JP2615391B2 - Enzyme analysis system and analysis method using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an analysis system using a biosensor and an analysis method thereof. In particular, the present invention relates to a high-speed, high-sensitivity, high-output biosensor analysis device using a biofunctional substance-immobilized microelectrode (microelectrode) and an analysis method using the biosensor.

[Prior Art] It is already known that a biosensor in which an enzyme, an antibody, a microorganism, or the like is immobilized on platinum or carbon surface can measure various chemical substances and biological substances quickly and continuously. In such a biosensor, the biofunctional substance is obtained by attaching a film containing the biofunctional substance on the electrode, or by applying an enzyme or the like to the electrode whose surface has been chemically treated, and then interposing the enzyme between the enzyme and the electrode surface. It has been immobilized by methods such as forming a covalent bond. However. The performance of biosensors is evaluated based on reproducibility, durability, high sensitivity, response speed, etc. However, the former method has difficulty in response speed, and the latter method has difficulty in increasing the immobilization density. Met. Also, 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 substance electrodes are mounted on one electrode.

The conventional immobilized enzyme electrode is a Clark-type oxygen electrode,
Alternatively, it has a structure in which an enzyme-immobilized membrane is mounted on a flat platinum surface. As a method for producing the same, there are a method of attaching a separately prepared immobilized enzyme film to a platinum electrode, and a method of applying and immobilizing an enzyme on a smooth electrode whose surface is chemically treated. However, miniaturization is difficult with such a method. On the other hand, a semiconductor integration technology has recently attracted attention as this miniaturization technology. In this method using semiconductor technology, an enzyme electrode of several mm in size can be manufactured, but since it is a potential detection method, satisfactory results are not obtained in terms of sensitivity and response. The following microsizing is also considered very difficult.

[Problems to be Solved by the Invention] Under the circumstances described above, the present inventors differed from the conventional biosensor system in that, for the purpose of overcoming the difficulties of the conventional sensors described above, By using a functional material electrode (hereinafter, referred to as a microelectrode), there is provided an analysis system capable of measuring a very small amount of a sample with high speed response and high sensitivity, and an analysis method using the same.

Another object of the present invention is to provide a high-performance biosensing system that can rapidly measure an extremely small amount of sample by using a microdevice using the above-mentioned microelectrode as a working electrode. That is. The present invention provides a biosensor analysis system and method capable of measuring a substance (eg, glucose) in a trace sample by detecting a current generated when a potential is applied to the trace sample. Aim. It is a further object of the present invention to provide an analysis system and method that do not depend on the amount of liquid to be measured. Further, the present invention provides a bioanalysis system in which a minute enzyme electrode (microelectrode) can be arranged on a minute surface, so that the sample amount can be extremely small (for example, a minimum amount can be measured within 1 μl). The purpose is to: Another object of the present invention is to provide a bioanalytical system capable of real-time measurement for directly detecting an electrode active substance produced by an enzyme molecule inside a microelectrode. Further, since the bioanalytical system of the present invention can apply various voltammetric detection methods, the measurement can be carried out irrespective of the sample being stationary. Since the bioanalytical method of the present invention is an electrode active substance detection method, it can be applied to important enzymes such as oxidase and dehydrogenase. Furthermore, the bioanalysis system according to the present invention can be used as an electrode for enzyme immunoassay.

[Constitution of the Invention] [Means for Solving the Problems] The gist of the present invention is to impregnate a biofunctional substance into a fine metal particle layer deposited by electrochemical or electroless plating. In this way, the biofunctional substance is taken into the inside of the fine particle layer or adhered to the surface of the fine metal particle, and the biofunctional substance-immobilized microelectrode having the manufactured fine metal particle substance surface layer is used as a working electrode, and a pulse potential applying means is provided. An analysis system comprising a current measurement device for measuring a generated transient response current, and measuring a concentration of a target substance from measurement of the generated current. And
Further, it is preferable to use an electrode in which an insoluble polymer thin film is formed on the surface of the metal fine particles and the biofunctional substance is stabilized. Further, the present invention provides a method in which a biofunctional substance is impregnated into a metal fine particle layer deposited by electrochemical or electroless plating, thereby incorporating the biofunctional substance into the inside of the fine particle layer or adhering to the metal fine particle surface. At the very least, the fabricated biofunctional material-immobilized microelectrode having a surface layer of metal fine particle material was used as the working electrode, and when a constant potential was applied, the current value generated was measured to determine the concentration of the target substance. An analysis method is provided. Furthermore, a suitable analysis method can be provided by forming an insoluble polymer thin film on the surface of the metal fine particles and stabilizing the biofunctional substance. One of the processing methods to integrate this biofunctional material with the conductive material particles is to adsorb the biofunction material while electrochemically depositing the conductive material particles on the surface of the microconductive material. This is done in one step. The other is to immerse electrochemically deposited conductive metal fine particles in a solution of the biofunctional substance, to take the biofunctional substance into the microparticles, or to attach them to the surface. This is accomplished by a two-step process in which the biofunctional substances are directly crosslinked or coated by forming a polymer thin film on the surface of the fine particles to thereby prevent the elution of the biofunctional substances. The bioelectrode produced by integrating the biofunctional substance and the fine particle electrode can be formed in a very fine size and has the same function as a large bioelectrode. It is a microelectrode having a fine particle conductive material surface layer and immobilized biofunctional material. This bioelectrode is referred to as a microelectrode in the present invention. That is, the present invention measures the current value generated when a constant potential is applied to the microelectrode immobilized by using such a biofunctional substance-immobilized microelectrode, and determines the concentration of the trace target substance from the value. Can be determined.

The present inventors have previously succeeded in producing a small and efficient enzyme electrode (microelectrode) by utilizing a platinum blackening technique and an electrochemical adsorption technique of an enzyme, and have applied for a patent. No. 222256 of 1988 and 223556 of 1988
issue). This microelectrode has high-speed response and high sensitivity.

The present inventors have further studied and found a bioanalytical system and an analytical method capable of analyzing a trace substance in an extremely small amount of a sample by using a microelectrode having such excellent characteristics. That is, using this microelectrode as a working electrode,
A biosensing system that can measure extremely quickly by a constant potential application method has been obtained.

The microelectrode having a biological function used in the present invention is an electrode having a structure in which a conductive material fine particle layer impregnated with a biological function material such as an enzyme is provided on the surface of a fine electrode made of platinum or the like. For this electrode, as in the present invention, platinum black has not been used as a carrier of a biofunctional substance, and it has not been conventionally used (a platinum plate is made porous by corrosion and an enzyme or the like is connected with a cross-linking agent. However, there has not been a method of electrochemically controlling the size of platinum black fine particles to cover and immobilize a biofunctional substance.

That is. In this microelectrode fabrication, the method for directly immobilizing a biofunctional substance is not the carrier binding method which had to use a chemical reagent (crosslinking agent) in the past, but the biofunctional substance can be directly immobilized without chemical treatment. Things.

One manufacturing method of the microelectrode used in the present invention is, for example, electrodeposition of a biofunctional substance directly on a conductive substance such as a cross section of a platinum wire, and at the same time, electrolytic reduction (electrodeposition) of a metal salt to form a conductive material. It is intended to precipitate fine particles (e.g., metal) together with biological functional substances. For example, it can be manufactured by incorporating a biofunctional substance into pores in the fine particles while forming platinum fine particles on the fine platinum electrode. The size of the pores and the amount of the immobilized biofunctional substance can be controlled by adjusting the current density, electrodeposition time, and the like. The function of the immobilized biofunctional substance electrode thus obtained can be maintained for a long time.

Another method of fabricating a microelectrode is to fabricate a microelectrode by a two-step process. For example, a conductive fine particle layer such as platinum black is formed on a thin platinum wire (generally a conductive material). It is to manufacture and immobilize the biofunctional substance in it. Specifically, the electrode on which the conductive substance fine particle layer is formed is immersed in an aqueous solution of a biological functional substance, or
By applying it, it is impregnated. Then, in order to prevent slight elution of biofunctional substances and to provide antithrombotic properties, albumin is impregnated with high-molecular substances such as heparin and cross-linked. It is insolubilized by an agent to form an insoluble polymer thin film. The microelectrode fabricated in this way entraps and immobilizes the enzyme.
It can be considered that the inclusive method is applied to the metal electrode system. However, the formed polymer film can be formed into a thin film with a thickness of several hundred mm or less. It does not become a factor inhibiting the expression of the function.

Therefore, the size of the microelectrode manufactured as described above depends on the diameter of the platinum wire, and if a fine platinum wire is used, an extremely small bioelectrode that cannot be considered conventionally can be obtained. It is easy to produce bioelectrodes of micro order size. In this case, it is convenient for medical applications, for example, when implanted in the body, and very small electrodes make very small analysis of the sample chamber. Equipment can be manufactured, analysis of a small amount of sample becomes possible, rapid measurement,
A highly responsive analyzer can be obtained. Further, when an electrode having a relatively large size is manufactured, an analyzer capable of obtaining a large detection output can be manufactured.

In the fabrication of the microelectrode described above, the first conductive material and the fine-particle (ie, porous) conductive material to be deposited thereon, that is, the fine-particle metal, need not be the same material. A structure in which platinum black is deposited thereon may be used. In addition, instead of platinum, a conductive material such as gold or rhodium can be used as the porous conductive material, and a fine particle layer of the conductive material can be formed on the surface of the conductive material. The present invention can be suitably used as long as there is no obstacle.

Furthermore, since the platinum electrode of the microelectrode can be formed into various shapes such as a disk shape, a spherical shape, and a tube shape, it can be formed into a shape according to measurement and other requirements as a biosensor. It can also be used for applications.

Further, the microelectrodes utilized according to the present invention may be proteins,
It is also possible to apply a high molecular substance such as a polysaccharide and form a thin film cross-linked with a cross-linking agent to impart biocompatibility, maintain performance, and minimize elution of a biofunctional substance.

Here, the "biofunctional substance" is represented by enzymes and antibodies, and includes various catalysts, microbial cells, proliferating microorganisms, organelles, antigens, antibodies, haptens, and the like.

In addition, examples of the polymer substance that can be additionally used to coat the bioelectrode used in the present invention include proteins such as albumin; ion exchange resins; and polysaccharides such as heparin. As the cross-linking agent, there is a cross-linking agent suitable for the polymer substance to be used. For example, for albumin, glutaraldehyde, carbodiimide, maleimide cross-linking agent and the like are used.

In the above, the deposition method of the porous (fine particle) conductive material in the microelectrode was explained by electrolytic deposition.
Obviously, it can be performed by an electroless method.

The analysis method according to the present invention has a fast response, high sensitivity, and
A small biosensing system has been put into practical use. It is a very important technology for clinical chemistry analysis, which requires multi-item measurement such as microscopic and multifunctional biosensors, and the development of a portable health monitoring system. Let's be.

FIG. 1 shows a microenzyme (glucose oxidase) device 1 in which a microelectrode having a diameter of, for example, about 1 μm to 500 μm is defined as a working electrode 1, and a counter electrode 2 of a platinum wire and a silver electrode.
A micro device 6 having a configuration using silver chloride for the reference electrode 3 is shown. The above three electrodes, microelectrode 1, counter electrode 2 and reference electrode 3
Is fixed with resin 4 in the hole of Teflon resin 5 and fixed. Since such a micro device 6 has a structure in which only three thin metal wires are enclosed and fixed, a device having a very small size can be obtained.

For this microelectrode, for example,
It can be measured even with μl of sample. That is, a substance in the trace sample can be detected by a method of applying a pulse potential after dropping the trace sample and detecting a current value generated at this time.

Therefore, in the present invention, real-time measurement can be performed in order to directly detect a biologically functional substance, for example, an active substance in an electrode produced by an enzyme molecule inside the microelectrode.

Further, since various voltammetric detection methods can be applied to the electrode having the above structure, the sample can be measured irrespective of the stationary state.

In addition, since it is a method for detecting an active substance in an electrode, it is a bioanalysis system that can be applied to important biological functional substances such as oxidase and dehydrogenase.

The present invention will be described in detail with reference to examples below, but the present invention is not limited thereto.

[Example 1] Manufacturing method Various fine platinum wires having a diameter of 1 µm to 500 µm, 200 µm
After encapsulating a platinum wire having a diameter of m for the counter electrode and a silver wire having a diameter of about 500 μm to 1 mm with the resin 4, the surface of the fine platinum wire was polished with alumina powder. The enzyme was immobilized on the polished platinum surface by the following two methods.

(A) In a 1 ml solution (pH 3.5) containing chloroplatinic acid (33 mg), lead acetate (0.6 mg) and glucose oxidase (10 mg) at a constant potential (-0.2 V) or a constant current (-5 μA). The mixture was electrolyzed for a minute to precipitate glucose oxidase-impregnated platinum black.

(B) immersed in a 3% chloroplatinic acid solution containing 300 ppm of lead acetate, and electro-deposited platinum black at a current value of −50 μA for 10 minutes;
Platinum black having a thickness of about several μm was obtained. Next, after the obtained platinum black deposition electrode was dried at 25 ° C. for 60 seconds, it was kept at −0.3 V for 30 minutes in a 0.5 M aqueous sulfuric acid solution to generate hydrogen from the platinum black deposition electrode. Next, after air-drying at 20 ° C. for 60 seconds, 5 ml of glucose oxidase-containing phosphate buffer (pH 6.8) was added to 1 ml.
Immerse for 20 minutes and air dry again.

Next, in the microdevice 6 having the microelectrode obtained as described above, the silver wire was used as a silver / silver chloride reference electrode, and the microdevice 6 composed of the three electrodes thus prepared was replaced with 0.1 M The mixture was stirred and washed for 24 hours in a phosphate buffer to obtain a three-electrode microdevice used in the present invention.

[Example 2] Measurement of glucose concentration A glucose sample of 20 µl was added to this three-electrode micro device.
After dropping, a potential of 0.6 V was applied. The relationship between the peak current value generated at this time and the glucose concentration was examined. Next, the relationship between the peak current value and the solution volume was examined by changing the volume of the sample containing 10 mM glucose. That is. Various concentrations of glucose samples were dropped, and a potential of 0.6 V was applied. As a result, a response as shown in FIG. 2 was observed.

That is, when a potential of 0.6 V was applied, a current as shown in the right diagram of FIG. 2 was generated. When glucose is not contained, a current as shown in the left diagram of FIG. 2 is generated. When a phosphate buffer solution (50 mM, pH 7.0, 50 mM NaCl) containing 10 mM glucose is used as a standard sample, the current shown in FIG. The response current as shown in the right figure occurred.

That is, as is apparent from FIG. 2, a response current is generated immediately with the application of the potential, and rapidly attenuates. This indicates that the hydrogen peroxide generated by the glucose oxidase inside the electrode is immediately oxidized with the application of the potential. It was found that the peak current became constant from several times when the potential application was repeated.

On the other hand, the same behavior was observed with the phosphate buffer, but the peak current value was small. Therefore, the peak current value for each glucose concentration was measured, and the difference from the peak current value (ie, blank value) obtained with the phosphate buffer containing no glucose was determined to examine the relationship with the glucose concentration. The result is shown in FIG. That is, it was found that when the glucose concentration was changed in the range of 1 to 100 mM, a measurement curve as shown in FIG. 3 was obtained between the glucose concentration and the peak current value.

Further, it has been shown that the enzyme analysis system according to the present invention can measure the concentration of a small amount of a stationary sample in real time (a specific substance can be measured from a small amount of a sample in the order of milliseconds).

[Example 3] Liquid volume dependency test After taking 2,5,10,15,20 µl of a 10 mM glucose standard sample and dropping it into a microbiosensor system, the potential was measured.
The generated current when a constant potential of 0.6 V was applied was measured.
As a result, the one shown in FIG. 4 was obtained. That is. It was found that the obtained peak current did not depend on the liquid volume of the sample.

The above results show that in the analysis system and method according to the present invention, the measurement can be performed by detecting only a part of the measurement sample in the measurement, and the information of the whole sample in a stationary state can be extracted.

As described above, in the analysis system and method according to the present invention, it is not necessary to stir the sample, and therefore, it is possible to use it as an in-vivo bisensing system or a portable biosensor.

It goes without saying that in the bioanalytical system according to the present invention, measurement can be performed quickly and with high sensitivity in a convection vessel or in a batch system, with a high-speed response as in the stationary system.

[Effects of the Invention] The analysis system and method of the present invention have the following remarkable technical effects.

First, it is possible to provide a high-performance biosensing which has a very quick response, high sensitivity, and can measure and detect even a stationary sample.

Secondly, since a minute enzyme electrode (ie, microelectrode), counter electrode and reference electrode can be arranged on a minute surface, the amount of sample can be extremely small, and therefore, even a very small amount of sample can be quickly and responsively. Analytical systems and methods that can be measured are provided.

Third, it is possible to provide a system capable of real-time measurement for directly detecting an electrode active substance produced by an internal enzyme molecule.

Fourth, since various voltammetric detection methods can be applied, an analysis system capable of detecting a sample with high sensitivity even when the sample is in a stationary state can be provided.

Fifth, since it is an electrode active substance detection method, it can be applied to important enzymes such as oxidase and dehydrogenase, and biological functional substances.

Sixth, it is possible to provide a bioanalysis system that can be used as an in-vivo bisensing system or a portable biosensor.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram illustrating the structure and manufacturing method of a three-electrode microdevice used in the analysis system according to the present invention. FIG. 2 is a graph showing a current generated when a constant potential is applied according to the present invention. FIG. 3 is a graph showing the relationship between the glucose concentration and the generated current value by the micro analysis system of the present invention. FIG. 4 is a graph showing the relationship between the capacity of a glucose sample and the peak current value by the analysis system according to the present invention. [Explanation of Signs of Main Parts] 1 working electrode (micro electrode) 2 counter electrode 3 reference electrode 4 resin 5 Teflon (registered trademark) 6 micro device

Claims (4)

(57) [Claims] 1. A biofunctional substance is impregnated into a metal fine particle layer deposited electrochemically or by electroless plating so that the biofunctional substance is taken into the fine particle layer or adhered to the surface of the metal fine particles. And a pulse potential applying means, and a current measuring device for measuring a generated transient response current. An analytical system characterized by measuring the concentration of a substance. 2. The analysis system according to claim 1, further comprising forming an insoluble polymer thin film on the surface of the metal fine particles to stabilize the biofunctional substance. 3. A biofunctional substance is impregnated in a metal fine particle layer deposited electrochemically or by electroless plating, so that the biofunctional substance is taken into the fine particle layer or adhered to the surface of the metal fine particles. The microelectrode with a biofunctional substance immobilized on it has a surface layer made of metal microparticles, which is used as the working electrode.When a constant potential is applied, the current value generated is measured to determine the concentration of the target substance. Analysis method. 4. The analysis method according to claim 3, further comprising forming an insoluble polymer thin film on the surface of the metal fine particles to stabilize the biofunctional substance.