ES2319059A1 - Brushita as an enzyme immobilization system and its use in biocompatible amperometric biosensors. (Machine-translation by Google Translate, not legally binding) - Google Patents
Brushita as an enzyme immobilization system and its use in biocompatible amperometric biosensors. (Machine-translation by Google Translate, not legally binding) Download PDFInfo
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- ES2319059A1 ES2319059A1 ES200702403A ES200702403A ES2319059A1 ES 2319059 A1 ES2319059 A1 ES 2319059A1 ES 200702403 A ES200702403 A ES 200702403A ES 200702403 A ES200702403 A ES 200702403A ES 2319059 A1 ES2319059 A1 ES 2319059A1
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 36
- XAAHAAMILDNBPS-UHFFFAOYSA-L calcium hydrogenphosphate dihydrate Chemical compound O.O.[Ca+2].OP([O-])([O-])=O XAAHAAMILDNBPS-UHFFFAOYSA-L 0.000 claims abstract description 17
- 238000001179 sorption measurement Methods 0.000 claims abstract description 12
- 238000004132 cross linking Methods 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 9
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000012620 biological material Substances 0.000 claims description 10
- 239000012736 aqueous medium Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 102000004316 Oxidoreductases Human genes 0.000 claims description 2
- 108090000854 Oxidoreductases Proteins 0.000 claims description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 claims 3
- 239000011159 matrix material Substances 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 239000004568 cement Substances 0.000 abstract description 5
- 239000003125 aqueous solvent Substances 0.000 abstract description 2
- 239000001506 calcium phosphate Substances 0.000 abstract description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 abstract description 2
- 235000011010 calcium phosphates Nutrition 0.000 abstract description 2
- 229910010272 inorganic material Inorganic materials 0.000 abstract description 2
- 239000011147 inorganic material Substances 0.000 abstract description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 abstract description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 239000000872 buffer Substances 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 102000003425 Tyrosinase Human genes 0.000 description 5
- 108060008724 Tyrosinase Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012491 analyte Substances 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 239000012457 nonaqueous media Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000002041 carbon nanotube Substances 0.000 description 2
- 229910021393 carbon nanotube Inorganic materials 0.000 description 2
- 238000010382 chemical cross-linking Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WOAHJDHKFWSLKE-UHFFFAOYSA-N 1,2-benzoquinone Chemical compound O=C1C=CC=CC1=O WOAHJDHKFWSLKE-UHFFFAOYSA-N 0.000 description 1
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical compound CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000004082 amperometric method Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940094522 laponite Drugs 0.000 description 1
- XCOBTUNSZUJCDH-UHFFFAOYSA-B lithium magnesium sodium silicate Chemical compound [Li+].[Li+].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[Na+].[Na+].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3 XCOBTUNSZUJCDH-UHFFFAOYSA-B 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000007784 solid electrolyte Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000009834 vaporization Methods 0.000 description 1
- 230000008016 vaporization Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B25/00—Phosphorus; Compounds thereof
- C01B25/16—Oxyacids of phosphorus; Salts thereof
- C01B25/26—Phosphates
- C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Inorganic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Brushita como sistema de inmovilización de enzimas y su uso en biosensores amperométricos biocompatibles. La presente invención se refiere a un nuevo sistema de inmovilización enzimático que utiliza un material inorgánico biocompatible (Brushita, un cemento fraguado de fosfato cálcico) como matriz para la adsorción de enzimas y a un biosensor amperométrico preparado con dicho sistema de inmovilización. Comprende el procedimiento de fabricación del biosensor y su aplicación en medios acuosos y predominantemente orgánicos. En el proceso de fabricación del biosensor, primero se deposita una mezcla brushita-enzima sobre la superficie electródica y se deja secar al aire, a temperatura ambiente. Posteriormente se procede a la reticulación intermolecular del enzima adsorbido a la matriz, manteniendo el electrodo modificado en vapor de glutaraldehido. El biosensor resultante se utiliza para la determinación de analitos en disolventes acuosos y medios predominantemente orgánicos.Brushite as an enzyme immobilization system and its use in biocompatible amperometric biosensors. The present invention refers to a new enzyme immobilization system that uses a biocompatible inorganic material (Brushite, a calcium phosphate set cement) as a matrix for enzyme adsorption and to an amperometric biosensor prepared with said immobilization system. It includes the manufacturing procedure of the biosensor and its application in aqueous and predominantly organic media. In the biosensor manufacturing process, a brushite-enzyme mixture is first deposited on the electrode surface and allowed to air dry at room temperature. Subsequently, the intermolecular crosslinking of the enzyme adsorbed to the matrix is carried out, keeping the modified electrode in glutaraldehyde vapor. The resulting biosensor is used for the determination of analytes in aqueous solvents and predominantly organic media.
Description
Brushita como sistema de inmovilización de enzimas y su uso en biosensores amperométricos biocompatibles.Brushita as immobilization system of enzymes and their use in biocompatible amperometric biosensors.
La presente invención se refiere a un nuevo sistema de inmovilización enzimático que utiliza un material inorgánico biocompatible (Brushita, un cemento fraguado de fosfato cálcico) como matriz para la adsorción de enzimas, y a un biosensor para la determinación de analitos en medios acuosos y no acuosos basado en un sistema de inmovilización de enzimas sobre brushita.The present invention relates to a new enzyme immobilization system that uses a material inorganic biocompatible (Brushite, a phosphate cement set calcium) as a matrix for the adsorption of enzymes, and to a biosensor for the determination of analytes in aqueous and non-aqueous media based on an enzyme immobilization system on Little brush
Los biosensores constan de un transductor y un material biológico y uno de los aspectos más relevantes en la aplicabilidad de un biosensor es el tipo y calidad del material biológico. En este sentido se han propuesto distintos sistemas de inmovilización, entre ellos la adsorción de las biomoléculas sobre distintos materiales. Se trata de un método sencillo que consiste en poner en contacto las moléculas de enzima con el material adsorbente durante el tiempo suficiente para que se produzcan las interacciones entre la biomolécula y la matriz.The biosensors consist of a transducer and a biological material and one of the most relevant aspects in the Applicability of a biosensor is the type and quality of the material biological. In this sense, different systems of immobilization, including adsorption of biomolecules on different materials It is a simple method that consists in contacting the enzyme molecules with the material adsorbent for long enough for the interactions between the biomolecule and the matrix.
En la patente FR 2667077 se detalla el proceso de inmovilización de una proteína, preferentemente una enzima, por adsorción en un soporte sólido de un electrodo (vidrio, metal, polímero, componente de tela, fibra óptica, etc.) y posterior reticulación por vaporización de un agente reticulante (glutaraldehído) por pulverización, para hacer más estable la enzima. En esta patente se reivindica también un biosensor que utiliza un electrodo enzimático obtenido a través de este proceso de inmovilización para la determinación de analitos en disoluciones acuosas que presenta tiempos de respuesta cortos (del orden de 5-10 segundos).The process is detailed in FR 2667077 of immobilization of a protein, preferably an enzyme, by adsorption on a solid support of an electrode (glass, metal, polymer, fabric component, fiber optic, etc.) and later vaporization crosslinking of a crosslinking agent (glutaraldehyde) by spraying, to make the enzyme. This patent also claims a biosensor that uses an enzymatic electrode obtained through this process of immobilization for the determination of analytes in solutions aqueous that presents short response times (of the order of 5-10 seconds)
Se han descrito distintos biosensores enzimáticos amperométricos que utilizan como soporte para la adsorción de la enzima matrices inorgánicas como arcillas del tipo laponita (Cosnier and col., Mater. Sci. Eng., C., 2006, 26, 442-447; Li and Hu., J. Electroanal. Chem., 2003, 558, 155-165) e hidróxidos de doble capa (Mousty et al., Biosens. Bioelectron., 2007, 22, 1733-1738), nanopartículas de carbonato cálcico (Shan et al., Biosens. Bioelectron., 2007., 22, 1612-1617), nanopartículas de oro (Zhang et al., Biosens. Bioelectron., 2005, 21, 337- 345), nanotubos de carbono (Zhao et al., Electrochem. Commun., 2003, 5, 825-829) e híbridos de nanopartículas de oro con nanotubos de carbono (Chen et al., Biosens. Bioelectron., 2007, 22, 1268-1274) utilizándose para la determinación de analitos en medios acuosos. Entre las ventajas más importantes de estos biosensores destacan, en algunos casos, la biocompatibilidad de las matrices de inmovilización, en otros la conductividad eléctrica de los materiales utilizados, en otros la estabilidad de los biosensores y en otros su sencilla fabricación.Different amperometric enzyme biosensors have been described that use inorganic matrices as support for the enzyme adsorption as laponite clays (Cosnier and col., Mater. Sci. Eng., C., 2006, 26, 442-447; Li and Hu., J. Electroanal. Chem., 2003, 558, 155-165) and double layer hydroxides (Mousty et al ., Biosens. Bioelectron., 2007, 22, 1733-1738), calcium carbonate nanoparticles (Shan et al ., Biosens. Bioelectron., 2007., 22, 1612-1617), gold nanoparticles (Zhang et al ., Biosens. Bioelectron., 2005, 21, 337- 345), carbon nanotubes (Zhao et al ., Electrochem. Commun., 2003, 5, 825-829) and gold nanoparticle hybrids with carbon nanotubes (Chen et al ., Biosens. Bioelectron., 2007, 22, 1268-1274) being used for the determination of analytes in media aqueous. Among the most important advantages of these biosensors are, in some cases, the biocompatibility of the immobilization matrices, in others the electrical conductivity of the materials used, in others the stability of the biosensors and in others their simple manufacturing.
En los últimos años se han desarrollado los llamados biosensores de fase orgánica, que son dispositivos capaces de trabajar en disolventes orgánicos, pudiéndose utilizar para la determinación de analitos en muestras de muy distinta naturaleza y ampliando así el campo de aplicación de los biosensores (Sánchez-Paniagua et al., 2006., Biomol. Eng, 23, 135-147; Sánchez-Paniagua et al., Biosens. Bioelectron., 2006, 21, 2320-2328).In recent years, the so-called organic phase biosensors have been developed, which are devices capable of working in organic solvents, being able to use analytes in samples of very different nature and thus expanding the field of application of biosensors (Sánchez- Paniagua et al ., 2006., Biomol. Eng, 23, 135-147; Sánchez-Paniagua et al ., Biosens. Bioelectron., 2006, 21, 2320-2328).
La presente invención, según se expresa en el enunciado de esta memoria descriptiva, se refiere a un nuevo sistema de inmovilización enzimático que combina adsorción y entrecruzamiento y utiliza como matriz para la adsorción de enzimas un material inorgánico la brushita (cemento fraguado de fosfato cálcico) que es biocompatible (Kumta et al., Acta Biomater., 2005., 1, 65-83) y posee, además, características de electrolito sólido (Tortet y col., J. Solid State Chem., 1997., 132, 6-16). Así mismo se refiere a un biosensor amperometrico cuyo componente biológico consiste en uno o más enzimas inmovilizadas en dicha matriz, para la determinación de analitos en medios acuosos y predominantemente orgánicos.The present invention, as expressed in the statement of this specification, refers to a new enzyme immobilization system that combines adsorption and cross-linking and uses as a matrix for the adsorption of enzymes a brushite inorganic material (calcium phosphate cement set) which is biocompatible (Kumta et al ., Acta Biomater., 2005., 1, 65-83) and also has solid electrolyte characteristics (Tortet et al., J. Solid State Chem., 1997., 132, 6 -16). It also refers to an amperometric biosensor whose biological component consists of one or more enzymes immobilized in said matrix, for the determination of analytes in aqueous and predominantly organic media.
Las aportaciones más importantes de este nuevo dispositivo combinan la sencilla y rápida fabricación del biosensor como consecuencia de la inmovilización por adsorción, la ampliación del campo de aplicación del biosensor a estudios "in vivo", como consecuencia de utilizar una matriz biocompatible, así como a muestras de naturaleza hidrofóbica, ya que permite realizar medidas en medios no acuosos, y a la determinación de analitos a niveles traza por la elevada sensibilidad de estos dispositivos.The most important contributions of this new device combine the simple and rapid manufacture of the biosensor as a result of adsorption immobilization, the extension of the field of application of the biosensor to " in vivo " studies, as a result of using a biocompatible matrix, as well as to samples of hydrophobic nature, since it allows measurements in non-aqueous media, and the determination of analytes at trace levels due to the high sensitivity of these devices.
Brushita como sistema de inmovilización de enzimas y su uso en biosensores amperométricos biocompatibles.Brushita as immobilization system of enzymes and their use in biocompatible amperometric biosensors.
La presente invención se refiere a un nuevo sistema de inmovilización enzimático que utiliza un material inorgánico biocompatible (Brushita, un cemento fraguado de fosfato cálcico) como matriz para la adsorción de enzimas, y a un biosensor amperométrico basado en dicho sistema de inmovilización. Comprende el procedimiento de fabricación del biosensor y su aplicación en medios acuosos y predominantemente orgánicos.The present invention relates to a new enzyme immobilization system that uses a material inorganic biocompatible (Brushite, a phosphate cement set calcium) as a matrix for the adsorption of enzymes, and to a biosensor amperometric based on said immobilization system. Understands the biosensor manufacturing process and its application in aqueous and predominantly organic media.
La fabricación del biosensor es un proceso sencillo que requiere los siguientes pasos: (1) adsorción de la enzima en la brushita y (2) entrecruzamiento intermolecular enzimático con un agente entrecruzante. En primer lugar, se deposita una mezcla brushita-enzima sobre la superficie de un electrodo y se deja secar al aire, a temperatura ambiente. El electrodo modificado se mantiene en vapor de glutaraldehido para llevar a cabo la reticulación enzimática, que evita en gran medida la pérdida de enzima aumentando la estabilidad del biosensor. En la Figura 1 se muestra una micrografía de la superficie electródica después de producirse la adsorción y reticulación enzimática, observándose pequeñas partículas en buen contacto con la superficie del metal.Biosensor manufacturing is a process simple that requires the following steps: (1) adsorption of the Brushite enzyme and (2) intermolecular crosslinking Enzymatic with a crosslinking agent. First, it deposit a brushite-enzyme mixture on the surface of an electrode and allowed to air dry, at temperature ambient. The modified electrode is kept in steam of glutaraldehyde to carry out enzymatic crosslinking, which largely prevents enzyme loss by increasing stability of the biosensor. Figure 1 shows a micrograph of the electrode surface after adsorption occurs and enzymatic crosslinking, observing small particles in good contact with the metal surface.
El material biológico que se encuentra inmovilizado en brushita puede ser una o más enzimas oxidorreductasas. Para el entrecruzamiento químico se puede utilizar cualquier reactivo polifuncional que sirva como agente reticulante enzimático. El tipo de electrodo (transductor) utilizado dependerá del analito a determinar.The biological material found immobilized in brushite may be one or more enzymes oxidoreductases. For chemical crosslinking can be used any polyfunctional reagent that serves as a crosslinking agent enzymatic The type of electrode (transducer) used will depend of the analyte to be determined.
La proporción enzima-brushita en la mezcla puede variar entre 0,25 y 1,5; la cantidad de mezcla enzima-brushita depositada en la superficie electródica de área 0,07 cm^{2}, puede variar entre 12,5 \mug-100 \mug y tiempo de entrecruzamiento enzimático entre 5 y 25 minutos.The enzyme-brushite ratio in the mixture can vary between 0.25 and 1.5; the amount of mixture Brushite enzyme deposited on the surface electrode area 0.07 cm2, may vary between 12.5 \ mug-100 \ mug and cross-linking time Enzymatic between 5 and 25 minutes.
La determinación de los analitos con el biosensor propuesto se lleva a cabo mediante amperometría, en agitación constante, en una celda electroquímica termostatizada, que contiene el biosensor, un electrodo auxiliar y un electrodo de referencia, manteniendo el biosensor al potencial adecuado constante. Como consecuencia de la reacción entre la enzima inmovilizada y el analito, se produce la transformación química del sustrato. En la superficie del electrodo de trabajo (biosensor) tiene lugar la reacción redox de alguna de las especies que participan en la reacción enzimática, generándose una intensidad de corriente que estará relacionada con el analito presente en la muestra. El biosensor resultante permite la determinación de analitos en disolventes acuosos y medios predominantemente orgánicos.The determination of analytes with the Proposed biosensor is carried out by amperometry, in constant agitation, in a thermostated electrochemical cell, which it contains the biosensor, an auxiliary electrode and an electrode of reference, keeping the biosensor at the right potential constant. As a consequence of the reaction between the enzyme immobilized and analyte, chemical transformation of the substratum. On the surface of the working electrode (biosensor) the redox reaction of some of the species that they participate in the enzymatic reaction, generating an intensity of current that will be related to the analyte present in the sample. The resulting biosensor allows the determination of analytes in aqueous solvents and media predominantly organic
La Figura 1 muestra la micrografía de las muestras tirosinasa/brushita con entrecruzamiento con glutaraldehido sobre una superficie metálica.Figure 1 shows the micrograph of the samples tyrosinase / brushite with crosslinking with glutaraldehyde on a metal surface.
La Figura 2 muestra la curva de calibrado de catecol en medio acuoso obtenida con el biosensor de tirosinasa/brushita en disoluciones tampón fosfato 0,1 M pH 6,0.Figure 2 shows the calibration curve of catechol in aqueous medium obtained with the biosensor of tyrosinase / brushite in 0.1 M phosphate buffer solutions pH 6.0.
La Figura 3 muestra las curvas de calibrado de catecol en medios predominantemente orgánicos obtenidas con el biosensor de tirosinasa/brushita en mezclas de disolvente orgánico:tampón (98,5:1,5): (a) etanol:tampón y (b) acetonitrilo:tampón.Figure 3 shows the calibration curves of catechol in predominantly organic media obtained with the tyrosinase / brushite biosensor in solvent mixtures organic: buffer (98.5: 1.5): (a) ethanol: buffer and (b) acetonitrile: buffer.
La Figura 4 muestra la estabilidad del biosensor propuesto en (a) medio acuoso y (b) mezclas acetonitrilo:tampón (98,5:1,5).Figure 4 shows the stability of the biosensor proposed in (a) aqueous medium and (b) acetonitrile mixtures: buffer (98.5: 1.5).
La presente invención se ilustra mediante los siguientes ejemplos, los cuales no son limitativos de su alcance.The present invention is illustrated by following examples, which are not limiting their scope.
Ejemplo 1Example one
Para la fabricación del biosensor se prepara una suspensión de cemento de brushita (2 mg mL^{-1}) por dispersión en agua desionizada mediante agitación durante, al menos, 12 horas y una disolución de enzima (2 mg mL^{-1}) en agua desionizada. Una cantidad de la mezcla acuosa de tirosinasa/brushita (25 \mug:25 \mug) se deposita sobre la superficie (0,07 cm^{2}) de un electrodo de carbón vitrificado. Se mantiene el electrodo al aire durante 2 horas para que se produzca el secado total de la película. Posteriormente, se produce un entrecruzamiento químico de las moléculas de enzima, manteniendo el electrodo durante 15 minutos en vapor de glutaraldehído.For the manufacture of the biosensor a preparation is prepared Brushite cement suspension (2 mg mL -1) by dispersion in deionized water by stirring for at least 12 hours and an enzyme solution (2 mg mL -1) in deionized water. A amount of the aqueous tyrosinase / brushite mixture (25 mug: 25 mug) is deposited on the surface (0.07 cm 2) of a vitrified carbon electrode. The electrode is kept in the air for 2 hours for the total drying of the movie. Subsequently, chemical crosslinking of Enzyme molecules, holding the electrode for 15 steam minutes of glutaraldehyde.
Ejemplo 2Example 2
El biosensor preparado según se describe en el ejemplo 1 se utiliza para la determinación de catecol disuelto en disolución tampón fosfato (Figura 2) y en medios predominantemente orgánicos, como son mezclas acetonitrilo-tampón (98,5:1,5) y etanol- tampón (98,5:1,5) (Figura 3), mediante medidas amperométricas con agitación constante, aplicando al electrodo de trabajo un potencial de -0,1 V vs electrodo de calomelanos saturado, potencial al cual se reduce la o-quinona producida en la reacción enzimática.The biosensor prepared as described in the example 1 is used for the determination of catechol dissolved in phosphate buffer solution (Figure 2) and predominantly in media organic, such as acetonitrile-buffer mixtures (98.5: 1.5) and ethanol-buffer (98.5: 1.5) (Figure 3), by measurements amperometric with constant agitation, applying to the electrode of Work a potential of -0.1 V vs. saturated Calomelan electrode, potential to which the o-quinone produced is reduced In the enzymatic reaction.
Las características analíticas obtenidas en la determinación de catecol en disolución tampón fosfato son sensibilidad 46,57 A M^{-1} cm^{-2}, intervalo lineal 3 10^{-9} - 3 10^{-6} M y límite de detección, correspondiente a 3 veces la señal del ruido, 1 nM. La concentración máxima admisible para el contenido total de fenoles en aguas de bebida según la legislación de la Unión Europea (Directiva 80/778/EEC), es de 5 10^{-9} M. Teniendo en cuenta el límite de detección obtenido con este biosensor (1nM), estos dispositivos surgen como una interesante alternativa para este tipo de análisis.The analytical characteristics obtained in the Catechol determination in phosphate buffer solution are sensitivity 46.57 A M <-1> cm <2>, linear range 3 10 - 9 - 3 10 - 6 M and detection limit, corresponding to 3 times the noise signal, 1 nM. The maximum allowable concentration for the total phenolic content in drinking water according to the European Union legislation (Directive 80/778 / EEC), is 5 10-9 M. Taking into account the detection limit obtained with this biosensor (1nM), these devices arise as an interesting Alternative for this type of analysis.
Cuando el catecol se encuentra disuelto en un
disolvente predominantemente orgánico, como es la mezcla
acetonitrilo:tampón fosfato (98,5-1,5), se obtiene
una sensibilidad de 3,3 A M^{-1} cm^{-2}, un intervalo lineal
de 4 10^{-8} - 7 10^{-6} M
y un límite de detección de
40 nM. Estos resultados demuestran que el biosensor se puede
utilizar en el análisis de muestras orgánicas.When the catechol is dissolved in a predominantly organic solvent, such as the acetonitrile: phosphate buffer (98.5-1.5) mixture, a sensitivity of 3.3 AM -1 cm cm -2 is obtained , a linear range of 4-10-8-710-6 M
and a detection limit of 40 nM. These results demonstrate that the biosensor can be used in the analysis of organic samples.
Los tiempos de respuesta obtenidos con estos biosensores fueron siempre menores de 12 segundos, característica que permite su uso como detectores en sistemas de flujo continuo.The response times obtained with these biosensors were always less than 12 seconds, characteristic which allows its use as detectors in flow systems continuous.
Ejemplo 3Example 3
El biosensor, en medio acuoso, es muy estable durante los cinco primeros días (las señales obtenidas se mantienen por encima del 95% de la señal inicial), descendiendo la señal un 20% el séptimo día y un 60% al transcurrir 9 días. En medio no acuoso el biosensor es muy estable durante 3 días, comenzando el descenso de la señal a partir del cuarto día, tal y como muestra la Figura 4.The biosensor, in aqueous medium, is very stable during the first five days (the signals obtained are maintained above 95% of the initial signal), lowering the signal a 20% on the seventh day and 60% after 9 days. In between no aqueous the biosensor is very stable for 3 days, starting on decrease in signal from the fourth day, as shown by the Figure 4
Ejemplo 4Example 4
Para evaluar la precisión del método analítico se estudió la repetibilidad y precisión intermedia en medios acuoso y predominantemente orgánicos, obteniéndose los siguientes resultados. Se midió la corriente generada por una disolución de catecol en tampón de 100 nM y una disolución de catecol 0,8 \muM en acetonitrilo-tampón fosfato (98,5:1,5). En medio acuoso se obtiene un coeficiente de variación (CV) en el estudio de repetibilidad de 2,37% (n=10) y de 3,87% (n=20) para la precisión intermedia. Los CV obtenidos en disoluciones no acuosas para repetibilidad y precisión intermedia fueron 3,62% (n=10) y 5,55% (n=20) respectivamente.To evaluate the accuracy of the analytical method intermediate repeatability and accuracy in aqueous media was studied and predominantly organic, obtaining the following results. The current generated by a solution of catechol in 100 nM buffer and 0.8 µM catechol solution in acetonitrile-phosphate buffer (98.5: 1.5). In the middle aqueous a coefficient of variation (CV) is obtained in the study of repeatability of 2.37% (n = 10) and 3.87% (n = 20) for accuracy intermediate. The CVs obtained in non-aqueous solutions for repeatability and intermediate accuracy were 3.62% (n = 10) and 5.55% (n = 20) respectively.
Claims (8)
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| ES200702403A ES2319059B1 (en) | 2007-09-07 | 2007-09-07 | BRUSHITA AS A SYSTEM OF IMMOBILIZATION OF ENZYMES AND ITS USE IN BIOCOMPATIBLE AMPEROMETRIC BIOSENSORS. |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4812404A (en) * | 1985-09-20 | 1989-03-14 | Dental Kagaku Kabushiki Kaisha | Apatite immobilized glucanase |
| US6004786A (en) * | 1996-05-28 | 1999-12-21 | Toyo Denka Kogyo Co., Ltd. | Inorganic carrier containing bound silane coupling agent having carboxylic-ester group for immobilizing lipase |
| US20060204580A1 (en) * | 2002-04-18 | 2006-09-14 | Gower Laurie B | Biomimetic organic/inorganic composites, processes for their production, and methods of use |
-
2007
- 2007-09-07 ES ES200702403A patent/ES2319059B1/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4812404A (en) * | 1985-09-20 | 1989-03-14 | Dental Kagaku Kabushiki Kaisha | Apatite immobilized glucanase |
| US6004786A (en) * | 1996-05-28 | 1999-12-21 | Toyo Denka Kogyo Co., Ltd. | Inorganic carrier containing bound silane coupling agent having carboxylic-ester group for immobilizing lipase |
| US20060204580A1 (en) * | 2002-04-18 | 2006-09-14 | Gower Laurie B | Biomimetic organic/inorganic composites, processes for their production, and methods of use |
Non-Patent Citations (5)
| Title |
|---|
| HJERTÉN, S. et al. "Immobilization of Enzymes on Columns of Brushite". Journal of Chromatography, 1981, Volumen 215, páginas 25-30. Ver página 25, resumen. * |
| MOUSTY, C. et al. "Rutin Determination at an Amperometric Biosensor". Electroanalysis, Enero 2007, Volumen 19, Números 2-3, páginas 253-258. Ver página 253, resumen. * |
| SHAN, D. et al. "{}A New Polyphenol Oxidase Biosensor Mediated by Azure B in Laponite Clay Matrix". Electroanalysis, 2003, Volumen 15, Número 19, páginas 1506-1512. Ver página 1506, resumen. * |
| SHAN, D. et al. "A New Polyphenol Oxidase Biosensor Mediated by Azure B in Laponite Clay Matrix". Electroanalysis, 2003, Volumen 15, Número 19, páginas 1506-1512. Ver página 1506, resumen. * |
| SHAN, D. et al. "Layered Double Hydroxides: An Attractive Material for Electrochemical Biosensor Design". Analytical Chemistry, 2003, Volumen 75, Número 15, páginas 3872-3879. Ver página 3872, resumen; página 3873, Sección Experimental. * |
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