PL220926B1 - Thiophene derivative molecularly printed polymer formed by polymerization of thiophene derivatives and the use of this polymer for selectively determined and controlled release of adenosine 5'-triphosphate(ATP) - Google Patents

Abstract

A thiophene compound of structural formula (formula 1) (formula 1) Where (formula 1a) OR (formula lb); a polymer prepared using the thiophene compound and use of the polymer in a chemo-sensor for determination of adenosine-5-triphosphate or as a material for controlled release of adenosine-5-triphosphate.

Description

Description of the invention

The invention relates to (a) novel thiophene derivatives, a molecularly imprinted polymer containing a polymerized thiophene derivative, the use of this polymer as the recognition element of a chemical sensor for the selective determination of adenosine 5'-triphosphate (ATP), and (d) the use of this polymer as a material for controlled adenosine-5'-triphosphate (ATP) release.

Adenosine-5'-triphosphate (ATP) (hereinafter referred to as 1, see Fig. 8) was independently discovered by two laboratories [1, 2], ATP was recognized as the main energy transfer factor in living cells [3]. It is considered the monetary unit of energy conversion in living organisms. The energy released during the hydrolysis of high-energy bonds of phosphate groups enables the performance of a number of biological activities, such as muscle contraction, nerve stimulation or active transport, as well as the synthesis of a number of substances, including proteins and nucleic acids [4]. In living organisms, ATP cannot be stored because it is rapidly broken down. Therefore, in order to meet the current energy needs of the cell, it must be continuously produced. Therefore, the detection of the presence of ATP in a biological system is an indirect test of whether the system is alive or not. The concentration of ATP in the cytoplasm of living cells is relatively low; it amounts to ~ 1.0 μΜ [5]. Therefore, the detection of ATP at this concentration level is important for the study of biological systems. Advantageously, from the point of view of selectivity of ATP determinations, its concentration in the cell is a thousand times higher than the concentration of adenosine-5'-diphosphate (ADP), the product of the enzymatic dephosphorylation of ATP [6].

There are three main components in the structure of the ATP molecule, ie adenine, ribofuranose and triphosphate. Together, these three fragments possess 15 binding sites [7]. In nature, all these sites are involved in ATP recognition by non-covalently binding amino acids such as Glu156, His155, Phe28, Val35, Thr61, Tbr60, Lys59, His215, Ser55, Ser158, Gln161, Leu 157, Arg152 of the purinergic receptor. Such numerous interactions lead to the specific ATP recognition under these conditions.

A number of different sensors have been developed and manufactured for the recognition and determination of ATP in synthetic systems. Initially, biochemical sensors (biosensors) were used for this purpose. One of them used the ability of ATP to counteract glucose oxidation catalysed by glucooxidase [8]. However, the limit of detection (LOD) of this biosensor achieved by 10 µM chronoamperometry was too high to allow the determination of ATP in biological systems. Although the LOD value of another biosensor using the chemiluminescence of the ATP triphosphate luminol reaction product to process the detection signal was preferably much lower (10 nM ATP), this determination required the use of too many different expensive chemical reagents [9]. Recently, aptamers, ie functional oligonucleotides selected in vitro [10, 11], were used to build biosensors for ATP determination. By their nature, aptamers are characterized by high selectivity and affinity in many ways with their advantages overcoming natural ATP recognition receptors. However, the main disadvantage of ATP biosensors is the low durability and reproducibility of the determinations, which disqualifies them when used in routine determinations. Therefore, a number of attempts were made to produce synthetic ATP receptors aimed at building chemical sensors (chemosensors).

In recent years, the use of molecularly imprinted polymers (MIPs) for biomimetic recognition for selective determination has increased [12-19].

Thus, it would be desirable to develop a sensor with increased selectivity for ATP by using a recognition element specially developed for their determination.

Therefore, it is an object of the present invention to develop and produce a chemical that, once polymerized, could be used to build a chemical sensor for the determination of ATP. Another object of the present invention is to develop and produce a chemical that could be used to produce materials for the controlled release of ATP.

In the present application, four different electroactive thiophene derivatives were used as three functional monomers and one cross-linking monomer to build a chemosensor for ATP determination. The functional monomers include uracylphenyl-4- [bis (2,2'-bitienyl) methane] (hereinafter 2, see Fig. 8), (3-thiophene-3-yl) boric acid (hereinafter 3, see Fig. 8) and 1-methylamido-4- [bis (2,2'-bitienyl] methane] (hereinafter 4, see Fig. 8), and the role of the crosslinking monomer is 3,3'-bis [2,2'-bisPL 220 926 B1

- (2,2'-bitiophene-5-yl)] thianaphthene (hereinafter 5). The functional monomers used make it possible to prepare the complex in a solution in which ATP is bound at eleven points. That is, its eleven binding sites are involved in the formation of this complex. Preparation of the electrochemical polymerization solution required, on the one hand, dissolving all its five components with different polarity in one solution, and, on the other hand, ensuring electropolymerization conditions such that the thiophene cation radicals produced during it would not disappear too quickly. For this purpose, a procedure for the preparation of this solution was developed. It uses three different organic solvents and water. The polymer layers synthesized in this study with molecular cavities imprinted with ATP molecules are designated as MIP-ATP. The formation of the polymerization complex was modeled by quantum chemical calculations. Potentiodynamic electropolymerization was then performed to deposit MIP layers filled with ATP on the surfaces of two different molecular recognition signal to analytical electrical signal converters. After the ATP template was extracted from these layers, the recognition elements of these chemosensors were obtained. Piezoelectric microgravimetry (PM) and capacitive impedimetry (Cl) were selected as the signal processing techniques. The signal of ATP detection in the test solution, related to filling and emptying the molecular gaps of the polymer by the molecules determined with ATP, in the former was the change of the resonance frequency proportional to the changes in the polymer mass, and in the latter - the change in the electrical capacity of the MIP-ATP layer.

The subject of the invention is a chemical compound of structural formula (1).

where or

The invention also includes a molecularly imprinted polymer containing the polymerized chemical.

PL 220 926 B1

The invention also relates to the use of this molecularly imprinted polymer as the recognition element of a chemical sensor for the selective determination of adenosine 5'-triphosphate and its use as a controlled release material for adenosine-5'-triphosphate.

The invention is illustrated in more detail below in preferred embodiments with reference to the accompanying drawings.

Fig. 1 Potentiodynamic curves for 0.1 mM 1, 0.1 mM 2, 0.1 mM 3, 0.3 mM 4 and 0.5 mM 5 in 0.1 M (TBA) ClO4, in a solution by volume ratio water to acetonitrile to toluene to propanol like 1.5: 6: 1: 1.5, recorded with a platinum disc electrode with a diameter of 1 mm for (1) the first, (2) the second and (3) the third cycle of potential changes in the range potentials from 0.50 to 1.25 V vs Ag / AgCI. The rate of change of potential was 50 mV / s.

Fig. 2 Curves of the potential dependence of (a) current as well as changes in (b) resonance frequency and (c) dynamic resistance simultaneously recorded during the deposition of the MIP-ATP layer by potentiodynamic electropolymerization on the gold electrode of a quartz-crystal resonator with a fundamental resonance frequency of 10 MHz . The composition of the solution was the same as that described in the description of Fig. 1.

Fig. 3 Infrared reflection absorption spectroscopy (IRRAS) for the layer (1) of ATP, (2) unprinted polymer (NIP), as well as MIP-ATP (3) before and (4) after ATP extraction deposited on a gold-plated microscope slide with 0.1 M HCI for 5 h. at 60 ° C.

Fig. 4 High resolution X-ray photoelectron spectroscopy (XPS) spectra in the P 2p electron binding energy range for the MIP-ATP layer (a) before and (b) after ATP extraction with 0.1 M HCl for 5 h. at 60 ° C. For comparison, the XPS spectrum for the NIP layer is shown by the dashed curve in panel (a).

Fig. 5. Differential pulse voltamperograms for 0.1 M K4Fe (CN) 6 in 0.1 M KNO3, recorded with a 1 mm diameter platinum disc electrode covered with a MIP-ATP layer (1) before and (2) after extraction ATP template with 0.1 M HCI for 5 h. at 50 ° C. The MIP-ATP layer was deposited under potentiodynamic conditions during three cycles of potential changes in the potential range from 0.50 to 1.25 V vs Ag / AgCl, with a potential change rate of 50 mV / s.

Fig. 6 Capacity change over time for different ATP concentrations in 0.1 M KF (pH = 8.3). This change was measured under flow injection analysis (FIA) conditions at a potential of 0.50 V vs Ag / AgCl and a frequency of 20 Hz for the MIP-ATP layer with the ATP pattern extracted overlapping a 1 mm diameter platinum disc electrode. 0.1 M KF pumped at a rate of 20 µL / min served as the carrier solution. The inset shows the calibration curve for the ATP determination.

Fig. 7. Change in resonance frequency with time for different ATP concentrations (pH = 7.8). This change was measured under flow injection analysis (FIA) conditions for the MIP-ATP layer with the ATP template extracted, covering the gold electrode vaporized on a quartz resonator with a fundamental resonance frequency of 10 MHz. Water pumped at a rate of 20 µL / min served as the carrier liquid. The inset shows the calibration curve for the ATP determination.

Fig. 8 Scheme of "eleven point recognition 1. (a) Proposed structural formula of complex 1 with functional monomers 2, 3 and 4 and (b) structure of this complex optimized by DFT approximately B3LYPy6-31g (d).

Bibliography

[1] C.H. Fiske, Y. Subbarow, Science, 70 (1929) 381.

[2] K. Lohmann, Naturwissenschaften, 17 (1929) 624.

[3] F. Lipmann, Adv. Enzymol., 1 (1941) 99.

[4] K. Maruyama, J. Hist. Biol., 24 (1991) 145.

[5] M.W. Gorman, E.O. Feigl, C.W. Buffington, Clin. Chem., 53 (2007) 318.

[6] D.G. Nicholls, SJ. Ferguson, Bioenergetics 3, Academic Press, San Diego, USA, 2002.

[7] A.-M. Brandt, W. Raksajit, P. Mulo, A. Incharoensakdi, T.A. Salminen, P. Maenpaa, Arch.

Microbiol., 191 (2009) 561.

[8] D. Compagnone, G.G. Guilbault, Anal. Chim. Acta, 340 (1997) 109.

[9] T. Fujiwara, K. Kurahashi, T. Kumamaru, Anal. Chim. Acta, 349 (1997) 159.

[10] Y. Du, B. Li, H. Wei, Y Wang, E. Wang, Anal. Chem., 80 (2008) 5110.

[11] X. Zuo, 5. Song, J. Zhang, D. Pan, L. Wang, C. Fan, J. Am. Chem. Soc., 129 (2007) 1042.

[12] K. Haupt, K. Mosbach, Chem. Rev., 100 (2000) 2495.

PL 220 926 B1

[13] B. Sellergren, TrAC-Trends Anal. Chem., 16 (1997) 310.

[14] C. Malitesta, E. Mazzotta, R.A. Pieca, A. Poma, I. Chianella, S.A. Piletsky, Anal. Bioanal. Chem., 402 (2012) 1827.

[15] S. Suriyanarayanan, P. J. Cywinski, A. J. Moro, G. J. Mohr, W. Kutner, Top. Curr. Chem., 325 (2012) 165.

[16] A. Pietrzyk-Le, C.K.C. Bikram, F. D'Souza, W. Kutner, in: H.-J. Schneider (Ed.), Supramolecular Chemistry for 21st Century Technology, Taylor & Francis / CRC Press, London, UK, 2012, pp. 105-128.

[17] P.S. Sharma, F. D'Souza, W. Kutner, in: D.P. Nikolelis (Ed.), Portable chemical sensors: weapons against bioterrorism, NATO science for peace and security series A: chemistry and biology, Springer, Dordrecht, Netherland, 2012, pp. 63-94.

[18] P.S. Sharma, F. D'Souza, W. Kutner, TrAC-Trends Anal. Chem., 34 (2012) 59.

[19] P.S. Sharma, A. Pietrzyk-Le, F. D'Souza, W. Kutner, Anal. Bioanal. Chem., 402 (2012)

3177.

[20] A. Pietrzyk, W. Kutner, R. Chitta, M.E. Zandler, F. D'Souz, F. Sannicolo, P.R. Mussini, Anal. Chem., 81 (2009) 10061.

[21] F. Sannicolo, S. Rizzo, T. Benincori, W. Kutner, K. Noworyta, J.W. Sobczak, V. Bonometti, L. Falciola, P.R. Mussini, M. Pierini, Electrochim. Acta, 55 (2010) 8352.

[22] T.-P. Huynh, Chandra Bikram K. C, W. Lisowski, F. D'Souza, W. Kutner,

Bioelectrochemistry, 93 (2013) 37.

[23] MJ. Frisch, Gaussian, Inc., Wallingford CT, et al., 2009.

[24] A. Kochman, A. Krupka, J. Grissbach, W. Kutner, B. Gniewinska, L. Nafalski, Electroanalysis, 18 (2006) 2168.

[25] B. Soucaze-Guillous, W. Kutner, Electroanalysis 9 (1997) 32.

[26] R. Marczak, V.T. Hoang, K. Noworyta, M.E. Zandler, W. Kutner, F. D'Souz, J. Mater. Chem., 12 (2002) 2123.

[27] R. Cini, G. Giorgi, F. Lasch and, M. Sabat, A. Sabatini, A. Vacca, J. Chem. Soc. Dalton Trans. (1989) 575.

[28] H. Takeuchi, H. Murata, I. Harada, J. Am. Chem. Soc., 110 (1988) 392.

[29] A. Jaramillo, L.D. Spurlock, V. Young, A. Brajter-Toth, Analyst, 124 (1999) 1215.

[30] J.-L. Gong, F.-C. Gong, G.-M. Zeng, G.-L. Shen, R.-Q. Yu, Talanta, 61 (2003) 447.

[31] D.A. Buttry, M.D. Ward, Chem. Rev., 92 (1992) 1355.

Preferred Embodiments of the Invention

Example 1

Preparation of functional monomers

The functional monomer 3 (Fig. 8) and the crosslinker 5 were prepared according to the previously developed procedures [20, 21], and the procedures for the synthesis of functional monomers 2 (Fig. 8) and 4 (Fig. 8) were developed in the present study as follows.

1-Methylamido-4- [bis (2,2'-bitienyl) methane] 4. [Bis (2,2'-bitienyl) - (4-carboxyphenyl) methane [22] (500 mg, 1.10 mmol) was stirred for ~ 15 min under nitrogen in a 100 mL round bottom flask containing 25 mL of toluene followed by the addition of SOCl2 (1.85 mL, 25 mmol) and pyridine (2.02 mL, 25 mmol). The resulting solution was heated to reflux for 3 h. Its solvents were then evaporated to give a green acyl chloride derivative. At this stage, toluene (25 ml) was added and the resulting solution was stirred for 15 min under nitrogen. Then methylamine (0.05 mL, 1.10 mmol) and pyridine (6.52 mL, 80.625 mmol) were added and the resulting solution was stirred overnight at room temperature. After evaporation of the solvents, a green solid remained. This precipitate was purified by liquid chromatography on a silica gel column, eluting with a chlorophore ₁ m / m-methanol solution 95:05 by volume. Yield: 180 mg (35%). ¹ H NMR (CHCl3-d):

δ (in ppm) 8.10 (d, 2H, phenyl H), 7.45 (d, 2H, phenyl H), 7.20 (dd, 2H, bitiophene H), 7.10 (dd, 2H, bitiophene H), 7.00 (m, 4H, bitiophene H), 6.75 (dd, 2H, bitiophene, H) 5.82 (s, 1H, -CH-), 2.18 (s, 1H, methyl H ).

UracyIphenyl-4- [bis (2,2'-bitienyl) methane] 2. Via a 100 ml round bottom flask containing 1-methylamido-4- [bis (2,2'-bitienyl) methane (400 mg, 0.813 mmol), 5-iodouracil (150 mg, 0.633 mmol), anhydrous K2CO3 (750 mg, 5.42 mmol) tetrakis (triphenylphosphine) palladium (0) (105 mg, 0.09 mmol), nitrogen flushed for 15 min. Then toluene (15 ml) and tetrahydrofuran (15 ml) were added and the solution was heated to reflux for 16 h. at 90 ° C. The solution was then cooled at 6

Room temperature and filtered. After evaporation of the solvents from the filtrate, a light yellow-red solid remained. It was purified by liquid chromatography on a silica gel column using a 90:10 chloroform-methanol solution as eluent. The light red _{and the} fragrance (third) on the column contained the desired product. Yield: 180 mg (55%). ¹ H NMR (CHCl-d): δ (ppm) 8.01 (s, 1H, -CH uracil), 7.20-7.14 (m, 4H, phenyl H), 7.08 (dd, 2H, bitiophene H), 7.02-6.92 (m, 4H, bitiophene H), 6.84 (d, 2H, bitiophene H), 6.74 (dd, 2H, bitiophene H), 5.68 ( s, 1H, -CH-).

Example 2

Preparation of layers of molecularly imprinted polymers with ATP

The MIP-ATP layers were prepared by electrochemical polymerization under potentiodynamic conditions in the potential range from 0.50 to 1.25 V vs Ag / AgCl at the rate of change of the potential of 50 mV / s. The growth of the MIP-ATP layer, both on the platinum disc electrode and on the gold electrode of the quartz crystal resonator (QCR), Au-QCR, with a fundamental resonance frequency of 10 MHz, was controlled by the number of cycles of potential change. After electropolymerization was completed, the MIP-ATP layers were thoroughly washed with acetonitrile to remove excess stock electrolyte solution. The ATP template was then extracted from the layers with 0.1 M HCl for 5 h. at 60 ° C. A solution of this acidity was used for the extraction due to the rapid decomposition of the ATP triphosphate group in solutions with higher acidity.

Non-imprinted polymer (NIP) control layers were deposited from an electropolymerization solution containing no ATP template using the same procedure as the procedure used to deposit the MIP-ATP layers.

Calculations and measurements

The structure of the ATP complex with functional monomers 2, 3, and 4 was optimized (Fig. 8) and the values of the thermodynamic functions of its formation were calculated using the density functional theory (DFT) approximately B3LYP / 6-31g (d) from using Gaussian 2009 software [23].

The depleted MIP-ATP layers covering Au-QCRs were examined by PM under flow-injection analysis (FIA) conditions. The water used as the carrier liquid was pumped at a rate of 20 μΐ / min through a QCR holder model EQCM 5610 manufactured by the Institute of Physical Chemistry of the Polish Academy of Sciences (Warsaw) [24] using a syringe pump model NE-500 by New Era Pump Systems (Farmingdale NY, USA) . A model 7725 six-position rotary metering valve from Rheodyne (Cotati CA, USA) was used to inject samples of test solutions. The volume of each sample was 200 µΗ. The injected substances were dissolved in a solution with the same composition as the vehicle liquid, ie in water in the PM tests and in 0.1 M KF in the Cl tests.

The conditions of FIA for ATP determination in the C1 tests were the same as in the PM tests, only the OCR flow holder was replaced with a flow electrochemical vessel with a large volume and thin-film radial flow with a 1 mm diameter platinum disc electrode attached in a PTFE insulator [25]. The applied constant AC potential and frequency were held at 0.50 V vs Ag / AgCI and 20 Hz, respectively. At this potential value on the electrode, no Faraday process was carried out.

Deposition of MIP-ATP layers on various test electrodes

According to the optimized structure of the ATP complex with functional monomers (Fig. 8b), a polymerization solution was prepared with a weight ratio of components of 1: 2: 3: 4: 5 as 1: 1: 1: 3: 5. Preferably, a high excess of monomer was used. crosslinking 5 to immobilize the molecular voids permanently in the polymer network. ATP is soluble and the resulting solutions are stable if an aqueous solution with an acidity in the range 7.7 <pH <8.3 is used for dissolution. Therefore, a solution of three organic solvents and water was used for dissolution, in which the volume concentration of water was 15%. The presence of 10% toluene in this solution ensured complete dissolution 4. However, toluene does not dissolve in water. Therefore, 15% propanol made it possible to prepare a homogeneous solution. Finally, a large excess of acetonitrile was added to this solution, which along with the base electrolyte, 0.1 M (TBA) ClO4, was necessary for the electropolymerization to be carried out. The final composition of the solution was determined by a volume ratio of water to acetonitrile to toluene to propanol of 1.5: 6: 1: 1.5. Depending on the type of subsequent tests, the MIP-ATP layer was deposited on a platinum disk, on Au-QCR, or on a microscope slide with an evaporated layer of gold.

PL 220 926 B1

Deposition of the MIP-ATP layer on a platinum disc electrode

In order to deposit the MIP-ATP layer on a platinum disc electrode with a diameter of 1 mm, potentiodynamic electropolymerization was used with three cycles of potential changes ranging from 0.50 to 1.25 V vs Ag / AgCl at a polarization speed of 50 mV / s (Fig. 1) ). Despite the presence of water, an electropolymerization inhibitor, the cumulative anode peak of electropolymerization of functional monomers and cross-linking monomers - thiophene derivatives - appeared on the current-potential curve at potential 0.95 V vs Ag / AgCl, and a dark brown layer adhering to its surface was deposited on the electrode. MIP-ATP. However, the current of this peak in subsequent cycles was lower and lower. This drop in current was most likely related to the reverse action of the MIP-ATP layer to block the charge transfer necessary for electropolymerization.

The ATP electro-oxidation was also tested with a thiophene-free solution. As could be expected from our previous research [26], the voltammetric anode peak for the oxidation of the adenine fragment of ATP occurred at a potential of ~ 1.0 V. However, it was no longer there after one cycle of potential change, because the ATP oxidation product was most likely adsorbed on the electrode surface. . This adsorption prevented further electrooxidation of ATP in the potential range from 0.50 to 1.25 V. Due to this adsorption and electrical resistance of the MIP-ATP layer, the adenine fragment of ATP was not electro-oxidized in the subsequent electropolymerization cycles.

Deposition of the MIP-ATP layer on the gold electrode of a quartz resonator (Au-QCR)

To deposit the MIP-ATP layer on the Au-QCR to perform PM measurements (Fig. 2), the same procedure was used as the procedure described above used to deposit such a layer on the platinum disc electrode. In this electrodeposition (Fig. 2a), the anode current higher than the corresponding anode current of platinum deposition on the platinum electrode was due to the much larger area of the gold electrode (5 mm in diameter). The deposition of the MIP-ATP layer on the Au-QCR resulted in a decrease in the resonance frequency, indicating an increase in the Au-QCR mass (Fig. 2b). This frequency drop was smaller in each successive cycle of the potential change, consistent with the progressive lower anode current recorded in each of these successive cycles. Moreover, the simultaneously measured dynamic resistance increased (Fig. 2c) indicating that the viscoelasticity of the MIP-ATP layer increased with the increase of its thickness.

Deposition of the MIP-ATP layer on a microscope slide with an evaporated layer of gold

To characterize the MIP-ATP layer by infra-red reflection-absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS) and spectroscopic ellipsometry, both the NIP layer and MIP-ATP were deposited on gold electrodes deposited on microscope slides using the same procedure as described above. For comparison, an ATP layer was also deposited on such a gold electrode by evaporation from a drop of 0.1 M ATP solution.

Efficiency of printing and extraction of ATP from MIP-ATP layers

After electropolymerization, the ATP template was extracted from the MIP-ATP layer. Extraction progress was monitored by IRRAS, XPS and differential pulse voltammetry (DPV).

Using IRRAS measurements, spectra were recorded for four samples, including the ATP layer (curve 1 in Fig. 3), NIP (curve 2 in Fig. 3) and MIP-ATP before (curve 3 in Fig. 3) and after (curve 3 in Fig. 3). 4 in Fig. 3) ATP template extraction. In the spectrum for the MIP-ATP layer before the extraction one can distinguish peaks corresponding to the vibration of the bonds in the ATP template, despite the significant overlap of these peaks with the peaks corresponding to the vibration of the polymer bonds. These are the peaks with the wavenumber 1102 and 875 cm ^-1 corresponding to the stretching vibrations of the bonds of the PO ₂ and POP moieties in complexed ATP, respectively [27, 28]. These peaks were significantly lower after extraction of the ATP template. The above changes in the spectrum were significantly enhanced after the normalization of the peak heights in relation to the high peak with a wavenumber of 800 cm ^-1 corresponding to the vibration of the bonds in the polythiophene molecule.

Removal of the ATP template from the MIP-ATP layer was confirmed by XPS studies in the field of electron binding energy of a phosphorus atom. In the XPS spectrum for the layer before extraction, there were two peaks with binding energies of 134 and 134.9 eV (Fig. 4a) corresponding to the electrons 2p3 / 2 and 2p1 / 2 of the phosphorus atom, respectively [29]. They confirm the presence of ATP in the MIP-ATP layer, as only ATP contains phosphorus in the tested system. There were no phosphorus peaks in the spectrum after ATP extraction (Fig. 4b), which confirms the complete removal of ATP from this layer.

The removal of the ATP template from the MIP-ATP layer was also evidenced by the results of DPV studies (Fig. 5). In these studies, Fe was used as a redox probe. The occupying molecular void of the polymer

The template particles hinder diffusion of the probe to the electrode surface [30]. Therefore, the Fe electrooxidation peak (CN) was suppressed (curve 1 in Fig. 5). However, after ATP extraction, this peak was significantly higher (curve 2 in Figure 5) because the evacuation of the imprinted molecular voids allowed the probe to diffuse freely to the electrode surface.

The thickness of the MIP-ATP layer determined by means of spectroscopic ellipsometry, deposited by means of potentiodynamic electropolymerization during three cycles of potential changes, was ~ 100 nm.

Determination of ATP under the conditions of flow injection analysis. (FIA)

ATP was determined under FIA conditions using the MIP-ATP layer with the extracted ATP template and two different methods to quantify the ATP recognition signal into an analytical signal, i.e. C1 and PM.

Example 3

Determination of ATP under FIA conditions using a C1 chemosensor with a MIP-ATP recognition layer

For the determination of ATP with a chemical sensor with signal processing with Cl, a platinum disc electrode with a diameter of 1 mm was covered with a ~ 100 nm thick MIP-ATP layer and an ATP template was extracted therefrom. The electric capacitance of the double layer C was used to determine ATP. This capacity was determined from the measured value of the imaginary component of impedance, Z, at a constant frequency of the alternating current, ω, and the potential applied to the electrode, according to equation (1).

Z ”= - æt) C

As expected, after each additional sample of ATP solution was injected, the capacity initially increased as ATP penetrated the MIP-ATP layer. Thereafter, this capacity decreased to its original value as ATP was washed out of the layer by excess carrier solution - the base electrolyte (0.1 M KF) (Fig. 6). This behavior indicates the complete reversibility of ATP binding in the layer. The measured changes in capacity were most likely responsible for the changes in the capacity of the rigid part of the electric double layer due to the negligible contribution of the capacity of its fuzzy part to the total capacity of the layer at such a high concentration of the base electrolyte with specifically non-adsorbable 0.1 M KF ions. Therefore, the rigid part of the layer was approximated using the Helmholtz model. In this model, the capacitance of the layer is directly proportional to its electrical permittivity. This permeability increases with the increasing filling of the molecular gaps in the MIP-ATP layer by the ATP analyte.

The height of the capacity change peak was proportional to the ATP concentration in the test solution. The linear concentration range extended from at least 10 to 100 µΜ (inset in Figure 6), satisfying the following linear regression equation: C = 0.44 + 0.04 CATP. In this equation, CATP is the concentration

ATP in test solution. With a signal to noise ratio of S / N = 3, the LOD value and the sensitivity of it

The -2 -1 chemosensor was 0.5 µM ATP and 0.04 nF cm ^- µM ', respectively, with a correlation coefficient of 0.99. These analytical parameters show that the produced chemical sensor is suitable for the determination of ATP in biological systems. Preferably, for these ATP assays, the analytical Cl signal was much more stable than the PM signal in the assays described below.

Example 4

Determination of ATP under FIA conditions using a PM chemosensor with a MIP-ATP recognition layer

For the determination of ATP under FIA conditions using the PM chemosensor, the Au-QCR transducer covered with a MIP-ATP layer with a thickness of ~ 100 nm, with the ATP template extracted, was used. This transducer was mounted in the EQCM 5610 flow holder. For sufficiently rigid layers mounted on the resonator, its resonance frequency change, Δ /, is opposite to mass changes, Δη, according to the Sauerbry equation, equation (2) [31].

In this equation, f ₀ is the fundamental resonance frequency of Au-QCR (equal to 10 MHz for the resonators used in the present tests), A is the acoustically active area ₂

Au-QCR (equal to 0.1963 cm for the resonators used in this study), μ is the module

PL 220 926 B1

-2 -1 shear quartz single crystal with AT cut (μ = 2.947x10 gs ^- cm ^- ) ap _q is the density of the quartz (p _q = 2.648 g cm ^-3 ). For such thin MIP-ATP layers as used in this study, changes in their viscoelastic properties can be neglected [20].

After each injection of the ATP solution, the resonance frequency initially decreased (Fig. 7). The increase in the mass of the layer due to ATP penetration into it was responsible for this decrease in frequency. The excess carrier fluid (water) then flushed the ATP away from the layer, lowering its mass. Thereby the frequency increased until it reached the value of the background frequency confirming the reversible ATP binding in the layer, shown above in the C1 measurements.

The height of the peak of frequency changes was opposite to the concentration of ATP in the test solution at least in the range from 10 to 100 μΜ, satisfying the following linear regression equation: Δ / = -3.17-0.32

CATP. For S / N = 3, the LOD value of the chemical sensor with recognition layer thus produced

MIP-ATP and with PM signal processing was 0.1 μΜ with a sensitivity of 0.6 Hz μΜ ^- with a correlation coefficient of 0.99. The LOD value of the FIA-PM chemosensor is therefore preferably an order of magnitude lower than that of the FIA-CI chemosensor described above. However, PM measurements are experimentally more demanding.

Example 5

Controlled release of ATP

Figures 6 and 7 simultaneously illustrate the controlled release of ATP. After ATP is concentrated in the polymer, it is released in a controlled manner. Therefore, in both plots (Figs. 6 and 7) there are no grades (grades with a well-formed plateau would be if ATP were irreversibly bound) after injection of the next ATP solution and the peaks (minima or maxima), i.e. concentration of ATP in the polymer after injection, and then decrease of this signal due to excess carrier solution. Excess of this solution washes ATP from the polymer.

Conclusions

Molecular modeling proved to be an efficient tool to optimize the structure and study the stability of the ATP complex with three different functional monomers, which were bis (2,2'-bitienyl) methane derivatives, used before laboratory measurements. The synthesis of two new derivatives with different binding substituents opened up new possibilities for the molecular imprinting of ATP. Solving the problem of dissolving components of very different polarity in one solution and their electropolymerization in the presence of a small amount of water resulted in the development of a new procedure for the preparation of MIP layers printed with a water-soluble template, such as ATP.

Potentiodynamic electropolymerization in the range of positive potentials is excellent for imprinting ATP into a polymer made of thiophene derivatives due to (i) short preparation time of the M1P layer, which is only a few minutes needed to deposit the layer with only three cycles of potential changes, (ii) good adhesion MIP layers to a substrate with sufficient roughness, (iii) easy control of the layer thickness by the number of potential cycles, and (iv) easy control of the porosity of the layer by selecting the appropriate components of the electropolymerization solution. Analytical detection signals recorded as peaks rather than grades indicate that the binding of ATP molecules in the imprinted MIP-ATP molecular gaps is fully reversible. This behavior is beneficial for the manufacture of reusable chemical sensors for ATP determination. The lowest LOD value for the MIP-ATP layer with a thickness of ~ 100 nm was 0.1 μΜ ATP. It was achieved using the FIA-PM sensor. This sensor is the preferred sensor for the determination of ATP in biological systems because it uses water as a carrier fluid. The detection of the FIA-CI chemosensor of 0.5 pM ATP is, disadvantageously, lower than that of the FIA-PM chemosensor. However, it is still high enough for the determination of ATP in biological systems. Controlled release of ATP is also possible, for example by the methods described in Examples 3 and 4 described above.

Claims (4)

1. A chemical compound of structural formula (formula 1), where 2. A molecularly imprinted polymer comprising a polymerized chemical compound as defined in claim 1; 1. 3. Use of a molecularly imprinted polymer as defined in claim 1; 2 as the recognition element of a chemical sensor for the selective determination of adenosine-5'-triphosphate. 4. Use of a molecularly imprinted polymer as defined in claim 1; 2 as a controlled release material for adenosine-5'-triphosphate.