JPH01155244A - Measuring method of concentration of substrate of organism and oxygen sensor used therefor - Google Patents
Measuring method of concentration of substrate of organism and oxygen sensor used thereforInfo
- Publication number
- JPH01155244A JPH01155244A JP31410887A JP31410887A JPH01155244A JP H01155244 A JPH01155244 A JP H01155244A JP 31410887 A JP31410887 A JP 31410887A JP 31410887 A JP31410887 A JP 31410887A JP H01155244 A JPH01155244 A JP H01155244A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- concentration
- oxygen
- luminescent material
- substrate
- 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.)
- Granted
Links
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 51
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 239000001301 oxygen Substances 0.000 title claims abstract description 49
- 239000000758 substrate Substances 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 47
- 102000004190 Enzymes Human genes 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 239000000463 material Substances 0.000 claims description 29
- 239000012528 membrane Substances 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 13
- 150000001875 compounds Chemical class 0.000 claims description 12
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims description 10
- 229920000642 polymer Polymers 0.000 claims description 10
- 230000005284 excitation Effects 0.000 claims description 3
- 239000003446 ligand Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 229910052707 ruthenium Inorganic materials 0.000 claims description 3
- 125000002524 organometallic group Chemical group 0.000 claims 6
- 229910052723 transition metal Inorganic materials 0.000 claims 4
- 150000003624 transition metals Chemical class 0.000 claims 4
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims 2
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 claims 2
- 238000004020 luminiscence type Methods 0.000 abstract description 24
- 230000005281 excited state Effects 0.000 abstract description 7
- 150000004696 coordination complex Chemical class 0.000 abstract description 4
- 238000005259 measurement Methods 0.000 abstract description 4
- 230000036632 reaction speed Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 34
- 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 20
- 239000008103 glucose Substances 0.000 description 20
- 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 19
- 238000010799 enzyme reaction rate Methods 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 10
- 238000006911 enzymatic reaction Methods 0.000 description 10
- 108010015776 Glucose oxidase Proteins 0.000 description 7
- 239000004366 Glucose oxidase Substances 0.000 description 7
- 229940116332 glucose oxidase Drugs 0.000 description 7
- 235000019420 glucose oxidase Nutrition 0.000 description 7
- 239000010408 film Substances 0.000 description 6
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 5
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 230000036284 oxygen consumption Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 108010022355 Fibroins Proteins 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 239000012327 Ruthenium complex Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 229920002818 (Hydroxyethyl)methacrylate Polymers 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- QXYRRCOJHNZVDJ-UHFFFAOYSA-N 4-pyren-1-ylbutanoic acid Chemical compound C1=C2C(CCCC(=O)O)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 QXYRRCOJHNZVDJ-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 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 2
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 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 2
- 235000012209 glucono delta-lactone Nutrition 0.000 description 2
- 229960003681 gluconolactone Drugs 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229920005615 natural polymer Polymers 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 241000255794 Bombyx mandarina Species 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 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
- 150000001298 alcohols Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052741 iridium Inorganic materials 0.000 description 1
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920005597 polymer membrane Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- DLOBKMWCBFOUHP-UHFFFAOYSA-N pyrene-1-sulfonic acid Chemical compound C1=C2C(S(=O)(=O)O)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 DLOBKMWCBFOUHP-UHFFFAOYSA-N 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- YAYGSLOSTXKUBW-UHFFFAOYSA-N ruthenium(2+) Chemical compound [Ru+2] YAYGSLOSTXKUBW-UHFFFAOYSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、生体の基質濃度や酵素反応速度を測定する方
法及びそれに使用する酵素センサーに関し、特に酵素の
系に発光体（金属錯体）を共存させ。Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for measuring the substrate concentration and enzyme reaction rate of a living body, and an enzyme sensor used therein, and particularly relates to a method of measuring the substrate concentration and enzyme reaction rate of a living body, and in particular to a method for measuring the substrate concentration and enzyme reaction rate of living organisms, and in particular to a method for measuring the substrate concentration and enzyme reaction rate of living organisms, and in particular to a method of measuring the substrate concentration and enzyme reaction rate of living organisms, and an enzyme sensor used therein. Let them coexist.
該発光体の酵素反応にもとずく発光が酸素により消光さ
れることを利用して発光強度測定を行なうことにより生
体の基質濃度を測定する方法及びそれに使用する酵素セ
ンサーに関する。The present invention relates to a method for measuring a substrate concentration in a living body by measuring the luminescence intensity by utilizing the fact that the luminescence caused by the enzymatic reaction of the luminescent material is quenched by oxygen, and an enzyme sensor used therefor.
（従来の技術） 従来、基質濃度や酵素反応速度を測定する場合。(Conventional technology) Traditionally, when measuring substrate concentration or enzyme reaction rate.
次の反応による。Due to the following reaction.
グルコース
したがって、Ｈ２０□やグルコノラクトンの発生量から
酸素消費量によりグルコース濃度を測定でき、これらの
測定にはＨ２Ｏ２電極、ＰＲ膜電極酸素電極などの電極
で行っている。Therefore, the glucose concentration can be measured from the amount of generated H20□ and gluconolactone to the amount of oxygen consumed, and these measurements are performed using electrodes such as a H2O2 electrode, a PR membrane electrode, and an oxygen electrode.
しかしながら、これらの方法には （１）グルコノラクトンによるｐＨ変化は小さい。However, these methods (1) The pH change caused by gluconolactone is small.
（２）酸素消費量は酸素電極上に到達した酸素分子のみ
を測定するのに限定され、又消費量を測定するので面倒
である。また、電極を用いて微小ながら電流を流さない
といけないので、生体系の測定には好ましくない。(2) Oxygen consumption is limited to measuring only the oxygen molecules that have reached the oxygen electrode, and it is troublesome to measure the consumption. Furthermore, since it is necessary to apply a small current using an electrode, it is not preferable for measuring biological systems.
（３）過酸化水素（ｎｚｏ＊）量は膜電極を使用し、膜
透過するＨ、Ｏ□のみを測定するので界面上のＨ，Ｏ，
濃度が律速となる、という欠点があった。(3) The amount of hydrogen peroxide (nzo*) uses a membrane electrode and measures only the H, O□ that permeates the membrane, so the H, O,
The drawback was that the concentration was rate-limiting.
（発明が解決しようとする問題点）
本発明は上記従来技術の問題点を解決すべく種々検討し
た結果１本発明を完成したもので、本発明の目的は、酵
素反応時の酸素消費量を周りの妨害物質に邪魔されずに
、酵素系の共存発光体の励起状態からの発光が酸素によ
り消光することを利用し、その時の発光強度測定により
基質濃度又は酵素反応速度を測定する生体基ＩＪｆｖＡ
度の測定方法及びそれに使用する酵素セシサーを提供す
る。(Problems to be Solved by the Invention) The present invention was completed as a result of various studies to solve the above-mentioned problems of the prior art.The purpose of the present invention is to reduce the amount of oxygen consumed during enzyme reactions. A biological group IJfvA that measures the substrate concentration or enzyme reaction rate by measuring the luminescence intensity at that time by utilizing the fact that the luminescence from the excited state of the coexisting luminescent material in the enzyme system is quenched by oxygen without being disturbed by surrounding interfering substances.
Provided are a method for measuring oxidation and an enzyme secessor used therefor.
（問題点を解決する手段）
すなわち、本発明は生体基質を含む溶液中において、生
体基質と溶存酸素を酵素の下で反応させ。(Means for Solving the Problems) That is, the present invention reacts a biological substrate with dissolved oxygen in the presence of an enzyme in a solution containing the biological substrate.
紫外領域または可視光領域の光を該溶液中に浸漬した発
光体に照射し、前記反応により消費されて低減した溶存
酸素濃度に応じて該発光体から発生する光の強度により
生体基質濃度を測定することを特徴とする生体基質濃度
の測定方法であり、また、基板上に酵素および発光体を
固定化したことを特徴とする酵素センサーである。Irradiate the luminescent material immersed in the solution with light in the ultraviolet region or visible region, and measure the biological substrate concentration based on the intensity of the light generated from the luminescent material according to the dissolved oxygen concentration consumed and reduced by the reaction. The present invention is a method for measuring the concentration of a biological substrate, and an enzyme sensor is characterized in that an enzyme and a luminescent material are immobilized on a substrate.
すなわち、換言すれば、本発明は、酵素の系に発光体（
金属錯体）を共存させ、酵素反応に伴う酸素消費量を該
発光体励起状態と酸素との反応による発光強度変化によ
りｉ青濃度を求め、基質濃度又は酵素反応速度を測定す
る生体基質濃度の測定方法であり、また、その際、使用
する酵素センサーである。この場合、酵素反応系として
の酵素は酸素と直接または間接的に反応して酸素を消費
する酵素反応物質である。このような反応系の基質の例
としては、グルコース、尿酸、コレステロール、資化糖
、その他の糖（スクロース、マルトース、ガラクトース
）、ホスファチジルコリン、乳酸、ニコチン酸アミドジ
ヒドロキシナーゼ（ＮＡＤＨ）、蓚酸、アミノ酸、アル
コール類、モノアミン、アスコルビン酸、ピルビン酸な
どが挙げられる。In other words, the present invention provides an enzyme system with a luminescent material (
Measurement of biological substrate concentration in which the i-blue concentration is determined by the change in luminescence intensity due to the reaction between the excited state of the luminescent material and oxygen, and the substrate concentration or enzyme reaction rate is measured by coexistence of a metal complex) and the oxygen consumption accompanying the enzyme reaction. The method and enzyme sensor used in the process. In this case, the enzyme used as the enzyme reaction system is an enzyme reaction substance that reacts directly or indirectly with oxygen and consumes oxygen. Examples of substrates for such reaction systems include glucose, uric acid, cholesterol, assimilated sugars, other sugars (sucrose, maltose, galactose), phosphatidylcholine, lactic acid, nicotinamide dihydroxynase (NADH), oxalic acid, amino acids, Examples include alcohols, monoamines, ascorbic acid, and pyruvic acid.
従来の方法は前述したように、（１）式に示す例のよう
な酵素反応を生成Ｈ，Ｏ□やグルコノラクトンの発生量
、酸素消費量などよりグルコース濃度を各々電極法で（
Ｈｚ Ｏｓ電極、ｐＨ電極、酸素電極など）測定するの
に対し、本発明では酵素の系に発光体（金属錯体）を共
存させ、酵素反応に伴う酸素消費量を該発光体励起状態
と酸素との反応による発光強度変化により酸素濃度を求
めるものであって、酵素の化学反応（酵素反応に直接又
は間接的に伴う酸素消費量）を周りの妨害物質に邪魔さ
れずに。As mentioned above, in the conventional method, the enzyme reaction as shown in equation (1) is carried out, and the glucose concentration is measured using the electrode method (
Hz Os electrode, pH electrode, oxygen electrode, etc.), whereas in the present invention, a luminescent material (metal complex) is made to coexist in the enzyme system, and the oxygen consumption accompanying the enzyme reaction is measured based on the excited state of the luminescent material and oxygen. The oxygen concentration is determined by the change in luminescence intensity caused by the reaction, and the chemical reaction of the enzyme (oxygen consumption directly or indirectly associated with the enzyme reaction) is not interfered with by surrounding interfering substances.
共存発光体（金属錯体）励起状態と該酸素の化学反応を
利用し、酸素濃度を発光強度により測定してその時の酸
素濃度、すなわち基質濃度又は酵素反応速度を測定を算
出することができるのである。Using the excited state of the coexisting luminescent material (metal complex) and the chemical reaction between the oxygen, the oxygen concentration can be measured by the luminescence intensity, and the oxygen concentration at that time, that is, the substrate concentration or enzyme reaction rate, can be calculated. .
次に、本発明における発光体および酵素センサーについ
て説明する。Next, the luminescent material and enzyme sensor in the present invention will be explained.
発光体：
発光体とは、光励起状態からケイ光又はリン光を発して
元の基底状態に戻る化合物をいう６本発明においては、
その光励起状態で酸素と反応することにより発光強度が
減少することを利用し、発光強度を測定して酸素濃度を
求めることが原理となっているので、その光励起状態か
ら発光し且つその発光強度が酸素の共存により減少する
ような発光体なら何でも用いることができる０本発明で
使用できる発光体としては例えば２．２１−ビピリジン
やその誘導体を配位子とする金属錯体、例えばトリス（
２，２ξビピリジン）ルテニウム錯体やその他イリジウ
ムなどの錯体、トリス（０−フェナントロリン）ルテニ
ウム錯体のピレンヤその誘導体（ピレンスルホン酸やピ
レン酪酸など）のような有機化合物などが挙げられる。Luminescent substance: A luminescent substance refers to a compound that emits fluorescence or phosphorescence from a photoexcited state and returns to its original ground state.6 In the present invention,
The principle is to use the fact that the luminescence intensity decreases due to a reaction with oxygen in the photoexcited state and measure the luminescence intensity to determine the oxygen concentration. Any luminescent material that decreases when oxygen is present can be used. Examples of luminescent materials that can be used in the present invention include metal complexes having 2,21-bipyridine and its derivatives as ligands, such as tris(
Examples include organic compounds such as 2,2ξbipyridine) ruthenium complex and other complexes such as iridium, and derivatives thereof (pyrenesulfonic acid, pyrenebutyric acid, etc.) of tris(0-phenanthroline)ruthenium complex.
酵素センサー系：
酵素又は酵素群と基質および発光体を均一溶液中で共存
させて用いてもよい。あるいはこれらを合成または天然
の高分子膜中に固定化した膜を用いることができる。使
用できる合成高分子膜としては、アクリル酸、メタクリ
ル酸、ヒドロキシエチルメタクリレート、スチレン、Ｎ
−ビニルピロリドン、などの単独又は共重合体が挙げら
れる。また天然高分子膜としては絹、セルロース、ゼラ
チンなどの膜が挙げられる。本発明で用いられる酵素セ
ンサー膜とするためには、これらの高分子膜中に酵素お
よび発光体を物理的にあるいは化学的に固定する。固定
手段として物理的に固定する方法は、単に膜に吸着させ
るか、被吸着物質（酵素、発光体など）を含む膜成分の
溶液からキャスト法などにより成膜するか、あるいは被
吸着物質と単量体の混合物を光線などにより重合するこ
とにより膜化する。化学的に固定する方法は、高分子化
合物に予め酵素や発光体などを共有結合で導入して、後
に成膜するか、あるいは成膜した後に酵素や発光体など
を化学反応により導入、結合する。Enzyme sensor system: An enzyme or a group of enzymes, a substrate, and a luminescent material may be used together in a homogeneous solution. Alternatively, a membrane in which these are immobilized in a synthetic or natural polymer membrane can be used. Synthetic polymer membranes that can be used include acrylic acid, methacrylic acid, hydroxyethyl methacrylate, styrene, N
-Vinylpyrrolidone, etc. alone or as a copolymer. Examples of natural polymer membranes include membranes made of silk, cellulose, gelatin, and the like. In order to obtain the enzyme sensor membrane used in the present invention, enzymes and luminescent materials are physically or chemically immobilized in these polymer membranes. Physical fixing methods include simply adsorbing the adsorbed substance to the membrane, forming a film from a solution of membrane components containing the adsorbed substance (enzyme, luminescent material, etc.) by a casting method, or simply attaching the adsorbed substance to the membrane. A film is formed by polymerizing a mixture of polymers using light or the like. For chemical fixation, enzymes, luminescent materials, etc. are introduced into a polymer compound in advance through covalent bonds, and then a film is formed, or after the film is formed, enzymes, luminescent materials, etc. are introduced and bonded through a chemical reaction. .
高分子化合物に発光体を化学的に共有結合で固定する例
としては、トリス（２，２−ビピリジン）ルテニウム錯
体（Ｒｕ（ｂｐｙ）ａ２＋と略す）構造をペンダント基
として有する次の重合体が挙げられる。An example of chemically fixing a light emitter to a polymer compound through a covalent bond is the following polymer having a tris(2,2-bipyridine)ruthenium complex (abbreviated as Ru(bpy)a2+) structure as a pendant group. It will be done.
Ｒｕ［Ｐ（Ｍ−Ｖｂｐｙ）］（ｂＰｙ）ａ”÷（ＮＮは
２，２ξビピリジンを表わす。）但し、Ｘ−はＣｌ２−
１ＣＱ Ｏ，−などのアニオン、には４−メチル−４′
−ビニル−２，２２−ビピリジンまたはアクリル酸、メ
タクリル酸、ヒドロキシエチルメタクリレート、スチレ
ン、Ｎ−ビニルピロリドンなどの単量体単位、Ｘ、Ｙ、
Ｚは各繰返し単位のモル分率でＸ、Ｙは０〜０，９９．
Ｚは０．０１〜１の範囲から選ばれる。この高分子ペン
ダント型発光体は単独で又は他の高分子化合物と一緒に
膜化し、その膜中には酵素または酵素群を共存させて酵
素センサーとして用いることができる。この高分子型発
光体は膜から溶出し難いので、安定な酵素センサーとし
て用いることができる。Ru[P(M-Vbpy)](bPy)a”÷(NN represents 2,2ξbipyridine), where X- is Cl2-
Anions such as 1CQ O,- are 4-methyl-4'
- vinyl-2,22-bipyridine or monomer units such as acrylic acid, methacrylic acid, hydroxyethyl methacrylate, styrene, N-vinylpyrrolidone, X, Y,
Z is the mole fraction of each repeating unit, and Y is 0 to 0.99.
Z is selected from the range of 0.01 to 1. This polymer pendant light emitter can be formed into a film alone or together with other polymer compounds, and an enzyme or enzyme group can coexist in the film to be used as an enzyme sensor. Since this polymer type luminescent material is difficult to elute from the membrane, it can be used as a stable enzyme sensor.
これらの膜材料のうち、絹膜は生体成分になじむので、
生体系に直接挿入して用いられる酵素センサー用膜とし
て用いるのに極めて適している。Among these membrane materials, silk membranes are compatible with biological components, so
It is extremely suitable for use as a membrane for enzyme sensors that are directly inserted into biological systems.
次に実施例をもって本発明の詳細な説明する。Next, the present invention will be explained in detail with reference to Examples.
実施例１
共栓付全面透明石英セル（光路長１ｃｍ）中に０．１Ｍ
グルコース、５０μＭ（マイクロモル）、トリス（２，
２’ −ビピリジン）ルテニウム（ＩＩ）ｔｉｌｔ体の
塩化物のリン酸緩衝水溶液（ＰＨ７，０）３ｃｃを加え
た。Example 1 0.1M in a completely transparent quartz cell with a stopper (light path length 1cm)
Glucose, 50 μM (micromoles), Tris(2,
3 cc of a phosphate buffered aqueous solution (PH 7,0) of 2'-bipyridine)ruthenium (II) tilt chloride was added.
該セルを分光蛍光光度計（日本分光（製）製ＦＰ５５０
Ａ）にセットし、励起光波長４６０ｎｍ、発光側分光器
波長６０５ｎｍにて発光強度工０を測定した。The cell was measured using a spectrofluorometer (FP550 manufactured by JASCO Corporation).
A), and the emission intensity was measured using an excitation light wavelength of 460 nm and an emission side spectrometer wavelength of 605 nm.
このように、発光側分光器波長６０５ｎｍを一定にして
おき、上記セルのなかにグルコースオキシダーゼＩＩ！
１ｇ／１１１１２濃度の水溶液を１〜５μ阿添加する。In this way, the emission side spectrometer wavelength is kept constant at 605 nm, and glucose oxidase II!
Add 1 to 5 μm of an aqueous solution having a concentration of 1 g/11112.
この時の酵素反応（グルコースの酵素酸化反応）により
酸素分子が消費されるので発光強度が増加するし、発光
強度の時間変化を測、定する。At this time, oxygen molecules are consumed by the enzymatic reaction (enzymatic oxidation reaction of glucose), so the luminescence intensity increases, and the change in luminescence intensity over time is measured.
グルコースとグルコースオキシダーゼを共存させた系で
はグルコースの酸化により酸素が消費され発光強度が増
加する。酸素濃度と発光強度との関係を第１図に示す。In a system in which glucose and glucose oxidase coexist, oxygen is consumed by oxidation of glucose and luminescence intensity increases. FIG. 1 shows the relationship between oxygen concentration and luminescence intensity.
この第１図より任意の時間における酸素濃度を知ること
ができ、したがって、酵素反応速度を知ることも可能で
ある。因に本条件下ではグルコースオキシダーゼ１μｇ
当り１分当りの発光強度増加は反応前の１．０４倍で、
これは酸素濃度変化にして１．０４＋＋＋Ｍ／ｗｉｎ／
μｇグルコースオキシダーゼに相当することになる。From FIG. 1, it is possible to know the oxygen concentration at any given time, and therefore it is also possible to know the enzyme reaction rate. In fact, under these conditions, 1 μg of glucose oxidase
The increase in luminescence intensity per minute was 1.04 times that before the reaction,
This is a change in oxygen concentration of 1.04+++M/win/
This corresponds to μg glucose oxidase.
このように基質（ここではグルコース）濃度が酸素濃度
より過剰に共存する系を用いれば酵素（ここではグルコ
ースオキシダーゼ）の活性や反応速度を求めることがで
き、また逆に基質濃度が酸素濃度より充分小さい条件下
では、発光強度の測定により基質濃度を測定することが
可能である。In this way, by using a system in which the substrate (glucose in this case) coexists in excess of the oxygen concentration, it is possible to determine the activity and reaction rate of the enzyme (in this case, glucose oxidase). Under low conditions, it is possible to determine the substrate concentration by measuring the luminescence intensity.
以上述べたように、まず、”（ｂｐｙ）ａ”十水溶液に
おいて、 Ｒｕ錯体励起状態からの発光（６０５ｎｍ）
相対強度を溶液中の酸素濃度に対してプロットすると第
１図のような直線となり、これから発光の相対強度から
酸素濃度を求めることが可能である。As mentioned above, first, in a 10-aqueous solution of "(bpy)a", the emission (605 nm) from the excited state of the Ru complex is detected.
When the relative intensity is plotted against the oxygen concentration in the solution, a straight line as shown in FIG. 1 is obtained, and from this it is possible to determine the oxygen concentration from the relative intensity of the emitted light.
実施例２
絹フィブロインの４％水溶液にグルコースオキシダーゼ
を絹に対して０．２％＋Ｒｕ（ｂｐｙ）ｓ”十を０．１
ｍＭ濃度になるように添加し、アクリル板上にキャスト
して風乾し、厚さ約１μｍの組込を得た。まず検量線を
作成するため、この組込を既知濃度の酸素を含む水溶液
に浸漬して、酸素濃度と相対発光強度の関係を求めると
第２図のような関係を得た。Example 2 Glucose oxidase was added to a 4% aqueous solution of silk fibroin at a concentration of 0.2% relative to silk + 0.1% Ru(bpy)s''
It was added to a concentration of mM, cast on an acrylic plate, and air-dried to obtain an incorporation with a thickness of about 1 μm. First, in order to create a calibration curve, this assembly was immersed in an aqueous solution containing oxygen at a known concentration, and the relationship between oxygen concentration and relative luminescence intensity was determined, and the relationship shown in FIG. 2 was obtained.
このような特性を持つ酵素センサーをグルコースを０．
１〜１ｍＭ含み且つ空気を飽和させた水溶液中に浸漬し
、４５０ｎｍ光で励起して６０５ｎｍの発光を測定した
。その発光強度の減少量から酸素濃度変化量を求め、従
って系中のグルコース濃度を定量することができた。An enzyme sensor with such characteristics can be used to absorb glucose at 0.
It was immersed in an aqueous solution containing 1 to 1 mM and saturated with air, excited with 450 nm light, and emitted at 605 nm was measured. The amount of change in oxygen concentration was determined from the amount of decrease in the luminescence intensity, and it was therefore possible to quantify the glucose concentration in the system.
実施例３
実施例２においてグルコースオキシダーゼの他にインベ
ルターゼを絹に対して０．２％用い、他は実施例２と同
様にしてグルコース濃度を測定できるバイオセンサーを
作った。グルコースを０．０５〜１ｄ含みかつ空気を飽
和させた水溶液にこの酵素センサーを浸漬し、発光強度
の減少量と第２図の関係からグルコース濃度を定量する
ことができた。Example 3 A biosensor capable of measuring glucose concentration was produced in the same manner as in Example 2 except that in addition to glucose oxidase, invertase was used at 0.2% based on silk. This enzyme sensor was immersed in an aqueous solution containing 0.05 to 1 d of glucose and saturated with air, and the glucose concentration could be determined from the decrease in luminescence intensity and the relationship shown in FIG. 2.
実施例４
実施例２においてＲｕ（ｂｐｙ）３”＋の代わりに構造
式１においてＭがアクリル酸、Ｘが（４１’、 Ｘ＝０
．９２４． Ｙ＝ｔ、０．０５４、Ｚ＝０．０２２の高
分子ペンダント型Ｒｕ（ｂｐｙ）ａ”十を１ｄ錯体単体
濃度が０．１ｄになるように細膜水溶液に添加し、他は
実施例２と同様にしてグルコースセンサーを作り、その
検量線として第２図と同様な結果を得た。このグルコー
スセンサーにより水溶液中のグルコース濃度を発光法に
より測定できた。Example 4 In Example 2, instead of Ru(bpy)3''+ in structural formula 1, M is acrylic acid and X is (41', X=0
．． 924. Polymer pendant type Ru(bpy)a'' with Y=t, 0.054, Z=0.022 was added to the thin film aqueous solution so that the concentration of 1d complex alone was 0.1d, and the other conditions were as in Example 2. A glucose sensor was made in the same manner as above, and the results obtained as a calibration curve were similar to those shown in Figure 2.With this glucose sensor, the glucose concentration in an aqueous solution could be measured by the luminescence method.
実施例５
実施例２においてＲｕ（ｂｐｙ）３”＋の代わりにピレ
ン酪酸を用いた他は実施例２と同様にしてグルコースセ
ンサーを作った。このセンサーを既知濃度の酵素を含む
水中に浸漬し、３３８ｎｍ光で励起し、３８０ｎｍの発
光の相対強度を測定したところ、第３図の関係を得た。Example 5 A glucose sensor was made in the same manner as in Example 2 except that pyrenebutyric acid was used instead of Ru(bpy)3''+ in Example 2. This sensor was immersed in water containing enzyme at a known concentration. , 338 nm light and the relative intensity of 380 nm emission was measured, and the relationship shown in FIG. 3 was obtained.
この関係を用い、未知のグルコースを含む水溶液中のグ
ルコース濃度を定量することができた。Using this relationship, it was possible to quantify the glucose concentration in an aqueous solution containing unknown glucose.
また、ＰＭＭＡ、石英などの光ファイバーの先端を基板
とし、酵素及び発光体を固定化させることにより、直径
数μｍの微小酵素センサーが得られる。Further, by using the tip of an optical fiber such as PMMA or quartz as a substrate and immobilizing an enzyme and a luminescent substance, a micro enzyme sensor with a diameter of several μm can be obtained.
本実施例では、生体基質としてグルコースを例にあげた
が、これに限られるものではなく前述の他の生体基質に
ついても同様である。また、組込は。In this example, glucose is used as an example of the biosubstrate, but the present invention is not limited to this, and the same applies to the other biosubstrates mentioned above. Also, the built-in.
実施例に限られるものではなく家蚕再生絹フイブロイン
膜、野蚕再生絹フイブロイン膜を用いることもできる。The present invention is not limited to the examples, and a regenerated silk fibroin membrane from domestic silkworms and a regenerated silk fibroin membrane from wild silkworms can also be used.
（効果）
以上、述べたように本発明は酵素反応系において酸素と
共に発光体を存在させ、共存発光体の励起状態からの発
光可視光線作成が酸素により消光することを利用し、そ
の時の発光強度測定により基質濃度又は酵素反応速度で
測定する酵素センサー及びそれを用いた生体基質濃度の
測定方法であるので電流を流す必要がなく、生体系の測
定に対しても極めて安全であり、又、溶液中の他の生体
基質、或は他の妨害イオンの影響をうけることなく高精
度の測定ができる等の効果を奏するのである。(Effects) As described above, the present invention allows a luminescent substance to exist together with oxygen in an enzyme reaction system, and takes advantage of the fact that visible light is quenched by oxygen from the excited state of the coexisting luminescent substance, and the emission intensity at that time is Since the enzyme sensor measures the substrate concentration or enzyme reaction rate, and the method uses it to measure biological substrate concentration, there is no need to apply an electric current, making it extremely safe for measuring biological systems. This has the effect of enabling highly accurate measurements without being affected by other biological substrates or other interfering ions.
第１図は本発明の実施例１の酸素濃度に対する相対発光
強度の関係図、第２図は本発明の実施例２の酸素濃度に
対する相対発光強度の関係図及び第３図は実施例５の場
合の酸素濃度に対する相対発光強度の関係図である。FIG. 1 is a diagram showing the relationship between relative luminescence intensity and oxygen concentration in Example 1 of the present invention, FIG. 2 is a diagram showing the relationship between relative luminescence intensity and oxygen concentration in Example 2 of the present invention, and FIG. FIG. 3 is a relationship diagram of relative luminescence intensity with respect to oxygen concentration in the case of FIG.
Claims (1)
素を酵素の下で反応させ、紫外領域または可視光領域の
光を該溶液中に浸漬した発光体に照射し、前記反応によ
り消費されて低減した溶存酸素濃度に応じて該発光体か
ら発生する光の強度により生体基質濃度を測定すること
を特徴とする生体基質濃度の測定方法。 ２、発光体は紫外領域、可視光領域の励起光により発光
し且つその発光が酸素により消光されることを特徴とす
る特許請求の範囲第１項記載の生体基質濃度の測定方法
。 ３、発光体が遷移金属の有機金属錯化合物であることを
特徴とする特許請求の範囲第１項ないし第２項記載の生
体基質濃度の測定方法。 ４、有機金属錯化合物は金属がルテニウムで、配位子が
２．２’−ビピリジン、ｏ−フェナントロリンまたはそ
れらの誘導体であることを特徴とする特許請求の範囲第
３項記載の生体基質濃度の測定方法。 ５、発光体が遷移金属の有機金属錯化合物を一成分とす
る高分子化合物である特許請求の範囲第２項ないし第４
項のいずれかに記載の生体基質濃度の測定方法。 ６、基板上に酵素および発光体を固定化したことを特徴
とする酵素センサー。 ７、発光体は紫外領域、可視光領域の励起光により発光
し且つその発光が酸素により消光されるものであること
を特徴とする特許請求の範囲第６項記載の酵素センサー
。 ８、発光体が遷移金属の有機金属錯化合物であることを
特徴とする特許請求の範囲第６項記載または第７項記載
の酵素センサー。 ９、有機金属錯化合物は金属がルテニウムで、配位子が
２．２’−ビピリジン、ｏ−フェナントロリンまたはそ
れらの誘導体であることを特徴とする特許請求の範囲第
８項記載の酵素センサー。 １０、発光体が遷移金属の有機金属錯化合物を一成分と
する高分子化合物であることを特徴とする特許請求の範
囲第７項ないし第９項のいずれかに記載の酵素センサー
。 １１、酵素及び発生体の固定化物質が絹膜であることを
特徴とする特許請求の範囲第７項ないし第１０項のいず
れかに記載の酵素センサー。[Claims] 1. In a solution containing a biological substrate, the biological substrate and dissolved oxygen are reacted under the presence of an enzyme, and a luminescent material immersed in the solution is irradiated with light in the ultraviolet region or visible region, A method for measuring biological substrate concentration, characterized in that the biological substrate concentration is measured by the intensity of light generated from the luminescent material in accordance with the dissolved oxygen concentration that is consumed and reduced by the reaction. 2. The method for measuring biological substrate concentration according to claim 1, wherein the luminescent material emits light by excitation light in the ultraviolet region or visible light region, and the light emission is quenched by oxygen. 3. The method for measuring biological substrate concentration according to claims 1 or 2, wherein the luminescent material is an organometallic complex compound of a transition metal. 4. In the organometallic complex compound, the metal is ruthenium, and the ligand is 2,2'-bipyridine, o-phenanthroline, or a derivative thereof. Measuring method. 5. Claims 2 to 4, wherein the luminescent material is a polymer compound containing an organometallic complex compound of a transition metal as one component.
The method for measuring biological substrate concentration according to any one of paragraphs. 6. An enzyme sensor characterized by having an enzyme and a luminescent material immobilized on a substrate. 7. The enzyme sensor according to claim 6, wherein the luminescent material emits light by excitation light in the ultraviolet region or visible light region, and the light emission is quenched by oxygen. 8. The enzyme sensor according to claim 6 or 7, wherein the luminescent material is an organometallic complex compound of a transition metal. 9. The enzyme sensor according to claim 8, wherein the metal in the organometallic complex compound is ruthenium, and the ligand is 2,2'-bipyridine, o-phenanthroline, or a derivative thereof. 10. The enzyme sensor according to any one of claims 7 to 9, wherein the luminescent material is a polymer compound containing an organometallic complex compound of a transition metal as one component. 11. The enzyme sensor according to any one of claims 7 to 10, wherein the enzyme and generator immobilized substance is a silk membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31410887A JPH0659236B2 (en) | 1987-12-14 | 1987-12-14 | Method for measuring biological substrate concentration and oxygen sensor used therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31410887A JPH0659236B2 (en) | 1987-12-14 | 1987-12-14 | Method for measuring biological substrate concentration and oxygen sensor used therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01155244A true JPH01155244A (en) | 1989-06-19 |
| JPH0659236B2 JPH0659236B2 (en) | 1994-08-10 |
Family
ID=18049338
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP31410887A Expired - Lifetime JPH0659236B2 (en) | 1987-12-14 | 1987-12-14 | Method for measuring biological substrate concentration and oxygen sensor used therefor |
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| Country | Link |
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| JP (1) | JPH0659236B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2699170A1 (en) * | 1992-12-15 | 1994-06-17 | Asulab Sa | 2,2'-Bipyridine ligand transition metal complexes substituted with at least one alkylammonium radical, process for their production and their use as a redox mediator |
| US5580527A (en) * | 1992-05-18 | 1996-12-03 | Moltech Corporation | Polymeric luminophores for sensing of oxygen |
| WO2003057904A1 (en) * | 2001-12-28 | 2003-07-17 | Arkray, Inc. | Method of colorimetry and reagent for use therein |
| US7550273B2 (en) | 2001-12-28 | 2009-06-23 | Arkray, Inc. | Colorimetric method and reagent used for the same |
-
1987
- 1987-12-14 JP JP31410887A patent/JPH0659236B2/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5580527A (en) * | 1992-05-18 | 1996-12-03 | Moltech Corporation | Polymeric luminophores for sensing of oxygen |
| FR2699170A1 (en) * | 1992-12-15 | 1994-06-17 | Asulab Sa | 2,2'-Bipyridine ligand transition metal complexes substituted with at least one alkylammonium radical, process for their production and their use as a redox mediator |
| EP0602488A1 (en) * | 1992-12-15 | 1994-06-22 | Asulab S.A. | Complexes of a transition metal with 2,2-bipyridine ligands substituted by at least one ammonium alkyl radical, process for their fabrication and their use as redox mediator |
| WO2003057904A1 (en) * | 2001-12-28 | 2003-07-17 | Arkray, Inc. | Method of colorimetry and reagent for use therein |
| US7550273B2 (en) | 2001-12-28 | 2009-06-23 | Arkray, Inc. | Colorimetric method and reagent used for the same |
| US8574896B2 (en) | 2001-12-28 | 2013-11-05 | Arkray, Inc. | Colorimetric method and reagent used for the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0659236B2 (en) | 1994-08-10 |
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