JPH02102203A - Water-soluble polymeric compound containing immobilized chemiluminescent reagent and method for immobilization - Google Patents
Water-soluble polymeric compound containing immobilized chemiluminescent reagent and method for immobilizationInfo
- Publication number
- JPH02102203A JPH02102203A JP25545988A JP25545988A JPH02102203A JP H02102203 A JPH02102203 A JP H02102203A JP 25545988 A JP25545988 A JP 25545988A JP 25545988 A JP25545988 A JP 25545988A JP H02102203 A JPH02102203 A JP H02102203A
- Authority
- JP
- Japan
- Prior art keywords
- water
- chemiluminescent reagent
- polymer compound
- soluble polymer
- substance
- 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
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 44
- 229920003169 water-soluble polymer Polymers 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims description 17
- 239000000126 substance Substances 0.000 claims abstract description 43
- 150000001875 compounds Chemical class 0.000 claims abstract description 38
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 29
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229920000642 polymer Polymers 0.000 claims abstract description 12
- 239000001257 hydrogen Substances 0.000 claims abstract description 10
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 10
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000003377 acid catalyst Substances 0.000 claims abstract description 6
- 230000004048 modification Effects 0.000 claims abstract description 6
- 238000012986 modification Methods 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 238000007127 saponification reaction Methods 0.000 claims abstract description 5
- 239000010409 thin film Substances 0.000 claims description 19
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 17
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 13
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 13
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 11
- 239000007795 chemical reaction product Substances 0.000 claims description 8
- 230000003100 immobilizing effect Effects 0.000 claims description 7
- 230000035699 permeability Effects 0.000 abstract description 2
- 238000011156 evaluation Methods 0.000 abstract 1
- 239000003054 catalyst Substances 0.000 description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000004020 luminiscence type Methods 0.000 description 9
- 102000003992 Peroxidases Human genes 0.000 description 8
- 108040007629 peroxidase activity proteins Proteins 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 6
- 108010015776 Glucose oxidase Proteins 0.000 description 5
- 239000004366 Glucose oxidase Substances 0.000 description 5
- 102000004316 Oxidoreductases Human genes 0.000 description 5
- 108090000854 Oxidoreductases Proteins 0.000 description 5
- 239000012467 final product Substances 0.000 description 5
- 229940116332 glucose oxidase Drugs 0.000 description 5
- 235000019420 glucose oxidase Nutrition 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 2
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000009435 amidation Effects 0.000 description 2
- 238000007112 amidation reaction Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 238000006193 diazotization reaction Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000004539 Acyl-CoA Oxidase Human genes 0.000 description 1
- 108020001558 Acyl-CoA oxidase Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- 108010000659 Choline oxidase Proteins 0.000 description 1
- 102000005870 Coenzyme A Ligases Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 description 1
- 238000006683 Mannich reaction Methods 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000011420 Phospholipase D Human genes 0.000 description 1
- 108090000553 Phospholipase D Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 108010042687 Pyruvate Oxidase Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108010060059 Sarcosine Oxidase Proteins 0.000 description 1
- 102000008118 Sarcosine oxidase Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000000019 Sterol Esterase Human genes 0.000 description 1
- 108010055297 Sterol Esterase Proteins 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 108010090622 glycerol oxidase Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- YSZIOXAEADAJLX-UHFFFAOYSA-N phthalazine-1,4-dione Chemical compound C1=CC=C2C(=O)N=NC(=O)C2=C1 YSZIOXAEADAJLX-UHFFFAOYSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002689 polyvinyl acetate Polymers 0.000 description 1
- 239000011118 polyvinyl acetate Substances 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は化学発光を利用した薄膜の物質透過性能を評価
する方法において使用する化学発光試薬、及びその製法
に関し、特に薄膜が生体膜である場合の使用に適した化
学発光試薬、及びその製法に関する。[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a chemiluminescent reagent used in a method for evaluating the substance permeation performance of a thin film using chemiluminescence, and a method for producing the same, particularly when the thin film is a biological membrane. This invention relates to chemiluminescent reagents suitable for use in various cases, and methods for producing the same.
ルミノール（５−ａｍｉｎｏ−２，３−ｄｉｈｙｄｒｏ
１，４−ｐｈｔｈａｌａｚｉｎｅｄｉｏｎｅ ）等の
化学発光試薬をアミノ酸、また蛋白質等に固定化（ラベ
ル化）し、高速液体クロマトグラフィーでの化学発光を
用いた高感度検出や抗原抗体反応等に利用する試みは、
従来から数多くの研究がなされ、主としてジアゾ化法、
カルボジイミド法によるカップリング反応が利用されて
いる（例えばＡｎｔｈｏｎｙ Ｋ、 Ｃａｍｐｂｅｌｌ
ａｎｄ Ｊ。Luminol (5-amino-2,3-dihydro
Attempts have been made to immobilize (label) chemiluminescent reagents such as 1,4-phthalazinedione) on amino acids, proteins, etc., and use them for high-sensitivity detection using chemiluminescence in high-performance liquid chromatography, antigen-antibody reactions, etc. ,
A lot of research has been done to date, mainly on diazotization method,
Coupling reactions using the carbodiimide method have been used (for example, Anthony K., Campbell et al.
and J.
５ｔｅｖｅｎ Ａ、 Ｓｉｍｐｓｏｎ 、 Ｔｅｃｈｎ
ｉｑｕｅｓ ｉｎ ＭｅｔａｂｏｌｉｃＲｅｓｅａｒｃ
ｈ、 Ｂ２１３、ＰＩ〜Ｐ５６）。5teven A., Simpson, Techn.
iques in MetabolicResearch
h, B213, PI-P56).
しかしながら従来のカップリング反応を利用した化学発
光試薬のアミノ酸、また蛋白質等への固定化方法は、例
えばルミノールを直接ジアゾ化したり、またはアミド化
するとルミノールのπ電子系に太き（影響を与えるため
に、−旦ルミノール誘導体とする必要があり、一般に反
応工程が複雑である。ルミノールにおける芳香族アミノ
基の電子供与性は、ルミノールの発光機構に重要な寄与
をしており、そのアミノ基をジアゾ化したり、アミド化
すると、ルミノールを固定化しえても著しく発光効率が
減少したり或いは全く発光しなくなるという問題があり
、ルミノール誘導体を経由せざるを得なかった。However, conventional methods for immobilizing chemiluminescent reagents onto amino acids, proteins, etc. using coupling reactions do not allow direct diazotization or amidation of luminol, which may affect the π-electron system of luminol. To do this, it is necessary to convert the amino group into a luminol derivative, and the reaction process is generally complicated.The electron-donating property of the aromatic amino group in luminol makes an important contribution to the luminescence mechanism of luminol, or amidation, there is a problem that even if luminol can be immobilized, the luminous efficiency is significantly reduced or no luminescence is emitted at all, so it has been necessary to use luminol derivatives.
本発明者等は化学発光を利用した薄膜の物質透過性能を
評価する方法を出願している（特開昭６３−１３３０３
９号、特願昭６３−１０３６００号、特願昭６３−１３
８１０８号、特願昭６３２０２４７２号）が、薄膜とし
て生体膜を対象とする場合、ルミノールは水に対してあ
まり溶解しないために、例えばジメチルスルフオキシド
等と水との混合溶媒に完全に溶解させて使用されている
が、ジメチルスルフオキシドは生体膜を変質させるため
にその使用は避けなければならないという問題がある。The present inventors have applied for a method for evaluating the substance permeation performance of thin films using chemiluminescence (Japanese Patent Application Laid-Open No. 13303-1983).
No. 9, Patent Application No. 1983-103600, Patent Application No. 1983-13
8108, Japanese Patent Application No. 63202472), when a biological membrane is targeted as a thin film, since luminol is not very soluble in water, it must be completely dissolved in a mixed solvent of water and dimethyl sulfoxide, etc. However, there is a problem in that dimethyl sulfoxide deteriorates biological membranes and its use must be avoided.
そのため本発明は、化学発光試薬を水溶性とすることを
目的とし、化学発光試薬を固定した水溶性高分子化合物
、及び化学発光試薬の固定方法、更にこの水溶性高分子
化合物を使用し、生体膜における物質透過性能を評価す
る方法の提供を課題とする。Therefore, the present invention aims to make a chemiluminescent reagent water-soluble, and provides a water-soluble polymer compound on which a chemiluminescent reagent is immobilized, a method for immobilizing a chemiluminescent reagent, and a method for immobilizing a chemiluminescent reagent. Our objective is to provide a method for evaluating the substance permeation performance of membranes.
本発明における化学発光試薬を固定した水溶性高分子化
合物は、一級アミノ基を有する化学発光試薬における一
級アミノ基がメチロール化された化合物により変性され
た水溶性高分子化合物であり、その変性高分子化合物の
分子量が５０００〜１０００００、変性率が０．０１〜
５％である、化学発光試薬を固定した水溶性高分子化合
物であることを特徴とするものである。The water-soluble polymer compound on which a chemiluminescent reagent is immobilized in the present invention is a water-soluble polymer compound modified with a compound in which the primary amino group of a chemiluminescent reagent having a primary amino group is methylolated, and the modified polymer The molecular weight of the compound is 5000 to 100000, and the modification rate is 0.01 to
It is characterized by being a water-soluble polymer compound on which a chemiluminescent reagent of 5% is immobilized.
分子量が５０００以下であると、この水溶性高分子化合
物を使用する生体膜における物質透過性能を評価する方
法において、被試験用薄膜を介した透過物質を含有する
水溶液側への拡散を抑制しえなくなる。また分子量が１
０００００以上であると化学発光試薬を固定した高分子
化合物の水溶液が高粘度となり、透過側での自由拡散に
支障をきたしてしまう。また化学発光試薬による変性度
は、０．０１以下であると薄膜での物質透過が発光量に
比例するという仮定が成立しえなくなり、物質透過性能
の定量的評価に問題が生じる。また変性度が５以上であ
ると水への溶解度が小さくなる。When the molecular weight is 5,000 or less, in the method of evaluating substance permeation performance in a biological membrane using this water-soluble polymer compound, diffusion of the permeated substance to the aqueous solution side through the thin membrane under test may be suppressed. It disappears. Also, the molecular weight is 1
If it is more than 00,000, the aqueous solution of the polymer compound on which the chemiluminescent reagent is immobilized will have a high viscosity, which will impede free diffusion on the permeation side. Furthermore, if the degree of modification by the chemiluminescent reagent is 0.01 or less, the assumption that substance permeation through a thin film is proportional to the amount of luminescence no longer holds true, causing problems in quantitative evaluation of substance permeation performance. Moreover, when the degree of modification is 5 or more, the solubility in water becomes low.
また化学発光試薬の水溶性高分子への固定方法は、一級
アミノ基を有する化学発光試薬とホルムアルデヒドとを
加熱して反応させ、次いでその反応生成物を酸触媒の存
在下、活性水素を有する高分子化合物と反応させること
を特徴とするものである。In addition, a method for immobilizing a chemiluminescent reagent to a water-soluble polymer involves heating and reacting the chemiluminescent reagent having a primary amino group with formaldehyde, and then converting the reaction product into a polymer having active hydrogen in the presence of an acid catalyst. It is characterized by reacting with molecular compounds.
ここで上記一級アミノ基を有する化学発光試薬としては
ルミノール、イソルミノール等であることができ、また
活性水素を有する水溶性高分子化合物としてはポリビニ
ルアルコール、デキストラン、寒天等を使用することが
できる。ポリビニルアルコールとしてはポリビニルアセ
テートの７５〜９０％鹸化率のものを使用するとよい。Here, the chemiluminescent reagent having a primary amino group can be luminol, isoluminol, etc., and the water-soluble polymer compound having active hydrogen can be polyvinyl alcohol, dextran, agar, etc. As the polyvinyl alcohol, it is preferable to use one having a saponification rate of 75 to 90% of polyvinyl acetate.
また、本発明における該薄膜の物質透過性能を評価する
方法は、一級アミノ基を有する化学発光試薬とホルムア
ルデヒドとの反応生成物を酸触媒の存在下活性水素を有
する高分子化合物と反応させることにより得られる、化
学発光試薬を固定した水溶性高分子化合物と、相互に混
合されると化学発光を生じる物質を含有する水溶液とを
、生体薄膜を介して分離して配置し、前記化学発光を生
じる物質が前記薄膜への浸透、透過により化学発光試薬
を固定した水溶性高分子化合物からなる物質と混合させ
発光を生じさせ、その発光を光検出器により計測するこ
とを特徴とするものである。Furthermore, the method of evaluating the substance permeation performance of the thin film in the present invention involves reacting a reaction product of a chemiluminescent reagent having a primary amino group with formaldehyde with a polymer compound having active hydrogen in the presence of an acid catalyst. The resulting water-soluble polymer compound having a chemiluminescent reagent immobilized thereon and an aqueous solution containing a substance that produces chemiluminescence when mixed with each other are separated and arranged via a biological thin film to produce the chemiluminescence. This method is characterized in that the substance permeates and permeates the thin film and mixes with a substance made of a water-soluble polymer compound on which a chemiluminescent reagent is immobilized to generate luminescence, and the luminescence is measured by a photodetector.
即ち第１の物質を含有する溶液を、被試験用薄膜を介し
て化学発光試薬を固定した水溶性高分子化合物を含有す
ると共に第１の触媒の存在下前記第１の物質と反応し、
その反応生成物が該化学発光物質と第２の触媒の存在下
更に反応して化学発光させる第２の物質を含有する混合
溶液とを対向させて配置させることにより、前記第１の
物質をして該薄膜を浸透、透過せしめて混合溶液と接触
させることにより発光させるものである。That is, a solution containing a first substance containing a water-soluble polymer compound having a chemiluminescent reagent immobilized thereon via a thin film to be tested is reacted with the first substance in the presence of a first catalyst;
By arranging a mixed solution containing a second substance whose reaction product reacts with the chemiluminescent substance in the presence of a second catalyst to cause chemiluminescence, the first substance is produced. When the thin film is permeated and brought into contact with a mixed solution, it emits light.
上記第１の物質がグルコースの場合は、第１の触媒はグ
ルコース酸化酵素、以下、コレステロールの場合はコレ
ステロール酸化酵素、コレステロールエステルの場合は
コレステロールエステルヒドラーゼ、コレステロールエ
ステル酸化酵素、中性脂肪の場合はリパーゼ、およびグ
リセロール酸化酵素、リン脂質の場合はホスフォリパー
ゼＤ、およびコリン酸化酵素、遊離脂肪酸の場合はアシ
ルＣｏＡ合成酵素、およびアシルＣｏＡ酸化酵素、尿素
の場合はウレアーゼ、グルタミン酸脱水素酵素、および
グルタミン酸酸化酵素、アンモニアの場合はグルタミン
酸脱水素酵素、およびグルタミン酸酸化酵素、尿酸の場
合はウリカーゼ、クレアチンの場合はタレアチニナーゼ
、タレアチナーゼ、およびサルコシン酸化酵素、乳酸の
場合は乳酸酸化酵素、ピルビン酸の場合はピルビン酸酸
化酵素、アミノ酸の場合はアミノ酸酸化酵素、無機リン
の場合はピルビン酸酸化酵素が好ましく使用できる。When the first substance is glucose, the first catalyst is glucose oxidase; hereafter, in the case of cholesterol, cholesterol oxidase; in the case of cholesterol ester, cholesterol ester hydrolase; in the case of cholesterol ester oxidase; in the case of neutral fat are lipase and glycerol oxidase, phospholipase D and choline oxidase for phospholipids, acyl-CoA synthetase and acyl-CoA oxidase for free fatty acids, urease, glutamate dehydrogenase for urea, and glutamate oxidase, glutamate dehydrogenase and glutamate oxidase for ammonia, uricase for uric acid, taleatininase, taleatinase, and sarcosine oxidase for creatine, lactate oxidase for lactate, and glutamate oxidase for pyruvate. In the case of amino acids, amino acid oxidase can be preferably used, and in the case of inorganic phosphorus, pyruvate oxidase can be preferably used.
またこれらの第１物質の前駆体、例えばグルコースの場
合、その前駆体である澱粉等も同様に使用することがで
き、その透過性能を評価することができることは勿論で
ある。これらの物質は水溶液の状態で使用され、第１の
物質と第１の触媒の存在下反応する第２の物質は水溶液
中の溶存酸素である。この場合、第１物質と第２物質は
反応して過酸化水素を発生するものであればよく、また
生じた過酸化水素は第２の触媒であるペルオキシダーゼ
を触媒としてルミノールを酸化して発光させるものであ
る。Further, in the case of precursors of these first substances, such as glucose, starch, etc., which are the precursors thereof, can also be used in the same manner, and it goes without saying that the permeation performance thereof can be evaluated. These substances are used in the form of an aqueous solution, and the second substance that reacts with the first substance in the presence of the first catalyst is dissolved oxygen in the aqueous solution. In this case, the first substance and the second substance need only react to generate hydrogen peroxide, and the generated hydrogen peroxide oxidizes luminol using the second catalyst, peroxidase, to emit light. It is something.
またグルコースオキシダーゼを第１物質とし、グルコー
ス、ペルオキシダーゼ、及びルミノールとの混合溶液を
使用して触媒であるグルコースオキシダーゼの透過性能
を測定することができ、更にペルオキシダーゼを第１物
質としてグルコースオキシダーゼ、グルコース、及びル
ミノールとの混合溶液を使用してペルオキシダーゼの透
過性能を測定することも可能である。Furthermore, using glucose oxidase as the first substance, the permeation performance of glucose oxidase, which is a catalyst, can be measured using a mixed solution of glucose, peroxidase, and luminol. It is also possible to measure peroxidase permeation performance using a mixed solution with luminol and peroxidase.
または第１の物質として化学発光を触媒する物質を使用
し、薄膜を介して配置する物質として該触媒により化学
発光する物質を使用することにより、その触媒の透過量
を測定することができる。Alternatively, by using a substance that catalyzes chemiluminescence as the first substance and using a substance that emits chemiluminescence due to the catalyst as the substance disposed through the thin film, the amount of permeation of the catalyst can be measured.
この場合、触媒なしの条件でも一般にはわずかながら化
学発光が生じバックグラウンドレベルが上昇してしまう
が、微量の金属イオンの透過性能を知るためには有効で
ある。このような化学発光物質としてはルミノール、イ
ソルミノール等と過酸化水素水との混合溶液であり、触
媒としてはＦｅハあるいはＣｒ’″１、Ｃｏ”、Ｃｕ”
、Ｍｎ”、Ｃｕト、Ｚｎ”等がある。In this case, even in the absence of a catalyst, a small amount of chemiluminescence generally occurs and the background level increases, but it is effective for understanding the permeation performance of trace amounts of metal ions. Such a chemiluminescent substance is a mixed solution of luminol, isoluminol, etc. and hydrogen peroxide solution, and the catalyst is Fe, Cr'''1, Co'', Cu''.
, Mn'', Cu, Zn'', etc.
上記発光量を検出する光検出器としては、光電子増倍管
、または像増強管、赤外センサー、１次元センサー等を
使用することができる。As a photodetector for detecting the amount of light emitted, a photomultiplier tube, an image intensifier tube, an infrared sensor, a one-dimensional sensor, etc. can be used.
本発明における一般アミノ基を有する化学発光試薬をホ
ルムアルデヒドとまず反応させ、次いでその反応生成物
を酸触媒の存在下、活性水素を有する高分子化合物と反
応させる反応は、マンニッヒ反応における反応機構と同
様の反応機構で進むものと考えられる。この反応を利用
することにより従来のようにルミノール誘導体を経由し
て固定する必要がなく、化学発光試薬を簡便に高分子化
合物に固定することができる。また本発明は活性水素を
有する高分子化合物としてポリビニルアルコール等を使
用することにより、その反応生成物を水溶性とすること
ができ、また上記反応により化学発光試薬における発光
機構に重要な働きをするπ電子系に殆ど影響を与えない
ことを見出したものである。The reaction mechanism of the present invention in which a chemiluminescent reagent having a general amino group is first reacted with formaldehyde and then the reaction product is reacted with a polymer compound having active hydrogen in the presence of an acid catalyst is similar to the reaction mechanism in the Mannich reaction. It is thought that the reaction proceeds according to the following reaction mechanism. By utilizing this reaction, a chemiluminescent reagent can be easily immobilized on a polymer compound without the need for immobilization via a luminol derivative as in the conventional method. Furthermore, by using polyvinyl alcohol or the like as a polymer compound having active hydrogen, the reaction product of the present invention can be made water-soluble, and the above reaction plays an important role in the luminescence mechanism of chemiluminescent reagents. It was discovered that this has almost no effect on the π-electron system.
このように化学発光試薬である例えばルミノールを固定
化させ、水溶性化させることにより、例えば生体膜にお
けるグルコース、アミノ酸等の透過量を生体膜を損傷す
ることなく測定することができるものである。By immobilizing a chemiluminescent reagent such as luminol and making it water-soluble in this manner, the amount of permeation of glucose, amino acids, etc. through a biological membrane can be measured without damaging the biological membrane.
以下、実施例により本発明を説明する。The present invention will be explained below with reference to Examples.
（以下、余白）
〔実施例１〕
Ｈ
ルミノール４．２ ｇ Ｓ溶媒として水５０ｍ Ｉｌを
５００１のナス型フラスコに入れ、室温条件下で撹拌し
つつ１．８ ｍｌのホルマリン水溶液を滴下した。(Hereinafter, blank spaces) [Example 1] H Luminol 4.2 g S Water 50 ml Il was placed in a 5001 eggplant-shaped flask, and 1.8 ml of formalin aqueous solution was added dropwise while stirring at room temperature.
滴下後５０℃で１時間加熱後、濃塩酸２ｍｌ、及び温水
３００ ｖａｎに溶解させたポリビニルアルコール（鹸
化率８５％、重合度約１５００）を滴下した。滴下後、
３５°Ｃで一晩反応させた。反応終了後、全容量１．２
！まで水を加え、５０’Ｃまで加熱し、その反応生成物
をできるだけ溶解した後、ひだ折り濾過を行い、濾液を
冷却後、２日間多量の水に対して透析し、未反応のルミ
ノールを除去した。透析後透析内液から溶媒である水を
除去して乾燥させ、ルミノールが置換したポリビニルア
ルコール７．８ｇを得た。After heating at 50° C. for 1 hour after dropping, 2 ml of concentrated hydrochloric acid and polyvinyl alcohol (saponification rate: 85%, degree of polymerization: about 1500) dissolved in 300 vans of warm water were added dropwise. After dripping,
The reaction was allowed to proceed overnight at 35°C. After the reaction is complete, the total volume is 1.2
! Add water to 50°C and heat to 50'C to dissolve as much of the reaction product as possible, then perform fold filtration, cool the filtrate, and dialyze against a large amount of water for 2 days to remove unreacted luminol. did. After dialysis, water as a solvent was removed from the dialysate and dried to obtain 7.8 g of polyvinyl alcohol substituted with luminol.
またルミノールがポリビニルアルコールに物理吸着して
いる可能性があるので、ポリビニルアルコールは難溶で
、ルミノールが可溶のテトラヒドロフラン（ＴＨＦ）で
最終生成物を充分洗浄し、洗浄後のＴＨＦを触媒である
ペルオキシダーゼ水溶液に混合し、上記同様３％過酸化
水素水溶液を添加したが、全く発光は観察されず、ルミ
ノールはポリビニルアルコールに化学結合していること
を確認しえた。In addition, since luminol may be physically adsorbed to polyvinyl alcohol, polyvinyl alcohol is poorly soluble and the final product is thoroughly washed with tetrahydrofuran (THF), in which luminol is soluble, and the washed THF is used as a catalyst. It was mixed with a peroxidase aqueous solution and a 3% hydrogen peroxide aqueous solution was added in the same manner as above, but no luminescence was observed, confirming that luminol was chemically bonded to polyvinyl alcohol.
次に最終生成物であるルミノール置換ポリビニルアルコ
ールの一部を試験管に採取し、極微量の触媒であるペル
オキシダーゼを添加し、両者を水に溶解させた。暗室中
で３％の過酸化水素水を滴下したところ、青白いルミノ
ール発光を肉眼で観察することができ、生体膜の物質透
過性能評価に使用しうることがわかった。Next, a portion of the final product, luminol-substituted polyvinyl alcohol, was collected in a test tube, a trace amount of peroxidase, a catalyst, was added, and both were dissolved in water. When 3% hydrogen peroxide solution was dropped in a dark room, pale luminol emission could be observed with the naked eye, indicating that it could be used to evaluate the substance permeation performance of biological membranes.
最終生成物の、水ニジメチルスルホキシド９：１中での
ＵＶ吸収スペクトルを第１図に示す。The UV absorption spectrum of the final product in water and dimethyl sulfoxide 9:1 is shown in FIG.
第１図において、最終生成物のＵＶ吸収スペクトルはル
ミノール自身の吸収スペクトルと一致しており、また最
終生成物とルミノール自身のモル吸光係数（ε−５，Ｉ
ＸＩＯ’ 、３５０ｎｍ）は等しいと仮定して最終生
成物の吸光度測定から、ルミノールは約０．１４％、ポ
リビニルアルコールに結合していることがわかった。In Figure 1, the UV absorption spectrum of the final product is consistent with the absorption spectrum of luminol itself, and the molar extinction coefficient (ε-5, I
Absorbance measurements of the final product, assuming equal XIO', 350 nm), showed that approximately 0.14% of the luminol was bound to the polyvinyl alcohol.
〔実施例２〕
第２図は本発明の方法を実施するための、被試験用薄膜
の物質透過性能測定装置の略図である。[Example 2] FIG. 2 is a schematic diagram of an apparatus for measuring substance permeation performance of a thin film to be tested for carrying out the method of the present invention.
１は生体膜、２．３はセル、４は第１物質水溶液、５は
混合水溶液、６は光検出器、７は暗箱を示す。1 is a biological membrane, 2.3 is a cell, 4 is a first substance aqueous solution, 5 is a mixed aqueous solution, 6 is a photodetector, and 7 is a dark box.
第１物質溶液としては０．０１％グルコース水溶液、混
合物水溶液としてはグルコース酸化酵素５０ｍｇ、上記
実施例１で調製したルミノール置換ポリビニルアルコー
ル１ｇ、ベルオキシターゼ１０ｍｇを５０ｃｃのＯ，１
Ｍ燐酸緩衝液（ｐＨ７）に含有させた水溶液を使用し、
被試験用薄膜はヘアレスマウス、及び毛を除去したラッ
トの腹部皮膚（１０〜３０μｍ膜厚）を採取し使用した
。The first substance solution was 0.01% glucose aqueous solution, the mixture aqueous solution was 50 mg of glucose oxidase, 1 g of luminol-substituted polyvinyl alcohol prepared in Example 1, and 10 mg of peroxidase in 50 cc of O, 1
Using an aqueous solution containing M phosphate buffer (pH 7),
The thin films to be tested were obtained from the abdominal skin (10 to 30 μm film thickness) of hairless mice and rats from which hair had been removed.
まず暗箱７内に、第１物質水溶液４を入れたセル２を、
生体膜１を介して混合物水溶液５を開口部まで満たした
セル３上に、各開口部を対向させて静置させ、その上部
に光検出器６を配置し、暗箱を暗くした。First, the cell 2 containing the first substance aqueous solution 4 is placed in the dark box 7.
A cell 3 filled with the aqueous mixture solution 5 up to the openings via the biological membrane 1 was left standing with the openings facing each other, and a photodetector 6 was placed above the cell 3 to darken the dark box.
セル２中のグルコースは、生体膜１を透過して混合物水
溶液中に移行し、そこでグルコースオキシターゼ、およ
び水溶液中の溶存酸素と反応し、過酸化水素を発生し、
生じた過酸化水素はベルオキシターゼを触媒としてルミ
ノールが酸化され発光が生じる。この発光を光検出器６
により計測し、生体膜１を透過したグルコース量の経時
変化及び生体膜における透過位置等の２次元像を得た。Glucose in the cell 2 passes through the biological membrane 1 and moves into the aqueous mixture solution, where it reacts with glucose oxidase and dissolved oxygen in the aqueous solution to generate hydrogen peroxide,
The generated hydrogen peroxide oxidizes luminol using peroxidase as a catalyst, producing luminescence. This luminescence is detected by a photodetector 6.
A two-dimensional image of the change over time in the amount of glucose that permeated through the biological membrane 1 and the permeation position in the biological membrane was obtained.
一級アミノ基を有する化学発光試薬とホルムアルデヒド
とを加熱して反応させ、次いでその反応生成物を酸触媒
の存在下、活性水素を有する高分子化合物と反応させて
水溶性高分子化合物とすることにより極めて容易に化学
発光試薬を水溶性高分子化合物に固定することが可能で
あり、また得られた高分子化合物は水溶性であるので、
生体膜の物質透過性能を化学発光を利用して評価する際
に不可欠な化学発光試薬を提供するものである。By heating and reacting a chemiluminescent reagent having a primary amino group with formaldehyde, and then reacting the reaction product with a polymer compound having active hydrogen in the presence of an acid catalyst to form a water-soluble polymer compound. Since it is possible to immobilize a chemiluminescent reagent to a water-soluble polymer compound very easily, and the obtained polymer compound is water-soluble,
The present invention provides chemiluminescent reagents that are essential for evaluating the substance permeability of biological membranes using chemiluminescence.
第１図は実施例１において得られた化学発光試薬を固定
した水溶性高分子化合物のＵＶ吸収スペクトル図、第２
図は本発明における薄膜の物質透過性能測定装置の概略
図である。
■は生体膜、２．３はセル、４は第１物質水溶液、５は
混合水溶液、６は光検出器、７は暗箱を示す。Figure 1 is a UV absorption spectrum diagram of the water-soluble polymer compound on which the chemiluminescent reagent obtained in Example 1 was immobilized;
The figure is a schematic diagram of an apparatus for measuring substance permeation performance of a thin film according to the present invention. 3 is a biological membrane, 2.3 is a cell, 4 is a first substance aqueous solution, 5 is a mixed aqueous solution, 6 is a photodetector, and 7 is a dark box.
Claims (6)
アミノ基がメチロール化された化合物により変性された
水溶性高分子化合物であり、その変性高分子化合物の分
子量が５０００〜１０００００、変性率が０．０１〜５
％である、化学発光試薬を固定した水溶性高分子化合物
。(1) A water-soluble polymer compound modified with a compound in which the primary amino group in a chemiluminescent reagent having a primary amino group is methylolated, and the modified polymer compound has a molecular weight of 5,000 to 100,000 and a modification rate of 0. 01-5
%, a water-soluble polymeric compound with immobilized chemiluminescent reagents.
性高分子化合物が鹸化率７５〜９０％のポリビニルアル
コールである請求項１記載の化学発光試薬を固定した水
溶性高分子化合物。(2) The water-soluble polymer compound having a chemiluminescent reagent immobilized thereon according to claim 1, wherein the chemiluminescent reagent is luminol and the water-soluble polymer compound is polyvinyl alcohol with a saponification rate of 75 to 90%.
デヒドとを加熱して反応させ、次いでその反応生成物を
酸触媒の存在下、活性水素を有する水溶性高分子化合物
と反応させ、水溶性高分子化合物を化学発光試薬により
変性することを特徴とする化学発光試薬の固定方法。(3) A chemiluminescent reagent having a primary amino group is reacted with formaldehyde by heating, and the reaction product is then reacted with a water-soluble polymer compound having active hydrogen in the presence of an acid catalyst to form a water-soluble polymer. A method for immobilizing a chemiluminescent reagent, which comprises denaturing a compound with a chemiluminescent reagent.
水素を有する水溶性高分子化合物が鹸化率７５〜９０％
のポリビニルアルコールである請求項３記載の化学発光
試薬の固定方法。(4) The chemiluminescent reagent is luminol, and the water-soluble polymer compound having active hydrogen has a saponification rate of 75 to 90%.
The method for immobilizing a chemiluminescent reagent according to claim 3, wherein polyvinyl alcohol is used.
アミノ基がメチロール化された化合物により変性された
水溶性高分子化合物であり、その変性高分子化合物の分
子量が５０００〜１０００００、変性率が０．０１〜５
％である、化学発光試薬を固定した水溶性高分子化合物
と、相互に混合されると化学発光を生じる物質を含有す
る水溶液とを薄膜を介して分離して配置し、前記化学発
光を生じる物質を前記薄膜へ浸透、透過させることによ
り、化学発光試薬を固定した水溶性高分子化合物からな
る物質と混合させて発光させ、その発光を光検出器によ
り計測することにより該薄膜の物質透過性能を評価する
方法。(5) A water-soluble polymer compound modified with a compound in which the primary amino group in a chemiluminescent reagent having a primary amino group is methylolated, and the modified polymer compound has a molecular weight of 5,000 to 100,000 and a modification rate of 0. 01-5
%, a water-soluble polymer compound having a chemiluminescent reagent fixed thereon, and an aqueous solution containing a substance that generates chemiluminescence when mixed with each other are separated via a thin film, and the substance that generates chemiluminescence is placed. By infiltrating and permeating the thin film, the chemiluminescent reagent is mixed with a substance made of a water-soluble polymer compound to which it is immobilized, emitting light, and the light emission is measured with a photodetector to evaluate the substance permeation performance of the thin film. How to evaluate.
質透過性能を評価する方法。(6) The method for evaluating the substance permeation performance of a thin film according to claim 5, wherein the thin film is a biological membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25545988A JPH02102203A (en) | 1988-10-11 | 1988-10-11 | Water-soluble polymeric compound containing immobilized chemiluminescent reagent and method for immobilization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25545988A JPH02102203A (en) | 1988-10-11 | 1988-10-11 | Water-soluble polymeric compound containing immobilized chemiluminescent reagent and method for immobilization |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02102203A true JPH02102203A (en) | 1990-04-13 |
Family
ID=17279060
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25545988A Pending JPH02102203A (en) | 1988-10-11 | 1988-10-11 | Water-soluble polymeric compound containing immobilized chemiluminescent reagent and method for immobilization |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02102203A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8183060B2 (en) | 2001-09-19 | 2012-05-22 | Daiichi Pure Chemicals Co., Ltd. | Luminescent polymer and use thereof in bioassay |
-
1988
- 1988-10-11 JP JP25545988A patent/JPH02102203A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8183060B2 (en) | 2001-09-19 | 2012-05-22 | Daiichi Pure Chemicals Co., Ltd. | Luminescent polymer and use thereof in bioassay |
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