JPH0814563B2 - Enzyme electrode manufacturing method and enzyme electrode manufacturing apparatus - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing an enzyme electrode used as an enzyme sensor and the like and an apparatus for producing the enzyme electrode, and in particular, partially deactivating an enzyme by heat. The present invention relates to a method for manufacturing an enzyme electrode and a manufacturing apparatus for the same using the method.

[Conventional technology]

In the differential enzyme electrode, one working electrode has no responsiveness to the substrate, the response of the active substance other than the substrate in the sample is detected, and it is specified by determining the current difference from the other working electrode. The substrate concentration of is calculated. Therefore, it is necessary to give the enzyme activity to only one of the two working electrodes. As a method for satisfying this need, (a) a method of immobilizing an enzyme only on one working electrode, (b) immobilizing an enzyme on two working electrodes in advance and then one of the enzyme immobilization parts There is a method of inactivating, that is, inactivating the enzyme.

Here, in the differential enzyme electrode, unless the diffusion of the reactant on the surfaces of both working electrodes is made uniform, the difference in the current between the two working electrodes due to the reactant becomes inaccurate, and therefore accurate measurement cannot be performed. Become.

Of the above two methods, the method (a) requires the formation of an enzyme immobilizing substance of the same quality as the enzyme-immobilized electrode side in the absence of enzyme on the non-enzyme-immobilized electrode side, Difficult and difficult to achieve product homogeneity, it is rarely used for electrodes other than specific applications. On the other hand, the method (b) is advantageous in terms of process because homogeneity can be obtained relatively easily by the partial deactivation method.

[Problems to be Solved by the Invention]

Among the above methods, in view of the advantage of the method (b), several methods have been proposed in the past, but the invention described in Japanese Patent Publication No. 62-14276 is particularly manufactured. It can be said that it is excellent in terms of electrode performance and the like. According to the method of the present invention, an enzyme-immobilized membrane having an enzyme is preliminarily formed on both electrodes, and a photomask is arranged so as to cover portions other than the inactivated portion, and in this state, electromagnetic waves such as ultraviolet rays and X-rays are irradiated. , A method of deactivating these electromagnetic wave irradiation portions. In this method, although the performance of the electrode manufactured as described above is good, a large-scale device such as a masking device and an electromagnetic wave irradiation device is required. Further, there is a problem in terms of productivity such that deactivation requires a relatively long time, and if the enzyme-immobilized portion is thick, it is necessary to further extend irradiation time.

[Means for solving the problem]

The present invention is configured in view of the above points, as a method of deactivating the enzyme immobilization portion of a predetermined portion of the electrode, a heat source is directly or indirectly arranged with respect to the deactivation target portion, A method for partially deactivating an enzyme by heat, a method for producing an enzyme electrode based on this method, and further applying this method to an apparatus for producing an enzyme electrode, deactivates each of a plurality of enzyme electrodes connected in series. It is an enzyme electrode manufacturing apparatus in which a heat source is accurately positioned with respect to a portion.

[Action]

When a heat source is placed for a few seconds on the part of the enzyme-immobilized membrane formed over the pair of working electrodes that is to be deactivated, the enzyme in the heat source placement part is deactivated and a differential enzyme electrode is obtained. be able to.

[Specific Description of Invention Structure]

First, the following method is included in the deactivation method by heat that constitutes the present invention.

(I) When the thickness of the enzyme-immobilized part is smaller than the distance between the two working electrodes, a heat source is placed in direct contact with the upper part (surface) of the enzyme-immobilized part, and this heat source causes the enzyme to A method of heating to a temperature at which the carrier is deactivated and the carrier is not denatured.

(Ii) When the thickness of the substrate is smaller than the distance between the two working electrodes, a heat source is brought into contact with the back side of the substrate and the substrate and the conductive pattern (the electrode located at the portion to be deactivated) are used. The method of indirectly deactivating the enzyme immobilization part.

(Iii) Taking advantage of the fact that the working electrode is a conductor and this working electrode has better thermal conductivity than the substrate, contact the heat source with a part of the conductive pattern connected to the working electrode. ,
A method of heating the enzyme-immobilized portion to the enzyme inactivation temperature via the conductive pattern and the working electrode.

(Iv) A method in which a current is applied to a portion of the conductive pattern to be deactivated, and this portion is heated to deactivate it.

(V) A method of appropriately combining and using the methods shown in (i) to (iv) above.

First, in the case of the methods (i) and (ii), a heating member matched with the shape of the deactivating portion is formed of a substance having good thermal conductivity, and this heating member is heated to a predetermined temperature in advance with a heater or the like. There is a method of deactivating by arranging a heating member having a predetermined temperature in the deactivating portion, or forming a heater itself according to the shape of the deactivating portion and deactivating with the heater.

In the method (iii), the shape of the heat source is not particularly limited, but the size of the heat source is set to a size that is located in the pattern connected to the working electrode, and the heat source protrudes from this pattern to generate heat in areas other than the inactivated portion. It is desirable to prevent the influence of.

At the contact area between the enzyme immobilization part and the heat source, in order to avoid mechanical destruction of the enzyme immobilization part such as peeling due to contact with the heat source, a protective film such as silicone rubber is placed, and indirect contact is made via this protective film. You may make it heat.

FIG. 1 shows a plan view of an enzyme electrode which is usually used as a disposable electrode.

This enzyme electrode is a main body side connecting portion A connected to the measuring apparatus main body, an insulating portion B located in the central portion and having a conductive pattern formed under the insulating film, application of a sample, penetration into a sample. It is composed of a detection unit C that performs pickling and the like. Of these, reference numerals 1 and 2 in the detection unit C are working electrodes, 3 are reference electrodes, 4
Is a counter electrode, each of which is patterned with copper on the substrate 5 made of glass epoxy resin, and its end is electrically connected to the main body side connecting portion A through the insulating portion B. The working electrode 1, the working electrode 2 and the reference electrode 3 are plated with Pt.

Reference numeral 6 denotes an immobilized enzyme film (hereinafter simply referred to as “enzyme film”) formed so as to cover these working electrode 1, working electrode 2 and reference electrode 3.
It is called). The portion of the enzyme film that covers the working electrode 1 retains the enzyme activity after being deactivated by the method described below. Reference numeral 6a indicates an enzyme-immobilized portion in which this enzyme activity is retained. In this way, the working electrode 1 retains the enzyme activity and the working electrode 2 is deactivated, so that the concentration of the specific substance in the sample can be obtained by obtaining the differential output between both working electrodes. Is possible.

 FIG. 2 shows an example of a method for partially deactivating the enzyme film.

Of the enzyme film 6 formed by applying GOD (glucose oxidase) to photosensitive PVA (polyvinyl alcohol), the heat source 7 is brought into contact with the back surface portion of the substrate 5 at the position corresponding to the deactivation target portion. . The heat source 7 is preferably a heat source that utilizes heat generated by electric resistance in order to easily adjust the temperature.

The heat from the heat source 7 is transferred to the enzyme film 6 side through the substrate 5 and a part of the working electrode 2 which is a conductive pattern, and this heat causes the deactivating portion 6b to center around the working electrode 2 forming portion. It is formed. On the other hand, the part including the working electrode 1 retains the enzyme activity without being affected by the heat from the heat source 7, and thus the enzyme immobilization part
6a.

 FIG. 3 shows another configuration.

Below the insulating layer of the insulating portion B, a conductive portion for electrically connecting the working electrode 1, 2, the reference electrode 3 and the counter electrode 4 and the terminal portion forming the main body side connecting portion A is patterned. . Of these, the heating portion 8 is formed integrally with the conductive portion pattern 2 ′ in the middle of the conductive portion pattern 2 ′ connected to the working electrode 2 to be deactivated. For example, a heat source similar to the heat source is directly or indirectly arranged with respect to the heating unit 8. As a result, the heat of the heat source is transmitted to the working electrode 2 through the heating portion 8 and part of the conductive portion pattern 2 ', and the temperature of the working electrode 2 rises. Due to this temperature rise of the working electrode 2, the enzyme in the enzyme film 6 around the working electrode 2 is deactivated, and the deactivating portion 6b is formed so as to cover the working electrode 2.

Example 1 The electrode configuration is as shown in FIG. 1, the substrate 5 has a width of 5 mm,
The total length is 40 mm and the thickness is 0.5 mm. The working electrode 1, working electrode 2 and reference electrode 3 are printed on the substrate with copper by the printed circuit technology, and the thickness of Pt is 0.3 μm. It is plated and is for use as an H ₂ O ₂ detection electrode.

Enzyme coating is PVA-SbQ (JP-A-56-5576) 2wt% glucose oxidase [EC (1,1,3,4), Oriental Yeast Co., Ltd., 370nnits / mg] to 2.0mg / g The adjusted part was coated on the detection part C. After further drying,
The copper coating portion was cured by UV irradiation for 500 w (340 nm) for 10 minutes to form the enzyme coating 6.

Next, as shown in FIG. 2, the heat source 7 is arranged on the lower surface of the working electrode 2 through the substrate 5, and the heat source temperature is set to 200 ° C.
The heating part was deactivated by heating for 2 seconds to obtain a H ₂ O ₂ detection electrode.

[Test 1] In order to test the performance of the above electrodes, a phosphate buffer solution 0.1
M, pH 7.2, glucose 10 mM (180 mg / dl) and volume 10 μ were used as samples.

As a performance test, a detection method using a cyclic voltammogram is used, and the sweep range is -600mV to 1000mV,
The sweep speed was 100 mV / sec, and the current value of 600 mV was calculated for the electrode before heat treatment and the electrode after heat treatment, and the performance of both electrodes was compared.

4 and 5 show cyclic voltammograms, of which the one shown in FIG. 4 shows the cyclic voltammograms of the electrodes before the heat treatment. As is clear from the figure, there is of course no significant difference between the working electrode 1 and working electrode 2 currents, and the measured voltage 600m
At V, working electrode 2 showed the same 13 μA as working electrode 1.

On the other hand, in the electrode in which the enzyme film on the working electrode 2 is deactivated by the method described in the above-mentioned embodiment, the working electrode 2 becomes about 5.6 μA at the measurement target voltage of 600 mV as shown in FIG.
It was confirmed that the enzyme in working electrode 2 was completely inactivated. On the contrary, the current of the working electrode 1 at the same measurement voltage shows 14 μA, which is almost the same as that of the electrode not subjected to the heat treatment, and it is confirmed at the same time that the heat treatment on the working electrode 2 does not affect the working electrode 1 at all. I was able to.

Next, a method and an apparatus for manufacturing an electrode using the method shown above will be described.

First, when the above-mentioned electrode is used as a disposable sensor, it is a major technical issue to reduce the manufacturing cost and provide it at a low cost in addition to the performance of the sensor.

The sensor manufacturing method and apparatus described below are also configured in consideration of this point, and are premised on having both mass production and high product accuracy.

First, FIG. 6 shows a substrate assembly. This substrate assembly is formed with 25 parts to be disposable sensors S,
The entire part that should be this sensor is 40 mm wide and 150 mm long
The size has become. A working electrode 1, 2, a reference electrode 3, and a counter electrode 4 are formed on the detection portion C of each sensor S, and an enzyme film 6 is pre-coated on these portions. 9a and 9b are positioning holes formed in the four corners of the substrate assembly 9, respectively.

FIG. 7 shows a heating unit on which the substrate assembly 9 is mounted and a predetermined portion of the substrate assembly 9, that is, each sensor S.
The heat sources for heating the enzyme deactivating part are arranged in the number corresponding to the number of the sensors S. Reference numeral 10 denotes the heating unit main body, and positioning pins 10a, 10b are provided at four corners of the heating unit main body 10 at positions corresponding to the positioning holes 9a, 9b of the substrate assembly 9.
Is erected. An arrow 11 in FIGS. 8 and 9 is a heating unit including heating members arranged in a number corresponding to the number of sensors formed in the substrate assembly with respect to the heating unit.

The configuration of this heating unit is shown in FIG. 9. Reference numeral 12 is a heating member that forms the center of this heating unit, and the heating unit 10 is inserted therethrough, and the upper end 12a thereof is the enzyme of each sensor. It has a size and shape corresponding to the deactivation target portion [see FIG. A coil spring 13 is arranged in the heating knit insertion portion of the heating member 12, and the upper end portion 12a of the heating member 12 is positioned so as to project from the groove portion 10c of the heating unit 10 due to its elastic force. The upper end portion 12a is configured to be slightly projecting from the surface of the flat portion 10d of the heating unit 10. The shape of the upper end surface of the heating member 11 is the ninth
As shown in FIG. 3B, the shape corresponds to the shape of the deactivated portion of the enzyme film on the substrate side, and is formed in a rectangular shape of 2 mm × 1 mm, for example.

On the other hand, a portion (hereinafter referred to as "heat receiving portion") 12b projecting to the back surface side of the heating unit 10 is positioned so as to be inserted into a ring-shaped heat source (an electric heater shown in the drawing) 14 and receives heat from the heat source 14. Has become.

Next, a working process for partially deactivating the enzyme film using this apparatus will be shown.

First, the positioning holes 9a and 9b of the substrate assembly 9 are set in the heating unit.
The substrate assembly 9 is placed on the heating unit 10 by inserting it into the predetermined 10 positioning pins 10a and 10b. Next, the pressing member 15 having the holes 15a and 15b for pin insertion at the positions corresponding to the positioning pins 10a and 10b of the heating unit 10 is formed.
The pins 10a and 10b are arranged so as to be inserted into the pins 15a and 15b. As a result, the substrate assembly 9 is pressed against the heating unit 10 by the pressing member 15. In this state, each heating member 12 slightly protruding from the flat surface 10d of the heating unit 10.
The upper end surface 12a of the above is closely attached to the portion corresponding to the deactivating portion on the back surface of each substrate by the elastic force of the coil spring 13. Subsequently, each heat source 14 is heated, and the heat of the heat source reaches the upper end portion 12a via the heat receiving portion 12b. Furthermore, this heat reaches the deactivated part of the enzyme film through the substrate, and deactivates the enzyme in this part. In this case, the groove 10c on the heating unit 10 side and the groove 15c on the pressing member 15 side form a space for heat dissipation in the portion where the heating member 11 is located, and the heating time required for deactivation is several seconds. Since it is very short, the heat of the heating member does not affect anything other than the deactivated part. As a method for setting the heating start timing, in addition to the above-described method, electric power may be automatically supplied to each heat source 14 by attaching the pressing member 15. When the deactivation of the enzyme film is completed, the substrate assembly 9 is removed from the heating unit 10 and each sensor portion S is separated to obtain a product.

Although the above steps are configured based on the method of indirectly heating the deactivation object via the substrate, the deactivation section may be directly brought into contact with the heating member to deactivate it. Generally, the method of directly contacting the heat source with the deactivating portion is strict in terms of temperature control of the heat source, but on the other hand, the amount of heat dissipated through the substrate is small, so that the deactivating portion and the non-deactivating portion are reduced. It becomes possible to form the boundary with and more clearly.

 FIG. 10 shows another configuration example of the heating unit.

Reference numeral 16 denotes a heating member having a configuration different from that described above. The upper end portion 16a of the heating member 16 is always arranged at a position lower than the flat portion 10d of the heating unit 10. Reference numeral 16b is a heat receiving portion forming a part of the heating member 16, and the upper end portion 16a side is locked in the groove portion 10c of the heating unit 10 so that it is always suspended via the center shaft portion 16c. . Reference numeral 17 denotes a buffer portion formed on the lower surface of the heat receiving portion 16b, which includes a tubular portion 17a slidable in the axial direction of the heat receiving portion 16b and a coil spring 18 housed in the tubular portion 17a. There is. Next, an arrow 19 is an elevating device that elevates and lowers the heating member 16, and in the illustrated configuration, a cam 20 that contacts the bottom surface of the buffer portion 17, and a cam shaft 21 that rotates the cam 20,
It is composed of a motor 22 for rotating the cam shaft 21.

Next, 23 is a position sensor that detects the presence or absence of the substrate assembly 9 in the heating unit 10, 24 is a position sensor that detects the presence or absence of the pressing member 15, and 25 is a temperature sensor that detects the temperature near the upper end 16a of the heating member 16. Is. Reference numeral 26 is a control device for automatically controlling the illustrated device.

 The operating state of the above device will be described below.

First, when the device is not in operation, the cam 20 serves as a buffer member.
The heating member 16 is not in contact with the bottom surface of the heating unit 16, and therefore the upper end 16a side is locked to the groove 10c plane of the heating unit 10 as shown in the drawing, and the heat receiving unit 16b is hung by its own weight. Has become. Therefore, the upper end 16 of the heating member 16 is
The position a is lower than the flat portion 10d of the heating unit 10.

Next, the controller 26 turns on the heat source 14 to heat the heating member 16. During this time, the control device 26 controls the temperature of the upper end portion 16a of the heating member 16 that directly or indirectly contacts the deactivating portion by the temperature sensor 2
We are monitoring at 5. When the upper end 16a reaches a predetermined temperature, the substrate assembly 9 is placed on the heating unit 10 and the pressing member 15 is further placed thereon. When the position sensors 23 and 24 detect that the board assembly 9 and the pressing member 15 are installed, the control device 26 issues a signal for rotating the motor 22 and also has a built-in timer 26a.
Works. The rotation of the cam 20 raises the entire heating member 16 via the buffer portion 17, and the upper end portion 16a of the heating member 16 directly contacts the deactivating portion of each sensor portion of the substrate assembly or indirectly through the substrate, and a predetermined range is set. Deactivate. At this time, the heating member 16 comes into contact with the substrate assembly 19 at a predetermined pressure due to the elasticity of the coil spring 18 housed in the buffer portion 17.

The contact time is set in advance in the timer 26a described above, and the motor 22 is operated again with the lapse of this predetermined time,
The heating member 16 is lowered to complete one deactivating step.

It should be noted that the heating member in the cooled state is brought into contact with the substrate assembly in advance, then the process is organized to heat the heating member, and the temperature information from the temperature sensor 25 is used as a correction value for the operation of the timer 26a. It is also possible to take a method. Further, as a matter of course, instead of the control device, it is possible to manually control each part. Further, although the heating member elevating / lowering device shown in the drawing is a cam mechanism, it is not intended to limit the mechanism structure, and it may be operated by using an air cylinder or the like.

Although the present invention has been described above by taking an enzyme electrode as an example, the present invention is not limited to the enzyme electrode, and it is necessary to use an ion selective field effect transistor (ISFET), etc. It is possible to apply the invention.

〔effect〕

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a method for deactivating an enzyme-immobilized part of a predetermined part of an electrode, in which a heat source is placed directly or indirectly in contact with the part to be deactivated. Since the heat is transferred to the specific part of the enzyme immobilization part by heat conduction to partially deactivate the enzyme, a large machine for generating electromagnetic waves and a precise photomask are unnecessary. Thus, the device can be formed inexpensively and compactly.

In addition, the heating time required for deactivation is only a few seconds, and it is possible to easily deactivate a predetermined part in a surprisingly short time of only a few hundredth compared to the deactivation method using electromagnetic waves. The productivity of can be greatly improved.

Furthermore, the method using heat conduction does not raise the temperature of the object to be heated to a temperature above the heat source, so the control of deactivation time and the like has a higher degree of control tolerance than the method using electromagnetic waves, and the heat source depends on electrical resistance. Since it is possible to use an easily controlled one such as heat generation, by using a configuration in which these heat sources are accurately positioned with respect to each inactivated portion of a large number of enzyme electrodes connected in series,
It becomes possible to provide a large amount of high-performance sensors at low cost.

[Brief description of drawings]

FIG. 1 is a plan view of an enzyme electrode showing an embodiment of the method of the present invention,
FIG. 2 is a diagram showing an example of a method for deactivating the enzyme electrode shown in FIG. 1 by using a cross section taken along the line I-I in FIG. 1, and FIG. 3 is a plan view of the enzyme electrode showing another example of the heating section configuration. Partial view, FIG. 4 is a cyclic voltammogram of the electrode without deactivation, FIG. 5 is a cyclic voltammogram of the electrode when one working electrode is deactivated, and FIG. 6 is a plan view of the substrate assembly. FIG. 7 is a plan view of the heating unit, FIG. 8 is a cross-sectional view of each member showing the arrangement state of the pressing member, the substrate assembly and the heating unit, and FIG. 9 is a detailed view of a part of FIG. First
FIG. 10 is a partial sectional view of a heating unit showing another configuration example of the heating member. 1, 2 ... Working electrode, 2 '... Conductive pattern 3 ... Reference electrode, 5 ... Substrate 6 ... Enzyme film, 6a ... Enzyme immobilization part 6b ... Deactivation part, 7 ... Heat source 8 ... … Heating part, 9 …… Substrate assembly 10 …… Heating unit, 11 …… Heating part 12, 16 …… Heating member 12a, 16a …… Heating member upper end 14 …… Heat source, 15 …… Pressing member 17 …… Cushioning member, 18 ... Coil spring 25 ... Control device, A ... Main body side connection part B ... Insulation part, C ... Detection part

Claims (2)

[Claims] 1. A reference electrode and a pair of working electrodes formed on a substrate are covered with an enzyme film, and an enzyme film covering one working electrode of a pair of working electrodes is heated to form an enzyme film of the heated portion. In the method of producing an enzyme electrode by deactivating, directly against the enzyme film to be deactivated,
Alternatively, by indirectly arranging the heat source in contact with the substrate, the heat from the heat source is transferred to the enzyme film portion where the heat source is arranged by heat conduction to heat, thereby deactivating the heated portion of the enzyme film. A method for producing an enzyme electrode, which is characterized in that 2. A heating unit comprising a heating unit having a plurality of heating members and a pressing member engaging with the heating unit, the substrate assembly having a plurality of portions serving as enzyme sensors. On the other hand, by arranging and fixing using a pressing member, by directly or indirectly contacting the heating member with each of the portions of the substrate assembly that are to be enzyme sensors and are to be inactivated of the enzyme film. An apparatus for manufacturing an enzyme electrode, which is configured to transfer heat of a heating member to the contact portion by heat conduction.