JP2569404B2 - Method for immobilizing biofunctional substance and electrode using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for immobilizing a biofunctional substance and an electrode for a biosensor using the same. In particular, the present invention relates to a technique for miniaturizing an electrode for rapidly, highly sensitively and continuously measuring a specific substance, and a biosensor obtained by this technique.

[Prior Art] It is already known that a biosensor in which an enzyme or an microorganism is immobilized on a platinum or carbon surface with an enzyme can quickly and continuously measure various chemical substances and biological substances. In biosensors, one of the biofunctional substances is to separately prepare a film containing the biofunctional substance and paste it on the electrode, and to apply the enzyme or the like to the electrode whose surface has been chemically treated. It has been immobilized by a method of forming a covalent bond between the surface and the like. However, the performance of the biosensor is evaluated based on reproducibility, durability, high sensitivity, response speed, etc. However, the former method has difficulty in terms of response speed, and the latter method requires an increased immobilization density. Was difficult. Further, in any of the methods, immobilization requires several complicated steps, and it is difficult to obtain a multifunctional sensor in which several types of biofunctional material electrodes are mounted on one sensor.

Further, the conventional immobilized enzyme electrode has a structure in which an enzyme immobilized film is mounted on a flat platinum surface. Examples of the production method include a method in which a separately prepared immobilized enzyme membrane is bonded to a platinum electrode, and a method in which an enzyme is applied to a smooth platinum electrode whose surface is chemically treated to immobilize the enzyme. However, miniaturization is difficult with such a method. On the other hand, a semiconductor integration technology has recently attracted attention as this miniaturization technology. With this method using semiconductor technology, an enzyme electrode with a size of several mm can be manufactured, but since it is a potential detection method, satisfactory results in terms of sensitivity and response cannot be obtained. The following micronization is also considered quite difficult.

[Problems to be Solved by the Invention] In the above-described situation, the present inventors differed from the conventional sensor electrode structure in that the purpose of the present invention was to overcome the above-mentioned drawbacks of the conventional sensor and to minimize the size of the living body. Provided is a biosensor electrode capable of quick and high-sensitivity measurement by a functional material electrode preparation method. In addition, the present invention provides a biosensor electrode capable of increasing the detection sensitivity in spite of the fact that the electrode surface is several hundred times larger than the apparent electrode surface area by atomizing the microelectrode surface. The purpose is to provide. It is another object of the present invention to provide a biosensor having a quick response since a sufficient amount of a biofunctional substance can be immersed and fixed to a deep part of the electrode because the electrode surface is in the form of fine particles.

[Constitution of the Invention] [Means for Solving the Problems] The present invention has a noble metal fine particle layer electrochemically deposited and formed on an electrode surface in order to solve the above technical problem. And, the noble metal fine particle layer is immersed in a solution containing a biofunctional substance represented by an enzyme or an antibody,
Alternatively, there is provided a bioelectrode having a molecular discriminating power, wherein the biofunctional substance is directly taken into the noble metal fine particle layer by a contacting treatment.
It is preferable that the biofunctional substance taken into the noble metal fine particle layer is further cross-linked. Further, on the surface of the noble metal fine particle layer, in order to further stabilize the biofunctional substance and the noble metal fine particles,
A structure in which a polymer thin film is formed is preferable. Also,
The present invention electrochemically deposits noble metal fine particles on an electrode surface, and forms a layer of the noble metal fine particles on the electrode surface,
Next, the obtained noble metal fine particle layer is immersed or brought into contact with a solution containing a biofunctional substance represented by an enzyme or an antibody, and the biofunctional substance is directly in the noble metal fine particle layer. Provided is a method for producing a bioelectrode having a molecular discriminating ability characterized by taking in.

The microelectrode having a biological function according to the present invention is an electrode having a structure in which a surface of a fine electrode (for example, diameter: 100 to 100 μm) made of platinum or the like and having a conductive material fine particle layer impregnated with a biological functional material such as an enzyme is provided on the surface. It is. In particular, an electrode having a platinum black surface layer formed by electrodeposition of platinum is known to have a high catalytic activity for hydrogen reduction. However, as in the present invention, it is difficult to use platinum black as a carrier for a biofunctional substance. Not done conventionally. There is an immobilization method in which a platinum plate is made porous by corrosion and an enzyme or the like is connected with a cross-linking agent, but the porous body produced by this method usually has a specific surface area of only several m ² / g. Further, there has been no method of controlling and controlling the size of the platinum black fine particles of the present invention to cover and immobilize the biofunctional substance. That is, the method for immobilizing a biofunctional substance according to the present invention is not a carrier binding method which had to use a conventional chemical reagent (crosslinking agent), but can directly immobilize a biofunctional substance without chemical treatment. It is. In the present invention, the polymer thin film used for coating is mainly used for completely preventing elution of the enzyme and imparting biocompatibility. If the enzyme inside the microparticles is linked by a cross-linking agent, the enzyme will not be completely eluted. In this case, the main purpose of thinning the surface is to provide biocompatibility.

The method for immobilizing a biofunctional substance such as an enzyme of the present invention is to immobilize the biofunctional substance in a conductive fine particle layer such as platinum black. Specifically, the metal electrode having the fine particle layer is immersed in an aqueous solution of a biofunctional substance, or impregnated by coating the metal electrode, and then a slight elution of the biofunctional substance is prevented, and In order to impart antithrombotic properties, a polymer substance such as albumin or heparin is impregnated thereon, and this is insolubilized with a crosslinking agent to form an insoluble polymer thin film. Such an immobilization method of the present invention can be considered as applying the so-called entrapment method of entrapping and immobilizing an enzyme in a gel or a polymer to a metal electrode system. Hundreds
It can be made into a thin film of a degree or less, and the presence of the polymer film does not become a hindrance to the expression of the biological function of the intrasexually functional substance.

Here, the “biofunctional substance” is represented by enzymes and antibodies, and includes various enzymes, microbial cells, proliferating microorganisms, organelles, antigens, antibodies, haptens, and the like. In the present invention, instead of platinum, a "conductive substance" such as rhodium, gold, or carbon can be used.
Those capable of forming a fine particle layer of “conductive substance” on the surface of the conductive substance can be suitably used in the present invention, unless there is another obstacle.

Examples of the polymer substance that can be used in the present invention include proteins such as albumin and polysaccharides such as heparin. As the crosslinking agent, there is a crosslinking agent suitable for the polymer used. For example, for albumin, glutaraldehyde, carbodiimide, maleimide crosslinking agent, etc. are used.

The method for immobilizing a biofunctional substance according to the present invention is an extremely important technology for the development of a portable chemical health monitoring system and clinical chemistry analysis that requires multi-item measurement such as micronization of biosensors and multifunctionality. One. That is, recently, various multi-biosensors using integrated circuit technology have been devised. In this regard, the method of immobilizing an enzyme or the like on the surface of the microelectrode according to the present invention is also important. Furthermore, it is clear that the enzyme electrode obtained by the present invention has high sensitivity and shows a quick response.

That is, the structure of the conductive material layer on which the biological functional material is immobilized according to the present invention is as shown in FIG. As shown in the figure, the biofunctional substance is uniformly incorporated into the conductive fine particles. For example, platinum particles deposited on a substrate precipitate as black particles with high bulk depending on the electrodeposition conditions, and the deposited platinum black layer does not easily peel off even when vigorously stirred in an aqueous solution. Like the substrate platinum, it becomes a part of the electrode, so to speak, is a shape in which a biofunctional substance such as an enzyme is integrated in the electrode. The platinum black layer can be impregnated with a polymer such as albumin and cross-linked with a cross-linking agent such as glutaraldehyde to prevent elution of enzymes and the like.

If a conductive material having a biofunctional substance immobilized at a high density is used, a highly sensitive biosensor electrode can be obtained. That is, for example, an electrode prepared by immobilizing an enzyme or the like inside or on the surface of a platinum black fine particle of a platinum electrode having a platinum black surface layer is used as a biosensor electrode by an amperometry method as shown in the next embodiment. , High sensitivity. By using the above-described immobilization method according to the present invention, a biosensor comprising a microelectrode system can be formed.

The size of the fine particles or the distance between the particles used in the present invention can be controlled by changing the forming conditions.

Such electrodes include, in addition to platinum, rhodium,
Gold, carbon, or graphite as a substrate on which a fine particle layer of rhodium fine particles, gold fine particles, platinum black, carbon graphite fine powder, or conductive metal oxide fine particles is formed. The substance is immobilized.

[Example] Glucose is decomposed into gluconic acid and hydrogen peroxide in the presence of glucose oxidase. By combining the enzyme with a platinum electrode, the oxidation current of hydrogen peroxide corresponding to the glucose concentration is measured. The glucose concentration can be determined. From this point, an experiment was next performed using the electrode manufactured according to the present invention.

First, a platinum wire having a diameter of 100 μm was sealed in a soda glass tube. This platinum wire was polished to obtain a circular fine platinum electrode. Then, in a 0.5 M aqueous sulfuric acid solution, the silver / silver chloride electrode was used as a reference electrode, and the platinum electrode was subjected to potential scanning at a potential of +1.3 V to -0.25 V at a scanning speed of 100 mV / sec for 30 minutes. The above electrode was immersed in a 3% chloroplatinic acid solution containing 300 ppm of lead acetate, and electroplating of platinum black was performed at a current value of −50 μA for 10 minutes. The thickness of the deposited platinum black layer was about several μm. Next, the obtained platinum black deposition electrode is air-dried at 25 ° C for 60 seconds, and then the platinum black deposition electrode is kept at -0.3 V for 30 minutes in a 0.5 M aqueous sulfuric acid solution using the silver / silver chloride electrode as a reference electrode. Then, hydrogen was generated from a platinum black deposition electrode.

The platinum black electrode was air-dried at 20 ° C. for 60 seconds, and then phosphate buffer containing 5500 units of glucose oxidase (pH 6.
8) Dipped in 1 ml for 20 minutes. After air drying again at 20 ℃ for 30 seconds,
The glucose concentration was measured in a phosphate buffer using the obtained enzyme-immobilized microelectrode. That is, glucose was measured using a reference electrode (reference electrode), a counter electrode (platinum), and a working electrode, that is, a glucose oxidase-immobilized platinum black electrode prepared according to the present invention. Each electrode is connected to a potentiostat, and while the working electrode is maintained at +0.6 V with respect to the reference electrode, glucose is injected, and the oxidation current value due to hydrogen peroxide flowing between the counter electrode and the working electrode is measured. did. As a result, this electrode has a 100% response within 3 seconds and can measure glucose at a concentration of 0.01 mg / dl as shown in Fig. 2. The measurement range is 0.01 mg / dl to 100 mg / dl. Indicates linearity.

The sensor output of the present invention accompanying the addition of glucose (0.9 mg / dl), ie. The change in the oxidation current of hydrogen peroxide was very responsive, as shown in FIG. As described above, it took less than 3 seconds to reach 100% response. The response of the sensor was extremely fast and reached a steady state value immediately. As described above, the biosensor using the method for immobilizing a biologically functional substance according to the present invention is
It has been shown that a microbiosensor can be produced by a simple method, showing a quick response, high sensitivity, and a simple method.

[Effects of the Invention] The method for immobilizing a biologically functional substance of the present invention and the biosensor using the same have firstly provided an electrode that can be detected with high sensitivity, and secondly, easily include a biologically functional substance such as an enzyme. The electrode can be immobilized and the biofunctional material can be immobilized without damaging the biofunctional material, so that an electrode with almost no loss of activity can be obtained. Third, high-density biofunctional material can be immobilized and rapid Remarkable technical effects were obtained, such as the provision of an electrode capable of obtaining a high response.

[Brief description of the drawings]

FIG. 1A is a sectional view showing the structure of a biosensor electrode manufactured according to the present invention. FIG. 1B is a cross-sectional view in which a part of FIG. 1A is enlarged, and shows the structure of the surface of the fine particles impregnated with a biofunctional substance. FIG. 2 is a graph showing the relationship between the response output measured by the biosensor electrode of the present invention and the glucose concentration. FIG. 3 is a graph showing the responsiveness of the biosensor according to the present invention. [Explanation of Signs of Main Parts] 1... Fine particles of conductive substance 2... Fine particles of biofunctional substance 3.

Claims (6)

(57) [Claims] 1. A precious metal fine particle layer electrochemically deposited and formed on an electrode surface, and the noble metal fine particle layer is immersed in a solution containing a biofunctional substance represented by an enzyme or an antibody. A bioelectrode having a molecular discriminating power, wherein the biofunctional substance is directly incorporated into the noble metal fine particle layer by a process of causing or contacting the bioelectrode. 2. The bioelectrode according to claim 1, wherein said biofunctional substance taken into said noble metal fine particle layer is further subjected to a crosslinking treatment. 3. The surface of the noble metal fine particle layer is further provided on the surface of the noble metal fine particle layer in order to secure stabilization of the biofunctional substance and the noble metal fine particles.
3. The bioelectrode according to claim 1, wherein a polymer thin film is formed. 4. Precious metal fine particles are electrochemically deposited on an electrode surface to form a layer of the noble metal fine particles on the electrode surface, and then the obtained noble metal fine particle layer is typified by an enzyme or an antibody. A method for producing a bioelectrode having molecular discriminating power, characterized by immersing or contacting a solution containing a biofunctional substance into the noble metal fine particle layer. 5. The method for producing a bioelectrode according to claim 4, further comprising a step of performing a crosslinking treatment. 6. The method according to claim 4, further comprising a step of performing a polymer thin film forming process.
The method for producing a bioelectrode according to the above item.