JPH0723882B2 - Biosensor and sensor plate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention particularly relates to a biosensor and a sensor plate in which peeling of an enzyme-immobilized film is prevented.

[Conventional technology]

Biosensors generally include enzymes, antibodies, functional organic molecules,
Using a molecular recognition membrane in which biological materials such as microorganisms are immobilized on a polymer carrier, changes in current, potential, heat, light, etc. caused by selective reaction in the membrane are changed by pH electrode, peroxide electrode,
An oxygen signal, a semiconductor electrode, a phototransistor, a thermistor, or other transducer is used to convert the signal into an electric signal, and the ion concentration in the sample can be measured by display based on this signal.

Specifically, for example, a pH sensitive film is formed on the gate electrode of a field effect transistor (FET) formed in a semiconductor,
Furthermore, by forming an enzyme-immobilized film on it, the chemical substance in the sample solution is decomposed by an enzyme, and the concentration of this chemical substance can be measured by measuring the hydrogen ion concentration generated by this, a so-called ion-sensitive electric field. Effect transistor (IS
FET) is known. This ISFET utilizes the fact that the conductivity near the semiconductor surface changes in response to the change in the electric field generated at the interface between the pH sensitive film and the hydrogen ion solution, and this can be detected by an external circuit.

The method for forming the enzyme-immobilized membrane is roughly classified into a direct immobilization method in which the enzyme is directly immobilized on the pH electrode surface, and an entrapping immobilization method in which the enzyme is entrapped in a polymer carrier. Compared to the direct fixation method, the entrapping fixation method is characterized in that the treatment is milder, the enzyme is less likely to change, and the activity is easily maintained for a long time.

With the enzyme-immobilized membranes produced by either method, the pH electrode is expensive in the past, so if the activity of the enzyme-immobilized membrane decreases, a jig or net is used on the electrode surface so that only this enzyme-immobilized membrane can be replaced with a new one. It was fixed to the electrode.

On the other hand, since it is troublesome to replace the enzyme-immobilized membrane as described above, it became possible to form an inexpensive electrode, so the development of a disposable biosensor in which all electrodes with the enzyme-immobilized membrane are discarded Is being promoted. Again, the pH
As the method of immobilizing the enzyme on the electrode, both the direct immobilization method and the entrapping immobilization method are used as in the above, but the latter is preferable because the activity can be retained for a long time as in the above.

To fix the enzyme to the pH electrode by the entrapping immobilization method, conventionally,
The use of a photopolymerizable polymer, agar, gelatin, polyacrylamide, or another polymer as the polymer carrier has been studied. In particular, since the action of the enzyme is suitable for the aqueous solution sample, the hydrophilic polymer is also preferable as the polymer carrier.

[Problems to be Solved by the Invention]

However, in the case of hydrophilic polymers, carrying the enzyme,
Although it is effective for immobilizing on a pH electrode, when a polymer aqueous solution containing an enzyme is applied to the electrode to form an enzyme-immobilized membrane, the swollen enzyme-immobilized membrane containing water is dried during work and Due to natural drying during storage of the finished product, the product shrinks and simply adheres to the electrode surface, so that the problem of peeling from the electrode surface frequently occurs.

Specifically, for example, on the pH electrode surface formed on the surface of a PVC (polyvinyl chloride) resin film, a photopolymerizable polymer (a vinyl group at both ends of a prepolymer having a basic skeleton of polyethylene glycol and polypropylene glycol) is formed. When an enzyme-immobilized membrane carrying glycosyl oxidase (GOD) is formed in an enzyme-immobilized membrane, the enzyme-immobilized membrane often peels from the pH electrode surface.

An object of the present invention is to provide a biosensor in which peeling of the enzyme-immobilized membrane is prevented.

[Means for Solving the Problems]

The present invention, in order to solve the above problems, in a biosensor capable of electrically detecting the sensitive value of a sample liquid by using an electrode having a sensitive film in which an ion sensitive film and an enzyme-immobilized film are sequentially laminated, Provided is a biosensor characterized in that each of a sensitive membrane and an enzyme-immobilized membrane contains a polymer or prepolymer having reactivity with a crosslinking agent, and the polymers or prepolymers of both membranes are chemically bonded by the crosslinking agent. To do.

At this time, the ion-sensitive membrane is a pH-sensitive membrane containing a polyvinyl chloride polymer having a hydroxyl group, the enzyme-immobilized membrane is a prelimer having a polymerizable double bond, and the cross-linking agent is a hydroxyl group or a polymerizable double bond. It is also preferable that the electrode is a silane compound having reactivity with both of the above and the electrode has a laminated structure of silver and silver chloride.

Further, the present invention provides a separation electrode, which is used by being connected to an input electrode of the electric amplification circuit, and a separation comparison electrode, on an insulating substrate separate from the substrate of the electric amplification circuit, An ion-sensitive membrane and an enzyme-immobilized membrane are sequentially laminated, and each of these ion-sensitive membrane and enzyme-immobilized membrane contains a polymer or prepolymer having reactivity with a crosslinking agent. The present invention provides a sensor plate characterized in that a polymer is chemically bonded by the above-mentioned cross-linking agent.

At this time, the ion-sensitive membrane is a pH-sensitive membrane containing a polyvinyl chloride polymer having a hydroxyl group, the enzyme-immobilized membrane is a prepolymer having a polymerizable double bond, and the cross-linking agent is a hydroxyl group or a polymerizable double bond. It is also preferable that the electrode is a silane compound having reactivity for both bonds and that the electrode has a laminated structure of silver and silver chloride.

Next, the present invention will be described in detail.

In the present invention, an ion-sensitive membrane and an enzyme-immobilized membrane are sequentially laminated on an electrode, and a polymer or a prepolymer is contained in these layers, and the type thereof is one that reacts with a crosslinking agent. In this case, the ion-sensitive membrane and the enzyme-immobilized membrane may have different reactive groups such as a hydroxyl group and a polymerizable double bond, or may have the same reactive group.

Examples of the polymer having a hydroxyl group include polymers having vinyl alcohol as a copolymerization component, and specific examples thereof include polyvinyl chloride-based polymers. Further, a partially saponified product of vinyl acetate copolymer and a modified product thereof may also be mentioned. Further, examples of those having a polymerizable double bond include those which are polymerized by light, heat or the like under a polymerization initiator.

Examples of the cross-linking agent having reactivity with both the hydroxyl group and the polymerizable double bond include compounds having the following structures.

(In the formula, R ₁ , R ₂ and R ₃ may be the same or different and each represents an alkyl group such as methyl or ethyl, a phenyl group or the like.) Note that a saponified product of this compound is also used.

[Action]

Since the polymer or prepolymer contained in each of the enzyme-immobilized membrane and the ion-sensitive membrane is chemically bonded with the cross-linking agent, the enzyme-immobilized membrane firmly adheres to the ion-sensitive membrane and is difficult to peel off.

〔Example〕

Next, an embodiment of the present invention will be described with reference to FIGS.

The copper foil adhered to the paper polyester substrate 1 was patterned by the photographic method and then the surface was polished to form the smooth copper electrodes 1a and 1b.

Next, using a commercially available cyan-based silver strike plating bath containing 1 g / constant and a constant current power source, the copper electrodes 1a and 1b were used as cathodes, and platinum-plated titanium mesh was used as an anode, and the cathode current density was 0.
The above substrate was immersed in a bath for 5 seconds in a state of being set to 5 A / dm ² and then taken out and washed with water.

Then, it was immersed in a commercially available cyan-based electrolytic silver bright plating solution containing 20 g / silver while maintaining the temperature at 50 ° C.
Using a and 1b as cathodes and platinum-plated titanium mesh as an anode, electrolytic plating was performed at a cathode current density of 1.2 A / dm ² for 1 minute and 30 seconds to form 15 μm silver layers 2a and 2b on the copper electrodes 1a and 1b, respectively. .

Then, in 0.1N (N) hydrochloric acid (HCl), the substrate is used as an anode, and a platinum-plated titanium mesh electrode is used as a cathode.
Electrolytic treatment was performed at an anode current density (0.2 A / dm ² ) for 2 minutes and 40 seconds to form silver chloride layers 3a and 3b on the surfaces of the silver layers 2a and 2b. The surface roughness was 200 nm as measured by a stylus film thickness meter (Tencor Corporation thin film surface profiler Alpha Step 200).

Next, except for a part of the silver chloride layer 3a, that is, a part forming a pH sensitive film which will be described later, a part of the silver chloride layer 3b serving as a reference electrode connected to this part with a sample liquid, and a terminal part for external contact. The epoxy resin layer 4 was formed.

The pH sensitive film 6 is formed on the part of the silver chloride layer 3a which is not covered with the epoxy resin layer 4. The pH sensitive film 6 has a weight composition ratio of vinyl chloride: vinyl acetate: vinyl alcohol.
It is composed of 32% polyvinyl chloride polymer consisting of 9: 3: 6, 67% dioctyl adipate, and 1% tridodecylamine.

As shown in FIG. 1, a bank 5 made of an adhesive tape of polyethylene terephthalate was formed so as to surround the pH sensitive film 6 and a portion of the silver chloride layer 3b which was not covered with the epoxy resin layer 4.

Then, 10 μl of a solution containing 10% triethoxyvinylsilane, 10% water, and 80% methanol is dropped onto the pH sensitive membrane 6 surrounded by the bank 5, and left for 5 minutes. After that, the remaining liquid which was not absorbed by the pH sensitive film 6 was removed with Bencotton and left at room temperature for 2 hours to react the vinylsilyl group with the vinyl chloride polymer.

Next, the enzyme-immobilized membrane 7 is formed on the pH-sensitive membrane 6 as follows.

Gel base (ENTG3800 manufactured by Kansai Paint Co., Ltd.) 1 g Polymerization initiator 0.05 g 50 mg / ml Glucose oxidase (GOD) solution 0.5 ml was mixed in a test tube with a composition of 5 to 10 μm.
Is dropped on the pH sensitive membrane 6 and is irradiated with an ultraviolet lamp for 3 minutes to form an enzyme-immobilized membrane 7.

In this way, the vinyl chloride-based polymer of the ion-sensitive membrane and the main agent of the enzyme-immobilized membrane are crosslinked by triethoxyvinylsilane, and when the relationship between them is schematically shown,
It looks like this:

Finally, the whole was immersed in a phosphate buffer solution to extract the unreacted material of the above composition, which was then washed and dried to obtain a glucose biosensor plate. This was enclosed in an aluminum pack.

After 100 pieces of the enclosed glucose biosensor plates were left for one day and night, they were opened to visually determine whether or not the enzyme-immobilized membrane 7 was peeled off, and the frequency was examined, and the results are shown in Table 1.

In addition, the glucose biosensor plate was connected to an electrical amplifier circuit as described below, and the relative sensitivity was obtained.
Shown in.

Comparative Example A glucose biosensor was prepared in the same manner as in the above example except that the treatment with triethoxyvinylsilane was not performed, and the frequency of peeling of the enzyme-immobilized membrane was measured in the same manner as in the above example. Further, the relative sensitivity was obtained in the same manner as in the above example. The results are shown in Table 1.

From the above results, it is shown that in the examples, the frequency of peeling of the enzyme-immobilized membrane is extremely low, and the sensitivity of the pH-sensitive membrane is not affected by the treatment with triethoxyvinylsilane.

The above glucose biosensor plate is composed of an electrode and an Ag / AgCl that are sequentially laminated with an ion-sensitive membrane and an enzyme-immobilized membrane.
Connect the electric potential between the reference electrode and the electric amplification circuit device,
By measuring the concentration of the glucose contained in the enzyme-fixed membrane surrounded by the bank and the exposed portion of the silver chloride layer 3b as the reference electrode, the concentration of the glucose is measured as the output value of the ion sensor. be able to.

In the above, the pH-sensitive membrane contained triethoxyvinylsilane, but it may be contained in the coating solution for the enzyme-immobilized membrane, and the coating membrane may be applied to the pH-sensitive membrane and polymerized. A triethoxyvinylsilane layer may be formed between the films by coating the liquid.

Further, GKT Sharp 2000 (manufactured by Denki Kagaku Kogyo Co., Ltd.) is a specific example of the polyvinyl chloride-based polymer used for the pH sensitive membrane. As a polymer or prepolymer used for the enzyme-immobilized membrane, when a photocurable one mainly having polyethylene glycol (PEG) or polypropylene glycol (PPG) as a skeleton and having ethylenically unsaturated groups at both ends is used, The thing of a structure is mentioned. In the table, numbers with ENT-, ENYG-, and ENTP- indicate product names (manufactured by Kansai Paint Co., Ltd.).
Can also be mentioned. The ENTP system is hydrophobic.

[Effect of the Invention] According to the present invention, the polymer or prepolymer contained in both the enzyme-immobilized membrane and the ion-sensitive membrane is chemically bonded by a cross-linking agent, so that the adhesive strength thereof can be increased and the enzyme-immobilized membrane can be immobilized. The frequency of peeling of the film can be significantly reduced.

[Brief description of drawings]

FIG. 1 is a plan view of a sensor plate of the present invention, and FIG. 2 is a II-II sectional view thereof. In the figure, 2a and 2b are silver layers, 3a and 3b are silver chloride layers, 6 is a pH sensitive membrane, and 7 is an enzyme immobilization membrane.

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Claims (6)

[Claims] 1. A biosensor capable of electrically detecting a sensitive value of a sample liquid by using an electrode having a sensitive membrane in which an ion sensitive membrane and an enzyme immobilized membrane are sequentially laminated, and a biosensor comprising the ion sensitive membrane and the enzyme immobilized membrane. A biosensor characterized in that a polymer or prepolymer having reactivity with a crosslinking agent is contained in each, and the polymers or prepolymers of both of these films are chemically bonded by the crosslinking agent. 2. The ion sensitive membrane is a pH sensitive membrane containing a polyvinyl chloride polymer having a hydroxyl group, and the enzyme immobilization membrane is an enzyme holding membrane containing a prepolymer having a polymerizable double bond and an enzyme. The biosensor according to claim 1, wherein the cross-linking agent is a silane compound having reactivity with each of the hydroxyl group and the polymerizable double bond. 3. An electrode in which an ion-sensitive membrane and an enzyme-immobilized membrane are sequentially laminated has a laminated structure composed of an upper layer containing silver chloride as a main component and a lower layer containing silver as a main component. The biosensor according to claim 1 or 2. 4. A separation electrode and a separation comparison electrode, which are used by being connected to an input electrode of the electric amplification circuit, are provided on an insulating substrate which is separate from the substrate of the electric amplification circuit, and ions are provided on the separation electrode. A sensitive membrane and an enzyme-immobilized membrane are sequentially laminated, and a polymer or prepolymer having reactivity with a cross-linking agent is contained in each of the ion-sensitive membrane and the enzyme-immobilized membrane. A sensor plate in which the above is chemically bonded by the cross-linking agent. 5. The ion sensitive membrane is a pH sensitive membrane containing a polyvinyl chloride polymer having a hydroxyl group, and the enzyme immobilization membrane is an enzyme holding membrane containing a prepolymer having a polymerizable double bond and an enzyme. The sensor plate according to claim 4, wherein the crosslinking agent is a silane compound having reactivity with each of the hydroxyl group and the polymerizable double bond. 6. An electrode in which an ion-sensitive membrane and an enzyme-immobilized membrane are sequentially laminated has a laminated structure composed of an upper layer containing silver chloride as a main component and a lower layer containing silver as a main component. The sensor plate according to claim 5.