JPS63139249A - Sensor composed of immobilized enzyme composite - Google Patents
Sensor composed of immobilized enzyme compositeInfo
- Publication number
- JPS63139249A JPS63139249A JP61286364A JP28636486A JPS63139249A JP S63139249 A JPS63139249 A JP S63139249A JP 61286364 A JP61286364 A JP 61286364A JP 28636486 A JP28636486 A JP 28636486A JP S63139249 A JPS63139249 A JP S63139249A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- immobilized
- oxidase
- peroxidase
- immobilized enzyme
- 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
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 14
- 239000002131 composite material Substances 0.000 title abstract 2
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 108090000790 Enzymes Proteins 0.000 claims abstract description 25
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 21
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 12
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 12
- -1 heme compound Chemical class 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims description 10
- 239000000758 substrate Substances 0.000 abstract description 10
- 150000002978 peroxides Chemical class 0.000 abstract description 4
- 239000007795 chemical reaction product Substances 0.000 abstract description 2
- 238000006911 enzymatic reaction Methods 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 abstract 2
- 230000002255 enzymatic effect Effects 0.000 abstract 2
- 229940088598 enzyme Drugs 0.000 description 20
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 18
- 239000000126 substance Substances 0.000 description 18
- 239000000852 hydrogen donor Substances 0.000 description 10
- 238000004020 luminiscence type Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 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 7
- 239000008103 glucose Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 5
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 5
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 5
- OOIOHEBTXPTBBE-UHFFFAOYSA-N [Na].[Fe] Chemical compound [Na].[Fe] OOIOHEBTXPTBBE-UHFFFAOYSA-N 0.000 description 5
- 229960003767 alanine Drugs 0.000 description 5
- 229940099898 chlorophyllin Drugs 0.000 description 5
- 235000019805 chlorophyllin Nutrition 0.000 description 5
- 238000005206 flow analysis Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 229940076788 pyruvate Drugs 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 4
- IWYDHOAUDWTVEP-UHFFFAOYSA-N mandelic acid Chemical compound OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001917 fluorescence detection Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 235000020778 linoleic acid Nutrition 0.000 description 3
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- BMUDPLZKKRQECS-UHFFFAOYSA-K 3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid iron(3+) hydroxide Chemical compound [OH-].[Fe+3].[N-]1C2=C(C)C(CCC(O)=O)=C1C=C([N-]1)C(CCC(O)=O)=C(C)C1=CC(C(C)=C1C=C)=NC1=CC(C(C)=C1C=C)=NC1=C2 BMUDPLZKKRQECS-UHFFFAOYSA-K 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000006866 deterioration Effects 0.000 description 2
- 239000000386 donor Substances 0.000 description 2
- 229940109738 hematin Drugs 0.000 description 2
- 229940025294 hemin Drugs 0.000 description 2
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000013307 optical fiber Substances 0.000 description 2
- 239000005373 porous glass Substances 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LEGPZHPSIPPYIO-UHFFFAOYSA-N 3-Methoxyphenylacetic acid Chemical compound COC1=CC=CC(CC(O)=O)=C1 LEGPZHPSIPPYIO-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000003820 Lipoxygenases Human genes 0.000 description 1
- 108090000128 Lipoxygenases Proteins 0.000 description 1
- 102100030856 Myoglobin Human genes 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- LUNQZVCDZKODKF-PFVVTREHSA-L copper acetic acid (2S)-6-amino-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-(1H-imidazol-5-yl)propanoyl]amino]hexanoate (2S)-6-amino-2-[[(2S)-2-[(2-amino-1-oxidoethylidene)amino]-3-(1H-imidazol-5-yl)propanoyl]amino]hexanoate hydron Chemical compound [Cu+2].CC(O)=O.CC(O)=O.NCCCC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1.NCCCC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 LUNQZVCDZKODKF-PFVVTREHSA-L 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 235000012209 glucono delta-lactone Nutrition 0.000 description 1
- 229960003681 gluconolactone Drugs 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000021312 gluten Nutrition 0.000 description 1
- 108010038983 glycyl-histidyl-lysine Proteins 0.000 description 1
- 108010055756 hemalin Proteins 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011289 tar acid Substances 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、酸化酵素、該酸化酵素と連鎖的に反応する酵
素若しくは微生物と、ヘム化合物および／またはペルオ
キシダーゼとを同一担体上に固定化した固定化酵素複合
体からなるセンサーに関し、さらに詳しくは、シー材中
に含まれる着目基質を水素供ｔ３４体存在下において定
ユするのに、ａ効な酵素センサーに関する。Detailed Description of the Invention (Field of Industrial Application) The present invention provides an oxidizing enzyme, an enzyme or microorganism that reacts with the oxidizing enzyme in a chain reaction, and a heme compound and/or peroxidase, which are immobilized on the same carrier. The present invention relates to a sensor comprising an immobilized enzyme complex, and more particularly to an enzyme sensor that is effective in determining a substrate of interest contained in a sheet material in the presence of hydrogen donor T34.
（従来の技術）
酵素及び水素供与体を用いた各種物質の測定としては、
グルコースオキシターゼ（８１ｕｃｏｓｅ ｏｘｉ−ｄ
ａｓｅ）、アミノ酸オキシグーゼ（ａｍｉｎｏ ａｃｉ
ｄ ｏｘｉ−ｄａｓｅ）５の酸化酵素により生じた過酸
化水素を赤血塩やペルオキシダーゼ（ｐｅｒｏｘｉｄａ
ｓｅ）により水素供与体の一つであるルミノール（ｌｕ
ｍｉｌＩｏｌ）とｆｆＺさせ生じる発尤や、！−ヒドロ
キシフェニル酢酸（ｐ−１＋ｙｄｒｏｘｙｐｌ＋ｅｎｙ
ｌ ａｃｅＬｉｃ ａｃｉｔｌ ）と反応させ生じる蛍
光物質を測定することにより、基質であるグルコース（
ｇｌｕｃｏｓｃ）やアミノ酸を定量する方法が知られて
いる。(Prior art) Measurement of various substances using enzymes and hydrogen donors includes:
Glucose oxidase (81ucose oxi-d
ase), amino acid oxyguse (amino aci)
Hydrogen peroxide produced by the oxidase of doxi-dase 5 is treated with red blood salt or peroxidase.
luminol (lu se), which is one of the hydrogen donors.
milIol) and ffZ and the explosion that occurs! -Hydroxyphenylacetic acid (p-1+ydroxypl+eny
By measuring the fluorescent substance produced by reacting with the substrate glucose (
Methods for quantifying glucosc) and amino acids are known.
そして、上記方法に基づく種々の分析システムが考案さ
れているが、その構成はほとんど同一である。すなわち
、一つの流路に配置された一つ以上の固定化酸化１１ｙ
索カラムを通過し、基質が反応した際に生じる過酸化水
素を含む流体と、別の流路より流れてくる水素供与体と
赤血塩若しくはペルオキシダーゼを含む流体を混合通路
に導き攪拌し、生ずる発光や蛍光物質を検出器により検
知するもの、若しくは過酸化水素を含む流体と、別の流
路を流れてくる水素供与体を含む流体をペルオキシダー
ゼを固定化した混合流路に導き攪拌、反応させ、生じる
発光や蛍光物質を検出器により検知するものである。Various analysis systems based on the above method have been devised, but their configurations are almost the same. That is, one or more immobilized oxidants 11y arranged in one flow path
A fluid containing hydrogen peroxide, which is generated when the substrate reacts after passing through the hydrogen column, and a fluid containing hydrogen donor and red blood salt or peroxidase flowing from another channel are introduced into a mixing channel and stirred. A fluid that detects luminescence or fluorescent substances with a detector, or a fluid containing hydrogen peroxide and a fluid containing a hydrogen donor flowing through a separate channel are introduced into a mixing channel in which peroxidase is immobilized, and are stirred and reacted. , the generated luminescence and fluorescent substances are detected by a detector.
（発明が解決しようとする問題点）
しかし、このようなシステムは複数の流路を持つため、
混合のための流路やｖｉ数の送液ポンプ、流路切り替え
装置等が必要となり、非常に複雑な構成となる。また、
さらに生じる過酸化水素によす酸化酵素自体が酸化、変
性し、活性が低下してしまうといった問題がある。(Problem to be solved by the invention) However, since such a system has multiple flow paths,
A flow path for mixing, a vi-number liquid feeding pump, a flow path switching device, etc. are required, resulting in a very complicated configuration. Also,
Furthermore, there is a problem in that the oxidizing enzyme itself that reacts to the generated hydrogen peroxide is oxidized and denatured, resulting in a decrease in activity.
（問題点を解決するための手段）
本発明は、上述した問題点を解消するためになされたも
ので、装置の簡略化と＃素話性低下の防止が可能なばか
りでなく、高い精度で着目した基質を測定可能とするも
のであり、酸化酵素、訊酸化酵素と連鎖的に反応する酵
素若しくは微生物から少なくとも酸化酵素を選択し、こ
れとヘム化合物および／又はペルオキシダーゼとを同一
担体上に固定化したことを特徴とする。(Means for Solving the Problems) The present invention has been made to solve the above-mentioned problems, and it not only simplifies the device and prevents deterioration of voice quality, but also enables high accuracy. This method enables the measurement of the substrate of interest, and involves selecting at least an oxidase from among oxidases, enzymes that react in a chain reaction with oxidases, or microorganisms, and immobilizing this and heme compounds and/or peroxidase on the same carrier. It is characterized by the fact that it has become
（作 用）
本発明者らは、過酸化物を産物として生成するような酸
化酵素若しくは該酸化酵素と連鎖的に反応する酵素若し
くは微生物と、過酸化物を分解、反応させるヘム化合物
および／またはペルオキシダーゼを同一担体上に固定し
ておけば、生成した反応産物は連鎖的に反応する酵素ま
たは微生物により次々と反応し、酵素反応が非常に速や
かに進仔し定量性が高まるばかりでなく、最終的に生じ
る過酸化物は上記ｆｌｕ素群を侵襲する前に速やかに分
解され、酵素の活性低下を防ぐことも可能となり、さら
に装置を７しく簡略化することも可能となることを見い
出したゆ
すなわち、本発明は、酸化酵素若しくは該酸化酵素と連
鎖的に反応する酵素若しくは微生物を含む酵素と、ヘム
化合物および／またはペルオキシダーゼとを同一担体上
に固定化した固定化酵素複合体からなるセンサーであり
、水素（Ａ与体存在下において試料中に含まれる着目基
質を定量することが出来る。(Function) The present inventors have developed an oxidase that produces peroxide as a product, or an enzyme or microorganism that reacts in a chain reaction with the oxidase, and a heme compound and/or that decomposes and reacts peroxide. If peroxidase is immobilized on the same carrier, the reaction products produced will be reacted one after another by enzymes or microorganisms that react in a chain reaction, and the enzymatic reaction will not only proceed very quickly and improve quantitative performance, but also improve the final We have discovered that the peroxides generated during the process are quickly decomposed before they attack the flu element group, making it possible to prevent a decrease in enzyme activity and furthermore making it possible to simplify the device by 70%. That is, the present invention provides a sensor comprising an immobilized enzyme complex in which an oxidase, an enzyme that reacts in a chain reaction with the oxidase, or an enzyme containing a microorganism, and a heme compound and/or peroxidase are immobilized on the same carrier. It is possible to quantify the target substrate contained in a sample in the presence of hydrogen (A donor).
酸化酵素及ゾ酸化酵素と連鎖的に反応する酵素の例を着
目基質と対応させて示すと表１、表２に示すとおりであ
る。Examples of enzymes that react in a chain manner with oxidases and zo-oxidases are shown in Tables 1 and 2 in association with substrates of interest.
表 １
又、酸化酵素と連鎖的に反応する微生物を着目基質と対
応させて例示すると表３に示すとおりである。Table 1 In addition, Table 3 shows examples of microorganisms that react with oxidases in a chain manner in association with substrates of interest.
本発明においては、上記の酵素若しくは微生物の内から
少なくとも酸化酵素を選択し、これに該酸化酵素と連鎖
的に反応する＃素若しくは微生物を必要に応じて組み合
ｔせで用いる。In the present invention, at least an oxidizing enzyme is selected from among the enzymes or microorganisms mentioned above, and an element or a microorganism that reacts in a chain manner with the oxidizing enzyme is used in combination as necessary.
ヘム化合物としては例えば、ヘマチン（ｈｅｍａｔｉｎ
）、ヘミン（ｈｅｉ＊ｉｎ）、クロロフィリン鉄ナトリ
ウム塩（ｃｂｌｏｒｏｐｈｙｌｌｉｎ Ｆ ｅ−Ｎ ａ
５ａｌＬ）、ヘモグロビン（ｂｅｍｏｇｌｏｂｉｎ）
、ミオグロビン（ｍｙｏｇｌｏｂｉｎ）、チトクローム
Ｃ（ｃｈｙｔｃｒｏｍｅ Ｃ）等が例示できる。Examples of heme compounds include hematin.
), hemin (hei*in), chlorophyllin iron sodium salt (cbrorophyllin F e-Na
5alL), hemoglobin (bemoglobin)
, myoglobin, cytochrome C, and the like.
本発明においては、上記化合物の内から任意の一種又は
二種以上を選んで用いる。In the present invention, one or more of the above compounds are selected and used.
上記の酵素ならびに化合物を固定化する担体、および固
定化方法は、従来公知のものでよい。例えば、多孔性が
ラスと−ズ、アガロース（ａｇａｒｏｓｅ）・デキスト
ラン（ｄｅｘＬｒａｎ）、ポリスチレン（ｐｏｌｙｓｔ
ｙ−ｒｅｎｅ）ｈ！？の担体上に、シップ塩基結合法、
ノアゾニウムカップリング法、インシアナート誘導体へ
の結合法、縮合試薬を用いる方法等を用いて固定化する
。The carrier and immobilization method for immobilizing the above enzymes and compounds may be conventionally known ones. For example, porous materials such as glass, agarose, dextran, polystyrene, etc.
y-rene) h! ? Ship base binding method,
Immobilization is performed using a noazonium coupling method, a method of binding to an incyanate derivative, a method using a condensation reagent, or the like.
水素供与体としては、ベルオキシグーゼ反応に用いられ
ているものを適用すればよく、酸化により発色する物質
、蛍光を発する物質、化学発光を生じる物質あるいは色
１．蛍光、発光を失う物質のいずれを用いてもよい１例
えば、発色する物質としては、４．４’−ジアミノノフ
ェニルアミン（４，４’−ｄｉａ輪１ｎｏｄｉｐｈｅｎ
ｙｌ ａｍｉｎｅ）、ノアニジノン（ｄｉａ−ｎｉｓｉ
ｃｌｉｎｅ）等、蛍光を発する物質としでは、戸−ヒド
ロキシフェニル酸Ｒ（ｆ） −ｈｙｄｒｏｘｙｐｈｅｎ
ｙｌ ａｃｅｔｉａｃｉｄ）、４−ヒトｔ７斗シー３−
メトキシフェニル酢酸（４−ｈｙｄｒｏｘｙ−３−ｍｅ
ｔｈｏｘｙｐｈｅｎｙｌ ａｃｅｔｉｃ ａｃｉｄ）等
、化学発光を生じる物質としては、ルミノール（ｌｕ−
ｍｉｎｏｌ）、ロフィン（Ｉｏｐｈｉｎｅ）ｌが例示さ
れる。As the hydrogen donor, those used in the peroxyguse reaction may be used, such as a substance that develops color upon oxidation, a substance that emits fluorescence, a substance that produces chemiluminescence, or a color 1. Either a substance that loses fluorescence or luminescence may be used. For example, as a substance that develops color, 4,4'-diaminophenylamine (4,4'-dia ring 1nodiphen
yl amine), noanidinone (dia-nisi
Among the substances that emit fluorescence, such as R(f) -hydroxyphenyl
yl acetiacid), 4-human t7 tocy 3-
Methoxyphenylacetic acid (4-hydroxy-3-me
Examples of substances that produce chemiluminescence include luminol (lu-
Examples include Iophine l.
（実施例） 以下本発明の実施例を添付図面に基づき詳述する。(Example) Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
（実施例１）
１１図は本発明に基づく酵素センサーを示しており、水
素供与体として化学発光物質を泪いる光検知型の一例で
ある。（１）は固定化酵素複合体、（２）は空気抜き用
の穴を備える固定酵素複合体支持管である。（３）は光
繊維、（４）は被覆材、（５）は光電子倍増管、（６）
は光検出器を示している。(Example 1) Figure 11 shows an enzyme sensor based on the present invention, which is an example of a photodetection type that uses a chemiluminescent substance as a hydrogen donor. (1) is an immobilized enzyme complex, and (2) is an immobilized enzyme complex support tube provided with air vent holes. (3) is an optical fiber, (4) is a coating material, (5) is a photomultiplier tube, (6)
indicates a photodetector.
第２図は上述したセイサーを用いた測定システムの一例
で、該センサーの先端部（７）は３０℃に維持された恒
温水槽（８）中に配置される遮光容器（９）に挿入され
、遮光ＩＩ（１０）により厳密に遮光される。FIG. 2 shows an example of a measurement system using the above-mentioned sacer, in which the tip (7) of the sensor is inserted into a light-shielding container (9) placed in a thermostatic water bath (8) maintained at 30°C. The light is strictly blocked by the light shielding II (10).
該遮光容ｎ（９）内の溶液中には回転子（１１）が入れ
られており回軟磁場発生装置！ （１２）により溶液が
攪拌される。 （１３）は試料を注入するマイクロシリ
ンノ、（１４）は記録計である。A rotor (11) is placed in the solution in the light-shielding volume n(9), and is a rotating soft magnetic field generator! (12) stirs the solution. (13) is a micro cylinder for injecting the sample, and (14) is a recorder.
上記測定システムによりグルコースセンサーの特性を調
べた。固定化酵素複合体としては、グルコースオキシグ
ーゼとヘマチン（ｈｅｍａＬｉｎ）をノンクロへキシル
カルボッイミド（ｄｉｃｙｃｌｏｈｅｘｙｌ ｅａｒ−
ｂｏｄｉｉｍｉｄｅ）により固定化したものを用いた０
反応液には１０μｇ７ｍｌのルミノール（Ｉｕｍｉｎｏ
ｌ）を含むｏ、ｉＭホウ１Ｍ！緩１［１ｌ（ｒ’１ｌＩ
Ｏ）を用いた。The characteristics of the glucose sensor were investigated using the above measurement system. As an immobilized enzyme complex, glucose oxyguse and hematin (hemaLin) were combined with non-chlorohexylcarboimide (dicyclohexyl ear-
0 using immobilized with bodiimide)
The reaction solution contained 10 μg and 7 ml of Luminol.
l) including o, iMhou 1M! Loose 1[1l(r'1lI
O) was used.
グルコースは、グルコースオキシグーゼにより酸化され
、グルコノラクトン（ｇｌｕｃｏｎｏｌａｃｔｏｎｅ）
と過酸化水素に分解される。ここで生じる過酸化水素が
水系供与体であるルミノールとヘミンにより反応し発光
する。Glucose is oxidized by glucose oxygose and converted to gluconolactone.
and decomposes into hydrogen peroxide. The hydrogen peroxide produced here reacts with the aqueous donor luminol and hemin to emit light.
第３図は、発光強度と試料中に含まれるグルツース濃度
との関係を示すグラフであり、十分な定量性が得られた
。なお、発光強度は試料注入後１０秒から４０秒までの
積算値で示した。FIG. 3 is a graph showing the relationship between the luminescence intensity and the concentration of gluten contained in the sample, and sufficient quantitative performance was obtained. Note that the luminescence intensity was expressed as an integrated value from 10 seconds to 40 seconds after sample injection.
（実施例２）
次に本発明に基づくセンサーを用いた流れ分析システム
について詳述する。(Example 2) Next, a flow analysis system using a sensor based on the present invention will be described in detail.
第４図は、本発明に基づき、水素供与体として化学発光
性物質を用いた光検出型流れ分析システムの一例を示す
、 （１５）はガラス製蛇管で、固定化酵素複合体カラ
ム（１６）により反応、発光した試料液が通過し、光検
出器である光電子倍増管（１７）により検出される。ガ
ラス製蛇管（１５）、固定化酵素複合体カラム（１６）
、該光電子倍増Ｗ （１７）は、暗箱（１８）により厳
密に遮光される。 （１９）は光電子倍増管入出力ｉ置
、（２０）は試料導入部、（２１）は送液用ポンプであ
る。Figure 4 shows an example of an optical detection type flow analysis system using a chemiluminescent substance as a hydrogen donor based on the present invention. The sample liquid reacted and emitted light passes through the tube, and is detected by a photomultiplier tube (17), which is a photodetector. Glass tube (15), immobilized enzyme complex column (16)
, the photoelectron multiplier W (17) is strictly shielded from light by a dark box (18). (19) is a photomultiplier tube input/output position, (20) is a sample introduction part, and (21) is a liquid feeding pump.
上記分析システムにより不飽和脂肪酸センサーの特性を
調べた。固定化酵素複合体にはりボキシダーゼ（ｌｉｐ
ｏｘｉｄａｓｅ）とクロロフィリン鉄ナトリウム塩（ｃ
ｈｌｏｒｏｐｂｙｌｌｉｎ Ｆ ｅ−Ｎ ａ ５ａｌｔ
）を多孔性〃ラスビーＸにグルタルアルデヒド（ＦＩｌ
ｕｔａｒａｌｄｅｈｙｄｅ）により固定化したものを用
いた０反応液には、１０μｇ／ｚｊ！ルミ／−ルを含む
０，１Ｍグリシン緩衝液（ＰＨ１０）を用いた。試料と
しては精製したリノール酸を０．０５％トリトンＸ −
１００（ｔｒｉＬｏｎ Ｘ−１００、和光純薬株式会社
製）が含まれる０、１Ｍグリシン緩衝液（ＰＨＩＯ）に
所定量超音波処理し可溶化したものを用いた０反応温度
は３０℃、流速はｌｚ１／ｓｉｎ、試料注入１は１００
μｌとした。The characteristics of the unsaturated fatty acid sensor were investigated using the above analysis system. The immobilized enzyme complex contains boxidase (lip
oxidase) and chlorophyllin iron sodium salt (c
hloropbyllin F e-N a 5alt
) to porous Rusby-X with glutaraldehyde (FIl
10 μg/zz! A 0.1M glycine buffer (PH10) containing Lumi/-L was used. As a sample, purified linoleic acid was mixed with 0.05% Triton
100 (triLon /sin, sample injection 1 is 100
It was taken as μl.
第５図は発光強度とリノール酸濃度との関係を示すグラ
フであり、十分な定量性を示した。FIG. 5 is a graph showing the relationship between luminescence intensity and linoleic acid concentration, which showed sufficient quantitative properties.
（実施例３）
第６図は、本発明に基づき、水素供与体として蛍光性物
質を用いｒ−蛍光検出型流れ分析システムの一例を示し
ている。試料導入部（２２）より注入された試料は送液
用ポンプ（２３）により送られ、検出用カラム（２４）
と参照ｍカラム（２５）に流入し、蛍光性物質が生成す
る。この蛍光性物質は石英セル（２６）に送られ、蛍光
検出Ｗ＃（２））１こより検知される。（２８）は蛍光
検出器入出力装置で、（２９）は記録計である。(Example 3) FIG. 6 shows an example of an r-fluorescence detection type flow analysis system based on the present invention using a fluorescent substance as a hydrogen donor. The sample injected from the sample introduction part (22) is sent by the liquid pump (23), and is sent to the detection column (24).
and the reference m column (25), and a fluorescent substance is generated. This fluorescent substance is sent to a quartz cell (26) and detected by fluorescence detection W# (2). (28) is a fluorescence detector input/output device, and (29) is a recorder.
上記分析システムによりＬ−７ラニン（Ｌ−ａｌａ−ｎ
ｉｎｅ）センサーの特性を調べた。検出用固定化酵素複
合体としては、ア）エンアミ／トランスフェラーゼ（ａ
ｌａｎｉｎｅ ａｍｉ＋＋ｏＬｒａｎｓｆｅｒａｓｅ）
とピルビン酸オキシグーゼ（ｐｙｒｕｖａＬｅ ｏｘｉ
ｄａｓｅ）とクロロフィリン鉄ナトリウム塩を多孔性ガ
ラスピーズにジシクロヘキシルカルボシイミドにより固
定化したものを用いた。参照用固定化１［複合体として
は、ピルビン酸オキシグーゼとクロロフィリン鉄ナトリ
ウム塩を多孔性ガラスピーズに固定化したものを用いた
０反応液は、０．ＩＭリンｒＲ緩衝液にα−ケトゲルタ
ール酸（α−ｋｃＬｏＨｌｕｔａｒｉｃ ａｃｉｄ）
５ ｚＭ 。Using the above analysis system, L-7 lanin (L-ala-n)
ine) The characteristics of the sensor were investigated. The immobilized enzyme complex for detection includes a) enzyme/transferase (a)
lanine ami++oLransferase)
and pyruvate oxyguse (pyruvaLe oxi
dase) and chlorophyllin iron sodium salt were immobilized on porous glass beads with dicyclohexylcarbosiimide. Reference immobilization 1 [0 reaction solution using pyruvate oxyguse and chlorophyllin iron sodium salt immobilized on porous glass beads as a complex was 0. α-kcLoHlutaric acid in IM phosphorR buffer
5zM.
チアミンニリン酸（ｔｌ＋ｉａｍｉｎ Ｌｒ１ｐｈｏｓ
ｐｂａＬｅ）０．２ａＭ ％Ｆ Ａ Ｄ （ｆ １ａｖ
ｉｎ −ａｄｅｎｉｎｅ ｄｉｎｕｃｌｅｏｔｉｄｅ）
０．０２ｚＭ 。Thiamine diphosphoric acid (tl+iamin Lr1phos
pbaLe)0.2aM %F A D (f 1av
in -adenine dinucleotide)
0.02zM.
硫酸マグネシウム５ｍＭを加えたものに蛍光性水素供与
体である戸−ヒドロキシフェニル酢酸ｒ−ｈｙｄｒ＊ｘ
ｙｐｌｔｅｎｙｌ ａｃｅｔｉｃ ａｃｉｄ）５ ｘＭ
を加え、ＰＨ８，０に調整したものを用いた。検出ｍカ
ラムでは、Ｌ−アラニンが７ラニンアミノトランス７エ
ラーゼによりａ−ケトゲルタール酸と反応し、ピルビン
酸とＬ−グルタミン酸（ｇｌｕＬａｍｉｃ ａｃｉｄ）
になる。ここで生成したピルビン酸はピルビン酸オキシ
グーゼにより酸化されてアセチルリン酸（ａｃｅｔｙｌ
ｐｈｏ−ｓｐｈａｔｅ）と二酸化炭素、過酸化水素に
分解され、この過酸化水素はクロロフィリン鉄ナトリウ
ム塩により？−ヒドロキシフェニル酢酸と反応し蛍光を
発する物質となる。一方、参照ｍカラムでは試料中にピ
ルビン酸や過酸化水素が含まれる場合にそれぞれ反応が
進行し、蛍光を発する物質を与え、測定値を補正する。Fluorescent hydrogen donor hydroxyphenylacetic acid r-hydr*x to which 5mM magnesium sulfate was added
ypltenyl acetic acid) 5 xM
was added to adjust the pH to 8.0. In the detection m-column, L-alanine reacts with a-ketogel tar acid by 7-lanine aminotrans 7-elase, resulting in the formation of pyruvate and L-glutamic acid (gluLamic acid).
become. The pyruvate produced here is oxidized by pyruvate oxyguse to acetyl phosphate (acetyl phosphate).
pho-sphate), carbon dioxide, and hydrogen peroxide, and this hydrogen peroxide is decomposed by chlorophyllin iron sodium salt. -Reacts with hydroxyphenylacetic acid to become a fluorescent substance. On the other hand, in the reference m column, when pyruvic acid or hydrogen peroxide is contained in the sample, reactions proceed, providing substances that emit fluorescence, and correcting the measured values.
ｔＡ７図に相対蛍光強度とＬ−アラニン濃度との関係を
示すグラフで、ＯはＬ−アラニンだけを含む試料を用い
た場合で、・は１０〜５０μＭのピルビン酸を混入させ
た場合の結果である。いずれの場合も高い精度でＬ−ア
ラニンを検出することが出来た。Figure tA7 is a graph showing the relationship between relative fluorescence intensity and L-alanine concentration, where O is the result when using a sample containing only L-alanine, and . be. In both cases, L-alanine could be detected with high accuracy.
（発明の効果）
以上詳述した如く、本発明によれば、固定化酵素複合体
を用いることにより、Ｗｃｒｌｌが簡略化でさ、＃素話
性の低下がなく、かつ着目した基質を高い精度で定量す
ることが出来る。(Effects of the Invention) As detailed above, according to the present invention, by using an immobilized enzyme complex, Wcrll can be simplified, there is no deterioration in speech performance, and the substrate of interest can be detected with high precision. It can be quantified by
ｔｊＳｉ図は本発明に基づ（酵素センサーの一例、！５
２図は３１ＳＦ素センサーを用いた測定システムの一例
を示している。第３図は本発明に基づくグルコースセン
サーによる発光強度とグルツース濃度との関係を示すグ
ラフである。第４図は本発明に基づく光検出型流れ分析
システムの一例、ｆｆ５５図は本発明に基づく不飽和脂
肪酸センサーによる発光強度とリノール酸濃度の関係を
示すグラフである。ｆｆ１６図は本発明に基づく蛍光検
出型流れ分析システムの一例、７７７図は該分析システ
ムによる相対蛍光強度とＬ−アラニン濃度の関係を示す
グラフである。
（１）・・・固定化酵素複合体（２）・・・支持管（３
）・・・光繊維 （４）・・・被覆材（５）・
・・光電子倍増管 （６）・・・光検出器（７）・・
・センサー先端部 （８）・・・恒温水措（９）・・・
遮光容器 （１０）・・・遮光蓋（１１）・・・
回転７− （１２）・・・回転磁場発生装置
（１３）・・・マイクロシリンノ（１４）・・・記録計
（１５）・・・〃ラス製蛇管
（１６）・・・固定化酵素複合体カラム（１７）・・・
光電子倍増管 （１８）・・・ｖＪ 箱（１９）・
・・光電子倍増管入出力装置（２０）（２２）・・・試
料導入部 （２１）（２３）・・・送液用ポンプ（２４
）・・・検出用カラム （２５）・・・参照用カラム
（２６）・・・石英セル （２７）・・・蛍光検
出器（２８）・・・蛍光検出器入出力装置
（２９）・・・記録計
特許出頴人 株式会社 資 生 堂〃
東京工業大学艮田中郁二
第１図
第２図
第ケ図
（８児ｉｔ ＆ ｘ ｔｏ’ ｃｐ３
町
園
４１ＩＣｔ 速ｔ Ｘ １０’ｃｏｕｎｔｓヨ
（〇
四The tjSi diagram is based on the present invention (an example of an enzyme sensor, !5
Figure 2 shows an example of a measurement system using a 31SF elementary sensor. FIG. 3 is a graph showing the relationship between luminescence intensity and glucose concentration by the glucose sensor based on the present invention. FIG. 4 is an example of the optical detection type flow analysis system based on the present invention, and FIG. ff55 is a graph showing the relationship between luminescence intensity and linoleic acid concentration by the unsaturated fatty acid sensor based on the present invention. Figure ff16 is an example of the fluorescence detection type flow analysis system based on the present invention, and Figure 777 is a graph showing the relationship between relative fluorescence intensity and L-alanine concentration using the analysis system. (1) Immobilized enzyme complex (2) Support tube (3
)...Optical fiber (4)...Coating material (5)・
...Photomultiplier tube (6)...Photodetector (7)...
・Sensor tip (8)... Constant temperature water treatment (9)...
Light-shielding container (10)... Light-shielding lid (11)...
Rotation 7- (12)... Rotating magnetic field generator (13)... Micro cylinder (14)... Recorder (15)... Glass flexible tube (16)... Immobilized enzyme complex Body column (17)...
Photomultiplier tube (18)...vJ box (19)
...Photomultiplier tube input/output device (20) (22) ...Sample introduction section (21) (23) ...Liquid feeding pump (24)
)...Detection column (25)...Reference column (26)...Quartz cell (27)...Fluorescence detector (28)...Fluorescence detector input/output device (29)...・Recorder patent issuer Shiseido Co., Ltd.
Tokyo Institute of Technology Ikuji Hastanaka Figure 1 Figure 2 Figure ke (8 children it & x to' cp3 Machizono 41 ICt Speed t
Claims (4)
しくは微生物から少なくとも酸化酵素を選択し、これと
ヘム化合物および／又はペルオキシダーゼとを同一担体
上に固定化した固定化酵素複合体からなるセンサー。(1) An immobilized enzyme complex consisting of an oxidase, an enzyme that reacts with the oxidase in a chain reaction, or at least an oxidase selected from microorganisms, and a heme compound and/or peroxidase immobilized on the same carrier. sensor.
ダーゼとを同一担体上に固定したことを特徴とする特許
請求の範囲第１項記載のセンサー。(2) The sensor according to claim 1, characterized in that an oxidase, a heme compound and/or a peroxidase are immobilized on the same carrier.
と、ヘム化合物および／又はペルオキシダーゼとを同一
担体上に固定化したことを特徴とする特許請求の範囲第
１項記載のセンサー。(3) The sensor according to claim 1, characterized in that an oxidase, an enzyme that reacts in a chain manner with the oxidase, and a heme compound and/or peroxidase are immobilized on the same carrier.
物と、ヘム化合物および／又はペルオキシダーゼとを同
一担体上に固定化したことを特徴とする特許請求の範囲
１項記載のセ ンサー。(4) The sensor according to claim 1, wherein an oxidase, a microorganism that reacts in a chain reaction with the oxidase, and a heme compound and/or peroxidase are immobilized on the same carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61286364A JPS63139249A (en) | 1986-12-01 | 1986-12-01 | Sensor composed of immobilized enzyme composite |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61286364A JPS63139249A (en) | 1986-12-01 | 1986-12-01 | Sensor composed of immobilized enzyme composite |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63139249A true JPS63139249A (en) | 1988-06-11 |
Family
ID=17703425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61286364A Pending JPS63139249A (en) | 1986-12-01 | 1986-12-01 | Sensor composed of immobilized enzyme composite |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63139249A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5641390A (en) * | 1993-05-20 | 1997-06-24 | Oxford Glycosystems Ltd. | Labelled sugars immobilized on a solid support |
| CN113249426A (en) * | 2021-05-11 | 2021-08-13 | 四川大学 | Preparation method of collagen-based glucose fluorescent sensitive membrane |
-
1986
- 1986-12-01 JP JP61286364A patent/JPS63139249A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5641390A (en) * | 1993-05-20 | 1997-06-24 | Oxford Glycosystems Ltd. | Labelled sugars immobilized on a solid support |
| CN113249426A (en) * | 2021-05-11 | 2021-08-13 | 四川大学 | Preparation method of collagen-based glucose fluorescent sensitive membrane |
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