JPH05232076A - Alpha-amylase activation measuring electrode and alpha-amyklase activation measuring method - Google Patents

Abstract

PURPOSE:To use all blood as tested solution without applying centrifugation or filtration in measuring alpha-amylase activation, to enhance a measurement limit and to increase measurement precision. CONSTITUTION:A hydrogen peroxide selectively permeating film 2, a fixed GOD film 3, a conjugation enzyme fixed film 4 and a diffusion limiting film 5 are arranged in sequence on the surface of a base electrode 1. By using the diffusion limiting film 5, the function of separating interfering material and the function of limiting the diffusion and permeation of alpha-amylase and enzyme reaction material are achieved.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an electrode device for measuring α-amylase activity and a method for measuring α-amylase activity. More specifically, it is suitable for use in measuring α-amylase activity in a test solution. The present invention relates to a method for measuring α-amylase activity based on electric signals obtained at mutually different timings using the electrode device and two kinds of enzymes described above.

[0002]

2. Description of the Related Art Conventionally, in 95% or more of medical facilities, blood for patients with a possibility of acute pancreatitis, pancreatic cancer, hepatobiliary disease, renal failure, salivitis, parotiditis, etc.
The α-amylase activity is measured using urine, saliva or the like as a test solution. Specifically, (1) an iodometric method (also referred to as an amyloclastic method) in which starch is gradually decomposed by α-amylase is followed by an iodostarch reaction, and (2) a decomposition reaction by α-amylase Saccharogenic method for measuring the reducing power of maltose, (3) Chromogenic method for colorimetrically measuring soluble pigment released by the action of α-amylase using insoluble colored starch cross-linked with pigment as a substrate There is.

However, these methods have the disadvantages that the reaction time is long, the coloration is unstable, the reproducibility and the selectivity are insufficient, and further centrifugation and filtration operations are required. However, this method is not suitable for measurement in the above medical facilities that are highly urgent and require high measurement accuracy. Further, in place of these methods, a so-called enzymatic method utilizing the substrate specificity of the enzyme has been proposed. Since the enzymatic method has advantages such as high selectivity and shortened reaction time, the above (1) and (2)
It is more suitable for measurement in medical facilities than the method (3).

[0004]

However, in the conventional enzymatic method, it is necessary to remove or convert glucose and maltose contained in the test solution, and the colorimetric method is adopted as a quantitative method. Therefore, α−
A test solution with high amylase activity has the disadvantages that it needs to be measured after dilution, the test solution may be contaminated by pipetting during dilution, and the enzyme electrode detector must be installed. The two measuring methods (see Japanese Patent Laid-Open No. 56-97863) have the disadvantages that maltose in the test solution gives a positive error, that both enzyme electrode detectors must be calibrated at the same time, enzyme electrode detection With the increase in the number of containers, the number of peripheral electronic circuit parts and the like increases remarkably, and the configuration becomes complicated. Further, in order to eliminate these disadvantages, an amylase substrate solution having a predetermined concentration is supplied by using an enzyme electrode obtained by sealingly depositing an immobilized GOD membrane on which glucose oxidase (hereinafter abbreviated as GOD) is immobilized. A test solution containing amylase and a decomposition auxiliary enzyme are injected into one of the immobilized GOD membrane electrode parts,
The current related to the amount of glucose existing from the beginning, which is the result of the degrading action by the immobilized GOD membrane, rises rapidly at the beginning and then becomes a substantially constant value, and thereafter gradually decreases due to the degrading action by the amylase and the auxiliary enzyme. Since it rises, a method of measuring amylase activity based on a gradually increasing amount of change in current has been proposed (Japanese Patent Publication No. 1-47).
No. 160). However, even when the method for measuring amylase activity described in JP-B-1-47160 is adopted, when whole blood is used as a test solution,
Since hemoglobin and the like in blood cells flow out into the test solution due to hemolysis, there is a disadvantage that the accuracy of the amylase activity measurement result finally obtained is lowered. Since the rate of decrease in measurement accuracy depends on the degree of hemolysis, even in the case of using whole blood as the test solution, for use in medical facilities where urgency is required. There has been a strong demand for a method for measuring amylase activity that can achieve highly accurate measurement of amylase activity.

Furthermore, not only when the α-amylase activity in the test solution is high but also when the amount of the substrate contained in the test solution is large, the amount of glucose reacted in the presence of GOD is significantly increased, As a result, the amount of dissolved oxygen consumed in the test solution is remarkably increased, and there is also a disadvantage that the measurable upper limit is restricted by the amount of dissolved oxygen.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and even when whole blood is used as a test solution, it is possible to easily perform a blood cell separation operation, a filtration operation, etc. And an α-amylase activity measuring electrode device capable of highly accurately measuring α-amylase activity, and α-
It is intended to provide a method for measuring amylase activity.

[0007]

In order to achieve the above object, the electrode device for measuring α-amylase activity according to claim 1 has a predetermined relative position with respect to the measurement surface of the electrode and holds the test solution. Α-amylase, a polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase, 2
A diffusion limiting membrane that limits permeation and diffusion of the sugar and / or polysaccharide is arranged, and a first enzyme that causes a hydrolysis reaction of the monosaccharide between the measurement surface and the diffusion limiting membrane, And a second enzyme that decomposes the disaccharide and / or polysaccharide into monosaccharides.

In the electrode device for measuring α-amylase activity according to claim 2, the diffusion limiting film is arranged so as to be removable from the electrode. In the electrode device for measuring α-amylase activity according to claim 3, the first enzyme is immobilized on the membrane and the second enzyme is also immobilized on the membrane, and the second enzyme-immobilized membrane is It is arranged at a predetermined position closer to the diffusion limiting film than the first enzyme-immobilized film.

In the electrode device for measuring α-amylase activity according to claim 4, the second enzyme-immobilized membrane is arranged so as to be removable from the electrode. The electrode device for measuring α-amylase activity according to claim 5 further includes electrode moving means for moving the electrode in a predetermined direction, and
The enzyme-immobilized membrane, the second enzyme-immobilized membrane, and the diffusion-limiting membrane are arranged in a relative positional relationship in which they adhere to each other following the movement of the electrode in a predetermined direction for a predetermined distance. is there.

In the method for measuring α-amylase activity according to a sixth aspect of the present invention, an electrode device comprising a first enzyme-immobilized membrane, a second enzyme-immobilized membrane and a diffusion limiting membrane is disposed on the electrode surface. The test solution and the reaction substrate obtained by modifying the non-reducing end with a protecting group are spotted in a predetermined order, and the interfering substances contained in the test solution are separated by the diffusion limiting membrane and interfered in the presence of the second enzyme. The test solution from which the substance is separated and the reaction substrate are decomposed to obtain a monosaccharide, and the monosaccharide is hydrolyzed in the presence of the first enzyme to hydrolyze the monosaccharide. The electric signal output from the electrode is measured at least twice at predetermined time intervals, and the α-amylase activity measurement signal in the test solution is obtained based on the obtained electric signals.

In the α-amylase activity measuring method according to the seventh aspect, the first measurement of the electric signal is performed by the endpoint method, and the second measurement of the electric signal is performed by the rate method. In these cases, the first
Examples of the enzyme include GOD, and examples of the second enzyme include α-D-glucosidase, β-D-glucosidase, exo-1,4-α-D-glucosidase, and β-.
Amylase (however, β-amylase needs to be used in combination with any of the above-mentioned enzymes) and the like can be exemplified, and as the reaction substrate, the non-reducing end is a protecting group that is not affected by the second enzyme. A modified substrate may be used, and examples of the protecting group include a benzyl group, an ethylidene group, an isopropylidene group, a benzylidene group, a methyl group, an ethyl group, a dimethyl group, a diethyl group, and a carboxymethyl group. Maltotetraose, maltopentaose, maltodextrin, starch, soluble starch,
Examples thereof include amylose and amylopectin, and examples of the diffusion limiting film include polycarbonate. Further, a hydrogen peroxide electrode or an oxygen electrode can be used as the electrode.

Further, the immobilization includes not only the case where the enzyme is bound to the membrane for a considerably long period of time when the measurement is carried out, but also the case where the enzyme is bound to the membrane to some extent only for a short time after the solution is spotted. Used as a concept.

[0013]

The electrode device for measuring α-amylase activity according to claim 1 holds the predetermined relative position with respect to the measurement surface of the electrode in the presence of α-amylase and α-amylase in the test solution. A diffusion limiting membrane that restricts permeation and diffusion of a disaccharide and / or a polysaccharide that is decomposed into a polysaccharide, a disaccharide and / or a polysaccharide is arranged, and between the measurement surface and the diffusion limiting membrane, Since the first enzyme for hydrolyzing the monosaccharide and the second enzyme for decomposing the disaccharide and / or the polysaccharide into the monosaccharide are arranged, the test solution and the α-amylase When a reaction substrate that is decomposed into a disaccharide and / or a polysaccharide by the method is spotted on the diffusion limiting membrane, it is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase or α-amylase. Penetration of polysaccharides, disaccharides and / or degraded polysaccharides Of α-amylase, which is restricted to reach the position of arrangement of each enzyme, a polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase, a disaccharide and / or a degraded polysaccharide, Not only can the concentration be greatly reduced, but the permeation of interfering substances contained in the test solution can be reliably prevented. Then, due to a large decrease in the concentration of α-amylase, a polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase, a disaccharide and / or a decomposed polysaccharide, The amount of monosaccharides that consume dissolved oxygen by hydrolysis can also be significantly reduced, the upper limit of measurable α-amylase activity can be significantly increased, and the accuracy of α-amylase activity measurement can be significantly increased. .. In addition, the test solution and the supply of the reaction substrate to the electrode device for measuring α-amylase activity need only be spotted, and blood cell separation operation, filtration work, etc. are not required, thus achieving a great improvement in operability. it can.

In the electrode device for measuring α-amylase activity according to claim 2, since the diffusion limiting film is arranged so as to be removable from the electrode, the α-amylase activity should be measured based on the test solution. It is possible to reliably prevent the inconvenience that clogging is caused by the interfering substance due to the above and the permeation diffusion limiting effect is changed due to the clogging by a simple operation of replacing the diffusion limiting film with a new one. Regarding the frequency of replacement, if the measurement based on the test solution that does not contain much interfering substance is repeated, the replacement may be performed every multiple times, but the measurement based on the test solution containing a large amount of interfering substance should be performed. When repeating, it is preferable to replace each measurement.

According to the electrode device for measuring α-amylase activity of claim 3, the first enzyme is immobilized on the membrane and the second enzyme is also immobilized on the membrane, and the second enzyme is immobilized. Since the immobilization membrane is arranged at a predetermined position closer to the diffusion limiting membrane than the first enzyme immobilization membrane, the electrode device for measuring α-amylase activity can be easily integrated, and transportation and storage are significantly facilitated. In addition, the activity of each enzyme can be easily retained.

According to the electrode device for measuring α-amylase activity of claim 4, since the second enzyme-immobilized membrane is arranged so as to be removable from the electrode, the activity of the second enzyme is lowered, Alternatively, when it is deactivated, only the second enzyme-immobilized membrane needs to be replaced with a new one, and the disadvantage that the first enzyme-immobilized membrane must be wastefully discarded can be reliably prevented.

The electrode device for measuring α-amylase activity according to claim 5 further comprises an electrode moving means for moving the electrode in a predetermined direction, and the first enzyme-immobilized membrane,
Since the second enzyme-immobilized membrane and the diffusion limiting membrane are arranged in a state of maintaining a relative positional relationship in which they adhere to each other following the movement of the electrode in the predetermined direction for the predetermined distance, The degree of freedom of arrangement can be increased, and considering that the exchange work of each membrane is performed during non-measurement, the exchange work of each membrane can be significantly simplified.

According to the method for measuring α-amylase activity of claim 6, if the test solution and the reaction substrate which is decomposed into α-amylase into disaccharide and / or polysaccharide are spotted on the diffusion limiting membrane. , Α-amylase, a polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase,
Α-amylase which reaches the position of each enzyme by limiting the permeation and diffusion of disaccharide and / or degraded polysaccharide.
The concentration of the polysaccharide, disaccharide and / or degraded polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase can be significantly reduced, and it can be contained in a test solution. Permeation of interfering substances can be reliably blocked. Then, the test solution and the supply of the reaction substrate to the electrode device for measuring α-amylase activity only need to be spotted, and the blood cell separation operation, the filtration work, etc. are not required, so that a great improvement in operability is achieved. it can.

As described above, the interfering substances are separated and decomposed into α-amylase and disaccharide and / or polysaccharide in the presence of α-amylase. Polysaccharide, disaccharide and / or degradation In the state where the concentration of the polysaccharide is significantly reduced, the test solution from which the interfering substance is separated and the reaction substrate are decomposed in the presence of the second enzyme to obtain a monosaccharide, The hydrolysis reaction of the monosaccharide is performed in the presence, and the electric signal output from the electrode by the hydrolysis reaction of the monosaccharide is measured at least twice at predetermined time intervals, and based on the obtained electric signals. Α in the test solution
Since the amylase activity measurement signal is obtained, the upper limit of measurable α-amylase activity can be significantly increased, and the accuracy of α-amylase activity measurement can be significantly increased.

According to the α-amylase activity measuring method of claim 7, since the first measurement of the electric signal is carried out by the endpoint method, the disaccharide and / or polysaccharide contained in the test solution can be measured. Since the electrical signal at the end of the resulting reaction and at that time can be accurately detected, and the second and subsequent electrical signals are measured by the rate method,
For the subsequent measurements, it is not necessary to wait until the end of the reaction, and the measurement of α-amylase activity can be achieved in a short time.

[0021]

Embodiments will now be described in detail with reference to the accompanying drawings showing embodiments. FIG. 1 is a vertical cross-sectional view schematically showing an embodiment of an electrode device for measuring α-amylase activity of the present invention, in which a hydrogen peroxide selective permeable film 2, a GOD-immobilized film 3, 2 are formed on the surface of a base electrode 1. A conjugated enzyme-immobilized membrane 4 and a diffusion limiting membrane 5 for decomposing sugar and / or polysaccharide into monosaccharides are laminated and integrated in this order.

The base electrode 1 is a hydrogen peroxide electrode,
The center electrode 1a made of platinum or the like and the counter electrode 1b made of silver or the like are integrated by a base body 1c made of an insulating material, and the end faces from which the center electrode 1a and the counter electrode 1b are exposed are formed into convex curved surfaces. Then, the above films are laminated on the convex curved surface and integrated by a cap 1d of a screw-in type, a press-fit type or the like, the central portion of which is opened. The diffusion limiting membrane 5 may be one whose main function is to separate interfering substances such as blood cells, for example, a polycarbonate membrane having a large number of micropores, but α-amylase, a polysaccharide, a disaccharide, It may have a main function of limiting the permeation diffusion of glucose and the like. Further, for example, a conjugate enzyme having 3.4 units of α-glucosidase and 1.6 units of glucoamylase is used, and the conjugate enzyme immobilized on the nonwoven fabric by cross-linking or the like is used as the conjugate enzyme-immobilized membrane 4.

The operation in the case of measuring the α-amylase activity in whole blood as a test solution using the electrode device for measuring α-amylase activity having the above structure will be described with reference to the flowchart shown in FIG. .. Whole blood as a test solution is spotted on the diffusion limiting membrane 5 in step SP1, and a reaction substrate solution {for example, 10 mM BES buffer (pH 7.0), 2 is deposited on the diffusion limiting membrane 5 in step SP2.
% Benzyl maltopentaoxide). When the spotting work in steps SP1 and SP2 is performed, the diffusion limiting film 5 completely blocks the permeation of interfering substances such as blood cells in whole blood in step SP3, and the α-amylase, disaccharide in whole blood is also blocked. And / or the conjugated enzyme-immobilized membrane 4 and the immobilized GOD membrane 3 are reached in this order with diffusion and permeation of the polysaccharide and the reaction substrate being restricted. Therefore, in step SP4, the disaccharide and / or polysaccharide is decomposed into the monosaccharide on the conjugated enzyme-immobilized membrane 4 and the immobilized G is immobilized.
The monosaccharide is hydrolyzed in the OD film 3 to generate hydrogen peroxide, and the hydrogen peroxide reaches the surface of the base electrode 1 through the hydrogen peroxide selective permeable film 2. As a result, step S
At P5, hydrogen peroxide is oxidized and reduced on the surface of the base electrode 1, and a current corresponding to the amount of hydrogen peroxide is output.

On the other hand, in step SP4, the reaction substrate is decomposed by α-amylase into a disaccharide and / or a polysaccharide, and after being decomposed into the disaccharide and / or the polysaccharide, the same disaccharide and / Or the decomposed polysaccharide is decomposed into a monosaccharide and the monosaccharide is hydrolyzed, and an electric current corresponding to the amount of hydrogen peroxide generated is output in step SP5.

However, the rate of decomposition reaction into a monosaccharide starting from a disaccharide and / or a degraded polysaccharide is α-
Since the decomposition reaction rate of polysaccharides by amylase is remarkably fast, the reaction that does not require the presence of α-amylase proceeds early, and the reaction that requires the presence of α-amylase gradually proceeds. The output current characteristics shown in FIG. 3 are obtained. Of this output current characteristic, the portion that rises steeply and is almost saturated is the portion due to the reaction that does not require the presence of α-amylase, and the portion that gradually increases thereafter requires the presence of α-amylase. Since it is the portion caused by the reaction, in steps SP6 and SP7, the output current value at the time of almost saturation and the output current value at the time when a predetermined time has passed after the saturation are measured, and both output currents are measured at step SP8. The measurement of the α-amylase activity in whole blood can be achieved by calculating the increment value (or differential value) of the output current per unit time based on the value and performing the conversion calculation based on the calculated increment value. The above-mentioned point of almost saturation can be detected, for example, by determining whether or not the amount of change in the output current value per unit time is below a predetermined threshold value. In addition, α-based on the increment value
Regarding the measurement of amylase activity, for example, a calibration curve is obtained by calculating an increment value using a reference solution having a known α-amylase activity as a test solution, and the α-amylase activity is increased based on an unknown test solution. The α-amylase activity may be measured based on the value and the calibration curve.

However, better measurement results were obtained by using the pH 5.7 BS buffer instead of the pH 7.0 BES buffer. Therefore, it is preferable to keep the reaction substrate solution slightly acidic. Specifically, in order to maintain the activity of each of the above-mentioned enzymes, the immobilized GOD membrane 3 is used at the time of non-measurement.
And the conjugated enzyme-immobilized membrane 4 is moistened with a solution having a pH of 7.0, and a slightly acidic reaction substrate solution is spotted at the time of measurement to ensure good measurement of α-amylase activity and good preservation of each enzyme. Can be achieved.

[0027]

[Embodiment 2] FIG. 4 is a vertical cross-sectional view schematically showing another embodiment of the electrode device for measuring α-amylase activity of the present invention. The difference from the embodiment of FIG. Cap 1 with membrane 2 and immobilized GOD membrane 3 stacked
It is attached only to the base electrode 1 by e, and is detachably attached to the immobilized GOD film 3 in a laminated state of the conjugated enzyme immobilization film 4 and the diffusion limiting film 5 with the cap 1f so as to be detachable.

Therefore, in the case of this embodiment, the exchange of the conjugated enzyme immobilization membrane 4 and the diffusion limiting membrane 5 can be easily achieved. As a result, it is possible to replace only the necessary membrane portion while leaving the immobilized GOD membrane 3 having a significantly longer life than the conjugated enzyme-immobilized membrane 4, and thus the immobilized GOD membrane 3 of the embodiment shown in FIG. Effective utilization can be achieved.

[0029]

[Embodiment 3] FIG. 5 is a vertical sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity according to the present invention. The difference from the embodiment of FIG. 1 g of a cap having a diffusion limiting membrane 6 having a main function of limiting the permeation and diffusion of α-amylase, a polysaccharide, a disaccharide, glucose and the like, and a separation of interfering substances such as blood cells. It is only the point that was detachably attached.

Therefore, in the case of this embodiment, the diffusion limiting membrane 6 is considerably clogged by measuring the α-amylase activity in a test solution containing a lot of interfering substances such as whole blood. For example, highly accurate measurement of α-amylase activity can be achieved by replacing the diffusion limiting film 6 with a new one for each measurement. Further, if the diffusion limiting film 6 is replaced, the diffusion transmittance of the diffusion limiting films 5 and 6 as a whole changes, but since the diffusion limiting film 5 is not replaced, the fluctuation of the diffusion transmittance is greatly changed. It can be reduced.

[0031]

[Embodiment 4] FIG. 6 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention. The difference from the embodiment of FIG. A point further provided with an electrode moving mechanism 7 such as a ball screw mechanism for reciprocating between the non-measurement position and the measurement position, and the cap 1f of the conjugated enzyme immobilization film 4 and the diffusion limiting film 5 with respect to the base electrode 1. Instead of removably attaching with,
In a non-measurement state, the conjugated enzyme immobilization membrane 4 and the diffusion limiting membrane 5 are arranged at predetermined positions apart from the immobilized GOD membrane 3.
The only difference is that the conjugated enzyme-immobilized membrane 4 and the diffusion limiting membrane 5 are brought into close contact with the immobilized GOD membrane 3 in this order in the measurement state.

Therefore, in the case of this example, since all the membranes are brought into close contact only in the measurement state, the measurement of α-amylase activity can be achieved without any inconvenience, and in the non-measurement state, the conjugated enzyme-immobilized membrane 4 and Since the diffusion limiting film 5 is separated from the immobilized GOD film 3, the replacement work of these films can be easily achieved. It is also possible to bring the immobilized GOD film 3 of the base electrode 1 into contact with a wetting liquid having a pH of 7.0 at the time of non-measurement. In this case, the GOD is maintained even when the non-measurement state continues for a long period of time. And the marked decrease in the amount of dissolved oxygen associated with the enzymatic reaction of glucose can be recovered in a short time.
-The time interval of amylase activity measurement can be shortened.

[0033]

[Embodiment 5] FIG. 7 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention. The difference from the embodiment of FIG. The only difference is that the film 4 and the diffusion limiting film 5 are attached to the thin plate member 8. More specifically, a thin plate 8 made of a material having resistance to the test solution and the reaction substrate solution has a through hole 8a formed at a predetermined position to allow passage of the test solution and the reaction substrate solution. The conjugated enzyme immobilization film 4 and the diffusion limiting film 5 are attached so as to cover the through holes 8a. Both films 4 and 5 are attached to different surfaces of the thin plate member 8 so that a predetermined clearance is formed between the films 4 and 5. Specifically, the conjugated enzyme-immobilized film 4 is attached to the side close to the base electrode 1.

Therefore, in the case of this embodiment, only by having the thin plate material 8, the conjugated enzyme immobilization film 4 and the diffusion limiting film 5 are provided.
Both membranes 4 and 5 can be replaced with new membranes without touching any of them, and the occurrence of an infection accident can be reliably prevented when body fluid is used as the test solution. Further, since the thin plate material 8 is handled not directly handling a film having a remarkably thin thickness,
Workability such as replacement can be significantly improved.

However, it goes without saying that both the films 4 and 5 may be attached to the same side of the thin plate member 8 in a laminated state in this embodiment. In this case, the adhered state of both membranes 4 and 5 can be stabilized, the accuracy of measurement of α-amylase activity can be improved, and the manufacturing work can be facilitated.

[0036]

[Embodiment 6] FIG. 8 is a vertical sectional view schematically showing still another embodiment of the electrode device for measuring .alpha.-amylase activity of the present invention. The difference from the embodiment of FIG. The only difference is that a remarkably long sheet is used, a plurality of through holes 8a are formed in a long thin plate member 8, and both films 4 and 5 are attached so as to cover each through hole 8a. In order to hold the long thin plate material 8 in a state where the space utilization efficiency is improved, one is used as a supply portion 9a for the thin plate material 8 and the other is used as a winding portion 9b for the thin plate material 8 and is wound with the supply portion 9a. Bridge part 9c connecting between the taking part 9b
The thin plate member 8 is housed in the cartridge 9 having the. Then, a spotting portion 9d that allows the test solution and the reaction substrate solution to be attached and an electrode penetration portion 9e that allows the undercoat electrode 1 to penetrate are formed at predetermined positions of the bridge portion 9c. Further, a thin plate driving mechanism (not shown) for moving the thin plate 8 in a predetermined direction is provided.

Therefore, in the case of this embodiment, the thin plate member 8 is exchanged until the α-amylase activity is measured using all the films 4 and 5 attached to the long thin plate member 8. There is no need. Therefore, the frequency of exchanging the thin plate member 8 can be remarkably reduced, and the operability can be remarkably enhanced. Furthermore, the thin plate material 8 that should be discarded along with the replacement
Since the is stored in the cartridge 9, it is possible to reliably prevent the occurrence of an infection accident due to the body fluid adhering to the thin plate material 8 to be discarded. Furthermore, the base electrode 1
Since the thin plate member 8 is moved in a predetermined direction in a state where the is lowered, it is possible to prevent the activity of the immobilized GOD film 3 from being lowered due to the rubbing.

[0038]

[Embodiment 7] FIG. 9 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention. The difference from the embodiment of FIG. Only the diffusion limiting film 5 is attached to the plate member 8, and the conjugated enzyme immobilization film 4 is attached so as to cover the electrode penetration portion 9e of the bridge portion 9c.

Therefore, in the case of this embodiment, only the diffusion limiting film 5 can be replaced with a new diffusion limiting film 5 by moving the thin plate member 8 in a predetermined direction, and all diffusions attached to the thin plate member 8 can be exchanged. While the measurement is performed a plurality of times using the limiting membrane 5, the use of one conjugated enzyme-immobilized membrane 4 is continued.
As a result, the amount of the coupled enzyme discarded along with the replacement of the cartridge 9 can be significantly reduced as compared with the embodiment of FIG. Further, in this example, since the conjugated enzyme-immobilized film 4 is adhered to the lower surface of the bridge portion 9c, the inner surface of the bridge portion 9c is rubbed with the movement of the thin plate member 8 and the activity is reduced. It is possible to prevent the occurrence of the inconvenience.

The present invention is not limited to the above-mentioned embodiment, and for example, immobilizing the conjugated enzyme on the membrane by freeze-drying or the like, and including the conjugated enzyme instead of immobilizing the conjugated enzyme on the membrane. It is possible to arrange a solution tank, it is possible to omit the hydrogen peroxide selective permeable membrane 2, and it is possible to use a mounting aid and a mounting assist mechanism other than a cap, and a coupled enzyme. It is possible to interpose a solution layer containing a conjugated enzyme instead of immobilizing the enzyme on the membrane, and further, a diffusion limiting membrane is arranged between the immobilized GOD membrane 3 and the conjugated enzyme (immobilized membrane). Besides, it is possible to make various design changes within a range not changing the gist of the present invention.

[0041]

As described above, the invention of claim 1 is characterized in that α-amylase, a polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase, a disaccharide and / or a degradation product. Α-amylase that reaches the position of each enzyme by limiting the permeation and diffusion of the polysaccharide, a polysaccharide that is decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase,
Not only can the concentration of disaccharides and / or decomposed polysaccharides be significantly reduced, but permeation of interfering substances contained in the test solution can be surely blocked, and in the presence of α-amylase and α-amylase, The amount of monosaccharide that consumes dissolved oxygen by hydrolysis is increased due to a large decrease in the concentration of the polysaccharide, which is decomposed into the sugar and / or the polysaccharide, the disaccharide, and / or the decomposed polysaccharide. Since the upper limit of measurable α-amylase activity can be significantly increased, the accuracy of α-amylase activity measurement can be significantly improved, and the electrode device for measuring α-amylase activity can be significantly reduced. The supply of the test solution and the reaction substrate only needs to be spotted, and the blood cell separation operation, the filtration operation, and the like are not required, so that a significant improvement in operability can be achieved.

The invention of claim 2 is an α-based solution based on a test solution.
Measure amylase activity. It is possible to reliably prevent the inconvenience that clogging occurs due to the interfering substances and the permeation diffusion limiting effect fluctuates due to the clogging with a simple operation of replacing the diffusion limiting film with a new one. Has a unique effect. The invention of claim 3 is
The electrode device for measuring α-amylase activity can be easily integrated, and transportation, storage, etc. can be significantly facilitated, and activity retention of each enzyme can be easily achieved.

According to the invention of claim 4, when the activity of the second enzyme is reduced or inactivated, only the second enzyme-immobilized membrane is replaced with a new one, and the first enzyme-immobilized membrane is used. The unique effect of reliably preventing the inconvenience of having to discard wastefully is achieved. The invention of claim 5 is
It is possible to increase the degree of freedom in arranging each film, and it is possible to significantly simplify the replacement work of each film which is performed at a timing commensurate with the life of each film.

According to the sixth aspect of the present invention, the test solution and the reaction substrate which is decomposed into a disaccharide by α-amylase are simply spotted on the diffusion limiting membrane in the presence of α-amylase and α-amylase. Of α-amylase and α-amylase that reach the position of each enzyme by limiting the permeation / diffusion of the disaccharide and / or the degraded polysaccharide The concentration of polysaccharides, which are decomposed into disaccharides and / or polysaccharides in the presence, can be significantly reduced, and permeation of interfering substances contained in a test solution can be achieved. Can be reliably prevented, and by extension, the upper limit of the measurable α-amylase activity can be significantly increased, and the accuracy of α-amylase activity measurement can be significantly increased. Since the test solution and the supply of the reaction substrate to the tester need only be spotted, the blood cell separation operation, the filtration operation, etc. are not required, so that a significant improvement in operability can be achieved.

According to the invention of claim 7, the electric signal at the end of the reaction due to the disaccharide contained in the test solution and its rotation can be accurately detected, and the electric signal after the second time can be measured by the rate method. Therefore, it is not necessary to wait until the end of the reaction for the second and subsequent measurements, and there is a unique effect that the measurement of α-amylase activity can be achieved in a short time.

[Brief description of drawings]

FIG. 1 is a longitudinal sectional view schematically showing an embodiment of an electrode device for measuring α-amylase activity of the present invention.

FIG. 2 is a flow chart for explaining the action when the α-amylase activity measuring electrode device having the configuration of FIG. 1 is used to measure the α-amylase activity in whole blood as a test solution.

FIG. 3 is a diagram showing a time change of an output current from a base electrode.

FIG. 4 is a vertical sectional view schematically showing another embodiment of the electrode device for measuring α-amylase activity of the present invention.

FIG. 5 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention.

FIG. 6 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention.

FIG. 7 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention.

FIG. 8 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention.

FIG. 9 is a longitudinal sectional view schematically showing still another embodiment of the electrode device for measuring α-amylase activity of the present invention.

[Explanation of symbols]

1 Base Electrode 3 Immobilized GOD Membrane 4 Coupling Enzyme Immobilized Membrane 5 Diffusion Restriction Membrane 7 Electrode Transfer Mechanism

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Claims (7)

[Claims] 1. A predetermined relative position with respect to the measurement surface of an electrode (1) is held and decomposed into a disaccharide and / or a polysaccharide in the presence of α-amylase or α-amylase in a test solution. A diffusion limiting membrane (5) for limiting permeation and diffusion of the polysaccharide, disaccharide and / or polysaccharide
Between the measurement surface and the diffusion limiting membrane (5), a first enzyme (3) for performing a hydrolysis reaction of a monosaccharide, a disaccharide and / or
Alternatively, an electrode device for measuring α-amylase activity, which is provided with a second enzyme (4) that decomposes a polysaccharide into a monosaccharide. 2. The electrode device for measuring α-amylase activity according to claim 1, wherein the diffusion limiting film (5) is detachably arranged with respect to the electrode (1). 3. The first enzyme (3) is immobilized on the membrane and the second enzyme (4) is also immobilized on the membrane, and the second enzyme-immobilized membrane (4) is The α-amylase activity measuring electrode device according to claim 1 or 2, which is arranged at a predetermined position closer to the diffusion limiting film (5) than the enzyme-immobilized film (3). 4. The second enzyme-immobilized membrane (4) has an electrode (1).
The α-amylase activity measuring electrode device according to claim 3, wherein the electrode device is detachably arranged. 5. A first enzyme-immobilized membrane (3), a second enzyme-immobilized membrane (4) and an electrode moving means (7) for moving the electrode (1) in a predetermined direction. The diffusion limiting film (5) is arranged in a state of maintaining a relative positional relationship in which the diffusion limiting film (5) follows the movement of the electrode (1) in a predetermined direction by a predetermined distance and is in a close contact with each other. An electrode device for measuring α-amylase activity according to item 1. 6. An electrode device comprising a first enzyme-immobilized membrane (3), a second enzyme-immobilized membrane (4) and a diffusion limiting membrane (5) arranged on the surface of an electrode (1). The reaction solution obtained by modifying the test solution and the non-reducing end with a protecting group is spotted in a predetermined order, and the interfering substance contained in the test solution is separated by the diffusion limiting membrane (5) to obtain the second enzyme. In the presence of (4), the test solution from which the interfering substance is separated and the reaction substrate are decomposed to obtain a monosaccharide, and the monosaccharide is hydrolyzed in the presence of the first enzyme (3). The electrical signal output from the electrode (1) by the hydrolysis reaction of the monosaccharide is measured at least twice at predetermined time intervals, and α- in the test solution is measured based on the obtained electrical signals. A method for measuring α-amylase activity, which comprises obtaining an amylase activity measurement signal. 7. The method for measuring α-amylase activity according to claim 6, wherein the first measurement of the electric signal is performed by the end point method, and the second and subsequent measurement of the electrical signal is performed by the rate method.