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Synthesis of an amphiphilic copolymer bearing rhodamine moieties and its self-assembly into micelles as chemosensors for Fe3+ in aqueous solution
Reactive & Functional Polymers 72 (2012) 169–175
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Synthesis of an amphiphilic copolymer bearing rhodamine moieties and its self-assembly into micelles as chemosensors for Fe3+ in aqueous solution Yu Wang, Haiqiang Wu, Jing Luo ⇑, Xiaoya Liu ⇑ School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, People’s Republic of China
a r t i c l e
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Article history: Received 2 November 2011 Received in revised form 25 December 2011 Accepted 26 December 2011 Available online 2 January 2012 Keywords: Amphiphilic copolymer Micelles Rhodamine 6G Chemosensor
a b s t r a c t We report on the fabrication of an amphiphilic random copolymer-based colorimetric and fluorescent chemosensor for Fe3+ ions that was prepared by free radical polymerization of a novel rhodamine-based Fe3+-recognizing monomer, R6GEM, with N-vinylpyrrolidone (NVP). Because of its amphiphilic property, the copolymer P(NVP-co-R6GEM) can self-assemble into micelles, which allows it to be used as a chemosensor in aqueous solution. Upon addition of Fe3+ ions to the micelle solution, visual color change and fluorescence enhancement were observed. Moreover, other metal ions did not induce obvious changes to the absorption and fluorescence spectra. The water dispersibility and biocompatibility of these polymer micelles could provide a new strategy for detecting analytes in environmental and biological systems. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The development of fluorescent sensors for the detection of environmentally and biologically relevant species [1–3], particularly for heavy metal and transition metal cations, is currently of great interest because these metal ions are involved in a variety of fundamental biological processes in organisms [4–8]. Of the various transition metal ions, the trivalent form of iron (Fe3+) is an essential element in the human body because it provides the oxygen-carrying capacity of heme and acts as a cofactor in many enzymatic reactions [9–11]. To date, numerous fluorescent chemosensors have been developed to detect Fe3+ [12–16]. Rhodamine is a molecule that is used extensively as a fluorescent labeling reagent and a dye laser source because of its excellent spectroscopic properties, including a large molar extinction coefficient, high fluorescence quantum yield, visible light excitation and long wavelength emission [17,18]. Moreover, rhodamine derivatives with a spirolactam structure are colorless and nonfluorescent, whereas the corresponding ring-opened amide provides both chromogenic and fluorogenic responses that facilitate ‘‘naked eye’’ analyte detection. Therefore, rhodamine-based dyes have been widely used as sensing materials [19–23]. However, these rhodaminebased small molecule chemosensors typically exhibit poor water solubility and usually only function in a medium of pure organic solvent or an aqueous solution containing at least 50% organic ⇑ Corresponding authors. Tel./fax: +86 510 85917763. E-mail addresses: [email protected] (J. Luo), [email protected] (X. Liu). 1381-5148/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2011.12.007
cosolvent [21,23]. The lack of water solubility greatly limits the potential applications of rhodamine-based small molecules in biological systems and for environmental analyses. Amphiphilic block copolymers can self-organize into core–shell micellar structures in aqueous solution, and certain water-insoluble organic dyes can self-assemble into the hydrophobic core of polymeric micelles. Therefore, the water dispersibility of the organic dyes could be enhanced [24–27]. However, because of the non-covalent incorporation of organic dyes, these micelles may encounter a dye leakage caused by dilution or temperature variation. To solve this problem, covalent methods have been developed for incorporating organic dyes into micelles by attaching organic dyes to the backbone of the amphiphilic copolymers [28–32]. However, the block copolymers mentioned above were synthesized by atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer polymerization (RAFT), which are normally complex and tedious processes and require a strict control of the polymerization conditions. Similar to block copolymers, amphiphilic random copolymers can also selfassemble into micelles. In addition, amphiphilic random copolymers are easy to synthesize and have much broader applications than amphiphilic block copolymers. However, to the best of our knowledge, there is no report on the use of micelles assembled from amphiphilic random copolymers as fluorescent probes. In this work, we report on the synthesis of an amphiphilic random copolymer P(NVP-co-R6GEM) that is easily prepared by the free-radical polymerization of a hydrophilic N-vinylpyrrolidone (NVP) and a hydrophobic rhodamine-base monomer R6GEM. P(NVP-co-R6GEM) could easily self-assemble into micelles in
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aqueous solution; a process that combines the advantages of the water dispersibility of the micelles and the metal-ion coordination ability of the rhodamine moiety to provide a novel type of metal ion chemosensor. Preliminary experiments showed that the P(NVP-co-R6GEM) micelle selectively and sensitively reported the presence of Fe3+ in aqueous solution via a fluorescence ‘‘turn-on’’ and a visible color change. 2. Experimental procedures 2.1. Materials Rhodamine 6G, methacryloyl chloride, N-vinylpyrrolidone (NVP) and tris(hydroxymethyl)aminomethane (Tris) were purchased from Aladdin Chemistry Co., Ltd., Shanghai, China and used as received. Triethylamine, 1,2-ethylenediamine, anhydrous Na2SO4, PVP and organic solvents were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. 2,20 -Azoisobutyronitrile (AIBN) was recrystallized from ethanol, and dichloromethane (CH2Cl2) was dried and distilled prior to use. Solutions of metal ions were prepared in distilled water from their corresponding nitrate salts (Ag+, Ba2+, Ca2+, Co2+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+), except for CdCl2. Double distilled water was used for all the experiments. 2.2. Instruments and measurements 1 H NMR and 13C NMR spectra were acquired on a Bruker– DMX500 spectrometer (Bruker, Germany) with TMS as an internal standard and CDCl3 as solvent. Mass spectra were obtained with an HP 1100 LC–MS (Agilent, US). FTIR spectra were recorded on an FTLA 2000-104 FTIR spectrophotometer (ABB Bomem, Canada). Gel permeation chromatography (GPC) was performed at room temperature on an Agilent-1100 instrument (Agilent, US) with N,N-dimethylformamide (DMF) as the eluent. The molecular weight was estimated by comparison to a polyethylene glycol (PEG) standard curve. The micelle size and size distribution were determined with a Zetasizer nano-zeta potential analyzer (Malvern, UK). Morphological examination of the micelles was conducted using a JEOL (Japan) JEM-2100(HR) transmission electron microscope (TEM). One drop of micelle solution (at a concentration of 0.1 mg/mL) was placed on a copper-mesh coated with carbon and then air-dried before measurement. Absorption spectra were recorded on a TU-1901 spectrophotometer (Pgeneral Co., Ltd., Beijing, China). Fluorescence spectra were acquired on a RF5301PC fluorescence spectrophotometer (Shimadzu, Japan), and both the excitation and the emission slit widths were 1.5 nm. All pH measurements were performed with a Sartorius basic pH-MeterPB-10 (Sartorius, Germany).
153.6, 151.7, 147.5, 132.5, 131.3, 128.3, 128.1, 123.8, 122.8, 118.0, 106.2, 96.5, 65.0, 44.0, 40.8, 38.4, 16.7, 14.7. ESI-MS: m/z 457.2 for [M+H]+, calc. for C28H32N4O2 = 456.6. 2.4. Synthesis of the R6GEM monomer The R6GEM monomer was prepared by the reaction between R6GEDA and methacryloyl chloride in an ice bath. To a 250-mL flask, R6GEDA (2.28 g, 5.0 103 mol) was dissolved in 80 mL of CH2Cl2, Et3N was added (0.84 mL, 6.0 103 mol) and the mixture was cooled in an ice bath. A solution of methacryloyl chloride (0.52 g, 5.0 103 mol) in CH2Cl2 (50 mL) was then added dropwise to the flask while stirring. All reagents and glass apparatuses were dried prior to use. After the addition of the reagents, the mixture was allowed stir in the cold ice bath for approximately 4 h. The reaction mixture was then washed with water (5 100 mL), and the organic layer was dried over anhydrous Na2SO4 and filtered. The solvent was removed under reduced pressure, and the resulting precipitate was purified by recrystallization from acetonitrile and then dried in vacuo to produce R6GEM as a white solid (1.83 g, 69.8% yield). 1H NMR (CDCl3), d (ppm): 7.96 (dd, 1H), 7.54 (s, 1H), 7.48 (dd, 2H), 7.07 (dd, 1H), 6.36 (s, 2H), 6.23 (s, 2H), 5.77 (s, 1H), 5.32 (s, 1H), 3.55 (s, 2H), 3.36 (t, 2H), 3.23 (q, 4H), 3.00 (t, 2H), 1.99 (s, 3H), 1.90 (s, 6H), 1.35 (t, 6H). 13C NMR (CDCl3), d (ppm): 170.1, 168.1, 153.9, 151.7, 147.6, 139.8, 132.9, 130.4, 128.2, 127.9, 123.9, 122.9, 119.5, 118.2, 105.1, 96.6, 65.8, 41.0, 39.6, 38.3, 18.7, 16.7, 14.7. ESI-MS: m/z 525.3 for [M+H]+, calc. for C32H36N4O3 = 524.7. 2.5. Synthesis of the amphiphilic copolymer P(NVP-co-R6GEM) The amphiphilic copolymer P(NVP-co-R6GEM) was prepared by radical copolymerization of the hydrophobic functional monomer R6GEM and the hydrophilic molecule N-vinylpyrrolidone (NVP). A mixture of R6GEM (0.32 g, 6.1 104 mol), NVP (1.33 g, 1.2
2.3. Synthesis of R6GEDA R6GEDA was synthesized as previously described [33]. To a 250mL flask, rhodamine 6G (4.80 g, 1.0 10–2 mol) was dissolved in 60 mL of ethanol. Then, 5 mL (an excess) of ethylenediamine was added dropwise with vigorous stirring at room temperature. After the addition, the stirred mixture was heated and refluxed for 12 h until the fluorescence of the solution had disappeared. The reaction was cooled, and the solvent was removed under reduced pressure. The resulting precipitate was collected and washed with cold ethanol (3 20 mL). The crude product was purified by recrystallization from acetonitrile and dried in vacuo to produce 3.45 g of R6GEDA in a 75.5% yield. 1H NMR (CDCl3), d (ppm): 7.95 (dd, 1H), 7.47 (dd, 2H), 7.08 (dd, 1H), 6.36 (s, 2H), 6.25 (s, 2H), 3.53 (t, 2H), 3.23 (q, 4H), 2.37 (t, 2H), 1.92 (s, 6H), 1.34 (t, 6H). 13C NMR (CDCl3), d (ppm): 168.6,
Scheme 1. Synthesis of (a) R6GEM and (b) P(NVP-co-R6GEM).
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102 mol) and AIBN (0.02 g, 1.2 104 mol) in 1,4-dioxane (15 mL) was added to a dry polymerization tube. The solution was deoxygenated by purging it with purified N2 gas. The tube was then sealed and stirred for 24 h in a thermostat bath regulated to 70 °C. After cooling to room temperature, the mixture was added
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dropwise into an excess of diethyl ether, and the precipitate was filtered and dissolved in CH2Cl2 (6 mL) and again precipitated into diethyl ether. The above dissolution-precipitation cycle was repeated three times. The final product was dried under vacuum to produce P(NVP-co-R6GEM) as a white powder (1.47 g, 89.1% yield).
Scheme 2. Proposed ring-opening mechanism in the presence of ferric ions (a) and a schematic illustration of the formation of the micelle and the ferric ion sensing of the micelle in aqueous solution (b).
Fig. 1. 1H NMR spectra of R6GEM and P(NVP-co-R6GEM).
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Fig. 2. Absorption spectra of (a) R6GEM (0.03 mg/mL), (b) P(NVP-co-R6GEM) (0.13 mg/mL) and (c) PVP (0.50 mg/mL) in DMF solution.
25 mL of DMF, and 8 mL of distilled water was then added dropwise (100 lL/min) to the polymer solution while stirring to initiate micellization. During this process, the solution had blue opalescence. After the addition of water, the obtained solution was stirred for 6 h and then added dropwise to 132 mL of distilled water to stabilize the micelles. The resulting micelle solution was placed into a dialysis bag (Spectrum, Mn cutoff of 3500) and dialyzed against distilled water at room temperature for 48 h; the water was replaced six times during the dialysis time period. Finally, the micelle solution was transferred into a 250-mL volumetric flask and diluted to the measurement mark with distilled water to obtain a stock solution of 2 mg/mL. All of the measurements were conducted according to the following procedure. Different amounts of metal ions were added with a micropipette to 10-mL volumetric tubes containing 5 mL of micelle solution and 2 mL of 25 mM Tris–HCl buffer (pH 7.0). The solutions were then diluted to 10 mL with water and mixed. After the reaction solutions had been kept at room temperature for 2 h, 3 mL of each solution were transferred to a 1-cm cell, and the absorbance and fluorescence spectra were recorded using an excitation wavelength of 520 nm. 3. Results and discussion 3.1. Synthesis of the monomer R6GEM
Fig. 3. Changes in the turbidity of the solutions of P(NVP-co-R6GEM) and PVP with changes in the water content; the initial polymer concentration was 20 mg/mL in DMF and k = 621 nm. The inset shows the Tyndall light scattering experiment for the final solutions of PVP (left) and P(NVP-co-R6GEM) (right).
2.6. P(NVP-co-R6GEM) self-assembled into micelles that were chemosensors in aqueous media P(NVP-co-R6GEM) copolymer micelles were prepared by the following procedure: P(NVP-co-R6GEM) (0.50 g) was dissolved in
As shown in Scheme 1a, the Fe3+-recognizing monomer R6GEM was synthesized in two steps using rhodamine 6G as the starting material. The intermediate R6GEDA was first prepared in a reaction between rhodamine 6G and ethylenediamine. R6GEDA was then reacted with methacryloyl chloride in an ice bath to produce the target monomer R6GEM. The structures of the R6GEDA and R6GEM compounds were confirmed by 1H NMR, 13C NMR and ESI-MS (see the Supporting information, Fig. S1). The monomer R6GEM was designed to chelate metal ions with its amide carbonyl oxygens [22,34,35]. Upon the addition of Fe3+ ions, the spirolactam moiety of the rhodamine group opened (the formation of an open-ring structure), which resulted in the appearance of a pink color and an orange fluorescence (Scheme 2a). 3.2. Synthesis and characterization of P(NVP-co-R6GEM) P(NVP-co-R6GEM) was synthesized by free radical polymerization of N-vinylpyrrolidone (NVP) and R6GEM at an NVP:R6GEM molar ratio of 20:1 (Scheme 1b). Because poly(N-vinylpyrrolidone)
Fig. 4. Particle size distribution (a) and TEM image (b) of the P(NVP-co-R6GEM) micelles.
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Fig. 5. UV–vis absorption (a) and fluorescence spectra (b) recorded for a P(NVP-co-R6GEM) micelle solution (1 mg/mL) in the presence of different concentrations of Fe3+ ions (0 lM, 5 lM, 10 lM, 20 lM, 30 lM, 40 lM, 50 lM, 60 lM, 80 lM, 100 lM and 120 lM). The excitation wavelength was 520 nm, and the slit width was 1.5 nm. Inset: (a) the plot of the A–A0 intensity at 533 nm, (b) the plot of the relative fluorescence intensity at 553 nm.
Fig. 6. Photographs recorded under visible light (a) and UV light at 365 nm (b) for micelle solutions of P(NVP-co-R6GEM) (1 mg/mL) in the presence of different concentrations of Fe3+ (0 lM, 10 lM, 20 lM, 40 lM, 60 lM and 80 lM).
(PVP) is a biocompatible, nonionic, water-soluble polymer with extremely low cytotoxicity [39,40], it has recently attracted attention as the hydrophilic block of amphiphilic block copolymers [36–38]. Thus, NVP was appropriately selected as the hydrophilic monomer. The structure of the obtained polymer was confirmed by FTIR spectral analysis and 1H NMR spectroscopy. In the FTIR spectra (as shown in Fig. S2 in the Supporting information), the prominent peak at 1671 cm1 was attributed to the stretching vibration of an amide carbonyl, and the wide absorption band at 3480 cm1 was indicative of the –NH and –OH functional groups. The 1H NMR spectra of R6GEM and P(NVP-co-R6GEM) in CDCl3 are shown in Fig. 1. The characteristic NMR signals corresponding to the vinyl groups of the monomers (d(H) 5.77 and 5.32) disappeared completely in the polymer, and the signals corresponding to the xanthene and benzene rings of R6GEM (d(H) 6.0–8.0) appeared in the 1H NMR spectrum of P(NVP-co-R6GEM). This observation confirmed that the hydrophobic functional monomer R6GEM had been successfully incorporated into the polymer. The signals at 1.2–3.5 ppm were assigned to the aliphatic CH3 and CH2 groups, and the signal at 3.7 ppm was assigned to the N–CH groups in the repeating PVP units. GPC analysis revealed that P(NVP-co-R6GEM) had a molecular weight (Mn) of 7100 and a polydispersity index (PDI) of 1.92. Fig. 2 shows the absorption spectra of R6GEM, P(NVP-co-R6GEM)
Fig. 7. Absorbance changes at 533 nm (a) and relative fluorescence intensity (b) recorded for the micelle solution of P(NVP-co-R6GEM) (1 mg/mL) upon addition of 50 lM of various metal ions (kex = 520 nm, kem = 553 nm; excitation and emission slit widths = 1.5 nm; pH = 7.0).
and PVP in DMF solution. The absorption spectra of P(NVP-coR6GEM) and R6GEM were similar; both the polymer and the monomer exhibited an absorption at 304 nm, which can be attributed to the absorption of the ring-closed tautomer of rhodamine
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Fig. 8. Reversible absorbance (a) and fluorescence (b) response of the micelle solution of P(NVP-co-R6GEM) to Fe3+. 1: 1 mg/mL P(NVP-co-R6GEM); 2: 1 mg/mL P(NVP-coR6GEM) with 50 lM of Fe3+; 3: 1 mg/mL P(NVP-co-R6GEM) with 50 lM of Fe3+ followed by the addition of 100 lM of ethylenediamine.
6G moieties [33]. In contrast, PVP showed no optical absorption in this region. The absorption of P(NVP-co-R6GEM) between 285 nm and 335 nm was clearly due to the rhodamine moieties of R6GEM that were incorporated into P(NVP-co-R6GEM), and the polymer chain did not change the optical characteristics of R6GEM. The absorption characteristics of the monomers and the polymer allow the use of a standard curve method (according to the Beer–Lambert Law equation) to estimate the percentage of monomers that are covalently bound to the polymer chain [41]. According to this method, the mass concentration of the monomer R6GEM immobilized to the copolymer was 17.53%. This concentration was calculated based on the absorbance of the polymer and the extinction coefficient of R6GEM (e = 11,793 M1 cm1 at 304 nm). 3.3. Preparation of the P(NVP-co-R6GEM) micelles The P(NVP-co-R6GEM) micelles were prepared by adding distilled water dropwise to the P(NVP-co-R6GEM) solution in DMF (20 mg/mL). Because of the amphiphilic property of the copolymer P(NVP-co-R6GEM), the micellar structure forms easily by selfassembly. As shown in Scheme 2b, when water was gradually added to the DMF solution of P(NVP-co-R6GEM), the hydrophobic moieties of R6GEM started to associate because of the enhanced hydrophobic interaction between the R6GEM moieties and the selected solvent mixture of DMF and H2O [42]. Meanwhile, the hydrophilic segments of PVP tended to be exposed to the aqueous phase to maintain and stabilize the formed hydrophobic microphase. The self-assembly behavior of P(NVP-co-R6GEM) was studied by the turbidity method [43]. The absorbance of a DMF solution of P(NVP-co-R6GEM) at 621 nm was measured for different water contents (by adding water dropwise), and the turbidity was calculated according to the following equation: turbidity = 1–10–A, where A was the absorbance of the solution. Fig. 3 shows typical results of the turbidity changes when water was added to the solutions of P(NVP-co-R6GEM) and PVP in DMF. For P(NVP-co-R6GEM), when the water content reached 16.7 vol.%, a turbidity jump occurred. This water content is defined as the critical water content (CWC) at which the hydrophobic segments start to aggregate. In comparison, the PVP solution had no similar turbidity change. The inset in Fig. 3 shows a simple Tyndall scattering experiment for the obtained solutions. A stronger light scattering signal was observed in the solution of P(NVP-co-R6GEM) when compared with PVP, confirming the formation of micelles. The micelle size of P(NVP-co-R6GEM) in aqueous solution was investigated with a Zetasizer nano-zeta potential analyzer, and the result showed that the micelles had an average diameter of approximately 53 nm with
a narrow size distribution (Fig. 4a). Transmission electron microscopy (TEM) (Fig. 4b) showed that the micelles were mostly spherical in shape and that the diameter was approximately 40 nm (similar to the result obtained by dynamic light scattering (DLS)). 3.4. P(NVP-co-R6GEM) micelles as chemosensors for metal ions The application of P(NVP-co-R6GEM) micelles as chemosensors for metal ions were investigated by UV–vis absorption and fluorescence spectroscopies. As shown in Fig. 5, the micelle solution showed only a weak absorption above 500 nm and almost no fluorescence in the absence of Fe3+ ions, indicating that the spirolactam structure of rhodamine is predominant. However, upon addition of Fe3+ ions, a new absorption band appeared, centered at 533 nm, and its intensity increased with increasing concentrations of Fe3+ ions (Fig. 5a). Concomitant with the appearance of the new absorption band, a visible color change occurred from colorless to pink (Fig. 6a). The enhancement in absorbance clearly suggests the formation of the delocalized xanthene moiety of the rhodamine group. Furthermore, the fluorimetric response of a micelle solution with Fe3+ ions was also studied (Fig. 5b). The emission spectrum was obtained by excitation at 520 nm. With the addition of Fe3+ ions, a significant enhancement of fluorescence (with an emission maximum at 553 nm) was observed that corresponded to the fluorescence emission of rhodamine 6G. Fig. 6b shows the color change from colorless to orange that occurred when the micelle solutions were irradiated with UV light at 365 nm. The absorption and emission changes are associated with the Fe3+-induced spirolactam ring opening of the rhodamine group (Scheme 2a). The size and sizedistribution of P(NVP-co-R6GEM) micelles after the addition of ferric ions were also measured by DLS. The average size was approximately 58 nm, which was a slightly increased value compared with the micelles before the addition of ferric ions (see the Supporting information, Fig. S3). The selectivity of the P(NVP-co-R6GEM) micelle solution for common metal ions was investigated by absorbance and fluorescence measurements. As shown in Fig. 7a, among a series of cations, including Ag+, Ba2+, Ca2+, Co2+, Cu2+, Cd2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+, only Fe3+ gave a considerable absorption enhancement. The presence of Hg2+ ions gave a small absorbance increase, while other metal ions did not induce obvious changes compared with a blank sample. Next, the detection selectivity was investigated by fluorescence measurement. Fig. 7b shows the fluorescence intensity change of the micelle solution in the presence of various ions; Fe3+ induced a dramatic emission enhancement (37-fold), whereas other metal ions produced a
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much smaller or no fluorescence increase under identical conditions, with the exception of Hg2+, which had a slight enhancing effect (15.7-fold) [44]. These results suggested that P(NVP-coR6GEM) micelles can serve as a ‘‘naked-eye’’ and fluorescence chemosensor for Fe3+. The response of P(NVP-co-R6GEM) micelles to Fe3+ is reversible when ethylenediamine is added [22]. As shown in Fig. 8, upon addition of an excess of ethylenediamine to the mixture of P(NVPco-R6GEM) and Fe3+ in aqueous solution, both the color and the fluorescence disappeared instantly, indicating the decomplexation of Fe3+ by ethylenediamine followed by the spirolactam ring closure reaction (Scheme 2). Further addition of Fe3+ still resulted in similar absorption and fluorescence changes. Thus, the polymer nanoparticles can be classified as reversible chemosensors for Fe3+. 4. Conclusion In conclusion, an amphiphilic random copolymer P(NVP-coR6GEM) was synthesized by free radical polymerization, and the copolymer self-assembled into micelles in aqueous solution. The micelles served as water-soluble chemosensors that selectively bind Fe3+ ions over other common metal ions, leading to an obvious color change and a prominent fluorescence enhancement. In addition, the water dispersibility and biocompatibility of the micelles may provide a new approach for measuring ferric ions in environmental and biological milieus. Acknowledgements This work was supported by the Natural Science Foundation of China (Grants 20974041, 21174056 and 51103064). Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.reactfunctpolym.2011.12.007.
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Synthesis of an amphiphilic copolymer bearing rhodamine moieties and its self-assembly into micelles as chemosensors for Fe3+ in aqueous solution Yu Wang, Haiqiang Wu, Jing Luo ⇑, Xiaoya Liu ⇑ School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 25 December 2011 Accepted 26 December 2011 Available online 2 January 2012 Keywords: Amphiphilic copolymer Micelles Rhodamine 6G Chemosensor
a b s t r a c t We report on the fabrication of an amphiphilic random copolymer-based colorimetric and fluorescent chemosensor for Fe3+ ions that was prepared by free radical polymerization of a novel rhodamine-based Fe3+-recognizing monomer, R6GEM, with N-vinylpyrrolidone (NVP). Because of its amphiphilic property, the copolymer P(NVP-co-R6GEM) can self-assemble into micelles, which allows it to be used as a chemosensor in aqueous solution. Upon addition of Fe3+ ions to the micelle solution, visual color change and fluorescence enhancement were observed. Moreover, other metal ions did not induce obvious changes to the absorption and fluorescence spectra. The water dispersibility and biocompatibility of these polymer micelles could provide a new strategy for detecting analytes in environmental and biological systems. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The development of fluorescent sensors for the detection of environmentally and biologically relevant species [1–3], particularly for heavy metal and transition metal cations, is currently of great interest because these metal ions are involved in a variety of fundamental biological processes in organisms [4–8]. Of the various transition metal ions, the trivalent form of iron (Fe3+) is an essential element in the human body because it provides the oxygen-carrying capacity of heme and acts as a cofactor in many enzymatic reactions [9–11]. To date, numerous fluorescent chemosensors have been developed to detect Fe3+ [12–16]. Rhodamine is a molecule that is used extensively as a fluorescent labeling reagent and a dye laser source because of its excellent spectroscopic properties, including a large molar extinction coefficient, high fluorescence quantum yield, visible light excitation and long wavelength emission [17,18]. Moreover, rhodamine derivatives with a spirolactam structure are colorless and nonfluorescent, whereas the corresponding ring-opened amide provides both chromogenic and fluorogenic responses that facilitate ‘‘naked eye’’ analyte detection. Therefore, rhodamine-based dyes have been widely used as sensing materials [19–23]. However, these rhodaminebased small molecule chemosensors typically exhibit poor water solubility and usually only function in a medium of pure organic solvent or an aqueous solution containing at least 50% organic ⇑ Corresponding authors. Tel./fax: +86 510 85917763. E-mail addresses: [email protected] (J. Luo), [email protected] (X. Liu). 1381-5148/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2011.12.007
cosolvent [21,23]. The lack of water solubility greatly limits the potential applications of rhodamine-based small molecules in biological systems and for environmental analyses. Amphiphilic block copolymers can self-organize into core–shell micellar structures in aqueous solution, and certain water-insoluble organic dyes can self-assemble into the hydrophobic core of polymeric micelles. Therefore, the water dispersibility of the organic dyes could be enhanced [24–27]. However, because of the non-covalent incorporation of organic dyes, these micelles may encounter a dye leakage caused by dilution or temperature variation. To solve this problem, covalent methods have been developed for incorporating organic dyes into micelles by attaching organic dyes to the backbone of the amphiphilic copolymers [28–32]. However, the block copolymers mentioned above were synthesized by atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer polymerization (RAFT), which are normally complex and tedious processes and require a strict control of the polymerization conditions. Similar to block copolymers, amphiphilic random copolymers can also selfassemble into micelles. In addition, amphiphilic random copolymers are easy to synthesize and have much broader applications than amphiphilic block copolymers. However, to the best of our knowledge, there is no report on the use of micelles assembled from amphiphilic random copolymers as fluorescent probes. In this work, we report on the synthesis of an amphiphilic random copolymer P(NVP-co-R6GEM) that is easily prepared by the free-radical polymerization of a hydrophilic N-vinylpyrrolidone (NVP) and a hydrophobic rhodamine-base monomer R6GEM. P(NVP-co-R6GEM) could easily self-assemble into micelles in
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aqueous solution; a process that combines the advantages of the water dispersibility of the micelles and the metal-ion coordination ability of the rhodamine moiety to provide a novel type of metal ion chemosensor. Preliminary experiments showed that the P(NVP-co-R6GEM) micelle selectively and sensitively reported the presence of Fe3+ in aqueous solution via a fluorescence ‘‘turn-on’’ and a visible color change. 2. Experimental procedures 2.1. Materials Rhodamine 6G, methacryloyl chloride, N-vinylpyrrolidone (NVP) and tris(hydroxymethyl)aminomethane (Tris) were purchased from Aladdin Chemistry Co., Ltd., Shanghai, China and used as received. Triethylamine, 1,2-ethylenediamine, anhydrous Na2SO4, PVP and organic solvents were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. 2,20 -Azoisobutyronitrile (AIBN) was recrystallized from ethanol, and dichloromethane (CH2Cl2) was dried and distilled prior to use. Solutions of metal ions were prepared in distilled water from their corresponding nitrate salts (Ag+, Ba2+, Ca2+, Co2+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+), except for CdCl2. Double distilled water was used for all the experiments. 2.2. Instruments and measurements 1 H NMR and 13C NMR spectra were acquired on a Bruker– DMX500 spectrometer (Bruker, Germany) with TMS as an internal standard and CDCl3 as solvent. Mass spectra were obtained with an HP 1100 LC–MS (Agilent, US). FTIR spectra were recorded on an FTLA 2000-104 FTIR spectrophotometer (ABB Bomem, Canada). Gel permeation chromatography (GPC) was performed at room temperature on an Agilent-1100 instrument (Agilent, US) with N,N-dimethylformamide (DMF) as the eluent. The molecular weight was estimated by comparison to a polyethylene glycol (PEG) standard curve. The micelle size and size distribution were determined with a Zetasizer nano-zeta potential analyzer (Malvern, UK). Morphological examination of the micelles was conducted using a JEOL (Japan) JEM-2100(HR) transmission electron microscope (TEM). One drop of micelle solution (at a concentration of 0.1 mg/mL) was placed on a copper-mesh coated with carbon and then air-dried before measurement. Absorption spectra were recorded on a TU-1901 spectrophotometer (Pgeneral Co., Ltd., Beijing, China). Fluorescence spectra were acquired on a RF5301PC fluorescence spectrophotometer (Shimadzu, Japan), and both the excitation and the emission slit widths were 1.5 nm. All pH measurements were performed with a Sartorius basic pH-MeterPB-10 (Sartorius, Germany).
153.6, 151.7, 147.5, 132.5, 131.3, 128.3, 128.1, 123.8, 122.8, 118.0, 106.2, 96.5, 65.0, 44.0, 40.8, 38.4, 16.7, 14.7. ESI-MS: m/z 457.2 for [M+H]+, calc. for C28H32N4O2 = 456.6. 2.4. Synthesis of the R6GEM monomer The R6GEM monomer was prepared by the reaction between R6GEDA and methacryloyl chloride in an ice bath. To a 250-mL flask, R6GEDA (2.28 g, 5.0 103 mol) was dissolved in 80 mL of CH2Cl2, Et3N was added (0.84 mL, 6.0 103 mol) and the mixture was cooled in an ice bath. A solution of methacryloyl chloride (0.52 g, 5.0 103 mol) in CH2Cl2 (50 mL) was then added dropwise to the flask while stirring. All reagents and glass apparatuses were dried prior to use. After the addition of the reagents, the mixture was allowed stir in the cold ice bath for approximately 4 h. The reaction mixture was then washed with water (5 100 mL), and the organic layer was dried over anhydrous Na2SO4 and filtered. The solvent was removed under reduced pressure, and the resulting precipitate was purified by recrystallization from acetonitrile and then dried in vacuo to produce R6GEM as a white solid (1.83 g, 69.8% yield). 1H NMR (CDCl3), d (ppm): 7.96 (dd, 1H), 7.54 (s, 1H), 7.48 (dd, 2H), 7.07 (dd, 1H), 6.36 (s, 2H), 6.23 (s, 2H), 5.77 (s, 1H), 5.32 (s, 1H), 3.55 (s, 2H), 3.36 (t, 2H), 3.23 (q, 4H), 3.00 (t, 2H), 1.99 (s, 3H), 1.90 (s, 6H), 1.35 (t, 6H). 13C NMR (CDCl3), d (ppm): 170.1, 168.1, 153.9, 151.7, 147.6, 139.8, 132.9, 130.4, 128.2, 127.9, 123.9, 122.9, 119.5, 118.2, 105.1, 96.6, 65.8, 41.0, 39.6, 38.3, 18.7, 16.7, 14.7. ESI-MS: m/z 525.3 for [M+H]+, calc. for C32H36N4O3 = 524.7. 2.5. Synthesis of the amphiphilic copolymer P(NVP-co-R6GEM) The amphiphilic copolymer P(NVP-co-R6GEM) was prepared by radical copolymerization of the hydrophobic functional monomer R6GEM and the hydrophilic molecule N-vinylpyrrolidone (NVP). A mixture of R6GEM (0.32 g, 6.1 104 mol), NVP (1.33 g, 1.2
2.3. Synthesis of R6GEDA R6GEDA was synthesized as previously described [33]. To a 250mL flask, rhodamine 6G (4.80 g, 1.0 10–2 mol) was dissolved in 60 mL of ethanol. Then, 5 mL (an excess) of ethylenediamine was added dropwise with vigorous stirring at room temperature. After the addition, the stirred mixture was heated and refluxed for 12 h until the fluorescence of the solution had disappeared. The reaction was cooled, and the solvent was removed under reduced pressure. The resulting precipitate was collected and washed with cold ethanol (3 20 mL). The crude product was purified by recrystallization from acetonitrile and dried in vacuo to produce 3.45 g of R6GEDA in a 75.5% yield. 1H NMR (CDCl3), d (ppm): 7.95 (dd, 1H), 7.47 (dd, 2H), 7.08 (dd, 1H), 6.36 (s, 2H), 6.25 (s, 2H), 3.53 (t, 2H), 3.23 (q, 4H), 2.37 (t, 2H), 1.92 (s, 6H), 1.34 (t, 6H). 13C NMR (CDCl3), d (ppm): 168.6,
Scheme 1. Synthesis of (a) R6GEM and (b) P(NVP-co-R6GEM).
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102 mol) and AIBN (0.02 g, 1.2 104 mol) in 1,4-dioxane (15 mL) was added to a dry polymerization tube. The solution was deoxygenated by purging it with purified N2 gas. The tube was then sealed and stirred for 24 h in a thermostat bath regulated to 70 °C. After cooling to room temperature, the mixture was added
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dropwise into an excess of diethyl ether, and the precipitate was filtered and dissolved in CH2Cl2 (6 mL) and again precipitated into diethyl ether. The above dissolution-precipitation cycle was repeated three times. The final product was dried under vacuum to produce P(NVP-co-R6GEM) as a white powder (1.47 g, 89.1% yield).
Scheme 2. Proposed ring-opening mechanism in the presence of ferric ions (a) and a schematic illustration of the formation of the micelle and the ferric ion sensing of the micelle in aqueous solution (b).
Fig. 1. 1H NMR spectra of R6GEM and P(NVP-co-R6GEM).
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Fig. 2. Absorption spectra of (a) R6GEM (0.03 mg/mL), (b) P(NVP-co-R6GEM) (0.13 mg/mL) and (c) PVP (0.50 mg/mL) in DMF solution.
25 mL of DMF, and 8 mL of distilled water was then added dropwise (100 lL/min) to the polymer solution while stirring to initiate micellization. During this process, the solution had blue opalescence. After the addition of water, the obtained solution was stirred for 6 h and then added dropwise to 132 mL of distilled water to stabilize the micelles. The resulting micelle solution was placed into a dialysis bag (Spectrum, Mn cutoff of 3500) and dialyzed against distilled water at room temperature for 48 h; the water was replaced six times during the dialysis time period. Finally, the micelle solution was transferred into a 250-mL volumetric flask and diluted to the measurement mark with distilled water to obtain a stock solution of 2 mg/mL. All of the measurements were conducted according to the following procedure. Different amounts of metal ions were added with a micropipette to 10-mL volumetric tubes containing 5 mL of micelle solution and 2 mL of 25 mM Tris–HCl buffer (pH 7.0). The solutions were then diluted to 10 mL with water and mixed. After the reaction solutions had been kept at room temperature for 2 h, 3 mL of each solution were transferred to a 1-cm cell, and the absorbance and fluorescence spectra were recorded using an excitation wavelength of 520 nm. 3. Results and discussion 3.1. Synthesis of the monomer R6GEM
Fig. 3. Changes in the turbidity of the solutions of P(NVP-co-R6GEM) and PVP with changes in the water content; the initial polymer concentration was 20 mg/mL in DMF and k = 621 nm. The inset shows the Tyndall light scattering experiment for the final solutions of PVP (left) and P(NVP-co-R6GEM) (right).
2.6. P(NVP-co-R6GEM) self-assembled into micelles that were chemosensors in aqueous media P(NVP-co-R6GEM) copolymer micelles were prepared by the following procedure: P(NVP-co-R6GEM) (0.50 g) was dissolved in
As shown in Scheme 1a, the Fe3+-recognizing monomer R6GEM was synthesized in two steps using rhodamine 6G as the starting material. The intermediate R6GEDA was first prepared in a reaction between rhodamine 6G and ethylenediamine. R6GEDA was then reacted with methacryloyl chloride in an ice bath to produce the target monomer R6GEM. The structures of the R6GEDA and R6GEM compounds were confirmed by 1H NMR, 13C NMR and ESI-MS (see the Supporting information, Fig. S1). The monomer R6GEM was designed to chelate metal ions with its amide carbonyl oxygens [22,34,35]. Upon the addition of Fe3+ ions, the spirolactam moiety of the rhodamine group opened (the formation of an open-ring structure), which resulted in the appearance of a pink color and an orange fluorescence (Scheme 2a). 3.2. Synthesis and characterization of P(NVP-co-R6GEM) P(NVP-co-R6GEM) was synthesized by free radical polymerization of N-vinylpyrrolidone (NVP) and R6GEM at an NVP:R6GEM molar ratio of 20:1 (Scheme 1b). Because poly(N-vinylpyrrolidone)
Fig. 4. Particle size distribution (a) and TEM image (b) of the P(NVP-co-R6GEM) micelles.
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Fig. 5. UV–vis absorption (a) and fluorescence spectra (b) recorded for a P(NVP-co-R6GEM) micelle solution (1 mg/mL) in the presence of different concentrations of Fe3+ ions (0 lM, 5 lM, 10 lM, 20 lM, 30 lM, 40 lM, 50 lM, 60 lM, 80 lM, 100 lM and 120 lM). The excitation wavelength was 520 nm, and the slit width was 1.5 nm. Inset: (a) the plot of the A–A0 intensity at 533 nm, (b) the plot of the relative fluorescence intensity at 553 nm.
Fig. 6. Photographs recorded under visible light (a) and UV light at 365 nm (b) for micelle solutions of P(NVP-co-R6GEM) (1 mg/mL) in the presence of different concentrations of Fe3+ (0 lM, 10 lM, 20 lM, 40 lM, 60 lM and 80 lM).
(PVP) is a biocompatible, nonionic, water-soluble polymer with extremely low cytotoxicity [39,40], it has recently attracted attention as the hydrophilic block of amphiphilic block copolymers [36–38]. Thus, NVP was appropriately selected as the hydrophilic monomer. The structure of the obtained polymer was confirmed by FTIR spectral analysis and 1H NMR spectroscopy. In the FTIR spectra (as shown in Fig. S2 in the Supporting information), the prominent peak at 1671 cm1 was attributed to the stretching vibration of an amide carbonyl, and the wide absorption band at 3480 cm1 was indicative of the –NH and –OH functional groups. The 1H NMR spectra of R6GEM and P(NVP-co-R6GEM) in CDCl3 are shown in Fig. 1. The characteristic NMR signals corresponding to the vinyl groups of the monomers (d(H) 5.77 and 5.32) disappeared completely in the polymer, and the signals corresponding to the xanthene and benzene rings of R6GEM (d(H) 6.0–8.0) appeared in the 1H NMR spectrum of P(NVP-co-R6GEM). This observation confirmed that the hydrophobic functional monomer R6GEM had been successfully incorporated into the polymer. The signals at 1.2–3.5 ppm were assigned to the aliphatic CH3 and CH2 groups, and the signal at 3.7 ppm was assigned to the N–CH groups in the repeating PVP units. GPC analysis revealed that P(NVP-co-R6GEM) had a molecular weight (Mn) of 7100 and a polydispersity index (PDI) of 1.92. Fig. 2 shows the absorption spectra of R6GEM, P(NVP-co-R6GEM)
Fig. 7. Absorbance changes at 533 nm (a) and relative fluorescence intensity (b) recorded for the micelle solution of P(NVP-co-R6GEM) (1 mg/mL) upon addition of 50 lM of various metal ions (kex = 520 nm, kem = 553 nm; excitation and emission slit widths = 1.5 nm; pH = 7.0).
and PVP in DMF solution. The absorption spectra of P(NVP-coR6GEM) and R6GEM were similar; both the polymer and the monomer exhibited an absorption at 304 nm, which can be attributed to the absorption of the ring-closed tautomer of rhodamine
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Fig. 8. Reversible absorbance (a) and fluorescence (b) response of the micelle solution of P(NVP-co-R6GEM) to Fe3+. 1: 1 mg/mL P(NVP-co-R6GEM); 2: 1 mg/mL P(NVP-coR6GEM) with 50 lM of Fe3+; 3: 1 mg/mL P(NVP-co-R6GEM) with 50 lM of Fe3+ followed by the addition of 100 lM of ethylenediamine.
6G moieties [33]. In contrast, PVP showed no optical absorption in this region. The absorption of P(NVP-co-R6GEM) between 285 nm and 335 nm was clearly due to the rhodamine moieties of R6GEM that were incorporated into P(NVP-co-R6GEM), and the polymer chain did not change the optical characteristics of R6GEM. The absorption characteristics of the monomers and the polymer allow the use of a standard curve method (according to the Beer–Lambert Law equation) to estimate the percentage of monomers that are covalently bound to the polymer chain [41]. According to this method, the mass concentration of the monomer R6GEM immobilized to the copolymer was 17.53%. This concentration was calculated based on the absorbance of the polymer and the extinction coefficient of R6GEM (e = 11,793 M1 cm1 at 304 nm). 3.3. Preparation of the P(NVP-co-R6GEM) micelles The P(NVP-co-R6GEM) micelles were prepared by adding distilled water dropwise to the P(NVP-co-R6GEM) solution in DMF (20 mg/mL). Because of the amphiphilic property of the copolymer P(NVP-co-R6GEM), the micellar structure forms easily by selfassembly. As shown in Scheme 2b, when water was gradually added to the DMF solution of P(NVP-co-R6GEM), the hydrophobic moieties of R6GEM started to associate because of the enhanced hydrophobic interaction between the R6GEM moieties and the selected solvent mixture of DMF and H2O [42]. Meanwhile, the hydrophilic segments of PVP tended to be exposed to the aqueous phase to maintain and stabilize the formed hydrophobic microphase. The self-assembly behavior of P(NVP-co-R6GEM) was studied by the turbidity method [43]. The absorbance of a DMF solution of P(NVP-co-R6GEM) at 621 nm was measured for different water contents (by adding water dropwise), and the turbidity was calculated according to the following equation: turbidity = 1–10–A, where A was the absorbance of the solution. Fig. 3 shows typical results of the turbidity changes when water was added to the solutions of P(NVP-co-R6GEM) and PVP in DMF. For P(NVP-co-R6GEM), when the water content reached 16.7 vol.%, a turbidity jump occurred. This water content is defined as the critical water content (CWC) at which the hydrophobic segments start to aggregate. In comparison, the PVP solution had no similar turbidity change. The inset in Fig. 3 shows a simple Tyndall scattering experiment for the obtained solutions. A stronger light scattering signal was observed in the solution of P(NVP-co-R6GEM) when compared with PVP, confirming the formation of micelles. The micelle size of P(NVP-co-R6GEM) in aqueous solution was investigated with a Zetasizer nano-zeta potential analyzer, and the result showed that the micelles had an average diameter of approximately 53 nm with
a narrow size distribution (Fig. 4a). Transmission electron microscopy (TEM) (Fig. 4b) showed that the micelles were mostly spherical in shape and that the diameter was approximately 40 nm (similar to the result obtained by dynamic light scattering (DLS)). 3.4. P(NVP-co-R6GEM) micelles as chemosensors for metal ions The application of P(NVP-co-R6GEM) micelles as chemosensors for metal ions were investigated by UV–vis absorption and fluorescence spectroscopies. As shown in Fig. 5, the micelle solution showed only a weak absorption above 500 nm and almost no fluorescence in the absence of Fe3+ ions, indicating that the spirolactam structure of rhodamine is predominant. However, upon addition of Fe3+ ions, a new absorption band appeared, centered at 533 nm, and its intensity increased with increasing concentrations of Fe3+ ions (Fig. 5a). Concomitant with the appearance of the new absorption band, a visible color change occurred from colorless to pink (Fig. 6a). The enhancement in absorbance clearly suggests the formation of the delocalized xanthene moiety of the rhodamine group. Furthermore, the fluorimetric response of a micelle solution with Fe3+ ions was also studied (Fig. 5b). The emission spectrum was obtained by excitation at 520 nm. With the addition of Fe3+ ions, a significant enhancement of fluorescence (with an emission maximum at 553 nm) was observed that corresponded to the fluorescence emission of rhodamine 6G. Fig. 6b shows the color change from colorless to orange that occurred when the micelle solutions were irradiated with UV light at 365 nm. The absorption and emission changes are associated with the Fe3+-induced spirolactam ring opening of the rhodamine group (Scheme 2a). The size and sizedistribution of P(NVP-co-R6GEM) micelles after the addition of ferric ions were also measured by DLS. The average size was approximately 58 nm, which was a slightly increased value compared with the micelles before the addition of ferric ions (see the Supporting information, Fig. S3). The selectivity of the P(NVP-co-R6GEM) micelle solution for common metal ions was investigated by absorbance and fluorescence measurements. As shown in Fig. 7a, among a series of cations, including Ag+, Ba2+, Ca2+, Co2+, Cu2+, Cd2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+, only Fe3+ gave a considerable absorption enhancement. The presence of Hg2+ ions gave a small absorbance increase, while other metal ions did not induce obvious changes compared with a blank sample. Next, the detection selectivity was investigated by fluorescence measurement. Fig. 7b shows the fluorescence intensity change of the micelle solution in the presence of various ions; Fe3+ induced a dramatic emission enhancement (37-fold), whereas other metal ions produced a
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much smaller or no fluorescence increase under identical conditions, with the exception of Hg2+, which had a slight enhancing effect (15.7-fold) [44]. These results suggested that P(NVP-coR6GEM) micelles can serve as a ‘‘naked-eye’’ and fluorescence chemosensor for Fe3+. The response of P(NVP-co-R6GEM) micelles to Fe3+ is reversible when ethylenediamine is added [22]. As shown in Fig. 8, upon addition of an excess of ethylenediamine to the mixture of P(NVPco-R6GEM) and Fe3+ in aqueous solution, both the color and the fluorescence disappeared instantly, indicating the decomplexation of Fe3+ by ethylenediamine followed by the spirolactam ring closure reaction (Scheme 2). Further addition of Fe3+ still resulted in similar absorption and fluorescence changes. Thus, the polymer nanoparticles can be classified as reversible chemosensors for Fe3+. 4. Conclusion In conclusion, an amphiphilic random copolymer P(NVP-coR6GEM) was synthesized by free radical polymerization, and the copolymer self-assembled into micelles in aqueous solution. The micelles served as water-soluble chemosensors that selectively bind Fe3+ ions over other common metal ions, leading to an obvious color change and a prominent fluorescence enhancement. In addition, the water dispersibility and biocompatibility of the micelles may provide a new approach for measuring ferric ions in environmental and biological milieus. Acknowledgements This work was supported by the Natural Science Foundation of China (Grants 20974041, 21174056 and 51103064). Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.reactfunctpolym.2011.12.007.
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