JP6823436B2 - Measurement method of target component and measurement device of target component - Google Patents
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
Description
本発明は、目的成分の測定方法および目的成分の測定装置に関する。 The present invention relates to a method for measuring a target component and a device for measuring the target component.
血中グルコース濃度の電気化学的測定方法として、グルコース酸化酵素またはグルコース脱水素酵素、電子伝達物質および電極を使用し、前記電子伝達物質の酸化電位以上の電圧を前記電極に印加し、得られた測定値に基づきグルコース濃度を算出する方法が用いられている。 As a method for electrochemically measuring the blood glucose concentration, a glucose oxidase or glucose dehydrogenase, an electron transfer substance and an electrode were used, and a voltage equal to or higher than the oxidation potential of the electron transfer substance was applied to the electrode to obtain the result. A method of calculating the glucose concentration based on the measured value is used.
しかしながら、前記測定方法により算出されるグルコース濃度は、血中のヘマトクリット、アスコルビン酸等の干渉物質により影響を受ける。このため、ヘマトクリット、アスコルビン酸等の干渉物質についてグルコース測定用電極とは異なる電極で測定し、これらの測定値よりグルコース量を補正する必要がある（特許文献１および２）。 However, the glucose concentration calculated by the above measurement method is affected by interfering substances such as hematocrit and ascorbic acid in the blood. Therefore, it is necessary to measure interfering substances such as hematocrit and ascorbic acid with an electrode different from the glucose measuring electrode and correct the glucose amount from these measured values (Patent Documents 1 and 2).
したがって、新たなグルコースの測定方法が求められている。 Therefore, a new method for measuring glucose is required.
そこで、本発明は、例えば、グルコースをはじめとする目的成分を測定するための新たな目的成分の測定方法および目的成分の測定装置の提供を目的とする。 Therefore, an object of the present invention is to provide, for example, a new method for measuring a target component for measuring glucose and other target components, and a device for measuring the target component.
前記本発明の課題を解決するために、本発明の目的成分の測定方法（以下、「測定方法」ともいう。）は、試料、目的成分に対する酸化還元触媒、および電子伝達物質の存在下、第１の電圧（Ｖ１）を電極系に印加する第１の印加工程と、
第２の電圧（Ｖ２）を前記電極系に印加する第２の印加工程と、
前記第２の印加工程において、前記電極系によりシグナルを取得するシグナル取得工程とを含み、
前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、
前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であることを特徴とする。
In order to solve the above-mentioned problem of the present invention, the method for measuring the target component of the present invention (hereinafter, also referred to as “measurement method”) is the first in the presence of a sample, an oxidation-reduction catalyst for the target component, and an electron transfer substance. The first application step of applying a voltage of 1 (V 1 ) to the electrode system, and
A second application step of applying a second voltage (V 2 ) to the electrode system, and
The second application step includes a signal acquisition step of acquiring a signal by the electrode system.
The first voltage is a voltage equal to or higher than the oxidation potential (E) of the electron transmitter.
The second voltage is a voltage lower than the oxidation potential (E) of the electron transmitter.
本発明の目的成分の測定装置（以下、「測定装置」ともいう。）は、電極系と、
試料、目的成分に対する酸化還元触媒、および電子伝達物質の存在下、第１の電圧（Ｖ１）および第２の電圧（Ｖ２）を前記電極系に印加する電圧印加手段と、
前記第２の電圧（Ｖ２）印加において、前記電極系からシグナルを取得するシグナル取得手段とを含み、
前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、
前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であり、
前記本発明の目的成分の測定方法に使用することを特徴とする。
The device for measuring the target component of the present invention (hereinafter, also referred to as “measurement device”) includes an electrode system and
A voltage applying means for applying a first voltage (V 1 ) and a second voltage (V 2 ) to the electrode system in the presence of a sample, a redox catalyst for a target component, and an electron transport chain.
Including the signal acquisition means for acquiring a signal from the electrode system when the second voltage (V 2 ) is applied.
The first voltage is a voltage equal to or higher than the oxidation potential (E) of the electron transmitter.
The second voltage is a voltage lower than the oxidation potential (E) of the electron transmitter.
It is characterized by being used in the method for measuring the target component of the present invention.
本発明によれば、グルコースをはじめとする目的成分を測定できる。 According to the present invention, a target component such as glucose can be measured.
本発明の測定方法および測定装置は、例えば、前記第１の電圧（Ｖ１）が、Ｅ≦Ｖ１≦Ｅ＋１．１５の範囲の電圧である。 In the measuring method and measuring device of the present invention, for example, the first voltage (V 1 ) is a voltage in the range of E ≦ V 1 ≦ E + 1.15.
本発明の測定方法および測定装置は、例えば、前記第２の電圧（Ｖ２）が、Ｅ−０．２５≦Ｖ２＜Ｅの範囲の電圧である。 In the measuring method and measuring device of the present invention, for example, the second voltage (V 2 ) is a voltage in the range of E-0.25 ≦ V 2 <E.
本発明の測定方法および測定装置は、例えば、前記シグナルが、前記第２の電圧印加時を基準として、０．４秒以内に取得したシグナルである。 In the measuring method and measuring device of the present invention, for example, the signal is a signal acquired within 0.4 seconds with reference to the time when the second voltage is applied.
本発明の測定方法および測定装置は、例えば、前記シグナルが、１時点で取得したシグナルである。 In the measuring method and measuring device of the present invention, for example, the signal is a signal acquired at one time point.
本発明の測定方法および測定装置は、例えば、前記シグナルが、連続的に取得したシグナルである。 In the measuring method and measuring device of the present invention, for example, the signal is a continuously acquired signal.
本発明の測定方法および測定装置は、例えば、前記シグナルが、複数の時点で取得したシグナルである。 In the measuring method and measuring device of the present invention, for example, the signal is a signal acquired at a plurality of time points.
本発明の測定方法および測定装置は、例えば、前記目的成分が、グルコースである。 In the measuring method and measuring device of the present invention, for example, the target component is glucose.
本発明の測定方法および測定装置は、例えば、前記酸化還元触媒が、酸化還元酵素または脱水素酵素である。 In the measuring method and measuring apparatus of the present invention, for example, the redox catalyst is an oxidoreductase or a dehydrogenase.
本発明の測定方法および測定装置は、例えば、前記酸化還元触媒が、グルコース酸化酵素またはグルコース脱水素酵素である。 In the measuring method and measuring apparatus of the present invention, for example, the redox catalyst is glucose oxidase or glucose dehydrogenase.
本発明の測定方法および測定装置は、例えば、前記試料が、生体試料であり、好ましくは、血液である。 In the measuring method and measuring device of the present invention, for example, the sample is a biological sample, preferably blood.
本発明の測定方法は、例えば、前記シグナルから前記試料中の目的成分量を算出する算出工程を含む。 The measuring method of the present invention includes, for example, a calculation step of calculating the amount of the target component in the sample from the signal.
本発明の測定方法は、例えば、前記電極系により補正シグナルを取得する補正シグナル取得工程と、
前記補正シグナルに基づき、前記シグナルを補正する補正工程とを含み、
前記算出工程において、前記補正後のシグナルから前記試料中の目的成分量を算出する。
The measuring method of the present invention includes, for example, a correction signal acquisition step of acquiring a correction signal by the electrode system.
A correction step of correcting the signal based on the correction signal is included.
In the calculation step, the amount of the target component in the sample is calculated from the corrected signal.
本発明の測定方法および測定装置は、例えば、前記第１の電圧（Ｖ１）が、Ｅ＜Ｖ１である。 In the measuring method and measuring device of the present invention, for example, the first voltage (V 1 ) is E <V 1 .
本発明の測定装置は、例えば、前記シグナルから前記試料中の目的成分量を算出する算出手段を含む。 The measuring device of the present invention includes, for example, a calculation means for calculating the amount of a target component in the sample from the signal.
本発明の測定装置は、例えば、前記電極系により補正シグナルを取得する補正シグナル取得手段と、
前記補正シグナルに基づき、前記シグナルを補正する補正手段とを含み、
前記算出手段において、前記補正後のシグナルから前記試料中の目的成分量を算出する。
The measuring device of the present invention includes, for example, a correction signal acquisition means for acquiring a correction signal by the electrode system.
Including a correction means for correcting the signal based on the correction signal.
In the calculation means, the amount of the target component in the sample is calculated from the corrected signal.
＜目的成分の測定方法＞
本発明の目的成分の測定方法は、前述のように、試料、目的成分に対する酸化還元触媒、および電子伝達物質の存在下、第１の電圧（Ｖ１）を電極系に印加する第１の印加工程と、第２の電圧（Ｖ２）を前記電極系に印加する第２の印加工程と、前記第２の印加工程において、前記電極系によりシグナルを取得するシグナル取得工程とを含み、前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であることを特徴とする。本発明の測定方法は、前記第１の電圧が、前記電子伝達物質の酸化電位以上の電圧であり、前記第２の電圧が、前記電子伝達物質の酸化電位未満の電圧であることを特徴とし、その他の工程および条件は、特に制限されない。
<Measurement method of target component>
As described above, the method for measuring the target component of the present invention is a first application in which a first voltage (V 1 ) is applied to the electrode system in the presence of a sample, a redox catalyst for the target component, and an electron transfer material. The first step includes a step, a second application step of applying a second voltage (V 2 ) to the electrode system, and a signal acquisition step of acquiring a signal from the electrode system in the second application step. The voltage of 1 is a voltage equal to or higher than the oxidation potential (E) of the electron transfer material, and the second voltage is a voltage lower than the oxidation potential (E) of the electron transfer material. The measuring method of the present invention is characterized in that the first voltage is a voltage equal to or higher than the oxidation potential of the electron transfer material, and the second voltage is a voltage lower than the oxidation potential of the electron transfer material. , Other steps and conditions are not particularly limited.
本発明者は、鋭意研究の結果、前記電子伝達物質の酸化電位以上の第１の電圧の印加後、前記電子伝達物質の酸化電位未満の第２の電圧の印加時に取得したシグナルが、前記試料中の目的成分量に応じて変動するとの知見、具体的には、例えば、前記第１の電圧の印加後、前記第２の電圧の印加開始後に得られる過渡電流が、前記試料中の目的成分量と負の相関関係を示すとの知見を見出し、本発明を確立するに至った。また、本発明の測定方法は、メカニズムは不明であるが、前記目的成分の測定において、例えば、前記ヘマトクリット、アスコルビン酸等の干渉物質の影響を低減できる。このため、本発明の測定方法は、例えば、前記干渉物質を測定するための電極を含まない簡易な構造のバイオセンサでも実施できる。さらに、本発明の測定方法は、例えば、前記干渉物質の影響が低減されているため、前記干渉物質の影響を補正するための補正シグナルを取得せずに実施できる。このため、本発明の測定方法は、例えば、シグナルの取得回数を低減でき、シグナルを取得する際に生じる測定誤差の影響を低減できるため、再現性よく目的成分を測定できる。また、前記特許文献１および２は、前記電極系への電圧の印加後、コットレル電流が安定した状態でシグナルを取得するが、本発明の測定方法は、例えば、前記コットレル電流が安定する前に実施できる。このため、本発明の測定方法によれば、例えば、測定時間を短縮できる。さらに、本発明の測定方法は、例えば、前記電極系に印加する電圧と、前記シグナルの取得時とを調整することで、公知の電気化学的測定方法に使用する電極系を含むバイオセンサで実施できる。このため、本発明の測定方法によれば、例えば、公知のバイオセンサにもコストをかけずに導入できる。 As a result of diligent research, the present inventor obtained the signal obtained when a first voltage equal to or higher than the oxidation potential of the electron transfer material was applied and then a second voltage lower than the oxidation potential of the electron transfer material was applied to the sample. Findings that the amount of the target component in the sample varies, specifically, for example, the transient current obtained after the application of the first voltage and the start of application of the second voltage is the target component in the sample. We have found that it shows a negative correlation with the amount, and have established the present invention. Further, although the mechanism of the measuring method of the present invention is unknown, the influence of interfering substances such as hematocrit and ascorbic acid can be reduced in the measurement of the target component. Therefore, the measuring method of the present invention can be carried out, for example, by a biosensor having a simple structure that does not include an electrode for measuring the interfering substance. Further, the measuring method of the present invention can be carried out without acquiring a correction signal for correcting the influence of the interfering substance, for example, because the influence of the interfering substance is reduced. Therefore, the measuring method of the present invention can reduce, for example, the number of times a signal is acquired and the influence of a measurement error that occurs when the signal is acquired, so that the target component can be measured with good reproducibility. Further, in Patent Documents 1 and 2, after applying a voltage to the electrode system, a signal is acquired in a state where the Cottrell current is stable, but the measuring method of the present invention is, for example, before the Cottrell current is stable. Can be implemented. Therefore, according to the measuring method of the present invention, for example, the measuring time can be shortened. Further, the measuring method of the present invention is carried out by a biosensor including an electrode system used in a known electrochemical measuring method by adjusting, for example, a voltage applied to the electrode system and a time when the signal is acquired. it can. Therefore, according to the measuring method of the present invention, for example, it can be introduced into a known biosensor at no cost.
本発明において、「シグナルを取得する」は、例えば、「シグナルを測定する」ということもできる。また、「シグナル」を取得または測定するとは、例えば、シグナルの大きさまたは強さ等を表す「シグナル値」を取得または測定することを意味する。また、シグナルの変動は、例えば、シグナル値の変動を意味する。 In the present invention, "acquiring a signal" can also be referred to as "measuring a signal", for example. Further, acquiring or measuring a "signal" means, for example, acquiring or measuring a "signal value" indicating the magnitude or strength of a signal. Further, the fluctuation of the signal means, for example, the fluctuation of the signal value.
本発明において、「酸化電位」は、例えば、前記電子伝達物質と前記電極系とを含む酸化反応系における前記電極系の電位である。前記酸化電位は、例えば、前記電子伝達物質を含む酸化電位の測定系と、作用極、対極および参照電極を含む電極系または前記作用極および前記参照電極を含む電極系とを用い、前記対極または前記作用極と前記参照電極とが連結された状態で、下記酸化電位の測定条件で測定し、得られた電流値における正方向のピークの電流値を示す電位として測定できる。前記酸化電位は、例えば、１回の測定で得られた電位に基づき、設定してもよいし、複数回の測定で得られた電位に基づき、設定してもよい。後者の場合、前記酸化電位は、例えば、複数回の測定で得られた電位における平均、最小または最大の電位としてもよいし、複数回の測定で得られた電位における最小の電位から最大の電位までの範囲としてもよい。前記酸化電位は、例えば、予め測定した酸化電位でもよいし、前記目的成分の測定時に測定した酸化電位でもよい。具体例として、前記電子伝達物質がルテニウム錯体であり、前記電極系の材料がカーボンである場合、前記酸化電位は、例えば、３０〜５０ｍＶである。 In the present invention, the "oxidation potential" is, for example, the potential of the electrode system in the oxidation reaction system including the electron transport chain and the electrode system. For the oxidation potential, for example, the measurement system of the oxidation potential containing the electron transfer substance and the electrode system including the working electrode, the counter electrode and the reference electrode or the electrode system including the working electrode and the reference electrode are used, and the counter electrode or the electrode is used. With the working electrode and the reference electrode connected to each other, it can be measured under the following measurement conditions of the oxidation potential, and can be measured as a potential indicating the current value of the peak in the positive direction in the obtained current value. The oxidation potential may be set, for example, based on the potential obtained in one measurement, or may be set based on the potential obtained in a plurality of measurements. In the latter case, the oxidation potential may be, for example, the average, minimum or maximum potential at the potentials obtained by the plurality of measurements, or the minimum to maximum potentials at the potentials obtained by the plurality of measurements. It may be in the range up to. The oxidation potential may be, for example, the oxidation potential measured in advance, or the oxidation potential measured at the time of measuring the target component. As a specific example, when the electron transfer substance is a ruthenium complex and the material of the electrode system is carbon, the oxidation potential is, for example, 30 to 50 mV.
（酸化電位の測定条件）
測定系における電子伝達物質の濃度：１０〜５００ｍｍｏｌ／Ｌ
測定系に用いる試料：血液検体
測定系の温度：２５℃
開始電位：−８００ｍＶ
折り返し電位：８００ｍＶ
掃引速度：２０ｍＶ／ｓ
(Measurement condition of oxidation potential)
Electron transmitter concentration in the measurement system: 10-500 mmol / L
Sample used for measurement system: Blood sample Measurement system temperature: 25 ° C
Starting potential: -800 mV
Folding potential: 800 mV
Sweep speed: 20 mV / s
本発明の測定方法は、例えば、前記試料の目的成分の有無を測定する定性分析であってもよいし、前記試料の目的成分量を測定する定量分析であってもよい。 The measuring method of the present invention may be, for example, a qualitative analysis for measuring the presence or absence of a target component of the sample, or a quantitative analysis for measuring the amount of the target component of the sample.
本発明において、前記第１の印加工程、前記第２の印加工程、および前記シグナル取得工程は、前記試料、前記酸化還元触媒、および前記電子伝達物質の存在下で実施される。このため、本発明の測定方法は、例えば、前記試料、前記酸化還元触媒、および前記電子伝達物質を含む反応系に対し、前記第１の印加工程、前記第２の印加工程、および前記シグナル取得工程を実施するということもできる。前記反応系は、例えば、液体系であることが好ましく、前記試料、前記酸化還元触媒、および前記電子伝達物質を含む反応液または酸化還元液ということもできる。 In the present invention, the first application step, the second application step, and the signal acquisition step are carried out in the presence of the sample, the redox catalyst, and the electron transmitter. Therefore, in the measuring method of the present invention, for example, the first application step, the second application step, and the signal acquisition are applied to the reaction system containing the sample, the redox catalyst, and the electron transport chain. It can also be said that the process is carried out. The reaction system is preferably, for example, a liquid system, and may also be a reaction solution or a redox solution containing the sample, the redox catalyst, and the electron transfer substance.
本発明において、前記試料は、特に制限されず、例えば、液体試料である。前記液体試料は、例えば、生体由来の検体（生体試料）があげられ、前記生体試料の希釈液、懸濁液等でもよい。前記生体試料は、例えば、血液、尿、胃液、喀痰、羊水、腹膜液、間質液等の体液、大腸、肺等の組織、口腔内細胞、生殖細胞、爪、毛等の細胞等があげられる。前記血液は、例えば、全血、血漿、血清、溶血液等があげられる。前記試料は、例えば、前記目的成分を含む試料でもよいし、前記目的成分を含むか否か不明な試料でもよい。 In the present invention, the sample is not particularly limited, and is, for example, a liquid sample. Examples of the liquid sample include a biological sample (biological sample), and a diluted solution or suspension of the biological sample may be used. Examples of the biological sample include body fluids such as blood, urine, gastric fluid, sputum, amniotic fluid, peritoneal fluid, and interstitial fluid, tissues such as colon and lung, cells in the oral cavity, germ cells, nails, and hair. Be done. Examples of the blood include whole blood, plasma, serum, and lysed blood. The sample may be, for example, a sample containing the target component, or a sample for which it is unknown whether or not the target component is contained.
本発明において、前記目的成分は、特に制限されず、例えば、グルコース等の糖、Ｃ反応性タンパク質（ＣＲＰ）、ＨｂＡ１ｃ、甲状腺刺激ホルモン（ＴＳＨ）、ＦＴ３、ＦＴ４、ｈＣＧ、ＨＢｓ抗原、ＨＢｃ抗体、ＨＣＶ抗体、ＴＹ抗原、アンチ−ストレプトリシン Ｏ（ＡＳＯ）、ＩＶ型コラーゲン、マトリックスメタロプロテイナーゼ−３（ＭＭＰ−３）、ＰＩＶＡＫ−ＩＩ、α１マイクログロブリン、β１マイクログロブリン、アミロイドＡ（ＳＡＡ）、エラスターゼ１、塩基性フェトプロテイン（ＢＦＰ）、カンジダ抗原、子宮頸管粘液中顆粒球エラスターゼ、ジゴキシン、シスタチンＣ、第ＸＩＩＩ因子、尿中トランスフェリン、梅毒、ヒアルロン酸、フィブリンモノマー複合体（ＳＦＭＣ）、フォン・ウィルブランド因子（第ＶＩＩＩ因子様抗原）、プロテインＳ、リウマチ因子（ＲＦ）、ＩｇＤ、α１アシドグリコプロテイン（α１ＡＧ）、α１アンチトリプシン（α１ＡＴ）、α２マクログロブリン、アルブミン（Ａｌｂ）、セルロプラスミン（Ｃｐ）、ハプトグロビン（Ｈｐ）、プレアルブミン、レチノール結合蛋白（ＲＢＰ）、β１Ｃ／β１Ａグロブリン（Ｃ３）、β１Ｅグロブリン（Ｃ４）、ＩｇＡ、ＩｇＧ、ＩｇＭ、βリポ蛋白（β−ＬＰ）、アポ蛋白Ａ−Ｉ、アポ蛋白Ａ−ＩＩ、アポ蛋白Ｂ、アポ蛋白Ｃ−ＩＩ、アポ蛋白Ｃ−ＩＩＩ、アポ蛋白Ｅ、トランスフェリン（Ｔｆ）、尿中アルブミン、プラスミノーゲン（ＰＬＧ）およびリポ蛋白（ａ）（ＬＰ（ａ））、乳酸、低密度脂質、高密度脂質、トリグリセリド等があげられる。本発明において、測定対象の目的成分は、例えば、１種類でもよいし、２種類以上でもよい。 In the present invention, the target component is not particularly limited, and for example, sugar such as glucose, C-reactive protein (CRP), HbA1c, thyroid stimulating hormone (TSH), FT3, FT4, hCG, HBs antigen, HBc antibody, HCV antibody, TY antigen, anti-streptricin O (ASO), type IV collagen, matrix metalloproteinase-3 (MMP-3), PIVAK-II, α1 microglobulin, β1 microglobulin, amyloid A (SAA), elastase 1 , Basic Fetoprotein (BFP), Candida Antigen, Cervical Mucus Granulocyte Elastase, Digoxin, Cystatin C, Factor XIII, Urinary Transfectin, Pyramid, Hyaluronic Acid, Fibrin Monomer Complex (SFMC), von Willbrand Factor (Factor VIII-like antigen), protein S, rheumatic factor (RF), IgD, α1 acid glycoprotein (α1AG), α1 antitrypsin (α1AT), α2 macroglobulin, albumin (Alb), celluloplasmin (Cp), haptoglobin (Hp), prealbumin, retinol binding protein (RBP), β1C / β1A globulin (C3), β1E globulin (C4), IgA, IgG, IgM, β lipoprotein (β-LP), apoprotein AI, apo Protein A-II, Apoprotein B, Apoprotein C-II, Apoprotein C-III, Apoprotein E, Transtransferase (Tf), Urinary albumin, Plasminogen (PLG) and Lipoprotein (a) (LP (a) )), Lactobacillus, low-density lipid, high-density lipid, triglyceride and the like. In the present invention, the target component to be measured may be, for example, one type or two or more types.
本発明において、前記酸化還元触媒は、例えば、前記目的成分に対する酸化還元反応を触媒する触媒であり、酸化反応および還元反応の一方を触媒してもよいし、両反応を触媒してもよい。前記酸化還元触媒は、特に制限されず、例えば、前記目的成分に応じて、適宜決定できる。前記酸化還元触媒は、例えば、酸化酵素、還元酵素、酸化還元酵素、脱水素酵素等があげられる。具体例として、前記目的成分がグルコースである場合、前記酸化酵素は、例えば、グルコース酸化酵素等があげられ、前記脱水素酵素は、例えば、グルコース脱水素酵素等があげられる。前記目的成分が乳酸である場合、前記酸化還元酵素は、例えば、乳酸酸化還元酵素等があげられ、前記脱水素酵素は、例えば、乳酸脱水素酵素等があげられる。前記酸化還元触媒（例えば、酸化還元酵素）は、例えば、さらに、補因子を含んでもよい。前記補因子は、特に制限されず、例えば、前記酸化還元触媒（例えば、酸化還元酵素）の種類に応じて、適宜決定できる。前記補因子は、例えば、フラビンアデニンジヌクレオチド（ＦＡＤ）、ピロロキノリンキノン（ＰＱＱ）、ニコチンアミドアデニンジヌクレオチド（ＮＡＤ）、ニコチンアミドアデニンジヌクレオチドリン酸（ＮＡＤＰ）等があげられる。前記酸化還元触媒（例えば、酸化還元酵素）は、例えば、１種類を単独で用いてもよいし、２種類以上を併用してもよい。 In the present invention, the redox catalyst is, for example, a catalyst that catalyzes a redox reaction with respect to the target component, and may catalyze one of the oxidation reaction and the reduction reaction, or may catalyze both reactions. The redox catalyst is not particularly limited, and can be appropriately determined, for example, according to the target component. Examples of the redox catalyst include oxidase, reductase, oxidoreductase, dehydrogenase and the like. As a specific example, when the target component is glucose, the oxidase may be, for example, glucose oxidase, and the dehydrogenase may be, for example, glucose dehydrogenase. When the target component is lactic acid, the oxidoreductase includes, for example, lactate oxidoreductase, and the dehydrogenase includes, for example, lactate dehydrogenase. The redox catalyst (eg, oxidoreductase) may further contain, for example, a cofactor. The cofactor is not particularly limited and can be appropriately determined, for example, depending on the type of the redox catalyst (for example, oxidoreductase). Examples of the cofactor include flavin adenine dinucleotide (FAD), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP). As the redox catalyst (for example, oxidoreductase), for example, one type may be used alone, or two or more types may be used in combination.
本発明において、前記電子伝達物質は、例えば、前記目的成分と前記酸化還元触媒との酸化還元反応により生じた電子を受容し、前記電極系へ供与する物質でもよいし、前記電極系から電子を受容し、前記目的成分と前記酸化還元触媒との酸化還元反応に供与する物質でもよい。前記電子伝達物質は、特に制限されず、例えば、前記酸化還元触媒の種類に応じて、適宜決定できる。具体的に、前記電子伝達物質は、例えば、ルテニウム錯体、フェリシアン化合物、オスミウム錯体、鉄錯体、他の有機金属錯体、有機金属錯体ポリマー、導電性イオン塩、ベンゾキノン等の有機化合物、導電性酸化還元ポリマー等があげられる。前記電子伝達物質は、例えば、１種類を単独で用いてもよいし、２種類以上を併用してもよい。 In the present invention, the electron transfer substance may be, for example, a substance that receives electrons generated by a redox reaction between the target component and the redox catalyst and donates them to the electrode system, or transfers electrons from the electrode system. It may be a substance that receives and contributes to the redox reaction between the target component and the redox catalyst. The electron transfer substance is not particularly limited, and can be appropriately determined, for example, depending on the type of the redox catalyst. Specifically, the electron transfer substance is, for example, a ruthenium complex, a ferrician compound, an osmium complex, an iron complex, another organic metal complex, an organic metal complex polymer, a conductive ion salt, an organic compound such as benzoquinone, or conductive oxidation. Examples include reduced polymers. As the electron transmitter, for example, one type may be used alone, or two or more types may be used in combination.
本発明において、前記電極系は、例えば、前記目的成分および前記酸化還元触媒の酸化還元反応によるシグナルを検出する電極である。前記電極系は、例えば、前記シグナルの検出部位である作用極と対極とを含む少なくとも１セットの電極群を備える。前記電極系において、前記電極群の数は、例えば、１セットでもよく、２セット以上でもよい。前記電極系において、前記作用極は、例えば、１個でもよく、２個以上でもよい。後者の場合、前記２個以上の作用極は、例えば、異なる作用極があげられる。前記異なる作用極は、例えば、電極の材料が異なるもの、検出する目的成分が異なるもの等があげられる。前記作用極には、例えば、前記酸化還元触媒と前記電子伝達物質とが、予め配置されてもよく、前記酸化還元触媒と前記電子伝達物質とが、前記作用極にコーティングされてもよい。この場合、例えば、前記電極系と前記試料とを接触させることで、前記目的成分の測定ができ、より簡便に目的成分の測定ができる。前記電極系において、前記対極は、例えば、１個でもよいし、２個以上でもよい。前記電極系において、各電極群は、例えば、それぞれ別個の対極を有してもよいし、１つの電極群における対極が、他の電極群の対極を兼ねてもよい。前記電極系は、例えば、必要に応じて、参照電極等の他の電極を含んでもよい。前記電極系の各電極の材料は、特に制限されず、例えば、前記目的成分、前記酸化還元触媒、前記電子伝達物質の種類に応じて、適宜決定できる。 In the present invention, the electrode system is, for example, an electrode that detects a signal due to a redox reaction of the target component and the redox catalyst. The electrode system includes, for example, at least one set of electrodes including an working electrode and a counter electrode, which are detection sites for the signal. In the electrode system, the number of the electrode groups may be, for example, one set or two or more sets. In the electrode system, the number of working electrodes may be, for example, one or two or more. In the latter case, the two or more working poles may be, for example, different working poles. Examples of the different working electrodes include those having different electrode materials and those having different target components to be detected. For example, the redox catalyst and the electron transfer material may be arranged in advance on the working electrode, or the redox catalyst and the electron transmitting material may be coated on the working electrode. In this case, for example, by bringing the electrode system into contact with the sample, the target component can be measured, and the target component can be measured more easily. In the electrode system, the number of counter electrodes may be, for example, one or two or more. In the electrode system, each electrode group may have a separate counter electrode, for example, or the counter electrode in one electrode group may also serve as the counter electrode of another electrode group. The electrode system may include, for example, other electrodes such as a reference electrode, if desired. The material of each electrode of the electrode system is not particularly limited, and can be appropriately determined depending on, for example, the type of the target component, the redox catalyst, and the electron transmitter.
前記電極系は、例えば、さらに、シグナル取得手段に連結され、前記シグナル取得手段が、前記作用極に連結されていることが好ましい。この場合、前記作用極は、例えば、端子を介して、前記シグナル取得手段と連結されてもよい。前記シグナル取得手段は、例えば、検出器等があげられる。前記検出器は、特に制限されず、例えば、電流検出器、電位検出器、電圧検出器等があげられる。前記検出器は、例えば、電流／電圧変換器、Ａ／Ｄ変換器、演算器等を含んでもよい。 It is preferable that the electrode system is further connected to, for example, a signal acquisition means, and the signal acquisition means is connected to the working electrode. In this case, the working electrode may be connected to the signal acquisition means, for example, via a terminal. Examples of the signal acquisition means include a detector and the like. The detector is not particularly limited, and examples thereof include a current detector, a potential detector, and a voltage detector. The detector may include, for example, a current / voltage converter, an A / D converter, an arithmetic unit, and the like.
前記電極系としては、例えば、前記目的成分を測定可能な公知の電気化学的測定方法に使用するバイオセンサの電極系を用いてもよい。前記電極系として前記バイオセンサを用いる場合、前記バイオセンサは、例えば、前記作用極と前記対極とを含む少なくとも１セットの電極群を備える電極系と前記検出器とを有し、前記検出器が、前記作用極に連結されている。前記バイオセンサにおいて、前記検出器は、例えば、前記バイオセンサ内に配置されてもよいし、前記バイオセンサ外に配置されてもよい。後者の場合、例えば、前記シグナルの取得前から、予め、前記バイオセンサと前記検出器とが連結されてもよいし、取得時に、前記バイオセンサと前記検出器とが連結されてもよい。 As the electrode system, for example, an electrode system of a biosensor used in a known electrochemical measurement method capable of measuring the target component may be used. When the biosensor is used as the electrode system, the biosensor has, for example, an electrode system including at least one set of electrodes including the working electrode and the counter electrode, and the detector. , Connected to the working electrode. In the biosensor, the detector may be arranged, for example, inside the biosensor or outside the biosensor. In the latter case, for example, the biosensor and the detector may be connected in advance before the acquisition of the signal, or the biosensor and the detector may be connected at the time of acquisition.
前記バイオセンサは、例えば、さらに、前記試料を導入する導入部、前記試料を前記電極系に移動させる移動部等を有してもよい。前記バイオセンサは、例えば、前記酸化還元触媒と前記電子伝達物質とが配置されていてもよい。前記酸化還元触媒と前記電子伝達物質の配置箇所は、例えば、前記作用極、前記導入部、前記移動部等があげられる。前記酸化還元触媒と前記電子伝達物質とは、例えば、１箇所に配置されてもよいし、２箇所以上に配置されてもよい。前記作用極に配置される場合、前記作用極が、例えば、前記酸化還元触媒と前記電子伝達物質にコーティングされている。また、前記バイオセンサが２個以上の作用極を有する場合、各作用極に配置される前記酸化還元触媒および前記電子伝達物質は、それぞれ、同じでもよいし、異なってもよい。 The biosensor may further include, for example, an introduction section for introducing the sample, a moving section for moving the sample to the electrode system, and the like. In the biosensor, for example, the redox catalyst and the electron transmitter may be arranged. Examples of the location where the redox catalyst and the electron transfer substance are arranged include the working electrode, the introducing portion, and the moving portion. The redox catalyst and the electron transmitter may be arranged at one place or at two or more places, for example. When placed on the working electrode, the working electrode is, for example, coated on the redox catalyst and the electron messenger. Further, when the biosensor has two or more working electrodes, the redox catalyst and the electron transmitter arranged in each working pole may be the same or different.
本発明において、前記シグナルは、前記電極系により取得可能なシグナルである。前記シグナルは、特に制限されず、例えば、前記目的成分および前記酸化還元触媒の酸化還元反応に基づくシグナルであり、具体例として、電気シグナルがあげられる。前記電気シグナルは、例えば、電流、電流から変換された電圧、電流および時間から算出された電気量および、これらに応じたデジタル信号等でもよい。これらの電気シグナルは、例えば、公知の方法によって相互に変換できる。具体例として、前記電極系により電流を測定した場合、前記電流は、例えば、電流／電圧変換器等によって電圧に変換でき、また、アナログ信号である電圧は、例えば、Ａ／Ｄ（アナログ／デジタル）変換器によってデジタル信号に変換できる。 In the present invention, the signal is a signal that can be obtained by the electrode system. The signal is not particularly limited, and is, for example, a signal based on the redox reaction of the target component and the redox catalyst, and specific examples thereof include an electric signal. The electric signal may be, for example, a current, a voltage converted from the current, an amount of electricity calculated from the current and time, a digital signal corresponding thereto, or the like. These electrical signals can be converted to each other, for example, by known methods. As a specific example, when the current is measured by the electrode system, the current can be converted into a voltage by, for example, a current / voltage converter, and the voltage which is an analog signal is, for example, A / D (analog / digital). ) It can be converted to a digital signal by a converter.
前記第１の印加工程は、前述のように、前記試料、前記目的成分に対する酸化還元触媒、および前記電子伝達物質の存在下、前記第１の電圧を前記電極系に印加する。前記第１の印加工程は、例えば、前記試料、前記酸化還元触媒、および前記電子伝達物質を含む反応系に、前記電極系を接触させ、前記電極系に前記第１の電圧を印加することにより実施できる。前記反応系は、例えば、前記試料に、前記酸化還元触媒および前記電子伝達物質を添加することにより調製できる。前記電極系として、前記バイオセンサの電極系を用いる場合、前記反応系は、例えば、前記酸化還元触媒と前記電子伝達物質とがコーティングされた前記電極系に対し、前記試料を接触させることにより調製してもよい。前記電極系への第１の電圧の印加は、例えば、電圧印加手段により実施できる。前記電圧印加手段は、特に制限されず、例えば、前記電極系に電圧を印加できればよく、公知の手段として電圧器等が使用できる。 In the first application step, as described above, the first voltage is applied to the electrode system in the presence of the sample, the redox catalyst for the target component, and the electron transmitter. In the first application step, for example, the electrode system is brought into contact with a reaction system containing the sample, the redox catalyst, and the electron transfer substance, and the first voltage is applied to the electrode system. Can be implemented. The reaction system can be prepared, for example, by adding the redox catalyst and the electron transmitter to the sample. When the electrode system of the biosensor is used as the electrode system, the reaction system is prepared, for example, by bringing the sample into contact with the electrode system coated with the redox catalyst and the electron transfer substance. You may. The application of the first voltage to the electrode system can be carried out by, for example, a voltage applying means. The voltage applying means is not particularly limited as long as a voltage can be applied to the electrode system, and a voltage device or the like can be used as a known means.
前記第１の印加工程において、前記第１の電圧（Ｖ１）は、前記酸化電位（Ｅ）以上の電圧、すなわち、Ｅ≦Ｖ１（Ｖ）であればよく、その上限は、特に制限されない。前記酸化電位が複数回の測定で得られた電位に基づき設定される場合、前記酸化電位は、前記複数回の測定で得られた電位における最大の電位であることが好ましい。前記第１の電圧は、例えば、前記シグナル取得時のノイズを低減できることから、好ましくは、Ｅ＋０．１５≦Ｖ１（Ｖ）、Ｅ＋０．２５≦Ｖ１（Ｖ）である。前記第１の電圧の上限は、例えば、前記反応系における水の加水分解が起きない電圧の最大値が好ましく、前記干渉物質の影響を低減できることから、より好ましくは、Ｖ１≦Ｅ＋１．１５（Ｖ）、Ｖ１≦Ｅ＋１．０５（Ｖ）、Ｖ１≦Ｅ＋１（Ｖ）、Ｖ１≦Ｅ＋０．９５（Ｖ）である。前記第１の電圧の範囲は、前記シグナル取得時のノイズを低減でき、且つ前記干渉物質の影響を低減できることから、好ましくは、Ｅ≦Ｖ１≦Ｅ＋１．１５（Ｖ）の範囲、Ｅ＋０．１５≦Ｖ１≦Ｅ＋１．０５（Ｖ）の範囲、Ｅ＋０．２５≦Ｖ１≦Ｅ＋０．９５（Ｖ）の範囲である。具体例として、前記電子伝達物質がルテニウム錯体であり、前記電極系の材料がカーボンである場合、前記第１の電圧は、例えば、５０〜１２００ｍＶの範囲、２００〜１１００ｍＶの範囲、３００〜１０００ｍＶの範囲である。前記第１の電圧は、例えば、一定であってもよいし、変動してもよいが、好ましくは、前者である。 In the first application step, the first voltage (V 1 ) may be a voltage equal to or higher than the oxidation potential (E), that is, E ≦ V 1 (V), and the upper limit thereof is not particularly limited. .. When the oxidation potential is set based on the potentials obtained in the plurality of measurements, the oxidation potential is preferably the maximum potential in the potentials obtained in the plurality of measurements. The first voltage is preferably E + 0.15 ≦ V 1 (V) and E + 0.25 ≦ V 1 (V) because noise at the time of acquiring the signal can be reduced, for example. The upper limit of the first voltage is preferably, for example, the maximum value of the voltage at which water does not hydrolyze in the reaction system, and the influence of the interfering substance can be reduced. Therefore, V 1 ≤ E + 1.15 (more preferably). V), V 1 ≤ E + 1.05 (V), V 1 ≤ E + 1 (V), V 1 ≤ E + 0.95 (V). The first voltage range preferably has a range of E ≤ V 1 ≤ E + 1.15 (V) and E + 0.15 because the noise at the time of acquiring the signal can be reduced and the influence of the interfering substance can be reduced. The range is ≦ V 1 ≦ E + 1.05 (V) and E + 0.25 ≦ V 1 ≦ E + 0.95 (V). As a specific example, when the electron transfer substance is a ruthenium complex and the material of the electrode system is carbon, the first voltage is, for example, in the range of 50 to 1200 mV, in the range of 200 to 1100 mV, and in the range of 300 to 1000 mV. The range. The first voltage may be constant or variable, for example, but is preferably the former.
前記第１の印加工程において、前記第１の電圧の印加時間は、特に制限されず、例えば、０．１〜１０秒、０．５〜８秒、１〜５秒である。前記電極系への電圧印加は、例えば、連続的に印加してもよいし、非連続的に印加してもよい。前記第１の印加工程において、前記電極系の電流は、例えば、前記反応系に応じて適宜設定できる。 In the first application step, the application time of the first voltage is not particularly limited, and is, for example, 0.1 to 10 seconds, 0.5 to 8 seconds, and 1 to 5 seconds. The voltage applied to the electrode system may be, for example, continuously applied or discontinuously applied. In the first application step, the current of the electrode system can be appropriately set according to, for example, the reaction system.
前記第２の印加工程は、前述のように、前記第２の電圧を前記電極系に印加する。前記第２の印加工程は、例えば、前記反応系と接触する前記電極系に前記第２の電圧を印加することにより実施できる。前記電極系への第２の電圧の印加は、例えば、前述の電圧印加手段により実施できる。 In the second application step, as described above, the second voltage is applied to the electrode system. The second application step can be carried out, for example, by applying the second voltage to the electrode system in contact with the reaction system. The application of the second voltage to the electrode system can be carried out by, for example, the voltage application means described above.
前記第２の印加工程は、例えば、前記第１の印加工程と連続的に行ってもよいし、非連続的に行ってもよいが、好ましくは、前者である。前者の場合、前記第２の印加工程は、前記第１の印加工程の終了と同時に前記第２の印加工程を行う。 The second application step may be performed continuously or discontinuously with, for example, the first application step, but the former is preferable. In the former case, the second application step is performed at the same time as the end of the first application step.
前記第２の印加工程において、前記第２の電圧（Ｖ２）は、前記酸化電位（Ｅ）未満の電圧、すなわち、Ｖ２＜Ｅ（Ｖ）であればよく、その下限は、特に制限されない。前記酸化電位が複数回の測定で得られた電位に基づき設定される場合、前記酸化電位は、前記複数回の測定で得られた電位における最小の電位であることが好ましい。前記第２の電圧は、例えば、前記酸化電位に近づくにつれて、前記シグナル取得時のノイズをより低減できることから、前記酸化電位に近い電位が好ましい。前記第２の電圧の下限は、例えば、前記干渉物質の影響を低減できることから、好ましくは、Ｅ−０．２５≦Ｖ２（Ｖ）、Ｅ−０．２≦Ｖ２（Ｖ）、Ｅ−０．１５≦Ｖ２（Ｖ）、Ｅ−０．１≦Ｖ２（Ｖ）である。前記第２の電圧の範囲は、例えば、前記シグナル取得時のノイズを低減でき、且つ前記干渉物質の影響を低減できることから、好ましくは、Ｅ−０．２５≦Ｖ２＜Ｅ（Ｖ）の範囲、Ｅ−０．２≦Ｖ２＜Ｅ（Ｖ）の範囲、Ｅ−０．１５≦Ｖ２＜Ｅ（Ｖ）の範囲、Ｅ−０．１≦Ｖ２＜Ｅ（Ｖ）の範囲である。具体例として、前記電子伝達物質がルテニウム錯体であり、前記電極系の材料がカーボンである場合、前記第２の電圧は、例えば、−２００ｍＶ以上、５０ｍＶ未満の範囲、−１００ｍＶ以上、５０ｍＶ未満の範囲、−５０ｍＶ以上、５０ｍＶ未満の範囲である。前記第２の電圧は、例えば、一定であってもよいし、変動してもよいが、好ましくは、前者である。 In the second application step, the second voltage (V 2 ) may be a voltage lower than the oxidation potential (E), that is, V 2 <E (V), and the lower limit thereof is not particularly limited. .. When the oxidation potential is set based on the potential obtained by the plurality of measurements, the oxidation potential is preferably the smallest potential among the potentials obtained by the plurality of measurements. The second voltage is preferably a potential close to the oxidation potential, for example, because noise at the time of acquiring the signal can be further reduced as the voltage approaches the oxidation potential. The lower limit of the second voltage is preferably E-0.25 ≦ V 2 (V), E-0.2 ≦ V 2 (V), E- because the influence of the interfering substance can be reduced, for example. 0.15 ≦ V 2 (V) and E-0.1 ≦ V 2 (V). The second voltage range is preferably a range of E-0.25 ≦ V 2 <E (V) because, for example, noise at the time of acquiring the signal can be reduced and the influence of the interfering substance can be reduced. , E-0.2≤V 2 <E (V) range, E-0.15≤V 2 <E (V) range, E-0.1≤V 2 <E (V) range. .. As a specific example, when the electron transfer substance is a ruthenium complex and the material of the electrode system is carbon, the second voltage is, for example, in the range of −200 mV or more and less than 50 mV, -100 mV or more and less than 50 mV. The range is -50 mV or more and less than 50 mV. The second voltage may be constant or variable, for example, but is preferably the former.
前記第２の印加工程において、前記第２の電圧の印加時間は、特に制限されず、後述するシグナル取得工程におけるシグナルの取得時点に応じて、適宜設定できる。前記第２の電圧の印加時間は、例えば、０．００１〜１０秒、０．００１〜２秒、０．００５〜１．５秒、０．０１〜１秒である。前記電極系への電圧印加は、例えば、連続的に印加してもよいし、非連続的に印加してもよい。前記第２の印加工程において、前記電極系の電流は、例えば、前記反応系に応じて適宜設定できる。 In the second application step, the application time of the second voltage is not particularly limited and can be appropriately set according to the signal acquisition time point in the signal acquisition step described later. The application time of the second voltage is, for example, 0.001 to 10 seconds, 0.001 to 2 seconds, 0.005 to 1.5 seconds, and 0.01 to 1 second. The voltage applied to the electrode system may be, for example, continuously applied or discontinuously applied. In the second application step, the current of the electrode system can be appropriately set according to, for example, the reaction system.
前記シグナル取得工程は、前述のように、前記第２の印加工程において、前記電極系によりシグナルを取得する。前記シグナルは、例えば、前記電極系に連結された前記シグナル取得手段（例えば、前記検出器等）により取得できる。前記電極系が複数の作用極を有する場合、前記シグナルは、前記複数の作用極で取得してもよい。 In the signal acquisition step, as described above, in the second application step, a signal is acquired by the electrode system. The signal can be acquired, for example, by the signal acquisition means (for example, the detector or the like) connected to the electrode system. When the electrode system has a plurality of working electrodes, the signal may be acquired by the plurality of working electrodes.
前記シグナル取得工程において、前記シグナルの取得回数は、特に制限されず、例えば、１回でもよいし複数回でもよい。前者の場合、前記シグナルは、例えば、１時点で取得したシグナルでもよいし、所定時間、連続的に取得したシグナルでもよい。前記シグナルが連続的に取得したシグナルである場合、前記シグナルは、例えば、前記シグナルの積分値ということもできる。後者の場合、前記シグナルは、例えば、複数の時点で取得したシグナルでもよいし、複数の作用極で取得したシグナルでもよい。 In the signal acquisition step, the number of times the signal is acquired is not particularly limited, and may be, for example, once or a plurality of times. In the former case, the signal may be, for example, a signal acquired at one time point or a signal acquired continuously for a predetermined time. When the signal is a continuously acquired signal, the signal can be, for example, an integral value of the signal. In the latter case, the signal may be, for example, a signal acquired at a plurality of time points or a signal acquired at a plurality of working poles.
前記複数の時点で取得したシグナルは、例えば、経時的に複数回取得したシグナルを意味する。前記時点の数、すなわち、シグナル取得の回数は、複数時点であればよく、例えば、２時点以上であり、好ましくは、２〜１０時点、２〜５時点である。 The signal acquired at the plurality of time points means, for example, a signal acquired a plurality of times over time. The number of time points, that is, the number of times of signal acquisition may be a plurality of time points, for example, 2 time points or more, preferably 2 to 10 time points and 2 to 5 time points.
前記シグナル取得工程において、前記シグナルを取得するタイミングは、特に制限されず、例えば、前記目的成分、前記酸化還元触媒、前記電子伝達物質、前記第１の電圧、前記第２の電圧等によって、適宜決定できる。具体例として、前記シグナルは、前記干渉物質の影響を低減できることから、好ましくは、前記第２の電圧印加時を基準（０秒）として、０．４秒以内、０．２秒以内、０．１秒以内、０．０５秒以内に取得したシグナルであり、より好ましくは、前記第２の電圧印加時を基準として、０〜０．４秒、０．０５〜０．４秒、０〜０．２秒、０．０５〜０．２秒、０〜０．１秒、０．０５〜０．１秒、０〜０．０５秒に取得したシグナルである。前記シグナルが１時点で取得したシグナルである場合、前記シグナルを取得するタイミングは、例えば、前記第２の電圧印加時を基準として、０．２秒以内、０．１５秒以内、０．１秒以内に取得したシグナルであり、好ましくは、前記第２の電圧印加時を基準として、０〜０．２秒、０．０５〜０．２秒、０〜０．１５秒、０．０５〜０．１５秒、０〜０．１秒、０．０５〜０．１秒に取得したシグナルである。前記シグナルが前記シグナルの積分値である場合、前記シグナルを取得するタイミングは、例えば、前記第２の電圧印加時を基準として、０〜０．２秒、０．０５〜０．２秒、０〜０．１５秒、０．０５〜０．１５秒、０〜０．１秒、０．０５〜０．１秒に取得したシグナルである。前記シグナルが前記複数の時点で取得したシグナルである場合、前記複数のシグナルのうち最初に取得するシグナルは、例えば、０．５秒以内、０．３秒以内、０．１秒以内に取得したシグナルであり、好ましくは、前記第２の電圧印加時を基準として、０〜０．２秒、０．０５〜０．２秒、０〜０．１５秒、０．０５〜０．１５秒、０〜０．１秒、０．０５〜０．１秒に取得したシグナルである。また、前記複数のシグナルのうち最後に取得するシグナルは、例えば、０．５秒以内、０．３秒以内、０．１秒以内に取得したシグナルであり、好ましくは、前記第２の電圧印加時を基準として、０〜０．２秒、０．０５〜０．２秒、０〜０．１５秒、０．０５〜０．１５秒、０〜０．１秒、０．０５〜０．１秒に取得したシグナルである。具体例として、前記目的成分がグルコースであり、前記酸化還元触媒がグルコース脱水素酵素であり、前記電子伝達物質がルテニウム錯体である場合、前記シグナルを取得するタイミングは、例えば、前記第２の電圧印加時を基準として、０〜０．２秒、０．０５〜０．２秒、０〜０．１５秒、０．０５〜０．１５秒、０〜０．１秒、０．０５〜０．１秒に取得したシグナルである。 In the signal acquisition step, the timing of acquiring the signal is not particularly limited, and is appropriately determined by, for example, the target component, the redox catalyst, the electron transmitter, the first voltage, the second voltage, and the like. Can be decided. As a specific example, since the influence of the interfering substance can be reduced, the signal is preferably within 0.4 seconds, within 0.2 seconds, or 0, based on the time when the second voltage is applied (0 seconds). It is a signal acquired within 1 second and within 0.05 seconds, and more preferably 0 to 0.4 seconds, 0.05 to 0.4 seconds, and 0 to 0 based on the time when the second voltage is applied. . Signals acquired at 2 seconds, 0.05 to 0.2 seconds, 0 to 0.1 seconds, 0.05 to 0.1 seconds, and 0 to 0.05 seconds. When the signal is a signal acquired at one time point, the timing of acquiring the signal is, for example, within 0.2 seconds, within 0.15 seconds, or 0.1 seconds with reference to the time when the second voltage is applied. It is a signal acquired within the range, preferably 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0, based on the time when the second voltage is applied. It is a signal acquired in .15 seconds, 0 to 0.1 seconds, and 0.05 to 0.1 seconds. When the signal is an integral value of the signal, the timing of acquiring the signal is, for example, 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 with reference to the time when the second voltage is applied. It is a signal acquired in ~ 0.15 seconds, 0.05 to 0.15 seconds, 0 to 0.1 seconds, 0.05 to 0.1 seconds. When the signal is a signal acquired at the plurality of time points, the signal acquired first among the plurality of signals is acquired within, for example, within 0.5 seconds, within 0.3 seconds, and within 0.1 seconds. It is a signal, preferably 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0.15 seconds, based on the time when the second voltage is applied. It is a signal acquired in 0 to 0.1 seconds and 0.05 to 0.1 seconds. The signal acquired last among the plurality of signals is, for example, a signal acquired within 0.5 seconds, within 0.3 seconds, or within 0.1 seconds, and preferably the second voltage application. Based on time, 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0.15 seconds, 0 to 0.1 seconds, 0.05 to 0. It is a signal acquired in 1 second. As a specific example, when the target component is glucose, the redox catalyst is glucose dehydrogenase, and the electron transfer substance is a ruthenium complex, the timing for acquiring the signal is, for example, the second voltage. 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0.15 seconds, 0 to 0.1 seconds, 0.05 to 0 based on the time of application . It is a signal acquired in 1 second.
前記シグナルが前記複数回のシグナルである場合、前記シグナルを取得する時間の間隔は、特に制限されず、例えば、前記シグナルの種類、前記目的成分、前記第１の電圧、前記第２の電圧等によって、適宜決定できる。具体例として、前記複数のシグナルは、例えば、０．０１〜１０秒、０．０５〜５秒、０．１〜３秒の間隔で取得したシグナルである。 When the signal is the plurality of signals, the time interval for acquiring the signal is not particularly limited, and for example, the type of the signal, the target component, the first voltage, the second voltage, and the like. Can be determined as appropriate. As a specific example, the plurality of signals are signals acquired at intervals of, for example, 0.01 to 10 seconds, 0.05 to 5 seconds, and 0.1 to 3 seconds.
本発明の測定方法は、前記シグナルから前記試料中の目的成分量を算出する算出工程を含んでもよい。前記算出工程において、前記目的成分量は、例えば、得られたシグナルおよびシグナルと試料中の前記目的成分量との相関関係に基づき、算出できる。前記相関関係は、例えば、検量線、回帰直線、検量表等で表わすことができる。前記相関関係は、例えば、前記目的成分量が既知である標準試料について、前記本発明の測定方法により得られたシグナルと、前記標準試料の前記目的成分量とをプロットすることにより求めることができる。前記標準試料は、前記目的成分の希釈系列が好ましい。このように算出を行うことによって、信頼性の高い定量が可能となる。前記算出工程は、例えば、ハードウェアであるデータ処理装置等の算出手段により実施でき、具体的には、前述の前記バイオセンサにより実施できる。前記データ処理装置は、例えば、ＣＰＵ等を備えてもよい。 The measuring method of the present invention may include a calculation step of calculating the amount of the target component in the sample from the signal. In the calculation step, the target component amount can be calculated based on, for example, the obtained signal and the correlation between the signal and the target component amount in the sample. The correlation can be represented by, for example, a calibration curve, a regression line, a calibration table, or the like. The correlation can be obtained, for example, by plotting the signal obtained by the measurement method of the present invention and the target component amount of the standard sample for a standard sample whose target component amount is known. .. The standard sample is preferably a dilution series of the target component. By performing the calculation in this way, highly reliable quantification becomes possible. The calculation step can be carried out by, for example, a calculation means such as a data processing device which is hardware, and specifically, can be carried out by the above-mentioned biosensor. The data processing device may include, for example, a CPU or the like.
本発明の測定方法は、例えば、前記干渉物質の影響を低減し、より信頼性の高い測定が可能になることから、前記シグナルを補正シグナルにより補正してもよい。具体的に、本発明の測定方法は、前記電極系により補正シグナルを取得する補正シグナル取得工程と、前記補正シグナルに基づき、前記シグナルを補正する補正工程とを含み、前記算出工程において、前記補正後のシグナルから前記試料中の目的成分量を算出することが好ましい。 In the measuring method of the present invention, for example, the signal may be corrected by a correction signal because the influence of the interfering substance is reduced and more reliable measurement is possible. Specifically, the measurement method of the present invention includes a correction signal acquisition step of acquiring a correction signal by the electrode system and a correction step of correcting the signal based on the correction signal, and in the calculation step, the correction It is preferable to calculate the amount of the target component in the sample from the subsequent signal.
前記補正シグナルは、例えば、前記干渉物質による影響を補正するシグナルである。前記干渉物質は、例えば、ヘマトクリット、アスコルビン酸、糖、ＥＤＴＡ（エチレンジアミン四酢酸）等の血液凝固防止剤等があげられる。具体的に、前記補正シグナルは、例えば、前記電極系により取得した前記干渉物質由来のシグナル、前記電極系により取得した前記目的成分由来のシグナルがあげられ、より具体的には、前記電極系に連結されたシグナル取得手段により取得した前記目的成分由来のシグナルがあげられる。 The correction signal is, for example, a signal that corrects the influence of the interfering substance. Examples of the interfering substance include hematocrit, ascorbic acid, sugar, and anticoagulants such as EDTA (ethylenediaminetetraacetic acid). Specifically, the correction signal includes, for example, a signal derived from the interfering substance acquired by the electrode system and a signal derived from the target component acquired by the electrode system, and more specifically, the electrode system. Examples thereof include signals derived from the target component acquired by the linked signal acquisition means.
前記補正シグナルが前記干渉物質由来のシグナルの場合、前記補正シグナルの測定に用いる電極群は、例えば、前記目的成分を測定する電極群と同じもよいし、異なってもよい。後者の場合、前記電極系は、例えば、前記目的成分を測定する電極群に加えて、前記干渉物質を測定する電極群を含む。また、前記反応系は、例えば、前記干渉物質の種類に応じて、前記干渉物質由来のシグナルの取得に必要な試薬等を含んでもよい。前記電極系による補正シグナルの取得方法は、特に制限されず、例えば、前記干渉物質の種類に応じて、公知の方法により実施できる。前記干渉物質由来のシグナルによる前記シグナルの補正方法は、特に制限されず、例えば、前記干渉物質由来のシグナルと、前記シグナルの変動との相関関係に基づき、補正する方法があげられる。前記相関関係は、例えば、前記目的成分量が既知であり、且つ異なる前記干渉物質を含む標準試料について、前記本発明の測定方法により得られたシグナルと、前記標準試料の前記目的成分量とをプロットすることにより求めることができる。 When the correction signal is a signal derived from the interfering substance, the electrode group used for measuring the correction signal may be, for example, the same as or different from the electrode group for measuring the target component. In the latter case, the electrode system includes, for example, an electrode group for measuring the interfering substance in addition to the electrode group for measuring the target component. Further, the reaction system may contain, for example, a reagent necessary for obtaining a signal derived from the interfering substance, depending on the type of the interfering substance. The method of acquiring the correction signal by the electrode system is not particularly limited, and for example, it can be carried out by a known method depending on the type of the interfering substance. The method of correcting the signal by the signal derived from the interfering substance is not particularly limited, and examples thereof include a method of correcting the signal based on the correlation between the signal derived from the interfering substance and the fluctuation of the signal. The correlation is, for example, the signal obtained by the measurement method of the present invention and the target component amount of the standard sample for a standard sample in which the target component amount is known and contains different interfering substances. It can be obtained by plotting.
前記補正シグナルが前記目的成分由来のシグナルの場合、前記補正シグナルは、例えば、前記シグナル取得工程において取得された複数のシグナルでもよい。前記複数のシグナルは、例えば、前述の説明を援用できる。前記複数のシグナルによるシグナルの補正方法は、特に制限されず、例えば、前記目的成分量と、それに応じた複数のシグナルとの関係に基づき、補正する方法があげられる。具体例として、前記シグナルの補正方法は、例えば、米国特許第８７６０１７８号明細書を参照できる。前記補正工程は、例えば、前述のハードウェアであるデータ処理装置等の補正手段により実施でき、具体的には、前述の前記バイオセンサにより実施できる。 When the correction signal is a signal derived from the target component, the correction signal may be, for example, a plurality of signals acquired in the signal acquisition step. The plurality of signals can be referred to, for example, the above description. The method of correcting the signal by the plurality of signals is not particularly limited, and examples thereof include a method of correcting based on the relationship between the target component amount and the plurality of signals corresponding thereto. As a specific example, the method for correcting the signal can be referred to, for example, US Pat. No. 8,760,178. The correction step can be carried out by, for example, a correction means such as the above-mentioned hardware data processing device, and specifically, the above-mentioned biosensor.
そして、前記目的成分量は、例えば、前記補正後のシグナルおよび前記シグナルと試料中の前記目的成分量との相関関係に基づき、算出できる。 Then, the target component amount can be calculated based on, for example, the corrected signal and the correlation between the signal and the target component amount in the sample.
＜目的成分の測定装置＞
本発明の目的成分の測定装置は、前述のように、電極系と、試料、目的成分に対する酸化還元触媒、および電子伝達物質の存在下、第１の電圧（Ｖ１）および第２の電圧（Ｖ２）を前記電極系に印加する電圧印加手段と、前記第２の電圧（Ｖ２）印加において、前記電極系からシグナルを取得するシグナル取得手段とを含み、前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であり、前記本発明の目的成分の測定方法に使用することを特徴とする。
<Measuring device for target component>
As described above, the device for measuring the target component of the present invention has a first voltage (V 1 ) and a second voltage (V 1 ) in the presence of an electrode system, a sample, an oxidation-reduction catalyst for the target component, and an electron transfer substance. The first voltage includes a voltage applying means for applying V 2 ) to the electrode system and a signal acquiring means for acquiring a signal from the electrode system when applying the second voltage (V 2 ). The voltage is equal to or higher than the oxidation potential (E) of the electron transfer material, and the second voltage is lower than the oxidation potential (E) of the electron transfer material, which is used in the method for measuring the target component of the present invention. It is characterized by that.
本発明の測定装置は、前記電圧印加手段により印加する前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であり、前記本発明の目的成分の測定方法に使用することを特徴とし、その他の構成および条件は、特に制限されない。本発明の測定装置によれば、例えば、本発明の測定方法を簡便に実施できる。本発明の測定装置は、例えば、前記本発明の測定方法の説明を援用できる。 In the measuring device of the present invention, the first voltage applied by the voltage applying means is a voltage equal to or higher than the oxidation potential (E) of the electron transfer material, and the second voltage is the oxidation of the electron transfer material. The voltage is less than the potential (E), and is characterized by being used in the method for measuring the target component of the present invention, and other configurations and conditions are not particularly limited. According to the measuring device of the present invention, for example, the measuring method of the present invention can be easily carried out. The measuring device of the present invention can refer to, for example, the description of the measuring method of the present invention.
つぎに、本発明の実施例について説明する。なお、本発明は、下記の実施例により何ら制限されない。 Next, examples of the present invention will be described. The present invention is not limited by the following examples.
［実施例１］
前記酸化還元触媒、前記電子伝達物質、および前記電極系を備えるバイオセンサを用いて、酸化電位を測定し、さらに、本発明の測定方法により、目的成分が測定できることを確認した。
[Example 1]
The oxidation potential was measured using the redox catalyst, the electron transport chain, and the biosensor provided with the electrode system, and it was confirmed that the target component could be measured by the measuring method of the present invention.
（１）バイオセンサの作製
前記バイオセンサは、図１に示すように、以下の手順で作製した。まず、図１（Ａ）に示すように、グルコースセンサの絶縁性基板１１として、ＰＥＴ（ポリエチレンテレフタレート）製基板（長さ５０ｍｍ、幅６ｍｍ、厚み２５０μｍ）を準備し、その一方の表面に、スクリーン印刷により、リード部をそれぞれ有する作用極１２および対極１３からなるカーボン電極系を形成した。
(1) Preparation of biosensor The biosensor was manufactured by the following procedure as shown in FIG. First, as shown in FIG. 1 (A), a PET (polyethylene terephthalate) substrate (length 50 mm, width 6 mm, thickness 250 μm) was prepared as the insulating substrate 11 of the glucose sensor, and a screen was formed on one surface thereof. By printing, a carbon electrode system composed of a working electrode 12 and a counter electrode 13 having lead portions was formed.
つぎに、図１（Ｂ）に示すように、前記電極上に絶縁層１４を形成した。まず、絶縁性樹脂ポリエステルを、７５％（ｗｔ）となるように溶媒カルビトールアセテートに溶解させて絶縁性ペーストを調製し、これを前記電極上にスクリーン印刷した。印刷条件は、３００メッシュスクリーン、スキージ圧４０ｋｇであり、印刷する量は、電極面積１ｃｍ２あたり０．００２ｍＬとした。なお、検出部１５上と、リード部１２ａ、１３ａ上には、スクリーン印刷を行わなかった。そして、１５５℃で２０分間、加熱処理し、絶縁層１４を形成した。 Next, as shown in FIG. 1 (B), an insulating layer 14 was formed on the electrode. First, the insulating resin polyester was dissolved in the solvent carbitol acetate so as to be 75% (wt) to prepare an insulating paste, which was screen-printed on the electrode. The printing conditions were a 300 mesh screen and a squeegee pressure of 40 kg, and the printing amount was 0.002 mL per 1 cm 2 of the electrode area. No screen printing was performed on the detection unit 15 and the lead units 12a and 13a. Then, it was heat-treated at 155 ° C. for 20 minutes to form an insulating layer 14.
さらに、図１（Ｃ）に示すように、絶縁層１４を形成しなかった検出部１５に、無機ゲル層１６を形成した。まず、０．３％（ｗｔ）の合成スメクタイト（商品名「ルーセンタイトＳＷＮ」、コープケミカル社製）、６．０％（ｗｔ）のルテニウム化合物（［Ｒｕ（ＮＨ3）6］Ｃｌ3、同仁化学研究所社製）、酢酸ナトリウム、およびコハク酸を含む無機ゲル形成液（ｐＨ７．５）を調製した。１．０μＬの無機ゲル形成液を、検出部１５に分注した。なお、検出部１５の表面積は約０．６ｃｍ２であり、前記検出部１５における電極１２、１３の表面積は約０．１２ｃｍ２であった。そして、これを、３０℃で乾燥させて、無機ゲル層１６を形成した。 Further, as shown in FIG. 1C, an inorganic gel layer 16 was formed on the detection unit 15 which did not form the insulating layer 14. First, 0.3% (wt) synthetic smectite (trade name "Lucentite SWN", manufactured by Corp Chemical Co., Ltd.), 6.0% (wt) ruthenium compound ([Ru (NH 3 ) 6 ] Cl 3 , Dojin An inorganic gel-forming solution (pH 7.5) containing sodium acetate and succinic acid (manufactured by Chemical Research Institute) was prepared. 1.0 μL of the inorganic gel-forming solution was dispensed into the detection unit 15. The surface area of the detection unit 15 was about 0.6 cm 2 , and the surface area of the electrodes 12 and 13 in the detection unit 15 was about 0.12 cm 2 . Then, this was dried at 30 ° C. to form an inorganic gel layer 16.
つぎに、図１（Ｄ）に示すように、前記無機ゲル層１６の上に、酵素層１７を形成した。まず、２．７Ｕのアスペルギルス・オリゼ型ＦＡＤ−ＧＤＨ（商品名「Glucosedehydrogenase (FAD-dependent)(GLD-351)」、東洋紡社製）、２５％（ｗｔ）の１-メトキシＰＭＳ（同仁化学研究所社製）、およびＡＣＥＳ緩衝液（ｐＨ７．５）を含む酵素液を調製した。１．０μＬの酵素液を、検出部１５の前記無機ゲル層１６の上に分注して、３０℃で乾燥させて酵素層１７を形成した。 Next, as shown in FIG. 1 (D), an enzyme layer 17 was formed on the inorganic gel layer 16. First, 2.7U Aspergillus oryzae type FAD-GDH (trade name "Glucose dehydrogenase (FAD-dependent) (GLD-351)", manufactured by Toyobo Co., Ltd.), 25% (wt) 1-methoxy PMS (Dojin Chemical Laboratory) An enzyme solution containing ACES buffer (pH 7.5) was prepared. 1.0 μL of the enzyme solution was dispensed onto the inorganic gel layer 16 of the detection unit 15 and dried at 30 ° C. to form the enzyme layer 17.
図１（Ｅ）に示すように、開口部を有するスペーサー１８を絶縁層１４上に配置した。さらに、図１（Ｆ）に示すように、スペーサー１８上に空気孔となる貫通孔２０を有するカバー１９を配置してバイオセンサを作製した。カバー１９と絶縁層１４とに挟まれたスペーサー１８の開口部の空間が、キャピラリー構造となるため、これを試料供給部２１とした。 As shown in FIG. 1 (E), a spacer 18 having an opening is arranged on the insulating layer 14. Further, as shown in FIG. 1 (F), a cover 19 having a through hole 20 serving as an air hole was arranged on the spacer 18 to prepare a biosensor. Since the space of the opening of the spacer 18 sandwiched between the cover 19 and the insulating layer 14 has a capillary structure, this is used as the sample supply unit 21.
（２）酸化電位の測定
前記バイオセンサに全血（Ｈｔ値４２％）を導入し、前述の酸化電位の測定条件により、電流値を測定した。なお、電流値の測定は、自家調製した電気化学測定器を用い、３回行った。
(2) Measurement of Oxidation Potential Whole blood (Ht value 42%) was introduced into the biosensor, and the current value was measured under the above-mentioned measurement conditions of the oxidation potential. The current value was measured three times using an electrochemical measuring instrument prepared in-house.
この結果を図２に示す。図２は、前記酸化電位の測定条件における電流値を示すサイクリックボルタモグラムである。なお、図２においては、−０．２〜０．２Ｖにおける電流値を示している。図２において、横軸は、前記電極系に印加した電位を示し、縦軸は、応答電流値を示す。図２に示すように、正の電流値において、３０〜５０ｍＶにピークが観察された。このため、前記バイオセンサにおいて、前記電子伝達物質の酸化電位は、３０〜５０ｍＶであることがわかった。 The result is shown in FIG. FIG. 2 is a cyclic voltammogram showing a current value under the measurement conditions of the oxidation potential. In addition, in FIG. 2, the current value in the range of −0.2 to 0.2V is shown. In FIG. 2, the horizontal axis represents the potential applied to the electrode system, and the vertical axis represents the response current value. As shown in FIG. 2, a peak was observed at 30 to 50 mV at a positive current value. Therefore, in the biosensor, it was found that the oxidation potential of the electron transmitter was 30 to 50 mV.
（３）目的成分の測定１
つぎに、所定濃度（０、６７、１３４、３３６、または６００ｍｇ／ｄＬ）となるようにグルコースを含む、ヘマトクリット（Ｈｔ）値４２％のグルコース試料１を調製した。
(3) Measurement of target component 1
Next, a glucose sample 1 having a hematocrit (Ht) value of 42% containing glucose so as to have a predetermined concentration (0, 67, 134, 336, or 600 mg / dL) was prepared.
そして、各グルコース試料について、前記バイオセンサおよび前記電気化学測定器を用い、実施例の条件として、下記（１−１）〜（１−６）の電圧印加条件で前記第１の電圧および前記第２の電圧を印加し、前記第２の電圧印加時を基準として、０．０５または０．２秒における電流値を測定した。 Then, for each glucose sample, using the biosensor and the electrochemical measuring instrument, the first voltage and the first voltage under the following voltage application conditions (1-1) to (1-6) as the conditions of the example. The voltage of 2 was applied, and the current value at 0.05 or 0.2 seconds was measured with reference to the time when the second voltage was applied.
（電圧印加条件）
（１−１）第１の電圧印加：１Ｖ１秒、第２の電圧印加：０Ｖ５秒
（１−２）第１の電圧印加：１Ｖ２秒、第２の電圧印加：０Ｖ５秒
（１−３）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：０Ｖ５秒
（１−４）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：１０ｍＶ５秒
（１−５）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：−１００ｍＶ５秒
（１−６）第１の電圧印加：２００ｍＶ２秒、第２の電圧印加：０Ｖ５秒
(Voltage application conditions)
(1-1) First voltage application: 1V 1 second, second voltage application: 0V 5 seconds (1-2) First voltage application: 1V 2 seconds, second voltage application: 0V 5 seconds (1-3) First 1 voltage application: 350 mV 2 seconds, 2nd voltage application: 0 V 5 seconds (1-4) 1st voltage application: 350 mV 2 seconds, 2nd voltage application: 10 mV 5 seconds (1-5) 1st voltage application: 350 mV2 Seconds, second voltage application: -100 mV 5 seconds (1-6) First voltage application: 200 mV 2 seconds, second voltage application: 0 V 5 seconds
これらの結果を図３に示す。図３は、前記グルコース試料で得られた電流値を示すグラフである。図３（Ａ）〜（Ｆ）は、それぞれ、前記条件（１−１）〜（１−６）の結果を示す。図３において、横軸は、グルコース濃度を示し、縦軸は、電流値を示し、図中の菱形（◇）は、０．０５秒の結果を示し、四角（□）は、０．２秒の結果を示す。図３（Ａ）〜（Ｆ）に示すように、いずれの電圧条件およびシグナル取得時間においても、グルコース試料中のグルコース濃度と、電流値とは、負の相関関係を示すことがわかった。グルコース濃度と、電流値とが、負の相関関係を示すことから、試料から得られた電流値により、試料中のグルコース濃度を算出できるといえる。したがって、本発明の測定方法により、グルコースをはじめとする目的成分を測定できることがわかった。 These results are shown in FIG. FIG. 3 is a graph showing the current value obtained from the glucose sample. 3 (A) to 3 (F) show the results of the above conditions (1-1) to (1-6), respectively. In FIG. 3, the horizontal axis indicates the glucose concentration, the vertical axis indicates the current value, the diamond (◇) in the figure indicates the result of 0.05 seconds, and the square (□) indicates the result of 0.2 seconds. The result of is shown. As shown in FIGS. 3A to 3F, it was found that the glucose concentration in the glucose sample and the current value showed a negative correlation under any voltage condition and signal acquisition time. Since the glucose concentration and the current value show a negative correlation, it can be said that the glucose concentration in the sample can be calculated from the current value obtained from the sample. Therefore, it was found that the target component such as glucose can be measured by the measuring method of the present invention.
（４）目的成分の測定２
所定濃度（０、６７、３３６、または６００ｍｇ／ｄＬ）となるようにグルコースを含む、ヘマトクリット（Ｈｔ）値４２％のグルコース試料２を調製した。そして、各グルコース試料について、実施例の条件として、前記条件（１−１）〜（１−６）に代えて、下記（２−１）〜（２−３）の電圧印加条件とした以外は、前記（３）と同様にして、０．０５、０．２、または０．４秒における電流値を測定した。
(4) Measurement of target component 2
A glucose sample 2 having a hematocrit (Ht) value of 42% was prepared containing glucose so as to have a predetermined concentration (0, 67, 336, or 600 mg / dL). Then, for each glucose sample, except that the conditions of the examples were the following voltage application conditions (2-1) to (2-3) instead of the above conditions (1-1) to (1-6). , The current value at 0.05, 0.2, or 0.4 seconds was measured in the same manner as in (3) above.
（電圧印加条件）
（２−１）第１の電圧印加：１Ｖ２秒、第２の電圧印加：０Ｖ５秒
（２−２）第１の電圧印加：１Ｖ２秒、第２の電圧印加：−２００ｍＶ５秒
（２−３）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：−２００ｍＶ５秒
(Voltage application conditions)
(2-1) First voltage application: 1V 2 seconds, second voltage application: 0V 5 seconds (2-2) First voltage application: 1V 2 seconds, second voltage application: -200 mV 5 seconds (2-3) First voltage application: 350 mV 2 seconds, second voltage application: -200 mV 5 seconds
これらの結果を図４に示す。図４は、前記グルコース試料で得られた電流値を示すグラフである。図４（Ａ）〜（Ｃ）は、それぞれ、前記条件（２−１）〜（２−３）の結果を示す。図４において、横軸は、グルコース濃度を示し、縦軸は、電流値を示し、図中の菱形（◇）は、０．０５秒の結果を示し、四角（□）は、０．２秒の結果を示し、三角（△）は、０．４秒の結果を示す。図４（Ａ）〜（Ｃ）に示すように、いずれの電圧条件およびシグナル取得時間においても、グルコース試料中のグルコース濃度と、電流値とは、負の相関関係を示すことがわかった。グルコース濃度と、電流値とが、負の相関関係を示すことから、試料から得られた電流値により、試料中のグルコース濃度を算出できるといえる。したがって、本発明の測定方法により、グルコースをはじめとする目的成分を測定できることがわかった。 These results are shown in FIG. FIG. 4 is a graph showing the current value obtained from the glucose sample. 4 (A) to 4 (C) show the results of the above conditions (2-1) to (2-3), respectively. In FIG. 4, the horizontal axis indicates the glucose concentration, the vertical axis indicates the current value, the diamond (◇) in the figure indicates the result of 0.05 seconds, and the square (□) indicates the result of 0.2 seconds. The result of is shown, and the triangle (Δ) shows the result of 0.4 seconds. As shown in FIGS. 4 (A) to 4 (C), it was found that the glucose concentration in the glucose sample and the current value showed a negative correlation under any voltage condition and signal acquisition time. Since the glucose concentration and the current value show a negative correlation, it can be said that the glucose concentration in the sample can be calculated from the current value obtained from the sample. Therefore, it was found that the target component such as glucose can be measured by the measuring method of the present invention.
［実施例２］
本発明の測定方法により、目的成分の測定におけるヘマトクリットによる影響が低減されていることを確認した。
[Example 2]
It was confirmed that the measurement method of the present invention reduced the influence of hematocrit on the measurement of the target component.
３３６または６００ｍｇ／ｄＬのグルコースを含む、所定のＨｔ値（０、２０、４２、または７０％）のグルコース試料３を調製した。そして、前記グルコース試料１に代えて、前記グルコース試料３を用いた以外は、前記実施例１（３）の条件（１−２）と同様にして、前記第２の電圧印加時を基準として、０．２秒における電流値を測定した。そして、下記式（１）に基づき、Ｈｔ値４２％のグルコース試料３の電流値を基準とした際のＨｔ値の影響度合いを算出した。また、コントロールは、前記第１の電圧および前記第２の電圧に代えて、前記バイオセンサに、比較例の条件として、２００ｍＶの電圧を５秒印加し、前記電圧印加開始時を基準として、５秒における電流値を測定した以外は、同様にして、Ｈｔ値の影響度合いを算出した。 A glucose sample 3 having a predetermined Ht value (0, 20, 42, or 70%) containing 336 or 600 mg / dL of glucose was prepared. Then, in the same manner as in the condition (1-2) of the first embodiment (3) except that the glucose sample 3 was used instead of the glucose sample 1, the second voltage was applied as a reference. The current value at 0.2 seconds was measured. Then, based on the following formula (1), the degree of influence of the Ht value when the current value of the glucose sample 3 having an Ht value of 42% was used as a reference was calculated. Further, in the control, instead of the first voltage and the second voltage, a voltage of 200 mV is applied to the biosensor for 5 seconds as a condition of the comparative example, and 5 with reference to the start of the voltage application. The degree of influence of the Ht value was calculated in the same manner except that the current value in seconds was measured.
Ｅｈ＝（Ｓｇ／Ｂｇ）×１００−１００ ・・・（１）
Ｅｈ：Ｈｔ値の影響度合い
Ｓｇ：グルコース試料の電流値
Ｂｇ：Ｈｔ値４２％のグルコース試料の電流値
E h = (S g / B g ) × 100-100 ・ ・ ・ (1)
E h : Degree of influence of Ht value S g : Current value of glucose sample B g : Current value of glucose sample with Ht value 42%
これらの結果を図５に示す。図５は、Ｈｔ値の影響度合いを示すグラフである。図５において、（Ａ）は、実施例における３３６ｍｇ／ｄＬのグルコースを含むグルコース試料の結果を示し、（Ｂ）は、実施例における６００ｍｇ／ｄＬのグルコースを含むグルコース試料の結果を示し、（Ｃ）は、コントロールの結果を示す。また、図５において、横軸は、Ｈｔ値を示し、縦軸は、Ｈｔ値の影響度合いを示す。図５（Ｃ）に示すように、一定の電圧を印加した場合、Ｈｔ値が４２％から離れるにつれて、得られた電流値に対する前記Ｈｔ値の影響度合いが、増加した。これに対し、図５（Ａ）および（Ｂ）に示すように、前記酸化電位以上の第１電圧および前記酸化電位未満の第２電圧を印加した場合、いずれのＨｔ値においても、得られた電流値に対するＨｔ値の影響度合いは、低かった。以上のことから、本発明の測定方法は、ヘマトクリットによる影響が低減されていることがわかった。 These results are shown in FIG. FIG. 5 is a graph showing the degree of influence of the Ht value. In FIG. 5, (A) shows the result of the glucose sample containing 336 mg / dL glucose in the example, (B) shows the result of the glucose sample containing 600 mg / dL glucose in the example, and (C) ) Indicates the result of the control. Further, in FIG. 5, the horizontal axis indicates the Ht value, and the vertical axis indicates the degree of influence of the Ht value. As shown in FIG. 5C, when a constant voltage was applied, the degree of influence of the Ht value on the obtained current value increased as the Ht value deviated from 42%. On the other hand, as shown in FIGS. 5A and 5B, when a first voltage equal to or higher than the oxidation potential and a second voltage lower than the oxidation potential were applied, they were obtained at any Ht value. The degree of influence of the Ht value on the current value was low. From the above, it was found that the measurement method of the present invention reduced the influence of hematocrit.
［実施例３］
本発明の測定方法における第１の電圧および第２の電圧の条件を満たす、種々の第１の電圧および第２の電圧を印加した場合において、目的成分の測定におけるヘマトクリットによる影響が低減されていることを確認した。
[Example 3]
When various first and second voltages satisfying the conditions of the first voltage and the second voltage in the measuring method of the present invention are applied, the influence of hematocrit on the measurement of the target component is reduced. It was confirmed.
３３６ｍｇ／ｄＬのグルコースを含むグルコース試料３を用い、実施例の条件として、下記（３−１）〜（３−１１）の電圧印加条件で前記第１の電圧および前記第２の電圧を印加した以外は前記実施例１（３）と同様にして、前記第２の電圧印加時を基準として、０．０５または０．２秒における電流値を測定した。そして、各測定時間について、前記実施例２と同様にして、Ｈｔ値の影響度合いを算出した。また、コントロールは、比較例の条件として、下記（３−１２）〜（３−１４）の条件で電圧を印加した以外は、同様にしてＨｔ値の影響度合いを算出した。なお、前記条件（３−４）、（３−５）、（３−１３）、および（３−１４）では、前記第２の電圧印加時を基準として、０．４秒における電流値を測定し、同様にしてＨｔ値の影響度合いを算出した。 Using glucose sample 3 containing 336 mg / dL of glucose, the first voltage and the second voltage were applied under the following voltage application conditions (3-1) to (3-11) as the conditions of the example. Except for the above, the current value at 0.05 or 0.2 seconds was measured in the same manner as in Example 1 (3) with reference to the time when the second voltage was applied. Then, for each measurement time, the degree of influence of the Ht value was calculated in the same manner as in Example 2. Further, the control calculated the degree of influence of the Ht value in the same manner except that the voltage was applied under the conditions (3-12) to (3-14) below as the conditions of the comparative example. Under the conditions (3-4), (3-5), (3-13), and (3-14), the current value in 0.4 seconds is measured with reference to the time when the second voltage is applied. Then, the degree of influence of the Ht value was calculated in the same manner.
（電圧印加条件）
（３−１）第１の電圧印加：１Ｖ２秒、第２の電圧印加：０Ｖ２秒
（３−２）第１の電圧印加：５００ｍＶ２秒、第２の電圧印加：０Ｖ２秒
（３−３）第１の電圧印加：１Ｖ５秒、第２の電圧印加：０Ｖ２秒
（３−４）第１の電圧印加：１Ｖ２秒、第２の電圧印加：０Ｖ７秒
（３−５）第１の電圧印加：１Ｖ２秒、第２の電圧印加：１０ｍＶ７秒
（３−６）第１の電圧印加：１Ｖ１秒、第２の電圧印加：０Ｖ５秒
（３−７）第１の電圧印加：１Ｖ２秒、第２の電圧印加：０Ｖ５秒
（３−８）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：−１００ｍＶ５秒
（３−９）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：０Ｖ５秒
（３−１０）第１の電圧印加：３５０ｍＶ２秒、第２の電圧印加：１０ｍＶ５秒
（３−１１）第１の電圧印加：２００ｍＶ２秒、第２の電圧印加：０Ｖ５秒
（３−１２）第１の電圧印加：１Ｖ２秒、第２の電圧印加：２００ｍＶ７秒
（３−１３）第１の電圧印加：１Ｖ２秒、第２の電圧印加：１００ｍＶ７秒
（３−１４）第１の電圧印加：−２００ｍＶ２秒、第２の電圧印加：０Ｖ７秒
(Voltage application conditions)
(3-1) First voltage application: 1V2 seconds, second voltage application: 0V2 seconds (3-2) First voltage application: 500mV2 seconds, second voltage application: 0V2 seconds (3-3) First 1 voltage application: 1V 5 seconds, 2nd voltage application: 0V 2 seconds (3-4) 1st voltage application: 1V 2 seconds, 2nd voltage application: 0V 7 seconds (3-5) 1st voltage application: 1V2 Second, second voltage application: 10 mV 7 seconds (3-6) First voltage application: 1 V 1 second, second voltage application: 0 V 5 seconds (3-7) First voltage application: 1 V 2 seconds, second voltage Application: 0V 5 seconds (3-8) First voltage application: 350 mV 2 seconds, second voltage application: -100 mV 5 seconds (3-9) First voltage application: 350 mV 2 seconds, second voltage application: 0 V 5 seconds ( 3-10) First voltage application: 350 mV 2 seconds, second voltage application: 10 mV 5 seconds (3-11) First voltage application: 200 mV 2 seconds, second voltage application: 0 V 5 seconds (3-12) 1st Voltage application: 1V2 seconds, 2nd voltage application: 200mV7 seconds (3-13) 1st voltage application: 1V2 seconds, 2nd voltage application: 100mV7 seconds (3-14) 1st voltage application: -200mV2 Second voltage applied: 0V 7 seconds
これらの結果を図６および７に示す。図６および７は、Ｈｔ値の影響度合いを示すグラフである。図６において、（Ａ）〜（Ｋ）は、それぞれ、前記条件（３−１）〜（３−１１）の結果を示し、図７において、（Ａ）〜（Ｃ）は、それぞれ、前記条件（３−１２）〜（３−１４）の結果を示す。また、図６および７において、横軸は、Ｈｔ値を示し、縦軸は、Ｈｔ値の影響度合いを示し、図中の菱形（◇）は、０．０５秒の結果を示し、四角（□）は、０．２秒の結果を示し、三角（△）は、０．４秒の結果を示す。図６（Ａ）〜（Ｋ）に示すように、前記酸化電位以上の第１電圧および前記酸化電位未満の第２電圧を印加した条件（３−１）〜（３−１１）の場合、いずれのＨｔ値および測定時間においても、得られた電流値に対するＨｔ値の影響度合いは少なかった。これに対し、図７（Ａ）〜（Ｃ）に示すように、前記酸化電位以上の第１電圧および第２電圧を印加した条件（３−１２）および（３−１３）、ならびに前記酸化電位未満の第１電圧および第２電圧を印加した条件（３−１４）の場合、Ｈｔ値が４２％から離れるにつれて、得られた電流値に対するＨｔ値の影響度合いが大きくなった。すなわち、Ｈｔ値の影響を大きく受けた。また、図６（Ｉ）および（Ｋ）に示すように、第１の電圧を前記電子伝達物質の酸化電位に近い電圧とした場合、ヘマトクリットによる影響をより低減できた。さらに、図６（Ｈ）〜（Ｊ）に示すように、第２の電圧を前記電子伝達物質の酸化電位に近い電圧とした場合、ヘマトクリットによる影響をより低減できた。以上のことから、本発明の測定方法における第１の電圧および第２の電圧の条件を満たす、種々の第１の電圧および第２の電圧を印加した場合においても、目的成分の測定におけるヘマトクリットによる影響を低減でき、また、第１の電圧および第２の電圧を前記電子伝達物質の酸化電位により近い電圧とすることにより、ヘマトクリットによる影響をより低減できることがわかった。 These results are shown in FIGS. 6 and 7. 6 and 7 are graphs showing the degree of influence of the Ht value. In FIG. 6, (A) to (K) show the results of the above conditions (3-1) to (3-11), respectively, and in FIG. 7, (A) to (C) are the above conditions, respectively. The results of (3-12) to (3-14) are shown. Further, in FIGS. 6 and 7, the horizontal axis indicates the Ht value, the vertical axis indicates the degree of influence of the Ht value, the diamond (◇) in the figure indicates the result of 0.05 seconds, and the square (□). ) Indicates the result of 0.2 seconds, and the triangle (Δ) indicates the result of 0.4 seconds. As shown in FIGS. 6A to 6K, any of the conditions (3-1) to (3-11) in which the first voltage equal to or higher than the oxidation potential and the second voltage lower than the oxidation potential are applied. The degree of influence of the Ht value on the obtained current value was also small in the Ht value and the measurement time. On the other hand, as shown in FIGS. 7A to 7C, the conditions (3-12) and (3-13) in which the first voltage and the second voltage higher than the oxidation potential were applied, and the oxidation potential. In the case of the condition (3-14) in which the first voltage and the second voltage less than were applied, the degree of influence of the Ht value on the obtained current value increased as the Ht value deviated from 42%. That is, it was greatly affected by the Ht value. Further, as shown in FIGS. 6 (I) and 6 (K), when the first voltage was set to a voltage close to the oxidation potential of the electron transmitter, the influence of hematocrit could be further reduced. Further, as shown in FIGS. 6 (H) to 6 (J), when the second voltage was set to a voltage close to the oxidation potential of the electron transmitter, the influence of hematocrit could be further reduced. From the above, even when various first and second voltages satisfying the conditions of the first voltage and the second voltage in the measuring method of the present invention are applied, hematocrit in the measurement of the target component is used. It was found that the influence can be reduced, and the influence of hematocrit can be further reduced by setting the first voltage and the second voltage to voltages closer to the oxidation potential of the electron transfer material.
［実施例４］
本発明の測定方法により、目的成分の測定におけるアスコルビン酸による影響が低減されていることを確認した。
[Example 4]
It was confirmed that the measuring method of the present invention reduced the influence of ascorbic acid on the measurement of the target component.
測定前日に採血した静脈血を用いて、３３６ｍｇ／ｄＬのグルコースおよび０または６ｍｇ／ｄＬのアスコルビン酸を含む血液試料を調製した。前記血液試料の酸素分圧は、３０−７０ｍｍＨｇであった。 A blood sample containing 336 mg / dL of glucose and 0 or 6 mg / dL of ascorbic acid was prepared using venous blood collected the day before the measurement. The oxygen partial pressure of the blood sample was 30-70 mmHg.
そして、前記グルコース試料１に代えて、前記血液試料を用い、前記第２の電圧印加時を基準として、０．０５または０．２秒における電流値を測定した以外は、前記実施例１（３）の条件（１−２）と同様にして、電流値を測定した。前記電流値の測定は、各血液試料について、１０サンプルずつ行った。そして、下記式（２）に基づき、０ｍｇ／ｄＬのアスコルビン酸を含む血液試料の電流値を基準とした際のアスコルビン酸の影響度合いを算出した。また、前記１０サンプルの電流値に基づき、変動係数（ＣＶ値）を算出した。さらに、コントロールは、前記第１の電圧および前記第２の電圧に代えて、前記バイオセンサに、２００ｍＶの電圧を２０秒印加し、前記電圧印加開始時を基準として、５秒における電流値を測定した以外は、同様にして、アスコルビン酸の影響度合いおよびＣＶ値を算出した。 Then, the blood sample was used instead of the glucose sample 1, and the current value at 0.05 or 0.2 seconds was measured with reference to the time when the second voltage was applied. ), The current value was measured in the same manner as in (1-2). The current value was measured by 10 samples for each blood sample. Then, based on the following formula (2), the degree of influence of ascorbic acid when the current value of the blood sample containing 0 mg / dL of ascorbic acid was used as a reference was calculated. Further, the coefficient of variation (CV value) was calculated based on the current values of the 10 samples. Further, the control applies a voltage of 200 mV to the biosensor for 20 seconds instead of the first voltage and the second voltage, and measures the current value in 5 seconds with reference to the start of the voltage application. Except for the above, the degree of influence of ascorbic acid and the CV value were calculated in the same manner.
Ｅａ＝（Ｓａ／Ｂａ）×１００−１００ ・・・（２）
Ｅａ：アスコルビン酸の影響度合い
Ｓａ：血液試料の電流値
Ｂａ：アスコルビン酸０ｍｇ／ｄＬの血液試料の電流値
E a = (S a / B a ) x 100-100 ... (2)
E a : Degree of influence of ascorbic acid S a : Current value of blood sample B a : Current value of blood sample of 0 mg / dL of ascorbic acid
アスコルビン酸の影響度合いの結果を図８に示す。図８は、アスコルビン酸の影響度合いを示すグラフである。図８において、（Ａ）は、実施例の結果を示し、（Ｂ）は、コントロールの結果を示す。また、図８において、横軸は、アスコルビン酸濃度（ＡｓＡ（ｍｇ／ｄＬ））を示し、縦軸は、アスコルビン酸の影響度合いを示す。図８（Ａ）に示すように、前記酸化電位以上の第１電圧および前記酸化電位未満の第２電圧を印加した場合、アスコルビン酸の有無によらず、得られた電流値に対するアスコルビン酸の影響度合いは、低かった。これに対し、図８（Ｂ）に示すように、一定の電圧を印加した場合、アスコルビン酸を含むと、得られた電流値に対する前記アスコルビン酸の影響度合いが、増加した。 The result of the degree of influence of ascorbic acid is shown in FIG. FIG. 8 is a graph showing the degree of influence of ascorbic acid. In FIG. 8, (A) shows the result of the example, and (B) shows the result of the control. Further, in FIG. 8, the horizontal axis represents the ascorbic acid concentration (AsA (mg / dL)), and the vertical axis represents the degree of influence of ascorbic acid. As shown in FIG. 8A, when a first voltage equal to or higher than the oxidation potential and a second voltage lower than the oxidation potential are applied, the effect of ascorbic acid on the obtained current value regardless of the presence or absence of ascorbic acid. The degree was low. On the other hand, as shown in FIG. 8B, when a constant voltage was applied, the degree of influence of the ascorbic acid on the obtained current value increased when ascorbic acid was included.
ＣＶ値の結果を図９に示す。図９は、ＣＶ値を示すグラフである。図９において、（Ａ）は、実施例の結果を示し、（Ｂ）は、コントロールの結果を示す。また、図９において、横軸は、アスコルビン酸濃度を示し、縦軸は、ＣＶ値（ＣＶ（％））を示す。図９（Ａ）に示すように、前記酸化電位以上の第１電圧および前記酸化電位未満の第２電圧を印加した場合、得られた電流値のＣＶ値は、アスコルビン酸の有無によらず一定であった。これに対し、図９（Ｂ）に示すように、一定の電圧を印加した場合、アスコルビン酸を含むと、得られた電流値のＣＶ値は増加した。 The result of the CV value is shown in FIG. FIG. 9 is a graph showing the CV value. In FIG. 9, (A) shows the result of the example, and (B) shows the result of the control. Further, in FIG. 9, the horizontal axis represents the ascorbic acid concentration, and the vertical axis represents the CV value (CV (%)). As shown in FIG. 9A, when a first voltage equal to or higher than the oxidation potential and a second voltage lower than the oxidation potential are applied, the CV value of the obtained current value is constant regardless of the presence or absence of ascorbic acid. Met. On the other hand, as shown in FIG. 9B, when a constant voltage was applied, the CV value of the obtained current value increased when ascorbic acid was included.
以上のことから、本発明の測定方法は、アスコルビン酸による影響が低減されていることがわかった。また、本発明の測定方法は、再現性よく目的成分を測定できることがわかった。 From the above, it was found that the measuring method of the present invention reduced the influence of ascorbic acid. Further, it was found that the measuring method of the present invention can measure the target component with good reproducibility.
以上、実施形態および実施例を参照して本発明を説明したが、本発明は上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解し得る様々な変更をすることができる。 Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of the present invention.
この出願は、２０１５年１２月７日に出願された日本出願特願２０１５−２３８５３３を基礎とする優先権を主張し、その開示の全てをここに取り込む。 This application claims priority on the basis of Japanese application Japanese Patent Application No. 2015-238533 filed on December 7, 2015, and the entire disclosure thereof is incorporated herein by reference.
本発明によれば、グルコースをはじめとする目的成分を測定できる。このため、本発明は、分析分野、臨床分野等において、極めて有用であるといえる。 According to the present invention, a target component such as glucose can be measured. Therefore, it can be said that the present invention is extremely useful in the fields of analysis, clinical practice, and the like.
Claims (13)
第２の電圧（Ｖ２）を前記電極系に印加する第２の印加工程と、
前記第２の印加工程において、前記電極系により電流値を取得する電流値取得工程と
前記電流値から前記試料中の目的成分量を算出する算出工程を含み、
前記電流値が、前記第２の電圧印加時を基準として、０．４秒以内に取得した電流値であり、
前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、
前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であることを特徴とする、目的成分の測定方法。 A first application step of applying a first voltage (V 1 ) to the electrode system in the presence of a sample, a redox catalyst for the target component, and an electron transmitter.
A second application step of applying a second voltage (V 2 ) to the electrode system, and
In the second application step, the current value acquisition step of acquiring a current value by the electrode system
Including a calculation step of calculating the amount of the target component in the sample from the current value .
The current value is a current value acquired within 0.4 seconds with respect to the time when the second voltage is applied.
The first voltage is a voltage equal to or higher than the oxidation potential (E) of the electron transmitter.
A method for measuring a target component, wherein the second voltage is a voltage lower than the oxidation potential (E) of the electron transmitter.
一項に記載の目的成分の測定方法。 The method for measuring a target component according to any one of claims 1 to 7 , wherein the redox catalyst is an oxidoreductase or a dehydrogenase.
前記補正シグナルに基づき、前記電流値を補正する補正工程とを含み、
前記算出工程において、前記補正後の電流値から前記試料中の目的成分量を算出する、請求項１から１０記載の目的成分の測定方法。 A correction signal acquisition step of acquiring a correction signal by the electrode system and
Including a correction step of correcting the current value based on the correction signal.
The method for measuring a target component according to claims 1 to 10 , wherein in the calculation step, the amount of the target component in the sample is calculated from the corrected current value .
試料、目的成分に対する酸化還元触媒、および電子伝達物質の存在下、第１の電圧（Ｖ１）および第２の電圧（Ｖ２）を前記電極系に印加する電圧印加手段と、
前記第２の電圧（Ｖ２）印加において、前記電極系から電流値を取得する電流値取得手段と
前記電流値から前記試料中の目的成分量を算出する算出手段を含み、
前記電流値が、前記第２の電圧印加時を基準として、０．４秒以内に取得した電流値であり、
前記第１の電圧が、前記電子伝達物質の酸化電位（Ｅ）以上の電圧であり、
前記第２の電圧が、前記電子伝達物質の酸化電位（Ｅ）未満の電圧であり、
請求項１から１１のいずれか一項に記載の目的成分の測定方法に使用することを特徴とする、目的成分の測定装置。 With the electrode system
A voltage applying means for applying a first voltage (V 1 ) and a second voltage (V 2 ) to the electrode system in the presence of a sample, a redox catalyst for a target component, and an electron transport chain.
In the second voltage (V 2) applied, the current value obtaining means for obtaining a current value from the electrode system
A calculation means for calculating the amount of the target component in the sample from the current value is included.
The current value is a current value acquired within 0.4 seconds with respect to the time when the second voltage is applied.
The first voltage is a voltage equal to or higher than the oxidation potential (E) of the electron transmitter.
The second voltage is a voltage lower than the oxidation potential (E) of the electron transmitter.
A device for measuring a target component, which is used in the method for measuring a target component according to any one of claims 1 to 11 .
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