JPH102875A - Enzyme reaction sensor and manufacture thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme reaction sensor with a small size by which glucose concentration existing in blood and urine can be measured many times and provide a manufacturing method for which a semiconductor processing technique with high productivity as its advantage is employed. SOLUTION: A glucose sensor comprises a silicon substrate 1, a plurality of grooves 2 formed in one surface of the silicon substrate 1, a pair of platinum electrode films 3, 4 formed in a plurality of grooves 2, and a Pyrex glass plate 5. Glucose oxidase as an enzyme is immobilized in the inside of a capillary 6 formed between the grooves 2 and the Pyrex glass plate 5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor utilizing an enzymatic reaction, such as a portable multi-type glucose sensor for clinical use, and more particularly to a sensor for measuring the concentration of glucose in blood or urine using an enzymatic reaction. And its manufacturing method.

[0002]

2. Description of the Related Art Diabetic diseases, one of the adult diseases, require measurement of blood glucose several times a day to control insulin administration and diet. Currently, glucose measurement methods include a colorimetric method and an enzyme reaction method.

The colorimetric method is a reduction method (Somogyi-Nels).
on method) is common. This method is measured through the following procedure. First, use zinc sulfate and barium hydroxide to remove the interfering proteins (deproteinization). Thereafter, the mixture is centrifuged and filtered to cause a reduction reaction and a color reaction. As another colorimetric method, there is a condensation method (o-toluidine / boric acid method).
This method utilizes the fact that when a sugar is heated together with o-toluidine under acidic conditions, the sugar turns blue-green.
Each of these colorimetric methods is a method of measuring the absorbance by applying absorption light having a specific wavelength to the reaction substance, and measuring the glucose concentration from the results.

On the other hand, as a measurement method using an enzyme reaction,
A method for quantifying glucose by measuring a product such as hydrogen peroxide generated when glucose reacts with an enzyme such as glucose oxidase (GOD) or quantifying a substance (enzyme) that decreases. There is.
Actually, as a detecting means, there are a method using an electrode (cyclic volt ammetry) and a colorimetric measuring method.

[0005]

The colorimetric method generally requires pretreatment and uses a spectroscopic measuring method, so that the detection unit becomes large. In addition, as described above, in the absorptiometry, an accurate measurement value may not be obtained because it is affected by another substance having a wavelength close to the intrinsic wavelength of the measurement substance.

On the other hand, the current apparatus using an enzymatic method does not require pretreatment because it utilizes the substrate selectivity of the enzyme. Therefore, the size of the device becomes large, and it is difficult to reduce the cost and mass production. In addition, there remains a problem in that the glucose concentration of the measurement sample is immediately detected and the condition of portability and measurement of a large number of samples is satisfied.

Therefore, the technical problems of the present invention are, for example,
Provided is an enzyme reaction sensor and a method for producing the same, which enable a large number of measurements of the glucose concentration in blood and urine with a small device and utilize the high mass productivity that is an advantage of semiconductor processing technology. Is to do.

[0008]

According to the present invention, for example, a large number of grooves and platinum electrode films are radially formed on a semiconductor substrate such as a circular silicon wafer using a micromachine technique.
Thereafter, the semiconductor substrate and a glass plate such as Pyrex glass are integrated to form a capillary, and an enzyme such as glucose oxidase is immobilized inside. As described above, by creating many capillaries and electrodes on a single semiconductor substrate and constructing an enzyme sensor such as a multi-type glucose sensor, the above-mentioned problems of miniaturization, multiple sample measurement, and mass productivity were solved. I have.

That is, (1) According to the present invention, a semiconductor substrate, a plurality of grooves formed on one surface of the semiconductor substrate,
A pair of electrode films made of a noble metal formed in each of the plurality of grooves; and a glass plate covering the grooves, wherein an enzyme is immobilized in a capillary formed by the grooves and the glass plate. An enzymatic reaction sensor is obtained.

[0010] (2) According to the present invention, in the enzyme reaction sensor, the enzyme reaction sensor is characterized in that the enzyme is glucose oxidase.

(3) According to the present invention, in any one of the enzyme reaction sensors, the semiconductor substrate is formed of a circular silicon wafer, and the plurality of grooves are formed on one surface of the silicon wafer along a radial direction. Thus, an enzyme reaction sensor characterized in that the enzyme reaction sensor is formed.

(4) According to the present invention, in any of the above enzyme reaction sensors, the noble metal is made of platinum, and the pair of electrode films has an electrode extraction end near the center of the silicon wafer. Thus, an enzyme reaction sensor is obtained.

(5) According to the present invention, in any one of the enzyme reaction sensors, the glass plate is made of Pyrex glass, and the glass plate other than the groove and the semiconductor substrate are bonded to each other. An enzyme reaction sensor characterized by the above is obtained.

(6) According to the present invention, a plurality of grooves are formed on a semiconductor substrate by etching using a micromachine technique, and a pair of electrode films made of a noble metal are formed to cover the grooves. Thus, a glass plate is joined and integrated to form a capillary, and an enzyme is immobilized inside the capillary, thereby producing a method for producing an enzyme reaction sensor.

(7) According to the present invention, in the method for manufacturing an enzyme reaction sensor, the semiconductor substrate is formed of a circular silicon wafer, and the groove is formed in a radial direction. A method for manufacturing a sensor is obtained.

(8) According to the present invention, in any one of the above-mentioned methods for producing an enzyme reaction sensor, the noble metal is made of platinum, and the pair of electrode films are formed by a sputtering method and an ion milling method. A method for manufacturing an enzyme reaction sensor, characterized in that the silicon wafer is formed so as to have an electrode extraction end near the center of the silicon wafer, is obtained.

(9) According to the present invention, in any one of the above-described methods for producing an enzyme reaction sensor, the glass plate is made of Pyrex glass, and is integrated with the silicon wafer by an anodic bonding method. Thus, a method for producing an enzyme reaction sensor can be obtained.

(10) According to the present invention, in any one of the above-mentioned methods for producing an enzyme reaction sensor, glucose oxidase is immobilized as the enzyme by a chemical bonding / inclusive method. Is obtained.

(11) According to the present invention, a pair of mutually overlapping substrates, a plurality of grooves formed on one surface of at least one of the pair of substrates,
At least two electrode films made of a noble metal formed on one of the pair of substrates corresponding to the plurality of grooves, wherein an enzyme is fixed inside a capillary formed by the grooves and the substrate; Thus, an enzyme reaction sensor characterized in that it has been converted into an enzyme reaction is obtained.

(12) According to the present invention, in the enzyme reaction sensor, the enzyme reaction sensor is characterized in that the enzyme is glucose oxidase.

(13) According to the present invention, in any one of the enzyme reaction sensors, the substrate is made of plastic having a circular shape, and the plurality of grooves are formed on one surface of the plastic substrate in a radial direction. Thus, an enzyme reaction sensor characterized by being formed along the line is obtained.

(14) According to the present invention, in any of the enzyme reaction sensors, the noble metal is made of platinum, and the electrode film has an electrode extraction end near the center of the plastics substrate. Thus, an enzyme reaction sensor is obtained.

(15) According to the present invention, there is provided the enzyme reaction sensor according to any one of the enzyme reaction sensors, wherein the plurality of electrode films are formed in three pieces.

(16) According to the present invention, in any one of the enzyme sensors, an enzyme reaction sensor is obtained in which the pair of substrates are bonded to each other.

(17) According to the present invention, in any one of the enzyme sensors, the pair of substrates may be provided with a plasma-polymerized film on a joint surface, and the pair of substrates may be welded by the plasma-polymerized film. An enzyme reaction sensor characterized by being joined to each other is obtained.

(18) According to the present invention, in any one of the enzyme sensors, the enzyme is immobilized at least by the amino group of the plasma polymerized film of a monomer having an amino group present on the inner wall surface of the capillary. An enzyme reaction sensor is obtained, wherein the amino group present in the enzyme is linked with a crosslinking reagent.

(19) According to the present invention, a plurality of grooves are formed on one substrate by using a micro-machine technology, and furthermore, the other substrate is made of a noble metal corresponding to the grooves. At least one pair of electrode film groups are formed, and the pair of substrates are overlapped and joined together so that the groove and the noble metal correspond to each other to form a capillary, and an enzyme is immobilized inside the capillary. Thus, a method for producing an enzyme reaction sensor is obtained.

(20) According to the present invention, in the method for manufacturing an enzyme reaction sensor, the substrate is made of a circular plastic, and the groove is formed in a radial direction. A manufacturing method is obtained.

(21) According to the present invention, in any one of the above methods for producing an enzyme reaction sensor, the noble metal is made of platinum, and the pair of electrode films are formed by sputtering and ion milling. A method for producing an enzyme reaction sensor, characterized in that the substrate is formed so as to have an electrode extraction end near the center of the substrate.

(22) According to the present invention, in any one of the above-described methods for producing an enzyme reaction sensor, the substrate is made of acrylic resin, and is bonded to and integrated with each other. A manufacturing method is obtained.

(23) According to the present invention, in any one of the above methods for producing an enzyme reaction sensor, an electrode group may be formed on the one substrate after forming a groove and on the other substrate. Before the formation, a method for producing an enzyme reaction sensor is provided, wherein a plasma polymerized film containing an amino group is formed on the surfaces of the surfaces to be bonded to each other of the substrates.

(24) According to the present invention, in any one of the above methods for producing an enzyme reaction sensor, the enzyme reaction sensor is characterized in that glucose oxidase is immobilized by a chemical bonding / inclusive method as the enzyme. Is obtained.

(25) According to the present invention, in the method for producing an enzyme reaction sensor, the enzyme has an amino group, and the immobilization of the enzyme is performed by combining the amino group of the plasma polymerized membrane with the amino group of the enzyme. A method for producing an enzyme reaction sensor is provided, which comprises binding by the action of a crosslinking reagent with a group.

[0034]

Embodiments of the present invention will be described below with reference to the drawings. In the embodiment of the present invention, a glucose sensor is described as an example of the enzyme reaction sensor, but it is obvious that the present invention is not limited to these.

FIG. 1A is an exploded perspective view showing a glucose sensor according to a first embodiment of the present invention, and FIG.
Is a plan view. Referring to FIGS. 1A and 1B,
The glucose sensor 10 is an integrated sensor device, and a groove 2 is formed on one surface of a silicon substrate 1 in a radial direction. Platinum electrode films 3 and 4 are formed in the groove 2 and are covered with a Pyrex glass plate 5. The Pyrex glass plate and the silicon substrate 1 are joined and integrated, and the groove 2 is covered with the Pyrex plate to form a capillary 6. Glucose oxidase is immobilized on the capillary 6.

The glucose sensor shown in FIG. 1 (b) was prepared by the following procedure.

First, a 2 inch diameter, 100 crystal lattices,
After cleaning the single-sided mirror-finished silicon wafer 1 in accordance with the method of RCA, an oxide film of about 0.8 Å was formed by thermal oxidation using a hydrogen combustion method. This is spin-coated with a photoresist, patterned with a photomask, etched with a hydrofluoric acid and ammonium fluoride solution, and etched using an EPW solution (ethylenediamine-pyrocatechol-water). Sixteen radial grooves 2 having a length of 12 mm, a width of 1 mm, and a depth of 56 μm were formed by wet etching. The silicon wafer 1 is left in the air or directly heated to form an insulating coating on the surface. A platinum film is formed to a thickness of about 200 angstroms using a sputtering apparatus on the entire surface of the silicon wafer 1 in which many grooves 2 are formed, a photoresist is spin-coated, and the platinum film is patterned. Etching was performed to form a pair (two electrodes) of platinum electrode films 3 and 4 for each groove (electrode area per unit: 1 mm ² ). Further, a three-dimensional structure was formed by anodically bonding the silicon wafer 1 to a glass substrate 5 made of Pyrex and having an outer diameter of 2 inches and an inner diameter of 1.2 inches. Through the above operation, the fine tube structure-capillary 6 and platinum electrode films 3 and 4 were formed. The electrode extraction portions 3a and 4a are formed to be exposed inside the capillary.

Next, the enzyme immobilization method will be described. Glucose oxidase (GOD) as an enzyme was immobilized by aminating the surface of glass or silicon and using glutaraldehyde (GA) as a cross-linking agent to crosslink the amino group of the enzyme. The prepared capillary 6 has 3-aminopropyltrietoxysilane (aminopropyltrietoxysilane).
10 μl of a toluene solution of silane, γ-APTES) was passed at room temperature and heated to 115 ° C. overnight. 2.5% (v /
v) Fill the column with a phosphate buffer (pH 7.1) containing glutaraldehyde (GA) and hold for 1 hour. After discharging, wash the column by passing it through a phosphate buffer and 10% (w /
v) The column was filled with a phosphate buffer containing GOD (Type II, Sigma) and held overnight, and glucose oxidase was immobilized inside.

Next, the measurement principle of the glucose sensor according to the first embodiment of the present invention will be described.

In the multi-type glucose sensor according to the first embodiment of the present invention, the glucose solution and the test sample are introduced into the inside of the silicon wafer from the capillary 6 at the tip of the silicon wafer by capillary action. The introduced glucose solution is converted into gluconolactone and hydrogen peroxide by the catalytic action of GOD immobilized inside the capillary as shown in the following formula 1.

[0041]

Embedded image

The generated hydrogen peroxide is electrochemically detected on a platinum electrode film in the capillary according to the following electrode reaction.

[0043]

Embedded image

In the electrochemical detection, substrates and products of many enzymatic reactions such as oxygen and hydrogen peroxide are electrochemically active, and each chemical species can be electrochemically separated and quantified. The detection device becomes an effective measurement device. In general, the number of detection targets in spectroscopic detection decreases in proportion to the cube of size, whereas in electrochemical detection, the detection target decreases in proportion to the square of size. In the detection method using the glucose sensor according to the present invention, since the electrochemical detection method is employed, the efficiency of the electrochemical detection method is relatively increased with miniaturization. Furthermore, the miniaturization can reduce the consumption of reagents and samples, that is, can shorten the measurement time. The generated hydrogen peroxide is oxidized by one electrode of the platinum electrode film and the anode electrode, and an anode current is observed. From the above, it was possible to quantify glucose by observing a change in the current value of the hydrogen peroxide concentration.

Next, an example of measuring glucose using the glucose sensor according to the first embodiment of the present invention will be described.

When measuring glucose using GOD, the redox species usually produced is hydrogen peroxide. In order to confirm whether hydrogen peroxide can be quantitatively detected by the manufactured device, an electrochemical reaction was performed by introducing a hydrogen peroxide solution prior to detection of glucose by the device. The capillary cell was filled with the hydrogen peroxide solution of each concentration adjusted, and cyclic voltammetry was performed. 0 to 10 between the two electrodes in the device
FIG. 2 shows a change in current value when scanning a potential up to 00 mV. As shown in FIG. 2, an oxidation current derived from hydrogen peroxide was observed at a current value around 800 mV. A relative relationship was observed between the concentration of hydrogen peroxide and the current value, indicating that the fabricated device functions as an electrochemical cell for detecting hydrogen peroxide. In the same manner, a glucose solution is introduced through a sample introduction hole. GOD in the capillary oxidizes glucose, and at this time, dissolved oxygen becomes hydrogen peroxide. Table 1 below shows response values when these samples were introduced.

[0047]

[Table 1]

FIG. 3 is a plan view showing the glucose sensor according to the second embodiment of the present invention from which the glass plate has been removed. As shown in FIG. 3, on a silicon substrate 1,
A groove 12 is formed, and platinum electrode films 13 and 14 are respectively formed. In the first embodiment of the present invention, the number of grooves, that is, the number of capillaries is 16, but in the second embodiment, the density is increased to 72 grooves. To this end, a pair of electrode extraction portions are also formed side by side.

When using the glucose sensor according to the first and second embodiments of the present invention, a glucose sensor having a measuring device equipped with a voltammeter connected to a platinum electrode membrane is prepared. The capillary may be sucked into the capillary by capillary action and measured.

In collecting blood, for example, the fingertip may be disinfected with a cotton swab and dried, and then punctured using a blood collecting device or the like to squeeze out one drop of blood.

Similarly to the glucose sensor according to the first embodiment, the glucose sensor according to the second embodiment can be used even after the glass plates are stacked and anodically bonded and integrated, and then immobilized with an enzyme.

FIG. 4 is an exploded perspective view showing a glucose sensor according to a third embodiment of the present invention.
(A) shows a first acrylic plate, and (b) shows a second acrylic plate. Referring to FIG. 4A, the first acrylic plate 21 has a hole 22 in the center, and a groove 24 having a rectangular cross section in the radial direction is formed on one surface 23 from the outer periphery toward the hole 22. A plurality is provided.

Referring to FIG. 4B, the second acrylic plate 25 has a platinum electrode 2 having a front end bent at a right angle on the surface.
7, 28 and 29 are provided, each extending stepwise in the radial direction. These platinum electrodes 27, 28, 29
Is formed by sputtering, and the base of the electrode 28 of the three electrodes 27, 28, 29 is
Are connected to the center electrode 30 near the center of the acrylic plate 25.

These first and second acrylic plates 21 and 25 are treated on one side as shown in the figure by plasma polymerization of ethylenediamine, and are bonded to each other. The enzyme is immobilized in the formed capillary.

FIGS. 5 (a), 5 (b) and 5 (c)
FIG. 4 is a perspective view showing a part of the first and second acrylic plates 21 and 25 of FIGS. 4A and 4B.

As shown in FIG. 5A, a plurality of grooves 24 having a depth of 1 mm and a width of 1 mm are formed on one surface of a donut-shaped disk having a thickness of 2 mm, a radius of 30 mm and an inner diameter of 20 mm. I have.

As shown in FIG. 5B, one surface of a disk having a thickness of 2 mm and a radius of 30 mm is formed so as to correspond to the groove 24.
Electrodes 27, 28 and 29 are formed in the radial direction. The groove 24 has a tip 27b, 28 which is long in the circumferential direction of the electrode.
b and 29b cross each.

As shown in FIG. 5C, the first acrylic plate shown in FIG. 5A and the second acrylic plate shown in FIG. The groove 24 of the first acrylic plate 21 is covered by one surface of the second acrylic plate 25, and the capillary 31 is radially oriented.
Is formed.

Next, a method for manufacturing an enzyme reaction sensor according to a third embodiment of the present invention will be described with reference to FIGS.

FIG. 6 schematically shows an apparatus for performing plasma polymerization. As shown in FIG. 6, the apparatus is provided with a sample stage 60 at an upper part in an apparatus main body 50 and a plasma generator 53 provided thereon. Plasma generator 5
3 is connected to an oscillator 52 and a matching device 51.
In a chamber below the sample stage, a reservoir 59 for storing a raw material such as ethylenediamine, a flow path 62 connected to the reservoir 59 and having a valve, and a flow path 62 are provided.
An inlet pipe 55 is connected to the inside of the chamber via a flow rate adjuster 58 provided at one end of the chamber 2.

On the other hand, the chamber is provided with an exhaust pipe 54 connected to a diffusion pump 56 and a rotary pump 57 in order to exhaust gas in the chamber. This apparatus uses ethylenediamine as a monomer material for a plasma polymerized film to introduce amino groups. Table 2 shows the conditions of the plasma polymerization.

[0062]

[Table 2]

Under the conditions shown in Table 2, the film formation rate of the plasma polymerized film is 1000 Å / min, and the thickness of the plasma polymerized film is 1000 Å.

Next, a specific procedure will be described.

First, as shown in FIGS. 7A and 8A, an acrylic substrate having an outer diameter of 30 mm and a thickness of 2 mm and an acrylic plate having an outer diameter of 30 mm, an inner diameter of 20 mm and a thickness of 2 mm are prepared. As shown in FIG. 7B, a groove 24 having a length of 10 mm, a depth of 1 mm, and a width of 1 mm is radially formed on the surface of the acrylic plate 21 by machining.
Are formed. As shown in FIG. 7 (c), an amino group is introduced into the surface of the acrylic substrate by ethylenediamine plasma treatment on one surface of the acrylic plate. Here, the production of the ethylenediamine plasma polymerized film will be described in detail. The plasma is a state of a substance in which electrons and positive ions formed by ionization have almost the same density and become neutral as a whole. The plasma polymerization method is a method of polymerizing in this plasma to synthesize a polymer. The film obtained by this method or this method is (a) that any monomer can be formed, (a) it is pinhole-free amorphous, and (c) thin film formation (（10 nm)
Possible and homogeneous, (d) not only film formation,
By changing the type of plasma gas, surface modification and modification (for example, introduction of a functional group) are possible. (E) Since it is a dry process, it has advantages such as easy integration with semiconductor technology. .

On the other hand, as shown in FIG. 8B, an amino group is introduced into one surface of the acrylic plate by an ethylenediamine plasma treatment using the apparatus shown in FIG.

As shown in FIG. 8C, a platinum electrode pattern 32 is formed by sputtering on an acrylic substrate on which no groove is formed. Here, the sputtering is a technique of bringing a metal or the like into a plasma state and attaching it to an opposing substrate, and is a process mainly used for a metal thin film for forming an electrode. The deposition rate of the platinum film is several angstroms per second, and the film thickness can be controlled by time. Here, a platinum electrode is formed by sputtering on the acrylic substrate that has been subjected to the plasma polymerization treatment. A metal mask is used to form an electrode pattern, and three electrodes are simultaneously formed for each pair of electrode patterns. Table 3 shows the sputtering conditions.

[0068]

[Table 3]

Under the conditions shown in Table 2 above, the deposition rate of the platinum film was 1
00 Å / min, and the thickness of the platinum film is 1000 Å.

Next, as shown in FIG. 9A, in order to integrate the capillary and the electrode pair, 1,2-dichloroethane is applied between the substrates, and the acrylic substrates are welded to each other. Subsequently, as shown in FIG. 9B, the enzyme is immobilized in the capillary 31 formed by glutaraldehyde.

The enzyme is immobilized by using glutaraldehyde, a divalent crosslinking reagent, between the amino group on the surface of the plasma polymerized membrane and the amino group present in the enzyme. This reaction is represented by the following chemical formula 3.

[0072]

Embedded image

The detailed procedure for immobilizing the enzyme is carried out as follows.

First, (i) a phosphate buffer containing 2.5% (v / v) glutaraldehyde (GA) (pH 5.
6) is prepared, applied to the capillary which has been subjected to the plasma treatment, and left for 30 minutes. (Ii) After washing this with a phosphate buffer solution, 10% (W / V) GOD (Type I
(I, Sigma), and then kept at room temperature for 30 minutes. (Iii) Wash away the physically adsorbed enzyme with phosphate buffer.

FIG. 10 is a cyclic voltammogram showing the electrode response of hydrogen peroxide by the capillary electrode according to the third embodiment of the present invention. In FIG.
The horizontal axis shows the potential E (mV), and the vertical axis shows the current value (A).

In the state (a), no current flows at all, that is, there is no short circuit.

In the state (b), no remarkable current change is observed at 400 to 800 mV.

When hydrogen peroxide of (c) is added, 300 to
An oxidation current was observed at 800 mV, indicating that hydrogen peroxide could be detected. As shown in FIG. 10, the measurement conditions were CV measurement with a phosphate buffer (pH 5.6) of 20 mM and a scan rate of 100 mV / sec.
It is performed at room temperature (27 ° C.).

FIG. 11 is a cyclic voltammogram of the electrode response of glucose by the capillary electrode according to the third embodiment of the present invention. The measurement conditions are the same as in FIG. 10, and the measurement is started three minutes after the introduction of the solution.

As shown in FIG. 11, no remarkable current value is observed when only the buffer solution is used, but (d) glucose oxidase (GOD) converts it into gluconolactone and hydrogen peroxide. It can be seen that was detected. Further, by measuring the degree of the current value at 600 mV in the CV diagram, it is possible to quantify glucose.

FIG. 12 is a diagram showing a current value change (calibration curve) depending on the glucose concentration of the enzyme reaction sensor according to the third embodiment of the present invention. The glucose at the optimum potential (600 mV) obtained in FIG. The current change when the concentration is changed is shown. Referring to FIG. 12, in the range of glucose 0 to 10 mM, 0 to 0.8.
A change in the current value of μA was observed.

Here, this calibration curve is represented by the following equation 1 when the current value is Y.

[0083]

(Equation 1)

FIG. 13 shows the dependence of the glucose concentration of each capillary on any four capillaries (G, H, I, and J, respectively) provided in the device according to the third embodiment of the present invention. The result of having measured the property is shown. The measurement conditions were the same as in FIG.

As shown in FIG.
It can be seen that in the glucose solution of M, the change is in the range of 0 to 1.5 μA.

[0086]

As described above, according to the present invention, a microenzyme reaction capillary composed of a silicon substrate and glass and an electrode for electrochemical measurement are manufactured, integrated and integrated by using micromachine technology. Enzyme that can be miniaturized, integrated, integrated, and mass-produced for biosensors, which have the advantage of being quick and simple, and that can be applied to a wider range. A reaction sensor and a method for manufacturing the same can be provided.

In the present invention, a measuring apparatus such as a liquid chromatography requires a transport system such as a pump for introducing a sample solution. However, all devices in the system require a capillary structure. In order to have a structure, a silicon capillary obtained by bonding a silicon substrate with a groove by anisotropic etching and glass is adopted, and the silicon capillary is also used as an enzyme reaction column, so the capillary itself is miniaturized. Capable of transporting the sample by capillary action and immobilizing the enzyme by chemical modification of the inner surface, and showed a sufficient response even to a small amount of glucose solution. An enzyme reaction sensor can be provided.

Further, according to the present invention, by arranging each device radially on a single silicon wafer and incorporating a large number of sensors, each device can be used as a disposable type sensor. It is possible to provide an enzyme reaction sensor that can be used continuously by the number of devices incorporated as a unit.

Further, according to the present invention, since the plastic substrate is used, it is possible to provide an enzyme reaction sensor which can be easily processed mechanically and can be manufactured at low cost.

Further, according to the present invention, since the plasma polymerization treatment is performed, it is possible to provide an enzyme reaction sensor in which the substrates can be easily bonded and the enzyme can be more easily immobilized.

[Brief description of the drawings]

FIG. 1A is an exploded perspective view showing a state before bonding a glass plate and a silicon wafer of a glucose sensor according to a first embodiment of the present invention. (B) is a plan view of the glucose sensor.

FIG. 2 is a graph showing the characteristics of the concentration of hydrogen peroxide and the amount of increase in the current value, and the table shows the current value for a buffer solution and a glucose solution.

FIG. 3 is a plan view showing a state before bonding a glass plate and a silicon wafer of an enzyme reaction sensor according to a second embodiment of the present invention.

FIG. 4 is an exploded perspective view showing an enzyme reaction sensor according to a third embodiment of the present invention, wherein (a) shows a first acrylic plate and (b) shows a second acrylic plate.

FIGS. 5 (a), (b) and (c) are FIGS.
It is a perspective view showing a part of acrylic board 21 and 25 shown in (b).

FIG. 6 is a diagram schematically showing an apparatus for performing plasma polymerization.

FIGS. 7 (a), (b), and (c) are diagrams illustrating a method for manufacturing an enzyme reaction sensor according to a third embodiment of the present invention.

FIGS. 8 (a), (b) and (c) are diagrams showing a method for manufacturing an enzyme reaction sensor according to a third embodiment of the present invention.

FIGS. 9A and 9B are diagrams showing a method for manufacturing an enzyme reaction sensor according to a third embodiment of the present invention.

FIG. 10 is a cyclic voltammogram showing an electrode response of hydrogen peroxide by a capillary electrode of an enzyme reaction sensor according to a third embodiment of the present invention.

FIG. 11 is a cyclic voltammogram showing an electrode response of hydrogen peroxide by a capillary electrode of an enzyme reaction sensor according to a third embodiment of the present invention.

FIG. 12 is a diagram showing a current value change (calibration curve) according to a glucose concentration of an enzyme reaction sensor according to a third embodiment of the present invention.

FIG. 13 is a graph showing the results of measuring the dependence of the enzyme reaction sensor according to the third embodiment on the glucose concentration of the capillaries (G, H, I, J).

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Silicon substrate 2 Groove 3, 4 Platinum electrode film 3a, 4a Electrode taking-out part 5 Glass plate 6 Capillary 10 Glucose sensor 11 Silicon substrate 12 Groove 13, 14, Platinum electrode film 13a, 14a Electrode taking-out part 21 First acrylic plate 22 hole Part 23 One surface 24 Groove 25 Second acrylic plate 27, 28, 29 Electrode 30 Center electrode 50 Chamber 51 Matching device 52 Oscillator 53 Plasma generator 54 Exhaust pipe 55 Inlet pipe 56 Diffusion pump 57 Rotary pump 58 Flow rate regulator 59 Reservoir 60 Sample stage 61 Conductor 62 Flow path

Claims (25)

[Claims] A semiconductor substrate, a plurality of grooves formed on one surface of the semiconductor substrate, a pair of noble metal films respectively formed in the plurality of grooves, and a glass plate covering the grooves. An enzyme reaction sensor comprising an enzyme immobilized in a capillary formed by the groove and the glass plate. 2. The enzyme reaction sensor according to claim 1, wherein said enzyme is glucose oxidase. 3. The enzyme reaction sensor according to claim 1, wherein the semiconductor substrate is formed of a circular silicon wafer, and the plurality of grooves are formed on one surface of the silicon wafer along a radial direction. An enzyme reaction sensor characterized by the following. 4. The enzyme reaction sensor according to claim 1, wherein the noble metal is made of platinum, and the pair of electrode films has an electrode extraction end near the center of the silicon wafer. An enzyme reaction sensor characterized in that: 5. The enzyme reaction sensor according to claim 1, wherein the glass plate is made of Pyrex glass, and the glass plate other than the groove and the semiconductor substrate are bonded to each other. An enzyme reaction sensor characterized in that: 6. Using a micromachine technology, a plurality of grooves are formed on a semiconductor substrate by etching, a pair of noble metal films is formed, and a glass plate is bonded and integrated so as to cover the grooves. Forming a capillary, and immobilizing an enzyme in the capillary. 7. The method according to claim 6, wherein the semiconductor substrate is formed of a circular silicon wafer and the groove is formed in a radial direction. 8. The method for manufacturing an enzyme reaction sensor according to claim 6, wherein the noble metal is made of platinum, and the pair of electrode films are formed near a center of the silicon wafer by a sputtering method and an ion milling method. A method for producing an enzyme reaction sensor, characterized in that the enzyme reaction sensor is formed so as to have an electrode extraction end. 9. The method for manufacturing an enzyme reaction sensor according to claim 6, wherein the glass plate is made of Pyrex glass, and is integrated with the silicon wafer by an anodic bonding method. A method for producing an enzyme reaction sensor. 10. The method for producing an enzyme reaction sensor according to claim 6, wherein glucose oxidase is immobilized as the enzyme by a chemical bonding / inclusive method. Manufacturing method. 11. A pair of mutually overlapping substrates, a plurality of grooves formed on one surface of at least one of the pair of substrates, and a plurality of grooves corresponding to the plurality of grooves.
An enzyme reaction comprising: at least two electrode films made of a noble metal formed on one of the pair of substrates; and an enzyme immobilized in a capillary formed by the groove and the substrate. sensor. 12. The enzyme reaction sensor according to claim 11, wherein the enzyme is glucose oxidase. 13. The enzyme reaction sensor according to claim 11, wherein the substrate is made of plastic having a circular shape, and the plurality of grooves are formed on one surface of the plastic substrate along a radial direction. An enzyme reaction sensor characterized in that: 14. The enzyme reaction sensor according to claim 11, wherein the noble metal is made of platinum, and the electrode film has an electrode extraction end near the center of the plastics substrate. An enzyme reaction sensor. 15. The enzyme reaction sensor according to claim 11, wherein three of said plurality of electrode films are formed. 16. The enzyme reaction sensor according to claim 11, wherein said pair of substrates are joined to each other. 17. The enzyme sensor according to claim 11, wherein the pair of substrates has a plasma-polymerized film on a bonding surface, and the pair of substrates is formed by welding the plasma-polymerized film. An enzyme reaction sensor that is joined to each other. 18. The enzyme sensor according to claim 11, wherein the enzyme is immobilized by using the amino group of a plasma polymerized film of a monomer having an amino group at least on the inner wall surface of the capillary and the amino group present in the enzyme. An enzyme reaction sensor comprising a group and a group bonded by a crosslinking reagent. 19. A plurality of grooves are formed on one substrate using a micromachine technique, and at least a pair of electrode film groups made of a noble metal are formed on another substrate corresponding to the grooves. An enzymatic reaction sensor, wherein the groove and the noble metal are made to correspond to each other, and the pair of substrates are overlapped, joined and integrated to form a capillary, and an enzyme is immobilized in the capillary. Manufacturing method. 20. The method according to claim 19, wherein the substrate is made of circular plastic, and the groove is formed in a radial direction. 21. The method for manufacturing an enzyme reaction sensor according to claim 19, wherein the noble metal is made of platinum, and the pair of electrode films are formed near the center of the substrate by a sputtering method and an ion milling method. A method for producing an enzyme reaction sensor, characterized in that the sensor is formed so as to have an extraction end. 22. The method for manufacturing an enzyme reaction sensor according to claim 19, wherein the substrate is made of an acrylic resin, and is bonded to and integrated with each other. Production method. 23. The method for manufacturing an enzyme reaction sensor according to claim 19, wherein a groove is formed on the one substrate and an electrode group is formed on the other substrate. A method for producing an enzyme reaction sensor, comprising: forming a plasma polymerized film containing an amino group on the surface of the surfaces of the substrates to be joined to each other before forming. 24. The method for producing an enzyme reaction sensor according to claim 19, wherein glucose oxidase is immobilized by a chemical bonding / inclusive method as the enzyme. Manufacturing method. 25. The method for producing an enzyme reaction sensor according to claim 23, wherein the enzyme has an amino group, and the enzyme is immobilized by a crosslinking reagent between the amino group of the plasma polymerized film and the amino group of the enzyme. A method for producing an enzyme reaction sensor, comprising binding by the action of