JPH06138080A - Biosensor - Google Patents

Abstract

PURPOSE:To provide a highly reliable biosensor. CONSTITUTION:After providing an electrode system composed mainly of a working electrode 4 and counter electrode 5 on an insulating substrate 1, a reactive layer 7 containing a phosphate, enzyme, electron acceptor, and hydrophilic high polymer is formed on the electrode system. In addition, a pH buffer agent (a substance having a pH buffering function in a solution state) is arranged on the substrate 1. Since the optical isomer mobile equilibrium reaction of pyranose is accelerated by the phosphate, a quick sensor response can be obtained. Because of the action of the pH buffer agent, in addition, highly accurate measurement can be performed without adjusting the pH of a sample solution.

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 quickly quantifying a specific component in a sample.

[0002]

2. Description of the Related Art A sucrose sensor will be described as an example of a biosensor having a system involving an equilibrium transfer reaction of optical isomers of pyranose in an enzymatic reaction process.

As a method for quantifying sucrose using an enzyme electrode, invertase (EC 3.2.1.26: I
NV), mutarotase (EC5.1.3.3: hereinafter abbreviated as MUT), glucose oxidase (EC1.
1.3.4: A method in which three enzymes (hereinafter abbreviated as GOD) and an oxygen electrode or a hydrogen peroxide electrode are combined is generally known (for example, Shuichi Suzuki "Biosensor" Kodansha). The above method will be described. Sucrose in the sample is hydrolyzed to α-glucose and fructose by INV. Next, MUT promotes the isomerization of α-glucose to β-glucose, and this β
-Glucose is selectively oxidized by GOD. In the presence of oxygen, oxygen is reduced to hydrogen peroxide in the oxidation reaction process by the GOD. At this time, the amount of oxygen decrease is measured by an oxygen electrode, or the amount of increase of hydrogen peroxide is measured by a hydrogen peroxide electrode to quantify sucrose.

[0004]

The enzyme reagent is often relatively expensive, but the biosensor having the above structure requires three kinds of enzymes, so that the cost is high and it is disadvantageous in practical use. I was received. Further, since this measurement system has a three-step enzyme reaction, the time required for measurement tended to be relatively long.

Further, the specific activity of the enzyme has a property of depending on the hydrogen ion concentration (pH), and the pH at which the highest activity is obtained (optimum pH) is different for each enzyme. Therefore, it is difficult to obtain a constant sensor response when the pH of the sample solution is different. Measurement without pretreatment of the sample solution makes the measurement operation extremely simple, but when the sensor response is affected by the pH of the sample solution, pretreatment to keep the pH constant is required, which is not convenient. Be damaged.

[0006]

In order to solve the above problems, the present invention provides an electrode system having at least a working electrode and a counter electrode on an insulating substrate, and a phosphate and an enzyme on the electrode system. The present invention provides a biosensor having a reaction layer mainly composed of an electron acceptor and a hydrophilic polymer. Further, a substance having a pH buffering action in a solution state (referred to as "pH buffer" in the present invention) is arranged on the electrode system.

[0007]

[Function] Phosphate ion (PO ₄ ^3- , HPO ₄ ^2- , H ₂ PO ₄
^- ) Accelerates the equilibrium transfer reaction of the optical isomers of pyranose, so that a rapid sensor response can be obtained.
Furthermore, an enzyme (MU that catalyzes the equilibrium transfer of optical isomers)
, Etc.) can be excluded from the biosensor component. As a result, an inexpensive and highly reliable biosensor can be obtained.

Further, the action of the pH buffer allows the enzymatic reaction to proceed within a certain pH range, and as a result, a highly accurate sensor response can be obtained regardless of the pH of the sample solution.

[0009]

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

(Example 1) As an example of a biosensor,
The sucrose sensor will be described. FIG. 1 is a cross-sectional view of a sucrose sensor manufactured as an example of the biosensor of the present invention, FIG. 2 is an exploded perspective view of the sucrose sensor shown in FIG. It is a figure which shows an example of the response of a sucrose sensor.

The method for producing the sucrose sensor will be described below. Silver paste was printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Next, a conductive carbon paste containing a resin binder was printed to form the working electrode 4. The working electrode 4 is in contact with the lead 2.

Next, an insulating paste was printed to form an 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. Further, the insulating layer 6 partially covers the leads 2 and 3.

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 5.

Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was dropped on the electrode system (working electrode 4, counter electrode 5) and dried to form a CMC layer. Formed. Subsequently, INV, GOD as an enzyme and potassium ferricyanide as an electron acceptor were added to the CMC layer in a phosphate buffer solution (0.2M: KH ₂ P).
O ₄ −0.2 M: Na ₂ HPO ₄ ; pH = 7.4) was added dropwise to the mixed solution, and the mixture was heated in a warm air dryer at 50 ° C. for 10 minutes.
It was dried for a minute to form the reaction layer 7.

When a mixed solution of a phosphate, an enzyme and an electron acceptor is dropped, the CMC layer made of a hydrophilic polymer is once dissolved, and the reaction layer 7 is formed in a form of being mixed with the enzyme in the subsequent drying process. To do. However, since it is not accompanied by stirring or the like, it is not in a completely mixed state, and the electrode system surface is in a state of being covered only with CMC. That is, since the enzyme and the electron acceptor do not come into contact with the surface of the electrode system, it is possible to prevent the adsorption of proteins on the surface of the electrode system.

After forming the reaction layer 7 as described above,
The cover 9 and the spacer 8 were adhered in a positional relationship as shown by the one-dot chain line in FIG. 2 to produce a sucrose sensor.

To this sucrose sensor, 3 μl of an aqueous sucrose solution was supplied as a sample solution from the sample supply hole 10. The sample solution reached the air holes 11 and the reaction layer 7 on the electrode system was dissolved.

In order to make the supply of the sample solution smoother, an organic solvent solution of lecithin (for example, toluene) is further spread over the reaction layer from the sample supply section (sensor tip) and dried. After forming the lecithin layer, the cover 9 and the spacer 8 may be adhered.

A voltage of +0.5 V is applied between the counter electrode 5 and the working electrode 4 of the electrode system after a fixed time from the supply of the sample liquid,
When the current value after the second was measured, a value proportional to the sucrose concentration in the sample solution was obtained.

FIG. 3 shows the measurement time-dependent characteristics of the sensor response of the sucrose sensor of the present invention and of the sucrose sensor prepared by removing the phosphate in the reaction layer. In FIG. 3, a is the sucrose sensor of the present invention, b is the sucrose sensor of the present invention from which phosphate is excluded, and c is a.
2 is a response of the sensor having a constitution in which MUT is further added to the reaction layer of the sucrose sensor of Example 1 to the sucrose aqueous solution (20 mM).

The sucrose sensor (a) of the present invention is MU
Although it is slightly inferior to the sensor (c) containing T, it can be seen that the sensor response becomes constant in a shorter time than the sensor (b) without phosphate.

Sucrose is hydrolyzed by INV to produce α-glucose, and this α-glucose is rapidly converted to β-glucose by phosphate, and this β-glucose is oxidized by GOD. The electrons transferred by the oxidation reaction by GOD reduce potassium ferricyanide to potassium ferrocyanide. Next, by applying the above voltage, an oxidation current of the produced potassium ferrocyanide is obtained, and this current value corresponds to the concentration of sucrose as a substrate.

(Example 2) As an example of a biosensor,
The sucrose sensor will be described.

In the same manner as in Example 1, the leads 2, 3 were formed on the insulating substrate 1 made of polyethylene terephthalate.
An electrode system (working electrode 4, counter electrode 5) and an insulating layer 6 were formed. Further, a CMC layer was formed on the electrode system.

Next, INV, GOD, potassium ferricyanide, and dipotassium phosphate were added to the CMC layer, and a maleic acid-tris (hydroxymethyl) aminomethane buffer solution (pH = 7.5: maleic acid-Tris buffer) was used. A mixed solution dissolved in a liquid) was dropped and dried in a warm air drier to form a reaction layer 7. Further, in the same manner as in Example 1, the sucrose sensor was manufactured by integrating it with the cover 9 and the spacer 8.

Although the method of forming the reaction layer 7 in contact with the surface of the electrode system has been described, it is not always necessary. When integrated with the cover 9 and the spacer 8 as shown in FIG. 2, a space is formed in the upper part of the electrode system by the cover 9, the spacer 8 and the insulating substrate 1. The reaction layer can be formed at an appropriate place as long as it is a portion corresponding to the wall of the space of the cover 9, the spacer 8 and the insulating substrate 1. Since the sample solution supplied to the sensor fills the space, the reaction layer can be dissolved.

3 μl of a sucrose aqueous solution as a sample solution was supplied from the sample supply hole 10 to the sucrose sensor manufactured as described above and the sensor response current was measured. As a result, a value proportional to the sucrose concentration was obtained. It was

(Example 3) As an example of a biosensor,
The sucrose sensor will be described.

In the same manner as in Example 1, the leads 2, 3 were formed on the insulating substrate 1 made of polyethylene terephthalate.
An electrode system (working electrode 4, counter electrode 5) and an insulating layer 6 were formed.

Next, a 0.5 wt% hydroxyethyl cellulose (hereinafter abbreviated as HEC) aqueous solution was dropped onto the electrode system and dried to form an HEC layer. Continuing, H
INV, MUT, GOD, potassium ferricyanide, and disodium phosphate on the EC layer [N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid]
-Sodium hydroxide buffer (pH = 7.2: below, HE
A mixed solution dissolved in a PES-NaOH buffer solution) was added dropwise and dried in a warm air dryer to form a reaction layer 7.

To the sucrose sensor manufactured as described above, 10 μl of a sucrose aqueous solution was dropped onto the reaction layer as a sample solution to dissolve the reaction layer, and after a fixed time, a constant voltage was applied between the working electrode and the counter electrode as in Example 1. The voltage was applied and the response current was measured.

Isomerization of glucose from α-form to β-form is M
Facilitated by the action of both UT and phosphate ions. Like the sucrose sensor of this example, by having both MUT and phosphate ions in the reaction layer, the rate of isomerization of glucose from α-form to β-form can be further accelerated. As a result, the sensor response can be obtained in a shorter time.

Example 4 As an example of a biosensor,
The glucose sensor will be described.

In the same manner as in Example 1, the leads 2, 3 were formed on the insulating substrate 1 made of polyethylene terephthalate.
An electrode system (working electrode 4, counter electrode 5) and an insulating layer 6 were formed.

Next, a 0.5 wt% hydroxypropylcellulose (hereinafter abbreviated as HPC) aqueous solution was dropped onto the electrode system and dried to form an HPC layer. Subsequently, a mixed solution prepared by dissolving GOD, potassium ferricyanide, and dipotassium phosphate in a sodium dihydrogen citrate-sodium hydroxide buffer (pH = 5.5) was dropped on the HPC layer, and the mixture was heated at 50 ° C. The reaction layer 7 was formed by drying for 10 minutes in a warm air dryer. Further, the cover and the spacer were adhered in the same manner as in Example 1 to manufacture a glucose sensor.

A glucose solution prepared as described above was supplied with 10 μl of an aqueous glucose solution as a sample solution to dissolve the reaction layer, and after a certain period of time, a constant voltage was applied between the working electrode and the counter electrode to give a response. The current was measured.

GOD has a function of specifically oxidizing only the β-form among glucose isomers, but glucose has an α-form and a β-form in an equilibrium state in an aqueous solution. When the glucose sensor of this example is used, the α-form of glucose is converted to β-form by the action of phosphate ions added in advance in the reaction layer.
Since the isomerization to the body is carried out rapidly, a response current proportional to the total glucose amount in the sample solution can be obtained in a short time.

Furthermore, when MUT is added to the reaction layer,
The measurement time can be further shortened and the measurement accuracy can be improved.

The optimum pH of the GOD used in this example is also
Is around 5, and the enzymatic reaction is performed at pH 5 regardless of the pH of the sample solution due to the buffering action of the pH buffer (sodium dihydrogen citrate-sodium hydroxide) added in the reaction layer.
You can proceed in the vicinity. Therefore, highly accurate measurement is possible.

As the buffer solution, sodium dihydrogen citrate-NaOH (pH = 5.5) was used, but other than this, citric acid-trisodium citrate, citric acid-tripotassium citrate, citric acid. Potassium dihydrogen-NaO
Similar effects were obtained even when the combination of H, sodium hydrogen maleate-NaOH, potassium hydrogen phthalate-NaOH, and succinic acid-sodium tetraborate was adjusted to a pH value of about 5 to 6.

Example 5 As an example of the biosensor,
The sucrose sensor will be described. FIG. 4 is a sectional view of the sucrose sensor of this embodiment.

In the same manner as in Example 1, the leads 2, 3 were formed on the insulating substrate 1 made of polyethylene terephthalate.
An electrode system (working electrode 4, counter electrode 5) and an insulating layer 6 were formed, and a CMC layer was formed on the electrode system.

Next, the INV, MUT,
GOD and potassium ferricyanide and phosphate buffer _{(0.2M: KH 2 PO 4 -0.2M} : Na 2 HPO 4; p
The mixed solution dissolved in H = 7.4) was added dropwise and dried in a warm air dryer at 50 ° C. for 10 minutes to form a reaction layer 7.

Next, CMC was added to phosphate buffer (0.5M:
_{KH 2 PO 4 -0.5M: Na 2} HPO 4; pH = 7.4)
The solution dissolved in was dropped onto the inside of the cover 9 and dried to form the pH buffer layer 20. The cover 9 on which the pH buffer layer 20 was formed and the spacer 8 were bonded in the same manner as in Example 1 to fabricate a sucrose sensor (the cross section is shown in FIG. 4).

A sucrose standard solution having a pH of 3 to 7 was supplied to this sucrose sensor as a sample solution, and the response was measured in the same manner as in Example 1. As a result, a value proportional to the sucrose concentration was obtained without depending on the pH of the sample solution. was gotten.

Example 6 As an example of a biosensor,
The sucrose sensor will be described.

In the same manner as in Example 1, the leads 2, 3 were formed on the insulating substrate 1 made of polyethylene terephthalate.
An electrode system (working electrode 4, counter electrode 5) and an insulating layer 6 were formed. Further, a CMC layer was formed on the electrode system.

Then, a mixed aqueous solution of INV, GOD, potassium ferricyanide and dipotassium phosphate was dropped on the CMC layer and dried to form a reaction layer 7. Further, as in the first embodiment, the cover 9 and the spacer 8 are formed.
A sucrose sensor was manufactured by integrating with the above.

3 μl of an aqueous sucrose solution was used as a sample solution in the sucrose sensor in the same manner as in Example 1 to supply the sample supply hole 10
Then, the sensor response current was measured and the value was proportional to the sucrose concentration.

Example 7 As an example of a biosensor,
The sucrose sensor will be described.

In the same manner as in Example 1, the leads 2, 3 were formed on the insulating substrate 1 made of polyethylene terephthalate.
An electrode system (working electrode 4, counter electrode 5) and an insulating layer 6 were formed. Further, a CMC layer was formed on the electrode system.

Next, INV and MUT are formed on the CMC layer.
And GOD and potassium ferricyanide in a phosphate buffer (0.6 M: KH ₂ PO ₄ -0.6 M: Na ₂ HPO ₄ ; p
The mixed solution dissolved in H = 7.4) was added dropwise and dried in a warm air dryer at 50 ° C. for 10 minutes to form a CMC-enzyme-potassium ferricyanide layer. Next, the CMC-
2 wt% of polyvinylpyrrolidone (hereinafter abbreviated as PVP) as a hydrophilic polymer on the enzyme-potassium ferricyanide layer
% Ethanol solution was developed and then dried to form a reaction layer 7.

By thus forming the structure in which the surface of the reaction layer is covered with the hydrophilic polymer layer, the smoothness of the surface can be enhanced. As a result, when the sample liquid is supplied to the sensor, it is possible to suppress the generation of bubbles generated by the air left in the undulating portion of the surface, and it is possible to reduce the variation in the response.

In particular, when the concentration of the pH buffering agent in the reaction layer is high as in this example, many irregularities are formed on the surface of the reaction layer due to crystal precipitation, so that the reaction layer formed by the hydrophilic polymer is used. Smoothing the surface is effective.

Although the fructose sensor and the glucose sensor are shown in the above embodiments, the present invention can be applied to a glucose-6-phosphate sensor, a maltose sensor, a lactose sensor and a cellulose sensor. In this case, as the enzyme, alkaline phosphatase, maltase, β-galactosidase, and cellulase may be used in order.

In the sucrose sensor of the above example, KH ₂ PO ₄ -Na ₂ HPO ₄ (pH = 7.4) and HEPES-NaOH (pH = 7.2) and maleic acid-Tris (pH = 7) were used as buffer solutions. .5) was used, but in addition to these, KH ₂ PO ₄ —K ₂ HPO ₄ , NaH ₂ PO ₄ —K ₂
HPO ₄ , NaH ₂ PO ₄ —Na ₂ HPO ₄ , Citric acid-N
a ₂ HPO ₄ , citric acid-K ₂ HPO ₄ , Tris-Tri
s hydrochloride, [N-tris (hydroxymethyl) methyl-
2-Aminoethanesulfonic acid] -NaOH, [piperazine-N, N'-bis (2-ethanesulfonic acid)]-Na
The same effect was obtained even when the combination of OH was adjusted to a value near pH 7 to 8.

Further, although CMC, HEC, HPC and PVP were used as the hydrophilic polymer in the above examples, the present invention is not limited to these and other cellulose derivatives, specifically, methyl cellulose, ethyl cellulose and ethyl. Hydroxyethyl cellulose, carboxymethyl ethyl cellulose may be used, and further, 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 can be used. Also obtained the same effect.

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

Further, although the method of dissolving the enzyme and the electron acceptor in the sample solution has been shown in the above-mentioned examples, the invention is not limited to this, and the invention can be applied to the case of being insolubilized in the sample solution by immobilization. be able to.

Further, in the above embodiment, the bipolar electrode system having only the working electrode and the counter electrode has been described, but the three-electrode system including the reference electrode enables more accurate measurement.

[0061]

As described above, according to the present invention, a highly reliable biosensor can be obtained at low cost.

[Brief description of drawings]

FIG. 1 is a sectional view of a sucrose sensor manufactured as an example of a biosensor of the present invention.

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

FIG. 3 is a diagram showing a response example of the sucrose sensor.

FIG. 4 is a sectional view of a sucrose sensor according to another embodiment of the present invention.

[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 Sample supply hole 11 Air hole 20 pH buffer layer

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Shiro Nankai 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (7)

[Claims] 1. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer formed on the electrode system, wherein the reaction layer is at least A biosensor comprising an enzyme, an electron acceptor, a hydrophilic polymer, and a phosphate. 2. The biosensor according to claim 1, wherein a pH buffer is formed on or in contact with an insulating substrate. 3. The bio according to claim 1, wherein the reaction layer is formed by sequentially stacking a first layer made of a hydrophilic polymer and a second layer containing at least an enzyme, an electron acceptor and a phosphate. Sensor. 4. The reaction layer according to claim 1, wherein the reaction layer comprises a first layer made of a hydrophilic polymer, a second layer containing at least an enzyme, an electron acceptor and a phosphate, and a hydrophilic polymer. A biosensor formed by sequentially stacking a third layer made of. 5. The biosensor according to claim 1 or 2, wherein the enzyme is a combination of invertase and glucose oxidase, or glucose oxidase. 6. The biosensor according to claim 1, wherein the enzyme is a combination of invertase, glucose oxidase and mutarotase, or a combination of glucose oxidase and mutarotase. 7. The biosensor according to claim 1 or 2, wherein the enzyme is selected from alkaline phosphatase, maltase, β-galactosidase, and cellulase.