Gold-Nanorod-Based Hybrid Cellular Probe with Multifunctional

ARTICLE pubs.acs.org/JPCC

Gold-Nanorod-Based Hybrid Cellular Probe with Multifunctional Properties SK Basiruddin, Amit Ranjan Maity, Arindam Saha, and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata-700032, India

bS Supporting Information ABSTRACT: Hybrid nanoparticles having multimodal imaging/detection and magnetic separation option are considered as a useful probe in understanding cellular function. Here we have synthesized gold-nanorod-based plasmonic-fluorescent, plasmonic-magnetic, and plasmonic-fluorescent-magnetic hybrid cellular probe and showed that they can be used as dualimaging or imaging-separation purpose. In the hybrid probes, the nanorod component acts as dark-field contrast agent, the quantum dot acts as a fluorescence probe, and the magnetic iron oxide offers magnetic separation. The hybrid nanoparticles have good colloidal stability under physiological condition and option for further functionalization with different affinity molecules. Oleyl- and glucose-functionalized hybrid nanoprobes have been synthesized and used as dual imaging probes and for magnetic separation. A variety of other multifunctional nanoprobes can be derived for specific cellular targeting, imaging, and separation application.

’ INTRODUCTION Hybrid nanoparticles composed of more than one nanocomponents are considered as a new class of multifunctional probe with multimodal imaging, imaging-detection, and imagingseparation options.1
6 A variety of hybrid nanoparticles composed of quantum dot (QD)/iron oxide/gold nanosphere are reported showing the advantage of multifunctional property.1
6 For example, QD-iron oxide has fluorescence imaging and magnetic resonance imaging (MRI) option as well as the advantage for magnetic separation;1
3 iron oxide-gold nanoparticle has magnetic separation and SERS-based detection option;4 and QDgold nanoparticle has dual imaging and SERS-based detection option.5,6 Gold-nanorod-based hybrid nanoparticles have attracted much attention because the unique optical property of nanorod can be coupled to the property of other component nanoparticles.7
14 Gold nanorod has length-dependent tunable absorption and scattering properties ranging from visible to nearinfrared.15
21 and this property has been extensively used for surface-enhanced Raman spectroscopy (SERS)-based ultrasensitive detection,18 dark-field imaging,17 and targeted photothermal therapy17,19,20 of cancer cell/bacteria. In nanorod-based hybrids, these properties can be coupled to fluorescence property of QD,14 magnetic property of iron oxide,8,9 and catalytic property of metal.11
13 For example, gold nanorod-magnetic nanoparticle shows the advantage of tunable optical property of nanorod along with the magnetic property.7
9 Gold-nanorodbased hybrids with palladium or platinum component possess plasmonic property of nanorod and catalytic property of palladium/platinum.11
13 Silver particle/layer deposition on gold nanorod modifies the optical property and SERS sensitivity.10,18 r 2011 American Chemical Society

Nanorod-QD hybrid has been used for quenching-based DNA detection.14 The synthetic approach of nanorod-based hybrids involves linking of component particles by conjugation chemistry,9 using nanorod- or polymer-coated nanorod as seed to grow other component particle7,8,11
13 and via specific interaction of oligonucleotide.14 Among them, the seeding-based approach has a very limited scope for the utilization of highquality component nanoparticles. This is because high-quality nanoparticles such as QD and iron oxides are prepared in high temperature where nanorod is unstable.7,8,11
13 The conjugation chemistry and oligonucleotide-based approach have to be optimized for different hybrid systems.9 Therefore, there is a need to develop an efficient approach for nanorod-based hybrids where different types of high-quality nanoparticle can be utilized. We work on the cellular-labeling application of functional nanoparticle,22 and toward this goal, we have developed various coating chemistry for nanoparticle,23 extended those coatings to different types nanoparticles,23
25 transformed those coated nanoparticles into functional nanoparticles via conjugation chemistry, and applied them as cell labels.22,23 We have also developed different approach to prepare multifunctional nanoparticle that includes magnetic-plasmonic and magnetic-fluorescent hybrid nanoparticles by ligands exchange method,26 plasmonic-fluorescent nanoparticle via incorporation of fluorophore on the polyacrylate backbone of the coated particle,24,27 and magnetic-fluorescent nanoparticle via the incorporation of more than one nanocomponent during polyacrylate coating.28 Received: July 13, 2011 Revised: September 2, 2011 Published: September 06, 2011 19612

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The Journal of Physical Chemistry C During those studies, we found that polyacrylate coating developed for hydrophobic nanoparticles can be applied to hydrophilic gold nanorod after appropriate modifications, and surfactantstabilized nanorod can be transformed into functional nanorod as well as fluorescent gold nanorod by incorporation of fluorescein on the coating backbone.24 In addition, the polyacrylate coating can be extended to produce various hybrid nanoparticles using hydrophobic/hydrophilic nanocomponents.28 In this report, we have synthesized gold-nanorod-based different hybrid nanoprobes using ligand exchange and polyacrylate coating approaches. We found that the choice of appropriate coating is necessary in successful preparation of nanorod-based plasmonicmagnetic, plasmonic-fluorescent, and plasmonic-magnetic-fluorescent hybrid nanoprobes with high colloidal stability. Goldnanorod-based different hybrids have been synthesized and used as dual imaging probe or imaging- and separation-based applications.

’ EXPERIMENTAL SECTION Materials. Sodium tetrachloroaurate(III) dihydrate (SigmaAldrich), cetyltrimethylammonium bromide (CTAB, SigmaAldrich), 11-mercaptoundecanol (Sigma-Aldrich), 11-mercaptoundecanoic acid (Sigma-Aldrich), Igepal CO-520 (Sigma), N-(3-aminopropyl) methacrylamide hydrochloride (Polysciences), poly(ethylene glycol) methacrylate (Mn ≈ 360, SigmaAldrich), N,N0 -methylenebisacrylamide (Sigma-Aldrich), ammonium persulfate (Sigma-Aldrich), N,N,N0 ,N0 -tetramethylethylenediamine (Alfa Aesar), D-glucosamine (Sigma-Aldrich), oleylamine (Sigma-Aldrich), glutaraldehyde (Sigma-Aldrich), and N-ethyl-N0 -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma-Aldrich) were used as-received without further purification. Gold Nanorod Synthesis and Thiol Coating. Gold nanorod was synthesized in aqueous CTAB according to the nonseeding method, using a reducing agent mixture consisting of borohydride and ascorbic acid.16 Here borohydride acts for particle nucleation and ascorbic acid helps for particle growth and nanorod length can be controlled by changing the ratio of borohydride and ascorbic acid.16 The ratio of borohydride and ascorbic acid was adjusted in such a way that it produces nanorod with long wavelength at ∼700
900 nm. The nanorods produced by this way have diameter of 6
8 nm and length variation from 25 to 40 nm depending on borohydride and ascorbic acid ratio. After synthesis, nanorods were separated from excess CTAB by centrifuging the solution at 16 000 rpm. The precipitated nanorods were then dissolved in fresh water and ligand exchanged with mercaptoundecanol and mercaptoundecanoic acid. Typically, 200 μL of ethanolic solution of mercaptoundecanol (10 mg dissolved in 1 mL) was added to the 1 mL of aqueous nanorod solution. Next, the solution was sonicated for 5 min, followed by separation of nanorods by centrifuging the solution at 16 000 rpm. Precipitated nanorods were dissolved in 1 mL of ethanol. Next, 200 μL of ethanolic solution of mercaptoundecanoic acid (5.5 mg dissolved in 1 mL of ethanol) was added and kept for overnight. Finally, nanorods were precipitated by centrifuging the solution at 16 000 rpm for 5 min and then dissolved in water or buffer solution. Preparation of Hydrophilic Quantum Dot and Iron Oxide Nanoparticle. Cadmium-based CdSe QD was synthesized by high-temperature pyrolysis of carboxylate precursors of Cd in octadecene.29 CdSe nanoparticles were purified from free ligands

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and coated with ZnS shell at 200 °C in octadecene via the alternate injection of Zn stearate dissolved in octadecene and elemental S dissolved in octadecene.29 Hydrophobic γ-Fe2O3 was prepared by high-temperature degradation of iron(II)-oleate in octadecene solvent, and particle size was controlled between 5 and 25 nm by using a different amount of fatty amine or fatty acid.28 After synthesis, the hydrophobic QD/iron oxide was purified from free ligands using the standard precipitation
redispersion procedure. These hydrophobic particles were then converted into the hydrophilic particle via polyacrylate coating, as previously described.27 The coated particle possesses primary amine and polyethylene glycol on the coating backbone, and the primary amine group has been used for conjugation chemistry.27 Synthesis of Hybrid Nanoparticle via One-Step Polyacrylate Coating. First stock solution of gold nanorod and hydrophobic QD/γ-Fe2O3 were prepared separately from the assynthesized particles. Concentrated gold nanorod solution was prepared from the as-synthesized nanorod solution. Typically, two microcentrifuge tubes having 1.5 mL of as-synthesized nanorod solution were centrifuged at 16 000 rpm; then, all precipitated nanorods were dissolved in 200 μL of 0.2 M of CTAB solution. Similarly, stock solutions of hydrophobic QD/ γ-Fe2O3 were prepared in cyclohexane after the excess surfactant was removed using the conventional precipitation
redispersion method. The amount of QD/γ-Fe2O3 varies between 30 and 70 mg/mL. Next, stock solutions of three different acryl monomers were prepared in Igepal-cyclohexane reverse micelle. Typically, 12 mg of N-(3-aminopropyl) methacrylamide hydrochloride was taken in a 2 mL microcentrifuge tube and dissolved in 100 μL of H2O; then, 0.5 mL of Igepal and 1.4 mL of cyclohexane were added. Optically clear solution of reverse micelle was formed with dissolved acryl monomer. Similarly, another two acryl monomer solutions were prepared separately in different microcentrifuge tubes. Typically, 36 μL of poly(ethylene glycol) methacrylate was dissolved in 100 μL of H2O, and 3 mg of methylenebisacryleamide was dissolved in 200 μL of H2O by sonication. Next, 0.5 mL of Igepal and 1.4 mL of cyclohexane were added to each tube and mixed thoroughly to produce optically clear solutions. Next, solutions of three acryl monomer were mixed and transferred to a three-necked flask and kept under magnetic stirring conditions. Then, 200 μL of concentrated gold nanorod solution was added, followed by 100 μL of N,N,N,N-tetramethylethylenediamine and stirred for ∼10 min. Next, 100
200 μL of hydrophobic QD, γ-Fe2O3, or both was added; then, the reaction mixture was purged with nitrogen for 15 min to make the reaction mixture completely O2-free. After that, ammonium persulfate solution (3 mg dissolved in 100 μL of H2O) was added as radical initiator. The reaction was continued for 1 h and then quenched by the addition of a small amount of ethanol that induced particle precipitation. The precipitate was washed with chloroform for three times and dried well. After that, precipitate was dissolved in double-distilled water. Synthesis of Hybrid Nanoparticle Using Thiol Coated Gold Nanorod. In this approach, thiol-coated gold nanorod and polyacrylate coated QD/γ-Fe2O3 were used for the preparation of nanorod-based hybrids. The carboxylate group on the surface of thiol-coated nanorod and primary amine group of polyacrylate-coated QD/γ-Fe2O3 surface were covalently linked via EDC-based coupling chemistry. Typically, 1 mL of QD/γFe2O3 solution was taken in phosphate buffer (pH 7.0); then, 19613

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Scheme 1. Different Synthetic Strategy for Gold-Nanorod-Based Hybrid Nanoprobe

nanorod solution was added dropwise. The addition of nanorod solution was stopped before any visible precipitation starts so that most of the QD/γ-Fe2O3 remained in solution. Next, 0.1 mL of NHS (3 mg/mL) and 0.1 mL of EDC (3 mg/mL) were added, and reaction mixture was kept for overnight. Next, high-speed (10 000
16 000 rpm) centrifuge was used to separate the nanorod hybrid from free QD/γ-Fe2O3. Because QD/γFe2O3 does not precipitate under such condition, unbound QD/ γ-Fe2O3 was removed by separating the precipitate from supernatant. Precipitate was dissolved in fresh water, and this precipitation
redispersion step was performed three times for complete separation of free QD/γ-Fe2O3. Finally, particles were solubilized in 1 mL of buffer solution. Functionalization and Cell Labeling. Nanorod-based hybrids were further functionalized with glucose and oleylamine. Glucose and oleyl functionalization were achieved via glutaraldehyde-based conjugation chemistry using glucosamine and oleylamine, respectively. Primary amine group of glucosamine/ oleylamine was covalently linked to primary amine-terminated hybrid nanoparticle using two terminal aldehyde groups of glutaraldehyde, as previously reported.27 The conjugation experiments were performed using a 0.1 M carbonate buffer of pH 10.0. First, 0.1 mM of glucosamine/oleylamine was mixed with an equivalent amount of glutaraldehyde in 0.5 mL of aqueous carbonate buffer solution of pH 10.0. After 15 min, 0.2 mL of this solution was mixed with a hybrid nanoparticle solution that was prepared by mixing 1.0 mL of hybrid nanoparticle solution with 0.2 mL of carbonate buffer solution. After 1 h, this solution was mixed with 200 μL of NaBH4 (0.2 M) solution to reduce the imine bond formed by reaction between aldehyde and amine. After 1 h, this whole solution was dialyzed overnight at 4 °C against deionized water using 12
14 kDa molecular weight cutoff (MWCO) membrane to remove unbound reagents. Finally, particle solution was mixed with phosphate buffer of pH 7.0 and preserved at 4 °C. HepG2 and H9C2 cells were grown in a tissue culture flask and then subcultured in a 24-well plate tissue culture plate, with

Figure 1. Optical property of gold-nanorod-QD and gold-nanorodQD-γ-Fe2O3 hybrid nanoparticle in aqueous PBS buffer of pH 7.4. Fluorescence spectrum is obtained by exciting at 380 nm.

0.5 mL of culture medium in each plate. In dark-field microscopic imaging study cells were cultured on a circular coverslip placed under tissue culture plate. After overnight, the cells were attached to the tissue culture plate/coverslip. Next, they were incubated 19614

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Figure 2. Transmission electron microscopic (TEM) images of polyacrylate-coated QD and γ-Fe2O3, thiol-coated gold nanorod, and different hybrid nanoparticles composed of gold nanorod. Small particles in composite are either QDs or γ-Fe2O3. Red and black arrows indicated QD and γ-Fe2O3, respectively.

with 10
100 μL of functionalized hybrid nanoparticle solution for 4 to 5 h. The cells were then washed with PBS buffer and used for imaging study. Instrumentation. UV
visible absorption spectra were recorded using Agilent 8453 spectrophotometer in a 1 cm quartz cell. Fluorescence spectra were measured using a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon). FEI Technai G2 transmission electron microscope was used for transmission electron microscopy (TEM) studies. Samples were prepared by placing a drop of the diluted particle solution on carboncoated copper grid. Time-correlated single photon counting (TCSPC) measurement was performed with 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 fluorescence decay was analyzed with IBH DAS6 software. Bright-field and fluorescence imaging were performed using Olympus microscope IX81 with DP70 digital camera. Dark-field imaging was performed using an inverted Zeiss microscope (Axio Observer. A1) with a dark field condenser and an oil immersion objective. In this setup, the condenser delivered a narrow beam of white light, and the oil immersion objective collected only the scattered light from the sample. The image was captured using a colored camera (ProgRess C3), and ProgRess Capture Pro 2.7.7 software was used for image acquisition and for adjusting the white light balance.

’ RESULTS Synthesis of Hybrid Nanoparticles. Different strategies for the synthesis of gold-nanorod-based hybrids are summarized in Scheme 1. Gold nanorod is first synthesized by a solution chemical nonseeding method in the presence of cetyltrimethyl ammonium bromide (CTAB) as shape-inducing surfactant.16 Next, CTAB-capped nanorod is transformed into thiol-coated nanorod by ligand exchange method. First, mercaptoundecanol is used for ligand exchange that replaces CTAB and produces ethanol-soluble nanorod. Next, mercaptoundecanols present on the nanorod surface are partially replaced with mercaptoundecanoic acid via similar ligand exchange method. The resultant nanorod is coated with mercaptoundecanol and mercaptoundecanoic acid having both the alcohol and the carboxylate functionality at the outer surface and offers water solubility. Similarly, high-quality hydrophobic ZnS-coated CdSe QD is synthesized via high-temperature organometallic route;29 then, it is converted to water-soluble QD using the polyacrylate coating method,23 which introduce both primary amine and polyethylene glycol at the outer surface. A similar approach has been used to prepare polyacrylate-coated γ-Fe2O3. Next, hybrid nanoparticles are prepared using CTAB-coated gold nanorod, thiol-coated gold nanorod, hydrophobic QD/γ-Fe2O3, and polyacrylate-coated hydrophilic QD/γ-Fe2O3. We have tried a different approach to derive gold-nanorodbased hybrid so that the method can be simple and efficient in 19615

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The Journal of Physical Chemistry C preparing high-quality hybrid nanoparticle (Figures 1 and 2). First, we have tried a simple one-step polyacrylate coating approach using the mixtures of component nanoparticle. This approach is very useful for nanorod-γ-Fe2O3 but not suitable for nanorod-QD. We found that nanorods react with QD during

Figure 3. Change of gold nanorod surface plasmon by successive addition of 5 μL of QD solutions in 2 mL of Au nanorod solution. CTAB-capped gold nanorods are dissolved in Igepal-cyclohexane reverse micelle; then, hydrophobic QD (top panel) or polyacrylate-coated QD (bottom panel) are added. Spectra are measured after 5 min of each successive addition of hydrophobic QD and after 24 h of each successive addition of polyacrylate-coated QD. Arrow indicates a decrease in nanorod plasmon band with increasing QD concentration. This type of reaction of nanorod by QD can be stopped if one of the component particles is coated with thiol.

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hybrid nanoparticle formation, which results in the loss of nanorod optical property as well as quenching of QD fluorescence. Figure 3 shows that gold nanorod surface plasmon is completely vanished when the nanorod and QD are present together in reverse micelle solution. The reactivity of nanorod is significantly slowed if the as-synthesized hydrophobic QD is converted to polyacrylate-coated QD and then mixed with nanorod or both QD and γ-Fe2O3 are used together to prepare nanorod-QD-γ-Fe2O3 hybrid. Interestingly, if one of the component particles is capped with thiol, then the nanorod reactivity to QD can be stopped almost completely. This observation prompted us for an alternative synthetic scheme that uses thiol-based coating for one of the component particles in the synthesis of nanorod-QD hybrid (Scheme 1). In this approach, we have prepared thiol-coated gold nanorods and polyacrylate-coated QD separately and then linked them by conjugation chemistry (EDC coupling). Alternatively, QD can be thiol-coated and then linked to polyacrylate-coated nanorod, but we have not tried this option because of known weaker binding of thiols with QD surface as compared with gold nanorod surface. There are three key steps in this synthetic design of hybrid nanoparticle. The first step is the preparation of thiolated nanorod with both surface carboxylic acid and hydroxyl groups that offer water solubility and colloidal stability of nanorod-QD conjugate. The second step is avoiding the polyvalent interaction-based precipitation reaction between the carboxylate-functionalized anionic nanorod and amine-functionalized QD during the EDC coupling step. This is achieved by dropwise mixing the nanorod solution into an excess of QD solution so that each nanorod is surrounded by a maximum number of QDs that minimize the nanorod-QD aggregate formation. In this mixture of nanorod and QD, both particles are colloidally stable, and EDC coupling is performed in this stage. The third step is removing unbound QD from nanorod-QD

Figure 4. Optically clear aqueous solution of different hybrid nanoparticles, which can be precipitated by adding salt and redispersed after removing salt by dialysis. Magnetic hybrids are separable using bar magnets. 19616

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The Journal of Physical Chemistry C hybrid by centrifuging the mixture at a speed where hybrid particles precipitate but QD remains in solution. The hybrid nanoparticles prepared by this method are highly water-soluble, have both primary amine and polyethylene glycol on their surface, and have the optical property of both the gold nanorod and QD. This approach can also be extended to preparation of nanorod-QD-γ-Fe2O3 using a mixture of polymer-coated γ-Fe2O3 and QD during the EDC coupling with thiolated nanorod. Property of Hybrid Nanoparticles. The absorption spectra of the hybrid nanoparticles show the dominant absorbance of gold nanorod, having both longitudinal and transverse surface plasmon band, which is usually observed for gold nanorod,20 and fluorescence spectra show the presence of a sharp band of QD (Figures 1 and 4). With the naked eye, the composite color appears violet because of the color of nanorod but appears green/ orange/red (depending on QD size) under the UV light. The nature of the fluorescence spectrum of the QD remains the same in hybrid nanoparticles compared with the pure QD. However, the UV
visible spectrum of Au nanorod is altered in the hybrid compared with the pure Au nanorod. Both the longitudinal and transverse plasmon band red shift and broaden. The transverse plasmon peak of Au nanorod red shifts by ∼10 nm, whereas the longitudinal plasmon peak red shifts by ∼25 nm in the hybrid nanoparticle compared with the pure Au nanorod. The hybrid nanoparticles have good colloidal stability in the PBS buffer of pH range 7
9 with salt concentration of 0.1 M. The hybrid nanoparticles can be precipitated in saturated solution of phosphate salt and by adjusting at pH 10. Using this condition, we can precipitate the hybrid nanoparticle and redisperse them through dialysis that removes salt. The composition of polymer and inorganic component in various hybrid nanoparticles has been determined using TGA analysis. Polymer present in hybrid particle varies from 40 to 60 wt %, which is evident from mass loss at 200
300 °C. This composition is similar to our previous observation of polyacrylate-coated gold nanorod and magnetic QD.24,28 Hybrid nanoparticles are observed as a rod shape in the TEM image, where only inorganic nanoparticles are visible without showing polyacrylate shell due to their low electron density. Because hybrid nanoparticles consist of Au nanorod of 40 nm  10 nm surrounded by spherical QD/γ-Fe2O3 of 4
8 nm, the final TEM size becomes rod-shaped. (Figure 2). However, as-synthesized and thiol-coated gold nanorods have CTAB/thiol coating (thickness