ELECTROCHEMICAL SENSOR FOR THE DETECTION OF XYLAZINE IN PREPARED BEVERAGES

Abstract

The present invention relates to an electrochemical sensor for detecting xylazine in prepared beverages, since administration of this drug causes central nervous system depression, respiratory depression, bradycardia, hypotension, and even death. Based on its detection principle, the electrochemical sensor was designed with a preventative approach to avoid cases of sexual assault or theft, as it is portable and occupies minimal space. Its use is geared towards detecting this compound in acidic, carbonated, and other substrates served at low temperatures in public places. This device can also be useful for resolving cases in forensic toxicology. The results have been favorable, detecting the presence or absence of xylazine in alcoholic prepared beverages such as whiskey and tequila, among others, in the presence of interfering substances such as sugary and carbonated drinks.

Description

The present invention relates to a device for detecting sedatives in solutions, more specifically to an electrochemical method for detecting xylazine in prepared beverages. Likewise, the present invention relates to the use of Nickel / Aluminium nanoparticles for the manufacture of a high sensitivity, selectivity and rapid response electrode for xylazine. Another aspect of the present invention relates to providing a device useful in the primary prevention of cases of sexual abuse caused by the use of sedatives. OBJECTIVES OF THE INVENTION The first objective of the present invention is to employ nanomaterials and provide a scalable electrode manufacturing methodology with sufficient sensitivity and selectivity to detect the presence of low concentrations of xylazine in liquids. The second objective of the present invention is to provide a reliable and easy-to-use solution for the detection of 7LCL LÍVZZnZ / B / YIAI xylazine in prepared beverages as a primary measure for the prevention of sexual abuse. BACKGROUND Electrochemical sensors have proven to be an important analytical tool due to their high sensitivity and selectivity, fast response time, simple operation, and miniaturization. The fundamental concept in analyte detection by electrochemical sensors involves the measurement of the electric current generated by chemical reactions in the electrochemical system composed of electrode arrays in intimate contact with an electrolyte solution. One of the recent trends in the development of electrochemical sensors is the use of nanomaterials, which leads to substantial improvements in their performance with respect to sensitivity, selectivity, and detection rate. The surface of the nanomaterial electrode can be significantly transformed through the functionalization process. Nanomaterials and functional electrode nanostructures can detect small molecules such as glucose.As an example, Sijo Francis (2022) in Design, Fabrication, and Characterization of Multifunctional Nanomaterials, points out that PtCu bimetallic nanochains can be used to detect glucose very specifically in the presence of other biomolecules such as ascorbic acid, uric acid, and dopamine. Ni / AI nanoparticles show good physical, chemical and biological properties intrinsic to their nanometric size. Since they can be produced in different sizes and shapes, they can be functionalized with a wide range of ligands. Layered double hydroxides (LDHs) are known as anionic clays. In their configuration, the metal cations are at the center of the octahedra with shared edges and their vertices have hydroxide ions connected to each other to form infinite two-dimensional sheets. Their electrical relationships allow an agile interaction towards detection through electrochemistry for the oxidation of agents in a solution. They also have biocompatible properties, are non-toxic, and have great hydrolytic and chemical stability, as cited by Ay, AN, et.al. (2019) in A simple mechanochemical route to layered double hydroxides: synthesis of hydrotalcite-like Mg-AI-NO3-LDH by Manual Grinding in a Mortar. In relation to the substrate to be analyzed, the present invention has focused on xylazine since it is used as a veterinary sedative; the administration of this drug causes central nervous system depression, respiratory depression, bradycardia, hypotension and even death. Xylazine is a clonidine derivative that was developed as a sedative, analgesic and muscle relaxant for use in animals and, according to Reyes, JC, et.al. (2012) in The emerging of Xylazine as a new drug of abuse and its health consequences among drug users in Puerto Rico, it was not approved by the FDA for human use. Paddleford, RR, et. al. (1999) in Alpha2 agonists and antagonists, states that xylazine is available in veterinary pharmaceutical formulations containing 20, 100, and 300 milligrams of xylazine for administration in doses ranging from 0.25 to 4 milligrams per pound intramuscularly or 0.5 milligrams per pound intravenously. Likewise, Saishahas, K, et. al. (2021) in A portable electrochemical sensor for detection of the veterinary drug xylazine in beverage simples, notes that this dose range supports analgesia for 15–30 min, but the sedative effect may continue for 1–2 h. There are reports of xylazine being used to facilitate sexual assaults and robbery, since victims are unable to recall the events that took place while under the influence of this substance. However, since xylazine can be rapidly eliminated from the body in a period of 4.9 hours, as reported by Gallanosa AG, et. al. (1981) in Human xylazine overdose: a comparative review with clonidine, phenothiazines, and tricyclic antidepressants, it is very difficult to detect in biological samples, therefore, the determination of xylazine in alcoholic beverages could help prevent this type of assault. Finding materials to detect xylazine is important since this is the key to successful detection. Current technologies for xylazine quantification are based on gas or liquid chromatography coupled with mass spectrometry. These methods are reliable but require expert analysis, lengthy preparation times before analysis, and large and expensive analytical instruments. Nanotechnology, combined with electrochemical detection techniques, is emerging as an interesting alternative due to its high sensitivity, rapid response, simplicity, and low cost. 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ Some xylazine sensing works with an operating principle similar to that of this invention are as follows: ElShal, et. al. (2019), developed an electrochemical method for the estimation of xylazine in medicines and human serum, using an electrode based on multi-walled carbon nanotubes (MWCNT) and ionic liquids with a detection limit of 4.80 nM, the results of this study were published in the article Highly Sensitive Voltammetric Sensor Using Carbon Nanotube and an Ionio Liquid Composite Electrode for Xylazine Hydrochloride, published in the journal Anal. Sci. In the same year, Mendes LF et. al., in their article Forensic~ electrochemistry: Electrochemical study and quantification of xylazine in pharmaceutical and urine samples, proposed a method for the determination of xylazine in pharmaceutical formulations and urine for forensic analysis purposes. The electrochemical study uses a glassy carbon electrode and differential pulse voltammetry (DPV), achieving a detection limit of 120 nM / L. These two works have an analytical and diagnostic focus, which does not make them candidates as a means of abuse prevention. However, there are reports from Saishas et. al. (2021 and 2022), who have developed a portable electrochemical sensor for the detection of xylazine in prepared beverages using absorption stripping voltammetry (AdSV) and a graphene nanoplatelet-modified electrode as the sensing phase. This sensor shows a sensitivity of 0.1 mg / L as the detection limit and 0.4 mg / L as the quantification limit. In 2022, the same research group developed an electrochemical sensor using paper-based carbon electrodes modified with polyaniline nanocoral, achieving a detection limit of 0.06 pg / mL and evaluating it in prepared beverages. However, these studies do not report the response time and reusability of the sensor, which are fundamental parameters for considering it as a preventive measure. The use of layered double hydroxides (LDHs) as a xylazine recognition base in working electrodes for electrochemical systems has not been described. LDHs are known as anionic clays; their electrical relationships allow for rapid interaction for electrochemical detection of oxidizing agents in solution. They also have biocompatible properties, are nontoxic, and exhibit high hydrolytic and chemical stability. Good results have been demonstrated in terms of response time sensitivity and long-term stability in device applications. Additionally, the works described in the state of the art do not report a response time, which is a fundamental parameter to be considered as a primary preventive measure in cases of sexual abuse caused by the use of sedatives, nor do they carry out a study on the effect of pH or temperature on the performance of xylazine detection in buffer solutions and different types of beverages. Regarding patents, the preliminary search has not yielded any results for work on the principle and application of this invention, but various technologies have been found for the detection of other substances used in sexual assault. The technology referred to in patent document US 8,241,575 B2 is characterized by the detection of ketamine, rohypnol and gamma hydroxybutyrate (GHB) in different beverages and differs from the present invention in that it is a device that uses molecularly imprinted polymers having a cross-linked core and several polymers adhered to the mineral to detect these analytes, wherein the core has molecular hexagonal cavities adapted to selectively receive and bind to the marker molecular structures and also uses a colorimetric indicator. This device comprises a housing having an inlet for receiving a flow of fluid, wherein the inlet is coated with a molecularly imprinted polymer having a cross-linked core which has molecular sized cavities adapted to receive and displace the molecular marker. US patent document 9,029,098 B1 relates to a date rape drug detector comprising an elongated shaft having an upper end and a lower end. The upper end of the shaft can be bent at a ninety-degree angle to grip the rim of a beverage container. Proximate the lower end is a chamber having a test strip that can be unfolded to a perpendicular position with respect to the shaft. Along the strip are several reactive dots that change color in the presence of a date rape drug (GHB). A first dot is exposed while the remaining dots are covered with a progressively thicker layer of water-soluble acetate. Adjacent to each of the covered dots is a rectangle that changes color in the presence of the liquid. Therefore, each 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ spot and associated rectangle require different immersion times than the others to be exposed. Once a rectangle changes color (preferably to red), the user is quickly alerted that the layer on the corresponding adjacent spot has dissolved. The user then removes the strip to allow the spot to air dry to complete the reaction. US Patent 7,238,533 B1 describes an illicit drug detection technology that includes a substance chemically reactive to a suspected drug. This substance can be mixed with nail polish, applied to a decal, or simply discreetly moistened with a suspicious liquid. It is presented as an alternative that can be easily used by the user. Another alternative for detecting date rape drugs in beverages is presented in US patent document 201 1 / 0039346A1 , which describes a stirring straw and a chemical reagent that coats the surface of the straw and changes color upon contact with a date rape drug (Gamma hydroxybutyrate or ROHYPNOL). This invention includes a method of detecting date rape drugs comprising the steps of coating a stirring device with a chemical reagent; inserting the stirring device into a beverage; and observing the stirring device for an indication of the presence of a date rape drug. The stirring straw can be easily stored in the user's purse and can be used when necessary. Also, the use of this device helps a victim of a possible rape alert the authorities about people who might use these types of drugs for sexual assault purposes. Another device for detecting this type of drug is presented in US patent document 201 3 / 0209325 A1. The object of this invention was to generate a discreet and concealable alternative, mounted on a fingernail to the drug recognition element (test strip), easily alerting the user to the presence of any of the date rape drugs. Unlike more cumbersome detection methods, a small test strip mounted under the fingernail places a small sample of the suspected beverage and, through a colorimetric change, indicates the presence or absence of these drugs. It should be noted that the works described in the state of the art do not report a response time, which is a fundamental parameter to be considered as a primary preventive measure in cases of sexual abuse caused by the use of sedatives, nor do they carry out a study on the effect of pH or temperature on the performance of xylazine detection in buffer solutions and different types of beverages. TECHNICAL PROBLEM TO BE SOLVED Based on the above, electrochemical sensors exist for detecting analytes in liquid media; however, the invention presented here addresses: 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ detection of xylazine in substrates such as prepared alcoholic beverages that support the presence of interferents such as sugary and carbonated beverages, • with the sensitivity required to detect the presence of xylazine at levels below 0.1 mg / L, using nanomaterials on the electrode surface for the construction of an electrochemical sensor, • a fast response time in the range of 5 seconds, and • the ability to reuse the electrodes. BRIEF DESCRIPTION OF THE INVENTION A simplified summary is presented below, intended to provide an overview of the invention, which should be complemented by the detailed description of the invention presented below. Unlike what is described in the state of the art, the present invention consists of an electrochemical sensor for detecting xylazine, which is designed with a preventive approach, since it is a portable device that occupies a minimum space, which allows it to be used to avoid cases of sexual abuse or theft, its use is oriented to the detection of this compound in acidic, carbonated, etc. substrates, which are served at low temperatures in public places. The results have been favorable, detecting the presence or absence of xylazine in prepared alcoholic beverages such as whiskey, tequila, and others, as well as beverages such as bottled water, coffee, and apple juice, in the presence of interfering agents such as sugary and carbonated drinks. Furthermore, this device may be useful for resolving forensic toxicology cases. Therefore, the manufacture of these electrochemical sensors may be an option. ZLCL Ln / Zznz / B / YIAI easy to operate requiring small sample amount for detection of xylazine in different kinds of beverages. ZLCL Ln / Zznz / B / YIAI BRIEF DESCRIPTION OF THE FIGURES The attached figures are explained as follows: Figure 1. Top view of the electrochemical sensor for the detection of xylazine in prepared beverages, which is composed of a working electrode (Ai), a reference electrode (A2) and a counter electrode (A3), where its four layers are shown. Figure 2. Side view of the electrochemical sensor for the detection of xylazine in prepared beverages, showing its four layers. Figure 3. Cyclic voltammetry of the electrochemical sensor for the detection of xylazine in prepared beverages, evaluated in the presence and absence of xylazine. Figure 4. Differential pulse voltammetry electrochemical sensor for the detection of xylazine in prepared beverages, evaluated at different xylazine concentrations. Figure 5. Evaluation of reuse of the electrochemical sensor for detection of xylazine in prepared beverages. Figure 6. Differential pulse voltammetry of the electrochemical sensor for the detection of xylazine in prepared beverages, evaluated at different concentrations of xylazine in different beverages. DETAILED DESCRIPTION OF THE INVENTION 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ The present invention consists of the manufacture of an electrochemical sensor for the detection of xylazine in prepared beverages designed with a preventive approach, since it is a portable device that occupies minimal space. In a first step of the present invention, a catalytic ink was prepared containing layered nickel / aluminum double hydroxide nanoparticles as a xylazine recognition element, which has a composition between 0.5-1.0% layered nickel / aluminum double hydroxides, together with Vulcan carbon (0.5-1.0%) and a perfluorosulfonic acid compound (7%), isopropyl alcohol (90%) and a quaternary salt of tetrabutylammonium bromide (2%). These components are mixed at room temperature for 5 minutes by means of a vortex stirrer. Then, 2 pL of the catalytic ink is manually deposited on the working electrode, allowing it to dry at room temperature for 5 minutes. This catalytic ink was used for the evaluation of glassy carbon electrodes, where the developed ink was deposited, where first an electrochemical cleaning of the surface of the glassy carbon electrode is carried out, using the cyclic voltammetry technique in acidic conditions and subsequently a cleaning is done with distilled water to proceed to the deposit of the catalytic ink that contains nanoparticles of Nickel / Aluminum double hydroxide lamellar. Several evaluations were made to obtain the calibration curve using different concentrations of xylazine in buffer solutions with acidic pH and temperature of 25°C. An Ag / AgCl electrode was used as the reference electrode and graphite as the auxiliary electrode. As shown in Figures 1 and 2, the electrochemical sensor for the detection of xylazine in prepared beverages comprises: 0. A plastic base or non-conductive material, which can be paper or cloth; 1. A first or initial layer (A), as shown in Figure 1A, which belongs to a three-electrode system, which are divided into three sections: working electrode (Ai), reference electrode (A2) and graphite counter electrode (A3); this first or initial layer corresponds to a carbon material. 2. A second layer or recognition surface (B), as shown in Figure 1B, in which both the reference electrode with Ag / AgCI (B1) and the working electrode with the catalytic ink containing Nickel / Aluminum double hydroxide lamellar nanoparticles deposited on the working electrode (B2) are functionalized. 3. A third or insulating layer (C), as shown in Figure 1C, in which the insulating material is deposited on a section of the electrodes located above the work area and the electrical contacts (C1). This insulating material is of the plastic or polymer type. 4. A fourth layer or metallic layer (D), as shown in Figure 1 D, where a conductive material, preferably gold or copper, is deposited on the terminals of each electrode as an electrical contact (Di). Once the electrochemical sensor for the detection of xylazine in prepared beverages has been manufactured, its use consists of introducing it vertically into the beverage for a minimum of 5 seconds, in order to obtain a reading that allows the presence and concentration of xylazine to be discerned up to a detection limit of 0.1 mg / L. The electrochemical sensor for the detection of xylazine in prepared beverages can be reused for up to 7 cycles while maintaining a reading efficiency above 80%, as indicated in Figure 5. Likewise, detection evaluations were carried out using xylazine in different beverages, such as mezcal, whiskey, wine, cola, coffee and bottled water to observe the signal behavior using the differential pulse voltammetry technique, obtaining favorable results for its detection, as shown in Figure 6. BEST WAY TO CARRY OUT THE INVENTION For the purpose of illustrating the technology and process of the present invention, the following examples are provided. These examples are intended to illustrate both the system (device) and the method and are not intended as limiting conditions of the invention. EXAMPLE 1 . The electrochemical sensor for the detection of xylazine in prepared beverages was fabricated as described above. For the preparation of the catalytic ink containing nanoparticles of Nickel / Al aluminum double hydroxide layered, we worked with containing 5mg of this material, 5mg of carbon ZLCL Ln / Zznz / B / YIAI Vulcan, 20mg of the quaternary salt of tetrabutylammonium bromide, 70μI_ of a perfluoroisobutyric acid compound and 900μI_ of isopropyl alcohol. All these components were mixed in a vortex mixer to homogenize the catalytic ink for 5 minutes. 2μI_ of this catalytic ink was manually deposited on the working electrode, allowed to dry for 5 minutes and then evaluated in a potentiostat / galvanostat equipment. A 10mgL'1 sample of xylazine was used in a buffer solution of boric acid, glacial acetic acid and phosphoric acid, also known, and hereafter referred to, as Britton-Robinson Buffer. The following tests were performed to verify the operation of the electrochemical sensor for the detection of xylazine in prepared beverages. a) Evaluation for the detection of xylazine. Xylazine detection was evaluated by cyclic voltammetry using a pH 3 buffer solution, as shown in Figure 3. This sensor is based on the use of Ni / AI double layered hydroxides. Figure 3 shows that in the presence of xylazine, two oxidation peaks occur at 0.416 V and 1.017 V, which do not appear when the analyte is not present using the cyclic voltammetry technique, using a drop of sample that can. A response time of 5 seconds was used for detection. b) Determination of the detection limit. A drop of xylazine solution in Britton-Robinson pH 3 buffer was used at different xylazine concentrations with a range of 0 to 100 mgL'1. The evaluation was carried out using the differential pulse voltammetry technique with a scan rate of 20 mV s1; amplitude 100 mV; press width 2000 ms. Figure 4 shows the differential pulse voltammetry of the evaluation of the Nickel / Aluminum nanoparticle electrode for the detection of xylazine in prepared beverages where an increase in current is presented with an increase in xylazine concentration where a detection limit of 0.00561 mgL-1 is obtained. 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ c) Evaluation of the reuse of the electrochemical sensor for the detection of xylazine in prepared beverages. The reuse cycles were evaluated, where each cycle is defined as the detection when the xylazine sample placed on the Nickel / Aluminum nanoparticle electrode was used for the detection of xylazine in prepared beverages and the detection was carried out by differential pulse voltammetry, a response time of 5 seconds was taken and the current density value was recorded. Subsequently, the electrode was cleaned by applying distilled water by means of a squeezing bottle on the sensor surface to eliminate any traces of xylazine from the sample applied in the previous detection. Then the same electrode was evaluated using the same conditions as the previous detection. Figure 5 shows the results of this reuse evaluation, where the current does decrease with each cycle where number 5 the current decreases by 8.1364% compared to the initial cycle.There are no reports of reuse tests in previous studies, which is one of the benefits of the nickel / aluminum nanoparticle electrode developed for the detection of xylazine in prepared beverages. After cycle 11, there is a 38.3428% decrease. 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ d) Evaluation of the efficiency of xylazine detection in different beverages. The selected beverages were tequila, whiskey, cider, apple juice, bottled water, and coffee. The xylazine concentration selected was 0.1 mg / L, and the sample temperature was 25°C. The results obtained are summarized in Table 1, and Figure 6 shows the differential pulse voltammogram for the xylazine detection evaluation in beverages. A scan rate of 20 mV s1; amplitude of 100 mV; and press width of 2000 ms were used. Of the selected samples, only the xylazine sample in coffee could not be detected by the Nickel / Aluminum nanoparticle electrode for the detection of xylazine in prepared beverages, attributed to the complexity of the sample. Table 1 Substrate Xylazine Concentration (mg / L) Operation Time Result Tequila 0.1 5 sec. Detected Whiskey 0.1 5 sec. Detected Cider 0.1 5 sec. Detected Apple Juice 0.1 5 sec. Detected Bottled Water 0.1 5 sec. Detected Coffee 0.1 5 sec. Not Detected 7LCL ίη / ΖΖΠΖ / Β / ΥΙΛΙ The invention has been sufficiently described to enable a person of ordinary skill in the art to reproduce it and obtain the results recited herein. However, any person skilled in the art to which this invention pertains may be able to make modifications not described herein. However, if the material claimed in the following claims is required for the application of these modifications to the composition or manufacturing process thereof, said compositions or processes shall be included within the scope of this invention.

Claims

1. Electrochemical sensor for the detection of xylazine in prepared beverages characterized by having a plastic base or non-conductive material, a first layer or initial layer (A), a second layer or recognition surface (B), a third layer or insulating layer (C) and a fourth layer or metallic layer (D).

2. Electrochemical sensor for the detection of xylazine in prepared beverages, according to that described in claim 1, characterized in that the first layer or initial layer (A) corresponds to a carbon material and consists of a system of three electrodes, which are divided into three sections: working electrode (Ai), reference electrode (A2) and graphite counter electrode (A3).

3. Electrochemical sensor for the detection of xylazine in prepared beverages, according to what is described in claim 1, characterized in that in the third layer or insulating layer (C), the plastic or polymer type insulating material is deposited on the section of the electrodes located on the work area and the electrical contacts (Ci).

4. Electrochemical sensor for the detection of xylazine in prepared beverages, according to what is described in claim 1, characterized in that in the fourth layer or metallic layer (D), a conductive material, preferably gold or copper, is deposited on the terminals of each electrode as an electrical contact (D1).

5. Electrochemical sensor for the detection of xylazine in prepared beverages, according to that described in claim 1, characterized in that in the second layer or recognition surface (B), both the reference electrode is functionalized with Ag / AgCI (Bi), and the working electrode with the catalytic ink containing Nickel / Aluminum double hydroxide laminar nanoparticles deposited on the working electrode (B2).

6. Electrochemical sensor for the detection of xylazine in prepared beverages, according to that described in claims 1 and 5, characterized in that the catalytic ink contains nanoparticles of Nickel / Aluminum double layered hydroxide, which has a composition between 0.5-1.0% of Nickel / Aluminum double layered hydroxides, together with Vulcan carbon (0.5-1.0%) and a compound of perfluorosulfonic acid (7%), isopropyl alcohol (90%) and a quaternary salt of tetrabutylammonium bromide (2%).

7. Use of the electrochemical sensor for the detection of xylazine in beverages prepared according to that described in claim 1, characterized in that it must be introduced vertically into the beverage for a minimum time of 5 seconds, to obtain a reading that allows discerning the presence and concentration of xylazine up to a detection limit of 0.1 mg / L.

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