JPH0750058B2 - Enzyme-immobilized electrode and method for producing the same - Google Patents
Enzyme-immobilized electrode and method for producing the sameInfo
- Publication number
- JPH0750058B2 JPH0750058B2 JP1164797A JP16479789A JPH0750058B2 JP H0750058 B2 JPH0750058 B2 JP H0750058B2 JP 1164797 A JP1164797 A JP 1164797A JP 16479789 A JP16479789 A JP 16479789A JP H0750058 B2 JPH0750058 B2 JP H0750058B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- film
- immobilized
- electrode
- interference removal
- 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
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 102000008186 Collagen Human genes 0.000 claims description 22
- 108010035532 Collagen Proteins 0.000 claims description 22
- 229920001436 collagen Polymers 0.000 claims description 22
- 239000000126 substance Substances 0.000 claims description 10
- 239000004020 conductor Substances 0.000 claims description 6
- 230000002452 interceptive effect Effects 0.000 claims description 5
- 239000012528 membrane Substances 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 230000035945 sensitivity Effects 0.000 description 12
- 239000012298 atmosphere Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 10
- 239000003431 cross linking reagent Substances 0.000 description 10
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 10
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 108010010803 Gelatin Proteins 0.000 description 7
- 238000004132 cross linking Methods 0.000 description 7
- 229920000159 gelatin Polymers 0.000 description 7
- 239000008273 gelatin Substances 0.000 description 7
- 235000019322 gelatine Nutrition 0.000 description 7
- 235000011852 gelatine desserts Nutrition 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000003020 moisturizing effect Effects 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000002585 base Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 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 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 229910052697 platinum Inorganic materials 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 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 4
- 238000001035 drying Methods 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- 229920000083 poly(allylamine) Polymers 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 3
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 108010089254 Cholesterol oxidase Proteins 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229920006289 polycarbonate film Polymers 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 229920003169 water-soluble polymer Polymers 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 239000007772 electrode material Substances 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 この発明は、グルコース等の基質検出用の電極などとし
て用いられる酵素固定化電極（「酵素電極」とも言う）
およびその製造方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to an enzyme-immobilized electrode (also referred to as "enzyme electrode") used as an electrode for detecting a substrate such as glucose.
And a manufacturing method thereof.
酵素を白金等の電極本体（または電極素材。以下同様）
表面上に固定化した酵素固定化電極として、ゼラチンお
よび架橋剤としてのグルタルアルデヒドを含む希薄な酵
素溶液を白金電極に塗布し、電極本体表面上で製膜を行
うとともに、酵素を共有結合的に固定化して電極本体表
面上に酵素固定化膜を形成したものがある。ここで、ゼ
ラチンは、酵素固定化膜のマトリックス成分となるもの
であって、希薄な酵素溶液と架橋剤としてのグルタルア
ルデヒドだけでは架橋反応により得られる酵素固定化膜
の強度が弱いので、膜強度を高めるために使用される。
たとえば、グルコースオキシダーゼの固定化膜では、酵
素に対して５〜10倍程度のゼラチンを加えてグルタルア
ルデヒドで架橋させて製膜するようにしている。The enzyme is an electrode body such as platinum (or electrode material, and so on)
As an enzyme-immobilized electrode immobilized on the surface, a dilute enzyme solution containing gelatin and glutaraldehyde as a cross-linking agent was applied to a platinum electrode to form a film on the surface of the electrode body and covalently bind the enzyme. There is one in which an enzyme-immobilized film is formed on the surface of an electrode body by immobilization. Here, gelatin is the matrix component of the enzyme-immobilized membrane, and the strength of the enzyme-immobilized membrane obtained by the cross-linking reaction is weak only with a dilute enzyme solution and glutaraldehyde as the cross-linking agent. Used to increase the
For example, in the case of a glucose oxidase-immobilized film, gelatin is added in an amount of about 5 to 10 times that of the enzyme and cross-linked with glutaraldehyde to form a film.
このような酵素固定化電極を用いるに際しては、試料溶
液中における被測定物質以外の多種多様の成分の妨害か
ら酵素固定化電極を保護する必要がある。そのため、電
極本体表面と酵素固定化膜の間などに妨害物質を除去す
る妨害物質除去膜（以下「妨害除去膜」と言う）を形成
した妨害不感型酵素固定化電極が提案されている。When using such an enzyme-immobilized electrode, it is necessary to protect the enzyme-immobilized electrode from interference with various components other than the substance to be measured in the sample solution. Therefore, an interference-insensitive enzyme-immobilized electrode has been proposed in which an interference-substance removal film (hereinafter referred to as "interference removal film") that removes an interference substance is formed between the surface of the electrode body and the enzyme-immobilized film.
たとえば、特開昭62−88953号公報では、電極本体表面
にグルコースオキシダーゼの固定化膜を設けたものを、
ピロールおよびNaClを含む電解重合液中に浸して電解重
合処理を行い、前記固定化膜の上に電解重合膜を備えた
酵素固定化電極が記載されている。同電解重合膜が妨害
除去膜である。また、特開昭63−304150号公報では、電
極本体表面に内側膜、中間膜および外側膜をこの順に設
けた酵素固定化電極が記載されている。中間膜は、グル
コースオキシダーゼの固定膜である。外側膜は、ポリカ
ーボネートフィルムであり、未希釈の血液または血清中
のグルコース分子に比べて高分子物質の除去を行う。内
側膜は、酢酸セルロースからなる膜であり、ポリカーボ
ネートフィルムで除去されなかった妨害物質（たとえ
ば、アスコルビン酸および尿酸）を除去する。For example, in JP-A-62-88953, the one in which a glucose oxidase immobilization film is provided on the surface of the electrode body,
There is described an enzyme-immobilized electrode having an electrolytic polymerization film on the immobilization film, which is immersed in an electrolytic polymerization solution containing pyrrole and NaCl for electrolytic polymerization treatment. The electropolymerized film is the interference removal film. Further, JP-A-63-304150 describes an enzyme-immobilized electrode in which an inner membrane, an intermediate membrane and an outer membrane are provided in this order on the surface of the electrode body. The intermediate membrane is a fixed membrane of glucose oxidase. The outer membrane is a polycarbonate film that removes polymeric substances compared to glucose molecules in undiluted blood or serum. The inner membrane is a membrane composed of cellulose acetate, which removes interfering substances not removed by the polycarbonate film (eg, ascorbic acid and uric acid).
電極本体表面上で製膜された妨害除去膜を備えた妨害不
感型酵素固定化電極では、妨害除去膜が乾燥に弱いた
め、湿度の変化により膜の収縮が生じ、水溶液中での測
定および乾燥を繰り返せば、電極本体表面から妨害除去
膜および酵素固定化膜が剥離し、検出感度が低下するな
どして使用できなくなるという欠点がある。In the interference-insensitive enzyme-immobilized electrode equipped with the interference removal film formed on the surface of the electrode body, the interference removal film is weak against drying, so the film contracts due to changes in humidity, and measurement and drying in aqueous solution If the above procedure is repeated, the interference removal film and the enzyme-immobilized film are peeled off from the surface of the electrode body, and the detection sensitivity is lowered, which makes it unusable.
また、第３図(a)にみるように、酵素を固定化していな
い妨害除去膜５は、湿度が低すぎると、電極本体１より
剥離するため、酵素の固定化作業をたとえば湿度80％以
上の調湿器内で行わなければならない。Further, as shown in FIG. 3 (a), the interference removal film 5 on which the enzyme is not immobilized is peeled off from the electrode body 1 when the humidity is too low. Must be done in the humidity controller.
この発明は、このような事情に鑑みてなされたものであ
って、電極本体からの妨害除去膜の剥離が生じにくく、
出力感度の初期変動の小さい酵素固定化電極を提供する
ことを第１の課題とする。さらに、この発明は、妨害除
去膜の上に酵素固定化膜を形成する場合に、雰囲気の調
湿を行わなくてもよい酵素固定化電極の製造方法を提供
することを第２の課題とする。The present invention has been made in view of such circumstances, in which peeling of the interference removal film from the electrode body hardly occurs,
It is a first object to provide an enzyme-immobilized electrode with a small initial fluctuation in output sensitivity. Further, a second object of the present invention is to provide a method for producing an enzyme-immobilized electrode which does not require humidity control of the atmosphere when the enzyme-immobilized membrane is formed on the interference removal membrane. .
発明者らは、妨害除去膜の電極本体からの剥離を防いだ
り、雰囲気の調湿を行わずに妨害除去膜の上に酵素固定
化膜を形成したりするための手段を検討した。その結
果、妨害除去膜自体に保湿性を持たせればよいことを見
出し、この発明を完成させた。The inventors examined means for preventing the interference removal film from peeling from the electrode body and forming an enzyme-immobilized film on the interference removal film without controlling the humidity of the atmosphere. As a result, they have found that it is sufficient that the interference removing film itself has a moisturizing property, and completed the present invention.
したがって、上記第１の課題を解決するために、この発
明にかかる酵素固定化電極は、導電性材料からなる電極
本体に酵素固定化膜および妨害除去膜が設けられている
ものにおいて、前記妨害除去膜がコラーゲンを含んでい
ることを特徴とする。Therefore, in order to solve the first problem, the enzyme-immobilized electrode according to the present invention is one in which the enzyme-immobilized film and the interference removal film are provided on the electrode body made of a conductive material. The membrane is characterized in that it contains collagen.
上記第２の課題を解決するために、この発明にかかる酵
素固定化電極の製造方法は、導電性材料からなる電極本
体表面上に形成された妨害除去膜の上に酵素固定化膜を
形成するにあたり、前記妨害除去膜にコラーゲンを含め
ておくことを特徴とする。In order to solve the second problem, in the method for manufacturing an enzyme-immobilized electrode according to the present invention, the enzyme-immobilized film is formed on the interference removal film formed on the surface of the electrode body made of a conductive material. At this time, the interference removal film is characterized by including collagen.
この発明の酵素固定化電極は、電極本体表面上に少なく
とも妨害除去膜および酵素固定化膜を備えている。好ま
しくは、電極本体表面上に下地膜、妨害除去膜および酵
素固定化膜の３層を少なくとも備えている。The enzyme-immobilized electrode of the present invention comprises at least an interference removal film and an enzyme-immobilized film on the surface of the electrode body. Preferably, at least three layers of a base film, an interference removal film and an enzyme immobilization film are provided on the surface of the electrode body.
ここで、下地膜は、妨害除去膜の電極本体への密着性を
良くするために必要に応じて用いられる。下地膜は、た
とえば、ゼラチン水溶液などの生体高分子水溶液に架橋
剤を添加したものを電極本体表面に塗布し、架橋反応を
行って形成された膜である。下地膜のマトリックス成分
はゼラチンに限定されない。架橋剤としては、たとえば
グルタルアルデヒド等のジアルデヒドが用いられる。Here, the base film is used as necessary in order to improve the adhesion of the interference removal film to the electrode body. The base film is, for example, a film formed by applying a cross-linking agent to an aqueous solution of a biopolymer such as gelatin aqueous solution to the surface of the electrode main body and performing a cross-linking reaction. The matrix component of the base film is not limited to gelatin. As the cross-linking agent, for example, a dialdehyde such as glutaraldehyde is used.
妨害除去膜は、マトリックス成分の少なくとも一部が保
湿剤であり、たとえば、アルブミン等のタンパク、ポリ
アリルアミン等の水溶性高分子、保湿剤および架橋剤を
含む溶液を電極本体または前記下地膜または酵素固定化
膜上に所定量を塗布し、架橋反応を行わせて、電極表面
上で製膜を行う。保湿剤としては、妨害除去膜の水分を
保ったりあるいは高めたりするものであれば特に限定は
ないが、コラーゲン、界面活性剤、ポリビニルピロリド
ンなどの水溶性高分子があるが、妨害除去膜の保湿性を
保ち、かつ、妨害除去効果の低下が少ないという点から
は、コラーゲンを保湿剤として用いる。コラーゲンとし
ては、酸処理コラーゲン、アルカリ処理コラーゲン、酵
素可溶性コラーゲンなどのいずれも使用可能であり、コ
ラーゲンのタイプも特に限定はない。妨害除去膜を作製
するための溶液（以下「妨害除去膜作製液」と言う）で
は、コラーゲンの濃度は、特に限定されないが、0.01〜
0.1重量％の範囲が好ましい。0.1重量％を越えると、膜
の透過性が悪くなり、応答速度が悪くなる。この発明に
かかる酵素固定化電極の応答性は、たとえば20〜30秒で
ある。また、妨害除去膜の架橋の程度によって除去され
うる物質の種類を適宜変えることも可能である。妨害除
去膜のマトリックス成分としては、コラーゲンを含むの
であれば、上記のものに限定されない。架橋剤として
は、たとえばグルタルアルデヒド等のジアルデヒドが用
いられる。At least a part of the matrix component of the interference removal film is a moisturizing agent, and for example, a solution containing a protein such as albumin, a water-soluble polymer such as polyallylamine, a moisturizing agent and a cross-linking agent is added to the electrode body or the base film or enzyme A predetermined amount is applied on the immobilization film, a crosslinking reaction is performed, and a film is formed on the electrode surface. The moisturizer is not particularly limited as long as it retains or enhances the water content of the interference-removing film, but there are water-soluble polymers such as collagen, surfactants and polyvinylpyrrolidone. Collagen is used as a moisturizing agent from the viewpoints of maintaining the properties and reducing the interference removal effect little. As the collagen, any of acid-treated collagen, alkali-treated collagen, enzyme-soluble collagen and the like can be used, and the type of collagen is not particularly limited. The concentration of collagen in the solution for preparing the interference-eliminating film (hereinafter referred to as "interference-eliminating film-preparing solution") is not particularly limited, but it may range from 0.01 to
A range of 0.1% by weight is preferred. If it exceeds 0.1% by weight, the permeability of the membrane is deteriorated and the response speed is deteriorated. The responsiveness of the enzyme-immobilized electrode according to the present invention is, for example, 20 to 30 seconds. It is also possible to appropriately change the type of substance that can be removed depending on the degree of crosslinking of the interference removal film. The matrix component of the interference removal film is not limited to the above as long as it contains collagen. As the cross-linking agent, for example, a dialdehyde such as glutaraldehyde is used.
酵素固定化膜は、グルコースオキシダーゼおよびコレス
テロールオキシダーゼ等の酵素、ゼラチン等の生体高分
子、および、架橋剤を含む溶液（たとえば、水溶液）を
電極本体上または妨害除去膜上に所定量塗布し、架橋反
応を行わせて製膜を行うとともに固定化して作られる。
酵素は、１種類のみを用いるようにしてもよいし、失活
などの悪影響が生じないのであれば、２種類以上を用い
るようにしてもよい。酵素固定化膜のマトリックス成分
としては、酵素の働きを損なわないのであれば、上記の
ものに限定されない。架橋剤としては、たとえばグルタ
ルアルデヒド等のジアルデヒドが用いられる。The enzyme-immobilized membrane is crosslinked by applying a predetermined amount of a solution (for example, an aqueous solution) containing an enzyme such as glucose oxidase and cholesterol oxidase, a biopolymer such as gelatin, and a crosslinking agent onto the electrode body or the interference removal membrane. It is made by reacting to form a film and immobilizing it.
Only one type of enzyme may be used, or two or more types may be used as long as no adverse effects such as inactivation occur. The matrix component of the enzyme-immobilized membrane is not limited to the above as long as the function of the enzyme is not impaired. As the cross-linking agent, for example, a dialdehyde such as glutaraldehyde is used.
このようにして得られるこの発明にかかる酵素固定化電
極は、コラーゲンによって妨害除去膜の水分が保たれる
ため、保湿性を保つことができ、乾燥による妨害除去膜
の電極本体からの剥離を防ぐことができる。また、測定
溶液中での膨潤率、乾燥させたときの収縮率が小さくな
り、酵素固定化電極として使用したときの感度のバラツ
キがなく、安定した測定を行うことができる。In the enzyme-immobilized electrode according to the present invention thus obtained, the moisture of the interference removal film is retained by collagen, so that the moisture retention property can be maintained and peeling of the interference removal film from the electrode body due to drying is prevented. be able to. Further, the swelling rate in the measurement solution and the contraction rate when dried are small, and there is no variation in sensitivity when used as an enzyme-immobilized electrode, and stable measurement can be performed.
また、妨害除去膜作製用の水溶液の塗布性も良くなり、
電極表面上に膜厚が均一になるように製膜することがで
きる。Also, the coatability of the aqueous solution for forming the interference removal film is improved,
The film can be formed on the surface of the electrode so that the film thickness is uniform.
コラーゲンを含まない従来の妨害除去膜では、電極本体
に妨害除去膜作製液を塗布後、調湿器の外、たとえば、
湿度40〜60％程度の雰囲気下に５〜10分間放置すると、
第３図(a)にみるように、妨害除去膜５が電極本体１か
ら剥離してしまう。このため、調湿器内で湿度80％の雰
囲気下に保って第３図(b)にみるように妨害除去膜４を
作製する必要があった。これに対し、この発明にかかる
酵素固定化電極の製造方法では、妨害除去膜の上に酵素
固定化膜を形成するにあたり、あらかじめ前記妨害除去
膜にコラーゲンを含ませておくので、調湿器の外、たと
えば、湿度40〜60％程度の雰囲気下に５〜10分間放置し
ても、第３図(b)にみるように妨害除去膜４が形成さ
れ、剥離が生じにくい。第１〜３図中、３は、セラミッ
ク、合成樹脂等からなる絶縁性基板である。電極本体１
は、たとえば、この基板３の上に形成されているが、こ
のようなものに限定されない。In the case of a conventional interference removal film that does not contain collagen, after applying the interference removal film preparation liquid to the electrode body, outside the humidity controller, for example,
If you leave it in an atmosphere with a humidity of 40 to 60% for 5 to 10 minutes,
As shown in FIG. 3 (a), the interference removal film 5 is peeled off from the electrode body 1. For this reason, it is necessary to keep the humidity inside the humidity controller at 80% in the atmosphere to fabricate the interference removing film 4 as shown in FIG. 3 (b). On the other hand, in the method for producing the enzyme-immobilized electrode according to the present invention, when the enzyme-immobilized film is formed on the interference-removing film, the interference-removing film is preliminarily made to contain collagen. Even if left outside, for example, in an atmosphere having a humidity of about 40 to 60% for 5 to 10 minutes, the interference removing film 4 is formed as shown in FIG. 3 (b), and peeling hardly occurs. In FIGS. 1 to 3, 3 is an insulating substrate made of ceramic, synthetic resin or the like. Electrode body 1
Is formed on the substrate 3, for example, but is not limited to such.
なお、この発明にかかる酵素固定化電極は、1000回以上
の連続測定も可能である。The enzyme-immobilized electrode according to the present invention is capable of continuous measurement 1000 times or more.
妨害除去膜がコラーゲンを含んでいることにより、乾燥
・湿潤による収縮・膨潤の変化が小さくなり、剥離しに
くくなる。Since the interference removal film contains collagen, the change in shrinkage / swelling due to drying / wetting becomes small and peeling becomes difficult.
また、妨害除去膜がコラーゲンを含んでいることによ
り、同妨害除去膜の上に酵素固定化膜を形成するときの
雰囲気の調湿を行わずにすみ、妨害除去膜が電極本体か
ら剥離するのが防がれるという利点も得られる。In addition, since the interference removal film contains collagen, it is not necessary to control the humidity of the atmosphere when the enzyme immobilization film is formed on the interference removal film, and the interference removal film is peeled from the electrode body. The advantage of being prevented is also obtained.
以下に、この発明の具体的な実施例および比較例を示す
が、この発明は下記実施例に限定されない。Specific examples and comparative examples of the present invention will be shown below, but the present invention is not limited to the following examples.
−実施例１〜５および比較例− 第１表にみるような組成の下地膜作製溶液、妨害除去膜
作製液および酵素固定化溶液を用いた。妨害除去膜のマ
トリックス成分としてポリアリルアミンおよびアルブミ
ンを、架橋剤としてグルタルアルデヒドを、保湿剤とし
てコラーゲンをそれぞれ用いた。酵素固定化膜のマトリ
ックス成分は、ゼラチン、ポリアリルアミン、アルブミ
ンであり、架橋剤はグルタルアルデヒドである。下地膜
作製液を第１図にみるように、リード部２を有し、基板
３上に設けられた電極本体（白金電極）１表面上に所定
量塗布したのち架橋反応を行わせて下地膜を製膜した。
この下地膜の表面に第１表に示す組成の妨害除去膜作製
液６を塗布して（第２図参照）架橋反応を行わせ、妨害
除去膜４を形成した（第３図(b)参照）。この妨害除去
膜４の表面に第１表に示す組成の酵素固定化溶液を塗布
して架橋反応を行わせ、酵素固定化膜を形成した。-Examples 1 to 5 and Comparative Example- An underlayer film forming solution, an interference removal film forming solution and an enzyme immobilization solution having the compositions shown in Table 1 were used. Polyallylamine and albumin were used as matrix components of the interference removal film, glutaraldehyde was used as a cross-linking agent, and collagen was used as a moisturizing agent. The matrix component of the enzyme-immobilized membrane is gelatin, polyallylamine, albumin, and the cross-linking agent is glutaraldehyde. As shown in FIG. 1, the undercoat film preparation liquid has a lead portion 2 and is applied on a surface of an electrode body (platinum electrode) 1 provided on a substrate 3 in a predetermined amount, and then a cross-linking reaction is performed to perform the undercoat film. Was formed into a film.
The interference removing film preparation liquid 6 having the composition shown in Table 1 was applied to the surface of the base film (see FIG. 2) to cause a crosslinking reaction to form an interference removing film 4 (see FIG. 3 (b)). ). An enzyme-immobilized solution having the composition shown in Table 1 was applied to the surface of the interference removal film 4 to cause a crosslinking reaction to form an enzyme-immobilized film.
実施例および比較例において、電極面積は１×１mm2、
溶液塗布量は0.5μｌとした。In the examples and comparative examples, the electrode area is 1 × 1 mm 2 ,
The solution application amount was 0.5 μl.
なお、比較例では、妨害除去膜作製液を第２図にみるよ
うに塗布してから湿度40〜60％の雰囲気下に５〜10分間
放置したところ、第３図(a)にみるように、妨害除去膜
５が電極本体１から剥離してしまった。このため、妨害
除去膜作製液を塗布して調湿器内に入れ、湿度80％以上
の雰囲気下で妨害除去膜の作製（第３図(b)参照）およ
び酵素固定化膜の作製を行った。In the comparative example, the interference removal film preparation liquid was applied as shown in FIG. 2 and then left for 5 to 10 minutes in an atmosphere with a humidity of 40 to 60%, as shown in FIG. 3 (a). The interference removing film 5 has peeled off from the electrode body 1. For this reason, the interference removal film preparation liquid is applied and placed in a humidity controller to prepare an interference removal film (see Fig. 3 (b)) and an enzyme-immobilized film in an atmosphere with a humidity of 80% or more. It was
上記実施例および比較例で用いたポリアリルアミンは平
均分子量約60000であり、実施例で用いたコラーゲンは
（株）高研製の牛真皮・可溶性タイプＩコラーゲン溶液
（pH3.0、濃度0.3％）のものであった。The polyallylamine used in the above Examples and Comparative Examples had an average molecular weight of about 60,000, and the collagen used in the Examples was a bovine dermis soluble type I collagen solution (pH 3.0, concentration 0.3%) manufactured by Koken Co., Ltd. It was a thing.
実施例１〜５および比較例の各酵素固定化電極について
検出感度（測定感度）、保湿性および感度変動を調べて
結果を第１表に示した。感度は、酵素固定化電極をポー
ラロアナライザ等の測定装置に接続し、電圧0.6V（対Ag
電極）、サンプル量を20μｌとして測定した。サンプル
は、150mg/dlのグルコース溶液、100mg/dlのコレステロ
ール溶液、妨害物質として、アスコルビン酸（濃度20mg
/dl）、血液の凝固防止などに使用されるNaF（濃度50mg
/dl）を用いた。保湿性は、妨害除去膜作製液を塗布
後、湿度40〜60％の雰囲気下で５〜10分間程度のうちに
妨害除去膜が剥離しない場合を○、剥離した場合を×で
示した。感度変動は、得られた酵素固定化電極を用いて
標準液で20回測定したときの１回目の感度の測定値と20
回目の感度の測定値との変化を下式により求めて示し
た。The detection sensitivities (measurement sensitivities), the moisturizing properties and the sensitivity fluctuations of the enzyme-immobilized electrodes of Examples 1 to 5 and Comparative Example were examined, and the results are shown in Table 1. Sensitivity is determined by connecting the enzyme-immobilized electrode to a measuring device such as a polaro-analyzer and measuring a voltage of 0.6 V (against Ag
Electrode) and the sample amount was 20 μl. Samples are 150 mg / dl glucose solution, 100 mg / dl cholesterol solution, ascorbic acid (concentration 20 mg
/ dl), NaF used for blood coagulation prevention (concentration 50 mg
/ dl) was used. The moisturizing property is indicated by ◯ when the interference removal film was not peeled off within 5 to 10 minutes in the atmosphere of 40 to 60% humidity after applying the interference removal film preparation liquid, and by X when peeled. Sensitivity fluctuations are measured by using the obtained enzyme-immobilized electrode and measuring the standard solution 20 times
The change from the measured value of the sensitivity of the second time was obtained and shown by the following formula.
第１表からわかるように、実施例１〜３の酵素固定化電
極は、いずれもグルコースを感度良く検出することがで
き、実施例４および５の酵素固定化電極は、いずれもコ
レステロールを感度良く検出することができた。また、
実施例１〜５では、妨害物質はほとんどまたは全く検出
されなかった。これに対し、比較例の酵素固定化電極で
は、感度変動が実施例のもの（±２％以内）よりも大き
かった（±４〜５％）。また、比較例のものは、湿度40
〜60％RHの雰囲気下では、第３図(a)にみるように、妨
害除去膜５が電極本体１から剥離したが、実施例のもの
では、同じ条件下で剥離しなかった。 As can be seen from Table 1, all of the enzyme-immobilized electrodes of Examples 1 to 3 can detect glucose with high sensitivity, and the enzyme-immobilized electrodes of Examples 4 and 5 both detect cholesterol with high sensitivity. Could be detected. Also,
In Examples 1-5, little or no interfering substances were detected. On the other hand, in the enzyme-immobilized electrode of the comparative example, the sensitivity variation was larger than that of the example (within ± 2%) (± 4 to 5%). The humidity of the comparative example is 40
In an atmosphere of -60% RH, as shown in Fig. 3 (a), the interference removal film 5 was peeled from the electrode body 1, but in the example, it was not peeled off under the same conditions.
なお、上記実施例では、固定化する酵素として、グルコ
ースオキシダーゼおよびコレステロールオキシダーゼを
用いたが、この発明において使用できる酵素はこれらの
２者に限定されるものではない。また、測定用の液も血
液に限るものではない。Although glucose oxidase and cholesterol oxidase were used as the enzymes to be immobilized in the above examples, the enzymes usable in the present invention are not limited to these two. The measurement liquid is not limited to blood.
この発明にかかる酵素固定化電極は、導電性材料からな
る電極本体に酵素固定化膜および妨害除去膜が設けられ
ているものにおいて、前記妨害除去膜がコラーゲンを含
んでいることを特徴とするので、妨害除去膜の剥離がな
く、感度の良い、安定した測定を行うことができる。The enzyme-immobilized electrode according to the present invention is characterized in that the enzyme-immobilized film and the interference removal film are provided on the electrode body made of a conductive material, and the interference removal film contains collagen. In addition, the interference removal film is not peeled off, and stable and stable measurement can be performed.
この発明にかかる酵素固定化電極の製造方法は、導電性
材料からなる電極本体表面上に形成された妨害除去膜の
上に酵素固定化膜を形成するにあたり、前記妨害除去膜
にコラーゲンを含めておくことを特徴とするので、妨害
除去膜の上に酵素固定化膜を形成するときに、調湿する
必要がある。The method for producing an enzyme-immobilized electrode according to the present invention, in forming the enzyme-immobilized film on the interference-removing film formed on the surface of the electrode body made of a conductive material, the interference-removing film contains collagen. Since it is characterized by being set, it is necessary to control the humidity when the enzyme-immobilized film is formed on the interference removal film.
第１図は、実施例および比較例に用いた白金電極の概略
を表す斜視図、第２図は、同白金電極に妨害除去膜作製
液を塗布した状態を表す概略断面図、第３図(a)は、比
較例の酵素固定化電極における妨害除去膜形成後で酵素
固定化膜形成前の状態を表す概略断面図、第３図(b)
は、実施例の酵素固定化電極における妨害除去膜形成後
で酵素固定化膜形成前の状態を表す概略断面図である。 １……電極本体、４……妨害除去膜FIG. 1 is a perspective view showing the outline of platinum electrodes used in Examples and Comparative Examples, and FIG. 2 is a schematic cross-sectional view showing a state in which an interference removal film preparation liquid is applied to the platinum electrodes, FIG. FIG. 3 (b) is a schematic cross-sectional view showing a state after the formation of the interference removal film and before the formation of the enzyme-immobilized film in the enzyme-immobilized electrode of Comparative Example.
FIG. 4 is a schematic cross-sectional view showing a state after formation of an interference removal film and before formation of an enzyme-immobilized film in the enzyme-immobilized electrode of the example. 1 ... Electrode body, 4 ... Interference removal film
Claims (2)
膜および妨害物質除去膜が設けられている酵素固定化電
極において、前記妨害物質除去膜がコラーゲンを含んで
いることを特徴とする酵素固定化電極。1. An enzyme-immobilized electrode in which an enzyme-immobilized film and an interfering substance removing film are provided on an electrode body made of a conductive material, wherein the interfering substance removing film contains collagen. Fixed electrode.
された妨害物質除去膜の上に酵素固定化膜を形成する酵
素固定化電極の製造方法において、前記妨害物質除去膜
にコラーゲンを含めておくことを特徴とする酵素固定化
電極の製造方法。2. A method for producing an enzyme-immobilized electrode comprising forming an enzyme-immobilized film on an interfering substance-removing film formed on the surface of an electrode body made of a conductive material, wherein the interfering-substance removing film contains collagen. A method for producing an enzyme-immobilized electrode, which comprises:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1164797A JPH0750058B2 (en) | 1989-06-27 | 1989-06-27 | Enzyme-immobilized electrode and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1164797A JPH0750058B2 (en) | 1989-06-27 | 1989-06-27 | Enzyme-immobilized electrode and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0328752A JPH0328752A (en) | 1991-02-06 |
| JPH0750058B2 true JPH0750058B2 (en) | 1995-05-31 |
Family
ID=15800119
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1164797A Expired - Lifetime JPH0750058B2 (en) | 1989-06-27 | 1989-06-27 | Enzyme-immobilized electrode and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0750058B2 (en) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT397513B (en) * | 1992-12-15 | 1994-04-25 | Avl Verbrennungskraft Messtech | AMPEROMETRIC ENZYME ELECTRODE |
| US5741319A (en) * | 1995-01-27 | 1998-04-21 | Medtronic, Inc. | Biocompatible medical lead |
| US8974386B2 (en) | 1998-04-30 | 2015-03-10 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US8480580B2 (en) | 1998-04-30 | 2013-07-09 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US8688188B2 (en) | 1998-04-30 | 2014-04-01 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US6338790B1 (en) | 1998-10-08 | 2002-01-15 | Therasense, Inc. | Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator |
| US6591125B1 (en) | 2000-06-27 | 2003-07-08 | Therasense, Inc. | Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator |
| US6560471B1 (en) | 2001-01-02 | 2003-05-06 | Therasense, Inc. | Analyte monitoring device and methods of use |
| US7381184B2 (en) | 2002-11-05 | 2008-06-03 | Abbott Diabetes Care Inc. | Sensor inserter assembly |
| WO2004061420A2 (en) | 2002-12-31 | 2004-07-22 | Therasense, Inc. | Continuous glucose monitoring system and methods of use |
| US8771183B2 (en) | 2004-02-17 | 2014-07-08 | Abbott Diabetes Care Inc. | Method and system for providing data communication in continuous glucose monitoring and management system |
| USD914881S1 (en) | 2003-11-05 | 2021-03-30 | Abbott Diabetes Care Inc. | Analyte sensor electronic mount |
| US9788771B2 (en) | 2006-10-23 | 2017-10-17 | Abbott Diabetes Care Inc. | Variable speed sensor insertion devices and methods of use |
| US7766829B2 (en) | 2005-11-04 | 2010-08-03 | Abbott Diabetes Care Inc. | Method and system for providing basal profile modification in analyte monitoring and management systems |
| US8226891B2 (en) | 2006-03-31 | 2012-07-24 | Abbott Diabetes Care Inc. | Analyte monitoring devices and methods therefor |
| US7620438B2 (en) | 2006-03-31 | 2009-11-17 | Abbott Diabetes Care Inc. | Method and system for powering an electronic device |
| US8123686B2 (en) | 2007-03-01 | 2012-02-28 | Abbott Diabetes Care Inc. | Method and apparatus for providing rolling data in communication systems |
| US20100213057A1 (en) | 2009-02-26 | 2010-08-26 | Benjamin Feldman | Self-Powered Analyte Sensor |
| WO2011026147A1 (en) | 2009-08-31 | 2011-03-03 | Abbott Diabetes Care Inc. | Analyte signal processing device and methods |
| CN110461217B (en) | 2017-01-23 | 2022-09-16 | 雅培糖尿病护理公司 | Systems, devices, and methods for analyte sensor insertion |
| JP2023540275A (en) | 2020-08-31 | 2023-09-22 | アボット ダイアベティス ケア インコーポレイテッド | Systems, devices, and methods for analyte sensor insertion |
Family Cites Families (1)
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-
1989
- 1989-06-27 JP JP1164797A patent/JPH0750058B2/en not_active Expired - Lifetime
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| JPH0328752A (en) | 1991-02-06 |
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