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EP-4245857-B1 - METHOD FOR ELECTROCHEMICALLY MEASURING OXIDOREDUCTASE USING A BIOSENSOR

EP4245857B1EP 4245857 B1EP4245857 B1EP 4245857B1EP-4245857-B1

Inventors

  • SHINOZAKI, KOTARO
  • OGA, Misaki
  • MIKAMI, TOSHIYUKI

Dates

Publication Date
20260506
Application Date
20230316

Claims (10)

  1. A method for electrochemically measuring an amount of an oxidoreductase in a sample containing the oxidoreductase, using a biosensor, wherein the biosensor comprises: a conductive part including two or more electrodes (11, 12, 13, 14); and a reagent layer (15) that is arranged in contact with the conductive part, the reagent layer (15) including a substrate of the oxidoreductase and two or more types of mediators, wherein the mediators include a first mediator and a second mediator, wherein the first mediator is a mediator for transferring, to the second mediator, electrons generated by a reaction between the substrate and the oxidoreductase, and the second mediator is a mediator for transferring, to the electrodes (11, 12, 13, 14), the electrons transferred from the first mediator, and wherein the substrate of the oxidoreductase is glucose, the method comprising: bringing the sample containing the oxidoreductase into contact with the reagent layer (15); performing incubation for a predetermined time after the contact, thereby causing the second mediator to accumulate the electrons; applying a voltage across the electrodes (11, 12, 13, 14) after the incubation; measuring an electric signal generated by the application of the voltage; and calculating the amount of the oxidoreductase based on the electric signal.
  2. The method according to claim 1, further comprising setting the incubation time within a time range in which the substrate contained in the reagent layer (15) is not exhausted by the reaction between the substrate and the oxidoreductase.
  3. The method according to claim 1 or 2, wherein the amount of the substrate in the reagent layer (15) is 4.4 nmol or more per 1 cm 3 of the reagent layer (15).
  4. The method according to any one of claims 1 to 3, wherein the time for the incubation is 1 minute or more.
  5. The method according to any one of claims 1 to 4, wherein the oxidoreductase is glucose dehydrogenase.
  6. The method according to any one of claims 1 to 5, wherein the combination of the first mediator and the second mediator is a combination of 1-methoxy-5-ethylphenazinium ethylsulfate (1-mPES) and a ruthenium compound, or a combination of 1-methoxy-5-methylphenazinium methylsulfate (1-mPMS) and a ruthenium compound.
  7. The method according to any one of claims 1 to 6, wherein the electric signal used in the calculation of the amount of the oxidoreductase is an electric signal measured within 10 seconds after the application of the voltage.
  8. The method according to any one of claims 1 to 7, further comprising converting an analysis object into an oxidoreductase whereby to prepare the sample containing the oxidoreductase.
  9. The method according to claim 8, wherein the biosensor includes a second reagent layer (16) containing a substance that can convert the analysis object into the oxidoreductase.
  10. The method according to any one of claims 1 to 9, wherein the method comprises: bringing the sample containing an oxidoreductase into contact with the reagent layer (15) of the biosensor; performing a first application by applying a voltage across the electrodes (11, 12, 13, 14) after the contact; performing a first measurement by measuring an electric signal generated by the first application; calculating a time for incubation based on the electric signal obtained by the first measurement; performing incubation for the incubation time determined by the calculation; performing a second application by applying a voltage across the electrodes (11, 12, 13, 14) after the incubation; performing a second measurement by measuring an electric signal generated by the second application; and calculating an amount of the oxidoreductase based on the electric signal obtained by the second measurement.

Description

BACKGROUND 1. Field The present disclosure relates to a method for electrochemically measuring an amount of an oxidoreductase using a biosensor. 2. Description of Related Art A biosensor is a molecular measuring device using a detection unit in which a biological material such as an enzyme is arranged and a transducer (signal conversion device) such as electrodes. The biosensor is used for measuring various biological analysis objects such as glucose in a living body. Electrochemical measurement with an enzyme is generally performed as a method for measuring a concentration of glucose using a biosensor. As one type of method with which a concentration of glucose can be measured with accuracy, various methods that apply the Cottrell equation have been reported (JP-A-3(1991)-293556, Japanese Patent No. 2651278, Japanese Patent No. 2702286, Japanese Patent No. 2901678, Japanese Patent No. 4018748, Japanese Patent No. 5244116). The Cottrell equation is one of the diffusion equations that depends on the electrode size and shape. As is well known, since the thickness of an electrode used in a biosensor such as a glucose sensor is relatively small as compared with the range in which diffusion occurring with electron transfer occurs (up to several hundreds of micrometers), the current response arising when a potential is applied to an electrode is not constant, and attenuates in inverse proportion to a square root of time (attenuates in proportion to t-1/2) according to the Cottrell equation. Japanese Patent No. 2901678 discloses a method in which, in order to measure an amount of an analysis object while preventing a difference from occurring in measured values depending on a user, the analysis object and a reagent (oxidizer and oxygen, etc.) are mixed in a measurement cell having electrodes to react with each other, incubation is performed until the end of the enzyme reaction, and then a voltage is applied to the electrodes to measure a Cottrell current. On the other hand, for the Cottrell current (having attenuation in line with the Cottrell equation) to be established, it is necessary that there should be no substantial change in the concentration of the materials after the application of a voltage, that is, the enzyme reaction should be substantially complete. Therefore, the method disclosed in Japanese Patent No. 2901678 has a problem that it is necessary to ensure a certain reaction time, which means that the method requires time before the measurement. To solve this problem, Japanese Patent No. 5244116 proposes to measure glucose by using an electrochemical process without Cottrell attenuation. Cottrell attenuation has an attenuation coefficient of -0.5 (constant). Japanese Patent No. 5244116 discloses a configuration in which an attenuation coefficient in such a short reaction (incubation) time that does not allow the enzyme reaction to finish is initially determined, and a concentration of an analysis object in a sample is determined based on the attenuation coefficient and an output signal having transition attenuation. In recent years, a method for electrochemically measuring a concentration of a biological material other than glucose has been reported (Yu Xiang et al., Nature Chemistry, 2012, 3(9): 697-703, Tian Lan et al., Methods Mol Biol. 2015, 1256, 99-109, Naveen K. Singh et al., Biosensors and Bioelectronics, 2021, 180, 113111, Tian Lan et al., Biotechnology Advances, 2016, 34(3) 331-341, Jinhee Lee et al., Electrochemistry, 2015, 83(12) 1085-1090, Yoshihiko Nonaka et al., Electrochemistry, 2012, 80(5) 363-366, Koichi Abe et al., Electrochemistry, 2012, 80(5) 348-352, Nasa Savory et al., Urakami Foundation memoirs, 2014, Vol. 21 136-142). Yu Xiang et al, Tian Lan et al. (2015), Naveen K. Singh et al., and Tian Lan et al. (2016) disclose a configuration in which sucrose is converted by an oxidoreductase into glucose, and is measured by a glucose meter (Blood Glucose Meter, BGM) or the like. On the other hand, however, this configuration has a problem that, when an analysis object in a sample has a lower concentration relative to that of glucose, or an extremely low concentration, a sufficient sensitivity cannot be obtained as compared with a case of measuring glucose. In view of this, a method of modifying electrodes, a method of localizing multiple electron transfer enzymes, and the like, for enhancing the sensitivity are reported in the disclosure of Jinhee Lee et al. A method that uses glucose dehydrogenase (GDH) as a detection enzyme to measure a response current of GDH and converts a concentration of an analysis object into GDH, for measuring a concentration of a vascular endothelium cell growth factor (VEGF) in blood is reported in the disclosures of Yoshihiko Nonaka et al. and Koichi Abe et al. US5122244 A discloses a method for electrochemically measuring an amount of an oxidoreductase in a sample using a biosensor, wherein the biosensor comprises a conductive part including electrodes and