GB2548540A

GB2548540A - New bis(2, 2'-bithienyl)methane derivative and method of producing thereof, molecularly imprinted polymer film, method of producing therof and its use

Info

Publication number: GB2548540A
Application number: GB1409820.6A
Authority: GB
Inventors: Huynh Tan-Phat; Sosnowska Marta; Sobczak Janusz; Bikram K C Chandra; Nesterov Vladimir; Kutner Wlodzimierz
Original assignee: INST CHEMMII FIZYCZNEJ POLSKIEJ AKADEMII NAUK
Current assignee: INST CHEMMII FIZYCZNEJ POLSKIEJ AKADEMII NAUK
Priority date: 2013-06-03
Filing date: 2014-06-03
Publication date: 2017-09-27
Anticipated expiration: 2034-06-03
Also published as: GB201409820D0; PL223094B1; PL404175A1; GB2548540B

Abstract

(I) A bis (2,2-bithienyl)methane derivative of formula (I) (bis(2,2-bithienyl)-(4-amino-phenyl)methane). A method of producing the bis (2,2-bithienyl)methane derivative of formula (I) is provided wherein the initial solution of 2,2-biothiophene and 4-acetaminobenaldehyde in ethylene glycol is reacted with perchloric acid at a temperature ranging from 40 to 90oC, which results in bis(2,2-bithienyl)-(4-aminophenyl)methane of formula 1. A method for fabricating a molecularly imprinted polymer and a molecularly imprinted polymer film produced by that method consisting of a polymer molecularly imprinted with nitroaromatic compounds, in particular those selected from the group comprising 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT) are also outlined. The molecularly imprinted polymer film is useful as a chemical sensor for the selective detection and determination of nitroaromatic compounds.

Description

New bis(2,2'-bithienyl)methane derivative and method of producing thereof, molecularly imprinted polymer film, method of producing thereof and its use for selective detection and determination of nitroaromatic compounds

The invention relates to a new bis(2,2'-bithienyl)methane derivative and a method of producing thereof, a molecularly imprinted polymer film, a method of producing thereof and its use for selective detection and determination of nitroaromatic compounds.

Nitro compounds are active components of explosives commonly used not only for military (bombs, mines, grenades), but also for civil purposes (blasting devices, propellants, fireworks, etc.). For that, over 100 different nitro compounds are used. Among them, nitro-substituted hydrocarbons account for one of the largest groups. In compounds belonging to this group, the nitro substituents are directly attached to the carbon atoms of the hydrocarbon (an alkane chain or an aromatic ring). The more nitro groups on the molecule of the explosive, the higher is the energy released during the detonation.

Therefore, nitroaromatic compounds (NTs) carrying three nitro groups per molecule, such as 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4,6-trinitrophenol (TNP) (Scheme 1), are among those most highly explosive. NTs are toxic -they contaminate soil, water, or air posing a serious threat to natural environment. The concentrations of NTs permitted for humans range between 2 to 7 nM [Patnaik, P., Chap. 40, in A Comprehensive guide to the hazardous properties of chemical substances. 2007, John Wiley & Sons: New Jersey, p. 691-702], Therefore, chemical sensors for determination of NTs on a so low level have been developed, fabricated and commercialised. The research is, however, still being pursued to develop chemosensors of low manufacturing cost, high sensitivity, and high selectivity [Toal, S. J. and Trogler, W. C. J., Polymer sensors for nitroaromatic explosives detection, Mater. Chem. 2006, 16, 2871-2883, and Salinas, Y. et al., Optical chemosensors and reagents to detect explosives, Chem. Soc. Rev. 2012, 41, 1261-1296],

Molecularly imprinted polymers (MIPs) are very promising when used for selective determination of explosive NTs. This is because size, shape, and special orientation of recognition sites produced in imprinted cavities of MIPs accurately match up those of binding sites of target analytes. [K. Haupt, Molecularly imprinted polymers in analytical chemistry, Analyst 2001, 126, 747-756; A. McCluskey et al., Molecularly imprinted polymers (MIPs): sensing, an explosive new opportunity? Org. Biomol. Chem. 2007, 5, 3233-3244; P. S. Sharma, F. D'Souza, and W. Kutner, Chap. 4, in Portable chemical sensors - weapons against bioterrorism, NATO science for peace and security, Series A: Chemistry and biology, D. P. Nikolelis, Editor, Springer, Berlin, 2012, pp. 63-94, and P. S. Sharma et al., Molecular imprinting for selective chemosensing of environmental hazards and drugs of abuse, TRAC-Trends Anal. Chem. 2012, 34, 59-77]. For example, MIP films in fluorescent chemosensors prepared by polymerisation of methacrylic acid (MAA) and ethylene glycol dimethylacrylate (EGDMA), were used to modify the surface of CdSe quantum dots [Stringer, R. C. et al., Detection of nitroaromatic explosives using a fluorescent-labeled imprinted polymer, Anal. Chem. 2010, 82, 4015-4019, and Stringer, R. C., et al., Comparison of molecular imprinted particles prepared using precipitation polymerisation in water and chloroform for fluorescent detection of nitroaromatics, Anal. Chim. Acta 2011, 703, 239-244]. The fluorescence of these quantum dots, emitted at the wavelength 605 nm, was quenched due to binding of DNT and TNT (Scheme 1) by the MIP films coating the quantum dots. However, the limit of detectability (LOD) of these chemosensors was relatively low, ranging from 0.1 to 0.5 μΜ. Therefore, the chemosensors were applicable only for determination of NTs in solutions.

Scheme 1. Structural formulae of four most common explosive nitroaromatic compounds. TNP - 2,4,6-trinitrophenol, TNT - 2,4,6-trinitrotoluene, TNB - 1,3,5-trinitrobenzene, and DNT- 2,4-dinitrotoluene.

In another study, a hierarchically imprinted silica film of a chemosensor was prepared using 510-nm diameter polystyrene particles to increase porosity of the film. Making use of the TNT fluorescence quenching at the 235-nm excitation wavelength, LOD of 8.8 nM TNT was reached. [Zhu, W. et al., Hierarchically imprinted porous films for rapid and selective detection of explosives, Langmuir 2011, 27, 8451-8457].

Electrochemical methods were also used to process a recognition signal of the MIP film complexation of butanethiol, and DNT or TNT complexation in ethanol. The signal was transduced to the charge-transfer resistance signal of the K4Fe(CN)6 redox probe [Apodaca, D. C. et al., Detection of 2,4-dinitrotoluene (DNT) as a model system for nitroaromatic compounds via molecularly imprinted short-alkyl-chain SAMs, Langmuir 2011, 27, 6768-6779], The interaction of the NT compounds and butanethiol in a polar protic solvent (ethanol), used for formation of a prepolymerisation complex, was weak. Hence, the LOD value reached for this sensor (~40 μΜ) was relatively high. This LOD could, however, be reduced to as low value as 13 nM TNT with the use of differential pulse voltammetry (DPV) and by replacing this protic solvent (ethanol) by that aprotic acetonitrile and butanethiol by self-assembled 1-dodecanethiol monolayer. [Nie, D. et al., Two-dimensional molecular imprinting approach for the electrochemical detection of trinitrotoluene, Sens. Actuators, B 2011,156, 43-49],

For gas-phase NTs detection, a polysilane recognition film was integrated with a light refracting interferometric component. A measurement of the refractive index of the MIP film allowed determining the LOD of 2.4 ppt TNT. [Edmiston, P. L. et al., Detection of vapor phase trinitrotoluene in the parts-per-trillion range using waveguide interferometry, Sens. Actuators, B 2010,143, 574-582], In another study aiming at the development of a TNT determination procedure in the gas phase, an acrylamine (AA) MIP, imprinted with a TNT template, was spin-coated on a quartz crystal resonator of a microbalance. The experimentally determined TNT uptake rate by the MIP was ~150 pg per pg MIP per hour [Bunte, G. et al., Gas phase detection of explosives such as 2,4,6-trinitrotoluene by molecularly imprinted polymers, Anal. Chim. Acta 2007,591, 49-56].

Molecular modeling was used to estimate the energy gain (~90 kJ mol'1) reached due to TNT complexation by six acrolein functional monomers [Saloni, J. M. et al., Theoretical investigation on monomer and solvent selection for molecular imprinting of nitrocompounds, J. Phys. Chem. A 2013,117,1531-1534].

The present patent application provides a novel MIP, developed and fabricated for selective detection, by τι-π interactions, and determination of NTs, including 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB) and 2,4-dinitrotoluene (DNT) (Scheme 1). For that, a new functional monomer, bis(2,2'-bithienyl)-(4-aminophenyl)methane 1, was designed and synthesised.

Therefore, the present invention relates to a new bis(2,2'-bithienyl)methane derivative, i.e., bis(2,2'-bithienyl)-(4-aminophenyl)methane 1.

In addition, the invention provides for a method for producing the aforementioned bis(2,2'-bithienyl)methane derivative, so that the initial solution of 2,2'-bithiophene and 4-acetaminobenzaldehyde in ethylene glycol is reacted with perchloric acid, at a temperature ranging from 40 to 90 °C, which results in bis(2,2'-bithienyl)-(4-aminophenyl)methane 1.

Preferably, the reaction is carried out in a 70% perchloric acid.

Preferably, the reaction is carried out at 60 °C.

The invention relates also to a method for fabricating molecularly imprinted polymer such that it comprises the following steps: (a) a solution is prepared, containing bis(2,2'-bithienyl)-(4-aminophenyl)methane as a functional monomer 1, tris([2,2'-bithiophen]-5-yl)methane as a cross-linking monomer 2, and imprinted nitroaromatic compound (NT), in solvent or solvent mixture, preferably in acetonitrile and dichlorobenzene mixed in a 1:1 (v:v) ratio, in which a π-π prepolymerisation complex of the aminophenyl substituent of the functional monomer 1 with aromatic ring of the nitroaromatic compound (NT) is formed, and subsequently (b) the prepolymerisation complex obtained in step (a) is electropolymerised, preferably under potentiodynamic conditions with potentials cycled linearly, deposited on the electrode from the aforementioned (step (a)) solution, 0.1 M in (TBAJCIO^ as a molecularly imprinted polymer film with imprinted template of a nitroaromatic compound (MIP-NT), and then (c) the imprinted, in the step (b) above, template of the nitroaromatic compound (NT) is extracted from the MIP-NT film, to result in an MIP film with emptied imprinted molecular cavities.

Preferably, the nitroaromatic compounds (NT) used in the method according to the invention are those selected from the group comprising 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT).

Preferably, in the method according to the invention, a functional monomer 1, a nitroaromatic compound (NT), and a cross-linking monomer 2, are used in step (a) in a 1:1:3 molar ratio, respectively.

Preferably, in the method according to the invention, the potentiodynamic electropolymerisation comprises from 1 to 20, preferably 9 linear cycles of potential changes ranging from 0.50 to 1.50 V vs. Ag/AgCI, at a scan rate from 5 to 500 mV s'1, preferably 50 mV s'1.

Preferably, in the method according to the invention, a metal or carbon electrode is used in step (b) as the electrode, preferably platinum disk electrode.

The invention provides also a film of molecularly imprinted polymer, such that it consists of a polymer molecularly imprinted using nitroaromatic compounds, in particular those selected from the group comprising 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT), produced with the method described above.

In addition, the invention comprises the use of an imprinted polymer film as defined above as a recognition unit of a chemical sensor for selective detection of nitroaromatic compounds, in particular such as 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT).

The invention comprises also the use of an imprinted polymer film as defined above as a recognition unit of a chemical sensor for determination of nitroaromatic compounds, in particular such as 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT).

The films of polymers imprinted with the above mentioned NTs (MIP-NTs) were deposited by means of potentiodynamic electropolymerisation on a platinum disk electrode from a solution containing 1 and various NTs, in a 1:1 1:NT molar ratio. Initially, these NTs were used as imprinting templates. Both the molecular modeling with density functional theory (DFT) at the M062X/3-21G* level and the fluorescence titration showed a 1:1 stoichiometry of the prepolymerisation complex of 1 with NT, in vacuum and in solution, respectively. To fabricate a chemosensor, MIP-NTs films with extracted NTs templates were prepared. DPV measurements using a redox probe and NT electroreduction were performed to confirm that the NT template was entirely removed from the MIP-NT film. The recognition signal of NT by the MIP-NT film, after extraction of the template, was transduced into the analytical signal of changes of the electric resistance of the film with electrochemical impedance spectroscopy (EIS), in the absence of faradaic processes, in connection with the injection analysis under steady-state conditions. The LOD of fabricated chemosensors ranged from 8 to 30 nM, indicating that they are suitable for NTs determination at the level of toxic concentrations (—10 nM) in natural environment contaminated with these analytes. The selectivity of the chemosensors with respect to interferents, as determined by molecular cross-imprinting, was in the range of 1.7 to 9.1.

In continuation of our former research on devising and fabricating MIP chemosensors for determination of biologically relevant compounds, as well as the procedures for these determinations [Pietrzyk, A. et al., Selective histamine piezoelectric chemosensor using a recognition film of the molecularly imprinted polymer of bis(bithiophene) derivatives, Anal. Chem. 2009, 81, 2633-2643; Pietrzyk, A. et al., Melamine acoustic chemosensor based on molecularly imprinted polymer film, Anal. Chem. 2009, 81, 10061-10070; Pietrzyk, A. et al., Molecularly imprinted poly[bis(2,2'-bithienyl)methane] film with built-in molecular recognition sites for a piezoelectric microgravimetry chemosensor for selective determination of dopamine, Bioelectrochemistry 2010, 80, 62-72; Pietrzyk, A. et al., Molecularly imprinted polymer(MIP) based piezoelectric microgravimetry chemosensor for selective determination of adenine, Biosen. Bioelectron. 2010, 25, 2522-2529, and Huynh, T.-P. et al., Electrochemically synthesized molecularly imprinted polymer of thiophene derivatives for flow-injection analysis determination of adenosine-5'-triphosphate (ATP), Biosens. Bioelectron. 2013, 41, 634-641], herein we present a functional monomer of the series of bis(2,2'-bithienyl)methane derivatives, i.e., bis(2,2'-bithienyl)-(4-aminophenyl)methane 1, designed and synthesised as a part of the invention for determination of some common NTs, such as TNT, TNP, TNB, and DNT. Recognition of these analytes was due to the π-π interactions between the aromatic rings of the NTs and that of the phenylamine group of 1. The occurrence of these interactions was confirmed with molecular modeling. Fluorescence titration of 1 with NT solutions allowed for determination of stability constants of prepolymerisation complexes in solutions. Then, MIP-NT films were deposited on platinum electrodes using potentiodynamic electropolymerisation. In the subsequent step, NT templates were extracted from MIP-NT films, as confirmed with DPV. Following the extraction, chemical recognition signals of NTs were transduced into analytical signals of electric resistance of MIP films in the absence of faradaic processes.

The present invention is now explained more in detail in preferred embodiment, with reference to the accompanying figures, wherein:

Fig. 1 shows a *H NMR spectrum of monomer 1 in deuterated chloroform;

Fig. 2 shows an electrospray ionisation mass spectrometry (ESI-MS) spectrum of i;

Fig. 3 shows emission spectra of 0.25 mM (1) 1, (2) TNP, (3) DNT, (4) TNT, and (5) TNB in toluene; excitation at Λ = 350 nm. The spectra 2 to 5 are expanded by 50 times;

Fig. 4 shows fluorescence of 1 quenched by (a) TNP, (b) TNT, (c) TNB, and (d) DNT in toluene; excitation at λ = 350 nm. Initial concentration 1 was c° = 0.25 mM, NT concentrations were in the range: 0.017 < cNt ^ 0,3 mM;

Fig. 5 shows cyclic voltammograms recorded with the potential scan rate 50 mV s'1 at the 1-mm diameter Pt disk electrode, in the acetonitrile (ACN) and 1,2-dichlorobenzene (DCB) solution (1:1, v:v), 0.1 M in (TBA)CI04 and 1 mM in (1) DNT, (2) TNT, (3) TNB, (4) TNP, and (5) 1; (6) a blank supporting electrolyte solution;

Fig. 6 shows cyclic voltammograms recorded with the potential scan rate 50 mV s'1 at the 1-mm diameter Pt disk electrode, for (1) 1 mM 1 and 0.1 M (TBA)CI04 in ACN and DCB solution (1:1, v:v), and after addition of (2) DNT, (3) TNT, (4) TNB, and (5) TNP to reach concentration of 1 mM in the tested solution;

Fig. 7 shows current curves as a function of potential recorded during potentiodynamic electropolymerisation for deposition of an MIP-TNP film on the 1 mm diameter Pt disk electrode for 0.5 mM TNP, 0.5 mM 1, 1.5 mM 2, and 0.1 M (TBA)CI04 in the ACN and DCB (1:1, v:v) solution; the numbers of consecutive cycles of the linear potential changes are given at the curves;

Fig. 8 shows differential pulse voltammograms recorded at (1) the 1-mm diameter Pt disk electrode immersed in 2 mM TNP, 0.1 M NaCI, and for an MIP-TNP film coated electrode (2) before and (3) after TNP extraction. The initial volume of 0.1 M NaCI in the electrochemical cell was 3 ml;

Fig. 9 shows an electrochemical impedance spectroscopy (EIS) plot of (a) the imaginary impedance component vs. real impedance component; the inset shows magnified semicircles obtained for the range of high frequencies, and (b) the impedance phase angle, φ, as a function of frequency logarithm; the inset shows a magnified plot in the B frequency range for (1) the MIP-TNP film after extraction of the TNP template, recorded after addition of the TNP analyte, to reach the final TNP concentration of (2) 0.13, (3) 0.38, (4) 0.75, (5) 2.5, (6) 4.5, (7) 7, and (8) 12 μΜ in the analysed 0.1 M KCI water and acetone (ACE) solution (1:1, v:v). The initial volume of 0.1 M KCI was 2 ml. The frequency was changed in the range of 500 kHz to 1 Hz. The measurements were performed at a constant potential that was equal to the open circuit potential for the analysed solution, as measured vs. Ag/AgCI electrode. The A, B, and C frequency ranges are described in text;

Fig. 10 shows calibration curves for electric resistance of the MIP-NTs films covering Pt disk electrodes as a function of NT concentration for films, after extraction of the NT template, (a) MIP-TNP, (b) MIP-TNT, (c) MIP-TNB, and (d) MIP-DNT, determined by injection of 0.1 M KCI solutions containing (a 1, c3, and d2) TNP, (bl, a3, and d3) TNT, (cl, o2, and b2) TNB, and (dl, b3, and c2) DNT. The initial volume of the 0.1 M KCI solution was 2 ml. The frequency was changed in the range of 500 kHz to 1 Hz. The measurements were performed at a constant potential that was equal to the open circuit potential for the analysed solution, as measured vs. Ag/AgCI electrode;

Fig. 11 shows the change of the real impedance component during 200-μΙ injections of water and ACE solutions (1:1, v:v), 0.1 M in KCI and different TNP concentrations (given in the Figure). The measurements were performed at a constant potential that was equal to the open circuit potential for the analysed solution, as measured vs. Ag/AgCI electrode, at 20 kHz frequency, for an MIP-TNP film coated Pt disk electrode after extraction of the TNP template. The inset shows the calibration curve for an MIP-TNP film coated electrode. The flow rate of the carrier liquid, 0.1 M KCI in the water and ACE solution (1:1, v:v), was 20 μΙ/min. 2. Experimental 2.1 Reagents and materials

Acetonitrile (ACN), 1,2-dichlorobenzene (DCB), TNB, TNP, DNT, and all reagents for synthesis were purchased from Sigma Aldrich.

Tetra-n-butylammonium perchlorate, (TBA)CI04, was supplied by SACHEM. Hydrochloric acid (HCI) and potassium chloride (KCI) were purchased from Fisher. Toluene and methanol were supplied by EMD.

Acetone (ACE) and TNT were supplied by, PHARMCO-AAPER and CHEM Service, respectively.

Indium-tin oxide (ITO) coated glass slides of electric resistance 10 Q cm"2 were supplied by Delta Technologies.

The cross-linking monomer, tris([2,2'-bithiophen]-5-yl)methane 2, was prepared according to an earlier reported procedure [Huynh, T.-P. et al., Molecularly imprinted polymer of bis(2,2'-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry 2012, DOI: 10.1016/j.bioelechem.2012.1007.1003]. 2.2 Preparation of functional monomer 1 (Scheme 2)

Bis(2,2'-bithienyl)-(4-aminophenyl)methane 1. First, 2,2'-bithiophene (2.54 g, 15.3 mmole) and 4-acetaminobenzaldehyde (1.0 g, 6.13 mmole) were mixed with ethylene glycol (80 ml) and the resulting mixture was stirred for 30 min under nitrogen. Then, 70% HCI04 (16 ml, 245.2 mmole) was added and the solution obtained was stirred for another 16 h at 60 °C. Subsequently, the mixture was cooled to room temperature, and then excess of dichloromethane was added in order to completely extract the product of the reaction. Afterwards, an excess acid of the mixture was neutralized with the saturated Na2C03 solution. The collected organic liquid layer was washed with water, and then dried with anhydrous Na2S04, and concentrated by solvent evaporation under reduced pressure. An oily product was purified by liquid chromatography on a silica gel column using hexane:dichloromethane (4:1, v:v) eluent. 1.53 g bis(2,2'-bithienyl)-(4-aminophenyl)methane was obtained (yield 57%). The *H NMR spectrum for d-CDCI3 solution (δ, ppm) (Fig. 1): 7.18-7.16 (dd, 2H, bithiophene Η), 7.16-7.13 (d, 2H, bithiophene H), 7.11-7.08 (dd, 2H, bithiophene H), 7.03-7.00 (m, 2H, bithiophene H), 6.98-6.95 (dd, 2H, phenyl H), 6.77-6.74 (dd, 2H, phenyl H), 6.68-6.64 (d, 2H, bithiophene H), 5.66 (s, 1H, -CH-), 3.7-3.3 (s, 2H, -NH2). ESI-MS analysis, [M+] molecular ion signal, m/z, calculated 435.7, found 436.0 (Fig. 2).

For crystallisation, 1 was dissolved in dichloromethane and spiked with a few drops of concentrated HCI, in a 5 ml vial. Then, the vial was tightly capped so that the solvent only slowly evaporated. After few weeks, dark-green needle-shape crystals were formed in the solution. The crystal structure determined herein is shown in Scheme 2. This crystal structure has been deposited at the Cambridge Crystallographic Data Centre (CCDC) and allocated the deposition number CCDC 923045.

Scheme 2. Crystal structure of bis(2,2'-bithienyl)-(4-aminophenyl)methane 1. 3. Results and discussion 3.1 Complexation of NT and 1 in toluene

In view of low polarity of both NTs and 1, a toluene non-polar solvent was chosen for fluorescence titration. This choice was advantageous also because of high solubility of both NTs and 1 in toluene. Moreover, in view of a low electric permittivity of toluene, ε = 2.374, intermolecular interactions of NT and 1 in that solvent were enhanced as compared with analogous interactions in solvents of higher polarity. An additional benefit of toluene was low volatility due to its high boiling point (110.6 °C), which allowed for maintaining concentration of both 1 and NT constant throughout the titration. The emission spectra of 1 were recorded with excitation at 350 nm. Based on analysis of the emission spectra of 1 and NTs (Fig. 3), the emission band of 1 observed during titration NTs (Fig. 4) was ascribed to fluorescence of its bis(2,2'-bithienyl)methane moiety. [Finden, J. et a I., Reversible and amplified fluorescence quenching of a photochromic polythiophene, Adv. Mater. 2008, 20, 1998-2002]. Addition of consecutive portions of the NT titrant solution resulted in gradual decrease of the emission intensity of 1 (Fig. 4). That was presumably due to the electron transfer from the photoexcited bis(2,2'-bithienyl)methane moiety of 1 to NT, an electron acceptor (more information are given below). By plotting the emission intensity against the NT concentration (insets in Fig. 4a, 4b, 4c, and 4d), the 1:1 stoichiometry was determined for 1-NT complexes. Therefore, the complexation equilibrium of 1 and NT can be expressed with Equation (1).

The stability constant, K, of the formed complex 1-NT can be calculated using the Benesi-Hildebrand relationship, Equation (2), [Atwood, J. L., Davies, J. E. D., Macnicol, D. D. and Vogtle, F, in Comprehensivesupramolecular chemistry, 1st ed., Davies, J. E. D., Ripmeester, J. A., Eds., Pergamon: Oxford, 1999, Vol. 8, pp 425-444]

where, /0 and / are fluorescence intensities, before and after addition of the NT titrant, respectively, whereas cf and %T stand for initial concentrations of 1 (0.25 mM) and the NT titrant, respectively. The difference of absorption coefficients is expressed as, ^T-iCF = si.MT ~ εί ~sm> where sl7 and %r are molar absorption coefficients of 1-NT, complex, monomer 1, and NT, respectively.

Table 1. Shift of anodic half-peak potentials (AEpa/2), stability constant (/(), and Gibbs energy change (AG) accompanying 1:1 complex formation by different nitroaromatic compounds (NTs) and monomer 1, determined with cyclic voltammetry, fluorescence quenching, and calculated with DFT molecular modeling at the M602X/3-21G* level.

Table 2. Linear regression equations obtained by fitting their parameters to the fluorescence titration curves shown in Fig. 4a, 4b, 4c, and 4d.

By fitting experimental data obtained for each NT in the concentration range of 0 to 0.25 mM (inset in Fig. 4) with straight lines, we obtained linear regression equations (Table 2). Next, the values of complex stability constants were obtained by dividing the straight line intercepts by the slopes (Table 1). The stability constant of the 1-TNP complex is the highest, being almost one-and-a-half times higher than that of the 1-TNT complex, twice that of the 1-TNB complex, and thrice that of the 1-DNT complex. Differences in the stability constants of 1-NT complexes originate from differences in molecular structures of these complexes - the presence of different benzene-ring substituents (see below). Moreover, the stability constant of the 1-TNB complex determined herein was higher than that of the bisporphyrins-TNB complex [Jeyakumar, D. and Krishnan, V., Intermolecular complexes of singly linked bisporphyrins with trinitrobenzene, Spectrochim. Acta, A 1992, 48, 1671-1682]. The higher value obtained in the present study was due to the use of a low electric permittivity solvent, such as toluene, rather than a much higher electric permittivity solvent, such as 1% pyridine in methanol, used for the bisporphyrins-TNB complex [Jeyakumar, D. and Krishnan, V., Intermolecular complexes of singly linked bisporphyrins with trinitrobenzene, Spectrochim. Acta, A 1992, 48, 1671-1682]. Hence, it was concluded herein that the solvent plays an important role in the complexation. 3.2 Molecular modeling of the 1-NT complexes

To determine stoichiometry and optimal structure of the complexes of 1 with each NT, the structures of these complexes were DFT modelled at the M062X/3-21G* level (Scheme 3). The M062X method was selected because of its high accuracy in optimisation of noncovalent bonds [Hohenstein, E. G. et al., Assessment of the performance of the M05-2X and M06-2X exchange-correlation functionals for noncovalent interactions in biomolecules, Chem. Theory Comput. 2008, 4, 1996-2000]. Expectedly, the π-π interactions, with their small deflection angle between the NT plane and that of the phenylamine moiety of the monomer 1, were responsible for complexation of one NT molecule with one molecule of 1. This result of the complex structure optimisation was in accord with the crystal structure of the complex of 4-(4-aminophenylsulfonyl)aniline with TNB, as determined earlier [Smith, G. and Wermuth, U. D., 4-(4-Aminophenylsulfonyl)aniline-l,3,5-trinitrobenzene (1/2), Acta Crystallogr. E 2012, 68, 494], The distances between interacting molecules in the complex of 1 and different NTs were in the range of 0.29 to 0.31 nm. These distances are thus shorter than that of ~0.36 nm determined for the complex of 4-(4- aminophenylosulfonylo)aniline with TNB [Smith, G. and Wermuth, U. D., 4-(4-Aminophenylsulfonyl)aniline-l,3,5-trinitrobenzene (1/2), Acta Crystallogr. E 2012, 68, 494], This indicates stronger binding of 1 with NT in the complexes reported herein.

Differences between the experimentally determined distances between the plane of the phenylamine moiety of 1 and that of the TNB ring, and those obtained in molecular modeling in the present patent application originate from the solvent (water and ethanol) effect, weakening the π-π interaction.

Scheme 3. Structures of the 1:1 complexes of 1 and (a) TNP, (b) TNT, (c) TNB, (d) DNT, optimised with the density functional theory (DFT) at the M062X/3-21G* level.

In the present study, values of the Gibbs energy changes accompanying formation of each of the four complexes were calculated at the same M062X/3-21G* level. The changes correlate well with the complex stability constants determined by the fluorescence titration (Table 1). However, differences between the energy gain due to complexation are not as large as those between the complex stability constants because of at least two reasons, i.e., (i) vacuum rather than a solvent was used in the fluorescence titration as the complexation medium for molecular modeling, and (ii) apart from interaction of the complex components no steric effect was taken into account for optimization of the complex structure.

Scheme 4. The HOMO (left) and LUMO (right) energy levels of the complexes of 1 and (a) TNP, (b) TNT, (c) TNB, and (d) DNT, calculated with molecular modeling using the density functional theory (DFT) at the M062X/3-21G* level; The Eg symbol stands for the energy difference between the HOMO and the LUMO levels of the complexes;

Differences in the complex stability constants determined with the fluorescence titration can be ascribed to the energy difference between the HOMO and LUMO energy levels of the complexes, Eg (Scheme 4). The HOMO level of the complex is localised on the bis(2,2'-bithienyl)methane moiety of 1 and, therefore, electron from this moiety is excited. The LUMO level of the complex is localised on the NT molecule, and hence the excited electron is transferred on that level. Therefore, fluorescence of the bis(2,2'-bithienyl)methane moiety of 1 is quenched by the NT. Apparently, stability constants of the complexes are inversely proportional to Eg because the higher the Eg energy gap, the less probable is the electron transfer from the HOMO level of the photoexcited bis(2,2'-bithienyl)methane moiety of 1 to the LUMO level of the NT molecule.

The same DFT method and basis set (M062X/3-21G*) were also used to optimise structure of the complex where the TNB nitro group interacted via hydrogen bonding with the amino group of 1 (Scheme 5), to compare the complex enthalpy of formation with that of the complex formed by τι-π interactions. It turned out that the complex formed due to hydrogen bonding shows a positive change of Gibbs energy (10,15 kJ mol1). This Gibbs energy gain was positive because the enthalpy change due to hydrogen bond formation was small (-30.15 kJ mol1) compared with high entropy change (-135.68 J mol1 K1) of so large moiety as bis(2,2'-bithienyl)methane. Conclusively, formation of a complex where 1 binds to TNB by hydrogen bonding is un likely.

Scheme 5. Structure of a complex of 1 and TNB, obtained by hydrogen bonding between amino moiety of 1 and TBN nitro group, optimised with molecular modeling using the density functional theory (DFT) at the M062X/3-21G* level. 3.3 Cyclic voltammetry of NTs and 1

Cyclic voltammetry (CV) measurements were performed for 1, each NT, and each 1-NT complex in 0.1 M (TBA)CI04 in the ACN and DCB (1:1, v:v) solution. The ACN and DCB solution prepared in the above mentioned volume ratio was found most appropriate for (i) dissolution of 1-NT, (ii) formation of stable prepolymerisation complexes, and (iii) subsequent electrochemical polymerisation of these complexes [Huynh, T.-P, Pieta, P, D'Souza, F., Kutner, W., Molecularly imprinted polymer for recognition of 5-fluorouracil by the RNA-type nucleobase pairing, Anal. Chem., 2013,85, 8304-8312],

For all NTs used in this study, no anodic CV peak was observed in the positive potential range. However, at negative potentials an irreversible cathodic peak appeared for each NT (curves 1-4 in Fig. 5). These peaks were assigned to electroreduction of a dimer formed by two NT molecules [Kemula, W. and Krygowski, T. M., Chapter 2: Nitro compounds, in Encyclopedia of electrochemistry of the elements, Bard, A. J. and Lund, H., Eds., Marcel Dekker: New York, 1979, Vol. 13, pp 78-130, and Gallardo, I. and Guirado, G. Oxygen carriers based on electrochemically reduced trinitroarenes, Phys. Chem. Chem. Phys. 2008, 10, 4456-4462], Passing N2 through solution during electroreduction resulted in binding of the NT molecules by the azo bond, -N=N-. Cathodic peak potentials were different for each NT, being equal -1.15, -0.80, -0.65, and -0.42 V vs. Ag/AgCI for DNT, TNT, TNB, and TNP, respectively. The differences in potential values for these peaks are due to different number of substituents and regioisomerism of substitution of the nitrophenyl ring [Toal, S. J. and Trogler, W. C. J., Polymer sensors for nitroaromatic explosives detection. Mater. Chem. 2006, 16, 2871-2883], For example, DNT has only two nitro (electron-withdrawing) groups compared to those three of TNT. So, the centre of the benzene ring of DNT is less positively charged than that of TNT. In effect, the most negative potential is required to reduce DNT (curve 1 in Fig. 5). On the other hand, TNP has one more electron withdrawing group (-OH). Therefore, its electroreduction is energetically the least demanding one, and its cathodic peak potential is most positive (curve 4 in Fig. 5). Hence, the experimental complex stability constants and the calculated Gibbs energy gain of the complex formation decreased from 1-TNP to 1-DNT (Table 1). Because NTs are electron acceptors, the better electron acceptor the NT is, the lower is the potential necessary to reduce it. Hence, the electron is faster transferred from the bis(2,2'-bithienyl)methane moiety to the TNP molecule than to any other NT molecule.

Unlike the voltammograms recorded for NTs, the voltammograms recorded for 1 show two anodic peaks, at 1.00 and 1.25 V (curve 5 in Figure 5). The peaks can be ascribed to electrooxidation of the amino group [Jannakoudakis, A. D. et al., Electrooxidation of aniline and electrochemical behaviour of the produced polyaniline film on carbon-fibre electrodes in aqueous methanolic solutions, Electrochim. Acta 1992, 38, 1559-1566] and the bis(2,2'-bithienyl)-methane moiety, respectively. The shifts of the anodic half-peak potential of the electrooxidation of the amino group of 1, leading to formation of a cation radical at the nitrogen atom position, allowed for estimation of the relative binding strength of 1 to different NTs. For that, the shifts of the anodic half-peak potential (A£pa/2), recorded after addition of NTs to reach the concentration of 1 mM were determined in the solution of 1 mM of 1 and 0.1 M (TBA)CI04 in ACN/DCB (1:1, v;v) (Table 1). The change of the A£pa/2 values was consistent with the changes in complex stability contants determined from the fluorescence titration, i.e., the A£pa/2 value for the 1-TNP complex was much higher than those for the other were, and the AEpa/2 value for 1-DNT was the lowest. The more stable the complex, the easier was its electrooxidation. 3.4 Deposition of the MIP-NT films by electropolymerisation

Molecular modeling, fluorescence titration, and measurements of the shifts of the electrooxidation anodic half-peak potentials of 1 in the presence of NTs have proven the formation of the 1-NT prepolymerisation complexes in solutions. Next, these complexes were immobilized in the form of MIP films on electrodes by potentiodynamic electropolymerisation. The solutions for the electropolymerisation were made 0.5 mM in one of the NT analytes, 0.5 mM in 1, 1.5 mM in 2, and 0.1 M in (TBA)CI04 in ACN/DCB (1:1, v:v) solution. The ratio of 1 to NT was selected to meet the 1:1 stoichiometry of the 1-NT complexes. In preparation of the solution for electropolymerisation, the cross-linking monomer 2 was added in a large excess in order to fix the MIP-NT matrix so as to maintain the shapes of molecular cavities unchanged after template extraction. The presence of the CI04” anions increased conductivity of the deposited MIP-NT film, and the presence of large-size TBA+ cations contributed to the development of porosity of the films [Roncali, J., Conjugated poly(thiophenes): synthesis, functionalization, and applications, Chem. Rev. 1992, 92, 711-738], In the course of electropolymerisation, anodic current decreased in each consecutive potential cycle, because each subsequent deposited polymer layer acted as a resistive barrier (Fig. 7). With growing thickness of the barrier, the anodic peak potential of oxidation of the -NH2 group and that of the bis(2,2'-bithienyl)methane moiety of 1 were shifted toward more positive potentials, or the peaks completely disappeared after the first potential cycle. Therefore, the MIP-TNT film deposited in nine potential cycles played the role of the recognition film with non-oxidised -NH2 groups of 1. All other MIP-NT films were deposited by potentiodynamic electropolymerisation on the Pt electrodes from similarly prepared solutions. 3.5 Extraction of the NT templates

Similarly to the previously reported TNT template extraction with ethanol [Bianchi, F. et al., Solid-phase microextraction of 2,4,6-trinitrotoluene using a molecularly imprinted-based fiber, Anal. Bioanal. Chem. 2012, 403, 2411-2418], a mixed solution of methanol and ACN (1:1, v:v) was used herein for the extraction of NTs with simultaneous vigorous stirring. This solvent selection was dictated by high affinity of the methanol -OH group to the —N02 group of the NT template on the one hand and high solubility of the template in ACN on the other.

The DPV peak current of the TNP electroreduction was used as a signal verifying the TNP extraction from the MIP-TNP film. Therefore, first, in a control experiment, the DPV curves for 2 mM TNP in 0.1 M NaCI were recorded in the potential range of -0.1 to -0.6 V. The DVP peak potential of the TNP electroreduction was —0.4 V (curve 1 in Fig. 8) [Lu, X. et al., Determination of explosives based on novel type of sensor using porphyrin functionalized carbon nanotubes, Colloids Surf., B 2011, 88, 396-401]. Next, the DPV curve for the MIP-TNP film in 0.1 M NaCI was recorded. Under these conditions, the peak of TNP electroreduction was clearly seen at —0.35 V (curve 2 in

Fig. 8). A small positive potential shift of the peak for the MIP-TNP film may be due to complexation of 1 and TNP. After TNP extraction (curve 3 in Fig. 8), the peak of the electroreduction disappeared indicating that TNP was completely removed from the film. 3.6 Capacitive impedimetry (Cl) chemosensors for NT determination

Indirect determination of analytes by measuring changes of capacity of the electrical double layer using MIP films is related to serious drawbacks. They are due to significant effect of ion concentration on the measured capacity, which reduces sensitivity and selectivity of capacitive chemosensors [Huynh, T.-P. et al., Electrochemically synthesized molecularly imprinted polymer of thiophene derivatives for flow-injection analysis determination of adenosine-5'-triphosphate (ATP), Biosens. Bioelectron. 2013, 41, 634-641], Therefore, the change of resistance of an MIP-NT film, Rf, determined from EIS measurements, was used herein as the analytical signal. This is a new method of transducing a chemical recognition signal into the analytical one. The method, as applied for systems studied herein, consists in recording of an EIS spectrum, in the absence of faradaic processes, for an MIP-NT film coated electrode in 0.1 M KCI in the water and ACE mixed solvent (1:1, v:v). The method allows for minimisation of the ion concentration effect on the analytical signal. The Rf is affected only by the presence of the analyte in the MIP-NT film. Moreover, unlike conductometric chemosensors with electric contacts for resistance measurement connected to MIP films and occupying significant part of the film surface area [Suedee, R. et al., Molecularly imprinted polymer-modified electrode for on-line conductometric monitoring of haloacetic acids in chlorinated water, Anal. Chim. Acta 2006, 569, 66-75], the chemosensors provided by the present invention do not require so large surface area as the contacts are not needed.

Fig. 9a shows the imaginary component of impedance as a function of the real impedance component for an MIP-TNP film coated Pt electrode, immersed in TNP solutions of different concentrations. Scheme 6 shows an electric equivalent circuit for the electrochemical cell with the MIP-NT film coated electrode film used herein under non-faradaic conditions [Freger, V. and Bason, S. J., Characterization of ion transport in thin films using electrochemical impedance spectroscopy I. Principles and theory, Membr. Sci. 2007, 302,1-9].

The applicability of the equivalent circuit (Scheme 6) for description of the electrochemical cell under study was verified using Bode plots, i.e., the dependence of the impedance phase angle, φ, on frequency,/, (Fig. 6b). The φ values were calculated with Equation (B).

where Z' and Z" stand for the real and the imaginary impedance components, respectively.

Scheme 6. The equivalent circuit of a non-faradaic electrochemical cell, for an MIP-NTfilm coated electrode in 0.1 M KCI in the water and ACE solution (1:1, v:v), used for determination of electric resistance of the film, Rf. The Rs symbol stands for electric resistance of the solution. The Cf and Cdi symbols stand for capacity of the MIP-NT film, and the electrical double layer, respectively. The W symbol stands for the Warburg impedance.

Three frequency ranges A, B, and Cof the Bode plot (Fig. 9b) correspond to the three sections A, B, and C of the equivalent circuit (Scheme 6). The A range in Fig. 9b is related to the resistance of solution, Rs, in the A section in Fig. 9a, measured at high frequencies, i.e., from 500 kHz to 50 kHz. The resistance is low (lower than -100 Ω), because of high (0.1 M) KCI concentration. The B range (intermediate frequencies) from 50 kHz to 30 Hz in Fig. 9b corresponds to a small semicircle in Fig. 9a, in the range of low values of the real impedance component. The semicircle represents the impedance of an MIP-NT film of finite thickness. The B section of the equivalent circuit, composed of a resistor and capacitor in parallel connection (Scheme 6), can be used to represent electric properties of the film. The C range in Fig. 9b, which corresponds to the range of linear curves of positive slopes in Fig. 9a, corresponds to a linearly connected capacity of the electrical double layer, Q\, and the Warburg impedance, W. This impedance is a measure of ion diffusion through the MIP-NT film in the C range (Fig. 9b) of low frequencies of 30 Hz to 1 Hz [Orazem, Μ. E. and Tribollet, B., Electrochemical impedance spectroscopy, John Wiley & Sons: New Jersey, USA, 2008].

The Rf values were determined by fitting the parameters of the equivalent circuit (Scheme 6) to the experimental data shown in Nyquist plots (Fig. 9a) for the MIP-NT films after template extraction. The films were deposited in 10 cycles of linear potential changes on Pt electrodes immersed in solutions, to which NT solutions of various concentrations were injected. The experimental Rf values were in the range of 10 < Rf < 20 kQ, thus indicating semiconducting properties of the MIP-NT films. The idea of using Rf as an analytical signal for NT determination originates indirectly from our earlier studies on the enhanced MIP film resistance with increasing analyte concentration in solution [Huynh, T.-P. et al., Molecularly imprinted polymer of bis(2,2'-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry 2012, DOI: 10.1016/j.bioelechem.2012.1007.1003]. In these studies, two different methods for transducing chemical recognition signal into analytical signal were used. The first one consisted in measuring the DPV peak current decrease for electrooxidation of a redox probe, K4[Fe(CN6)], with increasing analyte concentration in solution, while the other - the increase of capacity of the electrical double layer, Cdi, in line with this increase. In the present patent application, an increase in the Rf value is the higher the more NT molecules reach the molecularly imprinted MIP-NT films. The chemosensor's concentration response, as determined from the calibration curves for 0.1 M KCI and TNP of different concentrations, is linear in the range of 0.1 to 0.5 μΜ (Table 2 and Fig. 10a). Similarly constructed calibration plots for 0.1 M KCI, as well as TNT, TNB, DNT, and their corresponding MIP-NT recognition films are shown in Fig. 10b, 10c, and lOd, respectively. The range of chemosensor concentration linear response (0.1 to 3.0 μΜ) was narrower than in that for the MIP-TNP film (Table 2). The LOD values, determined for the signal-to-noise ratio of 3, for chemosensors for NT determination, were in the range of tens of nM (Table 3), allowing for determination of these NTs in a neutral solution, as the NT concentration under these conditions was ~10 nM. From among the fabricated chemosensors, the highest LOD value, i.e., the lowest detectability was found for the DNT chemosensor. This is because of weaker interaction of 1 with DNT (Table 1), and hence less DNT molecules bound to the MIP-DNT film. Therefore, the measurable analytical signal required higher DNT concentration in solution.

Selectivity of the fabricated NT chemosensors was determined by molecular cross-imprinting. In this imprinting technique, the template of one MIP-NT film was used as an interference in determinations using remaining chemosensors (Fig. 10). Selectivity of the MIP-NT films was determined with respect to two different NTs used as interferences, and the determined selectivity coefficients are compiled in Table 4. The majority of chemosensors fabricated in the present study showed high selectivity with respect to interferences, except for the MIP-DNT chemosensor because of weak binding of 1 and DNT, as discussed above. Hence, the selectivity of the MIP-DNT chemosensor with respect to DNT is only twice that with respect to TNP, and four-times higher with respect to TNT. Unexpectedly, the MIP-TNB chemosensor showed the highest selectivity (almost nine-times) with respect to DNT. The selectivity of MIP-TNT chemosensor with respect to TNT was almost twice that with respect to TNB.

Table 3. Analytical parameters of chemosensors for NT determination, determined for different MIP-NT films, under steady-state conditions, by injecting NT solutions of different concentrations. Linear regression equations for film resistance, Rf, were obtained by fitting to the experimental data (Fig. 9).

Moreover, it has been shown that the change of the film resistance is due to analyte binding by the film. For that, an MIP-TNP film was deposited on the Pt disk electrode, and after TNP extraction, it was used for measurement of the real impedance component, Z’, under flow-injection analysis (FIA) conditions. The dependence of the real impedance component on electric resistance of the film for parallelly connected resistor, Rf, and capacitor, Cf, of the equivalent circuit (range B in Scheme 6) is given by Equation (4).

(4)

The angular frequency, ω (ω = 2π/), was kept at a constant low level, / = 20 kHz, i.e., at the frequency corresponding to the impedance range of the film, where Z' is proportional to Rf only, because τ= RfCf is the time constant.

The real component of impedance increased with each subsequent injection of the solution of higher TNP concentration, thus indirectly reflecting the increase in film resistance (Fig. 11). The calibration curve for TNP determined using an MIP-TNP film deposited on a Pt electrode, constructed by plotting the dependence of the real impedance component on the TNP concentration, obeys the following linear regression equation, Z'(kQ) = 4.23(±0.05) + 0.75(±0.10) / cTNp (curve 1 in inset in Fig. 11). A Pt electrode was coated with a control NIP film, using the same procedure, but in the absence of NTs. A calibration curve for TNP was also constructed for this film. The curve obeys the following linear regression equation, Z'(kQ) = 4.01(±0.02) + 0.26(±0.03) / cTNp (curve 2 in inset in Fie. Ill

Table 4. Selectivity of MIP-NT chemosensors determined for 0.1 M KCI in the water and ACE mixed solvent (1:1, v:v) by injecting interferences of different concentrations.

4. Conclusions A one-pot procedure for synthesis of a functional monomer, bis(2,2'-bithienyl)-(4-aminophenyljmethane 1 was developed. The monomer was used for preparation of polymers imprinted with four different nitroaromatic explosive compounds. The polymers were used for detection, by τι-π interactions, and for selective determination of these compounds. The bis(2,2'-bithienyl)methane moiety 1 revealed significant advantages with respect to determination of NTs. They included (i) playing a fluorophore role for the fluorescence titration of 1 with each NT, (ii) decreasing polarity of 1, to dissolve it in low polarity solvents, and (iii) enabling potentiodynamic electropolymerisation to result in an MIP-NT film deposited on the electrode surface. Besides, the phenylamine group of 1 played a role of a τι-τι recognizing site of NTs, as demonstrated by the fluorescence titration, molecular modeling, and the cyclic voltammetry anodic half-peak potential shift of the phenylamine moiety electrooxidation. The DFT M062X method with the 3-21G* basis set appeared extremely suitable for molecular modeling of the τι-τι interactions, which well correlated with the experimental complexation data. Moreover, these results helped to understand more deeply the nature of the interactions of the phenylamine moiety of 1 with NTs at the 1:1 stoichiometry, as well as the fluorescence quenching of the bis(2,2'-bithienyl)methane moiety of 1 by electron transfer to NTs, in line with earlier findings [Smith, G. and Wermuth, U. D., 4-(4-Aminophenylsulfonyl)aniline-l,3,5-trinitrobenzene (1/2), Acta Crystallogr. E 2012, 68, 494],

An additional advantage of the solution provided herein is that the determination is related to a simple frequency scan at constant potential, at which no faradaic processes occur. The LOD values for the fabricated chemosensors were in the range of 8 to 29 nM, i.e., sufficiently low to use them for NT determination in soil and water. Moreover, selectivity studies, performed by cross-imprinting of NTs structurally similar to each other, revealed that the chemosensors were prevailingly sensitive to the analytes used for polymer templating. 5. Acknowledgements

The research was financially supported by: - the Foundation for Polish Science (MPD/2009/l/stypl9) to TPH, and (MPD/2009/l/stypl5) to MS,

- European Union under the European Regional Development Fund, Innovative Economy Programme grant, POIG.Ol.01.02-00-008/08 to WK

- US National Science Foundation (Grant No. 1110942) to FD - 0 European Union under the 7th FP. Grant REGPOT-CT-2011-285949-NOBLESSE to PP.

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

1. A new bis(2,2'-bithienyl)methane, comprising bis(2,2'-bithienyl)-(4-amino-phenyl)methane 1.

2. A method for producing the aforementioned bis(2,2'-bithienyl)methane derivative, characterised in that the initial solution of 2,2'-bithiophene and 4-acetaminobenzaldehyde in ethylene glycol is reacted with perchloric acid, at a temperature ranging from 40 to 90 °C, which results in bis(2,2'-bithienyl)-(4-aminophenyl)methane 1.

3. Method, according to claim 2, characterised in that a 70% perchloric acid is used.

4. Method, according to claim 2 or 3, characterised in that the process is carried out at 60 °C.

5. A method for fabricating molecularly imprinted polymer characterised in that it comprises the following steps: (a) a solution is prepared, containing bis(2,2'-bithienyl)-(4-aminophenyl)methane as a functional monomer 1, tris([2,2'-bithiophen]-5-yl)methane as a cross-linking monomer 2, and imprinted nitroaromatic compound (NT), in solvent or solvent mixture, preferably in acetonitrile and dichlorobenzene mixed in a 1:1 (v:v) ratio, in which a π-π prepolymerisation complex of the aminophenyl substituent of the functional monomer 1 with aromatic ring of the nitroaromatic compound (NT) is formed, and subsequently (b) the prepolymerisation complex obtained in step (a) is electropolymerised, preferably under potentiodynamic conditions with potentials cycled linearly, deposited on the electrode from the aforementioned (step (a)) solution, 0.1 M in (TBA)CI04, as a molecularly imprinted film with imprinted template of a nitroaromatic compound (MIP-NT), and then (c) the imprinted, in the step (b) above, template of the nitroaromatic compound (NT) is extracted from the MIP-NT film, to result in an MIP film with emptied imprinted molecular cavities.

6. Method according to claim 5, characterised in that the nitroaromatic compounds (NT) used are those selected from the group comprising 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT)..

7. Method, according to claim 5 or 6, characterised in that a functional monomer 1, a nitroaromatic compound (NT) and a cross-linking monomer 2, are used in step (a) in a 1:1:3 molar ratio, respectively.

8. Method, according to any claim from 5 to 7, characterised in that the potentiodynamic electropolymerisation comprises 1 to 20, preferably 9 linear cycles of potential changes ranging from 0.50 to 1.50 V vs. Ag/AgCI, at a scan rate of 5 to 500 mV s'1, preferably 50 mV s'1.

9. Method, according to any claim from 5 to 8, characterised in that a metal or carbon electrode is used in step (b) as the electrode, preferably a platinum disk electrode.

10. A molecularly imprinted polymer film, characterised in that it consists of a polymer molecularly imprinted with nitroaromatic compounds, in particular those selected from the group comprising 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT), produced with the method described in any claim from 3 to 7.

11. The use of an imprinted polymer film, according to claim 10, as a recognition unit of a chemical sensor for selective detection of nitroaromatic compounds, in particular such as 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT).

12. The use of an imprinted polymer film, according to claim 10, as a recognition unit of a chemical sensor for determination of nitroaromatic compounds, in particular such as 2,4,6-trinitrophenol (TNP), 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), and 2,4-dinitrotoluene (DNT).