JP2007256069A - Measuring method of biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem, wherein since the oxidation current value of a ferrocene derivative is also measured at the same time, when the ferrocence derivative is a reducing-type oxidizable and reducible substance in normal state is used as a mediator in an enzyme electrode type sensor of amperometric method, using the mediator, the reaction current due to enzymatic reaction, corresponding to the concentration of a measuring target substance cannot be obtained. <P>SOLUTION: In this three-electrode type biosensor having an acting electrode, a counter electrode and a reference electrode, a second potential is applied across the working electrode and the reference electrode, after first potential has been applied across both electrodes; and the concentration of the measuring target substance in a sample solution is measured at the application of the second potential, and the first potential is at a potential higher than the second potential. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biosensor measurement method, and more particularly to a measurement method for quantifying a trace amount of a measurement target substance in an enzyme electrode biosensor that quantifies the measurement target substance contained in a biological sample.

  Using an oxidoreductase that reacts specifically with the measurement target substance in the biological sample, the substance in the sample solution generated by the enzyme reaction due to electron transfer between the measurement target substance and the oxidoreductase is oxidized and reduced at the electrode, and the flowing current An enzyme electrode type biosensor that measures the concentration of a substance to be measured according to the amount has been developed. For example, in the case of a glucose sensor, there is a method of measuring glucose concentration by oxidizing hydrogen peroxide generated by the reaction of glucose oxidase and glucose at an electrode.

  However, since the oxidation potential of hydrogen peroxide is high, an enzyme electrode type that uses an oxidation-reduction substance with a low oxidation-reduction potential as a mediator that exchanges electrons with an oxidized or reduced enzyme produced by an enzymatic reaction. Many sensors are used.

  As the mediator, potassium ferricyanide, a benzoquinone compound, an osmium complex, a ferrocene derivative, or the like is used.

  Among the above mediators, potassium ferricyanide is used in many glucose sensors, but it is known that although it has a cost advantage, it has a low electron transfer property with an enzyme.

  On the other hand, ferrocene derivatives are known to have high electron transfer properties with enzymes, and various sensor forms have been shown (for example, see Patent Document 1).

JP-A-2-240555 (FIG. 1)

  The sensor using the ferrocene derivative of the above-mentioned patent document is formed by fixing dimethylferrocene to the electrode as a ferrocene derivative with a photocurable resin, and is +200 mV above the oxidation potential of dimethylferrocene between the reference electrode and the working electrode. The glucose concentration is obtained from the current value obtained by applying.

  When a ferrocene derivative is used as the redox substance, the ferrocene derivative that can exchange electrons with the enzyme that has reacted with the substrate is an oxidized ferrocene derivative. However, most ferrocene derivatives exist as reduced ferrocene derivatives under normal conditions.

  Therefore, using a ferrocene derivative as a mediator, the current value that can be applied to a potential higher than the oxidation potential of the ferrocene derivative is the oxidation current of the reduced ferrocene oxidized at the electrode and the oxidized ferrocene contained before the potential application. It is the sum of the reaction current between the substance to be measured and the enzyme mediated by the oxidized ferrocene derivative produced by oxidizing the reduced ferrocene derivative.

To accurately measure the reaction current using a ferrocene derivative as a mediator, immerse the sensor chip in an electrolyte solution, apply a potential higher than the oxidation potential of the ferrocene derivative for a long time, and then select the substance to be measured when the current value stabilizes. Although there is a method of measuring a current value change by adding a sample solution containing it, there is a problem that it cannot be used as a disposable sensor as used in many self blood glucose measuring devices.

  An object of the present invention is to provide a measurement method useful for a disposable enzyme electrode biosensor using a ferrocene derivative as a mediator.

  The biosensor measurement method of the present invention is a biosensor measurement method used in a tripolar biosensor having a working electrode, a counter electrode, and a reference electrode using a redox substance as a mediator, and is an oxidation used as a mediator. A step of applying a first potential between the working electrode and the reference electrode, and at least a part of the reducing material is a reduction-type redox material in a state before the potential is applied; Applying a second potential that is lower than the first potential between the electrodes and measuring the concentration of the substance to be measured in the sample solution.

The first potential and the second potential are both equal to or higher than the oxidation potential of the redox substance used as a mediator, and the first potential is a half-wave potential E obtained from the oxidation potential and reduction potential of the redox substance. The potential is preferably within a range of +200 mV to +600 mV with respect to _1/2 . However, the potential of the working electrode with respect to the reference electrode is preferably +1.0 V or less at which water decomposition occurs. Furthermore, the second potential is preferably a potential within +50 mV to +300 mV with respect to the half-wave potential E _1/2 of the redox substance.

  In the biosensor measurement method of the present invention, the enzyme and the mediator are preferably immobilized on at least the working electrode among the working electrode, the reference electrode, and the counter electrode.

  As a method for immobilizing the enzyme and the mediator, conventionally used water-soluble polymers such as polyvinyl alcohol and carboxymethyl cellulose, and hydrophobic polymers such as polyvinyl butyral and Nafion can be used. Moreover, the enzyme and the mediator may be immobilized in a mixed state or may be individually laminated. Glutaraldehyde may be used for immobilization of the enzyme.

  Furthermore, in the biosensor measurement method of the present invention, the redox substance that is a mediator is preferably a ferrocene derivative.

  As the ferrocene derivative to be used, in addition to ferrocene, ferrocenecarboxylic acid 1,1′-ferrocenedicarboxylic acid, ferrocenemethanol, 1,1′-ferrocenedimethanol, ferroceneethanol, methylferrocene, 1,1′-dimethylferrocene, etc. are used. However, it is preferable to use ferrocenemethanol, 1,1′-ferrocenedimethanol or the like having a low oxidation potential and high solubility in a polymer upon immobilization.

  In the biosensor measurement method of the present invention, it is preferable that the step of applying the first potential includes a step of changing the mediator to an oxidized type.

The ferrocene derivative immobilized on the electrode is changed to an oxidized type by application of the first potential, so that the reaction current can be accurately measured in the second potential application step. The concentration of the ferrocene derivative immobilized on the electrode varies depending on the electrode area, the concentration of the substance to be measured in the measurement sample, the enzyme activity, etc., but is 0.02 to 1 μmol / cm ² , and 0.05 to 0.4 μmol / cm with respect to the electrode. ² is preferred. When the concentration of the ferrocene derivative immobilized on the electrode is lower than the above range, the responsiveness of the substance to be measured in the high concentration range is low, and when the concentration of the ferrocene derivative is higher than the above range, the first potential application step In this case, the ferrocene derivative is not sufficiently oxidized, and the oxidation current of the ferrocene derivative is continuously observed when the second potential is applied, and an error occurs in the actual reaction current.

  In the biosensor measurement method of the present invention, it is preferable that the step of applying the first potential includes a step of oxidizing a reducing substance present in the sample.

  Examples of the reducing substance include ascorbic acid, bilirubin, uric acid, acetaminophen and the like present in a biological sample. These reducing substances have the effect of reducing the redox substances that are mediators, or the inhibitory effect of increasing the apparent reaction current value when they themselves are oxidized by applying a potential.

(Function)
According to the method of the present invention, the first potential application step oxidizes the mediator that is a redox substance and also converts the reducing substance contained in the biological sample into an oxidized form, whereby the second potential application step. This makes it possible to measure a more accurate reaction current measurement.

  According to the present invention, in an enzyme electrode biosensor using a ferrocene derivative as a mediator, the oxidation current of a mediator, which is a measurement error factor of a reaction current value, and the oxidation current due to a reducing substance in a biological sample can be ignored. Therefore, it is possible to quantify the substance to be measured present at a low concentration in the biological sample and improve the measurement accuracy.

  FIG. 1 shows an example of a sensor chip of an enzyme electrode type biosensor using a plan view and a cross-sectional view. 1A is a plan view of the sensor chip, and FIG. 1B is a cross-sectional view of the plan view of FIG. 1A taken along line A-A ′. An electrode and a wiring substrate are formed on the insulating substrate 1 with a silver foil 10. Wirings that lead to the working electrode 2, the counter electrode 3, and the connection terminal 5 are formed by screen printing using a carbon paste 11. Similarly, the wiring which leads to the reference electrode 4 and the connection terminal 5 is formed by screen printing using the silver-silver chloride paste 12. Wirings from the working electrode, the counter electrode, and the reference electrode are covered with an insulating material 13.

Hereinafter, using the sensor chip, for the measurement method of the biosensor of the present invention, glucose as a measurement target substance, glucose oxidase as an enzyme, and 1,1′-ferrocene dimethanol as a ferrocene derivative as a mediator Will be described in more detail. The half-wave potential E _1/2 obtained from the oxidation potential and reduction potential of 1,1′-ferrocenedimethanol was +140 mV, the first potential was +500 mV, and the second potential was +350 mV.

Example 1
3 μL of a 1 wt% carboxymethylcellulose aqueous solution in which 4 mg / mL glucose oxidase and 2 mM 1,1′-ferrocenedimethanol were dissolved was dropped on the 7 mm ² working electrode and dried to obtain an enzyme mediator immobilized electrode.

  Using the sensor chip thus prepared, 10 μL of PBS solutions having various glucose concentrations were added so as to cover the working electrode, the counter electrode, and the entire reference electrode. Immediately after the addition, +500 mV was applied between the working electrode and the reference electrode for 30 seconds, immediately switched to +350 mV, and the current value after 10 seconds was measured. The results are shown in FIG.

  FIG. 2 shows that a linear relationship is obtained from a glucose concentration range of 200 μM to a concentration range of 10 mM.

(Comparative Example 1)
Using a sensor chip produced in the same manner as in Example 1, 10 μL of PBS solutions having various glucose concentrations were added so as to cover the working electrode, the counter electrode, and the entire reference electrode. Immediately after the addition, a potential of +350 mV was applied between the working electrode and the reference electrode, and the current value after 10 seconds was measured. The results are shown in FIG.

  It can be seen from FIG. 3 that the data particularly in the low concentration region of the glucose concentration deviates from the straight line. Furthermore, when the reproducibility of measured values in the glucose concentration range from 200 μM to 1 mM was examined, it was revealed that the data varied greatly.

(Example 2)
Ascorbic acid was dissolved in a PBS solution of 1 mM glucose to a concentration of 100 μM to prepare a glucose-ascorbic acid solution. Using the sensor chip produced in the same manner as in Example 1, 10 μL of the ascorbic acid solution was added so as to cover the working electrode, the counter electrode, and the entire reference electrode. Immediately after the addition, a potential of +500 mV was applied between the working electrode and the reference electrode for 30 seconds, immediately after switching the potential to +350 mV and measuring the current value after 10 seconds, the current value was 8.43 μA. There was little change in results.

(Comparative Example 2)
Using the same glucose-ascorbic acid solution as in Example 2 and a sensor chip prepared in the same manner as in Example 1, 10 μL of the ascorbic acid solution was added so as to cover the working electrode, the counter electrode, and the entire reference electrode. Immediately after the addition, a potential of +350 mV was applied between the working electrode and the reference electrode, and the current value after 10 seconds was measured. As a result, it was 25.3 μA, and a very high current value was observed as compared with Example 2.

  From Example 1 and Comparative Example 1, it can be seen that the measurement method of the present invention maintains a linear relationship between the measurement target substance concentration and the current value in a wide concentration range of the measurement target substance. In the conventional measurement method as in Comparative Example 1, there is a mediator concentration variation on the electrode that occurs during the manufacture of the sensor chip, or a mediator that is an oxidized redox substance depending on the storage state of the sensor chip and a reduced redox substance. The measured current value varies greatly due to the change in the mediator abundance ratio.

  On the other hand, according to the measurement method of the present invention, most of the mediators immobilized on the electrode are oxidized in the first potential application step, so that the reaction current between the enzyme and the substance to be measured can be accurately measured.

  Further, from the results of Example 2 and Comparative Example 2, according to the measurement method of the present invention, the influence of the reducing substance in the sample solution, which is an inhibitory substance in measuring the concentration of the substance to be measured, can be greatly reduced. I understand that.

  In the above embodiment, the second potential is applied immediately after the application of the first potential, but a waiting time may be provided between them in an open circuit state. The above embodiment is only one example for explaining the present invention, and the biosensor measurement method of the present invention sets various conditions depending on the type of substance to be measured, the selection of the corresponding enzyme, and the selection of the mediator. Is possible.

FIG. 2 is a plan view and a cross-sectional view showing an example of a sensor chip used in the biosensor measurement method of the present invention. It is a figure which shows the measurement result of Example 1 using the measuring method of the biosensor of this invention. It is a figure which shows the measurement result of the comparative example 1 using the measuring method of the conventional biosensor.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Insulating board | substrate 2 Working electrode 3 Counter electrode 4 Reference electrode 5 Connection terminal 10 Silver foil 11 Carbon paste 12 Silver silver chloride paste 13 Insulation material

Claims (5)

  A biosensor measurement method used in a tripolar biosensor having a working electrode, a counter electrode, and a reference electrode using a redox substance as a mediator, wherein at least a part of the redox substance has a potential. A reduction-type redox substance in a state before being applied, a step of applying a first potential between the working electrode and the reference electrode; and the first potential between the working electrode and the reference electrode Applying a second potential that is lower than the first potential and measuring the concentration of the substance to be measured in the sample.   The biosensor measurement method according to claim 1, wherein the enzyme and the mediator are immobilized on the working electrode.   The biosensor measurement method according to claim 1, wherein the redox substance is a ferrocene derivative.   4. The method according to claim 1, wherein in the step of applying the first potential, the redox substance changes from the reduced redox substance to an oxidized redox substance. The biosensor measurement method according to claim 1.   5. The biosensor measurement method according to claim 1, wherein, in the step of applying the first electric potential, a reducing substance present in the sample is oxidized.

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