Anal. Chem. 2007, 79, 6507-6514
Synthesis, Characterization, and Biological Applications of Multifluorescent Silica Nanoparticles Michihiro Nakamura,*,† Masayuki Shono,‡ and Kazunori Ishimura†
Department of Anatomy and Cell Biology, Medical Informatics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan, and Support Center for Advanced Medical Sciences, The University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan
Multifluorescent silica nanoparticles were synthesized by the Sto1 ber method using conjugates of (3-aminopropyl)triethoxysilane and fluorescent dye-N-hydroxysuccinimide esters. The nanoparticles containing the fluorescent dyes were well dispersed and showed high, stable, and tunable fluorescence intensities. In addition, we prepared multifluorescent silica nanoparticles containing two kinds of fluorescent dyes and used the nanoparticles in biological applications. Flow cytometry analysis showed high and tuned fluorescence and multiple fluorescences from single nanoparticles with diameters of ∼400 nm. Fluorescence microscopy analysis also showed high and tuned fluorescence, as well as multiple fluorescences from single nanoparticles and from cells labeled with multifluorescent silica nanoparticles. The intracellular distribution of nanoparticles was evaluated by confocal microscopy and electron microscopy. We discuss the advantages and demonstrate the usefulness of our nanoparticles in relation to commercially available fluorescent nanoparticles including quantum dots. Fluorescent nanoparticles are useful for bioimaging, bioassay, and nanomedicine.1-4 Traditional fluorescent dyes such as fluorescein, rhodamine, Cy3, and Cy5 have long been used in bioimaging applications, but their low fluorescence intensity and low photostability make them unsuitable for high-sensitivity detection and real-time monitoring. Recently, new nanoparticles, including quantum dots, fluorescent latex particles, and fluorescent silica particles, have been developed and have shown great utility. Quantum dots have several advantages over traditional dyes: broad excitation spectra, size-tunable fluorescence properties, long fluorescence time, and photostability. Quantum dots have interest* To whom correspondence should be addressed. Phone: +81-88-633-9220. Fax: +81-88-633-9426. E-mail:
[email protected]. † The University of Tokushima Graduate School. ‡ The University of Tokushima. (1) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47-51. (2) Lingerfelt, B. M.; Mattoussi, H.; Goldman, E. R.; Mauro, J. M.; Anderson, G. P. Anal. Chem. 2003, 75, 4043-4049. (3) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauso, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684-688. (4) Santra, S.; Xu, J.; Wang, K.; Tan, W. J. Nanosci. Nanotechnol. 2004, 4 (6), 590-599. 10.1021/ac070394d CCC: $37.00 Published on Web 07/21/2007
© 2007 American Chemical Society
ing optical properties that can be exploited in a range of photonic applications, including biological fluorescence imaging5-10 and optoelectronic devices,11-17 and are becoming popular as a new material in biological fluorescence imaging. However, quantum dots have the following disadvantages: poor solubility, agglutination, blinking, low quantum yield, and toxicity in biological systems. For example, Derfus et al. showed that CdSe quantum dots without a ZnS shell were toxic to liver cells after exposure to UV light.18 In addition, the emission wavelength of quantum dots depends on the particle size; when the size of quantum dots is shifted to a bigger size, the peak of emission is shifted to a longer wavelength. Fluorescent latex nanoparticles, such as fluorescent polystyrene nanoparticles and fluorescent polymethacrylic nanoparticles, have also been employed in some biological applications.19,20 However, because of their hydrophobicity, agglutination, the drawbacks of large size (>100 nm), swelling, and dye leakage, these latex nanoparticles are not suitable for bioanalysis. Silica nanoparticles are highly hydrophilic and easy to prepare, separate, surface modify, and label. Fluorescent silica nanoparticles possess several advantages: high fluorescence intensity, good photostability due to the exclusion of oxygen by silica encapsulation, and good potential for surface modification with various (5) Chan, W. C.; Nie, S. Science 1998, 281, 2016-2018. (6) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (7) Green, M. Angew. Chem., Int. Ed. Engl. 2004, 43, 4129-4131. (8) Wu, A.; Liu, H.; Liu, J.; Haley, K. N.; Tradway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41-45. (9) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (10) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science 2003, 300, 1434-1436. (11) Bakueva, L.; Konstantatos, G.; Levina, L.; Musikhin, S.; Sargent, E. H. Appl. Phys. Lett. 2004, 84, 3459-3461. (12) McDonald, S. A.; Cyr, P. W.; Levina, L.; Sargent, E. H. Appl. Phys. Lett. 2004, 85, 2089-2091. (13) Coe, S.; Woo, W.; Bawendi, M.; Bulovic, V. Nature 2002, 420, 800-803. (14) Tsutsui, T. Nature 2002, 420, 752-755. (15) Huynh, W. U.; Dittmer, J. J.; Alivisatos, A. P. Science 2002, 295, 24252427. (16) Hikmet, R. A. M.; Talapin, D. V.; Weller, H. J. Appl. Phys. 2003, 93, 35093514. (17) Sun, B.; Marx, E.; Greenham, N. C. Nano Lett. 2003, 3, 961-963. (18) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11-18. (19) Bourel, D.; Rolland, A.; Leverge, R.; Genetet, B. J. Immunol. Methods 1988, 106, 161-167. (20) Adler, J.; Jayan, A.; Melia, C. D. J. Pharm. Sci. 1999, 88, 371-377.
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biomolecules.21,22 Fluorescent silica nanoparticles are prepared by (a) incorporation of a fluorescent dye during particle formation23-32 or by (b) coupling of a fluorescent dye to a particle surface.33-36 Recently, another type of fluorescent silica nanaoparticle, coreshell fluorescent silica nanoparticles (CU dots), was reported.37 A fluorescent dye can be incorporated into particles by simple doping of the dye during particle formation reaction (doping method)23-27,31,32 or by imposition of the dye on the silica network via the formation of bonds between the dye and a silane coupling reagent (imposition method).28-32 For example, silica nanoparticles containing fluorescein or rhodamine conjugated with (3-aminopropyl)triethoxysilane (APS) have been prepared by means of reaction between the amino residue of APS and isothiocyanates conjugated with the fluorescent dye. By means of the imposition method, fluorescent dyes that are difficult to incorporate into silica nanoparticles by the doping method can be efficiently incorporated into nanoparticles. In this paper, we describe a Sto¨ber synthesis of fluorescent silica nanoparticles using more reactive and stable chemical residues, succinimidyl esters conjugated with fluorescent dyes.38 We prepared fluorescent nanoparticles containing various kinds of fluorescent dyes. Multifluorescent silica nanoparticles containing two fluorescent dyes and fluorescent-tuned silica nanoparticles were also prepared and used in biological applications. We characterized our nanoparticles and compared them with commercially available fluorescent nanoparticles such as quantum dots, and we discuss some advantages of our multifluorescent silica nanoparticles. EXPERIMENTAL SECTION Materials. APS and tetraethoxysilane (TEOS) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). 5(6)-Carboxyfluorescein-N-hydroxysuccinimide ester was obtained from Roche Molecular Biochemicals (Tokyo, Japan), DY-495-X/5-N-hydroxysuccinimide ester and DY-635-N-hydroxysuccinimide ester were purchased from Dyomics GmbH (Jena, Germany), and rhodamine red-N-hydroxysuccinimide ester, Q-dot 525, and Q-dot 605 were obtained from Invitrogen (Carlsbad, CA). Fluoresbrite Plain YG (21) Tan, W.; Wang, K.; He, X.; Zhao, X. J.; Drake, T.; Wang, L.; Bagwe, R. P. Med. Res. Rev. 2004, 24, 621-638. (22) Yao, G.; Wang, L.; Wu, Y.; Smith, J.; Xu, J.; Zhao, W.; Lee, E.; Tan, W. Anal. Bioanal. Chem. 2006, 385, 518-524. (23) Vanzo, E. U.S. Patent 4,077,804, 1978. (24) Peterson, J. I. U.S. Patent 4,194,877, 1980. (25) Yabuuchi, N.; Otsuka, C.; Kashihara, A. U.S. Patent 5,367,039, 1994. (26) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Anal. Chem. 2001, 73, 4988-4993. (27) Zhao, X.; Bagwe, R. P.; Tan, W. Adv. Mater. 2004, 16, 173-176. (28) Van Blaaderen, A.; Vrij, A. Langmuir 1992, 8, 2921-2931. (29) Verhaegh, N. A. M.; van Blaaderen, A. Langmuir 1994, 10, 1427-1438. (30) Imhof, A.; Megens, M.; Engelberts, J. J.; de Lang, D. T. N.; Sprik, R.; Vos, W. L. J. Phys. Chem. B 1999, 103, 1408-1415. (31) Lian, W.; Litherland, S. A.; Badrane, H.; Tan, W.; Wu, D.; Baker, H. V.; Gulig, P. A.; Lim, D. V.; Jin, S. Anal. Biochem. 2004, 334, 135-144. (32) Rossi, L. M.; Shi, L; Quina, F. H.; Rosenzweig, Z. Langmuir 2005, 21 (10), 4277-4280. (33) Schwartz, A.; Williams, J.; Stevens, R. D. U.S. Patent 4,609,689, 1986. (34) Bele, M.; Siiman, O.; Matijevic, E. J. Colloid Interface Sci. 2002, 254, 274282. (35) Eiden-Assmann, S.; Lindlar, B.; Maret, G. J. Colloid Interface Sci. 2004, 271, 120-123. (36) Santra, S.; Xu, J.; Wang, K.; Tan, W. J. Nanosci. Nanotechnol. 2004, 4, 590599. (37) Ow, H.; Larson, D. R.; Srivastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U. Nano Lett. 2005, 5, 113-117. (38) Sto ¨ber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62-69.
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Table 1. Synthesis and Evaluation of Fluorescent Silica Nanoparticles
sample
fluorescent dye
dye solution volume (µL)a
diameter (nm)
standard deviation (nm)
F1 F2 F3 F4 R1 R2 R3 R4 D4-1 D4-2 D4-3 D4-4 D6-1 D6-2 D6-3 D6-4 F/D6 T
fluorescein fluorescein fluorescein fluorescein rhodamine rhodamine rhodamine rhodamine DY-495 DY-495 DY-495 DY-495 DY-635 DY-635 DY-635 DY-635 fluorescein/DY-635 none
50 25 12 6 50 25 12 6 50 25 12 6 50 25 12 6 6 0
364 447 424 377 463 413 387 450 307 364 383 394 440 450 433 434 360 417
106 86 84 108 66 25 78 31 57 72 52 83 64 57 87 55 50 66
a The volume of 10 mM APS-fluorescent dye conjugate that was mixed with a mixture of 21 µL of TEOS, 325 µL of ethyl alcohol, 36 µL of NH4OH, and 68 µL of distilled water.
0.2-µm microspheres and Fluoresbrite Calibration Grade YG 0.5µm microspheres were purchased from Polysciences, Inc. (Warrington, PA). Ethyl alcohol and 30% NH4OH were obtained from Wako Fine Chemicals Inc. (Osaka, Japan). OsO4 and paraformaldehyde were purchased from Merck (Darmstadt, Germany), and Epon-812, dodecenyl succinic anhydride, methyl nadic anhydride, and 2,4,6-tris[(dimethylamino)methyl]phenol were purchased from TAAB (Reading, UK). Preparation of Fluorescent Silica Nanoparticles. APSfluorescent dye conjugates were prepared by gently stirring a mixture of 10 mM APS and 10 mM fluorescent dye-N-hydroxysuccinimide ester (5(6)-carboxyfluorescein-N-hydroxysuccinimide ester, DY-495-X/5-N-hydroxysuccinimide ester, DY-635-Nhydroxysuccinimide ester, or rhodamine red-N-hydroxysuccinimide ester) in 100 µL of DMSO for 1 h. The various volumes of APS-fluorescent dye conjugates were mixed with a mixture of 21 µL of TEOS, 325 µL of ethyl alcohol, 36 µL of NH4OH, and 68 µL of distilled water using Sto¨ber’s method38 and incubated with gentle mixing for 1 day. After incubation, the reaction mixture was centrifuged to remove the remaining reagents. The particles were washed thoroughly with 70% ethyl alcohol and water. Electron Microscopy of Fluorescent Silica Nanoparticles. The nanoparticles were fixed on a 400-mesh copper grid coated with nitrocellulose, and transmission electron microscopy images were obtained with a Hitachi H500 or H800 electron microscope (Hitachi, Tokyo, Japan) or a JEOL JEM-1200EXII electron microscope (JEOL Ltd., Tokyo, Japan). Fluorescence and Light Microscopic Analysis of Fluorescent Silica Nanoparticles. Aliquots of the nanoparticle solutions were placed on glass slides and allowed to dry at room temperature; in addition, peritoneal cells containing fluorescent silica nanoparticles were prepared as described below. We analyzed the nanoparticles attached to the glass slides and those contained in the peritoneal cells by means of a fluorescence and light microscopy system consisting of an inverted fluorescence micro-
Figure 1. Transmission electron microscopy images of fluorescent silica nanoparticles. Silica nanoparticles without fluorescent dye (a) and particles containing fluorescein (b), DY-495 (c), or rhodamine red (d) were observed. Scale bars are 500 nm.
scope (TE 2000, Nikon, Kanagawa, Japan) equipped with a 100-W mercury lamp as a light source and a CCD camera (Digital Sight DS-L1, Nikon, Kanagawa, Japan) or by means of a confocal laser scanning microscope with a 400-mW krypton-argon laser (Leica TCS NT, Heidelberg, Germany). To evaluate the photostability of the fluorescence intensity, a fixed area of dye or a single fluorescent silica nanoparticle or Q-dot 605 particle attached to a glass slide was viewed through a CCD camera (Rolera-XR Mono Fast 1394 Cooled, Qimaging, Burnaby, BC, Canada) with continuous excitation and analyzed by Image-Pro Plus software (MediaCybernetics, Silver Spring, MD). Absorbance and Fluorescence Studies. Absorbance and fluorescence spectra of 0.01-0.2 mg/mL solutions of the fluores-
cent nanoparticles were obtained with a U-3000 spectrophotometer (Hitachi) and an F-2500 fluorescence spectrophotometer (Hitachi), respectively. Flow Cytometry. Flow cytometry analysis was performed on FACSCalibur flow cytometers (Becton Dickinson, San Jose, CA) with 488- and 635-nm excitation lasers. Green fluorescence was detected on the FL1 channel (530/30-nm band-pass filter), and red fluorescence was detected on the FL4 channel (661/16-nm band-pass filter). Fluorescent nanoparticles were analyzed, and data were obtained without compensation. Preparation and Transmission Electron Microscopy of Mouse Peritoneal Macrophages Containing Fluorescent Silica Nanoparticles. Male C57 BL/6J mice were injected Analytical Chemistry, Vol. 79, No. 17, September 1, 2007
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Table 2. Comparison of Fluorescence Intensities of Fluorescein-Containing Silica Nanoparticles (Silica NPs-F) and Quantum Dots 525 (Q-dot 525)
concentration average diameter (nm) particle count (counts/mL) measurement (Ex/Em) (nm) intensity intensity/particleb ratio specific intensityc ratio Figure 2. Fluorescence photostability of fluorescent silica nanoparticles, quantum dots, and rhodamine red. Fluorescence microscopy was used to compare the fluorescence stabilities of quantum dots (solid boxes), fluorescent nanoparticles (open circles), and rhodamine red (solid triangles).
intraperitoneally with 0.1 mL of a solution containing 10 mg/mL fluorescent silica nanoparticles per day for 3 days. After 1 week, mice were sacrificed, and the peritoneal cells were harvested and fixed. A portion of the cells was used for fluorescence and light microscopy. Mice peritoneal cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 4 h and then rinsed sequentially with phosphate-buffered saline containing 10, 15, and 20% sucrose for 4 h. The cells were washed, treated with 2% OsO4 and uranyl acetate, dehydrated in a graded series of ethanol solutions, and embedded in Epon epoxy resin. Ultrathin sections (thickness, 80 nm) were cut with a Reichert Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany) and examined with a JEOL JEM-1200EXII electron microscope (JEOL Ltd.). RESULTS AND DISCUSSION Preparation of Fluorescent Silica Nanoparticles. We prepared fluorescent silica nanoparticles by using a new method
silica NPs-F
Q-dot 525
Q-dot 525
0.18 mg/mL 290 6.1 × 109 a
40 nM 20 2.4 × 1013
40 nM 20 2.4 × 1013
495/519
495/519
276/525
7.6 1.2 × 10-9 1 4.2 × 101 1
12.7 5.3 × 10-13 4.4 × 10-4 1.3 × 102 3.0 × 100
200.1 8.3 × 10-12 6.9 × 10-3 2.0 × 103 4.8 × 101
a Concentration (0.18 mg/mL) divided by weight of one particle (2.93 × 10-11 mg). The weight of one particle was calculated from the volume of one particle: 4π(0.000290/2)3/3 (mm3) × 2.3 (specific gravity). b Intensity divided by particle count. c Intensity divided by particle count and then by the volume of one particle.
to impose various fluorescent dyes on a silica network via a succinimidyl ester reaction (Table 1). The yields of final products from starting materials ranged from 30 to 40% (TEOS weight basis). No aggregation of the nanoparticles was observed either visually and by electron microscopy. Various fluorescent dyes including fluorescein, rhodamine red, DY-495, and DY-635 could be imposed in the nanopaticles and showed fluorescent on fluorescent microscopy. Transmission electron microscopy showed that the size distributions and shapes of fluorescent silica nanoparticles containing fluorescein, rhodamine red, and DY-635 were similar to those of fluorescent silica nanoparticles without dye under the same conditions (Table 1 and Figure 1). However, nanoparticles containing a high concentration of DY-495 showed a small size distribution. To rule out the influence of remaining unreacted reagents, we prepared nanoparticles with just DY-495X/5-N-hydroxysuccinimide ester or APS. Neither APS nor DY-
Figure 3. Fluorescence microscopy of fluorescent silica nanoparticles. (A) Fluorescent-tuned silica nanoparticles containing fluorescein (R1R4) were observed under the same conditions. (B) Multifluorescent silica nanoparticles containing DY-635 and fluorescein (a, b), fluorescein (c, d), and DY-635 (e, f) were observed under the conditions for fluorescein (a, c, e) and DY-635 (b, d, f). The gain for observation of DY-635 was twice that of fluorescein. 6510 Analytical Chemistry, Vol. 79, No. 17, September 1, 2007
495-X/5-N-hydroxysuccinimide ester influenced the size of the nanoparticles within the same range of concentrations (data not shown). These results indicate that some APS-fluorescent dye conjugates can affect nanoparticle size. Evaluation of Fluorescence Intensity. The fluorescence intensity of multifluorescent silica nanoparticles containing fluorescein was evaluated and compared with that of Q-dot 525 particles. The fluorescence intensity of Q-dot 525 particles was evaluated under two sets of conditions: the first set of conditions was the same as that used to evaluate the fluorescence intensity of the silica nanoparticles containing fluorescein (excitation, and emission wavelengths were 495 and 519 nm, respectively); the second set was the optimum condition for Q-dot 525 particles (excitation and emission wavelengths were 276 and 525 nm, respectively). The fluorescence intensity of the silica nanoparticles containing fluorescein was higher than that of the Q-dot 525 particles because of the larger size of the silica nanoparticles (Table 2). Next we calculated specific fluorescence intensity, which is the theoretical fluorescence intensity divided by the particle volume. The specific fluorescence intensity of the silica nanoparticles was 1/3 that of the quantum dots under the same conditions and 1/48 that of the quantum dots under the optimum condition. In a previous study,37 core-shell fluorescent silica nanoparticles (CU dots) were prepared and the fluorescence intensity of CU dots containing tetramethylrhodamine isothiocyanate was compared with that of water-soluble CdSe/ZnS quantum dots using two-photon fluorescence correlation spectroscopy (excitation and emission wavelengths were 860 and 577 nm, respectively). The intensity of the CU dots was 1/2-1/3 that of the quantum dots. Our fluorescent particles have two advantages: nanoparticles of various sizes have the same emission wavelength, and nanoparticles of various emission wavelengths have the same size. Therefore, fluorescent silica nanoparticles would be used in experiments that require the same particle size but different emission wavelengths. Currently, the fluorescence intensity of silica nanoparticles is significantly lower than that of quantum dots under the optimum conditions for quantum dots, but improvements in the synthetic method, experimental conditions, and dye may increase the intensity. Using fluorescence microscopy, we compared the photostabilities of fluorescent silica nanoparticles containing rhodamine red, rhodamine red dye, and Q-dot 605 particles. The singleparticle fluorescence intensity of silica nanoparticles (111 351 from 175 889 au) was more photostable than the fluorescence intensity of a fixed area of rhodamine red (116 675 from 1 104 731 au) (Figure 2). Quantum dots showed high photostability, with occasional intensity changes due to photoblinking. After 250 s, the single-particle fluorescence intensities of Q-dot 605 particles and silica nanoparticles containing rhodamine red were 26 288 (from 25 562) and 111 351 (from 175 889) au, respectively, and both fluorescent Q-dot 605 particles and silica nanoparticles were visible. These results indicated that the fluorescent silica nanoparticles had high fluorescence intensity and good photostability and that they exhibited sustained emission without photoblinking. Thus, our nanoparticles may have an advantage in bioimaging experiments requiring high-speed photography. Photostabilities of fluorescent silica nanoparticles have been reported in previous articles. The photostability of CU dots was
Figure 4. Flow cytometry analysis of fluorescent-tuned silica nanoparticles and multifluorescent silica nanoparticles. (A) Fluorescent silica nanoparticles containing fluorescein (shaded peak) were compared with nanosized markers with 0.2- and 0.5-µm diameters (unshaded peaks). (B) Fluorescent-tuned silica nanoparticles containing fluorescein (F1-F4) and nanoparticles without dye (T) were analyzed. (C) Multifluorescent silica nanoparticles containing DY-635 and fluorescein (a), DY-635 only (b), and fluorescein only (c) were analyzed under the same conditions. These findings are superimposed in (d).
evaluated by fluorescence correlation spectroscopy and was greater than that of tetramethylrhodamine dye, but fluorescence decreased under continuous excitation.37 FloDots are dye-doped silica nanoparticles consisting of a luminescent dye dispersed within a silica matrix.22 The photostability of FloDots was evaluated by solid-state spectrofluorometry and was extremely good; that is, no significant change in fluorescence intensity was observed during continuous excitation. However, the evaluation method was different from that of previous reports, and the photostability of our fluorescent silica nanoparticles was similar to that of CU dots. We are currently developing our method to improve the photostability of our fluorescent silica nanoparticles. Analytical Chemistry, Vol. 79, No. 17, September 1, 2007
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Figure 5. Fluorescence microscopy of mice peritoneal cells containing multifluorescent silica nanoparticles. (A) Peritoneal cells labeled with multifluorescent silica nanoparticles containing fluorescein and DY-635 (a-c) and with fluorescent silica nanoparticles containing fluorescein only (d-f) were observed under the same conditions. Observation was performed with excitation at 480/15 nm (a, d), with excitation at 620/30 nm (b, e), or under bright field (c, f). (B) Peritoneal cells labeled with fluorescent silica nanoparticles containing rhodamine red were observed by confocal microscopy as a series of cell cross sections at various z-axis values.
Fluorescent-tuned silica nanoparticles containing various amounts of a fluorescent dye were attached to a glass slide and observed by fluorescence microscopy to evaluate the intensity of each particle. Fluorescent-tuned silica nanoparticles containing rhodamine red (R1-R4) showed different fluorescence intensities; the intensities depended on the amount of fluorescent dye (Figure 3A). These results suggest that the fluorescence intensities of silica nanoparticles are easy to tune. Moreover, fluorescent-tuned silica nanoparticles could be observed clearly by fluorescence microscopy even when their diameters were under 500 nm. Multifluorescent silica nanoparticles containing fluorescein and DY-635 showed fluorescence derived from both fluorescein and DY-635 (Figure 3B). Fluorescent silica nanoparticles containing only fluorescein or DY-635 showed a single fluorescence. Flow Cytometry Analysis. Flow cytometry measures the physical properties of cells or other particles and their fluorescence characteristics. Cells bound to fluorescent dye-conjugated antibod6512 Analytical Chemistry, Vol. 79, No. 17, September 1, 2007
ies were detected and characterized using flow cytometry. Fluorescent nanoparticles may turn out to be more useful than fluorescent dyes for labeling cells. Recently, the multiplexed flow cytometric assay has become popular as a rapid, highly sensitive, and accurate assay for multiple samples of small volume in the same tube.39 Microspheres have been used as a support in the multiplexed flow cytometric assay and also as calibrators. However, the use of nanoparticles for multiplexed flow cytometric assay and for calibrators is not common and might be a new field of nanotechnology in flow cytometry. In flow cytometry experiments, fluorescent silica nanoparticles containing fluorescein (average diameter 300 nm) were directly detectable by their fluorescence upon excitation at 480/15 nm, had high fluorescence intensity, and were uniform (Figure 4A). Compared with the fluorescence intensities of commercially (39) Kellar, K. L.; Iannone, M. A. Exp. Hematol. 2002, 30, 1227-1237.
Figure 6. Electron microscopy of mice peritoneal cells containing multifluorescent silica nanoparticles. Scale bars are 2 µm (a) and 200 nm (b).
available nanosized markers (Fluoresbrite Plain YG 0.2-µm microspheres and Fluoresbrite Calibration Grade YG 0.5-µm microspheres), the fluorescence intensity of silica nanoparticles containing fluorescein (GeoMean of intensity 1311) was closer to that of the marker with the 0.5-µm diameter (GeoMean of intensity 2322) than to that of the marker with the 0.2-µm diameter (GeoMean of intensity 61.8). These results indicate that the specific fluorescence intensity of our silica nanoparticles is higher than that of commercial flow cytometry because the ratio of fluorescence intensity to size of our nanoparticles was higher than that of the commercial markers. Flow cytometry analysis was applied to fluorescent-tuned silica nanoparticles containing fluorescein, multifluorescent silica nanoparticles containing fluorescein and DY-635, and fluorescent silica nanoparticles containing just fluorescein or DY-635. Fluorescenttuned silica nanoparticles containing fluorescein (F1-F4) were detected as peaks with different fluorescence intensities according to the concentrations of fluorescent dye and APS conjugate used to prepare the particles (Figure 4B). Silica nanoparticles containing fluorescein (F1) had almost maximal intensity with the same diameter under the new preparation condition. These results suggest that the fluorescence intensity of a nanoparticle can be tuned and can be detected clearly by flow cytometry analysis. Multifluorescent silica nanoparticles (average particle diameter ∼360 nm) containing fluorescein and DY-635 show two kinds of fluorescence signals upon excitations at 480/15 nm (FL1) and 620/ 30 nm (FL4) ((Figure 4C). Fluorescent silica nanoparticles containing just fluorescein showed a peak on FL1 but not FL4, whereas particles containing just DY-635 showed a peak on FL4 but not FL1. These results are the first findings that a single multifluorescent nanoparticle produces a distinct pair of signals and that a single fluorescent-tuned nanoparticle produces a single fluorescence signal, on flow cytometry analysis. Multifluorescent nanoparticles and fluorescent-tuned nanoparticles are good candidates for multiplexed flow cytometric assays using nanosized beads to measure proteins and DNA and for nanosized calibrators.
In addition, fluorescent nanoparticles with high intensity and multiple fluorescences would be useful for high-sensitivity detection and characterization of cells by flow cytometry. In Vivo Injection of Nanoparticles and Histological Analysis. Mice were intraperitoneally injected with a large amount of multifluorescent silica nanoparticles (3 mg/mouse). Injection resulted in no apparent toxicity, abnormal changes, or death for up to 1 month. Peritoneal cells were harvested after 1 week and examined by fluorescence and light microscopy. Positive cells showing the same fluorescence as that of the injected nanoparticles were observed clearly (Figure 5A). Cells from mice injected with fluorescent silica nanoparticles containing fluorescein showed a single fluorescence under excitation at 480/15 nm, whereas cells from mice injected with multifluorescent silica nanoparticles containing fluorescein and DY-635 showed two kinds of fluorescence under excitation at 620/30 nm. These two kinds of cells were clearly distinguished by their fluorescence patterns. These results suggested that multifluorescent silica nanoparticles could label cells and show specific fluorescence patterns from inside the cells. Peritoneal cells showing fluorescence due to fluorescent silica nanoparticles containing rhodamine red were examined by confocal fluorescence microscopy to determine the distribution of nanoparticles in the cells. A series of cell cross sections at various z-axis values showed the intracellular distribution of nanoparticles (Figure 5B). The nanoparticles were distributed on the surface of the cells and inside the cells, away from the nuclei. In addition, the fluorescence intensities of the nanoparticles of the top slice were not different from those of the bottom slice. These findings indicated that the photostability of the fluorescent silica nanoparticles was good and that these particles are applicable for intracellular distribution analysis by confocal fluorescence microscopy. The peritoneal cells were examined by electron microscopy. Individual cells had phagocytic vacuoles in the cytoplasm (Figure 6a and b) and an irregular surface with pleats and protrusions, a Analytical Chemistry, Vol. 79, No. 17, September 1, 2007
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well-developed Golgi complex, and a prominent rough endoplasmic reticulum (Figure 6a). These findings indicated that the positive cells were peritoneal macrophages and that macrophages performed nonspecific phagocytosis of multifluorescent silica nanoparticles and showed fluorescence from their cytoplasm. Other kinds of cells such as lymphocytes and eosinophils contained no vacuoles, indicating no endocytosis of silica nanoparticles. In addition, no indications of cell necrosis or apoptosis were observed histologically. These results indicate that multifluorescent silica nanoparticles would be useful for tracing two kinds of cells of the same cell type, such as macrophages derived from different mice with different stimulations. Furthermore, multifluorescent silica nanoparticles and fluorescent-tuned silica nanoparticles could be developed as bar code fluorescent silica nanoparticles. Such bar code nanoparticles could be used to prepare bar code cells for tracing various cells at the same time and for bioimaging. CONCLUSIONS We have described a new method for preparing fluorescent silica nanoparticles. In this method, a fluorescent dye is imposed on a silica network via a succinimidyl ester reaction. Our preparation method of fluorescent silica nanoparticles is simple, easy, safe, and cost-effective. We prepared both fluorescent-tuned and multifluorescent silica nanoparticles. Both types of nanoparticles fluoresced brightly, were well dispersed in solution, and had good photostability. In single silica nanoparticles, fluorescence
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wavelength and fluorescence intensity were tunable, and fluorescence was independent of particle size. The fluorescence intensity and photostability of our fluorescent silica nanoparticles were sufficient for detection of a single fluorescent particle; therefore, our nanoparticles are suitable for flow cytometry and fluorescence microscopy analyses. Our nanoparticles exhibited no toxicity on in vivo injection or in labeled cells. Our multifluorescent silica nanoparticles can be employed in many areas of in vitro and in vivo biological and medical analysis. We are currently attempting to improve the fluorescence intensities and photostabilities of our nanoparticles to be equal to those of quantum dots and hoping that these fluorescent nanoparticles will be useful materials for biomedical imaging, bioassay, and nanomedicine. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Younger Scientists (to M.N.), by a Grant for Practical Application of University R&D Results under the Matching Fund Method (to M.N.) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and by a Grant-in-Aid for Scientific Research (C) (to M.N.). The work is under patent pending (WO2006/070582).
Received for review February 26, 2007. Accepted May 26, 2007. AC070394D
