JP3437016B2 - Biosensor and method of quantifying substrate using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor and a method for quantifying a substrate using the biosensor for easily and rapidly quantifying a substrate in a sample.

[0002]

2. Description of the Related Art A glucose quantification method will be described as an example of a substrate quantification method. As an electrochemical quantification method of glucose, glucose oxidase (EC1.
1.3.4: Hereinafter, a method in which an oxygen electrode or a hydrogen peroxide electrode is combined with an oxygen electrode or a hydrogen peroxide electrode is generally known (for example, Shuichi Suzuki "Biosensor" Kodansha). GOD is a substrate β using oxygen as an electron acceptor.
-D-glucose is selectively oxidized to D-glucono-δ-lactone. With this reaction, oxygen is reduced to hydrogen peroxide. Glucose is quantified by measuring the oxygen consumption at this time with an oxygen electrode or by measuring the amount of hydrogen peroxide produced with a hydrogen peroxide electrode.

However, in the above method, as can be inferred from the reaction process, the measurement result is greatly affected by the dissolved oxygen concentration, and the measurement becomes impossible under the condition of no oxygen. Therefore, a new type glucose sensor has been developed which does not use oxygen as an electron acceptor but uses an organic compound such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or a metal complex as an electron acceptor. In this type of sensor, the glucose concentration can be obtained from the oxidation current by oxidizing the reduced form of the electron acceptor generated as a result of the enzymatic reaction at the electrode.

Further, by using such an electron acceptor in place of oxygen, it may be possible to accurately carry a known amount of GOD and electron acceptor on the electrode in a stable state. In this case, the electrode system and the reaction layer can be integrated in a state close to a dry state. Disposable glucose sensors based on this technology have attracted much attention in recent years because glucose concentrations can be easily measured simply by introducing a specimen sample into a sensor chip inserted in a measuring instrument. . Such a method is applicable not only to the quantitative evaluation of glucose but also to the quantitative evaluation of other substrates,
It is currently the subject of many studies.

[0005]

As described above, the technique of using the electron acceptor and further integrating the electrode system and the reaction layer has enabled a simple electrochemical quantitative evaluation of the substrate.
Moreover, with the introduction of the three-electrode system, it has become possible to perform evaluation with higher accuracy. However, when performing quantitative determination with a three-electrode type disposable sensor, an error and variation may occur in the measurement result due to the selection of the electrode material of the reference electrode to be used and the manufacturing method.

[0006]

The present invention provides a biosensor comprising an electrode system having a working electrode, a counter electrode and a reference electrode made of silver formed on an insulating substrate, and a reaction layer containing at least an enzyme. A method for quantifying the substrate by detecting a change in the substance concentration during the reaction of the enzyme with the substrate in the sample based on the electrochemical response obtained by applying a potential to the working electrode using In the above, before applying the potential to the working electrode, the anode potential is applied to the reference electrode to react with the water-soluble chloride contained in the sample or the water-soluble chloride provided in the sensor, and silver /
A silver chloride electrode is produced.

Further, the biosensor used here comprises an electrode system having a working electrode, a counter electrode and a reference electrode formed on an insulating substrate, and a reaction layer containing at least an enzyme, and the reference electrode is made of silver. Therefore, the reaction layer contains water-soluble chloride. The reaction layer preferably contains an electron acceptor.
Further, the reaction layer preferably contains a hydrophilic polymer.

When a polarizable electrode such as carbon, silver, or platinum is independently introduced into the reference electrode of a three-electrode type disposable sensor, the electrode potential generated at the electrode interface, that is, the reference potential is the ion contained in the sample. Greatly depends on the activity of. Therefore, the electrode potential may change greatly depending on the dissolved species in the sample and the difference in concentration. Therefore, in the present invention, a commonly used silver / silver chloride electrode is introduced as the ideal non-polarizing electrode into the reference electrode of the sensor to stabilize the reference electrode potential and improve the sensor response characteristic.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to specific examples. [Example 1] As an example of a quantification method, quantification of glucose will be described. The structure of the electrode system used in this example is shown in FIG. On an insulating substrate 1 made of polyethylene terephthalate, silver paste was printed by screen printing to form leads 2, 3 and 4. Next, a conductive carbon paste containing a resin binder was printed to form a working electrode 5 and a counter electrode 8. The working electrode 5 is in contact with the lead 2 and the counter electrode 8 is in contact with the lead 4.
Next, the insulating layer 6 was formed by printing an insulating paste.
The insulating layer 6 covers the outer peripheral portion of the working electrode 5, thereby keeping the area of the exposed portion of the working electrode 5 constant. Further, by exposing the tips of the leads 3, the reference electrode 7
Was formed. Further, the insulating layer 6 partially covers the leads 2, 3, and 4.

Next, an aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) was dropped onto the electrode system and dried to form a CMC layer. Further, an aqueous solution containing GOD as an enzyme and potassium ferricyanide as an electron acceptor was dropped on the electrode system and dried to form a reaction layer. Next, in order to make the supply of the sample solution onto the reaction layer smoother, an organic solvent solution of lecithin, such as a toluene solution, is added to the sample supply section (the tip of the sensor).
To a lecithin layer by spreading over the reaction layer and drying. Then, the cover 9 and the spacer 10
In FIG. 1, the glucose sensor was manufactured by adhering to the substrate 1 in the positional relationship shown by the one-dot chain line.

3 μl of human blood was used as a sample solution in this sensor.
Was supplied from the sample supply hole 11. The sample solution has air holes 12
The reaction layer on the electrode system was dissolved up to the portion. Simultaneously with the supply of the sample liquid, a constant anode potential was applied to the reference electrode 7 with reference to the counter electrode 8. As a result, silver chloride produced by reacting with chlorine ions contained in human blood is deposited on the surface of silver of the reference electrode, and a silver / silver chloride electrode is formed. After the lapse of a predetermined time, the potential application to the reference electrode 7 was stopped. After a lapse of a predetermined time, a constant potential was applied to the working electrode 5 with reference to the reference electrode 7, and the current value after 5 seconds was measured. The ferricyanide ion, GOD, and glucose in human blood react to oxidize glucose to gluconolactone and reduce the ferricyanide ion to ferrocyanide ion. A current response is obtained by oxidizing this ferrocyanide ion at the working electrode 5. As a result, a current response depending on the glucose concentration in the sample solution was obtained. The coefficient of variation of this response was 2.4%. On the other hand, it was 4.0% in the comparative example measured without depositing silver chloride on the reference electrode 7. As described above, according to the present invention, it was confirmed that the coefficient of variation was smaller, that is, the variation in sensor response was reduced.

[Example 2] Similar to Example 1, an electrode system was prepared on an insulating substrate. Next, CMC is placed on the electrode system.
The CMC layer was formed by dropping the aqueous solution of and dropping it. Further, an aqueous solution containing GOD as an enzyme and sodium chloride as a chloride was dropped on the electrode system and dried to form a reaction layer. Next, in order to more smoothly supply the sample solution onto the reaction layer, an organic solvent solution of lecithin, such as a toluene solution, is spread over the reaction layer from the sample supply section (sensor tip) and dried to lecithin. After forming the layers, the cover 9 and the spacer 10 were adhered to the substrate in the positional relationship shown by the one-dot chain line in FIG. 1 to produce a glucose sensor.

3 μl of an aqueous glucose solution as a sample solution was supplied to this sensor through the sample supply hole 11. The sample solution reached the air holes 12 and the reaction layer on the electrode system was dissolved. Simultaneously with the supply of the sample liquid, a constant anode potential was applied to the reference electrode 7 with reference to the counter electrode 8. As a result, chlorine ions eluted from the reaction layer react with silver to deposit silver chloride, and a silver / silver chloride electrode is formed on the reference electrode 7. After the lapse of a predetermined time, the potential application to the reference electrode 7 was stopped. After a lapse of a predetermined time, a constant potential was applied to the working electrode 5 with reference to the reference electrode 7, and the current value after 5 seconds was measured. GOD, oxygen and glucose react to oxidize glucose to gluconolactone and reduce oxygen to hydrogen peroxide. A current response is obtained by oxidizing this hydrogen peroxide at the working electrode 5. As a result, a current response depending on the glucose concentration in the sample solution was obtained.

The coefficient of variation of this response was 2.4%.
On the other hand, it was 4.0% in the comparative example measured without depositing silver chloride on the reference electrode 7. By thus containing sodium chloride as a chloride in the reaction layer, the same measurement as in Example 1 could be performed even when the sample solution did not contain chloride. Similar results were obtained when sodium chloride was carried on the cover or the substrate.

In the above examples, the sensor having the CMC layer was described, but the sensor response which depends on the glucose concentration was obtained also in the sensor excluding the CMC layer.
Although CMC is used as the hydrophilic polymer, the hydrophilic polymer is not limited thereto, and polyamino acids such as polylysine, polyvinyl alcohol, and polystyrene sulfonic acid can also be used. Further, although the sensor using potassium ferricyanide as the electron acceptor is described, p-benzoquinone, phenazine methosulfate, methylene blue, ferrocene derivative and the like can be used in addition to potassium ferricyanide. A sensor response is also obtained when oxygen is the electron acceptor.

In addition to glucose oxidase shown in the above examples, lactate oxidase, cholesterol oxidase, xanthine oxidase and the like can be used as the enzyme. On the other hand, as the chloride to be added, potassium chloride, lithium chloride, calcium chloride or the like can be used in addition to sodium chloride shown in the examples.
In the above embodiment, an example of the electrode system is shown in FIG. 1, but the arrangement of electrodes and leads is not limited to these.
Further, in the above-mentioned embodiment, the sensor having the cover member adhered has been described, but the present invention is not limited to this, and a sensor response having a glucose concentration can be obtained even in a sensor having no cover member. Although the method of dissolving the reaction layer in the sample solution has been described in the above embodiment, the method is not limited to this and can be applied to the case where the reaction layer is insolubilized in the sample solution.

[0017]

As described above, according to the present invention, it is possible to quantify a substrate with high reliability.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a glucose sensor used in an embodiment of the present invention, excluding a reaction layer.

[Explanation of symbols]

1 Insulating substrate 2, 3, 4 leads 5 Working pole 6 insulating layers 7 reference pole 8 opposite poles 9 cover 10 Spacer 11 Sample supply hole 12 air holes

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-6-174679 (JP, A) JP-A-6-130023 (JP, A) JP-A-4-319658 (JP, A) JP-A-2- 19758 (JP, A) JP-A-8-50112 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/327 G01N 27/30 311 G01N 27/416

Claims (4)

(57) [Claims] 1. An electrode system having a working electrode, a counter electrode and a reference electrode formed on an insulative substrate, and a reaction layer containing at least an enzyme, which is used for the reaction of the enzyme with the substrate in the sample. Apply a potential to the working electrode to change the substance concentration
In the biosensor that electrochemically detects with the electrode system based on the obtained electrochemical response, the reference electrode is made of silver and the reaction layer contains water-soluble chloride.
Before applying a potential to the working electrode, the reference electrode is
By applying a node potential, the reference electrode and the counter electrode
Reacts with the water-soluble chloride provided in the reaction layer,
A biosensor, characterized in that a silver / silver chloride electrode is produced on . 2. The biosensor according to claim 1, wherein the reaction layer contains an electron acceptor. 3. The reaction layer contains a hydrophilic polymer.
Or the biosensor according to 2. 4. An enzyme and a sample using a biosensor comprising an electrode system having a working electrode, a counter electrode and a reference electrode made of silver formed on an insulating substrate, and a reaction layer containing at least an enzyme. In the method for quantifying the substrate, a potential is applied to the working electrode by detecting a change in the substance concentration during the reaction with the substrate in the substrate based on the electrochemical response obtained by applying the potential to the working electrode. Before
The method is characterized in that a step of producing a silver / silver chloride electrode on the reference electrode is provided by reacting the water-soluble chloride contained in the sample or the water-soluble chloride contained in the sensor by applying an anode potential to the reference electrode. Substrate quantification method.