JP2743535B2 - Integrated sensor and manufacturing method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Summary] The present invention relates to an integrated sensor comprising an oxygen electrode or an enzyme electrode, or a combination thereof, with the aim of putting the integrated sensor into practical use. A plurality of sensors including at least an electrode, a gel containing an electrolytic solution filled in the concave portion, and a gas permeable film covering the concave portion, are configured to be joined to each other on the back surface of the silicon substrate. And the manufacturing method
Forming a recess by etching the silicon substrate;
After the silicon substrate having the concave portions formed thereon is washed in a solution to make it hydrophilic, a drying step is performed, and a heating step is performed with the back surfaces of the hydrophilic silicon substrate attached to each other. To be configured.

The present invention relates to an integrated sensor and a method for manufacturing the same.

The small oxygen electrode is used for measuring the dissolved oxygen concentration.

For example, the amount of biochemical oxygen supply (abbreviated as BOD) in water is measured from the viewpoint of water quality conservation, and an oxygen electrode is used to measure the dissolved oxygen concentration.

In the fermentation industry, it is necessary to adjust the dissolved oxygen concentration in the fermenter to efficiently promote alcohol fermentation. An oxygen electrode is used for this measurement, and an enzyme electrode is used for the measurement of glucose and amino acids. Is used.

In the field of clinical medicine, an oxygen electrode or an enzyme electrode is used by being attached to a catheter (Katheter) and inserted into a body.

[Conventional technology]

Conventional oxygen and enzyme electrodes are made of glass or a polymer compound, and have an anode and a cathode in a cylindrical cell with an open end. An oxygen electrode is formed by providing an oxygen permeable film in the portion.

In the case of an enzyme electrode, an enzyme is further fixed on the oxygen permeable membrane.

However, as long as such a structure is adopted, miniaturization is limited and mass production is difficult.

In order to solve this problem, the present inventors performed anisotropic etching using a silicon (Si) substrate to form a large number of holes with good pattern accuracy, and then used photolithography technology (photolithography). A new type of small oxygen electrode is proposed in which one electrode is formed, an electrolyte-containing body is accommodated in the hole, and finally the upper surface of the hole is covered with a gas permeable membrane.

(Japanese Patent Application No. 62-71739, filed on March 27, 1987) This oxygen electrode is small in size, has little variation in characteristics, and is suitable for mass production.

And agarose gel (agar) was used as the electrolyte-containing body.

However, in the case of agarose gel, there is an inconvenience that it is necessary to repeatedly inject into the minute holes on the Si substrate one by one using a micropipette.

Therefore, the inventors used polyacrylamide gel,
We have found and applied for a method of injecting the gel at once only into the many micro holes provided on the Si substrate.

(Japanese Patent Application No. 62-148221, filed on June 15, 1987) An application for a calcium alginate gel has been filed in the same manner.

(Japanese Patent Application No. 63-176978, filed on July 18, 1988) The latter manufacturing method will be described. After forming a large number of holes using a photolithography technique and an anisotropic etching technique, an Si electrode is formed. The substrate is coated with a negative resist except for the holes, and the substrate is immersed in an aqueous solution of sodium alginate containing an electrolytic solution. This is a method in which a porous carrier of calcium alginate containing an electrolytic solution is precipitated in a hole by immersion in a calcium aqueous solution.

FIG. 2 is a perspective view of the small oxygen electrode 1 thus manufactured, FIG. 3 is a sectional view of the small oxygen electrode taken along the line XX 'of FIG. 2, and FIG. FIG.

Now, the structure and the manufacturing process will be described as follows.

2 and 3, a silicon (Si) substrate 2 having a (100) plane on its surface is subjected to anisotropic etching using a photolithography technique (photolithography) to form a square hole, and then subjected to heat. An oxide film 3 made of silicon dioxide (SiO ₂ ) is provided by oxidation and insulated, and a pattern is formed by facing the electrodes 4 and 4 ′ from the hole to the surface of the substrate 2.

Next, an electrolytic solution-containing gel 6 made of calcium alginate containing an electrolytic solution is put in the hole, and the hole is covered with a gas permeable membrane 7.

FIGS. 4 (A) to 4 (D) specifically show this manufacturing process.

That is, the (100) plane is used as the substrate surface, the Si substrate 2 having a thickness of 350 μm and a diameter of 2 inches is prepared by mixing a mixed solution of hydrogen peroxide (H ₂ O ₃ ) and ammonia (NH ₃ ) with concentrated nitric acid (HNO ₃ ). After that, the Si substrate 2 is wet-oxidized at about 1000 ° C. in the presence of water vapor, and a SiO 2 layer having a thickness of about 0.8 μm
_An oxide film 3 of ₂ is formed.

Next, a negative resist having a viscosity of about 60 cP (Oka Chemical Co., Ltd., O
After spin-coating MR-83) on the front and back of the Si substrate 2 to form a resist film, the resist film on the surface is projected and exposed.
Open the hole forming part.

Next, 50% hydrofluoric acid (HF): 40% ammonium fluoride (NH ₄ F) =
After immersing the Si substrate 2 in a 1: 6 aqueous solution to remove the exposed oxide film 3, the resist is subsequently removed using a sulfuric acid (H ₂ SO ₄ ): H ₂ O ₂ = 2: 1 solution. .

Next, potassium hydroxide (KO) with a liquid temperature of 80 ° C and a concentration of 35%
H) Anisotropic etching by immersion in aqueous solution
After etching to 00 μm, the Si substrate 2 is thoroughly washed with pure water.

After that, the SiO film used at the time of etching is
After removal using an aqueous solution of% HF: 40% NH ₄ F = 1: 6, wet oxidation is again performed to form an oxide film 3 made of SiO _{2 with} a thickness of 0.8 μm.

Next, chrome (C) is deposited on the Si substrate 2 by a vacuum deposition method.
r) is formed to 400 Å and gold (Au) to 4,000 、, and then a resist pattern for forming an electrode is formed by using the same negative resist (OMR-83). ) 4g
And a solution prepared by dissolving 1 g of iodine (I ₂ ) in 40 ml of water. The Cr film was composed of 0.5 g of caustic soda and potassium ferricyanide [K ₃ Fe (CN) ₆ ].
Etching is performed using a solution dissolved in 4 ml of water to form electrodes 4, 4 '. (FIG. 4A) Next, a hole is formed on the Si substrate 2 and the Si excluding the electrode lead portion is removed.
After the resist film 5 is formed by covering the substrate 2 with a negative resist (OMR-83), the part not covered with the resist is immersed in a 1M NaOH solution to make it hydrophilic.

Next, sodium alginate was added to 0.1 mol of potassium chloride (K
Cl) Dissolved in aqueous solution to make 0.2% concentration, 0.1M KCl containing 5% calcium chloride (CaCl ₂ )
A solution B comprising an aqueous solution is prepared.

First, the Si substrate is immersed in the solution A, and when slowly pulled up, the resist film 5 is hydrophobic, so that the solution A remains only in the holes. Then, when the substrate is immersed in the solution B, a gel containing an electrolyte solution composed of calcium alginate is obtained. You can do 6. Next, a small oxygen electrode is completed by coating a gas-permeable membrane 7 made of a resist (for example, OMR-83) or the like on the electrolyte-containing gel 6 so as to cover the gel. .

(The above figure D) The manufacturing method of the small oxygen electrode described above is a method suitable for mass production, and the cost can be reduced.

However, the use of biosensors is different from the case where only the dissolved oxygen concentration is measured alone, because the concentrations of different types of substances such as dissolved oxygen concentration and glucose concentration, and amino acid concentration and glucose concentration in a solution are different. There are many applications for simultaneous measurement.

Therefore, practical use of a biosensor capable of simultaneous measurement has been desired.

[Problems to be solved by the invention]

The small sensor proposed by the inventors is a single biosensor using a Si substrate, and uses multiple enzyme electrodes or a combination of this and an oxygen electrode to simultaneously measure multiple concentrations There is a need.

Therefore, it is an issue to develop an integrated biosensor in which these are integrated.

[Means for solving the problem]

The above object is achieved by a sensor including at least two electrodes provided in a concave portion formed in a silicon substrate, a gel containing an electrolytic solution filled in the concave portion, and a gas-permeable membrane covering the concave portion. A plurality is configured so that the back surfaces of the silicon substrate are bonded to each other, and the manufacturing method includes a step of etching the silicon substrate to form a concave portion, and the step of etching the silicon substrate having the concave portion in a solution. The problem can be solved by including a step of drying after cleaning to make the silicon substrate hydrophilic, and a step of heating the silicon substrate with the hydrophilic surfaces bonded to each other.

[Action]

A method of integrating multiple sensors is to create different types of sensors on the same substrate, and cut and separate multiple units.

A method of making different types of sensors on the front and back of a substrate, and cutting and separating this substrate.

A method in which two substrates made of different types of sensors are attached to each other, and this substrate is cut to make different sensors on the front and back.

and so on.

However, forming a large number of oxygen electrodes and enzyme electrodes or different types of enzyme electrodes on the matrix on one side of the Si substrate has problems with dead space and crosstalk, and the determination of the sensor arrangement requires trial and error. Need.

In addition, since a thin substrate with a thickness of several 100 μm is used as a Si substrate, there is a risk of penetrating each other if holes with a depth of about 300 μm are made from both sides, and the material of the substrate becomes brittle due to chemical treatment There is a problem that the manufacturing yield is extremely reduced.

 From the above, the laminated structure is suitable.

Next, as a bonding method, there is also a method of bonding the back surfaces of the substrates to each other using an adhesive, but this sensor is used in an aqueous solution, and the adhesive is deteriorated and peeled for a long time, Insulation may be reduced.

Therefore, in the manufacture of this integrated sensor,
A method is adopted in which the surface of the Si substrate is bonded after making it hydrophilic, and dehydrated and condensed by heating to a high temperature.

That is, when the Si substrate is made hydrophilic and adhered in a state where a hydroxyl group (OH group) is attached to the Si atom on the surface, and heated to a high temperature in a state where the OH groups are attracted to each other, 2OH → O + H ₂ O ... (1) take the H ₂ O by dehydration condensation reaction, firm bonding of the Si-O-Si is completed.

The present invention forms an integrated sensor by this method.

〔Example〕

The following process is an example of manufacturing an integrated sensor including an oxygen electrode and an enzyme electrode that can simultaneously measure oxygen concentration and glucose concentration.

First, two Si wafers having a thickness of 350 μm and a diameter of 2 inches with a (100) plane on the surface were prepared, and hydrogen peroxide (H ₂ O ₂ ) was used.
Washing was performed using a mixed solution of hydrogen and ammonia (NH ₃ ) and concentrated nitric acid (HNO ₃ ).

Next, the Si wafer was subjected to wet thermal oxidation to form a 0.8 μm thick SiO ₂ film on the entire surface.

Next, a negative photoresist (OMR-8, manufactured by Tokyo Ohka)
3. Apply to the front and back of the wafer using a viscosity of 60 cP)
After heating for 30 minutes, an etching resist pattern was formed on the surface of the wafer.

Next, 50% hydrofluoric acid (HF): 40% ammonium fluoride (NH
₄ F) The wafer is immersed in an aqueous solution of 1: 6, the exposed SiO ₂ film not covered with the photoresist is removed by etching, and subsequently, sulfuric acid (H ₂ SO ₄ ): H ₂ O ₂ = 2 The resist was removed using a 1: 1 solution.

Next, Si in a 35% potassium hydroxide (KOH) solution at 80 ° C
Was anisotropically etched to form a hole having a depth of 300 μm, and then the wafer was washed with pure water.

After the completion of the anisotropic etching, 50% HF: 40% NH ₄ F
The wafer was immersed in an aqueous solution of = 1: 6 to remove the SiO ₂ film used at the time of etching.

(FIG. 1A) Next, after washing in a solution of H ₂ SO ₄ : H ₂ O ₂ = 2: 1 for 5 minutes, thoroughly washing with boiling pure water, drying, and then purifying in a clean atmosphere. The wafers were firmly adhered to each other while being aligned and heated at 1000 ° C. for 3 hours in an N ₂ atmosphere. Next, the Si wafer was washed with a mixed solution of H ₂ O ₂ and NH ₃ and concentrated HNO ₃ , and then wet oxidized at 1000 ° C. to a thickness of 0.8.
A μm SiO ₂ film 11 was formed.

Next, 400mm thick chrome (C
r) A thin film of gold (Au) with a thickness of 4000 mm is formed on the film and an electrode is formed on the SiO ₂ film using a negative photoresist (OMR-83, manufactured by Tokyo Ohka, viscosity 60 cP). Resist pattern was formed.

Then, the exposed Au film is made of Au etching solution (4 g KI
The I ₂ of 1g in a liquid) was dissolved in H ₂ O in 40 ml, also Cr etchant of Cr (0.5 g of NaOH and 1g of K ₃ Fe (CN) _{solution 6} was dissolved in H ₂ O in 4ml) After removing using, after washing with pure water,
The resist was removed by dipping in a solution of H ₂ SO ₄ : H ₂ O ₂ = 2: 1,
Au electrodes 12 were formed on both surfaces of the bonded substrates.

Next, a negative photoresist (OMR-83) is used on the surface of the main body except for the holes and the pads for making electrical contact.
To form a resist film 13, and chips were cut out in this state.

Next, when the chip was immersed in a 1 mol aqueous solution of NaOH, portions not covered with the resist became hydrophilic.

Next, to fill the calcium alginate, the following A
Liquid and liquid B were prepared.

Solution A: A solution of sodium alginate dissolved in a 0.1 M KCl solution, wherein the concentration of sodium alginate is 0.1%.
Four%.

Solution B: CaCl ₂ dissolved in a 0.1 M aqueous KCl solution,
Here, the concentration of CaCl ₂ is 5%.

First, when the chip was dipped in the solution A and slowly pulled up, the negative resist film 13 was hydrophobic.
The liquid was repelled from the resist film 13 and remained only in the holes.

When this chip was immersed in the solution B, the solution A instantaneously gelled to form an electrolyte-containing gel 14.

Next, the chip is immersed in a silicone resin solution (KR-5240, manufactured by Shin-Etsu Silicone) to obtain an electrolyte-containing gel 14.
Was covered with a gas permeable membrane 15, thereby forming two oxygen electrodes 16.

Next, glucose oxidase 17 was immobilized on the gas permeable membrane 15 of one oxygen electrode 16.

In this fixing method, the sensitive part having a gas permeable membrane 15 on one oxygen electrode 16 is made of glucose oxidase 5 mg, 10% bovine serum albumin 200 μ, 25% glutaraldehyde 10 μ
After immersing in a mixed solution containing
This was performed by rinsing with pure water, whereby an enzyme electrode 18 was formed.

By the above method, an integrated sensor including the oxygen electrode 16 and the enzyme electrode 18 was completed. (The above figure F) [Effects of the Invention] By implementing the present invention, it is possible to easily manufacture a large number of small integrated sensors without crosstalk between sensors.

[Brief description of the drawings]

1 is a sectional view showing a manufacturing process of the integrated sensor, FIG. 2 is a perspective view of a small oxygen electrode, FIG. 3 is a sectional view of the small oxygen electrode taken along the line XX 'in FIG. 2, and FIG. FIG. 4 is a cross-sectional view illustrating a manufacturing process of the small oxygen electrode. In the figure, 11 is a SiO ₂ film, 12 is an Au electrode, 13 is a resist film, 14 is an electrolyte-containing gel, 15 is a gas-permeable membrane, 16 is an oxygen electrode, 17 is glucose oxidase, and 18 is an enzyme electrode. .

Continuation of the front page (72) Inventor Naomi Kojima 1015 Kamiodanaka, Nakahara-ku, Kawasaki-shi, Kanagawa Fujitsu Limited

Claims (2)

(57) [Claims] 1. A sensor comprising at least two electrodes provided in a concave portion formed in a silicon substrate, a gel containing an electrolytic solution filled in the concave portion, and a gas permeable film covering the concave portion. An integrated sensor comprising a plurality of silicon substrates joined together on the back surface of the silicon substrate. 2. A method for manufacturing an integrated sensor comprising a plurality of sensors having their back surfaces bonded to each other, the method comprising: etching a silicon substrate to form a concave portion; and placing the silicon substrate having the concave portion in a solution. And drying the silicon substrate after making the silicon substrate hydrophilic, and heating the silicon substrate with the back surfaces of the silicon substrate being hydrophilic bonded together.