JP3494398B2 - Biosensor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD 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 Conventionally, various biosensors have been proposed as a method for easily quantifying a specific component in a sample without diluting or stirring the sample solution. The following sensor is disclosed as an example of a biosensor (Japanese Patent Laid-Open No. 062952/1990). That is, an electrode system consisting of a working electrode, a counter electrode and a reference electrode is formed on the insulating substrate by a method such as screen printing, and on this electrode system,
An enzyme reaction layer containing a hydrophilic polymer, a redox enzyme, and an electron acceptor was formed in contact with the electrode system. A buffer is added to the enzyme reaction layer as needed. When the sample solution containing the substrate is dropped on the enzyme reaction layer of the biosensor thus manufactured, the enzyme reaction layer dissolves in the sample solution, and the enzyme reacts with the substrate, and the electron acceptor Is reduced. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate in the sample solution can be quantified from the oxidation current value obtained at this time.

In principle, the biosensor as described above can measure various substances by selecting an enzyme whose substrate is a substance to be measured. Enzymes are protein-based and are often purified before being incorporated into sensors. Depending on the conditions of this purification process, the metal ion, which is a constituent element of the enzyme and is the active center of the enzyme, may be removed, and the three-dimensional structure of the enzyme may change. As a result, the substrate specificity of the enzyme changes or the activity decreases. Further, while the sensor is stored, it may absorb moisture in the atmosphere to be denatured. Therefore, the amount of the enzyme thus modified must be taken into consideration in the amount of the enzyme contained in the reaction layer. On the other hand, when the enzymatic reaction depends on the amount of enzyme in the reaction layer, the obtained response current value is not proportional to the concentration of the substrate. Therefore, the reaction layer must contain the enzyme in an amount sufficient to react with the substrate in the sample solution.

[0004]

When manufacturing a sensor as described above, it is necessary to incorporate a considerably large amount of enzyme into the reaction layer in comparison with the amount of the substrate expected to be contained in the sample. Will be required. Therefore, a considerable amount of enzyme is required per sensor, and the unit price of the sensor becomes very high. It is an object of the present invention to provide an inexpensive biosensor having high storage stability by maximizing the activity of the enzyme in the sensor, reducing the amount of enzyme supported per sensor.

[0005]

The biosensor of the present invention comprises an insulating substrate, an electrode system having at least a working electrode and a counter electrode formed on the substrate, and at least formed on or near the electrode system. Supplemented with pyrroloquinoline quinone
It is characterized by comprising a reaction layer containing dehydrogenase as an enzyme and an electron mediator, and a water-soluble salt of a divalent metal placed in the reaction layer or in the vicinity of the reaction layer so as to be isolated from the reaction layer. A preferred biosensor of the present invention is an electrically insulating substrate, an electrode system having at least a working electrode and a counter electrode formed on the substrate, and a sample liquid supplied to the electrode system between the substrate and the substrate combined with the substrate. A cover member that forms a sample liquid supply path, and at least pyrroloquinoline quinone formed on or near the electrode system.
It comprises a reaction layer containing dehydrogenase as a coenzyme and an electron mediator, and a water-soluble salt of a divalent metal placed in a place isolated from the reaction layer or the reaction layer of the sample supply path. In the preferred biosensor of the present invention, the metal salt is isolated from the reaction layer.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The biosensor according to the present invention has a water-soluble salt of a divalent metal in or near the reaction layer as described above. When a sample solution such as blood is added to the sensor, if a metal salt is present in the sample solution supply path, the sample solution is introduced into the reaction layer while dissolving the metal salt. When the metal salt is contained in the reaction layer, the metal salt dissolves in the sample solution after the sample solution reaches the reaction layer. The dissolved metal salt dissociates to generate divalent metal ions. This metal ion is incorporated into the enzyme from which the metal ion has been lost due to purification and long-term storage. As a result, the three-dimensional structure of the enzyme is reconstructed and the activity is restored. by this,
Since the activity of the enzyme contained in the reaction layer is promoted and the amount of the enzyme carried by the sensor can be minimized, it is possible to manufacture an inexpensive sensor. As such a metal salt, at least one selected from a calcium salt, a cadmium salt, a manganese salt, a magnesium salt and a strontium salt is preferable. These salts are chlorides, nitrates,
Sulfate is preferred.

Enzymes whose structure and activity are restored by the above metal salts include dehydrogenase and oxidase. Among the dehydrogenases, particularly glucose dehydrogenase, when a divalent metal ion, especially calcium ion is added, the activity of the enzyme is improved, and particularly good response characteristics are obtained. Also, good response can be obtained with fructose dehydrogenase. As oxidase,
Glucose oxidase is preferably used. Many of the metal salts are hygroscopic. When a hygroscopic metal salt is supported in the reaction layer or on the surface of the reaction layer, the metal salt absorbs water in the air and dissolves during the storage period of the sensor, so part of the reaction layer may dissolve. . As a result, the enzyme contained in the reaction layer becomes instable and causes activity deterioration such as denaturation. Therefore, in such a case, the metal salt should be installed separately from the reaction layer. By installing the metal salt separately from the reaction layer, it is possible to improve the storage stability of the sensor in an atmosphere in which water is present. On the other hand, storage of the sensor in an atmosphere where it is not necessary to consider moisture, for example,
Attach the sensor to an airtight flap such as an aluminum laminated film.
When packaged in I Lum may the metal salt is contained in the reaction layer. As described above, it is preferable to determine whether to install the metal salt in the reaction layer, the place separated from the reaction layer, or both, depending on the storage atmosphere. In addition, from the reaction layer at or near the reaction layer
Divalent metal salts are included in other parts including the isolated place
Even if it does, the effect does not change.

In addition, since the metal salt is easily dissolved when the sample solution is supplied, it is preferable to install the metal salt in the sensor in a state of being carried in the hydrophilic polymer. As an example of such a hydrophilic polymer, carboxymethyl cellulose is preferable because it has very high water solubility. In addition, polyethylamino acids such as hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polylysine, polystyrene sulfonic acid, gelatin and its derivatives, acrylic acid and its salts, and methacrylic acid. And its salts, starch and its derivatives, maleic anhydride and its salts, agarose gel and its derivatives are also preferably used.

The amount of the water-soluble salt of a divalent metal in the present invention is such that when the salt is dissolved in the sample solution supplied to the sensor, the concentration of the divalent metal is in the range of 0.01 to 2 mg / ml. Is preferred. The lower limit of the concentration is the minimum concentration required for binding a metal such as calcium to an enzyme. Further, if the concentration exceeds 2 mg / ml, when the sample solution is injected into the sensor, it becomes difficult for the reaction layer to quickly dissolve in the sample solution. The sensor of the present invention is particularly directed to the quantification of substrates in blood. In such a sensor, the amount of enzyme carried per unit area (1 mm ² ) of the reaction layer is 1 μg to 50 μg / mm ² . Therefore, based on the unit area of the reaction layer, the amount of the water-soluble salt of a divalent metal is 0.003 to 0.6 μg / mm ² . On the other hand, as shown in the following examples, in a sensor in which the amount of sample liquid is about 3 μl, the amount of enzyme required for one sensor chip is 0.0
1 to 0.5 mg / chip. Therefore, the water-soluble salt of a divalent metal used per sensor chip is 0.03.
~ 6 μg.

The reaction layer according to the present invention contains an electron mediator. As the electron mediator, at least one selected from ferricyan ion, p-benzoquinone and its derivative, phenazine methosulfate, methylene blue, ferrocene and its derivative is preferably used. Further, the reaction layer may contain a hydrophilic polymer in addition to the above enzymes and electron mediators. By adding a hydrophilic polymer to the reaction layer, peeling of the reaction layer from the electrode system surface can be prevented. Further, the hydrophilic polymer also has an effect of preventing cracks on the surface of the reaction layer, and is effective in increasing the reliability of the biosensor. As such a hydrophilic polymer, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinylpyrrolidone,
Polymerization of polyvinyl alcohol, polyamino acids such as polylysine, polystyrene sulfonic acid, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride
The body and its salts, agarose gel and its derivatives are preferably used. As a method for measuring the oxidation current, there are a bipolar electrode method in which only a working electrode and a counter electrode are used, and a three-electrode method in which a reference electrode is added. The three-electrode method enables more accurate measurement.

[0011]

EXAMPLES The present invention will be described in more detail below with reference to specific examples. FIG. 1 is a schematic plan view of the biosensor according to the present invention with the reaction layer removed. On the electrically insulating substrate 1 made of polyethylene terephthalate, a silver paste is printed by screen printing to form the leads 2, 3
Is formed. Next, a carbon paste 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. Further, an insulating paste is printed on the substrate 1 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. Then, a carbon paste containing a resin binder is printed on the substrate 1 so as to come into contact with the leads 3 to form a ring-shaped counter electrode 5. A reaction layer is formed on the electrode system consisting of the electrodes 4 and 5. Then, the water-soluble salt 10 of the divalent metal is placed in at least one of the reaction layer and the place separated from the reaction layer.

FIG. 2 is an exploded perspective view of the biosensor from which the reaction layer and the water-soluble salt of divalent metal have been removed. A biosensor is manufactured by mounting the insulating substrate 1 of FIG. 1, the cover 9 having the air holes 11 and the spacer 8 in a positional relationship as shown by the one-dot chain line in FIG. At the slit 13 portion of the spacer 8, the substrate 1 and the cover 9
And the sample liquid supply path is formed. 12 is an opening of the sample liquid supply path. FIG. 3 is a perspective view of a cover member in which the spacer 8 and the cover 9 are superposed, and is arranged upside down with respect to FIG. Reference numeral 14 denotes a cover-side surface of the surface exposed in the space forming the sample liquid supply path. The reaction layer and / or the salt 10 may be provided on the surface of the cover member exposed to the sample liquid supply path. FIG. 4 is a vertical cross-sectional view of the main part of the biosensor according to the present invention. A reaction layer 7 containing an enzyme and an electron mediator is formed on an electrically insulating substrate 1 on which an electrode system is formed as shown in FIG. 1, and between the reaction layer 7 and the opening 12 of the sample liquid supply path. A water-soluble salt 10 of a divalent metal is supported separately from the reaction layer 7.

Example 1 5 μl of a mixed aqueous solution of pyrroloquinoline quinone-glucose dehydrogenase (hereinafter abbreviated as GDH) and potassium ferricyanide was dropped on the substrate 1 on which the electrode system was formed as shown in FIG. 1 and dried. Then, the reaction layer 7 was formed. The reaction layer 7 has a diameter of 3.6 m.
It was a circle of m and had a size that substantially covered the electrodes 4 and 5. A solution was prepared by dissolving 5 mg of calcium chloride in 10 ml of an aqueous solution of carboxymethyl cellulose (concentration: 0.5%). 5 μl of this solution was dropped on the substrate at a position indicated by 10 and dried in a warm air dryer at 50 ° C. for 10 minutes,
A water-soluble salt 10 of a divalent metal was supported on the substrate 1. A cover 9 and a spacer 8 were adhered to the above-mentioned substrate 1 in a positional relationship shown by a chain line in FIG. 2 to produce a biosensor.

As a glucose standard solution, the glucose concentration is 0, 200, 400, 600, and 1000 mg.
/ Dl was prepared, and this glucose standard solution was supplied to the sensor from the opening 12 of the sample solution supply path. Then, one minute after supplying the sample liquid, the working electrode 4 with the counter electrode 5 as a reference.
A pulse voltage of +0.5 V was applied to the sensor, and after 5 seconds, the current value flowing between the working electrode and the counter electrode was measured to measure the response characteristic of the sensor. The result is shown in FIG. As shown in FIG. 5, the obtained response current value was large, and the correlation between the response current value and the glucose concentration had excellent linearity. The prepared biosensor was stored in an atmosphere of 20% humidity for 6 months, and the response characteristics of the sensor were measured in the same manner. The result is shown in FIG. As shown in FIG. 5, the response characteristics of the biosensor after storage were almost the same as those immediately after the biosensor was manufactured.

Comparative Example 1 A sensor was manufactured in the same manner as in Example 1 except that the water-soluble salt of a divalent metal was not carried in the sensor. Then, in the same manner as in Example 1, the response characteristics of the biosensor were measured immediately after the sensor was manufactured and after storage for 6 months at a humidity of 20%. The result is shown in FIG. As shown in FIG. 5, the obtained response current value was reduced to about 60% of that of the biosensor manufactured in Example 1. Further, the response current value of the biosensor after being stored for 6 months was considerably lower than that of the sensor immediately after the sensor was manufactured, and the correlation between the response current value and the glucose concentration was also significantly reduced.

Example 2 A reaction layer was formed by dropping and drying a mixed aqueous solution of GDH and potassium ferricyanide and calcium chloride, and a water-soluble salt of a divalent metal was not carried alone in the sensor. A biosensor was produced in the same manner as in Example 1. Then, the response characteristics of the sensor were measured in the same manner as in Example 1 immediately after the sensor was manufactured and after the sensor was stored at a humidity of 20% for 6 months. The result is shown in Fig. 5.
Shown in. As shown in FIG. 5, the response current value of the biosensor after being stored for 6 months was about 85% of that of the sensor immediately after the sensor was manufactured.
%, And the correlation between the response current value and the glucose concentration also decreased slightly.

Example 3 Example 1 was repeated except that glucose oxidase (hereinafter abbreviated as GOX) was used as the enzyme.
A sensor was manufactured in the same manner as in. Then, in the same manner as in Example 1, the response characteristics of the sensor were measured immediately after the sensor was manufactured and after the sensor was stored at a humidity of 20% for 6 months. The result is shown in FIG. As shown in FIG. 6, the biosensor of this example had excellent response characteristics and storability.

Comparative Example 2 A sensor was prepared in the same manner as in Example 3 except that the water-soluble salt of divalent metal was not carried in the sensor. Then, in the same manner as in Example 3, the response characteristics of the sensor were measured immediately after the sensor was manufactured and after the sensor was stored at a humidity of 20% for 6 months. The result is shown in FIG. As shown in FIG. 6, the obtained response current value was reduced to about 60% of that of the biosensor manufactured in Example 3. Further, the response current value of the biosensor after being stored for 6 months was considerably lower than that of the sensor immediately after the sensor was manufactured, and the correlation between the response current value and the glucose concentration was also significantly reduced.

Example 4 A reaction layer was formed by dropping and drying a mixed aqueous solution of GOX and potassium ferricyanide and calcium chloride, and a water-soluble salt of a divalent metal was not supported alone in the sensor. A biosensor was produced in the same manner as in Example 3. Then, the response characteristics of the sensor were measured in the same manner as in Example 1 immediately after the sensor was manufactured and after the sensor was stored at a humidity of 20% for 6 months. The result is shown in Figure 6.
Shown in. As shown in FIG. 6, the response current value of the biosensor after being stored for 6 months was about 90% of that of the sensor immediately after the sensor was manufactured.
%, And the correlation between the response current value and the glucose concentration also decreased slightly.

Example 5 A sensor was produced in the same manner as in Example 1 except that fructose dehydrogenase (hereinafter abbreviated as FDH) was used as the enzyme. Then, in the same manner as in Example 1 except that the fructose standard solution was used as the standard sample solution, immediately after the sensor was manufactured and at a humidity of 20%, 6
The response characteristics of the sensor after storage for one month were measured. The result is shown in FIG. 7. As shown in FIG. 7, the obtained biosensor had excellent response characteristics and storability.

Comparative Example 3 A sensor was prepared in the same manner as in Example 5, except that the water-soluble salt of divalent metal was not carried in the sensor. Then, in the same manner as in Example 5, the response characteristics of the sensor were measured immediately after the sensor was manufactured and after the sensor was stored at a humidity of 20% for 6 months. The result is shown in FIG. 7. As shown in FIG. 7, the obtained response current value was reduced to about 65% of that of the biosensor manufactured in Example 5. Further, the response current value of the biosensor after being stored for 6 months was considerably lower than that of the sensor immediately after preparation of the sensor, and the correlation between the response current value and the fructose concentration was also significantly reduced. did.

Example 6 A reaction layer was formed by dropping and drying a mixed aqueous solution of FDH and potassium ferricyanide and calcium chloride, and a water-soluble salt of a divalent metal was not carried alone in the sensor. A biosensor was produced in the same manner as in Example 5. Then, the response characteristics of the sensor were measured in the same manner as in Example 1 immediately after the sensor was manufactured and after the sensor was stored at a humidity of 20% for 6 months. The result is shown in Fig. 7.
Shown in. As shown in FIG. 7, the response current value of the biosensor after storage for 6 months was about 85% of that of the sensor immediately after the sensor was manufactured.
%, And the correlation between the response current value and the glucose concentration also decreased slightly.

[0023]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to provide a biosensor that maximizes the activity of the enzyme in the sensor, reduces the amount of enzyme supported, and is inexpensive and has high storage stability. .

[Brief description of drawings]

FIG. 1 is a schematic plan view of a biosensor according to an embodiment of the present invention with a reaction layer removed.

FIG. 2 is an exploded perspective view of a biosensor according to another embodiment of the present invention from which a reaction layer and a water-soluble salt of a divalent metal are removed.

FIG. 3 is a perspective view of a cover member in which a cover and a spacer of the biosensor are overlapped with each other, which are arranged upside down with respect to FIG. 2.

FIG. 4 is a vertical cross-sectional view of a main part of the biosensor.

FIG. 5 is a response characteristic diagram of the biosensor in one example of the present invention with respect to a standard sample solution.

FIG. 6 is a response characteristic diagram of a biosensor in another example of the present invention with respect to a standard sample solution.

FIG. 7 is a response characteristic diagram of a biosensor in another example of the present invention with respect to a standard sample solution.

[Explanation of symbols]

1 Insulating Substrate 2, 3 Lead 4 Working Electrode 5 Counter Electrode 6 Insulating Layer 7 Reaction Layer 8 Spacer 9 Cover 10 Divalent Metal Water-Soluble Salt 11 Air Vent 12 Sample Liquid Supply Channel Opening 13 Slit 14 Sample Liquid Supply The surface on the cover side of the surface exposed in the space forming the road

Continuation of front page (56) Reference JP-A-9-43189 (JP, A) JP-A-8-278276 (JP, A) JP-A-2-245650 (JP, A) JP-A-2-102448 (JP , A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/327

Claims (5)

(57) [Claims] 1. An insulating substrate, an electrode system having at least a working electrode and a counter electrode formed on the substrate, and at least pyrroloquinoline formed on or near the electrode system.
A reaction layer containing a dehydrogenase using quinone as a coenzyme and an electron mediator, and a water-soluble salt of a divalent metal disposed in at least one of the reaction layer and a place separated from the reaction layer in the vicinity of the reaction layer. A biosensor characterized by: 2. An insulating substrate, an electrode system having at least a working electrode and a counter electrode formed on the substrate, and a sample liquid supply for supplying a sample liquid to the electrode system between the substrate and the substrate combined with the substrate. A cover member forming a channel, at least a pyrroloquinoline formed on or near the electrode system.
A water-soluble salt of a divalent metal, which is installed in at least one of a reaction layer containing dehydrogenase containing nonone as a coenzyme and an electron mediator, and a place separated from the reaction layer or the reaction layer of the sample solution supply path, A biosensor characterized by being provided. 3. The biosensor according to claim 1, wherein the water-soluble salt of a divalent metal is supported in a hydrophilic polymer film. 4. The water-soluble salt of a divalent metal is at least one selected from the group consisting of calcium salt, cadmium salt, manganese salt, magnesium salt and strontium salt.
The biosensor according to claim 1, which is a seed. 5. The amount of enzyme carried by the reaction layer is 1 to 50.
[mu] m / mm ^2, biosensor according to any one of claims 1-4 amount of the water-soluble salt is per unit area 0.003~0.6μg / mm ² of the reaction layer.