JPH0675054B2 - Direct immobilization of biofunctional substances - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for directly immobilizing a biofunctional substance and a biosensor electrode using the method. In particular, it relates to an electrode miniaturization technique for rapidly, highly sensitively and continuously measuring a specific substance and a biosensor obtained by this technique. Furthermore, in detail, a method of directly immobilizing a biofunctional substance such as an enzyme without using a cross-linking agent while forming a fine particle layer electrochemically on the surface of a minute electrode, and a high-performance microscopic method obtained by the method. Regarding biosensors.

[Prior Art] It is already known that a biosensor in which an enzyme, an antibody, a microorganism or the like is immobilized on the surface of platinum or carbon can measure various chemical substances and biological substances rapidly and continuously. In the biosensor, for the biofunctional substance, one is prepared by separately preparing a film containing the biofunctional substance and pasting it on the electrode, and the other is coating the surface of the electrode with an enzyme, etc. Have been immobilized by a method of forming a covalent bond between them and the surface. However, The performance of biosensors is evaluated by reproducibility, durability, high sensitivity, response speed, etc., but the former method is difficult in terms of response speed, and the latter method makes it difficult to increase the immobilization density. Met. Further, in any of the methods, immobilization requires several steps, and it is difficult to form a multifunctional sensor in which several kinds of biofunctional substance electrodes are mounted on one sensor.

In addition, the conventional immobilized enzyme electrode has a structure in which an enzyme-immobilized membrane is attached to a flat platinum surface. As a method for producing the same, there is a method in which a separately prepared immobilized enzyme membrane is attached to a platinum electrode, a method in which an enzyme is applied to a smooth platinum electrode whose surface is chemically treated and immobilized. However,
Miniaturization is difficult with such a method. On the other hand, the semiconductor integration technology has recently received attention as a miniaturization technology. With this method using semiconductor technology, an enzyme electrode with a size of several mm can be produced, but since it is a potential detection method, satisfactory results have not been obtained in terms of sensitivity and response, and it is currently available. It is considered quite difficult to make a micro-structure smaller than the size.

[Problems to be Solved by the Invention] In the above situation, the inventors of the present invention, unlike the conventional sensor electrode structure, aim to overcome the drawbacks of the above-described conventional sensor, and to reduce the size of the living body. With functional material electrode,
Provided is a biosensor electrode capable of quick and highly sensitive measurement. Further, according to the present invention, by forming fine particles on the surface of the microelectrode, the surface of the electrode is made several thousand times larger than the apparent surface of the electrode. An object is to provide a biosensor electrode capable of increasing the sensitivity.
A further object of the present invention is to provide a biosensor having a rapid response because a biofunctional substance in a sufficient amount can be immersed and immobilized in the deep part of the electrode because the surface of the electrode is in the form of fine particles. It is another object of the present invention to provide a biosensor electrode in which a biofunctional substance is directly fixed in the voids of the conductive fine particle substance. Therefore, it is an object of the present invention to provide a biofunctional substance immobilization method in which direct immobilization of a biofunctional substance is extremely easy.

[Structure of the Invention] [Means for Solving Problems] The present invention is directed to electrochemically depositing fine particles of a conductive substance on an electrode on a minute surface of a conductor made of a conductive substance. Containing the biofunctional substance on the surface of the minute electrode in a single step of adsorbing the biofunctional substance into the layer of the deposited fine particles of the conductive substance together with the electrochemical deposition treatment. The method for producing a biosensor electrode having a molecular discrimination ability is characterized by forming a surface layer of the fine particle conductive material. However, the “molecular recognition ability” is “the ability to specifically generate a signal only in a molecule determined by the biological functional substance”. For example,
When glucose oxidase is used as the biofunctional substance, a signal dependent only on glucose present in the sample is detected.

The microelectrode having a biological function according to the present invention has a structure in which a fine plate electrode made of platinum or the like (for example, diameter: 10 to 100 μm) has a conductive substance fine particle layer in which a biological functional substance such as an enzyme is impregnated on the surface. It is an electrode. In particular, an electrode having a platinum black surface layer formed by electrochemical deposition of platinum is known to have a high catalytic activity for hydrogen reduction, but it is not possible to use platinum black as a carrier for a biofunctional substance as in the present invention. , Which has not been performed conventionally (there is a method of immobilizing a platinum plate by making it porous by corrosion and connecting an enzyme or the like with a cross-linking agent), and further controlling the size of the platinum black fine particles according to the present invention to obtain a biofunctional substance. The method of encapsulating and immobilizing is unprecedented. Ie. The direct immobilization method of a biofunctional substance according to the present invention is not a carrier binding method which has conventionally required the use of a chemical reagent (crosslinking agent), but can directly immobilize a biofunctional substance without chemical treatment. is there.

According to the method for producing a biosensor electrode according to the present invention, for example, a biofunctional substance is directly electrodeposited on a conductive substance such as platinum, and at the same time, a metal salt is electrolytically reduced (electrodeposited) to obtain conductivity (eg, (Metal) A fine particle substance is deposited together with a biofunctional substance. Ie. It is produced by forming a fine particle of a conductive substance on a fine platinum electrode and taking in a biofunctional substance into a bore in the fine particle. The size of the bore and the amount of biofunctional substance to be immobilized can be controlled by adjusting the current density, the deposition time and the like. The function of the immobilized biofunctional substance electrode thus obtained can be maintained for a long period of time.

Furthermore, the electrode obtained according to the present invention forms a thin film obtained by coating a high molecular substance such as protein or polysaccharide and cross-linking with a cross-linking agent to give a biocompatible raw material, maintain performance, and dissolve a biofunctional substance to a minimum. You can also do it.

Here, the “biologically functional substance” is represented by an enzyme and an antibody, and includes various catalysts, microbial cells, proliferating microorganisms, organelles, antigens, antibodies, haptens and the like. In the present invention, instead of platinum, gold,
As long as there are no obstacles, a "conductive substance" such as rhodium that can form a fine particle layer of "conductive substance" on the surface of the conductive substance can be preferably used in the present invention.

In addition, the polymer substances that can be additionally used to coat the biosensor electrode of the present invention include proteins such as albumin and polysaccharides such as heparin. As the cross-linking agent, there is a cross-linking agent suitable for the polymer substance used. For example, for albumin, glutaraldehyde, carbodiimide, maleimide cross-linking agent, etc. are used.

INDUSTRIAL APPLICABILITY The method for immobilizing a biofunctional substance of the present invention is one of extremely important techniques for clinical chemistry analysis and development of a portable health monitoring system that requires multi-item measurement such as micro-sizing and multi-functionalization of biosensors. Is one. Ie. Recently, various multi-biosensors using integrated circuit technology have been proposed.
Also in this respect, the method of immobilizing an enzyme or the like on the surface of the microelectrode according to the present invention is important. Furthermore, it is clear that the enzyme electrode obtained by the present invention has a high sensitivity and a quick response.

Ie. The structure of the conductive substance layer on which the biofunctional substance is immobilized according to the present invention is shown in FIG. As shown in the figure, the fine particles of the biofunctional substance are uniformly incorporated in the fine conductive particles. Ie. By electrodepositing an enzyme or the like on a microelectrode and then electrolytically depositing a conductive substance in the form of fine particles, a biofunctional substance-immobilized electrode on which an enzyme or the like is directly immobilized is obtained.

By using a conductive substance in which a biofunctional substance is immobilized at a high density, a highly sensitive biosensor electrode can be obtained. That is, for example, an electrode prepared by immobilizing an enzyme or the like in a platinum black surface layer of a platinum electrode having a platinum black surface layer is highly expensive as a biosensor electrode by the amperometry method, as shown in the following examples. It becomes sensitive. By using the immobilization method according to the present invention described above, a biosensor including a microelectrode system can be constructed.

The size or pore size of the fine particles used in the present invention is
It can be changed by changing the formation conditions, that is, by adjusting the reduction current, the reduction time or the reduction voltage, or in the case of platinum black formation, by adjusting the lead acetate content in the precipitation solution.

In addition to platinum, such electrodes include gold, other precious metals, or carbon. There is one in which a layer of platinum black, fine particles of gold, fine particles of noble metal, and fine particles of conductive metal oxide is formed together with a biofunctional substance on a substrate made of glassy carbon, graphite or the like.

[Example] Glucose is converted into gluconic acid and hydrogen peroxide in the presence of glucose oxidase. By integrating the enzyme and a platinum electrode and measuring the oxidation current of hydrogen peroxide corresponding to the glucose concentration, The glucose concentration can be determined.

First, a method for producing the enzyme-immobilized electrode according to the present invention will be described.
Next, a glucose concentration measurement experiment was performed using the electrode. The results are shown below.

First, a platinum wire with a diameter of 100 μm was enclosed in a soda glass tube and polished with alumina powder.

Immobilization of the enzyme was performed by the following two methods.

(A) Sodium sulfate solution (0.2M, pH 3.2) containing chloroplatinic acid (1mg / ml) and glucose oxidase (1mg / ml)
Among them, the silver / silver chloride electrode was used as a reference electrode and the platinum electrode was used as a working electrode to carry out electrolysis at a constant potential (-0.2 V) for several tens of minutes to deposit platinum black while immobilizing the enzyme.

(B) Chloroplatinic acid (33 mg), lead acetate (0.6 mg) and glucose oxidase (10 mg) in 1 ml of a solution (pH 3.5), electrolyzed with a constant current (-5 μA) for 10 minutes, and glucose oxidase-impregnated platinum Black was deposited.

The electrode manufactured by the above method was stirred and washed in 0.1M phosphate buffer for 24 hours, and then used for the next glucose concentration measurement experiment.

The electrode manufactured by the above method can be easily adjusted in a one-step process, and a stable and quick response can be obtained as follows. Ie. The electrodeposited surface layer is
It is a sensing portion of the cross section shown in FIG.

In the above electrode production according to the present invention, positively charged glucose oxidase (isoelectric point = 4.2) is electrodeposited on the electrode, and platinum is electrolytically deposited. As a result, an enzyme is present in the pores of platinum black fine particles. Are fixed as shown in FIG. The enzyme can be immobilized by either of the above-mentioned two types of electrode preparation methods, but the amount of immobilized enzyme can be adjusted by the method (B), which promotes the enzyme electrode in the negative potential range and at the same time platinum at a constant current. Black can be grown and formed at a constant rate, and it is easy to obtain a homogeneous fine particle platinum black layer of enzyme precipitation. On the contrary,
The (A) method of depositing at a constant potential takes time because the current gradually decreases as the platinum black layer deposited increases.
It tends to be difficult to obtain a homogeneous precipitate.

Next, the responsiveness of the biosensor electrode according to the present invention is measured in a phosphate buffer with the measuring device shown in FIG. Ie. 1
Is a reference electrode (standard electrode), 2 is a counter electrode (platinum), and 3 is a working electrode. It is a glucose oxidase-immobilized platinum black electrode produced by the present invention. Each electrode was connected to a potentiostat as shown in the figure, and while the working electrode 3 was maintained at +0.6 V with respect to the reference electrode 1, glucose was injected while stirring and flowed between the counter electrode and the working electrode. The value of the oxidation current by hydrogen peroxide was measured.

The output increased immediately after glucose was added and reached a constant value within 3 seconds. The result of measuring the relationship between the glucose concentration and the output of the sensor is shown in FIG. Ie. With the biosensor according to the present invention, glucose can be measured even at a concentration of 0.1 mg / dl as shown in FIG.
Linearity was shown in the range of g / dl to 100 mg / dl.

The change of the sensor output of the present invention, that is, the oxidation current of hydrogen peroxide with the addition of glucose was very responsive as shown in FIG. As described above, 100% response was within 3 seconds. The response of the sensor was extremely fast and reached a steady value immediately. As described above, it has been clarified that the biosensor according to the method for immobilizing a biofunctional substance according to the present invention has a quick response and high sensitivity, and a microbiosensor can be manufactured by a simple method.

[Advantages of the Invention] With the method for immobilizing a biofunctional substance of the present invention and the biosensor using the same, firstly, an electrode capable of detecting with high responsiveness and high sensitivity was provided. The functional substance can be easily entrapped and immobilized, and the biofunctional substance electrode with almost no loss of activity can be obtained because the biofunctional substance is immobilized without being damaged. It was possible to provide an electrode that can be immobilized and obtain a quick response. Fourthly, it was possible to provide a method for easily immobilizing biofunctional substances such as enzymes on the surface of microelectrodes without chemical treatment. In Fig. 5, remarkable technical effects were obtained, such as the fact that a multifunctional enzyme sensor in which a plurality of enzymes were immobilized on the surface of a microelectrode could be made.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a glucose concentration measuring method using a biosensor according to the present invention. FIG. 2A is a schematic cross-sectional view showing the structure of the biosensor electrode produced according to the present invention. FIG. 2B is a schematic enlarged cross-sectional view enlarging and showing a circular dotted line portion of FIG. 2B,
1 shows the structure of a fine particle layer impregnated with a biofunctional substance. FIG. 3 is a graph showing the relationship between the response sensor output measured by the biosensor electrode of the present invention and the glucose concentration. FIG. 4 is a graph showing the response of the biosensor according to the present invention. [Explanation of Signs of Main Parts] 1 ... Reference electrode 2 ... Counter electrode 3 ... Working electrode (electrode of the present invention) 4 ... Conductive substance fine particles 5 ... Biofunctional substance 7 ... Substrate conductor

Claims (1)

[Claims] 1. A biofunctional substance is deposited on an electrode on a minute surface of a conductor made of a conductive substance while electrochemically depositing fine particles of the conductive substance, together with the electrochemical deposition treatment. In a single step of adsorbing in a layer of fine particles of a conductive substance, a surface layer of the fine particle conductive substance containing the biofunctional substance is formed on the surface of the minute electrode. A method for producing a biosensor electrode having the ability to identify molecules.