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Conjugated Polyelectrolyte-Grafted Silica Microspheres Katsu Ogawa,† Sireesha Chemburu,‡ Gabriel P. Lopez,‡ David G. Whitten,‡ and Kirk S. Schanze*,† Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611-7200, and Department of Chemical and Nuclear Engineering, UniVersity of New Mexico, Albuquerque, New Mexico 87131-1341 ReceiVed October 12, 2006. In Final Form: December 8, 2006 A direct method for preparation of conjugated polymer-grafted silica particles is reported. Silica particles (0.3 and 5 µm diameter) are treated with a 3-(trimethoxysilyl)propylamine derivative that is functionalized with an aryl iodide unit. A solution step-growth polymerization reaction is performed in solution that contains a dispersion of the aryl iodide-functionalized particles. The reaction is a Pd(0)-catalyzed (Sonogashira) A-B-type polymerization of an oligo(ethylene glycol)-fuctionalized diiodobenzene and a bis(propyloxy)sulfonate-substituted diethynylbenzene. The overall process affords silica particles that feature a surface graft layer of an anionic poly(phenylene ethynylene)-type conjugated polyelectrolyte. The particle surface modification process was monitored by infrared (FTIR) spectroscopy, and the polymer-grafted silica particles were characterized by thermogravimetric analysis, scanning and transmission electron microscopy, confocal fluorescence microscopy, and absorption and fluorescence spectroscopy. The conjugated polyelectrolyte-grafted silica particles are highly fluorescent, and a Stern-Volmer quenching study of the particles’ fluorescence with electron-transfer- and energy-transfer-type quenchers shows that the quenching response depends on the type of quenching mechanism.
Introduction Water-soluble conjugated polyelectrolytes (CPEs) based on poly(phenylene vinylene) (PPV) or poly(phenylene ethynylene) (PPE) backbones exhibit useful properties, such as strong photoluminescence and rapid transport of the singlet exciton along the π-conjugated backbone. Significant attention has been focused on amplified quenching of the CPE fluorescence by ionic quenchers.1-10 Amplified quenching effects have been demonstrated with quenchers that operate by either electrontransfer or energy-transfer mechanisms. In some CPE-quencher systems, Stern-Volmer constants for amplified quenching can be as large as 108 M-1.5,6 The highly efficient quenching in CPE-quencher systems has been explained by ion pairing between CPE chains and oppositely charged quenchers and by the rapid intrachain transport of the exciton.1-13 The large quenching response displayed by CPEs has led to their application in highly sensitive fluorescence-based sensors for biological * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (352) 392-9133. Fax: (352) 392-2395. † University of Florida. ‡ University of New Mexico. (1) Zhou, Q.; Swager, T. M. J. Am. Chem. Soc. 1995, 117, 7017-7018. (2) Zhou, Q.; Swager, T. M. J. Am. Chem. Soc. 1995, 117, 12593-12602. (3) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287-12292. (4) Kumaraswamy, S.; Bergstedt, T.; Shi, X. B.; Rininsland, F.; Kushon, S.; Xia, W. S.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7511-7515. (5) Wang, D. L.; Wang, J.; Moses, D.; Bazan, G. C.; Heeger, A. J. Langmuir 2001, 17, 1262-1266. (6) Harrison, B. S.; Ramey, M. B.; Reynolds, J. R.; Schanze, K. S. J. Am. Chem. Soc. 2000, 122, 8561-8562. (7) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. ReV. 2000, 100, 2537-2574. (8) Tan, C. Y.; Pinto, M. R.; Schanze, K. S. Chem. Commun. 2002, 446-447. (9) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505-7510. (10) Tan, C. Y.; Alas, E.; Muller, J. G.; Pinto, M. R.; Kleiman, V. D.; Schanze, K. S. J. Am. Chem. Soc. 2004, 126, 13685-13694. (11) Wang, J.; Wang, D. L.; Miller, E. K.; Moses, D.; Bazan, G. C.; Heeger, A. J. Macromolecules 2000, 33, 5153-5158. (12) Wang, J.; Wang, D.; Miller, E. K.; Moses, D.; Heeger, A. J. Synth. Met. 2001, 119, 591-592. (13) Swager, T. M. Acc. Chem. Res. 1998, 31, 201-207.
targets.3-5,14-18 Some assays also have utilized layer-by-layer (LbL) coated latex or silica19 nano- and microcolloids in combination with quencher-tether-ligand (QTL)4 or lipid bilayer assemblies.20 Examples of polymer-grafted colloids and surfaces have been reported which are fabricated by using surface-initiated living polymerization reactions, such as atom-transfer radical polymerization (ATRP)21-24 and nitroxide-mediated radical polymerization (NMRP).24,25 Although conjugated polymer LbL coated silica particles and conjugated polymer silica composites are known,19,26,27 a general method for preparation of silica particles that are surface-grafted with a conjugated polymer is not available. However, in related work Emrick and co-workers recently reported the preparation of CdSe nanoparticles that are surface-grafted with a PPV-based conjugated polymer. These materials were produced via palladium-mediated polymerization from the CdSe nanoparticle’s surface that is functionalized with a phosphine oxide derivative featuring aryl halide functionality.28 (14) Wang, D. L.; Gong, X.; Heeger, P. S.; Rininsland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 49-53. (15) DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir 2002, 18, 7785-7787. (16) Kushon, S. A.; Ley, K. D.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245-7249. (17) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896-900. (18) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954-10957. (19) Wosnick, J. H.; Liao, J. H.; Swager, T. M. Macromolecules 2005, 38, 9287-9290. (20) Zeineldin, R.; Piyasena, M. E.; Bergstedt, T. S.; Sklar, L. A.; Whitten, D.; Lopez, G. P. Cytometry, Part A 2006, 69A, 335-341. (21) Zhao, H.; Kang, X.; Liu, L. Macromolecules 2005, 38, 10619-10622. (22) Kizhakkedathu, J. N.; Norris-Jones, R.; Brooks, D. E. Macromolecules 2004, 37, 734-743. (23) Li, D. J.; Jones, G. L.; Dunlap, J. R.; Hua, F. J.; Zhao, B. Langmuir 2006, 22, 3344-3351. (24) Li, D. J.; Sheng, X.; Zhao, B. J. Am. Chem. Soc. 2005, 127, 6248-6256. (25) Zhao, B.; Li, D. J.; Hua, F. J.; Green, D. R. Macromolecules 2005, 38, 9509-9517. (26) Ravindranath, R.; Ajikumar, P. K.; Hanafiah, N. B. M.; Knoll, W.; Valiyaveettil, S. Chem. Mater. 2006, 18, 1213-1218. (27) Kim, K.; Webster, S.; Levi, N.; Carroll, D. L.; Pinto, M. R.; Schanze, K. S. Langmuir 2005, 21, 5207-5211.
10.1021/la0630108 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/08/2007
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Figure 1. Synthesis of compound 1 and PPE-SO3-OR11.
In another recent study, Carter and co-workers reported the preparation of a polyfluorene-based conjugated polymer-grafted surface based on a Ni(0)-mediated step-growth polymerization reaction.29 In their approach, a silica substrate was modified with a cross-linked polymethacrylate network containing an aryl halide group. The aryl halide units serve as grafting points for a polyfluorene produced by a Ni(0)-mediated step-growth polymerization reaction. In this paper we report the preparation and characterization of silica colloids that contain a surface graft layer of a poly(phenylene ethynylene)-based CPE. These colloids are of interest as signaling elements in biosensor schemes. Surface modification of 0.3 and 5 µm diameter silica particles was accomplished by first reacting the SiO2 surface with a trialkoxysilane bearing an aryl iodide group. The aryl iodide units are utilized as the grafting points for polymerization under Sonogashira conditions. Such direct modification of the surface gives some control over the surface density of the functional groups, thus allowing the number of polymer brushes on the surface to be adjusted accordingly. Herein, we describe the preparation and characterization of nanoand microscale silica particles that are surface-grafted with poly(phenylene ethynylene)-type CPEs utilizing Pd(0) catalysis. This widely applicable method gives access to silica-based colloids with useful properties of CPEs for possible applications in fluorescence sensing and antimicrobial action.30 Experimental Section General Synthetic Procedures and Source of Starting Materials. All chemicals used for synthesis were of reagent grade and purchased from Sigma-Aldrich Chemical Co. Uniformly sized silica microspheres were purchased from Bangs Lab (http://www. bangslabs.com) as a dry powder. Unless otherwise noted, chemicals and reagents were used without further purification. Reactions were carried out under a nitrogen atmosphere using freshly distilled solvents. 2,5-Diiodo-1,4-bis(3-sulfonatopropoxy)benzene (2) was synthesized according to the literature procedure.8 The procedure for preparation of 1,4-diethynyl-2,5-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benezene (3) is included in the Supporting Information. Characterization Methods. Unless otherwise noted, 1H and 13C NMR spectra were recorded on either a Varian Gemini 300 or a VXR 300 spectrometer, and chemical shifts are reported in parts per million relative to the peak for TMS. Infrared spectra were obtained using KBr pellets on a Perkin-Elmer Spectrum One FTIR spec(28) Skaff, H.; Sill, K.; Emrick, T. J. Am. Chem. Soc. 2004, 126, 1132211325. (29) Beinhoff, M.; Appapillai, A. T.; Underwood, L. D.; Frommer, J. E.; Carter, K. R. Langmuir 2006, 22, 2411-2414. (30) Lu, L. D.; Rininsland, F. H.; Wittenburg, S. K.; Achyuthan, K. E.; McBranch, D. W.; Whitten, D. G. Langmuir 2005, 21, 10154-10159.
trometer. Measurements were automatically corrected for water and carbon dioxide. Thermogravimetric analysis (TGA) data were obtained with a Perkin-Elmer 7 series thermal analysis system. UVvis absorption spectra were obtained on a Perkin-Elmer Lambda 25 dual-beam absorption spectrometer using 1 cm quartz cells. Steadystate fluorescence emission spectra were recorded on a SPEX TRIAX 180 spectrograph coupled with a Spectrum One CCD detector. Steady-state fluorescence excitation spectra were recorded on a SPEX FluoroMax spectrophotometer. 4-Iodo-N-[3-(trimethoxysilyl)propyl]benzamide (1). A solution of (3-aminopropyl)trimethoxysilane (1.78 mL, 10 mmol) in 10 mL of dichloromethane was added dropwise to a suspension of 4-iodobenzoyl chloride (1.33 g, 5 mmol) in 20 mL of dichloromethane at 0 °C (Figure 1). The mixture was stirred for 1 h, and the solvent was evaporated in vacuo. The residue was purified by column chromatography on silica using 2% methanol in chloroform. The solvent was removed by rotary evaporation to give a viscous oil. 1H NMR (300 MHz, CDCl3): δ 0.70 (t, J ) 8.1 Hz, 2H), 1.73 (m, 2H), 3.42 (m, 2H), 6.64 (br, 1H), 7.49 (d, J ) 8.7 Hz, 2H), 7.76 (d, J ) 8.7 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ 6.81, 22.78, 42.54, 50.89, 98.41, 128.78, 134.44, 137.91, 167.03. HRMS: m/z calcd for C13H20INO4Si 409.0206, found 410.0295 (MH+). PPE-SO3-OR11. Compound 2 (650 mg, 1 mmol), compound 3 (451 mg, 1 mmol), CuI (5.7 mg, 0.03 mmol), and Pd(PPh3)4 (35 mg, 0.03 mmol) were dissolved in a mixture of 30 mL of DMF, 20 mL of water, and 10 mL of triethylamine (Figure 1). The mixture was refluxed overnight. The reaction mixture was concentrated by rotary evaporation and added dropwise to 250 mL of acetone. The precipitate was dissolved in a small amount of Millipore water and treated with 50 mg of NaCN. The polymer was precipitated again in 250 mL of acetone, redissolved in water, and filtered through quantitative filter paper, followed by a 25 µm glass filter. The solution was dialyzed against water using a 6000-8000 MWCO cellulose membrane. The solution was concentrated via rotary evaporation, and the polymer was precipitated with acetone. The precipitate was collected by centrifugation and washed with acetone. The product was a bright yellow powder, and it was dried under vacuum for 5 h. 1H NMR (300 MHz, D2O): δ 2.21 (br), 3.06 (br), 3.26 (br), 3.41 (br), 3.50 (br), 3.72 (br), 4.15 (br), 6.84 (br). Viscometry. Polymer solutions with various concentrations ranging between 1.0 and 0.6 g/dL were prepared in DMSO. Following a literature procedure,31 the intrinsic viscosity of the polymer was found to be 0.34 dL/g. From this value, the molecular weight of the polymer was estimated to be ∼40000. Surface Modification of Silica Particles (SiO2-ArI). Silica particles (200 mg) and compound 1 (63 mg, 0.15 mmol) were mixed in 10 mL of toluene and refluxed for 4 h. The surface-modified silica particles SiO2-ArI were collected via centrifugation and washed with acetone several times. The particles were dried under vacuum overnight. (31) Tan, C. Y.; Pinto, M. R.; Kose, M. E.; Ghiviriga, I.; Schanze, K. S. AdV. Mater. 2004, 16, 1208-1212.
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Figure 2. Synthesis of polymer- and surface-grafted silica particles. SiO2-PPE. Compound 2 (119 mg, 0.184 mmol), compound 3 (84.7 mg, 0.188 mmol), surface-modified silica particles (SiO2ArI) (50 mg), CuI (1.07 mg, 5.6 µmol), and Pd(PPh3)4 (6.5 mg, 5.6 µmol) were dissolved in a mixture of 30 mL of DMF, 20 mL of water, and 10 mL of triethylamine. The reaction mixture was refluxed overnight. The polymer-grafted silica particles were collected via centrifugation and washed with methanol several times. The particles were washed successively with multiple portions of THF, methanol, and water. Methanol and water washes were repeated until the solution exhibited no yellow color with blue fluorescence under a UV lamp. Finally, the particles were washed with acetone. The particles were dried under vacuum overnight. A similar procedure was followed for the preparation of SiO2-PPE-4 using compound 2 (32.5 mg, 0.05 mmol), compound 3 (22.5 mg, 0.05 mmol), surface-modified silica particles (SiO2-ArI) (100 mg), CuI (0.3 mg, 1.5 µmol), and Pd(PPh3)4 (1.7 mg, 1.5 µmol).
Results and Discussion Synthesis and Method of Surface Modification. Conventional methods of silica surface modification involve reaction of surface hydroxyl groups with commercial silane coupling reagents, such as 3-(trimethoxysilyl)propylamine (APTS).32-34 A similar approach was taken to introduce reactive aryl iodide (ArI) functionality to silica nano- and microparticles. Prior to modification of the silica surfaces, APTS was treated with 4-iodobenzoyl chloride to form the corresponding amide (1), which as outlined in Figure 2 was subsequently used to introduce the ArI functionality onto the SiO2 surface. To estimate the stoichiometry when using 1 to surface modify the silica, it was necessary to determine the number of equivalents of accessible silanol groups in a colloid sample. The currently accepted value for the number of accessible silanol groups on a glass surface is approximately 5 groups/nm2.35 Using this value along with the diameter of the silica particles (300 nm or 5 µm), the relationship between the surface area and volume of a sphere, and the density of silica (1.96 g/mL), the number of silanol groups per gram of sample can be calculated. Such calculations for the silica particles afford estimates of 13000 (300 nm) and 197000 (5 µm) g of particles/mol of silanol groups. For the surface modification reactions, the number of equivalents of trialkoxysilane to silica particles was calculated on the basis of these equivalent weights. In an effort to investigate the effect of the surface graft density of the polymer on the properties of the resulting particles, surface modification reactions were carried out using a different number of equivalents of 1 relative to silanol. In particular, for preparation (32) Yan, M. D.; Ren, J. Chem. Mater. 2004, 16, 1627-1632. (33) Gil, R.; Fiaud, J. C.; Poulin, J. C.; Schulz, E. Chem. Commun. 2003, 2234-2235. (34) Choualeb, A.; Braunstein, P.; Rose, J.; Bouaoud, S. E.; Welter, R. Organometallics 2003, 22, 4405-4417. (35) Jal, P. K.; Patel, S.; Mishra, B. Talanta 2004, 62, 1005-1028.
Table 1. Vibrational Mode Assignment of Major Peaks Observed by FTIR Spectroscopy of Various Types of Silica Particles SiO2-OH SiO2-ArI SiO2-PPE-2 PPE-SO3-OR11 3432
3431 2932
1632
1641
1110 952 804 475
1111 ∼950 (s)a 804 474
a
3442 2922 2211 1643 ∼1210 (s)a 1108 950 803 476
3445 2933 2202 1601 1213
assignment OH str CH str conj CtC str OH str SdO str Si-O-Si asym str Si-OH str Si-O-Si sym str Si-O-Si bend
Shoulder peak.
of SiO2-PPE-1, >50 equiv of 1 was reacted with the silica particles, for SiO2-PPE-2, 10 equiv of 1 was used, and for SiO2PPE-3, 5 equiv of 1 and 5 equiv of APTS were used. In the latter sample, APTS was used to introduce nonreactive surface sites to reduce the surface density of the ArI functionality. Solution polymerization of 2 and 3 under Sonogashira conditions was carried out to synthesize PPE-SO3-OR11. The resulting polymer was characterized by 1H NMR and viscometry (see the Supporting Information). To graft the polymer onto the surface of the SiO2 particles, solution polymerization under the same conditions was carried out in the presence of ArI-modified silica particles (SiO2-ArI) (Figure 2). While this approach does not allow control of the chain length or polydispersity of the graft polymer, as outlined below it is evident that a substantial quantity of the CPE is grafted to the surface of the colloid particles. The SiO2 surface modification process was monitored by FTIR spectroscopy. Peaks observed in the FTIR spectra of the various modified silicas are summarized in Table 1. As seen in Figure 3a, the unmodified silica particles (SiO2-OH) exhibit a strong peak at 1110 cm-1 which is assigned to the Si-O-Si asymmetric stretch. In addition, the characteristic bands at 952, 804, and 475 cm-1 are assigned to the silanol Si-OH stretch, Si-O-Si symmetric stretch, and Si-O-Si bend, respectively. A broad band centered at 3400 cm-1 is due to the OH stretch originating from both silanol and adsorbed water. Another broad peak around 1600 cm-1 is also due to the OH stretch of adsorbed water.36 The FTIR spectrum of SiO2-ArI (Figure 3b) gives clear evidence for the presence of the ArI groups. In particular, in addition to the bands seen in the spectrum of SiO2, a series of weak bands at ∼2900 cm-1 are observed in the IR spectrum of the ArI-modified silica particle most likely due to the CH stretches from the APTS group. Also, multiple weak bands in the 1400-1600 region indicate the presence of sp3 and/or sp2 C-C bonds. These (36) Smith, B. C. Infrared Spectral Interpretation: A Systematic Approach; Springer: Berlin, Germany, 1999.
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Figure 4. Thermogravimetric analysis of unfunctionalized silica particles (SiO2-OH), aryl iodide-modified silica particles (SiO2ArI), and polymer-grafted silica particles with various surface graft densities (SiO2-PPE-1, -2, and -3).
Figure 3. Comparison of infrared spectra of silica particles: (a) unfunctionalized silica particles (SiO2-OH), (b) aryl iodide-modified silica particles (SiO2-ArI), (c) PPE-grafted silica particles (SiO2PPE-2), (d) free polymer (PPE-SO3-OR11).
observations support the premise that the surface modification with ArI was accomplished by the reaction of SiO2 with 1. To confirm the presence of PPE-SO3-OR11 on the surface of the silica particles following their reaction with the monomers and catalyst, IR spectra of polymer-grafted silica particles (SiO2PPE-2) and free polymer (PPE-SO3-OR11) were compared (Figure 3c,d). Although the weaker bands in the spectrum of SiO2-PPE-2 are suppressed by the strong band at 1108 cm-1, identification of important peaks is still possible. As seen in Figure 3d, the polymer PPE-SO3-OR11 gives a characteristic band at 1213 cm-1 associated with the sulfonate group on the polymer side chain. Although this band is obscured by the strong band at 1108 cm-1, an enhanced shoulder around 1210 cm-1 can be observed in the spectrum of SiO2-PPE-2 (Figure 3c). Also, a weak band at 2202 cm-1 is indicative of carbon-carbon triple bonds in the backbone of the polymer (Figure 3d). The expansion of the spectrum of SiO2-PPE-2 in this region shows a band at 2211 cm-1 (Figure 3c), confirming the presence of the ethynyl bonds in the polymer backbone. A control experiment was performed to validate the covalent attachment of the polymer to the silica surface. Thus, an identical polymerization reaction in the presence of unfunctionalized silica particles (SiO2-OH) instead of aryl iodide-modified silica particles (SiO2-ArI) gave free polymer and unmodified silica particles as assessed by the complete absence of the yellow color and fluorescence characteristic of the polymer. This result shows that the presence of the aryl iodide functionality is necessary for attachment of the polymer on the silica surface. From this result we conclude that the polymer is covalently attached to the surface rather than being physisorbed. Also, comparison of the loading levels of polymer on samples SiO2-PPE-2 and -3 indicates that the amount of aryl iodide on the surface is important for covalent attachment of polymer onto the silica surface (see below). Silica particles of 5 µm diameter (SiO2-PPE-4) were modified according to the same procedure used for the 300 nm particles. These larger size particles were convenient for analyses by scanning electron microscopy (SEM) and confocal fluorescence microscopy.
Characterization of Surface-Grafted Particles. TGA was used to estimate the loading levels of the polymer on the silica particles, and Figure 4 compares the TGA traces from unfunctionalized silica particles and the PPE-grafted particles. Several qualitative trends are evident in the TGA data. First, the unmodified particles exhibit approximately a 4% weight loss over the 250-700 °C temperature range. This weight decrease arises from loss of strongly adsorbed water and dehydration of residual silanol units. Each of the surface-modified samples exhibits a greater weight loss with temperature; this increased loss is associated with the presence of organic material on the particle surfaces. Note that the thermally induced weight loss in the TGA increases along the series SiO2-ArI < SiO2-PPE-3 < SiO2-PPE-2 < SiO2-PPE-1, indicating that the amount of surfaced-grafted organic (polymer) material increases along the same series. As mentioned previously, the three samples of SiO2PPE were prepared in an effort to vary the loading level of PPE on the surface. In particular, the reaction conditions were adjusted to increase the surface loading of the reactive ArI units in the order SiO2-PPE-3 < SiO2-PPE-2 < SiO2-PPE-1. Thus, the TGA results show that there is a direct correlation between the initial loading of ArI reactive groups and the amount of polymer that is grafted onto the particle surfaces. Calculations were carried out to estimate the thickness of the grafted polymer layer from the TGA data. This calculation was carried out only for SiO2-PPE-1, which was prepared using the SiO2-ArI sample with the highest surface concentration of reactive ArI units.37 On the basis of the TGA analysis, the thickness of the polymer graft layer on the SiO2-PPE-1 particles was estimated to be 12 nm. Since the length of a polymer repeat unit (PRU) of a similar conjugated polymer (PPE-SO3) has been estimated by molecular modeling to be ∼1.2 nm,10 the thickness of the polymer graft layer corresponds to approximately 10 PRUs. Electron microscopy was used to observe the changes in the surface texture of the particles caused by surface grafting of the polymer. As shown in Figure 5a, transmission electron microscopy (TEM) images of unfunctionalized silica particles (SiO2-OH) show the smooth surface of the particles. The TEM images of the 300 nm particles with the polymer surface graft layer are shown in Figure 5b,c. These particles clearly exhibit a rough (37) The calculation is carried out as follows. First, the mass of SiO2 in the sample is computed on the basis of the TGA mass loss compared to the mass of unmodified silica. Then the number of particles and the total surface area in the sample are estimated on the basis of the calculated total silica mass, given the density of SiO2 and assuming that the particles are all 300 nm in diameter. The volume of surface-grafted polymer is estimated by assuming a polymer density of 1 g cm-3, and finally the layer thickness is obtained by dividing the polymer volume by the total particle surface area.
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Figure 5. Electron microscope images of silica particles: (a) 300 nm unfunctionalized particles (TEM), (b) SiO2-PPE-2 (TEM), (c) SiO2PPE-2 (TEM), (d) 5 µm unmodified particles (SEM), (e) SiO2-PPE-4 (SEM).
surface texture, which we associate with the presence of the polymer on the surface. It is noted that the TEM images do not show a significant change in the size of the particles after grafting of the polymer onto the surface. This finding is consistent with the TGA results, which suggest that on average the graft layer is approximately 12 nm in thickness. Although the particle size was not affected, the TEM data suggest that the 300 nm particles are uniformly coated with a thin layer of polymer on the surface. Parts d and e of Figure 5 show SEM images of 5 µm SiO2 particles. The unmodified particles (Figure 5d) exhibit a very smooth uniform surface texture. By contrast, the particles which are surface-grafted (Figure 5e) exhibit an “orange peel”-like surface texture that we associate with the polymer. The image suggests that much of the surface is covered with the grafted layer; however, the covering is clearly nonuniform. There is evidence for large aggregates (thickness ∼50 nm) on the surface. The origin of this material is unclear. One possibility is that it is material that was initially produced in solution during the polymerization and then became chemically or physically adsorbed to the surface. Figure 6 shows confocal fluorescence microscope images of the 5 µm SiO2 particles with the surface-grafted polymer (SiO2PPE-4). These images clearly show the green fluorescence from the polymer on the surface of the particles, which confirms the electron microscopy results which suggest that the polymer is grafted to the surface of the particles. The images also indicate that although there is emission from the entire surface of the particle, there appear to be regions in which there is a “clustering” of the fluorescent material. This finding correlates with the SEM image of the surface of a 5 µm particle (Figure 5e), which suggests that the polymer graft layer has a nonuniform texture or thickness. In addition to the apparent nonuniformity of the fluorescence from the particle surfaces, the confocal images also suggest that the particles have a propensity to aggregate in the suspension, as evidenced by the predominance of particle clusters in the images (as opposed to isolated single particles). Absorption and Fluorescence Properties. In previous work it has been shown that the solution photophysical properties of PPE-type CPEs are strongly affected by the nature of the solvent. In a good solvent, such as methanol, the polymer chains are well solvated and they exhibit photophysical properties characteristic of excitons confined to a single chain. On the other hand, in a poor solvent, such as water, the polymer chains tend to aggregate.
Figure 6. Confocal fluorescence microscope images of 5 µm particles (SiO2-PPE-4). The length of horizontal scale bars is 5 µm, and the tilted scale bar is 14.3 µm. Table 2. Photophysical Properties of PPE-SO3-OR11 solvent
λmax (nm)
(M-1 cm-1)
λem (nm)
Φf
methanol water
439 468
22,100 21,900
467 484
0.39 0.26
Aggregation results in π-π stacking between chains, and due to the increased interchain interactions the aggregated polymers exhibit bathochromic shifts of both absorption and emission.8,10,31 To provide a point of reference for the properties of the surfacegrafted polymers, we first characterized the absorption and fluorescence of PPE-SO3-OR11 in methanol and water. The results are summarized in Table 2, and Figure 7 shows the optical spectra. As seen in Figure 7, a 30 nm bathochromic shift of the absorption maximum is observed upon switching of the solvent from methanol to water. This phenomenon has been reported for
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Figure 7. Absorption and emission spectra of PPE-SO3-OR11 in methanol and water and excitation and emission spectra of SiO2-PPE-2 as a suspension in methanol. Fluorescence spectra of the polymer are area normalized according to their relative quantum yields.
other PPE-type CPEs, and it is due to aggregation of the polymer chains in water. Aggregation in water has a more significant effect on the fluorescence properties of the polymer. In methanol, the fluorescence spectrum appears as a narrow band with λmax ) 467 nm with a shoulder at ∼500 nm. In water, on the other hand, the emission profile becomes broader and is red-shifted compared to that seen in methanol. In addition to the spectral shift, the fluorescence quantum efficiency decreases from 0.39 to 0.26. In previous work, it was shown that a similar polymer, PPE-SO3,10 exhibits a more significant decrease in fluorescence efficiency when the solvent is changed from methanol to water (0.78 and 0.1, respectively). In addition, upon switching of the solvent from methanol to water the fluorescence λmax for PPESO3 is red-shifted by almost 100 nm, whereas only a 17 nm shift is observed for PPE-SO3-OR11. Compared to PPE-SO3, PPESO3-OR11 has additional side chains that are expected to give rise to improved chain solubility and also to provide a steric barrier to interchain interactions. These effects are likely to contribute to the reduced aggregation of the polymer chains. Suspensions of CPE-grafted silica particles in methanol and water also exhibit strong fluorescence.38 As shown in Figure 7, the fluorescence of the surface-grafted particles (SiO2-PPE-2) appears as a very broad, structureless band, with λmax ) 535 nm. The fluorescence from the polymer-grafted particles is red-shifted compared to that of PPE-SO3-OR11 in water, a feature which suggests that the polymer is strongly aggregated on the surface of the silica. Attempts were made to measure the absorption spectra of suspensions of the polymer-grafted silica particles; however, the spectra were strongly distorted in the near-UV region due to light scattering. (The scattering also precluded measurement of the fluorescence quantum efficiency of the particles.) Although direct measurement of the absorption spectra of the grafted particles was not possible, the absorption profile could be approximated by the fluorescence excitation spectrum of a particle suspension in methanol. As seen in Figure 7, the excitation spectrum is very similar to the absorption spectrum of the free polymer. The λmax of excitation was found to be 415 nm, which is slightly blue-shifted from that of free polymer in methanol. This blue shift in the excitation spectrum may arise because on average the length of the surface-grafted polymer is lower compared to that of the solution-polymerized polymer. Alternatively, it is possible that the blue shift arises because the conformation of the conjugated backbone is disordered in the surface-grafted polymer layer. Fluorescence Quenching Properties. As noted in the Introduction, conjugated polyelectrolytes exhibit the property of (38) The measurements were performed on a suspension of particles in both water and methanol. However, the optical properties of the particles were the same in both solvents.
Figure 8. Structures of fluorescence quencher ions.
amplified quenching, whereby the fluorescence of the polymer is quenched by oppositely charged quencher ions with very high efficiency. Amplified quenching is attributed to very efficient static quenching which occurs within an ion pair complex formed between the quencher ion and the CPE chain(s).3,6 The quenching efficiency is also augmented by the ability of the fluorescent (singlet) exciton to diffuse rapidly within a CPE chain and among aggregated chains. Estimates based on ultrafast spectroscopy suggest that the singlet exciton is quenched by the cyanine energy acceptors with essentially unit efficiency when it is created within a radius of ca. 10 nm from a quencher ion.39 One objective of this work is to develop CPE-grafted silica colloids as signaling elements in fluorescence-based bioassays. As an initial step in this direction we have explored the fluorescence quenching properties of the PPE-grafted particles using five different cationic quencher ions including methyl viologen (MV2+), Cu2+, diethyldicarbocyanine (DEDCC), diethylcyanine (DEC), and diethylthiadicarbocyanine (DETDCC; structures of the quenchers are shown in Figure 8). Among these quenchers, MV2+ and Cu2+ act via a charge (electron)-transfer quenching mechanism, while the three cyanine dyes quench via the dipole-dipole (Fo¨rster) energy-transfer mechanism. An important distinction between the two quenching mechanisms is that, for charge-transfer quenching to be efficient, the exciton and quencher ion must be in close proximity (99 >99 >99
a Fraction of fluorophore accessible to the quencher. b SiO2-PPE-1. SiO2-PPE-3.
is supported by the quenching data for DETDCC, which are included in the Supporting Information. These data show that the DETDCC-induced quenching of the SiO2-PPE-2 fluorescence is accompanied by the appearance of the sensitized fluorescence from DETDCC at 700 nm. There are several interesting features with respect to the quenching studies of the SiO2-PPE-2 particles. First, all of the quencher ions give rise to very efficient quenching of the fluorescence from the particles; in each case the Stern-Volmer constants (Ksv) of quenching range from 105 to 107 M-1. This observation indicates that the surface-grafted CPEs retain the property of amplified quenching, which is a favorable property which may allow the materials to be used in sensor schemes. A second point of interest is that there is a distinct difference in the overall quenching seen for the quenchers that act by charge transfer (MV2+ and Cu2+) and by dipole-dipole energy transfer (the cyanine dyes). As exemplified in Figure 9a for quenching by MV2+, quenchers that act by the charge-transfer mechanism quench only a fraction of the total fluorescence from the SiO2PPE-2 particles. In particular, addition of MV2+ up to c ) 5 µM leads to quenching of approximately 60% of the SiO2-PPE-2
(1)
where Io and I are the fluorescence intensities in the absence and presence of quencher, fa is the fraction of total fluorescence that is quenched, and KSV is the Stern-Volmer quenching constant for the fluorescence component that is quenched. Use of this expression for charge-transfer quenching of SiO2-PPE-2 by MV2+ and Cu2+ affords KSV values in excess of 106 M-1, and fa ≈ 0.6, indicating that at the limit of high quencher concentration ca. 60% of the total fluorescence intensity is quenched. In contrast to the behavior of the charge-transfer quenchers, we find that the cyanine dye energy-transfer quenchers quench essentially 100% of the fluorescence emission from SiO2-PPE-2. As exemplified by the data for quenching by DEDCC in Figure 9b, in aqueous solution the dye quenches 90% of the SiO2-PPE-2 fluorescence at a concentration of ca. 2 µM with an overall KSV in excess of 106 M-1 (Table 3). We postulate that the difference in quenching behavior for the energy-transfer quenchers (cyanines) and the charge-transfer quenchers (MV2+ and Cu2+) arises because the PPE-SO3 graft layer on the surface of the colloids exists in a strongly aggregated state and that some fraction of the fluorescent polymer excitons are deeply trapped within these aggregates. The fact that the polymer layer exists in a strongly aggregated state presents a steric barrier that prevents the quencher ions from penetrating into some regions of the film. Since the polymer graft layer is on average 5 nm or greater in thickness, and the charge-transfer quenchers cannot access the “interior” regions of the graft film, the quenchers are only able to efficiently quench excitons that are in the “exterior” of the graft layer. Importantly, the excitons that are trapped deep within the aggregate structure are not able to be quenched by the charge-transfer quenchers, and emission from these trapped excitons is responsible for the “unquenched” fluorescence component. Different behavior is observed for the cyanine dye energy-transfer quenchers, because dipole-dipole energy transfer is active and efficient even when the excitonquencher separation distance is in excess of 5 nm. Thus, in this case, even though the aggregate excitons are trapped within the graft layer, long-distance dipole-dipole energy transfer still occurs, leading to effective quenching of the fluorescence from the PPE-grafted particles with increasing cyanine dye concentration. One final noteworthy point with respect to the quenching data is that it is seen that the Stern-Volmer quenching efficiencies for the cyanine dyes are an order of magnitude larger for aqueous suspensions compared to suspensions in methanol. This effect arises because the solubility of the cyanine dyes is considerably lower in water compared to methanol. As a result the dyeSiO2-PPE-2 association constant is considerably larger, which leads to the much larger quenching efficiency. (40) Gong, Y. K.; Miyamoto, T.; Nakashima, K.; Hashimoto, S. J. Phys. Chem. B 2000, 104, 5772-5778. (41) Lehrer, S. S. Biochemistry 1971, 10, 3254. (42) Lakowicz, J. R. Principles of Fluorescent Spectroscopy, 2nd ed.; Plenum Publishers: New York, 1999.
4548 Langmuir, Vol. 23, No. 8, 2007
Summary and Conclusions A method for preparation of silica particles that feature a surface graft layer of a fluorescent conjugated polyelectrolyte has been developed. The polymer is grafted to the surface of the particles via a Pd(0)-mediated cross-coupling reaction between a surfaceconfined aryl iodide moiety and the polymer chains which are synthesized in situ via a step-growth polymerization reaction. No direct evidence is available concerning the average degree of polymerization of the surface-grafted polymer; however, the available data suggest that the chains are on average 10 polymer repeat units in length. It has been demonstrated that the surface density of the polymer layer can be controlled to some extent by varying the loading of the aryl iodide on the surface of the particles. Analysis of the surface-grafted particles by FTIR, TGA, and electron and confocal fluorescence microscopy provides clear evidence that the poly(phenylene ethynylene) polyelectrolyte chains are confined to the surface of the silica particles. SternVolmer quenching studies show that cationic electron-transfer and energy-transfer quencher ions suppress the fluorescence of
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the surface-grafted polymer very efficiently. This observation suggests that the particles may be useful for applications as fluorescent sensors for biological targets. Work directed toward application of the particles in biosensor schemes is under way and will be reported in the future. Acknowledgment. Work at the University of Florida was supported by the Air Force Office of Scientific Research under Grant No. FA9550-04-1-0161. Work at the University of New Mexico was supported by the National Science Foundation (Grant No. EEC-0210835). We thank Dr. Mangesh T. Bore for help in obtaining the SEM images and Ms. Yan Liu for help with the TEM imaging. Supporting Information Available: A detailed synthetic procedure and characterization data of PPE-SO3-OR11 and a figure illustrating fluorescence spectra of SiO2-PPE-2 with added DETDCC quencher. This material is available free of charge via the Internet at http://pubs.acs.org. LA0630108
