JPS62171685A - Immobilized lypase enzyme membrane and production thereof - Google Patents
Immobilized lypase enzyme membrane and production thereofInfo
- Publication number
- JPS62171685A JPS62171685A JP61013901A JP1390186A JPS62171685A JP S62171685 A JPS62171685 A JP S62171685A JP 61013901 A JP61013901 A JP 61013901A JP 1390186 A JP1390186 A JP 1390186A JP S62171685 A JPS62171685 A JP S62171685A
- Authority
- JP
- Japan
- Prior art keywords
- lipase
- immobilized enzyme
- enzyme membrane
- membrane
- immobilized
- 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
- 239000012528 membrane Substances 0.000 title claims abstract description 68
- 108090000790 Enzymes Proteins 0.000 title claims description 68
- 102000004190 Enzymes Human genes 0.000 title claims description 68
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 239000000758 substrate Substances 0.000 claims abstract description 25
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000007864 aqueous solution Substances 0.000 claims abstract description 17
- 108090001060 Lipase Proteins 0.000 claims abstract description 16
- 102000004882 Lipase Human genes 0.000 claims abstract description 16
- 239000004367 Lipase Substances 0.000 claims abstract description 14
- 235000019421 lipase Nutrition 0.000 claims abstract description 14
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 12
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 11
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 11
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 11
- 238000010382 chemical cross-linking Methods 0.000 claims abstract description 8
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 5
- LHXSHHZTBFUYIS-UHFFFAOYSA-L disodium;5-azido-2-[[3-[(4-azido-2-sulfonatophenyl)methylidene]-2-oxocyclopentylidene]methyl]benzenesulfonate Chemical compound [Na+].[Na+].[O-]S(=O)(=O)C1=CC(N=[N+]=[N-])=CC=C1C=C(CC1)C(=O)C1=CC1=CC=C(N=[N+]=[N-])C=C1S([O-])(=O)=O LHXSHHZTBFUYIS-UHFFFAOYSA-L 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 8
- 238000004132 cross linking Methods 0.000 claims description 5
- NLYQILXYCSLRTN-UHFFFAOYSA-N cyclopentanone;sodium Chemical compound [Na].O=C1CCCC1 NLYQILXYCSLRTN-UHFFFAOYSA-N 0.000 claims description 5
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 abstract description 4
- 238000000576 coating method Methods 0.000 abstract description 4
- 239000011247 coating layer Substances 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000010408 film Substances 0.000 description 16
- 238000010586 diagram Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 108010088751 Albumins Proteins 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 125000003172 aldehyde group Chemical group 0.000 description 3
- BGTOWKSIORTVQH-UHFFFAOYSA-N cyclo-pentanone Natural products O=C1CCCC1 BGTOWKSIORTVQH-UHFFFAOYSA-N 0.000 description 3
- -1 cyclopentanone sodium salt Chemical class 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 238000000206 photolithography Methods 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000000016 photochemical curing Methods 0.000 description 1
- 229920005597 polymer membrane Polymers 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000004528 spin coating Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 230000002747 voluntary effect Effects 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
この発明は、＋Ｊ ／＜−ゼを連続使用するために必要
であシ、且つバイオセンサに適用できるリバー上固定化
酵素膜材料、同固定化酵素膜、及びその製造方法に関す
るものである。[Detailed Description of the Invention] [Industrial Application Field] The present invention provides an enzyme membrane material immobilized on a river, which is necessary for the continuous use of +J/<-ze, and which can be applied to a biosensor. The present invention relates to an enzyme membrane and a method for producing the same.
酵素を触媒として工業的に利用することは、発酵工業な
どを中心に昔から行なわれていた。しかし、従来、酵素
は水溶液中に溶解あるいは分散させて反応に用いられ、
反応終了後は酵素は回収されず、−回の反応ごとに捨て
られていた。ところが、最近、酵素を安定にくり返し使
用を可能にする酵素固定化技術の進展で、酵素の使用範
囲が急激に拡大し、発酵や化学物質の合成のみならず、
化学物質の計測にまで利用することができるようになっ
た。化学物質の計測手段としては、固定化酵素膜として
は膜厚が薄く、しかも膜面積が小さいものが利用される
。The industrial use of enzymes as catalysts has been practiced for a long time, mainly in the fermentation industry. However, conventionally, enzymes are dissolved or dispersed in an aqueous solution and used for reactions.
The enzyme was not recovered after the reaction was completed, and was discarded after every - reaction. However, with recent advances in enzyme immobilization technology that allows enzymes to be used repeatedly and stably, the scope of use of enzymes has expanded rapidly, extending beyond fermentation and the synthesis of chemical substances.
It can now be used to measure chemical substances. As a means for measuring chemical substances, an immobilized enzyme membrane that is thin and has a small membrane area is used.
バイオセンサは化学物質の計測に固定化酵素膜が利用さ
れた代表例である。このバイオセンサは固定化酵素膜と
、この膜内で消費あるいは生成する物質を検出し、これ
を電気信号に変換するトランスデユーサとから構成され
る。この場合、固定化酵素膜が果す役割は、測定したい
化学物質を識別することと、これをトランスデユーサが
検知できる物質の量的変化を生じさせることにある。Biosensors are a typical example in which immobilized enzyme membranes are used to measure chemical substances. This biosensor consists of an immobilized enzyme membrane and a transducer that detects substances consumed or produced within the membrane and converts them into electrical signals. In this case, the role of the immobilized enzyme membrane is to identify the chemical substance to be measured and to cause a quantitative change in the substance that can be detected by the transducer.
リパーゼを用いたバイオセンサとしては、ｐＨ電極上に
リパーゼ固定化酵素膜を形成したものがある。リパーゼ
は固定化酵素膜内で（１）式に従って中性脂質をグリセ
ロールと脂肪酸に分解する。As a biosensor using lipase, there is one in which a lipase-immobilized enzyme membrane is formed on a pH electrode. Lipase decomposes neutral lipids into glycerol and fatty acids according to formula (1) within an immobilized enzyme membrane.
ＣＨ，０ＣＯＲＣＨ，ＯＨ
この反応に伴うｐＨの低下をｐＨ電極で検知することに
よシ、中性脂質の濃度が測定できる。CH,0CORCH,OH By detecting the decrease in pH accompanying this reaction with a pH electrode, the concentration of neutral lipids can be measured.
従来の中性脂質測定用バイオセンサの固定化酵素膜とし
ては、リパーゼと牛血清アルブミンをグルタルアルデヒ
ドで化学架橋して形成する方法が一般的である。第２図
は、基板あるいはトランスデユーサ上に形成したリパー
ゼ固定化酵素膜の模式図である。図において、（１）は
表面にシランカップリング剤でアミノ基を導入した基板
あるいはトランスデユーサ、（コ）はリパーゼ、（、？
）は牛血清アルブミン、（’りはグルタルアルデヒドで
ある。The conventional immobilized enzyme membrane for a biosensor for measuring neutral lipids is generally formed by chemically crosslinking lipase and bovine serum albumin with glutaraldehyde. FIG. 2 is a schematic diagram of a lipase-immobilized enzyme membrane formed on a substrate or a transducer. In the figure, (1) is a substrate or transducer whose surface has an amino group introduced with a silane coupling agent, (co) is lipase, (,?
) is bovine serum albumin, ('ri is glutaraldehyde.
このリパーゼ固定化酵素膜の製造方法は、酵素う
であｌｉ ＩＪパーゼ（２）と牛血清アルブミン（３）
をあらかじめ水溶液中に溶解し、これを適当な濃度のグ
ルタルアルデヒド（りを含んだ溶液と混ぜ合せ、基板（
１）上に塗）付ける。そのまま放置しておくと、グルタ
ルアルデヒドによシ酵素−酵素、酵素−牛血清アルブミ
ン、牛血清アルブミン−牛血清アルプミ／、基板表面の
アミン基−酵素あるいは牛崩清アルブミンが化学架橋さ
れ、第２図に示したよりに三次元的な高分子膜の中に酵
素が捕捉され、水に不溶となり固定化される。The method for producing this lipase-immobilized enzyme membrane is based on the enzyme Udeali IJpase (2) and bovine serum albumin (3).
is dissolved in advance in an aqueous solution, mixed with a solution containing an appropriate concentration of glutaraldehyde, and the substrate (
1) Apply on top. If left as is, glutaraldehyde chemically crosslinks enzyme-enzyme, enzyme-bovine serum albumin, bovine serum albumin-bovine serum albumin, and amine group on the substrate surface-enzyme or bovine disintegrated albumin, resulting in a second As shown in the figure, the enzyme is captured in a more three-dimensional polymer membrane, becomes insoluble in water, and becomes immobilized.
バイオセンサは、最近、微小化が要求されることから、
リパーゼ固定化酵素膜としても微小加工が可能な材料及
び製造方法が必要である。従来のリパーゼ固定化酵素膜
は、上記のような材料と製造方法で形成されているので
、微小面積の固定化酵素膜を再現性良・く形成すること
が不可能であるという問題点があった。Recently, biosensors have been required to be miniaturized.
A material and manufacturing method that can be microfabricated as a lipase-immobilized enzyme membrane is also needed. Conventional lipase-immobilized enzyme membranes are formed using the materials and manufacturing methods described above, so there is a problem in that it is impossible to form immobilized enzyme membranes with a small area with good reproducibility. Ta.
この発明は、かかる問題点を解決するためになされたも
ので、フォトリソグラフィー技術により微小なリパーゼ
固定化酵素膜を形成することができるリパーゼ固定化酵
素膜を得ることを目的とする。The present invention has been made to solve these problems, and aims to obtain a lipase-immobilized enzyme membrane that can be formed using a photolithography technique.
また、この発明の別の発明は、上記目的に加えて、微小
面積で且つ膜強度の高い固定化酵素膜を再現性良く形成
することができるリパーゼ固定化酵素膜の製造方法を得
ることを目的とする。In addition to the above object, another object of the present invention is to provide a method for producing a lipase-immobilized enzyme membrane that can form an immobilized enzyme membrane with a small area and high membrane strength with good reproducibility. shall be.
この発明に係るリパーゼ固定化酵素膜は、ポリビニルピ
ロリドンと２．タービス（ｔ′−アジドーコ′−スルホ
ベンザル）シクロペンタノンナトリウム塩を含む水溶性
感光樹脂水溶液にリパーゼと牛血清アルブミンとを溶解
あるいは混合したものである。The lipase-immobilized enzyme membrane according to the present invention comprises polyvinylpyrrolidone and 2. Lipase and bovine serum albumin are dissolved or mixed in an aqueous solution of a water-soluble photosensitive resin containing Terbis(t'-azidoco'-sulfobenzal)cyclopentanone sodium salt.
また、この発明の別の発明に係るリパーゼ固定化酵素膜
の製造方法は、上記のリパーゼ固定化酵素膜を基板に塗
布して紫外線によシ光硬化した後、このリパーゼ固定化
酵素膜をグルタルアルデヒド水溶液で処理して酵素膜成
分をさらに化学架橋し、たものである。In addition, a method for producing a lipase-immobilized enzyme film according to another aspect of the present invention includes coating the above-mentioned lipase-immobilized enzyme film on a substrate and photocuring it with ultraviolet rays, and then coating the lipase-immobilized enzyme film with glutaric acid. The enzyme membrane components are further chemically crosslinked by treatment with an aldehyde aqueous solution.
この発明においては、水溶性感光樹脂の存在によりフォ
トリソグラフィー技術が適用できるので、微小な面積の
リパーゼ固定化酵素膜が形成される。In this invention, since photolithography technology can be applied due to the presence of a water-soluble photosensitive resin, a lipase-immobilized enzyme membrane with a minute area is formed.
また、この発明の別の発明においては、グルタルアルデ
ヒドによる化学架橋で酵素膜強度を高めたリパーゼ固定
化酵素膜を製造することができる。In another aspect of the present invention, it is possible to produce a lipase-immobilized enzyme membrane whose strength is increased by chemical crosslinking with glutaraldehyde.
この発明に、よるリパーゼ固定化酵素膜は、好適Ｋ Ｕ
ｔ” ＩＪビニルピロリドン２〜２０重童％、２゜タ
ービス（り′−アジドーコ′−スルホベンザル）シクロ
ペンタノンナトリウム塩０．２〜Ｑ、ｊ重量％、リパー
ゼ５〜１０重量％、十崩清アルブミンｊ〜ＩＱ重量％、
及び化学架橋剤例えばグルタルアルデヒドを含んだ水溶
液である。According to this invention, the lipase-immobilized enzyme membrane is preferably K U
t'' IJ vinylpyrrolidone 2-20% by weight, 2゜terbis(ri'-azidoco'-sulfobenzal) cyclopentanone sodium salt 0.2-Q, j% by weight, lipase 5-10% by weight, ten dissolved albumin j~IQ weight%,
and an aqueous solution containing a chemical crosslinking agent such as glutaraldehyde.
また、この発明によるリパーゼ固定化酵素膜の製造方法
では、基板の全体にリパーゼ固定化酵素膜を形成する場
合、１０〜２５％のグルタルアルデヒド水溶液が好適に
使用できる。一方、基板の一部のみにリパーゼ固定化酵
素膜を形成する場合には、紫外線をリパーゼ固定化酵素
膜を必要とする部分にのみ照射した後、／−ｆ％のグル
タルアルデヒド水溶液で現像して光架橋した部分の膜を
さらに化学架橋し、光架橋しない部分の膜を溶解して除
去することにより製造することができる。Furthermore, in the method for producing a lipase-immobilized enzyme membrane according to the present invention, when forming a lipase-immobilized enzyme membrane over the entire substrate, a 10-25% glutaraldehyde aqueous solution can be suitably used. On the other hand, when forming a lipase-immobilized enzyme film only on a part of the substrate, after irradiating ultraviolet rays only to the part that requires the lipase-immobilized enzyme film, develop with /-f% glutaraldehyde aqueous solution. It can be produced by further chemically crosslinking the photo-crosslinked portions of the film, and dissolving and removing the non-photocrosslinked portions of the film.
次に、この発明によるリパーゼ固定化酵素膜を図につい
て説明する。Next, the lipase-immobilized enzyme membrane according to the present invention will be explained with reference to the drawings.
第１Ａ図、第ｔＢ図、及び第１Ｃ図はこの発明によるリ
パーゼ固定化酵素膜の製造方法の一連の手順を示す模式
図でちる。第１Ａ図はリパーゼ固定化酵素膜を基板上に
塗布し２、特定の部分に紫外線を照射したリパーゼ固定
化酵素膜の模式図、第１Ｂ図は第１Ａ図で光架橋した後
に水溶液で現像したりパーゼ固定化酵素膜の模式図、第
１Ｃ図は第１Ｂ図で形成された固定化酵素膜をさらにグ
ルタルアルデヒドによシ化学架橋して強度を高めたリパ
ーゼ固定化酵素膜の模式図である。これらの図において
、（１）〜（＜ｔ、′）は上記従来のリパーゼ固定化酵
素膜におけるものと同一である。ＣＺ＞はポリビニルピ
ロリドン、 （Ａ）はコアタービス（ａ／ −アジド
ーユ′−スルホベンザル）シクロペンタノンナトリウム
塩、（７）はリパーゼ固定化酵素膜を光架橋させるため
の紫外線、（す）はフォトマスクである。FIG. 1A, FIG. tB, and FIG. 1C are schematic diagrams showing a series of steps in the method for producing a lipase-immobilized enzyme membrane according to the present invention. Figure 1A is a schematic diagram of a lipase-immobilized enzyme membrane in which a lipase-immobilized enzyme membrane is coated on a substrate 2 and specific portions are irradiated with ultraviolet rays. Figure 1B is a schematic diagram of a lipase-immobilized enzyme membrane in which a lipase-immobilized enzyme membrane is coated on a substrate and specific parts are irradiated with ultraviolet rays. Figure 1C is a schematic diagram of a lipase-immobilized enzyme membrane in which the immobilized enzyme membrane formed in Figure 1B is further chemically cross-linked with glutaraldehyde to increase its strength. . In these figures, (1) to (<t,') are the same as those in the conventional lipase-immobilized enzyme membrane. CZ> is polyvinylpyrrolidone, (A) is Coreturbis (a/-azidoyu'-sulfobenzal) cyclopentanone sodium salt, (7) is ultraviolet light for photocrosslinking the lipase-immobilized enzyme membrane, and (S) is a photomask. be.
実施例 １
分子量３６万のポリビニルピロリドンｆ１０重量％含ん
だ水溶液に、ポリビニルピロリドンに対して２重量％の
コアタービス（ｔｉｔ−アジドーコ′−スルホベ／サル
）シクロペンタノンナトリウム塩（東京応化工業株式会
社製）を含んだ水溶性感光樹脂を調製した。この水溶性
感光樹脂２００μノにリパーゼ／ｆｌＲ９、牛崩清アル
ブミン／Ｑｍ９を溶解し酵素溶液とした。ｒ−アミノプ
ロピルトリエトキシシラ／でアミノ基を表面に導入した
厚さ約５０００にの熱酸化膜がついたシリコンウェハー
基板上に、得られた酵素溶液をスピンナーを用いて２０
００ｒｐｍで２分間塗布した。この基板を２５０Ｗの超
高圧水銀灯で２分間露光し、２ｊ％グルメルアルデヒド
水溶液に５分間浸漬した後、０／Ｍグリシン溶液で／ｊ
分間未反応のアルデヒド基を・反応させてリパーゼ固定
化酵素膜を得た。このリパーゼ固定化酵素膜は、表面に
凹凸が多いが、平均膜厚が約コμｍという薄膜として形
成できた。Example 1 To an aqueous solution containing 10% by weight of polyvinylpyrrolidone f having a molecular weight of 360,000, 2% by weight of coreturbis (tit-azidoco'-sulfobe/sar) cyclopentanone sodium salt (manufactured by Tokyo Ohka Kogyo Co., Ltd.) relative to polyvinylpyrrolidone was added. A water-soluble photosensitive resin containing the following was prepared. Lipase/flR9 and bovine disintegrated albumin/Qm9 were dissolved in 200 μm of this water-soluble photosensitive resin to prepare an enzyme solution. Using a spinner, the obtained enzyme solution was placed on a silicon wafer substrate with a thermally oxidized film about 5,000 µm thick on which amino groups were introduced into the surface using r-aminopropyltriethoxysila/.
The coating was applied for 2 minutes at 00 rpm. This substrate was exposed for 2 minutes with a 250W ultra-high pressure mercury lamp, immersed in a 2j% glumeraldehyde aqueous solution for 5 minutes, and then immersed in a 0/M glycine solution.
The unreacted aldehyde groups were allowed to react for a minute to obtain a lipase-immobilized enzyme membrane. Although this lipase-immobilized enzyme membrane had many irregularities on the surface, it could be formed as a thin film with an average thickness of about 1 μm.
実施例 コ
実施例１と同様に調製した酵素溶液を、実施例１に示し
たものと同じ基板上に、スピンナーを用いてコｏｏｏｒ
ｖａで２分間回転塗布した。次に、この基板の特定の部
分のみに紫外線があたるようなフォトマスクを介して、
コｒＯｗの超高圧水銀灯を用いて紫外線を露光すること
により、基板上の膜の一部だけを光硬化した（第１Ａ図
）。次に、蒸留水にて光硬化していない未硬化の部分の
膜を溶解除去して微小なリパーゼ固定化酵素膜を得た（
第１Ｂ図）。次に、この微小な固定化酵素膜をさらに２
５％グルタルアルデヒド水溶液で５分間処理して化学架
橋することにより、この固定化酵素膜の強度を高めた（
第１Ｃ図）。未反応のアルデヒド基は０．７ Ｍグリシ
ンに／よ分間浸漬することにより反応させた。Example Co An enzyme solution prepared in the same manner as in Example 1 was placed on the same substrate as shown in Example 1 using a spinner.
Spin coating was carried out for 2 minutes using a vacuum cleaner. Next, through a photomask that allows ultraviolet rays to hit only specific parts of the substrate,
Only a portion of the film on the substrate was photocured by exposure to ultraviolet light using an ultra-high pressure mercury vapor lamp (FIG. 1A). Next, the uncured portion of the film that had not been photocured was dissolved and removed with distilled water to obtain a minute lipase-immobilized enzyme film (
Figure 1B). Next, this minute immobilized enzyme membrane was further
The strength of this immobilized enzyme membrane was increased by chemical crosslinking by treatment with a 5% glutaraldehyde aqueous solution for 5 minutes (
Figure 1C). Unreacted aldehyde groups were reacted by immersion in 0.7 M glycine for 1 minute.
実施例 ３
実施例１と同様に調製した酵素溶液を、実施例１に示し
たものと同じ基板上に、スピンナーを用いて２０００
ｆｉｌｌで一分間回転塗布した。次に、この基板の特定
の部分のみにｓｓｏｗの超高圧水銀灯の紫外線があたる
ようなフォトマスクを介して露光することにより、基板
上の膜の一部だけを光硬化した（第１Ａ図）。次に、こ
の基板を３％のグルタルアルデヒド水溶液に浸漬し、未
硬化の部分の膜を溶解除去すると同時に、光硬化した部
分の膜を化学架橋して微小なリパーゼ固定化酵素膜を形
成した（第１Ｃ図）。未反応のアルデヒド基は、０．
／ Ｍグリシンに１３分間浸漬することにより反応させ
た。Example 3 An enzyme solution prepared in the same manner as in Example 1 was placed on the same substrate as shown in Example 1 using a spinner for 2000 min.
Fill was applied by spinning for 1 minute. Next, only a portion of the film on the substrate was photocured by exposing only a specific portion of the substrate to ultraviolet rays from an SSOW ultra-high pressure mercury lamp through a photomask (FIG. 1A). Next, this substrate was immersed in a 3% glutaraldehyde aqueous solution to dissolve and remove the uncured portion of the film, and at the same time chemically crosslink the photocured portion of the film to form a minute lipase-immobilized enzyme film ( Figure 1C). Unreacted aldehyde groups are 0.
/M glycine for 13 minutes to react.
このようにして得たリパーゼ固定化酵素膜は、固定化膜
幅がわずかに太くなるが、−００３ｍピンチのラインア
ンドスペースパターンという線幅にまで微小化すること
ができた。Although the lipase-immobilized enzyme membrane thus obtained had a slightly thicker immobilized membrane width, it was possible to miniaturize the line width to a -003m pinch line-and-space pattern.
実施例 ダ
実施例３と同様に操作を行ない、基板を３％のグルタル
アルデヒド水溶液にて現像した後、さらに、２＆％グル
タルアルデヒド水溶液中に３分間浸漬し、一度化学架橋
している膜をさらに強固に化学架橋して、リパーゼ固定
化酵素膜を得た。Example 3 The same procedure as in Example 3 was carried out, and the substrate was developed with a 3% glutaraldehyde aqueous solution, and then immersed in a 2&% glutaraldehyde aqueous solution for 3 minutes to further remove the chemically crosslinked film. A lipase-immobilized enzyme membrane was obtained by strong chemical crosslinking.
なお、上記実施例では基板として表面にアミノ基を導入
した基板を用いたが、トランスデユーサを用いても同様
の動作を期待できる。In the above embodiment, a substrate having amino groups introduced into the surface was used as the substrate, but a similar operation can be expected even if a transducer is used.
この発明は以上説明したとおり、分子量数万〜数十万の
ポリビニルピロリドンと、コツ５−ビス（ｅｌ−アジド
ーコ′−スルホベンザル）シクロペンタノンナトリウム
塩と、リパーゼと、牛血清アルブミンと化学架橋剤とを
材料として構成したことによシ、フォトリソグラフィー
技術の適用が可能であり、微小なリパーゼ固定化酵素膜
が形成できる効果がある。As explained above, this invention consists of polyvinylpyrrolidone with a molecular weight of tens of thousands to hundreds of thousands, Kotsu-5-bis(el-azidoco'-sulfobenzal)cyclopentanone sodium salt, lipase, bovine serum albumin, and a chemical crosslinking agent. By using this material, it is possible to apply photolithography technology, which has the effect of forming a minute lipase-immobilized enzyme membrane.
また、この発明の別の発明は、上記のリパーゼ固定化酵
素膜を基板上に塗布、乾燥してリパーゼ固定化酵素膜を
形成し、得られたリパーゼ固定化酵素膜に紫外線を照射
することにより光架橋した後、このＩＪ ハーゼ固定化
酵素膜をグルタルアルデヒド水溶液でさらに化学架橋す
ることによシ、微小なリパーゼ固定化酵素膜が形成でき
、また、膜強度も高いことから、小形のバイオセンサが
得られる効果がある。Another invention of the present invention is to apply the above lipase-immobilized enzyme membrane onto a substrate, dry it to form a lipase-immobilized enzyme membrane, and irradiate the obtained lipase-immobilized enzyme membrane with ultraviolet rays. After photo-crosslinking, this IJ hase-immobilized enzyme membrane is further chemically cross-linked with an aqueous glutaraldehyde solution to form a minute lipase-immobilized enzyme membrane.The membrane's strength is also high, so it can be used as a compact biosensor. There is an effect that can be obtained.
第１Ａ図、第１Ｂ図、及び第１（’図はこの発明による
リパーゼ固定化酵素膜の製造方法の一連の手順を示す模
式図、第２図は従来のリパーゼ固定化酵素膜の模式図で
ある。
図において、（／’）は基板、（コ）はリパーゼ、（３
）は十崩潰アルブミン、（り）はグルタルアルデヒド（
りはポリビニルピロリドン、（６）はコツ５−ビス（り
′−アシドー２’−スルホベンザル）シクロペンタノン
ナトリウム塩、（り）は紫外線、（ｔ）はフォトマスク
である。
なお、各図中、同一符号は同一または相当部分を示す。
８ ； ７朴マスク
手続補正書（自発）
昭和６で千 ３月１つ日Figures 1A, 1B, and 1(') are schematic diagrams showing a series of steps of the method for producing a lipase-immobilized enzyme membrane according to the present invention, and Figure 2 is a schematic diagram of a conventional lipase-immobilized enzyme membrane. In the figure, (/') is the substrate, (co) is the lipase, and (3
) is decomposed albumin, (ri) is glutaraldehyde (
ri is polyvinylpyrrolidone, (6) is 5-bis(ri'-acido 2'-sulfobenzal)cyclopentanone sodium salt, (ri) is ultraviolet light, and (t) is a photomask. In each figure, the same reference numerals indicate the same or corresponding parts. 8; 7 Pak Mask Procedural Amendment (Voluntary) March 1st, 1938
Claims (5)
２，５−ビス（４′−アジド−２′−スルホベンザル）
シクロペンタノンナトリウム塩と、リパーゼと、牛血清
アルブミンと、化学架橋剤とを材料として構成したこと
を特徴とするリパーゼ固定化酵素膜。(1) Polyvinylpyrrolidone with a molecular weight of tens of thousands to hundreds of thousands,
2,5-bis(4'-azido-2'-sulfobenzal)
A lipase-immobilized enzyme membrane comprising cyclopentanone sodium salt, lipase, bovine serum albumin, and a chemical crosslinking agent.
重量％、２，５−ビス（４′−アジド−２′−スルホベ
ンザル）シクロペンタノンナトリウム塩０．２〜０、５
重量％、リパーゼ５〜１０重量％、及び牛血清アルブミ
ン５〜１０重量％を含んだ水溶液から作られたものであ
る特許請求の範囲第１項記載のリパーゼ固定化酵素膜。(2) The immobilized enzyme membrane contains polyvinylpyrrolidone 2 to 20%
Weight %, 2,5-bis(4'-azido-2'-sulfobenzal)cyclopentanone sodium salt 0.2-0.5
The lipase-immobilized enzyme membrane according to claim 1, which is made from an aqueous solution containing 5-10% by weight of lipase, and 5-10% by weight of bovine serum albumin.
求の範囲第１項記載のリパーゼ固定化酵素膜。(3) The lipase-immobilized enzyme membrane according to claim 1, wherein the chemical crosslinking agent is glutaraldehyde.
２，５−ビス（４′−アジド−２′−スルホベンザル）
シクロペンタノンナトリウム塩と、リパーゼと、牛血清
アルブミンとを含むリパーゼ固定化酵素膜を基板上に塗
布、乾燥してリパーゼ固定化酵素膜を形成し、得られた
リパーゼ固定化酵素膜に紫外線を照射することにより光
架橋した後、該リパーゼ固定化酵素膜をグルタルアルデ
ヒド水溶液でさらに化学架橋することを特徴とするリパ
ーゼ固定化酵素膜の製造方法。(4) polyvinylpyrrolidone with a molecular weight of tens of thousands to hundreds of thousands,
2,5-bis(4'-azido-2'-sulfobenzal)
A lipase-immobilized enzyme film containing cyclopentanone sodium salt, lipase, and bovine serum albumin is applied onto a substrate, dried to form a lipase-immobilized enzyme film, and the obtained lipase-immobilized enzyme film is exposed to ultraviolet light. A method for producing a lipase-immobilized enzyme membrane, which comprises photo-crosslinking the lipase-immobilized enzyme membrane by irradiation, and then further chemically cross-linking the lipase-immobilized enzyme membrane with an aqueous glutaraldehyde solution.
にのみ照射した後、１〜５％のグルタルアルデヒド水溶
液で現像して光架橋した部分の膜をさらに化学架橋し、
光架橋しない部分の膜を溶解して除去することを特徴と
する特許請求の範囲第４項記載のリパーゼ固定化酵素膜
の製造方法。(5) After irradiating ultraviolet rays only to the areas that require the lipase-immobilized enzyme membrane, the membrane is further chemically cross-linked in the photo-crosslinked areas by developing it with a 1-5% glutaraldehyde aqueous solution;
5. The method for producing a lipase-immobilized enzyme membrane according to claim 4, wherein the portion of the membrane that is not photo-crosslinked is dissolved and removed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61013901A JPS62171685A (en) | 1986-01-27 | 1986-01-27 | Immobilized lypase enzyme membrane and production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61013901A JPS62171685A (en) | 1986-01-27 | 1986-01-27 | Immobilized lypase enzyme membrane and production thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62171685A true JPS62171685A (en) | 1987-07-28 |
Family
ID=11846070
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61013901A Pending JPS62171685A (en) | 1986-01-27 | 1986-01-27 | Immobilized lypase enzyme membrane and production thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62171685A (en) |
-
1986
- 1986-01-27 JP JP61013901A patent/JPS62171685A/en active Pending
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