JPH0612351B2 - Enzyme electrode - Google Patents
Enzyme electrodeInfo
- Publication number
- JPH0612351B2 JPH0612351B2 JP56097871A JP9787181A JPH0612351B2 JP H0612351 B2 JPH0612351 B2 JP H0612351B2 JP 56097871 A JP56097871 A JP 56097871A JP 9787181 A JP9787181 A JP 9787181A JP H0612351 B2 JPH0612351 B2 JP H0612351B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- porous membrane
- immobilized
- electrode
- porous
- 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.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
【発明の詳細な説明】 本発明は、応答特性の改良された酵素電極に関する。The present invention relates to enzyme electrodes with improved response characteristics.
酵素電極は、酵素の有する特異性と電気化学的測定法が
結合して、特に生体微量成分を迅速簡便に測定できるも
のとして開発されてきている。The enzyme electrode has been developed as a device capable of quickly and easily measuring a trace amount of components in a living body by combining the specificity of the enzyme with an electrochemical measuring method.
酵素電極の一例としてグルコースオキシダーゼ酵素（以
下ＧＯＤと略す）を不溶固定化した酵素電極について説
明する。An enzyme electrode in which a glucose oxidase enzyme (hereinafter abbreviated as GOD) is insoluble and immobilized will be described as an example of the enzyme electrode.
この酵素電極は反応(1)式で示される酵素反応の生成物H
2O2 を電気化学的な反応(2)式によって基質であるグル
コースの濃度を測定することができる。This enzyme electrode is the product H of the enzyme reaction shown by reaction (1).
The concentration of glucose as a substrate can be measured by the electrochemical reaction (2) of 2 O 2 .
電気化学的に酵素反応生成物であるH2O2量を測定する場
合、血液等の生体中の電気化学的に活性な妨害物質、例
えばアスコルビン酸等の影響が大きいため、実際に酵素
電極を生体成分測定電極として用いる場合、種々の工夫
がなされている。 When measuring the amount of H 2 O 2 that is an enzymatic reaction product electrochemically, the effect of electrochemically active interfering substances in the living body such as blood, such as ascorbic acid, is large, so actually measuring the enzyme electrode When used as a biological component measuring electrode, various measures have been taken.
第１図は従来よく知られている酵素電極の例を示すもの
で、電極１の前面に酵素固定化多孔質膜２、その前面に
妨害物質除去用多孔質膜３を配した構成である。矢印被
検液の進入方向を示す。妨害物質除去用多孔質膜３はそ
の孔径の大きさによって被検液中の妨害物質を除去する
ものであり、酵素反応生成物のみを電極１によって測定
するのである。FIG. 1 shows an example of a well-known enzyme electrode, which has a structure in which an enzyme-immobilized porous membrane 2 is disposed on the front surface of the electrode 1 and an interfering substance-removing porous membrane 3 is disposed on the front surface thereof. Arrow indicates the approach direction of the test liquid. The interfering substance removing porous membrane 3 removes the interfering substance in the test liquid depending on the size of the pore size, and only the enzymatic reaction product is measured by the electrode 1.
第１図の改良として第２図に示す構成のものが提案され
ている。この電極は、導電性薄膜５を有する酸素固化多
孔質膜６と、絶縁性多孔質膜７、および導電性薄膜８を
有する多孔質膜９を積層したものである。１０は被検液
の進入方向を示す。As an improvement of FIG. 1, the structure shown in FIG. 2 has been proposed. This electrode is formed by laminating an oxygen-solidified porous film 6 having a conductive thin film 5, an insulating porous film 7, and a porous film 9 having a conductive thin film 8. Reference numeral 10 indicates the direction in which the test liquid enters.
なお、絶縁性多孔質膜７は、導電性薄膜５，８の電気的
接触を防止するものであり、例えば多孔質膜９に対する
導電性薄膜８の形成位置を図と反対側にするような場合
には、膜７を省略することもある。The insulating porous film 7 prevents electrical contact between the conductive thin films 5 and 8. For example, in the case where the conductive thin film 8 is formed on the porous film 9 on the opposite side of the drawing. In some cases, the film 7 may be omitted.
この酵素電極は、導電性薄膜８によって、被検液中の妨
害物質を電解除去し、目的とする測定物質を膜６の固定
化酵素と反応させ、反応生成物を導電性薄膜電極５によ
って検知する。This enzyme electrode electrolytically removes interfering substances in the test liquid by the conductive thin film 8, reacts the target measurement substance with the immobilized enzyme on the membrane 6, and detects the reaction product by the conductive thin film electrode 5. To do.
一般に、分析手段としての測定機器は、迅速，簡便に測
定できるものである程望ましい。酵素電極もその応答ス
ピードを速めるため、例えば第１図においては、電極１
の前に多孔質膜２，３と２枚あるよりも１枚の方が応答
スピードが速くなる。また、膜２，３の膜厚を薄くする
程、応答スピードが速くなる。In general, it is desirable that a measuring instrument as an analyzing means be capable of quick and easy measurement. In order to accelerate the response speed of the enzyme electrode as well, for example, in FIG.
The response speed is faster with one sheet than with two sheets of porous membranes 2, 3 before. Further, the thinner the films 2 and 3, the faster the response speed.
従来、応答スピードを速くするために、多孔質膜の厚さ
や、孔径に多くの注意が払われてきたが、本発明は、固
定化する酵素に注目することによって応答速度を改善す
るものである。Conventionally, much attention has been paid to the thickness and pore diameter of the porous membrane in order to increase the response speed, but the present invention improves the response speed by focusing on the enzyme to be immobilized. .
すなわち、本発明の酵素電極は、少なくとも妨害物質除
去用多孔質膜と導電性薄膜を有する酸素固定化多孔質膜
とからなる多孔質膜の積層体であって、妨害物質除去用
多孔質膜は酸素固定化多孔質膜に対して被検液側に位置
するように配置され、前記妨害物質除去用多孔質膜は被
検液側に導電性薄膜を有し酸素固定化多孔質膜と同一の
酸素を固定化したことを特徴とする。That is, the enzyme electrode of the present invention is a laminated body of a porous membrane comprising at least an interfering substance removing porous membrane and an oxygen-immobilized porous membrane having a conductive thin film, and the interfering substance removing porous membrane is It is arranged so as to be located on the side of the test liquid with respect to the oxygen-immobilized porous membrane, and the interfering substance-removing porous membrane has a conductive thin film on the side of the test liquid and is the same as the oxygen-immobilized porous membrane. It is characterized by fixing oxygen.
第１，２図に示す従来の酵素電極においては、各１枚の
多孔質膜２，６にのみ酵素を固定化しているが、本発明
では多孔質膜３，９あるいはさらに多孔質膜７などにも
酵素を固定化するのである。In the conventional enzyme electrode shown in FIGS. 1 and 2, the enzyme is immobilized only on each one of the porous membranes 2 and 6, but in the present invention, the porous membranes 3 and 9 or further the porous membrane 7 and the like. It also immobilizes the enzyme.
本発明によれば、導電性薄膜を形成した少なくとも２枚
の酵素固定化膜を積層して酵素電極を構成することによ
り酵素固定化量が増大し、基質との反応が円滑に進行し
応答スピードを改善することができる。また測定物質の
通過部分である多孔質膜の表面に水との親和性に富む酵
素を固定化することにより、その多孔質が固定化酵素に
よって水に濡れやすくなり、結果として多孔質膜中の拡
散抵抗が小さくなり応答スピードを改善することができ
る。さらに、多孔質膜がお互いに固定化酵素によって結
合している状態にある時も、２枚の間隙に空気など含ま
なくなるので応答スピードが速くなる。According to the present invention, at least two enzyme-immobilized membranes having a conductive thin film are laminated to form an enzyme electrode, whereby the amount of enzyme immobilized is increased, the reaction with the substrate proceeds smoothly, and the response speed is increased. Can be improved. In addition, by immobilizing an enzyme having a high affinity for water on the surface of the porous membrane, which is the passage part of the substance to be measured, the porosity of the enzyme becomes easily wet by the immobilized enzyme, and as a result, The diffusion resistance is reduced and the response speed can be improved. Further, even when the porous membranes are bound to each other by the immobilized enzyme, the gap between the two sheets does not contain air and the like, so that the response speed is increased.
なお、導電性薄膜としては、白金，金，銀などの金属、
酸化インジウム，酸化タングステン (WO3)などの金
属酸化物などの導電性材料を蒸着，スパッタリング，イ
オンプレーティング等の方法によって形成できる。As the conductive thin film, metals such as platinum, gold and silver,
Conductive materials such as metal oxides such as indium oxide and tungsten oxide (WO 3 ) can be formed by methods such as vapor deposition, sputtering and ion plating.
以下、本発明を実施例により説明する。Hereinafter, the present invention will be described with reference to examples.
孔径５μｍ，厚さ１０μｍのポリカーボネート多孔質膜
の片面に、白金を厚さ約１０００Åの厚さにスパッタリ
ングして導電性薄膜を形成したサンプルを２枚作成し、
その１枚の多孔質膜にグルコースオキシダーゼをグルタ
ルアルデヒドで固定化した。この酵素を固定化した多孔
質膜と、酵素を固定化してない他の１枚の多孔質膜との
間に、孔径０．２μｍ，厚さ１０μｍのポリカーボネー
ト多孔質膜を介在させ積層して酵素電極とした。Two samples were prepared by forming a conductive thin film by sputtering platinum to a thickness of about 1000Å on one surface of a polycarbonate porous film having a pore size of 5 μm and a thickness of 10 μm.
Glucose oxidase was immobilized on the single porous membrane with glutaraldehyde. The enzyme is immobilized by stacking a porous polycarbonate membrane having a pore diameter of 0.2 μm and a thickness of 10 μm between the porous membrane on which the enzyme is immobilized and another porous membrane on which the enzyme is not immobilized. It was used as an electrode.
この３枚の積層した多孔質膜を組み込んだ酵素電極測定
系を模式的に第３図に示した。１１は白金スパッタ薄膜
１２を有する酵素固定化多孔質膜、１３は白金スパッタ
薄膜１４を有する多孔質膜であり、１５は両者間に介在
した多孔質膜である。An enzyme electrode measurement system incorporating the three laminated porous membranes is schematically shown in FIG. Reference numeral 11 is an enzyme-immobilized porous film having a platinum sputtered thin film 12, 13 is a porous film having a platinum sputtered thin film 14, and 15 is a porous film interposed therebetween.
１６，１７はリード線であり、それぞれ多孔質膜１１，
１３の白金スパッタ薄膜１２，１４と接触している。Ag
／AgCl参照極１８，対極１９とリード線１６は、電極系
を構成し、同様に、Ag／AgCl参照極２０，対極２１，リ
ード線１７も電極系を構成している。Reference numerals 16 and 17 denote lead wires, which are porous membranes 11 and
It is in contact with the platinum sputtered thin films 12 and 14 of 13. Ag
The / AgCl reference electrode 18, the counter electrode 19, and the lead wire 16 form an electrode system, and similarly, the Ag / AgCl reference electrode 20, the counter electrode 21, and the lead wire 17 also form an electrode system.
２２は電極ケースであり、その中には、電解液２３が入
っている。また容器２４中に被検液２５があり、スター
ラー２６によって撹拌される。Reference numeral 22 denotes an electrode case, in which an electrolytic solution 23 is contained. Further, the test liquid 25 is contained in the container 24 and is stirred by the stirrer 26.
上記の構成で、白金薄膜１２と１４の電位をそれぞれ参
照極１８，２０に対して＋０．６Ｖに設定し、血漿２０
μｌを被検液１０ml中に注入したところ、第４図に示す
応答電流曲線Ａを得た。応答電流値が一定になるまで約
２０秒という応答スピードであった。With the above configuration, the potentials of the platinum thin films 12 and 14 are set to +0.6 V with respect to the reference electrodes 18 and 20, respectively, and the plasma 20
When 10 μl of the test solution was injected, a response current curve A shown in FIG. 4 was obtained. The response speed was about 20 seconds until the response current value became constant.
次に、第３図の多孔膜質１５にも酵素を固定化した場合
について同様に測定した応答曲線は、第４図のＢであ
り、応答スピードは約１０秒であった。上記の応答スピ
ードの違いは、基質であるグルコースと酵素反応生成物
である過酸化水素の多孔質膜中での拡散速度の差異によ
るものと思われる。Next, the response curve measured similarly when the enzyme was immobilized on the porous membrane 15 in FIG. 3 was B in FIG. 4, and the response speed was about 10 seconds. It is considered that the above difference in response speed is due to the difference in diffusion rate of glucose as a substrate and hydrogen peroxide as an enzymatic reaction product in the porous membrane.
一方、前記同様にして膜の片面側に白金層を形成したポ
リカーボネート多孔質膜を２枚作成し、次にグルコース
オキシダーゼを各々の膜に前記同様に固定化した。得ら
れた２枚の酵素固定化膜を各々の白金形成膜面を外側に
して積層して酵素電極とし、前記同様に第３図の酵素電
極測定系に組み込んで応答を測定したところ、１０秒以
内に定常値に達した。On the other hand, two polycarbonate porous membranes each having a platinum layer formed on one side of the membrane were prepared in the same manner as described above, and then glucose oxidase was immobilized on each membrane in the same manner as described above. The obtained two enzyme-immobilized membranes were laminated with each platinum-forming membrane surface facing outward to form an enzyme electrode, which was incorporated into the enzyme electrode measuring system shown in FIG. 3 and the response was measured. A steady value was reached within.
なお、実施例においては、グルコースオキシターゼ酵素
について説明したが、本発明は、この酵素に限定される
ことなく、他の酵素についても同様に適用できる。ま
た、固定化された酵素は、必要ならば、１種類以上、同
一の多孔質膜上あるいは他の多孔質膜上に固定化しても
よい。Although the glucose oxidase enzyme has been described in the examples, the present invention is not limited to this enzyme and can be similarly applied to other enzymes. If necessary, the immobilized enzyme may be immobilized on one or more kinds on the same porous membrane or on another porous membrane.
以上のように、本発明によれば、複数の多孔質膜を積層
した酵素電極の応答スピードを改善することができる。As described above, according to the present invention, it is possible to improve the response speed of the enzyme electrode in which a plurality of porous membranes are laminated.
【図面の簡単な説明】 第１図および第２図は酵素電極の構成例を示す断面模式
図、第３図は、酵素電極の測定系の模式図、第４図は酵
素電極の応答電流曲線である。 １，５，１２……導電性薄膜（電極）、２，６，１１…
…酵素固定化多孔質膜、３，９，１３……妨害物質除去
用多孔質膜、７，１５……絶縁用多孔質膜、８，１４…
…導電性薄膜。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 and FIG. 2 are cross-sectional schematic diagrams showing an example of the constitution of an enzyme electrode, FIG. 3 is a schematic diagram of an enzyme electrode measuring system, and FIG. 4 is a response current curve of the enzyme electrode. Is. 1, 5, 12 ... Conductive thin film (electrode), 2, 6, 11 ...
... Enzyme-immobilized porous membrane, 3,9,13 ... Interfering substance removing porous membrane, 7,15 ... Insulating porous membrane, 8,14 ...
... conductive thin film.
Claims (1)
性薄膜を有する酵素固定化多孔質膜とからなる多孔質膜
の積層体であって、妨害物質除去用多孔質膜は酵素固定
化多孔質膜に対して被検液側に位置するように配置さ
れ、前記妨害物質除去用多孔質膜は被検液側に導電性薄
膜を有し酵素固定化多孔質膜と同一の酸素を固定化した
ことを特徴とする酵素電極。1. A laminate of porous membranes comprising at least an interfering substance removing porous membrane and an enzyme-immobilized porous membrane having a conductive thin film, wherein the interfering substance removing porous membrane is an enzyme-immobilized porous membrane. The porous membrane for removing interfering substances has a conductive thin film on the side of the test solution and is fixed to the same side as the enzyme-immobilized porous membrane. The enzyme electrode characterized in that
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56097871A JPH0612351B2 (en) | 1981-06-24 | 1981-06-24 | Enzyme electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56097871A JPH0612351B2 (en) | 1981-06-24 | 1981-06-24 | Enzyme electrode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS57211542A JPS57211542A (en) | 1982-12-25 |
| JPH0612351B2 true JPH0612351B2 (en) | 1994-02-16 |
Family
ID=14203802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56097871A Expired - Lifetime JPH0612351B2 (en) | 1981-06-24 | 1981-06-24 | Enzyme electrode |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0612351B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3118015B2 (en) * | 1991-05-17 | 2000-12-18 | アークレイ株式会社 | Biosensor and separation and quantification method using the same |
-
1981
- 1981-06-24 JP JP56097871A patent/JPH0612351B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS57211542A (en) | 1982-12-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5916156A (en) | Electrochemical sensors having improved selectivity and enhanced sensitivity | |
| US5863400A (en) | Electrochemical cells | |
| US4431507A (en) | Enzyme electrode | |
| US5437973A (en) | Enzyme-electrode sensor | |
| CN1184471C (en) | Electrochemical detector | |
| Chen et al. | A biocompatible needle-type glucose sensor based on platinum-electroplated carbon electrode | |
| KR100741187B1 (en) | Electrochemical Sensor | |
| EP0048090A2 (en) | Substrate specific galactose oxidase enzyme electrodes | |
| JP2002195972A (en) | Biosensor | |
| CN1243953A (en) | Electrochemically analyzing component | |
| JPH01114746A (en) | Biosensor | |
| US20240085362A1 (en) | Method for improving stability of electrochemical sensor | |
| EP0231476A1 (en) | Selectively ion-permeable electrodes for analyzing selected ions in aqueous solution | |
| JP3528529B2 (en) | Biosensor | |
| JPH0612351B2 (en) | Enzyme electrode | |
| JP2977258B2 (en) | Biosensor | |
| Eggenstein et al. | Potentiometric biosensor in double matrix membrane technology | |
| JPS6212472B2 (en) | ||
| JPS585642A (en) | Enzyme electrode | |
| JPS5967452A (en) | Biosensor | |
| JP3483930B2 (en) | Biosensor | |
| JP2016529529A (en) | Electrochemical analytical test strip with ultrathin discontinuous metal layer | |
| Bridge et al. | Polydivinylbenzene/ethylvinylbenzene composite membranes for the optimization of a whole blood glucose sensor | |
| JP3591104B2 (en) | Biosensor manufacturing method | |
| JP4089975B2 (en) | Electrode for electrochemical measurement, method for producing the same, and electrochemical biosensor |