JP2000081407A - Biosensor, its manufacture and measuring method using biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a low-cost high-accuracy biosensor by simplifying its structure or manufacturing process. SOLUTION: This biosensor 1 is formed by stacking a working electrode 3, a reference electrode 4 and a reagent layer 5 on one surface of a substrate 2, and is reinforced with a hold member 6. An electrode part 7 consisting of the working electrode 3 and the reference electrode 4 is former at an end of integrated conductive members each made from the same material composed mainly of carbon. Lead parts 11, 12 for connecting the electrodes 3, 4 to terminals 9, 10 to be connected to a measuring device are sheathed with the hold member 6 made from polyethylene terephthalate in such a way that the terminals 9, 10 are exposed. A liquid containing at least an enzyme and low molecular compound as a hold-back agent is applied once and dried on the working electrode 3 and the reference electrode 4, so that the reagent layer 5 is formed thereon. During a steady level of blood sugar, a blood sample is dripped onto the reagent layer 5. It is also possible to disperse micro pieces with almost the same size as an erythrocyte into the reagent layer 5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for quantifying a specific component in a liquid sample in which various substances are mixed, and in particular, a small-sized planar type which can easily measure a small amount of sample, is easy to mass-produce, and is inexpensive. The present invention relates to a biosensor, a manufacturing method thereof, and a measuring method using the biosensor.

[0002]

2. Description of the Related Art Conventionally, biosensors have been proposed for quantifying specific components from biological liquid samples such as blood and urine.
For example, glucose in blood (hereinafter, referred to as a specific component)
It is called "blood sugar." ) Is used to quantify
In recent years, there has been a demand for a high-precision biosensor for blood glucose that is easy to operate, has low running costs, and is highly accurate so that individuals can check their own blood glucose. A disposable planar biosensor for blood glucose for each measurement has been proposed.

(First Prior Art) FIG. 12 shows an exploded perspective view of such a biosensor. FIG.
(A) to (e) show a method of manufacturing the biosensor 101.

First, an insulating substrate 102 is prepared (FIG. 13).
(A)), the terminal portions 109a and 110a and the electrode contact portion 10
9b and 110b are formed of a conductive paste containing a metal such as silver or gold (FIG. 13B), and then the electrode contact portions 1 of the leads 109 and 110 are formed.
09b and 110b on which electrode 10 is preferably made of carbon
3, 104 are formed by a screen printing method or the like (FIG. 13 (c)).

Next, lead portions 109 and 110 and electrode 1
03, 104 are covered with an insulating film 106, and the electrode portion 1
07 and the terminal portions 109a and 110a are exposed (FIG. 13
(D)). As the insulating film 106, a thermosetting type or an ultraviolet curable type is often used.

Next, the exposed electrode portion 107 is covered with a reagent layer 105 containing at least an enzyme or an enzyme and an electron transfer substance (FIG. 13E).
I had one.

As a general configuration of the reagent layer 105, a hydrophilic polymer layer represented by cellulose is formed as a first layer on the electrode portion 107 side, and then the first layer of the hydrophilic polymer layer is formed. As a second layer, an enzyme or a mixture of an enzyme and the same hydrophilic polymer as the first layer, or a mixture of an enzyme and an electron transfer substance as necessary, a mixture of the enzyme and the same hydrophilic polymer as the first layer A layer mixed with a transmitter is formed.

Further, an amphoteric lipid or polymer is applied as a third layer on the second layer, and an amphoteric lipid or polymer is applied to the second layer as the second layer. Those coated with substances, those containing enzymes and electron mediators in different layers, those further provided with a polymer layer between the enzymes and electron mediators, and other combinations of these were devised,
Four to five reagent layers have also been proposed. In forming these reagent layers, a solution in which a substance to be laminated is dissolved is prepared, and the solution is applied and dried.

The main purpose of forming such a hydrophilic polymer layer is to prevent foreign substances in a sample, such as blood cells and proteins in blood, from adsorbing on the electrode, or to delay the time until the adsorption. To make it happen.

(Second Prior Art) As such a biosensor, there is one described in JP-A-9-159644.

FIG. 14A is an exploded perspective view, and FIG.
14C is an external view, and FIG. 14C is a cross-sectional view taken along line FF of FIG. 14B.

The planar type biosensor 111 shown in FIG.
The two insulating substrates 112 and 113 form a space 116 facing the reagent layer 115 containing the enzyme via the spacer 114, and the lower layer of the reagent layer 115 has two pairs of comb-shaped interdigitated combs. An electrode 117 is provided and a lead 118 is provided.
And the connection terminal 119 exposed. Inlet 121
, A liquid sample such as blood is introduced into the space 116, and gas in the space 116 is discharged from the discharge hole 122.

FIG. 15 shows a method for manufacturing the biosensor 111.

First, as shown in FIG.
12 are prepared, and a pair of leads 118 which are also used as the connection terminals 119 are formed (FIG. 15B). Next, an electrode 117 is formed on the pair of leads 118 (FIG. 15).
(C)). Further, the insulating layer 120 (FIG. 15 (d)), the reagent layer 115 (FIG. 15 (d)) on the electrode 117, the spacer 114 (FIG. 15 (f)), and the insulating substrate 113 (FIG. g)) are sequentially formed and mounted to obtain the planar biosensor 11. The reagent layer 115 is formed in the same manner as the above-described biosensor.

(Third Prior Art) As such a biosensor, there is one described in Japanese Patent Publication No. 6-10662.

FIG. 16 is an exploded perspective view of the biosensor 131.

In the biosensor 131, the counter electrode 133a, the measurement electrode 134a, and the reference electrode 13 exposed from the insulating film 136 are provided.
5a is filled with a porous body 138 impregnated with an enzyme.
And a liquid sample such as blood
8 and quantified. FIG. 17 shows a method for manufacturing the biosensor 131.

First, an insulating substrate 132 is prepared (FIG. 17).
(A)) A conductive resin mainly composed of carbon is printed on the substrate 132 by screen printing, and the counter electrode 133a,
Measuring electrode 134a, reference electrode 135a and its lead 133
b, 134b, 135b, and terminal portions 133c, 13
4c and 135c are formed (FIG. 17B). Further, an insulating film 136 is formed on the electrode by screen printing, and a counter electrode 133a, a measuring electrode 134a,
A part of the electrode portion composed of the reference electrode 135a is exposed (FIG. 17C).

Next, a holding frame 137 having a hole on the electrode portion.
Is bonded to the insulating layer 136 (FIG. 17D), and the pores of the holding frame hold the porous body 138 impregnated with the enzyme to cover the electrode portion (FIG. 17E).

(Fourth prior art)
No. 662 also describes a biosensor 141.

FIG. 18 is an exploded perspective view of the biosensor 141, which has almost the same configuration as the biosensor 131, but differs in the configuration of the electrode section. The same components as those of the biosensor 131 are denoted by the same reference numerals, and description thereof is omitted.

FIG. 19 shows a method of manufacturing the biosensor 141.

First, an insulating substrate 132 is prepared (FIG. 19).
(A)) A counter electrode lead portion 133b, a measurement electrode lead portion 134b, and a reference electrode lead portion 135b are formed on a substrate 132 by screen printing using a conductive silver paste, and at the same time, a counter electrode terminal of an input / output terminal. 133c, the measurement electrode terminal 134c, the reference electrode terminal 135c, and the electrode connection part 1 of the counter electrode
33d, the electrode connection part 134d of the measurement electrode, and the electrode connection part 135d of the reference electrode are formed (FIG. 19B). As the electrode part, carbon paste is printed by screen printing,
The counter electrode 143, the measurement electrode 144, and the reference electrode 145 are formed (FIG. 19C).

An insulating film 136 is formed by screen printing, and a counter electrode 143 and a measuring electrode 14 are formed through a window of the insulating film 136.
4, exposing a part of the reference electrode 145 (FIG. 19D),
Thereafter, it is formed in the same manner as the biosensor (FIG. 19 (e),
(F)).

[0025]

However, the structure and manufacturing method of the reagent layer such as the biosensor 101 described above have the following problems.

First, the formation of a hydrophilic polymer layer, in which carboxycellulose is often used, takes a long time to dissolve uniformly because it is a polymer. Occasionally, bubbles are easily mixed, and once mixed, it is difficult to remove the bubbles. Also, even after drying, it is not a dense and flat `` film '' shape, but there is a feeling like a recrystallized state in which powder separates when touched, it is easy to peel off even after film formation, and subsequent handling is complicated is there. For these reasons, it is very difficult to control the film thickness, which has the effect of preventing foreign substances in the sample from adsorbing on the electrodes, and also causes a difference between sensors. Leads to deterioration of accuracy. Therefore, formation of such a reagent layer requires accumulation and skill of know-how for producing a uniform film thickness, and strict control is required at the time of drying and the like. Inspection costs for the process check of the reagent layer film were not negligible.

Further, if a two-electrode system of a measuring electrode and a counter electrode, in which an electrode portion and respective lead portions and terminal portions are integrally formed by screen printing of a conductive resin mainly composed of carbon on an insulating substrate, Since the resistance of the electrode is high and the potential actually applied to the electrode is likely to be unstable,
The counter electrode 133a and the measurement electrode 13 like the biosensor 131
The potential between the electrodes is corrected by adopting a three-electrode system composed of the reference electrode 4a and the reference electrode 135a, and the lead portion and the terminal portion are formed of a low-resistance material different from the electrode portion as in the biosensor 141.

However, if the three-electrode system is used, the size of the sensor itself is increased, and the circuit configuration is complicated due to the addition of a voltage correction amplifier and the cost is increased. On the other hand, even if the lead portion and the terminal are formed of a material different from that of the electrode portion, the cost increases due to the use of a different material or the addition of a step.

The biosensors 101, 111, 13
Each of the devices 141 and 141 has an indispensable configuration of an insulating film, and also has a process of forming an insulating film during the manufacturing process. The formation of the insulating film not only increases the cost but also complicates the manufacturing process of the sensor and requires careful attention. That is, since the insulating film defines the area of the electrode involved in the reaction, in order to obtain a high-precision biosensor with little variation in output between the electrodes, there is no difference in the reaction area between the electrodes. Not only must there be no chipping, "displacement" or pinholes in the insulating film at the time of formation, but also the lead must be covered without exposing even a small amount of the lead to prevent it from participating in the electrode reaction. No. Further, the insulating film is of a thermosetting type or an ultraviolet curing type, and strict control is required so that volatile substances such as gas generated at the time of formation do not adhere to the exposed portions of the electrodes. The check and inspection costs in the process of forming the insulating film were not negligible.

The present invention has been made to solve the problems of the prior art, and an object of the present invention is to provide a highly accurate and inexpensive biosensor by simplifying a manufacturing process and a configuration.

[0031]

According to a first aspect of the present invention, there is provided an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme. A biosensor for supplying a liquid sample to a sensitive portion including the reaction layer and detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system to measure a concentration of a specific component in the liquid sample. Wherein a low molecular weight compound is added to form the reaction layer.

If a low-molecular compound is added to form a reaction layer containing at least an enzyme as described above, the reaction layer can be easily formed into a layer or film. Therefore, not only the mass production can be facilitated and the efficiency can be improved by simplifying the manufacturing process, but also a high-precision biosensor with little difference between individuals can be produced and can be provided at a low cost. Also,
Low molecular compounds are easily dissolved in the liquid state before the reaction layer is solidified and formed, and even after the reaction layer is formed, the dissolution of liquid samples such as blood is faster than when a high molecular compound is added. Can be shortened.

The low molecular weight compound has a molecular weight of about 10
The compound can be appropriately selected from the following compounds in consideration of reactivity with the enzyme and the like.

According to a second aspect, in the first aspect, the reaction layer has at least a layer containing the low-molecular compound.

The reaction layer may be formed as a single layer containing a low molecular compound, or the reaction layer may be formed from a plurality of layers, and may include a layer containing a low molecular compound.

According to a third aspect, in the first or second aspect, the low molecular weight compound is a compound selected from the group consisting of a monosaccharide, a disaccharide, a trisaccharide and an amino acid.
It is a mixture of two or more kinds of low molecular compounds.

The low molecular weight compound may be appropriately selected depending on the enzyme, liquid sample and the like. As such, one kind selected from monosaccharides, disaccharides, trisaccharides and amino acids is used. There is a compound or a mixture of two or more low molecular weight compounds. However, the low-molecular compound that can be added to the reaction layer is not limited to this.

A fourth invention comprises an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and supplies a liquid sample to a sensitive part containing the electrode system and the reaction layer. And a method for producing a biosensor for measuring the concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system. Is formed in a single step.

As described above, the reaction layer containing the low molecular weight compound is
If it is formed by processes such as application and printing,
The manufacturing process can be simplified. If a low molecular compound is added, the reaction layer can be easily formed into a layer or film, so that the reaction layer can be formed in one process, and the mass production can be facilitated by simplifying the manufacturing process. Not only efficiency can be improved, but also a high-precision biosensor with little difference between individuals can be produced, and this can be provided at a low price. In addition, the low-molecular compound is easily dissolved in a liquid state before the reaction layer is solidified and formed, and even after the reaction layer is formed, the dissolution of a liquid sample such as blood is faster than when a high-molecular compound is added. Therefore, the time required for the measurement can be reduced.

According to a fifth aspect of the present invention, there is provided an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and a liquid sample is supplied to a sensitive portion containing the electrode system and the reaction layer. A biosensor for detecting the concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system, wherein the electrode system is open to at least the outside. In a method for producing a biosensor, wherein the space is provided with the electrode system, the space is provided with the electrode system, and the liquid sample is sucked into the space by capillary action from the opening. A liquid containing the components of the reaction layer is suctioned from the opening by capillary action and solidified to form a reaction layer.

As described above, if the opening is brought into contact with the liquid containing the components of the reaction layer and is sucked and solidified by capillary action, the reaction layer can be easily formed. Thus, an inexpensive biosensor can be provided. In addition, since the reaction layer can be formed after forming the electrode system, the space, and the like, the reaction layer is not affected by heat, chemicals, and the like, and a high-precision biosensor can be provided. .

As a planar type biosensor which can be manufactured by such a manufacturing method, a film-like member such as an insulating film is arranged to face each other via a space forming member with a minute gap therebetween. There is one in which an electrode system is provided on at least one of the facing surfaces. At this time, the space is formed between the opposing surfaces on which the electrode system is formed, and the end of the film-like member constituting the space forms an opening. Since the opening is formed of a film-like member opposed to each other with a minute gap therebetween, if the opening is brought into contact with a liquid, the liquid can be sucked into the space by capillary action. Therefore, a space including the electrode system is formed in advance by joining the film-shaped member and the space forming member, and the opening is brought into contact with the liquid containing the components of the reaction layer. Thus, by solidifying this, a sensitive portion composed of an electrode system and a reaction layer can be easily formed in the space.

According to a sixth aspect of the present invention, there is provided an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and a liquid sample is supplied to a sensitive portion containing the electrode system and the reaction layer. A biosensor for detecting the concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system, wherein the electrode system is open to at least the outside. In a biosensor which is disposed in a space having an opening, and aspirates the liquid sample into the space by capillary action from the opening, after forming a space provided with the electrode system, the reaction layer A reaction layer is formed by sucking and solidifying a liquid containing components from the opening by capillary action.

As described above, if the opening is brought into contact with the liquid containing the components of the reaction layer and is sucked and solidified by capillary action, the reaction layer can be easily formed. Thus, an inexpensive biosensor can be provided. In addition, since the reaction layer can be formed after forming the electrode system, the space, and the like, the reaction layer is not affected by heat, chemicals, and the like, and a high-precision biosensor can be provided. .

In the planar type biosensor having such a configuration, a film-like member such as an insulating film is arranged to face each other via a space forming member with a minute gap therebetween, and the surface of this film-like member is opposed to the film-like member. At least one of them is provided with an electrode system. At this time, the space is formed between the opposing surfaces on which the electrode system is formed, and the end of the film-like member constituting the space forms an opening. Since the opening is formed of a film-like member opposed to each other with a minute gap therebetween, if the opening is brought into contact with a liquid, the liquid can be sucked into the space by capillary action. Therefore, a space including the electrode system is formed in advance by joining the film-shaped member and the space forming member, and the opening is brought into contact with the liquid containing the components of the reaction layer. Thus, by solidifying this, a sensitive portion composed of an electrode system and a reaction layer can be easily formed in the space.

According to a seventh aspect of the present invention, there is provided an electrode system having a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer. Detecting a current between both electrodes when a predetermined voltage is applied to the working electrode with respect to the reference electrode, thereby detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme, and detecting the liquid sample. A biosensor for measuring the concentration of a specific component in the bioelectrode, wherein the working electrode and the reference electrode, a terminal for applying a voltage to the working electrode and the reference electrode, and extracting a current, the working electrode and the reference electrode And a lead connecting the respective terminals with a biosensor formed from a thin film member made of the same material, wherein the terminal and the lead are connected to a predetermined voltage of the working electrode with respect to the reference electrode. Department Adding a voltage drop caused by resistance, a measuring method using a biosensor, which comprises applying to said terminal portion.

If the working electrode, the reference electrode, and the respective lead portions and terminal portions are formed of a thin film member made of the same material, such as screen printing of carbon paste, the resistance of the lead portion and the terminal portion is high, and The potential applied to the pole and the reference pole becomes unstable, affecting measurement accuracy.
Instead of applying a predetermined voltage required to detect the electrochemical phenomenon caused by the reaction between the liquid sample and the enzyme to the terminals,
By adding a voltage drop generated by the resistance of the terminal portion and the lead portion to the predetermined voltage and applying the voltage drop to the terminal portion,
A predetermined voltage is stably applied between the working electrode and the reference electrode, and high-precision measurement can be performed without making the apparatus complicated and large. In addition, if the working electrode, the reference electrode, and the respective lead portions and terminal portions are formed of a thin-film member made of the same material, the manufacturing cost can be reduced, so that an inexpensive and highly accurate biosensor is provided. Can be.

According to an eighth aspect of the present invention, there is provided an electroconductive member having, on one end, an electrode system having at least a working electrode and a reference electrode on an insulating support member, and a reaction layer containing at least an enzyme. A liquid sample is supplied to a sensitive part including a system and the reaction layer, and an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme is detected by the electrode system to measure a concentration of a specific component in the liquid sample. In the biosensor, a holding member for reinforcing the supporting member is provided, and a portion of the conductive member adjacent to the electrode system is liquid-tightly covered by the holding member.

As described above, if the portion adjacent to the electrode system of the conductive member having the electrode system having at least the working electrode and the reference electrode at one end is covered with the holding member in a liquid-tight manner, the liquid sample can be formed. Even if it is supplied to the sensitive part, it does not come into contact with parts of the conductive member other than the electrode system and does not affect the detection of electrochemical phenomena. Can be reduced. Further, not only the mass production can be facilitated and the efficiency can be improved by simplifying the manufacturing process, but also a high-precision biosensor with little difference between individuals can be produced and can be provided at a low cost.

When the thickness of the insulating support member is reduced to reduce the size of the sensor or the like, the support member needs to be reinforced. Although it is not necessary to form an insulating film, a similar effect can be obtained by providing a member having another function with an insulating function according to the configuration of the biosensor.

According to a ninth aspect of the present invention, a conductive member having an electrode system having at least a working electrode and a reference electrode at one end and a reaction layer containing at least an enzyme are provided on an insulating support member. A liquid sample is supplied to a sensitive portion including the electrode system and the reaction layer, and a electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme is detected by the electrode system. And measuring the concentration of a specific component in the liquid sample, wherein the portion of the conductive member formed on the support member adjacent to the electrode system is liquid-tight by the holding member. It is characterized by covering.

As described above, if the portion adjacent to the electrode system in the conductive member having at least one electrode system including the working electrode and the reference electrode at one end is liquid-tightly covered by the holding member, the liquid sample can be removed. Even if it is supplied to the sensitive part, it does not contact the parts of the conductive member other than the electrode system and does not affect the detection of electrochemical phenomena. In addition, the manufacturing process can be simplified and the manufacturing cost can be reduced. This will make mass production easier,
It is possible to produce not only efficiency but also a high-precision biosensor with little difference between individuals.

When a portion adjacent to the electrode system is covered by the holding member, the portion is liquid-tightly covered by bonding with an adhesive, press-fitting the holding member to the supporting member, or pressing the supporting member into the holding member. Is possible, but is not limited to such a method.

A tenth invention comprises an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and supplies a liquid sample to a sensitive part containing the electrode system and the reaction layer. In a biosensor for detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system to measure a concentration of a specific component in the liquid sample, the reaction layer includes a small piece. And

In this way, by adding micro-pieces to the reaction layer, it is possible to uniformly prevent the foreign matter in the sample from adhering to the electrode system and to reduce the reaction current, and to reduce the reaction current proportional to the substrate concentration. The current can be accurately detected, and the difference between individuals can be extremely small.

The eleventh invention comprises an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and supplies a liquid sample to a sensitive part containing the electrode system and the reaction layer. Then, in a biosensor that detects an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system and measures the concentration of a specific component in the liquid sample, the reaction layer is provided with a small piece and a low-molecular compound. It is characterized by including.

If a low-molecular compound is added to form a reaction layer containing at least an enzyme as described above, the reaction layer can be easily and uniformly formed. In addition, the reaction layer to which the low-molecular compound is added dissolves faster in a liquid sample such as blood than when a high-molecular compound is added, so that the time required for measurement can be reduced. In addition, the addition of micro-pieces to the reaction layer can uniformly prevent the reaction current from decreasing due to foreign substances in the sample adhering to the electrode system, and can accurately control the reaction current proportional to the substrate concentration. Detection can be performed well, and the difference between individuals can be extremely small. Therefore, not only the mass production can be facilitated and the efficiency can be improved by simplifying the manufacturing process, but also a high-precision biosensor with little difference between individuals can be produced and can be provided at a low cost. As a low molecular weight compound, the molecular weight is approximately 10
The compound can be appropriately selected from the following compounds in consideration of reactivity with the enzyme and the like.

According to a twelfth aspect, in the tenth or eleventh aspect, the minute piece has a size substantially equal to that of a blood cell.

For example, by using a small piece having a size substantially the same as that of red blood cells in blood, it is possible to effectively prevent red blood cells in a blood sample from being adsorbed on the electrode system. However, it goes without saying that the size of the minute piece can be appropriately selected according to the size of the substance that is easily adsorbed on the electrode system in the liquid sample.

According to a thirteenth aspect, in the tenth or eleventh aspect, the minute piece has a spherical shape or a similar shape.

In this way, the size and shape of the individual minute pieces can be easily made uniform, and the effect of preventing foreign matter from adsorbing onto the electrode system can be further enhanced. Shapes similar to a sphere include a flat sphere, an elliptical sphere, and the like, but are not limited thereto.

According to a fourteenth aspect, in the tenth or eleventh aspect, the minute pieces have a specific gravity of 1.0 or more.

In this way, the fine pieces can be uniformly and stably dispersed in the reaction layer or on the electrode system.

According to a fifteenth aspect, in the tenth aspect or the eleventh aspect, at least two small pieces included in the reaction layer are provided.
It is characterized by more than types.

For example, by using two or more kinds of fine pieces having different sizes to close the gap between the fine pieces, or by forming a plurality of fine piece layers, foreign matter on the electrode system can be reduced. The effect of preventing adhesion can be further enhanced.

According to a sixteenth aspect, in the eleventh aspect,
The low molecular compound is a compound selected from one of monosaccharides, disaccharides, trisaccharides and amino acids, or a mixture of two or more low molecular compounds.

As described above, as the low molecular weight compound, the low molecular weight compound is a compound selected from one of monosaccharides, disaccharides, trisaccharides and amino acids, or a mixture of two or more low molecular weight compounds. Can be used, but the present invention is not limited to these.

A seventeenth invention comprises an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and supplies a liquid sample to a sensitive part containing the electrode system and the reaction layer. Then, in the biosensor manufacturing method of measuring the concentration of a specific component in the liquid sample by detecting the electrochemical phenomenon due to the reaction between the liquid sample and the enzyme in the electrode system, the reaction layer containing the fine pieces It is characterized by being formed in one process.

In this way, before the reaction layer is formed, the reaction layer can be formed after the fine pieces are uniformly dispersed in advance. Therefore, it is possible to form a reaction layer containing fine pieces in a single step, and to keep the number and distribution of the fine pieces contained in the reaction layer constant. The effect of preventing the adsorption on the upper side is also constant, and it is possible to efficiently provide an extremely high-precision biosensor in large quantities at low cost.

An eighteenth aspect of the present invention comprises an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and supplies a liquid sample to a sensitive portion containing the electrode system and the reaction layer. In the biosensor manufacturing method for detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system and measuring a concentration of a specific component in the liquid sample, a fine particle is previously placed on the electrode system. Is formed, and then the reaction layer is formed.

By forming the microscopic pieces on the electrode system in advance, the effect of preventing foreign substances in the sample from adsorbing on the electrode system can be improved, and a more accurate biosensor can be obtained. Can be supplied.

According to a nineteenth aspect, in the seventeenth aspect or the eighteenth aspect, the fine particles contained in the reaction layer are at least 2 pieces.
It is characterized by more than types.

For example, by using two or more kinds of minute pieces having different sizes to close the gap between the minute pieces, the effect of preventing foreign matter from adhering to the electrode system can be further enhanced.

A twentieth invention includes an electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, and supplies a liquid sample to a sensitive part containing the electrode system and the reaction layer. A method for producing a biosensor for detecting the concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system; After forming the layer containing the fine pieces, the reaction layer containing at least one kind of fine pieces different from the fine pieces is formed.

In this way, by forming a plurality of micro-piece layers, the effect of preventing foreign substances in the sample from adsorbing onto the electrode system can be further improved.

[0076]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on the illustrated embodiment.

(First Embodiment) FIG. 1 shows a planar type biosensor according to a first embodiment of the present invention. FIG.
1A is an exploded perspective view, FIG. 1B is an external view, and FIG.
FIG. 2 is a sectional view taken along the line AA of FIG.

The biosensor 1 is formed by laminating a working electrode 3, a reference electrode 4, and a reagent layer 5 on one side of a substrate 2 made of an insulating film made of polyethylene terephthalate.
It is reinforced by the holding material 6. The electrode portion (electrode system) 7 including the working electrode 3 and the reference electrode 4 is provided at the end of an integral conductive member made of the same carbon-based material. The terminals 9, 10 connected to the measuring device and the working electrode 3,
The lead portions 11 and 12 connecting to the reference electrode 4 are liquid-tightly covered with a holding material (holding member) 6 made of polyethylene terephthalate, and the terminal portions 9 and 10 are exposed. Working electrode 3
And a solution containing at least an enzyme and a low-molecular-weight compound as a holding agent is applied or dropped once on the reference electrode 4,
After drying, a reagent layer (reaction layer) 5 is formed. When quantifying blood glucose, a blood sample as a liquid sample may be dropped on the reagent layer 5. In the present embodiment, a sensitive part composed of the electrode part 7 and the reagent layer 5 is formed in a region exposed on the electrode part 7 side of the substrate 2 by the holding material 6.

Note that the shape of the biosensor 1 is not limited to the one shown in the figure, and the substrate 2, the working electrode 3, the reference electrode 4, the holding member 6
The material and forming method can be appropriately selected from those known from known materials and forming methods.

For example, as the material of the substrate 2 and the holding material 6, in addition to the above-mentioned polyethylene terephthalate, a resin sheet made of polyethylene naphthalate, polyethylene sulfide, polycarbonate, polyallyl acrylate, polyether sulfide, polyimide, etc.
It can be selected from plastic, ceramics, glass thin plate, paper, and the like.

The working electrode 3 and the reference electrode 4 are formed by a screen printing method suitable for mass production. However, electrode materials such as platinum, gold, silver, silver chloride, iron, zinc, nickel and palladium are deposited by vapor deposition and sputtering. It can also be manufactured by a thin film forming method such as a plating method, a plating method, and ion plating. As in this embodiment, the working electrode 3, the reference electrode 4, and the respective lead portions 11, 12
If the terminals 9 and 10 are formed of the same member, the forming process can be simplified and the manufacturing cost can be reduced.

The enzyme of the reagent layer 5 must be appropriately selected depending on the substance to be determined, and examples thereof include glucose oxidase, cholesterol oxidase, uricase, and pyruvate oxidase. The holding agent for easily forming the reagent layer 5 in a layered form is a low molecular weight compound, preferably one compound selected from monosaccharides, disaccharides and trisaccharides, a mixture of two or more types, or an amino acid. One compound or a mixture of two or more compounds.
It is needless to mention that substances involved in the enzymatic reaction, for example, glucose (glucose) when glucose oxidase is used as an enzyme cannot be used as a retention agent.
Depending on the electrode reaction, not only the enzyme but also potassium ferricyanide, ferrocene compound, p-
It is necessary to add an electron transfer substance such as benzoquinone. The formation of the reagent layer 5 is usually performed by drying the dropped reagent solution, but a screen printing method or the like can also be appropriately selected, and an appropriate low-molecular compound can be added for forming a stronger film. It is.

The holding member 6 is mounted with an adhesive in this embodiment. By covering the leads 11 and 12 with the holding material 6 in a liquid-tight manner, the leads 11 and 12 do not affect the measurement due to the electrochemical phenomena caused by the reaction between the enzyme and the sample.

As described above, in the present invention, the material, shape, thickness, and the like of the substrate 2, the holding member 13, and the electrode are not limited, and may be appropriately determined according to the use and usage of the planar enzyme electrode. It should be selected and set. At least one of the electrode portion 7, the lead portions 11, 12 and the terminal portions 9, 10 may be formed of another material.

(Example) As an example of the first embodiment, a biosensor for blood glucose will be described.

First, as the substrate 2, a length of 20 mm and a width of 6
A working electrode 3 and a reference electrode 4 are formed on one side of a polyethylene terephthalate having a thickness of 180 μm, which is cut into a substantially rectangular shape having a trapezoidal end at one end.

Next, the polyethylene terephthalate thickness 2
A holding material 6 of 50 μm is mounted on the substrate 2 with an adhesive to expose the electrode portions 7 and the terminal portions 9 and 10. The holding member 6 is cut out on the electrode portion 7 side, and the cut-out edge is
It is substantially parallel to each of the trapezoidal edges on the electrode portion 7 side of the substrate 2 and forms a substantially hexagonal space.

Next, at least 5 μm of the reagent solution is placed on the electrode portion 7.
1 was dropped and dried at 50 ° C. for 1 hour to form a reagent layer 5. The composition of the reagent solution was the enzyme glucose oxidase 0.1.
2%, electron mediator potassium ferricyanide 1.0%,
Support material trehalose 2.0%.

FIG. 2 is a block diagram showing a schematic configuration of a main part of a measuring device for performing measurement using the biosensor 1.

The terminal section 9 connected to the working electrode 3 returns an output to an inverting input terminal via a resistor 21 and an IV conversion section 23 comprising an operational amplifier 22 having a non-inverting input terminal grounded.
Connected to the inverting input terminal. The current detected by the working electrode is converted into a voltage by the IV converter 23. This voltage is converted into a digital signal by an AD conversion circuit 24 and input to a control circuit 25 including a CPU, a memory, and the like.

The terminal section 10 connected to the reference electrode 4 is connected to the output of a buffer circuit 27 composed of an operational amplifier 26 whose output is fed back to an inverting input terminal. 28a, 28b
It can be switched to. This switch 28
Performs voltage switching according to a signal from the control circuit 25.

The control circuit 25 is connected to a sensor detector 29 for detecting whether or not the biosensor 1 is attached to the measuring device, and a display unit 30 for displaying information such as measurement results.

The blood glucose planar biosensor 1 manufactured as described above is mounted on a measuring device, and the switch 28 is turned on.
Then, a voltage of 0.1 V is applied to the terminal 9 connected to the working electrode 3 with respect to the terminal 10 connected to the reference electrode 4 in advance, and blood is dropped on the reagent layer 5. When the enzymatic reaction starts and the electrode reaction current is detected, the switch 28 is switched so that no voltage is applied to the non-inverting input terminal. Ten seconds after the start of the enzymatic reaction, the terminal 9
Voltage of 0.6 V, 5 seconds after application or 10
Blood glucose is quantified by the output of the electrode after 2 seconds.

The measurement conditions can be appropriately set according to the enzyme and the liquid sample, and are not limited to the above conditions.

As described above, by adding a low molecular compound to a reagent solution, a reagent layer can be easily formed into a layer or a film, so that a dense and flat reagent layer can be easily formed even in a single step. This eliminates the need for the conventional control of the film thickness, which simplifies the manufacturing process. In addition, since a reagent layer having a uniform area can be easily formed, a high-precision biosensor with little variation among individuals can be provided. Also, by using a single reagent layer, a complicated and time-consuming step can be omitted as compared with the case of forming a plurality of reagent layers, so that the manufacturing process can be simplified and the manufacturing cost can be reduced. Mass production can be performed easily and efficiently. Furthermore, by adding a low molecular compound to the reagent solution, the lysis of blood is accelerated, so that the measurement time can be shortened.

(Second Embodiment) FIG. 3A is a top view of a planar biosensor 31 according to a second embodiment of the present invention, and FIG. 3B is a plan view of FIG. It is -B sectional drawing. FIG. 4 is a diagram illustrating a method of manufacturing the biosensor 31.

The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

First, the configuration and manufacturing method of the biosensor 31 will be described.

In this biosensor 31, a carbon paste is printed on the insulating film substrate 2 made of polyethylene terephthalate by a screen printing method and cured by heat drying or UV irradiation to form the electrode part 7, the terminal parts 9, 10 , And the lead portions 11 and 12 are integrally formed (FIG. 4A). The terminals 9 and 10 are connected to the measuring device to output a detection signal and the like. The electrode 7 including the working electrode 3 and the reference electrode 4 detects an electrochemical phenomenon caused by a reaction between the reagent and the liquid sample. And a lead portion 11,
Reference numeral 12 denotes a portion for connecting the terminal portions 9 and 10 and the electrode portion 7.

The substrate 2 has a substantially rectangular shape with one end formed in a semicircular shape. The electrode part 7 is formed near the semicircular end.

Next, the lead portions 11 and 12 excluding the electrode portion 7 and the terminal portions 9 and 10 were covered with an insulating film 32 to clarify each region (FIG. 4B). The insulating film 32 is formed by printing an insulating paste and curing it by heat drying or UV irradiation. The substrate surface is exposed in a semi-disc shape on the electrode portion side by the insulating film 32.

Next, a space forming film (space forming member) 33 and a cover film (film-like member) 34 are bonded on the insulating film 32 (FIG. 4C). FIG. 4D shows a state in which the space forming film 33 and the cover film 34 are bonded on the insulating film 32. The space forming film 33 has a substantially rectangular shape, and the cover film 34 has substantially the same shape and size as the substrate 2 and is formed of the same material as the substrate 2.
The space forming film 33 is bonded so that the electrode portion 7 is exposed in a semi-disc shape of the substrate 2. The cover film 34 is bonded so as to face the substrate 2. The terminal portion side end of the cover film 34 is cut along the end portion of the space forming film 33 to cut the terminal portions 9 and 1.
Expose 0. The end portions of the space forming film 33 and the cover film 34 on the terminal side may be appropriately formed so as to conform to the configuration of the measuring apparatus main body.

The front ends of the substrate 2 and the cover film 34 are both formed in substantially the same semicircular shape, and the space 2 formed by the substrate 2, the space forming film 33 and the cover film 34 is formed.
Reference numeral 5 denotes a flat, substantially semi-cylindrical space having a height of about 0.15 mm and a radius of 3 mm, and has an opening length of an opening 36 formed between the substrate 2 and the semicircular ends 2a, 34a of the cover film 34. Is about 10 mm, and the sample volume required for measurement is about 2 μl.

Next, a part of the opening 36 is brought into contact with the reagent solution 37 (FIG. 4E). The reagent liquid is sucked into the space 35 from the opening 36 by capillary action, and is filled in the space 35. The reagent layer 5 is formed by drying the reagent liquid filled in the space 35 in this manner. The reagent solution 37 is a mixed solution of 0.1% of glucose oxidase (glucose oxidase, GOD), 2% of potassium ferricyanide as an electron transfer substance, and 1.0% of fructose of a hydrophilic low molecular weight, at a temperature of 23 ° C. and a humidity of 50%. %). In the present embodiment, a sensitive part is constituted by the electrode layer 7 and the reagent layer 5 formed in the space 35.

In this way, the step of forming the reagent layer can be simplified, and an inexpensive biosensor can be provided. In addition, since the reagent layer is formed after assembling other portions of the biosensor, the reagent layer is not affected by heat, chemicals, and the like, and a highly accurate biosensor can be provided.

Hereinafter, a measurement procedure when measurement is performed using the biosensor 31 will be described. Fig. 2
A device similar to that of the first embodiment shown in FIG.

First, when the sensor detecting section 29 detects the attachment of the sensor 31, the switch 28 is connected to the side 28a by a signal from the control circuit 25, and a voltage of 0.1 V is applied between the terminal section 9 and the terminal section 10. When a small amount of a sample such as blood or body fluid is brought into contact with any part of the opening 13 in a state where this voltage is applied, the sample is aspirated by capillary action and immediately spreads into the space 12. The reagent layer 9 in contact with the liquid sample immediately dissolves and starts the enzymatic reaction. By the start of the enzymatic reaction, an electrode reaction current is detected, and potassium ferricyanide is reduced to start changing to potassium ferrocyanide.

When the control circuit 25 detects the electrode reaction current, it switches the switch 28 so that no voltage is applied to the non-inverting input terminal.

After 10 seconds from the start of the enzymatic reaction, the switch 28 is switched to the side 28b to apply a voltage of 0.6 V, which is a potassium ferrocyanide oxidation potential, between the terminal portions 9 and 10. 5 seconds after changing the voltage to 0.6 V, 1
The oxidation current amount after 0 seconds is measured. Since the amount of oxidation current is proportional to the amount of potassium ferrocyanide, the amount of potassium ferrocyanide is proportional to the amount of enzyme reaction, and the amount of enzyme reaction is proportional to the base mass (the amount of glucose in the sample), the glucose concentration in the sample is measured by measuring the amount of oxidation current. can do. The time from the start of the enzyme reaction to the application of 0.6 V is for waiting for the accumulation of potassium ferrocyanide, and may be set as appropriate according to the sample, the type of the enzyme, and the like. Further, the current measurement period after the voltage change may be appropriately set according to the reaction.

In this embodiment, the working electrode 3 and the reference electrode 4 are composed of two electrodes. However, a counter electrode may be further provided and composed of three electrodes. In this way, high-precision measurement is possible even when quantifying a high-resistance liquid sample such as blood. When a counter electrode is provided, it may be connected to the non-inverting input terminal side of the operational amplifier 26 in FIG. The working electrode and the reference electrode may be arranged so as to face the substrate and the cover film with a minute gap therebetween.

In the present embodiment, the ends of the substrate and the cover film on the opening side are formed in a semicircular shape. However, the present invention is not limited to such a shape, and the reagent is formed from the opening to the space where the electrode system is formed. The reagent layer can be formed by the above-described method as long as the liquid can be sucked by capillary action.
The substrate and the cover film constituting the opening may be arranged so as to be shifted in a direction parallel to either of them.

For the substrate, the working electrode, and the reference electrode, the same materials and the same forming method as in the first embodiment can be used. In addition, the same materials as in the first embodiment can be selected for the enzyme and the electron mediator. The insulating film 32 is not limited to the case where the insulating film 32 is formed as an independent member, but can also serve as an adhesive for the space forming film 33 or welding of the space forming film. Further, the method of forming the insulating film 32 is not limited to screen printing.

(Third Embodiment) FIG. 5 shows a biosensor 41 according to a third embodiment of the present invention.

FIG. 5A is an exploded perspective view, FIG. 5B is an external view, and FIG. 5C is a cross-sectional view taken along the line CC of FIG. 5B.

The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

The biosensor 41 is formed by laminating a working electrode 3, a reference electrode 4, and a reagent layer 5 as an electrode system on one surface of a substrate 2 made of an insulating film made of polyethylene terephthalate. Each of the working electrode 3 and the reference electrode 4 is made of the same material mainly composed of carbon,
And the terminal portions 9 and 10 are made of an insulating layer 4 made of an insulating paste.
2 to expose. A reagent solution containing an enzyme is applied on the electrode section 5 and dried to form a reagent layer 5. When quantifying blood glucose, a blood sample may be dropped on the reagent layer 5.

The shape of the biosensor 41 is not limited to the illustrated one, and the materials and forming methods of the substrate 2, the working electrode 3, the reference electrode 4, the insulating layer 42, and the reagent layer 5 may be any known materials and forming methods. A more suitable one can be appropriately selected.

For the substrate 2, the working electrode 3, and the reference electrode 4, the same materials and the same forming method as in the first embodiment can be used.

The insulating layer 42 is selected from an ultraviolet-curing or thermosetting insulating paste.

The enzyme of the reagent layer 5 needs to be appropriately selected depending on the substance to be determined.
The same substances as in the embodiment can be used. Further, as the electron transfer material, potassium ferricyanide or the like can be used as in the first embodiment.

As described above, in the present invention, the substrate,
The material, shape, thickness, etc. of the electrode are not limited.
The material, composition, shape, thickness, and forming method of the insulating layer and the reagent layer 5 can also be appropriately selected, and may be appropriately set according to the use and usage of the planar enzyme electrode.

(Example) As an example of the third embodiment, a blood glucose biosensor 41 will be described. FIG. 6 is a diagram illustrating a method of manufacturing the biosensor 41.

First, as the substrate 2, a length of 20 mm and a width of 6
A polyethylene terephthalate having a thickness of 180 μm, which is cut into a substantially rectangular shape whose one end has a substantially semicircular shape, is prepared (FIG. 6A). A carbon paste is applied on one surface of the substrate 2 by screen printing and baked to form the working electrode 3, the reference electrode 4, the leads 11, 12 and the terminals 9, 10 (FIG. 6B).

Next, an insulating layer 42 is formed from an ultraviolet-curable insulating paste, and the electrode section 7 and the terminal sections 9 and 1 are formed.
0 is exposed (FIG. 6 (c)). The insulating layer 42 has a substantially circular hole 42a near the substantially semicircular end.

Next, at least 5 μl of a reagent solution is dropped on the electrode portion 7 exposed from the hole 42a and dried at 50 ° C. for 1 hour to form a reagent layer 5 (FIG. 6D). The composition of the reagent solution is 0.2% of enzyme glucose oxidase, 1.0% of electron transfer substance potassium ferricyanide, and 2.0% of trehalose support material. In this embodiment, the sensitive portion is constituted by the electrode portion 7 exposed from the hole 42a and the reagent layer 5 formed thereon.

The blood glucose planar biosensor 41 manufactured as described above is mounted on a measuring device, and the switch 28 is turned on.
Then, a voltage of 0.1 V is applied to the terminal 9 connected to the working electrode 3 with respect to the terminal 10 connected to the reference electrode 4 in advance, and blood is dropped on the reagent layer 5. When the enzymatic reaction starts and the electrode reaction current is detected, the switch 28 is switched so that no voltage is applied to the non-inverting input terminal. Ten seconds after the start of the enzymatic reaction, the terminal 9
Voltage of 0.6 V, 5 seconds after application or 10
Blood glucose is quantified by the output of the electrode after 2 seconds.

In the case where a low-resistance material is used for the lead portion and the terminal portion, and the conventional technology in which the electrode portion, the lead portion, and the terminal portion are formed in a shape containing low resistance instead of screen printing with a material containing carbon instead of screen printing or the like. Similarly, a voltage of 0.6 V is applied to the terminal portion 9 with respect to the terminal portion 10 as compared with a case where 0.5 V is applied to the terminal portion connected to the working electrode with respect to the terminal portion connected to the reference electrode. If the voltage is applied, the high-resistance terminal section 9,
Since a voltage drop at 10 and the lead portions 11 and 12 is expected, a predetermined voltage can be applied between the working electrode 3 and the reference electrode 4, and accurate measurement can be performed.

When the working electrode 3 and the reference electrode 4 are formed of the same material in one manufacturing process, not only the manufacturing cost can be reduced and the manufacturing process can be simplified, but also mass production can be simplified and efficiency can be improved. A highly accurate planar biosensor with little difference can be produced, and an inexpensive planar biosensor can be provided. As described above, by applying a voltage to the terminals 9 and 10 by adding a voltage drop generated by the leads 11 and 12 and the terminals 9 and 10, high accuracy can be obtained even when an inexpensive planar biosensor is used. Measurement becomes possible.

[0129] The same apparatus as that of the first embodiment shown in FIG. 2 can be used for a measurement apparatus that performs measurement using the biosensor 41.

(Fourth Embodiment) FIG. 7 shows a planar biosensor 51 according to a fourth embodiment of the present invention.

FIG. 7 (a) is an exploded perspective view, FIG. 7 (b) is an external view, FIG. 7 (c) is an external view of a square capillary 54, FIG.
(D) is a diagram showing a state in which a blood sample is filled in the square capillary 54, FIG. 7 (e) is an external view of the biosensor 51 during measurement, and FIG. 7 (f) is a DD line in FIG. 7 (e). It is sectional drawing.

The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

In the present embodiment, as shown in FIG. 7A, the electrode portion 7, the lead portions 11, 12 and the terminal portion 9 are formed on a substantially rectangular substrate (support member) 52 by a material mainly composed of carbon. , 10 are integrally formed, the leads 11, 12 are covered with a holding material (holding member) 53 in a liquid-tight manner, and the electrode 7 and the terminals 9, 10 are exposed. The holding material 53 is connected to the substrate 5 together with the leads 11 and 12.
3 has a substantially U-shaped cross section that also covers the side surface.

The exposed electrode portion 7 as shown in FIG.
The reagent layer 5 is formed thereon. In this embodiment, a sensitive part is constituted by the electrode part 7 and the reagent layer 5 exposed by the holding material.

As shown in FIG. 7D, the rectangular capillary 54 has a substantially rectangular cross section and has two openings 54a and 54b.
Is a cylindrical body having a hollow interior 54c communicating with the outside.

In the present embodiment, the blood sample is not dropped onto the reagent layer 5 at the time of blood glucose quantification, but is separately filled in the hollow interior 54c of the rectangular capillary 54 as shown in FIG. As shown in FIG.
The blood sample 55 and the reagent layer 5 are brought into contact with each other by inserting the part of the reagent layer 5 from the opening 54b of the square capillary into the hollow interior 54c (FIG. 9E). The hollow interior 54c of the square capillary 54 is manufactured so as to match the size of the substrate 52 in the reagent layer 5 portion.
The length of the insertion portion of the reagent layer 5 in the insertion direction is defined by abutting on the reference numeral 3. The hollow sample 54 can be filled into the hollow interior 54c of the square capillary 54 by so-called capillary action by filling one of the openings 54a and 54b with the blood sample. Are easily filled.

(Example) As an example of the fourth embodiment, a blood glucose biosensor will be described.

FIGS. 8A to 8D are views for explaining a method of manufacturing the biosensor 51. FIG.

First, a polyethylene terephthalate having a thickness of 18 mm cut into a length of 20 mm and a width of 6 mm was used as the substrate 52.
0 μm is prepared (FIG. 8 (a)), and the working electrode 3, the reference electrode 4, the lead portions 11, 12, and the terminal portions 9, 10 are integrally formed on one surface by screen printing of a conductive member mainly made of carbon. (FIG. 8B).

Next, polyethylene terephthalate having a thickness of 2
A holding material 53 of 50 μm is mounted on the substrate 52 with an adhesive to expose the working electrode 3, the reference electrode 4, and the terminals 9, 10 (FIG. 8C).

Next, at least 5 μm of the reagent solution was placed on the electrode portion 7.
1 was dropped and dried at 50 ° C. for 1 hour to obtain a reagent layer 5 (FIG. 8D). The composition of the reagent solution is 0.2% of enzyme glucose oxidase, 1.0% of electron transfer substance potassium ferricyanide, and 2.0% of trehalose support material.

The thus-produced planar type biosensor for blood glucose 51 is mounted on a measuring device, and the switch 28 is turned on.
a, and a voltage of 0.1 V is applied to the terminal portion 9 connected to the working electrode 3 in advance with respect to the terminal portion 10 connected to the reference electrode 4, and the reagent layer 5 is filled with the blood sample 55 filled corner. It is inserted into the mold capillary 54. When the enzymatic reaction starts and the electrode reaction current is detected, the switch 28 is switched so that no voltage is applied to the non-inverting input terminal. 10 seconds after the start of the enzymatic reaction, a voltage of 0.6
V is applied, and blood glucose is quantified by an electrode output 5 seconds or 10 seconds after the application. For the measurement, the measuring device according to the first embodiment shown in FIG. 2 can be used.

As described above, by holding the leads 11 and 12 in a liquid-tight manner with the holding material 53, the leads 11 and 12 are involved in the electrochemical phenomenon caused by the reaction between the enzyme and the sample without providing an insulating layer. Does not affect the measurement. Therefore, it is possible to omit the printing of the insulating film and the step of forming the insulating layer or the insulating film for the heating and UV irradiation associated therewith. In addition, a high-precision planar biosensor with little difference between individuals can be produced, and an inexpensive planar biosensor can be provided.

(Fifth Embodiment) FIG. 8 shows a planar biosensor according to a fifth embodiment of the present invention. FIG.
8A is an exploded perspective view, FIG. 8B is an external view, and FIG.
FIG. 9 is a sectional view taken along line EE of FIG.

In the biosensor 61, a working electrode 63 and a reference electrode 64 are formed on one surface of a substrate 62 made of an insulating film made of polyethylene terephthalate, and an insulating layer 68 is further formed to define an electrode portion 67. The reagent layer 65 is formed on the portion 67 by lamination.

The electrode part 6 comprising the working electrode 63 and the reference electrode 64
Numerals 7 are provided at the ends of an integral conductive member made of the same carbon-based material. The lead portions 71, 72 connecting the terminal portions 69, 70 connected to the measuring device with the working electrode 63 and the reference electrode 64 are covered with an insulating layer 65, and the terminal portions 69, 70 are exposed. On the working electrode 64 and the reference electrode 64, a solution containing at least an enzyme, a low molecular compound as a holding material, and a small piece 73 for preventing foreign matter in the sample from adsorbing to the electrode portion 67 and lowering the output is used. The reagent layer 65 is formed by coating or dripping and drying.

At the time of quantifying blood sugar, a blood sample 74 as a liquid sample may be dropped on the reagent layer 65 as shown in FIG. In the present embodiment, a sensitive part composed of the electrode part 67 and the reagent layer 65 is formed in a region exposed on the electrode part 67 side of the substrate 62 by the insulating layer 68.

When the blood sample 74 is dropped on the reagent layer 65, the reagent components other than the small pieces 83 are dissolved by the blood sample as shown in FIG. Remains. In the blood glucose measurement, since the blood cells 75 in the blood sample generally adhere to the electrode section 67, there is a problem that the current value fluctuates depending on the amount of blood cells (ie, the level of hematocrit). The action of the minute pieces 73 prevents the blood cells 75 in the blood sample from adhering to the electrode portion 67, so that fluctuations in the current value due to the height of the hematocrit can be suppressed.

Note that the shape of the biosensor 61 is not limited to the one shown in the figure, and the substrate 62, the working electrode 62, the reference electrode 63,
The material and the formation method of the insulating layer 68 can be appropriately selected from known materials and a formation method.

For example, as a material of the substrate 62, in addition to the above-described polyethylene terephthalate, a resin sheet made of polyethylene naphthalate, polyethylene sulfide, polycarbonate, polyallylate, polyether sulfide, polyimide, etc., and further, plastic, ceramics, It can be selected from a thin glass plate, paper, or the like.

The working electrode 63 and the reference electrode 64 are formed by a screen printing method suitable for mass production. However, electrode materials such as platinum, gold, silver, silver chloride, iron, zinc, nickel, and palladium are formed by vapor deposition and sputtering. It can also be manufactured by a thin film forming method such as a plating method, a plating method, and an ion plating method. If the working electrode 63, the reference electrode 64, the respective lead portions 71, 72 and the terminal portions 69, 70 are formed of the same material as in the present embodiment, the forming process can be simplified and the manufacturing cost can be reduced. .

The enzyme of the reagent layer 65 must be appropriately selected depending on the substrate to be quantified, and examples thereof include glucose oxidase, cholesterol oxidase, uricase, and pyruvate oxidase. The holding material for easily forming the reagent layer 65 in a layered form is a low molecular weight compound, preferably one compound selected from monosaccharides, disaccharides and trisaccharides, or a mixture of two or more compounds. Alternatively, it is one kind of compound selected from amino acids or a mixture of two or more kinds. Needless to say, substances involved in the enzyme reaction or substances that inhibit the enzyme reaction, such as glucose when glucose oxidase is used, cannot be used as the holding material. Further, depending on the electrode reaction, it is necessary to add not only an enzyme but also an electron transfer substance such as potassium ferricyanide, a ferrocene compound, and p-benzoquinone to the reagent layer 65. The formation of the reagent layer 65 is generally performed by drying the dropped reagent solution, but a screen printing method or the like can also be appropriately selected, and an appropriate low-molecular compound can be added for forming a stronger film. It is.

Further, the minute pieces 7 added to the reagent layer 65
3 has a function of preventing a blood cell 75 in a blood sample from adhering to the electrode section 67, thereby suppressing a change in a current value due to a height of hematocrit. The material is divinylbenzene resin, nylon resin, urethane resin. And melamine resins. However, the present invention is not limited thereto, and an optimum material may be selected in consideration of the method of forming the reagent layer, the solvent, the reagent components, and the like. In addition, the shape of the minute piece 73 may be appropriately selected, such as a spherical shape or a flat spherical shape. Although the specific gravity of the minute piece 73 depends on the material, there is a need to select a material having a specific gravity suitable for the method and process of forming the reagent layer. Further, two or more kinds of different minute pieces may be added.

The insulating layer 68 was formed by screen-printing a resist paste. By covering the lead portions 71, 72 with the insulating layer 68, the lead portions 71, 72 are formed.
72 does not affect the measurement by participating in the electrochemical phenomenon caused by the reaction between the enzyme and the reagent.

As described above, in the present invention, the substrate 6
2. The material, shape, thickness, and the like of the electrode are not limited, and may be appropriately selected and set according to the use and usage of the planar enzyme electrode. Electrode part 67, lead parts 71, 7
2. At least one of the terminal portions 69 and 70 may be formed of a different material.

(Example) As an example of the fifth embodiment, a blood glucose biosensor 61 will be described. FIG.
FIG. 7 is a diagram illustrating a method for manufacturing the biosensor 61.

First, the substrate 62 is 20 mm long and 6 mm wide.
A polyethylene terephthalate having a thickness of 250 μm cut to a thickness of 250 mm was prepared (FIG. 11A), and a working electrode 6 was formed on one surface.
3. A reference electrode 64 is formed (FIG. 11B). Next, an insulating layer 68 is formed in order to define the areas of the working electrode 63 and the reference electrode 64 to form the electrode section 67.
(C)).

Next, at least 10 μl of a reagent solution is dropped onto at least the electrode section 67 and dried at 40 ° C. to form a reagent layer (reaction layer) 65 (FIG. 11D). The composition of the reagent solution is the enzyme glucose oxidase 200 U / m
1, 0.02 g of the electron mediator potassium ferricyanide /
ml, 0.55 g / ml of trehalose as a support material, and 0.05 g / ml of microbeads containing divinylbenzene resin as a main component are added and dispersed as minute pieces to this liquid.

The planar biosensor for blood glucose manufactured as described above is mounted on, for example, a measuring device having the schematic configuration shown in FIG. 2, and the switch 28 is switched to 28a. By applying an elastic pressure of 0.1 V to the terminal portion 69 connected to the working electrode 63, blood is dropped on the reagent layer 65. When the enzymatic reaction starts and the electrode reaction current is detected, the switch 28 is switched so that no voltage is applied to the non-inverting input terminal. 110 seconds after the start of the enzymatic reaction, a voltage of 0.degree.
8 V is applied, and blood glucose is quantified by an electrode output 10 seconds or 30 seconds after the application.

Note that the measurement conditions can be appropriately set according to the enzyme and the liquid sample, and are not limited to the above conditions.

In the present embodiment, the reagent layer 65 including the fine pieces is formed in one step, but the electrode system 67 and the insulating layer 6 are formed.
8 (FIG. 11 (c)), the small piece 3
5 is added or dispersed, and the solution is applied or dropped and dried to form a micro-piece layer 76 (FIG. 11E). A reagent solution containing an enzyme is applied or dropped on the micro-piece layer 76 and dried. To form the reagent layer 77 (FIG. 11).
(F)). At this time, two or more kinds of fine pieces may be added and dispersed in the fine piece layer 76, or a reagent liquid in which a different kind of fine pieces from the fine pieces in the fine pieces 76 are added and dispersed may be used. Can also.

[0162]

According to the first aspect of the present invention, if a low molecular compound is added to form a reaction layer containing at least an enzyme, the reaction layer can be easily formed into a layer or film. Therefore, not only the mass production can be facilitated and the efficiency can be improved by simplifying the manufacturing process, but also a high-precision biosensor with little difference between individuals can be produced and can be provided at a low cost. In addition, the low-molecular compound is easily dissolved in a liquid state before the reaction layer is solidified and formed, and even after the reaction layer is formed, the dissolution of a liquid sample such as blood is faster than when a high-molecular compound is added. Therefore, the time required for the measurement can be reduced.

According to the second aspect of the present invention, the reaction layer is formed as a single layer containing a low molecular compound, or the reaction layer is formed from a plurality of layers, and the first layer includes a layer containing a low molecular compound. The same effect as that of the invention is obtained.

According to the third aspect, as the low molecular weight compound, one compound selected from monosaccharides, disaccharides, trisaccharides and amino acids or a mixture of two or more low molecular weight compounds is selected. Thereby, an effect similar to that of the first or second invention can be obtained.

If the reaction layer containing the low molecular weight compound is formed by a single coating or printing process as in the fourth invention, the manufacturing process can be simplified. If a low molecular compound is added, the reaction layer can be easily formed into a layer or film, so that the reaction layer can be formed in one process, and the mass production can be facilitated by simplifying the manufacturing process. Not only efficiency can be improved, but also a high-precision biosensor with little difference between individuals can be produced, and this can be provided at a low price. In addition, the low-molecular compound is easily dissolved in a liquid state before the reaction layer is solidified and formed, and even after the reaction layer is formed, the dissolution of a liquid sample such as blood is faster than when a high-molecular compound is added. Therefore, the time required for the measurement can be reduced.

According to the fifth aspect of the present invention, the reaction layer can be easily formed by bringing the opening into contact with the liquid containing the components of the reaction layer and sucking and solidifying the liquid by capillary action. The process can be simplified and an inexpensive biosensor can be provided. In addition, since the reaction layer can be formed after forming the electrode system, the space, and the like, the reaction layer is not affected by heat, chemicals, and the like, and a high-precision biosensor can be provided. .

According to the sixth aspect of the present invention, the reaction layer can be easily formed by bringing the opening into contact with the liquid containing the components of the reaction layer and sucking and solidifying the liquid by capillary action. The process can be simplified and an inexpensive biosensor can be provided. In addition, since the reaction layer can be formed after forming the electrode system, the space, and the like, the reaction layer is not affected by heat, chemicals, and the like, and a high-precision biosensor can be provided. .

According to the seventh aspect, when the working electrode, the reference electrode, the respective lead portions and the terminal portions are formed by a thin film member made of the same material as in the case of screen printing of carbon paste, etc. By adding the voltage drop generated by the resistance of the terminal and lead to the voltage and applying the voltage to the terminal, a predetermined voltage is stably applied between the working electrode and the reference electrode. High-precision measurement is possible without increasing the size and size. In addition, if the working electrode, the reference electrode, and the respective lead portions and terminal portions are formed of a thin-film member made of the same material, the manufacturing cost can be reduced, so that an inexpensive and highly accurate biosensor is provided. Can be.

As in the eighth invention, a portion adjacent to the electrode system in a conductive member having an electrode system having at least a working electrode and a reference electrode at one end may be liquid-tightly covered by a holding member. Even if the liquid sample is supplied to the sensitive part, it does not affect the detection of electrochemical phenomena by contacting other parts of the conductive member than the electrode system.
Since an insulating film is not required, manufacturing cost can be reduced. Further, not only the mass production can be facilitated and the efficiency can be improved by simplifying the manufacturing process, but also a high-precision biosensor with little difference between individuals can be produced and can be provided at a low cost.

As in the ninth aspect, if a portion adjacent to the electrode system of the conductive member having at least one electrode system having at least a working electrode and a reference electrode is covered by the holding member in a liquid-tight manner, Even if the liquid sample is supplied to the sensitive part, it does not affect the detection of electrochemical phenomena by contacting other parts of the conductive member than the electrode system.
The process of forming the insulating film becomes unnecessary, simplifying the manufacturing process,
Manufacturing costs can also be reduced. In addition to facilitating mass production and improving efficiency, a high-precision biosensor with little difference between individuals can be produced.

According to the tenth aspect, by adding minute pieces to the reaction layer, it is possible to uniformly prevent the foreign matter in the sample from adhering to the electrode system and reduce the reaction current, and to reduce the reaction current in proportion to the substrate concentration. The detected reaction current can be accurately detected, and the difference between individuals can be made extremely small.

When a low molecular compound is added to form a reaction layer containing at least an enzyme as in the eleventh invention,
The reaction layer can be easily and uniformly formed. Also,
The reaction layer to which the low-molecular compound is added dissolves faster in a liquid sample such as blood than when a high-molecular compound is added, so that the time required for measurement can be reduced. In addition, the addition of micro-pieces to the reaction layer can uniformly prevent the reaction current from decreasing due to foreign substances in the sample adhering to the electrode system, and can accurately control the reaction current proportional to the substrate concentration. Detection can be performed well, and the difference between individuals can be extremely small. Therefore, not only the mass production can be facilitated and the efficiency can be improved by simplifying the manufacturing process, but also a high-precision biosensor with little difference between individuals can be produced and can be provided at a low cost.

According to the twelfth aspect, for example, by using a small piece having a size substantially equal to that of red blood cells in blood, it is possible to effectively prevent red blood cells in a blood sample from being adsorbed on the electrode system. can do.

According to the thirteenth aspect, the size and shape of the individual minute pieces can be easily made uniform, and the effect of preventing foreign matter from adsorbing on the electrode system can be further enhanced.

According to the fourteenth aspect, it is possible to form the fine pieces uniformly and stably dispersed in the reaction layer or on the electrode system.

According to the fifteenth aspect, for example, the gap between the fine pieces is made dense by using two or more kinds of fine pieces having different sizes, or by forming a plurality of fine piece layers. In addition, the effect of preventing foreign matter from adhering to the electrode system can be further enhanced.

As in the sixteenth aspect, the low molecular weight compound is a compound selected from monosaccharides, disaccharides, trisaccharides and amino acids, or two or more low molecular weight compounds. Mixtures of molecular compounds can be used.

According to the seventeenth aspect, before the reaction layer is formed, the reaction layer can be formed after the fine pieces are uniformly dispersed in advance. Therefore, it is possible to form a reaction layer containing fine pieces in a single step, and to keep the number and distribution of the fine pieces contained in the reaction layer constant. The effect of preventing the adsorption on the upper side is also constant, and it is possible to efficiently provide an extremely high-precision biosensor in large quantities at low cost.

By forming minute pieces on the electrode system in advance as in the eighteenth aspect, the effect of preventing foreign substances in the sample from adsorbing on the electrode system can be improved, and higher precision can be achieved. Biosensors can be supplied.

According to the nineteenth aspect, for example, by using two or more types of minute pieces having different sizes to close the gap between the minute pieces, the effect of preventing foreign matter from adhering to the electrode system can be reduced. Can be even higher.

According to the twentieth aspect, the effect of preventing the foreign matter in the sample from adsorbing onto the electrode system can be further improved by forming the plurality of fine piece layers.

[Brief description of the drawings]

FIG. 1A is an exploded perspective view of a biosensor according to a first embodiment of the present invention, FIG. 1B is an external view of the same, FIG.
(C) is an AA cross-sectional view of FIG. 1 (b).

FIG. 2 is a block diagram illustrating a schematic configuration of a main part of a measurement device that performs measurement using the biosensor according to the first embodiment of the present invention.

FIG. 3A is a top view of a biosensor according to a second embodiment of the present invention, and FIG. 3B is a cross-sectional view taken along line BB of FIG. 3A.

FIGS. 4A to 4E are diagrams illustrating a method for manufacturing a biosensor according to a second embodiment of the present invention.

5 (a) is an exploded perspective view of a biosensor according to a third embodiment of the present invention, FIG. 5 (b) is an external view thereof, and FIG.
FIG. 5C is a cross-sectional view taken along the line CC of FIG.

FIGS. 6A to 6D are diagrams illustrating a method for manufacturing a biosensor according to a third embodiment of the present invention.

7 (a) is an exploded perspective view of a biosensor according to a fourth embodiment of the present invention, FIG. 7 (b) is an external view thereof, and FIG.
FIG. 7C is an external view of a square capillary according to a fourth embodiment of the present invention, FIG. 7D is a diagram showing a state in which the same square capillary is filled with a blood sample, and FIG. FIG. 7F is a cross-sectional view taken along line DD of FIG. 7E.

FIGS. 8A to 8D are diagrams illustrating a method for manufacturing a biosensor according to a fourth embodiment of the present invention.

FIG. 9A is an exploded perspective view of a biosensor according to a fifth embodiment of the present invention, FIG. 9B is an overall perspective view of the same,
FIG. 9C is a sectional view taken along line EE of FIG. 9B.

FIG. 10 (a) is a diagram showing a state in which a blood sample is dropped on a biosensor according to a fifth embodiment of the present invention, and FIG. 10 (b) is a diagram in which a blood sample is supplied to a reagent layer of the biosensor. FIG. 5 is a diagram schematically showing the state of micro-pieces in a reagent layer and blood cells in a blood sample when performed.

FIG. 11 is a diagram illustrating a method for manufacturing a biosensor according to a fifth embodiment.

FIG. 12 is an exploded perspective view of a biosensor according to a first related art.

FIGS. 13A to 13E are diagrams showing a method for manufacturing a biosensor according to the first conventional technique.

14 (a) is an exploded perspective view of a biosensor according to a second conventional technique, FIG. 14 (b) is an external view thereof, and FIG.
FIG. 14C is a sectional view taken along line FF of FIG.

FIG. 15 is a view showing a method for manufacturing a biosensor according to a second conventional technique.

FIG. 16 is an exploded perspective view of a biosensor according to a third conventional technique.

FIG. 17 is a diagram showing a method for manufacturing a biosensor according to a third conventional technique.

FIG. 18 is an exploded perspective view of a biosensor according to a fourth conventional technique.

FIG. 19 is a diagram showing a method for manufacturing a biosensor according to a fourth conventional technique.

[Explanation of symbols]

 1,31,41,51,61 Biosensor 2,52,62 Substrate 3,63 Working electrode 4,64 Reference electrode 5,65,77 Reagent layer 6,53 Retaining material 7,67 Electrode part 9,10,69, 70 Terminal portion 11, 12, 71, 72 Lead portion 32 Insulating film 33 Space forming film 34 Cover film 35 Space portion 36 Opening 37 Reagent solution 42, 68 Insulating layer 54 Square capillary 55, 74 Blood sample 73 Small piece 75 Blood cell 76 small single layer

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Yusaku Sakota 24, Yamanouchi Yamanoshitamachi, Ukyo-ku, Kyoto-shi, Kyoto Prefecture Inside OMRON Life Science Research Institute Inc. (72) Inventor Masato Arai 24 Yamanouchi Yamanoshitamachi, Ukyo-ku, Kyoto, Kyoto Address Omron Life Science Research Inc. (72) Inventor Toshihiko Ogura 24 Yamanouchi Yamanoshita-cho, Ukyo-ku, Kyoto, Kyoto Prefecture 72 Omron Life Science Research Inc. (72) Inventor Muneo Tokita 24 Yamanouchi Yamanoshita-cho, Ukyo-ku, Kyoto, Kyoto Address: OMRON Life Science Research Inc. (72) Inventor Koichi Takizawa 24, Yamanouchi Yamanoshita-cho, Ukyo-ku, Kyoto-shi, Kyoto Prefecture OMRON Life Science Research Inc. (72) Inventor: Akihiro Fukao Yamanouchi Yamanouchi, Ukyo-ku, Kyoto-shi, Kyoto 24, Omron Life Science Research Inc. (72) Inventor Shinya Tanaka 24, Yamanouchi Yamanoshitacho, Ukyo-ku, Kyoto, Kyoto Prefecture

Claims (20)

[Claims] 1. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, In a biosensor that detects an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system and measures the concentration of a specific component in the liquid sample, a low molecular compound is added to form the reaction layer. A biosensor characterized by the following. 2. The biosensor according to claim 1, wherein the reaction layer has at least a layer containing the low-molecular compound. 3. The low molecular weight compound is a monosaccharide, a disaccharide,
The biosensor according to claim 1, wherein the biosensor is a compound selected from one of a trisaccharide and an amino acid or a mixture of two or more low-molecular compounds. 4. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, In a biosensor manufacturing method for detecting a concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system, the reaction layer containing a low-molecular compound may be used for one time. A method for producing a biosensor, which is formed in a process. 5. An electrode system comprising at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part comprising the electrode system and the reaction layer, A biosensor for detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system to measure a concentration of a specific component in the liquid sample, wherein the electrode system has at least an opening opened to the outside. A method for producing a biosensor, wherein the component is disposed in a space having the electrode system, and the liquid sample is sucked into the space by capillary action from the opening. A method for producing a biosensor, characterized in that a reaction layer is formed by sucking and solidifying a liquid containing a liquid through the opening by capillary action. 6. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, A biosensor for detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system to measure a concentration of a specific component in the liquid sample, wherein the electrode system has at least an opening opened to the outside. A biosensor that is disposed in a space having the liquid sample and that sucks the liquid sample into the space by capillary action from the opening. A biosensor wherein a reaction layer is formed by sucking and solidifying from the opening by capillary action. 7. An electrode system provided with a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, By detecting a current between the two electrodes when a predetermined voltage is applied to the working electrode, an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme is detected, and a specific component in the liquid sample is detected. A biosensor for measuring the concentration of: a working electrode and a reference electrode; a terminal portion for applying a voltage to the working electrode and the reference electrode and extracting a current; and a terminal for the working electrode and the reference electrode, respectively. In a measuring method using a biosensor in which a lead portion connecting a portion and a lead portion is formed by a thin film member made of the same material, a predetermined voltage of a working electrode with respect to the reference electrode, Yo Adding a voltage drop caused Te, a measuring method using a biosensor, characterized in that applied to the terminal portion. 8. An insulating support member, comprising: a conductive member having an electrode system having at least a working electrode and a reference electrode at one end; and a reaction layer containing at least an enzyme; A biosensor that supplies a liquid sample to a sensitive part including a layer and detects an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme with the electrode system to measure a concentration of a specific component in the liquid sample. A biosensor comprising a holding member for reinforcing the supporting member, wherein a portion of the conductive member adjacent to the electrode system is liquid-tightly covered by the holding member. 9. An insulating support member, comprising: a conductive member having an electrode system having at least a working electrode and a reference electrode at one end; and a reaction layer containing at least an enzyme, and reinforcing the support member. A liquid sample is supplied to a sensitive portion including the electrode system and the reaction layer, and an electrochemical phenomenon caused by a reaction between the liquid sample and the enzyme is detected by the electrode system, and the liquid sample is supplied to the sensitive portion. In the biosensor manufacturing method for measuring the concentration of a specific component in a sample, a portion of the conductive member formed on the support member, which is adjacent to the electrode system, is liquid-tightly covered by the holding member. A method for producing a biosensor. 10. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer. A biosensor for detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system and measuring a concentration of a specific component in the liquid sample, wherein the reaction layer includes a small piece. . 11. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, In a biosensor for detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system and measuring a concentration of a specific component in the liquid sample, the reaction layer includes a small piece and a low-molecular compound. Biosensor. 12. The biosensor according to claim 10, wherein the minute piece has a size substantially equal to that of a blood cell. 13. The biosensor according to claim 10, wherein the minute piece has a spherical shape or a shape similar thereto. 14. The biosensor according to claim 10, wherein the minute piece has a specific gravity of 1.0 or more. 15. The biosensor according to claim 10, wherein at least two types of fine pieces are included in the reaction layer. 16. The low molecular compound is a compound selected from one of monosaccharides, disaccharides, trisaccharides and amino acids or a mixture of two or more low molecular compounds. Item 12. The biosensor according to item 11, 17. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, In a biosensor manufacturing method for detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system and measuring a concentration of a specific component in the liquid sample, the reaction layer including fine pieces is subjected to one step. A method for producing a biosensor, comprising: 18. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, In a biosensor manufacturing method of detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system and measuring the concentration of a specific component in the liquid sample, a layer containing fine pieces in advance on the electrode system After forming
A method for manufacturing a biosensor, comprising forming the reaction layer. 19. The method for manufacturing a biosensor according to claim 17, wherein at least two types of fine pieces are included in the reaction layer. 20. An electrode system having at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, wherein a liquid sample is supplied to a sensitive part containing the electrode system and the reaction layer, In a biosensor manufacturing method for detecting an electrochemical phenomenon caused by a reaction between a sample and the enzyme with the electrode system and measuring a concentration of a specific component in the liquid sample, at least one kind of micro-piece is provided on the electrode system. A method for manufacturing a biosensor, comprising: forming a reaction layer including at least one kind of minute piece different from the minute piece after forming a layer including the minute piece.