JPH06109693A - Biosensor and measurement method using the same - Google Patents

Abstract

PURPOSE:To obtain a biosensor which can determine the specific constituent in a sample liquid rapidly and speedily by providing a reaction layer, a main electrode system, and a sub electrode system on an insulation substrate and further a hydrophilic macromolecular layer between both electrode systems. CONSTITUTION:A silver paste is printed on an insulation substrate 1 by screen printing, thus forming leads 2-5. Then, a conductive carbon paste containing resin binder is printed for forming a main electrode system measurement electrode 6 and a sub electrode system (a measuring electrode 8 and a counter electrode 9). The measurement electrode 6, the measurement electrode 8, and the counter electrode 9 contact the lead 2, the lead 4, and the lead 5, respectively. An insulation layer 10 is formed by printing an insulation paste, the insulation layer 10 covers the outer-periphery part of the measurement electrode 6, thus covering the leads 2-5 partially and the measurement electrode 8 and the counter electrode 9 completely. A conductive carbon paste containing the resin binder is printed so that it contacts the lead 3, thus forming a main electrode system counter electrode 7. A reaction layer 31 of hydrophilic macromolecule, oxygen, and electron receptor mixed liquid is formed on the main electrode system, a hydrophilic macromolecular layer 32 is formed between both electrodes, and then a cover 22 and a spacer 21 are adhered.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art The following biosensor has been proposed as a method for easily quantifying a specific component in a sample without diluting or stirring the sample solution (Japanese Patent Application Laid-Open No. 2-310457). ).

In this biosensor, an enzyme reaction layer composed of 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. It is a thing.

The operation of the biosensor will be described below by taking a glucose sensor as an example. When a sample solution containing glucose is supplied to the biosensor, the reducing substance existing in the sample solution is electrolytically oxidized at the electrode portion for removing interfering substances. After this, the enzyme reaction layer is dissolved, and glucose is oxidized by glucose oxidase which is a redox enzyme in the enzyme reaction layer. At this time, the electron acceptor in the enzyme reaction layer is reduced. Since the reducing substance in the sample solution is previously removed by the electrode portion for removing the interfering substance, it is possible to avoid interference such as reducing the electron acceptor.

When an appropriate constant voltage is applied between the measuring electrode and the counter electrode constituting the electrode system at the stage where all glucose in the sample solution has reacted, the reduced form of the electron acceptor is oxidized. By measuring this oxidation current value, the glucose concentration in the sample solution can be quantified.

[0006]

When quantifying a specific substrate in a sample solution containing a large amount of a reducing substance by using a biosensor having a conventional electrode for removing an interfering substance as described above, , Had the following problems.

When the concentration of the reducing substance in the sample solution is high, the sample solution reaches the enzyme reaction layer before the reducing substance is completely electrolytically oxidized in the electrode portion for removing the interfering substance, and the electron There is a problem that the sensor response is affected by reducing the receptor or directly oxidizing it at the electrode, and the measurement accuracy is reduced.

[0008]

In the present invention, a reaction layer, a main electrode system and a sub electrode system are provided on an insulating substrate, and a hydrophilic layer is provided between the main electrode system and the sub electrode system. A biosensor provided with a polymer layer is constructed.

Further, a measuring method is used in which the concentration of the reducing substance contained in the sample solution is quantified by the auxiliary electrode system and the concentration of the substrate is accurately quantified.

[0010]

With this configuration, when the sample liquid containing the substrate to be measured and the reducing substance is supplied to the biosensor, the reducing substance in the sample liquid can be quantified in the auxiliary electrode system. On the other hand, in the main electrode system, 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 acceptor is reduced. The electron acceptor is
At the same time, it is also reduced by the reducing substance in the sample solution.
Therefore, the amount of the reduced product of the electron acceptor produced on the main electrode system depends on the sum of the concentration of the substrate to be measured and the reducing substance.

According to the biosensor of the present invention, the hydrophilic polymer layer formed between the main electrode system and the sub electrode system is swollen by containing the sample liquid, and is swelled between the main electrode system and the sub electrode system by diffusion or the like. The effect of suppressing the mass transfer to the minimum can be obtained. If the enzyme forming the reaction layer is present on the sub-electrode system, the substrate to be measured reacts with the enzyme on the sub-electrode system, making it difficult to accurately quantify the reducing substance. Therefore, the hydrophilic polymer layer can maximally prevent the enzyme in the reaction layer from moving onto the auxiliary electrode system, and as a result, it becomes possible to obtain a highly accurate sensor response.

[0012]

EXAMPLES The present invention will be described below with reference to examples.

(Example 1) As an example of a biosensor,
The glucose sensor will be described.

FIG. 1 is an exploded perspective 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 glucose sensor without a reaction layer, and FIG. 3 is a base of the glucose sensor. It is a top view.

Silver paste was printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2, 3, 4, and 5. Next, a conductive carbon paste containing a resin binder was printed to form a measurement electrode 6 of the main electrode system and a sub electrode system (measurement electrode 8 and counter electrode 9). The measuring electrode 6 is in contact with the lead 2, the measuring electrode 8 is in contact with the lead 4, and the counter electrode 9 is in contact with the lead 5.

Next, the insulating layer 1 is printed by printing an insulating paste.
Formed 0. The insulating layer 10 covers the outer peripheral portion of the measurement electrode 6, and thereby keeps the area of the exposed portion of the measurement electrode 6 constant. Further, the insulating layer 6 includes the leads 2, 3,
It partially covers 4,5. The auxiliary electrode system (measurement electrode 8, counter electrode 9) is not completely covered by the insulating layer 10.

Next, a conductive carbon paste containing a resin binder was printed so as to come into contact with the leads 3 to form the counter electrode 7 of the main electrode system. As described above, the base 11 shown in FIG. 3 was produced.

Next, glucose oxidase (EC 1.1.3.4; hereinafter referred to as GOD) was used as an enzyme on the main electrode system.
And a ferricyanide potassium as an electron acceptor dissolved in a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer are added dropwise and dried to form the reaction layer 31. Formed.

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

Next, a 1 wt% CMC aqueous solution is dropped between the main electrode system and the sub electrode system on the insulating substrate 1 and dried to form C.
A hydrophilic polymer layer 32 made of MC was formed.

After forming the reaction layer as described above, the cover 22 and the spacer 21 were adhered to each other in a positional relationship shown by a chain line in FIG. When a transparent material such as a polymer is used for the cover and the spacer, the state of the reaction layer and the introduction state of the sample solution can be confirmed very easily from the outside.

Further, when the cover is attached, due to the capillarity of the space formed by the cover and the spacer, the sample liquid can be easily transferred to the reaction layer portion and the sub-electrode system portion by simply bringing the sample liquid into contact with the sample supply hole 23 at the sensor tip. be introduced.

In order to make the supply of the sample solution even smoother, an organic solvent solution of lecithin is spread over the reaction layer 31 from the sample supply section (sensor tip) before the cover and the spacer are bonded. Then, the lecithin layer may be formed by drying.

When the lecithin layer is provided, the sample liquid can be supplied even when the space formed by the insulating substrate 1, the cover 22 and the spacer 21 is of a size that cannot cause the capillary phenomenon. It will be possible.

3 μl of a mixed aqueous solution of glucose and ascorbic acid was supplied as a sample solution to the glucose sensor prepared as described above through the sample supply hole 23, and after 10 seconds, +1 V was applied to the measurement electrode 8 with the counter electrode 7 of the auxiliary electrode system as a reference. And apply 5
When the current value after the second was measured, a value corresponding to the ascorbic acid concentration in the sample solution was obtained.

Further, 1 minute after supplying the sample solution, +0.5 V was applied to the measuring electrode 6 with reference to the counter electrode 7 of the main electrode system, and the current value I was measured after 5 seconds. I is the sum of the oxidation current of potassium ferrocyanide produced by reducing potassium ferricyanide with ascorbic acid and the oxidation current of potassium ferrocyanide produced by reducing glucose when it is oxidized by GOD.

Since the ascorbic acid concentration is known by the auxiliary electrode system, the glucose concentration in the sample solution could be calculated from the oxidation current value obtained by the main electrode system.

When the sample solution is supplied to the glucose sensor,
The hydrophilic polymer layer 32 and the reaction layer 31 are dissolved. Since GOD and potassium ferricyanide in the reaction layer 31 are not particularly immobilized on the main electrode system, when the reaction layer dissolves in the sample solution, they diffuse into the sample solution. On the other hand, the hydrophilic polymer layer 32 of the present invention swells when dissolved in the sample liquid, and an effect of suppressing substance diffusion between the main electrode system and the sub electrode system is obtained. Therefore, it was possible to prevent the reaction layer constituents from moving onto the auxiliary electrode system in a short time, and as a result, the glucose concentration in the sample solution could be quantified with high accuracy.

(Example 2) On the insulating substrate 1 made of polyethylene terephthalate, the base 11 shown in FIG. 3 was formed by screen printing in the same manner as in Example 1.
A mixed aqueous solution of GOD, potassium ferricyanide, and CMC was dropped onto this main electrode system (measurement electrode 6, counter electrode 7) in the same manner as in Example 1.

Next, a mixed aqueous solution of potassium ferricyanide and CMC was dropped onto the sub electrode system (measurement electrode 8 and counter electrode 9), and polyvinyl pyrrolidone (hydrophilic polymer) was added as a hydrophilic polymer between the main electrode system and the sub electrode system. Hereafter, abbreviated as PVP) 2
A wt% aqueous solution is dropped and dried to form a reaction layer on the main electrode system.
A potassium ferricyanide-CMC layer was formed on the sub electrode system, and a hydrophilic polymer layer made of PVP was formed between the main electrode system and the sub electrode system.

Further, in the same manner as in Example 1, a glucose sensor was manufactured by integrating it with a cover and a spacer.

When 3 μl of a mixed aqueous solution of glucose and ascorbic acid was supplied as a sample solution to the glucose sensor prepared as described above through the sample supply hole, potassium ferricyanide-CMC layer on the sub-electrode system and hydrophilic polymer layer composed of PVP. , The reaction layers on the main electrode system were sequentially dissolved.

As in Example 1, the sample solution was supplied to
After 0.5 seconds, +0.5 V was applied to the working electrode of the sub electrode system with reference to the counter electrode of the sub electrode system, and the oxidation current value after 5 seconds was measured.
_{It was set to 0} .

Potassium ferricyanide on the secondary electrode system is reduced by ascorbic acid to produce potassium ferrocyanide. The oxidation current I ₀ obtained by applying +0.5 V is due to the oxidation of potassium ferrocyanide. Therefore, it is proportional to the ascorbic acid concentration in the sample solution.

Further, one minute after supplying the sample solution, +0.5 is applied to the working electrode of the main electrode system with reference to the counter electrode of the main electrode system.
V was applied and the current value I ₁ after 5 seconds was measured. I ₁ is an oxidation current of potassium ferrocyanide produced by reduction with ascorbic acid and a sum of potassium ferrocyanide produced by reduction when glucose is oxidized by GOD. Quantifying the ascorbic acid concentration from I ₀ ,
The glucose concentration in the sample solution could be calculated from the result and I ₁ .

The hydrophilic polymer layer made of PVP has the effect of preventing the GOD in the reaction layer from moving onto the sub-electrode system, as in Example 1.

Example 3 The base 11 shown in FIG. 3 was formed on the insulating substrate 1 made of polyethylene terephthalate by screen printing in the same manner as in Example 1.
GOD and CM on this main electrode system (measurement electrode 6, counter electrode 7)
The mixed aqueous solution of C was added dropwise. Further, an aqueous solution of hydroxyethyl cellulose (hereinafter abbreviated as HEC) as a hydrophilic polymer is dropped between the main electrode system and the sub electrode system and dried to form a reaction layer on the main electrode system, and a reaction layer of the main electrode system and the sub electrode system. In between
A hydrophilic polymer layer composed of was formed. Further, in the same manner as in Example 1, the glucose sensor was manufactured by integrating it with the cover and the spacer.

When 3 μl of a mixed aqueous solution of glucose and ascorbic acid was supplied as a sample solution to the glucose sensor prepared as described above through the sample supply hole, the hydrophilic polymer layer made of HEC and the reaction layer on the main electrode system were sequentially dissolved. did.

10 seconds after supplying the sample liquid, +1 V was applied to the working electrode of the sub-electrode system with reference to the counter electrode of the sub-electrode system, and 5
When the current value after the second was measured, a value corresponding to the ascorbic acid concentration in the sample solution was obtained.

Then, 1 minute after supplying the sample solution, +1 V was applied to the working electrode of the main electrode system with reference to the counter electrode of the main electrode system, and the current value I after 5 seconds was measured. Glucose in the sample solution reacts with oxygen by the action of GOD to generate hydrogen peroxide. The generated hydrogen peroxide is oxidized by applying the above-mentioned constant voltage, and an oxidizing current is obtained. Further, ascorbic acid in the sample solution is also oxidized by applying the above-mentioned constant voltage, and an oxidizing current is obtained. Therefore, the current value I of the main electrode system
Depends on both the glucose concentration and the ascorbic acid concentration, the glucose concentration could be calculated from the current value I of the main electrode system and the ascorbic acid concentration obtained by the auxiliary electrode system.

In the above measurement, if the GOD moves onto the sub-electrode system, accurate measurement cannot be performed.
The hydrophilic polymer layer made of C swelled with the sample solution and suppressed mass transfer between the main electrode system and the sub electrode system.
Is prevented from moving onto the auxiliary electrode system, and as a result, glucose can be quantified with high accuracy.

In the above embodiment, the reaction layer 31,
Hydrophilic polymer layer 32, potassium ferricyanide-CMC
Although the method of forming the layer in contact with the surface of the electrode system has been described, it is not always necessary. When integrated with the cover and the spacer, the cover, the spacer, and the insulating substrate form a space above the electrode system. The reaction layer, the hydrophilic polymer layer, and the potassium ferricyanide-CMC layer can be formed in appropriate places as long as they are the portions corresponding to the walls of the space of the cover, the spacer, and the insulating substrate. Since the sample liquid supplied to the sensor fills the space, the above effect can be obtained.

Although the glucose sensor is shown in the above embodiments, the present invention can be widely used in a reaction system involving an enzyme such as a sucrose sensor, a fructose sensor, a lactic acid sensor, a cholesterol sensor, an alcohol sensor, and an amino acid sensor.

Although carboxymethylcellulose, polyvinylpyrrolidone and hydroxyethylcellulose were used as the hydrophilic polymer in the above-mentioned examples, the hydrophilic polymer is not limited to them, and polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, and methacryl. Acid and its salt, starch and its derivative, maleic anhydride and its salt, and cellulose derivative, specifically, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, the same effect can be obtained. Was obtained.

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

Further, in addition to glucose oxidase, fructose dehydrogenase, invertase, mutarotase, lactate oxidase, cholesterol oxidase, alcohol oxidase, xanthine oxidase, amino acid oxidase and the like can be used as the enzyme.

Further, in the above embodiment, the bipolar electrode system having only the measuring electrode and the counter electrode was described, but more accurate measurement is possible by using the three-electrode system in which the reference electrode is added.

[0048]

As described above, according to the biosensor of the present invention, even a sample solution containing a reducing substance that interferes with the sensor response can be processed with high accuracy without pretreatment for removing the interferent. You can take measurements.

Further, according to the substrate concentration measuring method using the biosensor of the present invention, the variation in sensor response can be made extremely small.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a glucose sensor of an embodiment of a biosensor of the present invention.

FIG. 2 is an exploded perspective view of the glucose sensor excluding a reaction layer.

FIG. 3 is a base plan view of the glucose sensor.

[Explanation of symbols]

 1 Insulating substrate 2, 3, 4, 5 Lead 6 Measurement electrode of main electrode system 7 Counter electrode of main electrode system 8 Measurement electrode of auxiliary electrode system 9 Counter electrode of auxiliary electrode system 10 Insulating layer 11 Base 21 Spacer 22 Cover 23 Sample Supply hole 24 Air hole 31 Reaction layer 32 Hydrophilic polymer layer

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location 7235-2J G01N 27/46 336 B

Claims (4)

[Claims] 1. A reaction layer comprising an insulating substrate, a main electrode system and a sub electrode system formed on the insulating substrate, a hydrophilic polymer layer, and a reaction layer containing an enzyme. Is formed directly or indirectly on the main electrode system, and the hydrophilic polymer layer is arranged between the main electrode system and the sub electrode system. 2. The biosensor according to claim 1, wherein the reaction layer contains an electron acceptor. 3. The biosensor according to claim 2, further comprising an electron acceptor on the auxiliary electrode system. 4. A measuring method, which comprises using the biosensor according to claim 1 and quantifying a substrate concentration based on a difference in electric output between the main electrode system and the auxiliary electrode system.