JPH09304329A - Biosensor and substrate determination method using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly and highly precisely determine a substrate concentration without reducing detection sensitibity by using ferricynium ions derived from ferrocene electrolyte as an electron receptor contained in a reaction layer. SOLUTION: A conductive material and an insulating paste are printed on an insulating board 1, a working electrode 4 and an insulating layer 6 are formed respectively, and a counter electrode 5 is formed around the outer circumference of the working electrode 4, so as to be formed into an electrode system 13. Aqueous solution containing an oxidoreductase and ferrocene electrolyte is dripped and dried on a hydrophilic polymer layer which is formed by dripping and drying aqueous solution containing a hydrophilic high polymer, so that a reaction layer 7 containing the oxidoreductase and the electronic receptor is formed. This electronic receptor is ferricynium ions derived from the ferrocene electrolyte and preferably the concentration of the ferrocene electrolyte is 1-100mM in a sample liquid, when the reaction layer 7 is dissolved in the sample liquid. When the density is 1mM or less, measurable substrate concentration is narrow and when it is 100mM or higher, the reaction layer 7 is broken, so that the response current values are varied and the reliability is reduced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biosensor capable of quantifying a substrate (specific compound) contained in a sample such as blood, urine, and fruit juice with high accuracy, speedily and easily, and a substrate using the same. More specifically,
A biosensor capable of electrochemically quantifying a substrate concentration in a sample by reacting a substrate such as glucose and cholesterol contained in the sample with an oxidoreductase capable of reacting specifically with the biosensor and the biosensor. The method for quantifying the amount of the substrate that was used.

[0002]

2. Description of the Related Art In recent years, biosensors have been proposed which can easily quantify a specific compound (substrate) in a biological sample and food without diluting or stirring the sample.

Japanese Patent Laid-Open No. 3-202764 discloses that an electrode system is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer, a redox enzyme, and an electron acceptor are placed on the electrode system. Disclosed is a biosensor that includes a reaction layer that contains it, and a space that forms a sample supply path by combining a cover and a spacer. This biosensor can quantify the substrate concentration in the sample as follows: First, by dropping the sample solution on the space of the biosensor, the sample solution is supplied to the reaction layer by capillary action, and the reaction is performed. The layers dissolve. Next, an enzymatic reaction proceeds between the substrate in the sample solution and the redox enzyme in the reaction layer, and the electron acceptor in the reaction layer is reduced. After the completion of the oxidation reaction, the reduced electron acceptor is electrochemically oxidized to determine the substrate concentration in the sample solution from the oxidation current value obtained at this time.

In the above biosensor, potassium ferricyanide is often used as an electron acceptor.
A biosensor using potassium ferricyanide has excellent stability and low production cost, and is suitable for mass production. However, in a biosensor using potassium ferricyanide as an electron acceptor, the secondary reaction rate between the ferricyanide ion and the oxidoreductase is unstable, or the quinone derivative or other metal complex with high production cost is unstable. Is smaller than that of a biosensor using an electron acceptor such as. Therefore, such a biosensor has a problem that the enzyme reaction takes a long time and the substrate concentration cannot be rapidly quantified.

Further, several biosensors using ferrocene or its derivative having a second-order reaction rate higher than that of potassium ferricyanide as an electron acceptor are known.

Japanese Unexamined Patent Publication No. 2-240555 discloses a glucose sensor having a working electrode, which has a photocurable resin film containing a ferrocene compound on an electrode surface and a glucose oxidase-containing photocurable resin film in that order. ing. JP-A-2-99
Japanese Patent No. 851 discloses a glucose sensor provided with a working electrode having a layer in which a ferrocene compound is vapor-deposited on an electrode surface and a layer in which glucose oxidase is immobilized on the vapor-deposited layer. In these biosensors, ferrocene (that is, bis (cyclopentadienyl) iron (II)), 1,1′-dimethylferrocene, vinylferrocene and the like are used as the ferrocene compound.

Japanese Unexamined Patent Publication (Kokai) No. 5-256812 discloses a glucose sensor having a discriminating layer carrying glucose oxidase and a ferrocene compound on a working electrode, and a temperature maintaining means for maintaining a temperature near the working electrode at a predetermined temperature. are doing. In this biosensor, as a ferrocene compound,
Ferrocene and its derivatives are used.

However, since all of the ferrocene compounds used in these glucose sensors exist as reductants, in using the sensor, first, in order to achieve electron transfer from the substrate to the working electrode by the enzymatic reaction, , It was necessary to convert the ferrocene compound to the oxidant on the electrode.

Japanese Unexamined Patent Publication (Kokai) No. 6-3316 discloses a glucose sensor in which a hydrophobic redox substance (eg, ferrocene) ionized in an aqueous solution and a hydrophilic enzyme (eg, glucose oxidase) are modified on the surface of a conductive electrode. (Modified electrode) is disclosed. This glucose sensor
Ferrocene is electrolyzed in a phosphate buffer to form a solution containing ferricinium ions, glucose oxidase is then added to this solution, and the resulting mixed solution is applied or electrodeposited on the surface of a conductive electrode. It is produced by.

However, the above-mentioned modified electrode has a problem that phosphate ions and ferricinium ions form an ionic complex and inactivate the electrode surface. Further, the production of this modified electrode requires a step of electrolyzing ferrocene, resulting in a problem that both production time and cost are increased.

[0011]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above problems, and its object is to achieve a substrate concentration without causing a decrease in detection sensitivity because an enzymatic reaction takes place in a short time. It is intended to provide a biosensor capable of rapidly and highly accurately quantifying and a method for quantifying a substrate using the biosensor.

[0012]

The biosensor of the present invention comprises an insulating substrate, an electrode system formed on the substrate and having a working electrode and a counter electrode, an oxidoreductase and an oxidoreductase disposed on the substrate. And a reaction layer containing an electron acceptor, wherein the electron acceptor is a ferricinium ion derived from a ferrocene electrolyte. Thereby, the above object is achieved.

In a preferred embodiment, the ferrocene electrolyte is ferrocene hexafluorophosphate and ferrocene 4
In a preferred embodiment selected from the group consisting of fluoroborates, the reaction layer further contains at least one selected from the group consisting of hydrophilic polymers and surfactants.

In a preferred embodiment, the oxidoreductase is glucose oxidase; glucose dehydrogenase; lactate oxidase; lactate dehydrogenase; urigase; cholesterol oxidase; cholesterol oxidase and cholesterol esterase combination; glucose oxidase and invertase combination; glucose. Oxidase, invertase,
And a mutarotase combination; and a fructose dehydrogenase and invertase combination.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The biosensor of the present invention has an insulating substrate, an electrode system formed on the substrate and having a working electrode and a counter electrode, and a reaction layer arranged on the substrate.

A synthetic resin plate such as polyethylene terephthalate may be used as the electrically insulating substrate.

An electrode system having a working electrode and a counter electrode can be provided on this substrate using known methods. For example, after forming the leads on the substrate, a working electrode and a counter electrode are provided so as to be connected to the leads and insulate each other. A known conductive material can be used as the material of the lead and the electrode. Examples of such conductive materials include carbon, silver, platinum, gold, and palladium.

The reaction layer contains at least one oxidoreductase and at least one electron acceptor.

The electron acceptor used in the present invention is a ferricinium ion derived from a ferrocene electrolyte. As used herein, the term "ferrocene electrolyte"
The term means a salt that ionizes to produce ferricinium ion ([(C ₅ H ₅ ) ₂ Fe] ⁺ ) when an aqueous solution is prepared together with a redox enzyme described below. The anion that constitutes the ferrocene electrolyte is not particularly limited. Preferred examples of ferrocene electrolytes include ferrocene hexafluorophosphate and ferrocene tetrafluoroborate, which are commercially available from Aldrich Inc.

The concentration of the ferrocene electrolyte is not particularly limited, but it is preferably 1 mM to 100 mM in the sample solution when the reaction layer is dissolved in the sample solution. If the concentration of the ferrocene electrolyte is less than 1 mM, the measurable substrate concentration range may become extremely narrow. When the concentration of the ferrocene electrolyte exceeds 100 mM, the manufacturing cost of the biosensor increases, the reaction layer is cracked, the response current value varies, and the reliability during storage may decrease.

In the present invention, the redox enzyme may be an enzyme used in a conventional biosensor such as a glucose sensor, a cholesterol sensor, a lactate sensor, a uric acid sensor, and a sucrose sensor. Examples of redox enzymes include glucose oxidase (GOD), glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, urigase, cholesterol oxidase (C
hOD), cholesterol esterase (ChE), invertase, mutarotase, and fructose dehydrogenase, and combinations thereof. When the biosensor of the present invention is a glucose sensor, GOD or glucose dehydrogenase can be used as the redox enzyme. When the biosensor of the present invention is a lactate sensor, lactate oxidase or lactate dehydrogenase can be used as the redox enzyme. When the biosensor of the present invention is a uric acid sensor, urigase may be used as the redox enzyme. When the biosensor of the present invention is a cholesterol sensor, ChOD or a combination of ChOD and ChE can be used as the redox enzyme. When the biosensor of the present invention is a sucrose sensor,
As the redox enzyme, a combination of GOD and invertase; a combination of GOD, invertase, and mutarotase; or a combination of fructose dehydrogenase and invertase can be used.

The content of the oxidoreductase used in the biosensor of the present invention is not particularly limited, and an appropriate amount can be selected by those skilled in the art according to need. For example, when GOD is used, its content is 0.1 unit to 5 per sensor.
Units are preferred. When using a combination of ChOD and ChE, the content of ChOD is 0.1 unit to 5 per sensor.
A unit is preferable, and the content of ChE is preferably 0.1 unit to 5 units per sensor. here,
As used herein, the term “1 unit” means 1 μm.
It refers to the amount of oxidoreductase to be quantified to oxidize mol substrate in 1 minute.

The reaction layer further preferably contains a hydrophilic polymer and / or a surfactant.

Examples of hydrophilic polymers include carboxymethyl cellulose (CMC), hydroxyethyl cellulose,
Cellulose derivatives such as hydroxypropylcellulose, methylcellulose, ethylcellulose, ethylhydroxycellulose, and carboxymethylethylcellulose; polyvinylpyrrolidone; polyvinyl alcohol; gelatin and its derivatives; acrylic acid and its salts; methacrylic acid and its salts; starch and its derivatives; And maleic anhydride and its salts. CMC is particularly preferable.

Examples of surfactants include soybean-derived purified lecithin, octylthioglucoside, sodium cholate, dodecyl-β-maltoside, sodium deoxycholate, sodium taurodeoxycholate, Triton-X.
(Registered trademark), Lubrol PX (registered trademark), DK-ester (registered trademark), BIGCHAP (registered trademark), and DeoxyCHAP
(Registered trademark). Particularly, purified lecithin and octylthioglucoside are preferable.

When the above-mentioned ferricinium ion is used as an electron acceptor, ferrocene is produced through a series of enzymatic reactions. Since ferrocene has low solubility in water, ferrocene molecules generated by water contained in the sample solution may be deposited on the reaction layer. Therefore, by adding a surfactant to the reaction layer, the solubility of the generated ferrocene in water is improved.

The above-mentioned hydrophilic polymer and surfactant may be used in combination, and only either the hydrophilic polymer or the surfactant may be contained in the reaction layer as a further component. .

In the present invention, a layer made of the above-mentioned purified lecithin may be further formed on the reaction layer. When the lecithin layer is formed on the reaction layer, the sample liquid can be easily supplied to the reaction layer.

A preferred embodiment of the method for manufacturing a biosensor of the present invention will be described with reference to FIGS. 1 and 2.

First, a conductive material such as silver paste is printed on the insulating substrate 1 by screen printing, and the leads 2 are formed.
And 3 are formed. Then, a conductive material containing a resin binder is printed on the insulating substrate 1 to form the working electrode 4. The working electrode 4 is in contact with the lead 2.

Next, an insulating paste is printed on the insulating substrate 1 to form an insulating layer 6. The insulating layer 6 covers the outer peripheral portion of the working electrode 4, whereby the area of the exposed portion of the working electrode 4 is kept constant. As shown in FIG. 1, insulating layer 6 also covers a portion of leads 2 and 3. Further, a ring-shaped counter electrode 5 is formed on the outer peripheral portion of the working electrode 4 by using a conductive material containing a resin binder. Counter electrode 5
Is in contact with the lead 3. In this way, the electrode system 13 including the working electrode 4 and the counter electrode 5 is formed on the insulating substrate 1.

Alternatively, in the biosensor of the present invention, a three-electrode system including a reference electrode (not shown) in addition to the working electrode 4 and the counter electrode 5 may be formed on the insulating substrate 1. With this three-electrode system, a stable response current is obtained and the accuracy is further stabilized.

The reaction layer 7 can be arranged on the insulating substrate 1 as follows.

The aqueous solution containing the hydrophilic polymer is the electrode system 1
A hydrophilic polymer layer is formed by dropping on 3 and drying. On the other hand, a predetermined amount of oxidoreductase and ferrocene electrolyte are dissolved in water. Preferably, a few drops of surfactant are added to this aqueous solution. Then, an aqueous solution containing the obtained oxidoreductase and ferrocene electrolyte is added dropwise to the hydrophilic polymer layer. As a result, the hydrophilic polymer dissolves in this aqueous solution. Then, the dissolved hydrophilic polymer layer is dried to form the reaction layer 7 in which the redox enzyme and the electron acceptor are incorporated in the hydrophilic polymer layer. However, since no further operation such as stirring is performed in this incorporation, only the hydrophilic polymer exists at the interface between the reaction layer 7 and the electrode system 13. That is, since the redox enzyme and the electron acceptor are not in contact with the surface of the electrode system 13, it is possible to eliminate the case where the protein is adsorbed on the surface of the electrode system 13 to inactivate the surface of the electrode system 13. .

When the hydrophilic polymer layer is not used, an aqueous solution containing a redox enzyme and a ferrocene electrolyte is dropped directly onto the electrode system 13 and dried.

For repeated use of the biosensor of the invention, the redox enzyme and the ferrocene electrolyte can be immobilized on the hydrophilic polymer layer, for example by cross-linking with glutaraldehyde, or nitrocellulose or an ion exchange membrane. Can be immobilized on the hydrophilic polymer layer together with a polymeric material such as.

The reaction layer 7 formed as described above is
It covers the entire electrode system 13 as shown in FIG.

Next, if necessary, a solution of a predetermined amount of purified lecithin in an organic solvent such as toluene is spread on the reaction layer 7 and dried to form the lecithin layer 8. Finally, a spacer 10 having a sample supply path 12 and a cover 9 having a hole 11 are sequentially arranged on the reaction layer 7 (on which the lecithin layer 8 is formed, if any) by a known method. In this way, the biosensor of the present invention is manufactured.

The substrate concentration contained in the sample solution is quantified using the biosensor of the present invention, for example, as follows.

First, the sample solution containing the substrate is added to the reaction layer 7 via the sample supply path 12 or directly to the reaction layer 7. The reaction layer 7 is dissolved by this addition. After standing for a predetermined time, a pulse voltage (for example, +0.5 V) of a predetermined magnitude is applied to the working electrode 4 with respect to the counter electrode 5 in the anode direction, and the obtained response current value is measured by a known means. . Then, the obtained response current value is converted into the substrate concentration by using a calibration curve of the substrate concentration and the response current value prepared in advance using a substrate having a known concentration.

Examples of substrates that can be quantified as described above include glucose, cholesterol, lactic acid, uric acid, and sucrose.

The mechanism by which the response current value is obtained using the biosensor of the present invention will be described below.

For example, when the sample solution contains glucose as a substrate and GOD is used in the reaction layer 7, the reaction solution 7 is dissolved by the sample solution, and glucose in the sample solution is oxidized by GOD, It produces gluconolactone. At this time, the electrons generated from the oxidation reaction of glucose reduce ferricinium ions existing in the reaction layer 7 to ferrocene. Then, by applying the pulse voltage, an oxidation current of the generated ferrocene is obtained, and the magnitude of this current is measured as a response current value.
The obtained response current value is proportional to the glucose concentration existing in the sample solution.

The sample solution contains cholesterol ester and cholesterol as substrates, and in the reaction layer 7
When E and ChOD are used, the reaction solution 7 is dissolved by the sample solution and the cholesterol ester in the sample solution becomes ChE.
Is converted to cholesterol by. Then, all cholesterol in the sample solution is oxidized by ChOD,
Generates cholestenone. At this time, the electrons generated from the oxidation reaction of cholesterol reduce ferricinium ions existing in the reaction layer 7 to ferrocene. Then, by applying the pulse voltage, an oxidation current of the generated ferrocene is obtained, and the magnitude of this current is measured as a response current value. The obtained response current value is proportional to the total concentration of cholesterol ester and cholesterol present in the sample solution.

In this way, the substrate concentration contained in the sample solution can be quantified using the biosensor of the present invention.
Furthermore, since the ferricinium ion used as an electron acceptor has a higher secondary reaction rate than the ferricyanide ion conventionally used, the biosensor of the present invention can rapidly and highly increase the substrate concentration in a sample. It can be quantified with accuracy.

The biosensor of the present invention is useful for quantifying the concentration of a substrate contained in biological samples such as whole blood, plasma, serum, and urine, materials in the food industry, food products such as fruit juice, and the like. is there.

[0047]

EXAMPLES The present invention will be described below based on examples, but the present invention is not limited to these examples. In the drawings used to describe each embodiment,
The common elements are given the same numbers, and the description thereof is partially omitted as necessary.

Example 1 As an example of the biosensor of the present invention, a glucose sensor was produced as follows.

As shown in FIG. 1, a silver paste was printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3. Then, a conductive carbon paste containing a resin binder was printed on the insulating substrate 1 to form the working electrode 4.
The working electrode 4 was in contact with the lead 2.

Next, an insulating paste was printed on the insulating substrate 1 to form an insulating layer 6. The insulating layer 6 covers the outer peripheral portion of the working electrode 4, whereby the exposed area of the working electrode 4 is kept constant. Further, a conductive carbon paste containing a resin binder was printed on the insulating substrate 1 so as to come into contact with the leads 3 to form a ring-shaped counter electrode 5.

Then, GOD and ferrocene hexafluorophosphate (Aldri) were placed on the electrode system 13 (working electrode 4 and counter electrode 5).
ch) was added and dried to form a reaction layer 7. A lecithin toluene solution was dropped onto the reaction layer 7, spread over the entire reaction layer 7, and dried to form a lecithin layer 8. A spacer 10 is formed on the lecithin layer 8.
Then, the cover 9 was sequentially adhered to manufacture a glucose sensor.

3 μl of a 30 mg / dl glucose aqueous solution as a sample solution was added to the glucose sensor manufactured as described above through the sample supply path 12 in the spacer 10. The sample solution reached the height of the hole 11 provided in the cover 9, and the reaction layer 7 was dissolved. Then after addition
At 60 seconds, a pulse voltage of +0.5 V was applied to the working electrode 4 in the anode direction based on the counter electrode 5, and the response current value after 5 seconds was measured.

Further, regarding the response current values to the 45 mg / dl and 90 mg / dl glucose aqueous solutions, the response current values were measured in the same manner as above using a new glucose sensor for each measurement. The obtained response current value was proportional to the concentration of the glucose aqueous solution.

Example 2 Instead of ferrocene hexafluorophosphate, ferrocene tetrafluoroborate (Aldrich
A glucose sensor was produced in the same manner as in Example 1 except that the (produced product) was used. Using this glucose sensor, the response current values obtained from an aqueous glucose solution having the same concentration as in Example 1 were measured. The obtained response current value was proportional to the concentration of the glucose aqueous solution.

Example 3 The electrode system 13 was formed on the insulating substrate 1 in the same manner as in Example 1.

Then, a 0.5% by weight aqueous solution of CMC was dropped onto the electrode system 13 (working electrode 4 and counter electrode 5) and dried to form a CMC layer. An aqueous solution containing GOD and ferrocene hexafluorophosphate was added to the CMC layer,
The reaction layer 7 was formed by drying. Further, a toluene solution of lecithin was dropped on the reaction layer 7, spread on the entire reaction layer 7, and dried to form a lecithin layer 8. A spacer 10 and a cover 9 were sequentially adhered to the lecithin layer 8 to produce a glucose sensor.

3 μl of a 30 mg / dl glucose aqueous solution as a sample solution was added to the glucose sensor manufactured as described above through the sample supply path 12 in the spacer 10. The sample solution reached the height of the hole 11 provided in the cover 9, and the reaction layer 7 was dissolved. Then after addition
At 60 seconds, a pulse voltage of +0.5 V was applied to the working electrode 4 in the anode direction based on the counter electrode 5, and the response current value after 5 seconds was measured.

Further, regarding the response current values for the 45 mg / dl and 90 mg / dl glucose aqueous solutions, the response current values were measured in the same manner as above using a new glucose sensor for each measurement. The response current value obtained above was proportional to the concentration of the glucose aqueous solution.

Example 4 A glucose sensor was produced in the same manner as in Example 3 except that ferrocene tetrafluoroborate was used instead of ferrocene hexafluorophosphate. Using this glucose sensor, the response current values obtained from an aqueous glucose solution having the same concentration as in Example 3 were measured. The obtained response current value was proportional to the concentration of the glucose aqueous solution.

<Example 5> To the aqueous solution containing GOD and ferrocene hexafluorophosphate described in Example 3, purified soybean-derived lecithin (manufactured by SIGMA) was added as a surfactant, and this was added to CMC. A glucose sensor was produced in the same manner as in Example 3 except that the glucose sensor was dropped on the layer. Using this glucose sensor, the response current values obtained from an aqueous glucose solution having the same concentration as in Example 3 were measured. The obtained response current value was proportional to the concentration of the glucose aqueous solution.

Example 6 Ferrocene tetrafluoroborate was used in place of ferrocene hexafluorophosphate, and GOD was used.
Soybean-derived purified lecithin (SIGMA) as a surfactant in an aqueous solution containing ferrocene tetrafluoroborate.
Manufactured) was added dropwise, and this was added on the CMC layer,
A glucose sensor was produced in the same manner as in Example 3. Using this glucose sensor, the response current values obtained from an aqueous glucose solution having the same concentration as in Example 3 were measured. The obtained response current value was proportional to the concentration of the glucose aqueous solution.

Example 7 The electrode system 13 was formed on the insulating substrate 1 in the same manner as in Example 1.

Then, an aqueous solution containing 0.5% by weight of CMC was dropped onto the electrode system 13 (working electrode 4 and counter electrode 5) and dried to form a CMC layer. On the other hand, in an aqueous solution containing ChE, ChOD, and ferrocene hexafluorophosphate,
Octylthioglucoside was added dropwise as a surfactant.
The obtained aqueous solution was added onto the CMC layer and dried to form a reaction layer 7. Further, a toluene solution of lecithin was dropped on the reaction layer 7, spread on the entire reaction layer 7, and dried to form a lecithin layer 8. A spacer 10 and a cover 9 were sequentially adhered to the lecithin layer 8 to produce a cholesterol sensor.

The cholesterol sensor prepared as described above was added to the sample solution containing 50 mg / dl of cholesterol and 150
Standard solution 3 containing mg / dl cholesterol ester
μl was added via the sample feed channel 12 in the spacer 10. The sample solution reached the height of the hole 11 provided in the cover 9, and the reaction layer 7 was dissolved. Then, 180 seconds after the addition, a pulse voltage of +0.5 V was applied to the working electrode 4 in the anode direction based on the counter electrode 5 of the electrode system 13, and 5 seconds later, the cholesterol ester and cholesterol present in the sample solution The response current value corresponding to the total concentration of was measured.

Furthermore, the response current values for a standard solution containing 300 mg / dl cholesterol ester and 100 mg / dl cholesterol and a standard solution containing 450 mg / dl cholesterol ester and 150 mg / dl cholesterol were also measured for each measurement. A new cholesterol sensor was used to measure the response current value in the same manner as above. The response current value obtained above was proportional to the total concentration of cholesterol ester and cholesterol present in the sample solution.

<Example 8> A cholesterol sensor was prepared in the same manner as in Example 7 except that the reaction layer 7 did not contain ChE, and was prepared from the same standard solution containing cholesterol ester and cholesterol as in Example 7. The obtained response current value was measured. The obtained response current value was proportional to the concentration of cholesterol pre-existing in the standard solution.

Example 9 A cholesterol sensor was produced in the same manner as in Example 7 except that ferrocene tetrafluoroborate was used instead of ferrocene hexafluorophosphate. Using this cholesterol sensor, the response current values obtained from standard solutions containing cholesterol ester and cholesterol at the same concentrations as in Example 7 were measured. The obtained response current value was proportional to the total concentration of cholesterol ester and cholesterol present in the sample solution.

Example 10 Example 7 was repeated except that the reaction layer 7 did not contain ChE and ferrocene tetrafluoroborate was used instead of ferrocene hexafluorophosphate.
A cholesterol sensor was prepared in the same manner as in Example 7, and
The response current values obtained from the same standard solution containing cholesterol ester and cholesterol as in Example 1 were measured. The obtained response current value was proportional to the concentration of cholesterol pre-existing in the standard solution.

[0069]

According to the present invention, the enzyme reaction takes place in a short time, and the substrate concentration can be quantified rapidly and with high accuracy without lowering the detection sensitivity. The biosensor of the present invention can also be manufactured at low cost and does not deactivate the electrode surface due to the formation of ionic complexes. The biosensor of the present invention is a substrate (eg, glucose, cholesterol, lactic acid, uric acid, and the like) contained in biological samples such as whole blood, plasma, serum, and urine, materials in the food industry, food products such as fruit juice, and the like. It is useful for quantifying the concentration of sucrose.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a biosensor with a reaction layer removed, showing an example of the present invention.

FIG. 2 is a schematic cross-sectional view of a biosensor in which a reaction layer is arranged on an insulating substrate, showing an example of the present invention.

[Explanation of symbols]

 1 Insulating Substrate 2, 3 Lead 4 Working Electrode 5 Counter Electrode 6 Insulating Layer 7 Reaction Layer 8 Lecithin Layer 9 Cover 10 Spacer 11 Hole 12 Sample Supply Channel 13 Electrode System

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Shiro Nankai 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Shigeki Kamiko, 1006 Kadoma, Kadoma City, Osaka Matsushita Electric Industrial Co., Ltd. (72) Inventor Junko Iwata 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (4)

[Claims] 1. An insulating substrate, an electrode system formed on the substrate and having a working electrode and a counter electrode, and a reaction layer disposed on the substrate and containing a redox enzyme and an electron acceptor. A biosensor, wherein the electron acceptor is a ferricinium ion derived from a ferrocene electrolyte. 2. The ferrocene electrolyte is ferrocene 6
The biosensor according to claim 1, which is selected from the group consisting of fluorophosphate and ferrocene tetrafluoroborate. 3. The biosensor according to claim 1, wherein the reaction layer further contains at least one selected from the group consisting of a hydrophilic polymer and a surfactant. 4. The redox enzyme is glucose oxidase; glucose dehydrogenase; lactate oxidase; lactate dehydrogenase; urigase; cholesterol oxidase; combination of cholesterol oxidase and cholesterol esterase; combination of glucose oxidase and invertase; glucose oxidase, invertase, And a mutarotase combination; and 1 selected from the group consisting of a fructose dehydrogenase and invertase combination
The biosensor according to claim 1, which is a seed.