ES2319059B1 - BRUSHITA AS A SYSTEM OF IMMOBILIZATION OF ENZYMES AND ITS USE IN BIOCOMPATIBLE AMPEROMETRIC BIOSENSORS. - Google Patents

Abstract

Brushita as immobilization system of enzymes and their use in biocompatible amperometric biosensors.

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 a biosensor amperometric 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, first a brushite-enzyme mixture is deposited on the electrode surface and allowed to air dry, to room temperature. Subsequently, the crosslinking is carried out. intermolecular enzyme adsorbed to the matrix, maintaining the steam modified electrode of glutaraldehyde. The biosensor resulting is used for analyte determination in aqueous solvents and predominantly organic media.

Description

Brushita as immobilization system of enzymes and their use in biocompatible amperometric biosensors.

Object of the invention

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

State of the art

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.

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)

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.

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).

The present invention, as expressed in the statement of this specification, refers to a new enzyme immobilization system that combines adsorption and crosslinking 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.

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.

Description of the invention

Brushita as immobilization system of enzymes and their use in biocompatible amperometric biosensors.

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.

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.

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.

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.

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

Description of the figures

Figure 1 shows the micrograph of the samples tyrosinase / brushite with crosslinking with glutaraldehyde on a metal surface.

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.

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.

Figure 4 shows the stability of the biosensor proposed in (a) aqueous medium and (b) acetonitrile mixtures: buffer (98.5: 1.5).

Embodiment of the invention

The present invention is illustrated by following examples, which are not limiting their scope.

Example one

Preparation of tyrosinase biosensor immobilized in brushite on a vitrified carbon electrode

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.

Example 2

Determination of the concentration of a phenolic derivative (catechol) dissolved in aqueous medium and predominantly medium organic

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 amperometric measurements with constant agitation, applying to the working electrode a potential of -0.1 V vs electrode of saturated calomelanos, potential to which the o-quinone produced in the enzymatic reaction.

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. Concentration maximum allowable for the total phenolic content in water of drink according to the legislation of the European Union (Directive 80/778 / EEC), is 5 10 - 9 M. Taking into account the limit of detection obtained with this biosensor (1nM), these devices arise as an interesting alternative for this type of analysis.

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 -7 10-6 M
and a detection limit of 40 nM. These results demonstrate that the biosensor can be used in the analysis of organic samples.

The response times obtained with these biosensors were always less than 12 seconds, characteristic which allows its use as detectors in flow systems continuous.

Example 3

Biosensor Stability

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

Example 4

Accuracy of the analytical method

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)

1. Biological material with immobilized enzyme inside characterized in that it contains one or more enzymes adsorbed on brushite. 2. Biological material with immobilized enzyme according to claim 1, characterized in that the enzymes are oxidoreductases. 3. Method of obtaining the biological material with immobilized enzyme described in claims 1 and 2, characterized in that it comprises the following steps: (a) adsorption of the enzyme to brushite and (b) intermolecular crosslinking of the enzyme by glutaraldehyde vapor. 4. Use of biological material with enzyme immobilized, according to claim 1, as part of a biosensor 5. Use of biological material with enzyme immobilized according to claim 4, wherein the biosensor performs amperometric measurements in solutions by constant agitation. 6. Use of biological material with enzyme immobilized according to claims 4 and 5, wherein the biosensor It consists of an electrode on whose surface a mixture of Brushite enzyme. 7. Use of biological material with enzyme immobilized according to claims 4, 5 and 6, wherein the biosensor determines analytes in aqueous media. 8. Use of biological material with enzyme immobilized according to claims 4, 5 and 6, wherein the biosensor determines analytes in predominantly organic media.