JPH04122849A - Enzyme electrode - Google Patents
Enzyme electrodeInfo
- Publication number
- JPH04122849A JPH04122849A JP2244840A JP24484090A JPH04122849A JP H04122849 A JPH04122849 A JP H04122849A JP 2244840 A JP2244840 A JP 2244840A JP 24484090 A JP24484090 A JP 24484090A JP H04122849 A JPH04122849 A JP H04122849A
- Authority
- JP
- Japan
- Prior art keywords
- conductive
- enzyme
- substrate
- film
- electrode
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 62
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 62
- 239000000758 substrate Substances 0.000 claims abstract description 40
- 239000013078 crystal Substances 0.000 claims abstract description 22
- 229920006254 polymer film Polymers 0.000 claims abstract description 21
- 238000012546 transfer Methods 0.000 claims abstract description 16
- 238000000576 coating method Methods 0.000 claims description 9
- 239000011248 coating agent Substances 0.000 claims description 8
- 238000005259 measurement Methods 0.000 abstract description 9
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 abstract description 8
- 229910052802 copper Inorganic materials 0.000 abstract description 8
- 239000010949 copper Substances 0.000 abstract description 8
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 abstract description 8
- 239000004020 conductor Substances 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 230000003100 immobilizing effect Effects 0.000 abstract description 4
- 229910052697 platinum Inorganic materials 0.000 abstract description 4
- 229910052709 silver Inorganic materials 0.000 abstract description 4
- 239000004332 silver Substances 0.000 abstract description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 abstract description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052737 gold Inorganic materials 0.000 abstract description 2
- 239000010931 gold Substances 0.000 abstract description 2
- 229910052751 metal Inorganic materials 0.000 abstract description 2
- 239000002184 metal Substances 0.000 abstract description 2
- 239000000843 powder Substances 0.000 abstract description 2
- 238000007740 vapor deposition Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 54
- 238000000034 method Methods 0.000 description 21
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 18
- 229920000642 polymer Polymers 0.000 description 16
- 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 14
- 239000008103 glucose Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 13
- 239000001301 oxygen Substances 0.000 description 13
- 229910052760 oxygen Inorganic materials 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- 230000027756 respiratory electron transport chain Effects 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- 239000012528 membrane Substances 0.000 description 9
- 229920001940 conductive polymer Polymers 0.000 description 7
- 230000004043 responsiveness Effects 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- 239000007772 electrode material Substances 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 230000002452 interceptive effect Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010668 complexation reaction Methods 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 3
- 108010015776 Glucose oxidase Proteins 0.000 description 3
- 239000004366 Glucose oxidase Substances 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 229910021607 Silver chloride Inorganic materials 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- -1 ferricyanide ions Chemical class 0.000 description 3
- 238000005755 formation reaction Methods 0.000 description 3
- 229940116332 glucose oxidase Drugs 0.000 description 3
- 235000019420 glucose oxidase Nutrition 0.000 description 3
- 239000013081 microcrystal Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 229920002037 poly(vinyl butyral) polymer Polymers 0.000 description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000012047 saturated solution Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- LJCNDNBULVLKSG-UHFFFAOYSA-N 2-aminoacetic acid;butane Chemical compound CCCC.CCCC.NCC(O)=O LJCNDNBULVLKSG-UHFFFAOYSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108010015133 Galactose oxidase Proteins 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004952 Polyamide Substances 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
- 239000013543 active substance Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010292 electrical insulation Methods 0.000 description 1
- 239000012799 electrically-conductive coating Substances 0.000 description 1
- 238000003411 electrode reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- GNOIPBMMFNIUFM-UHFFFAOYSA-N hexamethylphosphoric triamide Chemical compound CN(C)P(=O)(N(C)C)N(C)C GNOIPBMMFNIUFM-UHFFFAOYSA-N 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- FRASJONUBLZVQX-UHFFFAOYSA-N naphthoquinone group Chemical group C1(C=CC(C2=CC=CC=C12)=O)=O FRASJONUBLZVQX-UHFFFAOYSA-N 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229950009506 penicillinase Drugs 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000003880 polar aprotic solvent Substances 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000767 polyaniline Polymers 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920006149 polyester-amide block copolymer Polymers 0.000 description 1
- 229920000128 polypyrrole Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 150000003518 tetracenes Chemical class 0.000 description 1
- FHCPAXDKURNIOZ-UHFFFAOYSA-N tetrathiafulvalene Chemical compound S1C=CSC1=C1SC=CS1 FHCPAXDKURNIOZ-UHFFFAOYSA-N 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、酵素電極に関し、特に、血液、尿等の体液成
分中に含有する微量の生体基質濃度を測定する酵素セン
サーとして適したものである。Detailed Description of the Invention (Field of Industrial Application) The present invention relates to an enzyme electrode, and is particularly suitable as an enzyme sensor for measuring the concentration of trace amounts of biological substrates contained in body fluid components such as blood and urine. be.
（従来の技術）
酵素の優れた基質特異性を利用した分析法が、臨床分析
化学、食品製造、環境化学等の分野で注目されている。(Prior Art) Analytical methods that utilize the excellent substrate specificity of enzymes are attracting attention in fields such as clinical analytical chemistry, food manufacturing, and environmental chemistry.
とりわけ、臨床分析化学の分野では、従来から、グルコ
ース、尿素、尿酸などを選択的に検出しうる酵素電極が
知られている。In particular, in the field of clinical analytical chemistry, enzyme electrodes that can selectively detect glucose, urea, uric acid, etc. have been known.
これら酵素電極は、一般には、電極と酵素固定膜とから
構成され、酵素反応による物質変化を電極により電気信
号の変化量として読み取ることにより、その酵素が特異
的に作用する基質の濃度を測定するものである。具体的
には、下記式に従い、生成または消費される過酸化水素
、酸素等の電極活性な物質を電極でモニターすることに
より、生体基質濃度を測定しようとするものがある。These enzyme electrodes generally consist of an electrode and an enzyme-immobilized membrane, and measure the concentration of the substrate on which the enzyme specifically acts by reading the substance change caused by the enzyme reaction as a change in electrical signal using the electrode. It is something. Specifically, some attempt to measure the biological substrate concentration by monitoring electrode-active substances such as hydrogen peroxide and oxygen that are produced or consumed using electrodes according to the following formula.
グルコースの定量を例にとると、酵素としてはグルコー
スオキシダーゼが一般に用いられ、生成した過酸化水素
を電気的にモニターすることで、グルコース濃度を測定
する。ここで過酸化水素をモニターする方法としては、
白金電極、炭素電極等を用い、一定電圧下で酸化するこ
とにより得られる酸化電流を計測することにより行われ
る。Taking the determination of glucose as an example, glucose oxidase is generally used as the enzyme, and the glucose concentration is measured by electrically monitoring the hydrogen peroxide produced. Here, the method for monitoring hydrogen peroxide is as follows:
This is carried out by measuring the oxidation current obtained by oxidizing under a constant voltage using a platinum electrode, a carbon electrode, or the like.
ところが、このような原理に基づく酵素電極は、次のよ
うな問題点を含んでいる。However, the enzyme electrode based on this principle has the following problems.
■上記式で明らかなように、基質が反応するためには、
化学量論的な酸素を必要とする。ところが、例えばグル
コースセンサーを例にとると、糖尿病患者の血中グルコ
ース濃度は、１５ｍＭ以上、健常者でも約７ｍＭ程度で
あるのに対し、溶存酸素量は、水の場合でも１ｍＭ、体
液ではそれ以下である。従って、糖尿病患者の血中グル
コース濃度を測定する場合、測定濃度範囲での直線性は
不可能である。■As is clear from the above formula, in order for the substrate to react,
Requires stoichiometric oxygen. However, taking a glucose sensor as an example, the blood glucose concentration of a diabetic patient is over 15mM, and even for a healthy person it is about 7mM, whereas the amount of dissolved oxygen is 1mM even in water and less than that in body fluids. It is. Therefore, when measuring the blood glucose concentration of diabetic patients, linearity is not possible in the measured concentration range.
そのため、試料血液を希釈したり、何らかの方法で酸素
を補給するといった手段が構じられている。For this reason, measures have been taken to dilute the sample blood or supplement oxygen by some method.
■過酸化水素を電気的にモニターする場合、試料溶液中
に過酸化水素と同様の電位で酸化される物質、例えばア
スコルビン酸のような還元性物質が存在すると、測定′
ｇｉ流にこれら妨害物質の酸化電流が上乗せされ、測定
誤差を生じる。そこで、これら誤差を取り除くため、酵
素を固定していない電極を補償極として補正したり、酸
素、過酸化水素分子と、測定基質は透過させるが、アス
コルビン酸の如く電極活性な緩衝物質を透過させないと
いった選択透過膜を装着する必要があった。■When monitoring hydrogen peroxide electrically, if there is a substance in the sample solution that is oxidized at the same potential as hydrogen peroxide, such as a reducing substance such as ascorbic acid, the measurement will fail.
The oxidation current of these interfering substances is added to the gi current, causing measurement errors. Therefore, in order to eliminate these errors, we correct the problem by using an electrode that does not have an immobilized enzyme as a compensation electrode, or allow oxygen, hydrogen peroxide molecules, and the measurement substrate to pass through, but do not allow electrode-active buffer substances such as ascorbic acid to pass through. It was necessary to install a selectively permeable membrane.
一方、これらの問題点を解決するため、導電性高分子を
利用した酵素電極、電子メデイエータ−を利用した酵素
電極が捉案されている。前者は、ポリピロール、ポリア
ニリン等の導電性高分子の電解重合時に、酵素をモノマ
ー溶液中に共存させ、重合時に重合膜中に酵素を捕捉す
るか、あるいはあらかじめ重合した導電性高分子膜上に
公知方法により酵素固定膜を設けることにより、導電性
の酵素固定膜を得るものである。この酵素電極では、例
えばグルコースを例にとると、
酵素
グルコース□グｌレコノラクトン＋２８”＋２ｅの反応
により生ずる電子を、導電性高分子のπ電子共役系を介
して移動させることにより、酵素の電子移動を行う。こ
の方式では、溶存酸素に影響されず、グルコース濃度を
測定することができる。On the other hand, in order to solve these problems, enzyme electrodes using conductive polymers and enzyme electrodes using electronic mediators have been proposed. The former method consists of allowing the enzyme to coexist in the monomer solution during electrolytic polymerization of conductive polymers such as polypyrrole and polyaniline, and capturing the enzyme in a polymer film during polymerization, or by placing the enzyme on a previously polymerized conductive polymer film. By providing an enzyme-immobilized membrane using this method, an electrically conductive enzyme-immobilized membrane is obtained. Taking glucose as an example, this enzyme electrode transfers electrons generated by the reaction of the enzyme glucose□glreconolactone+28''+2e via the π-electron conjugated system of the conductive polymer, thereby facilitating the electron transfer of the enzyme. With this method, glucose concentration can be measured without being affected by dissolved oxygen.
電子メデイエータ−を利用した酵素電極は、カーボンペ
ースト等の中にフェロセン類、ハンゾキノン、フェリシ
アン化イオン、Ｎ−メチルツェナジニウム等の電子メデ
イエータ−を封し込め、カーボンペースト電極表面に酵
素を固定化し、適当な高分子膜で被覆したものである。Enzyme electrodes using electron mediators encapsulate electron mediators such as ferrocenes, hanzoquinone, ferricyanide ions, N-methylzenadinium, etc. in carbon paste, and fix the enzyme on the surface of the carbon paste electrode. and coated with a suitable polymer film.
これは、グルコースセンサーでは次のような機構で濃度
測定を行うものである。即ち、グルコースが酸化される
と酵素が還元型となり、電子受容体であるメデイエータ
−へ電子移動することにより、酵素が酸化型に戻る。こ
の還元型メデイエータ−が電極反応により酸化型に戻る
際の電流により、グルコ−人濃度を測定するものである
。The glucose sensor measures concentration using the following mechanism. That is, when glucose is oxidized, the enzyme becomes a reduced form, and by electron transfer to a mediator, which is an electron acceptor, the enzyme returns to an oxidized form. The glucoconcentration is measured by the current generated when this reduced mediator returns to its oxidized form through an electrode reaction.
従って、溶存酸素量の影響を受けないという利点の他、
この時の電極電位は前出の過酸化水素をモニターする時
の電位（Ａｇ／ＡｇＣ１対比約０．７Ｖ） ニ比べ著し
く小さイ（Ａｇ／ＡｇＣｌ対比約０．１〜０．２Ｖ）た
め、妨害物質の酸化も起こりにくく、その結果、高精度
に測定できるという利点がある。Therefore, in addition to the advantage of not being affected by the amount of dissolved oxygen,
The electrode potential at this time is significantly lower than the potential used to monitor hydrogen peroxide (approx. 0.7 V compared to Ag/AgCl) (approximately 0.1 to 0.2 V compared to Ag/AgCl), so there is no interference. It has the advantage that oxidation of substances is less likely to occur, and as a result, measurement can be performed with high precision.
（発明が解決しようとする諜Ｂ）
上記の如く、酵素反応に伴い生成、あるいは消費される
物質の濃度を測定する原理に基づくセンサーは、溶存酸
素の影響、及び妨害物質の影響といった問題を有してい
る。また、酵素固定膜を酸素、過酸化水素電極に装着す
るという形態を必要とするため、微小化にも限界がある
。(Intelligence B to be solved by the invention) As mentioned above, sensors based on the principle of measuring the concentration of substances produced or consumed in enzymatic reactions have problems such as the influence of dissolved oxygen and the influence of interfering substances. are doing. Furthermore, since it is necessary to attach the enzyme-immobilized membrane to the oxygen and hydrogen peroxide electrodes, there is a limit to miniaturization.
一方、導電性高分子を利用して、酵素反応に伴う電子移
動を直接検知する酵素電極は、これらの問題を解決でき
る可能性はあるが、応答性が低く、応答時間が長い等の
問題がある。さらに、電解重合時に重合膜中に酵素を捕
捉するといった手法を取る場合は、固定化される酵素量
を制御することは難しく、また酵素電極として利用する
際、酵素の脱離による経時的な基質応答性の低下は避け
ることができない。On the other hand, enzyme electrodes that use conductive polymers to directly detect electron transfer associated with enzymatic reactions may be able to solve these problems, but they also have problems such as low responsiveness and long response times. be. Furthermore, when using methods such as capturing enzymes in a polymeric membrane during electropolymerization, it is difficult to control the amount of immobilized enzyme, and when used as an enzyme electrode, the substrate Decrease in responsiveness is unavoidable.
また、従来の電子メデイエータ−を利用した酵素電極で
は電導度が低く応答性、応答時間の点で不十分である他
、電子メデイエータ−をカーボンペースト中に分散させ
た形態をとるため、電子メデイエータ−の溶出、脱離に
伴う経時的な応答性の低下といった問題を含んでいる。Furthermore, enzyme electrodes using conventional electronic mediators have low conductivity and are insufficient in terms of response and response time. This includes problems such as a decrease in responsiveness over time due to elution and desorption.
本発明は、以上のことに鑑みて、溶存酸素の影響を受け
ず、また妨害物質の影響の少ない酵素電極とするために
、酵素反応に伴う電子移動を直接検知する方式をとり、
なおかつ、経時安定性に優れ、長期にわたり、高精度な
応答を与える酵素電極を提供することを目的とする。In view of the above, the present invention adopts a method of directly detecting electron transfer accompanying enzyme reactions in order to create an enzyme electrode that is not affected by dissolved oxygen and is less affected by interfering substances.
Furthermore, it is an object of the present invention to provide an enzyme electrode that has excellent stability over time and provides a highly accurate response over a long period of time.
（課題を解決するための手段）
本発明者らは、有機電荷移動錯体を含有する導電性材料
が、電気伝導性に優れ、安定な電極材料として作用する
ことを見い出し、この新規な電極材料に生体触媒を固定
化することにより、酵素反応に伴う電子移動を直接検知
する酵素電極を提供しうるとの着想に基づいて、本発明
の酵素電極を完成させた。(Means for Solving the Problems) The present inventors have discovered that a conductive material containing an organic charge transfer complex has excellent electrical conductivity and acts as a stable electrode material, and has developed this novel electrode material. The enzyme electrode of the present invention was completed based on the idea that by immobilizing a biocatalyst, it is possible to provide an enzyme electrode that directly detects electron transfer accompanying an enzyme reaction.
本発明は、導電性基体と、この基体表面に設けた有機電
荷移動錯体結晶を含有するｓｔ性被被膜を有し、この導
電性被膜中および／またはその表面上に酵素が固定化さ
れていることを特徴とする酵素電極、を要旨とする。The present invention comprises an electrically conductive substrate and a st-type coating containing an organic charge transfer complex crystal provided on the surface of the substrate, and an enzyme is immobilized in this electrically conductive coating and/or on the surface thereof. The subject matter is an enzyme electrode characterized by the following.
（作用）
本発明の酵素電極において使用する電極は、導電性基体
と、この基体表面に設けた有機電荷移動錯体結晶を含有
する導電性被膜とから構成される。(Function) The electrode used in the enzyme electrode of the present invention is composed of a conductive substrate and a conductive film containing organic charge transfer complex crystals provided on the surface of the substrate.
ｓｉｉ性基体としては、銅、銀、白金、金等の金属やカ
ーボン電極の他、これらの導電性材料からなる導電層を
蟇着等の手段により表面に設けた基体、あるいはこれら
の導電性材料の粉末を含有するペーストから作成した基
体等が使用できる。SII substrates include metals such as copper, silver, platinum, and gold, carbon electrodes, substrates with conductive layers made of these conductive materials provided on their surfaces by means such as deposition, or these conductive materials. Substrates made from pastes containing powders can be used.
有機電荷移動錯体結晶を含有する導電性被膜は、絶縁性
高分子フィルム内に、有ｍ電荷移動錯体結晶を、このフ
ィルムが導電性となるように含有させたものである。例
えば、絶縁性高分子フィルムの厚さ方向にこのフィルム
を貫通するように有機電荷移動錯体結晶を成長させた被
膜が導電性を示し、現時点では好ましい。A conductive film containing an organic charge transfer complex crystal is one in which an organic charge transfer complex crystal is contained in an insulating polymer film so that the film becomes electrically conductive. For example, a film in which organic charge transfer complex crystals are grown to penetrate an insulating polymer film in the thickness direction exhibits conductivity and is currently preferred.
ここで、有機電荷移動錯体（以下、有機ＣＴ錯体と称す
る）とは、有機電子受容体と電子供与体とから、両者の
間の電荷移動反応に伴い形成される化合物である。Here, an organic charge transfer complex (hereinafter referred to as an organic CT complex) is a compound formed from an organic electron acceptor and an electron donor due to a charge transfer reaction between the two.
この有機ＣＴ錯体の形成に用いる有機電子受容体として
は、特に制限されないが、シアノメチレン官能基を有す
る化合物が好ましく、中でもジシアノメチレン官能基と
、キノンあるいはナフトキノン骨格とを有する化合物が
好適である。このうちでも特に、７，７，８．８−テト
ラシアノキノジメタン（ＴＣＮＱ）はＣＴ錯錯体形成炉
強く、得られる有機ＣＴ錯体の電気伝導度が高いため応
答時間、応答性で有利である。また工業的にも比較的入
手が容易であることから好適である。The organic electron acceptor used to form this organic CT complex is not particularly limited, but a compound having a cyanomethylene functional group is preferable, and a compound having a dicyanomethylene functional group and a quinone or naphthoquinone skeleton is especially preferable. Among these, 7,7,8.8-tetracyanoquinodimethane (TCNQ) is particularly strong in the CT complex formation process, and the resulting organic CT complex has high electrical conductivity, so it is advantageous in terms of response time and responsiveness. . It is also suitable industrially because it is relatively easy to obtain.
有＠ＣＴ錯体の形成に用いる電子供与体としては、使用
する有機電子受容体と、導電性を有するＣＴ錯体を形成
しうるものであれば、特に制限されるものではなく、有
機、無機のいずれでもさしつかえない、具体的には、無
機材料としては銅、銀、コバルト、ニッケル、鉄、マン
ガンなど、また有機材料としては、テトラチアフルバレ
ン、テトラセレノフルバレン等のテトラセン類、及びそ
の誘導体、あるいは２，２゛−ビスピリジニウム、Ｎ−
メチルモノリニウム等、公知の電子供与体を使用するこ
とができる。The electron donor used to form the CT complex is not particularly limited as long as it can form a conductive CT complex with the organic electron acceptor used, and may be either organic or inorganic. Specifically, inorganic materials include copper, silver, cobalt, nickel, iron, manganese, etc., and organic materials include tetracenes such as tetrathiafulvalene and tetraselenofulvalene, and their derivatives. 2,2′-bispyridinium, N-
Known electron donors such as methyl monolinium can be used.
有機ＣＴＴ体結晶を含有させる絶縁性高分子としては、
有機電子受容体と電子供与体とのＣＴＴ体化反応を妨げ
ずに結晶を成長させることができ、適度な電気絶縁性を
有し、かつＣＴ錯体形成時に使用する有機電子受容体溶
液の溶剤に対して著しい溶解、膨潤等を起こすことのな
い、フィルム形成性のポリマーが望ましい。このような
ポリマーとしては、ポリビニルブチラール、ポリエステ
ル、ポリアミド、ポリエステルアミド等の熱可塑性ポリ
マーが例示され、これらの１種または２種以上を使用で
きる。As an insulating polymer containing organic CTT crystals,
It can grow crystals without interfering with the CTT formation reaction between the organic electron acceptor and the electron donor, has appropriate electrical insulation properties, and can be used as a solvent for the organic electron acceptor solution used when forming the CT complex. It is desirable to use a film-forming polymer that does not cause significant dissolution or swelling. Examples of such polymers include thermoplastic polymers such as polyvinyl butyral, polyester, polyamide, and polyesteramide, and one or more of these can be used.
上記絶縁性高分子としては、後述の酵素固定化方法に応
じ、また基質の拡散性等を考慮して適宜ポリマーを選択
でき、例えば、ポリビニルブチラールは、水不溶性であ
りながら親水性、吸水性を有し、しかも非常にミクロな
ボアを有するため、酵素の固定化に有利である。As the above-mentioned insulating polymer, an appropriate polymer can be selected depending on the enzyme immobilization method described below and considering the diffusibility of the substrate. For example, polyvinyl butyral is water-insoluble but has hydrophilic and water-absorbing properties. Moreover, since it has extremely microscopic pores, it is advantageous for immobilizing enzymes.
有機ＣＴｔｉＦ体結晶を上記絶縁性高分子フィルム内で
フィルム厚さ方向に成長させる方法としては、例えば以
下のような方法が可能である。As a method for growing organic CTtiF crystals in the film thickness direction within the above-mentioned insulating polymer film, the following method is possible, for example.
まず基体表面に電子供与体層を設けるか、あるいは電子
供与体としても機能する銅板等の基体を使用し、この電
子供与体上にポリマー被膜を形成させる。この場合の基
体は、後述するように導電性である必要はない。被膜の
形成は、一般の塗布方法によって行うことができ、例え
ば、ポリマーを適当な溶剤に溶解したポリマー溶液、あ
るいは溶融ポリマーをそのまま、ロールコータ−、ナイ
フコーター等により塗布することができる。First, an electron donor layer is provided on the surface of a substrate, or a substrate such as a copper plate that also functions as an electron donor is used, and a polymer film is formed on the electron donor. The substrate in this case does not need to be electrically conductive, as will be described later. The coating can be formed by a general coating method. For example, a polymer solution prepared by dissolving a polymer in a suitable solvent, or a molten polymer can be coated as it is using a roll coater, a knife coater, or the like.
電子供与体と有機電子受容体とのＣＴ錯体化反応時にお
ける反応速度、および得られる有１ＩＣＴ錯体微結晶を
含有するフィルムの電気伝導度を考慮すると、ポリマー
被膜の膜厚は通常０．０１〜５０μｍの範囲内であり、
好ましくは０．１〜１０μ−である。Considering the reaction rate during the CT complexation reaction between the electron donor and the organic electron acceptor, and the electrical conductivity of the resulting film containing the ICT complex microcrystals, the thickness of the polymer film is usually 0.01 to 1. within the range of 50 μm,
Preferably it is 0.1 to 10μ.
ポリマー被膜の膜厚が厚すぎると、続いて行うＣＴ錯体
化反応時において、有機電子受容体のポリマー膜中への
浸透速度が低下し、有機ＣＴ錯体結晶がポリマーフィル
ムを貫通するまで成長するのに長時間を要することにな
る。また、ポリマー被膜の膜厚が薄すぎると、ピンホー
ルの発生等を惹起することになり、その結果、電極材料
として使用する際、基体材料の溶出といった問題を生ず
ることがある。If the polymer film is too thick, the rate of penetration of the organic electron acceptor into the polymer film during the subsequent CT complexation reaction will decrease, and the organic CT complex crystals will grow until they penetrate the polymer film. It will take a long time. Furthermore, if the thickness of the polymer film is too thin, pinholes may occur, and as a result, when used as an electrode material, problems such as elution of the base material may occur.
ポリマー被膜の膜厚は、使用するポリマー溶液の量やポ
リマー濃度を変化させることにより、任意の厚さとする
ことができる。The thickness of the polymer film can be adjusted to any desired thickness by changing the amount of polymer solution used and the polymer concentration.
このようにして表面に電子供与体層を有する基体表面を
ポリマーで被覆した後、必要に応じて全体を乾燥する。After the surface of the substrate having the electron donor layer on the surface is coated with the polymer in this manner, the entire surface is dried if necessary.
次いで、このポリマー被膜表面の一部、ないし全部を有
機電子受容体を含有する溶液と接触させる。これにより
、溶液中の有機電子受容体は、ポリマー被膜の中に拡散
、浸透し、基体の表面を構成する電子供与体との間でｃ
ｔｔＷ体化反応化反応し、有＠ＣＴ錯体結晶がポリマー
フィルムの基体側から徐々に成長し、やがてポリマーフ
ィルムを貫通し、目的とする有機ＣＴｔｆ体含有フィル
ムが得られる。A portion or all of the surface of the polymer coating is then brought into contact with a solution containing an organic electron acceptor. As a result, the organic electron acceptor in the solution diffuses and permeates into the polymer film, and forms a c
The ttW formation reaction occurs, and @CT complex crystals gradually grow from the substrate side of the polymer film, eventually penetrating the polymer film, and the desired organic CTtf body-containing film is obtained.
有機電子受容体含有溶液の調製に使用する溶媒としては
、極性のある非プロトン溶削、例えばアセトニトリル、
ジオキサン、Ｎ、Ｎ−ジメチルホルムアミド、ジメチル
スルホキシド、ヘキサメチルホスホルアミド、メチルエ
チルケトン等が好適である。Solvents used to prepare organic electron acceptor-containing solutions include polar aprotic solvents such as acetonitrile,
Dioxane, N,N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide, methyl ethyl ketone, etc. are suitable.
この溶液における有機電子受容体の濃度は、溶剤１００
重量部に対して通常０．０１重量部〜飽和濃度、好まし
くは０．１重量部〜飽和濃度が適当である。The concentration of organic electron acceptor in this solution is 100%
The appropriate concentration is usually 0.01 part by weight to saturated concentration, preferably 0.1 part by weight to saturated concentration.
有機ＣＴ錯体の形成は、通常、１０〜３０°Ｃの温度で
行うが、用いる有機電子受容体と基体表面の電子供与体
の組み合わせによっては、ＣＴ錯体化反応が急激に進み
、緻密で均一な目的フィルムが得にくい場合がある。そ
のような場合は、必要に応して溶液、基体、雰囲気温度
を下げたり、溶液の濃度を低くすればよい、また逆に、
錯体化反応が遅く、有＠ＣＴ錯体結晶がフィルムを貫通
するまで成長するのに長時間を要する場合は、必要に応
じて、加熱することができる。The formation of an organic CT complex is usually carried out at a temperature of 10 to 30°C, but depending on the combination of the organic electron acceptor used and the electron donor on the substrate surface, the CT complexation reaction proceeds rapidly and forms a dense and uniform structure. It may be difficult to obtain the desired film. In such a case, the temperature of the solution, substrate, and atmosphere may be lowered, or the concentration of the solution may be lowered, as necessary.
If the complexation reaction is slow and it takes a long time for the @CT complex crystals to grow until they penetrate the film, heating can be performed as necessary.
有機電子受容体含有溶液の接触時間は、用いる有機電子
受容体と電子供与体との組み合わせや目的とするポリマ
ー被膜の膜厚に大きく依存するが、一般に１０秒から１
時間程度である。The contact time of the organic electron acceptor-containing solution depends largely on the combination of the organic electron acceptor and electron donor used and the thickness of the desired polymer film, but is generally between 10 seconds and 1 hour.
It takes about an hour.
このようにして、基体上に、絶縁性高分子被膜層内にそ
の厚さ方向に貫通するように有１１ＣＴ錯体結晶を成長
させた導電性被膜が形成される。導電性被膜は、必要に
応じ洗浄、乾燥する。In this way, a conductive film is formed on the substrate in which the 11 CT complex crystal is grown so as to penetrate through the insulating polymer film layer in the thickness direction. The conductive film is cleaned and dried as necessary.
使用した基体が導電性の場合は、上記導電性被膜が形成
された基体を、そのまま使用しても、この導電性被膜を
フィルムとして基体から剥離した後、他の導電性基体に
装着して電極材料とすることも可能である。使用した基
体がｉｔ性でない場合は、被膜を剥離して導電性基体に
装着して電極材料とする。If the substrate used is conductive, the substrate on which the conductive film is formed can be used as is, or the conductive film can be peeled off from the substrate as a film and then attached to another conductive substrate to form an electrode. It is also possible to use it as a material. If the substrate used is not IT-resistant, the coating is peeled off and attached to a conductive substrate to provide an electrode material.
上記のようにして得た電極の、導電性被膜中あるいはそ
の表面上に酵素を固定して、本発明の酵素電極を得る。The enzyme electrode of the present invention is obtained by immobilizing an enzyme in the conductive coating or on the surface of the electrode obtained as described above.
本発明の酵素電極において、導電性被膜中または表面に
固定する酵素としては、対象とする物質や目的とする化
学反応に応じ、酵素の基質特異性及び反応特異性を考慮
して適宜選択することができる。使用しうる酵素は、特
に制限されないが、例えばグルコースオキシダーゼ、ア
ルコールデヒドロゲナーゼ、ペルオキシダーゼ、カタラ
ーゼ、乳酸デヒドロゲナーゼ、ガラクトースオキシダー
ゼ、ペニシリナーゼ等が挙げられる。また、酸化還元酵
素と補酵素との組み合わせも可能である。In the enzyme electrode of the present invention, the enzyme to be immobilized in or on the surface of the conductive film should be selected appropriately depending on the target substance and the desired chemical reaction, taking into account the substrate specificity and reaction specificity of the enzyme. I can do it. Enzymes that can be used include, but are not particularly limited to, glucose oxidase, alcohol dehydrogenase, peroxidase, catalase, lactate dehydrogenase, galactose oxidase, penicillinase, and the like. Furthermore, a combination of an oxidoreductase and a coenzyme is also possible.
これらの酵素を導電性被膜に固定化するには、酵素を導
電性被膜に公知の固定化方法で直接固定化することがで
き、例えば、担体結合法、共有結合法、イオン結合法、
吸着法、架橋法などの固定化方法を用いることができる
。また、公知方法で得た酵素固定膜を導電性被膜に結合
させてもよい。In order to immobilize these enzymes on the conductive film, the enzymes can be directly immobilized on the conductive film by a known immobilization method, such as a carrier bonding method, a covalent bonding method, an ionic bonding method,
Immobilization methods such as adsorption and crosslinking methods can be used. Furthermore, an enzyme-immobilized membrane obtained by a known method may be bonded to the conductive coating.
この場合、酵素から有機ＣＴ錯体へのスムーズな電子移
動性を確保して応答性をよくするには、酵素固定膜は薄
い方がよく、また必ずしも均一な膜である必要はなく、
酵素と有機ＣＴ錯体が直接接触するようにすればよい。In this case, in order to ensure smooth electron transfer from the enzyme to the organic CT complex and improve responsiveness, the enzyme-immobilized membrane should be thinner, and it does not necessarily have to be a uniform membrane.
The enzyme and the organic CT complex may be brought into direct contact.
このようにして得られた本発明の酵素電極は、導電性基
体上に設けた導電性被膜上に酵素が接触するように固定
させた構造であり、従来の過酸化水素電極、酸素電極等
に比べ構造的に簡単であり、小型化が可能である。The enzyme electrode of the present invention thus obtained has a structure in which the enzyme is immobilized in contact with the conductive film provided on the conductive substrate, and is different from conventional hydrogen peroxide electrodes, oxygen electrodes, etc. It is structurally simpler and can be made smaller.
本発明の酵素電極で使用する有ＷＩＣＴ錯体結晶を含有
する導電性被膜は、酵素との間で電子移動が容易である
のみならず、従来電子メデイエータ−として使用されて
いたフェロセン類等と比較して、その結晶のポリマー分
散膜の電気伝導度は著しく大きい。これは、これら有機
ＣＴ錯体が発達した針状結晶を構成するため、同し含有
量でも膜中の導電パス数が多くなり、電子移動に有効に
寄与するためと考えられる。The conductive film containing the WICT-containing complex crystal used in the enzyme electrode of the present invention not only facilitates electron transfer between the enzyme and the enzyme, but also has a higher conductivity than ferrocenes, etc., which have been conventionally used as electron mediators. Therefore, the electrical conductivity of the crystalline polymer-dispersed film is extremely high. This is thought to be because these organic CT complexes constitute developed needle-like crystals, so even with the same content, the number of conductive paths in the film increases, contributing effectively to electron transfer.
更に、これらの錯体結晶を膜厚方向に結晶成長させるこ
とにより、より有効に電子移動がなされる。Furthermore, by growing these complex crystals in the film thickness direction, electron transfer can be carried out more effectively.
これらの観点から、本導電性被膜は酵素電極に使用する
電極材料として好適であり、従来の電子メデイエータ−
を利用した電極あるいは導電性高分子電極に比べて、応
答性、応答時間の点で優れている。From these points of view, the present conductive film is suitable as an electrode material for use in enzyme electrodes, and is suitable for use in conventional electronic mediators.
Compared to electrodes using conductive polymers or conductive polymer electrodes, they are superior in terms of responsiveness and response time.
また、従来の電子メデイエータ−を含む電極では電子メ
デイエータ−の溶出、脱離が避けられないが、本発明の
電極材料ではそのような欠点はない。Further, in conventional electrodes containing electronic mediators, elution and desorption of the electronic mediator is unavoidable, but the electrode material of the present invention does not have such drawbacks.
本発明の酵素電極で測定しうる物質としては、グルコー
ス等の糖分、乳酸、アルコール等の血液や尿中の微量生
体物質や、食品加工プロセスにおける糖分、アルコール
分等がある６本発明酵素電極を用いれば、上記のような
物質を選択的に高精度で、しかも長期にわたって繰り返
し分析することが可能である。また、物質の測定に限ら
ず、バイオリアクター等に使用することも可能である。Substances that can be measured with the enzyme electrode of the present invention include sugar such as glucose, trace biological substances in blood and urine such as lactic acid and alcohol, and sugar and alcohol in food processing processes. By using this method, it is possible to selectively analyze the above-mentioned substances with high precision and repeatedly over a long period of time. Moreover, it can be used not only for measuring substances but also for bioreactors and the like.
実施例１
厚み０．５１の銅板の片側に、電極部（３−麟×３１Ｉ
−）及び端子部を除いてエポキシ樹脂でモールドしたも
のを、５３１量％硫酸純水で洗浄し、乾燥した。Example 1 An electrode part (3-rin x 31I) was placed on one side of a copper plate with a thickness of 0.51
-) and the terminal portions were molded with epoxy resin and washed with 531% sulfuric acid pure water and dried.
ポリビニルブチラール樹脂（商品名：エスレックＢ、
ＢＸ−Ｌ 、槽水化学工業■製）の５重量％エチルアル
コール溶液を調製し、この溶液３０μｌを、上記銅板の
電極部に滴下し、乾燥し、ポリマー被覆銅電極を得た（
ポリマー膜厚：５μｍ）。Polyvinyl butyral resin (product name: S-LEC B,
A 5% by weight ethyl alcohol solution of BX-L (manufactured by Tansui Kagaku Kogyo ■) was prepared, and 30 μl of this solution was dropped onto the electrode part of the copper plate and dried to obtain a polymer-coated copper electrode (
Polymer film thickness: 5 μm).
７．７．８．８−テトラシアノキノジメタン（試薬、キ
シダ化学製、以下ＴＣＮΩと略す） １．ＯＪ！をアセ
トニトリル（試薬、スペクトル用）３０蒙！中に加えて
ＴＣＮＱの飽和溶液を調製した。7.7.8.8-tetracyanoquinodimethane (reagent, manufactured by Kishida Chemical Co., Ltd., hereinafter abbreviated as TCNΩ) 1. OJ! Acetonitrile (for reagents and spectra) 30 mo! A saturated solution of TCNQ was prepared.
このＴＣＮＱ飽和溶液中に、上記ポリマー被覆銅電極の
電極部を浸漬し、１０分間放置した。浸漬から１０分後
には、ポリマー表面に針状微結晶の析出が確認された。The electrode portion of the polymer-coated copper electrode was immersed in this TCNQ saturated solution and left for 10 minutes. Ten minutes after immersion, precipitation of acicular microcrystals was observed on the polymer surface.
こうして得られた有機ＣＴ錯体微結晶含有ポリマーが被
覆された電極をグルコースオキシダーゼ（Ａｓｐｅｒｇ
ｉｌｌｕｓ ｎｉｇｅｒ由来、Ｓｉｇｍａ社製、７ｙｐ
ｅ Ｖｌｌ）１０ｅｎｇ／ｍ ｌ水溶液に浸漬し、４℃
で２４時間放置して酵素固定化電極を得た。The electrode coated with the organic CT complex microcrystal-containing polymer obtained in this way was treated with glucose oxidase (Asperglucose oxidase).
derived from illus niger, manufactured by Sigma, 7yp
e Vll) immersed in a 10 eng/ml aqueous solution at 4°C.
This was left for 24 hours to obtain an enzyme-immobilized electrode.
上記電極を作用極とし、バッチ式測定装置の測定セルに
装着し、１００＋Ｍ リン酸緩衝液（ｐｈ ７．０）に
浸漬した。１ｃｍ２白金板を対向電極、銀／塩化銀電極
を参照電極とし、０．１５Ｖ（Ａｇ／ＡｇＣ］対比）の
電位を印加して、第１表に示す各グルコース濃度に対す
る応答電流を測定した。その結果を第１表に示す。この
ように、測定濃度範囲で良い直線関係が得られた。The above electrode was used as a working electrode, attached to a measurement cell of a batch type measurement device, and immersed in 100+M phosphate buffer (ph 7.0). Using a 1 cm 2 platinum plate as a counter electrode and a silver/silver chloride electrode as a reference electrode, a potential of 0.15 V (Ag/AgC] comparison) was applied to measure the response current for each glucose concentration shown in Table 1. The results are shown in Table 1. In this way, a good linear relationship was obtained over the measured concentration range.
次に、同じ測定装置、同し溶液を用い、窒素バブルを３
０分行った後、第１表に示す各グルコース濃度に対する
応答電流を測定した。その結果を第１表に示す。この結
果から、本酵素電極の応答電流は、溶存酸素濃度の影響
をほとんど受けていないことがわかる。Next, using the same measuring device and the same solution, three nitrogen bubbles were added.
After 0 minutes, the response current for each glucose concentration shown in Table 1 was measured. The results are shown in Table 1. This result shows that the response current of the present enzyme electrode is hardly affected by the dissolved oxygen concentration.
さらに、緩衝液を入れ換えて、０．１Ｍアスコルビン酸
を含む１００−一リン酸緩衝液（ｐ）ｌ ７．０）を用
い、同様に測定を行った。その結果を第１表に示す。Furthermore, the buffer solution was replaced with 100-monophosphate buffer (p)l 7.0) containing 0.1M ascorbic acid, and measurements were performed in the same manner. The results are shown in Table 1.
また、このセンサーをリン酸緩衝液中３０日室温放！後
においても、初期の約８０％の応答性を維持しており、
保存性に優れたものであった。Also, leave this sensor in phosphate buffer at room temperature for 30 days! Even after this, approximately 80% of the initial responsiveness was maintained.
It had excellent storage stability.
第１表
（発明の効果）
本発明の酵素電極は、酵素反応に伴う電子移動を直接検
知する方式をとるので、溶存酸素の多少に影響を受けず
、また電気化学的妨害物質に影響されることもない。し
かも応答寿命に優れた酵素電極である。従って、酵素セ
ンサーとして長期にわたり正確な測定を行うことができ
る。Table 1 (Effects of the Invention) The enzyme electrode of the present invention uses a method that directly detects the electron transfer accompanying the enzyme reaction, so it is not affected by the amount of dissolved oxygen and is not affected by electrochemical interfering substances. Not at all. Moreover, it is an enzyme electrode with excellent response life. Therefore, it is possible to perform accurate measurements over a long period of time as an enzyme sensor.
Claims (2)
動錯体結晶を含有する導電性被膜とを有し、この導電性
被膜中および／またはその表面上に酵素が固定化されて
いることを特徴とする酵素電極。(1) It has a conductive substrate and a conductive coating containing organic charge transfer complex crystals provided on the surface of the substrate, and an enzyme is immobilized in this conductive coating and/or on the surface thereof. An enzyme electrode featuring:
絶縁性高分子フィルム内に、このフィルムの厚さ方向を
貫通するように有機電荷移動錯体を結晶成長させたもの
から形成される請求項１記載の酵素電極。(2) A conductive film containing an organic charge transfer complex crystal,
2. The enzyme electrode according to claim 1, wherein the enzyme electrode is formed by crystal-growing an organic charge transfer complex within an insulating polymer film so as to penetrate through the thickness of the film.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2244840A JPH04122849A (en) | 1990-09-14 | 1990-09-14 | Enzyme electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2244840A JPH04122849A (en) | 1990-09-14 | 1990-09-14 | Enzyme electrode |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04122849A true JPH04122849A (en) | 1992-04-23 |
Family
ID=17124753
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2244840A Pending JPH04122849A (en) | 1990-09-14 | 1990-09-14 | Enzyme electrode |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04122849A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014002999A1 (en) * | 2012-06-25 | 2014-01-03 | 合同会社バイオエンジニアリング研究所 | Enzyme electrode |
-
1990
- 1990-09-14 JP JP2244840A patent/JPH04122849A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014002999A1 (en) * | 2012-06-25 | 2014-01-03 | 合同会社バイオエンジニアリング研究所 | Enzyme electrode |
| CN104520700A (en) * | 2012-06-25 | 2015-04-15 | 日本生物工程研究所有限责任公司 | Enzyme electrode |
| JPWO2014002999A1 (en) * | 2012-06-25 | 2016-06-02 | 合同会社バイオエンジニアリング研究所 | Enzyme electrode |
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