JPS6394993A - Dry analytical element containing oxidized form coenzyme - Google Patents
Dry analytical element containing oxidized form coenzymeInfo
- Publication number
- JPS6394993A JPS6394993A JP24028686A JP24028686A JPS6394993A JP S6394993 A JPS6394993 A JP S6394993A JP 24028686 A JP24028686 A JP 24028686A JP 24028686 A JP24028686 A JP 24028686A JP S6394993 A JPS6394993 A JP S6394993A
- Authority
- JP
- Japan
- Prior art keywords
- layer
- electron
- compound
- analytical element
- shielding layer
- 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
- 239000005515 coenzyme Substances 0.000 title claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 44
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229960003966 nicotinamide Drugs 0.000 claims abstract description 10
- 235000005152 nicotinamide Nutrition 0.000 claims abstract description 10
- 239000011570 nicotinamide Substances 0.000 claims abstract description 10
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 8
- 125000003831 tetrazolyl group Chemical group 0.000 claims abstract description 5
- 238000004458 analytical method Methods 0.000 claims description 18
- 230000027756 respiratory electron transport chain Effects 0.000 claims description 11
- 238000005259 measurement Methods 0.000 abstract description 4
- 239000003638 chemical reducing agent Substances 0.000 abstract 1
- 239000010410 layer Substances 0.000 description 97
- 239000003153 chemical reaction reagent Substances 0.000 description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 108010010803 Gelatin Proteins 0.000 description 14
- 239000012790 adhesive layer Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 14
- 229920000159 gelatin Polymers 0.000 description 14
- 239000008273 gelatin Substances 0.000 description 14
- 235000019322 gelatine Nutrition 0.000 description 14
- 235000011852 gelatine desserts Nutrition 0.000 description 14
- 239000004744 fabric Substances 0.000 description 12
- 238000003892 spreading Methods 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 102000004316 Oxidoreductases Human genes 0.000 description 10
- 108090000854 Oxidoreductases Proteins 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 10
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 8
- 229920001477 hydrophilic polymer Polymers 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 239000004094 surface-active agent Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 229930024421 Adenine Natural products 0.000 description 5
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 5
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 5
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 5
- 229960000643 adenine Drugs 0.000 description 5
- 239000003513 alkali Substances 0.000 description 5
- -1 etc.) Polymers 0.000 description 5
- 239000010419 fine particle Substances 0.000 description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 5
- 239000002759 woven fabric Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000003925 fat Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 229920000139 polyethylene terephthalate Polymers 0.000 description 4
- 239000005020 polyethylene terephthalate Substances 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 3
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 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 3
- 239000010452 phosphate Substances 0.000 description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 239000004305 biphenyl Substances 0.000 description 2
- 125000002529 biphenylenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3C12)* 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 125000002091 cationic group Chemical group 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
- 239000011247 coating layer Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 208000028659 discharge Diseases 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 238000010030 laminating Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- JZMJDSHXVKJFKW-UHFFFAOYSA-M methyl sulfate(1-) Chemical compound COS([O-])(=O)=O JZMJDSHXVKJFKW-UHFFFAOYSA-M 0.000 description 2
- 239000011325 microbead Substances 0.000 description 2
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 2
- 239000004745 nonwoven fabric Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 238000005375 photometry Methods 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- YZIXDSVRVOZUAZ-UHFFFAOYSA-N 2-(1,2-dihydrotetrazol-1-ium-3-yl)-4,5-dimethyl-1,3-thiazole;bromide Chemical compound [Br-].S1C(C)=C(C)N=C1N1N=C[NH2+]N1 YZIXDSVRVOZUAZ-UHFFFAOYSA-N 0.000 description 1
- JJMQRJKPLUACSO-UHFFFAOYSA-N 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyl-1,3-dihydrotetrazol-3-ium;chloride Chemical compound [Cl-].C1=CC([N+](=O)[O-])=CC=C1N1N(C=2C=CC(I)=CC=2)[NH2+]C(C=2C=CC=CC=2)=N1 JJMQRJKPLUACSO-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 1
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- 206010061619 Deformity Diseases 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- 102000002794 Glucosephosphate Dehydrogenase Human genes 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 102000057621 Glycerol kinases Human genes 0.000 description 1
- 108700016170 Glycerol kinases Proteins 0.000 description 1
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 description 1
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- GCYZRQQEBNFQTK-UHFFFAOYSA-N [2,3-dihydroxypropoxy(hydroxy)phosphoryl] phosphono hydrogen phosphate Chemical compound OCC(O)COP(O)(=O)OP(O)(=O)OP(O)(O)=O GCYZRQQEBNFQTK-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 108010023417 cholesterol dehydrogenase Proteins 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000003851 corona treatment Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000011344 liquid material Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 108010085346 steroid delta-isomerase Proteins 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
[発明の技術的分野]
本発明は、酸化型補酵素を含有する乾式分析要素に関す
るものである。DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to a dry analytical element containing an oxidized coenzyme.
[発明の背景]
デヒドロゲナーゼ系酵素と補酵素とを共役反応系に組合
わせた反応は臨床化学分析において広く用いられている
0例えばグリセリンデヒドロゲナーゼ、コレステロール
デヒドロゲナーゼ、乳酸デヒドロゲナーゼ、アルコール
デヒドロゲナーゼ。[Background of the Invention] Reactions in which a dehydrogenase enzyme and a coenzyme are combined in a coupled reaction system are widely used in clinical chemical analysis, such as glycerin dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase.
グルタメートデヒドロゲナーゼ、アルデヒドデヒドロゲ
ナーゼ、α−グリセロフォスフェートデヒドロゲナーゼ
、グルコース−6−燐酸デヒドロゲナーゼ等が関与する
反応系が、トリグリセリド、グリセリン、コレステロー
ル、乳酸、グルタメート、グリセリン−3=燐酸、グル
コース−6−燐酸等の基質や、アスパラギン酸アミノト
ランスフェラーゼ(AST)、アラニンアミノトランス
フェラーゼ(ALT)、乳酸デヒドロゲナーゼ(LDH
)、アミラーゼ、クレアチンキナーゼ(G K)等の酵
素の定量に用いられており、還元型補酵素の増加または
減少の直接測定によって定量分析ができる特徴がある。A reaction system involving glutamate dehydrogenase, aldehyde dehydrogenase, α-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. is used to react with triglyceride, glycerin, cholesterol, lactic acid, glutamate, glycerin-3=phosphoric acid, glucose-6-phosphate, etc. Substrates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH)
), amylase, creatine kinase (GK), and other enzymes, and is characterized by the ability to perform quantitative analysis by directly measuring the increase or decrease in reduced coenzymes.
しかし通常用いられるNADHにコチンアミドアデニン
ジヌクレオチド)またはNADPHにコチンアミドアデ
ニンンジヌクレオチドファスフェート)は、吸収極大が
340nm付近にあるため、その光学的定量のためには
、紫外域の測光装置が必要となり、機器が高価なものに
なる。また紫外域では多種の化合物が吸収を有するため
にそれらの干渉を受は易い。However, the absorption maximum of commonly used NADH (cotinamide adenine dinucleotide) or NADPH (cotinamide adenine dinucleotide phosphate) is around 340 nm, so a photometer in the ultraviolet region is required for optical quantitative determination. This makes the equipment expensive. In addition, in the ultraviolet region, various compounds have absorption, so they are easily interfered with.
NADH(またはNADPH)を紫外部吸収により直接
に定量する代りに、NADHの存在下で、電子伝達性化
合物を介して電子受容性化合物を還元し、可視領域にお
いて検出可能な化合物(染料)を形成する反応系が提案
されている。Instead of directly quantifying NADH (or NADPH) by ultraviolet absorption, an electron-accepting compound is reduced through an electron-transferring compound in the presence of NADH to form a compound (dye) that is detectable in the visible region. A reaction system has been proposed.
電子受容性化合物としてテトラゾリウム塩が用いられて
いるが、検体中からくる還元性物質と反応して、バック
グラウンドの着色(カブリということがある)を生じさ
せる。これを防ぐために、ブランク値を測定し補正した
り、アスコルビン酸などは、アスコルビン酸オキシダー
ゼを用いて分解して影響を除いているが、これらの方法
では測定に時間を要したり、費用が高くなる等の問題が
あった。Tetrazolium salts are used as electron-accepting compounds, but they react with reducing substances coming from the sample, causing background coloration (sometimes called fog). To prevent this, blank values are measured and corrected, and ascorbic acid is decomposed using ascorbic acid oxidase to remove the effects, but these methods take time to measure and are expensive. There were some problems.
[発明の要旨]
本発明は、酸化型ニコチンアミド補酵素、電子伝達性化
合物、および電子受容性染料形成性化合物を含む析出試
薬系を有する乾式分析要素において。SUMMARY OF THE INVENTION The present invention provides a dry analytical element having a precipitation reagent system comprising an oxidized nicotinamide coenzyme, an electron transfer compound, and an electron-accepting dye-forming compound.
検体中の還元性物質による電子受容性染料形成性化合物
の発色による正誤差を、光遮蔽層内か、あるいは光遮蔽
層よりも上の層に電子伝達性化合物を添加して影響を軽
減することを達成した乾式分析要素にある。To reduce the influence of errors caused by color development of electron-accepting dye-forming compounds caused by reducing substances in the specimen by adding an electron-transfer compound within the light-shielding layer or in a layer above the light-shielding layer. It is in the dry analysis element that achieved this.
[発明の詳細な記述1
本発明は公知の多種の乾式分析要素に適用することがで
きる。特にデヒドロゲナーゼ酵素系と還元型補酵素検出
系と被検液体がいずれも浸透し得る固体担体を含む分析
要素に適用することができる。[Detailed Description of the Invention 1 The present invention can be applied to various known dry analysis elements. In particular, it can be applied to analytical elements that include a solid carrier that can be permeated by a dehydrogenase enzyme system, a reduced coenzyme detection system, and a test liquid.
分析要素は、電子伝達性化合物を含む試薬層と該試薬層
よりも上層(支持体とは逆側の層)の微量の電子受容性
染料形成性化合物を含む光遮蔽層(その代わりに電子受
容性染料形成性化合物を含む層を光遮蔽層の上に設けて
もよい)で構成されていればよく、第二試薬層、反射層
、多孔質展開層、濾過層、検出層、(Registra
tion Layer)層、吸水層、支持体、下塗層お
よび業界公知のその他の層を含む多重層であってもよい
。The analytical element consists of a reagent layer containing an electron-transferring compound and a light-shielding layer (instead, an electron-accepting layer) containing a trace amount of an electron-accepting dye-forming compound above the reagent layer (a layer on the opposite side of the support). (a layer containing a dye-forming compound may be provided on the light shielding layer), a second reagent layer, a reflective layer, a porous development layer, a filtration layer, a detection layer, (Registra
It may be multiple layers, including a cation layer, a water absorption layer, a support, a subbing layer, and other layers known in the art.
支持体を用いる場合、実用的に好ましい構成は、
(1)支持体上に電子伝達性化合物、電子受容性染料形
成性化合物を含む呈色試薬層と電子受容性染料を含む光
遮蔽層を兼ねる展開層を有するもの、
(2)(1)の層構成に支持体と呈色試薬層の間に吸水
層を入れたもの、
(3)(2)の層構成に呈色試薬層と光遮蔽層と展開層
の間か光遮蔽層と展開層の間に濾過層を入れたもの、
(4)(2)の層構成に光遮蔽層と展開層の間に電子受
容性染料層を入れたもの、
などがある。When using a support, a practically preferable configuration is as follows: (1) The support has a color-forming reagent layer containing an electron-transfer compound and an electron-accepting dye-forming compound, and a light-shielding layer containing an electron-accepting dye. (2) A water-absorbing layer is added between the support and the color reagent layer in the layer structure of (1); (3) A color reagent layer and a light shielding layer are added to the layer structure of (2). (4) A filtration layer is inserted between the light shielding layer and the developing layer, or between the light shielding layer and the developing layer. (4) An electron-accepting dye layer is inserted between the light shielding layer and the developing layer in the layer configuration of (2). There are things, etc.
本発明の乾式分析要素において少なくとも一層の試薬層
に含まれる検出試薬系には、酸化型ニコチンアミド補酵
素が含まれる。酸化型ニコチンアミド補酵素とは、具体
的には、NAD+ にコチンアミド・アデニンジヌク
レオチド酸化型)またはNADP+ にコチンアミド・
アデニンジヌクレオチド・ホスフェート酸化型)を意味
する。In the dry analytical element of the present invention, the detection reagent system contained in at least one reagent layer contains an oxidized nicotinamide coenzyme. Specifically, oxidized nicotinamide coenzyme refers to cotinamide/adenine dinucleotide oxidized type for NAD+ or cotinamide/adenine dinucleotide oxidized type for NADP+.
adenine dinucleotide phosphate oxidized form).
NAD÷およびNADP+のうちどちらを使用するかは
、分析対象として、あるいは検出試薬として検出反応に
関与する酸化還元酵素の種類に応じて決定される。Which of NAD÷ and NADP+ to use is determined depending on the type of oxidoreductase involved in the detection reaction as an analysis target or as a detection reagent.
本発明の乾式分析要素において光遮蔽層よりも支持体側
に近い位置に少なくとも一層の試薬層に含まれる検出試
薬系には、電子伝達性化合物が含まれる。In the dry analytical element of the present invention, the detection reagent system contained in at least one reagent layer closer to the support than the light shielding layer contains an electron transfer compound.
本発明において電子伝達性化合物とは、被検物質の反応
により生成した還元型ニコチンアミド補酵素(電子供与
体)から電子を受は取って、後述する電子受容性染料形
成性化合物を還元する機能を有する化合物を意味する。In the present invention, the electron transfer compound refers to a compound that accepts and takes electrons from the reduced nicotinamide coenzyme (electron donor) produced by the reaction of the test substance, and reduces the electron-accepting dye-forming compound described below. means a compound having
上記電子伝達性化合物の具体例としては、5−メチルツ
ェナジニウム・メチルスルフェートあるいは1−メトキ
シ−5−メチルツェナジニウム・メチルスルフェート等
のN−メチルフェナジン・メトサルフェート類およびジ
アホラーゼ(ジヒドロリボアミドレダクターゼ、E C
1,6,4,3,)等を挙げるとかできる。これらの電
子伝達性化合物のうちではジアホラーゼが好ましい。Specific examples of the electron transporting compounds include N-methylphenazine methosulfates such as 5-methylzenazinium methylsulfate or 1-methoxy-5-methylzenazinium methylsulfate, and diaphorase (dihydro ribamide reductase, EC
1, 6, 4, 3,), etc. Among these electron transfer compounds, diaphorase is preferred.
本発明の乾式分析要素に用いられる検出試薬系には、さ
らに電子受容性染料形成性化合物が含まれる。電子受容
性染料形成性化合物は、上記電子伝達性化合物により還
元され、長波長領域において検出可能な化合物(染料)
を形成する物質である0本発明の乾式分析要素において
、電子受容性染料形成性化合物としては、テトラゾリウ
ム塩を用いることが好ましい。The detection reagent system used in the dry analytical element of the present invention further contains an electron-accepting dye-forming compound. The electron-accepting dye-forming compound is a compound (dye) that is reduced by the electron-transferring compound and can be detected in a long wavelength region.
In the dry analytical element of the present invention, which is a substance that forms 0, it is preferable to use a tetrazolium salt as the electron-accepting dye-forming compound.
テトラゾリウム塩の具体例としては、
3.3’−(3,3’−ジメトキシ−4゜4゛−ビフェ
ニレン)−ビス[2−(p−二トロフェニル)−2H−
テトラゾリウムクロリド](NET)。Specific examples of tetrazolium salts include 3.3'-(3,3'-dimethoxy-4゜4゛-biphenylene)-bis[2-(p-nitrophenyl)-2H-
Tetrazolium chloride] (NET).
3−(p−ヨードフェニル)−2−(p−二トロフェニ
ル)−5−フェニル−2H−テトラゾリウムクロリド(
INT)。3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (
INT).
3−(4,5−ジメチル−2−チアゾリル)−2H−テ
トラゾリウムプロミド(MTT)。3-(4,5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT).
3.3’−(4,4’−ビフェニレン)−ビス(2,5
−ジフェニル) −2H−テトラゾリウムクロリド;
3.3°−(3,3’−ジメトキシ−4,4“−ビフェ
ニレン)−ビス(2、5−ジフェニル)−2H−テトラ
ゾリウムクロリド;および3.3’−(3,3’−ジメ
トキシ−4,4゜−ビフェニレン)−ビス[2,5−ビ
ス(p−二トロフェニル)−2H−テトラゾリウムクロ
リド]を挙げることができる。3.3'-(4,4'-biphenylene)-bis(2,5
-diphenyl) -2H-tetrazolium chloride; 3.3°-(3,3'-dimethoxy-4,4"-biphenylene)-bis(2,5-diphenyl)-2H-tetrazolium chloride; and 3.3'- (3,3'-dimethoxy-4,4°-biphenylene)-bis[2,5-bis(p-nitrophenyl)-2H-tetrazolium chloride] can be mentioned.
光遮蔽層内か、光遮蔽層よりも上層に添加される電子受
容性染料形成化合物は、呈色試薬層に添加される電子受
容性染料形成性化合物と同種であっても、異なっていて
もよい、添加する量は5〜170 m g / rn’
テ使用でき、好ましくは7〜120 m g / m
’である。The electron-accepting dye-forming compound added within the light-shielding layer or in a layer above the light-shielding layer may be the same or different from the electron-accepting dye-forming compound added to the coloring reagent layer. Good, the amount to add is 5-170 mg/rn'
can be used, preferably 7-120 mg/m
'is.
なお、上記酸化型ニコチンアミド補酵素、電子伝達性化
合物および電子受容性染料形成性化合物を含む反応系の
詳細については、
A、 L、 Babson等によるCLINICA C
HIMICA AGTA、 Vol、12、210−2
15頁(1965) 。For details of the reaction system containing the oxidized nicotinamide coenzyme, electron transfer compound, and electron-accepting dye-forming compound, see CLINICA C by A. L. Babson et al.
HIMICA AGTA, Vol, 12, 210-2
15 pages (1965).
R,J、 Gay等ニヨルCLINICAL GHEM
ISTRYVol、14 (8)、740−753頁(
196B) ;および、R,D、 Capps II等
による0LINICAL CHEMISTRY、 Vo
l、12(7)、 406−413頁(1966) ;
等の文献に記載されている。R, J, Gay etc. CLINICAL GHEM
ISTRY Vol., 14 (8), pp. 740-753 (
196B) ; and 0LINICAL CHEMISTRY by R, D, Capps II, etc., Vo
l, 12(7), pp. 406-413 (1966);
It is described in the literature such as
本発明は測定すべき物質の酸化還元酵素を少なくとも一
層に含めることにより液体試料中における濃度を測定す
る目的に用いることができる0本発明の乾式分析要素は
酸化還元酵素(脱水素酵素)と反応し酸化型ニコチンア
ミド補酵素が電子受容体となる全ての物質の測定に使用
が可能である0例えばグリセリンを測定する場合には、
グリセリンデヒドロゲナーゼ、乳醜を測定する場合はラ
クテートデヒドロゲナーゼを添加することでそれぞれの
測定が可能となる。The present invention can be used for the purpose of measuring the concentration of a substance to be measured in a liquid sample by containing an oxidoreductase in at least one layer.The dry analytical element of the present invention reacts with an oxidoreductase (dehydrogenase). It can be used to measure all substances in which the oxidized nicotinamide coenzyme acts as an electron acceptor.
When measuring glycerin dehydrogenase and breast disfigurement, each measurement becomes possible by adding lactate dehydrogenase.
本発明の乾式分析要素に用いられる検出試薬系は、液体
試料中における様々な種類の酸化還元酵素活性の測定に
使用することもできる。すなわち、本発明の乾式分析要
素を用いれば、酸化型ニコチンアミド補酵素が電子受容
体となる全ての酸化還元酵素(脱水素酵素)の測定が可
能である。The detection reagent system used in the dry analytical element of the present invention can also be used to measure various types of oxidoreductase activities in liquid samples. That is, by using the dry analytical element of the present invention, it is possible to measure all oxidoreductases (dehydrogenases) in which oxidized nicotinamide coenzyme serves as an electron acceptor.
上記のように本発明の乾式分析要素を酸化還元酵素の活
性の測定に使用する場合には、上記検出試薬系には、さ
らに酸化還元酵素が触媒する酸化反応(脱水素反応)の
基質が加えられる0例えば、本発明の乾式分析要素を乳
酸デヒドロゲナーゼ活性測定用として用いる場合には、
上記検出試薬系にさらに乳酸が含まれる。グルコース−
6−燐酸デヒドロゲナーゼの場合には、基質としてグル
コース−6−燐酸が添加される。When the dry analytical element of the present invention is used to measure the activity of an oxidoreductase as described above, a substrate for the oxidation reaction (dehydrogenation reaction) catalyzed by the oxidoreductase is further added to the detection reagent system. For example, when using the dry analytical element of the present invention for measuring lactate dehydrogenase activity,
The detection reagent system further includes lactic acid. Glucose-
In the case of 6-phosphate dehydrogenase, glucose-6-phosphate is added as substrate.
光遮蔽層は、皮膜形成能を有する親木性ポリマーをバイ
ンダーとして、光反射性微粒子が分散されている水浸透
性の層であることが好ましし、光反射性微粒子は、試薬
層(または給水層)に生じた検出可能な変化(色変化、
発色等)を光透過性を有する支持体側から反射測光する
際に、展開層に点着供給された水性液体の色、特に試料
が全血である場合のヘモグロビンの赤色等を遮蔽すると
ともに光反射層または背景層としてもa溌する。The light-shielding layer is preferably a water-permeable layer in which light-reflecting fine particles are dispersed using a wood-philic polymer having film-forming ability as a binder. detectable changes (color change,
When performing reflection photometry from the light-transmissive support side for color development, etc., the color of the aqueous liquid dotted onto the developing layer, especially the red color of hemoglobin when the sample is whole blood, is blocked and the light is reflected. It can also be used as a layer or background layer.
光反射性を有する微粒子の例としては、二酸化チタン微
粒子、硫酸バリウム微粒子が好ましい。As examples of the light-reflecting fine particles, titanium dioxide fine particles and barium sulfate fine particles are preferable.
本発明において、必要に応じ展開層中にも上記のごとき
光遮蔽性微粒子を含有させてもよい。In the present invention, the above-mentioned light-shielding fine particles may also be contained in the spreading layer, if necessary.
本発明に用い得る吸水層、試薬層、濾過層、反射層に用
いられる親水性ポリマーは、一般には水吸収時の膨潤率
が30℃で約1.5〜20、好ましくは約2.5〜15
の範囲の天然または合成親水性ポリマーの例としては、
ゼラチン(例、アルカリ処理ゼラチン、醜処理ゼラチン
等)、ゼラチン誘導体(例、フタル化ゼラチン等)、ア
ガロース、ポリアクリルアミド、ポリビニルアルコール
、ポリビニルピロリドン等を挙げることができる。さら
に、必要に応じて界面活性剤(カチオン性、両性または
ン非イオン性界面活性剤)を含有させることもできる。The hydrophilic polymer used in the water absorption layer, reagent layer, filtration layer, and reflection layer that can be used in the present invention generally has a swelling rate of about 1.5 to 20 at 30°C, preferably about 2.5 to 2.5. 15
Examples of natural or synthetic hydrophilic polymers in the range of
Examples include gelatin (eg, alkali-treated gelatin, ugly-treated gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, etc.), agarose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like. Furthermore, a surfactant (cationic, amphoteric, or nonionic surfactant) can be included if necessary.
吸水層、光遮蔽層、濾過層、試薬層等の層の上には、展
開層を接着し積層するための接着層を設けてもよい、接
着層は水で湿潤してl;)るとき、または水を含んで膨
潤したときに展開層を接着することができるような親木
性ポリマーからなることが好ましい、接着層に用いるこ
とができる親水性ポリマーの例としては、吸水層に用い
られると同様な親水性ポリマーが挙げられる。これらの
うちではゼラチン、ゼラチン誘導体、ポリアクリルアミ
ド等が好ましい。接着層の乾燥膜厚は一般に約0.5絡
m〜約20ルm、好ましくは約1pm〜約10JLmの
範囲である。なお、接着層は検出層上以外にも、他の居
間の接着力を向上させるため所望の層上に設けてもよい
、接着層は親木性ポリマーと必要によって加えられる界
面活性剤等を含む水溶液を公知の方法で、試薬層等の上
に塗布する方法などにより設けることができる。An adhesive layer may be provided on the water absorption layer, light shielding layer, filtration layer, reagent layer, etc. for adhering and laminating the spreading layer. When the adhesive layer is moistened with water, , or a wood-philic polymer that can adhere to the spreading layer when swollen with water. Examples of hydrophilic polymers that can be used in the adhesive layer include those used in the water-absorbing layer. Examples include hydrophilic polymers similar to . Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer generally ranges from about 0.5 JLm to about 20 JLm, preferably from about 1 pm to about 10 JLm. In addition to the detection layer, the adhesive layer may be provided on other desired layers to improve adhesive strength.The adhesive layer contains a wood-philic polymer and a surfactant added as necessary. The aqueous solution can be provided by a known method, such as by applying the aqueous solution onto the reagent layer or the like.
吸水層、光遮蔽層、濾過層、試薬層等の層の上には、展
開層を接着し積層するための接着層を設けてもよい。接
着層は水で湿潤しているとき、または水を含んで膨潤し
たときに展開層を接着す、ることができるような親木性
ポリマーからなることが好ましい。接着層に用いること
ができる親木性ポリマーの例としては、吸水層に用いら
れると同様な親水性ポリマーが挙げられる。これらのう
ちではゼラチン、ゼラチン誘導体、ポリアクリルアミド
等が好ましい。接着層の乾燥膜厚は一般に約0.5JL
m〜約20pm、好ましくは約1gm〜約10終mの範
囲である。なお、接着層は検出層上以外にも、他の層間
の接着力を向上させるため所望の層上に設けてもよい、
接着層は親水性ポリマーと必要によって加えられる界面
活性剤等を含む水溶液を公知の方法で、試薬層等の上に
塗布する方法などにより設けることができる。An adhesive layer for adhering and laminating the spreading layer may be provided on the layers such as the water absorption layer, light shielding layer, filtration layer, and reagent layer. The adhesive layer is preferably made of a wood-philic polymer that is capable of adhering the spreading layer when wetted with water or swollen with water. Examples of wood-philic polymers that can be used in the adhesive layer include hydrophilic polymers similar to those used in the water-absorbing layer. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer is generally approximately 0.5JL.
m to about 20 pm, preferably from about 1 gm to about 10 gm. Note that the adhesive layer may be provided not only on the detection layer but also on a desired layer in order to improve the adhesion between other layers.
The adhesive layer can be provided by a known method such as applying an aqueous solution containing a hydrophilic polymer and a surfactant added if necessary onto the reagent layer.
展開層とは、その表面に点着供給された液体試料を、そ
の中に含有している成分を実質的に偏在させることなく
、横(水平)方向に単位面積当りほぼ一定量の割合で広
げる作用を有するものである。A spread layer is a layer in which a liquid sample is dotted onto its surface and spread out at a rate of approximately constant amount per unit area in the lateral (horizontal) direction without substantially unevenly distributing the components contained therein. It has an effect.
展開層のマトリックスを構成する材料としては、濾紙、
不織布、織物生地(例、ブロード、ボブリン等の平織等
)、織物生地(例、トリコット編、ダブルトリコット編
、ミラニーズ編等)、ガラス繊維症紙、プラッシュポリ
マーより形成されるメンブランフィルタ−1あるいはポ
リマーミクロビーズ等からなる三次元格子状構造物等を
用いることが好ましい、これらのうちでは、試薬類の保
持性の点で、織物生地および編物生地に代表される繊維
質層を用いることが特に好ましい。Materials constituting the matrix of the spreading layer include filter paper,
Membrane filter 1 or polymer formed from nonwoven fabric, woven fabric (e.g., plain weave such as broadcloth, boblin, etc.), woven fabric (e.g., tricot knit, double tricot knit, Milanese knit, etc.), glass fibrosis paper, plush polymer It is preferable to use a three-dimensional lattice structure made of microbeads, etc. Among these, it is particularly preferable to use a fibrous layer typified by woven fabrics and knitted fabrics in terms of retention of reagents. .
本発明の乾式分析要素に用いることができる織物生地ま
たは編物生地は水洗等の脱脂処理により少なくとも糸製
造時、織物製造時あるいは編物編成時に供給または付着
した油脂類を実質的に除去した織物または編物生地が好
ましい。The woven fabric or knitted fabric that can be used in the dry analysis element of the present invention is a woven fabric or knitted fabric that has been subjected to degreasing treatment such as washing with water to substantially remove at least oils and fats supplied or attached during yarn production, fabric production, or knitting. Fabric is preferred.
上記織物または編物生地を一体型多層分析要素の展開層
として用いる場合には、さらにその織物または編物生地
に特開昭57−66359号公報に開示の物理的活性化
処理(好ましくはグロー放電処理またはコロナ放電処理
等)を生地の少なくとも片面に施すか、あるいは特開昭
55−164356号、特開昭57−66359号公報
等に開示の親水性ポリマー含浸処理等の親木化処理また
はこれらの処理工程を逐次実施することにより織物また
は編物を親水化し、下側(支持体に近い側)の層との接
着力を強化することができる。When the above-mentioned woven or knitted fabric is used as a spreading layer of an integrated multilayer analytical element, the woven or knitted fabric is further subjected to physical activation treatment (preferably glow discharge treatment or corona discharge treatment, etc.) on at least one side of the fabric, or wood-filtering treatment such as hydrophilic polymer impregnation treatment disclosed in JP-A-55-164356, JP-A-57-66359, etc., or these treatments. By performing the steps sequentially, the woven or knitted fabric can be made hydrophilic and its adhesive strength with the lower layer (the side closer to the support) can be strengthened.
織物または編物生地からなる一体型多層分析要素の展開
層を前述また吸水層または接着層に接着、積層するには
、特開昭55−164356号および特開昭57−66
359号各公報等に開示の方法に従って作成することが
できる。すなわち、吸水層または接着層の塗布後未乾燥
のうちに、または乾燥後の層に水(または界面活性剤を
少量含む水)を実質的に均一に供給して層を膨潤させ、
ついで織物または編物生地を湿潤または膨潤している層
の上に実質的に均一に軽く圧力をかけながら接着、積層
し一体化する。In order to adhere and laminate the spread layer of an integrated multilayer analysis element made of woven or knitted fabric on the above-mentioned water absorbing layer or adhesive layer, Japanese Patent Application Laid-open Nos. 55-164356 and 57-66 are disclosed.
It can be created according to the method disclosed in each publication such as No. 359. That is, water (or water containing a small amount of surfactant) is substantially uniformly supplied to the water-absorbing layer or the adhesive layer while it is still wet after being applied, or after drying, to swell the layer.
The woven or knitted fabric is then bonded, laminated, and integrated onto the wet or swollen layer substantially uniformly and under gentle pressure.
また展開層がブテッシュボリマーまたはメンブランフィ
ルタ−からなる場合には特公昭53−21677号公報
等、ポリマーミクロビーズからなる三次元格子状構造物
である場合には特開昭55−90859号公報等、濾紙
または不織布からなる場合には特開昭57−14825
0号公報等にそれぞれ記載の方法に従って設けることが
できる。In addition, when the spreading layer is made of butech polymer or membrane filter, Japanese Patent Publication No. 53-21677, etc., and when it is a three-dimensional lattice structure made of polymer microbeads, Japanese Patent Application Laid-Open No. 55-90859 is disclosed. etc., when it is made of filter paper or non-woven fabric, Japanese Patent Application Laid-Open No. 57-14825
They can be provided according to the methods described in Publication No. 0 and the like.
展開層は展開i制御するために、親水性ポリマー、界面
活性剤を含浸することができる。The spreading layer can be impregnated with a hydrophilic polymer or a surfactant to control the spreading.
親木性ポリマーとしては、セルロース誘導体、ポリビニ
ルピロリドン、ポリビニルアルコール。Examples of wood-philic polymers include cellulose derivatives, polyvinylpyrrolidone, and polyvinyl alcohol.
ポリアクリルアミド等を挙げることができる。Examples include polyacrylamide.
また界面活性剤としては、非イオン、両性、カチオン、
アニオン界面活性剤から分析項目により適宜選択できる
。また塗布層を通過しずらい検出物質(親油性高分子量
物質)のために展開層に反応試薬、酵素を含ませること
もできる。In addition, surfactants include nonionic, amphoteric, cationic,
It can be appropriately selected from anionic surfactants depending on the analysis item. Furthermore, the developing layer may contain a reaction reagent or an enzyme for a detection substance (lipophilic high molecular weight substance) that is difficult to pass through the coating layer.
本発明の乾式分析要素は、一体型多層分析要素とした場
合には、−通約15mmから約30mmの正方形または
ほぼ同サイズの円形等の小片に裁断し、特開昭57−6
3452号、特開昭54−156079号、実開昭56
−142454号、実開昭58−32350号および特
開昭58−501144号各公報等に開示のスライド枠
等に納めて分析スライドとして用いるのが製造、包装、
輸送、保存および測定操作等の全ての観点で好ましい。When the dry analysis element of the present invention is made into an integrated multilayer analysis element, it is cut into small pieces such as squares of about 15 mm to about 30 mm or circular pieces of approximately the same size.
No. 3452, Japanese Unexamined Patent Publication No. 156079/1983, Japanese Unexamined Patent Publication No. 1983
Manufacturing, packaging,
It is preferable from all viewpoints such as transportation, storage, and measurement operations.
本発明の乾式分析要素の使用に際しては、約5ル交から
約30川見、好ましくは約8延文から約15ル見の水性
液体 料を展開層に点着供給し、必要に応じて約20℃
から約45℃の範囲の実質的に一定の温度でインクベー
ションする。そののち、一方の側から(一体型多層分析
要素においては光透過性支持体側から)乾式分析要素内
の色変化1発色等の検出可能な変化を反射測光し比色法
の原理により液体試料中の測定対象成分を分析する。When using the dry analysis element of the present invention, an aqueous liquid material of about 5 to about 30 degrees, preferably about 8 to about 15 degrees, is dotted onto the spreading layer, and if necessary, about 30 degrees of aqueous liquid is applied. 20℃
Incubation is carried out at a substantially constant temperature ranging from about 45°C to about 45°C. Thereafter, detectable changes such as color changes in the dry analytical element from one side (from the light-transmissive support side in the case of integrated multilayer analytical elements) are measured by reflectance photometry in the liquid sample using the principle of colorimetry. Analyze the component to be measured.
[実施例]
ゼラチン下塗りが施されである厚さ180 pmのポリ
エチレンテレフタレート(PET)フィルム上に下記処
方で乾燥膜厚が13μmになるように試薬発色層を塗布
した。[Example] A reagent coloring layer was coated on a 180 pm thick polyethylene terephthalate (PET) film with a gelatin undercoat so that the dry film thickness was 13 μm using the following formulation.
アルカリ処理ゼラチン 9.4g3.3’−(
3,3’−ジメトキシ−
4,4’ (ビフェニレン)−ビス
[2−(p−ニトロフェニル−
2H−テトラゾリウムクロライド)]
0.04g
ATP O,79gNAD
O、3gドータイトニト
ロ
テトラゾリウムブルー 0.2gクエン酸ナトリ
ウム 3g
ジアホラーゼ 2800Uグリセロー
ルキナーゼ 880Uグリセロール3燐酸
デヒドロゲナーゼ 5400U注ニド−タイ
トニトロテトラゾリウムブルーの化学名は、3.3’−
(3,3’−ジメトキシ−4,4′−ビフェニレン)ビ
ス[2−(p−ニトロフェニル)−5−フェニル−2H
−テトラゾリウム]ジクロリドである。Alkali-treated gelatin 9.4g3.3'-(
3,3'-dimethoxy-4,4' (biphenylene)-bis[2-(p-nitrophenyl-2H-tetrazolium chloride)] 0.04g ATP O, 79gNAD
O, 3g Dotite Nitrotetrazolium Blue 0.2g Sodium Citrate 3g Diaphorase 2800U Glycerol Kinase 880U Glycerol Triphosphate Dehydrogenase 5400U Note Nido-Tite Nitrotetrazolium Blue's chemical name is 3.3'-
(3,3'-dimethoxy-4,4'-biphenylene)bis[2-(p-nitrophenyl)-5-phenyl-2H
-tetrazolium] dichloride.
なお、上記試薬層は、アルカリ処理ゼラチンが17.5
g/1rrr’(支持体表面ff1)となる量にて塗布
された。Note that the reagent layer contains alkali-treated gelatin with a concentration of 17.5
It was applied in an amount of g/1rrr' (support surface ff1).
次に光遮蔽層を下記処方で7gmになるように塗布し、
乾燥した。Next, apply a light shielding layer with the following recipe to a thickness of 7gm.
Dry.
アルカリ処理ゼラチン 7gルチル型二酸化
チタン 35g3.3′−(3,3°−ジメト
キシ−
4,4” (ビフェニレン)−ビス
[2−(p−ニトロフェニル−
2H−テトラゾリウムクロライド)]
0.1g
水 50g
なお、上記光遮蔽層は、アルカリ処理ゼラチンが5.0
g/lrn’(支持体表面積)となる量にて塗布された
。Alkali-treated gelatin 7g Rutile titanium dioxide 35g 3.3'-(3,3°-dimethoxy-4,4" (biphenylene)-bis[2-(p-nitrophenyl-2H-tetrazolium chloride)] 0.1g Water 50g
Note that the light shielding layer contains alkali-treated gelatin with a content of 5.0%.
It was applied in an amount of g/lrn' (support surface area).
次に、塗布層上に30g/m’の割合で水を湿し水とし
て供給湿潤させたのち、グロー放電で親水化したポリエ
チレンテレフタレート紡績糸(50デニール)からなる
トリコット編物生地(平均厚さ250Bm)を圧着ラミ
ネートして展開層を設けた。この上に下記処方液を1r
rI″当り200m1塗布乾燥し中性脂肪検出用の多層
分析要素とした。Next, water was supplied as dampening water at a rate of 30 g/m' onto the coating layer to wet it, and then a tricot knitted fabric (average thickness 250 Bm) made of polyethylene terephthalate spun yarn (50 denier) made hydrophilic by glow discharge. ) was pressure-bonded and laminated to provide a development layer. Add 1r of the following prescription solution on top of this.
200 ml per rI'' was applied and dried to form a multilayer analytical element for neutral fat detection.
水 50
0mMポリビニルピロリドン(K2O) 20
gポリオキシエチレン
ノニルフェニルエーテル(n=40) 5 g2−
アミノ−2メチル−
1,3プロパンジオール 9gリボプロティン
リパーゼ 11400U[比較例]
光遮蔽層から電子受容性染料形成性化合物を除いたもの
を実施例と同様の方法で作成したものを比較サンプルと
した。完成した分析要素を1.5cmxt、scmの正
方形のチップに裁断し、特開昭57−63452号公報
に開示のプラスチックマウントに収めて中性脂肪定量用
化学分析スライドを完成させた。water 50
0mM polyvinylpyrrolidone (K2O) 20
g Polyoxyethylene nonylphenyl ether (n=40) 5 g2-
Amino-2-methyl-1,3-propanediol 9g Riboprotein lipase 11400U [Comparative example] A comparative sample was prepared using the same method as in the example except that the electron-accepting dye-forming compound was removed from the light-shielding layer. . The completed analysis element was cut into square chips of 1.5 cm x t and scm, and placed in a plastic mount disclosed in JP-A-57-63452 to complete a chemical analysis slide for quantifying neutral fat.
[分析スライドの評価]
各スライドに、ヘパリン採血後遠心分離した中性脂肪濃
度値、51.130.196.322゜421.523
mg/di(グリセロール3燐酸オキシダーゼ法を用い
た溶液分析法で求めた値)のヒト血漿をLOg1点着し
、37℃、6分間インキュベーションしたのち、中心波
長640nmの測定光でPET支持体側から発色の反射
光学濃度を測定し検量線を作成した。[Evaluation of analysis slides] Each slide shows the neutral fat concentration value after heparin blood collection and centrifugation, 51.130.196.322°421.523
One LOg of human plasma at mg/di (value determined by solution analysis using the glycerol triphosphate oxidase method) was applied, and after incubation at 37°C for 6 minutes, color was developed from the PET support side using measurement light with a center wavelength of 640 nm. The reflected optical density of the sample was measured and a calibration curve was created.
各個の分析スライドについて得られた検量線を第1表に
示す。The calibration curve obtained for each individual analysis slide is shown in Table 1.
第1表
実施例 比較例
51 0.420 0.418130
0.551 0.531196 0.672
0.681322 0.860 0.87
1421 0.996 0.990523
1.112 1.081次にビリルビン濃度の異
なるヒト検体を用いて検量線と同じ方法で発色させてビ
リルビンの影響を調べた。Table 1 Example Comparative Example 51 0.420 0.418130
0.551 0.531196 0.672
0.681322 0.860 0.87
1421 0.996 0.990523
1.112 1.081 Next, the influence of bilirubin was investigated by developing color in the same manner as the calibration curve using human samples with different bilirubin concentrations.
得られた結果を第2表に示す。The results obtained are shown in Table 2.
第2表の結果から明らかなように、ビリルビンの影響は
、比較例の分析スライドに比較して、実施例の分析スラ
イドの方が受けづらい。As is clear from the results in Table 2, the analysis slides of the Examples are less affected by bilirubin than the analysis slides of the Comparative Examples.
第2表
中性脂肪濃度 ビリルビン濃度 実施例 比較例54
2.0 54 54132 1
2.5 131 150210 3.2
211 211122 1.0 12
0 121次に、ヒト検体に、アスコルビン酸をO15
,15,20m g / d lそれぞれ添加したサン
プルを用いてその影響を各分析スライドについて調べた
。Table 2 Neutral fat concentration Bilirubin concentration Example Comparative example 54
2.0 54 54132 1
2.5 131 150210 3.2
211 211122 1.0 12
0 121 Next, ascorbic acid was added to the human sample using O15.
, 15, and 20 mg/dl, respectively, were used to examine the effects on each analytical slide.
その結果を第3表に示す。The results are shown in Table 3.
第3表
0 5 15 20IIg/d文実施例 75
78 80 83比較例 74 82 9
1 104本発明に従う構成の分析要素を用いた分析ス
ライドが優れた性能を示すことが明らかである。Table 3 0 5 15 20IIg/d sentence example 75
78 80 83 Comparative example 74 82 9
1104 It is clear that analytical slides using analytical elements configured according to the invention exhibit excellent performance.
Claims (1)
酵素、電子伝達性化合物および電子受容性染料形成性化
合物を含有する層を有する多層乾式分析要素において、 電子伝達性化合物を含む層よりも上側(電子伝達性化合
物を含む層と支持体とは反対側)に光遮蔽層を有し、さ
らに光遮蔽層か、あるいは光遮蔽層よりも上側の層に電
子受容性染料形成性化合物が含まれていることを特徴と
する分析要素。 2、電子伝達性化合物がジアホラーゼであることを特徴
とする特許請求の範囲第1項記載の分析要素。 3、酸化型ニコチンアミド補酵素がNADあるいはNA
DPであることを特徴とする特許請求の範囲第1項記載
の分析要素。 4、光遮蔽層か、光遮蔽層よりも上層に含まれている電
子受容性染料形成性化合物がテトラゾリウム塩であるこ
とを特徴とする特許請求の範囲第1項記載の分析要素。[Scope of Claims] 1. A multilayer dry analytical element having a layer containing a dehydrogenase, an oxidized nicotinamide coenzyme, an electron transfer compound, and an electron-accepting dye-forming compound on a support, comprising: It has a light-shielding layer above the layer containing the electron-transfer compound (on the opposite side from the layer containing the electron-transfer compound and the support), and further has an electron-accepting layer on the light-shielding layer or a layer above the light-shielding layer. An analytical element characterized in that it contains a dye-forming compound. 2. The analytical element according to claim 1, wherein the electron transfer compound is diaphorase. 3. Oxidized nicotinamide coenzyme is NAD or NA
The analysis element according to claim 1, characterized in that it is a DP. 4. The analytical element according to claim 1, wherein the electron-accepting dye-forming compound contained in the light-shielding layer or a layer above the light-shielding layer is a tetrazolium salt.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24028686A JPS6394993A (en) | 1986-10-09 | 1986-10-09 | Dry analytical element containing oxidized form coenzyme |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24028686A JPS6394993A (en) | 1986-10-09 | 1986-10-09 | Dry analytical element containing oxidized form coenzyme |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS6394993A true JPS6394993A (en) | 1988-04-26 |
Family
ID=17057222
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP24028686A Pending JPS6394993A (en) | 1986-10-09 | 1986-10-09 | Dry analytical element containing oxidized form coenzyme |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6394993A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0473189A2 (en) * | 1990-08-30 | 1992-03-04 | Kyoto Daiichi Kagaku Co., Ltd. | Multilayer analytical element for assaying fructosamine |
JP2008523415A (en) * | 2004-12-13 | 2008-07-03 | バイエル・ヘルスケア・エルエルシー | Size self-regulating composition and test apparatus for measuring analytes in biological fluids |
JP2009244014A (en) * | 2008-03-31 | 2009-10-22 | Cci Corp | Biosensor for measuring neutral fat |
-
1986
- 1986-10-09 JP JP24028686A patent/JPS6394993A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0473189A2 (en) * | 1990-08-30 | 1992-03-04 | Kyoto Daiichi Kagaku Co., Ltd. | Multilayer analytical element for assaying fructosamine |
JP2008523415A (en) * | 2004-12-13 | 2008-07-03 | バイエル・ヘルスケア・エルエルシー | Size self-regulating composition and test apparatus for measuring analytes in biological fluids |
US9982289B2 (en) | 2004-12-13 | 2018-05-29 | Ascensia Diabetes Care Holdings Ag | Size self-limiting compositions and test devices for measuring analytes in biological fluids |
JP2009244014A (en) * | 2008-03-31 | 2009-10-22 | Cci Corp | Biosensor for measuring neutral fat |
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