JP3794046B2 - Biosensor and biosensor for blood glucose level measurement - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biosensor capable of quickly and easily quantifying a substrate (specific component) contained in biological samples such as blood and urine, raw materials and products in the food industry, and also samples such as fruit juice.
[0002]
[Prior art]
As a quantitative analysis method for sugars such as glucose and sucrose, a photometric method, a colorimetric method, a reduction titration method, and methods using various chromatographies have been developed. However, none of these methods have low accuracy because the specificity for saccharides is not so high. For example, it is known that the light intensity meter method is greatly affected by the temperature during operation, although the operation is simple.
[0003]
In recent years, biosensors have been proposed that can easily quantify specific components (substrates) in biological samples and foods without diluting or stirring the sample liquid.
[0004]
Japanese Patent Laid-Open No. 3-202864 discloses a reaction layer in which an electrode system is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer, an oxidoreductase, and an electron acceptor are contained on the electrode system. Discloses a biosensor. This biosensor can quantify the substrate concentration in the sample as follows: First, by dropping the sample solution onto the reaction layer of the biosensor, the reaction layer is dissolved and the substrate in the sample solution is dissolved. An enzyme reaction proceeds with the oxidoreductase in the reaction layer, and then the electron acceptor in the reaction layer is reduced. After the completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, whereby the substrate concentration in the sample solution is quantified from the oxidation current value obtained at this time.
[0005]
US Pat. No. 5,192,415 discloses a hydrogen ion concentration control that can optimize the pH of a sample solution according to the type of oxidoreductase contained in the reaction layer without first adjusting the pH of the sample solution. A biosensor having a layer is disclosed.
[0006]
US Pat. No. 5,264,103 is formed on an electrically insulating substrate and has a main electrode system having a working electrode and a counter electrode; a reaction layer containing an oxidoreductase; and a distance from the main electrode system. And a sub-electrode layer having a working electrode and a counter electrode is disclosed.
[0007]
Each of the biosensors can be widely used, for example, as a glucose sensor, an alcohol sensor, a cholesterol sensor, and an amino acid sensor by selecting the oxidoreductase contained therein.
[0008]
[Problems to be solved by the invention]
Among such biosensors, glucose sensors are generally known to use glucose oxidase as an oxidoreductase. However, since glucose oxidase reacts only with β-glucose, which is present in an equilibrium state of approximately 63% of the isomers of glucose, this glucose sensor has a low response current value (ie, detection sensitivity), For example, when a very small amount of glucose is quantified, there is a problem that a measurement error associated therewith increases.
[0009]
Furthermore, when the polysaccharide is quantified using the above glucose sensor, since most of the glucose obtained by the hydrolase is an α-form, the α-glucose produced by the hydrolysis is once converted to mutarotase before quantification. The process of isomerizing to β-form by the above method was necessary.
[0010]
Japanese Patent Application No. 6-291401 discloses a biosensor using both the above-mentioned mutarotase and glucose oxidase. However, this biosensor cannot sufficiently improve the detection sensitivity of the glucose sensor if the reagent amount of the whole enzyme is small, and conversely, if the reagent amount of the whole enzyme is large, the manufacturing cost of the sensor becomes high. . When the substrate concentration in the sample solution is relatively high, the detection sensitivity of the biosensor containing mutarotase and glucooxidase is lower than that of the biosensor not containing mutarotase.
[0011]
[Means for Solving the Problems]
The biosensor of the present invention is a solution to the above-described drawbacks, and is a biosensor for detecting glucose, an electrically insulating substrate, and an electrode system formed on the substrate and having a working electrode and a counter electrode, A biosensor having a reaction layer disposed on the substrate or through the substrate and a space, wherein the reaction layer contains pyranose oxidase and glucose oxidase .
[0013]
Furthermore, in the present invention, the reaction layer contains pyranose oxidase and polysaccharide degrading enzyme.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a synthetic resin plate such as polyethylene terephthalate can be used as the electrically insulating substrate.
[0015]
An electrode system having a working electrode and a counter electrode can be provided on this substrate using a known method. For example, after forming a lead on the substrate, a working electrode and a counter electrode are provided so as to be connected to each lead and insulated from each other. As the material for the lead and the electrode, a known conductive material can be used. Examples include carbon, silver, platinum, gold, and palladium.
[0016]
The reaction layer contains pyranose oxidase. Pyranose oxidase is an enzyme that simultaneously oxidizes both α-glucose and β-glucose. An example of such a pyranose oxidase is pyranose oxidase (EC 1.1.3.10; PyOx).
[0017]
The content of PyOx is preferably 1 to 200 units, more preferably 2 to 50 units, per square centimeter of the reaction layer. As used herein, the term “unit” refers to the amount of oxidoreductase that oxidizes 1 μmol of substrate in 1 minute. When the content of PyOx is less than 1 unit per square centimeter of reaction layer, a time of several minutes or more is required at the time of measurement, and the response current value to be measured is easily influenced by evaporation of the sample liquid generated during the measurement. When the content of PyOx exceeds 200 units per square centimeter of the reaction layer, the manufacturing cost becomes high, and the reaction layer is easily broken when the reaction layer is formed, and the response current value tends to vary.
[0018]
In addition, the reaction layer may contain glucose oxidase (GOD) together with the pyranose oxidase in order to further improve the sensitivity of detecting glucose in the sample solution and to respond to a wider range of glucose concentrations. A suitable GOD content is preferably 1 to 200 units per square centimeter of the reaction layer. In this case, the content of PyOx is preferably 0.1 to 200 units, more preferably 0.2 to 40 units per square centimeter of the reaction layer.
[0019]
Furthermore, the reaction layer can contain various hydrophilic polymers. Examples include carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose, ethylcellulose, ethylhydroxyethylcellulose, carboxymethylethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, polylysine and other polyamino acids, polystyrene sulfonic acid Gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts. In particular, CMC is preferable.
[0020]
The reaction layer may contain a polysaccharide-degrading enzyme for hydrolyzing the polysaccharide to produce α-glucose. A polysaccharide-degrading enzyme is an enzyme having the ability to hydrolyze polysaccharides such as sucrose and maltose to produce glucose. Examples of polysaccharide degrading enzymes include sucrose hydrolase such as invertase (INV), maltose hydrolase such as maltase, lactose hydrolase such as β-galactosidase, and the like. The content of the polysaccharide degrading enzyme is preferably 1 to 400 units, more preferably 2 to 200 units, per square centimeter of the reaction layer.
[0021]
When a sample solution containing glucose is supplied to the reaction layer of the biosensor, α-glucose and β-glucose in the sample solution are each oxidized by the pyranose oxidase. At the same time, the dissolved oxygen in the sample solution is reduced to hydrogen peroxide. Here, when a voltage is applied, hydrogen peroxide is oxidized. Since the response current generated at this time is proportional to the generated hydrogen peroxide concentration, that is, the substrate concentration in the sample solution, the substrate concentration in the sample solution can be obtained by measuring the response current value.
[0022]
Instead of generating hydrogen peroxide simultaneously with the oxidation reaction of the substrate, an electron acceptor can be formed simultaneously with the enzyme reaction by containing an electron acceptor in the reaction layer. Examples of such electron acceptors include ferricyan ion, p-benzoquinone and derivatives thereof, phenazine methosulfate, methylene blue, ferrocene and derivatives thereof. One or more of these electron acceptors can be used. In particular, it is preferable to use ferricyan ion.
[0023]
The content of ferricyan ion is preferably 0.21 to 3.30 mg, more preferably 0.3 to 2.59 mg per square centimeter of the reaction layer. When the ferricyan ion content is less than 0.21 mg per square centimeter of the reaction layer, the measurable glucose concentration range tends to be extremely narrow. When the content of ferricyan ion exceeds 3.30 mg per square centimeter of the reaction layer, the reaction layer is cracked, resulting in variations in the response current value, and further, the reliability during storage tends to decrease.
[0024]
Next, a preferred embodiment of the biosensor manufacturing method of the present invention will be described with reference to FIGS. 1 and 2.
[0025]
First, a conductive material such as silver paste is printed on the electrically insulating substrate 1 by screen printing to form leads 2 and 3. Next, a conductive material containing a resin binder is printed on the substrate 1 to form the working electrode 4. The working electrode 4 is in contact with the lead 2.
[0026]
Next, an insulating paste 6 is formed on the substrate 1 by printing an insulating paste. The insulating layer 6 covers the outer periphery 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, the insulating layer 6 also covers a part of the leads 2 and 3. Further, a ring-shaped counter electrode 5 is formed on the outer periphery of the working electrode 4 using a conductive material containing a resin binder. The counter electrode 5 is in contact with the lead 3. In this way, an electrode system 8 having a working electrode 4 and a counter electrode 5 is formed on the substrate 1.
[0027]
Furthermore, in order to further stabilize the accuracy of the biosensor of the present invention, a three-electrode system having a reference electrode in addition to the working electrode 4 and the counter electrode 5 may be formed on the substrate 1.
[0028]
The reaction layer can be disposed on the substrate 1 as follows. First, an aqueous solution of the hydrophilic polymer is dropped on the electrode system 8 and dried to form a hydrophilic polymer layer. Next, an aqueous solution containing PyOx and, if necessary, the electron acceptor and / or polysaccharide-degrading enzyme is dropped onto the hydrophilic polymer layer and dried. The biosensor of the present invention can also be cross-fixed on the hydrophilic polymer layer using glutaraldehyde so that the pyranose oxidase and, if necessary, the polysaccharide-degrading enzyme can be used repeatedly. Alternatively, it may be fixed together with the polymer material. Furthermore, if necessary, the electron acceptor may be chemically fixed using a polymer material. In this way, the reaction layer 7 covering the entire electrode system 8 shown in FIG. 2 is formed.
[0029]
Alternatively, the reaction layer can be disposed on the substrate 1 via a space. In this case, as shown in FIG. 3, the biosensor includes a substrate 1 and a cover 30 disposed on the substrate 1 with a spacer 20 interposed therebetween. The cover 30 has a hole 31 and a reaction layer 37 on one surface thereof. The substrate 1 and the cover 30 can be arranged so that the reaction layer 37 and the electrode system 8 face each other. A method for forming the reaction layer 37 through the substrate 1 and the space is described in, for example, Japanese Patent Application Laid-Open No. 1-114747. In this biosensor, when the sample liquid supplied from the sample liquid supply port 38 reaches between the reaction layer 37 and the electrode system 8, the excess generated in the reaction layer 37 is similar to the biosensor shown in FIG. Hydrogen oxide or a reduced form of an electron acceptor can be measured by the electrode system 8.
[0030]
Example 1
As an example of the biosensor of the present invention, a glucose sensor was produced as follows. FIG. 1 is a schematic plan view in which a reaction layer of a glucose sensor produced as an example of the present invention is removed. FIG. 2 is a schematic sectional view of the glucose sensor.
[0031]
As shown in FIG. 1, a silver paste was printed by screen printing on an electrically insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3. Next, a conductive carbon paste containing a resin binder was printed on the substrate 1 to form the working electrode 4. This working electrode 4 was in contact with the lead 2.
[0032]
Next, an insulating paste 6 was printed on the substrate 1 to form an insulating layer 6. The insulating layer 6 covered the outer periphery of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant.
[0033]
Next, a conductive carbon paste containing a resin binder was printed on the substrate 1 so as to come into contact with the leads 3 to form a ring-shaped counter electrode 5.
[0034]
Next, an aqueous solution of 0.5 wt% carboxymethylcellulose (CMC) was dropped onto the electrode system 8 (working electrode 4 and counter electrode 5) and dried to form a CMC layer. On this CMC layer, a mixed solution of pyranose oxidase (PyOx) and potassium ferricyanide was dropped and dried to form a reaction layer 7. The amounts of PyOx and potassium ferricyanide contained in the reaction layer 7 were 10 units and 1.3 mg, respectively, per square centimeter of the reaction layer.
[0035]
A 90 mg / dl aqueous glucose solution was dropped as a sample solution on the reaction layer 7 of the glucose sensor produced as described above. Then, 1 minute after dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8, and the current value after 5 seconds was measured. Thus, the response current value with respect to the glucose aqueous solution was measured 12 times in total using a new glucose sensor for each measurement. The variation in the obtained response current value was small.
[0036]
Furthermore, the response current values for 180 and 360 mg / dl glucose aqueous solutions were also measured 12 times in the same manner as described above. The obtained response current value tended to increase depending on the increase in glucose concentration, and the rate of change was large.
[0037]
As a comparative experiment, the response current value with respect to the glucose concentration was measured 12 times in the same manner as above except that a glucose sensor containing glucose oxidase was prepared instead of PyOx. The obtained response current values varied between the same glucose concentrations. Furthermore, these response current values tended to increase depending on the increase in glucose concentration, but the rate of change was small.
[0038]
(Example 2)
As an example of the biosensor of the present invention, a sucrose sensor was produced as follows.
[0039]
In the same manner as in Example 1, the leads 2 and 3, the electrode system 8 (working electrode 4 and counter electrode 5) and the insulating layer 6 were formed on the electrically insulating substrate 1 made of polyethylene terephthalate. Next, an aqueous solution of 0.5 wt% carboxymethylcellulose (CMC) was dropped on the electrode system 8 having the working electrode 4 and the counter electrode 5 and dried to form a CMC layer.
[0040]
A mixed aqueous solution of PyOx, invertase (INV), and potassium ferricyanide was dropped onto the CMC layer and dried to form a reaction layer 7. The amounts of PyOx, INV and potassium ferricyanide contained in the reaction layer 7 were 10 units, 40 units and 1.3 mg, respectively, per square centimeter of the reaction layer.
[0041]
When a 171 mg / dl aqueous solution of sucrose was dropped as a sample solution on the reaction layer 7 of the sucrose sensor produced as described above, the reaction layer 7 was dissolved by the sample solution. Next, 3 minutes after the dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8, and the current value after 5 seconds was measured.
[0042]
Furthermore, the response current value for each of the 342 and 684 mg / dl sucrose aqueous solutions was measured in the same manner as described above using a new sucrose sensor for each measurement.
[0043]
The obtained response current value tended to increase depending on the increase in sucrose concentration, and the rate of change was large.
[0044]
As a comparative experiment, the same effect as described above was also obtained in a maltose sensor or lactose sensor prepared using maltose hydrolase or lactose hydrolase instead of INV.
[0045]
Example 3
As an example of the biosensor of the present invention, a glucose sensor was produced as follows. FIG. 4 is a graph showing the correlation between various glucose concentrations and response currents of a glucose sensor showing an example of the biosensor of the present invention; the curve (a) in FIG. 4 shows a pyranose oxidase in the reaction layer. (PyOx) and glucose oxidase (GOD) in the response current value change when containing, curve (b) represents the response current value change when containing only GOD, and curve (c) is , Represents a change in response current value when only PyOx is contained.
[0046]
In the same manner as in Example 1, the leads 2 and 3, the electrode system 8 (working electrode 4 and counter electrode 5), and the insulating layer 6 were formed on the electrically insulating substrate 1 made of polyethylene terephthalate. Next, an aqueous solution of 0.5 wt% carboxymethylcellulose (CMC) was dropped on the electrode system 8 having the working electrode 4 and the counter electrode 5 and dried to laminate a CMC layer.
[0047]
To this CMC layer, a mixed aqueous solution of PyOx, glucose oxidase (GOD), and potassium ferricyanide was dropped and dried to form a reaction layer 7. The amounts of PyOx, GOD and potassium ferricyanide contained in the reaction layer 7 were 1 unit, 10 units and 1.3 mg, respectively, per square centimeter of the reaction layer.
[0048]
A 90 mg / dl aqueous glucose solution was dropped as a sample solution on the reaction layer 7 of the glucose sensor produced as described above. Then, 1 minute after dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8, and the current value after 5 seconds was measured.
[0049]
Furthermore, response current values for 180 and 360 mg / dl glucose aqueous solutions were measured in the same manner as described above, using a new glucose sensor for each measurement. The response characteristic between the obtained glucose concentration and the response current value is shown in the curve (a) in FIG.
[0050]
As a comparative experiment, among the glucose sensors, sensors that did not contain PyOx and sensors that did not contain GOD were produced, and response current values were measured in the same manner as described above. These results are shown in curve (b) and curve (c) in FIG.
[0051]
As shown in FIG. 4, the glucose sensor containing only GOD as an enzyme (curve (b) in FIG. 4) reflects the concentration of only β-glucose contained in the sample solution, so the response current value is the highest. It was low. On the other hand, the glucose sensor containing only PyOx as an enzyme (curve (c) in FIG. 4) reflects the sum of the concentrations of α-glucose and β-glucose contained in the sample solution. In the low region, the response current was higher than the curve (c) in FIG. However, the response sensor value of the glucose sensor containing only PyOx was lower than the result of the curve (b) in FIG. 4 in the region where the glucose concentration was high. As a result, the glucose sensor including both PyOx and GOD (curve (a) in FIG. 4) always had a high response current value in the widest concentration range.
[0052]
(Example 4)
As an example of the biosensor of the present invention, a sucrose sensor was produced as follows.
[0053]
In the same manner as in Example 1, the leads 2 and 3, the electrode system 8 (working electrode 4 and counter electrode 5), and the insulating layer 6 were formed on the electrically insulating substrate 1 made of polyethylene terephthalate. Next, an aqueous solution of 0.5 wt% carboxymethylcellulose (CMC) was dropped on the electrode system 8 having the working electrode 4 and the counter electrode 5 and dried to form a CMC layer.
[0054]
To this CMC layer, a mixed aqueous solution of PyOx, GOD, invertase (INV) and potassium ferricyanide was dropped and dried to form a reaction layer 7. The amounts of PyOx, GOD, invertase, and potassium ferricyanide contained in the reaction layer 7 were 1 unit, 10 units, 40 units, and 1.3 mg, respectively, per square centimeter of the reaction layer.
[0055]
When a 171 mg / dl aqueous solution of sucrose was dropped as a sample solution on the reaction layer 7 of the sucrose sensor produced as described above, the reaction layer 7 was dissolved by the sample solution. Next, 3 minutes after the dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8, and the current value after 5 seconds was measured.
[0056]
Furthermore, the response current value for each of the 342 and 684 mg / dl sucrose aqueous solutions was measured in the same manner as described above using a new sucrose sensor for each measurement.
[0057]
The obtained response current value tended to increase depending on the increase of the sucrose concentration, and always had a high value in a wide concentration range.
[0058]
As a comparative experiment, a sucrose sensor was prepared in the same manner as described above except that the reaction layer 7 did not contain GOD, and the response current value was measured. The obtained response current value was always lower than that of the sucrose sensor including the GOD.
[0059]
In the maltose sensor or lactose sensor produced using maltose hydrolase or lactose hydrolase instead of INV, the same effect as described above was obtained.
[0060]
(Example 5)
A biosensor was produced in the same manner as in Examples 1 to 4, except that the reaction layer 7 did not contain potassium ferricyanide. Sample liquids having the same substrate concentration as those in Examples 1 to 4 were dropped onto these biosensors, and a voltage of +1.0 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8 after a predetermined time. The current value after 5 seconds was measured.
[0061]
The obtained response current value tended to increase depending on the increase of each substrate concentration.
(Example 6)
A glucose sensor was produced in the same manner as in Example 3. On the reaction layer 7 of this glucose sensor, whole blood having a glucose concentration of 95 mg / dl was dropped as a sample solution. One minute after the dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8, and the current value after 5 seconds was measured.
[0062]
Furthermore, response current values for whole blood having glucose concentrations of 170 and 320 mg / dl were measured in the same manner as described above using a new glucose sensor for each measurement. The response characteristic between the obtained glucose concentration and the response current value is shown by a curve d in FIG.
[0063]
As a comparative experiment, among the glucose sensors, a sensor that did not contain PyOx and a sensor that did not contain GOD were produced, and the response current value was measured in the same manner as described above. These results are shown in curve e and curve f in FIG. As shown in FIG. 5, a glucose sensor containing only GOD as an enzyme (curve e in FIG. 5 reflects only the β-glucose concentration in the whole blood, so the response current value was the lowest. On the other hand, a glucose sensor containing only PyOx as an enzyme (curve f in FIG. 5) always has a response current higher than that in FIG. 5e, reflecting the sum of the concentrations of α-glucose and β-glucose in whole blood. As a result, the glucose sensor containing both PyOx and GOD (curve d in FIG. 5) always had a high response current value in the widest concentration range.
[0064]
【The invention's effect】
As is clear from the above description, according to the present invention, a biological sample such as blood and urine, a raw material in food industry, a product, and a substrate (specific component) contained in a sample such as fruit juice with high accuracy, A biosensor that can be quickly and easily quantified can be obtained.
[Brief description of the drawings]
FIG. 1 is a schematic plan view of a biosensor according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view of a biosensor according to an embodiment of the present invention in which a reaction layer is arranged in direct contact with a substrate. FIG. 4 is a schematic cross-sectional view of a biosensor according to an embodiment of the present invention in which layers are arranged on a substrate via a space. FIG. 4 is a graph showing a correlation between glucose concentration and response current of the biosensor according to an embodiment of the present invention. 5. Graph showing the correlation between glucose concentration and response current in the biosensor example of the present invention.
DESCRIPTION OF SYMBOLS 1 Electrically insulating board | substrate 2, 3 Lead 4 Working electrode 5 Counter electrode 6 Insulating layer 7, 37 Reaction layer 8 Electrode system 20 Spacer 30 Cover 31 Hole 38 Sample supply hole a Pyranose oxidase (PyOx) and glucose oxidase (GOD) in the reaction layer B) Change in response current value when containing only GOD c Change in response current value when containing only PyOx d Pyranose oxidase (PyOx) and glucose oxidase in the reaction layer Change in response current value when containing (GOD) e Change in response current value when containing only GOD f Change in response current value when containing only PyOx

Claims (7)

A biosensor for detecting glucose, which is disposed on an electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, or on the substrate or through a space with the substrate. And a reaction layer, wherein the reaction layer contains pyranose oxidase and glucose oxidase . The biosensor according to claim 1, wherein the pyranose oxidase is pyranose oxidase (EC 1.1.3.10). It said reaction layer is, the biosensor of claim 1 or 2, wherein contains further the electron acceptor. The biosensor according to claim 1, 2 or 3, wherein the reaction layer further contains one selected from sucrose hydrolase, maltose hydrolase, and lactose hydrolase. The biosensor of claim 3 Symbol mounting the electron acceptor is a ferricyanide ion. A biosensor for detecting glucose, which is disposed on an electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, or on the substrate or through a space with the substrate. A reaction layer, wherein the reaction layer contains pyranose oxidase, glucose oxidase, and ferricyan ion , and the reaction layer has 0.1 to 200 units of the pyranose oxidase per square centimeter. Ze and the glucoamylase of 200 units from 1 unit - biosensor containing the ferricyanide ions 3.30mg from the scan oxidase and 0.21 mg. A biosensor for measuring blood glucose level, which is the configuration according to any one of claims 1 to 6 .

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