JP2000039416A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the solubility of a reagent for rapid measurement by forming a sample liquid supply path between an insulation substrate where an electrode system is provided on it and a cover member and arranging a carrier for carrying the reagent in the supply path. SOLUTION: A silver paste is printed on an insulation substrate 1 for forming leads 2 and 3 and the electrode system ground, and a conductive carbon paste including a resin binder is printed on the insulation substrate 1, thus forming an electrode system including a measurement electrode 4 and a counter electrode 5. Also, an insulation paste is printed to form an insulation layer 6. A space part for constituting a sample liquid supply path is formed at the part of a slit 12 of a spacer 8 between this sort of insulation substrate 1 and a cover 9, a carrier 13 is arranged at the space part, and a biosensor is created. In this case, the carrier 13 is made of fiber for carrying a reagent including an oxidation reduction enzyme and the sample liquid can be easily introduced into a part in the sensor simply by allowing the sample liquid to touch an opening part 10 that becomes a sample liquid supply path port.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor capable of easily performing a quick and accurate quantification of an object to be measured in a sample.

[0002]

2. Description of the Related Art Hitherto, the following biosensor has been proposed as a method for simply quantifying a specific component in a sample without diluting or stirring the sample solution (Japanese Patent Application Laid-Open No. 2-062952). Gazette). This biosensor is
An electrode system consisting of a measurement electrode, a counter electrode, and a reference electrode is formed on an insulating substrate by a method such as screen printing, and an enzyme reaction layer containing a hydrophilic polymer, a redox enzyme, and an electron mediator is formed on the electrode system. It is formed. A buffer is added to this enzyme reaction layer as needed.

[0003] When a sample solution containing a substrate is dropped onto the enzyme reaction layer of the biosensor thus produced, the enzyme reaction layer dissolves and the enzyme reacts with the substrate, whereby the electron mediator is reduced. You. After completion of the enzymatic reaction, the reduced electron mediator is electrochemically oxidized, and the substrate concentration in the sample solution can be determined from the oxidation current value obtained at this time. Such a biosensor can measure various substances in principle by selecting an enzyme using the substance to be measured as a substrate. For example,
If glucose oxidase is used as the oxidoreductase, a biosensor for measuring glucose concentration in blood can be configured. This sensor is widely used as a glucose sensor.

[0004] If cholesterol oxidase is used as the oxidoreductase, a biosensor for measuring serum cholesterol concentration can be constructed. Normally, the serum cholesterol level used as a diagnostic guideline in various medical institutions is the sum of the serum cholesterol concentration and the cholesterol ester concentration. Cholesterol ester cannot serve as a substrate for the oxidization reaction by cholesterol oxidase, so that a biosensor having a reaction layer containing cholesterol oxidase cannot measure serum cholesterol as a diagnostic guideline. Therefore, a process for converting cholesterol ester to cholesterol is required. Cholesterol esterase is known as an enzyme that catalyzes this process. By including this cholesterol oxidase together with cholesterol oxidase in the enzyme reaction layer, a biosensor for measuring the total cholesterol concentration in serum is constituted.

[0005]

The enzyme reaction layer of the biosensor constructed as described above is formed by dropping a mixed aqueous solution containing an oxidoreductase, an electron mediator and the like onto the electrode system and drying the mixture. Therefore, when the amount of the reagent is large, there is a problem that when the sample solution is dropped onto the reaction layer, the reaction layer does not rapidly dissolve and the measurement takes a long time. In particular, in the case of a sensor for measuring the total cholesterol concentration in serum as described above, since the enzyme reaction layer must contain two kinds of enzymes, cholesterol oxidase and cholesterol esterase, the amount of the reagent carried is extremely low. Therefore, it takes a very long time to dissolve the enzyme reaction layer after dropping the sample droplet, so that quick measurement cannot be performed.

When reagents such as oxidoreductase and electron mediator are carried in the enzyme reaction layer in a mixed state, the reagents may be denatured. In particular, when the sample solution is stored at a high temperature for a long period of time, there is a problem that the sensor response is deteriorated such that a large current value is observed even when the enzyme reaction substrate is not contained in the sample solution. The present invention has been made in view of the above circumstances, and has as its object to provide a biosensor capable of increasing the solubility of a reagent and enabling rapid measurement. It is another object of the present invention to provide a biosensor that maintains excellent response characteristics even after being stored for a long time.

[0007]

According to the present invention, there is provided a biosensor comprising an insulating substrate, an electrode system having at least a measurement electrode and a counter electrode provided on the substrate, and the above-mentioned substrate interposed between the substrate and the substrate in combination with the substrate. A cover member forming a sample liquid supply path for supplying a sample liquid to the electrode system, and a carrier made of fibers carrying a reagent containing at least an oxidoreductase, wherein the carrier is disposed in the sample liquid supply path. It is characterized by being. Another biosensor according to the present invention includes an insulating substrate, an electrode system having at least a measurement electrode and a counter electrode provided on the substrate, and a carrier made of fibers carrying a reagent containing at least an oxidoreductase, The carrier is fixed near the electrode system by an adhesive. Also, the carrier has at least 2
It consists of two carrier pieces, each carrying a different reagent.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION A biosensor according to the present invention carries a reagent such as an oxidoreductase dispersed and attached to the surface of a fiber constituting a carrier. With such a configuration, the surface area of the reagent in contact with the sample liquid is increased, and the solubility of the reagent in the sample liquid is improved.
To disperse and support the reagent on the surface of the carrier as described above,
The concentration of the reagent solution dropped onto the carrier may be appropriately adjusted.
Preferably, the carrier is itself inert to enzymatic and electrochemical reactions that occur within the biosensor,
It is preferable to use a sheet in which cellulose fiber, glass fiber, or fiber made of a polymer compound is laminated in a fleece or felt shape. Further, it is preferable to select a material having a porosity that allows the sample liquid to quickly penetrate into the carrier when the sample liquid is introduced into the sensor. For example, when filter paper made of glass fiber is used, the porosity is 70 to 95%.
It is good to use the one of the degree. In some cases, the carrier is fitted and held in the sample liquid supply path of the biosensor as described later, and therefore, the carrier preferably has some elasticity.

Another biosensor according to the present invention comprises preparing a plurality of carriers, supporting oxidoreductase, an electron mediator, etc. on separate carriers, and including each in a separated state in the biosensor. . With such a configuration, the solubility of the reagent in the sample solution is improved, and the denaturation of the reagent during storage can be suppressed.

[0010] Various modifications are possible for the arrangement of the carrier in the biosensor. One mode is to fit a carrier into the sample liquid supply path of the cover member,
In another mode, the carrier is fixed to the surface of the cover member on the side exposed to the sample liquid supply path or in the vicinity of the electrode system on the substrate using an adhesive. When fitting the carrier, the carrier is molded into the same shape as the sample liquid supply path of the cover member, and by fitting this into the sample liquid supply path,
Fix to the cover member. By using such a method, the process can be simplified and the productivity can be improved. When a plurality of carriers are used, it is preferable to prepare a carrier molded smaller than the sample liquid supply passage of the cover member, appropriately combine the individual carriers, and fit the carriers into the sample liquid supply passage. For example, a carrier having the same width as the sample liquid supply path and having a shorter length is arranged in order from the side where the sample liquid flows, so that the sample liquid permeates each carrier in order, or the length equal to the sample liquid supply path. A strip-shaped carrier having a short width may be arranged along the flow of the sample solution. In particular, the latter arrangement is preferable because the sample solution quickly penetrates and dissolves the reagent, and the dissolved reagent is easily mixed uniformly. Alternatively, a carrier having a small thickness may be prepared, and the carrier may be molded into the same shape as the sample liquid supply path of the cover member, and then may be overlapped with each other and inserted into the sample liquid supply path. It is preferable that the carriers supporting different reagents are divided into several parts, and then the carriers supporting different reagents are arranged adjacent to each other, so that the reagents dissolved in the sample liquid are easily mixed.

When the carrier is fixed using an adhesive, the carrier is fixed to a portion other than the electrode system so that the adhesive and the carrier do not affect the electrode reaction. The adhesive used is
It is preferable to use a highly viscous material that does not penetrate the carrier, for example, an adhesive for woodworking in the environment at the time of manufacturing the sensor. When the carrier is fixed by using such an adhesive, it is possible to prevent the carrier from moving and coming into contact with the electrode system to inhibit the electrode response due to swelling of the carrier itself when the sample liquid enters. it can. When a plurality of carriers are used, the individual carriers may be arranged separately.

In the case of a sensor without a cover member in which an electrode system and a reaction layer are simply formed on a substrate, carriers of various shapes can be used. However, it is preferable to fix the carrier at a position as close as possible to the electrode system. For example, a donut-shaped carrier whose inner circumference is larger than the outer circumference of the electrode system may be used. When the reagents are separately supported, as shown in FIG. 9 (a), different reagents are supported on the carrier 28 and the carrier 29, respectively, and fixed vertically.
Further, as shown in FIG. 9B, a carrier 30 and a carrier 31 obtained by dividing a donut-shaped carrier into half vertically are used.
As shown in (c), a carrier 32 and a carrier 33 having different inner diameters.
It is good to use.

Various oxidoreductases can be used as the oxidoreductase carried on the carrier. For example, glucose oxidase, lactate oxidase, cholesterol oxidase and the like can be mentioned. When measuring the serum cholesterol level, cholesterol oxidase and an enzyme having a cholesterol ester hydrolytic ability are used. Enzymes having cholesterol ester hydrolytic ability include cholesterol esterase and lipoprotein lipase. In particular, cholesterol esterase
The use of a suitable surfactant is advantageous because it can rapidly convert cholesterol esters to cholesterol.

When an enzyme having the ability to degrade cholesterol ester is used as the oxidoreductase, if a surfactant having an effect of improving the activity of the enzyme is contained in the reagent carried on the carrier, the time required for the enzyme reaction is reduced. Can be shortened. For example, surfactants that improve the activity of cholesterol esterase include n-octyl-β-D-thioglucoside, polyethylene glycol monododecyl ether, sodium cholate, dodecyl-β-maltoside, sucrose monolaurate,
Sodium deoxycholate, sodium taurodeoxycholate, N, N-bis (3-D-gluconamidopropyl) cholamide, N, N-bis (3-D-gluconamidopropyl) deoxycholamide and polyoxyethylene- pt-octylphenyl ether and the like can be optionally used.

[0015] Among the above-mentioned surfactants, a liquid which is a highly viscous liquid at room temperature and does not inhibit an enzymatic reaction is used as an adhesive for fixing a carrier. This has the advantage of facilitating the introduction of the sample liquid into the sensor. Such surfactants include:
Examples include polyethylene glycol monododecyl ether and polyoxyethylene-pt-octylphenyl ether.

When the electrode system of the biosensor is formed using an electrochemically stable metal such as platinum, the obtained oxidation current value does not include an error. However, since such a metal is expensive, in a disposable sensor, an electrode system is formed by forming a silver electrode using a silver paste or the like and then coating the silver electrode with a carbon paste. However,
When a surfactant is contained in the sample liquid, the sample liquid infiltrates between the carbon particles by the action of the surfactant. As a result, the activity of the carbon electrode may decrease. Also,
The sample liquid comes into contact with the silver electrode. For this reason, when a voltage is applied to the measurement electrode in this state, the silver electrode causes an oxidation reaction to generate a current, which may cause a positive error in the measured current value. In order to suppress such a phenomenon, there is a method of coating the electrode system surface with a hydrophilic polymer. The hydrophilic polymer forms a viscous layer even when the sample liquid is introduced, and suppresses the contact of the sample liquid with the electrode. Such hydrophilic polymers include carboxymethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, ethyl cellulose,
Examples include hydroxypropylcellulose, gelatin, polyacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts, polyacrylamide, methacrylate resins and poly2-hydroxyethyl methacrylate.

A solution in which an amphipathic substance such as lecithin is dissolved in an organic solvent is dropped to cover the above-mentioned hydrophilic polymer layer and dried to form an amphipathic substance layer. May be performed. Such amphiphilic substances include phospholipids such as lecithin.

When the electrode system of the biosensor is formed of platinum or the like, the substrate concentration can be measured using dissolved oxygen in the sample solution. However, when the electrode system is formed of silver and carbon, it is very difficult to measure the oxidation current value of hydrogen peroxide generated from dissolved oxygen. In addition, since the amount of dissolved oxygen is small, accurate values cannot be obtained when the substrate concentration is high. Therefore, when the electrode system of the biosensor is formed of silver and carbon, an electron mediator is contained in the reagent carried on the carrier. As such an electron mediator, a water-soluble compound capable of mediating electron transfer between an enzyme and an electrode, such as ferricyanide ion, p-benzoquinone, phenazine methosulfate and ferrocene, can be arbitrarily used.

The above-mentioned various reagents can suppress the denaturation of the reagent by being separately supported, rather than being supported on the carrier in a mixed state. In particular, when the electron mediator is carried on a carrier different from the carrier carrying the enzymes, the effect of suppressing the denaturation of the enzyme is remarkable. In addition, when an enzyme having a cholesterol ester hydrolytic ability is included in the reagent, a responsiveness of the sensor is improved by supporting a surfactant having a function of improving the activity of the enzyme on a carrier supporting the enzyme. . In addition, all of the above reagents may not be supported on the carrier, and a layer containing a part of the reagent may be formed at a position different from a certain position of the carrier and included in the reaction system. As a method of measuring the oxidation current, there are a two-electrode system including only a measurement electrode and a counter electrode, and a three-electrode method including a reference electrode.
More accurate measurement is possible with the three-electrode method.

[0020]

The present invention will be described in detail below with reference to specific examples. FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, from which a reaction layer is removed. 1
Denotes an insulating substrate made of polyethylene terephthalate. A silver paste is printed on the substrate 1 by screen printing to form the leads 2 and 3 and an electrode base. Then, an electrode system including the measuring electrode 4 and the counter electrode 5 is formed by printing a conductive carbon paste containing a resin binder on the substrate 1, and the insulating layer 6 is formed by printing the insulating paste. Each is formed. Measurement electrode 4 is on lead 2 and counter electrode 5 is on lead 3.
Connected to each other. The insulating layer 6 keeps the areas of the exposed portions of the measurement electrode 4 and the counter electrode 5 constant and partially covers the leads. The insulating substrate 1 on which the electrode system is formed in this manner, the cover 9 having the air holes 11, the spacer 8 and the carrier 13 carrying the reagent are placed in a positional relationship as shown by a dashed line in FIG. Adhering to produce a biosensor. In the biosensor having such a configuration, the slit 12 of the spacer 8 is provided between the substrate 1 and the cover 9.
A space constituting the sample liquid supply passage is formed in the portion, and the carrier 13 is disposed in this space. Then, the sample liquid is easily introduced into the inside of the sensor by a simple operation of only bringing the sample liquid into contact with the opening 10 serving as the sample liquid supply port.

FIG. 2 is a perspective view of a cover member in which a spacer 8 and a cover 9 are overlapped, and is arranged upside down from FIG. By combining the cover member and the substrate, a space that forms a sample liquid supply path is formed. Reference numeral 16 denotes a cover side of a surface exposed to a space constituting the sample liquid supply path.

FIG. 3 is a longitudinal sectional view of a biosensor according to one embodiment of the present invention. 1, an electrode system is formed on the insulating substrate 1, a hydrophilic polymer layer 7 is formed on the electrode system, and a lecithin layer 17 is formed so as to cover the layer 7. I have. The carrier 13 carrying the reagent is fitted and arranged in a space constituting the sample supply path. FIG. 4 is a longitudinal sectional view of a biosensor according to another embodiment of the present invention. 1, an electrode system is formed on the insulating substrate 1, and a hydrophilic polymer layer 7 and a lecithin layer 17 are formed on the electrode system. The carrier 13 supporting the reagent is fixed to the cover-side surface 16 of the sample liquid supply path with an adhesive 14.

FIG. 5 is an exploded perspective view of a biosensor according to another embodiment of the present invention, from which a reaction layer is removed. An insulating substrate 1 having an electrode system formed in the same manner as in FIG.
The biosensor is manufactured by adhering the cover 9, the spacer 8, and the carrier pieces 21 and 22 each carrying a different reagent to each other in a positional relationship shown by a dashed line in FIG.

Example 1 In this example, a biosensor having the structure shown in FIG. 3 was manufactured as follows. First, FIG.
A 0.5 wt% aqueous solution of a sodium salt of carboxymethyl cellulose (hereinafter abbreviated as CMC), which is a hydrophilic polymer, is dropped on the electrode system on the substrate 1 of above, and the mixture is heated in a 50 ° C. hot air drier for 10 minutes. After drying, the CMC layer 7 was formed. Then, the lecithin was added to the CMC layer 7 so as to cover the CMC layer 7.
3 μl of a 5% toluene solution was dropped and dried to form a lecithin layer 17. Next, an acrylic resin cover member as shown in FIG. 2 was prepared. Air hole 1 from opening 10
The length to the end of 1 was 4.5 mm, the width of the slit 12 was 2.0 mm, and the depth of the slit was 0.3 mm. Then, a felt made of glass fiber having a thickness of 0.2 mm (hereinafter, referred to as a glass filter) was cut into a size of 2 × 4.5 mm. Then, this glass filter was fitted into the slit 12 of the cover member and fixed.

This glass filter is provided with an interface having an action of activating cholesterol oxidase (hereinafter abbreviated as ChOD), cholesterol esterase (hereinafter abbreviated as ChE), potassium ferricyanide as an electron mediator, and cholesterol esterase. Activator polyoxyethylene-pt-octyl phenyl ether (hereinafter Triton X-100)
That. ) Was dissolved in water, and 5 μl of the mixed solution was added dropwise.
It was dried for 15 minutes in a warm air dryer at 50 ° C. to form a carrier 13 supporting the reagent. Then, the cover member and the substrate 1 were bonded in a positional relationship as shown by a dashed line in FIG. 1 to produce a biosensor.

The thus prepared biosensor was supplied with 3 μl of the sample solution from the opening 10 of the sample supply path. As the sample solution, a solution obtained by diluting a commercially available standard serum with physiological saline and changing the concentration of cholesterol contained therein in various ways was used. After 3 minutes, a pulse voltage of +0.5 V was applied to the measurement electrode in the anode direction based on the counter electrode, and a current value flowing between the measurement electrode and the counter electrode was measured after 5 seconds. As a result, the current value also increased with an increase in the total cholesterol concentration in serum, and excellent linearity was exhibited between the two.

Example 2 In this example, a biosensor having the configuration shown in FIG. 4 was manufactured as follows. In the same manner as in Example 1, the CMC was placed on the electrode system on the substrate 1 in FIG.
Layer 7 and lecithin layer 17 were sequentially formed. Next, the same glass filter as in Example 1 was cut into a size of 2 × 4.5 mm. Then, the surface 16 on the cover side of the sample liquid supply path
Then, a woodworking adhesive was applied as the adhesive 14, and then a glass filter was adhered and fixed. 5 μl of a mixed aqueous solution in which the same reagent as in Example 1 was dissolved in this glass filter
And dried in a hot air drier at 50 ° C. for 15 minutes,
A carrier 13 supporting the reagent was formed. And Example 1
A biosensor was prepared in the same manner as described above, and the current response to the same sample solution was measured. As a result, as shown in FIG. 10, favorable response values depending on the total cholesterol concentration in the serum were obtained.

Example 3 In this example, a biosensor having the structure shown in FIG. 4 was manufactured as follows. In the same manner as in the first embodiment, the CMC layer 7 is formed on the electrode system on the substrate 1 in FIG.
And a lecithin layer 17 were sequentially formed. Next, Trit, which is a surfactant, is placed on the surface 16 on the cover side of the sample liquid supply path.
An ethanol solution of onX-100 was added dropwise. And
By evaporating the ethanol, a paste-like Tri serving as an adhesive is formed on the surface 16 on the cover side of the sample liquid supply path.
A tonX-100 layer 14 was formed. And 2 × 4.
A felt made of cellulose fibers cut to 5 mm (hereinafter, referred to as a cellulose filter) was adhered and fixed on the layer 14. Triton X-100 is at room temperature (about 2
(5 ° C.), it was liquid, but did not infiltrate into the inside of the cellulose filter due to its high viscosity.

5 μl of a mixed aqueous solution in which the same reagent as in Example 1 was dissolved was added dropwise to this cellulose filter,
After drying for 15 minutes in a warm air dryer, a carrier 13 supporting the reagent was formed. Then, a biosensor was prepared in the same manner as in Example 1, and the current response value for the same sample solution was measured. As a result, a favorable response value depending on the total cholesterol concentration in the serum was obtained. In addition, since the carrier was adhered by the surfactant, the introduction of the sample liquid was very easy.

<< Embodiment 4 >> As shown in FIG.
The CMC layer 7 and the lecithin layer 17 were sequentially formed on the electrode system on the substrate 1 of FIG. Next, the same thickness as in the first embodiment is applied.
Two 2 mm glass filters were prepared. One sheet contains 0.6 of potassium ferricyanide, an electron mediator.
A mol / l aqueous solution was added dropwise to uniformly permeate. Then, it was dried in a hot air dryer at 50 ° C. for 15 minutes to carry 0.33 μmol of potassium ferricyanide per square millimeter. This glass filter is 1 × 4.5
The carrier piece 21 containing potassium ferricyanide was prepared by cutting into a size of mm. Next, Ch to the remaining one
OD 400units / ml, ChE 4000un
it / ml, and 6 wt% of Triton X-100
% Of the aqueous solution was added dropwise to uniformly permeate. Then, 5
Dry in a hot air drier at 0 ° C. for 15 minutes, 0.22 units ChOD, 2.2 units ChE and 0.2% Triton X-100 per square millimeter.
3 mg was carried. And this glass filter is 1 ×
The carrier piece 22 containing ChOD, ChE and Triton X-100 was cut to a size of 4.5 mm.

The carrier pieces 21 and 22 were used in Example 1
As shown in FIG. 6, it was arranged and fitted in the slit 12 of the cover member having the same shape as that of FIG. The cover member and the substrate 1 were adhered in a positional relationship as shown by a dashed line in FIG. 5 to produce a biosensor. Then, a current response value to the sample liquid similar to that in Example 1 was measured. As a result, a favorable response value depending on the total cholesterol concentration in the serum was obtained. Next, 5 biosensors produced in the same manner were used.
Stored at 0 ° C. for 1 week. Then, the response current value was measured in the same manner. As a result, a favorable response value depending on the total cholesterol concentration in the serum was obtained. The response value (blank value) to a sample containing no substrate (sample containing only physiological saline) was close to the value of the sensor immediately after the preparation.

<< Embodiment 5 >> As shown in FIG.
The CMC layer 7 and the lecithin layer 17 were sequentially formed on the electrode system on the substrate 1 of FIG. Next, the same thickness as in the first embodiment is applied.
Three 2 mm glass filters were prepared. The first glass filter carried potassium ferricyanide in the same manner as in Example 1. However, the concentration of the dropped aqueous solution of potassium ferricyanide was 0.9 mol / l, and the amount of potassium ferricyanide carried per square millimeter of the glass filter was 0.5 μmol. The second glass filter has a ChOD of 600 units / ml.
The aqueous solution was dropped and dried to carry 0.33 units of ChOD per square millimeter of the glass filter. ChE is 6000u for the third glass filter.
A mixed aqueous solution containing 9 wt% of nits / ml and Triton X-100 was dropped and dried, and 3.3 units of ChE per square millimeter of glass filter were added.
45 mg of Triton X-100 was loaded.

Each of these three glass filters was
It was cut into a size of 0.66 × 4.5 mm to prepare a carrier piece 23 containing potassium ferricyanide, a carrier piece 24 containing ChOD, and a carrier piece 25 containing ChE and Triton X-100. Then, after a woodworking adhesive was applied as an adhesive to the surface 16 of the cover member having the same shape as in Example 1, these carrier pieces were arranged and fixed as shown in FIG. Then, a biosensor was produced in the same manner as in Example 4, and the current response value of the sensor was measured immediately after the production and after storage at 50 ° C. for one week. As a result, a good response value depending on the total cholesterol concentration in the serum was obtained in each of the sensors immediately after the preparation and after the storage. The blank value of the sensor after storage was almost the same as the blank value of the sensor immediately after production. By carrying the electron mediator, ChOD, and the mixture of ChE and surfactant on separate carriers in this manner, better storage characteristics can be obtained.

<< Embodiment 6 >> As in Embodiment 1, FIG.
The CMC layer 7 and the lecithin layer 17 were sequentially formed on the electrode system on the substrate 1 of FIG. Next, an ethanol solution of Triton X-100 as a surfactant was dropped on the surface 16 on the cover side of the sample liquid supply path. Then, by evaporating the ethanol, the surface 16 on the cover side of the sample liquid supply path is formed.
In addition, a paste-like Triton X-100 that functions as an adhesive
A layer was formed. In addition, the same thickness as in the first embodiment, 0.2 mm
2 glass filters were prepared. The first glass filter carried 0.33 μmol / cm 2 of ferricyanide per 1 mm 2 in the same manner as in Example 4. The ChOD of the second glass filter was set to 0.22 per square millimeter in the same manner as in Example 4.
units, ChE to 2.2 units and Trit
0.3 mg of onX-100 was loaded. These two glass filters were cut into three pieces each having a size of 0.33 × 4.5 mm, and a carrier piece 2 containing potassium ferricyanide was cut.
6, a carrier piece 27 containing ChOD, ChE and Triton X-100 was prepared. Then, the carrier pieces 26 and the carrier pieces 27 are alternately arranged as shown in FIG. 8, and the Triton X-10 of the slit 12 of the cover member is arranged.
Fixed on layer 0.

Then, a biosensor was produced in the same manner as in Example 4, and the current response value of the sensor was measured immediately after the production and after storage at 50 ° C. for one week. As a result, a good response value depending on the total cholesterol concentration in the serum was obtained in each of the sensors immediately after the preparation and after the storage. Thus, by alternately arranging a plurality of carrier parts carrying different reagents and arranging them, it is possible to suppress denaturation of the reagents during storage and to easily mix the respective reagent components during the permeation of the sample solution. Thus, the response of the sensor is improved.

[0036]

As described above, according to the present invention, the concentration of a specific substance can be measured quickly and accurately.

[Brief description of the drawings]

FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, from which a reaction layer is removed.

FIG. 2 is a perspective view of a cover member in which a cover and a spacer of the biosensor are overlapped, and the cover member is arranged upside down from FIG.

FIG. 3 is a longitudinal sectional view of a main part of the biosensor.

FIG. 4 is a longitudinal sectional view of a main part of a biosensor according to another embodiment of the present invention.

FIG. 5 is an exploded perspective view of a biosensor according to another embodiment of the present invention, from which a reaction layer is removed.

FIG. 6 is a perspective view of a cover member of the biosensor in which a carrier piece is disposed in a sample liquid supply path.

FIG. 7 is a perspective view of a cover member of a biosensor according to another embodiment of the present invention, in which a carrier piece is disposed in a sample liquid supply path.

FIG. 8 is a perspective view of a cover member of a biosensor according to another embodiment of the present invention, in which a carrier piece is disposed in a sample liquid supply path.

FIG. 9 is a perspective view showing a configuration example of a carrier piece of the biosensor of the present invention.

FIG. 10 is a response characteristic diagram of the biosensor according to one embodiment of the present invention.

[Explanation of symbols]

Reference Signs List 1 Insulating substrate 2, 3 Lead 4 Measurement electrode 5 Counter electrode 6 Insulating layer 7 CMC layer 8 Spacer 9 Cover 10 Opening of sample liquid supply path 11 Air hole 12 Slit 13 Carrier 14 Adhesive 16 Configure sample liquid supply path Surface on the cover side of the surface exposed to the space 17 Lecithin layer 21, 23, 26 Carrier piece 22, 27 carrying potassium ferricyanide Carrier piece carrying ChOD, ChE and surfactant 24 Carrier carrying ChOD Piece 25 Carrier piece carrying ChE and surfactant 28, 29, 30, 31, 32, 33 Carrier piece

Claims (6)

[Claims] 1. An insulating substrate, an electrode system provided on the substrate and having at least a measurement electrode and a counter electrode, and a sample liquid supply which is combined with the substrate and supplies a sample liquid to the electrode system between the substrate and the substrate. A biosensor comprising: a cover member forming a passage; and a carrier made of a fiber carrying a reagent containing at least an oxidoreductase, wherein the carrier is arranged in the sample liquid supply passage. 2. An insulative substrate, an electrode system having at least a measurement electrode and a counter electrode provided on the substrate, and a carrier made of a fiber carrying a reagent containing at least an oxidoreductase, wherein the carrier is adhered. A biosensor, which is fixed in the vicinity of the electrode system by an agent. 3. The biosensor according to claim 1, wherein the carrier is composed of at least two carrier pieces, and each carrier piece carries a different reagent. 4. The biosensor according to claim 2, wherein the reagent includes at least cholesterol oxidase, cholesterol esterase, and an electron mediator, and the adhesive is a surfactant. 5. The biosensor according to claim 3, wherein the reagent contains at least cholesterol oxidase, cholesterol esterase, and an electron mediator, and the oxidoreductase and the electron mediator are supported on separate carrier pieces. 6. The method according to claim 3, wherein the reagent contains at least cholesterol oxidase, cholesterol esterase, a surfactant and an electron mediator, and the carrier piece containing cholesterol esterase contains a surfactant.
The biosensor according to any one of claims 1 to 5,