A novel ferrocene-based cyclotriphosphazene-mwcnt modified electrode with electrochemical properties used for the sensitive determination of ractopamine, and its production method

Abstract

The invention relates to a surface-imprinted combined modified electrode and its production method, comprising a novel chalcone-substituted cyclotriphosphazene (CBRG-Fe) and multi-walled carbon nanotube (MWCNT) composite, intended to be used as an electrochemically active sensor for the sensitive determination of ractopamine.

Description

[0001] A NOVEL FERROCENE-BASED CYCLOTRIPHOSPHAZENE-MWCNT MODIFIED ELECTRODE WITH ELECTROCHEMICAL PROPERTIES USED FOR THE SENSITIVE DETERMINATION OF RACTOPAMINE, AND ITS PRODUCTION METHOD

[0002] TECHNICAL FIELD

[0003] The invention relates to a surface-imprinted combined modified electrode and its production method, comprising a novel chalcone-substituted cyclotriphosphazene (CBRG-Fe) and multi-walled carbon nanotube (MWCNT) composite, intended to be used as an electrochemically active sensor for the sensitive determination of ractopamine.

[0004] PRIOR ART

[0005] Meat producers work to supply safe, nutritious, and palatable foods in a wide range of forms, from various fresh ground products made from beef, lamb, veal, and poultry to more highly processed products. Meat is presented to consumers using one or more processing technologies such as curing, smoking, fermentation, mixing, chopping, and cooking. The types of fresh meat offered to consumers differ in eating quality and composition depending on the age, sex, genetics (breed or species), and the specific cuts of the animal from which they are obtained. Nutrition, along with other production strategies or practices, also influences these characteristics. Management strategies used in meat animal production are selected to maximize production efficiency and meat quality, minimize environmental impact, and ensure animal health and welfare. Feed additives known as beta-agonists are used to improve growth efficiency, increase meat yield, and enhance carcass composition. Beta-agonists are naturally occurring and synthetic organic compounds that share a common core chemical structure characteristic of a class of compounds called phenethanolamines. Two naturally occurring beta-agonists are adrenaline (epinephrine) and noradrenaline (norepinephrine). However, neither of these compounds has beneficial effects on animal growth. Therefore, various synthetic beta-agonists — such as ractopamine, cimaterol, clenbuterol, L-644,969, salbutamol, and zilpaterol — are widely used for their muscle growth-promoting and anti-obesity effects.Ractopamine (RAC), an effective synthetically produced beta-agonist, is a compound that promotes animal growth by accelerating the increase in skeletal muscle mass and reducing body fat deposition. Because lipogenesis increases and the capacity for protein accretion decreases during the final 28 days before slaughter, RAC is frequently used in animal feed supplements during this period. The application of this additive reverses the metabolic profile by stimulating protein accretion and lipolysis while inhibiting lipogenesis. As a result, carcasses with lower fat content are obtained, along with an approximate 12% increase or a 20-40 kg gain in body weight. However, supplementing animals with RAC may cause them to become more stressed and hyperactive, which in turn negatively affects meat quality. Consequently, it can also lead to the production of meat containing high levels of RAC residues, resulting in quality deterioration and even rejection of the product by more selective markets. RAC residues in food products are transferred to consumers through the food chain. The potential effects and hazards of RAC in humans include symptoms such as palpitations, tachycardia, anxiety, confusion, muscle tremors, and dizziness. Because it poses a potential risk to public health, it has been identified as a source of public concern and has generated considerable debate. Long-term consumption of meat containing RAC has been associated with chromosomal aberrations and even the development of malignant tumors. For this reason, although RAC has been authorized as a feed additive to promote growth in fattening pigs and cattle in approximately 26 countries — including the United States, Australia, Canada, Japan, Mexico, Brazil, and Argentina (for pigs only) — many countries, including the European Union, China, Taiwan, Russia, India, and Turkiye, have banned its use on safety grounds. According to the Codex Alimentarius Commission, the maximum residue limits (MRLs) of RAC are set at 0.01 mg / kg in muscle meat, 0.04 mg / kg in liver, and 0.09 mg / kg in kidney for cattle and pigs. The U.S. Food and Drug Administration (US FDA) has established the acceptable daily intake at 1.25 mg / kg body weight per day. The World Health Organization (WHO) has set the permitted level of RAC in meat at 5 mg / kg. Considering the harmful effects of RAC on human health, it is critically important to develop reliable analytical methods for the sensitive monitoring, rapid detection, and accurate determination of RAC residue levels in animal-derived foods. For this purpose, high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), Raman spectroscopy, and chemiluminescence methods, as well as electrochemicaldetermination — which has emerged in recent years as an effective alternative technique — have been employed for the analysis of RAC. Despite the numerous advantages of these technologies, their widespread application is limited by factors such as complex sample preparation, extensive pretreatment time, high cost, complicated instrumentation, and other shortcomings. In comparison with other analytical methods, electrochemical determination offers several advantages, including high sensitivity toward the target analyte, rapid response time, low cost, and a relatively simple analytical approach.

[0006] Phosphazenes, which constitute an important class of inorganic compounds, are composed of repeating -P=N- units in their structures and feature linear or cyclic frameworks in which each phosphorus atom is bonded to organic or inorganic substituents (R). One prominent member of this group is cyclotriphosphazene (hexachlorocyclotriphosphazene, trimer, N3P3CI6), which exhibits a planar molecular geometry and demonstrates high chemical and thermal stability against heat, light, and various reaction conditions. Cyclotriphosphazenes enable the synthesis of novel compounds with diverse physical and chemical properties through reactions with aliphatic or aromatic reagents, depending on the nature of the substituents and the reaction conditions employed. This versatility allows cyclotriphosphazenes to be selected as core building blocks for use in a wide range of application areas. Among the functional groups that can be attached to phosphazene compounds via nucleophilic substitution reactions are chaicone derivatives. Chaicones belong to a class of flavonoids and exhibit a broad range of applications. Owing to the presence of two phenyl rings and an a,[3-unsaturated carbonyl system in their structures, chaicone compounds are highly reactive.

[0007] Previous studies conducted separately on ractopamine and on ferrocene-based chalcone-substituted cyclotriphosphazene compounds are presented below:

[0008] In the known technique, surface-imprinted electrodes were modified with multi-walled carbon nanotubes (MWCNTs) and molecularly imprinted membranes (MIMs) prepared on the electrode surface using thermal polymerization techniques. Ractopamine was determined under optimized conditions using Differential Pulse Voltammetry (DPV). The results demonstrated that the sensor response to ractopamine concentration exhibited a linear correlation in the range of 20 nM to 200 nM, with a detection limit of 6 nM, indicating suitable sensitivity and selectivity for ractopamine detection. Recoveries based on pig urine samples reached 87.7-96.9%. In another study, well-dispersed graphene / gold nanorod (G / GNR) composites were synthesized by the direct reduction of a mixture of graphene and gold solution using sodium borohydride. The synthesized composites were characterized by ultraviolet-visible spectroscopy and Fourier-transform infrared spectroscopy. Glassy carbon electrodes were modified with the G / GNR composites to fabricate an electrochemical ractopamine sensor. The peak currents varied linearly with ractopamine concentration in the range of 1 x 10’9to 2.7 x 10’6mol L"1, and a detection limit of 5.1 x 10’10mol L-1(S / N = 3) was achieved.

[0009] In a study conducted for the determination of ractopamine, an electrochemical sensor was fabricated by modifying a glassy carbon electrode (GCE) with carbon nanotubes (CNTs) and antimony tin oxide (ATO) nanoparticles. The surface layer was characterized using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and ATR FT-IR spectroscopy. Compared with an unmodified GCE and a CNT-modified GCE, the ATO NPs / CNTs / GCE exhibited catalytic activity toward the oxidation of ractopamine, showing a well-defined anodic peak at 600 mV. In another study aimed at the rapid determination of ractopamine (RAC), a novel disposable electrochemical sensor was developed based on the use of a magnetic surface-imprinted electrode (MSPE) modified with an iron oxide magnetic nanoparticle doped onto reduced graphene oxide (Fe3O4 / rGO). The morphology, structure, and composition of the nanocomposites were characterized by transmission electron microscopy (TEM), X-ray absorption spectroscopy (XANES), and X-ray photoelectron spectroscopy (XPS). The electrochemical properties of the modified MSPE under optimized conditions were recorded using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The peak currents obtained by DPV increased linearly with increasing RAC concentration, and the sensor exhibited two linear detection ranges of 0.05-10 pM and 10-100 pM, with a detection limit of 13 nM (S / N = 3). In another study for ractopamine determination, the surface-imprinted electrode was modified with ordered mesoporous carbon (OMC) and gold nanoparticles (AuNPs). For sensor characterization, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) were employed. Under optimized conditions, the DPV peak currents showed a validated linear relationship with the logarithmic (log) concentration of RAC in the range of 5 x 10’11to 1 x 10’9mol L-1(R2= 0.9911 ), and the detection limit was calculated as 4.23 x 10’11mol L’1(S / N = 3).For the rapid and highly sensitive electrochemical detection of ractopamine in meat, a dual nanostructured material consisting of graphitic carbon nitride (g-C3N4) and a bimetallic MOF-derived Cu@CoO / NC composite, denoted as g-C3N4 / CU@COO / NC, was prepared via a low-temperature pyrolysis process. The g-C3N4 / CU@COO / NC material was characterized using various techniques, including X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The surface-imprinted electrode fabricated for the detection of ractopamine (RAC) in meat samples exhibited electrochemical oxidation performance, providing a wide linear detection range (0.005 pmol L-1to 32.73 pmol L-1) and a low detection limit of 1.53 nmol L"1. Ferrocene chaicone (FAC)-based chromogenic, voltametric, and potentiometric sensors were developed for the selective quantitative determination of copper (II).

[0010] In UV / visible measurements, the addition of Cu (II) ions to FAC caused the disappearance of the low-energy band, accompanied by a 30 nm hypsochromic shift of the high-energy band. The cyclic voltammogram (CV) of FAC underwent electrochemical changes upon the addition of Cu(ll) ions, attributable to the formation of the FAC-Cu(ll) complex.

[0011] Patent applications related to ferrocene side-group-containing chaicone-substituted cyclotriphosphazene compounds and ractopamine are presented below:

[0012] In patent application No. CN113484377A, a graphene nanoscroll-based ractopamine electrochemical sensor was fabricated by spin-coating a glassy carbon electrode and subsequently drop-casting a ractopamine antibody solution onto the electrode surface to obtain the ractopamine electrochemical sensor.

[0013] Patent application No. CN113340960A relates to a method for preparing an electrochemical sensor for the detection of ractopamine based on a silica solution.

[0014] Patent application No. CN111239218A belongs to the field of electrochemiluminescence detection and, in particular, relates to an electrochemiluminescent aptamer sensor for ractopamine detection, as well as its preparation method and application method.

[0015] Patent application No. CN109813774A falls within the fields of analytical chemistry and photoelectrochemical sensors and specifically relates to a method for detecting ractopamine in lean meat extracts using a photoelectrochemical sensor.Patent application No. CN107132260A describes an electrochemical sensor for the detection of ractopamine using a nanomaterial-based glassy carbon electrode.

[0016] Patent application No. CN106198502A discloses a method for preparing an electrochemiluminescent ractopamine sensor and falls within the technical field of novel nanofunctional materials and biosensors.

[0017] Patent application No. CN105738352A describes a method for preparing a photoelectrochemical ractopamine sensor and belongs to the technical field of novel nanometer-scale functional materials and biosensors.

[0018] Patent application No. CN105445356A describes a portable ractopamine molecularly imprinted screen-printed electrochemical sensor.

[0019] Patent application No. CN104020197A relates to a ractopamine molecularly imprinted piezoelectric sensor and its method of preparation.

[0020] Patent application No. CN103983613A pertains to the field of SPR sensor chips and, in particular, relates to a ractopamine molecularly imprinted SPR sensor chip and its method of preparation.

[0021] In addition to all these considerations, existing sensor studies for the determination of ractopamine predominantly employ conventional three-electrode systems consisting of a reference, working, and counter electrode. Such three-electrode configurations suffer from several drawbacks, including difficulties in maintaining electrode surface cleanliness, the requirement for relatively large sample volumes, the lack of suitability for on-site or in situ analysis, and potential leakage issues at the electrodes. Furthermore, problems related to the stability and cost of modified electrodes after fabrication also represent significant challenges. Therefore, there is a clear need for the development of new and improved modified electrode systems for ractopamine determination.

[0022] BRIEF DESCRIPTION OF THE INVENTION

[0023] The invention relates to a surface-imprinted, combined modified electrode containing a novel chalcone-substituted cyclotriphosphazene (CBRG-Fe) and multiwalled carbon nanotube (MWCNT) composite, as well as its production method, intended for use as an electrochemically active sensor for the sensitive determination of ractopamine. In this context, on-site analysis is achieved using thishighly sensitive surface-imprinted modified electrode, developed through both a novel method and the synthesis of a new compound.

[0024] Our invention, instead of employing conventional three-electrode systems in which the reference, working, and counter electrodes are independent, utilizes a surface electrode that integrates the reference, working, and counter electrodes onto a single platform. This configuration enables low-cost analysis by depositing a very small amount of sample (at the microliter level) onto the sensor surface, offers excellent reproducibility, and provides compatibility with a portable device that allows in situ determination of target analytes. Consequently, when factors such as solvent consumption and column costs associated with chromatographic methods are taken into account, the surface-imprinted electrode of the present invention is highly cost-effective. In addition, the very low amounts of CBRG-Fe and MWCNT applied to the electrode surface for analysis further reduce the sensor cost, making the prepared sensor considerably more economical compared to other analytical methods. Moreover, the high stability of the synthesized CBRG-Fe compound, together with its low cost and time-efficient synthesis steps, constitutes a significant additional advantage.

[0025] LIST OF FIGURES

[0026] Figure 1. Structural representation of the CBRG-Fe compound (A), MALDI-TOF MS spectrum and31P-NMR spectrum (B),1H-NMR spectrum (C) Figure 2. SEM image of CBRG-Fe / SPE

[0027] Figure 3. SEM image of CBRG-Fe-MWCNT / SPE

[0028] Figure 4. CVs of SPE, CBRG-Fe / SPE, MWCNT / SPE, and CBRG-Fe- MWCNT / SPE (A); EIS of SPE, CBRG-Fe / SPE, and CBRG-Fe- MWCNT / SPE (B); DPVs of SPE, CBRG-Fe / SPE, and CBRG-Fe- MWCNT / SPE in the presence of RAC (C)

[0029] Figure 5. Graphical response obtained using the CBRG-Fe-MWCNT / SPE electrode for ractopamine (RAC)

[0030] Figure 6. Calibration curve of the CBRG-Fe-MWCNT / SPE sensor for ractopamine (RAC)

[0031] Figure 7. Selectivity of the proposed sensor for RAC analysis. Interfering substances: D-glucose (D-Glu), dopamine (Dop), glycine (Gly), ascorbic acid (AA), and uric acid (UA)

[0032] Figure 8. Flow diagram of CBRG-Fe-MWCNT / SPEDETAILED DESCRIPTION OF THE INVENTION

[0033] The novel surface-imprinted combined modified electrode of our invention, which is used as an electrochemically active sensor for the sensitive determination of ractopamine, comprises a chalcone-substituted cyclotriphosphazene (CBRG-Fe) and multi-walled carbon nanotube (MWCNT) composite.

[0034] For the CBRG-Fe compound used in our invention, at least 20 mL (optimally 40 mL) of acetone was added into a single-neck round-bottom flask. Subsequently, CBRG, synthesized according to the method described by Carriedo and co-workers, was added and the mixture was stirred. Then, at least 0.28 g (optimally 0.36 g) of the ferrocene-chalcone compound was introduced into the reaction medium. Finally, at least 0.14 g (optimally 0.35 g) of Cs2CO3was added, and the reaction mixture was stirred. The reaction was monitored, then terminated and purified. The formation of the reaction was confirmed, and the CBRG-Fe compound was successfully synthesized. Characterization studies of the synthesized CBRG-Fe compound and surface characterization studies of the electrode were carried out. Scanning electron microscopy (SEM) was employed to confirm the structures of the CBRG-Fe and MWCNT compounds and to investigate the surface morphological characteristics of the electrode. It was observed that the CBRG-Fe-MWCNT composite exhibited an irregular morphology on the electrode surface. These results demonstrate the formation of the CBRG-Fe-MWCNT composite on the electrode surface.

[0035] For the preparation of a sensor for ractopamine determination using the CBRG-Fe-MWCNT composite, the surface of the working electrode was first modified with CBRG-Fe and the CBRG-Fe-MWCNT composite. The CBRG-Fe and CBRG-Fe-MWCNT composites were prepared by sonication in ultrapure water for at least 1 hour. After the sonication process, at least 10 pL (optimally 12.5 pL) of the dispersion was drop-cast onto the surface of the working electrode and allowed to dry. Subsequently, cyclic voltammetry (CV) measurements were performed in the presence of 5 mM Fe(CN)63- / 4’, and the anodic and cathodic peak potentials and current signals were compared. From the CV results, the potential differences between the anodic and cathodic peaks of SPE, CBRG-Fe / SPE, and CBRG-Fe-MWCNT / SPE were determined. Compared to SPE and CBRG-Fe / SPE electrodes, the CBRG-Fe-MWCNT / SPE electrode exhibited a lower peak-to-peak potentialseparation (AEP). This observation confirms that the electron transfer kinetics between the analyte and the CBRG-Fe-MWCNT / SPE electrode are faster.

[0036] To investigate the electron transfer capability of the modified electrodes, Nyquist diagrams were obtained using the electrochemical impedance spectroscopy (EIS) technique. In Nyquist diagrams, the diameter of the semicircle corresponds to the charge-transfer resistance (Ret) at high frequency, which reflects the electron transfer capability of the modification material. The Ret values obtained for SPE, CBRG-Fe / SPE, and CBRG-Fe-MWCNT / SPE were determined. The Ret value of CBRG-Fe-MWCNT / SPE was lower than those of the other electrodes. This result indicates that the electrode possesses a higher electron transfer capability. Based on these findings, it can be stated that the results obtained from the EIS technique are consistent with, and show good agreement with, those obtained from the CV technique.

[0037] The DPVs of SPE, CBRG-Fe / SPE, and CBRG-Fe-MWCNT / SPE were examined in 0.1 M PBS (pH 7.4) in the presence of 200 pM RAC. Compared with the other electrodes, CBRG-Fe-MWCNT / SPE exhibited a higher current signal. This result indicates that the CBRG-Fe-MWCNT / SPE electrode has a strong sensing capability toward the electrooxidation of ractopamine.

[0038] The analytical performance of the CBRG-Fe-MWCNT / SPE sensor — including its linear concentration range, limit of detection, and sensitivity — was evaluated under optimized conditions using the differential pulse voltammetry (DPV) technique. The DPV responses of CBRG-Fe-MWCNT / SPE were examined in 0.1 M PBS (pH 7.4) containing various RAC concentrations ranging from 0.04 pM to 200 pM. The peak current corresponding to the oxidation reaction of RAC increased with increasing RAC concentration. From the resulting calibration curve, a linear regression equation of IQue (pA) (pA) = 0.4461 + 0.0327 CRAC (pM) was obtained, with a correlation coefficient of (R2 0,9939) 0.9939. Following the 3SD / m criterion, the limit of detection (LOD) was calculated to be 0.0012 pM. In addition, the sensitivity of CBRG-Fe-MWCNT / SPE for RAC determination was determined from the calibration curve as 0.0327 pA pM-1. To investigate the selectivity of the CBRG-Fe-MWCNT / SPE sensor, potential interfering substances — including D-glucose, dopamine, glycine, ascorbic acid, and uric acid — were selected. The peak currents showing changes in oxidation peak potential and current in an appropriate pH solution containing these interfering species (200 pM) were presented. The resultsdemonstrate that the CBRG-Fe-MWCNT / SPE sensor possesses a strong interference suppression capability in the presence of these compounds.

[0039] The reproducibility of the prepared sensor was investigated by monitoring the DPV signals of 25 pM RAC in 0.1 M PBS (pH 7.4) using five independently fabricated CBRG-Fe-MWCNT / SPE electrodes (2:1). The relative standard deviation (RSD) value obtained from five consecutive measurements performed with the same sensor was found to be 5.76%. In addition, the repeatability of the sensor was evaluated by recording five successive current responses of 25 pM RAC using the same electrode. The obtained signals exhibited an RSD value of 1.88%. The negligible RSD value (1.88%) observed for repeated measurements further demonstrates the stability and reliability of the sensor. The results of both tests indicate that the CBRG-Fe-MWCNT / SPE sensor exhibits good sensitivity for the determination of RAC.

Claims

CLAIMS1. A novel composite, surface-imprinted, combined modified electrode intended for use as an electrochemically active sensor for the sensitive determination of ractopamine, characterized by; comprising a chalcone-substituted cyclotriphosphazene (CBRG-Fe) and multi-walled carbon nanotubes (MWCNT).

2. A method for producing a surface-imprinted combined modified electrode comprising a novel chalcone-substituted cyclotriphosphazene (CBRG-Fe) and a multi-walled carbon nanotube (MWCNT) composite, intended for use as an electrochemically active sensor for the sensitive determination of ractopamine, characterized by;- adding the synthesized CBRG to at least 20 mL of acetone in a flask and stirring the mixture,- subsequently adding at least 0.28 g of a ferrocene-chalcone compound in an appropriate amount to the reaction medium, - adding Cs2CO3to the reaction and stirring the reaction mixture, - monitoring the reaction, followed by termination and purification of the reaction,- synthesizing the CBRG-Fe compound,- modifying the surface of the working electrode with CBRG-Fe and the CBRG-Fe-MWCNT composite,- preparing the CBRG-Fe and CBRG-Fe-MWCNT composite by sonication in ultrapure water for at least 1 hour,- after the sonication process, drop-casting at least 10 pL of the dispersion onto the surface of the working electrode and allowing it to dry.

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