JPH02228548A - Small l-lysine sensor and manufacture thereof - Google Patents

Abstract

PURPOSE:To obtain a sensor inexpensive and disposable by covering the surface of a semiconductor substrate having a hole formed by an anisotropic etching with an insulation film. CONSTITUTION:A semiconductor substrate 1 has a hole 5 formed by an anisotropic etching. The surface of the substrate 1 is covered with an insulation film 2. Two electrodes 3A and 3B reach the surface of the substrate 1 from the bottom of the hole 5 through the insulation film 2 to form a cathode/anode. The hole 5 is filled with a gel 6 containing an electrolyte and independent vegetative bacteria and the gel 6 is covered with a gas permeable film 14. Moreover, an enzyme immobilized film 15 having a decarbonating enzyme L-lysine decarboxylase immobilized thereon is formed on the gas permeable film 14. The electrolyte contained in the gel 6 herein usable is pottasium chloride or sodium sulfate. The cathode/anode of the electrodes 3A and 3B herein usable is a gold electrode or a platinum electrode.

Description

[Detailed Description of the Invention] [Summary] The present invention relates to an L-lysine sensor, and aims to provide an L-lysine sensor that is small and can be mass-produced at low cost, and a method for manufacturing the same. A semiconductor substrate having a groove (hole) formed by a semiconductor substrate, an insulating film covering the surface of the substrate, and a cathode/anode formed from the bottom of the liquid hole to the surface of the substrate via the insulating film. a main electrode, a gel containing an electrolyte and autotrophic bacteria filled in the hole, and a gas permeable membrane covering the gel;
A small 1-+7 gin sensor is constructed by having an enzyme-immobilized film on which the decarboxylase L-lysine decarboxylase is immobilized, which is formed on the gas-permeable film of the electrode sensitive part, and an anisotropic film is formed on the semiconductor substrate. a step of forming a groove (hole) by etching; a step of forming an insulating film covering the surface of the substrate;
A step of forming two electrodes that will become a cathode and an anode from the bottom of the liquid hole to the surface of the substrate via the insulating film, a step of covering the back side of the substrate with a hydrophobic insulating film, and a step of covering the part of the liquid hole. coating with a hydrophobic photoresist film;
The substrate is immersed in an aqueous alginate salt aqueous solution containing an electrolyte and autotrophic bacteria, and only the inside of the hole is filled with the aqueous solution.3 The substrate in this state is then immersed in an alkaline earth metal salt aqueous solution to form the alginate alkali metal salt aqueous solution. a step of gelling the gel, a step of forming a gas-permeable membrane covering the gel, and forming an enzyme-immobilized membrane on which the decarboxylase L-lysine decarboxylase is immobilized on the gas-permeable membrane of the electrode sensitive part. [Industrial Application Field] Zero-Hatsu'IQ is a small-sized L-lysine sensor that can be mass-produced at a low price. The present invention relates to a sensor and a method for manufacturing the same. To explain in more detail, 1 the present invention uses a combination of enzymatic and microbial functions, and further uses an electrochemical electrode to measure lysine and L-lysine.
This invention relates to a sensor for measuring IJ lysine. The L-lysine sensor of the present invention can measure L-lysine in amperometrin,
Moreover, it is revolutionary in that it is easy to miniaturize and mass-produce, and because it can be mass-produced in bulk, it is low cost. The present invention can be advantageously used in many measurement fields including medicine and fermentation.

[Conventional technology]

L-lysine can be measured by various methods, among which a particularly quick and easy method is Gui-1bau.
There is a lysine sensor developed by Lt et al. This was produced by immobilizing L-lysine decarboxylase on a potentiometric carbon dioxide electrode functional part. This sensor attempts to indirectly quantify L-lysine concentration by decomposing L-lysine with L-lysine decarboxylase and measuring the carbon dioxide generated at this time with a carbon dioxide electrode.

[Problem to be solved by the invention]

The Guilbaul L et al. lysine sensor uses a conventional carbon dioxide electrode as a transducer, but this carbon dioxide electrode is usually about the size of a fountain pen, making it difficult to measure small amounts of samples. It's inconvenient. Additionally, enzymes in such sensors are generally very unstable.

The enzyme membrane needs to be replaced. There is no problem if you can replace the entire electrode, but one carbon dioxide electrode costs 10
This is impossible as it is expensive at around 10,000 yen. Moreover, this L-lysine sensor is potentiometric,
It is easily affected by contaminants, and it is difficult to improve sensitivity.

[Means to solve the problem]

The above-mentioned problems have already been filed by the present inventors.

A solution is possible by using a small amperometric carbon dioxide sensor using autotrophic bacteria and a small oxygen electrode.

The present invention focuses on grooves (holes) formed by anisotropic etching.
an insulating film covering the surface of the substrate;
Two electrodes forming a cathode and anode formed from the bottom of the liquid hole to the surface of the substrate via the insulating film, a gel containing electrolyte and autotrophic bacteria filled in the hole, and covering the gel. A compact silica sensor comprising a gas-permeable membrane with a decarboxylase L-lysine decarboxylase formed on the gas-permeable membrane of an electrode sensitive part, and an anisotropically immobilized membrane on a semiconductor substrate. A process of forming grooves (holes) by chemical etching, a process of forming an insulating film covering the surface of the substrate, and a process of insulating the two electrodes that will become the cathode and anode from the bottom of the liquid hole to the surface of the substrate. A process of forming via a film, a process of coating the back side of the substrate with a hydrophobic insulating film, a process of covering the area except for the liquid hole with a hydrophobic photoresist film, and an alkali metal alginate salt containing electrolyte and autotrophic bacteria. A step of immersing the substrate in an aqueous solution to fill only the inside of the hole with the aqueous solution, and then immersing the substrate in this state in an aqueous alkaline earth metal salt solution to gel the alginate alkali metal salt aqueous solution; A small size L comprising the steps of forming a covering gas-permeable membrane and forming an enzyme-immobilized membrane on which a decarboxylase L-lysine decarboxylase is immobilized on the gas-permeable membrane of the electrode sensitive part.
- Achieved by a method for manufacturing a lysine sensor.

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

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

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

The L-lysine sensor may use potassium chloride as the electrolyte and silver/silver chloride reference ti as the anode.

The autotrophic bacteria of the L-lysine sensor are TIT/FJ-0001 bacteria that can selectively assimilate carbon dioxide (
@Koken Bokuyori No. 8473) can be used.

As the autotrophic bacteria of the L-lysine sensor, TIT/FJ-0002 bacteria (
8474) can be used.

According to one aspect of the present invention, there is provided a measurement sensor for measuring the concentration of lysine in a solution, the sensor being fixed near the cathode of an oxygen electrode, and comprising a sensor for measuring the concentration of lysine in a solution. A gel containing microorganisms and electrolytes that can selectively assimilate L-lysine, a gas-permeable membrane covering the gel, and a decarboxylase L-lysine decarboxylase that decomposes L-lysine and dissociates carbon dioxide. A sensor for measuring lysine is characterized by comprising:

According to another aspect of the present invention, there is provided a manufacturing method for miniaturizing an IJ lysine sensor and mass producing it.

The present inventors have filed a patent application regarding the miniaturization of the oxygen electrode, which is the basis for miniaturizing the L-lysine sensor (Japanese Patent Laid-Open No. 63
-23854Σ) There is a small oxygen electrode using photolithography. This oxygen electrode is made by forming two electrodes on a hole formed by anisotropic etching on a silicon substrate, with an insulating film interposed therebetween, and then accommodating an electrolyte-containing body inside the hole. 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.

It is small, has little variation in characteristics, and can be mass-produced in bulk, resulting in low cost.

By using such a small oxygen electrode, it is possible to easily downsize the carbon dioxide electrode that serves as the transducer of the L-lysine sensor.

However, this poses another problem: where and how to immobilize the microorganisms.

For the fixation of microorganisms, patent application No. 148221/1982
Based on the method disclosed in the above issue, it is considered to be very advantageous from both a process and safety standpoint. In addition, the inventors of the present invention found that fixing microorganisms on the surface of the small oxygen electrode disclosed in JP-A-63-238548 is prone to leakage of microorganisms and poses a safety problem. From a point of view.

A method was adopted in which microorganisms were immobilized in a gel together with an electrolyte inside the gas-permeable membrane of a small oxygen electrode.

In carrying out the second method of the present invention, a semiconductor substrate, a semiconductor substrate such as silicon, germanium, gallium arsenide, etc., 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. The silicon oxide film can be easily formed by thermally oxidizing the substrate, for example, when the substrate is silicon.

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. So 1, for example, 0.1? l
A polar mouth type using a neutral electrolyte such as an aqueous potassium chloride solution is preferable.

When manufacturing a carbon dioxide electrode based on a polar-mouth type oxygen electrode, one electrode is constructed from a metal electrode or a platinum electrode, and a voltage is applied between both electrodes during measurement. Formation of such an electrode can be advantageously performed by 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 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 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.
A suitable gas-permeable film material is a photoresist, preferably a negative film, which has good adhesion to the silicon substrate and the silicon oxide film as an insulator, and it is also essential that the electrolyte does not leak to the outside. Examples include 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 discovered that Pseudomonas ↑IT/FJ-00
01 bacterium (Feikoken Bacteria No. 8473) Unidentified T IT/
Particularly favorable results were obtained using the PJ-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 principle of the small-sized L-lysine sensor of the present invention will be explained with reference to FIG.

When L-lysine is added as a sample after stabilizing the output current value sufficiently with this L-lysine sensor immersed in a buffer solution, L-lysine is absorbed into the enzyme L-lysine that is immobilized on the enzyme-immobilized membrane 15. It is decomposed by the action of decarboxylase and 5 carbon dioxide is dissociated. This carbon dioxide reaches the vicinity of the immobilized microorganisms 8 after passing through the gas permeable membranes 11 and 14 as shown in Figure 2. Microorganisms 8 assimilate carbon dioxide, and at this time, respiratory activity increases and oxygen is consumed.
Therefore, the oxygen concentration reaching the cathode 3A decreases. If this amount of change in oxygen concentration can be understood as a change in electrochemical oxygen reduction current, the L-lysine concentration can be measured based on the resulting decrease in current value.

〔Example〕

Next, a preferred example of a method for manufacturing a small-sized L-lysine sensor according to the present invention will be explained 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 the gas permeability of the sensitive part is H14.
Enzyme 15 is fixed thereon, and a portion of electrodes 3A and 3B are exposed for connection to an attached device.

In the case of two 1i electrodes 3A3B, both the picture electrodes and the picture electrodes were made of gold electrodes in order to have a polar mouth type. The structure of the compact L-lysine sensor shown in FIG. 2 can be understood in detail from FIG. 3, which is a sectional view taken along line II-.

A silicon substrate 1 has holes formed by anisotropic etching, and a silicon oxide film 2 is deposited as an insulating film on 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 pieces m3A and 3B are deposited in pairs in the holes of the silicon substrate 1. Gold electrode 3A
and 3B, a portion of each extends to the outside of the groove, as shown in FIG. Furthermore, the holes in the silicon substrate 1 are filled with autotrophic bacteria and an electrolyte-containing gel 6. Furthermore, two gas permeable Ws are provided above the holes to cover the entire upper surface of the substrate 1 (excluding the exposed portion in FIG. 2).
i114 is coated, and the side and back surfaces are also covered as an insulating film.

The small L-lysine sensor shown in Figures 2 and 3 has two
For example, it can be advantageously manufactured using 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 L-lysine sensor is formed on one wafer, but in reality, many L-lysine sensors are formed on one wafer. It should be understood that the sensors 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.

Formation of 2.5 iOz film A silicon wafer was subjected to wet thermal oxidation to form a Si0g film with a thickness of 0.8 μm on the entire surface.

3. Formation of etching pattern A 5iOi etching resist pattern was formed on the wafer using a negative photoresist (TOKYO OHKA 0FIR-83, 60 cP).

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

Etching 5.5iOzB 50% hydrogen fluoride H:40% ammonium fluoride-1:
6 water? The wafer was immersed in an S solution, and the 5iO1 in exposed areas not covered with the photoresist was removed by etching.The resist was then removed with a sulfuric acid:31x hydrogen peroxide solution (2:1).

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

After completing this anisotropic etching, the SiO□ film used during etching was removed. This was carried out similarly to 5 in an aqueous solution of 50% hydrofluoric acid = 40% ammonium fluoride = 6.

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 with a thickness of 0.8 and l/11 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 type 0FIR-83° viscosity 60 cP
) was used to form a resist film for electrode formation on the gold thin film formed on 5 ift 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, 1 sulfuric acid: 311 times M hydrogen hydride water (2
:1)? 91! : More resist was removed.

■Gold etching liquid: 4gK! and 1 g +2 dissolved in 40 + ml of water ■Chrome etching solution: 0.5 g NaOH and 1 g KsFe(CN)a dissolved in 4 ml of water. Shown in Figure (A).

11. Hydrophobic insulating film formed on the back side of the substrate Hydrophobic insulating film (ES-10 made by Shin-Etsu Silicone) was formed on the back side of the substrate.
01) Apply 7 evenly and bake at 250°C.

12. Resist application (Figure 4 (B)) Apply negative photoresist (manufactured by Tokyo Ohka Co., Ltd.) to the surface of the main body except for the 5 holes 5 and the pads for electrical contact.
OMR-83, viscosity 60 cP) 4 This was done by applying a photoresist to the surface of the wafer, 6 pre-hake followed by exposure and development.

13. Cutting out 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 in a 1M 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 gels containing microorganisms and electrolyte.

Solution A: A solution in which wet TIT/FJ-0001 bacteria and sodium alginate were dissolved in a 0.1M potassium chloride aqueous solution. Bacterial count: 1.2 x 10” 7cm
”.Sodium alginate concentration 0.2m.

Solution B: Calcium chloride dissolved in 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 remained only in the hole 5. Ta. Next, when this chip was immersed in Solution B, Solution A instantaneously turned into a gel, and the T1/FJ-0001 bacteria were immobilized inside the gel.

16. Coating the gas-permeable membrane (FIG. 4(D)) The gas-permeable membrane 14 was coated with microorganisms and electrolyte on the gel 6 so as to cover the gel. In this example, KR-5240 manufactured by Shin-Etsu Silicone was applied as a gas permeable membrane by dip coating.

As a result, a gas permeable film and a sidewall insulating film were completed. This resist film is also formed on the back surface, which has an advantageous effect in further improving the insulation properties.

17. Formation of enzyme-immobilized membrane (Figure 4 (E)) L-lysine decarboxylase (manufactured by Sigma: type Vll:
from Escherichia coli) was fixed on the gas permeable membrane 14 of the small carbon dioxide electrode sensitive part, which will serve as the transducer, according to the following method.
L-lysine decarboxylase (manufactured by Sigma: ty
pe Vll: from Escherichia
coli) 2 mg to 10x bovine serum albumin (BSA)
)Water soluble? & Dissolve in 10 μl.

■Drop 10 μm of 10χ glutaraldehyde into the solution and mix well.

(2) Immerse the small carbon dioxide electrode sensitive part in this mixed solution, pull it out, and wait for the bovine serum albumin BSA to solidify.

After the reaction was completed, the unreacted portion of glutaraldehyde was removed by immersion in 10 mM phosphate buffer (pH 6,0) containing 0.1 M glycine for 30 minutes at room temperature after washing with pure water.

The completed lysine sensor can be placed in pure water or at 0.1 m.
10 containing M pyridoxal 5° phosphate (PLP)
Stored in mM phosphate buffer (PH6,0,4'c>).

This L-lysine sensor uses a buffer solution (for example, 10
After sufficiently stabilizing this sensor, which is used by immersing it in (a+M phosphate buffer), L-lysine can be quantified by adding a sample and checking the amount of change in current.

For measurement, first, the sensor sensitive part is immersed in a buffer solution, and then pyridoxal 5" phosphate (PLP) is added.
After stabilizing the output by appropriately adding L-lysine, a sample solution containing L-lysine was added, and the change in current value at this time was examined. If you wish to continue measurements, immerse the sensor sensitive part in 10mM phosphate buffer (pH 6.0) containing 1mM pyridoxal 5' phosphate (PLP) and wash thoroughly.
Just perform the above operation.

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

Further, FIG. 7 shows a calibration curve under the condition of 33"C02116,0.

The selectivity of this sensor for various L-amino acids was investigated. Here, the concentration of each amino acid was set to 2mM. L
-alanine, L-arginine, cystine, L-glutamine, L-glutamic acid - glycine, L-histidine, L-hydroxyproline, L-isoleucine, L-
Methionine L-phenyl-α-alanine, L-proline, L-serine, L-threonine, L-tryptophan,
This sensor did not show any noticeable response to L-tyrosine, L-valine, etc.

Although L-tyrosine, L-cystine, and L-1-lyptophan were not completely dissolved, the sensor still showed no response to these.

In the above example, the TIT/FJ-0001 bacterium (Feikoken Bacterial Reference No. 8473), which can selectively assimilate carbon dioxide, was used as an autotrophic bacterium. The S17 bacterium described in Publication No. 50-25551: Kaikoken Bacteria No. 1129 can be used.

〔Effect of the invention〕

According to the present invention, it is possible to produce a small, inexpensive L-lysine sensor that is disposable.

[Brief explanation of the drawing]

Fig. 1 is a principle diagram 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 of the present invention, and Fig. 3 is a line segment II of the electrode in Fig. 1. 4 (A) to (E) are sectional views sequentially showing the second 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. Figure 6 shows the PH characteristics of the small L-lysine sensor of the present invention. Figure 7 shows the calibration of the small L-lysine sensor of the present invention. Show the 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 microorganism and electrolyte-containing gel, 7 is a hydrophobic insulating film, 8 is a microorganism, 14 is a gas-permeable film, and 15 is an enzyme-immobilized film. δ Principle diagram of small-sized lysine resin No. 2 The entire area of small-sized L-lysine Sensor 4'jL-Short 1〆Kasaba 5p Small L-Lysine made by NsaQ Pro t! Susume L-resin pump/) Calibration Imine 7 Σ

Claims (1)

[Claims] 1. A semiconductor substrate having a groove (hole) formed by anisotropic etching, an insulating film covering the surface of the substrate, and a semiconductor substrate having a groove (hole) formed by anisotropic etching. Two electrodes forming the cathode and anode reach the surface, a gel containing electrolyte and autotrophic bacteria filled in the hole, a gas permeable membrane covering the gel, and a gas permeable membrane in the electrode sensitive part. 1. A small-sized L-lysine sensor comprising an enzyme-immobilized membrane formed on a sexual membrane and on which a decarboxylase L-lysine decarboxylase is immobilized. 2. A step of forming a groove (hole) on a semiconductor substrate by anisotropic etching, a step of forming an insulating film covering the surface of the substrate, and a step of forming a cathode and an anode from the bottom of the hole to the surface of the substrate. a step of forming two electrodes through the insulating film; a step of covering the back surface of the substrate with a hydrophobic insulating film;
A step of coating the substrate with a hydrophobic photoresist film except for the hole portion, and immersing the substrate in an alginate alkali metal salt aqueous solution containing an electrolyte and autotrophic bacteria to fill only the inside of the hole with the aqueous solution, and then the substrate in this state. a step of gelling the alginate alkali metal salt aqueous solution by immersing it in an alkaline earth metal salt aqueous solution, a step of forming a gas permeable membrane covering the gel, and a step of forming a gas permeable membrane on the electrode sensitive part. and forming an enzyme-immobilized membrane on which a decarboxylase L-lysine decarboxylase is immobilized.