JP2661240B2 - Small L-lysine sensor and method of manufacturing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Overview] The present invention relates to an L-lysine sensor, and an object of the present invention is to provide an L-lysine sensor that can be mass-produced in a small size at low cost and a method for manufacturing the same. A semiconductor substrate having a groove (hole) formed by the above, an insulating film covering the surface of the substrate, and a cathode / anode formed from the bottom of the hole to the surface of the substrate via the insulating film. An electrode, a gel containing an electrolyte and an autotrophic bacterium filled in the hole, a gas permeable membrane covering the gel, and a decarboxylase L- formed on the gas permeable membrane of the electrode sensitive part. Forming a small L-lysine sensor having an enzyme-immobilized membrane on which lysine decarboxylase is fixed, forming a groove (hole) on a semiconductor substrate by anisotropic etching, and covering the surface of the substrate Form insulating film Forming two electrodes serving as a cathode and an anode from the bottom of the hole to the surface of the base via the insulating film; and covering the back surface of the substrate with a hydrophobic insulating film. Coating the hydrophobic photoresist film except for the holes, and immersing the substrate in an aqueous solution of alkali metal alginate containing electrolyte and autotrophic bacteria to fill only the inside of the holes with the aqueous solution; A step of gelling the aqueous solution of the alkali metal alginate by immersion in an aqueous solution of an alkaline earth metal salt, a step of forming a gas-permeable membrane covering the gel, and Forming an enzyme-immobilized membrane on which decarboxylase L-lysine decarboxylase is immobilized, thereby constituting a method for producing a small L-lysine sensor.

[Industrial applications]

The present invention relates to an L-lysine sensor, and more particularly to an L-lysine sensor that can be mass-produced at a small size and at a low cost, and a method for manufacturing the same. More specifically, the present invention relates to a method for measuring L-lysine and a sensor for measuring L-lysine using an enzyme function and a microbial function in combination and further utilizing an electrochemical electrode. The L-lysine sensor of the present invention comprises:
L-lysine can be measured amperometrically, and it is revolutionary in that miniaturization and mass production are easy.
Low cost because batch mass production is possible. The present invention is used in many measurement fields including medical and fermentation.
It can be used advantageously.

[Conventional technology]

L-Lysine can be measured by various methods. Among them, a particularly quick and simple method is the L-lysine sensor developed by Guilbault et al. This was prepared by immobilizing L-lysine decarboxylase on a potentiometric carbon dioxide electrode functional part. In this sensor, L-lysine is decomposed by L-lysine decarboxylase, and the carbon dioxide generated at this time is measured by a carbon dioxide electrode to indirectly quantify the L-lysine concentration.

[Problems to be solved by the invention]

The L-lysine sensor of Geilbault et al. Uses a conventional carbon dioxide electrode as a transducer, but this carbon dioxide electrode is usually about the size of a fountain pen, which is inconvenient for measuring a small amount of sample. is there.
Generally, in such a sensor, the enzyme is very unstable, so that it is necessary to replace the enzyme membrane. There is no problem if the entire electrode can be replaced, but the carbon dioxide electrode is expensive at around 100,000 yen, which is impossible. Further, the L-lysine sensor is potentiometric, is easily affected by contaminants, and it is difficult to improve the sensitivity.

[Means for solving the problem]

The above problems can be solved by using a small amperometric carbon dioxide sensor using an autotrophic bacterium and a small oxygen electrode, which has already been filed by the present inventors.

The present invention relates to a semiconductor substrate having a groove (hole) formed by anisotropic etching, an insulating film covering the surface of the substrate, and forming from the bottom of the hole to the surface of the substrate via the insulating film. Two electrodes serving as a cathode and an anode to be filled, a gel containing an electrolyte and an autotrophic bacterium filled in the hole, a gas permeable membrane covering the gel, and an electrode sensitive part on the gas permeable membrane. A small L-lysine sensor having an enzyme-immobilized film on which the formed decarboxylase L-lysine decarboxylase is immobilized, and a step of forming a groove (hole) on the semiconductor substrate by anisotropic etching; A step of forming an insulating film covering the surface, a step of forming two electrodes serving as a cathode and an anode from the bottom of the hole to the surface of the substrate through the insulating film, and forming a back surface of the substrate with a hydrophobic insulating film. Coating with; Coating the hydrophobic photoresist film except for the holes, and immersing the substrate in an aqueous solution of alkali metal alginate containing electrolyte and autotrophic bacteria to fill only the inside of the holes with the aqueous solution; A step of gelling the aqueous solution of the alkali metal alginate by immersion in an aqueous solution of an alkaline earth metal salt, a step of forming a gas-permeable membrane covering the gel, and Forming an enzyme-immobilized membrane on which decarboxylase L-lysine decarboxylase is immobilized.

Potassium chloride or sodium sulfate can be used as the electrolyte of the L-lysine sensor.

A gold electrode as the cathode of the L-lysine sensor,
Alternatively, a platinum electrode can be used.

A gold electrode as the anode of the L-lysine sensor,
Alternatively, a platinum electrode can be used.

An L-lysine sensor using potassium chloride as the electrolyte of the L-lysine sensor and a silver / silver chloride reference electrode as the anode can be provided.

As the autotrophic bacterium of the L-lysine sensor, a TIT / FJ-0001 bacterium capable of selectively assimilating carbon dioxide (Microbial Laboratories No. 8473) can be used.

As the autotrophic bacterium of the L-lysine sensor, a TIT / FJ-0002 bacterium capable of selectively assimilating carbon dioxide (Microtechnological Laboratory No. 8474) can be used. According to one aspect of the present invention, there is provided a measurement sensor for measuring, for example, the concentration of L-lysine in a solution, the sensor being fixed near the cathode of an oxygen electrode, A gel containing a microorganism and an electrolyte capable of selectively assimilating lipase, a gas-permeable membrane coated with the gel, and a decarboxylase L-lysine decarboxylase that decomposes L-lysine and dissociates carbon dioxide. An L-lysine measurement sensor characterized by comprising:

According to another aspect, the present invention relates to such an L
-This is a manufacturing method for miniaturizing lysine sensors and mass-producing them at once.

The present inventors filed a patent application for miniaturization of an oxygen electrode, which is the basis of miniaturization of an L-lysine sensor (Japanese Patent Application Laid-Open No. 63-163).
There is a small oxygen electrode utilizing photolithography. This oxygen electrode is placed on a hole formed on a silicon substrate by anisotropic etching, with an insulating film interposed therebetween.
An oxygen electrode having a structure in which a book electrode is formed, an electrolyte-containing body is accommodated in the hole, and the upper surface of the hole is finally covered with a gas-permeable membrane. This oxygen electrode is small in size, has little variation in characteristics, and can be mass-produced in a batch, so that it is low in cost.

By utilizing such a small oxygen electrode, it is easy to miniaturize the carbon dioxide electrode serving as the transducer of the L-lysine sensor. However, for this purpose, where and how to fix the microorganisms further become a problem.

For fixing microorganisms, the method disclosed in Japanese Patent Application No. 148221/1987 is considered to be very advantageous in terms of process and safety. Further, the present inventors, when fixing microorganisms, disclosed in
The immobilization of microorganisms on the surface of the small oxygen electrode of -238548 makes it easy for the microorganisms to leak out and poses a safety problem.
A method of fixing in a gel together with an electrolytic solution was adopted.

In carrying out the method of the present invention, a semiconductor substrate, a semiconductor substrate of silicon, germanium, gallium arsenide or the like, in particular, a silicon substrate can be advantageously used as a base material of the electrode body. The insulating film can be formed from a silicon oxide film or the like. For example, when the substrate is silicon, the silicon oxide film can be easily formed by thermally oxidizing the substrate.

The two electrodes can be changed in various ways depending on whether the electrode to be manufactured is a polaro type or a galvanic type. In this carbon dioxide electrode structure, microorganisms and electrolyte coexist. Therefore, for example, it is preferable to use a polaro type using a neutral electrolyte such as a 0.1 M potassium chloride aqueous solution. When a carbon dioxide electrode is manufactured based on a polaro-type oxygen electrode, both electrodes are composed of a gold electrode or a platinum electrode, and a voltage is applied between both electrodes during measurement. Such an electrode can be advantageously formed by a film forming method such as vacuum evaporation and sputtering.

The photoresist to be applied on the substrate after forming the electrodes except for the holes and the portions that make electrical contact is preferably a negative photoresist, for example, OMR-83 manufactured by Tokyo Ohka.
It is. This type of photoresist is advantageous because it has the property of repelling an aqueous solution as a raw material when only the holes are filled with the 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, but initially they can be dip coated or spin coated with a liquid,
It is also an essential requirement that the adhesion between the silicon substrate and the silicon oxide film as the insulating film is good and the electrolyte does not leak out. Suitable gas permeable membrane materials include photoresists, preferably negative photoresists. Teflon (trade name) membranes must be avoided because they are permeable to oxygen but do not have adhesion.

Microorganisms that can be advantageously used in the practice of the present invention are those that can selectively assimilate carbon dioxide, such as those belonging to the genus Pseudomonas and other genera. The present inventors use Pseudomonas genus TIT / FJ-0001 bacteria (Microtechnical Laboratories No. 8473) and unidentified TIT / FJ-0002 bacteria (Microtechnical Laboratories No. 8474), and are particularly preferable. The results were obtained (the properties of these bacteria were described in
80549).

[Action]

In order to facilitate understanding of the operation of the present invention, the principle of the small L-lysine sensor of the present invention will be described with reference to FIG.

With this L-lysine sensor immersed in a buffer,
When L-lysine is added as a sample after sufficiently stabilizing the output current value, L-lysine is decomposed by the action of the enzyme L-lysine decarboxylase immobilized on the enzyme immobilization membrane 15 to dissociate carbon dioxide. The carbon dioxide reaches the vicinity of the immobilized microorganism 8 after passing through the gas-permeable membrane 14 as shown in the figure. Microorganism 8 assimilate carbon dioxide,
At this time, the respiratory activity becomes active and oxygen is consumed, and therefore, the oxygen concentration reaching the cathode 3A decreases. If the amount of change in the oxygen concentration can be regarded as a change in the electrochemical oxygen reduction current, the L-lysine concentration can be measured based on the resulting decrease in the current value.

〔Example〕

Next, a preferred example of a method for manufacturing a small L-lysine sensor 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 L-lysine sensor according to the present invention. The illustrated L-lysine sensor has a rectangular parallelepiped shape, and a gas-permeable membrane of the sensitive part.
Enzyme 15 is immobilized on 14, and a part of electrodes 3A and 3B is exposed for connection to an attached device. Electrode 3A,
In the case of 3B, both electrodes were constituted by gold electrodes in order to be a polaro type in this example. The structure of the small L-lysine sensor of FIG. 2 can be understood in detail from FIG. 3, which is a cross-sectional view taken along the line II-II. The silicon substrate 1 has a hole formed by anisotropic etching, and a silicon oxide film 2 is applied as an insulating film on the entire surface. Further, the back surface of the substrate is covered with a hydrophobic insulating film 7 that is difficult to tear. Two gold electrodes 3A and 3B are attached to the hole of the silicon substrate 1 in pairs. As shown in FIG. 2, the gold electrodes 3A and 3B partially extend to the outside of the groove. The holes of the silicon substrate 1 are filled with autotrophic bacteria and an electrolyte-containing gel 6. Further, the gas permeable film 14 is formed on the upper portion of the hole so as to cover the entire upper surface of the substrate 1 (excluding the exposed portion in FIG. 2).
Which is also covered on the side and back surfaces as an insulating film.

The small L-lysine sensor shown in FIGS. 2 and 3 can be advantageously manufactured, for example, by the manufacturing process shown in FIG. The main body after the electrodes are formed as shown in FIG. 4A can be manufactured through the following steps. In the following description, in order to facilitate understanding, a case where only one L-lysine sensor is formed on one wafer is described. However, in practice, a large number of L-lysine sensors are formed on one wafer. Is formed at the same time.

1. Wafer cleaning A 350-μm-thick (100) 2-inch silicon wafer was prepared and washed with a mixed solution of hydrogen peroxide and ammonia and concentrated nitric acid.

_2. Formation of SiO ₂ film The silicon wafer was subjected to wet thermal oxidation, and a SiO ₂ film with a film pressure of 0.8 μm was formed on the entire surface.

3.Formation of etching pattern Negative photoresist (OMR-83 manufactured by Tokyo Ohka, viscosity 60c)
Using P), a resist pattern for etching SiO ₂ was formed on the wafer.

4. Application of resist for backside protection The same negative photoresist as that used in the above process was applied to the backside of the wafer, and baked at 130 ° C. for 30 minutes.

5. Etching of SiO ₂ film The wafer was immersed in a 50% hydrofluoric acid: 40% ammonium fluoride = 1: 6 aqueous solution, and the exposed portions of the SiO ₂ that were not coated with the photoresist were removed by etching. Next, the resist was removed with a sulfuric acid: 31% aqueous hydrogen peroxide (2: 1) solution.

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

After the completion of the anisotropic etching, the SiO ₂ film used at the time of etching was removed. This was performed in a 50% hydrofluoric acid: 40% ammonium fluoride = 1: 6 aqueous solution as in the case of 5.

To grow an SiO ₂ film is formed a silicon wafer surface of 7.SiO ₂ film 1.
Subsequent to the cleaning step, the wafer was subjected to wet thermal oxidation. A film having a thickness of 0.8 μm was formed.

8. Formation of chromium and gold thin films Following the chromium thin film (400 mm, for adhesion between gold and substrate), a gold thin film (4000 mm) was formed by vacuum evaporation.

9.Formation of resist pattern for electrode formation Negative photoresist (OMR-83 manufactured by Tokyo Ohka, viscosity 60c)
Using P), a resist pattern for forming an electrode was formed on a gold thin film formed on SiO _{2 of} the wafer.

10. Etching of Gold and Chromium The substrate on which the resist pattern has been formed is immersed in the following etching solution for gold and chromium in this order, and the exposed gold and chromium portions are removed by etching.
After washing with pure water, sulfuric acid: 31% hydrogen peroxide solution (2:
1) The resist was removed by the solution.

Gold etching solution; 4 g KI and 1 g I ₂ dissolved in 40 ml of water Chromium etching solution: 0.5 g NaOH and 1 g K ₃ Fe (CN) ₄
Is dissolved in 4 ml of water. A state in which the gold electrode is formed is shown in FIG. 4 (A).

11. Hydrophobic insulating film formed on the back of the substrate Hydrophobic insulating film (ES-100 made by Shin-Etsu Silicone)
1) Apply 7 uniformly and bake at 250 ° C.

12. Resist application (Fig. 4 (B)) On the surface of the main body, except for the hole 5 and the pad part for making electrical contact, a negative photoresist (manufactured by Tokyo Ohka)
OMR-83, viscosity 60 cP) 4. This was performed by applying a photoresist on the surface of the wafer, and performing exposure and development after pre-baking.

13. Cutting of 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. Dip the tip of the chip in a 1M aqueous solution of sodium hydroxide. As a result, portions not covered with the resist became hydrophilic.

15. Filling of calcium alginate gel (FIG. 4 (C)) The following two types of solutions were prepared for forming a gel containing a microorganism and an electrolyte.

Solution A: A solution obtained by dissolving wet TIT / FJ-0001 bacteria and sodium alginate in a 0.1 M aqueous potassium chloride solution.
Number of bacteria: 1.2 × 10 ⁸ cells / cm ³ . Sodium alginate concentration 0.2
%.

Solution B: Calcium chloride dissolved in 0.1 M aqueous potassium chloride solution. Calcium chloride concentration 5%.

When the chip of FIG. 4 (B) is immersed in the solution A and slowly pulled up, the solution A is repelled from the resist film and remains only in the hole 5 because the negative type photoresist film 4 is hydrophobic. Was. Then, when this chip was immersed in solution B, solution A instantly gelled, and TIT / FJ-0001 bacteria were fixed inside the gel.

16. Coating of gas-permeable membrane (FIG. 4 (D)) A gas-permeable membrane 14 was coated on the gel 6 containing microorganisms and the electrolyte so as to cover the gel. In this example, KR-5240 made by Shin-Etsu Silicone was applied by dip coating as a gas permeable membrane. As a result, a gas permeable film and a side wall insulating film were completed. This resist film is also formed on the back surface, which advantageously acts to further improve the insulation.

17. Formation of enzyme-immobilized membrane (Fig. 4 (E)) L-lysine decarboxylase (Sigma: type VI)
I: from Escherichia coli) was immobilized on the gas permeable membrane 14 which is a small carbon dioxide electrode sensitive part as a transducer according to the following method.

L-Lysine decarboxylase (Sigma: type
VII: from Escherichia coli) is dissolved in 10 μl of 10% bovine serum albumin (BSA) aqueous solution.

Add 10 μl of 10% glutaraldehyde dropwise to the solution and mix well.

Immerse the small carbon dioxide electrode sensitive part in this mixed solution and lift it up, and wait for bovine serum albumin BSA to solidify.

After the reaction was completed, after washing with pure water, the unreacted portion of glutaraldehyde was removed by immersion in 10 mM phosphate buffer (pH 6.0) containing 0.1 M glycine at room temperature for 30 minutes.

Completed L-lysine sensor is pure water or 0.1mM
The cells were stored in a 10 mM phosphate buffer (pH 6.0, 4 ° C.) containing pyridoxal 5 ′ phosphate (PLP).

In this L-lysine sensor, the sensitive part is provided with a buffer solution (for example, 10
(mM phosphate buffer). After sufficiently stabilizing the sensor, L-lysine can be quantified by adding a sample and examining the amount of change in current.

In the measurement, first, the sensor sensitive part is immersed in a buffer solution, and the output is stabilized by appropriately adding pyridoxal 5 'phosphate (PLP), and then a sample solution containing L-lysine is added. The change in the current value at this time was examined. For subsequent measurements, the sensor-sensitive part should be 10 mM phosphate buffer (pH 6.0) containing 1 mM pyridoxal 5 'phosphate (PLP).
The above operation may be performed after immersion in the inside and sufficient washing.

5 and 6 show temperature characteristics and pH characteristics of the small L-lysine sensor.

FIG. 7 shows a calibration curve under the condition of 33 ° C. and pH 6.0.

The selectivity of this sensor for various L-amino acids was examined. Here, the concentration of various L-amino acids was 2 mM. L
-Alanine, L-arginine, L-cystine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-hydroxypropylin, L-isoleucine,
This sensor showed no noticeable response to L-methionine, L-phenyl-α-alanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc. .

L-tyrosine, L-cystine, and L-tryptophan did not completely dissolve, but the sensor again showed no response.

In the above example, a TIT / FJ-0001 bacterium capable of selectively assimilating carbon dioxide as an autotrophic bacterium (Microbial Lab. No. 8473)
No. 1129), but as an autotrophic bacterium, the S-17 bacterium described in Japanese Examined Patent Publication No. 50-25551: No. 1129 of B.I.

〔The invention's effect〕

According to the present invention, an inexpensive and disposable small and small L-lysine sensor can be manufactured.

[Brief description of the drawings]

FIG. 1 is a principle view of a small L-lysine sensor of the present invention, FIG. 2 is a perspective view showing a preferred example of a small L-lysine sensor according to the present invention, and FIG. 3 is a line segment II of the electrode of FIG. 4 (A) to 4 (E) are cross-sectional views sequentially showing the latter half of the manufacturing process of the small L-lysine sensor shown in FIGS. 2 and 3. The figure shows the temperature characteristics of the small L-lysine sensor of the present invention. FIG. 6 shows the pH characteristics of the small L-lysine sensor of the present invention. FIG. 7 shows the calibration of the small L-lysine sensor of the present invention. Indicates a line. 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 a gel containing microorganisms and an electrolyte, 7 is a hydrophobic insulating film, 8 is a microorganism, and 14 is a microorganism. Gas permeable membrane 15 is an enzyme immobilized membrane.

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Naomi Kojima 1015 Uedanaka, Nakahara-ku, Kawasaki-shi, Kanagawa Prefecture Inside Fujitsu Limited (56) References JP-A-63-238548 (JP, A) JP-A-63-238549 (JP, A) JP-A-62-214345 (JP, A)

Claims (2)

(57) [Claims] 1. A semiconductor substrate having a groove (hole) formed by anisotropic etching, an insulating film covering a surface of the substrate, and a hole extending from a bottom of the hole to a surface of the substrate via the insulating film. Two electrodes serving as a cathode and an anode to be formed, a gel containing an electrolyte and an autotrophic bacterium filled in the hole, a gas permeable membrane covering the gel, and an electrode sensitive part on the gas permeable membrane And an enzyme-immobilized membrane on which decarboxylase L-lysine decarboxylase is formed. 2. A step of forming a groove (hole) on a semiconductor substrate by anisotropic etching, a step of forming an insulating film covering a surface of the substrate, and a cathode extending from a bottom of the hole to a surface of the substrate. Forming two electrodes serving as anodes through the insulating film, covering the back surface of the substrate with a hydrophobic insulating film, and covering a hydrophobic photoresist film except for the holes. Immersed in an aqueous solution of an alkali metal alginate containing electrolytes and autotrophic bacteria to fill only the inside of the hole with the aqueous solution, and then immersed the substrate in this state in an aqueous solution of an alkaline earth metal salt to immerse the alkali metal alginate. Gelling the aqueous solution, forming a gas-permeable membrane covering the gel, and enzymatically immobilizing a decarboxylase L-lysine decarboxylase on the gas-permeable membrane of the electrode-sensitive part. Method of manufacturing a miniature L- lysine sensor characterized by having a step of forming.