JPH0564733B2 - - Google Patents

Description

[Detailed Description of the Invention] (a) Industrial Application Field This invention relates to a biosensor device. More specifically, the present invention relates to a biosensor device that combines an immobilized enzyme and a chemical substance-sensitive electrode and is useful in analytical fields such as clinical analysis, industrial processes, and environmental chemistry.

(b) Prior art Recently, functional membranes such as immobilized enzyme membranes and transducers such as electrodes, thermistors, photon counters, and sonic detectors have been combined to produce chemical substances caused by enzymatic reactions, etc. A variety of biosensors have been proposed, which use a transducer to convert changes in energy, heat generation, luminescence, sound waves, etc. into electrical signals, and analyze various components in a sample based on the output.

Among these biosensors, biosensors that use chemical substances produced or increased or decreased by enzymatic reactions as indicators usually use chemical-sensitive electrodes such as ion-selective electrodes or ion-selective field-effect transistors (IS FETs) as transducers. It is made by attaching an immobilized enzyme membrane to this sensitive surface. Membrane-like carriers for immobilizing enzymes include cellulose acetate, polyglutamate, ion exchange resin,
Organic polymers such as porous polyvinyl chloride are commonly used.

However, such a biosensor has a problem in that the adhesion between the membrane carrier and the sensitive surface is insufficient, and therefore stable performance cannot be obtained and it is not suitable for long-term use.

Regarding this point, the present inventors believe that the weak adhesion between the electrode and the immobilized enzyme membrane in conventional biosensors is due to the weak adhesion between the electrode sensitive surface and the immobilized enzyme membrane, which are made of inorganic solids mainly composed of aluminum oxide, silicon oxide, silicon nitride, etc. We considered that this was due to the incompatibility with the organic polymer that is the carrier of the enzyme, and conducted intensive research and examination based on this theory. As a result, by forming a metal hydroxide gel film on the sensitive surface on the spot and immobilizing the enzyme using this as a carrier, the immobilized enzyme has excellent adhesion to the sensitive surface and has excellent activity. We have discovered that a biosensor that is stable, durable, and sensitive can be obtained by forming a membrane.

(C) Purpose of the Invention The present invention provides a preferred biosensor device configured using the above-mentioned biosensor, and in particular, a biosensor that is not adversely affected by temperature, pH, interfering components, etc. during measurement. The aim is to provide equipment.

(d) Structure of the Invention According to the present invention, an immobilized enzyme membrane made of a metal hydroxide gel on which a predetermined enzyme is immobilized is placed on the sensitive surface of a chemical substance-sensitive electrode equipped with an inorganic solid sensitive surface. A biosensor portion is formed in close contact with a gel film on which enzymes are not immobilized and is made of the metal hydroxide gel described above and is formed in close contact with the sensitive surface of a chemical substance-sensitive electrode equipped with the same inorganic solid sensitive surface as above. Provided is a biosensor device characterized by comprising a reference electrode section.

One major feature of this invention is the use of a biosensor in which an immobilized enzyme using a metal hydroxide gel as a carrier is closely formed on the inorganic solid sensing surface of a chemically sensitive electrode. And other major features are
As a reference electrode for measurements using this biosensor, a metal hydroxide gel film with no enzyme immobilized is formed in close contact with the sensitive surface of the same chemical-sensitive electrode as the biosensor. The point is that it uses a combination of the following.

As the "chemical substance sensitive electrode with an inorganic solid sensitive surface" in this invention, various potentiometric solid electrodes, semiconductor electrodes, and their equivalents used in conventional biosensors can be applied. , more specifically, ion-selective electrodes, ion-selective field-effect transistors (IS
FET), platinum electrodes, etc. Typically, PH glass electrodes or PH-IS that detect PH changes due to enzyme reactions
It is preferable to use FET from the viewpoint of versatility.

The biosensor section of the present invention is characterized by in-situ metal hydroxide on the sensitive surface of the chemical substance-sensitive electrode (i.e., the end or the entire surface in the case of a glass electrode or a platinum electrode, or the gate surface in the case of an ISFET). It can be obtained by forming a physical gel membrane and immobilizing the enzyme on the gel membrane. On the other hand, the reference electrode portion in the present invention can be obtained in the same manner except that the enzyme described above is not immobilized.

The gel film formed at this time is required to be thin and in close contact with the sensitive surface. Such a gel film is produced by adding an acid to a solution of an easily volatile hydrophilic solvent (e.g., a lower alcohol solution such as methanol or ethanol) in which an organometallic chelate compound having a metal alkoxide or an enzyme as a ligand is dissolved. It can be formed by coating the above-mentioned sensitive surface and then leaving it as it is or under heating to advance hydrolysis and drying of the above-mentioned compound to convert it into the corresponding metal hydroxide.

The metal alkoxides used as raw materials for the gel film include Si(OCH ₃ ) ₄ , Si(OC ₂ H ₅ ) ₄ , Ti(OC ₃ H ₇ ) ₄ ,
V( _OC2H5 ) ₃ , Al( _OC3H7 ) ₃ , Co _{( OC2H5 ) 2} , _Ni
(OC ₂ H ₅ ), Fe(OC ₂ H ₅ ) ₃ , etc., and among these, lower alkoxysilane is preferred. Further, as the organometallic chelate compound having an enzyme as a ligand, metal chelate compounds of alkanediones or their derivatives are suitable, such as 2,4-pentanedione (acetylacetone), 3-phenyl-
2,4-pentanedione (3-phenylacetylacetone), 2,4-hexanedione, 2,4 (or 3,5)-heptanedione, 2,2,6,6-
Examples include calcium, aluminum, titanium, zinc, iron or potassium chelate compounds of lower alkanedions such as tetramethyl-3,5-heptanedione (dipiparoylmethane). Of course, these metal alkoxides and organometallic chelate compounds may be used in combination.

In addition, various mineral acids can be mentioned as acids to be added to the above-mentioned hydrophilic solvent solution, but it is preferable to use hydrofluoric acid. is most preferred. The amount of hydrofluoric acid added is expressed as a molar ratio to the metal.
Approximately 0.05 to 1.0 mol is appropriate. Note that a small amount of water may be added to this solution.

The metal hydroxide-based gel film that is formed directly on the sensitive surface in this way is a thin film (usually 0.1 to 0.5 μm) that adheres closely to the sensitive surface, and has a large number of particles that are ideal for enzyme immobilization. It has hydroxyl groups and is porous.

A chemical substance-sensitive electrode on which such a gel film is formed is used as a reference electrode in the present invention. On the other hand, the biosensor portion of the present invention can be obtained by immobilizing an enzyme on this gel membrane.

For the immobilization of the enzyme on the gel film formed on the sensitive surface, known methods such as a method using a silane coupling agent or a cyanogen bromide activation method can be applied, one of which is preferred. As an example, γ−
A coupling group is introduced by bringing a solution of aminopropyltriethoxysilane into contact with the above gel film,
then contacted with a solution of glutaraldehyde,
Finally, there is a method in which the enzyme is immobilized by Schiff base bonding by contacting with a desired enzyme solution.

As the enzyme to be immobilized, various enzymes can be used that are capable of carrying out an enzymatic reaction in which the component to be analyzed is used as a substrate and the chemical substance-sensitive electrode responds. Among these, it is preferable to apply at least an enzyme that can cause a PH change through an enzymatic reaction, and in this case, as a chemical substance-sensitive electrode, a PH glass electrode or a PH-
Preferably, IS FETs are used. Specific combinations of these enzymes and target components include glucose and glucose oxidase, urea nitrogen and urease, galactose and galactose oxidase,
Examples include neutral fat and lipase, penicillin and penicillinase, etc.

A specific example of the biosensor device of this invention is shown in FIG. The biosensor device 1 shown in FIG.
It is equipped with a biosensor section 10 and a reference electrode section 11, and an amplifier 14a,
It is configured to be detected by a differential amplifier 15 via a terminal 14b and to be indicated to a calibrated indicator 16.

The biosensor section 10 includes a MOS field effect transistor element (5; PH-
An immobilized enzyme membrane 4a in which an enzyme is immobilized on a metal hydroxide gel membrane is closely formed on the gate 55 of the ISFET. In addition, in the figure, 51 is SiO ₂ or
₅₂ is a semiconductor layer, 53 is a source region, 54 _is a drain region, 53' is a source wiring, and 54' is a drain wiring. 13 is a bias electrode for applying a reference voltage to the biosensor section 10 and the reference electrode section 11, respectively.

On the other hand, the reference electrode section 11 includes a metal hydroxide gel membrane 4 without enzyme immobilization instead of the immobilized enzyme membrane 4a.
Biosensor part 1 except that b is formed in close contact with
It is configured in the same way as 0. Gel film 4b at this time
has substantially the same composition as the gel membrane serving as a carrier for the immobilized enzyme membrane 4a.

Note that the measurement configuration of the biosensor device of the present invention is not limited to the above-mentioned specific example, and may be attached to a flow cell type sample channel, for example, and includes at least the biosensor section as described above and a reference electrode. It may be configured such that a specific component in a sample can be detected directly or indirectly potentiometrically.

(e) _Example PH _- ISFET (length _6.5 mm, width 0.6 mm,
The biosensor section of the present invention was fabricated using a gate area of approximately 0.1 mm ² ).

First, the above PH-
The solution was coated on the gate surface by applying it to the ISFET. Next, this PH-ISFET is
By leaving it in the air for 1 hour at ℃, hydrolysis of tetraethoxysilane and drying of the solvent etc. proceeded to form a Si(OH) ₄ gel film with a thickness of about 2000 Å on the gate surface.

The PH-ISFET with a gel film formed was treated with 5 wt% γ
- Immerse in a PH3.5 buffered aqueous solution of aminopropyltriethoxysilane and hold at 85℃ for 2 hours to bond and introduce a coupling agent through the hydroxyl groups in the gel film. After washing with water, PH of 2.5wt% glutaraldehyde.
7.0 buffer solution and degassed for 20 minutes at 30°C.
Schiff base having an aldehyde group was introduced at the end of the coupling agent by holding the mixture for 40 minutes.

The PH-ISFET treated in this way was immersed in an aqueous solution of 100 U of urease (dissolved in 5 ml of 0.1 M phosphate buffer at pH 7.0), degassed for 5 minutes, and then heated for 30 minutes.
The enzyme was immobilized by holding at 0.degree. C. for 3 hours, and a biosensor (for urea detection) of the present invention as shown in FIG. 2 was obtained.

On the other hand, a Si(OH) ₄ gel membrane (approximately 2,000
A reference electrode part was obtained by closely forming a PH-ISFET on the gate of the PH-ISFET, and a biosensor device as shown in FIG. 1 was constructed using the biosensor part and the reference electrode part.

This semiconductor biosensor device detects the PH increase caused by the enzymatic reaction between urea in the sample solution and urease immobilized on the biosensor.
It is configured so that it can be detected by ISFET,
Based on this output, the urea in the sample can be qualitatively and quantitatively determined.

The biosensor obtained above was used to measure the output (generated potential) of urea aqueous solutions of various concentrations (in 0.01M phosphate buffer, pH 7.0), and the results are shown in FIG.

Thus, it can be seen that the biosensor device of the present invention has good linearity from a urea concentration of 10 ^-4 g/ml to nearly 10 ^-1 g/ml, and has good sensitivity and accuracy.

On the other hand, as samples, the results of measuring the output of various blank solutions in which only the pH was changed are shown in the third section.
Shown in the figure.

In this way, the biosensor device of this invention
It was confirmed that this is an ideal system that is not affected by PH fluctuations in the entire system.

(F) Effects of the Invention As described above, the biosensor device of the present invention has excellent sensitivity and accuracy, and the metal hydroxide used as the base of the immobilized enzyme membrane of the biosensor part serves as the reference electrode part. Since it uses an electrode coated with a gel membrane, output fluctuations due to changes in measurement liquid conditions, such as temperature, pH, interfering components, etc., are substantially canceled out, making it possible to perform ideal measurements. be.

[Brief explanation of drawings]

FIG. 1 is a configuration explanatory diagram showing a specific example of the biosensor device of the present invention, FIG. 2 is a schematic sectional view of a gate forming part in the biosensor section of the same specific example, and FIG. FIG. 4 is a graph showing the relationship between the output and the system PH change in one embodiment of the sensor device, and FIG. 4 is a graph showing the responsiveness in the same embodiment. 1... Biosensor device, 4a... Immobilized enzyme membrane, 4b... Metal hydroxide gel membrane, 5... PH-
ISFET, 10...Biosensor part, 11...Reference electrode part, 55...Gate.

Claims (1)

[Claims] 1. An immobilized enzyme membrane made of a metal hydroxide gel on which a predetermined enzyme is immobilized is formed in close contact with the sensitive surface of a chemical substance-sensitive electrode equipped with an inorganic solid sensitive surface. a biosensor part, and a reference electrode part formed by closely forming a gel film made of the metal hydroxide gel without enzyme immobilization on the sensitive surface of a chemical substance-sensitive electrode having the same inorganic solid sensitive surface as above. A biosensor device comprising: 2. The biosensor device according to claim 1, wherein the metal hydroxide gel is a hydrolysis product of an organometallic chelate compound having a metal alkoxide or an enzyme as a ligand. 3. The biosensor device according to claim 1 or 2, wherein the chemical substance-sensitive electrode is an ion-selective electrode or an ion-selective field-effect transistor.