JPH03287064A - Method for measuring trace component by biosensor - Google Patents
Method for measuring trace component by biosensorInfo
- Publication number
- JPH03287064A JPH03287064A JP2088519A JP8851990A JPH03287064A JP H03287064 A JPH03287064 A JP H03287064A JP 2088519 A JP2088519 A JP 2088519A JP 8851990 A JP8851990 A JP 8851990A JP H03287064 A JPH03287064 A JP H03287064A
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- measurement
- counter electrode
- biosensor
- reaction layer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims description 6
- 238000005259 measurement Methods 0.000 claims abstract description 34
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 239000000523 sample Substances 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 108090000854 Oxidoreductases Proteins 0.000 claims description 7
- 102000004316 Oxidoreductases Human genes 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 6
- 229920001477 hydrophilic polymer Polymers 0.000 claims description 5
- 239000012488 sample solution Substances 0.000 claims description 4
- 239000008103 glucose Substances 0.000 abstract description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 14
- 238000003411 electrode reaction Methods 0.000 abstract description 12
- 230000001590 oxidative effect Effects 0.000 abstract description 5
- 238000007639 printing Methods 0.000 abstract description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 3
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- 238000001035 drying Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000010438 heat treatment Methods 0.000 abstract description 3
- 239000000370 acceptor Substances 0.000 description 11
- 230000003647 oxidation Effects 0.000 description 8
- 238000007254 oxidation reaction Methods 0.000 description 8
- -1 polyethylene terephthalate Polymers 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 7
- 108010015776 Glucose oxidase Proteins 0.000 description 3
- 239000004366 Glucose oxidase Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 229940116332 glucose oxidase Drugs 0.000 description 3
- 235000019420 glucose oxidase Nutrition 0.000 description 3
- 239000000276 potassium ferrocyanide Substances 0.000 description 3
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 229920000139 polyethylene terephthalate Polymers 0.000 description 2
- 239000005020 polyethylene terephthalate Substances 0.000 description 2
- 238000007650 screen-printing Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- KETQAJRQOHHATG-UHFFFAOYSA-N 1,2-naphthoquinone Chemical compound C1=CC=C2C(=O)C(=O)C=CC2=C1 KETQAJRQOHHATG-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 1
- 108010025188 Alcohol oxidase Proteins 0.000 description 1
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000001723 curing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 description 1
- 238000013007 heat curing Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical compound C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、バイオセンサを用いて生体試料中の微量の特
定成分の酸化還元電流を電気化学的に測定することによ
り、特定成分を定量する方法に係る。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for quantifying a specific component by electrochemically measuring the redox current of a minute amount of a specific component in a biological sample using a biosensor. Related.
従来の技術
従来、血液などの生体試料中の特定成分について、試料
液の希釈や攪拌などを行なうことなく簡易に定量できる
センサとして、第１図に示すようなバイオセンサが提案
されている。このバイオセンサは、ポリエチレンテレフ
タレートからなる絶縁性の基板１に５スクリーン印刷に
より導電性カーボンペーストを印刷し、加熱乾燥するこ
とによリ、対極２．測定極３からなる電極系を形成する
。BACKGROUND ART Conventionally, a biosensor as shown in FIG. 1 has been proposed as a sensor that can easily quantify a specific component in a biological sample such as blood without diluting or stirring the sample solution. This biosensor is manufactured by printing a conductive carbon paste on an insulating substrate 1 made of polyethylene terephthalate by five-screen printing, and heating and drying it to form a counter electrode 2. An electrode system consisting of measurement electrodes 3 is formed.
つき゛に、各々の電極の電気化学的に作用する部分とな
る対極反応部２′と１−の大きさの測定極反応部３′を
残して上記電極系を覆うように絶縁性ペーストを印刷被
着し、加熱硬化処理をして絶縁層４を形成する。その後
、対極反応部２′と測定極反応部３′を覆うように親水
性高分子と酸化還元酵素と電子受容体とからなる酵素反
応層５を形成したものである。このようにして形成され
た酵素反応層５へ試料液を滴下すると、酵素反応層５の
中に含憬れる酸化還元酵素と電子受容体が試料液に溶解
し、試料液中の基質との間で酵素反応が進行し電子受容
体が還元される。反応終了後、上記電極系２′と３′に
電圧を印加することにより、酵素反応により還元された
電子受容体が電気化学的に酸化され、このとき得られる
最大酸化電流値から試料液中の基質濃度を求めていた。Then, an insulating paste is printed to cover the electrode system, leaving a counter electrode reaction area 2' and a measurement electrode reaction area 3' of size 1-, which are the electrochemically active parts of each electrode. The insulating layer 4 is formed by applying heat and curing treatment. Thereafter, an enzyme reaction layer 5 made of a hydrophilic polymer, an oxidoreductase, and an electron acceptor is formed so as to cover the counter electrode reaction section 2' and the measurement electrode reaction section 3'. When the sample liquid is dropped onto the enzyme reaction layer 5 formed in this manner, the oxidoreductase and electron acceptor contained in the enzyme reaction layer 5 are dissolved in the sample liquid, and the bond between the oxidoreductase and the electron acceptor contained in the enzyme reaction layer 5 is An enzymatic reaction proceeds and the electron acceptor is reduced. After the reaction is completed, by applying a voltage to the electrode system 2' and 3', the electron acceptor reduced by the enzymatic reaction is electrochemically oxidized, and from the maximum oxidation current value obtained at this time, the I was looking for the substrate concentration.
発明が解決しようとする課題
このような従来のバイオセンサでは、被測定物質と酵素
とを予め反応させ、酵素反応の終了時点から測定を開始
するので、測定結果が得られる１でに長時間を要すると
いう問題があった。Problems to be Solved by the Invention In such conventional biosensors, the substance to be measured and the enzyme are reacted in advance, and the measurement is started from the end of the enzyme reaction, so it takes a long time to obtain the measurement results. There was a problem that it was necessary.
本発明はこのような課題を解決するもので、短時間に正
確に測定できるバイオセンサを提供することを目的とす
るものである。The present invention solves these problems, and aims to provide a biosensor that can perform accurate measurements in a short period of time.
課題を解決するための手段
このような課題を解決するために本発明測定方法は、絶
縁性の基板上の測定極と対極との間に酵素反応を設は酵
素反応上に試料液を置き、酵素反応中の酵素および電子
受容体と試料液との反応の結果生ずる物質の濃度変化を
電気化学的に前記電極系で検知し、試料液中の基質濃度
を測定する方法の乾燥状態にある酵素反応層上に定電圧
を印加しつつ、試料液を滴下し、流れる電流を測定する
ものである。Means for Solving the Problems In order to solve these problems, the measurement method of the present invention involves setting up an enzyme reaction between a measurement electrode and a counter electrode on an insulating substrate, placing a sample solution on the enzyme reaction, An enzyme in a dry state in a method for electrochemically detecting a change in the concentration of a substance resulting from a reaction between an enzyme and an electron acceptor with a sample liquid during an enzyme reaction using the electrode system and measuring the substrate concentration in a sample liquid. A sample solution is dropped onto the reaction layer while applying a constant voltage, and the flowing current is measured.
作 用
本発明によれば、試料液の滴下開始時期が正確に検出で
き、さらに、短時間に基質濃度の測定が可能となった。Effects According to the present invention, it is possible to accurately detect the timing of starting dropping of a sample liquid, and furthermore, it has become possible to measure the substrate concentration in a short time.
また、−度電圧を印加することにより、試料中に含！れ
ている還元性の物質を電解酸化して除去することが可能
なため、測定の精度を向上させることが可能となった。In addition, by applying a -degree voltage, it is possible to reduce the amount of contaminants contained in the sample. Since it is possible to remove the reducing substances that are contained in the sample by electrolytic oxidation, it has become possible to improve the accuracy of measurement.
実施例
以下、本発明の一゛実施例のバイオセンサであるグルコ
ースセンサについて説明する。EXAMPLE A glucose sensor, which is a biosensor according to an example of the present invention, will be described below.
（実施例１）
第１図ふ・よび第２図は、グルコースセンサの構成につ
いて示したものである。ポリエチレンテレフタレートか
らなる絶縁性の基板１に、スクリーン印刷により導電性
カーボンペーストを印刷し、加熱乾燥することにより、
対極２．測定極３からなる電極系を形成する。つぎに、
各々の電極の電気化学的に作用する部分となる対極反応
部２′と１−の大きさの測定極反応部３’（１ｄ）を残
して導電性ペーストを焼付けて形成した電極系を覆うよ
うに絶縁性ペースト印刷し、加熱硬化処理をして絶縁層
４を形成する。この対極反応部２′と測定極反応部３′
の表面を覆うようにセルロース系の親水性高分子の一種
であるカルボキシメチルセルロース（ＣＭＣ）の０．６
重量％水溶液を塗布乾燥し、さらに、ＣＭＣの０．６重
量％水溶液１ｇに酸化酵素としてグルコースオキシダー
ゼ（ＣＯＤ）１０ηと電子受容体のフェリシアン化カリ
ウム２０ηを溶かした溶液を滴下し、４ｏでで１０分間
乾燥して酵素反応層５を形成した。対極を基準にして測
定極に＋〇、５Ｖの定電圧を印加しつつ、酵素反応層５
にグルコース水溶液を滴下すると酸化電流が流れる。こ
の電流は試料中のグルコースがグルコースオキシダーゼ
により酸化される際フェリシアン化カリウムがフェロシ
アン化カリウムニ還元され、このフェロシアン化カリウ
ムが電極上で電解酸化されるために流れるものである。(Example 1) Figures 1 and 2 show the configuration of a glucose sensor. By printing conductive carbon paste by screen printing on an insulating substrate 1 made of polyethylene terephthalate and drying it by heating,
Opposite 2. An electrode system consisting of measurement electrodes 3 is formed. next,
A conductive paste is baked to cover the electrode system, leaving a counter electrode reaction part 2' and a measurement electrode reaction part 3' (1d) of size 1-, which are the electrochemically acting parts of each electrode. The insulating layer 4 is formed by printing an insulating paste on the substrate and subjecting it to heat curing treatment. The counter electrode reaction section 2' and the measurement electrode reaction section 3'
0.6% of carboxymethylcellulose (CMC), a type of cellulose-based hydrophilic polymer, is used to cover the surface of
A wt % aqueous solution was applied and dried, and then a solution of 10 η of glucose oxidase (COD) as an oxidizing enzyme and 20 η of potassium ferricyanide as an electron acceptor was added dropwise to 1 g of a 0.6 wt % aqueous solution of CMC, and the mixture was heated at 4o for 10 minutes. It was dried to form an enzyme reaction layer 5. While applying a constant voltage of +〇, 5V to the measurement electrode with reference to the counter electrode, the enzyme reaction layer 5
When an aqueous glucose solution is dropped into a cell, an oxidation current flows. This current flows because potassium ferricyanide is reduced to potassium ferrocyanide when glucose in the sample is oxidized by glucose oxidase, and this potassium ferrocyanide is electrolytically oxidized on the electrode.
流れる酸化電流のピーク値とグルコース濃度の間に相関
関係が得られ、グルコース濃度が５００■／ｄ ｌ ｌ
で良好な直線性を示した。印加電圧は＋ｏ、ｓＶに設定
したが、電子受容体の還元体が酸化され、かつ水素発生
などを伴わない電圧であればよい。酸化電流のピークは
遅くとも６秒以内に得られるので。A correlation was obtained between the peak value of the flowing oxidation current and the glucose concentration, and the glucose concentration was 500 μ/d l l
It showed good linearity. Although the applied voltage was set to +0, sV, any voltage may be used as long as it oxidizes the reduced form of the electron acceptor and does not involve hydrogen generation. The peak of oxidation current can be obtained within 6 seconds at the latest.
従来のバイオセンサで反応給了に１〜２分必要としてい
たθ１ｊ定が、大幅に測定時間を短縮できた。The measurement time of θ1j, which required 1 to 2 minutes to complete the reaction with conventional biosensors, has been significantly shortened.
なふ・、ピーク電流以外に、電流が最大になる筐で流れ
た総電気量を測定した結果もグルコース濃度と相関しセ
ンサへの試料の供給の仕方がバラついても総電気量の再
現性は良好であった。Nafu... In addition to the peak current, the measurement result of the total amount of electricity flowing in the case where the current is maximum also correlates with the glucose concentration, and even if the method of supplying the sample to the sensor varies, the reproducibility of the total amount of electricity is It was good.
（実施例２）
実施例１と同様にセンサを形成し、対極を基準にして測
定極にアノード方向へ＋〇、５ Ｖの定電圧を印加する
。グルコース水溶液を滴下し、酸化電流の立ち上がりを
検出後、電圧の印加をやめ、１０秒後にもう一度十ｏ、
ｓ Ｖの電圧を０．１秒印加し、酸化電流の最大値を測
定した。さらに、５０秒後、すなわち、測定開始から１
分後に＋ｏ、ｓ Ｖの電圧を同様に印加し５秒後の最大
電流値を測定した。(Example 2) A sensor is formed in the same manner as in Example 1, and a constant voltage of +〇, 5 V is applied to the measurement electrode toward the anode with the counter electrode as a reference. After dropping the glucose aqueous solution and detecting the rise of the oxidation current, stop applying the voltage, and after 10 seconds again
A voltage of s V was applied for 0.1 seconds, and the maximum value of the oxidation current was measured. Furthermore, after 50 seconds, that is, 1
After a minute, a voltage of +o, s V was applied in the same manner, and the maximum current value was measured after 5 seconds.
１０秒後の最大電流値と１分後の最大電流値は両者とも
グルコース濃度と相関があり、１０秒後の応答は７００
η／ｄｌ、１分後の応答は１０００η、／ｄ＃の高濃度
１で直線性があった。The maximum current value after 10 seconds and the maximum current value after 1 minute are both correlated with glucose concentration, and the response after 10 seconds is 700.
η/dl, the response after 1 minute was linear at a high concentration 1 of 1000 η,/d#.
実施例１の場合よシも直線性がのびたのは、フェリシア
ン化カリウムが溶解しながら反応しているため、６秒で
は高濃度のグルコースに対応するフェリシアン化カリウ
ムが供給できなかったのを１０秒待つことにより、より
高濃度のフェリシアン化カリウムを供給できたためと考
えられる。さらに、血液のような粘度の高い試料に対し
ては。The linearity was even better than in Example 1 because the potassium ferricyanide reacted while being dissolved, so the potassium ferricyanide corresponding to the high concentration of glucose could not be supplied in 6 seconds, but by waiting 10 seconds. This is thought to be because a higher concentration of potassium ferricyanide could be supplied. Furthermore, for highly viscous samples such as blood.
酸化電流の立ち上がりがばらつき、実施例１の測定方法
では再現性が悪かった。これは、酵素反応層の溶解速度
が粘度の高い試料によりばらついたためと考えられる。The rise of the oxidation current varied, and the measurement method of Example 1 had poor reproducibility. This is considered to be because the dissolution rate of the enzyme reaction layer varied depending on the sample with high viscosity.
しかし、実施例２のように１０秒間測定を待つことによ
り、かなり再現性を向上させることができた。However, by waiting 10 seconds for measurement as in Example 2, the reproducibility could be significantly improved.
１０秒後のデータをその１１グルコ一ス濃度に換算して
もよいが、５００η／ｄｌ以上と検知した場合は、反応
終了１で１分３０秒必要なため、測定時間をのばすよう
にすると、より精度の高い測定が可能となる。さらに、
試料中にアヌコルビン酸のような還元性の物質が含１れ
ている場合、電極上でフェロシアン化カリウムと同様酸
化されて誤差の要因となるが、１０秒後に一度電圧を印
加して酸化することにより、１分後の測定への影響を除
去することが可能となった。The data after 10 seconds can be converted to the 11-glucose concentration, but if it is detected as 500η/dl or more, it will take 1 minute and 30 seconds to complete the reaction 1, so if you try to extend the measurement time, More accurate measurements are possible. moreover,
If the sample contains a reducing substance such as anucorbic acid, it will be oxidized on the electrode like potassium ferrocyanide and cause errors, but by applying a voltage once after 10 seconds and oxidizing it. , it became possible to eliminate the influence on the measurement after 1 minute.
このように、試料を滴下する前に電圧を印加しておくこ
とにより、試料がいつセンサに供給されたかを検知でき
、１０秒間の待機時間も正確にできるため、スタート時
を指示する必要もなく測定の精度が向上した。In this way, by applying voltage before dropping the sample, it is possible to detect when the sample is supplied to the sensor, and the waiting time of 10 seconds can be set accurately, so there is no need to indicate when to start. Measurement accuracy has been improved.
なお、本発明のバイオセンサは上記実施例に示したグル
コースセンサに限らず、アルコールセンサやコレステロ
ールセンサなど、酸化還元酵素の関与する系に用いるこ
とができる。酸化還元酵素として、実施例では、グルコ
ースオキシダーゼを用いたが、他の酵素、たとえばアル
コールオキシダーゼ、コレステロールオキシダーゼ、キ
サンチンオキシダーゼなども用いることができる。筐た
、電子受容体として、上記実施例に用いたフェリシアン
化カリウムが安定に反応するので測定精度を高めるのに
適しているが％Ｐ−ベンゾキノンを使えば、反応速度が
大きいので高速化に適している。The biosensor of the present invention is not limited to the glucose sensor shown in the above embodiments, but can be used in systems involving redox enzymes, such as alcohol sensors and cholesterol sensors. In the examples, glucose oxidase was used as the oxidoreductase, but other enzymes such as alcohol oxidase, cholesterol oxidase, xanthine oxidase, etc. can also be used. As an electron acceptor, potassium ferricyanide used in the above example reacts stably and is suitable for increasing measurement accuracy, but using %P-benzoquinone has a high reaction rate and is therefore suitable for increasing speed. There is.
甘た、２．６−シクロロフエノールインドフエノール、
メチレンブルー、フェナジンメトサルフェート、β−ナ
フトキノン４−スルホン酸カリウム。Sweet, 2,6-cyclophenol indophenol,
Methylene blue, phenazine methosulfate, potassium β-naphthoquinone 4-sulfonate.
フェロセンなどが電子受容体として使用できる。Ferrocene and the like can be used as electron acceptors.
発明の効果
以上の実施例の説明からも明らかなように、本発明によ
れば、絶縁性の基板上に電極系を印刷し、酸化還元酵素
と親水性高分子と電子受容体とからなる酵素反応層を形
成したセンサにおいて、乾燥状態のセンサに定電圧を印
加しつつ、試料液をセンサに供給したときに流れる電流
を測定するもので、測定時間を１０秒間と大幅に短縮で
きる。さらに、乾燥状態が電圧を印加しているため、試
料の供給時を電流の立ち上がシで検知でき、正確に時間
が検知できるため、測定の精度を高め、操作を簡易化で
きた。また、乾燥状態のセンサに電圧を印加しつつ試料
液の供給時を検知後、一旦電圧の印加を止め再度電圧を
印加して、流れる電流を測定することにより高濃度の試
料や血液のような粘度の高い試料に対しても１０秒で測
定が可能となった。あるいは、１０秒後の応答値をもと
に、測定時間の調整をすることにより試料供給１分後の
測定の精度を高めたり、試料中に存在して測定の際誤差
要因となる還元性の物質を１０秒後に電圧を印加して電
解除去することも可能となった。Effects of the Invention As is clear from the description of the embodiments above, according to the present invention, an electrode system is printed on an insulating substrate, and an enzyme consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is produced. In a sensor formed with a reaction layer, a constant voltage is applied to the sensor in a dry state, and the current flowing when a sample liquid is supplied to the sensor is measured, and the measurement time can be significantly shortened to 10 seconds. Furthermore, since a voltage is applied during the dry state, it is possible to detect when the sample is being supplied by the rise of the current, and the time can be detected accurately, increasing measurement accuracy and simplifying operations. In addition, after detecting when a sample liquid is being supplied while applying a voltage to a dry sensor, the voltage can be stopped and applied again, and the flowing current can be measured to detect highly concentrated samples such as blood. It became possible to measure even highly viscous samples in 10 seconds. Alternatively, by adjusting the measurement time based on the response value after 10 seconds, it is possible to increase the accuracy of the measurement after 1 minute of sample supply, or to reduce the It became possible to electrolytically remove the substance by applying a voltage after 10 seconds.
第１図は本発明の一実施例に用いるバイオセンサの斜視
図、第２図は第１図のバイオセンサのＡ−Ｂ線縦断面図
である。
１・・・・・・基板、２・・・−・・対極、２′・・・
・・・対極反応部、３・・・・・・測定極、３′・・・
・・・測定極反応部、４・・・・・・絶縁層、５・・・
・・・酵素反応層。FIG. 1 is a perspective view of a biosensor used in an embodiment of the present invention, and FIG. 2 is a longitudinal cross-sectional view taken along line AB of the biosensor shown in FIG. 1...Substrate, 2...--Counter electrode, 2'...
...Counter electrode reaction section, 3...Measurement electrode, 3'...
...Measurement electrode reaction part, 4...Insulating layer, 5...
...Enzyme reaction layer.
Claims (2)
系を設け、前記電極系の表面に酸化還元酵素と親水性高
分子及び電子受容体からなる酵素反応層を設け、前記酵
素と前記電子受容体と試料液との反応により生成する物
質の濃度変化を電気化学的に前記電極系で検知して前記
基質濃度を測定するバイオセンサにおいて、前記測定極
と対極との間に一定電圧を印加しつつ前記酵素反応層上
に試料液を滴下し、流れる電流を測定するバイオセンサ
による微量成分の測定方法。(1) An electrode system consisting of a measurement electrode and a counter electrode is provided on an insulating substrate, an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is provided on the surface of the electrode system, and the enzyme In the biosensor, the substrate concentration is measured by electrochemically detecting a change in the concentration of a substance generated by the reaction between the electron acceptor and the sample liquid using the electrode system, and a constant voltage is provided between the measurement electrode and the counter electrode. A method for measuring trace components using a biosensor, in which a sample solution is dropped onto the enzyme reaction layer while applying a voltage, and the flowing current is measured.
系を設け、前記電極系の表面に酸化還元酵素と親水性高
分子及び電子受容体からなる酵素反応層を設け、前記酵
素と前記電子受容体と試料液の反応により生成する物質
の濃度変化を電気化学的に前記電極系で検知して前記基
質濃度を測定するバイオセンサにおいて、前記測定極と
対極との間に一定電圧を印加しつつ、前記酵素反応層上
に試料液が滴下されたことを検知後一旦電圧の印加を止
め、一定時間後に再度定電圧を印加し流れる電流を測定
するバイオセンサによる微量成分の測定方法。(2) An electrode system consisting of a measurement electrode and a counter electrode is provided on an insulating substrate, an enzyme reaction layer consisting of an oxidoreductase, a hydrophilic polymer, and an electron acceptor is provided on the surface of the electrode system, and the enzyme In a biosensor that measures the substrate concentration by electrochemically detecting a change in the concentration of a substance generated by the reaction between the electron acceptor and the sample liquid using the electrode system, a constant voltage is applied between the measurement electrode and the counter electrode. A method for measuring trace components using a biosensor, in which the application of voltage is temporarily stopped after detecting that a sample liquid has been dropped onto the enzyme reaction layer, and after a certain period of time, a constant voltage is applied again and the flowing current is measured. .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2088519A JP2517151B2 (en) | 1990-04-03 | 1990-04-03 | Measuring method of substrate concentration by biosensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2088519A JP2517151B2 (en) | 1990-04-03 | 1990-04-03 | Measuring method of substrate concentration by biosensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03287064A true JPH03287064A (en) | 1991-12-17 |
| JP2517151B2 JP2517151B2 (en) | 1996-07-24 |
Family
ID=13945078
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2088519A Expired - Lifetime JP2517151B2 (en) | 1990-04-03 | 1990-04-03 | Measuring method of substrate concentration by biosensor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2517151B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05340915A (en) * | 1991-10-18 | 1993-12-24 | Matsushita Electric Ind Co Ltd | Biosensor and measuring method using the same |
| WO2003025558A1 (en) * | 2001-09-14 | 2003-03-27 | Arkray, Inc. | Method, tool and device for measuring concentration |
| WO2005040784A1 (en) * | 2003-10-29 | 2005-05-06 | Arkray, Inc. | Specimen analysis method and specimen analysis device |
| CN100427937C (en) * | 2006-01-24 | 2008-10-22 | 南京师范大学 | Online detecting method for concentration of high concentration acid |
| US7504020B2 (en) | 2002-10-31 | 2009-03-17 | Panasonic Corporation | Determination method for automatically identifying analyte liquid and standard solution for biosensor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01219661A (en) * | 1987-02-24 | 1989-09-01 | Imperial Chem Ind Plc <Ici> | Analysis method and apparatus using enzyme electrode type sensor |
-
1990
- 1990-04-03 JP JP2088519A patent/JP2517151B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01219661A (en) * | 1987-02-24 | 1989-09-01 | Imperial Chem Ind Plc <Ici> | Analysis method and apparatus using enzyme electrode type sensor |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05340915A (en) * | 1991-10-18 | 1993-12-24 | Matsushita Electric Ind Co Ltd | Biosensor and measuring method using the same |
| WO2003025558A1 (en) * | 2001-09-14 | 2003-03-27 | Arkray, Inc. | Method, tool and device for measuring concentration |
| US7390391B2 (en) * | 2001-09-14 | 2008-06-24 | Arkray, Inc. | Concentration measuring method, concentration test instrument, and concentration measuring apparatus |
| USRE44522E1 (en) | 2001-09-14 | 2013-10-08 | Arkray, Inc. | Concentration measuring method, concentration test instrument, and concentration measuring apparatus |
| USRE45764E1 (en) | 2001-09-14 | 2015-10-20 | Arkray, Inc. | Concentration measuring method, concentration test instrument, and concentration measuring apparatus |
| US7504020B2 (en) | 2002-10-31 | 2009-03-17 | Panasonic Corporation | Determination method for automatically identifying analyte liquid and standard solution for biosensor |
| WO2005040784A1 (en) * | 2003-10-29 | 2005-05-06 | Arkray, Inc. | Specimen analysis method and specimen analysis device |
| US7763468B2 (en) | 2003-10-29 | 2010-07-27 | Arkray, Inc. | Specimen analysis method and specimen analysis device |
| CN100427937C (en) * | 2006-01-24 | 2008-10-22 | 南京师范大学 | Online detecting method for concentration of high concentration acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2517151B2 (en) | 1996-07-24 |
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