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Fluorescent detection of heparin by a cationic conjugated polyfluorene probe containing aggregation-induced emission units
Polymer 53 (2012) 490e494
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Fluorescent detection of heparin by a cationic conjugated polyfluorene probe containing aggregation-induced emission units Bowei Xu a, b, Xiaofu Wu a, Haibo Li a, b, Hui Tong a, *, Lixiang Wang a, * a b
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2011 Received in revised form 21 October 2011 Accepted 27 November 2011 Available online 3 December 2011
A novel conjugated cationic polyfluorene containing aminated tetraphenylethene (ATPE) unit is developed as a sensitive and selective fluorescence probe for heparin detection based on the combination of aggregation-induced emissive property of the ATPE units and the FRET process from the blue-emissive polyfluorene segments to the yellow-emissive ATPE units. The addition of anionic heparin will lead to the formation of heparin/polymer complexes, and turn on the aggregation-induced yellow emission of the ATPE units. A good linear relationship is found between the yellow emission intensity of the ATPE units and the heparin concentrations with a limit of detection of 30 nM in aqueous buffer. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Conjugated polymer Fluorescent probe Aggregation-induced emission
1. Introduction Heparin, known as the highest negative charged biomacromolecule, is widely used for surgical procedures and extracorporeal therapies [1e5]. Although heparin can effectively prevent the formation of clots within the blood, overdose of heparin during medical process will induce critical complication such as hemorrhage or thrombocytopenia [6e8]. Thus, careful monitoring of heparin level must be performed routinely to maintain accurate therapeutic concentrations and avoid potential bleeding risk. Traditional clinical procedures including activated clotting time (ACT) or activated partial thromboplastin time (aPTT) are inaccurate, expensive and time-consuming [9e11]. Thus, many fluorescent, colorimetric, and electrochemical methods have been developed for the detection of heparin. Among these methods, fluorescent probes have been proved to be a very promising way [12e15]. For example, Anslyn et al. designed a novel fluorescent sensor with cavity structure showing strong binding to heparin with good selectivity [16]. However, most of them are based on fluorescence quenching processes, which is not very desirable for direct visual monitoring of heparin [17,18]. Recently, Zhang et al. reported a fluorescence turn-on heparin chemical sensor based on a cationic silole derivative with aggregation-induced emission (AIE)
* Corresponding authors. E-mail addresses: [email protected] (H. Tong), [email protected] (L. Wang). 0032-3861/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2011.11.058
feature [19]. The probe was weakly luminescent in aqueous solution due to the free intramolecular rotation, and turned to be highly emissive because of the restriction of the intramolecular rotation after the formation of aggregates with negative charged heparin through electrostatic interactions [20e25]. In recent years, the application of conjugated polyelectrolytes (CPEs) as optical probe in biosensors has attracted considerable interests [26e29]. Compared to small molecule counterparts, the highly conjugated backbone of the CPEs will facilitate the rapid exciton migration and result in amplification of fluorescence signals [30e36]. Liu et al. have introduced 2,1,3-benzothiadiazole (BT) units into polyfluorene derivatives to develop several CPE turn-on or ratiometric fluorescent probes for heparin quantification [37e40]. It is believed that fluorescence resonance energy transfer (FRET) from the polyfluorene segments to the BT units occurs more efficiently via interchain in aggregates than that via the intrachain process within isolated polymer chains [41,42]. Thus, the formation of aggregates of the cationic polyfluorene derivatives and the anionic heparin will enhance the emission signal from the BT units. Therefore, the CPEs can be employed for the fluorescence detection of heparin. In this contribution, we designed and synthesized a novel cationic conjugated polyfluorene (PF-ATPE) containing aggregation-induced emissive aminated tetraphenylethene (ATPE) unit as a highly selective and sensitive probe for heparin detection (Scheme 1) [21,22]. Similar to the above-mentioned works, the blue-emissive cationic PF-ATPE will complex with heparin through the electrostatic interactions and lead to the formation of the
B. Xu et al. / Polymer 53 (2012) 490e494
491
Scheme 1. Synthetic route of PF-ATPE.
aggregates. In the aggregates, both aggregation-induced yellow emission of the ATPE unit and the more efficient FRET from the blue-emitting polyfluorene segments to the yellow-emitting ATPE units via interchain are expected to happen. The combination of these two fluorescence enhancement processes at the same time is hoped to amplify the fluorescence signal from the ATPE units greatly, which can be used for detection of heparin with high sensitivity. 2. Experimental 2.1. Materials and characterization 1
H and 13C NMR spectra were recorded in deuterated solvents at 298 K. Chemical shifts were reported as values (ppm) relative to internal tetramethylsilane. Number-average (Mn) and weightaverage (Mw) molecular weights were determined by GPC System using polystyrene as the standard in THF. UVevisible absorption spectra were taken using a Perkin Elmer Lambda 35 UVevis spectrometer. Photoluminescence (PL) spectra were recorded on a Perkin Elmer LS50B Luminescence spectrometer. All the PL and UV experiments were carried out at room temperature. Photographs of the polymer solutions were taken using a Canon EOS 400D digital camera under a hand-held UV lamp with lmax ¼ 365 nm. Milli-Q water (18.2 MU) was used for all the experiments. Synthetic procedures were carried out under an inert atmosphere. All solvents were purified according to standard procedures. Heparin, hyaluronate acid sodium (HA), and other chemicals were purchased from SigmaeAldrich Chemical Co. and were used as received. The molecule weight of heparin is 20 kD, and that of sodium hyaluronate (HA) is more than 1000 kD. 1,1-Bis(4-(diethylamino)phenyl)2,2-bis(4-bromophenyl)ethylene (ATPE) was synthesized by McMurry reaction according to our previous reports [22]. 2,7dibromo-9,9-bis(3-bromopropyl)fluorene (1) and the corresponding dioxaborolane monomer (2) were synthesized according to the literature [42]. 2.2. Synthesis 2.2.1. Poly[9,9-bis(60 -bromopropyl)fluorene-co-1,1-bis(4(diethylamino)phenyl)-2,2-bis(4-bromophenyl)ethylene] (P1) The neutral polymers were prepared by a palladium-catalyzed Suzuki cross-coupling reaction. The purified monomers 1, 2, and
3 were mixed in the appropriate ratios with Pd(PPh3)4 (1 mol %) in a mixture of 2 M K2CO3 (2 ml) and toluene (5 ml) under argon. The reaction mixture was degassed by three “freeze-pump-thaw” cycles and then heated to 95 C for 24 h. After cooling to room temperature, the polymer was purified by precipitation in methanol and air-dried overnight. The neutral polymer was obtained as bright-yellow powders with a yield of 80%. 1H NMR (300 MHz, CDCl3), d (TMS, ppm): 7.76e7.40 (m, 26H), 6.90 (br, 4H), 6.40 (br, 4H), 3.24 (br, 8H), 3.05 (m, 12H), 2.28 (br, 10H), 1.19e1.07 (m, 26H); GPC: Mn ¼ 2.1 104 g/mol, PDI ¼ 2.5. 2.2.2. Poly[9,9-bis((N,N,N-trimethylammoniumbromide)propyl) fluorene-co-1,1-bis(4-(diethylamino)phenyl)-2,2-bis(4bromophenyl)ethylene] (PF-ATPE) Condensed trimethylamine (8 mL) was added dropwise to a solution of the neutral precursor polymer (100 mg) in 20 mL of THF at 78 C. The mixture was then allowed to warm up to room temperature for 4 h. The precipitate was redissolved by addition of methanol (20 mL). After the mixture was cooled down to 78 C, more trimethylamine (6 mL) was added and the mixture was stirred for 24 h at room temperature. After most of the solvent was removed, the polymer was precipitated in acetone and dried at 50 C in vacuum. The product was obtained as a light orange powder with a yield of 60%. 1H NMR (300 MHz, CDCl3), d (TMS, ppm): 7.76e7.40 (m, 26H), 6.90 (br, 4H), 6.40 (br, 4H), 3.14 (s, 54H) 3.32 (br, 8H), 3.06 (m, 12H), 2.28 (br, 10H), 1.31e1.09 (m, 26H). 3. Results and discussion 3.1. Synthesis and characterization The synthetic route for PF-ATPE is shown in Scheme 1. The synthesis of ATPE by the McMurry reaction has been described in our previous paper [22]. Monomer 1 and 2 were prepared according to the published procedures in excellent yield [42]. By the Suzuki coupling reactions of 1, 2, and ATPE in an argon atmosphere with Pd(PPh3)4 as the catalyst, the PF-ATPE precursor (P1) was obtained. The comonomer feed molar ratio of ATPE, 1 and 2 was 1:1:2 to confirm that the molar ratio between the dibromides and the diboronic acid ester was 1:1. The formation of cationic conjugated polyelectrolyte PF-ATPE was achieved by the reaction of P1 with condensed trimethylamine in THF/Methanol (3:1) mixture. P1 was readily soluble in common organic solvents such as DCM,
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emission from ATPE units, suggesting the good water solubility of PF-ATPE [38]. As a result, there were no aggregates of PF-ATPE in the aqueous solution, which would suppress the aggregationinduced emission from the ATPE units. Considering there was an obvious spectra overlap between the blue emission of the polyfluorene segments and the absorption of ATPE units (Fig. 1), the energy transfer from the former to the latter should happen. However, the almost non-emissive ATPE units may only serve as energy traps for the blue emission. The fluorescence quantum yield of PF-ATPE is determined to be 0.20 relative to 9,10diphenylanthracene in cyclohexane (Ff ¼ 0.90) [43]. 3.3. Effect of pH
Fig. 1. Absorption and fluorescence spectra of PF-ATPE in aqueous solution and absorption spectra of ATPE in THF solution.
THF and Toluene, while PF-ATPE could be easily dissolved in polar solvents including DMF, DMSO, water and alcohols. 1H NMR spectra of P1 and PF-ATPE were in good agreement with their structures. The calculated molar ratio of ATPE units based on the 1H NMR spectra was also consistent with the feed ratio. Gel permeation chromatography (GPC) of P1 revealed the number-average molecular weight (Mn) of 2.1 104 with a polydispersity index (Mw/Mn) of 2.5. 3.2. Photophysical properties Absorption and emission spectra of PF-ATPE in aqueous solution (the polymer concentration based on repeat unit [RU] ¼ 3 106 M) are shown in Fig. 1. The absorption spectrum of PFATPE exhibited one main absorption band at 375 nm, which was typically attributed to the pep* transition of the polyfluorene backbone. To our surprised, the absorption peak around 393 nm of ATPE segments could not be found, perhaps because it is too close to the absorption of polyfluorene and the low amounts of ATPE units. Meanwhile, its emission maximum was at 419 nm with a broad tail from 500 to 600 nm, and a Stokes shift of 44 nm. The strong blue emission could be easily attributed to the polyfluorene segments. It was obvious that there was no significant yellow
For the practical application, the effects of pH on the fluorescence response of the PF-ATPE must be consideration. A series of absorption and PL spectra of PF-ATPE buffer solutions ([RU] ¼ 3 10e6 M) at a pH range from 3 to 11 were recorded in 2 mM HEPES buffer solutions (pH intervals of 1.0). Although the absorption peak of PF-ATPE was still around 375 nm, the absorption band from 380 nm to 500 nm gradually increased when the pH value of the buffer solution increased from 3 to 11. Considering only the absorption band of ATPE units covered this range, the phenomena could be explained by the protonation and deprotonation processes of ATPE units when the solution changed from acidic to basic environment. At the same time, the blue emission around 419 nm of PF-ATPE decreased significantly with increasing the solution pH value, which was consistent with the above-mentioned enhancement of the absorption band of ATPE units. The phenomena also support the existence of the energy transfer from polyfluorene segments to the non-emissive ATPE units. Compared with the significant change of the blue emission band, the variation of the broad tail from 500 to 600 nm could be negligible (Fig. 2). 3.4. Heparin quantification The quantitative analytical behavior of PF-ATPE for the heparin detection was examined by spectrophotometric titration experiment which was conducted by adding heparin into the polymer solution at [RU] ¼ 3 106 M in 2 mM HEPES aqueous buffer at pH ¼ 7.4. The absorption spectra of the PF-ATPE solution were practically unchanged. On the other hand, the PL spectra of the PFATPE solution changed significantly upon the heparin concentration (heparin concentration was calculated based on a sugar dimer as the repeat unit) increasing from 0 to 2.0 mM. There was a progressive increase of the yellow emission band around 563 nm
Fig. 2. (A) Normalized absorption spectra and (B) PL spectra of PF-ATPE in water as the pH is incrementally increased from 3 to 11.
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Fig. 3. (A) Response of fluorescence spectra of PF-ATPE in the presence of different concentrations of heparin; (B) Fluorescence intensity at 563 nm as a function of heparin concentration in 2 mM HEPES (pH ¼ 7.4).
and a concomitant decrease of the blue emission band at 419 nm with gradually increasing the heparin concentration. An isosbestic point was found at 504 nm. When the heparin concentration reached 1.0 mM, a 7.5-fold PL quenching at 419 nm and a 3.2-fold PL enhancement at 563 nm were observed. The emission color change from bright blue emission to yellow emission could also be clearly observed by naked eye. At the same time, the emission maximum changed from 419 nm to 563 nm with a large Stokes shift of 188 nm. In the range of 0e0.7 mM heparin concentration, the plot of the PL intensity at 563 nm as a function of the heparin concentration shows a good linear relationship with a coefficient of determination (R2) of 0.998, which is much closed to 1. This means that the fluorescent intensities at 563 nm are linearly related to the heparin concentration. Based on the fluorescence turn-on titration, the detection limit was 30 nM, and the slope of the calibration curve for PF-ATPE was determined as 2.3 106 M1, both indicating that PFATPE could really be a sensitive fluorescence probe for heparin (Fig. 3). Regarding the origin of the emission behavior upon the addition of heparin, both the aggregation-induced emission of the ATPE units and the energy transfer from the polyfluorene segments to ATPE units must be considered. Upon the addition of heparin, the cationic PE-ATPE will form stable inter-polyelectrolyte complexes with the negatively charged heparin through the strong electrostatic attraction interactions, which will lead to polymer aggregates
Fig. 4. PL spectra of PF-ATPE buffer solution before and after addition of heparin and HA.
[37e40]. Thus, the intramolecular rotation in ATPE units will be restricted in the aggregates, and the yellow emission of ATPE units will increase significantly [19,21,22]. At the same time, more efficient interchain energy transfer from the polyfluorene segments to the ATPE units can happen, which may quench the original blue emission and further enhance the yellow emission from the ATPE units [26,41]. Thus, the high sensitivity of PE-ATPE toward heparin can be easily understood. To investigate the selectivity of PF-ATPE toward heparin, the fluorescence spectrum of PF-ATPE in the presence of an analogue of heparin, sodium hyaluronate (HA), was studied as well. As shown in Fig. 4, the addition of HA led to much smaller response on the PFATPE emission spectra than that of heparin, which proved the highly selectivity of PF-ATPE to heparin over HA. The formation of the heparin/polymer complex is based on the electrostatic attraction between negatively charged heparin and cationic polyelectrolyte. Thus, the good selectivity is mainly derived from the different charge density between heparin and sodium hyaluronate. Considering that heparin possesses four negatively charged side groups per repeat unit and HA has only one, HA has much weaker electrostatic interaction with cationic polyelectrolyte compared to heparin. Therefore, the selectivity behavior of PF-ATPE was also very reasonable. 4. Conclusions In summary, we successfully developed a novel water-soluble cationic conjugated polyfluorene containing aminated tetraphenylethene units for heparin detection in buffer solutions. In buffer solution, PF-ATPE exhibits only blue emission from the polyfluorene segments. The ATPE units are almost non-emissive due to its aggregation-induced emission feature, which just serve as energy quenching traps for the blue emission from the polyfluorene segments. The addition of anionic heparin will lead to the formation of heparin/polymer complexes, and turn-on the aggregation-induced yellow emission of the ATPE units. A progressive increase of the yellow emission band around 563 nm and a concomitant decrease of the blue emission band at 419 nm are observed with gradually increasing the heparin concentration. PF-ATPE shows a good linear relationship (R2 ¼ 0.998) between the fluorescence intensity of the ATPE units and the heparin concentrations with a limit of detection of 30 nM. Furthermore, PF-ATPE exhibits good selectivity to distinguish heparin from its analogue HA. Easy synthesis of this water-soluble fluorescence polyelectrolyte and the robust aqueous detection condition make PFATPE a good candidate for the development of applicable heparin sensors.
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Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 20904055 and 21074130), the Science Fund for Creative Research Groups (No. 20921061), and the 973 Project (2009CB623601). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
Rabenstein DL. Nat Prod Rep 2002;19:312e31. Huang X, Rezaie AR, Broze GJ, Olson ST. J Biol Chem 2011;286:8740e51. Capila I, Linhardt RJ. Angew Chem Int Ed 2002;41:390e412. Freedman MD. J Clin Pharmacol 1992;32:584e96. Wallis DE, Lewis BE, Messmore H, Wehrmacher WH. Clin Appl Thromb Hemostasis 1998;4:160e3. Warkentin TE, Levine MN, Hirsh J, Horsewood P, Roberts RS, Gent M, Kelton JG. New Engl J Med 1995;332:1330e5. Girolami B, Girolami A. Semin Thromb Hemostasis 2006;32:803e9. Despotis GJ, Gravlee G, Filos K, Levy J. Anesthesiology 1999;91:1122e51. Raymond PD, Ray MJ, Callen SN, Marsh NA. Perfusion 2003;18:269e76. Boneu B, de Moerloose P. Semin Thromb Hemost 2001;27:519e22. Murray DJ, Brosnahan WJ, Pennell B, Kapalanski D, Weiler JM, Olson J. J Cardiothorac Vasc Anesth 1997;11:24e8. Wright AT, Zhong Z, Anslyn EV. Angew Chem Int Ed 2005;44:5679e82. Sun W, Bandmann H, Schrader T. Chem Eur J 2007;13:7701e7. Sauceda JC, Duke RM, Nitz M. Chem Bio Chem 2007;8:391e4. Zeng L, Wang P, Zhang H. Org Lett 2009;11:4294e7. Zhong Z, Anslyn EV. J Am Chem Soc 2002;124:9014e5. Chen Q, Cui Y, Cao J. Polymer 2011;52:383e90. Egawa, Y.; Hayashida, R.; Seki, T. Talanta 2008,76, 736e741.
[19] Wang M, Zhang DQ, Zhang G, Zhu DB. Chem Commun; 2008:4469e71. [20] Hong Y, Lam JWY, Tang BZ. Chem Commun; 2009:4332e53. [21] Yuan W, Zhao H, Shen XY, Mahtab F, Lam JWY, Sun JZ, Tang BZ. Macromolecules 2009;42:9400e11. [22] Xu BW, Wu XF, Li HB, Tong H, Wang LX. Macromolecules 2011;44:5089e92. [23] Li Z, Dong YQ, Lam JWY, Sun J, Qin A, Dong Y, Tang BZ. Adv Funct Mater 2009; 19:905e17. [24] Dong YQ, Lam JWY, Qin A, Sun J, Liu J, Li Z, Sun J, Sung HHY, Williams ID, Kwokc HS, Tang BZ. Chem Commun; 2007:3255e7. [25] Tong H, Hong Y, Dong YQ, Lam JWY, Li Z, Guo Z, Tang BZ. Chem Commun; 2006:3705e7. [26] Liu B, Bazan GC. J Am Chem Soc 2004;126:1942e3. [27] Ho H, Boissinot M, Bergeron MG, Corbeil G, Dore K, Boudreau D, Leclerc M. Angew Chem Int Ed 2002;41:1548e51. [28] Duan X, Li Z, He F, Wang S. J Am Chem Soc 2007;129:4154e5. [29] Gu Z, Chen XY, Shen QD, Ge HX, Xu HH. Polymer 2010;51:902e7. [30] Thomas SW, Joly GD, Swager TM. Chem Rev 2007;107:1339e86. [31] Kim TH, Swager TM. Angew Chem Int Ed 2003;42:4803e6. [32] Wong WY, Wang XZ, He Z. Nat Mater 2007;6:521e7. [33] Aly SM, Ho CL, Fortin D, Wong WY, Abd-El-Aziz AS, Harvey PD. Chem Eur J 2008;14:8341e52. [34] Yamada SH, Park SJ, Song SH, Heo MH, Shim JY, Jin YG, Kim JY, Suh HS. Polymer 2010;51:6174e81. [35] Wang WZ, Wang R, Zhang C, Lu S, Liu TX. Polymer 2009;50:1236e45. [36] Jin Y, Xu YB, Qiao Z, Peng JB, Wang BZ, Cao DR. Polymer 2010;51:5726e33. [37] Pu KY, Liu B. Adv Funct Mater 2009;19:277e84. [38] Pu KY, Liu B. Macromolecules 2008;41:6636e40. [39] Shi J, Pu KY, Zhan R, Liu B. Macromol Chem Phys 2009;210:1195e200. [40] Zhan R, Fang Z, Liu B. Anal Chem 2010;82:1326e33. [41] Hong JW, Hemme WL, Keller GE, Rinke MT, Bazan GC. Adv Mater 2006;18: 878e82. [42] Chi C, Mikhailovsky A, Bazan GC. J Am Chem Soc 2007;129:11134e45. [43] Crosby GA. J Phys Chem 1971;75:991e1024.
Contents lists available at SciVerse ScienceDirect
Polymer journal homepage: www.elsevier.com/locate/polymer
Fluorescent detection of heparin by a cationic conjugated polyfluorene probe containing aggregation-induced emission units Bowei Xu a, b, Xiaofu Wu a, Haibo Li a, b, Hui Tong a, *, Lixiang Wang a, * a b
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2011 Received in revised form 21 October 2011 Accepted 27 November 2011 Available online 3 December 2011
A novel conjugated cationic polyfluorene containing aminated tetraphenylethene (ATPE) unit is developed as a sensitive and selective fluorescence probe for heparin detection based on the combination of aggregation-induced emissive property of the ATPE units and the FRET process from the blue-emissive polyfluorene segments to the yellow-emissive ATPE units. The addition of anionic heparin will lead to the formation of heparin/polymer complexes, and turn on the aggregation-induced yellow emission of the ATPE units. A good linear relationship is found between the yellow emission intensity of the ATPE units and the heparin concentrations with a limit of detection of 30 nM in aqueous buffer. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Conjugated polymer Fluorescent probe Aggregation-induced emission
1. Introduction Heparin, known as the highest negative charged biomacromolecule, is widely used for surgical procedures and extracorporeal therapies [1e5]. Although heparin can effectively prevent the formation of clots within the blood, overdose of heparin during medical process will induce critical complication such as hemorrhage or thrombocytopenia [6e8]. Thus, careful monitoring of heparin level must be performed routinely to maintain accurate therapeutic concentrations and avoid potential bleeding risk. Traditional clinical procedures including activated clotting time (ACT) or activated partial thromboplastin time (aPTT) are inaccurate, expensive and time-consuming [9e11]. Thus, many fluorescent, colorimetric, and electrochemical methods have been developed for the detection of heparin. Among these methods, fluorescent probes have been proved to be a very promising way [12e15]. For example, Anslyn et al. designed a novel fluorescent sensor with cavity structure showing strong binding to heparin with good selectivity [16]. However, most of them are based on fluorescence quenching processes, which is not very desirable for direct visual monitoring of heparin [17,18]. Recently, Zhang et al. reported a fluorescence turn-on heparin chemical sensor based on a cationic silole derivative with aggregation-induced emission (AIE)
* Corresponding authors. E-mail addresses: [email protected] (H. Tong), [email protected] (L. Wang). 0032-3861/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2011.11.058
feature [19]. The probe was weakly luminescent in aqueous solution due to the free intramolecular rotation, and turned to be highly emissive because of the restriction of the intramolecular rotation after the formation of aggregates with negative charged heparin through electrostatic interactions [20e25]. In recent years, the application of conjugated polyelectrolytes (CPEs) as optical probe in biosensors has attracted considerable interests [26e29]. Compared to small molecule counterparts, the highly conjugated backbone of the CPEs will facilitate the rapid exciton migration and result in amplification of fluorescence signals [30e36]. Liu et al. have introduced 2,1,3-benzothiadiazole (BT) units into polyfluorene derivatives to develop several CPE turn-on or ratiometric fluorescent probes for heparin quantification [37e40]. It is believed that fluorescence resonance energy transfer (FRET) from the polyfluorene segments to the BT units occurs more efficiently via interchain in aggregates than that via the intrachain process within isolated polymer chains [41,42]. Thus, the formation of aggregates of the cationic polyfluorene derivatives and the anionic heparin will enhance the emission signal from the BT units. Therefore, the CPEs can be employed for the fluorescence detection of heparin. In this contribution, we designed and synthesized a novel cationic conjugated polyfluorene (PF-ATPE) containing aggregation-induced emissive aminated tetraphenylethene (ATPE) unit as a highly selective and sensitive probe for heparin detection (Scheme 1) [21,22]. Similar to the above-mentioned works, the blue-emissive cationic PF-ATPE will complex with heparin through the electrostatic interactions and lead to the formation of the
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Scheme 1. Synthetic route of PF-ATPE.
aggregates. In the aggregates, both aggregation-induced yellow emission of the ATPE unit and the more efficient FRET from the blue-emitting polyfluorene segments to the yellow-emitting ATPE units via interchain are expected to happen. The combination of these two fluorescence enhancement processes at the same time is hoped to amplify the fluorescence signal from the ATPE units greatly, which can be used for detection of heparin with high sensitivity. 2. Experimental 2.1. Materials and characterization 1
H and 13C NMR spectra were recorded in deuterated solvents at 298 K. Chemical shifts were reported as values (ppm) relative to internal tetramethylsilane. Number-average (Mn) and weightaverage (Mw) molecular weights were determined by GPC System using polystyrene as the standard in THF. UVevisible absorption spectra were taken using a Perkin Elmer Lambda 35 UVevis spectrometer. Photoluminescence (PL) spectra were recorded on a Perkin Elmer LS50B Luminescence spectrometer. All the PL and UV experiments were carried out at room temperature. Photographs of the polymer solutions were taken using a Canon EOS 400D digital camera under a hand-held UV lamp with lmax ¼ 365 nm. Milli-Q water (18.2 MU) was used for all the experiments. Synthetic procedures were carried out under an inert atmosphere. All solvents were purified according to standard procedures. Heparin, hyaluronate acid sodium (HA), and other chemicals were purchased from SigmaeAldrich Chemical Co. and were used as received. The molecule weight of heparin is 20 kD, and that of sodium hyaluronate (HA) is more than 1000 kD. 1,1-Bis(4-(diethylamino)phenyl)2,2-bis(4-bromophenyl)ethylene (ATPE) was synthesized by McMurry reaction according to our previous reports [22]. 2,7dibromo-9,9-bis(3-bromopropyl)fluorene (1) and the corresponding dioxaborolane monomer (2) were synthesized according to the literature [42]. 2.2. Synthesis 2.2.1. Poly[9,9-bis(60 -bromopropyl)fluorene-co-1,1-bis(4(diethylamino)phenyl)-2,2-bis(4-bromophenyl)ethylene] (P1) The neutral polymers were prepared by a palladium-catalyzed Suzuki cross-coupling reaction. The purified monomers 1, 2, and
3 were mixed in the appropriate ratios with Pd(PPh3)4 (1 mol %) in a mixture of 2 M K2CO3 (2 ml) and toluene (5 ml) under argon. The reaction mixture was degassed by three “freeze-pump-thaw” cycles and then heated to 95 C for 24 h. After cooling to room temperature, the polymer was purified by precipitation in methanol and air-dried overnight. The neutral polymer was obtained as bright-yellow powders with a yield of 80%. 1H NMR (300 MHz, CDCl3), d (TMS, ppm): 7.76e7.40 (m, 26H), 6.90 (br, 4H), 6.40 (br, 4H), 3.24 (br, 8H), 3.05 (m, 12H), 2.28 (br, 10H), 1.19e1.07 (m, 26H); GPC: Mn ¼ 2.1 104 g/mol, PDI ¼ 2.5. 2.2.2. Poly[9,9-bis((N,N,N-trimethylammoniumbromide)propyl) fluorene-co-1,1-bis(4-(diethylamino)phenyl)-2,2-bis(4bromophenyl)ethylene] (PF-ATPE) Condensed trimethylamine (8 mL) was added dropwise to a solution of the neutral precursor polymer (100 mg) in 20 mL of THF at 78 C. The mixture was then allowed to warm up to room temperature for 4 h. The precipitate was redissolved by addition of methanol (20 mL). After the mixture was cooled down to 78 C, more trimethylamine (6 mL) was added and the mixture was stirred for 24 h at room temperature. After most of the solvent was removed, the polymer was precipitated in acetone and dried at 50 C in vacuum. The product was obtained as a light orange powder with a yield of 60%. 1H NMR (300 MHz, CDCl3), d (TMS, ppm): 7.76e7.40 (m, 26H), 6.90 (br, 4H), 6.40 (br, 4H), 3.14 (s, 54H) 3.32 (br, 8H), 3.06 (m, 12H), 2.28 (br, 10H), 1.31e1.09 (m, 26H). 3. Results and discussion 3.1. Synthesis and characterization The synthetic route for PF-ATPE is shown in Scheme 1. The synthesis of ATPE by the McMurry reaction has been described in our previous paper [22]. Monomer 1 and 2 were prepared according to the published procedures in excellent yield [42]. By the Suzuki coupling reactions of 1, 2, and ATPE in an argon atmosphere with Pd(PPh3)4 as the catalyst, the PF-ATPE precursor (P1) was obtained. The comonomer feed molar ratio of ATPE, 1 and 2 was 1:1:2 to confirm that the molar ratio between the dibromides and the diboronic acid ester was 1:1. The formation of cationic conjugated polyelectrolyte PF-ATPE was achieved by the reaction of P1 with condensed trimethylamine in THF/Methanol (3:1) mixture. P1 was readily soluble in common organic solvents such as DCM,
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emission from ATPE units, suggesting the good water solubility of PF-ATPE [38]. As a result, there were no aggregates of PF-ATPE in the aqueous solution, which would suppress the aggregationinduced emission from the ATPE units. Considering there was an obvious spectra overlap between the blue emission of the polyfluorene segments and the absorption of ATPE units (Fig. 1), the energy transfer from the former to the latter should happen. However, the almost non-emissive ATPE units may only serve as energy traps for the blue emission. The fluorescence quantum yield of PF-ATPE is determined to be 0.20 relative to 9,10diphenylanthracene in cyclohexane (Ff ¼ 0.90) [43]. 3.3. Effect of pH
Fig. 1. Absorption and fluorescence spectra of PF-ATPE in aqueous solution and absorption spectra of ATPE in THF solution.
THF and Toluene, while PF-ATPE could be easily dissolved in polar solvents including DMF, DMSO, water and alcohols. 1H NMR spectra of P1 and PF-ATPE were in good agreement with their structures. The calculated molar ratio of ATPE units based on the 1H NMR spectra was also consistent with the feed ratio. Gel permeation chromatography (GPC) of P1 revealed the number-average molecular weight (Mn) of 2.1 104 with a polydispersity index (Mw/Mn) of 2.5. 3.2. Photophysical properties Absorption and emission spectra of PF-ATPE in aqueous solution (the polymer concentration based on repeat unit [RU] ¼ 3 106 M) are shown in Fig. 1. The absorption spectrum of PFATPE exhibited one main absorption band at 375 nm, which was typically attributed to the pep* transition of the polyfluorene backbone. To our surprised, the absorption peak around 393 nm of ATPE segments could not be found, perhaps because it is too close to the absorption of polyfluorene and the low amounts of ATPE units. Meanwhile, its emission maximum was at 419 nm with a broad tail from 500 to 600 nm, and a Stokes shift of 44 nm. The strong blue emission could be easily attributed to the polyfluorene segments. It was obvious that there was no significant yellow
For the practical application, the effects of pH on the fluorescence response of the PF-ATPE must be consideration. A series of absorption and PL spectra of PF-ATPE buffer solutions ([RU] ¼ 3 10e6 M) at a pH range from 3 to 11 were recorded in 2 mM HEPES buffer solutions (pH intervals of 1.0). Although the absorption peak of PF-ATPE was still around 375 nm, the absorption band from 380 nm to 500 nm gradually increased when the pH value of the buffer solution increased from 3 to 11. Considering only the absorption band of ATPE units covered this range, the phenomena could be explained by the protonation and deprotonation processes of ATPE units when the solution changed from acidic to basic environment. At the same time, the blue emission around 419 nm of PF-ATPE decreased significantly with increasing the solution pH value, which was consistent with the above-mentioned enhancement of the absorption band of ATPE units. The phenomena also support the existence of the energy transfer from polyfluorene segments to the non-emissive ATPE units. Compared with the significant change of the blue emission band, the variation of the broad tail from 500 to 600 nm could be negligible (Fig. 2). 3.4. Heparin quantification The quantitative analytical behavior of PF-ATPE for the heparin detection was examined by spectrophotometric titration experiment which was conducted by adding heparin into the polymer solution at [RU] ¼ 3 106 M in 2 mM HEPES aqueous buffer at pH ¼ 7.4. The absorption spectra of the PF-ATPE solution were practically unchanged. On the other hand, the PL spectra of the PFATPE solution changed significantly upon the heparin concentration (heparin concentration was calculated based on a sugar dimer as the repeat unit) increasing from 0 to 2.0 mM. There was a progressive increase of the yellow emission band around 563 nm
Fig. 2. (A) Normalized absorption spectra and (B) PL spectra of PF-ATPE in water as the pH is incrementally increased from 3 to 11.
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Fig. 3. (A) Response of fluorescence spectra of PF-ATPE in the presence of different concentrations of heparin; (B) Fluorescence intensity at 563 nm as a function of heparin concentration in 2 mM HEPES (pH ¼ 7.4).
and a concomitant decrease of the blue emission band at 419 nm with gradually increasing the heparin concentration. An isosbestic point was found at 504 nm. When the heparin concentration reached 1.0 mM, a 7.5-fold PL quenching at 419 nm and a 3.2-fold PL enhancement at 563 nm were observed. The emission color change from bright blue emission to yellow emission could also be clearly observed by naked eye. At the same time, the emission maximum changed from 419 nm to 563 nm with a large Stokes shift of 188 nm. In the range of 0e0.7 mM heparin concentration, the plot of the PL intensity at 563 nm as a function of the heparin concentration shows a good linear relationship with a coefficient of determination (R2) of 0.998, which is much closed to 1. This means that the fluorescent intensities at 563 nm are linearly related to the heparin concentration. Based on the fluorescence turn-on titration, the detection limit was 30 nM, and the slope of the calibration curve for PF-ATPE was determined as 2.3 106 M1, both indicating that PFATPE could really be a sensitive fluorescence probe for heparin (Fig. 3). Regarding the origin of the emission behavior upon the addition of heparin, both the aggregation-induced emission of the ATPE units and the energy transfer from the polyfluorene segments to ATPE units must be considered. Upon the addition of heparin, the cationic PE-ATPE will form stable inter-polyelectrolyte complexes with the negatively charged heparin through the strong electrostatic attraction interactions, which will lead to polymer aggregates
Fig. 4. PL spectra of PF-ATPE buffer solution before and after addition of heparin and HA.
[37e40]. Thus, the intramolecular rotation in ATPE units will be restricted in the aggregates, and the yellow emission of ATPE units will increase significantly [19,21,22]. At the same time, more efficient interchain energy transfer from the polyfluorene segments to the ATPE units can happen, which may quench the original blue emission and further enhance the yellow emission from the ATPE units [26,41]. Thus, the high sensitivity of PE-ATPE toward heparin can be easily understood. To investigate the selectivity of PF-ATPE toward heparin, the fluorescence spectrum of PF-ATPE in the presence of an analogue of heparin, sodium hyaluronate (HA), was studied as well. As shown in Fig. 4, the addition of HA led to much smaller response on the PFATPE emission spectra than that of heparin, which proved the highly selectivity of PF-ATPE to heparin over HA. The formation of the heparin/polymer complex is based on the electrostatic attraction between negatively charged heparin and cationic polyelectrolyte. Thus, the good selectivity is mainly derived from the different charge density between heparin and sodium hyaluronate. Considering that heparin possesses four negatively charged side groups per repeat unit and HA has only one, HA has much weaker electrostatic interaction with cationic polyelectrolyte compared to heparin. Therefore, the selectivity behavior of PF-ATPE was also very reasonable. 4. Conclusions In summary, we successfully developed a novel water-soluble cationic conjugated polyfluorene containing aminated tetraphenylethene units for heparin detection in buffer solutions. In buffer solution, PF-ATPE exhibits only blue emission from the polyfluorene segments. The ATPE units are almost non-emissive due to its aggregation-induced emission feature, which just serve as energy quenching traps for the blue emission from the polyfluorene segments. The addition of anionic heparin will lead to the formation of heparin/polymer complexes, and turn-on the aggregation-induced yellow emission of the ATPE units. A progressive increase of the yellow emission band around 563 nm and a concomitant decrease of the blue emission band at 419 nm are observed with gradually increasing the heparin concentration. PF-ATPE shows a good linear relationship (R2 ¼ 0.998) between the fluorescence intensity of the ATPE units and the heparin concentrations with a limit of detection of 30 nM. Furthermore, PF-ATPE exhibits good selectivity to distinguish heparin from its analogue HA. Easy synthesis of this water-soluble fluorescence polyelectrolyte and the robust aqueous detection condition make PFATPE a good candidate for the development of applicable heparin sensors.
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Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 20904055 and 21074130), the Science Fund for Creative Research Groups (No. 20921061), and the 973 Project (2009CB623601). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
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