JPH1019832A - Measuring method for concentration by using oxidoreductase immobilized biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method, for a concentration, in which an influence excerted on the measuring output of a measurement obstruction substance can be removed by installing one set of electrodes at an enzyme sensor and which uses an oxidoreductase immobilized biosensor. SOLUTION: A substance solution to be tested is brought into contact with, and reacted with, a biosensor wherein a working electrode A in which an oxidoreductase is immobilized on an identical substrate, a working electrode B in which at least one kind out of a deactivation oxidoreductase and an albumin is immobilized or a working electrode B which does not comprise an immobilized substance and a common counter electrode C for them are installed. After that, an identical voltage is applied simultaneously across the electrodes A, C and across the electrodes B, C, and the output difference after a deinite time in a current generated between the electrodes is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring concentration using an oxidoreductase-immobilized biosensor. More specifically, the present invention relates to a concentration measuring method using an oxidoreductase-immobilized biosensor capable of eliminating the influence of an interfering substance contained in a test substance solution on the measurement output.

[0002]

2. Description of the Related Art An enzyme sensor is known in which an oxidoreductase such as glucose oxidase, pyruvate oxidase, lactate oxidase, or alcohol oxidase is immobilized on an electrode. For example, in the case of a glucose sensor, it does not contain glucose. A potentiostatic current measurement method of immersing in a buffer solution, adding glucose thereto while a voltage is applied to the electrode, and measuring a current value flowing in proportion to the glucose concentration is generally used.

[0003] However, such a potentiostatic current measuring method has a problem that it is generally difficult to remove the influence on the measurement output of a measurement interfering substance such as uric acid and ascorbic acid contained in the test substance. Can be

[0004] In order to solve such problems, the present applicant has previously proposed the following enzyme sensor. (1) An enzyme in which an enzyme-immobilized film immobilized on at least the anode electrode is provided for at least one of two combinations of an anode electrode and a cathode electrode composed of a metal thin film formed on the same insulating substrate. Sensor (JP-A-62-32351
(2) In the enzyme sensor of the above (1), an enzyme sensor provided with a deactivated enzyme-immobilized membrane immobilized on at least the anode electrode is provided on the other set of combination electrodes (see JP-A-62-75346). Gazette,
No. 62-237348) (3) Three or more pairs of anode electrodes and cathode electrodes formed of a metal thin film formed on the same insulating substrate are provided, and different enzymes (at least one Is an inactivated enzyme) immobilized conjugated enzyme sensor (JP 62-235556 A)

[0005] In each of these measuring methods, although the object of eliminating the influence of the measuring interfering substance on the measurement output, which has been a problem of the constant potential current measuring method, can be achieved, two or more sets of combination electrodes must be provided. In this case, there is a problem in manufacturing the sensor.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide an oxidoreductase-immobilized biosensor which can eliminate the influence of a measurement interfering substance on the measurement output even if only one set of combination electrodes is provided in the enzyme sensor. An object of the present invention is to provide a method for measuring the concentration to be used.

[0007]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
A working electrode A having oxidoreductase immobilized thereon, a working electrode B having at least one of inactivated oxidoreductase and albumin immobilized thereon, a working electrode B having no immobilized substance, and a common counter electrode C thereof were provided on the same substrate. After a test substance solution was brought into contact with the biosensor to cause a reaction,
This is achieved by a method of measuring the concentration of a test substance using an oxidoreductase, in which the same voltage is simultaneously applied between the C electrodes and the output difference of the current generated between these electrodes after a certain time is measured.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a potential step differential measurement system is used in order to provide selectivity to a measurement interfering substance in a test substance. For this purpose, a biosensor having two working electrodes and one common counter electrode is used.

An oxidoreductase is immobilized on the working electrode A. For example, when glucose oxidase (GOD) is immobilized as an oxidoreductase by any commonly used method, not only glucose but also ascorbic acid or the like as a measurement interfering substance is used as a test substance in the working electrode A. They are also sensitive to electrodes when contained within.
The output at this time is the sum of the output O _AI to the output O _AG and interfering substances on the glucose.

On the other working electrode B, for example, inactivated GOD is immobilized. Such a dead GOD
, The glucose does not respond, but only the interfering substance responds. If the output in this case is O _BI , then O _AI = O _BI .

Therefore, if the output of the working electrode B is subtracted from the output of the working electrode A, the output difference O _AG becomes only the output for glucose, and the output for the interfering substance is cancelled.
This gives the selectivity of the measurement.

An example of a combination of those fixed to the working electrodes A and B is as follows. In this case, the inactivated oxidoreductase can be considered as a protein that has lost its function, and can be replaced with albumin. In immobilizing the enzyme, glutaraldehyde, photocrosslinkable PVA (polyvinyl alcohol) and the like are also used. Furthermore, mediators such as potassium ferricyanide and parabenzoquinone
(Electronic carrier), the linear calibration range
It can be expanded from 0 to 100 mg / dl to about 0 to 1000 mg / dl.

Working electrode A / B immobilized membrane oxidoreductase / inactivated oxidoreductase oxidoreductase / albumin oxidoreductase-mediator / inactivated oxidoreductase-mediator oxidoreductase-mediator / albumin-mediator oxidoreductase -Glutaraldehyde / inactivated oxidoreductase-glutaraldehyde oxidoreductase-glutaraldehyde-mediator / inactivated oxidoreductase-glutaraldehyde-mediator oxidoreductase-glutaraldehyde-albumin / inactivated oxidoreductase-glutaraldehyde-albumin Oxidoreductase-albumin / inactivated oxidoreductase-albumin oxidoreductase-albumin-glutaraldehyde-mediator / inactivated oxidoreductase-albumin-glutaraldehyde-mediator oxidoreductase-albumin / albumin oxidoreductase-albu Min-glutaraldehyde-mediator / albumin-glutaraldehyde-mediator oxidoreductase-glutaraldehyde / albumin-glutaraldehyde oxidoreductase-photocrosslinkable PVA / deactivated oxidoreductase-photocrosslinkable PVA oxidoreductase-photocrosslinking PVA / albumin-photocrosslinkable PVA oxidoreductase-photocrosslinkable PVA-mediator / inactivated oxidoreductase-photocrosslinkable PVA-mediator oxidoreductase-photocrosslinkable PVA-mediator / albumin-photocrosslinkable PVA -Mediator oxidoreductase-glutaraldehyde-mediator / albumin-glutaraldehyde-mediator oxidoreductase-glutaraldehyde-albumin / albumin-glutaraldehyde oxidoreductase-glutaraldehyde-albumin / inactivated oxidoreductase-glutaraldehyde Oxidation Reductase-albumin-glutaraldehyde-mediator / inactivated redox enzyme-glutaraldehyde-mediator

In the case where the working electrode B has no immobilized substance,
Although the characteristics are slightly reduced, they can be used. In this case, the following are fixed to the working electrode A. Oxidoreductase oxidoreductase-albumin oxidoreductase-mediator oxidoreductase-glutaraldehyde oxidoreductase-glutaraldehyde-mediator oxidoreductase-glutaraldehyde-albumin oxidoreductase-photocrosslinkable PVA oxidoreductase-photocrosslinkable PVA-mediator oxidoreductase-albumin-glutaraldehyde-mediator

As the insulating substrate on which the working electrodes A and B are provided, ceramics, glass, plastic, paper, and biodegradable materials (eg, microbial polyester) are used. On these insulating substrates, two working electrodes A and B and one common counter electrode C are formed by platinum, screen printing, vapor deposition, sputtering, or the like.
It is formed from gold, carbon, or the like. Thereafter, the central portion of each electrode is covered with a resin insulating film or the like.

A method for immobilizing glucose oxidase (GOD), which is a typical example of an oxidoreductase, will be described. For example, in the case of GOD of 165800 units / g, about 1 to 50 m
g, preferably about 1-20 mg, is dissolved in 1 ml of buffer or distilled water, and about 0.5-10 μl, preferably about 0.5-2 μl, of the solution is dissolved.
1 is dropped on the working electrode by a dropping method, a spin coating method, or the like, and dried at room temperature to form a layer having a thickness of about 0.05 to 10 μm, preferably about 0.1 to 2 μm.

At the time of immobilizing GOD, glutaraldehyde or photocrosslinkable PVA can be used to make the immobilization more complete, as seen in the above-mentioned combination examples. In the case of glutaraldehyde: glutaraldehyde is GO
It is used by being added in such an amount that the final concentration in the D solution is about 0.001 to 0.4% by weight, preferably about 0.1 to 0.3% by weight. In the case of photocrosslinkable PVA: PVA containing a photocrosslinkable group such as a stilbazolium group and a styrylpyridinium group in an amount of about 0.5 to 3.5 mol% (a saponification value of about 70 to 100 and a polymerization degree of about 500 to 400)
0) is used as a GOD solution added in an amount to give a concentration of about 5 to 50% by weight, preferably about 10 to 20% by weight,
After applying the GOD solution on the working electrode, at about 10-30 ° C. for about 0.
Dry for about 5 to 2 hours, then apply ultraviolet light for a predetermined time (about 0.5
(Eg, about 10 minutes) to form a photocrosslinked film having a thickness of about 0.05 to 10 μm, preferably about 0.1 to 2 μm, by irradiating with light or the like, washing and developing as necessary.

The GOD solution may be further added with albumin or a mediator, as in the above-mentioned combination examples, to further improve the linear calibration property thereof. In the case of albumin: Bovine serum albumin is used by further adding about 1 to 100 mg, preferably about 1 to 20 mg, to the GOD solution. For mediators: about 1-100 mg, preferably about 40-60 mg, for potassium ferricyanide, and about 1-200 mg, preferably about 100-2, for parabenzoquinone.
00 mg is used in addition to the GOD solution.

On the working electrode B side, when used as an inactivated oxidoreductase, the immobilized oxidoreductase is treated at about 40 to 70 ° C. for about 1 to 30 minutes, preferably at about 60 ° C. for about 10 minutes. An oxidoreductase solution that has been immobilized by heat treatment or that has been heat-treated in advance under such heat treatment conditions is dropped onto the working electrode B, and immobilization is performed by the method described above.

The measurement of the concentration of a test substance using a biosensor provided with these working electrodes A and B and their common counter electrode C is carried out by bringing a test substance solution into contact with the biosensor and causing a reaction between the AC electrodes. , And the same voltage is applied simultaneously between the BC electrodes, and the output difference of the current generated between these electrodes after a certain time is measured.

Generally, about 5 to 100 μl of the test substance solution is dropped on the biosensor so as to come into contact with the working electrodes A and B and the common counter electrode C, depending on the area of each electrode and the distance between the electrodes. After reacting for about 1 to 120 seconds, about 0.
A voltage of 1 to 0.9 V is applied simultaneously between the AC electrodes and between the BC electrodes, and measurement is made in a differential form about 1 to 120 seconds after the application.

[0022]

According to the method of the present invention, not only can the effect of measurement interfering substances on the measurement output be eliminated in the concentration measurement by the oxidoreductase-immobilized biosensor,
Also, a significant improvement in selectivity is achieved without a substantial reduction in the coefficient of variation.

[0023]

Next, the present invention will be described by way of examples.

EXAMPLE 1 Working electrodes A and B (area 0.16 cm ² ) and their common counter electrode C, both made of carbon, were screen-printed on a polyethylene terephthalate film (thickness 0.25 mm).
Was formed with a thickness of 5 μm so that the working electrodes A and B were located on both sides of the common counter electrode C.

GOD 10 mg in 1 ml of pH 7.0 phosphate buffer
And a solution in which 48 mg of potassium ferricyanide is dissolved 1.
5 μl was dropped on the working electrode A and dried at room temperature to form a 1 μm-thick GOD-immobilized membrane containing a mediator. Thereafter, the same GOD-potassium ferricyanide solution was heat-treated at 60 ° C. for 10 minutes to inactivate the mixture, and 1.5 μl of the deactivated GOD-potassium ferricyanide solution was added thereto.
The solution was dropped on the working electrode B and dried at room temperature.
Was formed thereon.

On each electrode surface of the GOD-immobilized biosensor having the two working electrodes A and B and the common counter electrode C, 40 μl of a predetermined concentration of an aqueous glucose solution was dropped and reacted for 30 seconds. Thereafter, a voltage of 0.6 V was simultaneously applied between the AC electrodes and between the BC electrodes, and the current value 10 seconds after the application was measured in a differential format.

As a result, a result indicated by a circle in the graph of FIG. 1 was obtained. From this result, it can be seen that the calibration was obtained within the range of the aqueous glucose solution concentration of 0 to 1000 mg / dl. Each sensor was disposable every time one sample was measured. In addition, the coefficient of variation indicating the reproducibility (n = 10) and selectivity (output of ascorbic acid when the output of the aqueous solution of glucose 100 mg / dl is 100) is also measured when the concentration of the aqueous glucose solution is 100 mg / dl. Was done.

Example 2 In Example 1, bovine serum albumin 10 was added on the working electrode B.
mg and 48 mg of potassium ferricyanide in phosphate buffer 1
1.5 μl of the solution dissolved in ml was dropped and dried at room temperature to form a 1 μm-thick mediator-containing albumin-immobilized membrane. The current value measured in the differential format is indicated by △ in the graph of FIG.

Example 3 In Example 1, no fixed film was formed on the working electrode B. The current value measured in the differential format is indicated by ▽ in the graph of FIG.

COMPARATIVE EXAMPLE In Example 3, the measurement method without using the common counter electrode C was performed, that is, the measurement was not of the differential type, and only the output of the working electrode A was measured. The measured current value is indicated by ◇ in the graph of FIG.

The coefficient of variation and selectivity in each of the above Examples and Comparative Examples are shown in the following table.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between glucose aqueous solution concentration and output in Examples 1 to 3 and Comparative Example.

Claims (2)

[Claims] 1. A biosensor provided with a working electrode A on which an oxidoreductase is immobilized on the same substrate, a working electrode B on which at least one of inactivated oxidoreductase and albumin is immobilized, and a common counter electrode C thereof, After the test substance solution is brought into contact with and reacted, the same voltage is simultaneously applied between the AC electrode and the BC electrode, and the output difference between the electrodes after a certain period of time is measured. A method for measuring the concentration of a test substance using a reductase. 2. A test substance solution is brought into contact with a biosensor provided with a working electrode A having an oxidoreductase immobilized thereon, a working electrode B having no immobilized substance, and a common counter electrode C on the same substrate to cause a reaction. And then applying the same voltage between the AC electrodes and between the BC electrodes at the same time, and measuring the output difference of the current generated between these electrodes after a certain period of time. .