JPH05312761A - Biosensor and its manufacture - Google Patents

Abstract

PURPOSE:To obtain a biosensor wherein the responsiveness of a measuring operation in a solution under test can be measured in a short time and to obtain its manufacturing method. CONSTITUTION:A discrimination layer 7 which has carried and held a biological substance is formed in such a way that it is deposited on a partitioned space at the bottom part of a measuring chamber 21. As a result, it can be formed in a prescribed thickness having a small irregularity and in a wide surface area. Since the irregularity in the thickness of the discrimination layer 7 can be reduced, an action pole 5 and a counter pole 15 can be formed at a narrow interval without bringing the discrimination layer 7 into contact with the counter pole 15 when an insulating layer 11 is interposed between the action pole 5 and the counter pole 15 so as to ensure a prescribed distance. Thereby, the line of electric force of an electric double layer which is formed between the action pole 5 and the counter pole 15 can be made short. As a result, a noise current which flows at the early stage of a measuring operation can be converged in a short time, and the measuring time of a substance under test can be shortened.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for measuring a substance to be measured by measuring an amount of electrical change due to a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance, and a method for producing the biosensor. Is.

[0002]

2. Description of the Related Art Conventionally, as this type of biosensor, for example, a flat plate type biosensor disclosed in JP-A-61-50262 is known. That is, as shown in FIG. 4, the flat plate type biosensor 100 includes a ceramic substrate 101, a working electrode 103 and a counter electrode 105 formed on the ceramic substrate 101, and a space between the working electrode 103 and the counter electrode 105. An insulating layer 108 for insulation, an identification layer 107 formed by coating a gel-like substance carrying a biological substance such as an enzyme on the working electrode 103, and the terminal portions 109 and 111 of the working electrode 103 and the counter electrode 105, respectively. An electric measuring unit (not shown) that measures the current value between them
And the identification layer 107 side is the sensitive portion 113.

To measure a solution to be measured using this biosensor 100, the sensitive part 113 is immersed in the solution to be measured. As a result, the biological substance carried on the discrimination layer 107 undergoes a biochemical reaction with the substance to be measured contained in the solution to be measured to generate oxygen or hydrogen peroxide. The substance to be measured is measured by measuring the electric current associated with the redox caused by the generation of oxygen and the like in the electrical measuring section.

[0004]

In use of such a biosensor 100, it is required that the solution to be measured can be measured in the shortest possible time. However, the conventional biosensor 100 is difficult to measure in a short time because of the following reasons. That is, FIG. 5 shows the relationship between the current value and time when measured by the biosensor 100. In FIG. 5, the broken line indicates the initial current value A, the alternate long and short dash line indicates the measured current value B, and the solid line indicates the measured current value C.

Here, the initial current value A is a current value when the working electrode 103 and the counter electrode 105 are immersed in a buffer solution containing no substance to be measured. When the buffer solution is immersed in the dry working electrode 103 and the counter electrode 105, this initial current is applied to the working electrode 1
An electric double layer is formed between the electrode 03 and the counter electrode 105, and the electric current flows so as to cancel the electric double layer. The time t1 required for the initial current to converge is the working electrode 10
3 increases in proportion to the distance between the counter electrode 105 and the counter electrode 105. Also,
The measured current value B is a current value that flows based on the biochemical reaction between the substance to be measured and the biological substance when the substance to be measured is added to the buffer solution from the state where the initial current value A has converged. That is, the actually measured current value B is a current value accompanying only the biochemical reaction, and when the reaction is saturated, it converges to a predetermined value.
Further, the measured current value C is a current value when a sensitive solution 113 of the biosensor is immersed in a solution to be measured prepared by dissolving a material to be measured in a buffer solution, It is the sum of the current value A and the measured current value B.

By the way, for example, in order to measure glucose and the like contained in urine with the biosensor 100,
It is necessary to use the above measured current value C. The measured current value C includes the initial current value A, and the measurement is impossible while the initial current value A is large (time point t0 to time point t1).
It can be measured only after the initial current value A converges. The time t1 until the initial current value A converges is the working electrode 1
This can be reduced by shortening the length of the line of electric force between the electric double layers formed at 03 and the counter electrode 105. In order to shorten the length of the lines of electric force between the electric double layers, the distance between the working electrode 103 and the counter electrode may be shortened.

However, the flat plate type biosensor 100 described above is used.
In the above, the identification layer 107 is formed by coating and forming a sol-like substance carrying a biological material on the working electrode 103. However, when the distance between the working electrode 103 and the counter electrode 105 is reduced, the identification layer 107 is formed only on the working electrode 103. Cannot be formed, and the sol-like substance is applied even to the counter electrode 105, making measurement impossible. Therefore, the distance between the working electrode 103 and the counter electrode 105 is
It cannot be shorter than 20 μm and the measurement time is 30
It takes more than a second.

An object of the present invention is to solve the above-mentioned conventional techniques, and an object thereof is to provide a biosensor capable of measuring the response to be measured in a solution to be measured in a short time, and a manufacturing method thereof. To do.

[0009]

In order to solve the above-mentioned problems, the invention of claim 1 is to measure the amount of electrical change due to the biochemical reaction between the substance to be measured and the biological substance in the solution to be measured. In a biosensor for measuring a substance to be measured by, an insulating substrate, a working electrode laminated on the insulating substrate, an identification layer carrying the biological material, and laminated on the working electrode, and this identification layer An insulating layer laminated on the insulating substrate except for, a counter electrode laminated on the insulating layer, for measuring the amount of electrical change between the working electrode,
A measurement chamber for storing the solution to be measured and for removing a part of the insulating layer so as to immerse the stored solution to be measured in the discrimination layer and the counter electrode.

Further, the invention of claim 2 is a method for manufacturing a biosensor for measuring a substance to be measured by measuring an amount of electrical change accompanying a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance. , A step of forming an insulating substrate, a step of laminating a working electrode on the insulating substrate, a step of forming an insulating layer on the insulating substrate and the working electrode, and on the insulating layer,
A step of forming a counter electrode for measuring the amount of electrical change between the working electrode and the measurement chamber for storing the solution to be measured, and removing a part of the insulating layer so as to expose the working electrode. The method is characterized by including a step of forming and a step of depositing an identification layer carrying a biological substance on the working electrode exposed in the measurement chamber.

[0011]

In the invention of claim 1, when the measurement chamber is filled with the solution to be measured, the discrimination layer at the bottom of the measurement chamber is immersed in the solution to be measured. The substance to be measured in the solution to be measured reacts with the biological substance carried on the discrimination layer to generate oxygen or hydrogen peroxide. The generation of oxygen or the like causes an oxidation-reduction reaction on the working electrode, and a current flows between the working electrode and the counter electrode. The substance to be measured in the solution to be measured is measured based on this current.

Further, in the present invention, the identification layer is laminated on the working electrode provided at the bottom of the measurement chamber. Since the identification layer is formed so as to be deposited in the partitioned space at the bottom of the measurement chamber, it can be formed to have a predetermined thickness with little fluctuation and a large surface area. In this way, it is possible to reduce the variation in the thickness of the identification layer.Therefore, if an insulating layer is interposed between the working electrode and the counter electrode and a certain distance is secured, the working layer and the counter electrode do not come into contact with the identification layer. And can be formed at narrow intervals. Therefore, since the distance between the working electrode and the counter electrode can be shortened, the length of the electric force line between the electric double layers formed in both electrodes can be shortened.
As a result, the initial current can be converged in a short time,
The measurement time of the substance to be measured can be shortened. Further, in the present invention, since the measurement chamber is filled with the measurement target solution for measurement, the measurement target solution does not flow out from the measurement chamber, and the measurement can be accurately performed with a small amount of the measurement target solution.

According to the second aspect of the present invention, the measuring chamber is formed by laminating the insulating substrate and the insulating layer and removing a part of the insulating layer so as to expose the working electrode. The configuration of claim 1 can be preferably realized by such simple steps.

[0014]

Preferred embodiments of the present invention will be described below in order to further clarify the constitution and operation of the present invention described above.

FIG. 1 is a plan view of the sensitive portion of the biosensor, and FIG. 2 is a sectional view taken along line II-II of FIG.
The sensitive portion 1 of the biosensor includes an insulating substrate 3, a working electrode 5 formed on the insulating substrate 3, an identification layer 7 supporting the biological material and laminated on the working electrode 5. Insulating layer 11 formed on insulating substrate 3 and having window 9
And a counter electrode 15 formed in the insulating layer 11 and having an opening 13.
It has and.

The space formed on the side wall 9a of the window 9 and the upper surface of the discrimination layer 7 is a measurement chamber 21 for storing the solution to be measured and immersing it in the discrimination layer 7.

The discriminating layer 7 is a layer formed by supporting a gel-like substance on a biological substance that biochemically reacts with the substance to be measured in the solution to be measured. For example, a substance obtained by sol- zing glucose oxidase with cellulose is used. It is a dried and solidified layer.
Further, the working electrode 5 and the counter electrode 15 are the detection units 5a and 15a installed in the vertical direction of the measurement chamber 21, the connection terminal units 5b and 15b, the detection units 5a and 15a and the terminal unit 5b, respectively.
It is formed of wiring portions 5c and 15c which respectively connect to 15b. An electric measurement unit (not shown) is connected to the terminal portions 5b and 15b of the working electrode 5 and the counter electrode 15, respectively, and the terminal portions 5b and 1b are connected to the electric measurement unit.
The value of the current flowing between 5b is obtained to obtain the measurement result of the substance to be measured.

Next, a measuring method using the above biosensor will be described. The sensitive part 1 of the biosensor is immersed in the solution to be measured, and a constant voltage is applied between the working electrode 5 and the counter electrode 15 in this state. The substance to be measured in the solution to be measured undergoes a biochemical reaction by the action of the biological substance of the discrimination layer 7. A current flows along with this reaction, and the substance to be measured can be measured based on this current.

Next, the manufacturing process of the biosensor will be described. (1) Manufacturing process of insulating substrate 3 (FIG. 3A) First, the insulating substrate 3 is formed. As the step of forming the insulating substrate 3, a method of cutting out a plate material made of glass, resin, or a composite material thereof, or firing a ceramic green sheet can be adopted.

(2) Step of forming working electrode 5 (see FIG. 3)
(B)) The working electrode 5 is formed on the insulating substrate 3. As a process of forming the working electrode 5, a known thick film printing method, vapor deposition method, sputtering method or the like can be adopted. As the material of the working electrode 5, gold, platinum, palladium, silver, titanium and the like and alloys thereof can be used.

(3) Step of forming insulating layer 11 (FIG. 3)
(C)) An insulating layer 11 is laminated on the insulating substrate 3 and the working electrode 5.
As the step of forming the insulating layer 11, a method of forming a plate material made of an insulating material and adhering it, a method of applying a molten resin to a predetermined thickness to form a layer, and an insulating material by an evaporation method are used. It is possible to employ a method of depositing a predetermined thickness and forming the same. As the material of the insulating layer 11, for example, glass, ceramics, resin, or a composite material thereof can be used.

(4) Step of forming the counter electrode 15 (FIG. 3)
(D)) The counter electrode 15 is formed on the insulating layer 11. As the step of forming the counter electrode 15, the same method as the working electrode 5, that is, the thick film printing method, the sputtering method, the vapor deposition method, or the like can be adopted. The opening 13 of the counter electrode 15 may be formed in a circular shape, a polygonal shape, a slit shape, or a lattice shape other than the square shape as shown in FIG. As the material of the counter electrode 15, gold, platinum, palladium, copper, iron, silver, titanium, aluminum, zinc, nickel, tin, and alloys thereof can be used.

(5) Step of forming the measuring chamber 21 (see FIG. 3)
(E)) A part of the insulating layer 11 is removed through the opening 13 of the counter electrode 15 so as to expose the working electrode 5, and thus the measurement chamber 21
To form. As a process of forming the measurement chamber 21, for example, a method of forming a mask by a photoresist method and forming it by etching or the like can be adopted. Note that, as a process of forming the measurement chamber 21, a method may be adopted in which the window 9 becomes the measurement chamber 21 by forming the insulating layer 11 having the window 9 and stacking the insulating layer 11 on the insulating substrate 3. Good.

Further, as shown in FIG. 2, the side wall 9a of the measuring chamber 21 has a shape that widens from the opening 13 toward the inner side.
That is, it is desirable to etch the insulating layer 11 so that the peripheral edge of the opening 13 of the counter electrode 15 overhangs. In this case, from the peripheral edge of the opening 13 to the side wall 9
The distance L to a is preferably as long as possible within a range in which the counter electrode 15 can be supported by the insulating layer 11 and the counter electrode 15 can face the working electrode 5 via the measurement chamber 21. This is the measurement chamber 2 in which the counter electrode 15 and the working electrode 5 face each other in parallel.
The region of 1 is widened, and the lines of electric force between the electric double layers formed in the counter electrode 15 and the working electrode 5 can be formed perpendicularly to the respective surfaces of the counter electrode 15 and the working electrode 5 and can be made short. , Time t1 until the initial current value A converges
This is because (see FIG. 5) can be shortened.

(6) Step of forming discrimination layer 7 After the measurement chamber 21 is formed, the discrimination layer 7 is formed on the working electrode 5. The identification layer 7 is loaded with a biological substance that biochemically reacts with the substance to be measured to generate oxygen or hydrogen peroxide. As a step of supporting the biological material on the identification layer 7,
Well-known methods can be applied, for example, encapsulation method of encapsulating a biological substance in a polymer matrix, covalent binding method of immobilizing a biological substance by using a substance that covalently binds, adsorption of the biological substance on an insoluble membrane. An adsorption method or the like can be adopted. Here, since the identification layer 7 is formed on the bottom of the measurement chamber 21, a sol-like polymer body carrying a biological substance is formed, and the sol-like polymer body is cut down on the detection part 5a of the working electrode 5. By doing so, it can be suitably formed. As the biological substance, in addition to various enzymes, microorganisms and the like can be used, and the substance to be measured corresponding thereto can be measured.

In the above embodiment, the identification layer 7 is laminated on the working electrode 5 provided at the bottom of the measuring chamber 21. Since the identification layer 7 is formed so as to be deposited in the partitioned space at the bottom of the measurement chamber 21, it can be formed to have a predetermined thickness with little fluctuation and a large surface area.
In this way, since the variation in the thickness of the identification layer 7 can be reduced,
If the insulating layer 11 is interposed between the working electrode 5 and the counter electrode 15 and a predetermined distance is secured, the working electrode 5 and the counter electrode 15 can be formed at a narrow interval without bringing the identification layer 7 into contact with the counter electrode 15. it can. Therefore, since the distance between the working electrode 5 and the counter electrode 15 can be narrowed, the electric resistance between the two electrodes can be reduced, and the ratio of the distance between the two electrodes to the area of the working electrode 5 can be reduced. Therefore, the equipotential surface is formed parallel to the working electrode, and the lines of electric force between the electric double layers formed between the two electrodes can be shortened. As a result, the initial current can be converged in a short time, and the measurement time of the substance to be measured can be shortened.

Further, as described above, when the time until the initial current converges is shortened, the initial current value after a fixed time becomes small, and the S / N ratio to the current value associated with the actual reaction is improved. There is also an effect that various measurements can be performed.
Further, in the embodiment, since the measurement chamber 21 is filled with the solution to be measured for measurement, it is possible to accurately perform measurement with only a small amount of the measurement solution filled in the measurement chamber 21.

<Experimental example> Next, the biosensor according to the above-mentioned example was applied to a glucose sensor for measuring glucose, and its evaluation test was conducted. The sensitive section 1 of the biosensor was created by the following steps. First, the insulating substrate 3
Glass plate (made by Corning: product name 7059)
Was processed into a size of 50 mm length × 50 mm width × 0.5 mm thickness. Next, a mask was formed with a photoresist, and Pt was vapor-deposited to a thickness of 0.3 μm on the unmasked insulating substrate 3 by an evaporation method to form a working electrode 5. The size of the detection unit 5a of the working electrode 5 is 2 mm in length × 2 mm in width. Next, the insulating layer 11 was formed by applying a polyimide resin (manufactured by Toray Industries, Inc .: trade name Fortnis) on the insulating substrate 3 to a thickness of 3 μm. Then, the counter electrode 15 is formed by the vapor deposition method so that the opening 13 is formed on the insulating layer 11.
Was formed to a thickness of 0.5 μm. After that, a mask is formed on the counter electrode 15 and the insulating layer 11 by a photoresist method,
A part of the insulating layer 11 was etched through the opening 13 of the unmasked counter electrode 15 to form a measurement chamber 21. Next, glucose oxidase was dissolved in albumin to form a sol, and the solified substance was cut down and dried to form the discrimination layer 7.

Glucose was measured by connecting the sensitive part 1 of the biosensor produced by the series of steps to the electrical measuring part. That is, the buffer solution (0.1 M phosphate buffer, 2
A sample solution was prepared by adding 50 mg of glucose to (7 ° C). A constant voltage of 0.9 V was applied between the working electrode 5 and the counter electrode 15 of the sensitive part 1 of the biosensor. In this state, the sensitive part 1 was immersed in the sample solution.

As a result, the time until the initial current converges and the influence disappears was 5 seconds, which was shorter than 30 seconds of the conventional flat plate type biosensor. Therefore, the measurement time was 15 seconds, which could be shortened by 15 seconds as compared with the conventional case.

The present invention is not limited to the above-described embodiments, but can be carried out in various modes without departing from the scope of the invention, and the following modifications can be made.

In the above embodiment, the opening 13 of the counter electrode 15
When is narrow, it may be difficult for the solution to be measured to fill the measurement chamber 21. As a means for solving this, the opening 13
Hydrophilic polymer (eg, polyvinyl acetate) around the periphery of
Put on. As a result, the wettability is improved, and the solution to be measured can be reliably filled in the measurement chamber 21.

[0033]

As described above, according to the present invention of claim 1, since the discrimination layer is formed so as to be deposited in the partitioned space at the bottom of the measuring chamber, the predetermined thickness with little fluctuation is obtained. And can be formed with a large surface area. In this way, it is possible to reduce the variation in the thickness of the identification layer.Therefore, if an insulating layer is interposed between the working electrode and the counter electrode and a certain distance is secured, the working layer and the counter electrode do not come into contact with the identification layer. Can be formed at narrow intervals. Therefore, the distance between the working electrode and the counter electrode can be narrowed, so that the lines of electric force between the electric double layers formed between the two electrodes can be shortened. As a result, the initial current can be converged in a short time, and the measurement time of the substance to be measured can be shortened.

Further, in the present invention, since the measurement chamber is filled with the solution to be measured, the measurement chamber can be accurately measured with only a small amount of the solution to be measured.

Further, according to the present invention of claim 2, the measuring chamber is formed by laminating the insulating substrate and the insulating layer and removing a part of the insulating layer so as to expose the working electrode. The configuration of claim 1 can be preferably realized by such simple steps.

[Brief description of drawings]

FIG. 1 is a plan view showing a sensitive portion of a biosensor according to an embodiment of the present invention.

FIG. 2 is a sectional view taken along line II-II of FIG.

FIG. 3 is an explanatory view explaining a manufacturing process of a sensitive part of the biosensor according to the embodiment.

FIG. 4 is a perspective view showing a sensitive part of a conventional biosensor.

FIG. 5 is a graph showing a relationship between a current value flowing at the time of measurement by the biosensor and time.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Sensing part 3 ... Insulating substrate 5 ... Working electrode 5a ... Detecting part 5b ... Terminal part 5c ... Wiring part 7 ... Identification layer 9 ... Window part 9a ... Side wall 11 ... Insulating layer 13 ... Opening 15 ... Counter electrode 21 ... Measuring chamber

Claims (2)

[Claims] 1. A biosensor for measuring a substance to be measured by measuring an amount of electrical change caused by a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance, which comprises an insulating substrate and an insulating substrate. A working electrode laminated on the working electrode, an identification layer supporting the biological substance and laminated on the working electrode, an insulating layer laminated on the insulating substrate excluding the identification layer, and an insulating layer on the insulating layer. A counter electrode that is laminated and that measures the amount of electrical change between the working electrode and one of the insulating layers that stores the solution to be measured and soaks the stored solution to be measured in the identification layer and the counter electrode. A biosensor having a measurement chamber in which a part is removed and formed. 2. A method for manufacturing a biosensor for measuring a substance to be measured by measuring an amount of electrical change associated with a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance, wherein an insulating substrate is formed. A step for laminating the working electrode on the insulating substrate, a step for forming an insulating layer on the insulating substrate and the working electrode, and a step for measuring an electrical change between the working layer and the working electrode on the insulating layer. A step of forming a counter electrode, a step of storing a solution to be measured and forming a measurement chamber by removing a part of the insulating layer so as to expose the working electrode, and a step of forming a measurement chamber on the working electrode exposed in the measurement chamber. And a step of depositing an identification layer supporting a biological material, the method for manufacturing a biosensor.