JPH09243599A - Quantitative method of substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the more highly accurate quantitative method of a substrate. SOLUTION: A biosensor having the electrode system comprising an acting electrode 4 and a counter electrode 5 formed on an insulating substrate 1 and a reaction layer including at least oxidoreductase and an electron acceptor is used. The change in concentration of material in the reaction between the substrate in a sample and the oxidoreductase and the electron acceptor is detected, based on the current response obtained by applying a voltage across the acting electrode 4 and the counter electrode 5. In this method, the application of the voltage for obtaining the current response is started from a potential E1 of the acting electrode 4 with the counter electrode 5 as the reference, and the current value at a potential E2 satisfying the condition of E2>E1 is detected as the current response after the elapse of the specified time from the application of E1.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a quantification method for quantifying a substrate such as glucose in a sample using a biosensor.

[0002]

2. Description of the Related Art A glucose quantification method will be described as an example of a substrate quantification method. As a method of electrochemically determining glucose, glucose oxidase (EC
1.1.3.4) and an oxygen electrode or a hydrogen peroxide electrode are generally known (for example,
"Biosensor" by Shuichi Suzuki, Kodansha). Glucose oxidase uses oxygen as an electron acceptor,
-D-glucose is selectively oxidized to D-glucono-δ-lactone. With this reaction, oxygen is reduced to hydrogen peroxide. Glucose can be quantified by measuring the oxygen consumption at this time with an oxygen electrode or the production amount of hydrogen peroxide with a hydrogen peroxide electrode.

According to the above method, as can be inferred from the reaction process, the measurement result is greatly affected by the concentration of oxygen dissolved in the sample solution. Further, the measurement becomes impossible under the condition without oxygen.

Therefore, without using oxygen as an electron acceptor,
A new type of glucose sensor using an organic compound such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or a metal complex as an electron acceptor has been developed.
In this type of sensor, a reduced form of an electron acceptor generated as a result of an enzymatic reaction is oxidized at an electrode, and the glucose concentration is obtained from the oxidation current.

Further, when such an electron acceptor is used instead of oxygen, it becomes possible to accurately carry a known amount of glucose oxidase and the electron acceptor on the electrode in a stable state. In this case, the electrode system and the reaction layer can be integrated in a state close to a dry state. Disposable glucose sensors based on this technology have recently attracted much attention because glucose concentration can be easily measured simply by introducing a specimen sample into a sensor chip inserted in a measuring instrument. . Such a method is applicable not only to the quantification of glucose but also to the quantification of other specific compounds.

[0006]

The technique for integrating the electrode system and the reaction layer using the electron acceptor as described above has made it possible to carry out a simple electrochemical quantitative evaluation of the substrate.

In particular, as for the biosensor for measuring blood glucose level, a lot of sensors of the same type have been developed due to the development of the above-mentioned technology, and the market competition is intensifying. Diabetics, who are the primary users of sensors, are also on the rise.

Under such circumstances, there is a demand for higher performance of the sensor characteristics, and the development competition thereof is intensifying.

[0009]

In order to achieve the above high performance, the present invention provides an electrode system comprising a working electrode and a counter electrode formed on an insulating substrate, and at least a redox enzyme and an electron acceptor. By using a biosensor having a reaction layer containing, by applying a voltage between the working electrode and the counter electrode, the change in the substance concentration during the reaction of the substrate in the sample with the redox enzyme and the electron acceptor is performed. In the quantitative method of detecting based on the obtained current response, the applied voltage for obtaining the current response starts from the working electrode potential with the counter electrode as a reference, and after a certain time has passed from the application of the working electrode potential, the applied working electrode potential. And a method of quantifying a substrate characterized by detecting a current value at a potential under a specific condition as a current response.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The method for quantifying a substrate of the present invention is not limited to the following examples, and the values of E1, E2, E3, application time and combination method, the values of E4, E5, E6, application, etc. The time, the combination method, and the like are not limited to this.

In the embodiment, the potential sweep speed is 60 m.
Although V / s is set, it is not limited to this, and is 50 to
Good response characteristics can be obtained even at 200 mV / s.
Further, the potential sweep is not limited to the linear function.

Further, although glucose oxidase was used as the oxidoreductase in the examples, it is not limited to this. A lactate sensor using lactate oxidase or lactate dehydrogenase as a redox enzyme, a glucose sensor using glucose dehydrogenase, a cholesterol sensor using cholesterol oxidase or cholesterol dehydrogenase, a uric acid sensor using uricase, a glucose oxidase, a combination of invertase, Even in a sucrose sensor using a combination of glucose oxidase, invertase, mutarotase, fructose dehydrogenase, invertase, fructose dehydrogenase, invertase, mutarotase, etc., the same effect as the glucose sensor described in the examples can be obtained.

In the embodiment, CM is used as the hydrophilic polymer.
C was used, but not limited thereto, polyvinylpyrrolidone, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts. , And cellulose derivatives, specifically, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose,
The same effect can be obtained by using ethyl hydroxyethyl cellulose or carboxymethyl ethyl cellulose.

On the other hand, as the electron acceptor, potassium ferricyanide shown in Examples is excellent in stability and the like, but p-benzoquinone, ferrocene and the like can also be used in addition thereto. Furthermore, in the embodiment, the two-electrode system including the measurement electrode and the counter electrode is described, but the same effect can be obtained in the three-electrode system including the reference electrode.

Further, in the examples, the method of dissolving the reaction layer in the sample solution has been shown, but the present invention is not limited to this and can be applied to the case where the reaction layer is insolubilized in the sample solution by immobilization. .

The present invention will be described below with reference to examples. (Example 1) As an example of a quantification method, quantification of glucose will be described. First, the glucose sensor used in the examples will be described. FIG. 1 shows an exploded perspective view of the glucose sensor excluding the reaction layer. Polyethylene terephthalate
The insulative substrate 1 made of a resin has leads 2, 3 formed by screen-printing a silver paste on the surface thereof.
Having. After forming the leads 2 and 3, a conductive carbon paste containing a resin binder is printed to form the working electrode 4. The working electrode 4 is in contact with the lead 2.

Next, an insulating paste is printed by printing an insulating paste.
Is formed. The insulating layer 6 covers the outer peripheral portion of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant. Further, the insulating layer 6 partially covers the leads 2 and 3.

Next, a conductive carbon paste containing a resin binder is printed so as to be in contact with the lead 3, and the counter electrode 5 is formed.
Is formed. A CMC layer is formed by dropping an aqueous solution of carboxymethyl cellulose (hereinafter referred to as CMC) onto the above electrode system (working electrode 4, counter electrode 5) and drying. Further, an aqueous solution containing glucose oxidase as an enzyme and potassium ferricyanide as an electron acceptor is dropped on the electrode system and dried to form a reaction layer 7 partially mixed with the CMC layer.

Next, in order to make the supply of the sample solution onto the reaction layer 7 smoother, an organic solvent solution of lecithin, such as a toluene solution, is spread over the reaction layer from the sample supply section (sensor tip). The lecithin layer 8 is formed by drying. Finally, Cover-9 and Spacer-1
No. 0 is bonded in a positional relationship as shown by the one-dot chain line in FIG. In this way, the glucose sensor is manufactured. FIG.
FIG. 3 is a vertical sectional view of the glucose sensor.

As a sample solution, 3 μl of an aqueous glucose solution was supplied to the sensor having the structure shown in FIG.
The sample solution reached the air holes 11 and the reaction layer on the electrode system was dissolved. After 55 seconds from the supply of the sample, 200 mV (E1) was applied to the working electrode 4 with the counter electrode 5 as a reference, and simultaneously with the application, the potential was swept to the positive potential side at 60 mV / s.
When the current value at 500 mV (E2) was measured 5 seconds after the potential was applied and plotted against the glucose concentration,
Good linearity was obtained. The change with time of the applied potential is shown in FIG. Compared to the case where the current value after 5 seconds was measured with the applied voltage kept constant at 500 mV, the slope of the straight line increased by about 30% by sweeping the potential. Similar results were obtained when the CMC layer was not formed.

(Example 2) When the electric potential E1 was set to various values and E2 was set to 500 mV and the same measurement as in Example 1 was carried out, E1 was -300 mV or more, particularly 0 to 300.
Good response characteristics were observed at mV. E1 is -30
When it is set to the negative potential side from 0 mV, a considerable amount of the reduced form of the electron acceptor is produced on the working electrode regardless of the enzymatic reaction. This is considered to have affected the response characteristics.

(Embodiment 3) When the electric potential E2 was set to various values and E1 was set to 200 mV and the same measurement as in Embodiment 1 was carried out, E2 was 700 mV or less, particularly 400 to 60.
Good response characteristics were observed at 0 mV. E1 is 70
When it is set to the positive potential side from 0 mV, in addition to the oxidation reaction of the electron acceptor, decomposition reaction of other dissolved ions and solvent may occur, which is considered to have affected the response characteristics. .

Example 4 The same glucose sensor as in Example 1 was used.

As a sample solution, 3 μl of an aqueous glucose solution was supplied to the sensor having the structure shown in FIG.
The sample solution reached the air holes 11 and the reaction layer on the electrode system was dissolved. 55 seconds after the sample was supplied, 0 mV (E1) was applied to the working electrode 4 with the counter electrode 5 as a reference. The applied potential was set to 800 mV (E3) after 1 second, and the applied potential was set to 0 mV again after 100 milliseconds. At the same time as returning to 0 mV, the potential was swept to the positive potential side at 60 mV / s.
When the current value at 500 mV (E2) was measured and plotted against the glucose concentration, good linearity was obtained. FIG. 4 shows the changes over time in the applied potential. 800 mV (v
s. By applying the potential of the counter electrode 5), it is considered that the electrode surface was cleaned and the easily oxidizable interfering substance was removed, and thereby the response characteristics were improved.

Example 5 The same glucose sensor as in Example 1 was used. As a sample solution, 3 μl of an aqueous glucose solution was supplied to the sensor having the configuration shown in FIG. The sample solution reached the air holes 11 and the reaction layer on the electrode system was dissolved. 55 seconds after supplying the sample, working electrode 4
800 mV (E4) was applied to the counter electrode 5. 100
After a lapse of milliseconds, the applied potential was set to 200 mV (E5), and 6
It was swept to the positive potential side at 0 mV / s. 500 mV (E6)
When the current value was measured and plotted against the glucose concentration, good linearity was obtained. FIG. 5 shows the changes over time in the applied potential. It is considered that by applying a potential of 800 mV (vs. counter electrode 5), the electrode surface was cleaned and easily oxidizable interfering substances were removed, and thereby the response characteristics were improved.

(Example 6) The potential E5 was set to various values, and the same measurement as in Example 5 was carried out with E6 set to 500 mV. E5 was -300 mV or higher, particularly 0 to 300.
Good response characteristics were observed at mV. E5 is -30
When it is set to the negative potential side from 0 mV, a considerable amount of the reduced form of the electron acceptor is produced on the working electrode regardless of the enzymatic reaction. This is considered to have affected the response characteristics.

(Example 7) When the potential E6 was set to various values and E5 was set to 200 mV and the same measurement as in Example 5 was performed, E6 was 700 mV or less, particularly 400 to 60.
Good response characteristics were observed at 0 mV. E6 is 70
When it is set to the positive potential side from 0 mV, in addition to the oxidation reaction of the electron acceptor, decomposition reaction of other dissolved ions and solvent may occur, which is considered to have affected the response characteristics. .

[0028]

As described above, according to the present invention, it is possible to quantify a substrate with high reliability.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a glucose sensor used in an embodiment of the present invention, excluding a reaction layer.

FIG. 2 is a vertical cross-sectional view of a main part of the glucose sensor.

FIG. 3 is a diagram showing changes with time in applied potential in one embodiment of the present invention.

FIG. 4 is a diagram showing changes with time in applied potential according to an embodiment of the present invention.

FIG. 5 is a diagram showing changes with time in applied potential in one embodiment of the present invention.

[Explanation of symbols]

 1 Insulating Substrate 2, 3 Lead 4 Working Electrode 5 Counter Electrode 6 Insulating Layer 7 Reaction Layer 8 Lecithin Layer 9 Cover 10 Spacer 11 Air Vent 12 Sample Supply Vent

Claims (5)

[Claims] 1. A substrate in a sample using a biosensor comprising an electrode system comprising a working electrode and a counter electrode formed on an insulating substrate, and a reaction layer containing at least a redox enzyme and an electron acceptor. In the quantitative method of detecting a change in the substance concentration during the reaction between the redox enzyme and the electron acceptor based on the current response obtained by applying a voltage between the working electrode and the counter electrode, It is characterized in that an applied potential for obtaining is started from a working electrode potential E1 based on a counter electrode, and after a lapse of a certain time from application of E1, a current value at a potential E2 satisfying a condition of E2> E1 is detected as a current response. Method for quantifying the substrate. 2. The electric potential E3 satisfying the condition of E3 ≧ E1 is applied for a certain time at a certain point before the current value at E2 is detected as a current response.
The method for quantifying a substrate according to 1. 3. A substrate in a sample using a biosensor comprising an electrode system comprising a working electrode and a counter electrode formed on an insulating substrate, and a reaction layer containing at least a redox enzyme and an electron acceptor. In the quantitative method of detecting a change in the substance concentration during the reaction between the redox enzyme and the electron acceptor based on the current response obtained by applying a voltage between the working electrode and the counter electrode, The applied potential for obtaining is started from the working electrode potential E4 with the counter electrode as a reference, and after a lapse of a fixed time from the application of E4, changes to a potential E5 satisfying the condition of E5 <E4, and after a lapse of a fixed time from the application of E5, E6> A method for quantifying a substrate, which comprises detecting a current value at a potential E6 satisfying the condition of E5 as a current response. 4. The method for quantifying a substrate according to claim 1, wherein E1 or E5 is −300 mV or higher, preferably 0 to 300 mV. 5. The method for quantifying a substrate according to claim 1, wherein E2 or E6 is 700 mV or less, preferably 400 to 600 mV.