Organophosphate pesticides as harmful contaminants and toxic substances often exist in agriculture products and environment. The severe fatal damage to the human health as well as threat to the safety of poultry and domestic animals are caused by their harmful effect to inhibit the normal function of acetylcholinesterase,which is a critical enzyme in nervous system ^{[1, 2]}. Considerable concerns about the amount of organophosphate residue in environment and their threat to biological systems have stimulated intensive researches to develop sensitive and selective methods for detection of these compounds ^{[3, 4]}. Huge research effort has been devoted to develop various organophosphate detection methods. Most wellknownmethods include gas chromatography-mass spectrometry ^[5],surface acoustic wave ^[6],surface-enhanced Raman spectroscopy ^[7],fiber optics ^[8],colorimetric ^{[9, 10, 11]} and fluorimetric method ^{[12, 13, 14, 15]}.

Fluorescent detection is generally considered as one of the best options owing to its high selectivity,sensitivity,portability and operational simplicity ^{[16, 17]}. Recently,the emission properties of macromolecules containing tridentate aromatic scaffolds 2,2':6',2''-terpyridine (terpy) or 2,6-bis-(1'-methyl-benzimidazolyl) pyridine (MeBIP) complexing with trivalent lanthanides Eu(III) have been explored ^{[18, 19, 20, 21]}. These previous researches show that the systems are very sensitive to the organophosphates. The sensitivity and sub-second response of these metal-ligand functionalized polymers make them to be suitable candidates for organophosphates detection.

As a continuation of our previous investigations,this study concentrates on the synthesis and characterization of a novel bisbenzimidazolylpyridine-functionalized fluorescent epoxy resin/ Eu(III) complex,which is designed to be used as chemosensor for organophosphates detection.

Aniline,bisphenol-A diglycidyl ether,diethyl chlorophosphate (DCP) and Eu(NO₃)₃·6H₂O were purchased from Alfa Aesar. Sodium hydride (60 wt% dispersion in mineral oil) was purchased from Sigma-Aldrich. 4-Bromo-2,6-bis-(1'-methyl-benzimidazolyl)pyridine (Br-MeBIP) was prepared according to literature procedures ^[22]. Tetrahydrofuran (THF) was purified by distillation with sodium and benzophenone. Deionized water (resistivity >18 MΩcm) was provide by a Milli-Q water purification system. All other reagents and solvents were purchased commercially and used without further purification. ¹H NMR spectroscopy was performed on a JEOL JNM-ECA300 NMR spectrometer with dimethyl-d₆ sulfoxide as solvent and tetramethylsilane (TMS) as the internal standard. FT-IR spectra were collected on a Nicolet 560-IR spectrometer: the samples were mixed with KBr and then pressed into thin transparent disks. Fluorescence spectra were measured at room temperature on an F-4500 spectrophotometer equipped with a Xenon lamp excitation source. The molecular weights and molecular weight distributions were evaluated by a gel permeation chromatographic (GPC) instrument equipped with a PLgel 5 μm mixed-D column and a refractive index (RI) detector (Wyatt Optilab rEX). The measurement was carried out at 35 ℃ with polystyrene as the standards and THF as the eluent under a flow rate was 1.0 mL/min. Thermal analyses of the compounds were carried out using TA Instruments Q2000 system at a heating rate of ±10 ℃/min under a nitrogen purge.

The synthetic route of the target fluorescent polymers is outlined in Scheme 1 which mainly contains three steps as follows. Firstly,an epoxy-based precursor polymers (BP-AN) was synthesized through the step polymerization between Bisphenol-A diglycidyl ether (BADGE) and aniline. Then,the polymeric ligand (BP-AN-MeBIP) was prepared through the Williamson coupling reaction between BP-AN and Br-MeBIP. Finally,the epoxy resin/ Eu(III) complex (BP-AN-MeBIP-Eu) was obtained by the noncovalent binding reaction between BP-AN-MeBIP and Eu(III) ions in the THF solution. The synthetic details are described below.

Scheme 1

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Scheme 1. The synthetic route of the fluorescent polymer BP-AN-MeBIP-Eu.

The precursor polymer was prepared according to the literature method ^[23]. Bisphenol-A diglycidyl ether (18.81 g,0.1 mol) and aniline (4.66 g,0.05 mol) were homogeneously mixed and polymerized at 110 ℃ for 20 h. The crude product was dissolved in a CHCl₃/CH₃OH solvent mixture (4:1,200 mL) followed by precipitation in 800 mL of acetone. The polymer was collected by filtration and dried under vacuum for 24 h. IR (KBr,cm^-1): 3380 (O-H,s),1600,1510,1463 (Benz. ring,s),1250 (C-O,s). ¹H NMR (DMSO-d₆): δ 7.07 (d,6H),6.82 (d,4H),6.72 (d,2H),6.54 (m,1H), 4.03 (m,2H),3.87 (s,4H),3.34-3.75 (m,4H),1.55 (s,6H). GPC: M_n = 44,620,M_w/M_n = 1.624. DSC: T_g = 89.9 ℃.

Sodium hydride (0.28 g,10 mmol),Br-MeBIP (0.05 g, 0.12 mmol) and BP-AN (1.0 g,2.24 × 10^-5 mol) were add into dried THF (40 mL) and reacted at room temperature for 24 h. The obtained mixture was dropped into plenty of petroleum ether,and the precipitate was collected by filtration. After washing with plenty of water for several times and drying,the crude product was dissolved in tetrahydrofuran and precipitated in petroleum ether again. The white solid product was obtained by filtration and dried in vacuum at 40 ℃ for 24 h. IR (KBr,cm^-1): 3366 (O-H,s),2962, 2926,2860 (C-H,s),1663,(C=N,s),1600,1507,1459 (Benz. ring, s),1247 (C-O,s). ¹H NMR (DMSO-d₆): δ 8.54 (s,2H),7.75 (d,2H), 7.68 (d,2H),7.36 (t,2H),7.30 (t,2H),7.08 (m),6.84 (m),6.72 (m), 6.54 (m),5.37,5.18 (m),4.27 (s,6H),4.03 (m),3.87 (m),3.34-3.75 (m),1.55 (m); GPC: M_n = 46,760,M_w/M_n = 1.794; DSC: T_g = 94.3 ℃.

BP-AN-MeBIP (100 mg) was dissolved in THF (15 mL) and then Eu(NO₃)₃·6H₂O (45 mg,0.1 mmol) was added into the solution. After stirring at room temperature for 6 h,the solvent was removed with rotary evaporator. The crude product was carefully washed with ethanol (3 × 15 mL) to remove the excess of unreacted Eu(III) ions. The product was dried in vacuum oven at 40 ℃ for 4 h to yield white solid material.

The epoxy-based polymer (BP-AN) was synthesized through the step polymerization between bisphenol-A diglycidyl ether (BADGE) and aniline. The polymerization temperature was controlled to be 110 ℃ to avoid the possible side-reaction between the secondary hydroxyl groups generated by the ring-opening reaction and the unreacted epoxide rings. The abbreviation BP-AN denotes the polymer prepared by the reactions of BADGE with aniline. After the step polymerization,a large amount of hydroxyl groups is generated on the main-chain of BP-AN,which make it possible to be further modified to introduce the functional groups. The polymer shows good solubility in polar organic solvents such as tetrahydrofuran (THF),N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMAc). All of these characteristics ensure BP-AN to be a perfect precursor polymer. The nucleophilic substitution reaction between BP-AN and Br-MeBIP was carried out in THF and catalyzed by NaH. Through this approach,the polymeric ligand (BP-AN-MeBIP) can be feasibly obtained. Finally, the fluorescent polymer BP-AN-MeBIP-Eu was obtained by the coordination between BP-AN-MeBIP and Eu(III) ions.

Fig. 1 shows the ¹H NMR spectra of the precursor polymer BP-AN and the polymer ligand BP-AN-MeBIP in DMSO-d₆. The assignment of the ¹H NMR spectrum of BP-AN can be found in a previous article ^[23]. Comparing with BP-AN,the ¹H NMR spectrum of BP-AN-MeBIP shows the similar resonances signals for the protons on the main-chain of the polymer. On the other hand,an obvious decrease of the integrated area for the resonances of the proton from the hydroxyl group can be observed,which means a significant amount of the hydroxyl group was reacted with Br-MeBIP. The new resonance signals appeared at 4.27,7.30,7.36,7.68,7.75 and 8.54 ppm on the 1H NMR spectrum of BP-AN-MeBIP are from MeBIP moieties,which confirms the success of the nucleophilic substitution reaction between BP-AN and Br-MeBIP.

Fig. 1

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Fig. 1. 1H NMR spectra (in DMSO-d6) of BP-AN and BP-AN-MeBIP.

The polymer ligand BP-AN-MeBIP was also characterized by GPC,DSC and IR. Fig. 2 gives GPC traces of BP-AN and BP-AN-MeBIP. It can be observed that the GPC curve of BP-AN-MeBIP shifts to the higher molecular weight region compared to that of BP-AN after introducing the ligand groups. The molecular weight distribution of BP-AN-MeBIP becomes slightly broader after the reaction. Fig. 3 shows the DSC curves of BP-AN and BP-AN-MeBIP on the first cooling scans. Comparing with BP-AN (T_g = 89.9 ℃),the Tg of BPAN- MeBIP increases to 94.3 ℃. In the IR spectra,new absorption peaks from pyridine moieties can be observed in the spectrum of BP-AN-MeBIP compared with BP-AN. All the characterizations, including ¹H NMR,GPC,DSC and IR,verify the successful preparation of BP-AN-MeBIP.

Fig. 2

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Fig. 2. GPC curves of BP-AN (solid line) and BP-AN-MeBIP (dash line).

Fig. 3

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Fig. 3. DSC curves of BP-AN (solid line) and BP-AN-MeBIP (dash line),the first cooling scan.

Binding reaction of the Eu(III) ions to the BP-AN-MeBIP was investigated by fluorescence spectroscopy. Fig. 4 shows the emission spectra of BP-AN-MeBIP-Eu and BP-AN-MeBIP with an excitation of 367 nm. It shows the typical emission peaks of the Eu(III) ions centered at 578 nm,593 nm,617 nm,646 nm and 684 nm for BP-AN-MeBIP-Eu,while no such emission can be seen for BP-AN-MeBIP. This result indicates the successfully loading of Eu(III) ions onto the MeBIP-functionalized epoxy-based polymers, where MeBIP ligands act as ‘‘antenna’’ for the Eu(III) ion emission.

Fig. 4

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Fig. 4. Fluorescent emission spectra of BP-AN-MeBIP-Eu (solid line) and BP-ANMeBIP (dotted dash line) in THF solution,λex = 367 nm.

In fluorescence spectrum described above,strong emission bands at 617 nm and 593 nm observed for BP-AN-MeBIP-Eu can be used for diethyl chlorophosphate (DCP) detection. It was observed that the addition of DCP into the solutions of the polymeric lanthanide complexes resulted in quick quenching of the fluorescent emission (Fig. 5). The observed fluorescence quenching is a result of ligand displacement by competitive binding,which ‘‘switches off’’ the Eu(III)-based emission. As shown by previous study,this reaction is highly specific for organophosphate. A plot of I₀/I vs. the concentration of the DCP,where I₀ is the original complex emission intensity and I the intensity in the presence of quencher,yields a linear relationship as shown in the insert in Fig. 5. The Stern-Volmer quenching constants K_sv is determined to be 0.377 × 10³ L/mol. This high K_sv demonstrates that the novel epoxy-based fluorescent polymer can be a promising candidate for using as organophosphate sensors. Optimization and application development of this organophosphate-sensitive epoxy resin are under way.

Fig. 5

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Fig. 5. Fluorescent emission spectra of 1 mg/mL of Eu(III) ions-loaded epoxy-based polymers as a function of the DCP concentration,λex = 367 nm. (From top to the bottom: 0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5 and 6.0 × 10-3 mol/L). The inset is the Stern-Volmer plots of I0/I vs. DCP concentration

By exploiting the binding affinity of organophosphates to lanthanide ions,we develop a novel epoxy-based chemosensor for the detection of organophosphates. The bisbenzimidazolylpyridine- functionalized epoxy resin (BP-AN-MeBIP) was synthesized and characterized by FT-IR,¹H NMR,GPC,and DSC. The complex of BP-AN-MeBIP with Eu(III) ions (BP-AN-MeBIP-Eu) was prepared and studied by fluorescence spectroscopy. The BP-AN-MeBIP-Eu complex showed a strong fluorescence emission with an excitation of 367 nm light. The fluorescence emission was observed to be instantaneously quenched by exposure to trace diethyl chlorophosphate because of the competitive coordinating effect. The Stern- Volmer quenching constants K_sv for quenching at 617 nm was determined to be 0.377 × 10³ L/mol,which is the same order as our previous works ^{[18, 20]}. The sensing ability and easy processing nature of the polymeric support enable the resin to be a promising chemosensor candidate for the detection of organophosphates.

Financial supports from the Research Foundation of the General Armament Department (No. 2008 CD 012) are gratefully acknowledged.