JPH02245650A - Biosensor - Google Patents

Abstract

PURPOSE:To measure a density of a substrate in an organism sample while improving a measuring accuracy by forming an enzyme reaction layer comprising an oxidation/reduction enzyme, a hydrophilic high polymer and an electron acceptor on a surface of an electrode system. CONSTITUTION:For example, a glucose sensor comprises a print of conductive carbon paste on an insulating substrate 1 to form an electrode system comprising a counter electrode 2 and a measuring pole 3. Then, an insulating paste is printed to cover the electrode system partially to form an insulation layer 4 by an overheating processing. Then, the surfaces of electrochemically acting parts 2' and 3' work are coated with an aqueous solution of carboxymethyl cellulose as one kind of hydrophilic high polymer to cover the surfaces and after a drying, glucose oxidase as oxidation/reduction enzyme is dissolved into a phosphoric acid buffer liquid to coat and dry. Moreover, toluene mixed with a fine crystal of potassium ferricyanide as electron acceptor is dripped on the parts to obtain an enzyme reaction layer 5 by forming a potassium ferricyanide layer. A potassium periodate as interfering material removing part 6 is supported near the electrode 2 to make a sample supply section.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a biosensor that can quickly and easily quantify specific components in various minute amounts of biological samples without diluting the sample liquid.

BACKGROUND OF THE INVENTION In the past, a biosensor as shown in FIG. 5 was proposed as a method for easily quantifying a specific component in a biological sample such as blood without diluting or stirring the sample solution. In this biosensor, an electrode 2.3 made of carbon or the like is formed on an insulating substrate 1 by a method such as screen printing, and a hydrophilic polymer, an oxidoreductase, and an electron acceptor are placed on the electrode 2.3. An enzyme reaction layer 6 consisting of the following is formed. When the sample liquid is dropped onto the enzyme reaction layer 5, the oxidoreductase and the electron acceptor are dissolved in the sample liquid, and an enzymatic reaction proceeds with the substrate in the sample liquid to reduce the electron acceptor. After the reaction is completed, the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention In such a conventional configuration, when a reducing substance is contained in the sample liquid, the response varies due to the reaction with the electron acceptor or the influence of the electrode reaction during the reaction.

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention provides an electrode system consisting of at least m electrodes and a counter electrode on an insulating substrate, and a In a biosensor that electrochemically detects changes in substance concentration using the electrode system and measures the substrate concentration in a sample solution, the surface of the electrode system mainly consists of redox enzymes, hydrophilic polymers, and electron acceptors. In this method, an enzyme reaction layer is formed, and an interfering substance removing section is further added.

Effects According to the present invention, a disposable type biosensor including an electrode system can be constructed, and the substrate concentration can be measured extremely easily by adding a sample liquid to the sensor. Moreover, since reducing substances in the sample liquid are oxidized in the interfering substance removing section when adding the sample, there is no effect on the response, and a stable response can be obtained.

Example An embodiment of the present invention will be described below.

Example 1 A glucose sensor will be described as an example of a biosensor. FIGS. 1 and 2 show an example of a glucose sensor, and are a perspective view and a longitudinal sectional view of the biosensor. A conductive carbon paste is printed on an insulating substrate 1 made of polyethylene terephthalate by screen printing and dried by heating to form an electrode system consisting of a counter electrode 2 and a measurement electrode 3. Next, an insulating paste is printed and heat-treated in the same way as before, so as to partially cover the electrode system and leave the electrochemically active parts 2' and 3' of each electrode 2.3. An insulating layer 4 is formed. An aqueous solution of CMC (carboxymethylcellulose), which is a type of cellulose-based hydrophilic polymer, was applied to cover the surface of the portions 2' and 3', and dried at 45°C for 30 minutes. A solution of glucose oxidase (GOD) dissolved in a phosphate buffer with pEI 5.8 as an oxidoreductase was applied onto the obtained CMC layer, and then dried at room temperature. A mixture of toluene as an organic solvent and microcrystals of potassium ferricyanide, which is an electron normal substance, was added dropwise thereon, and the mixture was allowed to stand at room temperature to vaporize the toluene, thereby forming a potassium ferricyanide layer. In this way, the enzyme reaction layer 5
was formed. Further, an interfering substance removing section 6 is provided near the electrode 2.
Potassium periodate was supported as a sample supply section.

In the glucose sensor configured as described above, serum as a sample liquid was dropped into the supply part by 10 μm, and after 2 minutes, a pulse voltage of +0.8 V was applied to the measurement electrode 3 toward the anode with reference to the counter electrode 2, and after 5 seconds Measure current. When serum is added, ascorbic acid, which is a reducing substance in the serum, is oxidized by the potassium periodate supported as the interfering substance removing part 6, thereby preventing it from reacting with potassium ferricyanide in the enzyme reaction layer 6. Further, potassium ferricyanide is dissolved by the serum from which interfering substances have been removed, and when glucose in the serum is oxidized in the enzyme reaction layer 5, it is reduced to potassium ferrocyanide. Therefore, by applying the above-mentioned pulse voltage, an oxidation current based on the concentration of the generated potassium ferrocyanide can be obtained. This current value corresponds to the concentration of glucose, which is a substrate. When a standard solution of glucose was added dropwise and the response current was measured, it was 500 mg/d.
Good linearity was obtained up to a high concentration of 1.

Next, 10 mg/ml of ascorbic acid as a representative reducing substance was added to the glucose standard solution, and measurements were taken. As in this example, when potassium periodate was present, almost no effect of ascorbic acid was observed. In the absence of potassium periodate, glucose concentration 100 mg/
About 10% higher response was obtained in di. This is considered to be because ascorbic acid reacts with potassium ferricyanide to produce potassium ferrocyanide, resulting in an apparently positive error.

According to this example, by supporting potassium periodate as the interfering substance removing portion 8, it was possible to oxidize ascorbic acid in advance and remove its influence.

Example 2 After forming a CMC-GOD layer as shown in Example 1, lecithin (phosphatidylcholine) was added to toluene as a surfactant when forming a potassium ferricyanide layer.
was dissolved to prepare a 1 wt % solution, and a layer of potassium ferricyanide and lecithin was formed by mixing microcrystals of potassium ferricyanide with this solution. In this way, the enzyme reaction layer of this example was provided.

When the concentration of lecithin was 0.01 wt % or more, potassium ferricyanide was well dispersed in toluene, making it easy to drip, and a thin potassium ferricyanide-lecithin layer could be formed even with a trace amount of liquid of 3 μm. In the absence of lecithin, there were disadvantages in that the potassium ferricyanide layer was formed unevenly and peeled off when the substrate was bent, but by adding lecithin, a uniform potassium ferricyanide layer that was difficult to peel off could be easily formed. As the lecithin concentration increases, the potassium ferricyanide layer becomes difficult to peel off, but the dissolution rate of potassium ferricyanide also decreases, so 0.01-3 wt% is considered to be appropriate.

When a glucose standard solution was dropped onto the above sensor and the response was measured in the same manner as in Example 1, it was found that the glucose concentration was 500.
Linearity was obtained down to mg/dl. Furthermore, when blood was dropped, it quickly spread through the lecithin layer and a reaction began, so a response with good reproducibility was obtained even with a small sample amount of Bμl.

When the sample amount was small, the amount of potassium periodate supported as an interfering substance removing part was also small, and the effect was seen.

When polyethylene glycol alkyl phenyl ether (trade name Nitriton A potassium ferricyanide layer was formed. In addition to the examples mentioned above, surfactants that can disperse electron acceptors in organic solvents and do not affect enzyme activity, such as oleic acid, polyoxyethylene glycerin fatty acid ester, and cyclodextrin, can also be used. If so, there are no particular restrictions.

In addition to CMC, gelatin, methylcellulose, and the like can be used as hydrophilic polymers, and starch-based, carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based polymers are preferred.

Since these polymers can be easily made into an aqueous solution, a thin film of a required thickness can be formed on the electrode by applying an aqueous solution of an appropriate concentration and drying.

The organic solvent in which the electron acceptor is mixed may be any organic solvent that has little effect on GoD activity and printed electrodes, such as toluene and petroleum ether.

Screen printing as a method for forming the electrode system can produce disposable biosensors with uniform characteristics at low cost, especially when using carbon, which is a cheap and stable electrode material. This is a convenient method for forming electrodes.

In addition to potassium periodate, active manganese oxide, iron chloride, P-benzoquinone, and the like were effective as substances that oxidize reducing substances in the sample. Although the above-mentioned substances had oxidizing power, the reaction was mild and had little effect on the enzyme reaction layer.

Example 3 When blood or serum is used as a sample liquid, it is known that the main reducing substance is ascorbic acid. Therefore, ascorbate oxidase and catalase were loaded on the sample supply part of a sensor that measures blood sugar.

When serum is added dropwise, ascorbic acid in the sample is oxidized by ascorbic acid oxidase, hydrogen peroxide is generated, and this is decomposed by catalase.

Therefore, it has become possible to measure blood sugar without the influence of ascorbic acid. Because it uses enzymes, it is mild,
Ascorbic acid could be quickly and selectively removed, and the effect on the enzyme reaction layer was small. Ascorbic acid oxidase and catalase can be cross-linked and immobilized at the same time, so when serum or blood is dropped, they react without dissolving, and they do not interfere with the enzyme reaction layer or electrode part of the glucose sensor. There was no. The effect of uric acid could be removed by using uricase instead of ascorbate oxidase.

Example 4 A cover 7 was attached to the sensor having the configuration shown in Example 2 as shown in FIG. Ascorbic acid oxidase and catalase were immobilized and supported on the sample supply portion of this cover, and a surfactant was added thereto. When blood is supplied to the sample supply section of the cover 7, it is quickly introduced into the interfering substance removal section 6 by the surfactant, and after reacting with the immobilized enzyme, the blood is transferred to the electrode section 2.
．． 3, the reaction progressed in the enzyme reaction layer 5, and a reproducible response was obtained. By reducing the volume inside the cover 7,
The amount of sample and the amount of ascorbic acid oxidase and catalase supported could be reduced to minute amounts. Furthermore, by surrounding the sample with the cover 7, the sample inside the cover 7 could be prevented from evaporating because it could be isolated from the outside air.

Example 5 In the biosensor with a cover shown in Example 4, the sample supply port was covered with a nylon nonwoven fabric on which ascorbic acid oxidase and catalase were immobilized, as shown in FIG. When the blood adhered to the sample supply port, it passed through the nylon nonwoven fabric and flowed to the enzyme reaction layer 5. However, ascorbic acid in the sample was reacted and removed, and large molecules such as blood cells were adsorbed to the nylon nonwoven fabric and removed, reducing the viscosity of the sample and increasing the reaction rate. In addition to nylon nonwoven fabric, bulbs, glass fibers, porous polycarbonate membranes, and the like could be used as carriers for interfering substances.

The biosensor of the present invention is not limited to the glucose sensor shown in the above embodiments, but can be used in systems involving redox enzymes, such as alcohol sensors and cholesterol sensors. Although glucose oxidase was used as the oxidoreductase in the above examples, other enzymes such as alcohol oxidase, cholesterol oxidase, xanthine oxidase, etc. can be used. Further, as the electron acceptor, potassium ferricyanide used in the above examples is suitable because it reacts stably, but P-benzoquinone is suitable for increasing the reaction rate because it has a high reaction rate. In addition, the effects of the invention in which 2,6-cyclophenol indophenol, methylene blue, phenazine methosulfate, potassium β-naphthoquinone 4-sulfonate, ferrocene, etc. An electrode system is printed on the substrate to form an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic molecule, and an electron acceptor, and a substance that dissolves and undergoes an oxidation reaction is added as an interfering substance removal part. By supporting the substrate, reducing substances that are present in the biological sample can be removed in advance, and the substrate concentration in the biological sample can be measured very easily, improving measurement accuracy.

Furthermore, by adding a surfactant when forming the electron acceptor layer, a small amount of the electron acceptor can be uniformly supported on the thin film layer that is difficult to peel off, which has a great effect on storage stability and mass production.

[Brief explanation of drawings]

FIG. 1 is a perspective view of a biosensor according to an embodiment of the present invention, FIGS. 2, 3, and 4 are longitudinal sectional views of the same biosensor, and FIG. 5 is a longitudinal sectional view of a conventional biosensor. It is. 130. Substrate, 2. ,,Opposite,3. , , measurement pole, 4. ,
, insulating layer, 5. , , enzyme reaction layer, θ01. Interfering substance removal section, 79. , Name of cover agent Patent attorney Shigetaka Awano Haka 1 person III
Figure 2! -m- 3-°゛ -m- 5=... -m- Kure temporary pair i measurement positive latitude rope 1 tsuba l! Reaction layer nvww lung i! temporary

Claims (7)

[Claims] (1) An insulating substrate is provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and an enzyme reaction layer consisting mainly of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is provided on the surface of the electrode system. Further, an interfering substance removing section is added, and the substance concentration change during the reaction between the enzyme, the electron acceptor, and the sample liquid is electrochemically detected by the electrode system, and the substrate concentration is measured. A biosensor featuring: (2) An insulating substrate is provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and an electron acceptor containing an oxidoreductase, a hydrophilic polymer, and a surfactant is mainly formed on the surface of the electrode system. Further, an interfering substance removal section is added, and a change in substance concentration during the reaction between the enzyme, electron acceptor, and sample liquid is electrochemically detected by the electrode system, and the substrate concentration is measured. A biosensor characterized in that it is configured to. (3) The biosensor according to claim 1 or 2, wherein the interfering substance removing section is made of a substance having oxidizing power. (4) The biosensor according to claim 1 or 2, wherein the interfering substance removing portion carries an enzyme. (5) The biosensor according to claim 4, wherein the enzyme of the interfering substance removing portion is immobilized. (6) The biosensor according to claim 1 or 2, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate. (7) The biosensor according to claim 1 or 2, wherein a cover is installed on the electrode system so as to include the enzyme reaction layer and the interfering substance removal section inside.