JPH01239449A - Preparation of enzyme immobilizing film - Google Patents
Preparation of enzyme immobilizing filmInfo
- Publication number
- JPH01239449A JPH01239449A JP63069138A JP6913888A JPH01239449A JP H01239449 A JPH01239449 A JP H01239449A JP 63069138 A JP63069138 A JP 63069138A JP 6913888 A JP6913888 A JP 6913888A JP H01239449 A JPH01239449 A JP H01239449A
- Authority
- JP
- Japan
- Prior art keywords
- membrane
- enzyme
- film
- aminosilane
- side chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 30
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 30
- 230000003100 immobilizing effect Effects 0.000 title abstract 5
- 238000002360 preparation method Methods 0.000 title description 6
- 239000004743 Polypropylene Substances 0.000 claims abstract description 24
- 229920001155 polypropylene Polymers 0.000 claims abstract description 24
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims abstract description 24
- -1 polypropylene Polymers 0.000 claims abstract description 22
- 229940081735 acetylcellulose Drugs 0.000 claims abstract description 18
- 229920002301 cellulose acetate Polymers 0.000 claims abstract description 18
- SMEGJBVQLJJKKX-HOTMZDKISA-N [(2R,3S,4S,5R,6R)-5-acetyloxy-3,4,6-trihydroxyoxan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@@H](O1)O)OC(=O)C)O)O SMEGJBVQLJJKKX-HOTMZDKISA-N 0.000 claims abstract description 17
- 239000004094 surface-active agent Substances 0.000 claims abstract description 17
- 150000001412 amines Chemical class 0.000 claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 125000000524 functional group Chemical group 0.000 claims abstract description 10
- 239000012528 membrane Substances 0.000 claims description 108
- 238000000034 method Methods 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 15
- 125000003277 amino group Chemical group 0.000 abstract description 10
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 239000011159 matrix material Substances 0.000 abstract description 7
- 230000000903 blocking effect Effects 0.000 abstract description 5
- 229910000077 silane Inorganic materials 0.000 abstract description 5
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 abstract description 4
- 238000004132 cross linking Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 description 26
- 229940088598 enzyme Drugs 0.000 description 24
- 230000002452 interceptive effect Effects 0.000 description 19
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 238000004737 colorimetric analysis Methods 0.000 description 7
- VVJKKWFAADXIJK-UHFFFAOYSA-N Allylamine Chemical compound NCC=C VVJKKWFAADXIJK-UHFFFAOYSA-N 0.000 description 6
- 108010015776 Glucose oxidase Proteins 0.000 description 6
- 239000004366 Glucose oxidase Substances 0.000 description 6
- 229940116332 glucose oxidase Drugs 0.000 description 6
- 235000019420 glucose oxidase Nutrition 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 5
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 4
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 4
- 235000010323 ascorbic acid Nutrition 0.000 description 4
- 229960005070 ascorbic acid Drugs 0.000 description 4
- 239000011668 ascorbic acid Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229940116269 uric acid Drugs 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108010025188 Alcohol oxidase Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- LJCNDNBULVLKSG-UHFFFAOYSA-N 2-aminoacetic acid;butane Chemical compound CCCC.CCCC.NCC(O)=O LJCNDNBULVLKSG-UHFFFAOYSA-N 0.000 description 1
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- QBUATDNGCWHUGM-UHFFFAOYSA-N N'-(3-dimethoxysilylbutyl)ethane-1,2-diamine Chemical compound CC(CCNCCN)[SiH](OC)OC QBUATDNGCWHUGM-UHFFFAOYSA-N 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 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
- 150000004756 silanes Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、体液、体組織、食品に含まれるグルコース、
尿素、トリグリセリド、りん脂質、アルコールなどの微
量成分を酵素を利用して選択的に定量するのに使用され
る酵素固定膜の調製方法に関するものである。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to glucose contained in body fluids, body tissues, and foods.
The present invention relates to a method for preparing an enzyme-immobilized membrane used to selectively quantify trace components such as urea, triglycerides, phospholipids, and alcohols using enzymes.
酵素固定膜を備えた電極（酵素膜電極）を、被検液と接
触させると、酵素反応によって電極感知物質（例えば、
過酸化水素）が生成し、この感知物質を電流量として検
知することにより、被検液中の特定物質の量を測定する
ことができる。例えば、血液、尿や食品中のグルコース
を測定する場合、過酸化水素電極を下地センサとする酵
素膜電極を構成し、酵素固定膜を透過した過酸化水素（
グルコースの酵素反応により生成した電極感知物質）を
分解して、過酸化水素の量に比例した電流を発生させ、
この電流量からグルコース量を測定することができる。When an electrode equipped with an enzyme-immobilized membrane (enzyme membrane electrode) is brought into contact with a test liquid, the electrode sensing substance (e.g.
Hydrogen peroxide) is generated, and by detecting this sensing substance as an amount of current, it is possible to measure the amount of the specific substance in the test liquid. For example, when measuring glucose in blood, urine, or food, an enzyme membrane electrode is configured with a hydrogen peroxide electrode as the underlying sensor, and the hydrogen peroxide that has passed through the enzyme immobilization membrane is
It decomposes the electrode sensing substance (generated by the enzyme reaction of glucose) and generates a current proportional to the amount of hydrogen peroxide.
The amount of glucose can be measured from this amount of current.
ところで、酵素膜電極を用いて、血液、尿や食品中の特
定物質を正確に測定するにあたってはこれらの被検液に
含まれる尿酸、アスコルビン酸、グルタチオン、メルカ
プト酢酸、アルブミンなど測定対象物質以外の電極活性
物質（妨害物質）を排除することが必要である。By the way, when accurately measuring specific substances in blood, urine, and food using enzyme membrane electrodes, it is necessary to measure substances other than the target substances such as uric acid, ascorbic acid, glutathione, mercaptoacetic acid, albumin, etc. It is necessary to exclude electrode active substances (interfering substances).
そのための一般的な手法としては、複数の電極を設けて
妨害物質に起因する電流値を差し引いて電極出力とする
方法や電気的に妨害物質を分解させる方法の他、酵素固
定膜自体の妨害物質阻止効果を高める方法がある。General methods for this purpose include a method in which multiple electrodes are provided and the current value caused by the interfering substance is subtracted to obtain the electrode output, a method in which the interfering substance is electrically decomposed, and a method in which the interfering substance in the enzyme-immobilized membrane itself is decomposed. There are ways to increase the blocking effect.
妨害物質阻止効果を高めた酵素固定膜としては■測定物
質選択透過層ａ、固定化酵素膜層ｂ、高分子物質除去層
Ｃの三層ａ、ｂ、ｃを積層した酵素固定膜（第７図イ）
（特公昭５６−４８０７０号公報）■−枚の膜で、緻密
な膜層ａ“と、多孔質な膜層ｂ°を有する非対称型の酵
素固定膜（第７図口）（特公昭５９−２１５００号公報
）が既に知られている。Enzyme immobilization membranes with enhanced interfering substance blocking effects include: ■ Enzyme immobilization membranes (No. 7 Figure a)
(Japanese Patent Publication No. 56-48070) - An asymmetrical enzyme-immobilized membrane consisting of two membranes, consisting of a dense membrane layer a" and a porous membrane layer b° (Fig. 7) 21500) is already known.
しかしながら、上記■の場合、三層ａ、ｂ、ｃを均一か
つ精密に積層、接着することが非常に困難であるため、
酵素固定膜の作成が面倒であり、しかも三層ａ、ｂ、ｃ
では全体として膜厚が厚くなるので、電極の応答性が悪
いという欠点があった。However, in the case of (2) above, it is extremely difficult to stack and adhere the three layers a, b, and c uniformly and precisely.
It is troublesome to create an enzyme-immobilized membrane, and there are three layers a, b, and c.
However, since the overall film thickness is increased, there is a drawback that the response of the electrode is poor.
■の酵素固定膜においては、数置な膜層ａ゛では酵素反
応により生ずる過酸化水素の透過量が小さくなって電流
量も僅かになり、また被検液に接する多孔質膜層ｂ′で
は被検液中の不溶性物質などによる汚染やつまりが生じ
やすくて透過量が少なくなる欠点があった。In the enzyme-immobilized membrane (2), the amount of permeation of hydrogen peroxide generated by the enzyme reaction is small in the several membrane layers a', and the amount of current is small, and the porous membrane layer b' in contact with the test liquid is This method has the disadvantage that contamination and clogging due to insoluble substances in the test solution tend to occur, resulting in a small amount of permeation.
上記の従来欠点に鑑み、本発明は、妨害物質阻止効果の
高い酵素固定膜を容易に作成できる酵素固定膜の調製方
法を提案するものである。In view of the above-mentioned conventional drawbacks, the present invention proposes a method for preparing an enzyme-immobilized membrane that can easily produce an enzyme-immobilized membrane that is highly effective in inhibiting interfering substances.
上記の目的を達成するために、本発明が講じた技術的手
段は、次のとおりである。即ち、本発明による酵素固定
膜の調製方法は、アミン系界面活性剤で親水処理した多
孔質ポリプロピレン膜とその片面に積層されたアセチル
セルロース膜とからなる担体膜における多孔質ポリプロ
ピレン膜層中の界面活性剤の側鎖中官能基およびアセチ
ルセルロース膜層中の側鎖中官能基に酢酸酸性下でアミ
ノシラン剤を反応させることを特徴としている。The technical means taken by the present invention to achieve the above object are as follows. That is, the method for preparing an enzyme-immobilized membrane according to the present invention involves the preparation of an interface between a porous polypropylene membrane layer in a carrier membrane consisting of a porous polypropylene membrane hydrophilized with an amine surfactant and an acetyl cellulose membrane laminated on one side of the carrier membrane. It is characterized in that the functional groups in the side chains of the activator and the functional groups in the side chains in the acetylcellulose membrane layer are reacted with an aminosilane agent under acetic acid acidity.
〔発明の効果］
上記の構成によれば、アミノシラン剤が架橋反応を起こ
して、アミン基（ＮＨｚ基）をもったシランマトリック
スを形成するので、アミノ基による酵素の固定が可能で
あると共に、シランマトリックスによる妨害物質阻止効
果が発揮されることになる。つまり、アセチルセルロー
ス膜層での妨害物質阻止効果と、アミン系界面活性剤を
含む多孔質ポリプロピレン膜層での、酵素固定に必要な
アミノ基の添加効果とが、−工程の反応処理で実現され
ることになる。[Effects of the Invention] According to the above configuration, the aminosilane agent causes a crosslinking reaction to form a silane matrix having amine groups (NHz groups). The effect of the matrix to inhibit interfering substances will be exerted. In other words, the effect of inhibiting interfering substances in the acetylcellulose membrane layer and the effect of adding amino groups necessary for enzyme immobilization in the porous polypropylene membrane layer containing an amine surfactant are realized by the reaction treatment in step -. That will happen.
従って、妨害物質阻止効果の高い、しかも酵素固定に必
要なアミノ基をもった酵素固定膜を容易に作成できるの
である。Therefore, it is possible to easily create an enzyme-immobilized membrane that has a high effect of inhibiting interfering substances and also has amino groups necessary for enzyme immobilization.
以下、本発明に係る酵素固定膜の鋼製方法の実施例を図
面に基づいて説明する。DESCRIPTION OF THE PREFERRED EMBODIMENTS Examples of the method for making steel for enzyme-immobilized membranes according to the present invention will be described below with reference to the drawings.
第１図は、酵素固定膜の調製に使用する担体膜を示す。FIG. 1 shows a carrier membrane used in the preparation of an enzyme-immobilized membrane.
図示の担体膜は、アミン系界面活性剤で親水処理された
、厚さ２５−１孔径０．４Ｘ０．０４−の多孔質ポリプ
ロピレン膜ｌとその片面（電極側の面）にラミネートし
た厚さ２〜３−のアセチルセルロース膜２とから構成さ
れている。（商品名ジュラガードＫ ３０７）
前記担体膜における多孔質ポリプロピレン膜１層中の界
面活性剤の側鎖中官能基およびアセチルセルロース膜２
層中の側鎖中官能基（例えば、−ＮＩＩ２、−０１１）
に、酢酸酸性下で、アミノシラン剤（例えば、Ｔ−アミ
ノプロピルトリエトキシシラン、Ｎ−βアミノエチルＴ
アミノプロピルトリメトキシシラン、Ｎ−βアミノエチ
ル−α−メチル−γ−アミノプロピルジメトキシシラン
、Ｎ−ビス−βヒドロキシエチル−Ｔアミノプロピルト
リエトキシシラン）を反応させる。The carrier membrane shown in the figure is a porous polypropylene membrane with a thickness of 25 mm and a pore diameter of 0.4 x 0.04 mm, which has been hydrophilized with an amine surfactant, and a 2 mm thick porous polypropylene membrane laminated on one side (electrode side surface) of the porous polypropylene membrane. ~3- acetylcellulose membrane 2. (Product name DURAGUARD K 307) Functional groups in side chains of surfactant in one layer of porous polypropylene membrane and acetyl cellulose membrane 2 in the carrier membrane
Functional groups in side chains in the layer (e.g. -NII2, -011)
Then, under acetic acid acidity, an aminosilane agent (e.g., T-aminopropyltriethoxysilane, N-β-aminoethyl T
aminopropyltrimethoxysilane, N-β-aminoethyl-α-methyl-γ-aminopropyldimethoxysilane, N-bis-βhydroxyethyl-T-aminopropyltriethoxysilane).
反応条件は次のとおりである。即ち、前記担体膜を、ア
ミノシラン剤０．１〜５％（好ましくは、１．０％）を
添加したｐＨ２〜４の酢酸水溶／＆（好ましくはｐＨ３
、０、酢酸２％）中に、９０°Ｃで、１時間浸漬し、更
に、９０°Ｃで１時間乾燥させる。The reaction conditions are as follows. That is, the carrier film is prepared in an aqueous solution of acetic acid at pH 2 to 4 (preferably pH 3) to which 0.1 to 5% (preferably 1.0%) of an aminosilane agent is added.
, 0, 2% acetic acid) at 90°C for 1 hour, and further dried at 90°C for 1 hour.
シカル後、上記の処理膜（アミノシラン剤で処理した担
体膜）の水洗およびｐＨ７，２の緩衝液による洗浄を行
った後、酵素（例えば、グルコースオキシダーゼ、アル
コールオキシターゼ、ウリカーゼ等）と牛アルブミンを
２：１の割合で混合した溶液に、最終濃度１％のグルタ
ルアルデヒドを混合し、その混合液を、上記の処理膜上
に均一に浸透させ、反応後、１Ｍグリシン溶液で膜中の
未反応基をブロフクする。After washing, the above-mentioned treated membrane (carrier membrane treated with aminosilane agent) is washed with water and a buffer solution of pH 7.2, and enzymes (e.g., glucose oxidase, alcohol oxidase, uricase, etc.) and bovine albumin are : Mix glutaraldehyde with a final concentration of 1% to the mixed solution at a ratio of 1%, and let the mixed solution uniformly permeate the above treated membrane. After the reaction, remove unreacted groups in the membrane with 1M glycine solution. Blog.
上記のようにして調製した酵素固定膜および調製過程で
の担体膜およびその構成要素について、各種の実験を行
った。第２図は本実験に利用した電極測定装置の概念図
を示したものである。同図において、３は酵素電極、４
はミキシングコイル、５はサンプルインジェクター、６
はエアダンパー、７はキャリア緩衝液、８は廃液タンク
、９はデジタル微少電流計である。Various experiments were conducted on the enzyme-immobilized membrane prepared as described above, the carrier membrane during the preparation process, and its constituent elements. FIG. 2 shows a conceptual diagram of the electrode measuring device used in this experiment. In the same figure, 3 is an enzyme electrode, 4
is the mixing coil, 5 is the sample injector, 6 is the mixing coil.
7 is an air damper, 7 is a carrier buffer, 8 is a waste liquid tank, and 9 is a digital microcurrent meter.
本実験により次の結果を得た。The following results were obtained from this experiment.
第３図は、担体膜のアミノシラン処理に使用するアミノ
シラン濃度と、処理膜（アミノシラン剤で処理した担体
膜）を使用して測定した過酸化水素標準液の電極出力お
よび妨害物質であるアスコルビン酸、尿酸の電極出力と
の関係を示す。Figure 3 shows the aminosilane concentration used in the aminosilane treatment of the carrier membrane, the electrode output of the hydrogen peroxide standard solution measured using the treated membrane (carrier membrane treated with an aminosilane agent), and the interfering substance ascorbic acid. The relationship between uric acid and electrode output is shown.
第４図は、上記処理膜に固定する酵素としてグルコース
オキシダーゼを用いた場合のグルコースオキンダーゼ電
極出力とアミノシラン濃度との関係を示す。FIG. 4 shows the relationship between glucose oxidase electrode output and aminosilane concentration when glucose oxidase is used as the enzyme immobilized on the treated membrane.
表１は、次の膜Ａ−Ｄを使用して、過酸化水素標準液、
妨害物質であるアスコルビン酸・尿酸の混合液および健
常人の凍原を測定した際の電極出力を示す。この表１か
ら、膜Ｃと膜りに妨害物質阻止効果が認められる。この
妨害物質阻止効果はラミネートされるアセチルセルロー
ス膜２との反応によるγアミノブロピルトリエＩ・キシ
シラン（以下、γＡＰＴＥＳ）を用いたアミノシラン処
理効果である。Table 1 shows the hydrogen peroxide standard solution, using the following membranes A-D.
Shows the electrode output when measuring a mixed solution of ascorbic acid and uric acid, which are interfering substances, and the frozen ground of a healthy person. From Table 1, it can be seen that Membrane C and the membrane have an effect of inhibiting interfering substances. This interfering substance inhibiting effect is an effect of aminosilane treatment using γ-aminopropyltrie I xysilane (hereinafter referred to as γAPTES) through reaction with the acetylcellulose membrane 2 to be laminated.
Ａ・・・アミン系界面活性剤で親水処理された多孔質ポ
リプロピレン膜１とその片面
にラミネートしたアセチルセルロース
膜２とから構成されたアミノシラン未
処理の膜
Ｂ・・・アミン系界面活性剤で親水処理された多孔質ポ
リプロピレン膜ｌをアミノシ
ラン処理した膜
Ｃ・・・アセチルセルロース膜２をアミノシラン処理し
た膜
Ｄ・・・アミン系界面活性剤で親水処理された多孔質ポ
リプロピレン膜１とその片面
にラミネートしたアセチルセルロース
膜２とから構成された担体膜をアミノ
シラン処理した膜
表１
表２は、次の膜Ａ゛〜Ｅ”を使用して、各膜Ａ“〜Ｅ°
が酵素（グルコースオキシダーゼ）をどの程度固定でき
るかを調べたものである。この表２から、膜Ｃ°と膜Ｄ
°に、酵素の固定に必要な官能ｕ （ＮＨｚ基）の添加
効果が認められる。このアミノ基添加効果は、アミン系
界面活性剤で親水処理された多孔質ポリプロピレン膜１
との反応によるｒ ＡＰＴＥＳ処理効果である。A: Membrane not treated with aminosilane, consisting of a porous polypropylene membrane 1 treated with an amine surfactant to make it hydrophilic, and an acetyl cellulose membrane 2 laminated on one side of the porous polypropylene membrane 1.B: Hydrophilic with an amine surfactant. Membrane C in which the treated porous polypropylene membrane 1 was treated with aminosilane...Membrane D in which the acetylcellulose membrane 2 was treated with aminosilane...Porous polypropylene membrane 1 hydrophilically treated with an amine surfactant and laminated on one side thereof. Table 1 Table 2 shows that the following membranes A゛~E'' were used, and each membrane A''~E°
This study investigated the extent to which the enzyme (glucose oxidase) could be immobilized. From this Table 2, film C° and film D
The effect of adding the functional u (NHz group) necessary for enzyme immobilization is observed at . This effect of adding amino groups is due to the fact that porous polypropylene membrane 1 treated with hydrophilic amine surfactant
This is the effect of r APTES treatment due to the reaction with
Ａ′・・・多孔質ポリプロピレン膜Ｉ
Ｂ”・・・アミン系界面活性剤で親水処理された多孔質
ポリプロピレン膜Ｉ
Ｃ”・・・アミン系界面活性剤で親水処理された多孔質
ポリプロピレン膜１をアミノ、シラン処理した膜
Ｄ′・・・アミン系界面活性剤で親水処理された多孔質
ポリプロピレン膜１とその片面
にラミネートしたアセチルセルロース
膜２とから構成された担体膜をアミノ
ソラン処理した膜
Ｅ″・・・アセチルセルロースＨ’Ｊ２をアミノシラン
処理した膜
表２
上記の表１、表２から、アミン系界面活性剤で親水処理
された多孔質ポリプロピレン膜１とこれにラミネートさ
れたアセチルセルロース膜２をγＡＰＴＥＳ処理するこ
とによって、妨害物質阻止効果と、酵素の固定に必要な
アミン基の添加効果とが同時に達成されることが分かる
。A'...Porous polypropylene membrane I B''...Porous polypropylene membrane I that has been hydrophilically treated with an amine surfactant C''...Porous polypropylene membrane 1 that has been hydrophilically treated with an amine surfactant Membrane D' treated with amino and silane... Membrane E treated with aminosolane on a carrier membrane composed of a porous polypropylene membrane 1 treated with hydrophilic treatment with an amine surfactant and an acetylcellulose membrane 2 laminated on one side of the porous polypropylene membrane 1 ″...Membrane Table 2 in which acetylcellulose H'J2 was treated with aminosilane From Tables 1 and 2 above, the porous polypropylene membrane 1 treated with hydrophilic treatment with an amine surfactant and the acetylcellulose membrane 2 laminated thereon It can be seen that by treating with γAPTES, the effect of inhibiting interfering substances and the effect of adding amine groups necessary for enzyme immobilization can be simultaneously achieved.
尚、上記の表１では、妨害物質阻止効果が、アセチルセ
ルロース膜２のみに起因するものか、アセチルセルロー
ス膜２にｒ ＡＰＴＥＳ処理を施したことに起因するも
のかの判別ができない。In Table 1 above, it is not possible to determine whether the interfering substance blocking effect is due only to the acetylcellulose membrane 2 or to the rAPTES treatment performed on the acetylcellulose membrane 2.
そこで、γＡＰ丁ＥＳで処理する代わりに、アミノ基の
添加効果はあるが、反応によってマトリンクスを形成し
ないアリルアミンで処理して作成した酵素固定膜と、上
述のとおりｒ ＡＰＴＥＳ処理して作成した酵素固定膜
とを用いて酵素電極法により血清中のグルコース濃度を
測定し、比色定量法による測定結果と比較した。Therefore, instead of treatment with γAP-ES, we used an enzyme-immobilized membrane prepared by treating with allylamine, which has the effect of adding amino groups but does not form a matrix through reaction, and an enzyme-immobilized membrane prepared by treating r-APTES as described above. Glucose concentration in serum was measured by enzyme electrode method using a membrane and compared with the measurement results by colorimetric method.
これによって得られた酵素電極法と比色定量法との相関
を第５図に示す。The correlation between the enzyme electrode method and the colorimetric method obtained in this way is shown in FIG.
第５図から明らかなように、アリルアミン処理膜では、
γＡＰＴＥＳ処理した酵素固定膜よりもグルコース濃度
が高値に測定されている。これは、妨害物質による正誤
差によるもので、アミノシラン処理法が有効であること
を示す一例でもある。即ちアミノシラン処理した酵素固
定膜では、アミノシラン剤が、アミノ基を有するシラン
マトリックスを形成しており、このシランマトリックス
が妨害物質阻止に寄与していると思われる。As is clear from Fig. 5, in the allylamine-treated membrane,
The glucose concentration was measured to be higher than that of the enzyme-immobilized membrane treated with γAPTES. This is due to a positive error caused by interfering substances, and is an example of the effectiveness of the aminosilane treatment method. That is, in the aminosilane-treated enzyme-immobilized membrane, the aminosilane agent forms a silane matrix having amino groups, and this silane matrix is thought to contribute to blocking interfering substances.
第６図は、γＡＰＴＥＳ処理して作成した酵素固定膜を
用いて酵素電極法により尿中のグルコース濃度を測定し
、比色定量法による測定結果と比較し◆ で
得られた酵素電極法と、比色定量法との相関を示す。Figure 6 shows the enzyme electrode method obtained by measuring the glucose concentration in urine by the enzyme electrode method using an enzyme-immobilized membrane prepared by treating with γAPTES and comparing it with the measurement results by the colorimetric method. Correlation with colorimetric method is shown.
表３は、γＡＰＴＥＳ処理して作成した酵素固定膜を用
いて酵素電極法により数種類の食品中のグルコース濃度
を測定し、比色定量法との相関を調べたものである。Table 3 shows the results of measuring the glucose concentration in several types of foods by the enzyme electrode method using an enzyme-immobilized membrane prepared by γAPTES treatment, and examining the correlation with the colorimetric method.
表３から明らかなように、γＡＰＴＥＳ処理して作成し
た酵素固定膜は、血液、尿などと同様に複雑な妨害物質
を含む食品中でも、正確に測定可能である。As is clear from Table 3, the enzyme-immobilized membrane prepared by γAPTES treatment can accurately measure even foods containing complex interfering substances, such as blood and urine.
また上記の調製方法により作成され、固定する酵素とし
てアルコールオキシダーゼを使用した酵素固定膜を用い
て、清酒類のアルコール濃度を測定した結果、アルコー
ル濃度表示値とよく一致していた。Furthermore, the alcohol concentration of sake was measured using an enzyme-immobilized membrane prepared by the above preparation method and using alcohol oxidase as the enzyme to be immobilized, and the result was in good agreement with the alcohol concentration display value.
表３Table 3
第１図は本発明の酵素固定膜の調製方法に使用する担体
膜の概略断面図、第２回は実験に使用した電極測定装置
の概念図、第３図は担体膜のアミノシラン処理に使用す
るアミノシラン濃度と、処理膜（アミノシラン剤で処理
した担体膜）を使用して測定した過酸化水素標準液の電
極出力および妨害物質であるアスコルビン酸、尿酸の電
極出力との関係を示すグラフ、第４図は上記処理膜に固
定する酵素としてグルコースオキシダーゼを用いた場合
のグルコースオキシダーゼ電掻出力とアミノソラン濃度
との関係を示すグラフ、第５図はアリルアミンで処理し
て作成した酵素固定膜とｒＡＰＴＥＳ処理して作成した
酵素固定膜とを用いて行った血清中のグルコース濃度の
酵素電極法による測定結果と、比色定量法との相関を示
すグラフ、第６図は１ ＡＰＴＥＳ処理して作成した酵
素固定膜を用いて行った尿中のグルコース濃度の酵素電
極法による測定結果と、比色定量法との相関を示すグラ
フである。
第７図イ１ 口は従来例の説明図である。
１・・・多孔質ポリプロピレン膜、２・・・アセチルセ
ルロース膜。Figure 1 is a schematic cross-sectional view of the carrier membrane used in the enzyme-immobilized membrane preparation method of the present invention, Figure 2 is a conceptual diagram of the electrode measuring device used in the experiment, and Figure 3 is the carrier membrane used for aminosilane treatment. Graph showing the relationship between the aminosilane concentration and the electrode output of a hydrogen peroxide standard solution and the interfering substances ascorbic acid and uric acid measured using a treated membrane (carrier membrane treated with an aminosilane agent), 4th The figure is a graph showing the relationship between the glucose oxidase electrocuring force and the aminosolane concentration when glucose oxidase is used as the enzyme immobilized on the treated membrane. Figure 5 shows the relationship between the enzyme-immobilized membrane prepared by treating with allylamine and the rAPTES treatment. Figure 6 is a graph showing the correlation between the measurement results of glucose concentration in serum by the enzyme electrode method and the colorimetric method using the enzyme-immobilized membrane prepared by 1. 2 is a graph showing the correlation between the measurement results of glucose concentration in urine by an enzyme electrode method using a membrane and a colorimetric method. FIG. 7A1 is an explanatory diagram of a conventional example. 1... Porous polypropylene membrane, 2... Acetyl cellulose membrane.
Claims (1)
ン膜１とその片面に積層されたアセチルセルロース膜２
とからなる担体膜における多孔質ポリプロピレン膜層中
の界面活性剤の側鎖中官能基およびアセチルセルロース
膜層中の側鎖中官能基に酢酸酸性下でアミノシラン剤を
反応させることを特徴とする酵素固定膜の調製方法。A porous polypropylene membrane 1 that has been hydrophilized with an amine surfactant and an acetyl cellulose membrane 2 laminated on one side of the membrane.
An enzyme characterized by reacting an aminosilane agent with a functional group in a side chain of a surfactant in a porous polypropylene membrane layer and a functional group in a side chain in an acetylcellulose membrane layer in a carrier membrane consisting of Method for preparing fixed membranes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63069138A JPH07117519B2 (en) | 1988-03-19 | 1988-03-19 | Method for preparing enzyme-immobilized membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63069138A JPH07117519B2 (en) | 1988-03-19 | 1988-03-19 | Method for preparing enzyme-immobilized membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01239449A true JPH01239449A (en) | 1989-09-25 |
| JPH07117519B2 JPH07117519B2 (en) | 1995-12-18 |
Family
ID=13393988
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63069138A Expired - Lifetime JPH07117519B2 (en) | 1988-03-19 | 1988-03-19 | Method for preparing enzyme-immobilized membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07117519B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017061473A (en) * | 2007-11-07 | 2017-03-30 | ロイコケア・アクチェンゲゼルシャフト | Biocompatible three-dimensional matrix for immobilization of biological materials |
| US9926383B2 (en) | 2006-05-05 | 2018-03-27 | Leukocare Ag | Biocompatible three dimensional matrix for the immobilization of biological substances |
-
1988
- 1988-03-19 JP JP63069138A patent/JPH07117519B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9926383B2 (en) | 2006-05-05 | 2018-03-27 | Leukocare Ag | Biocompatible three dimensional matrix for the immobilization of biological substances |
| JP2017061473A (en) * | 2007-11-07 | 2017-03-30 | ロイコケア・アクチェンゲゼルシャフト | Biocompatible three-dimensional matrix for immobilization of biological materials |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07117519B2 (en) | 1995-12-18 |
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