SK281547B6 - Detection tube of cholinesterase inhibitors in air and water - Google Patents
Detection tube of cholinesterase inhibitors in air and water Download PDFInfo
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- SK281547B6 SK281547B6 SK736-94A SK73694A SK281547B6 SK 281547 B6 SK281547 B6 SK 281547B6 SK 73694 A SK73694 A SK 73694A SK 281547 B6 SK281547 B6 SK 281547B6
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- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 239000000544 cholinesterase inhibitor Substances 0.000 title claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 239000003593 chromogenic compound Substances 0.000 claims abstract description 10
- 239000011521 glass Substances 0.000 claims abstract description 10
- 239000001913 cellulose Substances 0.000 claims abstract description 7
- 229920002678 cellulose Polymers 0.000 claims abstract description 7
- 230000000052 comparative effect Effects 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims abstract 2
- 239000000872 buffer Substances 0.000 claims description 10
- 239000012916 chromogenic reagent Substances 0.000 claims description 4
- 108090000371 Esterases Proteins 0.000 claims description 3
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims 1
- 229960001231 choline Drugs 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 15
- 239000000243 solution Substances 0.000 abstract description 13
- 102100032404 Cholinesterase Human genes 0.000 abstract description 11
- 239000003795 chemical substances by application Substances 0.000 abstract description 9
- 108090000322 Cholinesterases Proteins 0.000 abstract description 6
- AWBGQVBMGBZGLS-UHFFFAOYSA-N butyrylthiocholine Chemical compound CCCC(=O)SCC[N+](C)(C)C AWBGQVBMGBZGLS-UHFFFAOYSA-N 0.000 abstract description 6
- 229940048961 cholinesterase Drugs 0.000 abstract description 6
- GFFIJCYHQYHUHB-UHFFFAOYSA-N 2-acetylsulfanylethyl(trimethyl)azanium Chemical compound CC(=O)SCC[N+](C)(C)C GFFIJCYHQYHUHB-UHFFFAOYSA-N 0.000 abstract description 5
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 abstract description 3
- 241000283690 Bos taurus Species 0.000 abstract description 3
- 210000004556 brain Anatomy 0.000 abstract description 3
- 150000003839 salts Chemical class 0.000 abstract description 3
- 102100033639 Acetylcholinesterase Human genes 0.000 abstract description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 abstract description 2
- 108010053652 Butyrylcholinesterase Proteins 0.000 abstract description 2
- 241000283073 Equus caballus Species 0.000 abstract description 2
- 229940022698 acetylcholinesterase Drugs 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 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 1
- 229910052708 sodium Inorganic materials 0.000 abstract 1
- 239000011734 sodium Substances 0.000 abstract 1
- 239000003112 inhibitor Substances 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 239000003708 ampul Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 101710083761 Cholinesterase Proteins 0.000 description 3
- 229940122041 Cholinesterase inhibitor Drugs 0.000 description 3
- QOGIOOCHOQQIGE-UHFFFAOYSA-N S(=O)(=O)(OC)OC.S1C(=NC=C1)N=NC1=C(C=CC(=C1)C)OC(C)=O Chemical compound S(=O)(=O)(OC)OC.S1C(=NC=C1)N=NC1=C(C=CC(=C1)C)OC(C)=O QOGIOOCHOQQIGE-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 2
- 229960004373 acetylcholine Drugs 0.000 description 2
- 239000002696 acid base indicator Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 125000001639 phenylmethylene group Chemical group [H]C(=*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- NTBLZMAMTZXLBP-UHFFFAOYSA-M 2-acetylsulfanylethyl(trimethyl)azanium;iodide Chemical compound [I-].CC(=O)SCC[N+](C)(C)C NTBLZMAMTZXLBP-UHFFFAOYSA-M 0.000 description 1
- GALNBQVDMJRFGJ-UHFFFAOYSA-M 2-butanoyloxyethyl(trimethyl)azanium;iodide Chemical compound [I-].CCCC(=O)OCC[N+](C)(C)C GALNBQVDMJRFGJ-UHFFFAOYSA-M 0.000 description 1
- 101000943288 Equus caballus Cholinesterase Proteins 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- TWMCBBMHCFACMM-UHFFFAOYSA-N S-quinolin-2-yl ethanethioate Chemical compound C(C)(=O)SC1=NC2=CC=CC=C2C=C1 TWMCBBMHCFACMM-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- QGLZXHRNAYXIBU-WEVVVXLNSA-N aldicarb Chemical compound CNC(=O)O\N=C\C(C)(C)SC QGLZXHRNAYXIBU-WEVVVXLNSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- OEBRKCOSUFCWJD-UHFFFAOYSA-N dichlorvos Chemical compound COP(=O)(OC)OC=C(Cl)Cl OEBRKCOSUFCWJD-UHFFFAOYSA-N 0.000 description 1
- 229950001327 dichlorvos Drugs 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000004457 water analysis 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 Materials By The Use Of Chemical Reactions (AREA)
Abstract
Riešenie sa týka detekčnej rúrky inhibítorov cholínesteráz vo vzduchu a vo vode, ktorá obsahuje dve ampulky s tlmiacim roztokom pH 6,0 až 9,0, porovnávaciu vrstvu z rozdrveného skla impregnovanou zmesou substrátu a chromogénneho činidla alebo chromogénnym substrátom a indikačnú vrstvu z granulovanej bielenej celulózy s imobilizovanou cholínesterázou. Porovnávacia vrstva z rozdrveného skla obsahuje ako substrát acetyltiocholín alebo butyryltiocholín a ako chromogénne činidlo kyselinu 5,5'-ditio-bis(2-nitrobenzoovú). Alebo porovnávacia vrstva obsahuje ako substrát acetyltiocholín alebo butyryltiocholín a ako chromogénne činidlo N-etyl-N-[4-[4-etyl-3-sulfofenyl [metylaminofenyl] fenylmetylén-2,5-cyklohexadién]-1-ylidén]-3- -sulfobenzénmetán aminiumnátriumhydroxid-vnútorná soľ. Alebo porovnávacia vrstva obsahuje chromogénny substrát, 1-[tiazolylazo]-2-acetoxy-5-metylbenzéndimetylsulfát. Indikačná vrstva výhodne obsahuje hovädziu mozgovú acetylcholínesterázu alebo plazmovú konskú butyrylcholínesterázu.ŕThe solution relates to a detection tube of cholinesterase inhibitors in air and in water, which contains two ampoules with buffer solution pH 6.0 to 9.0, a comparison layer made of crushed glass impregnated with a mixture of substrate and chromogenic agent or a chromogenic substrate, and an indicator layer made of granulated bleached cellulose with immobilized cholinesterase. The comparison layer made of crushed glass contains acetylthiocholine or butyrylthiocholine as a substrate and 5,5'-dithio-bis(2-nitrobenzoic acid) as a chromogenic agent. Or the comparative layer contains acetylthiocholine or butyrylthiocholine as a substrate and N-ethyl-N-[4-[4-ethyl-3-sulfophenyl [methylaminophenyl] phenylmethylene-2,5-cyclohexadiene]-1-ylidene]-3- as a chromogenic agent. sulfobenzenemethane aminium sodium hydroxide-internal salt. Or the comparison layer contains the chromogenic substrate, 1-[thiazolylazo]-2-acetoxy-5-methylbenzenedimethylsulfate. The indicator layer preferably contains bovine brain acetylcholinesterase or plasma equine butyrylcholinesterase.
Description
Oblasť technikyTechnical field
Vynález sa týka rúrky na detekcii inhibitorov cholínesteráz vo vzduchu a vo vode. Vynález rieši konštrukciu prostriedku na určenie extrémne toxických zlúčenín, významných v oblasti vojenskej a priemyselnej chémie, agrochémie, monitorovania životného prostredia, až po civilnú obranu a bezpečnosť štátu.The invention relates to a tube for detecting cholinesterase inhibitors in air and water. The invention solves the construction of a means for the determination of extremely toxic compounds, important in the field of military and industrial chemistry, agrochemistry, environmental monitoring, to civil defense and state security.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Na detekciu a stanovenie súborov inhibitorov cholínesteráz (ΑΜΜΟΝ R.-VOSS G., Pflugers Árch. ges. Physiol., 235 (1935), s. 393; MICHEZ O., H., J., Zab clin. Med., 34 (1949), s. 1564), je veľmi používaná v celom rade modifikácií a aplikácií biochemická cholínesterázová reakcia ako veľmi citlivá a selektívna metóda. Na cholínesterázovej reakcii sú založené rôzne typy technických prostriedkov na detekciu a stanovenie inhibitorov cholínesteráz, počnúc komplikovanými automatickými analyzátormi ovzdušia a vody, cez jednoduchšie uskutočnenie, ako sú detekčné rúrky (LEICHNITZ,K.: Prufŕohrchen-Taschenbuch. 5. Ausgabe, Drägenwerk AG, Lubeck 1982, s. 131), až po jednoduchšie, ale o to viac používané detekčné papiere (WP 85/04423, čs. autorské osvedčenie č. 2701).For the detection and assay of cholinesterase inhibitors (ΑΜΜΟΝ R.-VOSS G., Pflugers Árch. Ges. Physiol., 235 (1935), p. 393; MICHEZ O., H., J., Zab clin. Med., 34 (1949), p. 1564), the biochemical cholinesterase reaction is widely used in a number of modifications and applications as a very sensitive and selective method. Different types of technical means for the detection and determination of cholinesterase inhibitors are based on the cholinesterase reaction, starting with complicated automatic air and water analyzers, through simpler design, such as detection tubes (LEICHNITZ, K .: Prufrohrchen-Taschenbuch. 5. Ausgabe, Drägenwerk AG, Lubeck 1982, p. 131), up to the simpler but more used detection papers (WP 85/04423, Czech author's certificate No. 2701).
Dosiaľ známe typy rúrok založených na biochemickej cholínesterázovej reakcii majú bez ohľadu na konštrukčné riešenie podobnú citlivosť a selektivitu.The known types of tubes based on the biochemical cholinesterase reaction have a similar sensitivity and selectivity regardless of the design.
Komerčne dostupné detekčné rúrky sú konštrukčne zatavené sklené rúrky, obsahujúce oddelené vrstvy inertných nosičov a sorbentov s činidlami a ampulkami s kvapalnými činidlami, potrebnými na biochemickú enzýmovú reakciu. Bežne majú rúrky sorpčnú vrstvu, najčastejšie silikagélovú, na ktorej sa pri nasávaní vzduchu rúrkou sorbujú inhibitory. Potom sa na túto vrstvu uloží enzým a po inkubácii enzýmu s inhibítorom substrát alebo postupným rozbitím ampuliek s roztokmi činidiel, alebo spláchnutím činidiel z inertných nosičov rozpustením suchých činidiel po rozbití ampulky s tlmiacim roztokom. Napriek komplikovanému uskutočneniu je tento biochemický reakčný princíp jediný, ktorý je dostatočne citlivý na prítomnosť extrémne toxických inhibítorov cholínesteráz v ovzduší a stále používaný na konštrukciu jednoduchých technických prostriedkov. Najznámejším riešením rúrky na určenie inhibitorov cholínesteráz vo vzduchu je biochemická rúrka používaná v armáde a civilnej obrane ČR a SR. Rúrka obsahuje ampulku s pufrovaným roztokom butyrylcholínesterázy a ampulku s roztokom substrátu butyrylcholínjodidu a acidobázického indikátora fenolovej červene. Po rozbití ampulky s enzýmom a nasatím analyzovaného vzduchu cez vrstvu drveného skla namočeného roztokom butyrylcholínesterázy a po rozbití ampulky s roztokom substrátu a acidobázickým indikátorom sa vizuálne porovná zmena zafarbenia náplne skúšobnej a porovnávacej dôkazovej rúrky. Nevýhodou takýchto rúrok je krátky čas exspirácie následkom toho, že enzým aj substrát sú vo vodnom roztoku a tiež principiálna závislosť detekcie od kyslých a bázických látok zachytených z analyzovaného vzduchu v rúrke. Výhodnejším riešením biochemickej rúrky s dobrou stabilitou pri skladovaní je rúrka podľa NL 1568383. Rúrka obsahuje jednu ampulku s tlmiacim roztokom a lyofilizované preparáty enzýmu a chromogénneho substrátu s pridaním látok urýchľujúcich rozpúšťanie činidiel v tlmiacom roztoku a indikačnú zónu, na ktorej sa sorbujú inhibitory. Po otvorení rúrky a nasatím vzduchu sa rozbitím ampulky s pufrom obidva rozpustné preparáty, enzým aj chromogénny substrát, umyjú na indikačnú vrstvu so sorbovanými inhibítormi a vyhodnotí sa zafarbenie tejto vrstvy po vykonaní enzýmovej reakcie.Commercially available detection tubes are structurally sealed glass tubes containing separate layers of inert carriers and sorbents with reagents and ampoules of liquid reagents required for the biochemical enzyme reaction. Typically, the tubes have a sorption layer, most often silica gel, on which inhibitors are absorbed as air is drawn through the tube. The enzyme is then deposited on this layer and after incubation of the enzyme with the inhibitor substrate or sequentially breaking the vials with reagent solutions, or rinsing the reagents from inert carriers by dissolving the dry reagents after breaking the vial with the buffer solution. Despite a complicated embodiment, this biochemical reaction principle is the only one that is sufficiently sensitive to the presence of extremely toxic cholinesterase inhibitors in the air and still used to construct simple technical means. The best known solution for the determination of cholinesterase inhibitors in the air is a biochemical tube used in the military and civil defense of the Czech Republic and Slovakia. The tube contains an ampoule with a buffered butyrylcholine esterase solution and an ampoule with a butyrylcholine iodide substrate solution and a phenol red acid-base indicator. After breaking the enzyme ampoule and aspirating the analyzed air through a layer of crushed glass soaked with butyrylcholinesterase solution and breaking the ampoule with substrate solution and acid-base indicator, the color change of the test and comparative proof tube contents is visually compared. The disadvantage of such tubes is the short expiration time due to both the enzyme and the substrate being in aqueous solution, as well as the principal dependence of detection on acidic and basic substances trapped from the air analyzed in the tube. A more preferred solution of a biochemical tube with good storage stability is a tube according to NL 1568383. The tube comprises a single ampoule of buffer and lyophilized enzyme and chromogenic substrate preparations with addition of agents to dissolve the reagents in the buffer and an indicator zone to which the inhibitors are absorbed. After opening the tube and aspirating the air, by breaking the ampoule with buffer, both the soluble preparations, the enzyme and the chromogenic substrate, are washed on the indicator layer with sorbed inhibitors and the color of this layer is evaluated after the enzyme reaction has been carried out.
Používané rúrky neumožňujú analýzu vody a vodných roztokov.The tubes used do not allow the analysis of water and aqueous solutions.
Uvedené nedostatky, najmä nízka životnosť rúrok, rušivý vplyv kyslých a bázických látok, nižšia citlivosť, nemožnosť analýzy vody pri vysokých ekonomických nákladoch na výrobu sú odstránené detekčnou rúrkou inhibitorov cholínesteráz vo vzduchu a vo vode podľa vynálezu.These drawbacks, in particular the low tube life, the disruption of acidic and basic substances, the lower sensitivity, the impossibility of water analysis at high economic production costs are eliminated by the detection tube of cholinesterase inhibitors in the air and water of the invention.
Podstata vynálezuSUMMARY OF THE INVENTION
Podstatou vynálezu je detekčná rúrka inhibitorov cholínesteráz vo vzduchu a vo vode, ktorá obsahuje dve ampulky s tlmiacim roztokom pH 6,0 až 9,0, porovnávaciu vrstvu z rozdrveného skla impregnovanou zmesou substrátu a chromogénneho činidla alebo chromogénnym substrátom a indikačnú vrstvu z granulovanej bielenej celulózy s imobilizovanou cholínesterázou. Výhodne porovnávacia vrstva z rozdrveného skla obsahuje ako substrát acetyltiocholín alebo butyryltiocholín a ako chromogénne činidlo kyselinu 5,5-ditio-bis(2-nitrobenzoovú). Alebo výhodne porovnávacia vrstva obsahuje ako substrát acetyltiocholín alebo butyryltiocholín a ako chromogénne činidlo N-etyl-N-[4[4-etyl[3-sulfofenyl)metyl]amino]fenyl]fenylmetylén]-2,5-cyklohexadién]-l-ylidén]-3-sulfobenzén-metán aminiumnátrium hydroxid-vnútomá soľ. Alebo výhodne porovnávacia vrstva obsahuje chromogénny substrát, l-[tiazolylazo]-2-acetoxy-5-metylbenzén dimetylsulfát.The present invention provides a detection tube for cholinesterase inhibitors in air and water comprising two ampoules with buffer pH 6.0 to 9.0, a crushed glass comparative layer impregnated with a substrate / chromogenic reagent or chromogenic substrate, and a granulated bleached cellulose indicator layer with immobilized cholinesterase. Preferably, the comminuted glass layer comprises acetylthiocholine or butyrylthiocholine as the substrate and 5,5-dithiobis (2-nitrobenzoic acid) as the chromogenic agent. Or preferably, the comparison layer comprises as substrate substrate acetylthiocholine or butyrylthiocholine and as chromogenic agent N-ethyl-N- [4- [4-ethyl [3-sulfophenyl) methyl] amino] phenyl] phenylmethylene] -2,5-cyclohexadiene] -1-ylidene ] -3-Sulfobenzene-methane-ammonium hydroxide-inner salt. Or preferably, the comparison layer comprises a chromogenic substrate, 1- [thiazolylazo] -2-acetoxy-5-methylbenzene dimethyl sulfate.
Indikačná vrstva výhodne obsahuje hovädziu mozgovú acetylcholínesterázu alebo plazmovú konskú butyrylcholínesterázu. Použitie rúrky spočíva v nasávaní vzduchu obsahujúceho pary alebo aerosól inhibitorov cholínesteráz indikačnou vrstvou navlhčenou tlmiacim roztokom rozbitím ampulky alebo v namočení indikačnej vrstvy analyzovanou vodou. Po skončení nasávania alebo po vybratí z vody a po inkubácii imobilizovaného enzýmu na indikačnej vrstve so sorbovaným inhibítorom sa na indikačnú vrstvu zmyje z porovnávacej vrstvy substrát a chromogénne činidlo alebo chromogénny substrát rozbitím ampulky s tlmiacim roztokom a porovná sa zafarbenie indikačnej a porovnávacej vrstvy.The indicator layer preferably comprises bovine brain acetylcholine esterase or plasma equine butyrylcholine esterase. The use of the tube consists in sucking in air containing vapor or aerosol of cholinesterase inhibitors by an indicator layer moistened with buffer by breaking the vial or by soaking the indicator layer with the water to be analyzed. Upon completion of aspiration or withdrawal from water and after incubation of the immobilized enzyme on the indicator layer with the sorbed inhibitor, the indicator and chromogenic reagent or chromogenic substrate is washed onto the indicator layer from the reference layer by breaking the vial with buffer and comparing the color of the indicator and reference layers.
Rúrka podľa vynálezu má proti dosiaľ používaným rúrkam vyššiu citlivosť pri dôkaze inhibitorov cholínesterázy v dôsledku použitia špecifického enzýmu a substrátu a možnosti ďalšieho zvýšenia citlivosti predĺžením inkubácie enzýmu s inhibítorom, dlhšiu expiráciu a umožňuje dôkaz inhibitorov aj vo vode a vodných extraktoch zo vzoriek vo veľkom rozpätí pH a porovnaním rýchlosti vyfarbovania indikačnej a porovnávacej vrstvy umožňuje semikvantitatívne vyhodnotenie koncentrácie inhibítora.The tube according to the invention has a higher sensitivity to the hitherto used tubes in the detection of cholinesterase inhibitors due to the use of a specific enzyme and substrate and the possibility of further increasing the sensitivity by prolonging the enzyme incubation with the inhibitor, longer exposure and allows detection of inhibitors also in water and aqueous sample extracts over a large pH range and by comparing the staining rate of the indicator and reference layers, it allows semi-quantitative evaluation of the inhibitor concentration.
Prehľad obrázkov na výkresochBRIEF DESCRIPTION OF THE DRAWINGS
Na pripojenom výkrese je znázornený model rúrky.The attached drawing shows a tube model.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Rúrka podľa obrázku 1 pozostáva z dvoch ampuliek s tlmiacim roztokom 1 a 2, z porovnávacej vrstvy 3 impregnovanej substrátom a chromogénnym činidlom alebo chromogénnym substrátom, z indikačnej vrstvy 4 s imobilizovanou cholínesterázou. Vrstvy sú od seba oddelené a v rúrke utesnené polyetylénovými hviezdičkami 5, hviezdič2 ky 5 sú od vrstiev oddelené sieťkami z polyamidovej tkaniny 6.The tube of Figure 1 consists of two vials of buffer 1 and 2, a comparator layer 3 impregnated with a substrate and a chromogenic reagent or a chromogenic substrate, an indicator layer 4 with immobilized cholinesterase. The layers are separated from each other and sealed in the pipe with polyethylene stars 5, the stars 5 being separated from the layers by nets of polyamide fabric 6.
Príklad 1Example 1
Rúrka podľa obrázku 1 obsahuje 2 ampulky s fosfátovým tlmiacim roztokom pH 8,5, porovnávacia vrstva je 0,1 g žltej sklenej drviny, (kmax. 412nm) zrnitosti 1,0 až 1,2 mm impregnovanej etanolovým roztokom obsahujúcim 1 % acetyltiocholínjodidu a 0,3 % kyseliny 5,5'-dítio-bis(2-dinitrobenzoovej). Indikačná vrstva je 0,05 g bielej granulovanej celulózy zrnitosti 0,8 až 1,2 mm s imobilizovanou acetylcholínesterázou so špecifickou aktivitou 1,5 nkat na 1 mg celulózy. Po odlomení koncov rúrky a rozbití ampulky pod indikačnou vrstvou tak, aby roztok z ampulky namočil indikačnú vrstvu, sa rúrkou nasáva analyzovaný vzduch. Po ukončení nasávania sa rozbije ampulka s tlmiacim roztokom nad porovnávacou vrstvou a roztok sa pretrepe na indikačnú vrstvu. Za prítomnosti inhibítorov v analyzovanom vzduchu zostane indikačná vrstva biela, neinhibovaná indikačná vrstva sa zafarbí do 1 minúty nažlto. Pri nasávaní 1,4 1 vzduchu za minútu v priebehu 2 minút je možné zistiť 5.10'7 mg . ľ1 izopropylmetylfosfonofluoridatu, 3.10‘7 mg . ľ1 pinakolylmetylfosfonofluoridatu, 2.10'7 mg.ľ1 0-etyl-S-(2-diizopropylaminoetyl)metyltiofosfonatu a 1.107 mg . ľ1 2-imetylaminoetyldimetylamídofosfofluoridatu.The tube of Figure 1 contains 2 ampoules with phosphate buffer pH 8.5, the reference layer is 0.1 g of yellow glass chips (kmax. 412nm) of 1.0 to 1.2 mm grain size impregnated with ethanol solution containing 1% acetylthiocholine iodide and 0 3% 5,5'-dithio-bis (2-dinitrobenzoic acid). The indicator layer is 0.05 g of white granular cellulose of a grain size of 0.8 to 1.2 mm with immobilized acetylcholinesterase with a specific activity of 1.5 nkat per 1 mg of cellulose. After breaking the ends of the tube and breaking the vial under the indicator layer so that the solution from the vial soaks the indicator layer, the analyzed air is sucked through the tube. After completion of aspiration, the buffer vial is broken above the reference layer and the solution is shaken to the indicator layer. In the presence of inhibitors in the analyzed air, the indicator layer remains white, the uninhibited indicator layer turns yellow within 1 minute. Intake 1.4 1 of air per minute for 2 minutes can be determined 5:10 '7 mg. 1 izopropylmetylfosfonofluoridatu ', 10.3' 7 mg. '1 pinakolylmetylfosfonofluoridatu, 2.10 "7 1 0 mg · l-ethyl-S- (2-diisopropylamino) metyltiofosfonatu 7 and 1.10 mg. L 1 2-imetylaminoetyldimetylamídofosfofluoridatu.
Príklad 2Example 2
Rúrka podľa obrázka 1 obsahuje 2 ampulky s fosfátovým tlmiacim roztokom pH 7,6, porovnávacia vrstva je 0,1 g modrej sklenej drviny (Tmax 580nm) zrnitosti 1,0 ažThe tube of Figure 1 contains 2 ampoules of phosphate buffer pH 7.6, the reference layer being 0.1 g of blue glass pulp (Tmax 580nm) of 1.0 to 0.2 grit size.
1,2 mm impregnovanej lyofilizáciou s etanolovým roztokom obsahujúcim 0,6 % l-[2-tiazolylazo]-2-acetoxy-5-metylbenzén dimetylsulfátu. Indikačná vrstva je 0,05 g bielenej granulovanej celulózy zrnitosti 1 mm s imobilizovanou butyrylcholínesterázou so špecifickou aktivitou 3 nkat na 1 mg celulózy. Po odlomení koncov rúrky sa indikačná vrstva rúrky na 1 minútu ponorí do analyzovaného vodného roztoku, po vybratí a 30 minútach inkubácie sa rozbije ampulka s tlmiacim roztokom nad porovnávacou vrstvou a roztok sa pretrepe na indikačnú vrstvu. Východiskové žlté zafarbenie indikačnej vrstvy za prítomnosti inhibítorov v analyzovanom vodnom roztoku zostane bledožlté. Pri neprítomnosti inhibítorov vo vode indikačná vrstva do 2 minút zmodrie rovnako ako porovnávacia vrstva. Po 30 minútach inkubácie je možné zistiť 0, 08 mg . ľ1 dichlórvosu, 0,4 mg. ľ1 posdrinu, 0,04 mg. ľ1 carbofuranu, 0,3 mg.ľ1 aldicarbu.1.2 mm impregnated by lyophilization with an ethanol solution containing 0.6% 1- [2-thiazolylazo] -2-acetoxy-5-methylbenzene dimethyl sulfate. The indicator layer is 0.05 g of bleached granulated cellulose of 1 mm grain size with immobilized butyrylcholine esterase having a specific activity of 3 nkat per 1 mg of cellulose. After breaking the tube ends, the tube indicator layer is immersed in the analyzed aqueous solution for 1 minute, after removal and 30 minutes of incubation, the vial with buffer above the reference layer is broken and the solution is shaken to the indicator layer. The starting yellow color of the indicator layer in the presence of inhibitors in the aqueous solution analyzed remains pale yellow. In the absence of inhibitors in water, the indicator layer turns blue within 2 minutes as well as the reference layer. After 30 minutes of incubation, 0.08 mg can be detected. dichlorvos I '1, 0.4 mg. 11 posrine, 0.04 mg. I '1 carbofuran, aldicarb 0.3 mg.l 1.
2. Detekčná rúrka inhibítorov cholínesteráz vo vzduchu a vo vode podľa nároku 1, vyznačujúca sa t ý m , že porovnávacia vrstva z rozdrveného skla obsahuje ako substrát acetyltíocholín alebo butyryltiocholín a ako chromogénne činidlo kyselinu 5,5'-ditio-bis(2-nitrobenzoovú).2. The air and water cholinesterase inhibitor detection tube of claim 1, wherein the crushed glass comparative layer comprises acetylthioquinoline or butyrylthiocholine as the substrate and 5,5'-dithiobis (2-nitrobenzoic acid) as the chromogenic agent. ).
3. Detekčná rúrka inhibítorov cholínesteráz vo vzduchu a vo vode podľa nároku 1, vyznačujúca sa t ý m , že porovnávacia vrstva obsahuje ako substrát acetyltiocholín alebo butyryltiocholín a ako chromogénne činidlo N-etyl-N-[4-[4[etyl[3-sulfofenyl)metyl]amino]fenyl]fenylmetylén]-2,5-cyklohehexadién]-l-ylidén]-3-sulfo-benzénmetán amíniumnátrium hydroxid-vnútomá soľ.3. The air and water cholinesterase inhibitor detection tube of claim 1, wherein the comparison layer comprises acetylthiocholine or butyrylthiocholine as the substrate and N-ethyl-N- [4- [4 [ethyl [3-] [3] as the chromogenic agent. sulfophenyl) methyl] amino] phenyl] phenylmethylene] -2,5-cyclohehexadiene] -1-ylidene] -3-sulfobenzenemethanamine sodium hydroxide inner salt.
4. Detekčná rúrka inhibítorov cholínesteráz vo vzduchu a vo vode podľa nároku 1, vyznačujúca sa t ý m , že porovnávacia vrstva obsahuje chromogénny substrát 1 -[tiazolylazo]-2-acetoxy-5-metylbenzén dimetylsulfát.4. The air and water inhibitor tube of cholinesterase inhibitors according to claim 1, wherein the comparison layer comprises a chromogenic substrate 1- [thiazolylazo] -2-acetoxy-5-methylbenzene dimethyl sulfate.
5. Detekčná rúrka inhibítorov cholínesteráz vo vzduchu a vo vode podľa nároku 1, vyznačujúca sa tým, že indikačná vrstva obsahuje hovädziu mozgovú acetylcholínestcrázu alebo plazmovú konskú butyrylcholínesterázu.The air and water cholinesterase inhibitor detection tube of claim 1, wherein the indicator layer comprises bovine brain acetylcholine esterase or plasma horse butyrylcholine esterase.
výkresdrawing
Priemyselná využiteľnosťIndustrial usability
Detekčná rúrka podľa vynálezu je použiteľná na účely chemického prieskumu a kontroly a pri monitorovaní životného prostredia, v chemickom a farmaceutickom priemysle, v poľnohospodárstve a v lesnom hospodárstve.The detection tube according to the invention is useful for chemical research and control purposes and for environmental monitoring, in the chemical and pharmaceutical industries, in agriculture and forestry.
Claims (1)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CZ931179A CZ285242B6 (en) | 1993-06-17 | 1993-06-17 | Detection tube of cholinesterase inhibitors in air and water |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| SK73694A3 SK73694A3 (en) | 1995-01-12 |
| SK281547B6 true SK281547B6 (en) | 2001-04-09 |
Family
ID=5462878
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SK736-94A SK281547B6 (en) | 1993-06-17 | 1994-06-15 | Detection tube of cholinesterase inhibitors in air and water |
Country Status (2)
| Country | Link |
|---|---|
| CZ (1) | CZ285242B6 (en) |
| SK (1) | SK281547B6 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL3196315T3 (en) | 2016-01-19 | 2020-07-13 | Oritest Spol. S R.O. | Spherical pellets, manufacturing process of such pellets, use, and a detection tube comprising such pellets |
| CZ2016311A3 (en) * | 2016-05-26 | 2017-07-12 | Oritest Spol. S R.O. | Coated spherical pellets, their production and adjustment in detection tubes for the detection of cholinesterase inhibitors |
-
1993
- 1993-06-17 CZ CZ931179A patent/CZ285242B6/en not_active IP Right Cessation
-
1994
- 1994-06-15 SK SK736-94A patent/SK281547B6/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| SK73694A3 (en) | 1995-01-12 |
| CZ285242B6 (en) | 1999-06-16 |
| CZ117993A3 (en) | 1995-10-18 |
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