JPH0469570A - Test piece for analysis - Google Patents
Test piece for analysisInfo
- Publication number
- JPH0469570A JPH0469570A JP18055490A JP18055490A JPH0469570A JP H0469570 A JPH0469570 A JP H0469570A JP 18055490 A JP18055490 A JP 18055490A JP 18055490 A JP18055490 A JP 18055490A JP H0469570 A JPH0469570 A JP H0469570A
- Authority
- JP
- Japan
- Prior art keywords
- test piece
- ammonia
- detection reagent
- analytical test
- carrier
- 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
- 238000004458 analytical method Methods 0.000 title claims description 16
- 238000012360 testing method Methods 0.000 title abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 125000003396 thiol group Chemical class [H]S* 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 7
- QILSFLSDHQAZET-UHFFFAOYSA-N diphenylmethanol Chemical compound C=1C=CC=CC=1C(O)C1=CC=CC=C1 QILSFLSDHQAZET-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims description 28
- -1 sodium tetraphenylborate Chemical compound 0.000 claims description 19
- 239000003463 adsorbent Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 150000004945 aromatic hydrocarbons Chemical class 0.000 claims description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 abstract description 48
- 229910021529 ammonia Inorganic materials 0.000 abstract description 24
- 239000000872 buffer Substances 0.000 abstract description 9
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000011734 sodium Substances 0.000 abstract description 4
- 229910052708 sodium Inorganic materials 0.000 abstract description 4
- 239000004094 surface-active agent Substances 0.000 abstract description 4
- 239000004744 fabric Substances 0.000 abstract description 3
- 150000002500 ions Chemical class 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 abstract 3
- 230000003213 activating effect Effects 0.000 abstract 1
- 230000000274 adsorptive effect Effects 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 238000000151 deposition Methods 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 108090000604 Hydrolases Proteins 0.000 description 4
- 102000004157 Hydrolases Human genes 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 125000006323 alkenyl amino group Chemical group 0.000 description 4
- 125000003282 alkyl amino group Chemical group 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 125000001769 aryl amino group Chemical group 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 125000000542 sulfonic acid group Chemical group 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- WMAVHUWINYPPKT-UHFFFAOYSA-M (e)-3-methyl-n-[(e)-(1-methyl-2-phenylindol-1-ium-3-ylidene)amino]-1,3-thiazol-2-imine;chloride Chemical compound [Cl-].C12=CC=CC=C2N(C)C(C=2C=CC=CC=2)=C1N=NC=1SC=C[N+]=1C WMAVHUWINYPPKT-UHFFFAOYSA-M 0.000 description 1
- UPGSWASWQBLSKZ-UHFFFAOYSA-N 2-hexoxyethanol Chemical compound CCCCCCOCCO UPGSWASWQBLSKZ-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000000538 analytical sample Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- JCEBMROGCIEFRX-UHFFFAOYSA-L disodium 5-[(2-hydroxynaphthalen-1-yl)diazenyl]-2-[4-[(2-hydroxynaphthalen-1-yl)diazenyl]-2-sulfonatophenyl]benzenesulfonate Chemical compound OC1=C(C2=CC=CC=C2C=C1)N=NC=1C=C(C(=CC=1)C1=CC=C(C=C1S(=O)(=O)[O-])N=NC1=C(C=CC2=CC=CC=C12)O)S(=O)(=O)[O-].[Na+].[Na+] JCEBMROGCIEFRX-UHFFFAOYSA-L 0.000 description 1
- FPVGTPBMTFTMRT-UHFFFAOYSA-L disodium;2-amino-5-[(4-sulfonatophenyl)diazenyl]benzenesulfonate Chemical compound [Na+].[Na+].C1=C(S([O-])(=O)=O)C(N)=CC=C1N=NC1=CC=C(S([O-])(=O)=O)C=C1 FPVGTPBMTFTMRT-UHFFFAOYSA-L 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000003248 enzyme activator Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019233 fast yellow AB Nutrition 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- AJDUTMFFZHIJEM-UHFFFAOYSA-N n-(9,10-dioxoanthracen-1-yl)-4-[4-[[4-[4-[(9,10-dioxoanthracen-1-yl)carbamoyl]phenyl]phenyl]diazenyl]phenyl]benzamide Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C=CC=C2NC(=O)C(C=C1)=CC=C1C(C=C1)=CC=C1N=NC(C=C1)=CC=C1C(C=C1)=CC=C1C(=O)NC1=CC=CC2=C1C(=O)C1=CC=CC=C1C2=O AJDUTMFFZHIJEM-UHFFFAOYSA-N 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000004149 tartrazine Substances 0.000 description 1
- UJMBCXLDXJUMFB-GLCFPVLVSA-K tartrazine Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)C1=NN(C=2C=CC(=CC=2)S([O-])(=O)=O)C(=O)C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 UJMBCXLDXJUMFB-GLCFPVLVSA-K 0.000 description 1
- 235000012756 tartrazine Nutrition 0.000 description 1
- 229960000943 tartrazine Drugs 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000001043 yellow dye Substances 0.000 description 1
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
Description
【発明の詳細な説明】 〔産業上の利用分野 本発明は改良された分析用試験片に関する。[Detailed description of the invention] [Industrial application field The present invention relates to improved analytical test strips.
さらに詳しくは、検出試薬およびテトラフエニルホウ酸
ナトリウムを吸着性担体に担持させてなる分析用試験片
に関する。More specifically, the present invention relates to an analytical test piece in which a detection reagent and sodium tetraphenylborate are supported on an adsorbent carrier.
本発明の分析用試験片を使用することにより分析試料が
アンモニアを含有している場合でもその影響を受ｌりる
ことなく正確に試料中の成分を測定することができる。By using the analytical test piece of the present invention, even if the analytical sample contains ammonia, the components in the sample can be accurately measured without being affected by the ammonia.
検出試薬を吸着性担体に担持した分析用試験片において
は、分析試料にアンモニアが含有される場合、アンモニ
アの影響で分析結果に正又は負の誤差を生じる場合があ
る。そこで従来は試験片に酸性成分を含有させたり、可
能な限り酸性にした緩衝剤を用いるなどにより試料溶液
中のアンモニア分子をアンモニウムイオンに変化させる
方法やイオン交換性物質を試験片に担持させてアンモニ
アを除去する方法がとられていた。In an analytical test piece in which a detection reagent is supported on an adsorbent carrier, if the analysis sample contains ammonia, the influence of ammonia may cause a positive or negative error in the analysis result. Conventionally, methods have been used to convert the ammonia molecules in the sample solution into ammonium ions, such as by adding acidic components to the test piece or using a buffer made as acidic as possible, or by making the test piece support an ion-exchange substance. A method was used to remove ammonia.
しかしながら、試験片に酸成分を加えたり酸性の緩衝剤
を用いるなどにより試料溶液中のアンモニア分子をアン
モニウムイオンに変化させる方法においては、被測定成
分が酵素であったり、酵素を反応試薬として用いたりす
る場合には、その反応至適ｐＨの範囲が決まっており、
使用できる酸や酸性の緩衝剤の種類や量が太き（制限さ
れる。さらにイオン交換性物質と試験片に担持させてア
ンモニアを除去する方法においては、試験片の構造が複
雑になったり、検出の速度や感度が低下する可能性があ
る。However, in the method of changing ammonia molecules in the sample solution into ammonium ions by adding an acid component to the test piece or using an acidic buffer, the component to be measured is an enzyme, or the enzyme is used as a reaction reagent. In this case, the optimum pH range for the reaction is determined,
There are restrictions on the type and amount of acids and acidic buffers that can be used.Furthermore, in the method of removing ammonia by supporting it on an ion exchange material and a test piece, the structure of the test piece becomes complicated. Detection speed and sensitivity may be reduced.
本発明は、このような従来試験片の問題点を解決するた
めになされたものであり、検出反応系への影響がほとん
どなく、適用範囲が広く、簡単な構造でしかもアンモニ
アの影響を完全に回避できる試験片を提供せんとするも
のである。The present invention was made to solve these problems with conventional test pieces, and has a wide range of applicability, has a simple structure, and completely eliminates the effects of ammonia. The aim is to provide a test piece that can avoid this.
上記目的は、以下の本発明によって達成される。 The above objects are achieved by the present invention as described below.
即ち、本発明は、検出試薬およびテトラフェニルホウ酸
ナトリウムを吸着性担体に担持させてなる分析用試験片
である。That is, the present invention is an analytical test piece comprising a detection reagent and sodium tetraphenylborate supported on an adsorbent carrier.
また本発明は検出試薬を担持させた担体層と、テトラフ
ェニルホウ酸ナトリウムを担持させた担体層とを積層さ
せてなる上記分析用試験片である。The present invention also provides the above-mentioned analytical test piece, which is formed by laminating a carrier layer carrying a detection reagent and a carrier layer carrying sodium tetraphenylborate.
さらに本発明は、検出試薬が一般式（Ｉ）Ｈ
（式中Ｘは同−又は異なってアルキルアミノ基、アルケ
ニルアミノ基、アリールアミノ基、スルホン酸基又はヒ
ドロキシ基を示し、Ｒは水素原子又は置換されていても
よい芳香族炭化水素を示す、）
で表わされるベンツヒトロールである上記分析用試験片
である。Furthermore, the present invention provides a detection reagent of the general formula (I)H (wherein X is the same or different and represents an alkylamino group, an alkenylamino group, an arylamino group, a sulfonic acid group, or a hydroxyl group, and R is a hydrogen atom or This is the above-mentioned analytical test piece, which is benzhydrol represented by ), which represents an optionally substituted aromatic hydrocarbon.
さらに本発明は、検出試薬が被測定酵素の加水分解作用
によりチオール基を有する化合物を酵素基質および前記
一般式（Ｔ）で表わされるベンツヒトロールの混合物で
ある上記分析用試験片である。Furthermore, the present invention provides the analytical test piece described above, wherein the detection reagent is a mixture of a compound having a thiol group due to the hydrolytic action of the enzyme to be measured, an enzyme substrate, and benzhitrol represented by the general formula (T).
本発明において使用される検出試薬には特に制限はなく
、分析用検出試薬として通常使用されるもの、例えば、
呈色剤、呈色指示薬、基質、酵素、緩衝剤、界面活性剤
、湿潤剤等が単独又は組合せて用いられる。被測定物質
がチオール基を有する化合物である場合には、検出試薬
として前記式（Ｉ）を有するベンツヒトロールが好適に
使用される。The detection reagents used in the present invention are not particularly limited, and include those commonly used as analytical detection reagents, such as
A coloring agent, a coloring indicator, a substrate, an enzyme, a buffer, a surfactant, a wetting agent, etc. are used alone or in combination. When the substance to be measured is a compound having a thiol group, benzhitrol having the above formula (I) is preferably used as the detection reagent.
一般式Ｉにおいて、Ｘは同−又は異なってアルキルアミ
ノ基、アルケニルアミノ基、アリールアミノ基、スルホ
ン酸基又はヒドロキシ基を示す。In general formula I, X is the same or different and represents an alkylamino group, an alkenylamino group, an arylamino group, a sulfonic acid group or a hydroxyl group.
アルキルアミノ基としては、ジメチルアミノ、ジエチル
アミノ、ジー１ｓｏ−プロピルアミノのようなジ（低級
）アルキルアミノ基が好ましい。アルケニルアミノ基と
してはジビニルアミノ基のようなジ（低級）アルケニル
アミノ基が好ましい。−般式Ｉで示されるチオール基検
出指示薬の具体例を示すと次の通りである。As the alkylamino group, di(lower) alkylamino groups such as dimethylamino, diethylamino, and di-1so-propylamino are preferred. The alkenylamino group is preferably a di(lower) alkenylamino group such as a divinylamino group. - Specific examples of the thiol group detection indicator represented by general formula I are as follows.
■ ４，４゛−ビス（ジメチルアミノ）ベンツヒトロー
ル
■ ４．４−ビス（ジエチルアミノ）ベンツヒトロール
■ ３−Ｃ４，４’−ビス（ジメチルアミノ）ジフェニ
ルヒドロキシメチル〕 −４−ヒドロキシベンゼンスル
ホン酸
■ ４．４′−ビス（ジメチルアミノ）ジフェニル（２
，７−シヒドロキシナフチル）メタノール■ ４，４′
−ビス（ジベンジルアミノ）ジフェニル−（２−ヒドロ
キシフェニル）メタノール被検出物質が加水分解酵素で
ある場合、適する緩衝剤としては、ホウ酸緩衝剤、リン
酸緩衝剤、トリス緩衝剤、クエン酸緩衝剤等があげられ
、その濃度は０．０２〜０．４ｍＭ（好ましくは０．１
〜０．３ｄ）で、ｐｌｌは５〜９（好ましくはｐｌ＋６
〜７）で使用する。■ 4,4'-bis(dimethylamino)benzhytrol ■ 4,4-bis(diethylamino)benzhytrol ■ 3-C4,4'-bis(dimethylamino)diphenylhydroxymethyl] -4-hydroxybenzenesulfonic acid ■ 4.4'-bis(dimethylamino)diphenyl(2
,7-hydroxynaphthyl)methanol■ 4,4'
-Bis(dibenzylamino)diphenyl-(2-hydroxyphenyl)methanol When the substance to be detected is a hydrolase, suitable buffers include borate buffer, phosphate buffer, Tris buffer, citrate buffer. The concentration is 0.02-0.4mM (preferably 0.1mM).
~0.3d), pll is 5-9 (preferably pl+6
Used in ~7).
界面活性剤、湿潤剤としては、トリトン、アルキル硫酸
ナトリウム、スパン、トウィーン、ポリエチレングリコ
ール、高級アルコール類、エーテルアルコール類等があ
げられ、好ましくは炭素数８〜１２の直鎖のアルコール
、炭素数８〜１５のエーテル結合を含む直鎖のアルコー
ルが用いられる。Examples of surfactants and wetting agents include Triton, sodium alkyl sulfate, Span, Tween, polyethylene glycol, higher alcohols, and ether alcohols, preferably linear alcohols having 8 to 12 carbon atoms, and 8 to 12 carbon atoms. A straight chain alcohol containing ~15 ether linkages is used.
濃度は含浸液に対して、０．１〜４．０％（好ましくは
１．０〜２．０％）で使用する。The concentration used is 0.1 to 4.0% (preferably 1.0 to 2.0%) based on the impregnation liquid.
さらに、試験片に検出試薬と異なる波長を有する色素を
予め添加しておくことによって、検出試薬の呈色変化が
目視でとらえ易くなる場合もある。Furthermore, by adding a dye having a wavelength different from that of the detection reagent to the test piece in advance, it may become easier to visually detect the color change of the detection reagent.
例えば、４．４’−ビス（ジメチルアミノ）ベンツヒト
ロールをチオールの検出試薬に用いた場合は、呈色変化
が色素無添加時には青色から無色であるが、黄色色素を
添加することによって緑色から黄色への変化となり、目
視での定量が容易となる。For example, when 4,4'-bis(dimethylamino)benzhitrol is used as a thiol detection reagent, the color changes from blue to colorless when no dye is added, but from green to colorless when a yellow dye is added. The color changes to yellow, making visual quantification easier.
添加色素としては、赤色系でベーシックレッド２９、ア
シッドレッド９７、黄色系では、タートラジン、アシッ
ドイエロー２９等があげられ、含浸液に対して０．０１
〜０．５％の濃度で添加するのが好ましい。Examples of added pigments include Basic Red 29 and Acid Red 97 for red colors, and Tartrazine and Acid Yellow 29 for yellow colors.
Preferably it is added at a concentration of ~0.5%.
以上の成分を濾紙又は布帛等の吸若性担体に担持させる
ためには、それぞれの成分を適当な溶媒例えばアセトン
に溶解せしめ、担体に含浸後、乾燥することによって溶
媒を除去して得ることができる。このようにして得られ
た試験片は適当な大きさ（例えば５Ｘ５ｍ＋ｓ角）に切
断し、ポリスチレンシート等の支持体に、両面テープ等
の接着剤を用いて固定することによって、試料への浸漬
が容易となる。In order to support the above-mentioned components on a youth-absorbing carrier such as filter paper or cloth, each component can be dissolved in a suitable solvent such as acetone, impregnated into the carrier, and then dried to remove the solvent. can. The test piece obtained in this way is cut into an appropriate size (for example, 5 x 5 m + s square) and fixed to a support such as a polystyrene sheet using an adhesive such as double-sided tape, so that it cannot be immersed in the sample. It becomes easier.
本発明によれば、試料溶液中に存在するアンモニアは、
試験片中に予め含有させておいたテトラフェニルホウ酸
ナトリウム（ＩＩ）と不溶性のイオン対（ｍ）を生成す
る。このイオン対は、反応溶液に不溶性の白色固体であ
り、化学的にほとんど不活性であるので、アンモニアは
実質上検出反応系から除去されたことになる。この反応
は、検出反応に多用される酵素反応に比較して速いため
、アンモニアによる妨害反応が回避できる。According to the invention, ammonia present in the sample solution is
An insoluble ion pair (m) is generated with sodium tetraphenylborate (II) previously contained in the test piece. Since this ion pair is a white solid that is insoluble in the reaction solution and is almost chemically inert, ammonia has been substantially removed from the detection reaction system. This reaction is faster than the enzymatic reaction often used in detection reactions, so interference reactions caused by ammonia can be avoided.
（ＩＩ）
例えば、チオエステル結合を有する酵素基質を使用した
加水分解酵素の検出系においては、検出試薬がアンモニ
アと反応するため、その影響を除去しなければならない
。以下にその検出反応のメカニズムを示す。一般式■で
示されるチオエステルを有する基質は、エステラーゼ、
プロテアーゼ等の加水分解酵素の作用によって、次式に
示すごとくチオエステル結合が分解されて一般弐Ｖで示
される遊離のチオールを生じる。(II) For example, in a detection system for a hydrolase using an enzyme substrate having a thioester bond, the detection reagent reacts with ammonia, so its influence must be removed. The mechanism of the detection reaction is shown below. Substrates having a thioester represented by the general formula ■ are esterases,
By the action of a hydrolytic enzyme such as protease, the thioester bond is decomposed as shown in the following formula to generate a free thiol represented by the general formula 2V.
Ｒ’−３ＣＯ−Ｙ Ｒ′−５Ｈ＋ＨＯＯＣ−Ｙ（Ｉ
Ｖ） （Ｖ）
〔上記式中、Ｒ′は置換されていても良い芳香族基を示
し、Ｙは被測定酵素によって認諜されうる有機残基を示
す。〕この遊離のチオールの検出は、下式に示すごとく
、一般式Ｉで示されるベンツヒトロールを用いて行うこ
とができる。すなわち、ベンツヒトロール＜ｒ）は測定
系に存在する酸によってカチオンｌ）となり、極大吸収
波長６０６ｎ＋＋＋の青色を示す。これに、加水分解酵
素の作用によって生じた遊離のチオール（Ｖ）が反応し
て複合体（■）を形成する。この複合体は無色のため、
６０６ｎ■の吸光度の減少を測定することによって、チ
オールを定量することが可能である。R'-3CO-Y R'-5H+HOOC-Y(I
V) (V) [In the above formula, R' represents an optionally substituted aromatic group, and Y represents an organic residue that can be recognized by the enzyme to be measured. ] Detection of this free thiol can be carried out using benzhitrol represented by the general formula I, as shown in the following formula. That is, benzhydrol<r) becomes a cation l) by the acid present in the measurement system, and exhibits a blue color with a maximum absorption wavelength of 606n+++. Free thiol (V) generated by the action of a hydrolase reacts with this to form a complex (■). Since this complex is colorless,
Thiols can be quantified by measuring the decrease in absorbance at 606n.
０Ｈ
（Ｖｌ）
ＳＲ’
（■）
ところが、試料溶液中にアンモニアが存在すると、ベン
ツヒトロール（１）と反応して無色の複合体を生成する
。すなわち、検出目的の加水分解酵素の反応によって生
じる遊離のチオールと競合することになる。実際は、チ
オールの方がアンモニアより反応速度が速いので、顕著
な妨害にこそならないが、最終的には試験片の感度の低
下の原因となる。この反応系にテトラフェニルホウ酸ナ
トリウムが存在すると、前記反応式に従いアンモニアが
除去され、はとんど妨害を受けなくなる。従って本発明
の分析用試験片は尿や汚水のようにアンモニアを含む試
料の分析に好適に使用される。0H (Vl) SR' (■) However, when ammonia is present in the sample solution, it reacts with benzhydrol (1) to produce a colorless complex. That is, it will compete with free thiol produced by the reaction of the hydrolase for detection. In reality, thiols have a faster reaction rate than ammonia, so they do not cause significant interference, but they ultimately cause a decrease in the sensitivity of the test strip. When sodium tetraphenylborate is present in this reaction system, ammonia is removed according to the above reaction formula, and there is almost no interference. Therefore, the analytical test piece of the present invention is suitably used for analyzing samples containing ammonia such as urine and sewage.
加水分解酵素検出のための試験片は、前記酵素基質と遊
離チオールの指示薬のベンツヒトロール、緩衝剤そして
テトラフェニルホウ酸ナトリウムを濾紙又は布帛等の担
体に吸着させて製造する。また、検出試薬層と、テトラ
フェニルホウ酸ナトリウムを含有するアンモニア除去層
をそれぞれ別の担体に吸告させたものを、積層しても良
い。このような積層構造にすることは、担持される検出
試薬同士が反応を起こすような場合に特に望ましい。A test strip for detecting a hydrolytic enzyme is produced by adsorbing the enzyme substrate, benzhytrol, a free thiol indicator, a buffer, and sodium tetraphenylborate onto a carrier such as filter paper or cloth. Alternatively, a detection reagent layer and an ammonia removal layer containing sodium tetraphenylborate adsorbed on separate carriers may be laminated. Such a layered structure is particularly desirable when the supported detection reagents react with each other.
場合によっては、酵素活性化剤や指示薬の増感剤、ある
いは界面活性剤を検出試薬系に添加することにより、よ
り高い感度と正確な測定結果が得られる。In some cases, higher sensitivity and more accurate measurement results can be obtained by adding enzyme activators, indicator sensitizers, or surfactants to the detection reagent system.
次に実施例を示して本発明をさらに具体的に説明する。Next, the present invention will be explained in more detail with reference to Examples.
［実 施 例〕
濾紙（アトパンチツク社Ｎｏ、５１４Ａ）を、０．２Ｍ
ボラックスー塩酸緩衝液（ｐＨ−６，０）に浸し、次い
で６０℃で５０分通風乾燥した。さらにこの濾紙を、２
．０ｄ（Ｎ−トシル−し−アラニルチオ）ベンゼン、０
．３ＩＩＭ ４．４′−ビス（ジメチルアミノ）ベンツ
ヒトロール、２％エチレングリコールモノヘキシルエー
テルおよび種々の濃度のテトラフェニルホウ酸ナトリウ
ムを含有するアセトン溶液に浸し、４０℃で２０分通風
乾燥した。これを５Ｘ５ｃ＋ｎ角に切断し、検体を滴下
して１分後の呈色変化を試験紙表面の６００ｎｍの反射
吸光度を測定することによって観察した。Δ１１定には
、人尿電子製のＭＣＰＤ２００を用いた。[Example] Filter paper (Atopanczyk No. 514A) was
It was immersed in borax-hydrochloric acid buffer (pH-6,0) and then dried with ventilation at 60°C for 50 minutes. Furthermore, this filter paper, 2
．． 0d(N-tosyl-alanylthio)benzene, 0
．． 3IIM 4.4'-Bis(dimethylamino)benzhytrol, 2% ethylene glycol monohexyl ether, and various concentrations of sodium tetraphenylborate were immersed in an acetone solution and dried with ventilation at 40° C. for 20 minutes. This was cut into 5×5c+n squares, the specimen was dropped, and the color change 1 minute later was observed by measuring the reflected absorbance at 600 nm on the surface of the test paper. For the Δ11 constant, MCPD200 manufactured by Human Urine Denshi was used.
検体に種々の濃度のアンモニア水溶液を用い、アンモニ
アの反応系に及はす影響、およびテトラフェニルホウ酸
ナトリウムの試薬への添加によるアンモニアの影響の回
避を調べた結果を、表１に示す。Table 1 shows the results of investigating the effect of ammonia on the reaction system and avoiding the effect of ammonia by adding sodium tetraphenylborate to the reagent using ammonia aqueous solutions of various concentrations as specimens.
２ Ｘ １０’ｕｎｉｔ／ｍｌのキモトリプシン溶液に
種々の濃度のアンモニアを添加した検体を用い、同様の
実験を行った結果を表２に示す。Table 2 shows the results of a similar experiment using samples prepared by adding various concentrations of ammonia to a chymotrypsin solution of 2 x 10' units/ml.
テトラフェニルホウ酸ナトリウムを試験片に添加しない
場合、検体溶液中のアンモニア濃度によって吸光度が大
きく変化しているが、添加した場合はほとんどアンモニ
アの影響は受けていない。When sodium tetraphenylborate is not added to the test piece, the absorbance changes greatly depending on the ammonia concentration in the sample solution, but when sodium tetraphenylborate is added, it is hardly affected by ammonia.
表 １ 吸 光 度Table 1 Absorption light degree
Claims (4)
を吸着性担体に担持させてなる分析用試験片。(1) An analytical test piece comprising a detection reagent and sodium tetraphenylborate supported on an adsorbent carrier.
ウ酸ナトリウムを担持させた担体層とを積層させてなる
請求項１に記載の分析用試験片。(2) The analytical test piece according to claim 1, wherein a carrier layer carrying a detection reagent and a carrier layer carrying sodium tetraphenylborate are laminated.
ニルアミノ基、アリールアミノ基、スルホン酸基又はヒ
ドロキシ基を示し、Ｒは水素原子又は置換されていても
よい芳香族炭化水素を示す。） で表わされるベンツヒドロールである請求項１又は２に
記載の分析用試験片。(3) The detection reagent has the general formula (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. , R represents a hydrogen atom or an optionally substituted aromatic hydrocarbon. The analytical test piece according to claim 1 or 2, which is benzhydrol represented by:
ール基を有する化合物を酵素基質および前記一般式（
I ）で表わされるベンツヒドロールの混合物である請求
項１又は２に記載の分析用試験片。(4) The detection reagent converts a thiol group-containing compound into an enzyme substrate and the general formula (
The analytical test piece according to claim 1 or 2, which is a mixture of benzhydrol represented by I).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18055490A JPH0469570A (en) | 1990-07-10 | 1990-07-10 | Test piece for analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18055490A JPH0469570A (en) | 1990-07-10 | 1990-07-10 | Test piece for analysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0469570A true JPH0469570A (en) | 1992-03-04 |
Family
ID=16085310
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18055490A Pending JPH0469570A (en) | 1990-07-10 | 1990-07-10 | Test piece for analysis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0469570A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007218866A (en) * | 2006-02-20 | 2007-08-30 | Toyama Univ | Method of measuring formaldehyde |
| JP2011524526A (en) * | 2008-06-13 | 2011-09-01 | エーエルティー バイオサイエンス， エルエルシー． | Device for rapid measurement of disease related thiol compounds |
-
1990
- 1990-07-10 JP JP18055490A patent/JPH0469570A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007218866A (en) * | 2006-02-20 | 2007-08-30 | Toyama Univ | Method of measuring formaldehyde |
| JP2011524526A (en) * | 2008-06-13 | 2011-09-01 | エーエルティー バイオサイエンス， エルエルシー． | Device for rapid measurement of disease related thiol compounds |
| JP2013190443A (en) * | 2008-06-13 | 2013-09-26 | Alt Bioscience Llc | Device for rapid determination of disease-associated thiol compounds |
| US8815152B2 (en) | 2008-06-13 | 2014-08-26 | Alt Bioscience, Llc | Device for rapid determination of disease-associated thiol compounds |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5300439A (en) | Method, device, and composition for the assay of ions | |
| CA1129323A (en) | Diagnostic agent for the detection of components in liquids | |
| FI81119B (en) | TESTREMSA FOER BESTAEMNING AV ETANOL, ALCOHOL OXIDE COMPOSITION FOR FOERFARANDE FOER BESTAEMNING AV ETANOL. | |
| JP3718242B2 (en) | Preparation of diagnostic test strips containing tetrazolium salt indicator | |
| IE65537B1 (en) | Use of a sparingly soluble salt of a heteropoly acid for the determination of an analyte a corresponding method of determination as well as a suitable agent therefor | |
| US4753890A (en) | Analytical element and method for determination of magnesium ions | |
| JPS63258883A (en) | Compound, reagent and method for measuring cation | |
| AU617300B2 (en) | Reagent and procedure for determining cations | |
| US4871678A (en) | Agent and process for the determination of calcium | |
| EP0036563B1 (en) | Bilirubin-resistant composition for the determination of cholesterol, test device containing the composition and method of making the test device | |
| EP0198286B1 (en) | Analytical compositions, elements and methods utilizing reduction of ferric ion chelates to form detectable dyes | |
| Zare-Dorabei et al. | Design of a novel optical sensor for determination of trace gadolinium | |
| FI79697B (en) | VID DETEKTERING AV IONER ANVAENDBAR NY INDONAFTOLDERIVAT OCH FOERFARANDE FOER DESS FRAMSTAELLNING. | |
| JPS58178256A (en) | Calcium analysis element | |
| JPH0469570A (en) | Test piece for analysis | |
| JPS6152300A (en) | Composition for measuring hydrogen peroxide | |
| US4120754A (en) | Detection of alkylating compounds | |
| JPH04157365A (en) | Dry analysis element for analyzing iron ion | |
| GB2049180A (en) | Bilirubin-resistant determination of uric acid | |
| US5948631A (en) | Stable mixture for the detection of alkaline phosphatase containing a salt of o-cresolphthalein monophosphoric acid | |
| KR100379646B1 (en) | Reagent composition, test piece and measurement kit | |
| JPS589697A (en) | Production of kit for measuring uric acid in sample | |
| EP0553820A2 (en) | Composition and test strip for measuring peroxidatively active substances | |
| WO1998038263A1 (en) | Optical membrane films for polycation detection | |
| JPH0411900A (en) | Determination of hydrolase |