JP2515005B2 - Measuring method and apparatus using enzyme electrode - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to glucose and deoxy-D- in a sample.
The present invention relates to a measuring method and apparatus using an enzyme electrode for quantifying glucose.

2. Description of the Related Art Deoxyglucose is widely distributed in nature as a sugar component of plant glycosides. Therefore, in the fields of phytochemistry and biochemistry, there is a high demand for this measurement of deoxyglucose. As a method for quantifying deoxyglucose, conventionally known colorimetric quantification methods such as the thiobarbituric acid method, the diphenylamine acetic acid method, and the cysteine / sulfuric acid method.

However, in such a method, during the measurement operation,
The sample has to be left for about 0.5 to 20 hours, the measurement time becomes unnecessarily long, and deoxyglucose cannot be quantified rapidly. Further, since a strong acid which is inconvenient to use is used, there is a problem that the measuring operation becomes complicated. Moreover, in the above-mentioned conventional colorimetric quantification method, it is not possible to quantify the respective amounts of glucose and deoxyglucose at the same time.

Further, to date, no enzyme has been found that acts on deoxyglucose to catalyze a reaction that specifically produces an electrode reactant. Therefore, no attempt has been made to measure deoxyglucose using an enzyme electrode.

On the other hand, a measuring method for measuring glucose using an immobilized enzyme electrode has come to be used in many fields in recent years. Glucose oxidase is usually immobilized on the immobilized enzyme electrode used for quantifying glucose. Although glucose oxidase is an enzyme having a relatively high substrate specificity, it is known to catalyze not only the oxidation of glucose but also the oxidation of deoxyglucose. That is, deoxyglucose is several% to several tens% of glucose in the case of glucose due to glucose oxidase.
Is oxidized at the rate of. Therefore, in the method for measuring glucose using the above-mentioned immobilized enzyme electrode, not only glucose in the sample but also deoxyglucose is measured. Since the interference of the measurement with deoxyglucose is related to the essence of the enzymatic reaction, it is extremely difficult to eliminate its influence. Heretofore, an effective solution to such a problem has not been presented, and there is a strong demand for the development of an accurate method for quantifying glucose.

Therefore, the object of the present invention is to solve the above technical problems and use an enzyme electrode capable of simultaneously and rapidly measuring glucose and deoxy-D-glucose in a sample. A measuring method and device are provided.

Means for Solving the Problems The present invention relates to a first glucose oxidase-immobilized first
An enzyme electrode and a second enzyme electrode having glucose oxidase and mutarotase immobilized thereon are provided, and each of glucose and deoxy-D-glucose is quantified based on the outputs of the first enzyme electrode and the second enzyme electrode. Is a measuring method using an enzyme electrode.

Further, the present invention is a flow-type measuring device for measuring a sample in a buffer solution, which comprises a first enzyme electrode having glucose oxidase immobilized thereon, and a first enzyme electrode disposed downstream of the buffer solution flow, A second enzyme electrode on which glucose oxidase and mutarotase are immobilized; and an operation means for performing an operation on the outputs of the first enzyme electrode and the second enzyme electrode to determine each of glucose and deoxy-D-glucose. This is a measuring device using an enzyme electrode.

Operation According to the present invention, glucose oxidase immobilized on both the first enzyme electrode and the second enzyme electrode catalyzes the reaction represented by the following formula.

Β-D-glucose can be quantified based on the hydrogen peroxide produced in this reaction or the oxygen consumed.

However, when deoxy-D-glucose is contained in the sample, this deoxy-D-glucose is also oxidized by glucose oxidase.

Therefore, with the first enzyme electrode on which glucose oxidase was immobilized, β-D-glucose and deoxy-D-
The value which added each amount with glucose is measured.

On the other hand, mutarotase is generally an enzyme that catalyzes the phenomenon of epimer conversion in a solution of monosaccharides, that is, mutarotation. Therefore, when it is used in the measurement of glucose, it usually catalyzes the mutarotation of glucose as shown in the second equation.

In the second enzyme electrode on which mutarotase and glucose oxidase are immobilized, β-D produced by the second formula
-Glucose is quantified by oxidizing glucose with glucose oxidase as described above and detecting the decrease in hydrogen peroxide or oxygen produced. Thus, mutarotase is generally used to improve the measurement sensitivity in the quantification of glucose, and to prevent the quantification result from being influenced by the ratio of glucose epimers.

The present inventor discovered that mutarotase acted on deoxyglucose during the experiment for improving the selectivity of the enzyme electrode using glucose oxidase, and brought about a specific change in combination with glucose oxidase, and earnestly studied. As a result, the present invention has been completed.

That is, mutarotase generally catalyzes a reaction that mutually converts α-D-glucose and β-D-glucose in equilibrium in an aqueous solution, as shown in the second formula. Therefore, when β-D-glucose is consumed by glucose oxidase, α-D is relatively consumed.
-The glucose will increase and the conversion of α-D-glucose to β-D-glucose by mutarotase will be accelerated. Therefore, as a result, α-D in the sample
-Glucose is sequentially converted to β-D-glucose and efficiently oxidized by glucose oxidase.

However, when a similar experiment is performed on deoxyglucose, the rate of oxidation of deoxyglucose by glucose oxidase is decreased by mutarotase. That is, when deoxyglucose, the measurement value measured by the first enzyme electrode immobilized glucose oxidase, and the measurement value measured by the second enzyme electrode immobilized glucose oxidase and mutarotase, when compared under the same conditions, It was found that the former measurement value was large.

The cause of the decrease in the oxidation rate is considered as follows. For glucose equilibrated in aqueous solution, α
-It is said that the body occupies 37% and the β-body occupies 63%. On the other hand, when the β-form is oxidized by glucose oxidase, the concentration of β-form in the immobilized enzyme layer decreases, but if the mutarotase activity is sufficiently higher than the glucose oxidase activity in the same immobilized enzyme layer, α -Body is rapidly converted to β-body, resulting in the sensitivity of the electrode to glucose (100/63) due to the presence of mutarotase.
That is about 1.6 times.

On the other hand, since deoxyglucose has a structure very similar to glucose, it is oxidized by glucose oxidase and interacts with mutarotase. However, unlike glucose, mutarotase does not appear to catalyze the mutarotation of deoxyglucose. Therefore, when mutarotase coexists in the immobilized enzyme layer, deoxyglucose, which is a substrate for glucose oxidase, partially binds to mutarotase, resulting in a decrease in the effective concentration of deoxyglucose. Will be apparently reduced.

Although the above explanation is one inference and complete proof is difficult, the following facts are listed as side evidence.

Four immobilized enzyme electrodes were prepared in which the ratio of glucose oxidase activity to mutarotase activity was adjusted to 1: 0, 1: 0.1, 1: 1, 1:10. First, when the relative sensitivity of glucose was examined, when the activity ratio was 1: 0, the standard was 1.04 times in the case of 1: 0.1 and 1.3% in the case of 1: 1.
In the case of 1:10, the sensitivity was 1.59 times. On the other hand, when the case of deoxyglucose was examined, there was no change in the sensitivity up to the case of 1: 1 on the basis of the case of 1: 0, and in the case of 1:10, the sensitivity was 0.31 times.

A more detailed examination revealed that when immobilized, the mutarotase activity showed a gradual decrease in sensitivity from 1.5 times to 5 times that of glucose oxidase activity, and a large decrease in sensitivity was seen at a point exceeding 5 times, and from 5 times to 50 times, A certain decrease in sensitivity was observed.

In the case of glucose, the sensitivity increases as the ratio of the mutarotase activity increases, and when it is about 2 times, it becomes 1.59 times and becomes constant thereafter. In the case of glucose, it can be explained by considering that the amount of mutarotase required for making the rate-determining step of the reaction a step involving glucose oxidase is approximately 1: 2 in the solution used for immobilization.

On the other hand, in the case of deoxyglucose, it cannot be simply explained by the enzyme activity.
It is easy to explain that deoxyglucose is adsorbed and the substrate of glucose oxidase is subjected to a kind of masking.

We also compared the oxidation rates of both substrates by glucose oxidase in the presence of mutarotase in solution. When the mutarotase activity was changed to 0.1 to 2 times that of glucose oxidase activity, glucose showed an increase in the oxidation rate depending on the added amount of mutarotase, but deoxyglucose showed almost no change. This is considered to be because, unlike in the immobilized enzyme layer, the effect of masking deoxyglucose by mutarotase is low in a free enzyme system in which the probability of collision between the substrate and the enzyme molecule is low.

From the above phenomenon, in the immobilized enzyme electrode used in the present invention, the activity ratio of glucose oxidase and mutarotase is 1: 1.5 or more, 1:50 or less, more preferably 1: 5.
It is desirable that the ratio is 1 to 50 or less. It should be noted that the addition of 50 times or more mutarotase increases the protein concentration of mutarotase and increases the solution viscosity used for immobilization, which is not preferable. Further, the effect of the present invention is not affected even if other proteins or polymers are made to coexist for the purpose of increasing the crosslinking strength of the immobilized enzyme layer.

By utilizing such a phenomenon, a first enzyme electrode having glucose oxidase immobilized thereon and a second enzyme electrode having glucose oxidase and mutarotase immobilized thereon can be used. The concentrations of deoxyglucose and glucose in the sample can be obtained based on the change value of the output current of the sample.

That is, the relationship between the current change value G1 and the glucose concentration X1 is obtained as shown in the third equation by the glucose calibration curve at the first enzyme electrode.

G1 = A1 * X1 + B1 (3) Here, A1 and B1 are constants depending on the device, etc., and can be determined from the glucose calibration curve.

Similarly, in the second enzyme electrode, the relationship between the current change value G2 and the glucose concentration X1 is obtained as shown in the equation (4).

G2 = A2 · X1 + B2 (4) where A2 and B2 are constants depending on the device.

As described above, the second enzyme electrode also detects β-D-glucose converted from α-D-glucose. However, in the sample as an aqueous solution, α-D
Since -glucose and β-D-glucose are in an equilibrium state, the ratio of their concentrations is constant. Therefore, the current change value at the first and second enzyme electrodes
Between G1 and total glucose concentration X1, the third and fourth equations
The relationships shown by the equations are established. The same applies to deoxyglucose described below.

Next, in the case of injecting deoxyglucose into the measuring device, the relationship between the current change value D1 obtained from the calibration curve of deoxyglucose at the first enzyme electrode and the deoxyglucose concentration X2 is shown in the fifth formula. .

D1 = A3 · X2 + B3 (5) where A3 and B3 are constants depending on the device.

Similarly, the current change value D2 and the deoxyglucose concentration X2 obtained from the calibration curve of deoxyglucose at the second enzyme electrode
The relationship between and is shown in the sixth equation.

D2 = A4.X2 + B4 (6) where A4 and B4 are constants depending on the device.

Therefore, when a sample containing glucose and deoxyglucose is measured, the current change value (G1 + D1) at the first enzyme electrode becomes as shown in the seventh formula based on the third to sixth formulas. .

G1 + D1 = A1 * X1 + B1 + A3 * X2 + B3 (7) Similarly, current change value (G2 + D2) at the second enzyme electrode
Is shown in the eighth equation.

G2 + D2 = A2 * X1 + B2 + A4 * X2 + B4 (8) Based on the seventh and eighth equations, glucose concentration X1
Is expressed as in Expression 9.

Similarly, the deoxyglucose concentration X2 is also shown in the tenth formula.

In this way, if the calibration curves of glucose and deoxyglucose are prepared in advance for the first enzyme electrode and the second enzyme electrode, respectively, the constants A1 to A4, B1 to B4 can be obtained by this calibration value. After determination, the concentrations X1 and X2 of both glucose and deoxyglucose in the sample can be obtained by the equations 9 and 10, respectively.

Further, although the present invention can be carried out by both the batch system and the flow system, when the system is carried out by the flow system, the second enzyme electrode may be arranged downstream of the first enzyme electrode in the apparatus. desirable.

As described above, in the second enzyme electrode, α-glucose in the sample is converted into β-glucose by the action of mutarotase. Therefore, if the dilution rates of the sample in the buffer solution in the first enzyme electrode and the second enzyme electrode are equal, a higher current change value is observed in the second enzyme electrode. When the calibration curve is linear within the measurable range of the first enzyme electrode, that is, when the calibration curve for the second enzyme electrode is also within that range, the dilution rate of the sample is set higher than that of the first enzyme electrode. Must also have a high dilution rate in the second enzyme electrode.

By disposing the second enzyme electrode on the downstream side of the first enzyme electrode, the sample is diluted in the conduit, and the dilution rate of the second enzyme electrode can be increased. Further, in connection with the convenience of diluting the sample, the present invention can be more suitably carried out in the measurement by the flow method.

Hereinafter, the flow-type measuring apparatus according to the present embodiment will be described.
It will be described with reference to the drawings. This flow type measuring device is the first
It includes an enzyme electrode 6a, a second enzyme electrode 6b, and a potentiostat 9 which is a calculating means for calculating a current change value from the first enzyme electrode 6a and the second enzyme electrode 6b. Measuring cell
5a includes a first enzyme electrode 6a having glucose oxidase immobilized as a working electrode, and an Ag / AgCl electrode 8 as a reference electrode.
a is fixed respectively. Further, a stainless steel pipe 7a is arranged as a counter electrode, and this flow type measuring device is of a three-electrode type.

In the measurement cell 5b, a second enzyme electrode 6b having glucose oxidase and mutarotase immobilized as a working electrode,
The Ag / AgCl electrode 8b is fixed as a reference electrode. A stainless steel pipe 7b is provided in association with the measuring cell 5b.

The injector 3 described later is connected to the measuring cell 5a via the diluting conduit 4a. Further, the measuring cell 5a is connected to the measuring cell 5b via a diluting conduit 4b. Such diluting conduits 4a, 4b and measuring cells 5a, 5b are arranged in the thermostat 12.

The buffer solution 1 is sent by the pump 2 at a predetermined flow rate. Sample is injected from Injecta 3 and buffer 1
Is mixed in. The buffer solution in which the sample is mixed is guided to the measuring cell 5a via the diluting pipe 4a, and then the diluting pipe 4a.
It is led to the measuring cell 5b via 4b. The buffer solution that has passed through the measurement cell 5b is drained to the waste bottle 11 via the conduit 4c.

To create the above-mentioned calibration curve, Injecta 3
Standard solution containing only Glycose and Deoxy-D
-Injecting a standard solution containing only glucose, the first enzyme electrode
The current change value of 6a and the second enzyme electrode 6b is measured. Based on the obtained calibration curve, the constants A1 to
A4 and B1 to B4 are determined. When measuring a sample of unknown concentration, the current change value at the first enzyme electrode 6a is set to (G1 + D1) in the formulas 9 and 10 and the current change value at the second enzyme electrode 6b is set to ( G2 + D2), the glucose concentration X1 and deoxyglucose concentration X2 in the sample
Is obtained.

Examples The following will describe Examples of the present invention in more detail, but the present invention is not limited to these.

Example 1 (1) Preparation of electrode First enzyme electrode On a surface of a platinum electrode having a diameter of 2 mm, the surface of which was thoroughly cleaned,
Glucose oxidase (Sigma, Type II) 3 mg /
ml, bovine serum albumin (Sigma V, Fraction V) 7 mg / ml, and an aqueous solution containing 0.5% by weight of glutaraldehyde (5 μ) were added dropwise, and the mixture was treated at 40 ° C. for 30 minutes for immobilization. The electrode 6a is created.

Second enzyme electrode 0.1 ml mutarotase (Sigma, PORCINE KIDNE
Y-derived (25000 units / 1.1 ml) was dialyzed in 0.1 M sodium phosphate buffer of pH 7.0, glucose oxidase (Sigma, Type II) 5 mg, bovine serum albumin (Sigma, fraction V) ) Is added, and the pH is adjusted to 0.1 at pH 7.5.
It was dissolved in 1.0 ml of M phosphate buffer. Furthermore, glutaraldehyde was added so that the final concentration would be 0.2% by weight, and 3 μ was dropped on the surface of a platinum electrode having a diameter of 2 mm, the surface of which was thoroughly cleaned.
The enzyme electrode 6b was created.

(2) Measuring device The flow-type measuring device shown in Fig. 1 comprises an injector 3 for high performance liquid chromatograph, capable of injecting a sample of the order of μ, Ag / AgCl as the first enzyme electrode 6a and a reference electrode. A measuring cell 5a having an electrode 8a attached thereto and a stainless steel pipe 7a as a counter electrode, and the second enzyme electrode
6b and Ag / AgCl electrode 8b installed, stainless steel tubing 7
and a measurement cell 5b provided with b. For example, a Teflon dilution pipe with an inner diameter of 0.5 mm and a length of 1.5 mm
4a, 4b, 4c are connected between the injector 3 and the measuring cell 5a, between the measuring cell 5a and the measuring cell 5b, and between the measuring cell 5b and a waste liquid bottle 11 described later. It
The measurement cells 5a and 5b have an inner volume of 40 μ, and the first and second enzyme electrodes 6a and 6b and the Ag / AgCl electrodes 8a and 8b are arranged to face each other via a buffer passage. The first and second enzyme electrodes 6a and 6b have a potentiostat 9
A voltage of +0.60 V is applied to the g / AgCl electrodes 8a and 8b, respectively.

In such a structure, the temperature of the thermostat 12 is strictly controlled. The temperature inside the thermostat 12 is maintained at 37 ° C. A buffer 2 for high-performance liquid chromatography is used to feed the buffer solution 1, and 0.1 M sodium phosphate buffer solution having a pH of 6.0 as the buffer solution 1 is delivered at a flow rate of 1.0 ml / min. The buffer solution after the measurement is supplemented in the waste bottle 11. The measured value is recorded by the recorder 10.

(3) Sample measurement Glucose and 2-deoxy-D-glucose
A standard solution containing 0, 20, and 30 mM respectively was prepared and injected from the injector 3, and the first enzyme electrode 6a and the second enzyme electrode 6b were injected.
The current change value was measured. Based on this, the calibration curve shown in FIG. 2 was prepared.

It can be seen from the lines l1 to l4 in FIG. 2 that the calibration curve has linearity when the concentrations of glucose and 2-deoxy-D-glucose are 30 mM or less. Therefore, the concentration of glucose and 2-deoxy-D-glucose was 30 mM.
For the sample of, the first enzyme electrode 6a and the second enzyme electrode
It turns out that it is within the measurable range of 6b.

The calibration curve for the first enzyme electrode 6a when the standard solution containing glucose at 10, 20, and 30 mM, respectively, is injected is shown by the line l1. The constant A1 of the third equation was obtained from the slope of the line 11, that is, the tangent, and the constant B1 was obtained from the current change value of the line 11 at the glucose concentration of 0 mM. The calibration curve for glucose at the second enzyme electrode 6b is shown in line l2,
The constants A2 and B2 were obtained from the tangent of the line 12 and the current change value at a glucose concentration of 0 mM.

Further, a calibration curve of 2-deoxy-D-glucose at the first enzyme electrode is shown in a line l3, and from the tangent of the line l3 and the current change value at a 2-deoxy-D-glucose concentration of 0 mM, constants A3 and B3 are obtained. Each was asked. The second enzyme electrode of 2-deoxy-D-glucose
The calibration curve in 6b is shown by the line l4, and the constants A4 and B4 were obtained from the tangent of the line l4 and the current change value at the 2-deoxy-D-glucose concentration of 0 mM.

In this way, the constants A1 to A4 and B1 to B4 are determined in advance, and the sample is injected from the injector 3 when measuring the sample. At this time, the current change value of the first enzyme electrode 6a (G1 +
Since D1) and the current change value (G2 + D2) of the second enzyme electrode 6b are measured, the glucose concentration X1 and the 2-deoxy-D-glucose concentration X2 can be obtained by the ninth and tenth equations.

In order to examine the measurement accuracy in this example, measurement was performed on glucose and 2-deoxy-D-glucose composition samples shown in Table 1. Current change values (G1 + D1), (G2 + D) of the first enzyme electrode 6a and the second enzyme electrode 6b
2), and glucose concentration X1 determined by measurement
And the 2-deoxy-D-glucose concentration X2 are as shown in Table 1.

As described above, in this example, glucose and 2-deoxy-D-glucose can be simultaneously and rapidly quantified. Moreover, as shown in Table 1, it is possible to perform measurement with sufficient accuracy by a simple operation.

According to the present invention, the potentiostat 9 having an arithmetic function is used to automatically perform the arithmetic operations shown in the ninth and tenth equations to obtain the obtained glucose concentration X1 and 2-deoxy-D-glucose concentration X2. May be configured to be recorded by a recorder 10, or a normal potentiostat 9 may be used to record the first enzyme electrode 6a and the second enzyme electrode on the recorder 10.
The current change values (G1 + D1) and (G2 + D2) of 6b may be recorded, and then the glucose concentration X1 and the 2-deoxy-D-glucose concentration X2 may be obtained by the ninth and tenth equations.

EFFECTS OF THE INVENTION As described above, according to the present invention, the glucose concentration and deoxyglucose concentration in a sample can be measured accurately and quickly.

[Brief description of drawings]

FIG. 1 is a system diagram showing a configuration of a flow type measuring apparatus according to an embodiment of the present invention, and FIG. 2 is a graph showing glucose and 2-deoxy-D-glucose obtained by the flow type measuring apparatus shown in FIG. It is a graph which shows a calibration curve. 1 ... Buffer solution, 3 ... Injector, 4a, 4b ... Dilution conduit, 5a, 5b ... Measurement cell, 6a ... First enzyme electrode, 6b
…… Second enzyme electrode, 7a, 7b …… Stainless steel piping (counter electrode), 8a, 8b …… Ag / AgCl electrode (reference electrode), 9 …… Potentiostat, 12 …… Constant temperature bath

Continuation of front page (56) Reference JP 54-81177 (JP, A) JP 58-216947 (JP, A) JP 61-93948 (JP, A) JP 63-317757 (JP , A)

Claims (2)

(57) [Claims] 1. A first in which glucose oxidase is immobilized.
An enzyme electrode and a second enzyme electrode on which glucose oxidase and mutarotase are immobilized are provided, and based on the outputs of the first enzyme electrode and the second enzyme electrode,
A measuring method using an enzyme electrode, which comprises quantifying glucose and deoxy-D-glucose. 2. A flow-type measuring device for measuring a sample in a buffer solution, which comprises a first enzyme electrode having glucose oxidase immobilized thereon, and a first enzyme electrode disposed downstream of the buffer solution flow, A second enzyme electrode on which glucose oxidase and mutarotase are immobilized; and an operation means for performing an operation on the outputs of the first enzyme electrode and the second enzyme electrode to determine each of glucose and deoxy-D-glucose. A measuring device using an enzyme electrode.