JP4543163B2 - Latex microparticles carrying an enzyme and biotin anisotropically - Google Patents
Latex microparticles carrying an enzyme and biotin anisotropically Download PDFInfo
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- JP4543163B2 JP4543163B2 JP2003350034A JP2003350034A JP4543163B2 JP 4543163 B2 JP4543163 B2 JP 4543163B2 JP 2003350034 A JP2003350034 A JP 2003350034A JP 2003350034 A JP2003350034 A JP 2003350034A JP 4543163 B2 JP4543163 B2 JP 4543163B2
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- 239000004816 latex Substances 0.000 title claims description 64
- 229920000126 latex Polymers 0.000 title claims description 64
- 102000004190 Enzymes Human genes 0.000 title claims description 36
- 108090000790 Enzymes Proteins 0.000 title claims description 36
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims description 26
- 229960002685 biotin Drugs 0.000 title claims description 12
- 235000020958 biotin Nutrition 0.000 title claims description 12
- 239000011616 biotin Substances 0.000 title claims description 12
- 239000011859 microparticle Substances 0.000 title description 23
- 239000010419 fine particle Substances 0.000 claims description 58
- 239000002245 particle Substances 0.000 claims description 25
- 238000007720 emulsion polymerization reaction Methods 0.000 claims description 22
- 125000003700 epoxy group Chemical group 0.000 claims description 21
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 17
- 239000000178 monomer Substances 0.000 claims description 17
- 125000004057 biotinyl group Chemical group [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 claims description 16
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 11
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 11
- 125000003277 amino group Chemical group 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
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- 229920000642 polymer Polymers 0.000 claims description 8
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- 239000002904 solvent Substances 0.000 claims description 4
- 229920002818 (Hydroxyethyl)methacrylate Polymers 0.000 claims description 3
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 claims description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 8
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 8
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 8
- LXEKPEMOWBOYRF-UHFFFAOYSA-N [2-[(1-azaniumyl-1-imino-2-methylpropan-2-yl)diazenyl]-2-methylpropanimidoyl]azanium;dichloride Chemical compound Cl.Cl.NC(=N)C(C)(C)N=NC(C)(C)C(N)=N LXEKPEMOWBOYRF-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
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- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 5
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
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- CCTFAOUOYLVUFG-UHFFFAOYSA-N 2-(1-amino-1-imino-2-methylpropan-2-yl)azo-2-methylpropanimidamide Chemical compound NC(=N)C(C)(C)N=NC(C)(C)C(N)=N CCTFAOUOYLVUFG-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 150000001615 biotins Chemical class 0.000 description 2
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- 238000010538 cationic polymerization reaction Methods 0.000 description 2
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- 229940068840 d-biotin Drugs 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 229940076788 pyruvate Drugs 0.000 description 2
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- 229910001927 ruthenium tetroxide Inorganic materials 0.000 description 2
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- KFDVPJUYSDEJTH-UHFFFAOYSA-N 4-ethenylpyridine Chemical compound C=CC1=CC=NC=C1 KFDVPJUYSDEJTH-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
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- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 1
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Description
本発明は、複数の生体・化学物質（例えば酵素など）を異方性ラテックス微粒子の各々の官能基を介して異方的に固定化することにより、多段階酵素反応や微小空間における物質輸送を行うナノバイオマシンの要素デバイスを創製する。 In the present invention, a plurality of biological / chemical substances (for example, enzymes) are anisotropically immobilized through the functional groups of anisotropic latex fine particles, thereby enabling multi-stage enzyme reaction and mass transport in a minute space. Create elemental devices for nanobiomachines.
近年、単一の官能基を有するラテックス微粒子は、ラテックス診断薬、アフィニティー生体物質分離、ドラッグデリバリーシステム、酵素の担体などのバイオ分野へ幅広く応用されている。これまでに、例えばタンパク質、DNA、医薬分子などがラテックス微粒子に固定化されてきた。このように、官能性ラテックス微粒子はナノバイオテクノロジーの重要な基盤的要素技術のひとつとして注目されている。
一方、固定化酵素はバイオ材料、食品工業と分析化学などに広く利用されてきた。様々な酵素は物理的吸着、共有結合及びカプセル化によって、異なった形（例えば、膜、ビーズなど）を持つ担体に固定化できる。このような不溶材質に固定化した酵素は、フリーな（固定化されていない）酵素と比べると、分離が容易、連続操作が可能、操作が簡単、ある種の酵素では安定性が向上するなどの利点がある。
ラテックス微粒子は粒子径が小さいゆえに、平面担体と比べると比表面積が大きく、より大量の酵素が固定化でき、酵素担体としての応用が期待されている。
一方、ソープフリー乳化重合により、ラテックスを得ることは知られている（非特許文献１）。
本発明者は、グリシジルメタクリレートモノマーとジビニルベンゼンモノマーを重合開始剤の存在下でソープフリー乳化重合を行い、シードポリマー粒子を合成し、次いで、スチレン、2-メルカプトエタノール及び溶媒を加えて、水中でソープフリーシード乳化重合を行い、官能基としてエポキシ環と水酸基をそれぞれ微粒子の異なるドメインに持たせた異方性高分子微粒子（異方性ラテックス微粒子）に関する発明を完成させて出願している（特許文献１）。
さらに本発明者は、エポキシ環と水酸基をそれぞれ微粒子の異なるドメインに持たせた異方性ラテックス微粒子を用いて、水酸基側を活性エステル型ビオチン誘導体と反応させ、微粒子の片側のみをビオチン化した異方性ビオチン化ラテックス微粒子に関する発明を完成させて出願している（特許文献２）。
On the other hand, immobilized enzymes have been widely used in biomaterials, food industry and analytical chemistry. Various enzymes can be immobilized on carriers with different shapes (eg, membranes, beads, etc.) by physical adsorption, covalent bonding and encapsulation. Enzymes immobilized on such insoluble materials are easier to separate, continuous operation, easier to operate, and more stable with certain enzymes, compared to free (non-immobilized) enzymes. There are advantages.
Latex fine particles have a small particle size, and therefore have a larger specific surface area than that of a flat carrier, can immobilize a larger amount of enzyme, and are expected to be applied as an enzyme carrier.
On the other hand, it is known that latex is obtained by soap-free emulsion polymerization (Non-patent Document 1).
The present inventor performed soap-free emulsion polymerization of glycidyl methacrylate monomer and divinylbenzene monomer in the presence of a polymerization initiator to synthesize seed polymer particles, and then added styrene, 2-mercaptoethanol and a solvent in water. We have completed and filed an application for anisotropic polymer fine particles (anisotropic latex fine particles) that have undergone soap-free seed emulsion polymerization and have epoxy rings and hydroxyl groups as functional groups in different domains of the fine particles (patent) Reference 1).
Further, the present inventor used anisotropic latex fine particles having epoxy rings and hydroxyl groups in different domains of the fine particles, reacted with an active ester type biotin derivative on the hydroxyl side, and biotinylated only one side of the fine particles. Kataseibi punch line emission of latex invention was completed regarding microparticles are filed (Patent Document 2).
しかし、上記の発明により得られる異方性ビオチン化ラテックス微粒子の粒子径は、２００nmのサイズ径であり、もっと小さい微粒子の異方性ビオチン化ラテックス微粒子が望まれていた。
生体系の多くの反応や輸送は酵素反応によって進むが、その中にはアデノシン三リン酸(ATP)という高エネルギー物質を必要とするものがある。生体系に存在する分子モーターとよばれる生体ナノマシンも、運動するためにはATPのエネルギーを必要とする。しかしながら、分子モーターはそれ自身ではATPを作ることができず、外から供給しなければならない。そこで、本発明で解決しようとする課題は、分子モーターに結合することができ、かつATPを生産することができる新規微粒子を開発することである。すなわち、１００nm程度のサイズ径であり、微粒子の片側は分子モーターへの結合部位Aを持ち、他の片側はATPを作る酵素への結合部位Bを持ち、酵素とビオチンを異方的に担持したラテックス微粒子を作り出すことを目的とする。
However, the particle diameter of the anisotropic biotinyl emissions latex particles obtained by the invention is the size diameter of 200 nm, it has been desired anisotropic biotinyl emissions latex particulates of smaller particles.
Many reactions and transport in biological systems proceed by enzymatic reactions, some of which require a high-energy substance called adenosine triphosphate (ATP). Biological nanomachines called molecular motors in biological systems also require the energy of ATP to move. However, molecular motors themselves cannot make ATP and must be supplied from the outside. Therefore, the problem to be solved by the present invention is to develop new fine particles that can be bound to a molecular motor and can produce ATP. That is, it has a size diameter of about 100 nm, one side of the fine particle has a binding site A to the molecular motor, the other side has a binding site B to the enzyme that produces ATP, and carries the enzyme and biotin anisotropically. The goal is to create latex particulates.
本発明において、上記の課題を解決すべく、鋭意研究した結果、広い領域のドメインＡにある水酸基と狭い領域のドメインＢにあるエポキシ基を、それぞれ微粒子に持たせた異方性ラテックス微粒子であって、エポキシ側をアミノ基に変換し、微粒子の片側のみをビオチン化した異方性ビオチン化ラテックス微粒子とすることにより、上記課題を解決することに成功した。
本発明で用いる二つの官能性を有する異方性複合ラテックス微粒子は、ソープフリー乳化重合によって合成され、約100nmのサイズを持ち、種々の組み合わせの官能基を持つ微粒子を製造することができる。本発明者は、ヒドロキシエチルメタクリレートモノマーとメチルメタクリレートモノマーとエチレンジメタクリレートモノマーを重合開始剤の存在下でソープフリー乳化重合させ、シードポリマー粒子を合成し、次いで、スチレンモノマーとグリシジルメタクリレートモノマーとジビニルベンゼンモノマー及び溶媒を加えて、水中でソープフリーシード乳化重合を行い、官能基として水酸基とエポキシ環をそれぞれ微粒子の異なる大きさのドメインに持たせた異方性異方性ラテックス微粒子を調製できる。
本発明においては、水酸基とエポキシ基をそれぞれ微粒子の異なるドメインに持たせた異方性ラテックス微粒子の典型的な製造方法は、ヒドロキシエチルメタクリレートモノマーとメチルメタクリレートモノマーとエチレンジメタクリレートを重合開始剤の存在下でソープフリー乳化重合を行ってシードポリマー粒子を合成し、次いで、スチレンモノマーとグリシジルメタクリレートモノマーとジビニルベンゼンモノマー及び溶媒を加えて、水中でソープフリーシード乳化重合を行う方法である。エポキシ基はアミノ基に変換しビオチンを結合し、次に水酸基をエポキシ基へ変換する。この製造方法により得られた異方性ビオチン化ラテックス微粒子を用いて、２つの結合部位A、Bにそれぞれ独立に反応する性質をもつ異方性ビオチン化ラテックス微粒子を製造することができる。
また、本発明においては、異方性ビオチン化ラテックス微粒子の粒子径は、１００ｎｍであることが好ましい。
さらに、本発明においては、異方性ラテックス微粒子の水酸基側をエピクロルヒドリン等でエポキシ基に変換し、これにアミノ基、水酸基、チオール基などから選ばれる官能基を有する化合物を反応させて、２つの結合部位A、Bにそれぞれ独立に反応する性質をもつ異方性ビオチン化ラテックス微粒子を得ることもできる。
さらにまた、本発明は、異方性ビオチン化ラテックス微粒子のエポキシ基に、酵素を固定した異方性ビオチン化ラテックス微粒子とすることができる。
このとき、酵素が、ピルビン酸キナーゼ（pyruvate kinase）であることが好ましいが、原理的にはあらゆる酵素が可能である。
In the present invention, as a result of intensive studies to solve the above problems, anisotropic latex fine particles in which fine particles have a hydroxyl group in a wide domain A and an epoxy group in a narrow domain B are obtained. Te, converts the epoxy side to an amino group, by only one side of the fine particles and anisotropic biotinyl emissions latex microparticles biotinylated, it succeeded in solving the above problems.
The anisotropic composite latex fine particles having two functionalities used in the present invention are synthesized by soap-free emulsion polymerization, and have a size of about 100 nm and can produce fine particles having various combinations of functional groups. The inventor conducted soap-free emulsion polymerization of hydroxyethyl methacrylate monomer, methyl methacrylate monomer, and ethylene dimethacrylate monomer in the presence of a polymerization initiator to synthesize seed polymer particles, and then styrene monomer, glycidyl methacrylate monomer, and divinylbenzene. By adding a monomer and a solvent, soap-free seed emulsion polymerization is performed in water to prepare anisotropic anisotropic latex fine particles having hydroxyl groups and epoxy rings as functional groups in domains of different sizes.
In the present invention, a typical method for producing anisotropic latex fine particles having a hydroxyl group and an epoxy group in different domains of fine particles is the presence of a polymerization initiator comprising hydroxyethyl methacrylate monomer, methyl methacrylate monomer and ethylene dimethacrylate. In this method, soap-free emulsion polymerization is performed to synthesize seed polymer particles, and then styrene monomer, glycidyl methacrylate monomer, divinylbenzene monomer and solvent are added to perform soap-free seed emulsion polymerization in water. The epoxy group is converted to an amino group to bind biotin, and then the hydroxyl group is converted to an epoxy group. Using anisotropic biotinyl emissions latex particles obtained by this manufacturing method, the two binding sites A, is possible to manufacture the anisotropic biotinyl emissions latex particulates having a property that reacts independently to B it can.
In the present invention, the particle diameter of the anisotropic biotinyl emissions latex particles is preferably 100 nm.
Furthermore, in the present invention, the hydroxyl group side of the anisotropic latex fine particles is converted to an epoxy group with epichlorohydrin or the like, and a compound having a functional group selected from an amino group, a hydroxyl group, a thiol group and the like is reacted therewith, binding site a, anisotropic biotinyl emissions latex particles having a property of reacting each independently B can be obtained.
Furthermore, the present invention is the epoxy group of the anisotropic biotinyl emissions latex particulates can be anisotropic biotinyl emissions latex particulates with a fixed enzyme.
At this time, the enzyme is preferably pyruvate kinase, but in principle, any enzyme is possible.
本発明で開発した「酵素とビオチンを異方的に担持したラテックス微粒子」の出発原料である異方性ラテックス微粒子は二段階ソープフリー乳化重合によって合成される。ソープフリー乳化重合とは、モノマーと水の混合物にラジカル重合開始剤を導入し、ラテックス微粒子を作る方法であり、通常の乳化重合との違いは界面活性剤を使用しないので、粒子表面の電荷が、通常の乳化重合によって合成された微粒子の表面電荷よりも低く、また適度なサイズを持つので簡単に遠心分離できる。ラテックス微粒子の特徴は、得られた微粒子の単分散性（サイズの均一性）が高いことである。
また、カチオン性の重合開始剤2,2’-azobis(2-amidinopropane)2HCl(V-50)等を使用し、微粒子に正電荷を持たせた微粒子を用いて酵素を固定化すれば、酵素の固定化速度や固定化量が増加することができる。
さらに、アデノシン二リン酸(ADP)をATPに転換する酵素、ピルビン酸キナーゼ（pyruvate kinase）をエポキシ基を介して直接微粒子に固定化したものは、図３に示すピルビン酸の時間 生成曲線特性を有する。
The anisotropic latex fine particles, which are the starting materials for the “latex fine particles anisotropically supporting enzyme and biotin” developed in the present invention, are synthesized by two-stage soap-free emulsion polymerization. Soap-free emulsion polymerization is a method in which a radical polymerization initiator is introduced into a mixture of monomer and water to make latex fine particles. The difference from ordinary emulsion polymerization is that a surfactant is not used, so the charge on the particle surface is reduced. Since the surface charge of the fine particles synthesized by ordinary emulsion polymerization is lower than that of the fine particles and has an appropriate size, it can be easily centrifuged. The feature of latex fine particles is that the obtained fine particles have high monodispersity (size uniformity).
In addition, if a cationic polymerization initiator 2,2'-azobis (2-amidinopropane) 2HCl (V-50) or the like is used and the enzyme is immobilized using fine particles in which fine particles have a positive charge, the enzyme The immobilization speed and the amount of immobilization can be increased.
Furthermore, the enzyme that converts adenosine diphosphate (ADP) to ATP, pyruvate kinase, directly immobilized on the microparticles via an epoxy group, shows the time generation curve characteristics of pyruvate shown in FIG. Have.
本発明の異方性ビオチン化ラテックス微粒子は、表面の片側にエポキシ基ドメイン、他の片側に水酸基ドメインを有する異方性ラテックス微粒子（直径：約100nm程度）を用いて、まず、エポキシ基側をアミノ化しカルボン酸型ビオチン誘導体と反応させ、微粒子の片側のみをビオチン化する。ビオチンは緩やかな反応条件下でアビジンと特異的に結合するので、ビオチン化した微粒子はアビジン化したものに簡単に固定化できる。一方、ラテックス微粒子の水酸基側は、エピクロルヒドリン等によりエポキシ化し、アミノ基、水酸基、チオール基などを有する種々の物質を簡単に直接固定化、あるいは、エポキシ基にリンカーを導入してから様々な生体・化学物質を固定化することができる。
さらに、本発明では、異方性ラテックス微粒子は二段階ソープフリー乳化重合によって合成される。ソープフリー乳化重合とは、モノマーと水の混合物にラジカル重合開始剤を導入し、ラテックス微粒子を作る方法であり、通常の乳化重合との違いは界面活性剤を使用しない。したがって、粒子表面の電荷が、通常の乳化重合によって合成された微粒子の表面電荷よりも低く、また適度なサイズを持つので簡単に遠心分離できる。ラテックス微粒子の特徴は、得られた微粒子の単分散性（サイズの均一性）が高いことである。
この異方性ラテックス微粒子は、「酵素とビオチンを異方的に担持したラテックス微粒子」の出発原料となる。
また、固定化酵素に影響を与える重要な因子の一つは、担体表面の静電的性質である。多くの酵素やタンパク質の等電点はpH7以下であり、中性或いはその以上のpHではそれらは負の電荷を有する。本発明では、異方性ラテックス微粒子を調製する際に、カチオン性の重合開始剤2,2’-azobis(2-amidinopropane)2HCl(V-50)等を使用し、微粒子に正電荷を持たせた。このような正電荷を持った微粒子を用いて酵素を固定化すれば、酵素の固定化速度や固定化量が増加すると考えられる。
Anisotropic biotinyl emissions latex particulates of the present invention, an epoxy group domains on one surface, anisotropic latex particulates having a hydroxyl domain other side (diameter: about 100 nm) using a first, epoxy group The side is aminated and reacted with a carboxylic acid-type biotin derivative, and only one side of the microparticle is biotinylated. Since biotin specifically binds to avidin under mild reaction conditions, biotinylated microparticles can be easily immobilized on avidinized ones. On the other hand, the hydroxyl side of latex fine particles is epoxidized with epichlorohydrin or the like, and various substances having amino groups, hydroxyl groups, thiol groups, etc. are easily fixed directly, or various linkers are introduced into the epoxy group and various biological / Chemical substances can be immobilized.
Furthermore, in the present invention, the anisotropic latex fine particles are synthesized by two-stage soap-free emulsion polymerization. Soap-free emulsion polymerization is a method in which a radical polymerization initiator is introduced into a mixture of a monomer and water to form latex fine particles. A difference from normal emulsion polymerization is that a surfactant is not used. Therefore, since the surface charge of the particles is lower than the surface charge of the fine particles synthesized by ordinary emulsion polymerization and has an appropriate size, it can be easily centrifuged. The feature of latex fine particles is that the obtained fine particles have high monodispersity (size uniformity).
The anisotropic latex fine particles serve as a starting material for “latex fine particles carrying an enzyme and biotin anisotropically”.
One of the important factors affecting the immobilized enzyme is the electrostatic property of the support surface. The isoelectric point of many enzymes and proteins is below pH 7, and at neutral or higher pH they have a negative charge. In the present invention, a cationic polymerization initiator 2,2′-azobis (2-amidinopropane) 2HCl (V-50) or the like is used to prepare the anisotropic latex fine particles so that the fine particles have a positive charge. It was. If the enzyme is immobilized using such fine particles having a positive charge, it is considered that the enzyme immobilization speed and amount are increased.
１．異方性ラテックス微粒子の調製：
異方性ラテックス微粒子poly(2-hydroxylethylmethacrylate-co-methylmethacrylate)/poly-(styrene-co-glycidylmethacrylate)(P(HEMA-MMA)/P(St-GMA)は二段階ソープフリー乳化重合により合成した。その合成ルートは下に示す。
最初に、純水(285g)中、重合開始剤2,2’-azobis(2-amidinopropane)2HCl(V-50)(0.45g)の存在下、２−ヒドロキシメチルメタクリレート(HEMA)(0.6g)、メタクリル酸メチル(MMA)(11.4g)、架橋剤エチレンジメタクリレート(EGDMA)(3g)を80℃、200rpmの攪拌速度下で１５時間反応させた。得られたシード微粒子はセルロース膜を用いて、24時間水道水、24時間純水で透析を行い、未反応のモノマー、開始剤、オリゴマーを除去した。
次にEGDMAで架橋したP(HEMA-MMA)シード粒子を用い、ソープフリーシード乳化重合により多官能性ラテックス微粒子(P(HEMA-MMA)/P(St-GMA))を調製した。その方法と重合条件は以下の通りである。
純水(154g)中、P(HEMA-MMA)シードラテックス(40.6g、ポリマー微粒子の含量は2g)、スチレン(St)(1g)、メタクリル酸グリシジル(0.05g)、ジビニルベンゼン(DVB)(0.05g)、V-50(0.02g)、トルエン(4g)を反応させた。重合反応は200rpm、70℃で24時間行った。二段階の重合反応のモノマー変化率はすべて100%、得られた複合高分子ラテックスの固形分は1.5%、数平均直径は約100nmであった。ラテックス微粒子を四酸化ルテニウムによって染色して測定したTEM写真を図１に示す。
TEMの結果より、両側にそれぞれ水酸基ドメインとエポキシ基ドメインを有するラテックス微粒子が得られたことが示唆された。ここで、暗い部分はポリスチレンーメタクリル酸グリシジルドメイン（エポキシ基ドメイン）で、より明るい部分はポリＨＥＭＡ−ＭＭＡドメイン（水酸基ドメイン）を示す。
1. Preparation of anisotropic latex particulates:
Anisotropic latex microparticles poly (2-hydroxylethylmethacrylate-co-methylmethacrylate) / poly- (styrene-co-glycidylmethacrylate) (P (HEMA-MMA) / P (St-GMA)) were synthesized by two-stage soap-free emulsion polymerization. The synthesis route is shown below.
First, in pure water (285 g), 2-hydroxymethyl methacrylate (HEMA) (0.6 g) in the presence of a polymerization initiator 2,2′-azobis (2-amidinopropane) 2HCl (V-50) (0.45 g) Then, methyl methacrylate (MMA) (11.4 g) and a crosslinking agent ethylene dimethacrylate (EGDMA) (3 g) were reacted at 80 ° C. under a stirring speed of 200 rpm for 15 hours. The obtained seed microparticles were dialyzed using a cellulose membrane with tap water for 24 hours and pure water for 24 hours to remove unreacted monomers, initiators and oligomers.
Next, polyfunctional latex fine particles (P (HEMA-MMA) / P (St-GMA)) were prepared by soap-free seed emulsion polymerization using P (HEMA-MMA) seed particles crosslinked with EGDMA. The method and polymerization conditions are as follows.
In pure water (154 g), P (HEMA-MMA) seed latex (40.6 g, content of polymer fine particles is 2 g), styrene (St) (1 g), glycidyl methacrylate (0.05 g), divinylbenzene (DVB) (0.05 g), V-50 (0.02 g), and toluene (4 g) were reacted. The polymerization reaction was carried out at 200 rpm and 70 ° C. for 24 hours. The monomer change rates of the two-stage polymerization reactions were all 100%, the solid content of the obtained composite polymer latex was 1.5%, and the number average diameter was about 100 nm. FIG. 1 shows a TEM photograph obtained by staining latex fine particles with ruthenium tetroxide.
From the TEM results, it was suggested that latex microparticles having hydroxyl and epoxy groups on both sides were obtained. Here, a dark part shows a polystyrene-glycidyl methacrylate domain (epoxy group domain), and a brighter part shows a poly-HEMA-MMA domain (hydroxyl domain).
２．異方性ラテックス微粒子のエポキシ側のビオチン化：
まず、微粒子の表面のエポキシ基をアミノ基に転換させる。ラテックス微粒子(20g)に28%のアンモニア水溶液(5g)を加え、その混合物を攪拌しながら、65℃、24時間を反応させた。得られたアミノ化微粒子を純水で６回洗った、得られたアミノ基をニンヒドリンテストにより定量した結果、約593個/微粒子であった。その後、アミノ基をカルボジイミドの存在下で、カルボン酸型ビオチン、d-biotinと反応させ、ビオチン化した。反応式は下に示す。
微粒子のビオチン結合を確認するために、ビオチン化した微粒子をアビジン化した金微粒子（Streptavidin金微粒子：直径10nm）と結合させ、図２に示すようにTEM観察（四酸化ルテニウムで染色せず）によって金微粒子を確認した。
その結果、明らかに金微粒子がラテックス微粒子の一端（ポリSt-GMA側）に局在していて、ビオチンの分布が偏っていることが判明した。
2. Biotinylation of anisotropic latex fine particles on the epoxy side:
First, the epoxy group on the surface of the fine particles is converted to an amino group. A 28% aqueous ammonia solution (5 g) was added to the latex fine particles (20 g), and the mixture was reacted at 65 ° C. for 24 hours while stirring. The obtained aminated fine particles were washed 6 times with pure water. The obtained amino groups were quantified by the ninhydrin test, and as a result, the number was about 593 particles / fine particle. Thereafter, the amino group was biotinylated by reacting with carboxylic acid type biotin and d-biotin in the presence of carbodiimide. The reaction formula is shown below.
In order to confirm the biotin binding of the microparticles, biotinylated microparticles were combined with avidinized gold microparticles (Streptavidin gold microparticles: 10 nm in diameter) and TEM observation (not stained with ruthenium tetroxide) as shown in FIG. Gold fine particles were confirmed.
As a result, it was found that the gold fine particles were clearly localized at one end (poly St-GMA side) of the latex fine particles, and the biotin distribution was biased.
３．異方性ビオチン化ラテックス微粒子の水酸基側の酵素固定化：
アミノ基を有する酵素を直接共有結合により固定化するため、水酸基をアミノ基との反応性を富むエポキシ基へ変換した。5gのビオチン化微粒子を5gの1N NaOH水溶液で置換させ、5gのepichlorohydrinを添加し、室温で攪拌しながら、16時間を反応させた。反応式は下に示す。得られたエポキシ基の定量はエポキシ基をアミノ基に転換させ、ニンヒドリンテストにより行った。その値は約2.9×104個/微粒子であった。
In order to immobilize the amino group-containing enzyme directly by covalent bonding, the hydroxyl group was converted to an epoxy group rich in reactivity with the amino group. 5 g of biotinylated fine particles were replaced with 5 g of 1N NaOH aqueous solution, 5 g of epichlorohydrin was added, and the mixture was reacted at room temperature for 16 hours. The reaction formula is shown below. The obtained epoxy group was quantified by converting the epoxy group to an amino group and performing a ninhydrin test. The value was about 2.9 × 10 4 particles / fine particle.
４．異方性ビオチン化ラテックス微粒子に固定した酵素の活性測定：
固定化酵素の活性は下に示した二段階酵素反応により計測した。
ホスホエノールピルビン酸（PEP）(26.5mg)、ADP(50.3mg)と還元型ニコチンアミドアデニンジヌクレオチド(NADH)(70.9mg)を37mM KClと10mM MgSO4を含むpH7.4のリン酸緩衝液1Lに添加し、よく混合後、混合液の340nmでのUV吸収を測定した。その後、110mgの固定化ピルビン酸キナーゼ（1gのラテックス微粒子に約150mgのピルビン酸キナーゼを固定した微粒子）或いはフリーのピルビン酸キナーゼ(110mg)と100mgの乳酸脱水素酵素を添加し、二段階酵素反応を開始させた。反応は25℃の恒温水槽で攪拌しながら行った。ピルビン酸の時間 生成曲線を図３に示す。用いたピルビン酸キナーゼの活性は200units/mg、乳酸脱水素酵素の活性は1000 nits/mgであるので、一段目の酵素反応で生成したピルビン酸は速やかに乳酸へ転換させることができる。そのために、NADHの減少量はピルビン酸の生成量に一致すると考えられる。酵素の活性は基質PEP濃度の逆数対反応時間のプロットの傾きから計算され、固定化酵素の活性は約フリーな酵素の活性の20%であった。
4). Measurement of enzyme activity immobilized on anisotropic biotinylated latex microparticles :
The activity of the immobilized enzyme was measured by the two-stage enzyme reaction shown below.
Phosphoenol pyruvate (PEP) (26.5mg), ADP (50.3mg) and reduced nicotinamide adenine dinucleotide (NADH) (70.9mg) and 37 mM KCl and phosphate buffer 1L of pH7.4 containing 10 mM MgSO 4 After mixing well, the UV absorption at 340 nm of the mixture was measured. Then, add 110 mg of immobilized pyruvate kinase (fine particles of about 150 mg of pyruvate kinase immobilized on 1 g of latex microparticles) or free pyruvate kinase (110 mg) and 100 mg of lactate dehydrogenase, and perform a two-step enzyme reaction Was started. The reaction was carried out with stirring in a constant temperature water bath at 25 ° C. The time generation curve of pyruvic acid is shown in FIG. Since the pyruvate kinase used has an activity of 200 units / mg and lactate dehydrogenase has an activity of 1000 nits / mg, pyruvate generated in the first-stage enzyme reaction can be quickly converted to lactic acid. For this reason, the decrease in NADH is considered to coincide with the amount of pyruvic acid produced. The enzyme activity was calculated from the slope of the plot of the inverse of substrate PEP concentration versus reaction time, and the immobilized enzyme activity was approximately 20% of the free enzyme activity.
本微粒子のエポキシ基はほとんどの酵素と結合可能であり、またビオチンはアビジンを介して種々の物質と結合可能であるため、本微粒子は一種の「固定化酵素カセット」あるいは「固定化酵素デバイス」と考えられ、広い分野での応用が期待できる。 Since the epoxy group of this microparticle can bind to most enzymes and biotin can bind to various substances via avidin, this microparticle is a kind of "immobilized enzyme cassette" or "immobilized enzyme device". It can be expected to be applied in a wide range of fields.
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