JPH0783871A - Biosensor - Google Patents

Abstract

PURPOSE:To take a highly accurate measurement by providing a reaction layer containing enzyme for transforming an interfering substance involved in the reaction system of a measurement object to such a substance as not involved in the reaction system. CONSTITUTION:Glucose in sample liquid is oxidized by pyranose oxidase contained in a reaction layer 9. Thus, the glucose disappears from the liquid after the dissolution of the layer 9. Sucrose in the liquid is hydrolyzed due to invertase in a reaction layer 7, and alpha-glucose and fructose are thereby generated. Also, isomerization from alpha-glucose to beta-glucose is facilitated with mutaloze, and beta-glucose is oxidized by glucose oxidase. In this case, potassium ferricyanide coexistent in the layer 7 is reduced to generate potassium ferrocyanide. This generated amount is in proportion to a sucrose amount in the liquid. When voltage is applied across a counter electrode 5 and a working electrode 4, an oxidation current value is obtained in proportion to the generated amount of potassium ferrocyanide. The concentration of sucrose can be determined from the value.

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 and quickly quantifying a specific component in a sample.

[0002]

2. Description of the Related Art A sucrose sensor will be described as an example of a biosensor. As a method for quantifying sucrose using an enzyme electrode, invertase (EC 3.2.1.
26: hereinafter abbreviated as INV), mutarotase (EC5.
1.3.3: A method in which three enzymes of glucose oxidase (EC1.1.3.4: hereinafter abbreviated as GOD) and an oxygen electrode or a hydrogen peroxide electrode are combined is generally well known. (For example, Shuichi Suzuki "Biosensor" Kodansha). The above method will be described. Sucrose in the sample is hydrolyzed to α-glucose and fructose by INV. Next, MUT promotes isomerization of α-glucose to β-glucose, and this β-glucose is selectively oxidized by GOD. In the presence of oxygen, oxygen is reduced to hydrogen peroxide in the oxidation reaction process by the GOD. At this time, the amount of oxygen decrease is measured by an oxygen electrode, or the amount of increase of hydrogen peroxide is measured by a hydrogen peroxide electrode to quantify sucrose.

Next, a method of causing a redox compound to function as an electron acceptor instead of oxygen will be described.
As a biosensor of this system, a method has been proposed in which the sample solution can be easily quantified without diluting or stirring the sample solution (JP-A-3-54447). In this biosensor, an electrode system is formed on an insulating substrate by a method such as screen printing, and a layer containing a hydrophilic polymer and a redox enzyme, a hydrophilic polymer layer and an electron acceptor are formed on the electrode system. The layers containing the layers are sequentially formed. When the sample solution is dropped onto the enzyme reaction layer, the reaction layer is dissolved, the enzyme reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized,
The substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

[0004]

In the prior art, in order to quantify sucrose by hydrolyzing sucrose to be measured to generate β-glucose and oxidizing this β-glucose, a sample solution is used. When sucrose and β-glucose coexisted therein, it was difficult to accurately quantify only sucrose. Furthermore, in a biosensor in which a redox compound functions as an electron acceptor, when ascorbic acid is contained in the sample solution, an error may occur in the sensor response, which hinders accurate substrate quantification. Had the problem.

[0005]

In order to solve the above-mentioned problems, the present invention provides an electrically insulating substrate, an electrode system formed on the substrate and comprising at least a working electrode and a counter electrode, and at least a hydrophilic polymer. A first reaction layer containing an enzyme and an electron acceptor and provided in contact with the electrode system, and an interfering substance involved in the reaction system of the measurement object in the first reaction layer does not participate in the reaction system A second reaction layer that contains an enzyme that converts into a substance and that is provided in the vicinity of the electrode system; and a membrane that permeates the object to be measured but limits the permeation of the enzyme in the second reaction layer. The present invention provides a biosensor including an isolation layer that covers the reaction layer of 1.

Here, when a preferable combination of the enzymes and the like used in the first and second reaction layers is given, the enzyme contained in the first reaction layer is glucose oxidase and is contained in the second reaction layer. There are biosensors where the enzyme is pyranose oxidase. The enzyme contained in the first reaction layer is a combination of at least one selected from the group consisting of invertase, maltase, β-galactosidase and cellulase and glucose oxidase, and the enzyme contained in the second reaction layer. Is a hexokinase, and the second reaction layer further contains adenosine-5′-triphosphate, and the second reaction layer further comprises glucose-6-phosphate dehydrogenase and β.
-There are biosensors containing nicotinamide adenine dinucleotide phosphate. Further, there is a biosensor in which the enzyme contained in the second reaction layer is ascorbate oxidase.

[0007]

The enzyme contained in the second reaction layer can chemically change the response interfering substance coexisting in the sample liquid containing the measurement target to a substance not involved in the reaction system of the measurement target. Furthermore, the isolation layer can prevent the enzyme in the second reaction layer from participating in the enzyme reaction system in the first reaction layer. Therefore, the influence of coexisting interfering substances can be reduced.

[0008]

EXAMPLES The present invention will be described below with reference to examples. Example 1 A sucrose sensor will be described as an example of a biosensor. FIG. 1 is an exploded perspective view of a sucrose sensor manufactured as an example of the biosensor of the present invention, FIG. 2 is an exploded perspective view of the sucrose sensor excluding the first and second reaction layers and the isolation layer, and FIG. FIG. 3 is a schematic cross-sectional view of the sucrose sensor excluding a cover and a spacer.

The method for producing the sucrose sensor will be described below. Silver paste is printed on the electrically insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Next, a conductive carbon paste containing a resin binder is printed to form the working electrode 4. The working electrode 4 is in contact with the lead 2. Next, the insulating paste is printed to form the insulating layer 6. The insulating layer 6 covers the outer peripheral portion of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant. Further, the insulating layer 6 partially covers the leads 2 and 3.
Next, a conductive carbon paste containing a resin binder is printed so as to come into contact with the leads 3 to form the counter electrode 5.

Next, the electrode system (working electrode 4, counter electrode 5)
A 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer is dropped on the above and dried to form a CMC layer. Then, on the CMC layer, INV, MUT, GOD as an enzyme and potassium ferricyanide as an electron acceptor were added to a phosphate buffer solution (0.2
_{M: KH 2 PO 4 -0.2M:} Na 2 HPO 4; pH = 7.
The mixed solution dissolved in 4) is dropped and dried in a warm air dryer at 50 ° C. for 10 minutes to form a first reaction layer 7 containing a hydrophilic polymer, an enzyme and an electron acceptor. When a mixed aqueous solution of an enzyme and an electron acceptor is dropped on the CMC layer,
The CMC layer, which is a hydrophilic polymer, is once dissolved, and in the subsequent drying process, the first reaction layer 7 including the hydrophilic polymer, the enzyme, and the electron acceptor is formed in a state of being mixed with the enzyme and the like. However, since it is not accompanied by stirring, it is not in a completely mixed state, and the surface of the electrode system is in a state of being covered only with CMC. That is, it is possible to prevent protein adsorption on the electrode system surface.

Next, a 2 wt% methanol solution of poly (2-hydroxyethylmethacrylate) (hereinafter abbreviated as PHEMA) as a water-insoluble polymer is dropped on the first reaction layer 7 and dried to obtain a water-insoluble polymer. An isolation layer 8 made of molecules is formed. CMC, enzyme and potassium ferricyanide are almost insoluble in methanol. Therefore, the first reaction layer 7 is covered by a separate layer 8 of a PHEMA film.

Next, pyranose oxidase (EC
1.1.3.10: An aqueous solution of PyOD (hereinafter abbreviated as PyOD) is dropped on the insulating layer 6 and dried to form the second reaction layer 9. In this case, water was used as the solvent for dissolving PyOD, but if necessary, an appropriate buffer solution such as phosphoric acid was used as the solvent to form the second reaction layer containing PyOD and the buffer salt. You can also do it. Second reaction layer 9
Need not necessarily be formed at a position away from the first reaction layer 7 as described above. When the first and second reaction layers are formed at distant positions, it is possible to reduce deterioration in sensor storage characteristics due to the decrease in activity of each enzyme reagent.
On the other hand, it is also possible to form the second reaction layer 9 in contact with the first reaction layer 7. In this case, since the formation location of the second reaction layer coincides with that of the first reaction layer, it is possible to make the sensor shape smaller. After the first reaction layer 7 and the second reaction layer 9 are formed as described above, the cover 22 and the spacer 21 are adhered to each other in a positional relationship shown by a dashed line in FIG. To make.

To this sucrose sensor, 3 μl of a mixed aqueous solution of sucrose and glucose was used as a sample solution, and the sample supply hole 2
When supplied from 3, the sample solution reaches the air holes 24 part,
The second reaction layer 9 and the first reaction layer 7 are sequentially dissolved.
Three minutes after supplying the sample solution, a voltage of +0.5 V was applied between the counter electrode 5 and the working electrode 4 of the electrode system, and the current value after 5 seconds was measured, which was proportional to only the sucrose concentration in the sample solution. The obtained value was obtained.

The measuring principle will be described below. Glucose in the sample solution is oxidized by PyOD contained in the second reaction layer, but PyOD has no oxidizing effect on sucrose. Therefore, glucose is hardly present in the sample solution after the second reaction layer is dissolved. Sucrose in the sample solution is equivalent to INV in the first reaction layer.
Is hydrolyzed to produce α-glucose and fructose. This isomerization of α-glucose to β-glucose is promoted by MUT, and β-glucose is selectively oxidized by GOD. At this time, potassium ferricyanide coexisting in the first reaction layer is reduced to produce potassium ferrocyanide. The amount of potassium ferrocyanide produced is proportional to the amount of sucrose in the sample solution. By applying the voltage, an oxidation current value proportional to the amount of potassium ferrocyanide produced can be obtained. Therefore, the sucrose concentration can be quantified from the oxidation current value.

The isolation layer 8 allows sucrose in the sample solution to permeate but does not allow PyOD in the second reaction layer 9 to permeate,
Therefore, PyOD is the first by INV, MUT, GOD.
It has an effect of preventing participation in the enzyme reaction system in the reaction layer. This is a PHE formed from a methanol solution
This may be due to the fact that the membrane of MA has micropores with a pore size appropriate to prevent the permeation of enzyme molecules, which is two orders of magnitude larger than sucrose.

[Example 2] As an example of a biosensor,
The sucrose sensor will be described. In the same manner as in Example 1, the leads 2, 3 and the electrode system (working electrode 4, counter electrode 5) were formed on the insulating substrate 1 made of polyethylene terephthalate.
And the insulating layer 6 is formed. Further, in the same manner as in Example 1, the first reaction layer 7 made of CMC, INV, MUT, GOD, and hydrophilic polymer containing potassium ferricyanide as a main component-enzyme-electron acceptor, and the isolation layer made of PHEMA. 8 is formed. Next, hexokinase (EC
2.7.1.1: Hereinafter, abbreviated as HX) and adenosine-
A mixed aqueous solution of 5'-disodium triphosphate (hereinafter abbreviated as ATP) was dropped on the insulating layer 6 and dried to form the second reaction layer 9, and the cover was formed in the same manner as in Example 1. 22 and the spacer 21 were integrated to produce a sucrose sensor.

A sucrose sensor prepared as described above was used as a sample solution in a mixed aqueous solution of sucrose and glucose of 3 μm.
When 1 was supplied from the sample supply hole 23 in the same manner as in Example 1 and the sensor response current was measured, a value proportional to only the sucrose concentration was obtained without depending on the glucose concentration. The measurement principle will be briefly described below. Glucose in the sample solution is phosphorylated by HX contained in the second reaction layer to become glucose-6-phosphate. ATP is required for the phosphorylation of glucose by HX,
ATP becomes adenosine-5'-diphosphate. HX has no effect on sucrose. Therefore, the second
Almost no glucose is present in the sample solution after the reaction layer of (1) is dissolved. The sucrose contained in the sample solution is
In the same manner as above, it reacts with the enzyme in the first reaction layer to produce potassium ferrocyanide. Further, in the same manner as in Example 1, the sucrose concentration can be determined from the oxidation current of this potassium ferrocyanide.

[Example 3] As an example of a biosensor,
The sucrose sensor will be described. In the same manner as in Example 1, the leads 2, 3 and the electrode system (working electrode 4, counter electrode 5) were formed on the insulating substrate 1 made of polyethylene terephthalate.
And the insulating layer 6 is formed. Further, in the same manner as in Example 1, the first reaction layer 7 made of CMC, INV, MUT, GOD, and hydrophilic polymer containing potassium ferricyanide as a main component-enzyme-electron acceptor, and the isolation layer made of PHEMA. 8 is formed.

Next, HX, ATP, glucose-6-
Phosphate dehydrogenase (EC 1.1.1.49: hereinafter abbreviated as G6PDH), β-nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) and C
A mixed aqueous solution of MC is dropped on the insulating layer 6 and dried to form the second reaction layer 9. Further, similarly to the first embodiment, the sucrose sensor is manufactured by integrating the cover 22 and the spacer 21 together. CMC in the second reaction layer 9
By including such a hydrophilic polymer as described above, it is possible to prevent cracking of the second reaction layer 9 after formation and separation from the insulating layer 6. This effect is particularly effective when the amount of the reagent contained in the second reaction layer is large as in this example.

As a sample solution, 3 μl of a mixed aqueous solution of sucrose and glucose was added to the sucrose sensor prepared as described above.
When 1 was supplied from the sample supply hole 23 in the same manner as in Example 1 and the sensor response current was measured, a value proportional to the sucrose concentration was obtained without being affected by the glucose concentration. The measurement principle will be briefly described below. Glucose in the sample solution is phosphorylated by HX contained in the second reaction layer to become glucose-6-phosphate. This glucose-6-phosphate is oxidized by G6PDH to gluconolactone-6-phosphate. NADP is required for the oxidation reaction of glucose-6-phosphate by G6PDH, and NADP is reduced to NADPH. G6PD
As H oxidizes glucose-6-phosphate, the equilibrium of the reaction by HX shifts to the production of glucose-6-phosphate. Therefore, the phosphorylation reaction of glucose by HX is accelerated, and the effect of removing glucose existing in the sample solution in advance as an interfering substance can be enhanced. Sucrose in the sample solution reacts with the enzyme in the first reaction layer in the same manner as in Example 1 to produce potassium ferrocyanide. Furthermore, in the same manner as in Example 1,
The sucrose concentration can be determined from the oxidation current of potassium ferrocyanide.

Although the sucrose sensor is shown in Examples 1 to 3 above, the present invention uses a combination of maltase and GOD instead of the combination of INV, MUT and GOD as the enzyme contained in the first reaction layer. Use of β-galactosidase and GOD for maltose sensor
Can be applied to the lactose sensor by using the combination of and the cellulose sensor by using the combination of cellulase and GOD.

[Embodiment 4] As an example of a biosensor,
The glucose sensor will be described. In the same manner as in Example 1, the leads 2, 3 and the electrode system (working electrode 4, counter electrode 5) were formed on the insulating substrate 1 made of polyethylene terephthalate.
And the insulating layer 6 is formed. Further, a CMC layer was formed on the electrode system (working electrode 4, counter electrode 5) in the same manner as in Example 1, and subsequently, a mixed aqueous solution of GOD as an enzyme and potassium ferricyanide as an electron acceptor was dropped on the CMC layer. Then, it is dried in a warm air dryer to form a first reaction layer 7 composed of a hydrophilic polymer-enzyme-electron acceptor.

Next, PHEMA is used as the water-insoluble polymer.
2 wt% methanol solution is dropped onto the first reaction layer 7 and dried to form the isolation layer 8. Next, ascorbate oxidase (EC 1.10.3.3: hereinafter, A
An aqueous solution of abbreviated as sOD) is dropped on the insulating layer 6 and dried to form the second reaction layer 9. First as above
After forming the reaction layer 7, the isolation layer 8 and the second reaction layer 9 of FIG.
Inside, the glucose sensor is manufactured by adhering with a positional relationship as shown by the alternate long and short dash line.

To this glucose sensor, 3 μl of a mixed aqueous solution of glucose and ascorbic acid was supplied as a sample solution from the sample supply hole 23. The sample solution reached the air holes 24, and the second reaction layer 9 and the first reaction layer 7 were sequentially dissolved. One minute after supplying the sample solution, a voltage of +0.5 V was applied between the counter electrode 5 and the working electrode 4 of the electrode system, and the current value after 5 seconds was measured, it was hardly affected by ascorbic acid. A value proportional to the glucose concentration in the sample solution was obtained. The measurement principle will be briefly described below. Ascorbic acid in the sample solution is AsOD contained in the second reaction layer.
Oxidized to dehydroascorbic acid. Glucose in the sample solution reacts with GOD in the first reaction layer, and at the same time potassium ferricyanide is reduced to produce potassium ferrocyanide. Further, by applying the voltage, the oxidation current of potassium ferrocyanide can be obtained. Since the amount of potassium ferrocyanide produced is proportional to the amount of glucose in the sample solution, the glucose concentration can be quantified.

When AsOD is not contained in the second reaction layer, ascorbic acid in the sample solution interferes with the sensor response for the following reason. Ascorbic acid reacts with potassium ferricyanide in the first reaction layer, and potassium ferrocyanide is produced separately from the reaction between GOD and glucose. Therefore, in this case, since the amount of potassium ferrocyanide produced depends on both ascorbic acid and glucose, the glucose concentration cannot be accurately quantified from the reduction current of potassium ferrocyanide.

Although the glucose sensor was shown in Example 4 above, the present invention relates to other redox enzymes such as fructose, maltose, lactose sensor, cellulose sensor, lactate sensor, alcohol sensor and cholesterol sensor. Can be widely applied to the reaction system. As enzymes, besides invertase, mutarotase and glucose oxidase, fructose dehydrogenase, maltase, β-galactosidase, cellulase, lactate oxidase, alcohol oxidase, cholesterol oxidase, xanthine oxidase, amino acid oxidase and the like can be used. Although PHEMA was used as the water-insoluble polymer in the above Examples 1 to 4, the water-insoluble polymer is not limited to this, and one having the above-mentioned effect can be freely selected.

In the sucrose sensor of the above embodiment, KH ₂ PO ₄ —Na ₂ HPO ₄ (pH = 7.4) was used as the buffer solution. In addition to these, HEPES-NaOH, K
_{H 2 PO 4 -K 2 HPO 4} , NaH 2 PO 4 -K 2 HPO 4, N
_{aH 2 PO 4 -Na 2 HPO 4} , citric acid -Na ₂ HPO _4,
Citric acid-K ₂ HPO ₄ , Tris-Tris hydrochloride,
[N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid] -NaOH, [piperazine-N,
The same effect can be obtained by adjusting the combination of N'-bis (2-ethanesulfonic acid)]-NaOH to a value around pH 7-8. Furthermore, although CMC was used as the hydrophilic polymer in the above examples, the present invention is not limited to these, and other cellulose derivatives, specifically hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, Carboxymethyl ethyl cellulose may be used, and further polyvinylpyrrolidone, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts can be used. Also has the same effect.

On the other hand, as the electron acceptor, in addition to potassium ferricyanide shown in the above examples, p-benzoquinone, phenazine methosulfate, methylene blue, ferrocene derivative and the like can be used. Further, in the above-mentioned Examples, the method of dissolving the enzyme and the electron acceptor in the sample solution was shown, but the method is not limited to this and can be applied to the case where the enzyme and the electron acceptor are insolubilized in the sample solution by immobilization. . Further, in the above-mentioned embodiment, the bipolar electrode system having only the working electrode and the counter electrode is described, but the three-electrode system including the reference electrode enables more accurate measurement.

[0029]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to provide a biosensor capable of highly accurate quantification without being affected by a response interfering substance coexisting in a sample solution.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a sucrose sensor according to an embodiment of the present invention.

FIG. 2 is an exploded perspective view of the same sucrose sensor excluding a first reaction layer and a second reaction layer.

FIG. 3 is a schematic cross-sectional view of the sucrose sensor excluding a cover and a spacer.

[Explanation of symbols]

 1 Insulating Substrate 2 Lead 3 Lead 4 Working Electrode 5 Counter Electrode 6 Insulating Layer 7 First Reaction Layer 8 Isolation Layer 9 Second Reaction Layer 21 Spacer 22 Cover 23 Sample Supply Hole 24 Air Hole

Claims (6)

[Claims] 1. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, containing at least a hydrophilic polymer, an enzyme and an electron acceptor, and provided in contact with the electrode system. The first reaction layer, which contains an enzyme that converts an interfering substance involved in the reaction system of the measurement object in the first reaction layer into a substance not involved in the reaction system, is provided in the vicinity of the electrode system. A bioreactor comprising a second reaction layer and a membrane that allows a substance to be measured to permeate but limits the permeation of an enzyme in the second reaction layer, and comprises a separation layer covering the first reaction layer. Sensor. 2. The biosensor according to claim 1, wherein the second reaction layer further contains a hydrophilic polymer. 3. The biosensor according to claim 1, wherein the enzyme contained in the first reaction layer is glucose oxidase, and the enzyme contained in the second reaction layer is pyranose oxidase. 4. The enzyme contained in the first reaction layer comprises a combination of at least one selected from the group consisting of invertase, maltase, β-galactosidase and cellulase and glucose oxidase, and the enzyme contained in the second reaction layer. The enzyme used is hexokinase, and the second
The biosensor according to claim 1, wherein the reaction layer contains adenosine-5'-triphosphate. 5. The second reaction layer further comprises glucose-6.
The biosensor according to claim 4, which contains -phosphate dehydrogenase and β-nicotinamide adenine dinucleotide phosphate. 6. The biosensor according to claim 1, wherein the enzyme contained in the second reaction layer is ascorbate oxidase.