ES2725248B2 - DETECTION AND DETERMINATION PROCEDURE OF CHLORIDE ION IN LIQUID SAMPLES - Google Patents

Description

DESCRIPTION

CHLORIDE ION DETECTION AND DETERMINATION PROCEDURE IN

LIQUID SAMPLES

TECHNICAL FIELD OF THE INVENTION

The present invention is encompassed in the field of electrochemistry applied to chemical analysis, particularly in the analysis of detection of chloride ion present in a liquid sample.

The present invention relates to a detection method using a sensor of the type consisting of three electrodes obtained by screen printing, to detect chloride ion in liquid samples, as well as to the determination of chloride ion by means of said sensor.

Thus, in one aspect, the present invention describes a sensor of the type consisting of three electrodes, each electrode including a contact, a conductive section and an active area, one of the electrodes being a working electrode, another of the electrodes being an auxiliary electrode. or counter electrode and the other electrode a reference electrode, all consisting of silk-screen printing of an ink of conductive materials on a plastic sheet, where the conductive sections, the counter electrode and the working electrode are screen printed with a conductive platinum ink and the reference with an Ag / AgCl ink in the active area.

According to a second aspect, the invention provides a method for the determination of the chloride ion in a liquid sample using said sensor.

BACKGROUND OF THE INVENTION

Chloride ion concentration is an important parameter in various environmental applications, in the pharmaceutical and food industries (Hern, JA et al. Talanta 1983, 30 (9), 677-682; Morales, JA et al., J. Chromatogr. A 2000, 884 (1-2), 185-190, Galvis-Sánchez, AC et al. AOSS Food Control 2011, 22 (2), 277-282, Pérez-Olmos, R. et al., MCBSM Food Chem. 1997, 59 (2), 305-311).

This anion is also an essential electrolyte that maintains homeostasis within the body and its concentration can show the presence of various diseases.

Thus, for example, the determination of chloride in sweat samples is the most useful diagnostic test in cystic fibrosis (Sosnay, PR et al., J. Pediatr. 2017, 181, S52-S57). Depending on age, the concentration of chloride in sweat remains between 30 and 70 mmolT1, while levels above 70 or 80 mmolT1 are unequivocal proof that the disease is suffered (Barrio Gómez de Agüero, MI et al., An. Españoles Pediatr. 1999, 50 (6), 625-634; Farrell, PM et al., J. Pediatr. 2017, 181, S16-S26).

In addition, sweat chloride ion concentration is a potential biomarker of electrolyte loss, as it is the most abundant ion in perspiration (Latzka, WA; J. Clin. Sports Med. 1999, 18 (3), 513- 524; Harvey, CJ; Int. J. Cosmet. Sci.

2010, 32 (5), 347-355; Stefaniak, AB, J. Toxicol. Vitr. 2006, 20 (8), 1265-1283). Equally important is the determination of its concentration in the blood plasma, whose range is between 96 and 106 mmolT1. Lower or higher concentrations are indicative of hypo or hyperchloremia (Morrison, G., Clinical Methods: the history, physical, and laboratory Examinations .; Walker, HK, Hall, WD, Hurst, JW, Eds .; Boston: Butterworths, 1990; pp 890-894).

Also the amount of chloride ion present in other biological fluids, such as urine, in which the chloride ion ranges in a concentration range of 110 to 250 mmolT1, can provide information about kidney malfunction (Mutter, WP; Korzelius , CA, Hosp. Med. Clin. 2012, 1 (3); Schrier, RW, J. Am. Soc. Nephrol. 2011, 22 (9), 1610-1613).

It has also been observed that in patients suffering from amyotrophic lateral sclerosis the concentration of chloride in the cerebrospinal fluid has lower levels than usual (Watanabe, S. et al., HJ Neurol. Sci. 2009, 285 (1-2), 146-148).

Therefore, the control of chloride ion in these fields is of great importance.

Among the methods commonly used for the determination of chloride are known volumetric methods, such as the Mohr method, in which a titration of the chloride ion in the form of silver chloride is used, potentiometric methods using ion selective electrodes (Grubb , WT Chloride-selective electrode, US3740326, 1973), and more recently methods based on the use of ion chromatography (Pereira, JSF et al., J. Chromatogr. A 2008, 1213 (2), 249-252; Pimenta, AM et al., J. Pharm. Biomed. Anal. 2004, 36 (1), 49-55). Other optical techniques such as scattering of resonance light or turbidimetric methods have also been used in the determination of chloride.

Voltammetric methods using screen printed electrode systems (SPE) have already been described in the state of the art. This type of disposable platforms have clear advantages due to their low cost, the possibility of being used on site and their ease of modification (Renedo, OD et al., Talanta 2007, 73 (2), 202-219). Most of the works related to the determination of chloride with SPE are based on the formation of a stable compound between silver and chloride (Campos, I. et al., Sensors Actuators, B Chem. 2010, 149 (1), 71- 78; Chiu, MH et al., Biosens. Bioelectron. 2009, 24 (10), 3008-3013; Choi, DH et al., Sensors Actuators, B Chem. 2017, 250, 673-678). The nernstian shift of the voltammetric peak of a control species, which occurs in the presence of chloride when using pseudo SPE Ag / AgCl reference electrodes in the electrode system, has also been used for chloride analysis.

For example from document US10657760 an amperometric device is known that incorporates multiple sensors to obtain the analyte profile of a sample.

WO2007026152A1 describes an electrochemical sensor with an electrode arrangement printed on a support plate, the electrodes being isolated by a layer of insulating material provided with openings to expose the working area of each electrode arrangement.

From document ES201400313 a screen-printed sensor of the type described in the preamble of claim 1 is known, where a carbon conductive ink is used and where the presence of a control of ferrocenomethanol or ferricyanide is necessary, the chloride ion is determined indirectly from the oxidation of ferrocenomethanol or the reduction of ferricyanide.

Despite the fact that the existing sensors allow obtaining good results for some applications, they present several limitations and problems related fundamentally to the complexity in the preparation of the electrode systems and also allow very small ranges of use, so they lose applicability for the determination of chloride in biological media in general, its applications being useful in very limited fields.

Thus, for example sensors based on potentiometric methods are limited by the operability of the electrode membrane, which can accelerate its dysfunction when used in certain matrices. On the other hand, measurement with these methods normally requires high reaction times, since longer times are necessary to reach the required equilibrium characterized by a stable reading of the potential. Achieving such an equilibrium requires more time when the sample is more complex, so the analysis as a whole takes more time. Furthermore, for example to measure the chloride in any sample, for example in sweat, the addition of an electrolyte (such as KNO3) is necessary to adjust the ionic strength of the medium, which requires more preparation in the analysis.

As for the known screen-printed sensors, they generally imply the presence of another species in the system, a species that can interfere with certain matrices. In the particular case of the sensor described in ES201400313, the need for a control such as ferrocenomethanol or potassium ferricyanide directly influences the performance of the sensor. Since its use is essentially to determine chloride in aqueous samples, the presence of the control substances can act as interferers in more complex matrices. Likewise, said presence limits the operating range and its detection limit. In tests carried out with it using ferrocenomethanol, the detection capacity is 10 mM and the range of measurement concentrations is between 10 mM and 90 mM.

The present description provides a sensor of the type indicated above that It solves the disadvantages of the already known sensors, providing a very simple, easy-to-use and economic screen-printed sensor, in which a Pt-based ink has been used, compared to the carbon-based ones commonly used, for the construction of the electrode. work (SPPtE). The sensitivity and versatility of the proposed sensor presents notable advantages compared to the approaches described in the prior art for the detection of chloride ions in different types of food, environmental and pharmaceutical samples, being a very useful tool in the diagnosis of diseases related to low or high concentrations of chloride ion in the different biological fluids.

The screen-printed sensor described here could be used for a single use due to its low cost or it can be used in complex matrices more than 100 times without affecting its operation. On the other hand, the time necessary for the measurement is very short, obtaining results in just 20 seconds, a very short time compared to that necessary to obtain a response in the potentiometric method. Furthermore, the screen-printed sensor described here can directly measure chloride in sweat samples without the need to add any reagent, due to the absence of interferences. This parameter is key for the diagnosis of cystic fibrosis, which increases the interest of its possible applications. It would also be useful in the analysis of food, pharmaceutical, etc. samples.

The sensor described here by the invention, based on the electrochemical reaction that occurs on a Pt surface, catalyzes the electrochemical reaction of chloro-chloride oxidoreduction. Therefore, the observed signal originates directly from the electroactive species which is chloride. The material of the electrode for this reaction to take place is, therefore, the key to the sensor and, consequently, the detection limit reached is 0.76 mM, and the much wider range of use is 0.76 to 150 mM.

BRIEF DESCRIPTION OF THE FIGURES

The present specification is complemented with illustrative and non-limiting figures of exemplary embodiments of the invention.

An example of a sensor as described here is shown in Figure 1 showing a working electrode, an auxiliary or counter electrode and a reference electrode, including the electrodes a contact, a conductive section and an active area, screen printed on a sheet plastic, as well as an insulating element that, although shown in the figure as a separate element for clarity, is a screen printed insulator that covers the sensor except in the active areas of the electrodes and contacts.

Figure 2 shows a diagram of the procedure for using the sensor described here.

A linear scan voltampetogram obtained from known chloride ion concentration samples using the procedure of Figure 2 is shown in Figure 3A. Figure 3B shows the standard deviation associated with each curve.

DESCRIPTION OF THE INVENTION

The present invention is established and characterized in the independent claims, while the dependent claims describe other features thereof.

As indicated above, in one aspect the invention describes a chloride ion detection sensor present in a liquid sample, of the type consisting of a working electrode, an auxiliary or counter electrode and a reference electrode, the electrodes including a contact , a conductive section and an active area, all the electrodes screen printed with an ink of conductive materials on a plastic sheet, where the conductive sections, the counter electrode and the working electrode are screen printed with a conductive platinum ink and the reference one with a Ag / AgCl ink in the active area.

Thus, as shown in Figure 1, the sensor described here consists essentially of a plastic sheet (4), usually porous in polyester or ePTFE, on which the contacts (1.3,2.3,3.3) are silk-screened and the sections (1.2,2.2,3.2) of a working electrode (2), a counter electrode (1) and a reference electrode (3), as well as the active area (1.1) of the counter electrode (1) and the active area (2.1) of the working electrode (2), the active area (3.1) of the reference electrode (3) being screen printed with an Ag / AgCl ink. The sensor described here also includes a screen printed insulating material (5) that covers it except in the active areas of the electrodes (1.1,2.1,3.1) and the contacts (1.3,2.3,3.3).

Although the figure shows concrete shapes for the electrodes (1,2,3), active areas (1.1,2.1,3.1) and sections (1.2,2.2,3.2), in the example illustrated rectangular, arched or circular, said Shapes are not limiting, these elements may have other suitable shapes and be dimensioned in correspondence with the final shape desired for the sensor.

Regarding conductive ink, platinum (Pt) is a noble metal with extensive applications in the electrochemical field. The properties of Pt electrodes are notoriously influenced by the presence of chloride. In fact, the interactions between chloride anions and Pt surfaces have been described and several works have been published studying these interactions. This metal is shown to be especially susceptible to chloride when high anode potentials are applied during experiments (see eg Hall, SB et al., Electrnchim. Acta, 2000, 45, 3573-3579). Also at the anode limits, chloride ions are specifically adsorbed on platinum surfaces, blocking the adsorption of oxygen by the surface and causing the appearance of chlorine.

Thus, the sensor described here efficiently detects the chloride ion considering the stable reaction between this anion and the platinum ink screen printed surfaces:

Pt H2O - e- ^ PtOH H + (R1)

PtOH - e- ^ PtO H + (R2)

PtO Cl- H2O ^ PtCl-ads 2OH (R3)

PtCl-ads Cl- 2e- ^ PtCl2 (R4)

PtCl2 H2O ^ PtO OH 2Cl- 2e- (R5)

According to a second aspect, the invention provides a method for the determination of the chloride ion in a liquid sample using the sensor previously described, comprising the procedure:

- an activation stage of the working electrode (2), adding to cover the electrode surface 200 ^ l of a 0.1 M solution of KNO3 and subjecting it to 5 consecutive cyclical sweeps of potential between -0.2 and 1.5 V ;

- a calibration step consisting of coating the electrode surface with a standard phosphate buffer solution pH = 6.2 containing chloride ion (approx. 200 ^ l) and applying an anodic deposition of 1.50 V for 15 seconds; record the linear voltamperogram by performing a cathodic sweep; remove the solution, for example by adsorption with filter paper, washing with distilled water and drying by absorption and adding standard solutions of chloride ion prepared in phosphate buffer (200 ^ l) successively, repeating the same process of washing and drying the electrode after each measurement and construct the calibration line representing the voltammetric peak as a function of the chloride concentration; Y

- a measurement step consisting of adding a small volume (200 ^ l) of a sample solution containing chloride ion to be tested by coating the electrode system on the same electrode once it is clean and dry and recording the voltamperogram, transferring it to the calibration line to determine the concentration of chloride in liquid sample.

The proposed mechanism for the operation of the sensor described here is as follows: the activation step with 0.1 M KNO3 favors the formation of platinum oxide according to reactions R1 and R2. Thus, the complexation of the chloride ion with the surface of the working electrode (2) occurs, as indicated in reaction R3. The anode deposition step results in the formation of an oxidized state of the chlorine complexes (reaction R4), after which they are reduced by cathodic Linear Sweep Voltammetry (LSV), recycling the oxide surface (reaction R5). Reactions R2 to R4 can occur at 1.5V at the anode. The R5 reaction occurs due to the desorption of the chloride ions adsorbed to more negative potentials.

As shown in figure 2, the anode potentials lead to the formation of oxidized Cl- - Pt complexes on the screen-printed working electrode (2) of the sensor described here.

Examples

Sensor Manufacturing

For the manufacture of the previously described sensor, 0.5 mm thick polyester films (could also be ePTFE) and Ag / AgCl and platinum inks supplied by Gwent Group (Mamhilad, United Kingdom) were used. The sensor was manufactured by sequential deposition on the polyester film. Briefly, with the silver and silver / silver chloride inks, cured at 120 ° C for 20 minutes, the conductive sections (1.2,2.2,3.2) and the reference electrode (3) were printed respectively. To define the counter electrode (1), a carbon ink (supplied by Achenson Colloiden, Scheemda) was used and hardened at 60 ° C for 30 minutes. To define the working electrode (2), platinum ink was used, which hardened at 80 ° C for 30 minutes. Finally, a dielectric ink (supplied by Achenson Colloiden, Scheemda) was used to delimit the final geometry of the three electrodes (1,2,3), which hardened under the same conditions.

Comparative analyzes were performed using a conventional sensor with a 1.6mm diameter platinum electrode as the working electrode (2), a platinum wire counter electrode (1), and a conventional silver / silver chloride electrode as the electrode. reference (3).

Preparation of sample

Samples obtained from wholemeal biscuits were prepared from a chicken broth, both commercial, and human urine and sweat samples were collected from healthy volunteers, which were stored at -20 ° C until use. Briefly, the biscuits were ground and calcined for 15 minutes at 400 ° C, the resulting product being dissolved in 500 ml of Milli-Q Water (Millipore, Bedford, USA). The suspension was filtered and centrifuged to remove suspended particles. The broth of Chicken was also centrifuged, and the fatty and solid fractions were removed prior to analysis.

Cyclic voltammetric measurements

Before carrying out these measurements, each electrode (1,2,3) was activated with 200 ^ l of a 0.1 M KNO3 solution and subjecting it to 5 consecutive cyclical sweeps of potential between -0.2 and 1.5 V, after which a calibration was made with a standard solution of phosphate buffer 0.31 mM pH = 6.2 containing different concentrations of chloride ion from corresponding solutions of NaCl 5, 10, 20, 30, 60, 80 and 100 mM and applying a sweep from -0.70 V to 1.30 V (vs. Ag / AgCl electrode) at 0.1 Vs-1, and potential steps of 0.02 V.

Voltammetric measurements were carried out using a PalmSens® electrochemical potentiostat with PS Trace 4.2 software (PalmSens® Instruments BV, Houten).

Experiments with the conventional sensor were performed in a cell with 10 ml of reference electrolyte from a 3M NaCl solution.

Linear scanning voltammetry (LSV) for the determination of chloride

Prior to LSV, a deposition step of 1.50 V was applied for 15 seconds and cathodic measurements were immediately carried out at a potential of 1.11 to 0.10 V, potential steps of 0.002 V and velocity of sweep of 0.20 Vs-1, obtaining a reduction peak at 0.80 V. The measurements were obtained with 200 ^ l of the reference electrolyte containing different concentrations of NaCl (Figures 3A and 3B).

Determination of chloride in samples containing chloride

LSV tests were performed with 200 ^ l of synthetic sweat containing chloride concentrations in the range of 1 to 150 mM, and reduction peaks were used to construct the calibration curve. Different samples were tested containing chloride using the sensor and the method of the invention (A) and corroborated using a specific chloride specific sensor by the potentiometric method (B). The results are shown in the following table:

These results show that the procedure of the invention is applicable to all kinds of liquid samples, being useful in the food, biological, health, environmental and pharmaceutical fields.

Claims (3)

1.-Procedure for the determination of chloride ion in a liquid sample using a chloride ion detection sensor, of the type consisting of a working electrode (2), an auxiliary or counter electrode (1) and a reference electrode (3) , including the electrodes a contact (1.3,2.3,3.3), a conductive section (1.2,2.2,3.2) and an active area (1.1,2.1,3.1), all the electrodes screen printed with an ink of conductive materials on a plastic sheet (4), the conductive sections (1.2,2.2,3.2), the counter electrode (1) and the working electrode (2) being screen printed with a conductive platinum ink and the reference electrode (3) with an Ag / AgCl ink in the active area (3.1) and including the detection sensor a screen printed insulating material (5) that covers it except in the active areas (1.1,2.1,3.1) of the electrodes and contacts (1.3,2.3,3.3), comprising The procedure: - a step of activating the working electrode (2), adding it to coat the electrode surface with a 0.1 M solution of KNO3 and subjecting it to 5 consecutive cyclical sweeps of potential between -0.2 and 1.5 V; - a calibration step consisting of coating the electrode surface with a standard phosphate buffer solution pH = 6.2 containing chloride ion and applying an anodic deposition of 1.50 V for 15 seconds; record the linear voltamperogram by performing a cathodic sweep; eliminate the solution, and add standard chloride ion solutions prepared in phosphate buffer successively, and construct the calibration line representing the voltammetric peak as a function of the chloride concentration; Y - a measurement step consisting of adding a small volume of a sample solution containing chloride ion to be tested by coating the electrode system on the same electrode and recording the voltamperogram, transferring it to the calibration line to determine the concentration of chloride in the liquid sample . 2. -Procedure according to claim 1, characterized in that in the activation stage, platinum oxide is formed according to reactions R1 and R2, the chloride ion complexing with the surface of the working electrode (2) screen-printed with the conductive ink platinum according to reaction R3. Pt H2O - e- ^ PtOH H + (R1) PtOH - e- ^ PtO H ¹ (R2) PtO Cl- H2O ^ PtCl-ads 2OH (R3) 3. Procedure according to the preceding claims, characterized in that in the anodic deposition of the calibration step, the chlorine complexes are oxidized according to reaction R4, after which they are reduced by cathodic linear scanning voltammetry, recycling the surface oxide according to reaction R5. PtCl-ads Cl- 2e- ^ PtCl2 (R4) PtCl2 H2O ^ PtO OH 2Cl- 2e- (R5)