JP2658769B2 - Biosensor - 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 capable of quickly and accurately quantifying a specific component in a sample.

[0002]

2. Description of the Related Art The following biosensor has been proposed as a method for simply quantifying a specific component in a sample without diluting or stirring the sample solution (Japanese Patent Application Laid-Open No. 1-291153). ).

In this biosensor, an electrode system is formed on an insulating substrate by a method such as screen printing, and an enzyme reaction layer comprising a hydrophilic polymer, an enzyme, and an electron acceptor is formed on the electrode system. It is integrated with the spacer and cover.

[0004] The operation of such a biosensor will be described below. When the sample solution is introduced onto the enzyme reaction layer from the sample supply port, the reaction layer dissolves, the enzyme reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. After the enzymatic reaction, the reduced electron acceptor is oxidized electrochemically,
The substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

Further, a plurality of electrode systems and an enzyme reaction layer are provided on one surface of an insulating substrate, and the quantity of a plurality of substrates is determined by one.
An embodiment implemented with one biosensor is also disclosed.

On the other hand, as a method for removing an electrode reaction interfering substance in a sample solution, the following biosensor has been proposed (Japanese Patent Application Laid-Open No. 2-310457). An enzyme reaction layer comprising a hydrophilic polymer, an enzyme and an electron acceptor is formed on an electrode system formed on an insulating substrate, and an electrode portion for removing interfering substances is further added. When the sample liquid is supplied to the biosensor, the reducing substance present in the sample liquid is electrolytically oxidized at the electrode for removing interfering substances. Thereafter, the enzyme reaction layer dissolves, the enzyme reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. Since the reducing substance in the sample solution was removed in advance by the interfering substance removing electrode, a stable sensor response was obtained.

[0007]

However, such a conventional biosensor has the following problems when quantifying a plurality of components in a sample solution at a time. Since multiple electrode systems are formed on the same surface of an insulating substrate, when the enzyme and electron acceptor dissolve in the sample solution, they move to different electrode systems, enabling accurate substrate quantification. This could have been the case, especially when the enzyme and electron acceptor were not immobilized on the electrode system.

Further, the conventional method for removing interfering substances has the following problems. If the concentration of the reducing substance in the sample liquid is high, the sample liquid reaches the enzyme reaction layer before the reducing substance is all electrolytically oxidized in the electrode portion for removing the interfering substance, and affects the electrode reaction. give. As a result, there has been a problem that an error occurs in the sensor response and the measurement accuracy is reduced.

An object of the present invention is to solve the above-mentioned problems, and an object of the present invention is to provide a biosensor capable of easily performing quick and accurate quantification.

[0010]

According to the present invention, in order to achieve the above object, a plurality of sets of electrode systems are provided on different surfaces of an insulating substrate. Further, a reaction layer containing a different enzyme or a combination of enzymes is formed directly or indirectly on a plurality of sets of electrode systems provided on different surfaces of an insulating substrate.

[0011] Further, a part of the plurality of electrode systems is used to quantify a reducing substance which affects the sensor response.

[0012]

According to this configuration, when a sample liquid containing a reducing substance is supplied to the biosensor, the reducing substance in the sample liquid is applied to the electrode system formed on the surface where the reaction layer containing the enzyme is not formed. Can be determined.

In the electrode system formed on the surface on which the reaction layer having the enzyme is formed, the enzyme reaction proceeds between the enzyme in the reaction layer dissolved in the sample solution and the substrate to be measured, and the electron reaction proceeds. The receptor is reduced. On the other hand, the electron acceptor is also reduced by a reducing substance in the sample solution. Therefore,
The amount of the reduced form of the electron acceptor on the electrode system formed on the surface on which the reaction layer having the enzyme is formed depends on both the concentration of the substrate to be measured and the concentration of the reducing substance.

According to the present invention, a reducing substance can be quantified by an electrode system formed on a surface on which a reaction layer containing an enzyme is not formed. A biosensor capable of accurately quantifying the substrate concentration can be realized.

Further, by providing a reaction layer having different enzymes or a combination of enzymes and a plurality of electrode systems on different surfaces of an insulating substrate, a plurality of substrates in a sample solution can be simultaneously and highly accurately. A biosensor capable of quantification can be realized.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to the drawings.

Embodiment 1 In this embodiment, a glucose sensor will be described as an example of a biosensor.

FIG. 1 is a cross-sectional view of a glucose sensor manufactured as an embodiment of the biosensor of the present invention. FIG. 2 is an exploded perspective view of the same glucose sensor, except for a reaction layer, as viewed from above in FIG. FIG. 4 is an exploded perspective view of the glucose sensor excluding the reaction layer, as viewed from below in FIG. 1. FIG. 4 is a base plan view of the glucose sensor as viewed from above in FIG.

Hereinafter, a method of manufacturing the glucose sensor will be described. Silver paste was printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3. Next, the measuring electrode 4 was formed by printing a conductive carbon paste containing a resin binder. The measurement electrode 4 is in contact with the lead 2. Next, the insulating paste was printed to form the insulating layer 10. The insulating layer 10 covers the outer peripheral portion of the measurement electrode 4, thereby keeping the area of the exposed portion of the measurement electrode 4 constant.

Next, a conductive carbon paste containing a resin binder was printed so as to be in contact with the leads 3 to form a counter electrode 5.

On the back surface of the insulating substrate 1 on which the electrode pattern is printed as described above, the leads 6 and
7, the measurement electrode 8, the counter electrode 9, and the insulating layer 11 are formed by printing.
Was manufactured.

In addition to the above method, the base 30 can also be manufactured by laminating insulating substrates each having an electrode pattern formed on only one side thereof.

Further, the electrode pattern of the measuring electrode 8 and the counter electrode 9 does not necessarily have to be the same as the pattern of the measuring electrode 4 and the counter electrode 5, and for example, a pattern as shown in FIG. 5 can be used.

Next, glucose oxidase (EC1.1.
3.4; hereinafter abbreviated as GOD) and a mixed aqueous solution obtained by dissolving potassium ferricyanide as an electron acceptor in a 0.5% by weight aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was added dropwise. The reaction layer 20 was formed by drying in a hot air drier at 10 ° C. for 10 minutes.

As described above, the manufacturing process can be simplified by dropping and drying the mixed solution of the hydrophilic polymer, the enzyme and the electron acceptor at once.

After the reaction layer 20 was formed as described above, the covers 13 and 17 and the spacers 12 and 16 were adhered in a positional relationship shown by a dashed line in FIGS. When a transparent material such as a polymer is used for the cover and the spacer, the state of the reaction layer and the state of introduction of the sample solution can be extremely easily checked from the outside.

When the cover is mounted, the sample liquid is supplied to the sample supply holes 14 and 18 at the tip of the sensor due to the capillary action in the space formed by the cover, the spacer and the insulating substrate.
It is easily introduced into the space by a simple operation of contacting the space.

In order to make the supply of the sample liquid smoother, it is preferable that the lecithin layer is formed by further developing an organic solvent solution of lecithin from the sample supply section to the portion extending from the reaction layer to drying.

When the above-mentioned lecithin layer is provided, even if the space formed by the insulating substrate 1, the covers 13, 17 and the spacers 12, 16 is so large that no capillary phenomenon can be exhibited, The liquid can be supplied.

To the glucose sensor prepared as described above, 10 μl of a mixed aqueous solution of glucose and ascorbic acid was supplied as sample liquid from the sample supply holes 14 and 18. The sample liquid reaches the air holes 15 and 19, respectively, and the reaction layer 20
Dissolves.

Next, +1 V is applied to the measurement electrode 8 with reference to the counter electrode 9.
Was applied, and the current value I ₀ after 5 seconds was measured. Measuring pole 8,
Since there is no enzyme and electron acceptor on the counter electrode 9,
I ₀ is the oxidation current of ascorbic acid present in the sample solution. Further, since there is no hydrophilic polymer or the like on the measurement electrode 8 and the counter electrode 9 that suppresses substance diffusion, I ₀ can be obtained immediately after the supply of the sample liquid.

On the other hand, on the measurement electrode 4 and the counter electrode 5, glucose is oxidized by GOD in the dissolved reaction layer 20, and potassium ferricyanide is reduced to form potassium ferrocyanide. Further, the potassium ferricyanide is reduced by ascorbic acid.

One minute after supplying the sample solution, +0.5 V was applied to the measurement electrode 4 with the counter electrode 5 as a reference, and the current value I ₁ was measured 5 seconds later. I ₁ is an oxidation current when potassium ferrocyanide generated by the above two kinds of reduction reactions is oxidized. The glucose concentration calculated from I ₁ and I ₀ was in good agreement with the amount of glucose contained in the sample solution regardless of the ascorbic acid concentration.

By arranging the two kinds of electrode systems on different surfaces of the insulating substrate, the reaction layer constituents such as GOD do not move onto the measurement electrode 8 and accurate quantification is possible. Was.

Embodiment 2 FIG. 6 is a sectional view of a glucose sensor manufactured as another embodiment of the biosensor of the present invention.

A base 30 was formed on an insulating substrate 1 made of polyethylene terephthalate by screen printing in the same manner as in Example 1.

A mixed aqueous solution of GOD, potassium ferricyanide and CMC was dropped and dried on the measurement electrode 4 and the counter electrode 5 in the same manner as in Example 1 to form a reaction layer 20. Next,
Potassium ferricyanide and CMC on measuring electrode 8 and counter electrode 9
Was dropped and dried to form a potassium ferricyanide-CMC layer 21. Further, in the same manner as in Example 1, the glucose sensor was manufactured integrally with the covers 13 and 17 and the spacers 12 and 16.

10 μl of a mixed aqueous solution of glucose and ascorbic acid was supplied as sample liquid to the glucose sensor prepared as described above through the sample supply holes 14 and 18. The sample liquid reaches the air holes 15 and 19, and the reaction layer 20 and the potassium ferricyanide-CMC layer 21 dissolve.

On the measuring electrode 8 and the counter electrode 9, the potassium ferricyanide-CMC layer 21 is dissolved in the sample solution, and potassium ferricyanide is reduced by ascorbic acid in the sample solution. Ten seconds after the sample solution was supplied, +0.5 V was applied to the measurement electrode 8 based on the counter electrode 9 and the current value was measured five seconds later. The current value was proportional to the ascorbic acid concentration in the sample solution.

On the other hand, on the measurement electrode 4 and the counter electrode 5, the reaction layer 20 is dissolved in the sample solution. Potassium ferricyanide in the reaction layer 20 is reduced by ascorbic acid in the sample solution,
Potassium ferrocyanide is produced through two types of reduction reactions, that is, the reduction that occurs when glucose in a sample solution is oxidized by GOD.

One minute after supplying the sample solution, +0.5 V was applied to the measurement electrode 4 with the counter electrode 5 as a reference, and the current value I was measured 5 seconds later.

I is the sum of the oxidation current when potassium ferrocyanide generated by the reaction with ascorbic acid is oxidized and the oxidation current when potassium ferrocyanide generated by reducing glucose when oxidized by GOD is oxidized. It is.

The ascorbic acid concentration in the sample solution was quantified from the responses at the measurement electrode 8 and the counter electrode 9, and the glucose concentration in the sample solution could be calculated from the amount of potassium ferrocyanide obtained from I.

When the oxidation current is measured by the measurement electrode 8 and the counter electrode 9, accurate quantification cannot be performed if GOD exists on the measurement electrode 8 and the counter electrode 9. Therefore, by forming both electrode systems on different surfaces of the substrate as in the present invention, it is possible to completely prevent the GOD from moving onto the measurement electrode 8, and as a result, highly accurate measurement becomes possible. Embodiment 3 A sugar sensor will be described as another embodiment of the biosensor of the present invention.

Hereinafter, a method of manufacturing the sugar sensor will be described with reference to FIG. A base 30 shown in FIG. 4 was manufactured in the same manner as in Example 1. Next, a mixed aqueous solution of GOD, potassium ferricyanide and CMC was dropped on one of the two electrode systems (measurement electrode 4 and counter electrode 5) and dried to form a reaction layer 20.

Further, fructose dehydrogenase (EC1.1.99.11; hereinafter abbreviated as FDH) as an enzyme, potassium ferricyanide as an electron acceptor, and hydroxyethylcellulose (hereinafter abbreviated as HEC) as a hydrophilic polymer are used as a buffer solution (pH). = 4.5) was dropped on the other electrode system (measurement electrode 8, counter electrode 9) and dried to form an FDH reaction layer 22. Further, in the same manner as in Example 1, the sugar sensor was integrated with the cover and the spacer.

As a sample solution, 10 μl of a mixed aqueous solution of glucose and fructose was applied to the thus prepared sugar sensor.
Is supplied from the sample supply holes 14 and 18, and after 1 minute, +0.5 V is applied to the measurement electrode 4 based on the counter electrode 5, and after 2 minutes, +0.5 V is applied to the measurement electrode 8 based on the counter electrode 9. 5
The current value after 2 seconds was measured.

The electrode system on which the reaction layer 20 is arranged (measurement electrode 4,
At the counter electrode 5), a current value corresponding to the glucose concentration was obtained, and at the electrode system (measuring electrode 8, counter electrode 9) in which the FDH reaction layer 22 was arranged, a current value corresponding to the fructose concentration was obtained.

The reaction layer 20 and the FDH reaction layer 2 were added to the sample solution.
When 2 is dissolved, the substrate in the sample solution is finally oxidized by the enzyme. The electron transfer there reduces potassium ferricyanide to potassium ferrocyanide. Next, by applying the above-mentioned constant voltage, an oxidation current was obtained when the generated potassium ferrocyanide was oxidized, and this current value corresponded to the substrate concentration in the sample solution.

The GOD used for the reaction layer 20 and the GOD
The FDH used in Example 1 differs in the pH condition at which the maximum enzyme activity is obtained. As described above, the optimum pH condition generally differs depending on the type of the enzyme used.

When both reaction layers are arranged on the same surface of the substrate, the buffer component contained in the FDH reaction layer moves into the reaction layer containing GOD by diffusion into the sample solution, and the optimum pH condition is obtained. May not be obtained. Furthermore, since there is a possibility that the enzyme may move onto a plurality of electrode systems due to diffusion in the sample solution, it is necessary to set conditions under which the enzyme does not move due to immobilization or the like, which may limit the sensor configuration.

By arranging reaction layers containing different enzymes on different surfaces of an insulating substrate as in the present embodiment, when each reaction layer is dissolved in a sample solution, the constituent elements of each reaction layer are dissolved. Can be prevented from moving with each other,
The pH on each electrode system can be easily set to an optimum value, and the enzyme can be freely diffused in the sample solution.

When juice was supplied to the sugar sensor as a sample solution and the sensor response was measured, glucose and fructose in the juice could be quantified accurately.

In this embodiment, different enzymes are used,
Although a biosensor for quantifying a plurality of substrate components in a sample solution has been described, it is also possible to configure a biosensor having a reaction layer using the same enzyme on different surfaces of an insulating substrate. In this case, since the same substrate in the sample solution is quantified, for example, the substrate can be quantified based on the average value of the oxidation current value obtained in each electrode system, and more accurate measurement can be performed.

In the above embodiment, the reaction layer 20,
Although the method of forming the potassium ferricyanide-CMC layer 21 and the FDH reaction layer 22 in contact with the electrode system surface has been described, it is not always necessary. When integrated with the cover and the spacer, a space is formed above the electrode system by the cover, the spacer, and the insulating substrate. A reaction layer can be formed at an appropriate place as long as it corresponds to the wall surface of the space portion of the cover, the spacer, and the insulating substrate. Since the sample liquid supplied to the sensor fills the space, the reaction layer can be dissolved.

In the above embodiment, the method of quantifying glucose and fructose was described. However, the present invention can be widely applied to systems involving an enzyme reaction such as an alcohol sensor, a lactic acid sensor, a cholesterol sensor, and an amino acid sensor.

In the above embodiment, GOD and FDH were used as enzymes.
However, besides this, invertase, mutarotase, alcohol oxidase, lactate oxidase, cholesterol oxidase, xanthine oxidase, amino acid oxidase and the like can also be used.

In the above examples, carboxymethylcellulose and hydroxyethylcellulose were used as hydrophilic polymers. However, the present invention is not limited to these. Other cellulose derivatives, specifically, hydroxypropylcellulose, methylcellulose, ethylcellulose, ethylcellulose Hydroxyethylcellulose, carboxymethylethylcellulose 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 The same effect was obtained by using.

On the other hand, as the electron acceptor, p-benzoquinone, phenazine methosulfate, methylene blue, a ferrocene derivative and the like can be used in addition to the potassium ferricyanide shown in the above embodiment.

In the above embodiment, the method of dissolving the enzyme and the electron acceptor in the sample solution has been described. However, the present invention is not limited to this, and the present invention is also applicable to the case where the enzyme and the electron acceptor are insoluble in the sample solution by immobilization. be able to.

In the above embodiment, a bipolar electrode system having only a measurement electrode and a counter electrode has been described. However, if a three-electrode system including a reference electrode is used, more accurate measurement can be performed.

[0062]

As is clear from the above description, according to the present invention, even a sample solution containing a reducing substance which may interfere with the sensor response can be obtained without a pretreatment for removing the obstacle. Accurate measurement can be performed. Furthermore, high-precision quantification of a plurality of components in a sample solution can be performed simultaneously.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a glucose sensor according to one embodiment of the present invention.

FIG. 2 is an exploded perspective view of the glucose sensor, except for a reaction layer, viewed from above.

FIG. 3 is an exploded perspective view of the glucose sensor, viewed from below, excluding a reaction layer.

FIG. 4 is a top plan view of the glucose sensor viewed from above.

FIG. 5 is a plan view showing another example of the base used for the biosensor of the present invention.

FIG. 6 is a cross-sectional view of a glucose sensor manufactured as another embodiment of the biosensor of the present invention.

FIG. 7 is a cross-sectional view of a sugar sensor manufactured as still another embodiment of the biosensor of the present invention.

[Explanation of symbols]

 Reference Signs List 1 Insulating substrate 2, 3, 6, 7 Lead 4, 8 Measurement electrode 5, 9 Counter electrode 10, 12 Insulating layer 12, 16 Spacer 13, 17 Cover 14, 18 Sample supply hole 15, 19 Air hole 20 Reaction layer 21 Potassium ferricyanide-CMC layer 22 FDH reaction layer 30 Base

Claims (4)

(57) [Claims] An insulating substrate, a plurality of sets of electrode systems formed on different surfaces of the insulating substrate, and a reaction layer formed directly or indirectly on the insulating substrate. Wherein the reaction layer contains at least an enzyme, a hydrophilic polymer, and an electron acceptor. 2. The biosensor according to claim 1, wherein an electron acceptor is provided on a surface of the insulating substrate on which no reaction layer is formed. 3. An insulating substrate, a plurality of electrode systems formed on different surfaces of the insulating substrate, and a plurality of reaction layers formed directly or indirectly on the insulating substrate. Wherein each of the plurality of reaction layers contains at least an enzyme, a hydrophilic polymer, and an electron acceptor. 4. The biosensor according to claim 3, wherein enzymes or combinations of enzymes contained in the plurality of reaction layers are different from each other.