JPH0227251A - Small organism electrode and manufacture thereof - Google Patents

Abstract

PURPOSE:To enable selective fixing of a microorganism at a fine place by filling a hole formed on the surface of a substrate with a solution only containing material for formation of a gel to dip it into a solution containing a microorganism and a gelatinization promoting material for gelatinization. CONSTITUTION:A hole 5 is made on an Si substrate 1 by an anisotropic etching and gold electrodes 3A and 3B upto the surface of the substrate 1 from the bottom of the hole 5 are formed through an insulation film (SiO2) 2. A negative type photo resist 4 is covered excluding the part of the hole 5 and after the back of the substrate 1 is covered with a hydrophobic insulation film 7, the substrate 1 is immersed into an aqueous solution (liquid A) of sodium arginate containing an electrolyte to fill the inside of the hole 5 with the liquid A. Moreover, the substrate 1 in this condition is emmersed into a CaCl2 aqueous solution containing an independent nourishing germ with which the electrolyte and CO2 are made conductive to gelatinize 6 the liquid A. Then, after the CaCl2 aqueous solution unreacted and the microorganism are washed with water, the entire surface of the substrate 1 (except for a exposed part) is covered with a gas permeable film 14 thereby enabling the manufacture of a massproducible small CO2 electrode with a safe construction.

Description

[Detailed Description of the Invention] [Summary] Regarding a small carbon dioxide electrode, the purpose of the present invention is to provide a carbon dioxide electrode that is small and can be mass-produced at low cost. A drilled 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 microorganisms filled inside the hole. A small microbial electrode comprising an electrolyte-containing porous material and a gas-permeable membrane covering and closing the holes, wherein the electrolyte-containing porous material is a gel of alginate metal salt containing autotrophic bacteria. Configure a small microbial electrode as follows.

[Industrial application field]

The present invention relates to a small microbial electrode, and in particular to a small, low-cost, mass-producible carbon dioxide electrode and a method for manufacturing the same. It is revolutionary in that it can be easily mass-produced, and can be advantageously used in many measurement fields including medicine, the environment, and 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.

[Conventional technology]

In the field of measurement, it is well known that measuring carbon dioxide is important. Conventionally, carbon dioxide electrodes based on potentiometry 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 dioxide electrodes, see e.g.
J.J.

Severinghaus and ^, F, Brad
ley, J., Appl.

Physiol, 13.515 (1958)) However, this potentiometric type 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 influenced by the Nernst equation.

In order to solve this problem, the present inventors would like to develop an amperometric carbon dioxide electrode having a structure in which autotrophic bacteria are immobilized near the cathode of a Clark-type oxygen electrode (Japanese Patent Laid-Open No. 62-80549). However, since this carbon dioxide electrode uses an oxygen electrode as a transducer, the structure of this oxygen electrode makes it possible to achieve miniaturization, multifunctionalization, cost reduction, etc. using photolithography, for example. That was extremely difficult.

[Problem to be solved by the invention]

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, in producing a small carbon dioxide electrode using microorganisms and an oxygen electrode, the problem is how to selectively and efficiently immobilize microorganisms in minute locations. For this fixation method, the method originally disclosed in Japanese Patent Application No. 148221/1980 is applied, for example, by mixing microorganisms in an aqueous acrylamide solution and taking advantage of the difference between the hydrophilicity and hydrophobicity of the negative photoresist and the substrate. Then, we devised a way to fix it in a tiny spot. However, when we examine this method in detail, we find that if microorganisms are mixed into the acrylamide aqueous solution, this aqueous solution will adhere anywhere, regardless of whether it is hydrophilic or hydrophobic. It was found that this method could not be used to selectively immobilize microorganisms in minute locations.

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

Therefore, an object of the present invention is to place microorganisms inside a gas-permeable membrane of a small oxygen electrode together with an electrolyte in a structure that allows selective immobilization of microorganisms in minute locations, prevents leakage of microorganisms, and has a safe structure. The object of the present invention is to provide a microbial electrode fixed in a gel that is small, inexpensive, and mass-producible, and a method for manufacturing the same.

[Means to solve the problem]

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 carbon dioxide measurement method comprising a gel containing microorganisms and an electrolyte that are fixed to a carbon dioxide and can selectively assimilate the carbon dioxide, and a gas permeable membrane covering the same. According to another aspect of the present invention, there is provided a manufacturing method and a method for immobilizing microorganisms for miniaturizing such a carbon dioxide sensor and mass-producing it in bulk.

That is, a hole is made on a silicon substrate by anisotropic etching, two electrodes are formed from the bottom of the hole to the surface of the substrate via an insulating film, and a negative photoresist is formed except for the liquid hole. After coating the back side of the substrate with a hydrophobic insulating film, it is immersed in an electrolyte-containing sodium alginate aqueous solution, filling only the inside of the hole with this aqueous solution, and then the substrate in this state is coated with an electrolyte and autotrophic bacteria-containing calcium chloride. A small carbon dioxide electrode characterized in that the sodium alginate aqueous solution is gelled by immersion in an aqueous solution, unreacted calcium chloride aqueous solution and microorganisms are washed away, and then a gas permeable membrane is formed by dip coating. Regarding the miniaturization of oxygen electrodes, which is the basis of miniaturization of carbon dioxide electrodes in the manufacturing method, there is a small oxygen electrode using photolithography, which the present inventors have filed a patent application for (Japanese Patent Application No. 71739/1982). This oxygen electrode is made by forming two electrodes through an insulating film over a hole formed on a silicon substrate by anisotropic etching, further accommodating an electrolyte-containing body inside the hole, and finally This is an oxygen electrode with a structure in which 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 discovered a method that allows gel to be injected all at once (Patent application 1986-
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 create a negative mold on a silicon substrate on which electrodes are formed in each formula, except for the liquid hole part. After coating the resist, the substrate is immersed in a solution of a photopolymerizable monomer, preferably acrylamide, containing an electrolyte, and each hole is filled with the solution and cured by irradiation with ultraviolet rays. It is characterized by forming a porous carrier inside 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.

By using such a small oxygen electrode, the carbon dioxide electrode can be easily miniaturized. However, for this purpose, there is an additional problem of where and how to immobilize the microorganisms.

For the fixation of microorganisms, the above-mentioned patent application
It was thought that basing the method on the method disclosed in No. 221 would be very advantageous from both a process and safety standpoint. However, as mentioned in all sections, it was impossible to apply this method as is. Therefore, we investigated an alternative method, and first, we found an aqueous solution containing only gel raw materials without microorganisms. (first solution) is filled only in specific locations on a substrate by taking advantage of the difference between hydrophilicity and hydrophobicity, and the substrate in this state is placed in a solution (second solution) containing microorganisms and a gelling promoter. It has been found that if there is a method of immersing the microorganism in a gel solution, it is possible to selectively immobilize the microorganisms by washing away the second solution containing the remaining microorganisms with water. It has been found that for this purpose, alginate metal salt gel such as calcium alginate gel can be used according to the method described in the Examples.

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 a calcium aqueous solution to form a calcium alginate gel, and a sol-gel method 1 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.

Note that alginic acid is an organic polymer electrolyte and its carboxylic acid group (COOH) is highly active. Furthermore, alginic acid forms insoluble salts with divalent or higher valent metals (excluding magnesium and mercury).

In addition, the present inventors believe that when immobilizing microorganisms,
From the viewpoint that fixing microorganisms on the surface of a small oxygen electrode as disclosed in Japanese Patent Application No. 62-71739 would cause safety problems because the microorganisms would easily leak out, microorganisms were fixed inside the gas permeable membrane of the small oxygen electrode. We adopted a method of fixing it in a gel together with an electrolyte.

In implementing the method of the present invention, a semiconductor substrate, particularly a silicon 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 a silicon substrate, for example.

The two electrodes can be of different colors depending on whether the electrode is of the polar or galvanic type, but the structure of this carbon dioxide electrode does not allow for the coexistence of microorganisms and electrolyte. Therefore, for example, when manufacturing a carbon dioxide electrode based on a polar type oxygen electrode, it is preferable to use a polar type using a neutral electrolyte such as a 0.1M potassium chloride aqueous solution. Both are composed of gold or platinum electrodes, and a voltage is applied between both 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.

The photoresist to be applied onto the substrate after the electrodes are formed, except for the hole portions and the portions for making electrical contact, is preferably a negative type photoresist, such as Tokyo Ohka OMR-
It is 83. This type of photoresist is advantageous because it has the property of repelling the aqueous solution that is the raw material when only the hole portions are filled with an electrolyte-containing porous material.

Various electrolytes such as potassium chloride and sodium sulfate can be used as the electrolyte to be retained by the porous carrier.

Gas-permeable membranes are hydrophobic and do not allow aqueous solutions to pass through them, but they are initially liquid and can be dip-coated or spin-coated.
Suitable gas-permeable film materials that have good adhesion to the silicon substrate and the silicon oxide film as an insulating film and that the electrolyte does not leak outside include photoresists, preferably negative-type photoresists. Examples include resist. Teflon (trade name) membrane is oxygen i
! Although it is a pass-through, it has no turning power, so its use must be avoided.

Microorganisms that can be advantageously used in carrying out the present invention include microorganisms that can selectively assimilate carbon dioxide, such as microorganisms belonging to the genus Shademonas and other genera.
The present inventors have discovered that Pseudomonas sp.TIT/FJ-000
1 bacterium (Feikoken Bacteria No. 8473), unidentified T I T
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).

[Effect]

In order to facilitate understanding of the operation of the present invention, the attached
The principle of the small microorganism electrode of the present invention, particularly the small carbon dioxide electrode, 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 14. The microorganism 8, for example, a microorganism that can selectively assimilate carbon dioxide, assimilates carbon dioxide, and at this time, respiratory activity becomes active and oxygen is consumed, so that the concentration of oxygen that 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 was 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 in an amberometric manner. . On the other hand, using lithography technology and thin film formation technology used in the formation of semiconductor integrated circuits,
By miniaturizing the oxygen electrode, which is the transducer in the carbon dioxide electrode, and selectively immobilizing microorganisms in minute locations, we have made it possible to miniaturize the carbon dioxide electrode and mass-produce it 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 carbon dioxide electrode shown in FIG. , part of the terminals 13A and 3B are exposed for connection to the attached device. Electric i3A, 3B
In this example, both the picture electrode and the gold electrode were used to form a polar mouth type.

The structure of the small carbon dioxide electrode in Figure 2 is 11-11.
A more detailed understanding can be obtained from FIG. 3, which is a cross-sectional view along the line. A silicon substrate 1 has a hole 5 formed by anisotropic etching, and a silicon oxide film 2 is deposited as an insulating film over the entire surface thereof. (In addition,
Since individual chips are formed separately by slicing the wafer, no silicon oxide film is formed on the side surfaces of the slice cut portion in the illustrated example).Furthermore, the back surface of the substrate is covered with a hydrophobic insulating film 7 that is hard to tear. In the hole 5 of the silicon substrate 1, two pieces of gold ti3A and 3B are deposited as a pair. As shown in FIG. 2, a portion of each of the gold electrodes 3A and 3B extends to the outside of the groove. Furthermore, the holes 5 in the silicon substrate 1 are filled with autotrophic bacteria and an electrolyte-containing gel 6. moreover,
A gas permeable film 14 is coated on the upper part of the hole so as to cover the entire upper part of the substrate 1 (excluding the exposed part in FIG. 2), and this is also covered as an insulating film on the side and back surfaces. ing.

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 forming the electrodes 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! It should be understood that the poles are formed at the same time.

1. 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 was subjected to wet thermal oxidation, and an St island film having a thickness of 0.8 μm was formed on the entire surface.

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

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

5. SiO, film etching 50% hydrofluoric acid: 50% ammonium fluoride-1:
The wafer was immersed in a 6-aqueous solution, and exposed portions of S+02 not covered with photoresist were removed by etching. Subsequently, the resist was removed using a sulfuric acid/hydrogen peroxide solution (2:1).

6. Anisotropic etching of Si Anisotropic etching of silicon was performed in a 35% potassium hydroxide aqueous solution at 80°C. Etching depth 300
After completion of etching, the wafer was washed with pure water.

After completing this anisotropic etching, the SiO□ film used during etching was removed. This was carried out in a 1:6 aqueous solution of 50% hydrofluoric acid:50% ammonium fluoride in the same manner as in 5.

7. Formation of StOg film In order to grow SjOtM2 on the exposed surface of the anisotropically etched silicon wafer, 1. Following the cleaning step, the wafer was wet thermally oxidized. II! A film 2 with a thickness of 0.8 μ-〇 was formed.

Formation of 8° Chromium and Gold Thin Films Following the ROM thin film (400 people, for adhering the 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 Si0g wafer using the following method.

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: l
) The resist was removed using a solution.

■Total etching solution: 4gKr and 1g! 2 to 40
Sol dissolved in water■ Chrome etching solution a, o, s g NaOH and 1g K2Fe<CN)h dissolved in 411 water Figure 4 (A) shows the state in which gold etching is formed. .

11. Forming a hydrophobic insulating film on the back side of the substrate A hydrophobic insulating film (KR-28 made by Shin-Etsu Silicone) was formed on the back side of the substrate.
2) Apply 7 evenly and bake at 250°C.

12. Applying resist (Figure 4 (B)) Apply negative photoresist (Tokyo Ohka Co., Ltd.
OMR-83, viscosity 60 cP) 4 was applied to the surface of the wafer, followed by exposure and development after blivata.

]3. Cutting the Substrate A large number of carbon dioxide electrode bodies formed on the substrate were cut into chips.

14. Make the inside of the hole hydrophilic. Immerse the tip end of the tip in a 1M sodium/lium hydroxide aqueous 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 gels containing microorganisms and electrolyte.

Solution A: Sodium alginate dissolved in O, 1M potassium chloride aqueous solution, sodium alginate concentration 0
．． 2X.

Solution B: Moist TIT/FJ-0001 bacteria 5I1
g and calcium chloride dissolved in a 0.1 M potassium chloride aqueous solution, calcium chloride concentration 5%.

When the chip shown in FIG. 4(B) was immersed in liquid A and slowly pulled up, the negative photoresist film 4 was hydrophobic, so the liquid A was repelled from the resist film and flowed into the hole 5. When this chip was then immersed in Solution B, Solution A instantly turned into a gel and the TIT/FJ-0001 bacteria were immobilized inside the gel.

Microorganisms and unreacted calcium chloride aqueous solution adhere to the resist, but these can be easily removed by rinsing with pure water.

16. Coating of a gas permeable membrane (Fig. 4 (D)) A gas permeable membrane 114 is coated on the microorganism and electrolyte-containing gel 6 so as to cover the gel. In this example, as a gas permeable membrane, The same negative photoresist used in Grade 3 was used. That is, a negative photoresist (TOKYO OHKA OMI?-83 (trade name), viscosity 60 cP) was applied by dip coating. This resist is
Leave the small oxygen electrode body in pure water or saturated steam for several hours in a bright place without pre-baking.
It took a long time to remove the solvent thinner and expose it to light. As a result, a gas permeable film and a sidewall insulating film were completed. This resist film is also formed on the back side, but this is more t! It works advantageously to improve IA affinity.

〔Effect of the invention〕

According to the method of the present invention, it is easy to immobilize microorganisms in minute locations, and this technology can be used to create small microorganism electrodes using autotrophic bacteria and small oxygen electrodes. becomes possible.

[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. 1'1-11, and FIGS. 4(A) to 4(D) are cross-sectional views showing the manufacturing process of the small carbon dioxide electrode shown in FIGS. 1 and 2 in order. be. 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, 6 is an electrolyte-containing gel,
7 is a hydrophobic insulating film, 8 is a microorganism, and 14 is a gas permeable film.・), Type 2 Nishi 1 Tomoe Kuru f He Mme Teka C Kata f Rizu No. 1 Rono hq9Lt%%”x(?>i9p, 1.': J Mine 2
? Intoxicated Koma 2 Tohru 3 S / Kari, 11 Manufacturing process of fatigue-based electrodes 4 ■

Claims (6)

[Claims] (1) A substrate, a hole formed on the surface of the substrate by anisotropic etching, and a hole formed on the substrate via an insulating film, the hole being formed from the bottom of the hole to the surface of the substrate. A small microbial electrode comprising two electrodes, an electrolyte-containing porous material containing microorganisms filled in the holes, and a gas permeable membrane covering and closing the holes,
A small microbial electrode characterized in that the electrolyte-containing porous material is a gel of metal alginate containing autotrophic bacteria. (2) The small carbon dioxide electrode according to claim 1, wherein the microorganism is an autotrophic bacterium capable of assimilating carbon dioxide. (3) The small microorganism electrode according to claim 1 or 2, wherein gold electrodes are used as the two electrodes. (4) A hole is made on the surface of the substrate by anisotropic etching, two electrodes are formed from the bottom of the hole to the surface of the substrate via an insulating film, and microorganisms are contained inside the hole. A method for manufacturing a small microbial electrode comprising filling an electrolyte-containing porous material and covering and closing the hole with a gas-permeable membrane, the step of filling the electrolyte-containing porous material containing microorganisms into the hole. , filling only the inside of the hole on the substrate using the difference between hydrophilicity and hydrophobicity selectively formed on the surface of the substrate with a first solution that does not contain microorganisms and only contains raw materials for gel formation. 1. A method for producing a small microbial electrode, comprising the steps of: immersing the substrate in a second solution containing microorganisms and a gelling promoter to gel the substrate. (5) A hole is made on the surface of the substrate by anisotropic etching, two electrodes are formed from the bottom of the hole to the surface of the substrate via an insulating film, and microorganisms are contained inside the hole. In a method for manufacturing a small microbial electrode, which comprises filling a porous material containing an electrolyte and covering and closing the hole with a gas-permeable membrane, only the inside of the hole is filled with an aqueous solution of a water-soluble metal alginate containing an electrolyte. After that, the substrate is immersed in an aqueous solution of a divalent or more metal salt that contains microorganisms and forms an insoluble salt with alginic acid to gel the aqueous solution of the water-soluble alginate metal salt, and remove the unreacted divalent or more metal salt. A method for producing a small microbial electrode, which comprises forming the gas-permeable membrane after washing a metal salt aqueous solution with water. (6) The method for producing a small carbon dioxide electrode according to claims 4 and 5, wherein the microorganism is an autotrophic bacterium capable of assimilating carbon dioxide.