JP3393361B2 - Biosensor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention provides:
Shows an electrochemical response corresponding to the concentration of a specific enzyme, such as glutamic acid-oxaloacetyl transferase (GOT), and a specific substrate, such as cholesterol ester, contained in a liquid, particularly body fluid such as blood or urine, or a measurement sample. TECHNICAL FIELD The present invention relates to a biosensor and a biosensor that measures the amount of a specific enzyme and a specific protein in a measurement sample.

[0002]

2. Description of the Related Art A biosensor in which a biologically functional substance consisting of an organism such as an enzyme, an antibody or a microorganism is immobilized on the surface of a platinum or carbon electrode is used to rapidly and continuously measure the amount and concentration of various chemical substances and biological substances. We already know what we can do.

This biosensor is utilized in various fields by taking advantage of its features, but it is particularly actively used in the field of clinical examination. The measurement of chemical substances and the amount of enzymes in such a biosensor will be described in detail below.

When an electrode active substance is produced by using a single enzyme among chemical substances, a biosensor generally has a structure in which an immobilized enzyme membrane is attached to the surface of an electrode such as flat plate platinum. is doing. As a method for producing the same, a method of attaching a separately prepared immobilized enzyme membrane to the surface of an electrode such as platinum is often used. For example, in a glucose sensor, glucose oxidase is immobilized on an enzyme-immobilized membrane, and this enzyme specifically acts on glucose in body fluids such as blood and urine, resulting in glucose + O ₂ + H ₂ O --- → Causes the reaction of gluconic acid + H ₂ O ₂ (1). The amount and concentration of glucose in the body fluid are measured by measuring the amount of hydrogen peroxide generated by this reaction by an electrochemical method.

By the way, in the conventional enzyme sensor, the above-mentioned electrode detection method is effective in producing an electrode active material.
The electrode method cannot be adopted when the product cannot be detected by the electrode. Therefore, it has been attempted to adopt a so-called complex enzyme system, which is a method of further converting an intermediate product using another enzyme to obtain an electrode active material as a product. For example, there is a method of immobilizing a plurality of enzymes in a film shape and attaching this to an electrode.

[0006]

In view of the biosensors of the prior art as described above, the problem to be solved by the invention is to immobilize a plurality and a large amount of enzymes on a metal electrode having extremely high porosity mainly by covalent bonding, At least one of the enzymes at that time recognizes and decomposes a component in the body fluid, and at the same time, the immobilized enzyme converts the product into an electrode active material for detection.

[0007] Furthermore, by covalently binding an antibody that recognizes a specific enzyme to a metal electrode rich in porosity, and indirectly immobilizing the enzyme in the body fluid to the porous electrode, the product of this enzyme is activated. Even if it is not a substance, it is intended to be detected by being converted into an electrode active material by the enzyme immobilized at the same time.

A biosensor capable of measuring metabolic components and protein components in body fluids can be provided by such a method. Here, when the protein component has no catalytic activity, the specific protein can be measured by the competitive binding method of a certain amount of the enzyme-labeled protein and the specific protein in the sample.

[0009]

It has been found that the above problems can be solved by arranging a plurality of enzymes or enzymes and antibodies in a local space.

That is, in the first approach, the present invention comprises an electrode material such as gold black having a porous surface, a group of enzymes such as transferase and synthase, and an enzyme that produces an electrode active substance such as oxidoreductase. Become. Further, when the amount of transaminase, etc. in the body fluid is to be measured, an antibody comprising an antibody against the enzyme and an enzyme that oxidizes the product of the enzyme and a porous metal electrode on which the enzyme is immobilized are used.

In the present invention, the porous electrode is a metal compound such as chloroauric acid which is reductively (electrochemically applied with a negative potential) deposited on the surface of the electrode, and has a surface area with respect to that of a smooth electrode. It means that the number has increased from several hundred times to several thousand times.

In the present invention, a complex enzyme system is constructed. Here, the complex enzyme system comprises a direct complex enzyme system and an indirect complex enzyme system. The direct complex enzyme system refers to a case where an electrode active substance is produced from an electrode-inactive substrate A (measurement target) by two types of enzymes when immersed in a measurement sample such as body fluid.

Further, in the indirect complex enzyme system, by binding and immobilizing a specific enzyme in a measurement sample with an antibody,
For the first time a complex enzyme system is formed. That is, a complex enzyme system is formed, the amount of enzyme in the measurement sample is measured as described below, and the amount of enzyme is measured in the substrate A solution. Here, the measurement target is enzyme A. Immobilized specific antibody + enzyme A ---
→ Immunochemically immobilized enzyme A (3)

In the present invention, the enzyme (or antibody)
Immobilization means gold on a black surface. This is to introduce a functional group by the mercaptide binding method and to bind an enzyme or an antibody to this functional group, and to bind a large amount of the enzyme or antibody via a Schiff bond using glutaraldehyde as a cross-linking agent. Furthermore, the platinum black electrode and the carbon electrode are to directly immobilize proteins.

For example, in the case of measuring the amount of cholesterol ester as an example of measuring a metabolite in a body fluid, the above reaction is as follows. Since the reaction of (6) also occurs at the same time, the total cholesterol can be measured by measuring the produced hydrogen peroxide.

Although it is not a body fluid, it is possible to measure starch in agricultural products. In this case, the starch can be directly measured by using the following complex enzyme system and detecting the produced hydrogen peroxide at the electrode.

In the present invention, the following antibody-enzyme system is employed as an example to measure the amount of enzyme in a measurement sample. The antibody may be either a monoclonal antibody or a polyclonal antibody. For example, GOT
When the direct measurement of is carried out, the anti-GOT antibody is immobilized.
Therefore, by immersing the electrode in which the GOT of the measurement sample is indirectly immobilized by the antigen-antibody reaction in the substrate solution, G
The amount of OT will be measured. Anti-GOT antibody + GOT (in sample) ---> anti-GOT antibody-GOT complex (8)

In the present invention, the electrochemical measuring method is a method of measuring the amount of electric current (or the amount of electric charge) generated in the above-mentioned reaction and determining the amount of the substrate or the amount of the enzyme, that is, the concentration based on it. means.

The electrode of the present invention can be used as a working electrode (measuring electrode) in an electrochemical measuring method,
It is widely used in an electrochemical measurement method using the electrode (biosensor) of the present invention as a working electrode. A two-electrode system including a counter electrode that also functions as a reference electrode and a three-electrode system in which the reference electrode and the counter electrode are separated from each other are formed, and the amount of current generated by the reaction is measured. A predetermined potential is applied to the working electrode, but elements other than the biosensor of the present invention are well known and need not be described further.

In the present invention, the electrode active substance is a substance which can be easily oxidized or reduced electrochemically,
At this time, those which can be more easily oxidized and reduced by using an electron mediator are included.

[0002] In the present invention, the complex enzyme system employed is such that at least one enzyme converts a biological substance that is not an electrode active substance into another type of electrode inactive substance, and at least one enzyme finally converts into an electrode active substance. It consists of what you do. A gold black electrode composed of such a complex enzyme system is schematically shown in FIG. FIG. 1A shows a state in which two enzymes are immobilized inside a gold-black porous hole, and FIG. 1B schematically shows a state in which two kinds of enzymes are covalently bound by a gold mercaptide bond. Shown in.

In the present invention, among the antibody-enzyme systems employed, an antibody means one that binds to a specific enzyme to be measured in a measurement sample, and an enzyme converts a product of the specific enzyme into an electrode active substance. What you do. A schematic diagram of this antibody-enzyme system is shown in FIG. Both the antibody and the enzyme are immobilized here by covalent bonding. The antibody-enzyme system becomes a complex enzyme system only by binding to the target enzyme as shown in FIG.

[00023]

Example 1 (Starch sensor in which a complex enzyme system consisting of glucoamylase and glucose oxidase was immobilized on a gold black electrode by a covalent bond method) A smooth gold electrode (diameter 1.6 mm) was mixed with sulfuric acid (30%) monohydrogen peroxide. (70%) wash in a mixture,
The electrode surface was polished with alumina. When a negative potential was applied to this electrode in a chloroauric acid solution containing lead acetate, gold was deposited and a gold black electrode could be prepared. The amount of gold black deposited was controlled by the amount of electricity supplied. That is, the porosity of gold black can be controlled by controlling the amount of electricity. The prepared gold black electrode was immersed in a 10 mM aqueous solution of aminoethanethiol for 2 hours, and an amino group was introduced into the gold black surface via a gold-mercaptide bond. After washing with ethanol, add 30% to 1% glutaraldehyde aqueous solution.
Immersion was carried out for a minute to introduce an aldehyde group on the gold black surface. After washing with phosphate buffer, glucose oxidase (30 mg
/ Ml) solution for 30 minutes, washed and then immersed in a glucoamylase (30 mg / ml) solution to prepare a bienzyme sensor on the gold black surface. When it was necessary to make the immobilization of the two enzymes inside the pores firm, further immersion in an albumin solution (1%) was followed by a crosslinking treatment with glutaraldehyde.

The produced bienzyme sensor was used as a working electrode, a platinum plate electrode was used as a counter electrode, and a silver / silver chloride electrode was used as a reference electrode to clarify the sensor characteristics. A starch solution was used as a substrate. The potential applied to the sensor is +90
0 mV and a starch concentration of 0.01 to 2.5 (w /
w)%.

FIG. 4 shows the change with time when starch is added. It can be seen that the sensor output increases as the starch concentration increases. This is shown in FIG. 5 as a calibration curve. From this figure, it was found that a linear relationship was observed in the range of the starch concentration of 0.01 to 0.5 (w / w)%.

[00026]

Example 2 (Bienzyme Sensor for Measuring Total Cholesterol) In the same manner, a composite enzyme sensor composed of cholesterol esterase and cholesterol oxidase was prepared and evaluated as a total cholesterol sensor. Here, in order to solubilize cholesterol and cholesterol ester, T
Phosphoric acid (0.1%) containing rtonX-100 (10%)
M) buffer was used. The applied potential was 500 mV. FIG. 6 shows the change with time when cholesterol palmitic acid was added as a cholesterol ester. Further, FIG. 7 shows the relationship between the substrate concentration and the sensor output when cholesterol, cholesterol linoleic acid, and cholesterol palmitic acid were added to the same sensor. From this figure, it is understood that total cholesterol can be estimated by multiplying cholesterol and its derivative by a correction coefficient.

[00027]

Example 3 (Measurement Sensor for GOT Content in Measurement Sample) A gold black electrode having an aldehyde group introduced on the surface by the above-mentioned treatment was immersed in a glutamate oxidase solution (10 mg / mL) for 30 minutes, and then the anti-bovine GOT was prepared. 30 in antibody solution (10 mg / mL)
An antibody-enzyme system was constructed by immersion for a minute.

A gold black electrode consisting of the above-described antibody-enzyme system was immersed in GOT bovine solution (10 mg / mL), and an antigen-antibody reaction was carried out for 1 hour under stirring. At this time, add 1 mL, 2 mL, 4 mL, 6 mL, 8 mL and 10 mL of bovine GOT solution.
It was set to mL. After the reaction, the complex enzyme system immunochemically prepared in aspartic acid and 2-oxoglutamic acid (10 mM each) was evaluated. As a result, linearity was obtained between the volume of the bovine GOT solution and the sensor output and the output in the range of 6 mL or less.

The above shows that the antibody binds bovine GOT, and this GOT transfers the amino group between aspartic acid and 2-oxoglutamic acid to produce glutamic acid and oxaloacetic acid. It is shown that glutamate oxidase converts this glutamic acid into α-ketoglutamic acid, ammonia, and hydrogen peroxide, and this hydrogen peroxide is detected by the electrode.

Amount of solution containing bovine GOT (bovine GOT
Since there is a linearity between the amount) and the sensor output, it can be seen that the bovine GOT bound to the antibody depends on the amount of the measurement sample. That is, it shows that it depends on the bovine GOT concentration under a constant solution amount.

[00031]

EFFECTS OF THE INVENTION By using the biosensor of the present invention and the method for producing the same, it becomes possible to measure a metabolite and a marker enzyme or marker protein peculiar to a disease which could not be detected by an electrochemical device in the past. As a result, measuring devices for a wide variety of substances in the measurement sample can be realized.

[Brief description of drawings]

FIG. 1 shows a state in which a complex enzyme system (enzyme A and enzyme B) is immobilized on a porous surface (A), and a schematic diagram (B) in which each enzyme is immobilized by a gold mercaptide bond. is there.

FIG. 2 is a schematic diagram in which an antibody (Ab) and an enzyme (E) are immobilized on a porous gold surface by a gold mercaptide bond.

FIG. 3 is a diagram in which an antibody immobilized by a gold mercaptide bond recognizes and binds to the corresponding enzyme GOT.

FIG. 4 is a time-dependent change when a potential of 900 mV is applied to a gold black electrode in which a complex enzyme system is formed from glucoamylase and glucose oxidase and starch is added.

FIG. 5 is a diagram showing a relationship between a starch concentration and a sensor output obtained by the above method.

FIG. 6 is a diagram showing changes over time in cholesterol ester concentration and sensor response when a complex enzyme system consisting of cholesterol esterase and cholesterol oxidase was formed on a gold black electrode and cholesterol palmitic acid was added.

FIG. 7 is a diagram showing a relationship between a concentration (mM) and a sensor output (nA) when cholesterol, cholesterol linoleic acid, and cholesterol palmitic acid are applied to a cholesterol esterase-cholesterol oxidase system.

[Explanation of symbols]

1. Cholesterol measurement curve 2. Cholesterol linoleic acid measurement curve 3. Measurement curve of cholesterol palmitic acid

─────────────────────────────────────────────────── --- Continuation of the front page (56) References JP-A-4-69564 (JP, A) JP-A-3-293556 (JP, A) JP-A-1-147357 (JP, A) JP-A-63- 223556 (JP, A) JP-A 2-110363 (JP, A) JP-A 4-125461 (JP, A) JP-A-55-78242 (JP, A) Technical Digest of The 8th Sensor Symposium, Japan, 1989 May 08, 1999, 99-102 (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/327 JISST file (JOIS)

Claims (17)

(57) [Claims] 1. A complex enzyme sensor characterized in that a plurality of kinds of enzymes are immobilized on the surface of a noble metal electrode which is a porous surface obtained by electrolytically reducing a noble metal compound. 2. The complex enzyme sensor according to claim 1, wherein at least one of the plurality of types of enzymes is an enzyme that converts a product produced by another enzyme into an electrode active substance. 3. The composite enzyme sensor according to claim 1, wherein the noble metal electrode is a gold electrode, and the enzyme is chemically bonded mainly to the porous surface by a gold mercaptide bond. 4. The complex enzyme sensor according to claim 1, wherein the noble metal electrode is a platinum electrode, and the enzyme is chemically bound by surface treatment of the electrode. 5. The complex according to any one of claims 1 to 4, further comprising a structure in which an enzyme is cross-linked on the enzyme covalently immobilized on the surface rich in porosity. Enzyme sensor. 6. A method for measuring a component in a body fluid, which comprises electrochemically measuring the complex enzyme sensor according to any one of claims 1 to 5 by a pulse potential detection method. 7. An antibody for a target enzyme marker and an enzyme for metabolizing a substance produced by the marker enzyme bound to the antibody are simultaneously formed on a noble metal electrode having a porous surface by electrolytically reducing a noble metal compound. Immobilized enzyme immunoelectrode. 8. A noble metal electrode, which has a porous surface obtained by electrolytically reducing a noble metal compound, is bound to an antibody of a target marker protein and a second antibody that binds to the marker protein bound to the antibody. An enzyme immunoelectrode on which an enzyme that metabolizes a substance produced by the immobilized enzyme is simultaneously immobilized. 9. A product produced by a marker enzyme immobilized with an antibody is made into an electrode active material by allowing the enzyme-immunoelectrode according to claim 7 to coexist with a marker enzyme in serum or blood for a certain period of time. A method for measuring the amount of a marker enzyme, which comprises converting to a marker enzyme amount. 10. The enzyme immunoelectrode according to claim 8,
After immobilizing the marker enzyme on the enzyme immunoelectrode via the antibody by allowing it to coexist with the marker enzyme in serum or blood for a certain period of time, the enzyme coexists in a solution containing a complex of a second antibody against the marker enzyme and the enzyme. A method for measuring a marker enzyme amount, which comprises converting the product produced by the method into an electrode active material and measuring the marker enzyme amount. 11. A method for measuring a body fluid component, which comprises electrochemically measuring by a pulse potential detection method using the enzyme immunoelectrode according to claim 7 or 8. 12. A method for producing a composite enzyme sensor, which comprises electrolytically reducing a noble metal compound to form a porous surface on the surface of a noble metal electrode, and immobilizing a plurality of kinds of enzymes on the surface. 13. The method for producing a composite enzyme sensor according to claim 12, wherein the noble metal electrode is a gold electrode, and the enzyme is immobilized by a gold mercaptide bond. 14. The method for producing a composite enzyme sensor according to claim 12, wherein the noble metal electrode is a platinum electrode, and the enzyme is chemically bonded and immobilized by surface treatment of the electrode. 15. The composite enzyme sensor according to claim 12, further comprising cross-linking the enzyme on the enzyme covalently immobilized on the surface rich in porosity. Method. 16. An enzyme that metabolizes an antibody of a target enzyme marker and a substance produced by a marker enzyme bound to the antibody on the surface of a precious metal electrode which is a surface rich in porosity by electrolytically reducing a precious metal compound. A method for producing an enzyme immunoelectrode, which comprises simultaneously immobilizing 17. A surface of a precious metal electrode, which is a porous surface obtained by electrolytically reducing a precious metal compound, binds to an antibody of a target marker protein and a second antibody that binds to the marker protein bound to the antibody. A method for producing an enzyme immunoelectrode, comprising simultaneously immobilizing an enzyme that metabolizes a substance produced by the generated enzyme.