JPH0115018B2 - - Google Patents
Info
- Publication number
- JPH0115018B2 JPH0115018B2 JP56006961A JP696181A JPH0115018B2 JP H0115018 B2 JPH0115018 B2 JP H0115018B2 JP 56006961 A JP56006961 A JP 56006961A JP 696181 A JP696181 A JP 696181A JP H0115018 B2 JPH0115018 B2 JP H0115018B2
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- nad
- substrate
- enzyme electrode
- concentration
- 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
Links
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 108010042687 Pyruvate Oxidase Proteins 0.000 claims description 7
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims description 3
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 229950006238 nadide Drugs 0.000 claims description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 17
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 14
- 239000000758 substrate Substances 0.000 description 11
- 239000004310 lactic acid Substances 0.000 description 7
- 235000014655 lactic acid Nutrition 0.000 description 7
- 230000003100 immobilizing effect Effects 0.000 description 5
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 4
- 239000005515 coenzyme Substances 0.000 description 4
- 229940076788 pyruvate Drugs 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000010439 graphite Substances 0.000 description 2
- 229910002804 graphite Inorganic materials 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 239000006230 acetylene black Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000003487 electrochemical reaction Methods 0.000 description 1
- 108010067653 lactate dehydratase Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (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)
Description
【発明の詳細な説明】
本発明は、酵素の特異的触媒作用を受ける基質
に対して電気化学的活性を有し、基質である乳酸
の濃度を迅速かつ簡便に測定でき、しかも繰り返
し使用することのできる酵素電極に関するもので
ある。DETAILED DESCRIPTION OF THE INVENTION The present invention has electrochemical activity toward a substrate that is subjected to specific catalytic action of an enzyme, and can quickly and easily measure the concentration of lactic acid, which is a substrate, and can be used repeatedly. The present invention relates to an enzyme electrode that can be used for various purposes.
酵素の有する特異的触媒作用を工業的に利用す
る試みの一例として、酵素反応系と電気化学反応
系を結びつけることにより、酵素と特異的に反応
する物質である基質の濃度を測定することが試み
られている。この様な試みの対象となる酸化還元
酵素の中で、補酵素を必要とする乳酸脱水酵素
（以下LDHで表す）などの脱水素酵素系の酵素に
ついては、ニコチンアミドアデニンジヌクレオチ
ド（以下NADで表す）などの補酵素を直接電極
反応させることにより電流として検出し、基質濃
度に関連づける方法が知られている。すなわち、
以下の(1)式の酵素反応で生成したNADの還元体
（NADH）を(2)式に示すごとく電気化学的に酸化
し、この時流れる電流値で基質である乳酸の濃度
を知ることができる。 As an example of an attempt to industrially utilize the specific catalytic action of enzymes, an attempt was made to measure the concentration of a substrate, a substance that specifically reacts with enzymes, by linking an enzyme reaction system and an electrochemical reaction system. It is being Among the oxidoreductases that are the target of such efforts, dehydrogenase enzymes such as lactate dehydratase (hereinafter referred to as LDH), which require coenzymes, are treated with nicotinamide adenine dinucleotide (hereinafter referred to as NAD). A method is known in which a coenzyme such as (indicated by 1) is directly reacted with an electrode to be detected as an electric current and correlated to the substrate concentration. That is,
The reduced form of NAD (NADH) produced by the enzymatic reaction of equation (1) below is electrochemically oxidized as shown in equation (2), and the concentration of the substrate lactic acid can be determined from the current value flowing at this time. can.
乳酸＋NAD+LDH
―――→
ピルビン酸＋NADH＋H+
(1)
NADH→NAD+＋H+＋2e (2)
上記方法に基づき、繰り返し使用が可能な基質
濃度測定用電極を構成するには、関連する酵素、
補酵素を電極の集電体近傍に固定化すれば良い。
しかしながら、従来この様な固定化電極において
は必ずしも十分な応答電流は得られていない。こ
れは、固定化に伴うLDH、NADの活性低下に加
えて(1)式の反応の平衡が左に片寄つており、生成
するピルビン酸が阻害的な作用をしているものと
考えられる。 Lactic acid + NAD + LDH ―――→ Pyruvate + NADH + H +
(1) NADH→NAD + +H + +2e (2) Based on the above method, in order to construct a substrate concentration measurement electrode that can be used repeatedly, related enzymes,
The coenzyme may be immobilized near the current collector of the electrode.
However, conventionally, such fixed electrodes do not always provide a sufficient response current. This is thought to be because, in addition to a decrease in the activity of LDH and NAD due to immobilization, the equilibrium of the reaction of equation (1) is shifted to the left, and the generated pyruvic acid has an inhibitory effect.
そこで、本発明者らは、以上に述べた点につい
て種々検討を重ねた結果、優れた特性を有する酵
素電極を見い出した。本発明の酵素電極の特徴
は、カーボン等の集電体上にLDH、NADととも
にピルビン酸オキシダーゼを固定化した点にあ
る。すなわち(3)式に示すごとく、(1)式で生成する
ピルビン酸をピルビン酸オキシダーゼの作用によ
りアセチルリン酸に変換し、これにより(1)式の反
応を円滑に右方向へ進行させることができる。 Therefore, the present inventors conducted various studies regarding the above-mentioned points, and as a result, they discovered an enzyme electrode having excellent characteristics. The enzyme electrode of the present invention is characterized in that pyruvate oxidase is immobilized together with LDH and NAD on a current collector such as carbon. In other words, as shown in equation (3), the pyruvate produced in equation (1) is converted to acetyl phosphate by the action of pyruvate oxidase, which allows the reaction of equation (1) to proceed smoothly in the right direction. can.
ピルビン酸＋リン酸＋O2
→アセチルリン酸＋CO2＋H2O2 (3)
これに伴い、(2)式に示すNADHの酸化電流が
増大し、その結果、感度を上げることができる。 Pyruvate + phosphoric acid + O 2 → acetyl phosphate + CO 2 + H 2 O 2 (3) Along with this, the oxidation current of NADH shown in equation (2) increases, and as a result, sensitivity can be increased.
以下、本発明を実施例により説明する。 The present invention will be explained below with reference to Examples.
酵素、補酵素の固定化担体を兼ねる集電体とし
てのカーボン粉末（グラフアイト）と結着剤とし
てのフツ素樹脂粉末からなる混合物を加圧成形に
よりペレツト状とし、電極基体を得る。得られた
電極基体上に、NAD水溶液（濃度100mg／ml）を
30μ／cm2の割合で展開、乾燥する。続いて、乳
酸脱水素酵素とピルビン酸オキシターゼの混合水
溶液を50μ／cm2の割合で展開し、乾燥する。こ
の後、固定化試薬としてグルタルアルデヒドを作
用させ、固定化反応を行わせた後、十分に洗浄す
る。こうして得られた酵素電極をＡとする。 A mixture consisting of carbon powder (graphite) as a current collector that also serves as a carrier for immobilizing enzymes and coenzymes, and fluororesin powder as a binder is formed into a pellet by pressure molding to obtain an electrode base. NAD aqueous solution (concentration 100 mg/ml) was placed on the obtained electrode substrate.
Spread at a rate of 30 μ/cm 2 and dry. Subsequently, a mixed aqueous solution of lactate dehydrogenase and pyruvate oxidase is developed at a rate of 50 μ/cm 2 and dried. Thereafter, glutaraldehyde is applied as a fixing reagent to carry out a fixation reaction, followed by sufficient washing. The enzyme electrode thus obtained is designated as A.
比較のために、前記と同様にNADを展開、乾
燥し、次に乳酸脱水素酵素だけを含む水溶液を展
開、乾燥し、グルタルアルデヒドを作用させて得
られた酵素電極をＢとする。 For comparison, an enzyme electrode obtained by developing and drying NAD in the same manner as above, then developing and drying an aqueous solution containing only lactate dehydrogenase, and allowing glutaraldehyde to act on the electrode is referred to as B.
上記、Ａ、Ｂの電極についての基質（乳酸）濃
度変化に対する電流応答を第１図に示す測定系で
測定した。図中１は記録針、２はポテンシヨスタ
ツト、３は参照極、４は下端部に酵素電極を装着
した電極ホルダー、５は基質を含むリン酸緩衝
液、６は塩橋、７は対極である。酵素電極を浸漬
後、ポテンシヨスタツトで電極電位をNADの十
分な酸化電位に設定したた後、基質として乳酸を
注入して所定の濃度とし、酵素反応で還元された
NADの酸化電流の定常値を測定した。第２図に
乳酸濃度変化に伴うＡ、Ｂ各極の電流値の増加を
示す。 The current responses of the electrodes A and B to changes in substrate (lactic acid) concentration were measured using the measurement system shown in FIG. In the figure, 1 is a recording needle, 2 is a potentiostat, 3 is a reference electrode, 4 is an electrode holder with an enzyme electrode attached to the lower end, 5 is a phosphate buffer containing a substrate, 6 is a salt bridge, and 7 is a counter electrode. be. After immersing the enzyme electrode, the electrode potential was set to a sufficient oxidation potential for NAD using a potentiostat, and then lactic acid was injected as a substrate to a predetermined concentration, which was reduced by an enzymatic reaction.
The steady-state value of NAD oxidation current was measured. FIG. 2 shows the increase in current values at each pole, A and B, as the lactic acid concentration changes.
図から明らかなごとく、本発明による酵素電極
Ａは、従来の電極Ｂに比較して大幅な電流増加が
認められており、優れた感度を有することがわか
る。これは先述のごとく、ピルビン酸オキシダー
ゼLDH、NADとともに固定化し、いわゆる複合
酵素電極とすることにより、阻害要因となるピル
ビン酸を除去したことによるものと考えられる。 As is clear from the figure, the enzyme electrode A according to the present invention shows a significant increase in current compared to the conventional electrode B, indicating that it has excellent sensitivity. This is thought to be due to the removal of pyruvate, which is an inhibitory factor, by immobilizing it together with pyruvate oxidase LDH and NAD to form a so-called composite enzyme electrode, as described above.
ピルビン酸オキシダーゼの固定化方法として
は、LDHと別々の工程で固定化するより、実施
例に示した様に、LDHと混合した状態で固定化
した方がより一層良好な感度が得られた。また集
電体としては、担体を兼ねることができ、かつ
NADHを電気化学的に元のNADに酸化できるも
のでなければならず、これに適するものとして
は、グラフアイト、活性炭、アセチレンブラツク
などのカーボンが最も有効であつた。 Regarding the method of immobilizing pyruvate oxidase, better sensitivity was obtained by immobilizing pyruvate oxidase in a mixed state with LDH as shown in the example, rather than by immobilizing it in a separate step with LDH. In addition, as a current collector, it can also serve as a carrier, and
It must be possible to electrochemically oxidize NADH to the original NAD, and carbons such as graphite, activated carbon, and acetylene black have been the most effective for this purpose.
以上のごとく、本発明の酵素電極は優れた応答
特性を有しており、その工業的価値は大である。 As described above, the enzyme electrode of the present invention has excellent response characteristics and has great industrial value.
第１図は酵素電極を用いて基質濃度を測定する
場合の測定系を示す図、第２図は乳酸濃度に対す
る酵素電極の電流増加を示す図である。
FIG. 1 is a diagram showing a measurement system for measuring substrate concentration using an enzyme electrode, and FIG. 2 is a diagram showing an increase in current of the enzyme electrode with respect to lactic acid concentration.
Claims (1)
ドアデニンジヌクレオチドを固定化してなる酵素
電極において、さらにピルビン酸オキシダーゼを
固定化したことを特徴とする酵素電極。 ２ 集電体がカーボンである特許請求の範囲第１
項記載の酵素電極。[Scope of Claims] 1. An enzyme electrode comprising lactate dehydrogenase and nicotinamide adenine dinucleotide immobilized on a current collector, further comprising pyruvate oxidase immobilized thereon. 2 Claim 1 in which the current collector is carbon
Enzyme electrode as described in section.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56006961A JPS57120853A (en) | 1981-01-19 | 1981-01-19 | Enzyme electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56006961A JPS57120853A (en) | 1981-01-19 | 1981-01-19 | Enzyme electrode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS57120853A JPS57120853A (en) | 1982-07-28 |
| JPH0115018B2 true JPH0115018B2 (en) | 1989-03-15 |
Family
ID=11652806
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56006961A Granted JPS57120853A (en) | 1981-01-19 | 1981-01-19 | Enzyme electrode |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS57120853A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8523631D0 (en) * | 1985-09-25 | 1985-10-30 | Pena Ltd Paul De | Bioelectrochemical cell |
| US6740215B1 (en) | 1999-11-16 | 2004-05-25 | Matsushita Electric Industrial Co., Ltd. | Biosensor |
| DE60205702T2 (en) | 2001-05-15 | 2006-02-02 | Matsushita Electric Industrial Co., Ltd., Kadoma | BIOSENSOR |
-
1981
- 1981-01-19 JP JP56006961A patent/JPS57120853A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS57120853A (en) | 1982-07-28 |
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