ES2660516B1 - Electrode device for the detection of ascorbic acid, manufacturing process and use of said device. - Google Patents

Abstract

The present invention consists of an electrode device for the detection of ascorbic acid, with three electrodes, all electrodes obtained by screen printing, as well as its manufacturing process and its use in the aforementioned detection.

Description

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ELECTRICAL DEVICE FOR THE DETECTION OF ASCORBIC ACID, MANUFACTURING PROCEDURE AND USE OF SUCH

DEVICE

DESCRIPTION

OBJECT OF THE INVENTION

The present invention is framed in the field of electrochemistry applied to chemical analysis.

Thus, the present invention relates to a device consisting of an electrode system for the detection of ascorbic acid, with three electrodes, all electrodes obtained by screen printing, as well as its manufacturing process and conditions of use in the aforementioned detection.

BACKGROUND OF THE INVENTION

The development of reproducible and sensitive devices for electrochemical detection of antioxidant ascorbic acid, which helps in detoxification and improves iron absorption in the body (PJ O'Connell, C. Gormally, M. Pravda, GG Guilbault, Development of an amperometric L-ascorbic acid (Vitamin C) sensor based on electropolymerised aniline for pharmaceutical and food analysis, Analytica Chimica Acta, 431 (2001) 239), follow

being studied today, as evidenced by the more than 10,000 scientific publications that can be found in the different databases. It has also been shown that the concentration of ascorbic acid in biological fluids can be used to assess oxidative stress in human metabolism, which has in turn been related to liver disease, cancer

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or diabetes (A. Ambrosi, A. Morrin, M.R. Smyth, A.J. Killard, The application of conducting polymer nanoparticle electrodes to the sensing of ascorbic acid, Analytical Chimica Acta, 609 (2008) 37).

Traditionally, spectroscopic, chromatographic and electrochemical methods have been used for the detection of ascorbic acid. The latter techniques are characterized by their simplicity, speed and portability, which also makes possible the detection in situ of the analyte of interest (CA Fuenmayor, S. Benedetti, A. Pellicano, MS Cosio, S. Mannino, Direct In Situ Determination of Ascorbic Acid in Fruits by Screen-Printed Carbon Electrodes Modified with Nylon-6 Nanofibers, Electro- analysis, 26 (2014) 704). However, in the detection of ascorbic acid especially in complex matrices such as food products or biological samples, relatively high working potentials are used for electrochemical oxidation to occur, which is associated with poor selectivity, and deactivation of the working electrode caused mainly due to the accumulation of reaction products on the electrode surface, which causes a progressive deterioration of the electrode sensitivity (PR Roy, MS Saha, T. Okajima, T. Ohsaka, Electrocatalytic oxidation of ascorbic acid by [Fe (CN ) 6] 3- / 4- redox couple electrostatically trapped in cationic N, N-dimethylaniline polymer film electropolymerized on diamond electrode, Electrochimica Acta, 51 (2006) 4447) (CA Fuenmayor, S. Benedetti, A. Pelli- cano , MS Cosio, S. Mannino, Direct In Situ Determination of Ascorbic Acid in Fruits by Screen-Printed Carbon Electrodes Modified with Nylon-6 Nanofibers, Electro- analysis, 26 (2014) 704

These drawbacks have been tried to minimize by using simple disposable devices.

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bles manufactured by screen printing. Screen printing techniques have acquired great consideration, since they allow the manufacture of disposable electrodes at low cost (JP Hart, A. Crew, E. Crouch, KC Honeychurch, RM Pemberton, Some recent designs and developments of screen-printed carbon electrochemical sensors / biosensors for biomedical, environmental, and industrial analytics, Analytical Letters, 37 (2004) 789), (O. Domínguez-Renedo, MA Alonso-Lomillo, MJ Arcos-Martínez, Recent developments in the field of screen-printed electrodes and their related applications, Talanta, 73 (2007) 202), (BC Janegitz, J. Cancino, V. Zucolotto, Disposable Biosensors for Clinical Diagnosis, Journal of Nanoscience and Nanotechnology, 14 (2014) 378) and (SA Ozkan, J .-M. Kauffmann, P. Zuman, Electroanalysis in Biomedical and Pharmaceutical Sciences: Voltammetry, Amperometry, Biosensors, Applications, Springer Berlin Heidelberg, Berlin, Heidelberg, 2015), with good reproducibility, design flexibility and automation n of processes (J.M. Cooper, A.E.G. Cass, Biosensors: a practical approach, Oxford University Press, Oxford, 2004).

Screen printing is a direct printing method, also called penetration printing. The deposition of inks is done layer by layer on a substrate. The quality of the chemical sensors thus manufactured depends, to a large extent, on the materials used.

By means of the technology of screen-printed electrodes, the miniaturization of the sensors is possible, offering the advantage of being versatile, they can be manufactured with different electrode configurations and with different inks, and have a low cost and can thus be used for mass production of disposable electrodes They can also be used for analysis

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on-site.

The differential pulse voltammetry technique consists in the application of a potential sweep to the electrode system, over which it periodically superimposes a series of potential impulses. The signal that is recorded is the difference in intensity after and before applying the pulse against the base potential. This differential current measurement eliminates capacitive intensities and background noise, increasing the sensitivity of the analysis.

In screen-printed electrode systems, the reference electrode is usually made up of a conductive paste based on Ag, so it does not act as a true reference but as a pseudo-reference.

The electrochemical methods indicated above for the determination of ascorbic acid are poorly selective and do not allow quantification of this compound in complex matrices. The present invention solves the aforementioned disadvantages, and proposes a device that works at a lower potential avoiding the interference of other compounds in complex samples. The device has great simplicity, low cost and short analysis time, compared to the rest of the techniques commonly used for its determination, so it is a great advance in its use for this determination in different types of samples such as biological samples .

DESCRIPTION OF THE INVENTION

The object of the invention is an electrode device, its method of manufacturing and using the

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same for the detection of ascorbic acid. The problem to be solved is the selective determination of ascorbic acid with simpler techniques, with low cost and in a relatively short analysis time compared to known techniques for the determination of ascorbic acid.

In view of the foregoing, in one aspect, the present invention relates to an electrode device for the detection of ascorbic acid in different types of liquid samples, obtained by screen printing characterized in that it comprises three electrodes, each electrode consists of a contact, a section and an active area, being a working electrode, an auxiliary or counter electrode, and a reference electrode, all the electrodes constituted by screen printing of an ink of conductive materials on a plastic polyester sheet. The conductive paths constituted by the contacts also called terminals (1.3, 2.3, 3.3) and the sections (1.2, 2.2, 3.2) have been printed with an Ag-based ink, the active areas of the electrodes that constitute the counter electrode (3.1 ) and the working electrode (2.1), have been printed with a carbon conductive ink and the reference one with an Ag / AgCl ink in the active area.

In another aspect, the invention relates to the manufacturing method of the electrode device described above, whose spatial arrangement allows rapid "in situ" analysis of the sample by electrochemical techniques.

The manufacturing process, screen printing of the device is carried out with the help of a series of screens, also called molds or patterns

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of screen printing, which allow to define the different electrodes on the same support. The silkscreen construction process basically involves several stages consisting of the sequential deposition of the different inks and their subsequent curing.

Four different screens were designed for the manufacture of these devices, corresponding to each level of ink deposition, as follows:

- The first screen defines the conduit tracks

flush, consisting of contacts (1.3, 2.3, 3.3) and sections (1.2, 2.2, 3.2) printed with an ink based on Ag.

- The second screen defines the shape and position

of the reference electrode.

- The third screen defines the active area of the

counter electrode (3.1) and the active area of the working electrode (2.1)

- The fourth screen is designed for the depo

sition of an insulating material to prevent contact between the solution and the conductive pathways, constituted by the contacts and the sections.

The set constitutes a miniaturized sensor whose geometry is optimal for the analysis of real liquid samples of small size. The device can be introduced into an electrochemical cell in which the sample is in solution.

To operate the device, it

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connect the contacts or terminals of each of the three electrodes (1.3, 2.3, 3.3) with the output terminals of

a potentiostat through electrical connections.

The present invention relates in another aspect to the use of the aforementioned device comprising the following steps:

- Modification of the working electrode with gold nanoparticles. The deposition of the gold nanoparticles (AuNPs) on the working electrode is performed electrochemically by depositing in the electrode a drop of 100 pL of 0.1 mM solution of HAuCl4 prepared in 0.5 M H2SO4, and applying a constant potential of +0.18 V vs Ag / AgCl SPE for 50 seconds.

- Introduction of the device in the electrochemical cell.

- Adding 5 mL of the sample in the presence of a 100 mM buffer solution of KH2PO4 and 100 mM KCl of pH = 7.

- Application of a potential of +0.2 V vs Ag / AgClSPE.

- Performing amperometric measurements by recording the intensity in the different screen-printed devices.

DESCRIPTION OF THE FIGURES

This descriptive report is complemented,

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with a set of figures, illustrative of the preferred and never limiting example of the invention.

Figure 1 shows a scheme of the electrode reaction.

Figure 2 represents an elevation view of the electrodes without insulation.

Figure electrodes with

3 represents an insulating view.

in elevation

from

the

Figure 4 shows a type amperogram obtained in the determination of ascorbic acid.

DETAILED EXHIBITION OF THE INVENTION

In the embodiment detailed herein, the invention relates to an electrode device for the detection of ascorbic acid, with three electrodes (1, 2, 3),

obtained all the electrodes by screen printing, as well as its manufacturing procedure, and its use in the aforementioned detection.

As the screen printing technique involves the use of concrete materials and gives rise to the specific geometric configuration of the device, the exposure of said manufacturing technique also serves as an explanation of the configuration of the device, both of which can be linked, configuration and manufacturing technique or procedure , during the present detailed exposition of the invention.

The reaction mechanism proceeds according to the scheme shown in Figure 1. Ascorbic acid

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it is oxidized to dehydroascorbic acid, the electrons being transferred to the electrode in which the reaction takes place.

The invention relates to the device, method of manufacture and use of a disposable three electrode device (1, 2, 3), screen printed on a polyester support, in particular ethylene polyterephthalate (PET), whose spatial arrangement allows rapid analysis "in situ" of small sample volumes by electrochemical techniques.

The device consists of three serialized electrodes (1, 2, 3): reference electrode (1), working electrode (2) and auxiliary electrode (3) or counter electrode.

The electrodes have three distinct zones or parts: the contacts (1.3, 2.3, 3.3) or terminals, for connection to a known potentiostat; the active areas (1.1, 2.1, 3.1), in direct contact with the sample to be analyzed and the sections (1.2, 2.2, 3.2), as a union between contacts (1.3, 2.3, 3.3) and the active areas (1.1, 2.1, 3.1). Being the set of contacts (1.3, 2.3, 3.3) and the sections (1.2, 2.2, 3.2) the conductive routes.

The process of manufacturing, screen printing, of the device is carried out with the help of a series of screens, molds or screen printing patterns in which, on a porous surface, the scheme of the motif to be printed that will define the device appears.

- The first screen or pattern is used to form, conducting with conductive Ag commercial paste the conductive paths constituted by the contacts (1,3), (2,3), (3,3) and the sections (1,2) , (2,2), (3,2) of

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the electrodes (1, 2, 3). That is, the base is created

electrical conductor of the device. After application this layer should be cured for 30 minutes at 120 ° C.

- The second screen or pattern is designed to obtain the active area (1.1) of the reference electrode (1). For its formation, using this second screen, a commercial product based on Ag / AgCl is printed on the conductive base in the area of the active area of said electrode, which is subsequently subjected to a healing process under the same conditions, 20 minutes at 120 ° C.

- The third screen or pattern is designed to print together the active areas of the counter electrode (3.1) and the working electrode (2.1) using carbon ink.

- The fourth screen or pattern is designed to print the insulating layer that covers the device, leaving free the contacts (1.3, 2.3, 3.3) and active areas (1.1, 2.1, 3.1) of the electrodes, using an insulating ink. The correct formation of this insulating layer requires healing at 80 ° C for 30 minutes.

In this embodiment, the active area of the reference electrode is rectangular in geometry, the circular working one and the counter electrode has an arcuate envelope shape of the other two electrodes. The best dimensions of the active area (1.1) of the device reference electrode correspond to 2 mm in width and 4 mm in length.

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For the working electrode (2) the best tested dimension of its active area (2.1) corresponds to a circle of 4.5 mm in diameter.

To put the device into operation, the upper conductive parts (1.3, 2.3, 3.3) of each of the electrodes (1, 2, 3) are connected to the output terminals of a potentiostat through electrical connections.

100 microliters of a gold salt are deposited in the electrode device, and it is subjected to a potential of 0.18 V to allow the modification of the surface of the working electrode with gold nanoparticles. Next, the electrode device is immersed in a cell with the solution to be analyzed and according to the technique selected to carry out the amperometric determination, the electrode device is subjected to a potential difference of 0.2 V, recording the intensity that is originates in the circuit due to the electrochemical reactions that the potential disturbance causes, in the analyte ascorbic acid.

To carry out the determination of a test sample of ascorbic acid, the sample of ascorbic acid is added to the cell where the electrode device was introduced in the presence of a phosphate buffer solution of pH = 7.0 and subjected to dissolution at a constant stirring speed. Subsequently, successive additions of standard solutions of ascorbic acid are made and the intensities recorded after each addition are measured.

The corresponding calibration line is obtained

Tooth and the concentration of ascorbic acid in the test sample is calculated by extrapolation, according to the standard addition procedure, measuring on the abscissa axis of the representation the value of the concentration for a value of y = 0.

The process has a reproducibility of 4.6% (n = 3) calculated as the deviation of the slopes of the calibration lines obtained.

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The detection capacity is 2.3 ± 0.9 and the range of measurement concentrations is between 1.9 - 16.6 ^ M.

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Claims (7)

5 10 fifteen twenty 25 30 35 1. - Electrode device for the detection of ascorbic acid obtained by screen printing characterized in that it comprises three electrodes (1, 2, 3), each electrode consists of a contact or terminal (1.3, 2.3, 3.3), a section (1.2, 2.2, 3.2) and an active area (1.1, 2.1, 3.1), being one of reference (1), another working electrode (2), and another auxiliary or counter electrode (3), constituted the terminals and sections with a silver ink , the active areas of the counter electrode and the working electrode with a carbon ink and the active area of the reference electrode with a silver / silver chloride ink. 2. - Electrode device according to claim 1 characterized in that the contacts (1.3, 2.3, 3.3), the sections (1.2, 2.2, 3.2) and the active area of the reference electrode (1,1) are rectangular in shape. 3. - Electrode device according to claim 2, characterized in that the active area (1.1) of the reference electrode is rectangular in shape, 2 mm wide and 4 mm long. 4. Device 1 characterized by working electrode diameter. electrode according to claim - because the active area (2.1) of (2) is a circle of 4.5 mm. 5. Electrode device according to claim 1 characterized in that the active area (3.1) of the auxiliary electrode or counter electrode (3) has an arc shape. 5 10 fifteen twenty 25 30 35 6. Manufacturing process of the device according to claim 1 characterized in that it comprises the following steps: -Screen printing of a sheet of polyester with silver ink with the shapes of the contacts (1.3, 2.3, 3.3), and the sections (1.2, 2.2, 3.2) of the three electrodes (1, 2, 3).
-Screen printing of the active surfaces of the working electrode (2.1) and the counter electrode (3. 1) with carbon ink.
-Screen printing of the active area of the electrode reference (1.1) with Ag / AgCl.
-Screen printing of an insulator covering the device except the active areas (1.1, 2.1, 3.1) of
electrodes and contacts (1.2, 2.2, 3.2) of The electrodes 7. Use of the device according to claim 1 characterized in that it comprises the following steps: - union of the contacts (1.3, 2.3, 3.3) of the electrodes (1, 2, 3) to a potentiostat for apply the appropriate potential to the electrode system. modification of the working electrode with gold nanoparticles. The deposition of the gold nanoparticles (AuNPs) on the working electrode is performed electrochemically by depositing in the electrode a drop of 100 pL of 0.1 mM solution of HAuCl4 prepared in 0.5 M H2SO4, and applying a constant potential of +0.18 V vs Ag / AgCl SPE for 50 seconds. 5 10 fifteen twenty 25 30 - introduction of the electrode device in the electrochemical cell. - Adding 5 mL of the ascorbic acid sample in the presence of a 100 mM KH2PO4 and 100 mM KCl buffer solution of pH = 7, subjecting the solution to a constant stirring speed. - application of a potential of +0.2 V vs Ag / AgClSPE. - realization of successive additions of standard solutions of ascorbic acid by measuring the intensities recorded after each addition. - representation of the intensities against ascorbic concentrations to build the calibration line. - calculation of the concentration of the sample of ascorbic acid problem by measuring on the abscissa axis of the representation the value of the concentration for a value of y = 0 according to the known standard addition method.