JPH0690754A - Enzyme modified through spacer with mediator and sensor involving the same modified enzyme - Google Patents
Enzyme modified through spacer with mediator and sensor involving the same modified enzymeInfo
- Publication number
- JPH0690754A JPH0690754A JP4269552A JP26955292A JPH0690754A JP H0690754 A JPH0690754 A JP H0690754A JP 4269552 A JP4269552 A JP 4269552A JP 26955292 A JP26955292 A JP 26955292A JP H0690754 A JPH0690754 A JP H0690754A
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- enzyme
- dehydrogenase
- ferrocene
- modified
- 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
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 83
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 83
- 125000006850 spacer group Chemical group 0.000 title claims abstract description 13
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims abstract description 45
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims description 21
- 239000000126 substance Substances 0.000 claims description 15
- 229930091371 Fructose Natural products 0.000 claims description 12
- 239000005715 Fructose Substances 0.000 claims description 12
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 12
- 238000007650 screen-printing Methods 0.000 claims description 8
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims description 2
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 claims description 2
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 claims description 2
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 claims description 2
- 108010009384 L-Iditol 2-Dehydrogenase Proteins 0.000 claims description 2
- 102100026974 Sorbitol dehydrogenase Human genes 0.000 claims description 2
- 108010023417 cholesterol dehydrogenase Proteins 0.000 claims description 2
- 108010085346 steroid delta-isomerase Proteins 0.000 claims description 2
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims 1
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims 1
- 239000003575 carbonaceous material Substances 0.000 claims 1
- 150000002211 flavins Chemical class 0.000 claims 1
- 150000005480 nicotinamides Chemical class 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 23
- 238000000034 method Methods 0.000 description 30
- 238000006243 chemical reaction Methods 0.000 description 21
- 239000000243 solution Substances 0.000 description 17
- 238000005259 measurement Methods 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- GPRSOIDYHMXAGW-UHFFFAOYSA-N cyclopenta-1,3-diene cyclopentanecarboxylic acid iron Chemical compound [CH-]1[CH-][CH-][C-]([CH-]1)C(=O)O.[CH-]1C=CC=C1.[Fe] GPRSOIDYHMXAGW-UHFFFAOYSA-N 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 239000008363 phosphate buffer Substances 0.000 description 8
- 238000007639 printing Methods 0.000 description 8
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 8
- 125000003277 amino group Chemical group 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- MBVFRSJFKMJRHA-UHFFFAOYSA-N 4-fluoro-1-benzofuran-7-carbaldehyde Chemical compound FC1=CC=C(C=O)C2=C1C=CO2 MBVFRSJFKMJRHA-UHFFFAOYSA-N 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 239000010409 thin film Substances 0.000 description 5
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 4
- YPZRHBJKEMOYQH-UYBVJOGSSA-N FADH2 Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1COP(O)(=O)OP(O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C(NC(=O)NC2=O)=C2NC2=C1C=C(C)C(C)=C2 YPZRHBJKEMOYQH-UYBVJOGSSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000002484 cyclic voltammetry Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- IBVAQQYNSHJXBV-UHFFFAOYSA-N adipic acid dihydrazide Chemical compound NNC(=O)CCCCC(=O)NN IBVAQQYNSHJXBV-UHFFFAOYSA-N 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 150000001718 carbodiimides Chemical class 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-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
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical compound CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- -1 hydrogen ions Chemical class 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000006479 redox reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 2
- CCBICDLNWJRFPO-UHFFFAOYSA-N 2,6-dichloroindophenol Chemical compound C1=CC(O)=CC=C1N=C1C=C(Cl)C(=O)C(Cl)=C1 CCBICDLNWJRFPO-UHFFFAOYSA-N 0.000 description 1
- ADIKQLIPZCZVJA-UHFFFAOYSA-N 5-chlorocyclopenta-1,3-diene;cyclopenta-1,3-diene;iron(2+) Chemical compound [Fe+2].C=1C=C[CH-]C=1.ClC1=CC=C[CH-]1 ADIKQLIPZCZVJA-UHFFFAOYSA-N 0.000 description 1
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241001024304 Mino Species 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- IDCBOTIENDVCBQ-UHFFFAOYSA-N TEPP Chemical compound CCOP(=O)(OCC)OP(=O)(OCC)OCC IDCBOTIENDVCBQ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- RNSRNNYIVZDRFQ-UHFFFAOYSA-N cyclopenta-1,3-diene 2-cyclopenta-2,4-dien-1-ylethanol iron(2+) Chemical compound [Fe++].c1cc[cH-]c1.OCC[c-]1cccc1 RNSRNNYIVZDRFQ-UHFFFAOYSA-N 0.000 description 1
- HEWFKXVSWQSSAT-UHFFFAOYSA-M cyclopenta-1,3-diene;cyclopenta-2,4-dien-1-ylidenemethanolate;iron(2+) Chemical compound [Fe+2].C=1C=C[CH-]C=1.[O-]C=C1C=CC=C1 HEWFKXVSWQSSAT-UHFFFAOYSA-M 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000007607 die coating method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000008278 dynamic mechanism Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 239000007772 electrode material Substances 0.000 description 1
- 238000003411 electrode reaction Methods 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 238000007646 gravure printing Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229920000831 ionic polymer Polymers 0.000 description 1
- YTOVAWUSMUMHIM-UHFFFAOYSA-N iron(2+);5-methylcyclopenta-1,3-diene Chemical compound [Fe+2].C[C-]1C=CC=C1.C[C-]1C=CC=C1 YTOVAWUSMUMHIM-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- 150000002926 oxygen Chemical class 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- MMXZSJMASHPLLR-UHFFFAOYSA-N pyrroloquinoline quinone Chemical compound C12=C(C(O)=O)C=C(C(O)=O)N=C2C(=O)C(=O)C2=C1NC(C(=O)O)=C2 MMXZSJMASHPLLR-UHFFFAOYSA-N 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】化学物質を測定することは、医
療、工業プロセス、環境などをはじめとする広い範囲の
分野で行なわれている。これらの分野には種々の化学物
質が存在しており、各種の化学物質を測定するために、
さまざまな物理・化学装置が駆使されており、これらを
より迅速、簡単かつ直接測定できる方法や装置が強く要
望されている。しかしながら、化学物質は単独で存在す
ることはなく、たいてい何種類もの物質が混在してい
る。このような系から、特定の化学物質を選択し、これ
を測定するためには、これらを識別する材料や試薬が必
要である。例えば生体触媒である酵素は、生体内の反応
を選択的に進めている。酵素は触媒機能と同時に分子認
識能を有している。従って、この酵素を用いることによ
って特定の化学物質を識別し、その化学物質の濃度を測
定することができるのである。[Industrial application] Measurement of chemical substances is carried out in a wide range of fields including medical treatment, industrial processes, environment and the like. Various chemical substances exist in these fields, and in order to measure various chemical substances,
A variety of physical and chemical devices are used, and there is a strong demand for a method and a device that can directly and quickly measure these. However, chemical substances do not exist alone, and usually many kinds of substances are mixed. In order to select a specific chemical substance from such a system and measure it, a material and a reagent that identify these substances are necessary. For example, an enzyme that is a biocatalyst selectively promotes a reaction in a living body. The enzyme has a molecular recognition ability as well as a catalytic function. Therefore, by using this enzyme, a specific chemical substance can be identified and the concentration of the chemical substance can be measured.
【0002】本発明は、各種分野において用いられる測
定センサーとして有用であるだけではなく、臨床検査の
分野においても、種々の微量な生体試料中の特定成分に
ついて試料液を迅速かつ簡易に定量することのできる酵
素センサーに関するものである。INDUSTRIAL APPLICABILITY The present invention is not only useful as a measurement sensor used in various fields, but also in the field of clinical examination, it is possible to quickly and easily quantify a sample liquid for a specific component in various trace biological samples. It relates to an enzyme sensor that can be used.
【0003】[0003]
【従来の技術】各種化合物の測定において、酵素の高い
基質選択性を利用した酵素センサーの有用性が認められ
ており、殊にグルコースなどの定量分析においては、既
に実用化がなされている。現在実用化されている酵素セ
ンサーは、測定物質に対して高い基質特異性を有する酵
素を基材に固定化し、試料中の目的物質に触媒作用を司
る酵素が作用することにより、生成する水素イオン量又
は消費される酸素量を水素イオン電極又は酸素電極によ
り電気化学的に各々検出することにより定量している。
従って、このタイプの酵素センサーで利用できる酵素と
しては、反応により酸素を消費するか又は水素イオンを
生成する酸化酵素、いわゆるデヒドロゲナーゼのみ限定
されることになる。2. Description of the Related Art The usefulness of an enzyme sensor utilizing the high substrate selectivity of an enzyme has been recognized in the measurement of various compounds, and it has already been put to practical use particularly in the quantitative analysis of glucose and the like. The enzyme sensor currently in practical use is a hydrogen ion produced by immobilizing an enzyme having a high substrate specificity for a substance to be measured on a substrate, and the enzyme that controls the catalytic action of the target substance in the sample. The amount or the amount of oxygen consumed is quantified by electrochemically detecting with a hydrogen ion electrode or an oxygen electrode, respectively.
Therefore, the enzymes that can be used in this type of enzyme sensor are limited to oxidases that consume oxygen or generate hydrogen ions by the reaction, so-called dehydrogenases.
【0004】例えば、脱水素酵素を膜中に包括固定化し
隔膜酸素電極上に装着しセンサーを構成できる。この脱
水素の作用で基質が脱水素されるので、酵素膜に密着さ
せてある酸素電極で電流値の変化から基質の濃度を測定
することができる。また、この酸素電極の代わりに水素
イオン電極でこれを測定しても同様に基質濃度を求める
ことができる。For example, a sensor can be constructed by entrapping and immobilizing dehydrogenase in a membrane and mounting it on a diaphragm oxygen electrode. Since the substrate is dehydrogenated by the action of this dehydrogenation, the concentration of the substrate can be measured from the change in the current value with the oxygen electrode that is in close contact with the enzyme membrane. Also, the substrate concentration can be obtained in the same manner by measuring this with a hydrogen ion electrode instead of this oxygen electrode.
【0005】これらの電極反応は電気化学 (酸化還元)
的メカニズムに基づく。基質の種類によっては、酵素と
基質が容易に反応しない系もあり、メディエータ、補酵
素を必要とする場合もある。さまざまな系に関して研究
が進められている。例えば、人工のメディエータを電極
表面に薄膜状に塗布し、更にその上を半透膜で皮膜する
方法も提案されている (EP78636B1)。また、難水溶性の
メディエータを電極剤中に含有させたり (Agric. Biol.
Chem., 52, 1557(1988)) 、水溶性の高いメディエータ
の場合には、電極に予めメディエータを含有させた後、
イオン性高分子と酵素からなる混合物を電極表面に薄膜
として形成させることにより、電解液中にメディエータ
が溶出しないようにする方法 (Agric. Biol. Chem.,52,
3187(1988)) が提案されている。These electrode reactions are electrochemical (redox)
Based on the dynamic mechanism. Depending on the type of substrate, there are systems in which the enzyme and substrate do not react easily, and there are cases where mediators and coenzymes are required. Research is progressing on various systems. For example, a method has been proposed in which an artificial mediator is applied to the surface of the electrode in a thin film form, and the surface of the electrode is coated with a semipermeable membrane (EP78636B1). In addition, a poorly water-soluble mediator may be contained in the electrode material (Agric. Biol.
Chem., 52 , 1557 (1988)), in the case of a highly water-soluble mediator, after including the mediator in the electrode in advance,
By forming a mixture of an ionic polymer and an enzyme as a thin film on the electrode surface, a method of preventing the mediator from eluting in the electrolytic solution (Agric. Biol. Chem., 52 ,
3187 (1988)) has been proposed.
【0006】しかしながら、いずれの場合にも電極の調
製が煩雑であり、酵素はメディエータを含有させた後の
電極表面に薄膜を形成するように用いられている。However, in any case, the preparation of the electrode is complicated, and the enzyme is used so as to form a thin film on the surface of the electrode after containing the mediator.
【0007】[0007]
【発明が解決しようとする課題】前述のように、従来の
酵素修飾電極は調製方法が煩雑で測定機器として品質管
理された製品を量産するには困難な作業を必要とし、し
かも何回も繰り返して使用する際に電極自体の寿命が短
い上に、利用できる酵素が制限されるなど、種々問題が
あった。そこで電極表面を段階的にメディエータ薄膜
層、酵素薄膜層及び半透膜層で順次被膜する方法が従来
からとられてきたが、電極の調製が煩雑であるという問
題点を有していた。As described above, the conventional enzyme-modified electrode is complicated in its preparation method and requires a difficult work to mass-produce a quality-controlled product as a measuring instrument, and moreover, it is repeated many times. When used for a long time, there are various problems such as short life of the electrode itself and limitation of usable enzymes. Therefore, a method of coating the surface of the electrode step by step with a mediator thin film layer, an enzyme thin film layer and a semipermeable membrane layer has been conventionally used, but it has a problem that the preparation of the electrode is complicated.
【0008】そこで、これらの煩雑な方法に代えて、予
めスペーサーを介してメディエータを酵素と共有結合し
て形成された複合体を調製しておき、これを溶液成分と
し、共存する基質成分を作用電極、対向電極、参照電極
を組み合わせてなる電極系にて、試料液との反応に際し
て基質濃度変化を電気化学的に検知し、基質濃度を測定
することができるセンサーシステムを確立した。Therefore, instead of these complicated methods, a complex formed by previously covalently binding a mediator to an enzyme via a spacer is prepared in advance, and this is used as a solution component, and the coexisting substrate component acts. We have established a sensor system that can measure the substrate concentration by electrochemically detecting the change in the substrate concentration during the reaction with the sample solution in the electrode system that combines the electrode, the counter electrode and the reference electrode.
【0009】上記測定系では、可搬性以外にも簡便性、
即時性などに難点があるので我々の有する印刷技術を用
いて上記電極をコンパクトに作製すべく研究を行なっ
た。即ち、スペーサーを介してメディエータを酵素に共
有結合した複合体を電極成分とした酵素修飾電極を作用
電極とし、上記と同様に対向電極、参照電極と組み合わ
せてなる電極系にて試料液との反応に際して基質濃度変
化を電気化学的に電流値もしくは電位値を検知し、基質
濃度を測定することができるセンサーを確立した。ここ
で酵素修飾電極は、上記複合体 (メディエータ‐スペー
サー‐酵素) にグラファイト、カーボンペースト等のよ
うな通常の電極に利用されている導電体と適当なビヒク
ルを添加することにより均一組成物とし、この均一組成
物を電極基材 (例えばカーボン電極) として導電性を示
さない基材の表面にパターン化スクリーン印刷 (被覆)
するという簡便な方法による製法をとった。更に各種の
酵素についても検討を加え、酵素修飾電極の製造方法を
確立し、その酵素修飾電極を内蔵する優れた性能のセン
サーを提供することを目的として種々の研究を重ねた結
果、本発明を完成した。In addition to portability, the above-mentioned measurement system is simple,
Since there is a problem in immediacy, we conducted research to make the above electrodes compact by using our printing technology. That is, an enzyme-modified electrode having a complex in which a mediator is covalently bound to an enzyme via a spacer as an electrode component is used as a working electrode, and a reaction with a sample solution is performed in an electrode system that is combined with a counter electrode and a reference electrode as described above. At the same time, we established a sensor that can measure the substrate concentration by electrochemically detecting the current value or potential value of the change in substrate concentration. Here, the enzyme-modified electrode has a uniform composition by adding the above-mentioned complex (mediator-spacer-enzyme) to a conductor used in a normal electrode such as graphite and carbon paste, and an appropriate vehicle, This uniform composition is used as an electrode base material (for example, a carbon electrode) on the surface of a base material that does not show conductivity by patterned screen printing (coating).
The manufacturing method was adopted as a simple method. Furthermore, by studying various enzymes, the method for producing an enzyme-modified electrode was established, and various studies were conducted for the purpose of providing a sensor with excellent performance incorporating the enzyme-modified electrode. completed.
【0010】[0010]
【課題を解決するための手段】本発明の第1は、スペ−
サ−を介してメディエータとデヒドロゲナーゼを共有結
合させてなる修飾酵素である。本発明で用いるデヒドロ
ゲナーゼとしては、例えばフルクトースデビトロゲナー
ゼが挙げられる。かかるデヒドロゲナーゼとしては、好
ましくはデヒドロゲナーゼがアルコールデヒドロゲナー
ゼ、アルデヒドデヒドロゲナーゼ、グルコースデビドロ
ゲナーゼ、グルタミン酸デビドロゲナーゼ、コレステロ
ールデヒドロゲナーゼ、ソルビトールデヒドロゲナー
ゼ、グリセロールデヒドロゲナーゼが挙げられる。The first aspect of the present invention is to provide a space.
It is a modified enzyme obtained by covalently binding a mediator and dehydrogenase through a ser. Examples of the dehydrogenase used in the present invention include fructose devitrogenase. Examples of such dehydrogenase include alcohol dehydrogenase, aldehyde dehydrogenase, glucose devidogenase, glutamate devidogenase, cholesterol dehydrogenase, sorbitol dehydrogenase, and glycerol dehydrogenase.
【0011】また、メディエータとしては、例えばフェ
ロセン、フェロセン誘導体、p−ベンゾキノン、フェリ
シアン化カリウム、フェナジンメトサルフェート、2,
6−ジクロロフェノールインドフェノール、PQQ、F
AD、NAD、NADPが挙げられるが、特にフェロセ
ン、フェロセン誘導体が好ましい。かかるフェロセン誘
導体としては、例えば1,1'−ジメチルフェロセン、フ
ェロセン酢酸、ヒドロキシエチルフェロセン、1,1'−
ビス(ヒドロキシメチル)フェロセン、フェロセンモノ
カルボン酸、フェロセン1,1'−ジカルボン酸、クロロ
フェロセン、メチルトリメチルアミノフェロセンが挙げ
られる。Examples of mediators include ferrocene, ferrocene derivatives, p-benzoquinone, potassium ferricyanide, phenazine methosulfate, 2,
6-dichlorophenol indophenol, PQQ, F
Examples thereof include AD, NAD and NADP, with ferrocene and ferrocene derivatives being particularly preferred. Examples of the ferrocene derivative include 1,1′-dimethylferrocene, ferroceneacetic acid, hydroxyethylferrocene, and 1,1′-
Examples thereof include bis (hydroxymethyl) ferrocene, ferrocene monocarboxylic acid, ferrocene 1,1′-dicarboxylic acid, chloroferrocene, and methyltrimethylaminoferrocene.
【0012】酵素とメディエータとの組合せとしては、
特にフルクトースデヒドロゲナーゼとフェロセン又はフ
ェロセン誘導体との組合せが好ましい。本発明の修飾酵
素は、スペーサーを介してメディエータと酵素を共有結
合させることを特徴とする。スペーサーとしてはイソプ
レン、ブタジエン、アセチレン及びこれらの誘導体並び
に各々のコポリマー等が挙げられる。As a combination of the enzyme and the mediator,
A combination of fructose dehydrogenase and ferrocene or a ferrocene derivative is particularly preferable. The modified enzyme of the present invention is characterized in that the enzyme is covalently bound to the mediator via a spacer. Examples of the spacer include isoprene, butadiene, acetylene, their derivatives and their respective copolymers.
【0013】しかし、スペーサーとしては、酵素の化学
修飾の必要上次式で示されるジヒドラジト化合物が使用
され特に好ましいのはn=4のアジピルジヒドラジトで
ある。However, as the spacer, a dihydrazito compound represented by the following formula is used because of the necessity of chemical modification of the enzyme. Particularly preferred is n = 4 adipyl dihydrazito.
【0014】[0014]
【化2】 [Chemical 2]
【0015】本発明の第2は、前記本発明の修飾酵素を
含む溶液成分と、作用電極、対向電極及び参照電極から
なる電極系とからなるセンサーであり、本発明の第3
は、前記本発明の修飾酵素を電極成分とした酵素修飾電
極、対向電極及び参照電極からなる電極系からなるセン
サーである。本発明の第3である前記センサーとして
は、電極系が絶縁性基板上にスクリーン印刷で形成され
たカーボンを主体とする材料からなるセンサーが好まし
い。The second aspect of the present invention is a sensor comprising a solution component containing the modifying enzyme of the present invention and an electrode system comprising a working electrode, a counter electrode and a reference electrode.
Is a sensor including an electrode system including an enzyme-modified electrode having the above-described modified enzyme of the present invention as an electrode component, a counter electrode, and a reference electrode. The third sensor of the present invention is preferably a sensor whose electrode system is made of a material mainly composed of carbon formed by screen printing on an insulating substrate.
【0016】以下、酵素とメディエータおよびスペーサ
ーとの好ましい組合せであるフラクトースデヒドロゲナ
ーゼ(以下「FDH」という。)とフェロセン又はフェ
ロセン誘導体及びスペーサーとしてヒドラジドの組合せ
を例にとり、本発明を更に詳細に説明する。 この組合
せでは、下記の2点が重要である。 1. フェロセン又はフェロセン誘導体によるFDHの化
学修飾。The present invention will be described in more detail with reference to fructose dehydrogenase (hereinafter referred to as "FDH") which is a preferable combination of an enzyme, a mediator and a spacer, ferrocene or a ferrocene derivative and a hydrazide as a spacer. In this combination, the following two points are important. 1. Chemical modification of FDH with ferrocene or a ferrocene derivative.
【0017】2. フルクトースセンサー電極を用いる環
境のpHコントロール。 FDHは以下の反応を触媒する。:2. Environmental pH control using a fructose sensor electrode. FDH catalyzes the following reactions. :
【0018】[0018]
【化3】 [Chemical 3]
【0019】一方、フェロセンなどのメディエータは、
上式のアクセプターに置き代わって以下のように酸化還
元試薬として働く。On the other hand, mediators such as ferrocene are
It replaces the acceptor in the above formula and acts as a redox reagent as follows.
【0020】[0020]
【化4】 [Chemical 4]
【0021】バイオセンサーとして用いる際、酸化還元
試薬 (過酸化水素や他のメディエータ) というものは、
酵素のFADHサイトと電極の間の反応を往復し、酵素
から電極への測定が充分できる位の電流を生ずる程度の
電子を伝達を行なう。また、フェロセンは過酸化水素と
比べて以下に示す利点がある。 1) 溶液中の酸素濃度は変わりやすいにもかかわらず、
フェロセン濃度は容易にコントロール可能である。 2) フェロセンは過酸化水素と比べて低い電極電位でも
測定可能である。そのため、フェロセンを用いたバイオ
センサーでは、過酸化水素を用いた場合に比べて、類似
した電位で還元されるアスコルビン酸などの化合物から
の妨害に対して測定中に受ける影響が少ない。When used as a biosensor, a redox reagent (hydrogen peroxide or other mediator) is
The reaction between the FADH site of the enzyme and the electrode is reciprocated, and electrons are transferred from the enzyme to the electrode to such an extent that a sufficient current can be measured. In addition, ferrocene has the following advantages over hydrogen peroxide. 1) Although the oxygen concentration in the solution is variable,
The ferrocene concentration can be easily controlled. 2) Ferrocene can be measured even at a lower electrode potential than hydrogen peroxide. Therefore, a biosensor using ferrocene is less affected by interference from a compound such as ascorbic acid that is reduced at a similar potential during measurement, as compared with the case where hydrogen peroxide is used.
【0022】フェロセンは電極炭素へ吸着により固定化
できる。また、水に対して低い可溶性を示すために、電
極からそんなに早く溶けはしないが、多少のロスは避け
られない。更に、その固定化フェロセンへデヒドロゲナ
ーゼを接触させようというアプローチもあることはあ
る。しかしながら、もしデヒドロゲナーゼの炭素鎖(な
お、この炭素鎖は酵素より枝分かれしたアルキル鎖、糖
鎖、アミノ酸の側鎖などである。)へフェロセンが結合
できれば、その酵素活性は、蛋白質 (酵素) の活性部位
付近にフェロセンを結合させてその酵素反応を妨害して
しまう場合よりも影響が少なくて済む。酵素の炭素鎖に
フェロセンが結合していれば、そのフェロセンは酵素の
FADH反応部位と電極の間の反応を担うであろう。更
に充分 (もしくは過剰) な量のフェロセン分子が酵素及
びその酵素の持つ炭素鎖に結合 (修飾) できれば、逆に
その触媒能としての影響を与えることなく、また酵素の
FADHサイトと電極間の反応を往復することなく、酵
素自身が持つFADH反応部位から酵素の様々な異なる
部位に結合した数個のフェロセン分子あるいは電極へ、
電子の受け渡しが直接可能となろう。いわば、“導電性
酵素”を作製することができた。このメディエータ修飾
酵素を作製するに至った過程及び該修飾酵素自体が、本
発明の中心たる要点である。Ferrocene can be immobilized on the electrode carbon by adsorption. Also, since it has low solubility in water, it does not dissolve so quickly from the electrode, but some loss is unavoidable. Further, there may be an approach of contacting dehydrogenase with the immobilized ferrocene. However, if ferrocene can bind to the carbon chain of dehydrogenase (this carbon chain is an alkyl chain branched from the enzyme, sugar chain, side chain of amino acid, etc.), the enzyme activity will be the activity of the protein (enzyme). The effect is less than when ferrocene is bound near the site and interferes with the enzymatic reaction. If ferrocene is attached to the carbon chain of the enzyme, it will be responsible for the reaction between the FADH reaction site of the enzyme and the electrode. Furthermore, if a sufficient (or excess) amount of ferrocene molecule can bind (modify) to the enzyme and the carbon chain of the enzyme, it will not adversely affect its catalytic ability and also the reaction between the enzyme's FADH site and the electrode. Without going back and forth, from the FADH reaction site of the enzyme itself to several ferrocene molecules or electrodes bound to various different sites of the enzyme,
Electronic delivery will be possible directly. So to speak, "conductive enzyme" could be produced. The process leading to the production of the mediator modifying enzyme and the modifying enzyme itself are the main points of the present invention.
【0023】更に、実際の測定系にて予めスペーサーを
介してメディエータをFDHと共有結合して形成した複
合体を基質 (フルクトース)と反応させると、その複合
体が“導電性酵素”であることから、基質濃度に応じて
溶液系の電導度が大きく変わることが観測された。特に
FDHはPQQ型酵素であり補酵素(NADH)に直接
酵素反応が依存しない為に前記の現象を得るのには特に
好ましい酵素であった。この系をそのまま用い、更にカ
ーボンペーストを加え、スクリーン印刷法により電極パ
ターンを、ある基材 (プラスチックなど) 上に印刷す
る。このチップを使い捨て型バイオセンサーのアタッチ
メントとした。更に、機械本体 (リーダー) と該アタッ
チメントをセットでセンサーシステムとして構築した。Furthermore, when a complex formed by covalently binding a mediator to FDH via a spacer in advance in an actual measurement system is reacted with a substrate (fructose), the complex is a "conductive enzyme". From this, it was observed that the conductivity of the solution system greatly changed depending on the substrate concentration. In particular, FDH is a PQQ type enzyme, and since the enzyme reaction does not directly depend on coenzyme (NADH), it was a particularly preferable enzyme for obtaining the above phenomenon. Using this system as it is, carbon paste is further added, and an electrode pattern is printed on a certain substrate (plastic or the like) by a screen printing method. This chip was used as a disposable biosensor attachment. Furthermore, the machine body (reader) and the attachment were constructed as a set as a sensor system.
【0024】[0024]
【実施例】以下、実施例及び比較例により本発明を更に
具体的に説明するが、本発明の範囲はこれらの実施例に
限定されるものではない。EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples.
【0025】[0025]
【実施例1】 酵素の炭素鎖のみに修飾したフェロセン−FDHの調製
(図1(A)) (1)過ヨウ素酸ナトリウムによりFDHの炭素鎖を酸
化した。FDH (100mg)を0.1M、pH6.0のリン酸緩衝
液 (1ml) に溶かし、20mM過ヨウ素酸ナトリウム (予め
上記の緩衝液で溶かしておく) と混合した。酸化は、4
℃にて1時間で進み、続いてエチレングリコール (100
μl)を加えて反応を停止させた。その後、更に4℃、30
分してから、pH 6.0、0.1 Mリン酸緩衝液を4回交換し
ながら反応混合物を48時間以上かけて透析した。 (2)クロスリンキング試薬:アジピルジヒドラジドの
酸化炭素鎖への結合。Example 1 Preparation of ferrocene-FDH modified only on the carbon chain of the enzyme (FIG. 1 (A)) (1) The carbon chain of FDH was oxidized with sodium periodate. FDH (100 mg) was dissolved in 0.1 M pH 6.0 phosphate buffer (1 ml) and mixed with 20 mM sodium periodate (previously dissolved in the above buffer). Oxidation is 4
Proceed for 1 hour at ℃, followed by ethylene glycol (100
μl) was added to stop the reaction. Then, further 4 ℃, 30
After that, the reaction mixture was dialyzed for 48 hours or longer while the pH 6.0, 0.1 M phosphate buffer was changed 4 times. (2) Cross-linking reagent: Binding of adipyl dihydrazide to the oxidized carbon chain.
【0026】アジピルジヒドラジド (100mg 乾燥粉末)
を酸化したFDHの透析の済んだ液に溶かし、その溶液
を室温暗所にて一晩放置し反応させた。過剰のアジピル
ジヒドラジドを除くために、pH 6.0、0.1 Mリン酸緩衝
液を4回交換しながら反応混合物を透析した。 (3)酵素の炭素鎖に結合した未反応ヒドラジド基とフ
ェロセンカルボキシアルデヒドとの反応。Adipyl dihydrazide (100 mg dry powder)
Was dissolved in a dialyzed solution of oxidized FDH, and the solution was allowed to react overnight at room temperature in a dark place. The reaction mixture was dialyzed with four changes of pH 6.0, 0.1 M phosphate buffer to remove excess adipyl dihydrazide. (3) Reaction of unreacted hydrazide group bonded to carbon chain of enzyme with ferrocene carboxaldehyde.
【0027】フェロセンカルボキシアルデヒド (濃度1
mg/ml、pH 6.0、0.1 Mリン酸緩衝液2ml) をアジピル
ヒドラジド溶液 (2ml) に混合し、4℃暗所にて一晩反
応させた。シアノボロハイドライドナトリウムを10mMの
終濃度になるように混合し、1時間反応させ、そして、
pH 6.0、0.1 Mリン酸緩衝液を4回交換し、反応混合物
を透析して、過剰のフェロセンカルボキシアルデヒドを
除いた。Ferrocene carboxaldehyde (concentration 1
mg / ml, pH 6.0, 0.1 M phosphate buffer 2 ml) was mixed with an adipyl hydrazide solution (2 ml) and reacted overnight at 4 ° C. in the dark. Sodium cyanoborohydride was mixed to a final concentration of 10 mM, reacted for 1 hour, and
The pH 6.0, 0.1 M phosphate buffer was changed 4 times and the reaction mixture was dialyzed to remove excess ferrocene carboxaldehyde.
【0028】[0028]
【実施例2】 酵素の炭素鎖と酵素自身の両方に修飾したフェロセン−
FDHの調製(図1(B)) 二段階修飾酵素を調製するために下記の2手法を試みた
(方法A、方法B) 。 方法A 方法Aは、Y.DeganiとA.Heller (J. Physical Chemistr
y, 91, 1285-1289(1987))による手法を参考にした。本
方法では、フェロセンカルボン酸と蛋白質自身の遊離ア
ミノ基をカルボジイミド法にて反応させることにより、
FDH自身にフェロセンが結合される。2段階の修飾酵
素 (即ち蛋白質本体とその蛋白質炭素鎖の両方にフェロ
センを結合する) を得るために、実施例1と同様にして
炭素鎖の一部にアジピルジヒドラジドを初めに結合す
る。そして、フェロセンカルボン酸を蛋白質本体のアミ
ノ基と蛋白質炭素鎖の遊離ヒドラジド基の両方と反応さ
せる。 (1)実施例1(1)と同様にして過ヨウ素酸ナトリウ
ムでFDHの炭素鎖を酸化した。 (2)実施例1(2)と同様にしてアジピルジヒドラジ
ドを酸化したFDHに反応させた。 (3)蛋白質本体と蛋白質炭素鎖ヒドラジド基の両方の
アミノ基をフェロセンカルボン酸と反応させた。反応が
進むように、蛋白質をほどいてアミノ基を露出させるた
めに、反応混合物に尿素を混合した。尿素 (810mg)、水
溶性カルボジイミド (1−エチル−3−[3−ジメチル
アミノプロピル]カルボジイミドヒドロクロリド;100m
g)とフェロセンカルボン酸 (80mg) をpH 7.3、0.15M
N−2−ヒドロキシエチルピペラジン−N'−2−エタ
ンスルホン酸(以下「HEPES」という。)緩衝液 (4ml)
中でソニケーションし、pH 7.2ないし7.3 に再び合わ
せた。このpHでフェロセンカルボン酸を飽和させた。ア
ジピルヒドラジド溶液 (2ml)を加え、必要に応じてpH
を確認、調整し、反応物を0℃暗所にて一晩放置した。
過剰量の試薬は、pH 6.0、0.1 Mリン酸緩衝液を4回透
析交換して除いた。 方法B 方法Bでは、蛋白質側鎖の酸化した炭素鎖のアルデヒド
基 (-CHO) と蛋白質本体の遊離カルボキシル基 (-COOH)
の両方にアジピルジヒドラジドを反応させた。そしてフ
ェロセンアルデヒドは結合したヒドラジドと反応させ
た。 (1)実施例(1)と同様にして過ヨウ素酸ナトリウム
でFDH炭素鎖の一部を酸化した。 (2)カルボジイミド存在下で酸化FDHにアジピルジ
ヒドラジドを反応させた。 0.5Mアジピルジヒドラジ
ド、pH5に調整 (2ml) 、9mg塩化ナトリウム、酸化し
た酵素溶液 (2ml) と1M水溶性カルボジイミド (1m
l) および3M尿素を全て混ぜてpHを5に調整した。室
温暗所にて4時間放置して反応が進む:30分おきにpHを
5に調整した。4時間後、反応液をpH 6.0、0.1 Mリン
酸緩衝液で4回透析交換した。 (3)フェロセンカルボキシアルデヒドとの反応 FDHヒドラジド溶液 (2.5ml) を 0.4mg/mlフェロセ
ンカルボキシアルデヒド (2.5ml)と混ぜ、暗所4℃にて
4時間反応させた。終濃度0.1mMになるようにシアノボ
ロハイドライドナトリウムを加え、そしてpH 6.0、0.1
Mリン酸緩衝液で4回反応液を透析した。Example 2 Ferrocene-modified on both the carbon chain of the enzyme and the enzyme itself
Preparation of FDH (Fig. 1 (B)) The following two methods were tried in order to prepare a two-step modified enzyme.
(Method A, Method B). Method A Method A is based on Y. Degani and A. Heller (J. Physical Chemistr
The method by Y, 91 , 1285-1289 (1987) was referred to. In this method, by reacting ferrocenecarboxylic acid with the free amino group of the protein itself by the carbodiimide method,
Ferrocene is bound to the FDH itself. In order to obtain a two-step modification enzyme (that is, to attach ferrocene to both the protein body and the carbon chain of the protein), adipyl dihydrazide is first attached to a part of the carbon chain in the same manner as in Example 1. Then, the ferrocenecarboxylic acid is reacted with both the amino group of the protein body and the free hydrazide group of the protein carbon chain. (1) The carbon chain of FDH was oxidized with sodium periodate in the same manner as in Example 1 (1). (2) In the same manner as in Example 1 (2), the adipyl dihydrazide was reacted with oxidized FDH. (3) Both the amino groups of the protein body and the protein carbon chain hydrazide group were reacted with ferrocenecarboxylic acid. Urea was mixed into the reaction mixture to unwind the protein and expose the amino groups so that the reaction proceeded. Urea (810mg), water-soluble carbodiimide (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride; 100m
g) and ferrocenecarboxylic acid (80 mg) at pH 7.3, 0.15M
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (hereinafter referred to as "HEPES") buffer solution (4 ml)
Sonicated in and re-adjusted to pH 7.2-7.3. Ferrocenecarboxylic acid was saturated at this pH. Add adipyl hydrazide solution (2 ml) and adjust pH if necessary
Was confirmed and adjusted, and the reaction product was left overnight at 0 ° C. in the dark.
The excess amount of reagent was removed by dialysis exchange with a pH 6.0, 0.1 M phosphate buffer solution four times. Method B In method B, the aldehyde group (-CHO) of the oxidized carbon chain of the protein side chain and the free carboxyl group (-COOH) of the protein body
Both were reacted with adipyl dihydrazide. The ferrocene aldehyde was then reacted with the bound hydrazide. (1) A portion of the FDH carbon chain was oxidized with sodium periodate in the same manner as in Example (1). (2) Oxidized FDH was reacted with adipyldihydrazide in the presence of carbodiimide. 0.5M adipyl dihydrazide, adjusted to pH 5 (2ml), 9mg sodium chloride, oxidized enzyme solution (2ml) and 1M water-soluble carbodiimide (1m
The pH was adjusted to 5 by mixing all of 1) and 3M urea. The reaction proceeds by leaving it at room temperature in the dark for 4 hours: the pH is adjusted to 5 every 30 minutes. After 4 hours, the reaction solution was dialyzed and exchanged four times with 0.1 M phosphate buffer (pH 6.0). (3) Reaction with ferrocene carboxaldehyde FDH hydrazide solution (2.5 ml) was mixed with 0.4 mg / ml ferrocene carboxaldehyde (2.5 ml) and reacted at 4 ° C in the dark for 4 hours. Add sodium cyanoborohydride to a final concentration of 0.1 mM, and adjust the pH to 6.0, 0.1.
The reaction solution was dialyzed 4 times with M phosphate buffer.
【0029】また、方法AおよびBによって最終的に得
られたフェロセン−FDHの電気化学的応答から判断し
て、両法に大差はないことが確認された。Also, it was confirmed that there is no great difference between the two methods, judging from the electrochemical response of ferrocene-FDH finally obtained by methods A and B.
【0030】[0030]
【実施例3】 酵素自身のみに修飾したフェロセン−FDH(図1
(C)) 既に、“課題を解決するための手段”の項で言及した
が、上記修飾部位による違い (実施例1及び2 )と比較
参考するために、酵素自身のみにメディエータを修飾し
たフェロセン−FDHも調製した。作製方法は実施例2
の方法Aに準じているが、フェロセンカルボン酸と蛋白
質自身の遊離アミノ基がカルボジイミド法にて反応する
ことにより、FDH自身にフェロセンが結合できる。Example 3 Ferrocene-FDH (Fig. 1) modified only on the enzyme itself.
(C)) As already mentioned in the section “Means for solving the problem”, in order to compare with the difference due to the above modification site (Examples 1 and 2), ferrocene in which the mediator is modified only in the enzyme itself. -FDH was also prepared. The manufacturing method is Example 2
According to the method A, the ferrocene can be bound to FDH itself by reacting the ferrocenecarboxylic acid with the free amino group of the protein itself by the carbodiimide method.
【0031】蛋白質本体のアミノ基をフェロセンカルボ
ン酸と反応させた。反応が進むように、蛋白質をほどい
てアミノ基を露出させるために、反応混合物に尿素を混
合した。尿素 (810mg)、水溶性カルボジイミド (100mg)
とフェロセンカルボン酸 (80mg) をpH 7.3、0.15M HEP
ES緩衝液 (4ml) 中でソニケーションし、pH 7.2ないし
7.3 に再び合わせた。このpHでフェロセンカルボン酸を
飽和させた。アジピルヒドラジド溶液 (2ml) を加え、
必要に応じてpHを確認、調整し、反応物を0℃暗所にて
一晩放置した。過剰量の試薬は、pH 6.0、0.1 Mリン酸
緩衝液を4回透析交換して除いた。The amino group of the protein body was reacted with ferrocenecarboxylic acid. Urea was mixed into the reaction mixture to unwind the protein and expose the amino groups so that the reaction proceeded. Urea (810mg), water-soluble carbodiimide (100mg)
And ferrocenecarboxylic acid (80mg) at pH 7.3, 0.15M HEP
Sonicate in ES buffer (4 ml), pH 7.2 to
Retuned to 7.3. Ferrocenecarboxylic acid was saturated at this pH. Add adipyl hydrazide solution (2 ml),
The pH was checked and adjusted as needed and the reaction was left overnight in the dark at 0 ° C. The excess amount of reagent was removed by dialysis exchange with a pH 6.0, 0.1 M phosphate buffer solution four times.
【0032】〔比較例1〕 酵素を修飾せずにメディエータと基質を混在させた系 実施例1〜3の有意性を明らかにするため、これらの系
において用いたフェロセン修飾酵素に代えて、修飾しな
いFDHを用いた。更に、メディエータとしてフェロセ
ンを混在させて下記の電気化学的評価に供した。[Comparative Example 1] System in which a mediator and a substrate are mixed without modifying the enzyme In order to clarify the significance of Examples 1 to 3, instead of the ferrocene modifying enzyme used in these systems, modification was performed. No FDH was used. Further, ferrocene was mixed as a mediator and subjected to the following electrochemical evaluation.
【0033】各測定系のFDH及びフェロセンの濃度な
どの条件をなるべく同一に揃える目的で、実施例1記載
の試薬処方を参考にし、以下のように調製した。 条件 FDH(非修飾) 100mg フェロセン 1mg リン酸緩衝液(pH 6.0、0.1 M) 30ml カタラーゼ(実施例4参照) 3mg フルクトース(存在又は不在) 10mM 又は0mM 本測定系(比較例1)にて、応答が極めて低く観測され
た。この理由としては、メディエータたるフェロセン分
子が溶液中に均一に存在しており、FDHと酸化還元反
応に寄与するフェロセン分子の数がかなり少ないことが
考えられる。これに対して実施例1〜3の各系ではフェ
ロセン分子がFDHの表面又は周辺に局在化しているの
で本測定系と比べてフェロセン分子が相当効率的に酸化
還元反応に寄与しているために、これらの差異が認めら
れたものと考えられる。For the purpose of making the conditions such as FDH and ferrocene concentrations of each measurement system as uniform as possible, the reagent formulation described in Example 1 was referred to and prepared as follows. Conditions FDH (unmodified) 100 mg Ferrocene 1 mg Phosphate buffer (pH 6.0, 0.1 M) 30 ml Catalase (see Example 4) 3 mg Fructose (presence or absence) 10 mM or 0 mM Response in this measurement system (Comparative Example 1) Was observed to be extremely low. The reason for this may be that the ferrocene molecules that are mediators are uniformly present in the solution, and the number of ferrocene molecules that contribute to FDH and the redox reaction is considerably small. On the other hand, in each system of Examples 1 to 3, since the ferrocene molecule is localized on the surface of FDH or in the periphery thereof, the ferrocene molecule contributes to the redox reaction considerably efficiently as compared with the present measurement system. It is probable that these differences were recognized in the above.
【0034】[0034]
【実施例4】 修飾FDHの評価 金作用電極 (直径1mm) 、プラチナ対向電極、Ag/AgCl
参照電極を用いたサイクリックボルタモメトリでフェロ
セン修飾FDHを評価した。使用前に酸化アルミニウム
(0.075μm) で作用電極を研磨した。北斗電子ポテンシ
オシュタット/ガルバノシュタットモデル HAB-151とグ
ラフテックWX1200記録計で測定を行なった。Example 4 Evaluation of Modified FDH Gold working electrode (diameter 1 mm), platinum counter electrode, Ag / AgCl
The ferrocene-modified FDH was evaluated by cyclic voltammetry with a reference electrode. Aluminum oxide before use
The working electrode was polished with (0.075 μm). Hokuto Denshi Potentiostat / Galvanostat model HAB-151 and Graphtec WX1200 recorder were used.
【0035】修飾酵素溶液は実施例1〜3に従い、希釈
することなく用いた。過酸化水素の妨害を避けるため
に、終濃度で 0.1mg/mlのカタラーゼを加えた。最初
に、酵素のサイクリックボルタモグラムをフルクトース
不在下で測定した。そして最終濃度で10mMになるように
フルクトース溶液を加えてから、全く同一条件でサイク
リックボルタモグラムを再び計った。また酸素の影響を
除くため、窒素によるバブリングを充分に行なった。The modified enzyme solution was used without dilution according to Examples 1-3. Catalase at a final concentration of 0.1 mg / ml was added to avoid interference with hydrogen peroxide. First, the cyclic voltammogram of the enzyme was measured in the absence of fructose. Then, a fructose solution was added so that the final concentration was 10 mM, and the cyclic voltammogram was measured again under exactly the same conditions. Further, in order to eliminate the influence of oxygen, bubbling with nitrogen was sufficiently performed.
【0036】実施例1、実施例2(方法A)及び実施例
3で得られたフェロセン修飾FDHを用いた場合のサイ
クリックボルタモグラムを測定したところ基質(フルク
トース)を加えることにより導電率の向上がみられた。The cyclic voltammograms of the ferrocene-modified FDH obtained in Examples 1, 2 (method A) and 3 were measured, and the conductivity was improved by adding the substrate (fructose). It was seen.
【0037】[0037]
【実施例5】 スクリーン印刷法による電極を用いた測定系 大日本印刷(株)により、特開平3-28199 号「体液成分
検査用試験片」で代表されるような臨床検査材料が開発
されている。その他のものとしては、特開昭60-178356
号「体液検査体」、特開昭60-238763 号「体液検査
体」、特開昭61-247967 号「ブドウ糖検出用検査体及び
その製造方法」、特開昭61-284661 号「ブドウ糖検出用
検査体」及び特開昭62-263468 号「ブドウ糖検出用イン
キ組成物および検査体」がある。これらの発明のあるも
のは、酵素を複数種類含む組成物をインキとし、スクリ
ーン印刷法による尿検査紙の製造法に関して詳細に言及
している。その応用として、呈色指示薬など以下の過程
で必要としない成分を除く代わりに、導電性成分を混ぜ
るなどして表1に示すような新しいインキ処方を開発し
た。他の印刷手段としては、グラビア印刷、ディスペン
サーによる塗布、ダイコートの使用が可能である。[Example 5] Measuring system using electrodes by screen printing method Dai Nippon Printing Co., Ltd. has developed a clinical test material typified by JP-A-3-28199 "Test piece for testing body fluid components". There is. Others include JP-A-60-178356.
No. "Body fluid test body", JP-A-60-238763 "Body fluid test body", JP-A-61-247967 "Glucose detection test body and its manufacturing method", JP-A-61-284661 "Glucose detection body""Inspectionbody" and JP-A-62-263468 "Glucose detection ink composition and inspection body". Some of these inventions make detailed reference to a method for producing a urine test paper by a screen printing method using a composition containing a plurality of kinds of enzymes as an ink. As its application, a new ink formulation as shown in Table 1 was developed by mixing a conductive component instead of removing a component such as a color change indicator that is not required in the following process. As other printing means, gravure printing, coating with a dispenser, or die coating can be used.
【0038】[0038]
【表1】 [Table 1]
【0039】スクリーン印刷法は既存の技術であり、ス
クリーンはテトロン、120 メッシュ、150 μm (膜厚)
の版を作製((株)ミノグループ)し、通常の条件で印
刷を行なった(特開昭61-284661号参照)。印刷後、直
ちに乾燥(60℃、5分)し、次いで、この印刷物を断裁
し、電極(チップ)として電気化学的測定に供した。印
刷パターンは、図2に示した(パターンA、パターン
B)。The screen printing method is an existing technology. The screen is Tetron, 120 mesh, 150 μm (film thickness).
Was prepared (Mino Group Co., Ltd.) and printing was carried out under normal conditions (see JP-A-61-284661). Immediately after printing, it was dried (60 ° C., 5 minutes), and then this printed material was cut and subjected to electrochemical measurement as an electrode (chip). The print patterns are shown in FIG. 2 (pattern A, pattern B).
【0040】パターンAは、図2よりインキA(修飾酵
素入り)により形成される一電極チップであり、同様に
パターンBは、インキA(修飾酵素入り)を作用電極、
インキB(酵素なし)を対向電極とする二電極チップで
ある。初めに一電極系で測定を行なった。即ち、測定系
は、既述の3電極方式であるが、該金作用電極に代えて
一電極チップを装填し、表2に示す電流値を得た。更
に、作用電極、対向電極を有する二電極チップ単独での
測定も同様に行なった。資料溶液は、フラクトース水溶
液、0mM 、5mM 、10mMの3水準を測定点とした。印刷、
乾燥後の電極パターンのサイズは、幅 3mm、長さ30mm、
厚さ100 μm であり、接点を取り出さない側から約20mm
を試料に浸して測定を行なった。Pattern A is a one-electrode chip formed of ink A (containing a modifying enzyme) from FIG. 2, and pattern B is a pattern of ink A (containing a modifying enzyme) as a working electrode.
This is a two-electrode chip using ink B (without enzyme) as a counter electrode. First, the measurement was performed with a one-electrode system. That is, although the measurement system is the three-electrode system described above, one electrode tip was loaded instead of the gold working electrode, and the current values shown in Table 2 were obtained. Furthermore, the same measurement was carried out using a two-electrode chip having a working electrode and a counter electrode. For the sample solution, three levels of fructose aqueous solution, 0 mM, 5 mM, and 10 mM were used as measurement points. printing,
The size of the electrode pattern after drying is 3 mm wide, 30 mm long,
It is 100 μm thick and is about 20 mm from the side where the
Was immersed in the sample for measurement.
【0041】[0041]
【表2】 [Table 2]
【0042】表2では、修飾酵素として実施例2(方法
A)で得られたフェロセン修飾FDHを用いた。また、
測定した電流値は、電位値が+450mV の時の作用電極、
対向電極間を流れる電流値である。また、印刷インキに
含まれる酵素量を容易に制御できるのでセンサーのダイ
ナミックレンジ及び感度も自由に選ぶことができる。二
電極系(パターンB)にて、ポータブルのフルクトース
スセンサーを作製できることが確かめられたので、実用
モデルとして、使い捨て型の前記のチップ(電極)をペ
ンタイプ(デジタル表示)の先端に装填するシステムを
も考案することができた。図3にペンタイプの実用モデ
ルを示す。In Table 2, the ferrocene-modified FDH obtained in Example 2 (method A) was used as the modifying enzyme. Also,
The measured current value is the working electrode when the potential value is +450 mV,
It is a current value flowing between the opposing electrodes. Moreover, since the amount of enzyme contained in the printing ink can be easily controlled, the dynamic range and sensitivity of the sensor can be freely selected. It has been confirmed that a portable fructose sensor can be produced with a two-electrode system (Pattern B). As a practical model, a system in which the disposable tip (electrode) is loaded on the tip of a pen type (digital display) I was able to devise. FIG. 3 shows a practical pen-type model.
【0043】[0043]
【発明の効果】本発明において、酵素とメディエータを
共有結合させることにより両者を物理的に強固に固定化
することが可能になった。これは、通常のセンサーや電
極系、電気化学系に極めて効率的に酵素を反応させるこ
とが可能となっただけでなく、反応系外へメディエータ
の漏出を防ぐことができる。すなわち従来のセンサーは
メディエーター(毒性の強いものが多い)が溶出すると
いう理由で体内での使用を禁じられいたが、本発明によ
り体内での使用が可能となった上に、既述のように酵素
表面に導電性を付与したので酸素が存在しない環境下で
の利用も容易に行なうことができるなど、さまざまな分
野において極めて有用である。さらに酵素表面をメディ
エータで充分にの修飾をおこなうことのできる本方法
は、従来のセンサーよりも高い感度を示す。INDUSTRIAL APPLICABILITY In the present invention, it becomes possible to physically and firmly immobilize the enzyme and the mediator by covalently bonding them. This not only makes it possible to react the enzyme with the ordinary sensor, electrode system, and electrochemical system extremely efficiently, but also can prevent the leakage of the mediator to the outside of the reaction system. That is, the conventional sensor is prohibited from being used in the body because the mediator (often highly toxic) elutes, but the present invention enables the use in the body, and as described above. Since the enzyme surface is provided with conductivity, it can be easily used in an environment where oxygen does not exist, and is extremely useful in various fields. Furthermore, the present method, which allows the enzyme surface to be sufficiently modified with a mediator, exhibits higher sensitivity than conventional sensors.
【0044】以上より、酵素ーメディエータ複合体、基
質を含む溶液系をスクリーン印刷法にて作製した電極系
で、(1)本来の“メディエータ”を併用にないです
む、(2)メディエータの漏出がないので例えばin viv
o での使用が可能である、(3)応答の感度が高い、
(4)反応は可逆的であり、安定した応答が得られる、
(5)不安定な酵素に化学修飾を施しているので溶媒な
どの変性要因に対して抵抗性を得た、(6)スクリーン
版など、刷版を工夫することによりさまざまな微細かつ
複雑な印刷パターンが設計できる、等の効果が認められ
る。From the above, an electrode system prepared by a screen printing method containing a solution system containing an enzyme-mediator complex and a substrate does not require (1) the original "mediator" to be used in combination, and (2) leakage of the mediator. So there is no such thing as in viv
can be used in (3) high sensitivity of response,
(4) The reaction is reversible, and a stable response can be obtained.
(5) Since unstable enzymes are chemically modified, they are resistant to denaturing factors such as solvents. (6) Various fine and complicated printing by devising printing plates such as screen plates. The effect that the pattern can be designed is recognized.
【図1】フェロセン−FDHの模式図。FIG. 1 is a schematic diagram of ferrocene-FDH.
【図2】電極チップの印刷パターンを示す図である。FIG. 2 is a diagram showing a print pattern of an electrode chip.
【図3】ペンタイプの実用モデルを示す図である。FIG. 3 is a diagram showing a pen-type practical model.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 C12Q 1/32 6807−4B 1/54 6807−4B ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location C12Q 1/32 6807-4B 1/54 6807-4B
Claims (8)
ペーサーを介して共有結合させてなる修飾酵素。1. A modified enzyme obtained by covalently binding a mediator and a dehydrogenase via a spacer.
ゲナーゼ、グルタミン酸デヒドロゲナーゼ、コレステロ
ールデヒドロゲナーゼ、アルデヒドデヒドロゲナーゼ、
グルコースデヒドロゲナーゼ、フラクトースデヒドロゲ
ナーゼ、ソルビトールデヒドロゲナーゼ及びグリセロー
ルデヒドロゲナーゼからなる群から選ばれるものである
請求項1記載の修飾酵素。2. The dehydrogenase is alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase,
The modified enzyme according to claim 1, which is selected from the group consisting of glucose dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase and glycerol dehydrogenase.
である請求項1又は2記載の修飾酵素。 【化1】 3. The modified enzyme according to claim 1, wherein the spacer is a dihydrazide compound represented by the following formula. [Chemical 1]
ン誘導体、ニコチンアミド誘導体、フラビン誘導体、キ
ノン又はその誘導体である請求項1記載の修飾酵素。4. The modified enzyme according to claim 1, wherein the mediator is ferrocene or a ferrocene derivative, a nicotinamide derivative, a flavin derivative, a quinone or a derivative thereof.
飾酵素を含む溶液成分と、作用電極、対向電極及び参照
電極からなる電極系からなるセンサー。5. A sensor comprising an electrode system comprising a working component, a counter electrode and a reference electrode, and a solution component containing the modifying enzyme according to claim 1.
飾酵素を電極成分とした酵素修飾電極、対向電極及び参
照電極からなる電極系からなるセンサー。6. A sensor comprising an electrode system comprising an enzyme-modified electrode containing the modified enzyme according to any one of claims 1 to 4 as an electrode component, a counter electrode and a reference electrode.
飾酵素を電極成分とした修飾酵素固定化電極、対向電極
及び参照電極からなる電極系からなるセンサー。7. A sensor comprising an electrode system comprising a modified enzyme-immobilized electrode using the modified enzyme according to any one of claims 1 to 4 as an electrode component, a counter electrode and a reference electrode.
で形成されたカーボンを主体とする材料からなる請求項
6記載のセンサー。8. The sensor according to claim 6, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4269552A JPH0690754A (en) | 1992-09-10 | 1992-09-10 | Enzyme modified through spacer with mediator and sensor involving the same modified enzyme |
EP93104952A EP0563795B1 (en) | 1992-03-31 | 1993-03-25 | Enzyme-immobilized electrode, composition for preparation of the same and electrically conductive enzyme |
DE69319771T DE69319771T2 (en) | 1992-03-31 | 1993-03-25 | Immobilized enzyme electrode, composition for its production and electrically conductive enzymes |
US08/357,987 US5804047A (en) | 1992-03-31 | 1994-12-16 | Enzyme-immobilized electrode, composition for preparation of the same and electrically conductive enzyme |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4269552A JPH0690754A (en) | 1992-09-10 | 1992-09-10 | Enzyme modified through spacer with mediator and sensor involving the same modified enzyme |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0690754A true JPH0690754A (en) | 1994-04-05 |
Family
ID=17473971
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4269552A Pending JPH0690754A (en) | 1992-03-31 | 1992-09-10 | Enzyme modified through spacer with mediator and sensor involving the same modified enzyme |
Country Status (1)
Country | Link |
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JP (1) | JPH0690754A (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1707635A1 (en) | 2005-03-07 | 2006-10-04 | Seiko Epson Corporation | Electrode substrate, detection device having the substrate, kit having the detection device, and detection method using the kit |
WO2007102347A1 (en) * | 2006-02-27 | 2007-09-13 | Sumitomo Electric Industries, Ltd. | Biosensor chip, biosensor system, and measuring device thereof |
JP2007232378A (en) * | 2006-02-27 | 2007-09-13 | Sumitomo Electric Ind Ltd | Biosensor system and measuring instrument |
JP2007232379A (en) * | 2006-02-27 | 2007-09-13 | Sumitomo Electric Ind Ltd | Biosensor chip |
JPWO2006104077A1 (en) * | 2005-03-29 | 2008-09-04 | シーシーアイ株式会社 | Biosensor |
US8343333B2 (en) | 2005-03-07 | 2013-01-01 | Seiko Epson Corporation | Electrode substrate, detection device equipped with electrode substrate, detection device kit and detection method using the kit |
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1992
- 1992-09-10 JP JP4269552A patent/JPH0690754A/en active Pending
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1707635A1 (en) | 2005-03-07 | 2006-10-04 | Seiko Epson Corporation | Electrode substrate, detection device having the substrate, kit having the detection device, and detection method using the kit |
US8343333B2 (en) | 2005-03-07 | 2013-01-01 | Seiko Epson Corporation | Electrode substrate, detection device equipped with electrode substrate, detection device kit and detection method using the kit |
JPWO2006104077A1 (en) * | 2005-03-29 | 2008-09-04 | シーシーアイ株式会社 | Biosensor |
JP4700687B2 (en) * | 2005-03-29 | 2011-06-15 | シーシーアイ株式会社 | Biosensor |
WO2007102347A1 (en) * | 2006-02-27 | 2007-09-13 | Sumitomo Electric Industries, Ltd. | Biosensor chip, biosensor system, and measuring device thereof |
JP2007232378A (en) * | 2006-02-27 | 2007-09-13 | Sumitomo Electric Ind Ltd | Biosensor system and measuring instrument |
JP2007232379A (en) * | 2006-02-27 | 2007-09-13 | Sumitomo Electric Ind Ltd | Biosensor chip |
US8012321B2 (en) | 2006-02-27 | 2011-09-06 | Sumitomo Electric Industries, Ltd. | Biosensor chip, biosensor system and measuring instrument thereof |
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