JPH0566983B2 - - Google Patents
Info
- Publication number
- JPH0566983B2 JPH0566983B2 JP60217149A JP21714985A JPH0566983B2 JP H0566983 B2 JPH0566983 B2 JP H0566983B2 JP 60217149 A JP60217149 A JP 60217149A JP 21714985 A JP21714985 A JP 21714985A JP H0566983 B2 JPH0566983 B2 JP H0566983B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- electrode
- immobilized
- electrodes
- membrane
- 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
- 102000004190 Enzymes Human genes 0.000 claims description 57
- 108090000790 Enzymes Proteins 0.000 claims description 57
- 239000012528 membrane Substances 0.000 claims description 33
- 239000000758 substrate Substances 0.000 claims description 24
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 22
- 229920000642 polymer Polymers 0.000 claims description 15
- 239000010409 thin film Substances 0.000 claims description 10
- 239000010408 film Substances 0.000 claims description 9
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 229940088598 enzyme Drugs 0.000 description 51
- 239000000126 substance Substances 0.000 description 22
- 230000002452 interceptive effect Effects 0.000 description 20
- 238000000034 method Methods 0.000 description 17
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 14
- 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 9
- 239000008103 glucose Substances 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 8
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 8
- 229960005070 ascorbic acid Drugs 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 229920006037 cross link polymer Polymers 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 235000000069 L-ascorbic acid Nutrition 0.000 description 5
- 239000002211 L-ascorbic acid Substances 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000011521 glass Substances 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 229910052697 platinum Inorganic materials 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 3
- 239000004366 Glucose oxidase Substances 0.000 description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 description 3
- 239000004372 Polyvinyl alcohol Substances 0.000 description 3
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical group N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 3
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 229940116332 glucose oxidase Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229920006254 polymer film Polymers 0.000 description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 description 3
- 229940116269 uric acid Drugs 0.000 description 3
- YVXDRFYHWWPSOA-BQYQJAHWSA-N 1-methyl-4-[(e)-2-phenylethenyl]pyridin-1-ium Chemical group C1=C[N+](C)=CC=C1\C=C\C1=CC=CC=C1 YVXDRFYHWWPSOA-BQYQJAHWSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 229910052804 chromium Inorganic materials 0.000 description 2
- 239000011651 chromium Substances 0.000 description 2
- 238000001259 photo etching Methods 0.000 description 2
- 238000000206 photolithography Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000004528 spin coating Methods 0.000 description 2
- 238000007740 vapor deposition Methods 0.000 description 2
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- 108010025188 Alcohol oxidase Proteins 0.000 description 1
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 1
- 108010000659 Choline oxidase Proteins 0.000 description 1
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 1
- 108010015133 Galactose oxidase Proteins 0.000 description 1
- 102000057621 Glycerol kinases Human genes 0.000 description 1
- SXZYCXMUPBBULW-SKNVOMKLSA-N L-gulono-1,4-lactone Chemical compound OC[C@H](O)[C@H]1OC(=O)[C@@H](O)[C@H]1O SXZYCXMUPBBULW-SKNVOMKLSA-N 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 101710163410 Probable glycerol kinase Proteins 0.000 description 1
- 229910004298 SiO 2 Inorganic materials 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000007772 electroless plating Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 235000012209 glucono delta-lactone Nutrition 0.000 description 1
- 229960003681 gluconolactone Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000007769 metal material Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 238000000059 patterning Methods 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920002120 photoresistant polymer Polymers 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001721 polyimide Polymers 0.000 description 1
- 239000009719 polyimide resin Substances 0.000 description 1
- 229920005597 polymer membrane Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 238000007650 screen-printing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000007738 vacuum evaporation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、酵素センサーに関する。更に詳しく
は、絶縁基板上に信頼性の高い過酸化水素電極を
形成せしめることのできる酵素センサーに関す
る。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an enzyme sensor. More specifically, the present invention relates to an enzyme sensor that can form a highly reliable hydrogen peroxide electrode on an insulating substrate.
最近、酵素反応や免疫反応などの生体反応を利
用した種々のバイオセンサーが開発されており、
特に臨床分野では、更に小型、高性能で低価格な
ものが求められるようになつてきている。酵素セ
ンサーは、こうしたバイオセンサーの一種であ
り、例えばグルコースオキシダーゼの触媒作用を
利用したグルコースセンサーは、血液や尿中のグ
ルコース濃度を測定するのに用いられ、実用的に
は糖尿病患者に対する臨床検査用として重要であ
る。
Recently, various biosensors that utilize biological reactions such as enzyme reactions and immune reactions have been developed.
Particularly in the clinical field, there is a growing demand for smaller, higher-performance, and lower-priced devices. Enzyme sensors are a type of biosensor. For example, glucose sensors that utilize the catalytic action of glucose oxidase are used to measure glucose concentrations in blood and urine, and are practically used in clinical tests for diabetic patients. important as such.
しかしながら、こういつた酵素センサーで実用
化され、市販されているものの大半は、電極と酵
素固定化膜とが一体構造となつていないため、小
型化の達成および大量生産による低コスト化の妨
げとなつている。 However, in most of these enzyme sensors that have been put to practical use and are commercially available, the electrode and enzyme-immobilized membrane are not integrated, which hinders the achievement of miniaturization and cost reduction through mass production. It's summery.
こうした市販酵素センサーの問題点に鑑み、最
近では電極上に直接酵素を固定化させる研究が進
められ、それに伴つてかなり小型化されたものが
開発されるようになつてきている。しかしなが
ら、同一基板上に多数個のセンサーを同時に形成
させ、大量生産を可能とせんとする場合には、例
えば外部電極との接続部を露出させ、検出部のみ
に酵素を固定化するというように、電極基板上の
必要な部分のみに酵素を固定化させる必要があ
る。
In view of these problems with commercially available enzyme sensors, research into directly immobilizing enzymes on electrodes has recently progressed, and as a result, considerably smaller versions have been developed. However, if a large number of sensors are to be formed on the same substrate at the same time to enable mass production, for example, the connection part with the external electrode is exposed and the enzyme is immobilized only on the detection part. , it is necessary to immobilize the enzyme only on the necessary portions of the electrode substrate.
本発明者らは、こうした課題を解決するため
に、光架橋重合体を用いて酵素固定化膜を形成せ
しめることが有効であることを見出し、絶縁基板
上に形成させた金属薄膜よりなるアノード電極お
よびカソード電極の少くとも前者の電極に、光架
橋重合体で固定化された酵素固定化膜を設置した
酵素センサーを先に提案している（特願昭60−
103699号）。 In order to solve these problems, the present inventors found that it is effective to form an enzyme-immobilized film using a photocrosslinked polymer, and the anode electrode consists of a metal thin film formed on an insulating substrate. We have previously proposed an enzyme sensor in which an enzyme-immobilized membrane immobilized with a photocrosslinked polymer is installed at least on the former cathode electrode (Japanese Patent Application No. 1983-
No. 103699).
このように構成された酵素センサーは、同一基
板上に多数個の微小なセンサーを同時に形成させ
ることができ、大量生産を可能とさせるが、形成
された１個の組合せ電極が過酸化水素電極を構成
する場合には、この電極が過酸化水素以外の還元
性妨害物質にも感応してしまうという問題のある
ことが新たに見出された。 Enzyme sensors configured in this way can simultaneously form many microscopic sensors on the same substrate, making mass production possible. In this case, it has been newly discovered that there is a problem in that this electrode is sensitive to reducing interfering substances other than hydrogen peroxide.
通常、こうした問題の対策としては、酵素固定
化膜と電極面との間に過酸化水素選択透過膜を設
置する方法がとられているが、上記酵素センサー
の場合には、電極面上に直接酵素が固定化されて
いるため選択透過膜の設置は不可能である。 Normally, as a countermeasure to this problem, a hydrogen peroxide selectively permeable membrane is installed between the enzyme immobilized membrane and the electrode surface, but in the case of the enzyme sensor mentioned above, it is Since the enzyme is immobilized, it is impossible to install a selectively permeable membrane.
そこで、本発明者らは、かかる課題の新たな解
決方法を求めて種々検討の結果、同一基板上に２
組の組合せ電極を形成させ、その内の１組につい
て、少くともアノード電極上に固定化された酵素
固定化膜を形成させることにより、上記課題が有
効に解決されることを先に見出した（特願昭60−
171850号）。 Therefore, as a result of various studies in search of a new solution to this problem, the present inventors have found that two
We have previously discovered that the above problem can be effectively solved by forming a set of combination electrodes, and forming an enzyme-immobilized membrane immobilized on at least the anode electrode for one of the sets ( Special request 1986-
No. 171850).
ここに提案された酵素センサーは、同一絶縁基
板上に形成させた金属薄膜よりなるアノード電極
およびカソード電極の組合せ電極２組の内の１組
について、少くともアノード電極上に固定化され
た酵素固定化膜を設置してなる。１組の組合せ電
極上への酵素固定化膜の固定化は、一般に光架橋
重合体によつて行われ、その光架橋重合体で固定
化される酵素固定化膜の形成は、先の提案と同様
に、光架橋性重合体と酵素との水性混合物にフオ
トリソグラフ法を適用することによつて行われ
る。 The enzyme sensor proposed here has at least an enzyme immobilized on the anode electrode for one of two sets of combined electrodes consisting of an anode electrode and a cathode electrode, which are made of metal thin films formed on the same insulating substrate. A chemical membrane is installed. Immobilization of an enzyme-immobilized membrane onto a set of combination electrodes is generally performed using a photo-crosslinked polymer, and the formation of an enzyme-immobilized membrane immobilized with the photo-crosslinked polymer is based on the previous proposal. It is likewise carried out by applying photolithographic methods to aqueous mixtures of photocrosslinkable polymers and enzymes.
また、酵素固定化膜を設置しない他の１組の組
合せ電極は、少くともアノード電極上が光架橋重
合体膜によつて被覆されて用いられるが、この場
合の光架橋重合体膜の形成も、光架橋性重合体を
含有する水溶液にフオトリソグラフ法を適用する
ことによつて行われる。 In addition, another set of combination electrodes without an enzyme-immobilized membrane is used with at least the anode electrode covered with a photocrosslinked polymer film, and in this case, the formation of the photocrosslinked polymer film is also , by applying a photolithography method to an aqueous solution containing a photocrosslinkable polymer.
これら２組の組合せ電極において、各組毎にア
ノード電極およびカソード電極を形成させてもよ
いが、カソード電極を２組の組合せ電極について
共通して用いられる１個のカソード電極とし、そ
れを各組合せ電極毎に切換えて用いるようにする
こともできる。 In these two sets of combined electrodes, an anode electrode and a cathode electrode may be formed for each set, but the cathode electrode may be one cathode electrode that is commonly used for the two sets of combined electrodes, It is also possible to switch and use each electrode.
酵素センサーの作製に際しては、まずうガラス
板、塩化ビニル樹脂、ポリイミド樹脂などの硬質
樹脂板、SiO2、Si3N4などの絶縁被膜を表面に形
成させたシリコンウエハーなどの平らな絶縁基板
上に、アノード電極およびカソード電極の組合せ
電極２組を形成させることが行われる。電極の形
成は、金、白金（以上アノード電極用）または
銀、金、白金（以上カソード電極用）などの金属
材料を用い、図面の第１図に示されるようなリフ
トオフ法、基板上に蒸着された金属薄膜をエツチ
ング除去してパターニングするフオトエツチング
法、電極形状の窓が開いたマスクを基板に重ねマ
スクごしに電極形成物質を蒸着させるマスク蒸着
法、電極形成物質を導電材料とする導電性塗料を
電極形状に印刷するスクリーン印刷法あるいは上
記フオトエツチング法またはマスク蒸着法におい
て蒸着の代り電極形成材料の無電解メツキを行な
うメツキ法などによつて行なうことができる。 When making an enzyme sensor, first, it is made on a flat insulating substrate such as a glass plate, a hard resin plate such as vinyl chloride resin, or polyimide resin, or a silicon wafer with an insulating film such as SiO 2 or Si 3 N 4 formed on the surface. Next, two sets of combined electrodes of an anode electrode and a cathode electrode are formed. The electrodes are formed using metal materials such as gold, platinum (for anode electrodes) or silver, gold, platinum (for cathode electrodes), and are deposited on the substrate using a lift-off method as shown in Figure 1 of the drawings. The photo-etching method involves removing and patterning the metal thin film that has been etched, the mask evaporation method involves placing a mask with an electrode-shaped window on the substrate and depositing an electrode-forming material through the mask, and the conductive method uses a conductive material as the electrode-forming material. This can be carried out by a screen printing method in which a color paint is printed in the shape of an electrode, or a plating method in which electroless plating of the electrode forming material is performed instead of vapor deposition in the above-mentioned photoetching method or mask vapor deposition method.
第１図に示された態様では、リフトオフ法が用
いられている。まず、清浄された平らな絶縁基
板、例えばガラス板１上に電極部が基板露出面２
の範囲内に形成されるように、ポジ型フオトレジ
スト３をパターニングする（工程ａ）。次いで、
真空蒸着法により、この基板上にクロム薄膜４
（厚さ約500Å）および金または白金薄膜５（厚さ
約0.2μｍ）を順次形成させる。ここで、クロム薄
膜は、電極を形成する金または白金薄膜とガラス
板基板との密着性を高めるために設けられている
（工程ｂ）。その後、全体をアセトンなどのレジス
ト剥離液中に浸漬してレジストを除去し、基板露
出面２に残存する蒸着薄膜５を電極面とした（工
程ｃ）。 In the embodiment shown in FIG. 1, a lift-off method is used. First, an electrode section is placed on a cleaned flat insulating substrate, for example a glass plate 1, on the exposed surface 2 of the substrate.
The positive photoresist 3 is patterned so that it is formed within the range of (step a). Then,
A thin chromium film 4 is deposited on this substrate by vacuum evaporation.
(thickness approximately 500 Å) and gold or platinum thin film 5 (thickness approximately 0.2 μm) are sequentially formed. Here, the chromium thin film is provided to improve the adhesion between the gold or platinum thin film forming the electrode and the glass plate substrate (step b). Thereafter, the whole was immersed in a resist stripping solution such as acetone to remove the resist, and the deposited thin film 5 remaining on the exposed surface 2 of the substrate was used as an electrode surface (step c).
このようにして絶縁基板上の所定の個所に、カ
ソード電極が１個共通して用いられる態様で、金
属薄膜よりなるアノード電極およびカソード電極
となる３個の電極を形成させたら、その内の少く
ともアノード電極となる１個の電極に酵素固定化
膜を設置させるが、その設置はフオトリソグラフ
法を用いて、例えば第２図に示される如くにして
行なわれる。 In this way, if three electrodes are formed at a predetermined location on the insulating substrate in such a manner that one cathode electrode is used in common, the anode electrode and the cathode electrode made of the metal thin film are formed. An enzyme-immobilized membrane is installed on one electrode, which serves as an anode electrode, and the installation is carried out using a photolithography method, for example, as shown in FIG. 2.
まず、第１図に示された如くにして形成された
絶縁板２上の電極面５に、光架橋性重合体と酵素
との水性混合物をスピンコート法、スプレー法な
どにより均一にコーテイング６する（工程ｄ）。
光架橋性重合体としては、それが酵素水溶液と共
に水性混合物として分散されるため一般に水溶性
重合体が用いられ、例えば分子中に光架橋性基と
してスチルバゾリウム基、ジアゾ基などの感光性
基、好ましくはスチルバゾリウム基を有するポリ
ビニルアルコールなどが水溶液として用いられ
る。水性混合物は、上記光架橋性重合体水溶液
（濃度8.5〜12重量％）１ｇに対して、酵素３〜72
mgを蒸留水0.8mlに溶解させた酵素水溶液が添加
され、それを数分間程度撹拌、混合してコーテイ
ングに用いられる。 First, an aqueous mixture of a photocrosslinkable polymer and an enzyme is uniformly coated 6 on the electrode surface 5 on the insulating plate 2 formed as shown in FIG. 1 by spin coating, spraying, etc. (Step d).
As the photocrosslinkable polymer, a water-soluble polymer is generally used because it is dispersed as an aqueous mixture together with an aqueous enzyme solution. Polyvinyl alcohol having a stilbazolium group is used as an aqueous solution. The aqueous mixture contains 3 to 72% of the enzyme per 1 g of the photocrosslinkable polymer aqueous solution (concentration 8.5 to 12% by weight).
An enzyme aqueous solution prepared by dissolving mg of the enzyme in 0.8 ml of distilled water is added, and the mixture is stirred and mixed for several minutes before being used for coating.
コーテイング液を絶縁基板上の電極面上にコー
テイングし、それが自然乾燥したら、そこをネガ
またはポジの画像を有するフオトマスク７で覆
い、紫外線照射して光架橋性重合体を光架橋さ
せ、未架橋部分を純水で溶去して、光架橋部分に
光架橋重合体で固定化された酵素固定化膜８を形
成させる（工程ｅ）。これを再度紫外線照射して
から乾燥させた。ここで使用されるフオトマスク
は、２組の組合せ電極の内の１組のアノード電極
１１にのみ、酵素固定化膜８が形成されるような
画像を有するものが用いられる。 The coating liquid is coated on the electrode surface on the insulating substrate, and when it dries naturally, it is covered with a photomask 7 having a negative or positive image, and the photocrosslinkable polymer is photocrosslinked by irradiation with ultraviolet rays, leaving the photocrosslinkable polymer uncrosslinked. The portion is eluted with pure water to form an enzyme-immobilized membrane 8 immobilized with a photocrosslinked polymer on the photocrosslinked portion (step e). This was irradiated with ultraviolet light again and then dried. The photomask used here has an image such that the enzyme-immobilized film 8 is formed only on one set of anode electrodes 11 out of the two sets of combined electrodes.
次に、還元性妨害物質に対する２組の組合せ電
極の感応特性を等しくするため、酵素固定化膜を
設置しない他の１組の組合せ電極のアノード電極
１２に、第２図と同様の手法により、光架橋性重
合体を含有する水溶液にフオトリソグラフ法を適
用し、そこに光架橋重合体膜９を被覆させ、これ
を参照用電極とした（工程ｆ）。 Next, in order to equalize the sensitivity characteristics of the two sets of combination electrodes to reducing interfering substances, the anode electrode 12 of the other set of combination electrodes in which the enzyme-immobilized membrane was not installed was coated with the same method as shown in FIG. A photolithographic method was applied to an aqueous solution containing a photocrosslinkable polymer, and a photocrosslinkable polymer film 9 was coated thereon, and this was used as a reference electrode (step f).
これを再度紫外線照射してから乾燥し、各素子
毎に分割して、その電極露出面５，５′，５″にリ
ード線１０，１０′，１０″を取り付ける。このよ
うにして作製された酵素センサーの一態様が、平
面図として第３図に示されている。また、第４〜
５図には、他の態様の酵素センサーの平面図が示
されており、第４図の態様では、電極面５′から
２本のカソード電極１３，１３′を延長して形成
させ、各アノード−カソード間の電極間距離を短
縮させると共に、これらの各組合せ電極がそれぞ
れ酵素固定化膜８および光架橋重合体膜９で覆わ
れており、また第５図の態様では、カソード電極
１３の面積を電極１１，１２の面積より広く設定
することにより、アノードに対するカソード電流
を安定にし、また電極間距離を一定にしたまま両
電極間の対向幅を長くすることにより、電極の応
答特性の向上が図られている。 This is irradiated with ultraviolet rays again and then dried, divided into each element, and lead wires 10, 10', 10'' are attached to the exposed electrode surfaces 5, 5', 5''. One embodiment of the enzyme sensor produced in this manner is shown in FIG. 3 as a plan view. Also, the 4th ~
FIG. 5 shows a plan view of another embodiment of the enzyme sensor. In the embodiment of FIG. 4, two cathode electrodes 13, 13' are formed by extending from the electrode surface 5', and each anode - The interelectrode distance between the cathodes is shortened, and each of these combined electrodes is covered with an enzyme immobilized membrane 8 and a photocrosslinked polymer membrane 9, respectively, and in the embodiment of FIG. 5, the area of the cathode electrode 13 is reduced. By setting the area to be larger than the area of the electrodes 11 and 12, the cathode current to the anode is stabilized, and by increasing the facing width between both electrodes while keeping the distance between the electrodes constant, the response characteristics of the electrodes can be improved. It is planned.
この酵素センサーによつて検出可能な基質とこ
の基質に対して反応する触媒としての酵素との組
合せの例は次の如くであり、これらの場合両電極
は過酸化水素電極として作用する。被検出物質
酵 素
グルコース グルコースオキシダーゼ
ガラクトース ガラクトースオキシダーゼ
エタノール アルコールオキシダーゼ
リン脂質 ホスホリパーゼ
コリン コリンオキシダーゼ
グリセリン グリセロキナーゼ
（２種類の酵素 Ｌ−α−グリセロ−３−リ
を同時固定 ン酸オキシダーゼ
このような酵素センサーをグルコースセンサー
として用いた場合には、次のように作用する。ま
ず、グルコースセンサーをグルコースを含まない
緩衝液中に浸漬し、電極面に、例えば金電極の場
合0.8Vの電圧を印加しておき、これにグルコー
スを添加すると、酵素が固定化された方の過酸化
水素電極側で、グルコースがグルコースオキシダ
ーゼ酵素固定化膜に拡散し、固定化酵素の触媒作
用により次のように反応する。 Examples of combinations of a substrate detectable by this enzyme sensor and an enzyme as a catalyst reacting with this substrate are as follows, in which both electrodes act as hydrogen peroxide electrodes. Detected substance enzyme glucose Glucose oxidase galactose Galactose oxidase ethanol Alcohol oxidase phospholipid Phospholipase choline Choline oxidase glycerin Glycerokinase (simultaneously immobilizes two enzymes L-α-glycero-3-ly) When used as a sensor, it works as follows: First, the glucose sensor is immersed in a buffer solution that does not contain glucose, and a voltage of 0.8 V is applied to the electrode surface, for example, in the case of a gold electrode. When glucose is added to this, the glucose diffuses into the glucose oxidase enzyme-immobilized membrane on the side of the hydrogen peroxide electrode on which the enzyme is immobilized, and the following reaction occurs due to the catalytic action of the immobilized enzyme.
グルコース＋O2酵素
――→
グルコノラクトン＋H2O2
この反応に伴つて発生する過酸化水素は、アノ
ード電極上で次のように酸化され、発生した過酸
化水素量に比例した電流、即ちグルコース濃度に
比例した電流が流れる。Glucose + O 2 Enzyme - → Gluconolactone + H 2 O 2The hydrogen peroxide generated during this reaction is oxidized on the anode electrode as follows, and a current proportional to the amount of hydrogen peroxide generated, that is, glucose A current proportional to the concentration flows.
H2O2→2H+＋O2＋2e-
このとき、参照側電極には酵素が固定化されて
いないため、酵素反応による電流は流れないの
で、酵素固定化側と参照側との差動出力を検出し
ても、酵素固定化側の電流のみを検出することが
できる。この場合に、試料液中にＬ−アスコルビ
ン酸などの還元性妨害物質が含まれていても、妨
害物質は固定化酵素の有無によらず、両方の組合
せ電極で等しく酸化されるので、このときにこれ
ら両電極を流れる電流の差動出力を検出すれば、
妨害物質に起因する電流値分が相殺され、酵素反
応に起因する電流値のみを検出することができ、
例えはＬ−アスコルビン酸の場合、その濃度が
0.5〜４mg／dlの範囲内では、両電極で発生する
妨害物質に起因する電流を３％以下に軽減できる
ことが確認された。 H 2 O 2 →2H + +O 2 +2e -At this time, since no enzyme is immobilized on the reference side electrode, no current due to the enzyme reaction flows, so the differential output between the enzyme immobilized side and the reference side is Even when detected, only the current on the enzyme-immobilized side can be detected. In this case, even if the sample solution contains a reducing interfering substance such as L-ascorbic acid, the interfering substance is oxidized equally at both combination electrodes regardless of the presence or absence of the immobilized enzyme. If we detect the differential output of the current flowing through these two electrodes, we get
The current value caused by interfering substances is canceled out, and only the current value caused by the enzyme reaction can be detected.
For example, in the case of L-ascorbic acid, its concentration is
It was confirmed that within the range of 0.5 to 4 mg/dl, the current caused by interfering substances generated at both electrodes could be reduced to 3% or less.
しかるに、酵素固定化膜と参照側となる光架橋
重合体膜とを光学顕微鏡で観察すると、光架橋重
合体膜が透明でかつ均質な膜状体を形成している
のに対し、酵素固定化膜の方は白色で、その表面
に微小な凹凸が無数に形成されていることが判明
した。 However, when observing the enzyme-immobilized membrane and the photo-crosslinked polymer membrane on the reference side using an optical microscope, it was found that the photo-cross-linked polymer membrane formed a transparent and homogeneous film-like body, whereas the enzyme-immobilized membrane formed a transparent and homogeneous membrane. It was found that the film was white and had countless microscopic irregularities formed on its surface.
そこで、これら両者の膜状体をなるべく同等な
ものとし、これによつて差動出力をより正確に検
出する手段を求めて更に検討した結果、参照側と
なる膜状体にも失活酵素を固定化せしめる方法が
きわめて有効であることを見出した。 Therefore, as a result of further investigation to find a means to more accurately detect differential output by making these two membrane bodies as similar as possible, we found that we also added an inactivated enzyme to the membrane body that would serve as the reference side. We have found that a method of immobilization is extremely effective.
従つて、本発明は酵素センサーに係り、この酵
素センサーは、同一絶縁基板上に金属薄膜よりな
るアノード電極およびカソード電極の組合せ電極
を２組形成させ、その１組には少くともアノード
電極上に固定化された酵素固定化膜を、他の１組
には少くともアノード電極上に固定化された失活
酵素固定化膜をそれぞれ設置してなる。
Therefore, the present invention relates to an enzyme sensor, in which two sets of combined electrodes of an anode electrode and a cathode electrode each made of a metal thin film are formed on the same insulating substrate, and one set has at least one electrode on the anode electrode. An immobilized enzyme-immobilized membrane is installed on one set, and an inactivated enzyme-immobilized membrane immobilized on at least an anode electrode is installed in the other set.
かかる酵素センサーの作製は、上記で図面の第
１〜２図を用いて説明した方法において、工程(d)
で用いられた光架橋性重合体含有酵素水溶液をオ
ーブン中で酵素の種類に応じて約80〜100℃で約
５〜10分間程度加熱し、酵素を失活させた水溶液
が工程(f)で用いられる以外は、同様の方法によつ
て行われる。そして、例えば第３〜５図に示され
る態様において、符号８側に酵素固定化膜が、ま
た符号９側に失活酵素固定化膜がそれぞれ固定化
された酵素センサーが得られる。 The production of such an enzyme sensor is carried out by step (d) in the method explained above using FIGS. 1 and 2 of the drawings.
The photocrosslinkable polymer-containing enzyme aqueous solution used in step (f) is heated in an oven at about 80 to 100°C for about 5 to 10 minutes depending on the type of enzyme, and the enzyme is deactivated. The same method is used except that For example, in the embodiments shown in FIGS. 3 to 5, an enzyme sensor is obtained in which an enzyme-immobilized membrane is immobilized on the 8 side and an inactivated enzyme-immobilized membrane is immobilized on the 9 side.
〔作用〕および〔発明の効果〕
本発明に係る酵素センサーは、参照側にも失活
酵素を固定化させることにより、参照側電極の膜
質を酵素側電極の膜質と等しくさせ、これによつ
て２組の組合せ電極間の妨害物質に対する応答特
性を等しくし、妨害物質信号の差動除去特性が一
層改善された。具体的には、Ｌ−アスコルビン酸
を用いた妨害物質信号の差動除去特性の測定で
は、その濃度が１〜100mg／dlの範囲内で、両電
極で発生する妨害物質に起因する電流を２％以下
に軽減することができ、この前の提案の0.5〜４
mg／dlの範囲内で３％以下という濃度範囲および
電流値と比較して、妨害物質信号の差動除去特性
のなお一層の改善が達成された。また、このよう
な特性は、妨害物質がＬ−アスコルビン酸の場合
だけではなく、尿酸などの場合にも同様に有効に
発揮される。[Function] and [Effects of the Invention] The enzyme sensor according to the present invention immobilizes an inactivated enzyme on the reference side as well, thereby making the membrane quality of the reference side electrode equal to that of the enzyme side electrode. The response characteristics to interfering substances between the two sets of combined electrodes are made equal, and the differential rejection characteristics of interfering substance signals are further improved. Specifically, in the measurement of the differential rejection characteristics of interfering substance signals using L-ascorbic acid, the current caused by the interfering substance generated at both electrodes is 2. It can be reduced to less than 0.5% to 4% of the previous proposal.
Compared to concentration ranges and current values of less than 3% in the mg/dl range, even further improvements in the differential rejection properties of the interfering substance signals were achieved. Further, such characteristics are effectively exhibited not only when the interfering substance is L-ascorbic acid, but also when the interfering substance is uric acid.
次に、実施例について本発明を説明する。 Next, the present invention will be explained with reference to examples.
実施例 １
光架橋性ポリビニルアルコール（光架橋性スチ
ルバゾリウム基含有量1.4モル％、けん化度88％、
重合度1400）の11.7重量％水溶液0.5ｇに、グル
コースオキシダーゼ酵素30mgを溶解させた蒸留水
0.4mlを添加し、数分間程度撹拌、混合してコー
テイング液を調製する。このコーテイング液を、
ガラス板上に形成させた２組の過酸化水素電極の
上に、4000rpm、20秒間の条件下でスピンコート
する。コーテイング液が自然乾燥したら、２組の
過酸化水素電極の内、一方の組のアノード電極部
分のみを紫外線照射できるようなネガの画像を有
するフオトマスクで覆い、紫外線照射（出力
250W）を15秒間行ない、その後純水による洗浄
によつて現像し、再び紫外線照射した。Example 1 Photocrosslinkable polyvinyl alcohol (photocrosslinkable stilbazolium group content 1.4 mol%, saponification degree 88%,
Distilled water with 30 mg of glucose oxidase enzyme dissolved in 0.5 g of an 11.7% by weight aqueous solution with a degree of polymerization of 1400).
Add 0.4 ml and stir and mix for several minutes to prepare the coating liquid. This coating liquid
Spin coating is performed on two sets of hydrogen peroxide electrodes formed on a glass plate at 4000 rpm for 20 seconds. After the coating solution dries naturally, cover it with a photomask with a negative image that allows only the anode electrode part of one set of the two sets of hydrogen peroxide electrodes to be irradiated with ultraviolet rays (output
250W) for 15 seconds, then development was performed by washing with pure water, and UV irradiation was performed again.
次に、上記酵素を溶解させた蒸留水をオーブン
中で95℃に６分間加熱した後、光架橋性ポリビニ
ルアルコールに添加してコーテング液とし、上記
と同様の手法により、酵素を固定化させていない
方の過酸化水素電極のアノード電極上に適用し、
そこに失活酵素固定化膜を被覆させた。 Next, the distilled water in which the enzyme was dissolved was heated to 95°C for 6 minutes in an oven, and then added to photocrosslinkable polyvinyl alcohol to form a coating solution, and the enzyme was immobilized using the same method as above. Apply on the anode electrode of the hydrogen peroxide electrode, whichever is not,
A deactivated enzyme-immobilized membrane was coated thereon.
このようにして製造された酵素センサーを用
い、還元性アスコルビン酸について、妨害物質信
号の差動除去特性をPH7.0、温度37℃で測定する
と、第６図に示される如く、○で示される点（Ｌ
−アスコルビン酸濃度と固定化酵素側電極を流れ
る電流値との関係を示している）と×で示される
点（Ｌ−アスコルビン酸濃度と参照固定化失活酵
素側電極を流れる電流値との関係を示している）
との差を表わす△で示される点（Ｌ−アスコルビ
ン酸濃度とこれら両電極を流れる電流値の差動出
力値との関係を示している）の結果から、妨害物
質濃度１〜100mg／dlの範囲内では、両電極で発
生する妨害物質に起因する電流を２％以下に軽減
できることが確認された。 Using the enzyme sensor manufactured in this way, the differential removal characteristics of interfering substance signals for reduced ascorbic acid were measured at pH 7.0 and temperature 37°C, and as shown in Figure 6, the results were indicated by ○. Point (L
- shows the relationship between the ascorbic acid concentration and the current value flowing through the immobilized enzyme side electrode) and the point indicated by x (the relationship between the L-ascorbic acid concentration and the current value flowing through the reference immobilized inactivated enzyme side electrode) )
From the results of the point indicated by △ representing the difference between the It was confirmed that within this range, the current caused by interfering substances generated at both electrodes could be reduced to 2% or less.
実施例 ２
実施例１において、妨害物質として尿酸を用
い、妨害物質信号の差動除去特性をPH7.75、温度
37℃で測定すると、第７図に示される如く、○、
×および△（第６図の場合と同様の関係を示して
いる）の結果から、妨害物質濃度１〜100mg／dl
の範囲内では、両電極で発生する妨害物質に起因
する電流を３％以下に軽減できることが確認され
た。Example 2 In Example 1, uric acid was used as the interfering substance, and the differential removal characteristics of the interfering substance signal were determined at pH 7.75 and temperature.
When measured at 37℃, as shown in Figure 7, ○,
From the results of × and △ (showing the same relationship as in Figure 6), the interfering substance concentration is 1 to 100 mg/dl.
It was confirmed that within this range, the current caused by interfering substances generated at both electrodes could be reduced to 3% or less.
第１図は、絶縁基板上に電極を形成させる工程
を順次示した断面図である。第２図は、絶縁基板
上に形成させた２個のアノード電極の上に、それ
ぞれ光架橋重合体で固定化された酵素固定化膜お
よび光架橋重合体膜（または失活酵素固定化膜）
を設置させる工程を順次示した断面図であり、第
３図はこのようにして作製された酵素センサーの
一態様の平面図である。第４〜５図は、本発明に
係る酵素センサーの他の態様の平面図である。ま
た、第６〜７図は、それぞれ妨害物質としてＬ−
アスコルビン酸または尿酸を存在させたときの差
動出力特性を示すグラフである。
（符号の説明）、１……絶縁基板、２……基板
露出面、５……電極、６……光架橋性重合体含有
酵素水溶液、７……画像を有するフオトマスク、
８……酵素固定化膜、９……光架橋重合体膜（ま
たは失活酵素固定化膜）、１１……酵素固定化膜
側アノード電極、１２……参照側アノード電極、
１３……カソード電極。
FIG. 1 is a cross-sectional view sequentially showing the steps of forming an electrode on an insulating substrate. Figure 2 shows an enzyme-immobilized film and a photo-crosslinked polymer film (or inactivated enzyme-immobilized film) each immobilized with a photocrosslinked polymer on two anode electrodes formed on an insulating substrate.
FIG. 3 is a cross-sectional view sequentially illustrating the steps of installing the enzyme sensor, and FIG. 3 is a plan view of one embodiment of the enzyme sensor produced in this manner. 4 and 5 are plan views of other embodiments of the enzyme sensor according to the present invention. In addition, FIGS. 6 and 7 show L- as an interfering substance, respectively.
It is a graph showing differential output characteristics when ascorbic acid or uric acid is present. (Explanation of symbols), 1... Insulating substrate, 2... Substrate exposed surface, 5... Electrode, 6... Enzyme aqueous solution containing a photocrosslinkable polymer, 7... Photomask having an image,
8... Enzyme-immobilized membrane, 9... Photo-crosslinked polymer membrane (or inactivated enzyme-immobilized membrane), 11... Enzyme-immobilized membrane side anode electrode, 12... Reference side anode electrode,
13...Cathode electrode.
Claims (1)
電極およびカソード電極の組合せ電極を２組形成
させ、その１組には少くともアノード電極上に固
定化された酵素固定化膜を、他の１組には少くと
もアノード電極上に固定化された失活酵素固定化
膜をそれぞれ設置してなる酵素センサー。 ２ 失活酵素として熱処理された酵素が用いられ
た特許請求の範囲第１項記載の酵素センサー。 ３ 固定化が光架橋重合体によつて行われた特許
請求の範囲第１項記載の酵素センサー。 ４ カソード電極１個が２組の組合せ電極に共通
して用いられている特許請求の範囲第１項記載の
酵素センサー。 ５ 組合せ電極が過酸化水素電極を構成している
特許請求の範囲第１項記載の酵素センサー。[Claims] 1. Two sets of combined electrodes consisting of an anode electrode and a cathode electrode made of metal thin films are formed on the same insulating substrate, and one set has at least an enzyme-immobilized film immobilized on the anode electrode. , and the other set includes at least an inactivated enzyme-immobilized membrane immobilized on an anode electrode. 2. The enzyme sensor according to claim 1, wherein a heat-treated enzyme is used as the inactivated enzyme. 3. The enzyme sensor according to claim 1, wherein the immobilization is performed by a photocrosslinked polymer. 4. The enzyme sensor according to claim 1, wherein one cathode electrode is commonly used for two sets of combined electrodes. 5. The enzyme sensor according to claim 1, wherein the combined electrode constitutes a hydrogen peroxide electrode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60217149A JPS6275346A (en) | 1985-09-30 | 1985-09-30 | Enzyme sensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60217149A JPS6275346A (en) | 1985-09-30 | 1985-09-30 | Enzyme sensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6275346A JPS6275346A (en) | 1987-04-07 |
| JPH0566983B2 true JPH0566983B2 (en) | 1993-09-22 |
Family
ID=16699623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60217149A Granted JPS6275346A (en) | 1985-09-30 | 1985-09-30 | Enzyme sensor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6275346A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0623719B2 (en) * | 1986-04-08 | 1994-03-30 | エヌオーケー株式会社 | Method for manufacturing enzyme sensor |
| AT402452B (en) * | 1994-09-14 | 1997-05-26 | Avl Verbrennungskraft Messtech | PLANAR SENSOR FOR DETECTING A CHEMICAL PARAMETER OF A SAMPLE |
| IL145182A (en) | 2001-08-29 | 2005-11-20 | Yissum Res Dev Co | Self-powered biosensor |
| JP5702457B2 (en) * | 2013-12-06 | 2015-04-15 | パナソニックヘルスケアホールディングス株式会社 | Biosensor system, sensor chip and method for measuring analyte concentration in blood sample |
| CN111398386A (en) * | 2020-05-12 | 2020-07-10 | 山东省科学院生物研究所 | Immobilized enzyme electrode, immobilized enzyme sensor and enzyme membrane anti-interference detection method thereof |
-
1985
- 1985-09-30 JP JP60217149A patent/JPS6275346A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6275346A (en) | 1987-04-07 |
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