JPH0259655A - Small-sized microorganism electrode and production thereof - Google Patents

Abstract

PURPOSE:To prevent the leakage of microorganisms by fixing the microorganisms together with an electrolyte to the gel in the outside part of the gas permeable electrode of an oxygen electrode and in the gas permeable film of the small-sized microorganism electrode. CONSTITUTION:A silicon substrate 1 formed by anisotropic etching is coated with a silicon oxide film 2 as an insulating film over the entire surface of the trapezoidal hole thereof and further the rear surface is coated with a hydrophobic insulating film 7. Two pieces of gold electrodes 3A, 3B are deposited in the hole of the substrate 1 and an electrolyte-contg. gel 6 is filled therein. Further, the upper part of the substrate 1 is coated with the gas permeable film 14 of the oxygen electrode in the form of covering the entire surface of the upper part. There is a microorganism immobilized layer film 8 thereon and the film is coated with a gas permeable film 15.

Description

[Detailed Description of the Invention] [Summary] Regarding small microbial electrodes, the purpose of the present invention is to provide a small microbial electrode that is small and can be mass-produced at low cost. 2, which is formed through an insulating film and extends from the bottom of the liquid hole to the surface of the substrate.
It has a main electrode, a porous body containing an electrolytic solution filled inside the hole, and a gas permeable membrane that covers and closes the liquid hole and covers the porous carrier containing the electrolytic solution. a microorganism-immobilized membrane formed on the sensitive part of the oxygen electrode and formed of a porous carrier containing an electrolyte and microorganisms; and a second gas-permeable membrane covering the microorganism-immobilized membrane. constitute a small microbial electrode.

[Industrial Application Field] The present invention relates to a small carbon dioxide electrode, and more particularly to a small carbon dioxide electrode that can be mass-produced at low cost, and a method for manufacturing the same. The carbon dioxide sensor of the present invention is revolutionary in that it can measure carbon dioxide in an amberometrin link and is easy to miniaturize and mass produce.
It can be advantageously used in many metrology fields including fermentation industrial processes. The microorganism that can be used in the sensor of the present invention, that is, the microorganism electrode, is a microorganism that can assimilate carbon dioxide, preferably a microorganism belonging to the genus Pseudomonas.

[Prior Art] It is well known that in the field of measurement, measurement of carbon dioxide is important. Conventionally, carbon dioxide electrodes based on botensiometry have been developed and actually used as sensors for measuring carbon dioxide, especially for the purpose of measuring carbon dioxide components in body fluids (blood). For more information about carbon monoxide electrodes, see e.g.
J.W.

Severinghaus and A. F. Brad
ley SJ, 8pplPhysio1. ,13,51
5 (1958)).

However, this potentiometric carbon dioxide electrode has various problems that occur during measurement due to its configuration. For example, this method has problems such as being susceptible to the influence of contaminants and being unable to improve sensitivity beyond a certain level because sensitivity is affected by Nernst's second.

In order to solve this problem, the present inventors developed an amperometric carbon dioxide electrode having a structure in which autotrophic bacteria were fixed near the cathode of a Clark-type oxygen electrode (Japanese Patent Application Laid-open No. 80549/1983). .

However, since this carbon dioxide electrode uses an oxygen electrode as a transducer, due to the structure of this oxygen electrode,
For example, it has been extremely difficult to achieve miniaturization, multifunctionalization, cost reduction, etc. using photolithography.

[Problem that the invention seeks to solve]

The present invention utilizes the principle of JP-A No. 62-80549 to miniaturize a carbon dioxide electrode. Unexamined Japanese Patent Publication 1986~
In order to miniaturize a carbon dioxide electrode based on the principles disclosed in 80549, first of all, a miniaturization of the oxygen electrode used as a transducer within this carbon dioxide electrode must be realized.

Furthermore, as a structural problem, this carbon dioxide electrode,
For example, when used inside the body, the problem is how to prevent the leakage of microorganisms and create a safe structure.

The object of the invention is therefore to prevent the leakage of microorganisms,
Microorganisms are immobilized in a gel together with an electrolyte via a gas permeable membrane on the sensitive part of a small oxygen electrode with a safe structure, and the gel is covered with a second gas permeable layer 119, which is small, low cost, and can be produced in large quantities. An object of the present invention is to provide a microbial electrode that can be produced and a method for manufacturing the same.

[Means for solving problems]

According to one aspect, the present invention is a measurement sensor for measuring the concentration of carbon dioxide in a solution such as a body fluid or in the air, the sensor comprising an oxygen electrode and a cathode of the oxygen electrode. A gel containing microorganisms and an electrolyte that are fixed to the carbon dioxide and can selectively assimilate the carbon dioxide, and a gas permeable membrane covering the same. It is in the measurement sensor.

Another aspect of the present invention is a manufacturing method for miniaturizing and mass-producing such a carbon dioxide sensor, and a method for immobilizing microorganisms.

The present inventors have filed a patent application for the miniaturization of oxygen electrodes, which is the basis of miniaturization of carbon dioxide electrodes (Japanese Patent Application No. 1986-
No. 71739) There is a small oxygen electrode using photolithography. This oxygen electrode is placed over a hole formed by anisotropic etching on a silicon substrate.
! This oxygen electrode has a structure in which two electrodes are formed through the hole, an electrolyte-containing body is housed inside the hole, and finally the upper surface of the hole is covered with a gas permeable membrane. This oxygen electrode is small, has little variation in characteristics, and can be mass-produced in bulk, resulting in low cost.

In the above-mentioned type of oxygen electrode, the electrolyte-containing body is
Usually it was a gel impregnated with an electrolyte, such as an agarose gel. However, in the case of agarose gel, it was necessary to repeatedly inject it into many tiny holes on a silicon substrate one by one using a micropipette.

The present inventors conducted research to further improve this point and make it more suitable for mass production. Using polyacrylamide gel, -
We have discovered a method that allows gel to be injected all at once (Patent application 1982-
No. 148221). The manufacturing method of this small oxygen electrode is to form a plurality of holes using lithography technology and anisotropic etching technology, and then form a negative mold on a solicon substrate with electrodes formed in the container, except for the hole portions. The substrate is immersed in a solution of a photopolymerizable monomer, preferably acrylamide, containing an electrolytic solution, and each hole is filled with the solution and cured by irradiation with ultraviolet rays. It is characterized by forming a porous carrier containing a liquid in the holes. According to this manufacturing method, it is possible to form a porous carrier that selectively retains the electrolyte only in the minute holes that form the small oxygen electrode, thereby making mass production possible.

The photoresist to be applied onto the substrate after the electrodes are formed, except for the holes and the areas for electrical contact, is preferably a negative type photoresist, such as OMR- manufactured by Tokyo Ohka.
It is 83. This type of photoresist is advantageous because it has the property of repelling water and light at a ratio of 1:1 when only the holes are filled with an electrolyte-containing porous material.

By using such a small oxygen electrode, the carbon dioxide electrode can be easily miniaturized.

In implementing the method of the present invention, a semiconductor substrate, particularly a Noricon substrate, can be advantageously used as the base material of the electrode body. The insulating film can be formed from a silicon oxide film or other materials. A silicon oxide film can be easily formed by thermally oxidizing the substrate, for example, when the substrate is covered with glue.

The two electrodes can vary depending on whether the electrodes being made are polar or galvanic. In the case of manufacturing a carbon dioxide electrode based on a polar mouth type oxygen electrode, for example, both the picture electrode and the electrode are composed of a gold electrode or a platinum electrode, and a voltage is applied between the two electrodes during measurement.

Formation of such an electrode can be advantageously performed by, for example, a film forming method such as vacuum evaporation or sputtering.

As the electrolytic chamber to be held by a porous carrier, various materials such as potassium chloride, sodium chloride, etc. can be used.

The method for forming an electrolyte-containing alginate gel is to immerse a substrate coated with a photoresist in an electrolyte-containing sodium alginate aqueous solution, fill only the hole portions with the aqueous solution, and then immerse the substrate in this state into an electrolyte-containing chloride gel. A method of gelling the sodium alginate aqueous solution by immersing it in an aqueous solution of sodium alginate to form a calcium alginate gel, and a sol-gel method used in the process of synthesizing glass.

For example, a substrate coated with photoresist is coated with alkoxide,
It is advantageous to use a method in which the mixed solution is immersed in a mixed solution of water and an electrolytic solution, the mixed solution is filled only in the hole, the mixed solution is gelled by a sol-gel method, and the electrolytic solution is retained by the gel. It can be implemented.

In addition, alginic acid is an organic polymer electrolyte, and its carboxylic acid 5 (COOI+) has different activities. Furthermore, alginic acid forms insoluble salts with divalent or higher valent metals (excluding magnesium and mercury).

Gas permeable membranes are hydrophobic and do not allow water/8'a to pass through, but they are initially liquid and can be dip coated or spin coated, and can be used as electrode materials, silicon substrates, and insulating films. It is also essential that the adhesion to the silicon oxide film is good and that the electrolyte does not leak outside. Suitable gas permeable membrane materials include:
Examples include photoresists, preferably negative-type photoresists. Although Teflon (trade name) membrane is permeable to oxygen, it does not have adhesive strength and should be avoided.

Microorganisms that can be advantageously used in carrying out the present invention are microorganisms that can selectively assimilate carbon dioxide, such as microorganisms belonging to the genus Pseudomonas and other genera.

The present inventors have discovered that Pseudomonas sp. T IT/FJ-00
01 bacteria (Feikoken Bacteria No. 8473), unidentified TIT/
Particularly favorable results were obtained using the FJ-0002 bacterium (Feikoken Bacteria No. 8474) (for the properties of these bacteria, see JP-A-62-80549). ) In addition, the present inventors, in immobilizing microorganisms,
From the viewpoint that fixing microorganisms on the surface of the small oxygen electrode disclosed in Japanese Patent Application No. 62-71739 poses a safety problem because the microorganisms are likely to leak out, the microorganisms are fixed outside the gas-permeable membrane of the oxygen electrode. In addition, a method was adopted in which the microbial electrode was immobilized in a gel together with an electrolytic solution inside the gas-permeable membrane of the small microbial electrode.

[Effect]

In order to facilitate understanding of the operation of the present invention, the attached
The principle of the small carbon dioxide electrode of the present invention will be explained with reference to the drawings.

As shown in the figure, carbon dioxide and oxygen in the solution or in the air reach the vicinity of the immobilized microorganisms 8 after passing through the gas permeable membrane 15. The microorganisms 8 absorb carbon dioxide, and at this time, respiratory activity becomes active and oxygen is consumed. Therefore, the concentration of oxygen that passes through the gas permeable membrane 14 of the oxygen electrode and reaches the cathode 3A decreases. If this amount of change in oxygen concentration can be interpreted as a change in the electrochemical oxygen reduction current, the carbon dioxide concentration can be measured based on the resulting decrease in current value. Conventionally, it has been difficult to measure carbon dioxide concentration amperometrically, but in the present invention, by using an oxygen electrode as a transducer, it has become possible to quantify carbon dioxide amperometrically. On the other hand, by miniaturizing the oxygen electrode, which is the transducer in the carbon dioxide electrode, by using lithography technology and thin film formation technology that are used in the formation of semiconductor integrated circuits, we have succeeded in making the carbon dioxide electrode microscopic. This made it possible to integrate and mass produce at once.

〔Example〕

A preferred example of the method for manufacturing a small carbon dioxide electrode according to the present invention will be described with reference to the accompanying drawings.

FIG. 2 is a perspective view showing a preferred example of a small carbon dioxide electrode according to the present invention. The illustrated carbon dioxide electrode has a rectangular parallelepiped shape, and the sensitive part is covered with a gas permeable membrane 15, and part of the electrodes 3A and 3B are exposed for connection to an attached device. In this example, the electrodes 3A and 3B were made of gold electrodes as well as the picture electrodes in order to have a polar mouth type.

The structure of the small carbon dioxide electrode shown in FIG. 2 can be understood in detail from FIG. 3, which is a cross-sectional view taken along line IIIT. A silicon substrate 1 has a trapezoidal hole (groove) formed by anisotropic etching, and a silicon oxide film 2 is deposited as an insulating film over the entire surface thereof. Furthermore, the back surface of the substrate is covered with a hydrophobic insulating film 7 that is hard to tear. Two gold electrodes 3A are placed in the holes of the silicon substrate 1.
and 3B are deposited in pairs. As shown in FIG. 2, a portion of each of the gold electrodes 3A and 3B extends to the outside of the groove. Further, the holes in the silicon substrate 1 are filled with an electrolyte-containing gel 6. Further, in the upper part of the hole, gas of the oxygen electrode as a transducer is placed so as to cover the entire upper surface of the substrate l (excluding the exposed part where electrodes 3A and 3B in FIG. 2 are exposed). A transparent film 14 is covered, which also covers the side and back surfaces as an insulating film. On the gas permeable membrane 14 of such an oxygen electrode, there is further a microorganism fixed layer membrane 8,
It is further covered with a gas permeable membrane 15.

The miniature carbon dioxide electrodes shown in FIGS. 2 and 3 can be advantageously manufactured, for example, by the manufacturing process shown in sequence in FIG. Note that the main body after electrode formation shown in FIG. 4(A) can be manufactured through the following steps. In the following explanation, in order to make it easier to understand, we will describe the case where only one carbon dioxide electrode is formed on one wafer, but in reality, many small carbon dioxide electrodes are formed at the same time. I hope you understand that.

(2) Wafer Cleaning A 2-inch (100)-sided silicon wafer with a thickness of 350 μm was prepared and cleaned with a mixed solution of hydrogen peroxide and ammonia and concentrated nitric acid.

2. Formation of SiO□ film A silicon wafer is wet thermally oxidized, and a film 1 is formed on the entire surface.
! to form an 8 μm SiO□ film.

3. Formation of etching pattern Using a negative photoresist (Tokyo Ohka OMR-83, viscosity 60 cP), Si0g was formed on the wafer.
A resist pattern for etching was formed.

4. The same negative photoresist used in the above step was applied to the back side of the resist-coated wafer and baked at 130°C for 30 minutes.

5. Etching of SiO□ film 50% hydrofluoric acid: 50% ammonium fluoride-1:
The wafer is soaked in a 6 ml water solution, and the exposed areas not covered with photoresist are coated with SiO! was removed by etching. Then sulfuric acid/hydrogen peroxide (2:1)?
, the resist was removed with a solution.

6.3i Anisotropic Etching A silicon wafer was anisotropically etched in a 35% potassium hydroxide aqueous solution at 80°C. After etching was completed to a depth of 300 μm, the wafer was washed with pure water.

After completing this anisotropic etching, the 340 g film used during etching was removed. This was carried out in a 50% hydrofluoric acid:50% ammonium fluoride-1:6 aqueous solution as in 5.

7. Formation of SiO□ film To grow a SiO□ film on the silicon wafer surface,
1. Following the cleaning step, the wafer was wet thermally oxidized. A film 2 having a thickness of 0.8 μm was formed.

8. Formation of Chromium and Gold Thin Films Following the ROM thin film (400 people, adhesion of gold and substrate), a gold thin film (4000 people) was formed by vacuum evaporation.

9. Formation of resist pattern for electrode formation Negative photoresist (Tokyo Ohka OMR-83, viscosity 60 cP)
A resist pattern for electrode formation was formed on the SiO□ of the wafer.

10. Gold and chromium etching The substrate on which the resist pattern has been formed is immersed in the following gold and chromium etching solutions in this order, and the exposed gold and chromium parts are removed by etching. Furthermore, after washing with pure water, sulfuric acid/hydrogen peroxide solution (2:1)
The resist was removed using a solution.

■Gold etching solution: 4g KI and 1g Ig dissolved in 40ml water ■Chromium etching solution: Q, 5g NaOH and 1g K7e(CN)a dissolved in 4n+1 water Gold electrode 3A 3B Figure 4 (A) shows the formed state.
Shown below.

11. Hydrophobic insulating film formed on the back side of the substrate Hydrophobic insulating film (R-28 manufactured by Shin-Etsu Silicone) was formed on the back side of the substrate.
2) is coated in a diagonal pattern and subjected to baking at 250° C. to form a hydrophobic insulating film 7.

12. Resist coating (Fig. 4 (B)) on the main body surface,
The area other than the hole (trapezoidal groove) 5 and the exposed pad portion for making electrical contact was covered with a negative photoresist (manufactured by Tokyo Ohka, OMR-83, viscosity 60 cP) 4. This was done by applying a photoresist to the surface of the wafer, exposing and developing after brining.

13. Cutting out the substrate The oxygen electrode (carbon dioxide electrode) bodies formed in large numbers on the substrate were cut out into chimney shapes.

14. Immerse the tip end portion of the chip in which the hole (trapezoidal groove) 5 is formed to make the inside of the hole (trapezoidal groove) water-soluble in a 10% aqueous sodium hydroxide solution. As a result, the areas not covered with resist became hydrophilic.

15. Filling with calcium alginate gel (Figure 4 (C)
) The following two types of solutions were prepared for forming electrolyte-containing gels.

Solution A: Sodium alginate dissolved in 0.11 potassium chloride aqueous solution.

Sodium alginate concentration 0.4%.

Solution B: Calcium chloride dissolved in 0.1 part potassium chloride aqueous solution.

Calcium chloride concentration 5%.

When the chip shown in Figure 4 CB) was thoroughly cleaned in liquid A and slowly pulled up, the negative photoresist film 4 was hydrophobic, so the previous liquid A was peeled off from the resist film and only entered the hole 5. The remaining. Then, when this chip was immersed in liquid B, liquid A instantly gelled, and the calcium alginate gel 6 was filled into the holes 5.

16. Covering the gas-permeable membrane (Fig. 4 (D)) The gas-permeable membrane 14 is coated on the electrolyte-containing gel 6 so as to cover the gel.
coated. In this example, the same negative photoresist as used in step 3 was used as the gas permeable film. That is, a negative photoresist (manufactured by Tokyo Ohka Co., Ltd. OMR-
83 (trade name), viscosity 60 cP) was applied by dip coating. This resist film is also formed on the side walls and the back surface, which has an advantageous effect in further improving the insulation properties.

17. @Biological fixation (Figure 4 (E)) Two types of solutions were prepared as follows for forming gels containing microorganisms and electrolyte.

Solution C: 0.5ml of 0.4χ sodium alginate aqueous solution
inside? 5 mg of TIT/FJ-0001 bacteria in wet state
Dissolved.

Solution B: 5χ aqueous calcium chloride solution.

The sensitive part of the small oxygen electrode completed in steps up to step 16 is first immersed in liquid C, and then immediately immersed in liquid D to immobilize microorganisms in the calcium alginate gel.

18. Formation of gas-permeable membrane (FIG. 4(E)) A gas-permeable membrane was formed on the microorganism-immobilized membrane 8 formed in step 17. Here, as in No. 16, a negative photoresist film was formed by dip coating.

This resist can be applied to a small oxygen electrode (
The main body of the carbon dioxide electrode was placed in pure water or saturated steam and left in a bright place for several hours to remove the xylene solvent and expose it to light. Thereby, the gas permeable membrane 15 was completed.

[Effects of the Invention] According to the method of the present invention, the disadvantage of being easily affected by contaminants is eliminated by forming a gas permeable membrane on the microorganism immobilization membrane, and the method also eliminates the disadvantage of being susceptible to the influence of contaminants by applying autotrophic bacteria and a small oxygen electrode. This makes it possible to miniaturize amperometric carbon dioxide electrodes.

[Brief explanation of the drawing]

Fig. 1 is a principle diagram of the small carbon dioxide electrode of the present invention, Fig. 2 is a perspective view showing a preferred example of the small carbon dioxide electrode of the present invention, and Fig. 3 is a line segment of the electrode of Fig. 2. The sectional view along ILII and FIGS. 4(A) to 4(E) are step-by-step sectional views sequentially showing the manufacturing process of the small carbon dioxide electrode shown in FIGS. 2 and 3. In the figure, 1 is a substrate, 2 is an insulating film, 3A and 3B are electrodes,
4 is a photoresist film, 5 is a hole (trapezoidal groove), 6 is an electrolyte-containing gel, 7 is a hydrophobic insulating film, 8 is a microorganism immobilization film,
And 14.15 is a gas permeable membrane. Carbon dioxide T-sensor Zenhara FG! z th fU3 14, /j ~ ---Gas fLM raw order ff -1-ha J fixed, Ihio Tatsuo 1 Nakune ■-g Main landing gear JA-11 ligature electrode 1 canode

Claims (5)

[Claims] (1) A semiconductor substrate having a trapezoidal groove (hole), two electrodes formed on the substrate via an insulating film and extending from the bottom of the hole to the surface of the substrate, and inside the hole. An oxygen electrode comprising a porous carrier filled with an electrolytic solution, a gas permeable membrane that covers and closes the holes and covers the porous carrier containing the electrolytic solution, and a sensitive part of the oxygen electrode. A small-sized microorganism electrode comprising: a microorganism-immobilized membrane formed on a porous carrier containing an electrolytic solution and microorganisms; and a second gas-permeable membrane covering the microorganism-immobilized membrane. (2) The small microorganism electrode according to claim 1, wherein the microorganism is an autotrophic bacterium. (3) The small-sized microorganism electrode according to claim 2, wherein the autotrophic bacterium is a bacterial cell belonging to the genus Pseudomonas that can assimilate carbon dioxide. (4) The bacterial cells belonging to the genus Pseudomonas are TIT/
FJ-0001 bacterium (FEI Bacteria No. 8473), or TIT/FJ-0002 bacterium (FEK Bacteria No. 8474)
4. The small microorganism electrode according to claim 3, wherein the microorganism electrode is a small microorganism electrode. (5) A trapezoidal groove (hole) is formed on a semiconductor substrate by anisotropic etching of the semiconductor, and two electrodes are connected on the substrate from the bottom of the hole to the surface of the substrate via an insulating film. A porous carrier containing an electrolytic solution is filled only inside the holes, and a gas permeable membrane is formed to cover and close the holes to cover the porous carrier containing the electrolytic solution to form an oxygen electrode. The sensitive part of the oxygen electrode is immersed in an aqueous alginate metal salt solution containing autotrophic bacteria, and then immersed in an aqueous solution of a divalent or higher metal salt that forms an insoluble salt with alginic acid to gel the alginate and separate it. A method for manufacturing a small microorganism electrode, comprising the steps of forming a trophic bacteria-immobilized membrane and covering the autotrophic bacteria-immobilized membrane with a gas-permeable membrane.