Novel benzimidazole-based conjugated polyelectrolytes: synthesis, solution photophysics and fluorescent sensing of metal ions

Abstract Two benzimidazole-based conjugated polyelectrolytes (+)-PPBIPV and (-)-PPBIPV which have opposite charges on their side chains were synthesized via Heck coupling reaction and characterized by 1H-NMR, UV-vis and PL spectroscopy. These two polyelectrolytes are both consisted of benzimidazole derivatives and phenylenevinylene units. The absorption and emission spectra reveal that the polymers both have solvent-dependency and concentration-dependency, and they exhibit aggregation effect in aqueous solution. In the respect of ion detection, the aqueous solution of (+)-PPBIPV has excellent selectivity and sensitivity for Fe3+. Moreover, Pd2+ can almost completely quench the fluorescence of (+)-PPBIPV in methanol solution, and its quenching constant KSV is 5.93×104 M-1. For (-)-PPBIPV, Sn2+ can double the fluorescence intensity of its aqueous solution, while (-)-PPBIPV has good identification for Fe3+ in methanol with a KSV = 3.44×105 M-1. Hence, two polyelectrolytes have considerable potential to become effective fluorescent sensing materials for some specific metal ions. All of the stoichiometric relationships between metal ions and conjugated polyelectrolytes were calculated using Benesi-Hildebrand equation.


Introduction
Conjugated polyelectrolytes (CPEs) which are a kind of conjugated polymers with water soluble ionic groups have received considerable interests in the past decades. Their ionized side-chain groups make CPEs have excellent solubility in water and other polar solvents. CPEs are a versatile class of materials that have been applied in a variety of photoelectric devices including polymer light-emitting diodes (PLEDs) (1), photovoltaic cells (2) and chemical and biological sensors (3,4). In particular, the chemical structure of CPEs offers several advantages in sensor applications, such as the increasing sensitivity of detection and water solubility. Therefore, CPEs have been widely explored as optical probes for various targets, such as DNA (5), proteins (6), small molecules (7) and metal ions (7,8).
The conjugated polyelectrolytes can be classified into cationic and anionic types depending on the different charged groups introduced on the side chain. Specifically, few cationic conjugated polyelectrolytes were reported and almost all of them were not synthesized directly but prepared by quaternization after the polymerization. Since not all the side groups of the polymers are electrically charged during this process, fully charged cationic polyelectrolytes cannot be obtained by indirect method. In recent years, cationic CPEs have potential applications in biological imaging such as folate receptor (FR) over expressed cancer cells (9), selective recognition of mammalian cells (10) and anionic surfactants (11), monitoring the physical changes of lipid membrane (10). On the other hand, anionic CPEs have been widely applied in the biological and chemical analysis area with their excellent water solubility. Direct electron transfer over a longer working distance can be accomplished through the sulfonated conjugate polyelectrolyte, which has a strong signal in DNA detection (12). And a probe system as fluorescence turn-on assay of simple saccharides is based on three anionic conjugated polyelectrolytes and three quenched molecules substituted by boric acid (13). However, almost all the anionic CPEs are consisted of phenyl units (14), thiophene derivatives (15) and fluorene monomers (16), and there are few literatures focused on water soluble N-heterocyclic systems. This can be attributed to complexity of the synthesis and purification. Although the fluorescent sensing properties of conjugated polyelectrolytes in the field of biology and chemistry have been extensively studied, there are few reports on the detection of metal ions in both cationic and anionic conjugate polyelectrolytes.
Benzimidazole is a nitrogen-containing heterocyclic compound fused from an imidazole ring and a benzene ring. With chemical modification by using various specific chemical methods, derivatives of benzimidazole can interact with enzymes, receptors and metal ions in the living body (17). Therefore, they have a wide range of applications in medicine (18) and pesticides (19). In recent years, due to the structural characteristics of benzimidazole, its derivatives have also received extensive attention in the field of fluorescent sensors (20). The sp 2 hybridized nitrogen atom on benzimidazole has a lone pair of electrons that can coordinate with metal ions, resulting in changes in optical properties to achieve the purpose of detection (21). Thus, there have been a number of reports about small molecule probes containing benzimidazole (22,23). Related studies on benzimidazolecontaining conjugated polymers reported in the past have mainly focused on light-emitting diodes (24) and photovoltaic devices (25). However, few benzimidazolebased conjugated polymers have been utilized for fluorescent sensors, and they are basically oil soluble (26). Among these studies, most of the fluorescence sensing properties were investigated in non-aqueous system, such as tetrahydrofuran (27). Therefore, it is interesting to develop novel CPEs containing benzimidazole which can detect metal ions in the water effectively.

1
H NMR spectra were recorded on a Bruker Avance II-400MHz spectrometer (Bruker, Karlsruhe, Germany) with tetramethylsilane as an internal reference. UV-vis absorption spectra were performed on an AnalytikJena Specord 200Plus UV-vis spectrophotometer (AnalytikJena, Jena, Germany). Photoluminescence (PL) spectra were measured by using a Hitachi F-4600 luminescence spectrophotometer (Hitachi, Tokyo, Japan). All spectra were measured at room temperature.
The crude product was dried under vacuum at 60°C for 12 h and was purified by recrystallization from aqueous solution of potassium chloride to give a white solid. Finally this solid was dried under vacuum overnight again. Yield: 0.56 g, 56.4%. 1

(-)-PPBIPV
Under argon atmosphere, the mixture solution of monomer 2 (0.25 g, 0.5 mmol), C4-DVB (0.14 g, 0.5 mmol), palladiumacetate (0.011 g, 0.05 mmol), tri(o-tolyl) phosphine (0.061 g, 0.2 mmol) and diisopropylamine (2.5 mL) and DMF (3 mL) was stirred at 100°C for 6 h. The reaction mixture was poured into methanol (20 mL) with dimethylglyoxime (0.158 g) and stirred for 10 min. The mixture solution was filtered and the filtrate was poured into the mixed solvent (ethyl ether/acetone: 1/1). The precipitate was filtered and then redissolved in DMSO (10 mL). The resulting solution was dialyzed against deionized water for 3 days using a 4kD MWCO cellulose membrane. Finally, the solvent was removed by rotary evaporation and the crude product was further dried under vacuum at 60°C for 48 h to give a yellow-brown solid. Yield: 0.1387 g, 43.4%.

Synthesis and characterization of monomers and polymers
The synthetic schemes for the monomers and the polymers are outlined in Scheme 1. During the formation of the imidazole ring, the position of the bromine substituent on the benzimidazole ring will be at the 4th or 5th position due to the different positions of oxidative dehydrogenation, and therefore compound 3 is a mixture. Of course, monomer 1 and monomer 2 derived from the nitrogen alkylation of compound 3 are also mixtures. The polymerization of the two polymers were both carried out via Heck coupling reaction using Pd(OAc) 2 as catalyst and P(o-tolyl) 3 as the corresponding ligand (31). Taking into account the presence of water soluble Scheme 1: Synthetic routes of monomers and polymers. monomers and oil soluble monomers, we chose DMF that can dissolve most of the substances as solvent. During the procedure of the copolymerization, the conjugated length of (+)-PPBIPV and (-)-PPBIPV which was detected by the UV-vis spectrum was adjusted to be almost identical by controlling the polymerization time.
As shown in Figure 1, it can be seen that the characteristic peaks of polymer (+)-PPBIPV correspond well with the characteristic peaks of each monomer. Specifically, in the 1 H NMR spectrum of (+)-PPBIPV, the presence of the peaks at 0.81-1.65 ppm is attributed to the characteristic peak of the methyl group in the alkyl side chain of monomer 1 and (+)-DVB; the peaks at 3.51 ppm and 3.91 ppm are all attributed to the hydrogen of N-CH 2 on the quaternary ammonium group in (+)-DVB; the peaks at 4.24-4.84 ppm are respectively attributed to O-CH 2 of (+)-DVB unit and N-CH 2 of monomer 1 unit. Since the polymer is not a single pure material but a mixture of different molecular weights, a broad peak usually appears in the NMR spectrum of the polymer. Furthermore, the polymer is a compound with a large delocalized π bond, hydrogen atoms in the conjugated structure are often subjected to complex de-shielding effects, so the peaks at 7.34-8.16 ppm are the peaks of aromatic hydrogen atoms in the polymer backbone. Figure 2 shows the 1 H NMR spectra of monomer 2, C4-DVB and (-)-PPBIPV. In the 1 H NMR spectrum of (-)-PPBIPV, the presence of the peaks at 0.56-2.10 ppm is attributed to the signal peaks generated by hydrogen atoms not adjacent to oxygen and nitrogen atoms on the alkyl side chains, showing the monomer 2 and C4-DVB units have been introduced into the main chain of (-)-PPBIPV. These results can also be found in the spectrum of (+)-PPBIPV in Figure 1. Moreover, the hydrogen atoms adjacent to oxygen and nitrogen atoms in the alkyl side chain appear at 3.84-4.55 ppm, and the signals in the region of 6.97-8.09 ppm are due to the hydrogen atoms of benzene ring.
It is well-known that CPEs can aggregate in its solution and that aggregated polymers can be easily absorbed on the chromatographic column, so precise molecular weight information of CPEs is difficult to obtain from gel permeation chromatography (GPC). Another reason is that it is difficult to find a suitable solvent to conform to the measurement requests. Furthermore, owing to the aggregation phenomenon of CPEs, light scattering which is the common technique to measure the molecular weight as same as GPC is not available (32,33). Thus, the solutions of (+)-PPBIPV and (-)-PPBIPV were dialyzed by 4kD MWCO cellulose membrane, which ensured that the molecular weight of polymers were more than 4000 (at least eight repeating units). Although we can only get a general result in this way, but it is enough to prove that the polymerization of the two CPEs has been completed successfully and satisfied the requirement of photophysics and sensing measurement.

Basic photophysical properties of polymers
The absorption and emission data of monomer 1, monomer 2, (+)-DVB, C4-DVB, (+)-PPBIPV and (-)-PPBIPV are listed in Table 1. In the water, (+)-PPBIPV shows broad absorption band covered 300-500 nm visible light region and the absorption maximum at 415 nm. This phenomenon is associated with the increasing conjugate chain of the polymer, which decreases the energy from ground state to excited state. The fluorescence of (+)-PPBIPV is green in the solution, while the fluorescence of (+)-DVB and monomer 1 is purple, and its fluorescence intensity is significantly stronger than two monomers. In comparison with monomer 1, the PL emission maximum of (+)-PPBIPV at 501 nm showing red shifted 139 nm is attributed to its increasing conjugation degree. In addition, the maxima of absorption peak and fluorescence emission peak of (+)-PPBIPV are much larger than those of monomer 1 and (+)-DVB, which verifies the success of the polymerization. As same as (+)-PPBIPV, the UV-vis and PL maxima of (-)-PPBIPV both show large red shifts compared to its monomers. (-)-PPBIPV exhibits the absorption maximum at 408 nm and emission maximum at 613 nm in the water. There is almost no overlap between the ultraviolet absorption band and the fluorescence emission band of (-)-PPBIPV, and the Stokes shift reaches 205 nm, which can effectively prevent fluorescence self-absorption and exhibit the potential of fluorescence sensor materials.
The absorption and emission maxima of (+)-PPBIPV and (-)-PPBIPV are listed in Table 1, while the photos of conjugated polyelectrolytes in different solvents are shown in Figure 3. As shown in Figure 3a, the maximum absorption peak of (+)-PPBIPV is located in 403 nm in methanol. However, it exhibits a red shift in water and DMSO, which is located in 415 nm and 417 nm, respectively. Similarly, when the solvent is changed from methanol to DMSO or water, the emission maximum has been red shifted from 470 nm (in methanol) to 480 nm (in DMSO) or 501 nm (in water). In addition, the PL emission intensity of (+)-PPBIPV is significant in both methanol and DMSO, but weak in aqueous solution. In water, (+)-PPBIPV aggregates strongly by π-π stacking as same as other conjugated polyelectrolytes (34), which means that the conjugated layers of the polyelectrolytes are stacked mutually. This aggregation causes the conjugated polyelectrolytes to reach a higher degree of conjugation, making it easier for excitons to transfer between the backbones (35). Thus, the fluorescence quenching of (+)-PPBIPV in water may be attributed to energy dissipation caused by excitons transfer. Figure 3b shows that the maximum absorption peaks are located in 417 nm in DMSO, 408 nm in water, and 405 nm in methanol, respectively. In addition, the emission peak of (-)-PPBIPV is located in 500 nm in methanol, corresponding to the intrinsic emission (36). However, it is worth noting that the fluorescence of (-)-PPBIPV in aqueous solution is very weak, and the emission maximum has been red shifted from 408 nm to 613 nm, which is consistent with the reported anionic conjugated polyelectrolyte (36). In general, the peak at long wavelength corresponds to aggregation emission in water. The shape of emission peak and the fluorescence solvent dependence are also the same as other sulfonatesubstituted conjugated polyelectrolytes (36,37). Compared to (+)-PPBIPV, the emission maximum of (-)-PPBIPV exhibits a greater red shift in water. This phenomenon Table 1: Optical properties of (+)-DVB, C4-DVB, monomer 1, monomer 2, (+)-PPBIPV and (-)-PPBIPV.  can be attributed to the fact that (-)-PPBIPV has a stronger aggregation effect. The fluorescence quantum yields (Φ) of (+)-PPBIPV and (-)-PPBIPV were measured by using quinine sulfate in 0.1 N sulfuric acid as standard (n = 1.336, Φ = 55%) according to the following equation (38)

Monomers/Polymers Solvent UV-vis max (nm) PL max (nm) Stokes Shift (nm)/(cm -1 )
where the subscript P and S denote to polymer solution and standard, respectively. Φ is the fluorescence quantum yield, F is the intensity of fluorescence, and n is the refractive index of the solvent. The Φ of (+)-PPBIPV and (-)-PPBIPV in methanol are 33% and 46%, respectively, which are basically consistent to the reference (39). However, the Φ of benzimidazole-based conjugated polymers is nearly 80% in THF (24). As reference reported, conjugate polyelectrolytes have lower fluorescence quantum yields than the uncharged oil soluble conjugated polymers of the same main-chain units (39). This phenomenon can be attributed to the fact that (+)-PPBIPV and (-)-PPBIPV aggregate strongly in the solvent, resulting in relatively low fluorescence quantum yields. On the other hand, the benzimidazole units of (+)-PPBIPV and (-)-PPBIPV are fluorescent chromophores, so their fluorescence quantum yields are higher than other PPVtype conjugated polyelectrolytes (40).

Metal ions response of polymers
The fluorescence responses of (+)-PPBIPV and (-)-PPBIPV on various metal ions were investigated.  (Figure 4 and Figure 7), when the metal ion is added to the polymer solution and the fluorescence intensity of the solution no longer changes, the ion concentration is called the limiting concentration. Figure 4a shows the PL spectra of aqueous solution of (+)-PPBIPV with Fe 2+ , Zn 2+ , Cd 2+ , Cr 3+ , Hg 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Cu 2+ , Sn 2+ and Fe 3+ . The PL max (nm) and maxima of fluorescence intensity of (+)-PPBIPV with different metal ions are listed in Table 2. It can be found that Fe 3+ , Sn 2+ , Cr 3+ and Hg 2+ can reduce the fluorescence of (+)-PPBIPV, but the decrease occurs most notably upon the addition of Fe 3+ . On the contrary, there is a small increase in fluorescence intensity of (+)-PPBIPV in the presence of Fe 2+ and Cu 2+ . And other metal ions hardly lead to changes in PL spectrum of (+)-PPBIPV. The fluorescence-quenching ratios (I 0 /I 1 -1) of aqueous solution of (+)-PPBIPV in the presence of different metal ions are shown in Figure 4c. It should be mentioned that even if the fluorescence quenching ratios of Sn 2+ and Cr 3+ are 0.34 and 0.25, respectively, they are still small compared to 1.52 of Fe 3+ . These results show that (+)-PPBIPV has better fluorescent sensitivity to Fe 3+ in aqueous solution.

Sensing properties of (+)-PPBIPV
The PL spectra of methanol solution of (+)-PPBIPV with various metal ions (Fe 2+ , Zn 2+ , Cd 2+ , Cr 3+ , Hg 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Cu 2+ , Sn 2+ , Fe 3+ and Pd 2+ ) were also investigated ( Figure 4b). Pd 2+ exhibits the greatest effect on the fluorescence of (+)-PPBIPV methanol solution, which almost completely quenches the fluorescence of the polymer with a quenching rate as high as 99%. Moreover, Cu 2+ , Sn 2+ , Fe 2+ and Fe 3+ can partially quench the solution fluorescence and their quenching rates are 72%, 55%, 44% and 34%, respectively. As shown in Figure 4d, the fluorescence quenching ratio of Pd 2+ is much larger than other metal ions, indicating that (+)-PPBIPV has high selectivity and excellent sensitivity for Pd 2+ in the methanol solution. Figure 5a gives the fluorescence spectra of (+)-PPBIPV in water with different concentrations of Fe 3+ . As the concentration of Fe 3+ increases, the fluorescence intensity of (+)-PPBIPV is quenched from 2611 to 1121. The fluorescence quenching efficiency of (+)-PPBIPV can be described by the Stern-Volmer equation (41): I 0 /I 1 = 1 + K SV [Q], where I 0 and I 1 are the fluorescence intensities of the system in the absence and presence of Fe 3+ , respectively, K SV is the Stern-Volmer quenching constant, and [Q] is the Fe 3+ concentration. At the concentrations of Fe 3+ between 0 and 162 μM, a Stern-Volmer plot of the fluorescence intensity of (+)-PPBIPV versus the concentration of Fe 3+ is linear with a K SV value of 7.78×10 3 M -1 , as shown in Figure 5b. The I 0 /I 1 versus [Fe 3+ ] is a straight line, indicating that there is only a single quenching mechanism in this system. Investigating the fluorescence sensor based on benzimidazole, it is found that the quenching mechanism is generally forming a complex between the sp 2 nitrogen atom and the metal ions (42). Therefore, the mechanism of fluorescence Table 2: PL max (nm) and maxima of fluorescence intensity of (+)-PPBIPV with different metal ions.   (Figure 5c). Furthermore, the K obtained from the slope and intercept of the line is 1.47×10 4 M -1 for (+)-PPBIPV binding to Fe 3+ . Subsequently, we investigated the ability of methanol solution (+)-PPBIPV to detect Pd 2+ . As Figure 6a shows, the fluorescence intensity of (+)-PPBIPV decreases regularly as the concentration of Pd 2+ increases, from 0 to 60 μM. As shown in Figure 6b, a Stern-Volmer plot of the fluorescence intensity of (+)-PPBIPV versus the concentration of Pd 2+ is a upward curve instead of a straight line, which indicates the fluorophore is quenched by both dynamic quenching and static quenching with Pd 2+ . On the other hand, when the concentration of Pd 2+ is between 0 and 15 μM, a Stern-Volmer plot of the fluorescence intensity of (+)-PPBIPV versus the concentration of Pd 2+ is linear with a K SV value of 5.93×10 4 M -1 , indicating the mechanism of fluorescence quenching of methanol solution (+)-PPBIPV towards Pd 2+ is static quenching in the low concentration region. Meanwhile, Benesi-Hildebrand plots of the fluorescence titration combined with (+)-PPBIPV and Pd 2+ is yielded linear correlation coefficients of 0.99581, using the Benesi-Hildebrand equation of linear fitting based on 1:1 binding (n = 1) (Figure 6c). The strong correlation proves that there is 1:1 binding between (+)-PPBIPV and Pd 2+ , and the K is calculated as 1.02×10 4 M -1 .

(+)-PPBIPV in water (+)-PPBIPV in methanol
All of the measurement methods of (-)-PPBIPV are the same as (+)-PPBIPV. The PL max (nm) and maxima of   Table 3: PL max (nm) and maxima of fluorescence intensity of (-)-PPBIPV with different metal ions. fluorescence intensity of (-)-PPBIPV with different metal ions are listed in Table 3. As shown in Figures 7a and 7c, Cu 2+ can quench the fluorescence of aqueous solution of (-)-PPBIPV by nearly 50%. Zn 2+ , Cd 2+ , Cr 3+ , Hg 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ and Fe 3+ exhibit little or no effect on the fluorescence intensity of the polymer. Both Fe 2+ and Sn 2+ can increase the fluorescence, but Sn 2+ is more obviously, which doubles the fluorescence intensity of the polymer approximately. Figure 7b shows the PL spectra of methanol solution of (-)-PPBIPV with different metal ions. Metal ions response of methanol solution of (-)-PPBIPV is more complicated than aqueous solution of (-)-PPBIPV. According to Figure 7d, the methanol solution of (-)-PPBIPV has certain selectivity for Fe 3+ , Pd 2+ , Cr 3+ and Sn 2+ , especially Fe 3+ .
Moreover, the Stern-Volmer plot of the fluorescence intensity of (-)-PPBIPV versus the concentration of Sn 2+ is a downward curve, which can also be fitted as the equation approximatively, y = -0.01325x + 0.93436 (Figure 8b). In this system, the addition of Sn 2+ increases the fluorescence intensity of (-)-PPBIPV, so the Benesi-Hildebrand equation takes this form (44) Figure 9a shows the fluorescence spectra of the (-)-PPBIPV in methanol solution with the increasing concentration of Fe 3+ . Add 30 μM Fe 3+ , causing the fluorescence of (-)-PPBIPV to be quenched 97.5%. As shown in Figure 9b, a Stern-Volmer plot of the fluorescence intensity of (-)-PPBIPV versus the concentration of Fe 3+ is a non-liner, which is similar to the Stern-Volmer plot of the methanol solution of (+)-PPBIPV with Pd 2+ . In the low concentration region, the mechanism of fluorescence quenching of methanol solution (-)-PPBIPV towards Fe 3+ is static quenching with a K SV value of 3.44×10 5 M -1 . From Figure 9c, the stoichiometric ratio of (-)-PPBIPV to Fe 3+ in methanol solution is 1:1, and the association constant K is 5.39×10 4 M -1 , which is much larger than the association constant of (+)-PPBIPV and Fe 3+ in aqueous solution (K = 1.47×10 4 M -1 ). Except for the difference in solvent, the reason may be that the side chain of (-)-PPBIPV is a negatively charged sulfonic acid group, which has an electrostatic attraction with metal ions. This force causes a large amount of metal ions to aggregate around the polymer, so metal ions and polymer receptor units can be better combined.

Conclusions
In summary, two novel benzimidazole-based conjugated polyelectrolytes (+)-PPBIPV and (-)-PPBIPV have been designed, synthesized and characterized successfully. In metal sensing aspects, it is demonstrated that Fe 3+ can remarkably reduce the fluorescence of (+)-PPBIPV in aqueous solution. When the concentration of Fe 3+ is in the range of 6-66 μM, Fe 3+ can be detected by (+)-PPBIPV quantitatively. Furthermore, Pd 2+ can quench almost all the fluorescence intensity of the polymer in methanol solution, of which K SV is 5.93×10 4 M -1 , and the results show that there are both static quenching and dynamic quenching in this system. In the low concentration region, there is a single static quenching, and the stoichiometric ratio of (+)-PPBIPV and Pd 2+ in methanol solution is 1:1. On the other hand, as for (-)-PPBIPV, Sn 2+ can double the fluorescence of its aqueous solution, and the results shows that the stoichiometric relationship between (-)-PPBIPV and Sn 2+ also is 1:1. Almost each ion can affect the fluorescence of (-)-PPBIPV in methanol solution, especially Fe 3+ , of which the fluorescence quenching efficiency is 97.5%. The reason for this phenomenon may be attributed to the complexation of metal ions with (-)-PPBIPV and the electrostatic attraction between positive metal ions and negative charges in side-chain of the polymer. By analyzing and comparing their response to metal ions, the influence of side chain electrification on the sensing properties of conjugated polymers can be further discussed. All results show that these two conjugated polyelectrolytes could potentially be used as effective fluorescent sensors for detecting metal ions in biological and environmental media.