PL233295B1 - Molecularly imprinted polymer by means of p-synephrine and selective chemosensor for electrochemical marking of p-synephrine with a layer of the molecularly imprinted polymer as the recognizing unit - Google Patents

Abstract

The application relates to a layered molecularly imprinted polymer (MIP) used as a recognition unit in a selective chemo-sensor for the electrochemical determination of p-synephrine, characterized in that it is a polymer made using p-synephrine as a template, containing 2,2'- bitiophene-5-carboxylate as functional monomer and 2,3'-bitiophene as cross-linking monomer. The subject of the application is also a method of obtaining a molecularly imprinted polymer (MIP) recognizing p-synephrine, by means of molecular imprinting, in the form of a layer and its use as an analyte recognition unit, including p-synephrine, in a selective chemosensor for electrochemical detection and/or determination analytes, both in synthetic and biological samples, with the addition of a redox probe to the test solution.

Description

Description of the invention

The subject of the invention is a polymer molecularly imprinted with p-synephrine and a selective chemosensor for the electrochemical determination of p-synephrine with a layer of molecularly imprinted polymer as the recognition unit.

Preparations containing extracts of bitter orange (Citrus aurantium) and sweet orange (Citrus sinensis) have found widespread use in dietary supplements, herbal preparations and folk medicine. An example is the preparation "Zhishi" belonging to the canon of Chinese folk medicine. Since the FDA (United States Food and Drug Administration) banned the use of ephedrine-containing weight loss agents in 2004 (Arbo, MD; Schmitt, G. C; Limberger, MF; Charao, MF; Moro, AM; Ribeiro, G. L; Dallegrave, E .; Garda, SC; Leal, MB; Limberger, RP, Subchronic toxicity of Citrus aurantium L. (Rutaceae) extract and p-synephrine in mice. Regul. Toxicol. Pharmacol .: RTP 2009, 54, 114-117), these agents are replaced by bitter orange extracts. The main component responsible for their action is p-synephrine. It has the ability to block the β-3 adrenergic receptors responsible for the regulation of fat and carbohydrate metabolism (Arch, JR, beta (3) -Adrenoceptor agonists: potential, pitfalls and progress. Eur. J. Pharmacol. 2002, 440, 99-107 ).

The wide use of preparations containing, among others p-synephrine is accompanied by a serious debate regarding the safety of their use. Many studies indicate possible adverse effects of p-synephrine on the circulatory system, such as increased blood pressure and heart rate, and cardiac arrhythmias (Rossato, LG; Costa, VM; Limberger, RP; Bastos Mde, L .; Remiao, F., Synephrine : from trace concentrations to massive consumption in weight-loss. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 2011, 49, 8-16). However, other studies suggest that the use of p-synephrine is completely safe, and that m-synephrine, which binds to adrenergic α and β-1 and β-2 receptors, is responsible for its side effects (Stohs, SJ; Preuss, HG; Shara, M ., A review of the receptor-binding properties of p-synephrine as related to its pharmacological effects. Oxid. Med. Cell Longev. 2011, 2011, 482973). m-Synephrine is present in bitter orange extracts in trace amounts. These extracts also contain trace amounts of other biogenic amines that may affect heart function, such as octopamine and tyramine (Thevis, M .; Koch, A .; Sigmund, G .; Thomas, A .; Schanzer, W., Analysis of octopamine in human doping control samples. Biomed. Chromatogr. 2012, 26, 610-615). Therefore, there is a need to develop an efficient, selective method for the detection and determination of p- and m-synephrine in both synthetic and biological samples.

Synephrine speeds up metabolism. That is why it has become a popular ingredient of preparations increasing the fitness of athletes. Since 2009, it has been subject to a monitoring program conducted by the World Anti-Doping Agency (WADA), the purpose of which is to track the effects of substances such as caffeine or phenylpropanamine on the human body. The potential risks associated with the use of synephrine and the fact that its close analog - octopamine - was already recognized as an illegal doping in 2010, suggest that the use of synephrine will soon be banned by WADA (WADA, The 2009 Monitoring Program, available at website: https://www.wada-ama.org/sites/default/files/resources/files/WADA_Monitoring_Program_2009_EN.pdf).

The existing methods of determining biogenic amines, such as gas chromatography, capillary electrophoresis, or high performance liquid chromatography, are expensive. Moreover, they require preliminary quite complicated and time-consuming sample preparation. This highlights the need to develop simple and cheap assays for the detection and determination of p-synephrine. However, only single examples of polymers molecularly imprinted with p-synephrine as a template are known in the literature. The first example is the use of MIP imprinted with p-synephrine as a medium for SPE (Solid phase extraction) (Fan, JP; Zhang, L; Zhang, XH; Huang, JZ; Tong, S; Kong, T. ; Tian, ZY; Zhu, JH, Molecularly imprinted polymers for selective extraction of synephrine from Aurcmtii Fructus Immaturus. Anal. Bioanal. Chem. 2012, 402, 1337-1346). This bed allowed the selective extraction of p-synephrine from the extracts of bitter orange and its concentration up to 24 times. The extracted p-synephrine was recovered 85 to 90%. The molar ratio of template (p-synephrine) to functional monomer (methacrylic acid) and crosslinking monomer (ethylene glycol dimethacrylate) in the polymerization solution was 1: 4: 20. Also the use of 1-vinyl-3-carboxybutyl-imidazole bromide as a functional monomer, an ionic liquid in its structure

PL 233 295 B1 containing a double bond allowed to obtain a selective extraction bed with comparable sorption properties (Fan, JP; Tian, ZY; Tong, S; Zhang, XH; Xie, Y. L; Xu, R; Qin, Y .; Li, L .; Zhu, JH; Ouyang, XK, A novel molecularly imprinted polymer of the specific ionic liquid monomer for selective separation of synephrine from methanol-water media. Food Chem. 2013, 141, 3578-3585). A third example of p-synephrine imprinting is the synthesis of MIP in the form of a membrane (Fan, JP; Li, L .; Tian, ZY; Xie, CF; Song, FT; Zhang, XH; Zhu, JH, A novel freestanding flexible molecularly imprinted membrane for selective separation of synephrine in methanolwater media. J. Membrane Sci. 2014, 467,13-22). In this case, 2-methylpropenoic acid and 2-hydroxyethyl acrylate were used as functional monomers.

Molecularly imprinted polymers (MIPs) are an excellent example of intelligent materials that mimic biological recognition. They have found application, incl. as selective recognition layers for the construction of chemosensors (Cieplak, M .; Kutner, W., Artificial biosensors: How can molecular imprinting mimic biorecognition? Trends Biotechnol. 2016, 34, 922-941). Chemosensors with these recognizing layers show analytical parameters (sensitivity, selectivity, lower detection limit, etc.) not much inferior to biosensors, but they surpass the latter in terms of ease of manufacture and manufacturing costs, as well as durability and resistance to external conditions such as elevated temperature, acidic or alkaline the environment, or the presence of organic solvents.

Therefore, the object of the present invention is to propose a new polymer, molecularly imprinted with p-synephrine, applicable as a recognition layer in a selective chemical sensor for electrochemical determination of p-synephrine with the above-described improved analytical parameters and lower manufacturing costs.

Thus, according to the present invention, a molecularly imprinted polymer (MIP) in the form of a layer used as recognition unit in the selective chemosensor for the electrochemical determination of p-synephrine is characterized in that it is a polymer made using p-synephrine as a template, containing acid 2,2'-bitiophene-5-carboxylic acid as functional monomer and 2,3'-bitiophene as crosslinking monomer.

The invention also comprises a method of obtaining a molecularly imprinted polymer (MIP) recognizing p-synephrine as defined above by molecular imprinting in the form of a layer, characterized in that it comprises the following steps in which (a) a MIP layer is obtained with an electropolymerization solution containing a solution of p-synephrine (template), 2,2'-bitiophene-5-carboxylic acid (functional monomer), 2,3'-bitiophene (cross-linking monomer), in a molar ratio of 1: 1: 1 up to 1: 100: 100, preferably 1: 3: 30, in acetonitrile, in the presence of 0.1 M tetrabutylammonium chlorate (VII) (base electrolyte) which (b) is deposited as a layer on the surface of the electrodes, preferably platinum disc electrodes , by electropolymerization under potentiodynamic conditions, in the potential range from -2.0 V to 2.0 V vs Ag / AgCI, preferably from 0 to 1.30 V vs Ag / AgCI, with a potential change rate of 5 to 500 mV / s, preferably 50 mV / s, or under potentiostatic conditions potentials ranging from -2.0 V to 2.0 V vs Ag / AgCI, to form a MIP layer with a molecularly imprinted template, and then (c) remove this template (p-synephrine) from the deposited MIP- layer u with 0.1 M NaOH at room temperature, (20 ± 1) ° C, to obtain a p-synephrine selective MIP layer.

Preferably, in the process according to the invention, the deposition in step (b) is carried out in the range of 1 to 1000 current-potential cycles, preferably 5 cycles.

Preferably, in the process according to the invention, the removal of the template in step (c) is performed for 10 minutes to 10 hours, preferably for 1 hour.

The invention also encompasses the use of a molecularly imprinted polymer (MIP) in the form of a layer as defined above as an analyte recognition unit, including p-synephrine, in a selective chemosensor for the electrochemical detection and / or determination of analytes in both synthetic and biological samples.

The use of the present invention makes it possible to carry out electrochemical measurements with the addition of a redox probe to the test solution, preferably K4 [Fe (CN) 6] / Ks [Fe (CN) 6].

The above-mentioned molecularly imprinted polymer was prepared as follows. P-synephrine template, acid, was used to deposit the polymer (Procedure 3.2.1, below)

PL 233 295 B1

2,2'-bitiophene-5-carboxylic acid as functional monomer and 2,3'-bitiophene as crosslinking monomer. The electrode modified in this way was used for electrochemical determinations with the addition of a redox probe, ie K4 [Fe (CN) 6] / K3 [Fe (CN) 6] (Procedure 3.2.2, below).

The invention will now be illustrated in more detail in the preferred embodiments with reference to the accompanying drawings in which:

Fig. 1.shows the structural formulas of (a) p-synephrine, (b) 2,2'-bitiophene-5-carboxylic acid, (c) 2,3'-bitiophene, (d) the proposed structure of the p-synephrine complex with three molecules of 2,2'-bitiophene-5-carboxylic acid and (e) the structure of the above-mentioned the complex optimized with the use of the density functional theory (DFT) with the base B3LYP / 6-31g (d) at 25 ° C, in acetonitrile,

Fig. 2 shows (a) a potentiodynamic curve recorded during the deposition of a MIP layer; the composition of the electropolymerization solution was as follows: 10 mM p-synephrine, 30 mM 2,2'-bitiophene-5-carboxylic acid, 300 mM 2,3'-bitiophene and 100 mM tetrabutylammonium chlorate, in acetonitrile; during the deposition, five current-potential cycles in the range from 0 to 1.30 V vs Ag / AgCI were performed with a potential change rate of 50 mV / s; (b) DPV signal changes for 0.1 M K4 [Fe (CN) 6] / K3 [Fe (CN) 6] (redox probe) in PBS solution (pH = 7.4), recorded with a platinum disc electrode coated with MIP layer prepared according to Procedure 3.2.1 below, (1) before extraction and after (2) 20, (3) 40, (4) 60 and (5) 80 minutes of extraction in 0.1 M NaOH,

Fig. 3.shows (a) DPV signal changes recorded with a platinum disc electrode covered with a MIP layer prepared according to Procedure 3.2.1, (1) after extraction with 0.1 M NaOH, and then in the presence of p- synephrine with a concentration of (2) 0.10, (3) 0.29, (4) 0.48, (5) 0.74 and (6) 0.99 μM and (b) DPV calibration curves, constructed for a disc electrode platinum coated with (1-5) MIP or (6) NIP prepared according to Procedure 3.2.1, for (1, 6) p-synephrine, (2) creatinine, (3) adrenaline, (4) urea and (5) glucose in PBS (pH = 7.4) using K4 [Fe (CN) 6] / K3 [Fe (CN) 6] as redox probe.

Preferred Embodiments of the Invention

In order to achieve the object of the present invention, we have completed the following research tasks in the following embodiments.

2.1 We optimized the molecular structures of the pre-polymerization complexes by quantum-chemical computer modeling using the density functional theory (DFT) (Figure 1e).

2.2 We have prepared the MIP layer by potentiodynamic electropolymerization.

2.3 We have developed a chemosensor for the selective determination of p-synephrine and determined its analytical parameters.

3. Materials and procedures

3.1 Materials

3.1.1 Reagents and reagents

All chemicals and solvents used were purchased from Sigma-Aldrich, except for 2,2'-bitiophene-5-carboxylic acid purchased from Enamine.

P r z k ł a d 1

3.2 Procedures

3.2.1 Manufacture of the molecularly imprinted polymer (MIP) and its deposition on the electrode surface

The MIP layers were prepared and deposited on platinum disc electrodes by electropolymerization under potentiodynamic conditions, in the potential range from 0 to 1.30 V vs Ag / AgCl. 5 current-potential cycles were performed with a potential change rate of 50 mV / s. The dependence of the current on the onopotential recorded during the electropolymerization is shown in Fig. 3a. Acetonitrile electropolymerization solution was 10 μM versus p-synephrine (template), 30 μM versus 2,2'-bitiophene-5-carboxylic acid (functional monomer), 300 μM versus 2,3'-bitiophene (crosslinking monomer) and 0, 1 M tetrabutylammonium chlorate (VII) (primary electrolyte). Prior to electropolymerization, the electrode was cleaned with a pyranium (peroxosulfuric acid) solution, then polished with β-alumine, 0.05 μm grain diameter, to a mirror surface.

PL 233 295 B1

After electropolymerization, the template (p-synephrine) was removed from the MIP layer. For this purpose, the electrode covered with a MIP layer was immersed for 60 min in 0.1 M NaOH, at room temperature, (20 ± 1) ° C.

In order to record the XPS and PM-IRRAS spectra and take AFM photos, the MIP layers were deposited on gold-plated glass plates in the manner described above.

Example 2

3.2.2 Electrochemical measurements using K4 [Fe (CN) 6] / K3 [Fe (CN) 6] as redox probe.

DPV measurements were made at room temperature, (20 ± 1) ° C, using a V-shaped electrochemical glass three-necked mini-cup with a capacity of ~ 2.0 mL. Measurements were made with 0.1 M K3 [Fe (CN) 6] and 0.1 M K4 [Fe (CN) 6] (redox probe) in PBS solution (pH = 7.4).

In DPV measurements, the potential was changed in the range from 0 to 0.60 V every 5 mV. The 50-ms amplitude of the potential pulses was 25 mV.

After the measurement was completed, the electrode was immersed in 0.1 M NaOH for 20 to 60 min, at room temperature, (20 ± 1) ° C, to extract the analyte so thoroughly that the measured DPV signal reached a constant maximum value of ~ 40 pA.

4. Results and discussion

4.1 Selecting the appropriate functional monomers and estimating the stability of the p-synephrine complexes produced by them

In the p-synephrine molecule (Fig. 3a) and in the molecules of the selected functional monomer (Fig. 3b) there are groups of atoms capable of forming hydrogen bonds or electrostatic interactions. The proposed interactions in the template complex with two functional monomer molecules are represented by the structural formula in Fig. 3g. The hypothesis for these interactions was confirmed by quantum-chemical calculations. These calculations confirmed the high negative free enthalpy increase associated with the formation of the above-mentioned complex (AG = -54 kJ / mol) indicating that the complex formed is stable and should not undergo changes during electropolymerization leading to the formation of a MIP layer. The optimized structure of this complex is shown in Fig. 1e.

P r z k ł a d 3

4.2 Synthesis method and deposition of a molecularly imprinted polymer by potentiodynamic electropolymerization

In order to prepare the MIP layer printed with the template, p-synephrine, potentiodynamic electropolymerization was carried out in the potential range from 0 to 1.30 V vs Ag / AgCI at a potential change rate of 50 mV / s. The MIP layer was deposited on a platinum disc electrode with a diameter of 1 mm. After electropolymerization was completed, the deposited layers were washed with the electropolymerization solvent, acetonitrile, to remove excess base electrolyte. Then the template, p-synephrine, was extracted from the MIP layer thus prepared with 0.1 M NaOH in 60 min, at room temperature, (20 ± 1) ° C. Using the same procedure as that used to prepare the MIP layer and an electropolymerization solution with the same composition but no p-synephrine, a non-imprinted polymer (NIP) layer was prepared.

Example 4

4.3 Indirect determination of p-synephrine under steady-state conditions using differential pulse voltammetry, DPV, in the presence of a redox probe, K4Fe (CN) 6 / K3Fe (CN) 6 in solution

For the indirect determination of p-synephrine by the MIP chemosensor, the so-called "Gating effect". It consists in lowering the current detection signal in the presence of the analyte in the MIP as a result of binding the analyte molecules inside the molecular cavities of the MIP layer. Then, according to the commonly accepted mechanism, the polymer swells (Yoshimi, Y .; Narimatsu, A .; Nakayama, K .; Sekine, S .; Hattori, K .; Sakai, K., Development of an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer. J. Artif. Organs 2009, 12, 264-270), which leads to blocking the diffusion channels of the redox probe through the MIP layer to the electrode surface and thus lowering the Faraday probe current ( Fig. 1a). To monitor this effect, DPV measurements were used in the presence of a redox probe, K4Fe (CN) 6 / K3Fe (CN) 6, on a platinum disc electrode covered with a MIP layer in the test solution. In the subsequent steps of extraction of the template (p-synephrine) from the MIP layer, the molecular gaps were gradually emptied.

PL 233 295 B1

As a result, the diffusion of the redox probe to the electrode surface was faster and faster and the DPV peak current was increasingly higher (curves 1-5, Fig. 2b). On the other hand, as the concentration of p-synephrine in the redox probe solution in which the electrode covered with the extracted MIP layer was immersed, the gaps were again filled with analyte particles. As a result, the diffusion of the redox probe was made more and more difficult and the DPV peak current was increasingly lower (curves 1-6, Fig. 3a).

In the DPV measurements, the relative changes in the peak current were proportional to the changes in the concentration of p-synephrine in the solution in the range from 0.10 to 0.99 μ a (Fig. 3b) and the linear regression equation describing this relationship is (/ dpv, o - / dpv, s) // dpv, o = 11.83x10'2 + 3.21x10'4 c / nM, where c is the concentration of psynephrine in the solution, (/ dpv, o - / dpv, s) // dpv, o to the relative change in the DPV peak current for the extracted MIP layer a / dpv, oi / dpv are the peak current in the absence and presence of the analyte or interfering substance, respectively. The correlation coefficient was 0.979, and the limit of detection was 7.22 pM, with a signal to noise ratio of S / N = 3.

The selectivity ratios for creatinine, adrenaline, urea, and glucose were 2.08, 2.63, 2.05 and 2.46, respectively, and the apparent imprinting ratio was, IF = 2.4 (Fig. 3b). The determined analytical parameters of the produced MLP chemosensor indicate that the functional monomers and their concentration in the polymerization solution were properly selected. This made the molecular imprinting of p-synephrine effective.

5. Conclusions

The results of computer modeling showed that the pre-polymerization complex of p-synephrine with the selected functional monomer is stable. This is indicated by a high negative free enthalpy increase accompanying the formation of this complex, calculated by quantum-chemical modeling.

Potentiodynamic electropolymerization turned out to be very convenient for depositing a MIP layer with an imprinted pattern, p-synephrine. The produced chemosensor enabled selective determination of the analyte in the concentration range from 0.10 to 0.99 μΜ with the use of DPV in the presence of the redox probe, K4 [Fe (CN) 6] / K3 [Fe (CN) 6] in the tested solution.

Claims (6)

Patent claims 1. A molecularly imprinted polymer (MIP) in the form of a layer used as recognition unit in a selective chemosensor for the electrochemical determination of p-synephrine, characterized in that it is a polymer made using p-synephrine as a template containing 2,2'-bitiophene acid -5-carboxylic acid as functional monomer and 2,3'-bitiophene as crosslinking monomer. 2. A method of obtaining a molecularly imprinted polymer (MIP) recognizing p-synephrine as defined in claim 1; 1, by molecular imprinting in the form of a layer, characterized in that it comprises the following steps, wherein (a) a MIP layer is obtained using an electropolymerization solution containing a solution of p-synephrine (template), 2,2'-bitiophene acid 5-carboxylic acid (functional monomer), 2,3'-bitiophene (crosslinking monomer), in a molar ratio of 1: 1: 1 to 1: 100: 100, preferably 1: 3: 30, in acetonitrile, in the presence of 0.1 M tetrabutylammonium chlorate (VII) as the primary electrolyte, which (b) is deposited as a layer on the surface of the electrodes, preferably platinum disc electrodes, by electropolymerization under potentiodynamic conditions, in the potential range from -2.0 V to 2.0 V vs Ag / AgCI, preferably from 0 to 1.30 V vs Ag / AgCI, with a potential change rate from 5 to 500 mV / s, preferably 50 mV / s, or under potentiostatic conditions in the potential range from -2.0 V to 2 , 0V vs Ag / AgCI, to form a MIP layer with a molecularly imprinted template, and then (c) removes this template (p-synephrine) is removed from the deposited MIP layer with 0.1 M NaOH at room temperature, 20 ± 1 ° C, and a p-synephrine selective MIP layer is obtained. 3. The method according to p. The process of claim 2, characterized in that the deposition in step (b) is carried out in 1 to 1000 current-potential cycles, preferably 5 cycles. PL 233 295 B1 4. The method according to p. Method according to claim 2 or 3, characterized in that the removal of the template in step (c) is carried out for 10 minutes to 10 hours, preferably for 1 hour. 5. Use of a molecularly imprinted polymer (MIP) in the form of a layer as defined in claim 1. 1 as the recognition unit for an analyte, including p-synephrine, in a selective chemosensor for the electrochemical detection and / or determination of analytes, both in synthetic and biological samples. 6. Use according to claim 1 5. A method according to claim 5, characterized in that it enables electrochemical measurements to be carried out with the addition of a redox probe, preferably K4 [Fe (CN) 6] / K3 [Fe (CN) 6], to the test solution.