JPH07117519B2 - Method for preparing enzyme-immobilized membrane - Google Patents
Method for preparing enzyme-immobilized membraneInfo
- Publication number
- JPH07117519B2 JPH07117519B2 JP63069138A JP6913888A JPH07117519B2 JP H07117519 B2 JPH07117519 B2 JP H07117519B2 JP 63069138 A JP63069138 A JP 63069138A JP 6913888 A JP6913888 A JP 6913888A JP H07117519 B2 JPH07117519 B2 JP H07117519B2
- Authority
- JP
- Japan
- Prior art keywords
- membrane
- enzyme
- immobilized
- aminosilane
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000012528 membrane Substances 0.000 title claims description 90
- 238000000034 method Methods 0.000 title claims description 18
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims description 24
- -1 polypropylene Polymers 0.000 claims description 18
- 239000004743 Polypropylene Substances 0.000 claims description 17
- 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 description 17
- 229920001155 polypropylene Polymers 0.000 claims description 17
- 229940081735 acetylcellulose Drugs 0.000 claims description 16
- 229920002301 cellulose acetate Polymers 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 239000004094 surface-active agent Substances 0.000 claims description 14
- 150000001412 amines Chemical class 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 125000000524 functional group Chemical group 0.000 claims description 7
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 description 27
- 108090000790 Enzymes Proteins 0.000 description 24
- 102000004190 Enzymes Human genes 0.000 description 24
- 229940088598 enzyme Drugs 0.000 description 24
- 230000002452 interceptive effect Effects 0.000 description 18
- 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
- 230000000903 blocking effect Effects 0.000 description 10
- 230000000694 effects Effects 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
- 125000003277 amino group Chemical group 0.000 description 8
- 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
- 238000006243 chemical reaction Methods 0.000 description 6
- 229940116332 glucose oxidase Drugs 0.000 description 6
- 235000019420 glucose oxidase Nutrition 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000005259 measurement Methods 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
- 238000004737 colorimetric analysis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229910000077 silane Inorganic materials 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229940116269 uric acid Drugs 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 2
- 108010025188 Alcohol oxidase Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010586 diagram Methods 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
- 239000012466 permeate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- LJCNDNBULVLKSG-UHFFFAOYSA-N 2-aminoacetic acid;butane Chemical compound CCCC.CCCC.NCC(O)=O LJCNDNBULVLKSG-UHFFFAOYSA-N 0.000 description 1
- IMKMEPLAEXUGBU-UHFFFAOYSA-N 3-[3-aminopropyl(diethoxy)silyl]oxybutan-1-ol Chemical compound NCCC[Si](OCC)(OCC)OC(C)CCO IMKMEPLAEXUGBU-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
- 108010093096 Immobilized Enzymes Proteins 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
- 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
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution 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
- 238000005580 one pot reaction Methods 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 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)
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 [Field of Industrial Application] The present invention is directed to body fluids, body tissues, glucose contained in foods,
The present invention relates to a method for preparing an enzyme-immobilized membrane used for selectively quantifying trace components such as urea, triglyceride, phospholipid, and alcohol by utilizing an enzyme.
〔従来の技術〕 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。[Prior Art] When an electrode having an enzyme-immobilized membrane (enzyme membrane electrode) is brought into contact with a test solution, an electrode-sensing substance (for example,
Hydrogen peroxide) is generated, and the amount of the specific substance in the test liquid can be measured by detecting this sensing substance as the amount of electric current. For example, when measuring glucose in blood, urine, or food, an enzyme membrane electrode that uses a hydrogen peroxide electrode as a base sensor is configured, and hydrogen peroxide that permeates an enzyme immobilization membrane (electrode sensing generated by the enzymatic reaction of glucose material)
Can be decomposed to generate a current proportional to the amount of hydrogen peroxide, and 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、アルコルビン酸、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, using an enzyme membrane electrode, in accurately measuring a specific substance in blood, urine or food, uric acid contained in these test liquids, ascorbic acid, glutathione, mercaptoacetic acid, other than the measurement target substance such as albumin It is necessary to eliminate electrode active substances (interfering substances).
そのため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。Therefore, as a general method, in addition to the method of providing multiple electrodes and subtracting the current value due to the interfering substance to obtain the electrode output, and the method of electrically decomposing the interfering substance, blocking the interfering substance of the enzyme-immobilized membrane itself There are ways to increase the 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） 一枚の膜で、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図ロ）特公昭59−21
500号公報）が既に知られている。As an enzyme-immobilized membrane having an enhanced inhibitory effect on interfering substances, an enzyme-immobilized membrane obtained by laminating three layers a, b, and c, which are a selective substance permeation layer a, an immobilized enzyme membrane layer b, and a polymeric substance removal layer c (see FIG. 7). B) (Japanese Patent Publication No. 56-48070) An asymmetric enzyme-immobilized membrane having a dense membrane layer a'and a porous membrane layer b'with a single membrane (Fig. 7B) Japanese Patent Publication No. 59- twenty one
No. 500) 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 above case, it is very difficult to uniformly and precisely laminate and adhere the three layers a, b and c, so that it is troublesome to prepare the enzyme-immobilized membrane, and the three layers a, b and c Since the film thickness is increased as a whole, there is a drawback that the response of the electrode is poor.
の酵素固定膜においては、緻密な膜層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 of No. 2, in the dense membrane layer a ′, the permeation amount of hydrogen peroxide generated by the enzyme reaction is small and the current amount is also small, and in the porous membrane layer b ′ in contact with the test liquid, the test substance is not detected. There is a drawback that the amount of permeation is reduced because contamination or clogging with insoluble substances in the liquid is likely to occur.
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 capable of easily producing an enzyme-immobilized membrane having a high interfering substance blocking effect.
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 comprises an interface in a porous polypropylene membrane layer in a carrier membrane composed of a porous polypropylene membrane hydrophilically treated with an amine-based surfactant and an acetylcellulose membrane laminated on one side thereof. It is characterized in that the functional group in the side chain of the activator and the functional group in the side chain in the acetylcellulose membrane layer are reacted with an aminosilane agent under acidity of acetic acid.
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、アミノ基（−NH2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、一工程の反応処理で実現され
ることになる。According to the above configuration, the aminosilane agent causes a cross-linking reaction to form a silane matrix having an amino group (-NH 2 group). Therefore, the enzyme can be immobilized by the amino group, and the silane matrix prevents interference. The substance blocking effect will be exhibited. In other words, the interfering substance blocking effect in the acetyl cellulose membrane layer and the addition effect of the amino group necessary for the enzyme immobilization in the porous polypropylene membrane layer containing the amine-based surfactant are realized by the one-step reaction treatment. Will be.
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 prepare an enzyme-immobilized membrane having a high effect of blocking interfering substances and having an amino group necessary for enzyme immobilization.
以下、本発明に係る酵素固定膜の調製方法の実施例を図
面に基づいて説明する。An embodiment of the method for preparing an enzyme-immobilized membrane 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。
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25μｍ、孔径0.4×0.04μｍの多孔質ポリプロ
ピレン膜１とその片面（電極側の面）にラミネートした
厚さ２〜３μｍ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K307） 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、−NH2,−OH）に、酢酸酸
性下で、アミノシラン剤（例えば、γ−アミノプロピル
トリエトキシシラン、Ｎ−βアミノエチルγ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−γ−アミノプロピルトリエトキ
シシラン）を反応させる。FIG. 1 shows a carrier membrane used for preparing an enzyme-immobilized membrane.
The carrier film shown in the figure is a porous polypropylene film 1 having a thickness of 25 μm and a pore size of 0.4 × 0.04 μm, which has been hydrophilically treated with an amine-based surfactant, and has a thickness of 2 to 3 μm laminated on one surface (surface on the electrode side) thereof. It is composed of an acetyl cellulose membrane 2. (Brand name Duraguard K307) Functional group in side chain of surfactant and acetylcellulose film 2 in 1 layer of porous polypropylene film in the carrier film
Aminosilane agent (for example, γ-aminopropyltriethoxysilane, N-βaminoethyl γaminopropyltrimethoxysilane, or the like) is added to the functional group in the side chain in the layer (for example, —NH 2 , —OH) under acidic acetic acid. N-β aminoethyl-α-methyl-γ-aminopropyldimethoxysilane, N-bis-β hydroxyethyl-γ-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の酢酸水溶液（好ましくはpH3.0、酢酸２％）
中に、90℃で、１時間浸漬し、更に、90℃で１時間乾燥
させる。The reaction conditions are as follows. That is, the carrier film was added with an aminosilane agent 0.1 to 5% (preferably 1.0%) in an acetic acid aqueous solution of pH 2 to 4 (preferably pH 3.0, acetic acid 2%).
It is dipped therein at 90 ° C. for 1 hour and further dried at 90 ° C. for 1 hour.
しかる後、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
をブロックする。After that, the above treated membrane (carrier membrane treated with aminosilane agent) was washed with water and washed with a buffer solution of pH 7.2, and then the enzyme (eg glucose oxidase, alcohol oxidase, uricase, etc.) and bovine albumin 2 : 1
Glutaraldehyde at a final concentration of 1% was mixed into the solution mixed at the ratio of 1%, and the mixed solution was allowed to uniformly permeate on the above treated membrane, and after the reaction, unreacted groups in the membrane were blocked with 1M glycine solution. To do.
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 performed on the enzyme-immobilized membrane prepared as described above, the carrier membrane in the preparation process, and its constituent elements. FIG. 2 is a conceptual diagram of the electrode measuring device used in this experiment. In the figure, 3 is an enzyme electrode, 4
Is a mixing coil, 5 is a sample injector, 6
Is an air damper, 7 is a carrier buffer solution, 8 is a waste liquid tank, and 9 is a digital micro ammeter.
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。FIG. 3 shows the concentration of aminosilane used for the aminosilane treatment of the carrier film, the electrode output of the hydrogen peroxide standard solution measured using the treated film (the carrier film treated with the aminosilane agent), and ascorbic acid as an interfering substance. The relationship with the electrode output of uric acid 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シダーゼ電
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 the glucose oxidase electrode output and the aminosilane concentration when glucose oxidase was 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建
常人の原尿を測定した際の電極出力を示す。この表１か
ら、膜Ｃと膜Ｄ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アミノプロピルトリエトキシシ
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 that using the following membranes A to D, a hydrogen peroxide standard solution,
The electrode output when measuring the mixed liquid of ascorbic acid / uric acid which is an interfering substance and the raw urine of a normal person is shown. From Table 1, it can be seen that the film C and the film D have an interfering substance blocking effect. This interfering substance blocking effect is an aminosilane treatment effect using γ-aminopropyltriethoxysilane (hereinafter, γAPTES) by the reaction with the laminated acetylcellulose membrane 2.
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１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基（NH2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γAPTES処理効果である。A ... Aminosilane-untreated membrane composed of a porous polypropylene membrane 1 hydrophilically treated with an amine-based surfactant and an acetylcellulose membrane 2 laminated on one side thereof B ... Porous hydrophilically treated with an amine-based surfactant Aminosilane-treated membrane of high quality polypropylene membrane 1 C ... Aminosilane treatment of acetylcellulose membrane 2 D ... Consists of porous polypropylene membrane 1 hydrophilically treated with amine-based surfactant and acetylcellulose membrane 2 laminated on one side thereof Aminosilane-treated membrane Table 2 shows that each of the membranes A'-E 'using the following membranes A'-E'.
Is the extent to which the enzyme (glucose oxidase) can be immobilized. From this Table 2, it can be seen that the effect of adding the functional group (NH 2 group) necessary for immobilizing the enzyme is added to the membrane C ′ and the membrane D ′. This amino group addition effect is a γAPTES treatment effect due to the reaction with the porous polypropylene membrane 1 hydrophilically treated with an amine surfactant.
Ａ′…多孔質ポリプロピレン膜１ 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１ 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シラン処
理した膜 Ｅ′…アセチルセルロース膜２をアミノシラン処理した
膜 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
ノ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 1 B' ... Porous polypropylene membrane 1 hydrophilically treated with amine-based surfactant C '... Aminosilane-treated membrane polypropylene D obtained by hydrophilically treating amine-based surfactant D ′ ... A carrier membrane composed of a porous polypropylene membrane 1 hydrophilically treated with an amine-based surfactant and an acetylcellulose membrane 2 laminated on one side thereof with aminosilane treatment E ′ ... Acetylcellulose membrane 2 with aminosilane treatment film From Tables 1 and 2 above, by subjecting the porous polypropylene membrane 1 hydrophilically treated with an amine-based surfactant and the acetyl cellulose membrane 2 laminated to this to γAPTES treatment, an interfering substance blocking effect and immobilization of enzyme It can be seen that the effect of adding the amino group necessary for the above is 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γAPTES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 addition, in Table 1 above, it is not possible to determine whether the interfering substance blocking effect is due to only the acetylcellulose membrane 2 or due to the γAPTES treatment of the acetylcellulose membrane 2.
そこで、γAPTES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ックスを形成しない
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γAPTES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 treating with γAPTES, there is an effect of adding an amino group, but the enzyme-immobilized membrane prepared by treating with allylamine that does not form a matrix by the reaction, and the enzyme-immobilized membrane prepared by treating with γAPTES as described above. The glucose concentration in the serum was measured by using the enzyme electrode method and compared with the measurement result by the 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。Fig. 5 shows the correlation between the enzyme electrode method and the colorimetric method obtained by this.
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 film,
The glucose concentration is higher than that of the enzyme-immobilized membrane treated with γAPTES. This is due to the positive error due to the interfering substance, and is also an example showing that the aminosilane treatment method is effective. That is, in the enzyme-immobilized membrane treated with aminosilane, the aminosilane agent forms a silane matrix having an amino group, and it is considered that this silane matrix contributes to the blocking of 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て得られた酵素電極法
と、比色定量法との相関を示す。Fig. 6 shows the ratio of the enzyme electrode method obtained by measuring the glucose concentration in urine by the enzyme electrode method using the enzyme-immobilized membrane prepared by γAPTES treatment and comparing the measurement results with the colorimetric assay method. The correlation with the color quantitative 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、比式定量法との相関を調べたものである。Table 3 shows the glucose concentration in several kinds of foods measured by the enzyme electrode method using the enzyme-immobilized membrane prepared by the γAPTES treatment, and the correlation with the ratio quantitative method was investigated.
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 be accurately measured even in foods containing complicated interfering substances like 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。The alcohol concentration of sake was measured using the enzyme-immobilized membrane prepared by the above-mentioned preparation method and using alcohol oxidase as the enzyme to be immobilized. As a result, it was in good agreement with the displayed alcohol concentration value.
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図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極出力とアミノシラン濃度
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γAPTES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γAPTES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，ロ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。FIG. 1 is a schematic sectional view of a carrier membrane used in the method for preparing an enzyme-immobilized membrane of the present invention, FIG. 2 is a conceptual diagram of an electrode measuring device used in an experiment, and FIG. 3 is used for aminosilane treatment of the carrier membrane. Graph showing the relationship between the aminosilane concentration and the electrode output of a hydrogen peroxide standard solution and the electrode outputs of ascorbic acid and uric acid which are interfering substances, measured using a treatment film (a carrier film treated with an aminosilane agent). Figure is a graph showing the relationship between glucose oxidase electrode output and aminosilane concentration when glucose oxidase is used as the enzyme to be immobilized on the treated membrane. Figure 5 shows the enzyme immobilized membrane prepared by treatment with allylamine and γAPTES treatment. Fig. 6 is a graph showing the correlation between the measurement result of the glucose concentration in the serum by the enzyme electrode method using the enzyme-immobilized membrane prepared and the colorimetric method, and Fig. 6 shows γAPTES A measurement result by the enzymatic electrode method of glucose concentration in urine was performed with an enzyme fixed film created by sense, is a graph showing the correlation between colorimetric method. 7A and 7B are explanatory views of the conventional example. 1 ... Porous polypropylene membrane, 2 ... Acetylcellulose membrane.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｑ 1/00 Ｂ 6807−4Ｂ Ｃ 6807−4Ｂ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location C12Q 1/00 B 6807-4B C 6807-4B
Claims (1)
ポリプロピレン膜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。1. A side chain of a surfactant in a porous polypropylene membrane layer in a carrier membrane comprising a porous polypropylene membrane 1 hydrophilically treated with an amine surfactant and an acetylcellulose membrane 2 laminated on one surface thereof. A method for preparing an enzyme-immobilized membrane, which comprises reacting a functional group and a functional group in a side chain in an acetylcellulose membrane layer with an aminosilane agent under acidic conditions of acetic acid.
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 JPH01239449A (en) | 1989-09-25 |
| JPH07117519B2 true 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) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1852443A1 (en) | 2006-05-05 | 2007-11-07 | Leukocare AG | Biocompatible three dimensional matrix for the immobilization of biological substances |
| EP2058335B1 (en) * | 2007-11-07 | 2020-06-24 | 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
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01239449A (en) | 1989-09-25 |
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