Langmuir 2004, 20, 6071-6073
Encapsulation of Quantum Nanodots in Polystyrene and Silica Micro-/Nanoparticles Xiaotun Yang† and Yong Zhang*,†,‡ Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, and Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 117576 Received February 13, 2004. In Final Form: April 8, 2004
Introduction Semiconductor quantum dots (QDs) have good potential for use as fluorescent probes in biological staining and diagnostics.1-3 The ideal optical properties of QDs offer the possibility of using them to tag biomolecules in ultrasensitive biological detection based on optical coding technology. However, QDs themselves are not water soluble, not biocompatible and chemically stable, and do not have functional groups for covalent conjugation with biomolecules.4 Efforts have been made to do some surface modifications on single QDs to solve the above problems, but the surface modification is very dependent on the surface chemistry of QDs. Recently there are many successful reports on the surface modification of QDs such as conjugation of mercaptoacetic acid to ZnS-capped or -uncapped CdSe and coating of silica on ZnS-capped CdSe QDs.5-7 However, QDs capped with small molecules such as mercaptoacetic acid are easily degraded by hydrolysis or oxidation of the capping ligand.8 To overcome this problem, a thin silica layer was covalently bound on the surface of ZnS-capped CdSe QDs and capping the QDs with a silica layer did not degrade their optical properties.9,10 In addition to capping single quantum dots with a silica monolayer, multiple quantum dots were also encapsulated into silica nanospheres.11,12 However, because of the hydrophobicity of QDs, it is difficult to encapsulate QDs directly in silica unless QDs are surface modified or special silane surfactant with hydrophobic tails and hydrophilic headgroups are used. * To whom correspondence may be addressed: Division of Bioengineering, Faculty of Engineering, Blk, EA-03-12, National University of Singapore, 9 Engineering Drive 1, Singapore 117576. Phone: +65-68744871. Fax: +65-68723069. E-mail: biezy@ nus.edu.sg. † Nanoscience and Nanotechnology Initiative. ‡ Division of Bioengineering, Faculty of Engineering. (1) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016. (2) Zhang, C. Y.; Ma, H.; Nie, S. M.; Ding, Y.; Jin, L.; Chen, D. Y. Analyst 2000, 125 1029. (3) Goldman, E. R.; Balighian, E. D.; Mattoussi, H.; Kuno, M. K.; Mauro, J. M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 6378. (4) Chan, W. C. W.; Maxwell, D. J.; Gao, X. H.; Bailey, R. E.; Han, M. Y.; Nie, S. M. Curr. Opin. Biotechnol. 2002, 13, 40. (5) Rogach, A. L.; Kornowski, A.; Gao, M. Y.; Eychmuller, A.; Weller, H. J. Phys. Chem. B 1999, 103, 3065. (6) Lorenz, J. K.; Ellis, A. B. J. Am. Chem. Soc. 1998, 120, 10970. (7) Gerion, D.; Pinaud, F.; Willimas, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. 2001, 105, 8861. (8) Chen, Y. F.; Rosenzweig, Z. Nano Lett. 2002, 2, 1299. (9) Rogach, A. L.; Nagesha, D.; Ostrander, J. W.; Giersig, M.; Kotov, N. A. Chem. Mater. 2000, 12, 2676. (10) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861. (11) Rogach, A. L.; Nagesha, D.; Ostrander, J. W.; Giersig, M.; Kotov, N. A. Chem. Mater. 2000, 12, 2676. (12) Katagiri, K.; Hamasaki, R.; Ariga, K.; Kikuchi, J. J. Am. Chem. Soc. 2002, 124, 7892.
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Another successful attempt is to tag latex beads with different combinations of quantum dots of various colors to create QD bar codes and the use of six colors and 10 intensity levels can theoretically encode one million biomolecules.13 However, it is difficult to control the number of QDs, and QD-tagged beads are not uniform and reproducible. Recently, synthesis of monodisperse polymer microspheres has stimulated great interest, and incorporation of metal and other nanoparticles in these microspheres is particularly attractive.14,15 The main challenges are to control the incorporation of the nanoparticles into the microspheres and control of colloid stability and monodispersity of the polymer particles. In this work, we synthesized polystyrene particles of controlled sizes on both micro- and nanolevels, and incorporate QDs into these particles. Silica coatings were made on polystyrene particles encapsulating QDs to provide another suitable surface for further conjugation of biomolecules. The encapsulation of QDs will solve the above-mentioned problems associated with QDs such as biocompatibility and stability. The approaches can also be used to encapsulate other optical nanocrystals or magnetic nanoparticles. Experimental Section Chemicals. Tetradecylphosphonic acid (TDPA, 90%) was purchased from Alfa Aesar. Trioctylphosphine oxide (TOPO, 99%), tri-n-octylphosphine (TOP, 90%), selenium powder (99.999%), CdO (99.999%), and tetraethyl orthosilicate (TEOS) were purchased from Aldrich and used as received. Styrene, divinylbenzene, and methacrylic acid (MAA) from Aldrich were distilled under reduced pressure and stored at 4 °C. Cetyltrimethylammonium bromide (CTAB) and 2,2′-azobisisobutyronitrile (AIBN) from Aldrich were used as surfactant and initiator, respectively. Methanol, toluene, chloroform, and ethanol and HCl were purchased from Merck. Synthesis of CdSe QD. CdSe quantum dots were synthesized based on a method developed by Peng et al.,16 with modifications. A 0.0625 g portion of CdO, 0.5580 g of TDPA, and 9.4420 g of TOPO were loaded into a 25 mL flask. The mixture was heated to 300 °C under Ar flow, and CdO was dissolved to generate a colorless homogeneous solution. The temperature of the solution was cooled to 270 °C, and selenium solution with 0.1027 g of Se powder dissolved in 5 g of TOP was injected. After injection, nanocrystals grown at 250 °C for different time periods to reach desired size and the solution underwent a color change from clear to yellow or red. The solution was injected into cool chloroform. The CdSe powders were precipitated by adding dry ethanol and collected by centrifugation, washed with methanol several times, and dried in a vacuum. Encapsulation of QDs in Polystyrene Particles Grafted with Carboxyl Groups (PS@QD). A 0.25 g portion of CTAB and 80 mL of water were mixed by high shear dispersion for 0.5 h to form micelles in water. Two milliliters of QD/toluene solution with different concentrations (in order to achieve a different amount of QDs in polystyrene particles) was added dropwise into the solution for 2 h to get emulsion. A mixture of monomers, 2.08 g (0.02 mol) of styrene, 1.30 g (0.01 mol) of divinylbenzene, 0.87 g (0.01 mol) of MAA, and 0.02 g of AIBN, was added dropwise into the system for 2 h in an ice bath under an argon atmosphere. The system was heated to 70 °C. After 20 h of polymerization, the polystyrene particles in the emulsion were precipitated and washed with water and ethanol several times to remove the surfactant. The obtained PS@QD was treated with NaOH/ethanol (13) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. M. Nat. Biotechnol. 2001, 19, 631. (14) Obare, S. O.; Jana, N. R.; Murphy, C. J. Nano Lett. 2001, 1, 601. (15) Caruso, R. A.; Susha, A.; Caruso, F. Chem. Mater. 2001, 13, 400. (16) Peng, Z. A.; Peng, X. G. J. Am. Chem. Soc. 2001, 123, 183.
10.1021/la049610t CCC: $27.50 © 2004 American Chemical Society Published on Web 06/08/2004
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Langmuir, Vol. 20, No. 14, 2004
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Figure 1. UV-vis absorption spectra (a), fluorescence spectra (λex ) 350 nm) (b) of CdSe quantum dots synthesized at different reaction time, and TEM image (c) of CdSe quantum dots. The scale bar is 20 nm. in an aqueous solution containing 5 wt % sodium dodecyl sulfate (SDS) and 2% phosphotungstic acid (PTA). After thorough mixing, a drop of PS@QD solution was put on a copper grid coated with a thin layer of Formvar. Fluorescence microscopy was carried out using an Axiostar Plus Microscope (ZEISS) equipped with a Hg excitation lamp and a true-color digital camera. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on a Bio-Rad FTS156 spectrometer.
Results and Discussion Figure 2. Optical (a) and fluorescence (b) images of PS@QD particles. The scale bar is 10 µm.
Figure 3. FT-IR spectra of PS-COOH@QD (a) and PS@QD (b) particles. solution to get negatively charged polystyrene spheres, washed with ethanol several times, and collected by centrifugation. Coating of Silica on Surfaces of PS@QD. A solution of 8 mL of ethanol and 72 mL of water was prepared, and the pH was adjusted to 1.5 using HCl. Then 4 mL of TEOS was added into the solution with vigorous stirring. A small amount of Rhodamine B was added to the solution in order to label the silica coating on PS@QD. After the mixture was stirred for 1 h, 2 mL of a PS@QD particle solution in water was added and the mixture was stirred vigorously to coat silica onto the surfaces of PS@QD. The hydrolysis and condensation of TEOS were carried out at room temperature. The silica-coated PS@QD was collected by centrifugation and washed several times with ethanol. Characterization. UV-visible absorption spectra were obtained using a Unicam 300 UV-Vis recording spectrometer. Fluorescence measurements were performed at room temperature using a RF 5301 (Shimatsu) spectrofluorometer. Transmission electron microscopy (TEM) measurement was carried out on JEOL 2010 transmission electron microscope operating at an acceleration voltage of 200 kV for CdSe quantum dots. A small drop of QD solution in CHCl3 was put on a 50 Å thick carbon-coated copper grid (300 mesh) with the excess solution immediately removed. PS@QD was observed using the same TEM at an acceleration voltage of 100 kV. PS@QD solution was diluted
The UV-vis absorption and fluorescence spectra of CdSe nanocrystals are shown in parts a and b of Figure 1, respectively. When the reaction time is increased from 0.5 to 1 min, the UV-vis absorption peak shifts from 450 to 480 nm, and correspondingly, the emission red shifts from 550 to 580 nm, which is due to the quantum size effects.17 The band gap of CdSe nanocrystals increases as their size decreases, and thus the emission color of the band edge PL of the nanocrystals shifts continuously from red to blue as the size of the nanocrystal decreases.18 The TEM image in Figure 1c shows that the synthesized CdSe nanocrystals are very monodisperse with the average particle size of about 5 nm. Although the synthesis of monodisperse polystyrene microspheres or hollow particles by dispersion polymerization of styrene has been extensively reported,19,20 however, the method is not suitable for incorporating hydrophobic QDs into polystyrene particles. In this work, the emulsion polymerization method was employed, using CTAB as surfactant, divinylbenzene as co-monomer to improve the strength of polystyrene capsules, and AIBN as initiator. The amount of AIBN was carefully controlled to increase the molecular weight of polystyrene that can influence the physical properties of the particles. The size of PS@QD particles can be adjusted by changing the ratio of oil/water and the amount of surfactant. Smaller particles can be produced by increasing the amount of surfactant and decreasing the ratio of oil/water. The PS@QD particles of uniform size distribution can be collected via centrifugation, with sizes ranging from 300 nm to 20 µm. The optical and fluorescence images of PS@QD particles are shown in parts a and b of Figure 2, respectively. The particles look spherical and relatively uniform with the particle size of about 5 µm. Strong green fluorescence emission was observed, which showed that the fluorescence emission of QDs was not quenched by the polystyrene particles. To introduce carboxyl functional groups to the surfaces of polystyrene particles, MAA was used as a co-monomer (17) Reiss, P.; Bleuse, J.; Pron, A. Nano Lett. 2002, 2, 781. (18) Brus, L. E. J. Chem. Phys. 1986, 90, 2555. (19) Okubo, M.; Minami, H.; Morikawa, K. Colloid Polym. Sci. 2003, 281, 214. (20) Ahmad, H.; Tauer, K. Colloid Polym. Sci. 2003, 281, 476.
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Figure 4. Fluorescence images of PS-COOH@QD (a) and silica-coated PS-COOH@QD (b) particles and blended image (c). The scale bar is 5 µm.
Figure 5. TEM images of PS-COOH@QD (a) and silica-coated PS-COOH@QD (b) particles. The scale bar is 200 nm.
for the polymerization. The particles were characterized using FT-IR to demonstrate the existence of carboxyl groups. The FT-IR spectra of styrene-divinylbenzeneMAA copolymer-QD (PS-COOH@QD) and styrenedivinylbenzene copolymer-QD (PS@QD) are shown in Figure 3. The absorption peak at 1640 cm-1, assigned to the CdC stretching of styrene monomers, was not observed in the spectra, which suggested that the polymerization was complete. The peaks at 758 and 3024 cm-1, corresponding to the out-of-plane hydrogen deformation of a monosubstituted phenyl groups and aromatic C-H stretching, respectively, appeared in the spectra of both samples. The absorption peak at 1700 cm-1 attributed to the CdO stretching was observed only in the spectra of styrenedivinylbenzene-MAA copolymer, indicating the existence of COOH groups. The PS-COOH@QD particles were treated with NaOH solution to obtain more negative charges on the particle surfaces and coated with silica subsequently. Rhodamine B was embedded in silica during the synthesis in order to visualize the silica coating using fluorescence micro-
scope. The particles were excited at different excitation wavelengths of QDs and Rhodamine B to generate green fluorescence from QDs and red fluorescence from Rhodamine B, as shown in parts a and b of Figure 4, respectively. Imaging software, Painshop Pro, was used to blend the two images into one, as shown in Figure 4c, to visualize the silica coating (in red) on PS@QD particles (in yellow, overlap of red and green fluorescence). It was also demonstrated by TEM observations that silica was successfully coated on PS@QD particles. The TEM images in Figure 5 of the PS-COOH@QD and silica-coated PSCOOH@QD particles showed that silica was smoothly coated on the polystyrene particles (Figure 5b) and the thickness of the coating was around 75 nm. The size of PS-COOH@QD particles used for silica coating in this TEM image was about 600 nm. Conclusions Luminescent cadmium selenide (CdSe) quantum dots were synthesized and successfully incorporated into polystyrene particles with carboxyl groups using emulsion polymerization method. The PS@QD particles were subsequently coated with a thin layer of silica to form a coreshell structure. Both PS@QD and silica-coated PS@QD particles were highly luminescent. The particle sizes were adjustable and ranged from 300 nm to 20 µm. These luminescent particles are biocompitable and have good potential for use as fluorescent probes in biological staining and diagnostics. Acknowledgment. The authors thank Chen Liting, Huang Ning, and Cai Jianling for help with experiments. This research was supported by NUS Research Grants R-398-000-005-112 and R-397-000-009-112. LA049610T
