JP2001050926A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure enzyme activity with improved response property by composing a biosensor of an oxidation enzyme which is acted by a constituent that is generated by the activity of the enzyme to be measured and a substrate for generating the substrate of the oxidation enzyme by the enzyme to be measured. SOLUTION: For example, the biosensor for measuring alanineaminotransferase (ALT) is provided with an operation electrode 1, a counter electrode 2, and a reference electrode 3 formed on a glass epoxy insulation substrate or the like, and an insulation film is provided at a part other than the active electrode surface at the tip of each electrode. On the active electrode surface, specific enzyme layer liquid containing pyruvic acid oxidase (POP) and an electron acceptor is applied to the active electrode surface for drying and forming an enzyme layer, and a substrate layer is formed by substrate layer liquid containing L-alanine and α-ketoglutaric acid. Then, pyruvic acid, generated from the L-alanine and α-ketoglutaric acid by ALT in a sample, reacts with POP and the electron receptor and is detected as a current value or the like, thus obtaining ALT enzyme activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a biosensor for measuring the activity of an enzyme present in a sample.

[0002]

2. Description of the Related Art A biosensor has a mechanism for converting a reaction based on a biochemical principle typified by an enzymatic reaction or an immunological reaction into an electric signal based on some signal accompanying the reaction. It is a sensor. Among biosensors, those utilizing an enzyme reaction, which was relatively quickly put into practical use, have been applied to the measurement of various enzyme substrates. For example, a blood glucose measurement biosensor in which glucose oxidase (hereinafter abbreviated as GOD) is immobilized on the electrode surface can be measured with simple operation and with sufficient accuracy even when the measurement is performed by the patient himself. .

[0003] In the case of measuring an enzyme substrate such as glucose, a measurement system can be constructed by immobilizing an enzyme that acts on the substrate to generate a signal substance that can be detected by an electrode. For example, in a blood glucose measurement sensor using GOD that has already been put to practical use, GOD immobilized on an electrode directly oxidizes glucose in the presence of an electron acceptor. Next, the electron acceptor (reduced type) is oxidized on the electrode, thereby generating an oxidation current. Since the intensity of the oxidation current reflects the substrate concentration, the measurement of the oxidation current enables the measurement of blood glucose. In order to realize a biosensor that does not require specialized techniques for operation based on such a measurement principle with an inexpensive device configuration, it was necessary to solve many problems. For example, GOD must be stably immobilized on the electrode surface, and a quick response can be expected during measurement. Although biosensors are advantageous in terms of safety, reproducibility, or stability when they are disposable, it is necessary to supply the electrodes themselves as inexpensively as possible. In addition, a structure that is less likely to be interfered by a component that may coexist in blood and interferes with electrical measurement is required. The blood used as a sample for blood glucose measurement is made of GO, such as ascorbic acid, uric acid, or bilirubin.
Components that interfere with the detection of enzymatic reactions by D may always be present. The biosensor for measuring blood glucose that has been put to practical use has condensed large and small devices for achieving these many problems. Nevertheless, if a signal substance detectable by the electrode can be generated by such a basic combination between the substrate and the enzyme, the enzymatic reaction on the electrode is completed in one step. Therefore, it can be said that setting conditions for providing a quick and stable response is relatively easy. Further, the components constituting the reaction are simple. That is, taking a blood glucose measurement sensor as an example, an enzyme (GOD only), an electron acceptor (mediator) such as potassium ferricyanide, and a buffer component are only required as basic components. In addition, if an auxiliary component such as an enzyme stabilizer is added, the basic configuration of the biosensor is completed. This is in contrast to the case where enzyme activity is to be measured.

[0004] Measurement of enzyme activity often requires at least two steps of enzymatic reaction as shown in the following reaction formula. That is, the enzyme to be measured acts on the substrate prepared on the biosensor side to give a predetermined product, and the second enzyme acts on the product supplied in the first reaction to detect by the electrode. Two stages of a second reaction giving a possible signal substance.

[0005]

Embedded image Here, the second enzyme acts specifically on a given product. Therefore, no signal substance is produced unless a product is provided by the enzyme to be measured. As the number of reaction steps increases from one to two, the components required for the reaction become more complicated. That is, as a reaction component, an enzyme that catalyzes a reaction (second reaction) for deriving a signal substance from an enzyme substrate, a product generated by the action of an enzyme to be measured from the enzyme substrate, and more often a second reaction Electron acceptor (mediator) is required. In order to realize such a condition on which a complex reaction proceeds smoothly on a biosensor, it is necessary to solve more problems than a biosensor using a substrate as a measurement target. For example,
Excellent response characteristics must be achieved while performing an enzymatic reaction over two stages. It is not easy to ensure that the components involved in the two-step enzymatic reaction are dissolved by the sample on the electrode and that the enzymatic reaction occurs quickly.

In the blood, ALT (L-Alanine 2-Oxoglutarat)
e Aminotransferase; EC.2.6.1.2; GPT), AST (L-Aspartat
e 2-Oxoglutarate Aminotransferase; EC.2.6.1.1; GO
T), AMY (α-Amylase), LDH (Lactate Dehydrogenase),
Alternatively, various enzymes having clinical significance are known, such as γGTP (γ-Glutamyl Transpeptidase). The activity of these enzymes is generally measured using liquid reagents or dry type analysis films.
A measurement method based on a liquid reagent is excellent in measurement sensitivity, accuracy, and the like, but does not enable simple measurement in a small facility or at home. Dry-type analytical films provide accurate measurement results with a small measuring device only by spotting of a sample, but are generally expensive and unsuitable for use in small-scale facilities and homes. If the enzyme activity can be measured by a biosensor like a blood glucose measurement sensor, inexpensive and excellent operability can be measured at the bedside or at home. In order to measure enzyme activity with a biosensor, it is necessary to construct a biosensor optimized for a much more complicated reaction system than blood glucose measurement, but there are still many issues to be solved. I have.

[0007] For example, the present inventor has found that ALT, which is often cited as a test item as an index of liver function, uses a substrate or secondary ALT.
It was confirmed that a uniform enzyme-reacted material layer could not be formed only by immobilizing a reaction component composed of an enzyme or the like for the reaction on the electrode, and as a result, responsiveness was reduced and accurate measurement could not be performed. As described above, a biosensor for measuring an enzyme activity that requires the use of various reaction components is required to have a structure different from that of a biosensor for measuring an enzyme substrate that has been put to practical use.

[0008] In a biosensor, an attempt to divide a reaction component on an electrode into two layers is known (Japanese Patent No. 2550463). However, the aim of this attempt was to reduce the effect of fluctuations in the hydrogen ion concentration occurring during the reaction on the measured values.
Also, it does not enable measurement of enzyme activity.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a biosensor for enzyme activity measurement which can realize a quick response and can be manufactured at low cost. It is an object of the present invention to realize a biosensor that is easy to mass-produce and has excellent reproducibility even in the measurement of enzyme activity, like the biosensor for enzyme substrates that has been put to practical use.

[0010]

Means for Solving the Problems The present inventor attempted to manufacture a biosensor by mixing the following components necessary for measuring the enzyme activity of ALT, and fixing the mixture on an electrode to form an enzyme reactant layer. . L-alanine (substrate) α-ketoglutarate (substrate) pyruvate oxidase (second reaction enzyme A, Pyruvat
e: oxygen oxidoreductase (phosphorylating); EC.1.
2.3.3, hereinafter abbreviated as POP) However, H ₂ O ₂ generated in the second reaction by the action of POP
It was suggested that redox might occur by redox reaction with α-ketoglutaric acid or pyruvic acid coexisting as a sample or a reaction component. We thought that interference of these components could be avoided by using an electron acceptor (mediator). However, it became clear that even if an electron acceptor was combined, another problem was involved in the measurement of ALT. That is, if all the reaction components obtained by adding potassium ferricyanide, which is an electron acceptor, to the above reaction components are mixed and fixed to the electrode surface, the output of the biosensor is not stable, and accurate measurement cannot be expected. The present inventor has found that the cause is that some components are precipitated as crystals as the reaction components are mixed and dried, and as a result, a uniform enzyme reaction product layer is not obtained.
In fact, in a poorly responsive biosensor, crystals were present on the electrode enough to be discerned by the naked eye. Therefore, if a plurality of reaction components required for measuring the enzyme activity are fixed on the electrode in two steps, a more uniform enzyme reactant layer can be formed stably, and a bio-response with good responsiveness can be obtained. I thought it could be a sensor. Then, the effects of various combinations of reaction components on the responsiveness of the biosensor were observed, and a combination of components necessary for forming a uniform enzyme reactant layer was found, thereby completing the present invention. The term “enzyme reactant layer” used herein means the entire layer containing necessary reaction components on the electrode. Therefore, for example, when the “enzyme layer” and “substrate layer” described later are formed in order,
The entire final layer containing the reaction components formed on the electrode surface corresponds to the enzyme reactant layer.

That is, the present invention relates to the following biosensor for measuring enzyme activity, a method for measuring enzyme activity using this biosensor, and a method for producing the same. In this specification, "B" is used for the enzyme whose activity is to be measured, and "A" is used for the enzyme constituting the second reaction required for the measurement, so that the functions of the enzymes can be clearly distinguished. In some cases, it is attached. [1] A biosensor for measuring enzyme activity, comprising the following elements: a) an oxidase A disposed on the electrode surface, the oxidase A acting on a component generated by the activity of the enzyme B to be measured; b) an enzyme substrate laminated on the oxidase A; The substrate forms the substrate of the oxidase A according to the activity of the enzyme B to be measured. [2] The enzyme B to be measured for activity is alanine aminotransferase, the oxidase A is pyruvate oxidase, and the substrate is L The biosensor according to [1], which is -alanine and α-ketoglutaric acid. [3] The biosensor according to [1], comprising the following elements, wherein the enzyme reaction for measuring the enzyme activity is such that the enzyme B to be measured acts on a substrate to generate a reaction product. 1
A biosensor comprising a reaction and a second reaction for generating a signal substance by another enzyme A acting on the reaction product. a) a first layer containing a reaction component necessary for the second reaction on the electrode surface; b) a second layer containing a component necessary for the first reaction laminated on the first layer; [4] composed of the following elements: A method for measuring enzyme activity, comprising using a biosensor for measuring enzyme activity. a) an oxidase A disposed on the electrode surface, the oxidase A acting on a component generated by the activity of the enzyme B to be measured; b) an enzyme substrate laminated on the oxidase A; The substrate produces the substrate of the oxidase according to the activity of the enzyme B to be measured. [5] A method for producing a biosensor for measuring enzyme activity, comprising the following steps. a) a step of arranging oxidase A on the electrode surface, wherein the oxidase A acts on a component generated by the activity of the enzyme B to be measured b) an enzyme substrate is arranged on the electrode surface after step a) A biosensor produced by the method according to [6] or [5], wherein the enzyme substrate produces a substrate for the oxidase A according to the activity of the enzyme B to be measured.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The biosensor for measuring enzyme activity according to the present invention is manufactured by fixing components necessary for an enzyme reaction on an electrode several times. The condition required at this time is to independently dissolve and fix at least the substrate on which the enzyme (B) to be measured acts. Other components can be mixed in the same solution. This does not mean that no components other than the substrate are added to the solution containing the substrate. For example, if components constituting the first reaction by the enzyme to be measured, including the substrate, are dissolved together and fixed to the electrode, the components necessary for the first reaction are contained in the same fixed layer. From that
This is a desirable combination in which a quick response can be expected. In this case, the components such as the second enzyme (A) necessary for the enzyme reaction for producing the signal substance constitute the other solution. The signal substance in the present invention means a substance capable of exchanging electrons with an electrode by applying a potential difference between the electrode and a solution in which the substance is dissolved. Specifically, a reduced mediator, H ₂ O ₂ or the like is a signal substance. For example, when ALT is to be measured, a biosensor according to the present invention can be manufactured by preparing a fixing solution based on the following combinations. The components constituting the solution and the range of the general addition amount in the fixing solution are shown below.

Examples of fixing solutions for the production of a biosensor for measuring ALT activity according to the present invention Enzyme layer buffer Buffer component Electron acceptor such as potassium ferricyanide 0.1 mM to 100 mM (preferably 1 mM to 10 mM) Pyruvate oxidase (POP) 100 ~ 1000 U / ml Substrate layer buffer buffer component L-alanine 25-1000 mM α-ketoglutaric acid 5-100 mM

In a biosensor for measuring ALT activity, a phosphate of 0.1 to 100 mM is used as a reaction substrate of POP in an enzyme layer solution, and a coenzyme required for expression of enzyme activity of POP is 0.005 to 100 mM. 5 mM thiamine pyrophosphate (TP
P), and 0.01 to 1 mM of magnesium ions. Further, the pH range in which POP acts stably is from 6 to 8. However, the phosphate may be used as a buffer component to give a buffer to the solution, or the buffer may be added in the presence of other compounds to increase the buffer. You may have it.

An enzyme layer solution (containing the second enzyme A) on the electrode surface
Is applied and dried, and then a substrate layer solution (containing a substrate) is applied and dried, whereby the biosensor for measuring ALT (B) activity according to the present invention can be obtained. At this time, a known binder component can be added to each fixing solution in order to form a physically stable film. As the binder component, polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), acrylic acid, alginic acid, or the like can be used.

As the buffer component, a buffer component that gives a pH necessary for a target enzyme reaction can be used. For example, in the biosensor for measuring the ALT activity, a phosphate buffer (10 mM) for adjusting the pH in the enzyme layer to 7.0 and a phosphate buffer (0.1 M) for adjusting the pH in the substrate layer to 7.0 are respectively used. Examples can be given. To each solution, an inorganic salt or a surfactant necessary for the expression of the enzyme activity, a protective agent for a reaction component, or the like can be added. Alternatively, when the solvent in which the reaction components are to be dissolved is different, it is also possible to dissolve in two or more combinations and fix each of them. An enzyme layer and a substrate layer can be respectively formed by applying a fixation solution onto the electrode surface by dropping or spin coating and drying. The enzyme layer and the substrate layer constituting the biosensor of the present invention need not necessarily be clearly separated layers. For example, the biosensor of the present invention can be obtained by applying and drying the substrate layer solution after applying and drying the enzyme layer solution. In the biosensor manufactured through such a process, it is expected that a part of the components of the enzyme layer once dried at the boundary between the two layers is dissolved in the substrate layer liquid and the two are locally mixed. .
However, the biosensor obtained through such a manufacturing process clearly shows excellent response characteristics as shown in the examples. Therefore, a biosensor obtainable by such a manufacturing method is included in the present invention, even if it is expected that a clear boundary between the enzyme layer and the substrate layer may not be found.

The use of an electron acceptor is advantageous for detecting a signal substance produced by the second reaction without being affected by a reaction component or an interfering component coexisting in a sample. By sharing the electron acceptor in the second reaction site,
The signal substance can be detected without interference by a reducing interfering substance such as ascorbic acid or uric acid. In addition, loss of H ₂ O ₂ due to α-ketoglutaric acid and pyruvic acid can be prevented. Further, by utilizing an electron acceptor, not only is it difficult to receive interference from interfering substances, but also an enzymatic oxidation reaction that does not depend on dissolved oxygen can proceed. Usually, dissolved oxygen is utilized as an electron acceptor in the enzymatic oxidation reaction. By artificially supplying a sufficient amount of electron acceptors here, an oxidation reaction that does not depend on dissolved oxygen becomes possible. As the electron acceptor, potassium ferricyanide, sodium ferricyanide, p-benzoquinone, phenazine methosulfate (PMS), or a ferrocene derivative is known. Generally, electron acceptors
It is desirable to contain the product supplied in the first reaction, that is, the substrate of the second enzyme in a sufficiently large amount as compared with the assumed amount. When potassium ferricyanide K ₃ Fe (CN) ₆ is used together with the second enzyme POP, an oxidation current is generated by the following reaction.

[0018]

Embedded image

The electrodes constituting the biosensor of the present invention include:
There is no particular limitation as long as a signal substance generated by the second reaction can be detected. Specifically, it can be manufactured by forming a working electrode and a counter electrode, or a metal thin film to be a reference electrode on an appropriate insulating substrate. The metal thin film can be provided by a screen printing method, a metal evaporation method, a plating method, a sputtering method, or the like. As the metal, platinum, gold, silver, carbon, and the like are generally used. The electrodes are made of a low-cost and easily miniaturized material such as polyethylene terephthalate, and a micro-electrode pattern is provided on this surface, whereby the whole sensor can be made a small chip. Because it can be manufactured at low cost, it is an ideal disposable (dis
(posable) type chips. Further, the miniaturization of the biosensor chip enables measurement with a small amount of sample.

In the biosensor of the present invention, the activity of various enzymes can be measured by using electrodes having the same structure and changing only the reaction components to be fixed thereon. Since the measurement mechanism of the oxidation current can be common, it is possible to cope with various enzyme activity measurements by replacing only the biosensor chip.

The enzyme (B) whose activity can be measured by the biosensor according to the present invention includes, for example, the following. Applicable enzyme to be measured (B)
And components necessary for measuring the activity of the enzyme, and a reaction formula for measuring the activity.

[0022]

Embedded image ALT (L-Alanine 2-Oxoglutarate Aminotransferase; EC.2.6.1.2; GPT) L-alanine + α-ketoglutarate ALT → pyruvic acid + L-glutamic acid pyruvate + [H ₂ PO ₄ ] ⁻ + H ₂ O + O ₂ POP → acetyl phosphate + [HCO ₃ ] ^- + H ₂ O ₂

[0023]

L-alanine + α-ketoglutarate ALT → pyruvic acid + L-glutamic acid L-glutamic acid + H ₂ O + O ₂ GLO (*) → α-ketoglutarate + NH ₃ + H ₂ O ₂ * GLO: glutamate oxidase [L-Glutamate oxygen oxidoreductase (deaminating); EC.1.4.3.11)]

[0024]

AST (L-Aspartate 2-Oxoglutarate Aminotransferase; EC.2.6.1.1; GOT) Alanine sulfinic acid + α-ketoglutaric acid AST → pyruvic acid + L-glutamic acid pyruvic acid + [H ₂ PO ₄ ] ⁻ + H ₂ O + O ₂ POP → acetyl phosphate + [HCO ₃ ] ^- + H ₂ O ₂

[0025]

Embedded image

[0026]

Embedded image L-aspartic acid + α-ketoglutaric acid AST → oxaloacetic acid + L-glutamic acid oxaloacetic acid + H ₂ O ODC (*) → pyruvic acid + H ₂ CO ₃ pyruvic acid + [H ₂ PO ₄ ] ⁻ + H ₂ O + O ₂ POP → Acetyl phosphate + [HCO ₃ ] ^- + H ₂ O ₂ * ODC: Oxaloacetate decarboxylase [ODC: Oxalate Decarboxylase; EC.4.1.1.2]

[0027] In the method for measuring enzyme activity according to the present invention, the output current of the electrode is 1-
After incubation for 20 minutes, preferably 3-10 minutes, a voltage of + 0.2- + 0.7 V is applied to the electrodes and the oxidation current is measured. A calibration curve is prepared based on the measurement value of the oxidation current measured in advance with the standard sample, and the activity value of the target enzyme is calculated from the oxidation current of the sample. Furthermore, in the method for measuring enzyme activity according to the present invention, it is possible to apply a “rate assay” technique based on the concept of enzyme reaction rate. For example, in a biochemical ALT activity measuring method in a clinical test area, a rate assay in which all reagents are added and an enzyme activity is measured from an average reaction rate during 1 minute to 2/3 minutes of the reaction is generally used. It is used for In the biosensor of the present invention, when the incubation time is set to 10 minutes, the voltage is applied once to measure the oxidation current in 1 to 3 minutes of the reaction, and then the oxidation current is measured again in 10 minutes of the reaction. By dividing the difference current value by the elapsed time, a signal proportional to the average reaction speed can be obtained. By applying the rate assay technique, accurate measurement can be achieved in a short time. In addition, since a precise response can be obtained even when the activity of the enzyme to be measured is high, a wide measurement range can be expected.

[0028] The biosensor of the present invention can be used for measurement of any sample requiring enzyme activity measurement, such as a body fluid, food, or culture solution. Examples of the body fluid sample include whole blood, serum, plasma, urine, and the like. Next, the present invention will be described more specifically based on examples.

[0029]

Example: ALT was selected as the enzyme (B) to be measured,
A biosensor according to the present invention using OP as the second enzyme (A) was manufactured and its measurement performance was confirmed. [Example 1] Preparation of electrode chip Copper foil (thickness 18) on a glass epoxy insulating substrate 0.4 mm thick
1 μm), an electrode pattern shown in black in FIG. 1 was formed, and the working electrode (Working Electrod
e), counter pole (Counter Electrode), reference pole (Reference El
ectrode). Then, an insulating film having a thickness of about 35 μm was applied to a portion other than the active electrode surface on the left side of the drawing by a resist process (the active electrode surface and the lead electrode surface on the right side in the drawing were used in the gray and black portions in the drawing). The part of the extra-fine pattern connected). Next, platinum (Pt) plating was performed on the copper foil surface. Through the above operations, the three platinum-plated electrodes were produced.

Example 2 Preparation of Enzyme Layer and Substrate Layer (1) Preparation of Enzyme Layer Solution 10 mM containing 20.0 mg / ml of polyvinyl alcohol (PVA) and 1.0 mg / ml of sodium lauryl sulfate (SDS)
Phosphate buffer (pH 7.0) (composition of 10 mM phosphate buffer (pH 7.0): NaH ₂ PO ₄ 3.3 mM, K ₂ HPO ₄ 6.7 mM), magnesium chloride 0.1 mM, potassium ferricyanide 1.0 mM, thiamine Pyrophosphate (TPP) (1.0 mM) was added and stirred, and pyruvate oxidase (POP) was further added at 1000 Unit / ml (about 40 mg / ml). The mixture was dissolved with stirring to obtain an enzyme layer solution. (2) Preparation of Substrate Layer Solution A 2.0% (w / v) aqueous solution of sodium alginate and 0.2 M phosphate buffer (pH 7.0) (composition of 0.2 M phosphate buffer (pH 7.0): NaH ₂
PO ₄ 67 mM, and the solution was readjusted to K ₂ HPO ₄ 133mM) to L- alanine 1000mM and α- ketoglutaric 7.0 pH to dissolve the acid 60 mM was mixed in equal amounts. The mixed solution was gently stirred to obtain a substrate layer solution.

(3) Preparation of Enzyme Reactant Layer on Electrode An enzyme layer solution was applied to the entire surface of the active electrode by applying 1.0 μl of the enzyme layer solution, and the solution was applied for about 3 hours.
After drying for an hour, an enzyme layer was formed. Next, 1.0 μl of a substrate layer solution was applied so as to cover the enzyme layer, and dried at least overnight to form a substrate layer.

[Example 3] ALT addition evaluation test (1) Preparation of ALT solution Tris-HCl buffer (40 mM, pH7.
The ALT stock solution was diluted to about 0-1000 U / L by 0) to prepare an ALT dilution series. (2) ALT addition evaluation test procedure 10 μl of the ALT solution is dropped on the electrode and incubated for a specified time. After the elapse of the reaction time, the electrochemical oxidation current was measured. The oxidation current was measured by applying a ramp voltage from +0.0 V to +0.8 V to the electrode at a rate of 20 mV / sec over 40 seconds. At this time, the electrode potential was applied such that the relative potential of the working electrode with respect to the potential of the reference electrode became a prescribed voltage. The counter electrode was connected to a ground line. The response current measured the current flowing through the working electrode. FIG. 2 shows a time chart of the rate assay for measuring the response current twice. In addition, a measurement in which “measurement 1” in FIG. 2 was omitted was also performed as a basic performance evaluation test.

(3) Results of ALT addition evaluation test FIG. 3 shows an example of the enzyme reaction product layer formed on the active electrode surface. The white area with the highest brightness is the active electrode surface, the lower elongated electrode is the working electrode, the upper elongated electrode is the counter electrode, and the small square electrode on the right side of the center is the reference electrode. ) Electrode.
Both the enzyme layer solution and the substrate layer solution were applied so as to cover all electrode surfaces. All of the layers were thin and spread almost uniformly over the entire coated surface, and it can be said that the enzyme reaction product layer was successfully prepared.

First, ALT in a Tris-HCl buffer solution (40 mM, pH 7.0)
Regarding the addition evaluation test, a current-potential response curve (Voltammog) measured by a measurement process in which “Measurement 1” in FIG.
ram) patterns are shown in FIGS. ALT enzyme activity is 300U /
L (FIG. 4), 71U / L (FIG. 5), 18U / L (FIG. 6), and 0.
For the sample of 0 U / L (FIG. 7), after dropping on each electrode, and incubating for 1, 5, 10 and 20 minutes, a ramp voltage of +0.0 V → + 0.8 V was applied to the working electrode. The current pattern when the power is applied is displayed. Electrode potential is about +0.
From 2V to + 0.7V, there is a current pattern that seems to originate from the electrochemical oxidation of ferrocyan ions. In particular
When the ALT enzyme activity is 300 U / L, it is remarkable (FIG. 4). On the other hand, in the case where ALT was not added, almost no change in the current pattern due to the passage of the incubation time was observed (FIG. 7). Regarding the dependence on the incubation time, an increase in the current value almost proportional to the incubation time was observed for any concentration other than zero (FIGS. 4 to 6). For each of these current patterns, the current value when the electrode potential was +0.5 V was used as the representative value, and the horizontal axis was the incubation time, and the vertical axis was the current value. FIG. 8 is a plot. It is shown that the increasing range of the current value is proportional to the incubation time, and the time gradient of the current increase is proportional to the ALT concentration.

Next, FIGS. 9 to 12 show current-potential response curve (Voltammogram) patterns obtained by measuring the response current twice for the reaction 2 minutes and the reaction 10 minutes according to the measurement process of FIG. The ALT enzyme activity in the sample was 300 U / L (Fig. 9) and 7
1U / L (Fig. 10), 18U / L (Fig. 11), 0.0U / L (Fig. 12)
The current pattern is shown when a sample is dropped on the electrode and incubated, and a gradient voltage of +0.0 V ⇒ +0.8 V is applied to the working electrode 2 minutes and 10 minutes after the drop.
In all cases except for the case where ALT was not added (FIG. 12), a larger response current was obtained in 10 minutes of the reaction than in 2 minutes of the reaction. Then, when a difference curve between the two response current curves was drawn, a clear peak appeared near the electrode potential of +0.3 V in each case. FIG. 13 shows the correlation between the current value at the +0.3 V point of the difference curve and the ALT enzyme activity value. The correlation between the ^two was as good as R ² = 0.991.

[0036]

According to the present invention, a biosensor for measuring enzyme activity having excellent responsiveness can be manufactured at low cost. Measuring enzyme activity requires more complicated reaction components than a blood glucose measurement biosensor, but the present invention can provide an optimal reaction environment for an enzyme reaction system composed of complicated reaction components. The biosensor for measuring enzyme activity according to the present invention has a simple manufacturing process and a simple structure, and can be manufactured at low cost. When the configuration of the biosensor for measuring the enzyme activity of the present invention is applied to a biosensor for measuring the activity of ALT, excellent response characteristics are realized by forming a uniform enzyme reactant layer. ALT, along with AST, is known as a typical deviating enzyme, and all of them have increased activity in blood due to damage to organ tissue cells. In particular, ALT is a relatively characteristic enzyme in the liver, and it is said that an increase in blood ALT activity can be regarded as an indicator of liver dysfunction. In fact, ALT activity is one of the items frequently measured as a liver function marker. The significance of the present invention is that it is possible to easily and inexpensively measure the activity of such important enzymes by using a biosensor.

[Brief description of the drawings]

FIG. 1 is a diagram showing a structure of an electrode. The black part is the electrode pattern, the two large rectangles or squares on the left in the figure are the active surfaces on which the enzyme layer and the substrate layer are arranged, and the right rectangle in the figure is the lead surface connected to the electronic circuit. is there. The insulating film is formed in a portion of an ultrafine pattern connecting the active surface and the lead surface among the gray portion and the black portion.

FIG. 2 shows a time chart of a rate assay for measuring a response current twice. The horizontal axis represents the enzyme reaction time (minutes), the vertical axis represents the electrode potential in the upper row, and the current in the lower row.

FIG. 3 is a photograph of an active electrode surface covered with an enzyme layer and a substrate layer. The lower elongated electrode is a working electrode, the upper elongated electrode is a counter electrode, and the small square electrode at the center right is a reference electrode.

FIG. 4 is a graph showing a current-potential response curve pattern obtained by the measurement system of the present invention. ALT enzyme activity in the sample is 300 U / L
In the case of the above, 1 minute, 5 minutes, 10 minutes after dropping on the electrode
5 shows the current pattern when a working voltage of + 0.0V⇒ + 0.8V is applied to the working electrode after incubation for 15 minutes, 15 minutes, and 20 minutes. The horizontal axis is the electrode potential (V), and the vertical axis is the current value (μA)
Represents

FIG. 5 is a graph showing a current-potential response curve pattern obtained by the measurement system of the present invention. ALT enzyme activity in the sample is 71 U / L
In the case of the above, 1 minute, 5 minutes, 10 minutes after dropping on the electrode
After a 20 minute incubation, the working electrode
⇒ Indicates the current pattern when a + 0.8V ramp voltage is applied. The horizontal axis represents the electrode potential (V), and the vertical axis represents the current value (μA).

FIG. 6 is a graph showing a current-potential response curve pattern obtained by the measurement system of the present invention. ALT enzyme activity in the sample is 18 U / L
In the case of the above, 1 minute, 5 minutes, 10 minutes after dropping on the electrode
After a 20 minute incubation, the working electrode
⇒ Indicates the current pattern when a + 0.8V ramp voltage is applied. The horizontal axis represents the electrode potential (V), and the vertical axis represents the current value (μA).

FIG. 7 is a graph showing a current-potential response curve pattern by the measurement system of the present invention. ALT enzyme activity in sample is 0.0 U / L
In the case of the above, 1 minute, 5 minutes, 10 minutes after dropping on the electrode
After a 20 minute incubation, the working electrode
⇒ Indicates the current pattern when a + 0.8V ramp voltage is applied. The horizontal axis represents the electrode potential (V), and the vertical axis represents the current value (μA).

FIG. 8 is a graph plotting current values for each ALT enzyme activity value when the electrode potential is +0.5 V. The horizontal axis represents the enzyme reaction time (minutes), and the vertical axis represents the current value (μA).

FIG. 9 is a graph showing a current-potential response curve pattern obtained by measuring a response current twice in 2 minutes and 10 minutes in response to the measurement process shown in FIG. 2; ALT enzyme activity in the sample is 300
U / L. The horizontal axis is the electrode potential (V), and the vertical axis is the current value (μA)
Represents

FIG. 10 shows a reaction 2 according to the measurement process shown in FIG.
7 is a graph showing a current-potential response curve pattern obtained by measuring a response current twice in 10 minutes and 10 minutes in a reaction. ALT enzyme activity in the sample is 7
1U / L. The horizontal axis is the electrode potential (V), and the vertical axis is the current value (μA)
Represents

FIG. 11 shows a reaction 2 according to the measurement process shown in FIG.
7 is a graph showing a current-potential response curve pattern obtained by measuring a response current twice in 10 minutes and 10 minutes in a reaction. ALT enzyme activity in the sample is 1
8U / L. The horizontal axis is the electrode potential (V), and the vertical axis is the current value (μA)
Represents

FIG. 12 shows a reaction 2 according to the measurement process shown in FIG.
7 is a graph showing a current-potential response curve pattern obtained by measuring a response current twice in 10 minutes and 10 minutes in a reaction. ALT enzyme activity in the sample
0.0 U / L. The horizontal axis represents the electrode potential (V), and the vertical axis represents the current value (μ
A).

FIG. 13 shows a reaction 2 according to the measurement process shown in FIG.
7 is a graph showing the correlation between the current value at the +0.3 V point and the ALT enzyme activity value in the difference curve of the current-potential response curves obtained by measuring the response current twice for 10 minutes and 10 minutes of the reaction. The horizontal axis is the ALT enzyme activity value
(U / L), and the vertical axis represents the current increment width (μA).

[Explanation of symbols]

 (1) Working electrode (2) Counter electrode (3) Reference electrode

Claims (6)

[Claims] 1. A biosensor for measuring enzyme activity, comprising: a) an oxidase A disposed on the electrode surface, the oxidase A acting on a component generated by the activity of the enzyme B to be measured; b) an enzyme substrate laminated on the oxidase A; The substrate forms the substrate for the oxidase A according to the activity of the enzyme B to be measured. 2. The enzyme B whose activity is to be measured is alanine aminotransferase, the oxidase A is pyruvate oxidase, and the substrates are L-alanine and α-
The biosensor according to claim 1, which is ketoglutaric acid. 3. The biosensor according to claim 1, comprising an enzyme reaction for measuring enzyme activity, wherein the enzyme B to be measured acts on a substrate to produce a reaction product. The first reaction that takes place and another enzyme A acting on the reaction product
And a second reaction that generates a signal substance by the biosensor. a) a first layer containing components required for the second reaction on the electrode surface b) a second layer containing components required for the first reaction laminated on the first layer 4. A method for measuring enzyme activity, comprising using a biosensor for measuring enzyme activity, comprising the following elements. a) an oxidase A disposed on the electrode surface, the oxidase A acting on a component generated by the activity of the enzyme B to be measured; b) an enzyme substrate laminated on the oxidase A; The substrate forms a substrate for the oxidase according to the activity of enzyme B to be measured. 5. A method for producing a biosensor for measuring enzyme activity, comprising the following steps. a) a step of arranging oxidase A on the electrode surface, wherein the oxidase A acts on a component generated by the activity of the enzyme B to be measured b) an enzyme substrate is arranged on the electrode surface after step a) In this step, the enzyme substrate generates a substrate for the oxidase A according to the activity of the enzyme B to be measured. 6. A biosensor manufactured by the method according to claim 5.