GB2503064A - Thiophene compound and use thereof - 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

Thiophene derivatives, molecularly imprinted polymer formed by polymerization of thiophene derivatives and the use thereof for selective determination and controlled release of adenosine-5'-triphosphate (ATP) The invention relates to (a) new chemical compounds, thiophene derivatives, (b) molecularly imprinted polymer formed by polymerisation of thiophene derivatives, (c) the use of this polymer as recognition unit of a chemosensor for selective determination of adenosine-5'-triphosphate (ATP), and (d) the use of this polymer as material for controlled release of adenosine-5'-triphosphate (ATP).

Adenosine-5'-triphosphate (ATP) (hereinafter referred to as 1, see Fig. 8) was discovered independently by two laboratories [1, 2]. It is regarded as the primary energy transporter in living cells [3] and considered the "molecular unit of currency " in energy conversion within living organisms. When its high-energy phosphate bonds are hydrolysed, energy released becomes available for a number of biological activities, such as muscle contraction, nerve excitation, and active transport, as well as for syntheses of several different compounds, including proteins and nucleic acids [4]. ATP cannot be stored in living organisms, because it decomposes quickly. Hence, it has to be produced on an on-going basis to satisfy on-demand energy needs of a cell. Therefore, detecting the presence of AlP in a biological system is an indirect test verifying if it is alive or not. The ATP concentration in cytoplasm of living cells is relatively low; it is -1.0 uM [5]. For this reason, detection of ATP at this concentration level is important in studies of biological systems. Advantageously for selectivity of ATP determination, its concentration in a cell is thousand-fold higher than that of adenosine-5'-diphosphate (ADP), a product of enzymatic AlP dephosphorylation [6].

The structure of ATP molecule comprises three main components, i.e., adenine, ribofuranose and triphosphate. These three fragments offer in total 15 binding sites [7]. In ATP recognition in nature, all these sites are engaged in non-covalent bonding with amino acids, such as G1u156, His155, Phe28, Va135, Thr6l, ThrGO, Lys59, His2lS, SerSS, SerlS8, G1n161, LeulSJ, ArglS2 of a purinergic receptor. Under these conditions, so abundant interactions lead to specific ATP recognition.

For recognition and determination of AlP in artificial systems several different sensors were developed and fabricated. Initially, biochemical sensors (biosensors) were used for that purpose. One of them exploited the ATP ability to prevent glucose oxidation catalysed by glucose oxidase [8]. The chronoamperometric 10-jiM limit of detection (LOD) reached for that biosensor was, however, too high as to allow for ATP determination in biological systems. Although the LOD of another biosensor using chemiluminescence of the product of reaction of triphosphate of ATP with luminol was, preferably, much lower (10 nM ATP), too many various expensive chemicals had to be used for this determination [9]. Recently, aptamers, i.e., in vitro selected functional nucleotides, were employed to design biosensors for ATP determination [10, 11]. They feature inherent high selectivity and affinity, and in many respects their advantages make them superior to natural receptors for ATP recognition.

However, the major disadvantage of the biosensors for ATP recognition is their low stability and reproducibility of assays disqualifying them for application in routine assays. Therefore, several attempts have been made to fabricate artificial ATP receptors with the aim to produce chemical sensors (chemosensors).

For recent years the use of molecularly imprinted polymers (MIPs) for biomimetic recognition in selective assays has been increasing [12-19].

Therefore, it would be desirable to develop a sensor with enhanced selectivity for ATP by employing a specially designed recognition unit dedicated to ATP determination.

For this reason, the purpose of the present invention is to develop and produce a chemical substance that after polymerisation could be used for fabrication of a chemosensor for ATP determination. Another purpose of the present invention is to develop and produce a chemical substance that could be used for fabricating materials for controlled release of ATP.

In the present patent application, four different electroactive thiophene derivatives as three functional monomers and one cross-linking monomer are used for fabrication of a chemosensor for ATP determination. The functional monomers include uracilphenyl-4- [bis(2,2'-bithienyl)methane] (hereinafter referred to as 2, see Fig. 8), thiophen-3-ylboronic acid (hereinafter referred to as 3, see Fig. 8), and 1-methylamide-4-[bis(2,2'-bithienyl)methane] (hereinafter referred to as 4, see Fig. 8), and the cross-linking monomer is 3,3'-bis[2,2'-bis(2,2'-bithiophene-5-yl)]thianaphthene (hereinafter referred to as 5). The functional monomers allow for preparing a complex in solution where ATP is bound at eleven points. This means that eleven ATP binding sites are engaged in formation of the complex.

Preparation of the solution for electrochemical polymerisation required on the one hand that all five its components, each of different polarity, were dissolved in single solution, and on the other hand that electropolymerisation was carried out under such conditions that thiophene cation radicals generated in the course of electropolymerisation did not decay too quickly. Consequently, a dedicated procedure for preparing the solution was developed. In the procedure, three different organic solvents and water are used. Polymer films with molecular cavities imprinted using ATP molecules synthesized herein are denoted as MIP-ATR Complex formation for polymerisation was modelled with quantum chemical calculations.

Then, the complex was potentiodynamically electropolymerised to yield MIP films filled with ATP molecules deposited on the surfaces of two different transducers converting molecular recognition signal into electrical analytical signal. Recognition units of these chemosensors were obtained after extraction of the AlP template from these films. The signal conversion techniques of choice were piezoelectric microgravimetry (PM) and capacitive impedometry (Cl). The ATP detection in the tested solution was related to filling and emptying molecular cavities in the polymer by the assayed PIP molecules and signalled in the former technique by the resonant frequency change, proportional to the change of the polymer mass, and in the latter -by the change of electric capacitance of the MIP-ATP film.

The invention relates to compound of the structural formula (1).

(formula 1) where R = -c-ç H (formula la) or R=-Q--J)=0 (formula lb).

The invention comprises also molecularly imprinted polymer prepared by polymerisation with the use of the compound.

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

The present invention is now explained more in detail in preferred embodiments, with reference to the accompanying figures, wherein: 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)C104 in a solution of water-to-acetonitrile-to-toluene-to-propanol volume ratio 1.5: 6: 1: 1.5, recorded with 1 mm diameter platinum disk electrode for the (1) first, (2) second, and (3) third potential cycle in the 0.50 to 1.25 V potential range vs. Ag/AgCI. The potential scan rate was so mv/s.

Fig. 2 Simultaneously recorded curves for the potential dependence of (a) current, as well as (b) resonant frequency change, and (c) dynamic resistance change for deposition of the MIP-ATP film by potentiodynamic electropolynierisation on a gold electrode of a quartz crystal resonator with 10 MHz fundamental resonant frequency. The solution composition was the same as that given in the caption of Fig. 1.

Fig. 3 lnfrared-Reflection-Absorption-Spectroscopy (IRRAS) spectra for (1) ATP, (2) non-imprinted polymer (NIP) and the MIP-ATP film (3) before and (4) after AlP extraction with 0.1 M HCI at 60 °C for 5 hours. All the films were deposited on gold plated glass slides.

Fig. 4 High resolution XPS (X-ray photoelectron spectroscopy) spectra in the P 2p electron binding energy region for the MIP-ATP film (a) before and (b) after ATP extraction with 0.1 M HCI at 60 °C for 5 hours. For comparison, the dashed line in panel (a) shows the XPS spectrum for a NIP film.

Fig. 5. Differential pulse voltammograms for 0.1 M K4Fe(CN)6 in 0.1 M KNO3, recorded with a MIP-ATP film coated 1 mm diameter platinum disk electrode (1) before and after (2) ATP template extraction with 0.1 M HCI at 50 °C for 5 hours. The MIP-ATP film was deposited under potentiodynamic conditions during three potential cycles in the 0.50 to 1.25 V potential range vs. Ag/AgCI, at the potential scan rate 50 mV/s.

Fig. 6 Capacity change in time for various ATP concentrations in 0.1 M KF (pH = 8.3). The change was measured under the flow-injection analysis (FIA) conditions at potential 0.50 V vs. Ag/AgCI and frequency 20 Hz for the ATP template extracted MIP-ATP film coating a 1 mm diameter platinum disk electrode. 0.1 M KF pumped at 20 aL/mm was used as a carrier solution. The inset shows the calibration curve.

Fig. 7. Resonant frequency change in time for various ATP concentrations (pH = 7.8). The change was measured under the flow-injection analysis (FIA) conditions, for a AlP-extracted MIP-ATP film coating the gold electrode evaporated on a quartz resonator with 10 MHz fundamental resonant frequency. Water pumped at 20 RL/min was used asa carrier liquid. The inset shows the calibration curve.

Fig. 8 Scheme of eleven-point recognition 1. (a) Proposed structural formula of the complex 1 with functional monomers 2, 3 and 4, and (b) DFT-optimised structure of this complex in a B3LYP,t-31g(d) approximation.

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Preferred embodiments of the invention

Example 1

Syntheses of functional monomers The functional monomer 3 (Fig. 8) and the cross-linking monomer 5 were synthesised according to procedures developed earlier [20, 21], and the procedures for synthesis of functional monomers 2 (Fig. 8) and 4 (Fig. 8) were developed under the present study as follows: 1-Methylamide-4-[bis(2,2'-bithienyl)methane] 4: [Bis(2,2'-bithienyl)-(4-carboxyphenyl)methane [22] (500 mg, 1.10 mmol) was stirred for -45 mm under nitrogen in a 100 ml round-bottom flask containing 25 ml toluene, then SOCI2 (1.85 ml, 25 mmol) and pyridine (2.02 ml, 25 mmol) were added. The solution was refluxed for 3 h. After that, the solvents were evaporated and a green acylchloride derivative was obtained. At this stage, toluene (25 ml) was added and the solution stirred for 15 mm under nitrogen. Next, methylamine (0.05 ml, 1.10 mmol) and pyridine (6.52 ml, 80.625 mmol) were added and the solution was stirred overnight at room temperature. On evaporation of the solvents, a green crude compound remained. The crude compound was purified by liquid chromatography on a column packed with silica using a chloroform-methanol solution (95:05, v:v) as the eluent.

Yield: 180 mg (35%). 1H NMR (CHCI3-d): 5(in ppm) 8.10 (d, 2H, phenyl H), 7.45 (d, 2H, phenyl H), 7.20 (dd, 2H, bithiophene H), 7.10 (dd, 2H, bithiophene H), 7.00 (m, 4H. bithiophene H), 6.75 (dd, 2H, bithiophene, H), 5.82 (s, 1H, -CH-), 2.18 (s, 3H, methyl H).

Uraci/phenyl-4-[b!s(2,2'-b!th!eny/)methane] 2: Through a 100 ml round-bottom flask containing 1-methylamide-4-[bis(2,2'-bithienymethane] (400 mg, 0.813 mmol), 5-iodouracil (150 mg, 0.633 mmol), anhydrous K2C03 (750 mg, 5.42 mmol) and tetrakis(triphenylphosphine)palladium(0) (105 mg, 0.09 mmol) nitrogen was passed for 15 mm. Then, toluene (15 ml) and THF (15 ml) were added, and the solution was heated under reflux at 90 °C for 16 h. After that, the solution was cooled down at room temperature and filtered. After evaporation of the solvents from the filtrate, a light yellow reddish crude compound was obtained, which was purified by liquid chromatography on a silica gel column using a CHCI3:MeOH (90:10, v:v) eluent. Light red coloured (third) fraction in the column contained the desired product. Yield: 180 mg (55%). 1H NMR (CHCI3-d): 6 (in ppm) 8.01 (s, 1H, uracil -CH-), 7.20-7.14 (m, 4H, phenyl H), 7.08 (dd, 2H, bithiophene H), 7.02-6.92 (m, 4H, bithiophene H), 6.84 (d, 2H, bithiophene H), 6.74 (dd, 2H, bithiophene H), 5.68 (s, 1H, -CH-).

Example 2

Fabrication of molecularly imprinted polymer films using AlP MIP-AIP films were prepared by potentiodynamic electropolymerisation with potential ranging from 0.50 to 1.25 V vs. Ag/AgCI, at the potential scan rate 50 mV/s. The MIP-ATP film growth, both on the platinum disk electrode and on the gold electrode of the quartz crystal resonator (QCR), Au-QCR, with 10 MHz fundamental resonant frequency, was controlled by the number of potential cycles. After completing electropolymerisation, the MIP-ATP films were washed with abundant acetonitrile to remove excess of the supporting electrolyte solution. Then, the ATP template was extracted from the films with 0.1 M HCI at °C for S h. The solution with this acidity was used for extraction because of fast decomposition of the ATP triphosphate group in solutions of higher acidity.

The same procedure as that used for depositing the MIP-ATP films was used to deposit films of the control non-imprinted polymer (NIP) from a solution for electropolymerisation, which did not contain the AlP template.

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

The ATP-extracted MIP-ATP films coating the Au-QCR5 were examined with PM under flow injection analysis (FIA) conditions. Water used as a carrier liquid was pumped at the flow rate 20 jil/min through a model EQCM 5610 flow-through holder for OCR produced by the Institute of Physical Chemistry of the PAS (Warsaw) [24] with a NE-500 syringe pump from New Era Pump Systems (Farmingdale NY, USA). A model 7725i six port rotary metering valve from Rheodyne (Cotati CA, USA) was used to inject the samples of examined solutions.

The volume of each sample was 200 p.l. Injected substances were dissolved in a solution of the same composition as that of the carrier liquid, i.e., in water and 0.1 M KF for PM and Cl determination, respectively.

The FIA conditions for AlP determination in Cl experiments were the same as those in PM ones, except that the flow-through holder for OCR was replaced with a large-volume thin-layer radial-flow electrochemical cell with fixed 1 mm diameter platinum disk electrode in a PTFE insulator [25]. The applied constant potential and angular frequency were kept at 0.50 V vs. Ag/AgCI and 20 Hz, respectively. At this potential, no faradaic processes occurred.

Deposition of MIP-ATP films on different working electrodes In accordance with the optimised structure of the ATP complex with functional monomers (Fig. Sb), a solution for polymerisation in the mole ratio of components 1 2: 3:4: 5 equal to 1: 1 1:3 5 was prepared. Preferably, a high excess of the cross-linking monomer 5 was used in order to durably immobilise molecular cavities in the polymer matrix. Because AlP is soluble, and the prepared solutions are stable if an aqueous solution of the acidity of 7.7 «= pH «= 8.3 is used for dissolution, a solution of three organic solvents and water with a volume concentration of water equal to 15% was used for dissolution. The presence of 10% toluene in this solution provided for complete dissolution of 4. Toluene is, however, insoluble in water. Therefore, propanol with concentration of 15% in the solution allowed for preparing a homogeneous solution. Finally, high excess of acetonitrile was added to the solution, which -together with the supporting electrolyte, 0.1 M (TBA)C104, was necessary to carry out the electropolymerisation. The final solution composition was reflected by the volume ratio of water-to-acetonitrile-to-toluene-to- propanol equalling 1.5:6: 1: 1.5. Depending on the type of subsequent studies, the MIP-ATP film was deposited on the platinum disk, Au-QCR, or the glass slide with evaporated gold film.

Deposition of the MIP-ATP film on a 1 mm diameter platinum disk electrode For deposition of an MIP-ATP film on the 1 mm diameter platinum disk electrode potentiodynamic electropolymerisation with three potential cycles ranging from 0.50 to 1.25 V vs. Ag/AgCI was performed with at the scan rate of 50 mV/s (Fig. 1). Despite the presence of water, an electropolymerisation inhibitor, a cumulative anodic peak of electropolymerisation of the functional monomers and the cross-linking monomer -thiophene derivatives -appeared in the curve of the potential dependence of current at 0.95 V vs. Ag/AgCI, and a dark brown MIP-ATP film well attached to the electrode surface deposited on the electrode. However, the current of this peak decreased in consecutive cycles. This current decrease was presumably due to a resistive barrier effect of the MIP film, hindering charge transfer needed for electropolymerisation.

The electrooxidation of ATP was also studied for a thiophene-free solution.

Expectedly, in accordance with our earlier research [26], the voltamperometric anodic peak of the adenine moiety of ATP appeared at -4.0 V. However, it completely disappeared after only one potential cycle, presumably because the product of ATP oxidation adsorbed on the electrode surface. This adsorption prevented further electrooxidation of ATP in the potential range from 0.50 to 1.25 V. Because of the adsorption and the resistive effect of the MIP-ATP film, the adenine moiety was not electrooxidised in subsequent electropolymerisation cycles.

Deposition of the MIP-ATP film on the gold electrode of a 10 MHz quartz crystal resonator (Au-QCR) The same procedure as that described above for depositing the MIP-ATP film on the platinum disk electrode was used to deposit an MIP-ATP film on the Au-OCR for PM measurements (Fig. 2). In this electrodeposition, (Fig. 2a), significantly larger surface area of the gold electrode (diameter 5 mm) resulted in anodic current higher than analogous anodic current during film deposition on the platinum electrode. The electrodeposition of the MIP-ATP film on the Au-OCR caused a decrease of resonant frequency indicating the Au-OCR mass increase (Fig. 2b). This frequency decrease was lower in each consecutive potential cycle, in accordance with anodic current increasingly lower in each of these consecutive cycles.

Additionally, simultaneously measured dynamic resistance increased (Fig. 2c) indicating that the viscoelasticity of the MIP-ATP film increased in line with increasing film thickness.

Deposition of the MIP-ATP film on the glass slide coated with evaporated gold film For the characterisation of the MIP-ATP film with infra-red reflection-absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and spectrophotometric ellipsometry, both the NIP and the MIP-ATP films were deposited on the gold electrodes deposited by evaporation on glass slides using the same procedure as that described above.

For comparison, an ATP film was deposited on the same gold electrode by drop casting from a 0.1 M ATP solution.

Efficiency of AlP imprinting and extracting form MIP-ATP films After electropolymerisation, the AlP template was extracted from the MIP-ATP film.

The progress of the extraction was monitored with IRRAS, XPS and DPV (differential pulse voltammetry) measurements.

IRRAS spectra were recorded for four samples, including that for the ATP film (curve 1 in Fig. 3), the NIP film (curve 2 in Fig. 3), and the MIP-ATP film before (curve 3 in Fig. 3) and after (curve 4 in Fig. 3) extraction of the ATP template. In the spectrum recorded for the MIP-ATP film before extraction peaks can be distinguished that are characteristic for bond vibrations in the ATP template, in spite of significant overlap of these peaks with the peaks corresponding to bond vibrations of the polymer. This are peaks at 1102 and 875 cm' corresponding to stretching vibrations of the P02 and P-O-P moieties in complexed ATP, respectively [27, 28]. These peaks were markedly lower after extraction of the AlP template. The above spectral changes became significantly enhanced after the peaks had been normalised with respect to the strong peak at 800 cm' corresponding to bond vibrations in the polythiophene molecule.

The AlP template removal from the MIP-ATP film was confirmed by XPS studies in the range of binding energies of the phosphorus atom. In the XPS spectrum for the film before extraction, there were two peaks at binding energies of 134 and 134.9 eV (Fig. 4a) corresponding to the 2p and 2p1p electrons of phosphorus, respectively [29]. Because ATP is the only source of phosphorus in the system under study, the presence of these peaks confirms the presence of ATP in the MIP-ATP film. The peaks corresponding to phosphorus were absent in the spectrum recorded after ATP extraction (Fig. 4b), which confirms complete ATP removal from the film.

In addition, the ATP template removal from the MIP-ATP film was confirmed by the results of DPV experiments (Fig. 5). In these measurements, Fe(CN)64 was used as a redox probe. The template molecules occupying molecular cavities in the polymer prevented diffusion of the probe to the electrode surface [30]. Therefore, the peak corresponding to electrooxidation of Fe(CN)62 was attenuated (curve 1 in Fig. 5). After ATP extraction, however, the peak was significantly higher (curve 2 in Fig. 5), because the imprinted molecular cavities were then emptied which enabled the probe to freely diffuse toward the electrode surface.

The thickness of the MIP-ATP film deposited by potentiodynamic electropolymerisation during three potential cycles was determined with spectrophotometric ellipsometry to be 400 nm.

Determination of AlP under flow injection analysis (FIA) conditions The ATP under FIA conditions was determined using the MIP-ATP film with extracted ATP template and two different methods for quantitative transduction of the AlP recognition signal into the analytical signal, i.e., Cl and PM.

Example 3

FIA determination of ATP using a Cl chemosensor with a MIP-ATP recognition film For ATP determination using a chemosensor with Cl signal transduction, a 1 mm diameter platinum disk electrode was coated with a -100 nm thick MIP-ATP film and the ATP template was extracted from the film. The ATP was determined in the measurement of electric capacity of the double layer, C. The capacity was determined from the measured imaginary component of impedance, Z", at constant angular frequency, co, and potential applied to the electrode, according to the equation (1).

zfl= (1) coC Expectedly, after injection of each consecutive sample of the ATP solution the capacity initially increased in line with ATP penetration into the MIP-ATP film. Then, the capacity decreased returning to its initial level as ATP was washed out from the film by the excess of carrier solution -the supporting electrolyte (0.1 M KF) (Fig. 6). This behaviour indicates full reversibility of ATP binding in the film. Presumably, the measured changes in capacity originated from the changes of the capacity of the compact part of the electrical double layer, because of a minute contribution of the capacity of the diffuse layer to the total layer capacity at so high concentration of the supporting electrolyte with specifically non-adsorbing ions like 0.1 M KR Therefore, the compact layer was approximated with the Helmholtz model. In this model, the capacity of the layer is directly proportional to its electric permittivity. This permittivity increased when the ATP analyte entered molecular cavities of the MIP-ATP film.

The height of the capacity change peak was proportional to the ATP concentration in the solution under study. The linear concentration range extended at least from 10 to jiM (inset in Fig. 6) obeying the following regression equation: C = 0.44 + 0.04 CA-ft. In this equation, CATP is the ATP concentration in the solution under study. At the signal-to-noise ratio, S/N = 3, the LOU of this chemosensor was 0.5 jiM ATP, and the sensitivity 0.04 nF cm2 jiM', with correlation coefficient 0.99. These analytical parameters prove that the fabricated chemosensor is suitable for ATP determination in biological systems. Preferably for these ATP determinations, the analytical Cl signal was much more stable than the PM signal in the determinations described below.

Example 4

FIA determination of AlP using PM chemosensor with a MIP-ATP recognition film An Au-OCR transducer, coated with the ATP-template-extracted -100 nm thick MIP-ATP film was used for ATP determination under FIA conditions with a PM chemosensor. The transducer was mounted in an [0CM 5610 flow-through holder. According to the Sauerbrey relation [31], equation (2), for sufficiently rigid films deposited on the resonator, the change in its resonant frequency, Af is opposite to the mass change, Am. Af--2L2AIn (2) In this equation, ft is the fundamental resonant frequency of the Au-OCR (10 MHz for resonators used in the present study), A is the acoustically active Au-OCR surface (equal to 0.1963 cit2 for resonators used in the present study), q is the shear modulus of an AT-cut quartz single crystal (pq = 2.947x10" g 52 cm1), and q is the quartz density (pq = 2.648 g cm3). For so thin MIP-ATP films as those used in the present study, changes in their viscoelastic properties can be neglected [20].

After each consecutive injection of the AlP solution, the resonant frequency initially decreased (Fig. 7). This drop in frequency resulted from growing film mass related to film penetration by ATP. The excess of the flowing carrier liquid (water) resulted in washing out the ATP from the film, thus decreasing the film mass. As a result, the frequency increased.

This growth reached the background frequency level, confirming reversible ATP binding in the film, as shown for Cl measurements above.

The change in the height of the frequency change peak was opposite to that of the ATP concentration in solution, at least in the range from 0.1 to 100 tiM, obeying the following regression equation: Af = -3.17 -0.32 CATp. For S/N=3, the LOD of the chemosensor so fabricated, with a recognition MIP-ATP film and with signal transduction using PM was equal to 0.1 pM at the sensitivity level 0.6 Hz pM', with correlation coefficient 0.99. Therefore, the LOD for the FIA-PM chemosensor is, preferably, by one order of magnitude lower than for the FIA-CI chemosensor described above. The PM measurements are, however, more demanding.

ExampleS

Controlled ATP release Fig. 6 and 7 both illustrate controlled release of ATR After accumulating ATP in the polymer, it is then released in a controlled way. Therefore, after injecting consecutive portion of the ATP solution no step-like signals appear in both plots (Fig. 6 and 7) (steps would be observed if ATP would be bound irreversibly), but peak-like signals (minima or maxima), i.e., a growth of the signal indicating that ATP is being accumulated in the polymer after injection, followed by a drop of the signal caused by the excess of the carrier liquid. The excess of this solution washes out PIP from the polymer.

Conclusions

Molecular modelling turned out to be an efficient tool for optimising geometry and examining stability of the complex of ATP and three different functional monomers, bis(2,2'-bithienyl)methane derivatives, prior to laboratory measurements. The synthesis of two new thiophene derivatives with different binding substituents opened new opportunities for molecular imprinting of ATR Successful solubilisation of components with very different polarities in one solution and their subsequent electropolymerisation in the presence of small amount of water resulted in a development of a new procedure for preparation of the MIP films templated by water-soluble analyte, such as ATR Potentiodynamic electropolymerisation in the range of positive potentials proved to be excellent for ATP imprinting into a polymer made of thiophene derivatives. The advantages include (i) fast preparation of the MIP film within a few minutes needed for film deposition using three potential cycles, (ii) good adhesion of the MIP film to the substrate with sufficient roughness, (iii) easy control of the film thickness with the number of potential cycles, and (iv) easy control of the film porosity by selecting appropriate components of the solution for electropolymerisation. Analytical detection signals recorded as peak-like rather than step-like signals indicate that ATP binding in imprinted molecular cavities of MIP-ATP is fully reversible. This behaviour is preferred for fabrication of reusable chemosensors for AlP determination. The lowest LOD for a -100 nm thick MIP-ATP film was 0.1 jiM ATP, and was reached by the PM chemosensor under FIA conditions. This chemosensor is preferred for ATP determination in biological systems, because water is used as the carrier liquid. The detectability of the Cl chemosensor under FIA conditions (0.5 aM ATP) is, non-preferably, lower than that of the PM chemosensor under FIA conditions. It is, however, still sufficiently high for ATP determination in biological systems. It is also possible to control AlP release, for instance with methods described in Examples 3 and 4 presented above.

Acknowledgements The present work was financed in part by the Foundation for Polish Science (Project no. MPD/2009/1/stypl9, support to TPH), the European Regional Development Fund under the Project "Innovative Economy" (Grant no. ERDFJ POlG.01.01.02-00-004J8 2007-2013, support to WK, KRN and JWS) and by the US National Science Foundation (Grant no. 1110942, support to FD).

The fees related to protection of the invention were financed by the Project NanOtechnology, Biomaterials and aLternative Energy Source for ERA integration FP7-REGPOT-CT-2011-285949-NOBLESSE.

Claims (4)

1. Claims 1. A compound of the structural formula (formula 1), (formula 1) where R = H (formula la) or R=_Q-2J)=0 (formula ib).
2. 2. Molecularly imprinted polymer prepared by polymerisation using the compound according to claim 1.
3. 3. The use of molecularly imprinted polymer according to claim 2 as recognition unit of a chemosensor for selective determination of adenosine-5'-triphosphate.
4. 4. The use of molecularly imprinted polymer according to claim 2 as material for controlled release of adenosine-5'-triphosphate.