ES2244310B1 - 3-INDOXYL PHOSPHATE AS SUBSTRATE OF PEROXIDASES. - Google Patents
3-INDOXYL PHOSPHATE AS SUBSTRATE OF PEROXIDASES. Download PDFInfo
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- ES2244310B1 ES2244310B1 ES200400260A ES200400260A ES2244310B1 ES 2244310 B1 ES2244310 B1 ES 2244310B1 ES 200400260 A ES200400260 A ES 200400260A ES 200400260 A ES200400260 A ES 200400260A ES 2244310 B1 ES2244310 B1 ES 2244310B1
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- peroxidases
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- 239000000758 substrate Substances 0.000 title claims abstract description 31
- 102000003992 Peroxidases Human genes 0.000 title claims abstract description 23
- 108700020962 Peroxidase Proteins 0.000 title claims abstract description 19
- JTNGEYANGCBZLK-UHFFFAOYSA-N 1h-indol-3-yl dihydrogen phosphate Chemical compound C1=CC=C2C(OP(O)(=O)O)=CNC2=C1 JTNGEYANGCBZLK-UHFFFAOYSA-N 0.000 title 1
- COHYTHOBJLSHDF-UHFFFAOYSA-N Indigo Chemical compound N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 claims abstract description 33
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 22
- 239000010452 phosphate Substances 0.000 claims abstract description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 238000011002 quantification Methods 0.000 claims abstract description 7
- 230000002255 enzymatic effect Effects 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 6
- KHLVKKOJDHCJMG-QDBORUFSSA-L indigo carmine Chemical compound [Na+].[Na+].N/1C2=CC=C(S([O-])(=O)=O)C=C2C(=O)C\1=C1/NC2=CC=C(S(=O)(=O)[O-])C=C2C1=O KHLVKKOJDHCJMG-QDBORUFSSA-L 0.000 claims description 6
- 229960003988 indigo carmine Drugs 0.000 claims description 6
- 235000012738 indigotine Nutrition 0.000 claims description 6
- 239000004179 indigotine Substances 0.000 claims description 6
- PCKPVGOLPKLUHR-UHFFFAOYSA-N indoxyl Chemical group C1=CC=C2C(O)=CNC2=C1 PCKPVGOLPKLUHR-UHFFFAOYSA-N 0.000 claims description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 abstract description 23
- 102000004190 Enzymes Human genes 0.000 abstract description 16
- 108090000790 Enzymes Proteins 0.000 abstract description 16
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 10
- 238000011161 development Methods 0.000 abstract description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 abstract description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract description 4
- 238000005063 solubilization Methods 0.000 abstract description 4
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- 238000004458 analytical method Methods 0.000 abstract description 2
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- 229940088598 enzyme Drugs 0.000 description 14
- 239000000047 product Substances 0.000 description 12
- 238000001514 detection method Methods 0.000 description 11
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- 235000000177 Indigofera tinctoria Nutrition 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
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- 238000006911 enzymatic reaction Methods 0.000 description 6
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 2
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 2
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- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 2
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
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- NOFNCLGCUJJPKU-UHFFFAOYSA-N 1,7-dimethyluric acid Chemical compound N1C(=O)N(C)C(=O)C2=C1NC(=O)N2C NOFNCLGCUJJPKU-UHFFFAOYSA-N 0.000 description 1
- UYOPTJREJWOZDO-UHFFFAOYSA-N 4,6-diethoxy-1h-1,3,5-triazin-2-one Chemical compound CCOC=1N=C(OCC)NC(=O)N=1 UYOPTJREJWOZDO-UHFFFAOYSA-N 0.000 description 1
- KWNBDPJHEKVDAW-UHFFFAOYSA-N 4-(4-chlorophenyl)-2-(4-methylphenyl)-4-oxobutanoic acid Chemical compound C1=CC(C)=CC=C1C(C(O)=O)CC(=O)C1=CC=C(Cl)C=C1 KWNBDPJHEKVDAW-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- WXNZTHHGJRFXKQ-UHFFFAOYSA-N 4-chlorophenol Chemical compound OC1=CC=C(Cl)C=C1 WXNZTHHGJRFXKQ-UHFFFAOYSA-N 0.000 description 1
- VSMDINRNYYEDRN-UHFFFAOYSA-N 4-iodophenol Chemical compound OC1=CC=C(I)C=C1 VSMDINRNYYEDRN-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241000147041 Guaiacum officinale Species 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical group [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 102100038609 Lactoperoxidase Human genes 0.000 description 1
- 108010023244 Lactoperoxidase Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
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- 239000012190 activator Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
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- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229940091561 guaiac Drugs 0.000 description 1
- 150000003278 haem Chemical group 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
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- 150000002475 indoles Chemical class 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 230000003647 oxidation Effects 0.000 description 1
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- VZPGINJWPPHRLS-UHFFFAOYSA-N phenazine-2,3-diamine Chemical compound C1=CC=C2N=C(C=C(C(N)=C3)N)C3=NC2=C1 VZPGINJWPPHRLS-UHFFFAOYSA-N 0.000 description 1
- 239000002530 phenolic antioxidant Substances 0.000 description 1
- 125000001484 phenothiazinyl group Chemical class C1(=CC=CC=2SC3=CC=CC=C3NC12)* 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
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- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
3-indoxil fosfato como sustrato de las peroxidasas. El 3-indoxil fosfato es oxidado en presencia de la peroxidasa de rábano silvestre (HRP) por el agua oxigenada (H2O2) a azul de índigo, heterociclo aromático insoluble en medio acuoso. A través de la cuantificación, previa solubilización o no, del azul de índigo, se logra medir la actividad de la peroxidasa. El hecho de que la fosfatasa alcalina y la HRP puedan utilizar el mismo sustrato enzimático (3-indoxil fosfato) y generar el mismo producto (azul de índigo) implica el empleo de la misma instrumentación analítica para la cuantificación de ambas enzimas. Esta invención resulta particularmente útil en la unificación de los reactivos e instrumentación necesaria para el desarrollo de ensayos ELISA, de aplicación al sector de los análisis clínicos.3-indoxy phosphate as a substrate for peroxidases. The 3-indoxy phosphate is oxidized in the presence of horseradish peroxidase (HRP) by hydrogen peroxide (H2O2) to indigo blue, aromatic heterocycle insoluble in aqueous medium. Through the quantification, prior solubilization or not, of indigo blue, the peroxidase activity is measured. The fact that alkaline phosphatase and HRP can use the same enzyme substrate (3-indoxy phosphate) and generate the same product (indigo blue) implies the use of the same analytical instrumentation for the quantification of both enzymes. This invention is particularly useful in the unification of the reagents and instrumentation necessary for the development of ELISA assays, applicable to the clinical analysis sector.
Description
3-Indoxil fosfato como sustrato de las peroxidasas.3-Indoxil phosphate as substrate of peroxidases.
Las peroxidasas son un grupo de enzimas que catalizan reacciones de reducción-oxidación por ello son clasificadas como oxidorreductasas.Peroxidases are a group of enzymes that catalyze reduction-oxidation reactions by They are classified as oxidoreductases.
En su modo general de actuación, las peroxidasas reducen peróxido de hidrógeno a agua mientras oxidan una gran variedad de sustratos. Son, por tanto, oxidorreductasas que utilizan peróxido de hidrógeno como aceptor electrónico para catalizar diferentes reacciones de oxidación. El proceso es el siguiente:In its general mode of action, peroxidases reduce hydrogen peroxide to water while oxidizing a large variety of substrates. They are, therefore, oxidoreductases that they use hydrogen peroxide as an electronic acceptor to catalyze different oxidation reactions. The process is the next:
Dador + H_{2}O_{2} \rightarrow Dador \ oxidado + 2 H_{2}OGiver + H_ {2} O_ {2} \ giver \ oxidized + 2 H2O
Múltiples tipos o especies de peroxidasas han sido estudiadas. Todas ellas tienen una estructura básica similar, pero en su conjunto presentan marcadas diferencias. Dentro del amplio grupo de las peroxidasas se encuentra la peroxidasa de rábano silvestre (HRP) (EC 1.11.1.7) (R.Huang, N.Hu, Bioelectrochemistry, 2001, 54, 75), glicoproteina con peso molecular aproximado de 44 kDa (A. Johannsson, Principles and practice of immunoassay, Ed. C.P.Price, D.J.Newman, Stockton Press, 1991, New York, pp. 300). En el centro activo de la HRP se encuentra ubicado un grupo hemo, donde el átomo de hierro está covalentemente unido a un átomo de nitrógeno de un resto de histidina.Multiple types or species of peroxidases have been studied. All of them have a similar basic structure, but as a whole they show marked differences. Within the large group of peroxidases is wild horseradish peroxidase (HRP) (EC 1.11.1.7) (R. Huang, N. Hu, Bioelectrochemistry , 2001, 54, 75), glycoprotein with approximate molecular weight of 44 kDa ( A. Johannsson, Principles and practice of immunoassay , Ed. CPPrice, DJNewman, Stockton Press, 1991, New York, pp. 300). A heme group is located in the active center of the HRP, where the iron atom is covalently bound to a nitrogen atom of a histidine residue.
Estudios previos han mostrado que la HRP actúa mediante un mecanismo que se desarrolla en tres etapas, del siguiente modo:Previous studies have shown that HRP acts through a mechanism that develops in three stages, of following mode:
1) formación del complejo I1) formation of complex I
HRP + H_{2}O_{2} \longleftrightarrow Complejo I HRP + H 2 O 2 \ longleftrightarrow Complex I
2) transición del complejo I al complejo II2) transition from complex I to complex II
Complejo I + AH \longleftrightarrow Complejo II + AI + AH complex \ Longleftrightarrow Complex II + TO
3) recuperación del enzima por reducción del complejo II3) enzyme recovery by reduction of complex II
Complejo II + AH \longleftrightarrow HRP + A + 2H_{2}OComplex II + AH \ longleftrightarrow HRP + A + 2H 2 O
donde AH es el dador en la forma reducida y A es el dador en la forma oxidada.where AH is the giver in the form reduced and A is the giver in the form oxidized
4) formación del producto4) product formation
2A \longleftrightarrowA_{2}2A \ longleftrightarrowA_ {2}
La especificidad de la HRP por el sustrato para formar el complejo I es elevada y sólo el H_{2}O_{2}, MeOOH y el EtOOH son capaces de combinarse con la HRP para generar dicho complejo I.The specificity of HRP for the substrate for forming complex I is high and only H 2 O 2, MeOOH and the EtOOH are able to combine with the HRP to generate said complex I.
Sin embargo la especificidad del enzima (en su forma complejo I y II) por los dadores representados por AH, en las ecuaciones 2 y 3 es bastante baja. Además, existe una gran variedad de compuestos que pueden actuar como dadores. Se han utilizado una amplia gama de sustratos para este enzima aunque se pueden destacar algunos de los siguientes: antioxidantes fenólicos (S.M.Khopde, K.I.Priyadarsini, Biophysical Chemistry, 2000, 88, 103), compuestos como la hidroquinona y el catecol (S.Cosnier, I.C.Popescu, Analytica Chimica Acta, 1996, 319, 145), el ácido 1,7-dimetilúrico (R.N.Goyal, A.K.Jain, N.Jain, J.Chem.Soc, 1996, 2, 1154), ácido 1-metilúrico (R.N.Goyal, M.S.Verma, N.Kumar, Bioelectrochem.Bioenerg, 1997, 43, 205), p-benzoquinona (E.Casero, M.Darder, F.Pariente, E.Lorenzo, Analytica Chimica Acta, 2000, 403, 1), compuestos de la familia de los indoles (T.Hu, G.Dryhurst, J.Electroanalytical Chemistry, 1997, 432, 7), de las fenotiazinas (S.S.Razola, E.Aktas, J-C.Viré, J-M.Kauffmann, Analyst, 2000, 125, 79) y una multitud de variados compuestos orgánicos.However, the specificity of the enzyme (in its complex form I and II) by the donors represented by AH, in equations 2 and 3 is quite low. In addition, there is a wide variety of compounds that can act as donors. A wide range of substrates have been used for this enzyme although some of the following can be highlighted: phenolic antioxidants (SMKhopde, KIPriyadarsini, Biophysical Chemistry , 2000, 88, 103), compounds such as hydroquinone and catechol (S.Cosnier, ICPopescu , Analytica Chimica Acta , 1996, 319, 145), 1,7-dimethyluric acid (RNGoyal, AKJain, N.Jain, J.Chem.Soc , 1996, 2, 1154), 1-methyluric acid (RNGoyal, MSVerma, N. Kumar, Bioelectrochem Bioenerg, 1997, 43, 205), p-benzoquinone (E. Casero, M. Darder, F. Pariente, E. Lorenzo, Analytica Chimica Acta , 2000, 403, 1), family compounds of the indoles (T. Hu, G. Dryhurst, J. Electroanalytical Chemistry , 1997, 432, 7), of the phenothiazines (SSRazola, E. Aktas, JC.Viré, JM Kaufmann, Analyst , 2000, 125, 79) and a multitude of varied organic compounds.
Una de las principales utilidades de la HRP es
su capacidad para unirse como agente de marcaje a anticuerpos o
hebras de ADN. De este modo y con la ayuda de algún sustrato
adecuado se podrá seguir la reacción inmunológica o de hibridación
en toda su extensión. Idealmente para el desarrollo de
inmunoensayos con detección electroquímica se requieren sustratos
no electroactivos que se transformen en productos electroactivos,
que se oxiden o reduzcan a potenciales adecuados. En el caso de que
sustrato y producto sean electroactivos, deben serlo a diferentes
potenciales para evitar interferencias. Además, para analizar
muestras biológicas es preferible utilizar potenciales inferiores a
+0.5 V debido a la posible interferencia de sustancias
electroactivas que se encuentren en la
misma.One of the main utilities of HRP is its ability to bind as a labeling agent to antibodies or strands of DNA. In this way and with the help of some suitable substrate, the immunological or hybridization reaction can be followed in its entirety. Ideally, for the development of immunoassays with electrochemical detection, non-electroactive substrates are required that are transformed into electroactive products, which are oxidized or reduced to suitable potentials. In the event that the substrate and product are electroactive, they must be at different potentials to avoid interference. In addition, to analyze biological samples it is preferable to use potentials below +0.5 V due to the possible interference of electroactive substances found in the
same.
Entre los sustratos de la HRP que suelen ser utilizados para el revelado de ensayos de tipo ELISA se puede mencionar a la o- fenilendiamina (OPD) cuya oxidación es catalizada por la HRP generando 2,3-diaminofenazina. Se puede seguir el desarrollo de la reacción cuantificando el producto coloreado a 405 nm. El guayacol incoloro es también utilizado. Se genera tetraguayacol que es de color marrón y que se detecta a 470 nm.Among the HRP substrates that are usually used for the development of ELISA type tests can be mention o-phenylenediamine (OPD) whose oxidation is catalyzed by HRP generating 2,3-diaminophenazine. It can follow the development of the reaction by quantifying the product colored at 405 nm. Colorless guaiac is also used. Be generates tetraguayacol that is brown and is detected at 470 nm.
Finalmente otros sustratos que se emplean para revelar esta marca pueden ser: tetrametilbenzidina (TMB), o-dianisidina (ODA), 2,2'-azinobis-3-etilbenzotiazolin-6-sulfonato de diamonio (ABTS), 3,3'-diaminobenzidina (DAB).Finally other substrates that are used to Reveal this brand can be: tetramethylbenzidine (TMB), o-dianisidine (ODA), 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonate of diamonium (ABTS), 3,3'-diaminobenzidine (DAB)
En la presente invención se utiliza el 3-indoxil fosfato (3-IP) como sustrato de las peroxidasas, y en concreto de la HRP. Este compuesto aromático ha sido utilizado como sustrato electroquímico de la fosfatasa alcalina por nuestro grupo de investigación, llevando a cabo la detección electroquímica del producto enzimático generado, el azul de índigo (C.F. Sánchez, A. Costa García, Electroanalysis, 1998, 10, 249; C.F. Sánchez, M. B. González García, A. Costa García, Biosensors & Bioelectronics, 2000, 14, 917).In the present invention, 3-indoxy phosphate (3-IP) is used as a substrate for peroxidases, and in particular HRP. This aromatic compound has been used as an electrochemical substrate of alkaline phosphatase by our research group, carrying out the electrochemical detection of the generated enzymatic product, indigo blue (CF Sánchez, A. Costa García, Electroanalysis , 1998, 10, 249 ; CF Sánchez, MB González García, A. Costa García, Biosensors & Bioelectronics , 2000, 14, 917).
Al añadir ácido sulfúrico concentrado sobre el azul de índigo, heterociclo aromático insoluble en medios acuosos, se consigue un proceso de sulfonación que conlleva la generación de índigo carmín. El comportamiento electroquímico de esta molécula ya ha sido estudiado (M.D. González, C.F. Sánchez, A. Costa García, Electroanalysis, 2002, 14, 665) con anterioridad por nuestro grupo de investigación sobre dos tipos diferentes de electrodos serigrafiados comerciales, haciendo uso de varias técnicas voltamperométricas. Si se hace un barrido cíclico entre -0.4V y +0.1V de índigo carmín (generado enzimáticamente) en ácido sulfúrico 2.5 M, se observará una señal reversible a -0.15V (vs pseudo-referencia Ag).By adding concentrated sulfuric acid on indigo blue, aromatic heterocycle insoluble in aqueous media, a sulfonation process is achieved that leads to the generation of indigo carmine. The electrochemical behavior of this molecule has already been studied (MD González, CF Sánchez, A. Costa García, Electroanalysis , 2002, 14, 665) previously by our research group on two different types of commercial screen-printed electrodes, making use of several voltammetric techniques. If a cyclic scan is made between -0.4V and + 0.1V of carmine indigo (enzymatically generated) in 2.5M sulfuric acid, a reversible signal will be observed at -0.15V (vs pseudo-reference Ag).
La presente invención es de aplicación a la determinación de la actividad de enzimas del grupo de las peroxidasas. Se basa en el empleo del 3-indoxil fosfato como sustrato de las peroxidasas y más concretamente como sustrato de la peroxidasa de rábano silvestre. Este sustrato o algún otro derivado suyo como el 5-bromo-4-cloro-3-indoxil fosfato son utilizados como tales con enzimas del grupo de las fosfatasas y más concretamente con la fosfatasa alcalina. Esta invención presenta, el primer sustrato válido para la peroxidasa de rábano silvestre y para la fosfatasa alcalina, principales enzimas utilizadas como "marcas" en análisis clínicos. Esta invención resulta, por tanto, particularmente útil en la unificación de la instrumentación y reactivos necesarios para el desarrollo de ensayos ELISA.The present invention is applicable to the determination of the enzyme activity of the group of peroxidases It is based on the use of 3-indoxil phosphate as a substrate for peroxidases and more specifically as Wild radish peroxidase substrate. This substrate or some other derivative of his like the 5-bromo-4-chloro-3-indoxil phosphate are used as such with enzymes of the group of phosphatases and more specifically with alkaline phosphatase. This invention presents, the first valid substrate for peroxidase of wild radish and for alkaline phosphatase, main enzymes used as "brands" in clinical analysis. This invention It is therefore particularly useful in the unification of instrumentation and reagents necessary for the development of ELISA tests.
La presente invención emplea el 3-indoxil fosfato (3-IP) como sustrato de las peroxidasas en presencia de peróxido de hidrógeno. El producto enzimático insoluble en disolución acuosa es el azul de índigo.The present invention employs the 3-indoxy phosphate (3-IP) as Peroxidases substrate in the presence of hydrogen peroxide. The enzyme product insoluble in aqueous solution is the blue of indigo.
El 3-indoxil fosfato puede ser utilizado como sustrato tanto en forma de sal disódica preferentemente, como de sal di-p-toluidínica. Otros derivados indoxílicos como el 5-bromo-4-cloro-3-indoxil fosfato (BCIP) pueden ser utilizados, en su forma de sal disódica, dipotásica o di-p toluidínica, como sustratos de la peroxidasa. El BCIP puede ser adicionado al medio de reacción en cualquier formato líquido comercial. El 5-bromo-6-cloro-3-indoxil fosfato también puede ser utilizado como sustrato de las peroxidasas, así como cualquier otro derivado indoxílico que genere un compuesto de la familia del índigo como producto de la reacción enzimática.3-indoxy phosphate can be used as a substrate both in the form of disodium salt preferably, as salt di-p-toluidine. Other derivatives indoxyl as the 5-bromo-4-chloro-3-indoxil Phosphate (BCIP) can be used, in its disodium salt form, dipotassium or di-p toluidine, as substrates of the peroxidase The BCIP can be added to the reaction medium in Any commercial liquid format. He 5-Bromo-6-Chloro-3-Indoxyl Phosphate can also be used as a substrate for peroxidases, as well as any other indoxyl derivative that generates a compound of the indigo family as a reaction product enzymatic
La catálisis enzimática tiene lugar en presencia de peróxido de hidrógeno preferentemente, aunque otros peróxidos (o precursores suyos) como el peróxido de hidrógeno de urea pueden ser también empleados. Estos reactivos peróxidos son añadidos al medio de la reacción en forma líquida o sólida.Enzymatic catalysis takes place in the presence of hydrogen peroxide preferably, although other peroxides (or his precursors) such as urea hydrogen peroxide can be also employees. These peroxide reagents are added to the medium. of the reaction in liquid or solid form.
El enzima que cataliza la reacción ha de pertenecer a la familia de las peroxidasas, independientemente de su origen. De forma preferente se utiliza la peroxidasa de rábano silvestre así como la lactoperoxidasa. El enzima puede estar libre en disolución o actuando como agente de marcaje de reactivos inmunológicos, hebras de ADN o cualquier otro reactivo que intervenga en ensayos de afinidad.The enzyme that catalyzes the reaction must belong to the peroxidases family, regardless of its origin. Preferably, horseradish peroxidase is used wild as well as lactoperoxidase. The enzyme may be free. in solution or acting as reagent labeling agent immunological, strands of DNA or any other reagent that Intervene in affinity trials.
El producto de la catálisis enzimática es de la familia de los indigoides. Su detección se puede realizar en base a su insolubilidad en medio acuoso, preferentemente con técnicas con fundamento gravimétrico, electroquímico u óptico-espectroscópico. La detección a través de estas técnicas también se realiza previa solubilización del producto enzimático. La solubilización del índigo tiene lugar con ácidos concentrados o con disolventes orgánicos polares o apolares. Otra forma de solubilizarlo es a través de su sulfonación con la adición de ácido sulfúrico concentrado, de este modo se genera un derivado suyo denominado índigo carmín.The product of enzymatic catalysis is of the Indigoid family. Its detection can be done based on its insolubility in aqueous medium, preferably with techniques with gravimetric, electrochemical or optical-spectroscopic. The detection through These techniques are also performed after solubilization of the enzymatic product Indigo solubilization takes place with concentrated acids or with polar or nonpolar organic solvents. Another way to solubilize it is through its sulfonation with the addition of concentrated sulfuric acid, thus generating a derived from him called indigo carmine.
La reacción enzimática tiene lugar en medio acuoso y preferentemente en disolución reguladora a pH ligeramente ácido. Las disoluciones reguladoras más empleadas son de citrato y de acetato.The enzymatic reaction takes place in between aqueous and preferably in a slightly pH regulating solution acid. The most commonly used regulatory solutions are citrate and of acetate.
La reacción enzimática se lleva a cabo preferentemente a una temperatura de 37ºC o a temperatura ambiente. La catálisis está favorecida por la presencia de un pequeño porcentaje (0-1%) de alguna proteína inerte como la seroalbúmina bovina.The enzymatic reaction is carried out. preferably at a temperature of 37 ° C or at room temperature. Catalysis is favored by the presence of a small percentage (0-1%) of some inert protein such as bovine serum albumin.
La concentración de este reactivo así como de todos los anterior y posteriormente mencionados han de ser ajustados en cada ensayo particular. Activadores de la reacción enzimática como detergentes no fónicos (Tween 20, Triton X-100) o compuestos halogenados (p-iodofenol, p-clorofenol) pueden ser también adicionados al medio de reacción.The concentration of this reagent as well as of all of the above and later mentioned must be adjusted in each particular essay. Reaction Activators enzymatic as non-phonic detergents (Tween 20, Triton X-100) or halogenated compounds (p-iodophenol, p-chlorophenol) can also be added to the reaction medium.
El 3-indoxil fosfato o sus derivados y el producto enzimático, indigo, no son agentes carcinógenos ni mutagénicos como sucede con alguno de los sustratos comúnmente utilizados con la HRP.3-indoxy phosphate or its derivatives and the enzymatic product, indigo, are not agents carcinogens or mutagenic as with any of the substrates commonly used with HRP.
Ejemplo de realización de la invenciónExample of realization of the invention
La eficacia del procedimiento se ilustra adicionalmente mediante el siguiente ejemplo que no pretende ser limitativo de su alcance. Este ejemplo muestra el uso del 3-indoxil fosfato como sustrato para la medida de la actividad de la peroxidasa de rábano silvestre en disolución, caracterizado por la generación de azul de índigo como producto de la reacción enzimática. La detección se lleva a cabo a través de la generación de índigo carmín, tras la sulfonación del azul de índigo con ácido sulfúrico concentrado. El índigo carmín soluble en disolución acuosa es detectado electroquímicamente sobre electrodos de carbono.The effectiveness of the procedure is illustrated additionally by the following example that is not intended to be limiting its scope. This example shows the use of 3-indoxy phosphate as a substrate for the measurement of the horseradish peroxidase activity in solution, characterized by the generation of indigo blue as a product of The enzymatic reaction. The detection is carried out through the generation of indigo carmine, after the sulfonation of indigo blue with concentrated sulfuric acid. The indigo carmine soluble in aqueous solution is electrochemically detected on electrodes carbon
La metodología empleada consta de las siguientes etapas:The methodology used consists of the following stages:
- Bloqueo de la superficie del pocillo con albúmina. Para ello se añaden a cada uno de los pocillos 200 \muL de una disolución reguladora PBS 10 mM de pH 7.4 (8 g/L de NaCl, 2 g/L de KCI) con un 3% de albúmina de suero bovino (BSA) y se deja descansar toda la noche a 4ºC. De este modo se evita la posible adsorción del enzima al pocillo asegurándose que todos los centros activos del mismo están disponibles.- Well surface block with albumin. For this, 200 µL are added to each well of a 10 mM PBS regulatory solution of pH 7.4 (8 g / L NaCl, 2 g / L of KCI) with 3% bovine serum albumin (BSA) and left rest overnight at 4 ° C. This avoids the possible adsorption of the enzyme to the well making sure that all the centers assets thereof are available.
- Etapa de lavado. Los pocillos se vacían de su contenido y se lavan 3-4 veces con una disolución reguladora correspondiente al mismo medio en el que se realizará la siguiente etapa.- Washing stage. The wells are emptied of their content and washed 3-4 times with a solution regulator corresponding to the same medium in which the next stage
- Desarrollo de la reacción enzimática. Se mezclan 50 \muL de una disolución de 3-indoxil fosfato 12 mM (preparada en disolución reguladora de acetato 0.1 M de pH 5.0), 50 \muL de una disolución acuosa de H_{2}O_{2} 2\cdot10^{-4} M y 8 \muL de una disolución acuosa de HRP de la concentración deseada. La temperatura y el tiempo de reacción son 47ºC y 30 minutos respectivamente. La reacción enzimática se lleva a cabo a pH 5.0. Durante toda esta etapa se mantiene una ligera agitación.- Development of the enzymatic reaction. Be mix 50 µL of a 3-indoxy solution 12 mM phosphate (prepared in 0.1 M acetate buffer solution pH 5.0), 50 µL of an aqueous solution of H2O2 2 · 10-4 M and 8 µL of an aqueous HRP solution of the desired concentration The temperature and reaction time are 47 ° C and 30 minutes respectively. The enzymatic reaction is carried out at pH 5.0. During this whole stage a slight agitation.
- Parada de la reacción y formación de índigo carmín. Se realiza con la adición de 100 \muL de H_{2}SO_{4} concentrado a cada uno de los pocillos que serán agitados durante un tiempo de 5 minutos para lograr una buena homogeneización.- Stop reaction and indigo formation carmine. It is done with the addition of 100 µL of H 2 SO 4 concentrated to each of the wells that will be stirred during a time of 5 minutes to achieve a good homogenization.
- Detección del índigo carmín generado. Esta etapa puede realizarse de modos diferentes en función de la técnica empleada en la detección.- Detection of the generated indigo carmine. This stage can be performed in different ways depending on the technique used in detection.
1) Voltamperometría cíclica: se hace en discontinuo, con la adición de 15 \muL del contenido del pocillo deseado y 25 \muL de H_{2}O sobre el electrodo serigrafiado de carbono. Se lleva a cabo un barrido de potenciales cíclico desde -0.4V (potencial que se mantiene durante 45 segundos) hasta +0.1V.1) Cyclic voltammetry: it is done in discontinuous, with the addition of 15 µL of the well contents desired and 25 µL of H2O on the screen-printed electrode of carbon. A cyclic potential scan is carried out from -0.4V (potential that remains for 45 seconds) up to + 0.1V
2) Sistema de flujo con detección amperométrica. Se introducen 100 \muL del contenido del pocillo deseado para llenar el loop. Se cambia la posición de la válvula de inyección y el volumen de muestra mencionado es arrastrado por el fluido portador H_{2}SO_{4} 1 M pasando posteriormente por la celda de medida. Durante las medidas se mantendrá un potencial constante de -0.30 V (vs electrodo de pseudo-referencia de Ag).2) Flow system with amperometric detection. 100 µL of the desired well content is introduced to fill the loop The position of the injection valve is changed and the mentioned sample volume is carried by the fluid carrier H_ {2} SO_ {4} 1 M subsequently passing through the cell of measure. During the measurements a constant potential of -0.30 V (vs pseudo-reference electrode of Ag).
A través de la detección del índigo carmín generado tras la solubilización del azul de índigo se consigue cuantificar la peroxidasa de rábano silvestre. De modo muy sencillo con ayuda de un sistema de flujo y detección amperométrica se logra un límite de detección 27 pg/pocillo (0.25 ppb) de HRP. Con una detección en discontinuo y voltamperometría cíclica se obtiene un límite de detección de 32.4 pg/pocillo (0.30 ppb) de HRP.Through the detection of carmine indigo generated after solubilization of indigo blue is achieved quantify horseradish peroxidase. Very simple with the help of an amperometric flow and detection system, it is achieved a detection limit of 27 pg / well (0.25 ppb) of HRP. With a discontinuous detection and cyclic voltammetry is obtained detection limit of 32.4 pg / well (0.30 ppb) of HRP.
El 3-indoxil fosfato es, por tanto, un sustrato válido para la cuantificación de peroxidasas. El 3-indoxil fosfato puede ser utilizado para la medida electroquímica de la actividad de la fosfatasa alcalina. Por tanto, ambas enzimas, principales marcas utilizadas en los inmunoensayos, pueden ser cuantificadas electroquímicamente a través del mismo producto enzimático, aunque generados bajo diferentes condiciones de reacción enzimática (pH, temperatura, tiempo de reacción).The 3-indoxy phosphate is, by therefore, a valid substrate for the quantification of peroxidases. He 3-indoxy phosphate can be used for electrochemical measurement of alkaline phosphatase activity. By both enzymes, the main brands used in the immunoassays, can be quantified electrochemically through of the same enzyme product, although generated under different enzymatic reaction conditions (pH, temperature, time of reaction).
El 3-indoxil fosfato es totalmente soluble en disolución acuosa. Este sustrato, al igual que el producto enzimático, azul de índigo, no son agentes carcinógenos ni mutagénicos como sucede con alguno de los sustratos comúnmente utilizados con la HRP.3-indoxy phosphate is totally soluble in aqueous solution. This substrate, like that the enzyme product, indigo blue, are not agents carcinogens or mutagenic as with any of the substrates commonly used with HRP.
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