JPS62144061A - Manufacture of enzyme electrode - Google Patents
Manufacture of enzyme electrodeInfo
- Publication number
- JPS62144061A JPS62144061A JP60285144A JP28514485A JPS62144061A JP S62144061 A JPS62144061 A JP S62144061A JP 60285144 A JP60285144 A JP 60285144A JP 28514485 A JP28514485 A JP 28514485A JP S62144061 A JPS62144061 A JP S62144061A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- membrane
- film
- enzyme electrode
- cell
- 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.)
- Pending
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔（既要〕
測定液に接する酸素透過膜、酵素固定膜及び半透膜より
なる三層を単層として一体化することにより加工性を向
上した酵素電極膜をもつ酵素電極の製造方法。[Detailed Description of the Invention] [(Already required)] An enzyme electrode membrane with improved processability by integrating three layers consisting of an oxygen permeable membrane, an enzyme immobilization membrane, and a semipermeable membrane in contact with a measurement solution into a single layer. Method for manufacturing an enzyme electrode.
本発明は加工性のよい酵素電極の製造方法に関する。 The present invention relates to a method for manufacturing an enzyme electrode with good processability.
酵素は生体細胞から生産される高分子量の有機触媒であ
ってエンザイム（Ｅｎｚｙｍｅ）と呼称されており、化
学的には単純蛋白質または複合蛋白質である。Enzymes are high molecular weight organic catalysts produced from living cells and are called enzymes, and chemically they are simple proteins or complex proteins.
そして生体内で分解５合成、酸化、還元などの複雑な化
学反応が容易に行われるのは多種多様の酵素が適当に配
置されて作用することによる。Complex chemical reactions such as decomposition, synthesis, oxidation, and reduction can be carried out easily in living organisms because a wide variety of enzymes are appropriately arranged and act.
ざて、酵素は生体の化学物質識別能力が高く、特定の物
質とのみ反応することから、この性質を利用して酵素セ
ンサとして使用されており、微生物膜を使用した微生物
センサや抗原膜を使用した免疫センサなどと共にバイオ
センサと言われている。Since enzymes have a high ability to identify chemical substances in living organisms and react only with specific substances, this property is used as an enzyme sensor, and microbial sensors using microbial membranes and antigen membranes are used. It is called a biosensor along with other immunosensors.
ここで、酵素センサばグルコース、尿酸、コレステロー
ルなどの濃度測定用として醗酵工業や医療診断の部門で
広く使用されている。Enzyme sensors are widely used in the fermentation industry and medical diagnosis sectors for measuring concentrations of glucose, uric acid, cholesterol, and the like.
本発明はか＼る酵素センサを構成する酵素電極の改良に
関するものである。The present invention relates to improvements in enzyme electrodes constituting such enzyme sensors.
第４図は酵素センサの代表的な断面構造を示すもので、
プラスチックスなどからなるセル１の先端が開放されて
おり、この開放端の先端に周囲を樹脂被覆により絶縁し
、先端に板状の白金（ＰＬ）カソード２が露出している
カソード３が挿着されており、カソード３の周りには１
艮線（Ａｇ）からなるアノード４が巻回され、このセル
１の中に塩化カリ （ＫＣＩ）などからなる電解液５が
封入されている。Figure 4 shows a typical cross-sectional structure of an enzyme sensor.
The tip of a cell 1 made of plastic or the like is open, and a cathode 3 whose surroundings are insulated with a resin coating and a plate-shaped platinum (PL) cathode 2 is exposed at the tip is inserted into the tip of the open end. 1 around cathode 3.
An anode 4 made of wire (Ag) is wound around the cell 1, and an electrolyte 5 made of potassium chloride (KCI) or the like is sealed in the cell 1.
次に、セル１の開放端にはこれを包むような形で酵素電
極膜６が張られており、０リング７などを用いて固定さ
れている。Next, an enzyme electrode membrane 6 is stretched around the open end of the cell 1 so as to wrap it, and is fixed using an O-ring 7 or the like.
そして、酵素センサの使用法としては、がかる酵素セン
サ８を測定溶液に浸漬し、酵素電極膜６の触媒作用によ
り、測定物質が酸化され、この際に電解液５の中の溶存
酸素（０□）が消費することにより電導度が変化する現
象を利用して測定物質の含有量が測定されている。To use the enzyme sensor, the enzyme sensor 8 is immersed in a measurement solution, and the substance to be measured is oxidized by the catalytic action of the enzyme electrode membrane 6. At this time, dissolved oxygen (0□ ) The content of the substance to be measured is measured using the phenomenon that the conductivity changes due to consumption.
例えばグルコース（Ｃｓ ＩＩ １２０６ ）濃度の測
定は酵素電極膜６を構成する酵素例えばグルコースオキ
シダーゼ（以下略称Ｃ０Ｄ）の作用によりグルコースが
酸化してグルコノラクトン（Ｃｓ １１１００６）とな
る際の０２消費量よりグルコース濃度が測定されている
。For example, the concentration of glucose (Cs II 1206 ) can be measured based on the amount of 02 consumed when glucose is oxidized to gluconolactone (Cs 111006) by the action of an enzyme, such as glucose oxidase (hereinafter abbreviated as C0D), which constitutes the enzyme electrode membrane 6. Glucose concentration is being measured.
０Ｄ
Ｃ６Ｈ１２０，＋０２−’Ｃ，１１、，０６＋Ｈ２Ｏ２
ここで、酵素電極膜６は第３図に示すように酵素固定膜
９を半透膜１０と酸素透過膜１２により上下より包んだ
三層構造をして用いられている。0D C6H120,+02-'C,11,,06+H2O2
Here, the enzyme electrode membrane 6 is used in a three-layer structure in which an enzyme immobilization membrane 9 is wrapped from above and below by a semipermeable membrane 10 and an oxygen permeable membrane 12, as shown in FIG.
すなわち半透膜１０はセルロース、コーラゲンなどの繊
維状高分子の膜からなり、担体として作用する。That is, the semipermeable membrane 10 is made of a fibrous polymer membrane such as cellulose or collagen, and acts as a carrier.
また酸素透過膜１２は多孔質な弗素系の高分子膜からな
り、液中に溶存しているガス分子のみを選択的に透過さ
せるものである。 −
一方、酵素固定膜９は酵素（この例の場合はＣ０Ｄ）を
膜状に固定したもので、これには各種の形成法が知られ
ている。The oxygen permeable membrane 12 is made of a porous fluorine-based polymer membrane, and selectively allows only gas molecules dissolved in the liquid to permeate therethrough. - On the other hand, the enzyme-immobilized membrane 9 is a film in which an enzyme (in this example, C0D) is immobilized, and various formation methods are known for this.
第５図は代表的な形成法である架橋化法を説明するもの
で、酵素（Ｅと略称）と血清アルブミン（Ａと略称）に
官能基を二個もつグルタルアルデヒド１１を用いで橋か
けを行い、効率の良い酵素膜を作る方法である。Figure 5 explains the cross-linking method, which is a typical formation method, and involves cross-linking using enzyme (abbreviated as E) and serum albumin (abbreviated as A) with glutaraldehyde 11, which has two functional groups. This is a method for making highly efficient enzyme membranes.
また包括法はアクリルアミドゲルの中に酵素分子を閉じ
込めて寒天状としたものである。In addition, the entrapment method traps enzyme molecules in acrylamide gel to form an agar-like structure.
なお、その他に共有結合法や吸着法など各種の方法があ
る。In addition, there are various other methods such as a covalent bond method and an adsorption method.
然し、かかる酵素固定膜９はその侭では溶は易い形態で
あり、そのため第３図に示すように半透膜１０と酸素透
過膜１２で挟んで不溶性の構造としている。However, the enzyme-immobilized membrane 9 is in a form that is easily soluble, and therefore, as shown in FIG. 3, it is sandwiched between a semipermeable membrane 10 and an oxygen permeable membrane 12 to form an insoluble structure.
第２図は第４図の先端部を拡大して示すもので、従来使
用されている酵素電極膜の装着法を示すものである。FIG. 2 is an enlarged view of the tip of FIG. 4, and shows a conventional method of attaching an enzyme electrode membrane.
すなわら、セル１の先端の開口部には絶縁体で被覆され
、先端に白金カソード２を備えたカソード３があり、中
には電解液５が充填されている。That is, the opening at the tip of the cell 1 is covered with an insulator and has a cathode 3 having a platinum cathode 2 at the tip, and an electrolytic solution 5 is filled inside.
ここで、従来は例えば半透膜１０を担体とし、この上に
酵素固定膜を形成し、これを図に示すように白金カソー
ド２を含むセル１の先端に被せて、Ｏリング７で止め、
次に酸素透過１１ｉ １２をその上に被覆してＯリング
７′で止めることにより三層構造の酵素電極膜を形成し
ていた。Here, conventionally, for example, a semipermeable membrane 10 is used as a carrier, an enzyme-immobilized membrane is formed on this, and as shown in the figure, this is placed over the tip of a cell 1 containing a platinum cathode 2 and fixed with an O-ring 7.
Next, an oxygen permeable film 11i 12 was coated thereon and secured with an O-ring 7' to form a three-layered enzyme electrode membrane.
従来はこのように別個に積層して酵素電極膜が形成され
ていたが、酵素固定膜はゼラチン状のため加工性が悪く
、また取付は操作が面倒なことが問題である。Conventionally, enzyme electrode membranes have been formed by laminating them separately in this way, but the problem is that the enzyme-immobilized membrane is gelatinous and has poor processability, and is difficult to install.
以上記したように酵素センサ８の先端に装着されている
酵素電極膜６は半透膜１０．酵素固定膜９および酸素透
過膜１２から構成されており、従来はそれぞれ単独に層
構成するか、半透膜１０上に酵素固定膜９を置くか、或
いは半透膜１０を担体とし、これに酵素を固定させたも
のを用いるなど各種の方法が採られているが、何れも白
金カソード２を含むセル１の開口部に積層して固定して
おり、取付は工数が嵩み、また作業性の悪いことが問題
である。As described above, the enzyme electrode membrane 6 attached to the tip of the enzyme sensor 8 is a semipermeable membrane 10. It is composed of an enzyme-immobilized membrane 9 and an oxygen-permeable membrane 12, and conventionally, each has been constructed as a separate layer, or the enzyme-immobilized membrane 9 has been placed on a semipermeable membrane 10, or the semipermeable membrane 10 has been used as a carrier. Various methods have been adopted, such as using enzymes with immobilized enzymes, but all of them involve laminating and fixing the platinum cathode 2 into the opening of the cell 1, which requires a lot of man-hours for installation, and is difficult to work with. The problem is that it is bad.
上記の問題は７ノード及びカソードを内部に備え、電解
液を満したセルの一端が開放されており、該開放端に酸
素透過膜、酵素固定膜および半透膜を積層して形成され
る酵素型）へ膜において、前記の酸素透過膜、酵素固定
膜および半透膜を一体化し単層として形成することを特
徴とする酵素電極膜の製造方法により解決することがで
きる。The above problem is that the cell is equipped with seven nodes and a cathode, and one end of the cell filled with electrolyte is open, and the enzyme formed by laminating an oxygen permeable membrane, an enzyme immobilization membrane, and a semipermeable membrane on the open end. This problem can be solved by a method for producing an enzyme electrode membrane, which is characterized in that the oxygen permeable membrane, the enzyme immobilization membrane, and the semipermeable membrane are integrated into a single layer.
本発明は酵素電極膜６を構成する半透膜１０．酵素固定
１１９９および酸素透過膜１２を一体化成形することに
より従来の問題点を解決するものである。The present invention provides a semipermeable membrane 10 constituting the enzyme electrode membrane 6. The conventional problems are solved by integrally molding the enzyme immobilization 1199 and the oxygen permeable membrane 12.
すなわらスラブゲルドライヤーを用い、三層を積層した
状態で減圧乾燥することにより薄膜状の酵素電極膜を作
るもので、これは型抜き、切断など任意に加工できるの
で極めて作業性よくセル１に装着することができる。In other words, using a slab gel dryer, a thin enzyme electrode membrane is created by drying the three layers in a stacked state under reduced pressure.This can be processed as desired, such as by cutting or cutting, so it is extremely easy to work with and can be used in cell 1. Can be installed.
第１図はこのように形成した酵素電極膜１４の装着状態
を示すもので、酵素電極膜１４は一回の操作でＯリング
７により固定することができる。FIG. 1 shows the state in which the enzyme electrode membrane 14 formed in this manner is attached, and the enzyme electrode membrane 14 can be fixed with the O-ring 7 in a single operation.
なお、本発明に係る酵素電極１４の形成法として先に第
５図に示すように酵素固定膜９を酵素（略称Ｅ）とアル
ブミン（略称Ａ）とをグルタルアルデヒドを用いて架橋
させた場合について記したが、これ以外に酵素だけで形
成する場合もある。In addition, as a method for forming the enzyme electrode 14 according to the present invention, as shown in FIG. As mentioned above, there are other cases in which it is formed using only enzymes.
すなわら、酵素としてＣＯＤのように特性の強い酵素を
用いる場合にはグルタルアルデヒドを架橋剤として使用
してよいが、弱い酵素を使用する場合はグルタルアルデ
ヒドにより失活し易い。That is, when using an enzyme with strong properties such as COD, glutaraldehyde may be used as a crosslinking agent, but when using a weak enzyme, it is likely to be deactivated by glutaraldehyde.
例えば酵素としてグリコール酸オキシダーゼ。For example, glycolate oxidase is an enzyme.
２．５−ヒドロキシコレカルシフェロール１−モノオギ
シゲナーゼなどはグルタルアルデヒドにより失活が生じ
易い。2.5-Hydroxycholecalciferol 1-monogysigenase and the like are likely to be inactivated by glutaraldehyde.
そのため、かかる場合は酵素だけを使用して酵素電極膜
１４を作ればよい。Therefore, in such a case, the enzyme electrode membrane 14 may be made using only the enzyme.
実施例１：
ＣＯＤ １０ｍｇ、生血端アルブミン２０ｍｇを１ｍｊ
２の水に溶解し、これに１０％のグルタルアルデヒドを
１０μｍ滴下し攪拌した後、この液を厚ざが１００μｍ
の半透膜上に塗布した。Example 1: 1 mj of COD 10 mg, raw blood end albumin 20 mg
2 was dissolved in water, 10% glutaraldehyde was added dropwise to the solution in a thickness of 10 μm, and the mixture was stirred.
was applied onto a semipermeable membrane.
この半透膜の上に厚さが約１０μｍの弗素樹脂系の酸素
透過膜で覆った状態でスラブゲルドライヤにセットし、
室温で減圧乾燥させて酵素電極膜を作った。This semi-permeable membrane was covered with a fluororesin-based oxygen permeable membrane with a thickness of about 10 μm and set in a slab gel dryer.
An enzyme electrode membrane was prepared by drying under reduced pressure at room temperature.
このようにして作った酵素電極膜は完全に圧着されたサ
ンドインチ構造をし、刃物で自由に裁断加工することが
でき、容易にセルの先端に装着することができる。The enzyme electrode membrane thus produced has a completely crimped sandwich structure, can be cut freely with a knife, and can be easily attached to the tip of a cell.
そして、これを用いた酵素センサの応答特性は従来と同
様であり、何等変わった点は認められなかった。The response characteristics of the enzyme sensor using this were the same as the conventional ones, and no changes were observed.
実施例２：
セルロースよりなる半透膜（三光純薬製）の上にグリコ
ール酸オキシダーゼの３Ｑｍ（Ｈ／ｍ １！水溶液を塗
布し、この上に厚さが１２μｍの弗素樹脂製の酸素透過
膜を密着させた状態でスラブゲルドライヤにセットし、
室温で減圧乾燥して完全に一体化した酵素電極膜を作っ
た。Example 2: A 3Qm (H/m 1!) aqueous solution of glycolate oxidase was applied on a semipermeable membrane made of cellulose (manufactured by Sanko Pure Chemical Industries, Ltd.), and an oxygen permeable membrane made of fluororesin with a thickness of 12 μm was placed on top of this. Place it in the slab gel dryer with the
A completely integrated enzyme electrode membrane was prepared by drying under reduced pressure at room temperature.
このようにして得た酵素電極膜は実施例１と同様に自由
に加工でき、またセルの先端への装着も簡単で、従来と
全く同じ応答特性を得ることができた。The enzyme electrode membrane thus obtained could be freely processed in the same manner as in Example 1, and could be easily attached to the tip of the cell, and the response characteristics exactly the same as those of the conventional membrane could be obtained.
Ｃ発明の効果〕
以上記したように本発明により得られる酵素電極膜は従
来のアクリルアミドゲル固定膜などと異なり自由に加工
が可能であり、そのため装着が簡単なため使用効率が改
善される。C. Effects of the Invention] As described above, the enzyme electrode membrane obtained by the present invention can be freely processed, unlike conventional acrylamide gel fixed membranes, etc., and is therefore easy to attach, improving usage efficiency.
第１図は本発明を適用した酵素電極の断面図、第２図は
従来の酵素電極の断面図、
第３図は酵素電極の断面図、
第４図は酵素センサの断面図、
第５図は酵素の固定法を示す模式図、
である。
図において、
２は白金カソード、 ６，１４は酵素電極膜、７．７
′はＯリング、 ８は酵素センサ、９は酵素固定膜、
１０は半透膜、１２は酸素透過膜、
である。
昂２層
第５図Fig. 1 is a sectional view of an enzyme electrode to which the present invention is applied, Fig. 2 is a sectional view of a conventional enzyme electrode, Fig. 3 is a sectional view of an enzyme electrode, Fig. 4 is a sectional view of an enzyme sensor, and Fig. 5 is a schematic diagram showing the enzyme immobilization method. In the figure, 2 is a platinum cathode, 6 and 14 are enzyme electrode membranes, and 7.7
' is an O-ring, 8 is an enzyme sensor, 9 is an enzyme immobilization membrane,
10 is a semipermeable membrane, and 12 is an oxygen permeable membrane. Ngong 2nd layer Figure 5
Claims (1)
ルの一端が開放されており、該開放端に酸素透過膜、酵
素固定膜および半透膜を積層して形成される酵素電極に
おいて、前記の酸素透過膜、酵素固定膜および半透膜を
一体化し単層として形成することを特徴とする酵素電極
の製造方法。The enzyme electrode is formed by laminating an oxygen permeable membrane, an enzyme immobilization membrane, and a semipermeable membrane on the open end of a cell that is equipped with an anode and a cathode and filled with an electrolytic solution at one end. A method for manufacturing an enzyme electrode, characterized in that an oxygen permeable membrane, an enzyme immobilization membrane, and a semipermeable membrane are integrated to form a single layer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60285144A JPS62144061A (en) | 1985-12-18 | 1985-12-18 | Manufacture of enzyme electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60285144A JPS62144061A (en) | 1985-12-18 | 1985-12-18 | Manufacture of enzyme electrode |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62144061A true JPS62144061A (en) | 1987-06-27 |
Family
ID=17687666
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60285144A Pending JPS62144061A (en) | 1985-12-18 | 1985-12-18 | Manufacture of enzyme electrode |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62144061A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100998835B1 (en) | 2003-01-17 | 2010-12-06 | 혼다 기켄 고교 가부시키가이샤 | Frame structure for scooter-type vehicle |
-
1985
- 1985-12-18 JP JP60285144A patent/JPS62144061A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100998835B1 (en) | 2003-01-17 | 2010-12-06 | 혼다 기켄 고교 가부시키가이샤 | Frame structure for scooter-type vehicle |
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