Fluorescent Turn-On Detection and Assay of Protein Based on

Mar 26, 2009 - Syntheses and Properties of Two-Dimensional, Dicationic Nonlinear Optical Chromophores Based on Pyrazinyl Cores. Benjamin J. Coe , John...
3 downloads 7 Views 1MB Size
Download PDF
J. Phys. Chem. C 2009, 113, 6809–6814

6809

Fluorescent Turn-On Detection and Assay of Protein Based on Lambda (Λ)-Shaped Pyridinium Salts with Aggregation-Induced Emission Characteristics Chun-Xue Yuan,† Xu-Tang Tao,*,† Lei Wang,† Jia-Xiang Yang,‡ and Min-Hua Jiang† State Key Laboratory of Crystal Materials, Shandong UniVersity, Jinan 250100, People’s Republic of China, and Department of Chemistry, Anhui UniVersity, Hefei 230039, People’s Republic of China ReceiVed: December 16, 2008; ReVised Manuscript ReceiVed: February 26, 2009

The lambda (Λ)-shaped pyridinium salts 2,8-(6H,12H-5,11-methanodibenzo[b,f]diazocineylene)-di(p-ethenylN-methylpyridinium) ditosylate (DMDPS), 2,8-(6H,12H-5,11-methanodibenzo[b,f]diazocineylene)-di(p-ethenylN-methylpyridinium) diiodide (DMDPI), and 2,8-(6H,12H-5,11-methanodibenzo[b,f]diazocineylene)-di(pethenyl-N-methylpyridinium) dinitrate (DMDPN) based on Tro¨ger’s base exhibit a typical aggregation-induced emission (AIE) behavior that it is virtually nonemissive in solution but highly luminescent in the aggregate state. The possibility of utilizing the AIE effect for protein detection and quantification is explored by using bovine serum albumin (BSA) as a model protein. These bioprobes undergo a large spectral change upon binding to BSA, which can be determined by fluorometry. Furthermore, the plots of photoluminescence intensity versus BSA concentration (0-70 µg/mL) display a good linear relationship, indicating quantitative fluorimetric protein detection may be achieved. Introduction Fluorescence (FL) bioprobes for protein detection and quantification have received great attention due to their high sensitivity, low background noises, and wide dynamic ranges, and accordingly, they show potential applications in chemistry, materials science, biology, and medicine.1 Upon binding with proteins, FL of the bioprobes can be enhanced/quenched and/ or red/blue-shifted, thus enabling visual observation of the protein species. Thereinto, fluorescent turn-on probes with improved emission intensity upon association with proteins are more useful, because the binding event to the host molecule may be readily followed by FL enhancement.2 Several fluorescent reagents targeting proteins have been developed for protein assays based on FL enhancement in solution, such as fluorescamine,3 cyanine dyes,4 and in SDS-polyacrylamide gels, such as SYPRO Ruby,5 1,8-ANS,6 and Nile Red.7 However, some FL bioprobes require lengthy procedures with carefully timed steps, some show small Stokes’ shifts and nonlinear calibration curves, and some are even not environmentally stable (e.g., Nile Red and fluorescamine).3,7 More importantly, almost all conventional FL probes encountered a thorny problem, which is the aggregation of dyes.8 Organic dye molecules tend to aggregate when dispersed in aqueous media or bound to proteins in large quantities. The aggregation usually quenches FL, which limits the effective ranges of the probes. Thus, development of simple and stable FL bioprobes without aggregation-caused quenching (ACQ) would be very rewarding work. Recently, a phenomenon, namely aggregation-induced emission (AIE), which is exactly opposite to the ACQ, was discovered by Tang et al.9 These nonemissive dyes can be induced to emit efficiently by the aggregate formation. A large number of AIE-active dyes have since been developed, examples of which include siloles,10 CN-MBE,11 DPDSB derivatives,12 * To whom correspondence should be addressed. Phone: +86-53188364963. Fax: +86-531-88574135. E-mail: [email protected]. † Shandong University. ‡ Anhui University.

TABLE 1: Photophysical Properties of DMDPS, DMDPI, and DMDPN in Solution (solu),a Aggregate (aggr),b and Binding (bindg)c States λab,d nm DMDPS DMDPI DMDPN

λem,e nm

∆λ,f nm

solu

aggr

bindg

aggr

bindg

aggr

bindg

395 394 393

399 389 388

385 381 382

546 549 546

526 528 528

147 160 158

141 147 146

a In acetonitrile (10 µM). b In 90% toluene/acetonitrile mixture (10 µM). c In BSA solution of DMDPS, DMDPI, and DMDPN (10 µM) in PBS (pH 7.0) containing 0.05% w/v SDS. d Absorption maximum. e Emission maximum. f Stokes’ shift.

fluorenonearylamine derivatives,13 DPDBF derivatives,14 conjugated polymers15 and others.16 The discovery of AIE-active materials resolves primarily the problem of fluorescence quenching resulting from the aggregation. They are mainly used for the fabrication of efficient optical and photonic devices. However, only a few examples of employing them as bioprobes for detecting biomolecules were known.2c,d,10e,17 We have recently developed a water-soluble Λ-shaped pyridinium salt DMDPS with AIE characteristics.18 The AIE effect of DMDPS is probably caused by the restrictions of enantiomerization and/or intramolecular vibrational motion of the chromophoric molecules in the aggregate state. We expect that binding the AIE dyes to protein molecules can suppress the two power-dissipation processes mentioned above, and turn their light emission on. In addition, its absorption is in the nearUV region (λmax ≈ 400 nm, Table 1), well separated from that of the nucleic bases. Also a large Stokes’ shift (∆λ ≈ 147 nm, Table 1), allows unambiguous detection without reabsorption effects and interference with the background FL of protein. Such attractive photophysical properties indicate that compound DMDPS can be employed for the fluorescent turn-on detection of protein. Also, the other two new Λ-shaped pyridinium salts DMDPI and DMDPN based on Tro¨ger’s base19 were prepared and their

10.1021/jp8111167 CCC: $40.75  2009 American Chemical Society Published on Web 03/26/2009

6810 J. Phys. Chem. C, Vol. 113, No. 16, 2009 CHART 1: Molecular Structure of Compounds DMDPS, DMDPI, and DMDPN

FL properties upon binding with protein were investigated in our study. The large Stokes’ shift, interesting AIE behavior, and fluorescent “light-up” feature in the presence of protein indicate the three water-soluble compounds are excellent protein probes with high sensitivity. Experimental Section Materials, Methods, and Instrumentation. All chemicals were purchased from Aldrich or Acros and used as received without further purification. Solvents were purified and dried according to standard procedures. Bovine serum albumin (BSA) was purchased as lyophilized crystalline powders from Sigma and stored in a refrigerator before use. 2,8-Diformyl-6H,12H5,11-methanodibenzo[b,f][1,5]diazocine (1)20 and 4-methyl-Nmethylpyridinium iodide (2)21 were prepared according to the literature procedures. Phosphate buffer solution (PBS) with pH 7.0 was obtained by dissolving Na2HPO4 · 12H2O (12.4 mmol, 4.44 g) and NaH2PO4 · 2H2O (7.6 mmol, 1.19 g) in doubly distilled water (1 L). PBS containing 0.05% w/v sodium dodecyl sulfate (SDS) (pH 7.0) was prepared by dissolving SDS (0.50 g) in PBS (pH 7.0) (1 L). For measurement of photophysical properties, the stock solutions of DMDPI and DMDPN with a concentration of 1.0 mM were prepared first by dissolving the appropriate amount of the dye in acetonitrile, respectively. Then, 50 µL of each stock solution were diluted with acetonitrile or a 90% toluene/ acetonitrile mixture to obtain 5.0 mL solutions under vigorous stirring at room temperature. For FL titration experiments, the stock solutions of DMDPS, DMDPI, and DMDPN with a concentration of 1.0 mM were prepared by dissolving the dye in PBS (pH 7.0). Similarly, a stock solution of BSA with a concentration of 1 mg/mL was obtained by dissolving BSA in PBS (pH 7.0). For the FL detection, the stock solution of BSA was added to 4 mL of PBS containing 0.05% w/v SDS (pH 7.0) and mixed adequately. Thereafter, 50 µL of the dye stock solution was added, followed by adding the PBS containing 0.05% w/v SDS (pH 7.0) to obtain a 5.0 mL solution. The mixtures were shaken for 30 min prior to taking their absorption and FL spectra. The 1H and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer with tetramethylsilane (TMS; δ 0 ppm) as internal standard. Coupling constants J are given in hertz. Elemental analysis was performed with a PE 2400

Yuan et al. elemental analyzer. The UV-visible absorption spectra were measured on a TU-1800 spectrophotometer. The photoluminescence (PL) spectra were collected on a Hitachi F-4500 fluorescence spectrophotometer with a 150 W Xe lamp. All spectrophotometric measurements were performed in quartz sample cells having 1 cm path length at room temperature. Synthesis. 2,8-(6H,12H-5,11-Methanodibenzo[b,f]diazocineylene)-di(p-ethenyl-N-methylpyridinium) Diiodide (DMDPI).Amixtureof2,8-diformyl-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine 1 (0.278 g, 1 mmol), 4-methyl-N-methylpyridinium iodide 2 (0.564 g, 2.4 mmol), and piperidine (3 drops) in absolute methanol (10 mL) was refluxed for 10 h and cooled to room temperature. The yellow precipitate was filtered, washed with a little methanol, and dried under vacuum. Product DMDPI (0.60 g) was isolated in 84% yield. 1H NMR (400 MHz, DMSO) δ (ppm) 4.20 (s, 6H), 4.25(d, 2H, J ) 16.9 Hz), 4.31 (s, 2H), 4.73 (d, 2H, J ) 16.7 Hz), 7.26 (d, 2H, J ) 8.4 Hz), 7.34 (d, 4H, J ) 16.5 Hz), 7.54 (dd, 2H, J ) 8.4 Hz), 7.87 (d, 2H, J ) 16.3 Hz), 8.11 (d, 4H, J ) 6.8 Hz), 8.78 (d, 4H, J ) 6.8 Hz). 13 C NMR (100.57 MHz, DMSO) δ (ppm) 46.80, 58.10, 65.98, 121.82, 123.16, 125.38, 127.02, 128.74, 130.43, 140.31, 144.95, 150.46, 152.53. Anal. Calcd for C31H30I2N4 · 4H2O: C, 47.40; H, 5.00; N, 7.13. Found: C, 47.74; H, 4.99; N, 7.15. 2,8-(6H,12H-5,11-Methanodibenzo[b,f]diazocineylene)-di(pethenyl-N-methylpyridinium) Dinitrate (DMDPN). To a stirring solution of DMDPI (0.356 g, 0.5 mmol) in acetonitrile (80 mL) was added AgNO3 (0.169 g, 1 mmol) dissolved in acetonitrile (10 mL), then the mixture was refluxed for 1 h. The silver iodide precipitate was filtered off from the solution and the filtrate was evaporated to afford product DMDPN (0.15 g) in 97% yield. 1 H NMR (400 MHz, DMSO) δ (ppm) 4.22 (s, 6H), 4.26 (d, 2H, J ) 16.9 Hz), 4.32 (s, 2H), 4.74 (d, 2H, J ) 16.7 Hz), 7.27(d, 2H, J ) 8.4 Hz), 7.36 (d, 4H, J ) 16.5 Hz), 7.55 (dd, 2H, J ) 8.4 Hz), 7.87 (d, 2H, J ) 16.3 Hz), 8.12 (d, 4H, J ) 6.8 Hz), 8.79 (d, 4H, J ) 6.8 Hz). 13C NMR (100.57 MHz, DMSO) δ (ppm) 47.24, 58.59, 66.48, 122.32, 123.65, 125.85, 127.52, 129.21, 130.93, 140.80, 145.45, 150.93, 153.03. Anal. Calcd for C31H30N6O6 · 5H2O: C, 55.35; H, 5.99; N, 12.49. Found: C, 55.48; H, 5.97; N, 12.62. Results and Discussion Synthesis and Characterization. The chemical structures of the Λ-shaped pyridinium salts investigated and discussed in this study are shown in Chart 1 As outlined in Scheme 1, similar to the synthesis of DMDPS,18 compound DMDPI was prepared in 84% yield by direct condensation of 2,8-diformyl-6H,12H5,11-methanodibenzo[b,f][1,5]diazocine 1 with 4-methyl-Nmethylpyridinium iodide 2 in the presence of piperidine used as a catalyzer. And then exchanging anion with AgNO3 gives DMDPN. The structures of the product molecules were confirmed by 1H NMR, 13C NMR, and elemental analysis methods. They are all readily soluble in polar solvents such as

SCHEME 1: Synthetic Routes to Compounds DMDPI and DMDPN

Protein Based on (Λ)-Shaped Pyridinium Salts

J. Phys. Chem. C, Vol. 113, No. 16, 2009 6811

Figure 1. FL photos of the solutions of (a) DMDPI and (b) DMDPN (10 µM) in acetonitrile and 90% toluene/acetonitrile mixtures under illumination with a 365.0 nm UV lamp.

Figure 2. PL spectra of DMDPI (a) and DMDPN (b) in pure acetonitrile and 90% toluene/acetonitrile mixture. The final concentration was kept unchanged at 10 µM, λex ) 390 nm.

Figure 3. UV-visible absorption spectra of DMDPI (a) and DMDPN (b) in pure acetonitrile and 90% toluene/acetonitrile mixture. The final concentration was kept unchanged at 10 µM.

methanol, ethanol, DMF, water, etc., but completely insoluble in nonpolar solvents like ethyl ether, toluene, benzene, and so on. Photophysical and Aggregation-Induced Emission Properties. Similar to our previously reported pyridinium salt DMDPS,18 the solutions of both DMDPI and DMDPN are nonemissive, while the aggregate emits bright yellow light when illuminated with a 365 nm UV lamp, as shown in Figure 1. The AIE feature was also quantitatively characterized by the

PL spectra and UV-visible absorption spectra of DMDPI and DMDPN in acetonitrile and in acetonitrile/toluene mixture (the final concentrations being kept constant at 10 uM), respectively. Figure 2 is the PL spectra of DMDPI and DMDPN in pure acetonitrile solutions and in 90% toluene/acetonitrile mixtures. A dramatic change of the FL intensity from the nonfluorescent acetonitrile solutions to the strongly fluorescent suspensions in 90% toluene/acetonitrile mixtures can be observed. When dilute acetonitrile solutions of DMDPI and DMDPN were excited at

6812 J. Phys. Chem. C, Vol. 113, No. 16, 2009

Figure 4. Effect of BSA (400 µg/mL) and/or SDS (0.05% w/v) on the FL spectrum of a buffer solution of DMDPS (10 µM). λex ) 380 nm.

390 nm, almost no PL signal was recorded. However, when a large amount of toluene was added to the acetonitrile solutions, a dramatic enhancement of luminescence emission was observed in the toluene/acetonitrile mixture, then the PL of the two compounds were switched on. Since toluene is a nonsolvent of the pyridinium salts, the molecules thus should have aggregated into solid particles in the toluene/acetonitrile mixture. In fact,

Yuan et al. the systems of 90% toluene/acetonitrile “solutions” had become a little turbid. The emissions of the two pyridinium salts were apparently induced by aggregation, and the compounds were AIE active. As shown in the corresponding absorption spectra of DMDPI and DMDPN in pure acetonitrile solutions and 90% toluene/ acetonitrile mixtures (Figure 3), the main absorption bands located at 390 nm and the absorbance intensities of the two compounds in 90% toluene/acetonitrile mixtures were obviously weaker than those in the pure acetonitrile. This was coincident with our previous study on the AIE of DMDPS and was explained as the decrease of the “effective concentration” of the solutions.18 The similar photophysical properties of the three pyridimium salts DMDPS, DMDPI, and DMDPN (shown in Table 1) are attributed to the same cation framework, where the luminescence comes from. Fluorescent Turn-On Detection and Quantification of Protein with Λ-Shaped Pyridinium Salts. The interaction of the three water-soluble salts DMDPS, DMDPI, and DMDPN with proteins, using BSA as a model protein, was studied by spectrofluorimetric titrations. As shown in Figure 4, the solution of DMDPS in the PBS (pH 7.0) is almost nonfluorescent. When amounts of BSA were added, no obvious PL enhancement was observed. However, further investigation indicated that, as in the case of some fluorescent protein probes,2a,e,3 adding the anionic surfactant SDS into the BSA solution of DMDPS dramatically boosts its FL. Obviously, SDS shows a cooperative

Figure 5. (a) FL spectra (λex ) 380 nm) of compound DMDPS (10 µM in PBS containing 0.05% w/v SDS, pH 7.0) in the presence of different amounts of BSA (from 0 to 900 µg/mL); (b) FL photos of solution of DMDPS (10 µM in PBS containing 0.05% w/v SDS, pH 7.0) in the absence and presence of BSA (the concentration of BSA is 400 µg/mL) under illumination with a 365.0 nm UV lamp. (c) Plots of PL peak intensities of DMDPS in PBS vs concentration of BSA. (d) Linear regions of the binding isotherm of DMDPS to BSA.

Protein Based on (Λ)-Shaped Pyridinium Salts

J. Phys. Chem. C, Vol. 113, No. 16, 2009 6813

Figure 6. (a) Plots of PL peak intensities of DMDPI and DMDPN in PBS vs concentration of BSA. (b) Linear regions of the binding isotherm of DMDPI and DMDPN to BSA.

effect on the protein-induced FL enhancement. To quantify the positive effect, we measured the PL spectra of the dye DMDPS (10 µM) in PBS (pH 7.0) with different contents of SDS in the presence of excessive BSA (400 µg/mL) to determine an optimal concentration of SDS in a preliminary experiment. The concentration of SDS, which induced the most effective FL, was 0.05% w/v (Figure S1, Supporting Information). Then all further measurements were performed in the presence of 0.05% w/v SDS. It should be noted that, in the absence of BSA, SDS induces the aggregation of the dyes, which also causes changes in the FL spectra. Therefore, during the preparation of the testing samples, SDS and BSA were mixed sufficiently before the dye was added. Figure 5a shows the representative PL spectra of the dye DMDPS (10 µM) with different concentrations of BSA in PBS (pH 7.0) containing 0.05% w/v SDS. Compound DMDPS itself has a very weak emission, whereas the emission of the DMDPS · BSA complex showed a large Stokes’ shift (141 nm, Table 1) and dramatic increase in the fluorescence intensity centered at around 526 nm, the photograph of which is shown in Figure 5b. The plots of PL peak intensities of compound DMDPS in PBS (pH 7.0) containing 0.05% w/v SDS as a function of BSA concentration are depicted in Figure 5c. Remarkably, the binding reaches saturation at protein concentrations of 700-900 µg/mL, at this point the FL of DMDPS increases by a factor of approximately 20. Notably, the plot of the FL peak intensity versus BSA concentration is linear (R2 ) 0.997, Figure 5d) in a rather broad range (0-70 µg/mL), thus allowing direct fluorimetric detection of the protein concentration. For the other two pyridinium salts DMDPI and DMDPN, similar behaviors upon binding to the BSA were observed. Their PL spectra and the curves of PL peak intensities as a function of BSA concentration are described in Figures S2 and S3 (Supporting Information) and Figure 6, respectively. It is obvious that DMDPS, DMDPI, and DMDPN can be used as “lightup” or “turn-on” FL bioprobes for protein detection and quantification. Mechanism of the Interaction of Λ-Shaped Pyridinium Salts with Protein. The addition of BSA to solutions of Λ-shaped pyridinium salts DMDPS, DMDPI, or DMDPN cannot boost their FL intensively, the reason being that the special Λ-shaped molecular configuration inhibits the pyridinium salts molecules from entering into the probe-binding sites of protein to form aggregate. However, they exhibit a significant FL enhancement upon interaction with BSA in the presence of

moderate anionic surfactant (SDS), which is known to denature the proteins and form structures, in which the surfactant micelles are distributed along the unfolded protein molecules (necklaceand-beads model),22 whereas excess SDS leads to a decrease of the FL, since the surfactant begins to replace the proteinbound probe molecules. Therefore, it may be assumed that the role of SDS is denaturing the proteins to provide access to the probe-binding sites which otherwise cannot be occupied by the dye molecules. Binding of the Λ-shaped pyridinium salts to the protein, through noncovalent interaction, such as hydrophobic and electrostatic interaction, reduces the conformational freedom of the probe molecules, hence inducing them to emit as aggregate does. It is thus the AIE nature that makes the dyes useful as protein probes. Conclusion In summary, we have successfully developed an efficient protein fluorimetric assay system based on Λ-shaped pyridinium salts by taking advantage of the AIE phenomenon and their excellent photophysical properties, such as the absorption in the near-UV region, and the large Stokes’ shift. Their calibration curves have a very wide linear range (0-70 µg/mL), thus quantitative assay of protein in solution within this concentration range by fluorimetric method can be achieved. In addition, the detection and quantification of BSA by the three nonemissive ionic dyes are carried out in the presence of surfactant SDS, which can be used in the protein gel electrophoresis, and they may find application for protein detection in gel electrophoresis due to the low protein-to-protein variability. Moreover, the AIE nature allows the use of large fluorophore/protein ratios, enabling the detection of trace amounts of low-abundance proteins. Further studies of their applications in biological science and technology are currently in progress. Acknowledgment. . We gratefully acknowledge the financial support from the state National Natural Science Foundation of China (Grant Nos. 50721002, 50590433) and 973 program of China (Grant No. 2004CB619002) Supporting Information Available: FL enhancement plot of binding DMDPS (10 µM) to BSA (400 µg/mL) in PBS with different contents of SDS, PL spectra of DMDPI and DMDPN, and the curves of PL peak intensities as a function of BSA

6814 J. Phys. Chem. C, Vol. 113, No. 16, 2009 concentration. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Silva, A. P.; Nimal Gunaratne, H. Q.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515. (b) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Orden, A. V.; Werner, J. H.; Keller, R. A. Chem. ReV. 1999, 99, 2929. (c) Patton, W. F. Biotechniques 2000, 28, 944. (d) Wiskur, S. L.; Ait-Haddou, H.; Anslyn, E. V.; Lavigne, J. J. Acc. Chem. Res. 2001, 34, 963. (e) Pu, L. Chem. ReV. 2004, 104, 1687. (f) Wolfbeis, O., Ed. Optical Sensors; Springer-Verlag: Heidelberg, Germany, 2004. (g) Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley-VCH: Weinheim, Germany, 2002. (2) (a) Granzhan, A.; Ihmel, H. Org. Lett. 2005, 7, 5119. (b) Suzuki, Y.; Yokoyama, K. J. Am. Chem. Soc. 2005, 127, 17799. (c) Tong, H.; Hong, Y.; Dong, Y.; Ha¨ussler, M.; Lam, J. W. Y.; Li, Z.; Guo, Z.; Guo, Z.; Tang, B. Chem. Commun. 2006, 3705. (d) Tong, H.; Hong, Y.; Dong, Y.; Ha¨ussler, M.; Li, Z.; Lam, J. W. Y.; Dong, Y.; Sung, H. H.-Y.; Williams, I. D.; Tang, B. J. Phys. Chem. B 2007, 111, 11817. (e) Granzhan, A.; Ihmel, H.; Viola, G. J. Am. Chem. Soc. 2007, 129, 1254. (f) Suzuki, Y.; Yokoyama, K. Proteomics 2008, 8, 2785. (3) Jones, L.; Haugland, R.; Singer, V. BioTechniques 2003, 34, 850. (4) Bhown, A.; Cornelius, T.; Volanakis, J.; Bennett, J. Anal. Biochem. 1983, 131, 337. (5) Berggren, K.; Steinberg, T. H.; Lauber, W. M.; Carroll, J. A.; Lopez, M. F.; Chernokalskaya, E.; Zieske, L.; Diwu, Z.; Haugland, R. P.; Patton, W. F. Anal. Biochem. 1999, 276, 129. (6) (a) Hartman, B.; Udenfriend, S. Anal. Biochem. 1969, 30, 391. (b) Horowith, P.; Bowman, S. Anal. Biochem. 1987, 165, 430. (7) Daban, J.; Bartolome´, S.; Samso´, M. Anal. Biochem. 1991, 199, 169. (8) (a) Lakowicz, J.; Malicka, J.; D’Auria, S.; Gryczynski, I. Anal. Biochem. 2003, 320, 13. (b) Chan, C.; Ha¨ussler, M.; Tang, B.; Dong, Y.; Sin, K.; Mak, W.; Trau, D.; Seydack, M.; Renneberg, R. J. Immunol. Methods 2004, 295, 111. (c) Birks, J. Photophysics of Aromatic Molecules; Wiley: London, UK, 1970. (9) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z. Chem. Commun. 2001, 1740. (10) (a) Chen, J.; Xie, Z.; Lam, J. W. Y.; Law, C. C. W.; Tang, B. Z. Macromolecules 2003, 36, 1108. (b) Chen, J.; Peng, H.; Law, C. C. W.; Dong, Y.; Lam, J. W. Y.; Williams, I. D.; Tang, B. Z. Macromolecules 2003, 36, 4319. (c) Mi, B.; Dong, Y.; Li, Z.; Lam, J. W. Y.; Ha¨ussler, M.; Sung, H. H. Y.; Kwok, H. S.; Dong, Y.; Williams, I. D.; Liu, Y.; Luo, Y.; Shuai, Z.; Zhu, D.; Tang, B. Z. Chem. Commun. 2005, 3583. (d) Yu, G.; Yin, S.; Liu, Y.; Chen, J.; Xu, X.; Sun, X.; Ma, D.; Zhan, X.; Peng, Q.; Shuai, Z.; Tang, B. Z.; Zhu, D.; Fang, W.; Luo, Y. J. Am. Chem. Soc. 2005, 127, 6335. (e) Wang, M.; Zhang, D.; Zhang, G.; Zhu, D. Chem. Commun. 2008, 4469. (11) (a) An, B.-K.; Kwon, S.-K.; Jung, S.-D.; Park, S. Y. J. Am. Chem. Soc. 2002, 124, 14410. (b) Lim, S.-J.; An, B.-K.; Jung, S.-D.; Chung, M.A.; Park, S. Y. Angew. Chem., Int. Ed. 2004, 43, 6346. (c) Lim, S.-J.; An,

Yuan et al. B.-K.; Park, S. Y. Macromolecules 2005, 38, 6236. (d) Tong, X.; Zhao, Y.; An, B.-K.; Park, S. Y. AdV. Funct. Mater. 2006, 16, 1799. (12) (a) Xie, Z.; Yang, B.; Cheng, G.; Liu, L.; He, F.; Shen, F.; Ma, Y.; Liu, S. Chem. Mater. 2005, 17, 1287. (b) Xie, Z.; Yang, B.; Xie, W.; Liu, L.; Shen, F.; Wang, H.; Yang, X.; Wang, Z.; Li, Y.; Hanif, M.; Yang, G.; Ye, L.; Ma, Y. J. Phys. Chem. B 2006, 110, 20993. (c) Li, Y.; Li, F.; Zhang, H.; Xie, Z.; Xie, W.; Xu, H.; Li, B.; Shen, F.; Ye, L.; Hanif, M.; Ma, D.; Ma, Y. Chem. Commun. 2007, 231. (13) (a) Liu, Y.; Tao, X.; Wang, F.; Shi, J.; Yu, W.; Ren, Y.; Zou, D.; Jiang, M. J. Phys. Chem. C 2007, 111, 6544. (b) Liu, Y.; Tao, X.; Wang, F.; Dang, X.; Zou, D.; Ren, Y.; Jiang, M. J. Phys. Chem. C 2008, 112, 3975. (14) (a) Tong, H.; Dong, Y.; Ha¨ussler, M.; Lam, J. W. Y.; Sung, H. Y.; Williams, I. D.; Sun, J.; Tang, B. Z. Chem. Commun. 2006, 1133. (b) Dong, Y.; Lam, J. W. Y.; Qin, A.; Li, Z.; Sun, J.; Sung, H. H. Y.; Williams, I. D.; Tang, B. Z. Chem. Commun. 2007, 40. (c) Tong, H.; Dong, Y.; Hong, Y.; Ha¨ussler, M.; Lam, J. W. Y.; Sung, H. H.-Y.; Yu, X.; Sun, J.; Williams, I. D.; Kwok, H. S.; Tang, B. Z. J. Phys. Chem. C 2007, 111, 2287. (15) (a) Deans, R.; Kim, J.; Machacek, M. R.; Swager, T. M. J. Am. Chem. Soc. 2000, 122, 8565. (b) Belton, C.; O’Brien, D. F.; Blau, W. J.; Cadby, A. J.; Lane, P. A.; Bradley, D. D. C.; Byrne, H. J.; Stockmann, R.; Ho¨rhold, H.-H. Appl. Phys. Lett. 2001, 78, 1059. (c) Holzer, W.; Penzkofer, A.; Stockmann, R.; Meysel, H.; Liebegott, H.; Ho¨rhold, H.-H. Polymer 2001, 42, 3183. (16) (a) Ryu, S. Y.; Kim, S.; Seo, J.; Kim, Y.-W.; Kwon, O.-H.; Jang, D.-J.; Park, S. Y. Chem. Commun. 2004, 70. (b) Bhongale, C. J.; Chang, C.-W.; Lee, C.-S.; Diau, E. W.-G.; Hsu, C.-S. J. Phys. Chem. B 2005, 109, 13472. (c) Wang, Z.; Shao, H.; Ye, J.; Tang, L.; Lu, P. J. Phys. Chem. B 2005, 109, 19627. (d) Tong, H.; Hong, Y.; Dong, Y.; Ha¨ussler, M.; Lam, J. W. Y.; Li, Z.; Guo, Z.; Guo, Z.; Tang, B. Z. Chem. Commun. 2006, 3705. (e) Kim, S.; Zheng, Q.; He, G. S.; Bharali, D. J.; Pudavar, H. E.; Baev, A.; Prasad, P. N. AdV. Funct. Mater. 2006, 16, 2317. (f) Zeng, Q.; Li, Z.; Dong, Y.; Di, C.; Qin, A.; Dong, Y.; Ji, L.; Zhu, Z.; Jim, C. K. W.; Yu, G.; Li, Q.; Li, Z.; Liu, Y.; Qin, J.; Tang, B. Z. Chem. Commun. 2007, 70. (g) Tong, H.; Dong, Y.; Dong, Y.; Ren, Y.; Ha¨ussler, M.; Lam, J. W. Y.; Wong, K.; Tang, B. Z J. Phys. Chem. B 2007, 111, 2000. (h) Qian, Y.; Li, S.; Zhang, G.; Qang, Q.; Wang, S.; Xu, H.; Li, C.; Li, Y.; Yang, G. J. Phys. Chem. B 2007, 111, 5861. (i) Qin, A.; Jim, C. K. W.; Tang, Y.; Lam, J. W. Y.; Liu, J.; Mahtab, F.; Gao, P.; Tang, B. Z J. Phys. Chem. B 2008, 112, 9281. (j) Xu, J.; Liu, X.; Lv, J.; Zhu, M.; Huang, C.; Zhou, W.; Yin, X.; Liu, H.; Li, Y.; Ye, J. Langmuir 2008, 4231. (17) Wang, M.; Zhang, D.; Zhang, G.; Tang, Y.; Wang, S.; Zhu, D. Anal. Chem. 2008, 80, 6443. (18) Yuan, C.; Tao, X.; Ren, Y.; Li, Y.; Yang, J.; Yu, W.; Wang, L.; Jiang, M. J. Phys. Chem. C 2007, 111, 12811. (19) Tro¨ger, J. J. Prakt. Chem. 1887, 36, 225. (20) Jensen, J.; Tejler, J.; Wa¨rnmark, K. J. Org. Chem. 2002, 67, 6008. (21) Zhao, C. F.; He, G. S.; Bhawalkar, J. D.; Park, C. K.; Prasad, P. N. Chem. Mater. 1995, 7, 1979. (22) (a) Turro, N. J.; Lei, X.-G. Langmuir 1995, 11, 2525. (b) Santos, S. F.; Zanette, D.; Fischer, H.; Itri, R. J. Colloid Interface Sci. 2003, 262, 400.

JP8111167