NANO LETTERS
Water-Soluble Poly(acrylic acid) Grafted Luminescent Silicon Nanoparticles and Their Use as Fluorescent Biological Staining Labels
2004 Vol. 4, No. 8 1463-1467
Z. F. Li and E. Ruckenstein* Department of Chemical and Biological Engineering, State UniVersity of New York at Buffalo, Buffalo, New York 14260 Received May 19, 2004
ABSTRACT UV-induced graft polymerization of acrylic acid (AAc) on the surface of silicon nanoparticles was used to prepare a stable aqueous luminescent silicon nanoparticle solution. By grafting a water-soluble polymer on the particle surface, the dispersions in water of the silicon nanoparticles became very stable and clear aqueous solutions could be obtained. XPS and NMR spectroscopy confirmed that PAAc was covalently grafted to the silicon nanoparticles. The grafted PAAc on silicon particles increased not only the dispersibility but also improved the photoluminescence stability of the silicon nanoparticles against degradation by water. The surface-modified nanoparticles were used as biological labels for cell imaging. The Si quantum dot labels exihibited bright fluorescence images and provided higher resistance to photobleaching than the commonly used organic dyes.
Introduction. Since the discovery in 1990 of the red light emission of electrochemically etched nanoporous silicon upon UV excitation,1 new methods of producing nanosized Si particles, the characterization of their structure, the determination of their optoelectronic properties,2-4 and their surface modification to cater to various types of applications have attracted great interest. One benefit of the silicon nanoparticles is the potentiality of their integration within the existing silicon technologies for creating nanoscale optoelectronic devices.5-9 In addition, the quantum dots became a new class of fluorescent probes for many biological applications, particularly the cellular imaging. The tunable fluorescence signatures, the high photoluminescence quantum efficiency, and the stability against photobleaching10,11 make the silicon nanoparticles an ideal candidate for substituting the fluorescent dyes in some biological assays and fluorescence imaging techniques. Additional surface coatings of these nanostructured Si materials must be, however, employed in order to increase the photoluminescence (PL) stability and their dispersibility (solubility) in various solvents. Numerous surface modifications of the porous silicon films with various organic compounds, ranging from alkyl chains to macromolecules have been carried out.12-18 However, the above surface modifications were limited to porous silicon films attached * Corresponding author. Tel: (716) 645-2911 ext 2214; Fax (716) 6453822; E-mail:
[email protected]. 10.1021/nl0492436 CCC: $27.50 Published on Web 06/18/2004
© 2004 American Chemical Society
to macroscopic substrates. Recently, free-standing silicon nanoparticles that exhibit bright visible photoluminescence have been produced in relatively large amounts (hundreds of milligrams per day).19 They are prepared in a two-step process using the CO2 laser heating of SiH4/H2/He mixtures followed by etching of the nanoparticles with hydrofluoric acid/nitric acid mixtures to reduce their size and passivate their surface. However, like the porous silicon, the freestanding silicon nanoparticles thus obtained do not have high PL stability. In addition, they have poor dispersibility in water and common solvents. Water solubility with good PL stability in water solution is required for the nanocrystals to be employed for bioimaging.20 Poly(acrylic acid) (PAAc) is an anionic polyelectrolyte which was frequently employed as scaffold for immobilization of biologically active molecules. The high density of carboxylic acid moieties along its backbone can be used to immobilize molecules containing amine groups, such as proteins.21,22 Thus, the preparation of PAAc-bearing surfaces constitutes one of the promising approaches to the generation of biofunctional surfaces for the use of silicon nanocrystals in the biological fields. This paper describes a simple technique to prepare freestanding and highly bright luminescent water-soluble silicon nanoparticles by the graft polymerization of acrylic acid under UV irradiation. The grafted layer of PAAc on the silicon nanoparticles makes the latter water soluble and
increases the PL stability of the particles, while retaining a high quantum yield. Furthermore, PAAc-modified silicon surfaces with high PL stability were used for the first time as biological labels for cell imaging. The above quantum dots bioimaging have higher resistance to photobleaching than the conventional organic dyes. Experimental Section. Materials. Acrylic acid (99% grade), sulfuric acid (H2SO4, 98 wt %), ethanol (HPLC grade), methanol (HPLC grade), potassium bromide (KBr, IR grade), and ethylenediaminetetraacetic acid (EDTA) were purchased from Aldrich. Hydrogen fluoride (HF, 48-51 wt %, A.C.S. reagent grade) and nitric acid (HNO3, 68-70 wt %) were obtained from EMD Chemicals. Methods. Nanosized (around 3-5 nm) crystalline silicon particles were synthesized as described previously.19 The obtained nanoparticles were etched with a mixture of HNO3 and HF to reduce their size and passivate their surface. The resulting Si nanoparticles exhibited, after washing, a bright red photoluminescence under UV excitation. To increase the number of Si-H bonds on the surface of the silicon particles, they were introduced into a 5 wt % HF aqueous solution, sonicated for a few minutes, and then collected by filtration and washed with water and methanol. For the graft polymerization of AAc, the silicon particles were first introduced into a 10 wt % monomer acrylic acid in ethanol. The solution was first bubbled with N2 to remove the dissolved oxygen that could inhibit the radical-initiated reaction and then was kept with magnetic stirring under UV irradiation for 24 h for the acrylic acid to react with the Si-H sites of the surface and further to graft polymerize. A Rayonet Chamber Photochemical Reactor (model RPR-100, manufactured by Southern New England Ultraviolet Co., Connecticut) equipped with 16 RPR-3500 Å UV tubes was used, and the temperature of the reactor chamber was kept at about 40 °C. After reaction, a clear solution of nanoparticles with grafted polymers was obtained. The clear particle solution could easily pass through a 0.1 µm pore filter paper, indicating that the particles were well dispersed into the solvent. Finally, a water solution of polymer-grafted nanoparticles was obtained by dialysis in deionized water to remove the free polymers and the ethanol. The Chinese hamster ovary (CHO) cells were chosen to be stained with PAAc-grafted silicon particles for the bioimaging experiment. First, CHO cells were taken out from a cell culture flask using a 5mM EDTA aqueous solution, washed with a phosphate buffer solution (PBS) (pH)7.4), and then fixed in a 1 wt % paraformaldehyde water solution for 10 min. The fixed cells were again washed two times with PBS solution and then located into a PAAc-silicon nanocrystal aqueous solution. After incubation for 2 h at room temperature, the sample was centrifuged and washed with a PBS (pH)5.5) solution before taking pictures under a fluorescent microscope. The number of nanoparticles labeled on each cell could be estimated by measuring the fluorescence intensities of the nanoparticle solution before and after 20 million cells were immersed for 2 h into and then centrifuged from the solution. The average number of nanoparticles per cell thus obtained was around 800 000. 1464
Figure 1. XPS C1s core-level spectra of PAAc-grafted Si nanoparticles.
Characterization. The XPS measurements were carried out on a Surface Science model SSX-100 small spot ESCA equipment, possessing an Al KR monochromatized X-ray source (1.48 keV photons). Transmission electron microscopy (TEM) investigations were carried out using a JEM2010 electron microscope equipped with a tungsten gun operating at an accelerating voltage of 200 KV. 1H NMR specimens were prepared by dissolving the polymer-coated nanoparticles in deuterated water, and the NMR spectra were recorded using a Varian Unity-Nova 400 (400 MHz) spectrometer. The photoluminescence spectra were recorded using a SLM model 8100 spectrofluorometer possessing a 420 nm emission cutoff filter to suppress any scattered light from the source. The excitation wavelength was set at 360 nm, and the PL intensity was measured as a function of time. The fluorescent microscopy analysis for bioimaging was performed using a Nikon FXA wide-field fluorescence and brightfield microscope with a 100× N.A. 1.3 oil immersion objective, using continuous-wave excitation provided by a 100W CW Hg high-pressure arc lamp. The camera was a Xylix PMI-1400 cooled CCD digital connected to a computer with ImageJ software. Results and Discussion. Figure 1 shows the C1s XPS core-level spectra of Si nanoparticles after the graft polymerization in 10 wt % AAc monomer solution. The distinct high binding energy (BE) at about 288.7 eV is characteristic of the carboxylic acid group of the grafted AAc polymer. The presence of this peak confirms the existence of a surfacegrafted poly(AAc). To verify that a Si-C bond was formed between silicon and the CdC bond of PAAc, NMR spectra were employed to find that the chemical shift at 5.8-6.4 ppm due to the CdC bond of AAc disappeared and those at 0.7-1.1 ppm of the Si-C bond23 appeared after grafting. The NMR spectra confirmed that PAAc was grafted on the surface of the Si nanoparticles through a chemical bond. Neither the freshly prepared nor the HF treated Si particles could form stable colloidal solutions in water or alcohols where they easily aggregated. After the UV-induced graft polymerization of PAAc, the dispersed grafted particles became soluble and stable in water and alcohols. The solution was very clear and remained stable for several months. The above dispersion was dropped onto holey carbon-coated Cu TEM grids or spin coated on silicon wafers, and both the TEM and AFM pictures revealed a high dispersibility (Figure 2). For the PAAc-capped Si nanoparticles, the dispersibility Nano Lett., Vol. 4, No. 8, 2004
Figure 2. (a) Transmission electron micrographs of PAAc-grafted Si nanoparticles and (b) AFM image of spin-coated PAAc-grafted Si nanoparticle dilute aqueous solution on silicon wafer.
of the nanoparticles was very high and one can observe that the TEM picture shows individual particles with diameters less that 10 nm. In contrast, such a high dispersibility was not observed for the uncoated Si particles, which were highly aggregated. The lack of PL stability of the nanostructured silicon has been a major barrier to its commercial application. Neither the hydrogen termination of the freshly etched silicon particles nor the thin oxide layer that formed upon exposure of these particles to air for a few hours protected them from photoluminescence quenching by the molecules of the solution. This limited the potential use of Si nanoparticles in the fabrication of devices. In our experiments, the PL stability of the PAAc-capped Si nanoparticles was examined and compared to that of the freshly prepared Si nanoparticles after their immersion in deionized water for a predetermined time period. One can see from Figure 3 that the PAAc-capped Si nanoparticles maintained 80% of their initial PL even after 5 days (Figure 3b) of immersion in water. In contrast, the uncapped Si nanoparticles lost their PL almost completely after 3 days (Figure 3a) of immersion in water. These results suggest that the general increase and the long-term stability of the PL might be attributed to a favorable arrangement of the PAAc chains, leading to a more efficient insulating barrier between the quenching molecules and the Si nanoparticle surface. The absorption and emission spectra of PAAc-grafted silicon nanoparticles in aqueous solutions are presented in Figure 4. The absorbance above 290 nm is due to Si nanoparticles (PAAc does not absorb above 290 nm at all), while below 290 nm it can be attributed to both the nanoparticles and the grafted PAAc. The emission spectrum of the PAAc-coated silicon nanoparticle solution is similar to that of uncoated nanoparticles, with a 10 nm blue shift for the former. The quantum efficiency of the coated silicon nanoparticle solution under an excitation wavelength of 360 nm was determined by comparing its integrated emission to that of quinine sulfate in a 0.1 M HClO4 aqueous solution as a reference standard.24 The quantum yield of the PAAc grafted Si nanoparticle aqueous solution thus obtained was 0.24. The highly PL stable and nonaggregated water-soluble PAAc-Si nanoparticles can be used as fluorescent biological Nano Lett., Vol. 4, No. 8, 2004
Figure 3. The intensity of the photoluminescence spectrum after immersion in deionized water as a function of time: (a) untreated Si nanoparticles and (b) PAAc-coated Si nanoparticles.
Figure 4. Absorption (dotted line) and emission (solid line) spectra of PAAc-coated Si nanoparticle aqueous solution.
labels. After being stained with PAAc-Si nanoparticles, the photoluminescence of the nanocrystal-labeled CHO cells was very bright and could be clearly spectrally resolved to the 1465
Figure 6. Changes in fluorescence intensity of CHO cells imaged by surface-modified Si nanoparticles and by the organic dyes Alexa488, Cy5, FITC, and LDS751 during their continuous exposure to a 100 W mercury lamp. Excitation filters used: 470 ( 20 nm for Alexa 488 and FITC; 530 ( 25 nm for Cy5 and LDS751; 360 ( 20 nm for Si nanoparticles. Emission filters used: 520 nm for Alexa 488 and FITC; 590 nm for Cy5 and LDS-751; 420 nm for Si nanoparticles. The images were captured with a cooled CCD camera at 1 min intervals.
Figure 5. Fluorescent microscopy image of CHO cells labeled with PAAc-Si nanoparticles (b is a higher magnification of a).
eye and the Polaroid camera. Figure 5 presents examples of fluorescent images of CHO cells labeled with PAAc-Si nanoparticles under a fluorescent microscope. One can see that red nanocrystal silicon particles are present on the surface of the cells. Another advantage of the Si nanocrystals as bioimaging labels is their size-dependent emission-tunning properties, which remain to be exploited in the future multicolor, multiparameter immunolabeling of cells. Further, the photostabilities of Si nanoparticles and organic dyes used in biological cell imaging were compared. Even though the number of Si nanoparticles on each cell was more than 10 times larger than that of the organic dyes, one can still compare their normalized PL intensities with respect to their initial intensities during their exposure to the irradiation of a 100 W high-pressure mercury lamp. Figure 6 presents a quantitative analysis of the change of the fluorescence intensity of CHO cells imaged with grafted Si nanoparticles and with the organic dyes: Alexa 488, Cy5, fluorescein isothiocyanate (FITC), and laser dye styryl (LDS751).25 From Figure 6, one can see that the FITC-labeled CHO cells have lost half of their intensities in 1 min, and the more stable Alexa 488 labeled cells have lost half of their intensities in 6 min. In contrast, the coated Si-nanoparticle-labeled CHO 1466
cells maintained a very high fluorescence, and no obvious PL intensity change was observed during our experiments (2 h). The photostability of the PAAc-coated Si nanoparticles could be also demonstrated by using the UV reactor previously employed for the graft polymerization of AAc. The coated nanoparticle solution in water kept 95% of their initial PL intensity after 24 h of UV exposure. In contrast all the four types of organic dyes (for the same optical densities as the coated nanoparticle solution) have lost completely their PL after their exposure to the same UV radiation for 30 min. Consequently, the excellent photostable water-soluble Si nanoparticles may become valuable for fluorescent molecular detection. In conclusion, free-standing luminescent crystalline Si nanoparticles were grafted with PAAc under UV irradiation. These coated particles could be well dispersed in water to form clear and stable aqueous solutions. PAAc coating passivated the surface of Si particles and greatly stabilized the PL of the nanoparticles against quenching and degradation. The stabilized water-soluble Si nanoparticles can be used as a bioimaging material for cells or other biological molecules. In addition, these water-soluble quantum dots exhibited a bright PL and were more resistant to photobleaching than were the conventional organic dyes used for cellular labeling. Acknowledgment. We are grateful to Dr. M. T. Swihart, Mr. Xuegeng Li, and Ms. Yuanqing He for providing us unetched Si nanoparticles, to Dr. S. Neelemagen and Mr. Mark. E. Beauharnois for providing fixed CHO cells and organic dyed CHO cells. References (1) Canham, L. T. Appl. Phys. Lett. 1990, 57, 1046. (2) Heath, J. R. Science 1992, 258, 1131. Nano Lett., Vol. 4, No. 8, 2004
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(17) Anderson, R. C.; Muller, R. C.; Tobias, C. W. J. Electrochem. Soc. 1993, 140, 1393. (18) Bakker, J. W. P.; Arwin, H.; Wang, G.; Jarrendahl, K. Phys. Status Solidi A 2003, 197, 378. (19) Li, X.; He, Y.; Talukdar, S. S.; Swihart, M. T. Langmuir 2003, 19, 8490. (20) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (21) Li, Z. F.; Kang, E. T.; Neoh, K. G.; Tan, K. L. Biomaterials 1998, 19, 45-53. (22) Franchina, J. G.; Lackowski, W. M.; Dermody, D. L.; Crooks, R. M.; Bergbreiter, D. E. Anal. Chem. 1999, 71, 3133. (23) Bhacca N. S.; Hollis, D. P.; Johnson, L. F.; Pier, E. A. Eds. NMR Spectrum Catalog; Varian Associates, 1963, lithographed in U.S.A. by National Press. (24) Velapoldi, R. A.; Mielenz, K. D. 1980 Standard Reference Materials: A Fluorescence SRM: Quinine Sulfate Dihydrate (SRM 936). NBS Spec. Publ. 260-264. (25) The organic dyes were bound to the incubated CHO cells via the dyes conjugated goat abtimouse IgG antibody. The number of fluorescence labels on each cell was approximately 50 000.
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