Functionalized Plasmonic−Fluorescent Nanoparticles for Imaging and

Sep 30, 2009 - Arindam Saha, SK Basiruddin, Rupa Sarkar, Narayan Pradhan, and Nikhil R. Jana*. Centre for AdVanced Materials, Indian Association for t...
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Functionalized Plasmonic-Fluorescent Nanoparticles for Imaging and Detection Arindam Saha, SK Basiruddin, Rupa Sarkar, Narayan Pradhan, and Nikhil R. Jana* Centre for AdVanced Materials, Indian Association for the CultiVation of Science, Kolkata-700032, India ReceiVed: May 22, 2009; ReVised Manuscript ReceiVed: September 2, 2009

Plasmonic-fluorescent composite nanoparticles are considered as unique, multifunctional nanoprobes for plasmon- and fluorescence-based imaging and detection. However, their synthesis is challenging due to fluorescence quenching of the fluorophore by plasmonic particles and most of the successful methods produce composite particles of large size (diameter > 50 nm), which limit their wider applications. Here we report 20-30 nm diameter plasmonic-fluorescent composite nanoparticles with reasonable fluorescence quantum yield (12-16%). These particles are composed of 3-6 nm diameter Au/Ag cores and fluorescein-incorporated polymeric shells. They have high water solubility, good colloidal stability, stable fluorescence properties, and are amenable in deriving various functional nanoprobes. Different functional nanoprobes are derived from these composites and successfully used for fluorescence-based cell labeling as well as plasmon-based detection applications. Introduction Nanoparticle-based imaging and detection probes are emerging as alternative molecular probes.1,2 These nanoprobes have bright, stable, and tunable properties, which provide options for making fluorescence/dark field optical probes and the possibility of making composite nanoparticle-based multifunctional nanoprobes.3 Among them, CdSe-ZnS quantum dot-based fluorescent nanoprobes and Au/Ag nanoparticle-based plasmonic nanoprobes are widely studied. While quantum dots are predominantly used as cell labeling reagents,2 Au/Ag nanoparticles are used for plasmonic, SERS-based optical detection probes1b and dark field contrast agents.1c,d However, cytotoxicity of cadmiumbased quantum dot probes4 limit their application and the search for alternative noncadmium-based quantum dots5 is currently receiving intense focus. By contrast, Au nanoparticle-based probes do not have such toxic effects and their plasmonic property-based dark field imaging in cell labeling applications has attracted current attention.1c,d,6 Recently, various composite nanoparticle-based multifunctional nanoprobes have been reported having plasmonic-magnetic, magnetic-fluorescent, or fluorescent-plasmonic properties.3 Such nanoparticles are emerging as powerful probes for the simultaneous fluorescence/dark field/magnetic resonance-based imaging, detection, and magnetic separation. Here, we report on a nanoprobe having the advantage of a quantum dot-like stable fluorescence property as well as a Au/Ag-like plasmonic propertysuseful for fluorescence-based imaging and plasmonic-based detection applications. Plasmonic-fluorescent nanoparticles have been considered as unique multifunctional nanoprobes for plasmon and fluorescence-based imaging and detection.7,8 Two types of plasmonicfluorescent nanoparticles have been reported, Au/Ag nanoparticle-organic fluorophore-based composite7 and Au/Ag nanoparticle-qunatum dot composite.8 Common synthetic methods involve the preparation of core-shell-type composite nanoparticles, where the core consists of one type of nanoparticle and the polymeric shell incorporates another,7f-h,8d by inclusion of plasmonic and fluorescent nanoparticles via layer-by-layer * To whom correspondence should be addressed. E-mail: camnrj@ iacs.res.in.

polyelectrolyte coating8b or linking two types of material by a chemical7a,j and biochemical method.8f However, the fluorescence is often quenched in the composite nanoparticle, as the plasmonic particle acts as a strong quencher. In order to minimize such fluorescence quenching, the separation distance of >5 nm should be maintained between the plasmonic particle and the fluorophore/QD.7f-h,8d-f This critical requirement increases the overall diameter of the composite particle to >50 nm, which often decreases their water solubility and cellular entryslimiting their cell labeling application.7f-h,8d,e Thus, it is very challenging to prepare smaller diameter plasmonicfluorescent composite nanoparticles with good fluorescence quantum yields. We have reported a synthetic method for high quality nanomaterials, such as metal,9 metal oxide,10 and doped semiconductor nanocrystals,5b as well as developing suitable coating chemistry11 for deriving functional nanomaterials. Recently, we have reported a polyacrylate-based coating chemistry that can be used to prepare various functional nanoparticles.11g,h In this work, we have extended this coating chemistry to prepare smaller diameter plasmonic-fluorescent nanoparticles composed of Au/Ag nanoparticles and fluorescein. The synthetic method involves the preparation of 3-6 nm diameter hydrophobic Au/Ag nanoparticles, followed by conversion into polymer-coated water-soluble nanoparticles with the incorporation of fluorescein at the polymer backbone. As fluorescein attachment in the polymer is random, it results in a distribution of distance between Au/Ag and fluorescein and thus only a partial quenching of fluorescence occurs, with a resultant fluorescent particle. The composite particles are 20-30 nm in diameter and functionalized and therefore ideal for cellular imaging applications. Experimental Section Materials. AuCl3, Ag-acetate, tetrabutylammonium borohydride, didodecyldimethyl ammonium bromide, Igepal CO-520, poly(ethylene glycol) methacrylate (Mn ≈ 360), N,N′-methylenebisacrylamide, fluorescein-o-methacrylate, ammonium persulfate, N,N,N′,N′ tetramethyl ethylene diamine, concanavalin A (Con A), D-glucosamine, oleylamine, oleic acid, glutaralde-

10.1021/jp904791h CCC: $40.75  2009 American Chemical Society Published on Web 09/30/2009

Functionalized Plasmonic-Fluorescent NPs hyde and N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) were purchased from Sigma-Aldrich and N-(3-aminopropyl) methacrylamide hydrochloride was purchased from Polysciences. All chemicals were used as-received without further purification. TAT peptide with sequence CGRKKRRQRRR (MW-1499.8) was purchased from GL Biochem (Shanghai) Ltd. with 97% purity. Synthesis of Au and Ag Nanoparticle. Monodispersed Au and Ag nanoparticles of 3-6 nm sizes were prepared in toluene using oleic acid as a particle stabilizer, as previously reported.9b In brief, 10 mL toluene solution of AuCl3 (15 mg AuCl3 dissolved in presence of 25 mg didodecyl dimethyl ammonium bromide) or Ag-acetate (17 mg Ag-acetate dissolved in presence of 0.1 mL octylamine) was mixed with 0.2 mL of oleic acid and kept under magnetic stirring. Then tetrabutyl ammonium bromide solution (25 mg dissolved in 2 mL toluene in the presence of 0.1 mL of oleic acids Caution, rapid evolution of hydrogen gas) was added rapidly and all at once. Pink or yellow color Au/Ag particle formed within a minute. As synthesized nanoparticle solution was purified using precipitation-redispersion methods to remove the excess reagents. In a typical purification process, 2.5 mL of nanoparticle solution was mixed with 2.5 mL of ethanol and then the particles were precipitated by centrifugation at 6000 rpm for 2 min, and the supernatant was discarded. The precipitated particles were finally dissolved in 10 mL of Igepal-cyclohexane reverse micelle (prepared by dissolving 1 mL Igepal in 9 mL cyclohexane) prior to polymer coating. Polyacrylate Coating in Deriving Plasmonic-Fluorescent Nanoparticle. We followed the polyacrylate coating chemistry reported earlier.11g,h Briefly, 10 mL of reverse micelle solution of Au or Ag nanoparticle was mixed with 13 mg N-(3aminopropyl) methacrylamide hydrochloride (dissolved in 100 µL water), 36 µL poly (ethylene glycol) methacrylate (dissolved in 100 µL water), 3 mg fluorescein o-acrylate (dissolved in 100 µL of tetramethyl ethylene diamine) and 3 mg methylene bis acryleamide (dissolved in 200 µL water by 10 min sonication) and optically clear solution was formed. The solution was taken in a three naked flask and put under oxygen free atmosphere by purging nitrogen for 15 min. Finally, ammonium persulfate solution (3 mg dissolved in 100 µL water) was injected as a radical initiator to start the polymerization. The polymerization was continued at room temperature for 1 h and then particles were precipitated by adding few drops of ethanol. The particles were washed with chloroform and ethanol and finally dissolved in 5.0 mL of distilled water. A further purification step was performed to remove unbound fluorescein or polymer bound fluorescein, which was not attached to Au/Ag particle. In a 1-2 mL of aqueous particle solution, ∼100-200 mg solid Na2HPO4 was added and dissolved by shaking until visible particle precipitation was observed. The precipitate was separated from the supernatant that contained unbound fluorescein. The precipitate was dissolved in 1-2 mL of fresh water. This precipitation-redispersion was repeated 2 more times for complete removal of unbound fluorescein. Finally, the stock solution of fluorescein bound Au/ Ag nanoparticles was prepared by dissolving them in 2.0 mL of phosphate buffer solution with a pH of 7.5. A part of the purified sample solution was reacted with fluorescamine solution (prepared by mixing 2 mg in 1 mL acetone) for the confirmation of the presence of primary amine. The green fluorescence changes into bluish green within 1-2 min as observed under UV excitation (see Supporting Information Figure S2).

J. Phys. Chem. C, Vol. 113, No. 43, 2009 18493 Glucose, Oleyl, and TAT Peptide Functionalization of Plasmonic-Fluorescent Nanoparticle. Glucose and oleyl functionalization was achieved using glutaraldehyde as the conjugation reagent.12 Glutaraldehyde conjugates of glucosamine/ oleylamine were prepared first by mixing them in a 1:1 molar ratio and then treating them with an amine functionalized particle solution in a carbonate buffer of pH 10.00. In order to prepare the glutaraldehyde-glucosamine conjugate, 9 mg of glucosamine was mixed with 10 µL of glutaraldehyde in 0.5 mL of aqueous carbonate buffer solution. Similar to the preparation of the glutaraldehyde-oleylamine conjugate, 17 µL of oleylamine was mixed with 10 µL of 50% aqueous glutaraldehyde in 0.5 mL ethanol. After 15 min of preparing these conjugates, they were treated separately with polymer coated nanoparticle solution. The polymer coated nanoparticle solution was prepared in two separate vials by mixing 1.0 mL of polymer coated nanoparticle with 0.2 mL of carbonate buffer solution. In one vial, all of the solution of glutaraldehyde-glucosamine conjugate solution was added and in another vial, 100 µL of glutaraldehyde-oleylamine conjugate solution was added. After one hour, these solutions were mixed with 200 µL of 0.2 M NaBH4 solution to reduce the imine bond formed by reaction between the aldehyde and amine. After one hour, these solutions were dialyzed overnight against distilled water using a 12-14 kDa molecular weight cutoff (MWCO) membrane, to remove unbound reagents. Finally, the particle solution was mixed with a phosphate buffer of pH 7.5 and preserved at 4 °C. TAT peptide functionalization was performed using an earlier protocol.11h A 2 mL amine functionalized polymer coated particle solution was prepared in a phosphate buffer of pH 7.0 and mixed with SMCC solution (1 mg SMCC dissolved in 1 mL dimethylformamide) and incubated for 1 h. Next, nanoparticles were separated from free reagents via Na2HPO4 induced precipitation. Precipitated particles were separated from free reagents, dissolved in fresh phosphate buffer, and then mixed with 100 µL of TAT peptide solutions (2 mg TAT peptide dissolved in 1 mL phosphate buffer solution) and incubated at 4 °C for overnight. Then the solution was allowed for overnight dialysis with MWCO 14000 to separate particles from free peptide. After dialysis, the particle solution was diluted with tris buffer of pH 7.0 and preserved at 4 °C. Biochemical Activity Test of Glucose Functionalzed Nanoparticle. Biochemical activity of glucose conjugated nanoparticles was performed in 0.02 M phosphate buffer of pH 7.5. Glucose functionalized Au/Ag solution was diluted in a phosphate buffer and then 1.0 mL of this solution was placed in separate test tubes to make different sets. Next, varying volumes of Con A solution between 1-100 µL were added to different sets. Con A solution was prepared by dissolving the protein in a 0.2 M phosphate buffer of pH 7.5, which have 10 mM of Ca2+ and Mn2+ ions. In control experiments, BSA solution was used instead of Con A solution. In some additional control experiments, polymer-coated nanoparticles without any glucose were used and mixed with Con A solution. Selective binding of Con A and glucose leads to particle aggregation, which was observed a few minutes after mixing Con A. Cell Labeling. Two different cell lines (COS-7, Neuro2a) grown in a tissue culture flask were subcultured in 24-well tissue culture plates with 0.5 mL of culture medium in each plate. The cells were attached to the tissue culture plate after overnight incubation. Next, they were incubated with 10-100 µL of oleyl/ TAT functionalized plasmonic-fluorescent nanoparticle solution (∼0.1 mg/mL) for 1 h. The cells were then washed with PBS

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buffer, followed by cell culture media in order to remove the unbound probe. Next, labeled cells are used for imaging. Alternatively, Ehrlich ascites carcinoma cells (EAC) were collected from peritoneal cavity of adult female mice after 7 days of inoculation. The solution of cells was prepared with a concentration ∼107 cell/mL. Next, 1.0 mL of this solution was mixed with 10-100 µL of plasmonic-fluorescent nanoparticle solution and incubated for 30 min. Next, labeled cells were separated from free nanoparticle by centrifuging at 2000 rpm for 3 min. The precipitated cells were redispersed in buffer solution. This type of precipitation-redispersion was repeated two more times and finally cells are dispersed in buffer solution. A drop of this solution was placed in a glass slide for imaging experiment. Instrumentation. All UV-vis spectra were measured in Shimadzu UV-2550 UV-vis spectrophotometer using a quartz cell with a 1-cm path length. Fluorescence spectra were measured in Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon) with 1 cm path length quartz cell. Transmission electron microscopic (TEM) images were taken with a GEOL 2010 transmission electron microscope after putting a drop of particle solution in carbon coated copper grid followed by drying of the sample. Fourier transform infrared (FTIR) spectra were measured with a Nicolet 6700 FT-IR (Thermo Scientific) using KBr plates by putting a drop of sample dispersion in chloroform followed by drying of the sample. The fluorescence image of incubated cells was captured using Olympus IX71 fluorescence microscope with DP70 digital camera. Dynamic light scattering (DLS) study was performed using model BI-200SM instrument (Brookhaven Instrument Corporation), after filtering the sample solution with Milipore syringe filter (0.2 µm pore size). Timecorrelated single photon counting (TCSPC) measurement was performed with a Horiba Jobin Yvon IBH Fluorocube apparatus after exciting the sample with 405 nm picosecond diode laser (IBH Nanoled). The fluorescence decay was collected with a Hamamatsu MCP (R3809) photomultiplier, and the fluorescence decay was analyzed with IBH DAS6 software. Au-fluorescein, Ag-fluorescein, and polyacrylate bound fluorescein samples were used for lifetime measurements. Polyacrylate bound fluorescein was prepared in the same procedure described for nanoparticle coating, except that nanoparticles are not added during the polymerization. This polymer was extensively dialyzed against water to remove free fluorescein. Results Synthesis and Purification. The synthetic scheme involves reverse micelle-based polyacrylate coating chemistry for hydrophobic Au and Ag nanoparticle. (Scheme 1) Hydrophobic Au/Ag nanoparticle has been synthesized according to earlier report.9b As-synthesized particles are coated with long chain fatty acid/amine and are highly soluble in chloroform/toluene/ cyclohexane but not in water/ethanol/acetone. For polyacrylate coating, these hydrophobic particles are solubilized in Igepalcyclohexane reverse micelle. Then they are mixed with acrylate monomers and the polymerization has been initiated in oxygen free atmosphere using persulfate initiator. The advantage of using reverse micelle is that hydrophobic nanoparticle and hydrophilic acrylate monomers are solubilized in this medium and coating has been performed in a homogeneous solution. We have used different monomers in an optimum reaction condition so that coated particles have good water solubility, colloidal stability, and fluorescence property. Fluorescein oacrylate is used to have fluorophore conjugated polymer coated particles, aminopropyl methacrylamide is used to have primary

Saha et al. SCHEME 1: Synthesis Steps of Plasmonic-Fluorescent Nanoparticle

amine functionality and water solubility, polyethylene glycol methacrylate is used to have polyethylene glycol functionality and water solubility. A small amount of bis-acrylamide is used to have some percentage of cross-linking between polymers so that the polymer coating can be more stable. The polymer coating condition has been optimized in our earlier works in such a way that it would produce water-soluble coated particles of good colloidal stability.11g,h In the present case, we have followed similar conditions except that fluorescein o-acrylate is used in a small percentage. Using this coating condition, we have synthesized coated particles that have fluorescence coming from fluorescein. However, a significant amount of unbound fluorescein is also present, which is either unreacted fluorescein o-acrylate or polymer bound fluorescein that are not bound to the particle. Dialysis of the as-synthesized water-soluble particle only partially removes such unbound fluorescein. So we have used a salt-induced particle aggregation method for complete removal of unbound fluorescein. These coated particles are water-soluble and positively charged due to the presence of primary amine groups, but the solubility decreases in basic pH and in high salt concentration.11g,h This property of coated particles has been used for purification. We used salt Na2HPO4 for this purpose. When a sufficient amount of Na2HPO4 is added to the aqueous solution of coated particles, the particles precipitate due to the basic pH and high ionic strength of the medium, leaving unbound fluorescein in solution. Thus, a fluorescent supernatant was observed that does not have a plasmon band of Au/Ag. The aggregated particles are separated, and they are again solubilized in fresh water. This process of precipitation-redispersion is repeated 2 more times to remove traces of the unbound fluorescein. Property. The purified coated particle solution is examined by UV-visible and fluorescence spectroscopy to confirm the fluorescein binding to the coated particle (Figure 1). The optical

Functionalized Plasmonic-Fluorescent NPs

Figure 1. Optical property of plasmonic-fluorescent nanoparticle. Fluorescence spectra were obtained by exciting at 475 nm and arbitrary unit was used in fluorescence intensity axis.

property indicates that particle solutions have the signature of surface plasmon due to Au or Ag and fluorescence property of fluorescein, suggesting the formation of plasmonic-flourescent composite nanoparticles. The UV-vis spectra show the surface plasmon band of Au between 500-600 nm and surface plasmon band of Ag near 410 nm. The absorbance of bound fluorescein at ∼500 nm leads to a broadening of Au plasmon band and appears as hump near Ag plasmon band. In the solution of composite particles, this fluorescein absorbance at 500 nm becomes stronger in basic pH but disappears in acidic pH, which is a characteristic feature of pure fluorescein. A fluorescence study shows that the composite particle solution has a fluorescence spectrum similar to pure fluorescein. The digital image of the composite particle shows the color of surface plasmon but under UV-light, it shows the green fluorescence of fluorescein. We have studied the quantum yield (QY) of the composite nanoparticle in order to evaluate the quality of fluorescence in the composite particle. The quantum yield has been calculated assuming 95% QY of fluorescein-o-methacrylate as standard. The concentration of nanoparticle-bound fluorescein is determined from their respective UV-visible spectrum(Supporting Information Figure S1). In brief, the contribution of fluorescein absorbance is calculated by measuring the UV-visible absorption spectra in acidic and basic media and difference in absorbance value at 500 nm is assumed as representative of fluorescein concentration. Using this assumption, the QY is determined as 12% for Ag-fluorescein and 16% for Aufluorescein particles. These values show that there is only a partial fluorescence quenching of fluorescein in the composite particle. The metal nanoparticle usually quenches or enhances the fluorescence of nearby fluorophore, which is distance dependent. If the fluorophore is located at 5 nM concentration, almost all of the cells get labeled. Below 5 nM concentration, labeled cells showed weaker fluorescence, although all of the cells were also labeled. The positive surface charge and multiple oleyl/TAT functionality in each particle induce strong interactions of particles with cell membranes, and thus the nanomolar concentration of the particle is sufficient to observe such labeling under fluorescence microscope. Further experiment was conducted to investigate the fluorescence stability of the particle both in labeled cell and in solution. (Supporting Information Figures S5-S7). Sample solution or labeled cells were exposed under continuous UV irradiation. The result shows that the fluorescence decrease of the sample solution is insignificant and the fluorescence image of the labeled cells fades only partially, even after 5-10 min of UV light exposure. Usually, molecular fluorescecence of fluorescein bleach rapidly within minutes under continuous UV light, and for that reason, the fluorescence image faded upon long-term imaging. In our composite probe, several fluoresceins are attached in each particle and thus bleaching becomes partial and slow. A similar stable fluorescence of fluorescein and other fluorophore conjugated silica particles was observed earlier.14 This result suggests that the fluorescence property of the composite nanoprobe is stable enough for relatively long-term imaging applications. We have also tested the usefulness of the plasmonic property of this composite nanoparticle. Glucose functionalized plasmonic-fluorescent particles show their biochemical activity toward Con A. Con A is a glycoprotein that has four glucose binding sites at pH 7.5. As a result, it induces cross-linking between glucose functionalized particles.15 This effect is seen as visible precipitate formation of particles after Con A addition. The particle aggregation and precipitate formation can be monitored using the surface plasmon of the plasomnic-fluorescent particle (Figure 5). As the particle size is small, the formation of coupled plasmon at the long wavelength due to the particle aggregation is not dominant. However, optically clear solutions turn cloudy, and then a pink/yellow precipitate appears, leaving a colorless supernatant. This result shows that the plasmonic property of the composite particle can also be used for optical detection applications. Discussion The goal of this work was to develop a plasmonic-fluorescent nanoprobe that can be used for plasmon-based detection, as well as fluorescence-based imaging applications. Gold and silver nanoparticles are widely used in optical detection applications, SERS substrates, and as scattering-based dark field imaging contrast agents. This is because Au and Ag nanoparticles have tunable optical properties, due to the presence of surface plasmon.1 In all of these applications, the particle size plays a significant role in different ways. In the 10-100 nm diameter range, the abosrption and scattering from Au and Ag nanoparticles increases rapidly.16 In that respect, larger particles give better optical signals. However, the biolabeling performance of particles >25 nm in diameter is generally poor due to steric effects during biochemical interaction, size-dependent cellular uptake, and nonspecific cellular uptake.4d,17,18 Considering all of these factors, our designed particle with 20-30 nm overall diameter and 3-6 nm Au/Ag core has optimal size. The smaller

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Figure 5. Glucose functionalized plasmonic-fluorescent Au nanoparticles as a plasmon-based optical sensor. UV-vis absorption spectra of a glucose functionalized particle solution before (black) and after Con A addition (pink). Particle aggregates in the presence of Con A, as observed from the colored precipitate, leaving a colorless supernatant.

probe size provides good solubility, better protein binding capability, and a higher cellular uptake property. However, due to the small size of the Au/Ag core, the plasmonic signal is relatively low, but the probe has the option to grow the Au/Ag core after labeling, so that the plasmonic signal and detection sensitivity can be enhanced.11d The surface-plasmon of the Au/Ag nanoparticle is widely used as a sensitive local environmental probe.19 In our probe design, the Au/Ag core is capped with a polyacrylate layer, so that it helps to maintain a distance between the core nanoparticle and the fluorescein, and thus, the fluorescence quenching effect is lessened. However, this polyacrylate coating layer isolates the core from the local environment to some degree, and so the Au/Ag core may not be very sensitive to the local environment. However, this design helps the composite nanoparticle to preserve both the plasmonic and fluorescence properties. Although our designed probe nanoparticle performs well in both plasmon and fluorescence-based imaging and detection applications, it might have other limitations. Fluorescence of composite nanoparticles could be affected differently by an external quencher, because of the different binding environment of fluorescein. To investigate this effect, we studied the fluorescence quenching of composite nanoparticles in the presence of different heavy metals and other ions commonly present in biological environments, such as Ca2+, Mg2+, Fe2+, Fe3+, and Zn2+(Supporting Information Figure S7). We have not observed any significant quenching effect, suggesting that the influence of an external quencher is less important than other effects, such as the ong-term UV-exposure and the quenching effect by the metal core. Use of this probe in single particle imaging or nanometer length scale imaging may not be prudent. It is expected that

J. Phys. Chem. C, Vol. 113, No. 43, 2009 18497 distribution of the fluorescein molecules within the polyacrylate shell around the metal core should produce an inhomogeneous signal and would affect the imaging analysis, particularly at the single particle length scale. This is a clear disadvantage for this probe. However, it would not greatly affect cases in which the image is based on many particles with a micrometer size length scale (scale much larger than single particle). In most practical uses, including the cell imaging presented here, multiparticle and larger length scales would be used in the imaging application. The fluorescence quantum yield and fluorescence stability under UV light is another concern with this probe. Although the fluorescence quantum yield of the composite is 12-16%, and the fluorescence is fairly stable under longer UV exposure times, it is yet to be tested whether the fluorescence is stable under long time laser excitation, commonly used in single particle imaging. Although both the Au-fluorescein and Ag-fluorescein probes have the capability of detection and imaging, their application potential would differ to a certain extent. Ag-fluorescein probes would be the preferred choice over Au-fluorescein when high detection sensitivity is required. This is because Ag-based methods are generally more sensitive than Au-based methods, due to the higher molar extinction coefficient of Ag nanoparticles.20 However, Au-fluorescein probes would be preferred for cellular imaging applications, as Au has a lower toxic effect than that of Ag.1d,21 Conclusions We have synthesized nanoprobes having the advantage of quantum dot-like stable fluorescence properties as well as Au/ Ag-like plasmonic properties. The synthetic method involves the preparation of 3-6 nm diameter hydrophobic Au/Ag nanoparticles, followed by conversion into polymer-coated water-soluble nanoparticles with the incorporation of fluorescein at the polymer backbone. When compared to earlier reports, these plasmonic-fluorescent composite nanoparticles are small (20-30 nm) in diameter and thus ideal probes for cellular imaging applications. Acknowledgment. The authors would like to thank Dr. Nihar R. Jana of the National Brain Research Centre (NBRC), Gurgaon, India for providing the cellular imaging facility. This work is supported by the DST (SR/S5/NM-47/2005) and CSIR of the Government of India. Supporting Information Available: UV-visible spectra of Au-fluorescein and Ag-fluorescein particle solutions used for QY calculation, fluorescence spectra of fluorescamine reaction, DLS size distribution, FTIR data, influence of external fluorescence quencher, and fluorescence stability of the plasmonicfluorescent nanoparticles in solution and in labeled cells. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) (a) Eugenii, K.; Willner, I.Angew. Chem., Int. Ed. 2004, 43, 6042. (b) Rosi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547. (c) Jain, P. K.; Huang, X. H.; El-Sayed, I. H.; El-Sayed, M. A. Acc. Chem. Res. 2008, 41, 1578. (d) Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilani, A. M.; Goldsmith, E. C.; Baxter, S. C. Acc. Chem. Res. 2008, 41, 1721. (2) (a) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (b) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435.

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