, the volume is likely to change due to the unevenness of the base material, and the sample supply due to the capillary phenomenon becomes unstable. On the other hand, if it exceeds 500 μm, the sample may not flow uniformly to the reaction unit 140, and the sample may not flow to a part of the reaction unit 140.

In the present embodiment, the T-shaped flow path is illustrated, but the biosensor according to the present invention is not limited to this, and the letter “U” for introducing the sample into the second base material 1100 is used. A sample supply path having a notch portion may be formed, and a third base material 1200 having a through hole may be disposed at a position corresponding to the notch portion of the second base material 1100.

(Third substrate)
The third base material 1200 is a base material that functions as a lid member. As the third substrate 1200, for example, a resin substrate, a ceramic substrate, a glass substrate, a semiconductor substrate, a metal substrate, or the like can be used. The first base material 1200 may be a rigid body or an elastic body. Among them, it is preferable to use an electrically insulating elastic body. For example, a film of polyethylene terephthalate (PET) resin, vinyl chloride resin, polystyrene (PS) resin, polypropylene (PP) resin, or the like can be suitably used. The shape of the third base material 1200 is adopted in accordance with the first base material 170, but has a notch partly so that the wiring part 150 is exposed.

(Adhesive layer)
As shown in FIG. 4, an adhesive layer 185 a is formed on the lower surface of the second substrate 1100. In addition, an adhesive layer 185 b is further formed on the upper surface of the second base material 1100 in order to bond the second base material 1100 and the third base material 1200 together. The adhesive layer 185b may be formed on the lower surface of the third base material 1200. Examples of the material of the adhesive layer 185a and the adhesive layer 185b include acrylic adhesives, ester adhesives, vinyl adhesives, silicon adhesives, etc. as synthetic adhesives, glue, natural rubber, Starch glue such as sap and natural polymers can be used. The thickness of the adhesive layer 185a and the adhesive layer 185b is preferably 3 μm or more and 50 μm or less. When the adhesive layer 185a is formed with a thickness equal to or greater than the total thickness of the metal layer 101, the second electrode layer 105, and the reaction portion 140, for example, about 20 μm, the second base material 1100 and the first base material 170 are bonded to each other. It is possible to prevent the sample supplied from the sample supply path 1510 from flowing into the wiring unit 150 by being in close contact via the 185a. In addition, since the first base 170 and the adhesive layer 185a are in close contact with each other, rust prevention of the metal layer 101 of the wiring portion 150 can be realized.

The third base material 1200 is fixed to the second base material 1100 via the adhesive layer 185b and is fixed to the first base material 170. By forming the adhesive layer 185b on the lower surface of the third base material 1200, the second base material 1100, the counter electrode 120, and the reference electrode 130 may be embedded with the adhesive layer 185b. In addition, when the metal layer 101 according to the present embodiment is formed by etching a metal foil or depositing a metal, an adhesive layer formed by applying a dry laminating adhesive to the upper surface of the first base material 170 is used. It may be fixed via. For such an adhesive layer, a polyester-based two-component curing adhesive or the like can be used.

In addition, the biosensor 1000 according to the embodiment of the present invention is involved in enzymes such as a cholesterol sensor, an alcohol sensor, a scroll sensor, a lactate sensor, and a fructose sensor as well as a glucose sensor by changing the enzyme of the reaction unit 140. It can be widely used in reaction systems. As the enzyme used for each biosensor, those suitable for the reaction system such as cholesterol esterase, cholesterol oxidase, alcohol oxidase, lactate oxidase, fructose dehydrogenase, xanthine oxidase, amino acid oxidase and the like can be appropriately used.

(Biosensor manufacturing method)
Next, a method for manufacturing the biosensor 1000 described in the above embodiment will be described with reference to FIGS. FIG. 3A to FIG. 4D show the manufacturing process of the biosensor 1000 and correspond to the AA ′ cross section of FIG. FIG. 4E is a view corresponding to the BB ′ cross section of FIG.

(Electrode manufacturing process)
A metal layer having a predetermined pattern is formed on the first surface (upper surface) of the first base material 170 (FIG. 3A). The metal layer 101 according to the present embodiment is preferably formed by etching metal foil or metal deposition. When the metal foil is formed by etching of metal foil or metal deposition, the thickness can be made uniform and the width of the metal layer 101 can be made uniform as compared with printing of a conventional silver paste. By forming the metal layer 101 by etching of metal foil or metal vapor deposition, the metal layer 101 can be formed thinner than an electrode layer made of conventional silver paste, so that resistance of the electrode layer can be suppressed and sensitivity can be increased. it can. In addition, by using the above material, the first electrode layer 103 containing carbon can be formed without performing surface treatment of the metal layer 101.

A first coating liquid containing conductive carbon and a first resin is applied to a predetermined portion of the metal layer 101 by a printing method (FIG. 3B). In order to prevent the occurrence of pinholes, the first coating liquid is preferably applied at least a plurality of times. Thus, the first electrode layer 103 that covers at least the upper surface of the metal layer 101 is formed. More preferably, the upper surface and side surfaces of the metal layer 101 are covered with the first electrode layer 103.

After the first electrode layer 103 is dried for a predetermined time, a second coating liquid containing carbon and a second resin is applied so as to be positioned on the first electrode layer 103 (FIG. 3 (c)). In order to prevent the occurrence of pinholes, it is preferable to apply the second coating liquid at least a plurality of times. Thereby, the second electrode layer 105 covering at least the upper surface of the first electrode layer 103 is formed. More preferably, the second electrode layer 105 covers the upper surface and the side surface of the first electrode layer 103.

A solution containing an enzyme and an electron acceptor is applied to the upper surface of one second electrode layer 105 of the electrode thus formed with a dispenser, and then dried at 40 ° C. to remove the solvent component. In this way, the working electrode 110 is formed (FIG. 3D), and the electrode system 100 is manufactured.

In addition, the 1st coating liquid contains the 1st solvent for melt | dissolving 1st resin. Further, the second coating liquid contains a second solvent for dissolving the second resin. If a coating solution containing the same type of solvent is repeatedly applied, defects such as cracks and trapping due to erosion are likely to occur. For this reason, for example, a resin that dissolves in the ketone system is selected as the first resin, and a resin that dissolves in the alcohol system is used as the hydrophilic second resin. A first coating solution containing a ketone solvent is applied and dried to form the first electrode layer 103, and an alcohol solvent that does not dissolve the first resin contained in the first electrode layer 103 is included. By applying and drying the second coating liquid to form the second electrode layer 105, the first electrode layer 103 can be formed without erosion.

(Manufacturing process of the second base material)
4A to 4D, the second base material 1100 and the third base material 1200 are formed, the electrode system 100 formed on the first base material 170 is pasted, and the biosensor 1000 is attached. It is a figure which shows the process of manufacturing. An adhesive layer 185 is formed on the surface of the second substrate 1100 to which the first substrate 170 and the electrode system 100 are attached and the surface to which the third substrate 1200 is attached (FIG. 4A). The base material 1100 having the adhesive layer 185 formed on both the upper surface and the lower surface is punched to form the sample supply path 1510 and the air vent channel 1530, thereby manufacturing the second base material 1100 (FIG. 4B). ).

(Third substrate manufacturing process)
A predetermined punching process is performed on the base material 1200 to form a cutout portion where at least a part of the wiring portion 150 is exposed, and the third base material 1200 is manufactured.

(Bonding process)
The 3rd base material 1200 is bonded to one side of the 2nd base material 1100 (Drawing 4 (c)). Then, it arrange | positions so that the 2nd base material 1100 and the electrode system 100 may oppose the other surface of the 2nd base material 1100, and the 1st base material 170 is bonded (FIG.4 (d) and ( e)). FIG. 4D is a view corresponding to the AA ′ cross section of FIG. 2A, in which a sample supply path 1510 and an air vent channel 1530 are formed. In the AA ′ section, the air vent channel 1530 is external. Open to. FIG. 4E is a view corresponding to the BB ′ cross section of FIG. 2A, and both end portions of the first base material 170 and the second base material 1100 are pasted with an adhesive layer 185a. In addition, after bonding the 2nd base material 1100 and the 1st base material 170, the 3rd base material 1200 may be bonded to the 2nd base material 1100 as the order of bonding.

(Cutting process)
The above-mentioned process is performed by multiple imposition, and after the bonding is completed, cutting is performed to obtain individual biosensors 1000.

As described above, according to the biosensor manufacturing method of the present embodiment, the first electrode layer is formed by applying the first coating liquid containing carbon and the first resin on one end of the metal layer. And forming a second electrode layer by applying a second coating solution containing carbon and a second resin so as to be positioned on the first electrode layer. Is more hydrophobic than the second resin, thereby suppressing the moisture in the sample and the electron acceptor contained in the reaction part from entering the second electrode layer, and the redox reaction of the electron acceptor. Corrosion of the metal layer disposed under the second electrode layer can be suppressed.

(measuring device)
FIG. 5 is a schematic diagram showing a state in which the biosensor 1000 and the measuring device 10000 according to one embodiment of the present invention are connected, and FIG. 5A is an overall view. The measuring device 10000 is a known measuring device, and is a device that detects the object to be detected contained in the sample by connecting the biosensor 1000 according to the present invention. The measurement device 10000 includes, for example, a connection electrode 13000 for receiving an electrical signal generated by the biosensor 1000, a calculation unit (not shown), a power source (not shown), a display unit 11000, and an operation unit 12000.

FIG. 5B is a diagram for explaining the inside of the measuring device 10000 at the broken line portion to which the biosensor 1000 of FIG. 5A is connected. When the biosensor 1000 is attached to the attachment portion of the measurement device 10000, the end portions of the three metal wires 101 of the biosensor 1000 are connected to the connection electrodes 1300 of the measurement device 10000, respectively. By this connection, an electrical signal generated by the biosensor 1000 is transmitted to the measuring device 10000. In addition, when two metal layers 101 are disposed on the first base 170, and the working electrode 21 is disposed on one side and the counter electrode 23 and the reference electrode 25 are separately disposed on the other side, the connection of the measuring device 10000 is performed. There are two electrodes.

As a measurement method, for example, a measurer attaches the biosensor 1000 to the measurement apparatus 10000, introduces a sample from the tip of the biosensor 1000 to the sample supply path 1510 provided in the second base material 1100, and operates the operation unit 12000. To start measurement. If the sample introduced into the sample supply path 1510 includes an object to be detected, the object to be detected reacts with a biological substance disposed in the reaction unit 140, and the electrical signal is the second of the biosensor 1000. It is detected by the electrode layer 105 and transmitted from the end of the metal layer 101 via the first electrode layer 103 to the measuring device 10000 via the connection electrode 13000 of the measuring device 10000. The measuring device 10000 converts the electrical signal received from the biosensor 1000 into a measured value by the calculation unit. The obtained measurement value is displayed on the display unit 11000, and the measurer can visually recognize the measurement result.

In the following example, an example of the biosensor 1000 according to the present invention described in the above-described embodiment will be specifically described.

Example 1
An adhesive layer was formed on the upper surface of the substrate of a 100 μm thick PET film (Lumirror manufactured by Toray Industries, Inc.) using a dry laminating agent. A 7 μm thick aluminum film (manufactured by Toyo Aluminum Co., Ltd.) was bonded through the adhesive layer. A linear resist pattern having a width of 1.5 mm was formed on the surface of the aluminum film by a printing method. The laminated film on which the resist pattern was formed was immersed in an oscillating hydrochloric acid tank (2N) to etch aluminum. After washing with water, the laminated film was immersed in a sodium hydroxide bath (0.5N) to peel off the resist pattern. Then, after performing water washing again, the metal layer 101 was formed.

An ink transfer pattern with a width of 2.1 mm and a length of 3 mm is produced on a 1.5 mm width aluminum pattern of the metal layer 101 with 90 lines of gravure printing plate making, and is covered by about 0.3 mm on the left and right when centering in the width direction of the substrate. Aligned, carbon gravure ink (FDK) containing carbon black as a carbon pigment, a copolymer of vinyl chloride and vinyl acetate as a first resin, and ethyl acetate and methyl ethyl ketone (1: 1) as a first solvent Conductive black ink (manufactured by Godo Ink Co., Ltd.) was printed as the first coating liquid and dried at 120 ° C. for 10 minutes to form the first electrode layer 103. Here, the number of lines of the gravure printing plate making means the number of lines engraved on 1 inch of the gravure plate, and as the number of lines increases, the depth of the engraved groove becomes shallower and the amount of ink transferred becomes smaller. . Therefore, when inks having the same concentration are transferred with plates having different numbers of lines, the ink film thickness increases as the number of lines decreases. In Example 1, the first electrode layer 103 was 0.7 μm thick due to the difference in thickness between the surface of the first electrode layer 103 formed by carbon coating and the metal layer 101 of the aluminum pattern.

On the first electrode layer 103, the same gravure plate making, the same alignment, carbon black as the carbon pigment, butyral resin as the second resin, toluene, isopropyl alcohol, methyl ethyl ketone as the second solvent Carbon gravure ink containing (60:25:15) (conductive black ink for treated PET, manufactured by Joint Ink Co., Ltd.) is printed as the second coating liquid, dried at 120 ° C. for 10 minutes, and then the second electrode layer 105 was formed. The second electrode layer 105 was 1.2 μm thick due to the difference in the thickness of the surfaces of the first electrode layer 103 and the second electrode layer 105 formed by carbon coating and the metal layer 101 of the aluminum pattern. With respect to the longitudinal direction of the metal layer 101, the first electrode layer 103 is cut out from the tips of the first electrode layer 103 and the second electrode layer 105 so that the total length of the metal layer 101 at the exposed aluminum portion is 33 mm. The resistance value between the metal layer 101 and the metal layer 101 which is 30 mm away from the tip of the third electrode layer 105 and the second electrode layer 105 was 15Ω.

(Comparative Example 1)
As Comparative Example 1, the metal layer 101 was formed in the same manner as in Example 1. An ink transfer pattern with a width of 2.1 mm and a length of 3 mm is produced on a 50 mm gravure plate making line on a 1.5 mm width aluminum pattern of the metal layer 101, and it is covered by about 0.3 mm on the left and right when centering in the width direction of the substrate. Alignment, printing carbon gravure ink similar to the second coating liquid of Example 1 (treated PET ink for treatment PET, manufactured by Godo Ink Co., Ltd.), dried at 120 ° C. for 10 minutes, and an electrode layer containing carbon 907 was formed. The electrode layer 907 containing carbon was 2 μm thick due to the difference in thickness between the surface of the electrode layer 907 containing carbon formed by carbon coating and the surface of the metal layer 101 having the aluminum pattern. With respect to the longitudinal direction of the metal layer 101, the total length of the metal layer 101 at the exposed portion of the aluminum is cut out from the tip of the electrode layer 907 containing carbon to be 33 mm, and is 30 mm away from the tip of the electrode layer 907 containing carbon. The resistance value measured with the metal layer 101 was 20Ω.

(Comparative Example 2)
In the same manner as in Comparative Example 1, an ink transfer pattern having a width of 2.1 mm and a length of 3 mm was prepared on an aluminum pattern having a width of 1.5 mm of the metal layer 101 with 80 lines of gravure printing plate making, and the left and right sides were aligned with the center in the width direction of the substrate. Align to a state of covering about 3 mm, print the same carbon gravure ink as the second coating liquid of Example 1 (treated PET ink for treatment PET, manufactured by Godo Ink Co., Ltd.), and dry at 120 ° C. for 10 minutes. Thus, an electrode layer 907 containing carbon was formed. The electrode layer 907 containing carbon was 1 μm thick because of the difference in thickness between the surface of the electrode layer 907 containing carbon formed by carbon coating and the surface of the metal layer 101 having the aluminum pattern. With respect to the longitudinal direction of the metal layer 101, the total length of the metal layer 101 at the exposed portion of the aluminum is cut out from the tip of the electrode layer 907 containing carbon to be 33 mm, and is 30 mm away from the tip of the electrode layer 907 containing carbon. The resistance value measured with the metal layer 101 was 13Ω.

(Evaluation of water resistance and chemical resistance)
Using the electrodes of Example 1, Comparative Example 1, and Comparative Example 2 obtained as described above, the working electrode, the counter electrode, and the reference electrode are arranged at a pitch of 2.4 mm, and the electrode layer has a length of 3 mm. The top portion of 2.4 mm was left, and the top and bottom 0.3 mm were masked with water-resistant adhesive tape. 1 unit of glucose oxidase (GLO-201 manufactured by Toyobo Co., Ltd.) as an enzyme and 70 μg of potassium ferricyanide as an electron acceptor were dissolved in 2 μL of distilled water between the 2.4 mm width masks, and uniformly dropped to prepare a working electrode. . After drying this at room temperature for 4 hours, it was stored in a sealed glass bottle containing a desiccant for 1 day, and used as a standard enzyme electrode. Further, this sealed glass that was stored at 40 ° C. for 2 months was used as a comparative enzyme electrode having water resistance and chemical resistance. The enzyme electrode was evaluated by wiring to the working electrode, the counter electrode, and the reference electrode of a potentiostat (ALD 760 manufactured by ABS Co., Ltd.), and detecting the current 2 seconds after dropping 2.5 μL of the test subject with a 0.5 V voltage applied. The subject used was an artificial standard glucose solution prepared by adding glucose to physiological saline at a concentration of 100 mg / dL.

In Example 1, the current was detected in all 10 standard enzyme electrodes and comparative enzyme electrodes. On the other hand, in 10 standard enzyme electrodes of Comparative Example 1, current was detected, but in the comparative enzyme electrode, current was lost in 2 out of 10. In addition, 3 out of 10 currents were lost in the standard enzyme electrode of Comparative Example 2, and all 10 currents were lost in the comparative enzyme electrode. From these results, it is presumed that in the comparative example, the metal layer deteriorated due to oxidation or corrosion, and the electrical resistance at the interface with the carbon electrode layer increased. On the other hand, in the enzyme electrode of the biosensor provided with the two carbon electrode layers according to the present example, corrosion of the metal layer is suppressed immediately after production and after long-term storage for 2 months, and good electrical characteristics can be obtained. It became clear.

100: electrode system, 101: metal layer, 103: first electrode layer, 105: second electrode layer 105, 110: working electrode, 120: counter electrode, 130: reference electrode, 140: enzyme reaction section, 150: wiring Part, 170: first substrate, 185a: adhesive layer, 185b: adhesive layer, 900: conventional electrode system, 907: electrode layer containing carbon, 910: working electrode, 920: counter electrode, 930: reference electrode, 940 : Enzyme reaction unit, 1000: biosensor, 1100: second substrate, 1200: third substrate, 1510: sample supply channel, 1530: air vent channel, 10000: measuring device, 11000: display unit, 12000: Operation unit, connection electrode 13000

Claims (4)

A first substrate;
A metal layer disposed on the first surface of the first substrate;
A first electrode layer comprising carbon and a first resin disposed on one end of the metal layer;
A second electrode layer comprising carbon disposed to cover the first electrode layer;
A reaction portion disposed so as to be positioned on the second electrode layer,
The biosensor according to claim 1, wherein the first electrode layer includes a first resin having a lower water absorption rate than that of the second electrode layer. The biosensor according to claim 1, wherein the second electrode layer includes a second resin having higher hydrophilicity than the first electrode layer. Forming a metal layer having a predetermined pattern on the first surface of the first substrate;
On one end of the metal layer, a first coating solution containing carbon, a first resin, and a first solvent is applied to form a first electrode layer,
Applying a second coating liquid containing carbon, a second resin, and a second solvent so as to be located on the first electrode layer, to form a second electrode layer,
Forming a reaction part to be located on the second electrode layer;
The biosensor manufacturing method, wherein the first resin is a resin having a lower water absorption rate than the second resin. The biosensor manufacturing method according to claim 3, wherein the second resin has higher hydrophilicity than the first resin.

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