ES2315197B1 - NEW AMPEROMETRIC BIOSENSOR, MANUFACTURING PROCEDURE AND USES. - Google Patents

Abstract

New amperometric biosensor, procedure of Manufacturing and uses

It is based on the combination of the enzyme Lacasa and Sonogel-Carbon materials.

It is applicable to in situ measurements of analytes of interest in agrifood samples during the manufacturing, packaging, and / or storage processes. In addition, given the high selectivity due to the presence of the biological substances, it is possible to adapt the same design for a wide range of samples of interest in such an important field as the environment.

Description

New amperometric biosensor, procedure of Manufacturing and uses

Technical sector

The present invention describes:

one-

A new method of manufacturing biosensors material based amperometric Sonogel-Carbon and the enzyme Lacasa.

2-

The use of these biosensors for determination qualitative and quantitative of polyphenols in beer, in others drinks and plant extracts in general.

State prior to the technique

An amperometric biosensor can be defined as a measuring device that contains a biological substance intimately connected or integrated to a transducer: an electrode amperometric in this case. (IUPAC, 1996).

The novelty of the proposed biosensor can be Find in two main aspects.

First , the first use in the history of our Sonogel-Carbon electrode as a transducer part of a biosensor based on the Lacasa enzyme. At this point we want to underline that the Sonogel-Carbon material is a totally different compound from the Sol-gel carbon ceramic electrodes.

Second , the development of a new bio-electrochemical method based on the Lacasa enzyme as a biological recognition element to measure polyphenols in beer samples, as well as other beverages and plant extracts.

As defined by the IUPAC, cited above, The general scheme of a biosensor consists of three parts Main: the transducer, the recognition element Biological and immobilization matrix. The following invention communicates news in the three components of this scheme.

First, the possibility of using our Sonogel-Carbon material as an electrochemical transducer (electrode) in a biosensor based on the enzyme lacasa is communicated for the first time. This electrode demonstrated advantages over other similar electrodes when used as an electrochemical sensor or as the basis of a biosensor based on the enzyme tyrosinase (Hidalgo-Hidalgo-de-Cisneros, JL et al , Patent P200100556, Spain, March 2001; Cordero-Rando , MM et al . Analytical Chemistry 74 (2002) 2423-2427; Cordero-Rando, MM et al . Electroanalysis 17 (2005) 806-814; EL Kaoutit, et al . Biosensors & Bioelectronics 22 (2007) 2958-2966).

Generally in the classical processes that are usually used to achieve sol-gel materials (Collinson, MM et al , Analytical Chemistry 72, pp. 702A-709A; Jin, W. et al , Analytica Chimica Acta 461, pp. 1-36 ) or sol-gel ceramic compounds (Sampath, S. et al . Analytical Chemistry 68, pp. 2015-2021, Pankratov, I. et al , pp. Journal of Electroanalytical Chemistry 393, pp. 35-41) the use of Alcohol is essential to homogenize the two constituent phases of the sun, which are alkoxide and water, which leads to an inevitable phenomenon called skrinkage-fenomenon that prevents control of the volume of the material and the size of the pores in it (Lev , O. Analysis 20, pp. 543-553). This disadvantage is very limiting if it is intended to use the sol-gel material thus synthesized for the development of amperometric sensors or biosensors. An elegant way to achieve a compact sol-gel material with stable pore size and fine texture is the use of high-power ultrasound to homogenize the two phases. In our aforementioned patent, we have communicated the use of this technology to manufacture ceramic electrochemical sensors (electrodes) where electrical conductivity is ensured thanks to the percolation phenomenon between the carbon grains introduced into the synthesized sonogel material. The biocompatibility of this ceramic compound with the enzyme tyrosinase was reported in our article: El Kaoutit, et al . Biosensors & Bioelectronics 22 (2007) 2958-2966. The results were very competitive with other designs of enzymatic biosensors published in the literature. Our research in this regard led us to conclude that our sonogel-carbon material is more bio-compatible with the lacasa enzyme than with other enzymes.

The biosensor that we propose (Sonogel-Carbon based on Lacasa) has undergone tests to measure polyphenols in beer samples (samples characterized by its instability, complexity and even by its inhibitory effect of biological recognition elements) and has demonstrated its ability to obtain an index bio-electrochemical polyphenols very repetitive, very accurate and correlated with the classic index based on the use of Folin-Ciocalteu reagent.

Virtually all the works reported in the literature use tyrosinase as a basic biological recognition element to manufacture biosensors to measure the polyphenol index in beers. However, a thorough review of the biocatalytic mechanism of tyrosinase can lead us to conclude that this enzyme, in addition to not being sensitive to all polyphenols, is specific only to flavanol-3-oles and demonstrates a reduction in its catalytic power in presence of acid polyphenols. In addition, the optimal operating pH of the enzyme is around 7, which implies a change in the pH of the beer samples in order to be able to perform the measurement (pH of the beers near 4.5) and prevents the use of biosensors based on tyrosinase for measurements in situ . All these disadvantages were overcome by our biosensor based on laccase, since this enzyme catalyzes practically the conversion of all existing polyphenol families in beer and that the optimum pH of its operation is around the pH of those samples.

Explanation of the invention.

The main novelty of the procedure developed consists in the use of the enzyme lacasa for the first time as an element of biological recognition in a biosensor amperometric designed to determine the index Bio-electrochemical polyphenols in beers. The bio-compatibility of this enzyme with our sonogel-carbon material, with our matrix of totally new immobilization and with the measurement means, explains The advantages of this invention.

The advantages of the new biosensor here developed on the usual preparations are several and important:

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Unlike the biosensor based on tyrosinase and our sonogel-carbon material reported in: El Kaoutit, et al . Biosensors & Bioelectronics 22 (2007) 2958-2966, the biosensor based on the laccase and the same material showed great stability in the samples of interest thanks to the agreement between the isoelectric points of the two components of the biosensor: the sonogel-carbon made by acid catalysis and the enzyme with a pI about 4.

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The use of a mixture, of mass proportion Optimized, Glutaradialdehyde and Nafion to immobilize the enzyme on the surface of the Sonogel-Carbon, generates stability advantages and biocompatibility Due to the synergistic effect of the two compounds, the first as the perfect matrix of cross-linking and the second as a protective additive that generates, thanks to its hybrid structure between hydrophobic and hydrophilic, a perfect matrix for the free movement of the enzyme and its protection from derived phenolic products that damage the surface of the electrode and the active centers of the enzyme.

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The use of biosensors thus manufactured to measure polyphenols in beers, as a typical food sample complex and unstable, demonstrated the potential of the methodology developed to achieve the proposed objective.

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The comparison of the method developed here to measure polyphenols in beers with the classic Folin-Ciocalteu method demonstrates great advantages over that method. Time, cost, precision, possible adaptation to on-site measures, the possibility of automation, etc., are aspects to be highlighted.

Embodiment of the invention Apparatus, materials and reagents

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The synthesis of the sonosols was carried out by soundproofing with a high ultrasonic generator power, 20 kHz ultasonic processor (Misonix Inc., Farmingdale, NY), Model 600-W equipped with head titanium.

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The electrochemical measurements were made with a Ecochemie Autotab PGSTAT20 potentiostat / electroplating equipment. (Utrecht, the Netherlands) connected to a personal computer and a Stand module VA 663 of Metrom.

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As electrode holders were used 1.15 mm internal diameter glass capillaries, 70 mm length.

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To prepare the electrodes Sonogel-Carbon the following were used reagents:

quad

Methyltrimethoxysilane (MTMOS) from home Merck

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Hydrochloric acid 35% of the house Panreac

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House Spectroscopic Grade Graphite (GMBH) SGL Carbon

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To biologically modify the electrodes Sonogel-Carbon were used:

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The enzyme lacca of the fungus trametes versicolor supplied by Fluka. Glutaradialdehyde and Nafion of 25% and 5% concentration respectively and supplied by Sigma and Aldrich.

Biosensors preparation procedure

to)

Manufacture of electrochemical transducer "electrode" as detailed in the patent: Hidalgo-Hidalgo-de-Cisneros, J. L., Cordero-Rando, M. M., Naranjo-Rodriguez, I., Blanco, O. E., Esquivias, F. L., Patent P200100556, Spain, March 2001.

b)

Biological functionalization of the electrode obtained. This phase is carried out in several stages:

one-

By polarization at variable potential in a three electrode cell proceeds to an electrode cleaning applying several voltammetric cycles.

2-

The electrode is cleaned and allowed to dry, while dissolves the enzyme in the appropriate regulatory solution; this Dissolution is added Glutaradialdehyde and subjected to polymerization in an ultrasonic bath, Nafion is added and Mix manually.

3-

From this solution the appropriate amount is taken through a micro-syringe, it is deposited on the electrode and left  dry at room temperature. The mass proportion of Glutaradialdehyde and Nafion is finally optimized at 1% and 0.5% of deposited volume, respectively.

4-

After eight hours the biosensor is introduced, since finished its manufacturing process, in an electrochemical cell which contains a well stirred regulatory solution and is left there for 15min to remove enzyme residues not completely adsorbed on the electrode surface.

5-

Once this process is finished, it is removed, washed gently with the same regulatory solution and kept submerged  in the same solution in the refrigerator at 4 ° C when not in use.

Way in which the invention is applicable industrial

The biosensor developed is capable of industrial application since it can be perfectly adapted to in situ measurements of analytes of interest in agrifood samples during the manufacturing, packaging, and / or storage processes. In addition, given the high selectivity due to the presence of biological substances, it is possible to adapt the same design for a wide range of samples of interest in such an important field as the environment.

Claims (4)

1. New amperometric biosensor based on the combination of the enzyme Lacasa and materials Sonogel-Carbon. 2. Method of manufacturing a new amperometric biosensor based on the combination of the enzyme Lacasa and Sonogel-Carbon materials, characterized in that after the electrochemical transducer "electrode" is manufactured by sonocatalysis, the biological functionalization of the electrode is performed, which comprises the following stages: Dissolution of the Lacasa enzyme in the appropriate regulatory solution; Addition of glutaraldehyde in the amount previously calculated and ultrasonic bath polymerization; addition of Nafion in adequate quantity; homogenization of the mixture; deposition on the surface of the electrode by micro-syringe of the required amount of this mixture to obtain the desired coating percentages. 3. Use of the new amperometric based biosensor in the combination of the enzyme Lacasa and materials Sonogel-Carbon, according to claims 1 and 2, for determine the bio-electrochemical index of polyphenols in beers, beverages and plant extracts in general. 4. Use of the new amperometric based biosensor in the combination of the enzyme Lacasa and materials Sonogel-Carbon, according to claims 1 and 2, in sewage or wastewater treatment and in Bio-synthesis of high purity products.