Contrasting Effect of Gold Nanoparticles and Nanorods with Different

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Contrasting Effect of Gold Nanoparticles and Nanorods with Different Surface Modifications on the Structure and Activity of Bovine Serum Albumin Soumyananda Chakraborty,†,^ Prachi Joshi,‡,§,^ Virendra Shanker,‡ Z. A. Ansari,§ Surinder P. Singh,*,‡,|| and Pinak Chakrabarti*,† †

Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India § Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India Department of Engineering Science and Materials, University of Puerto Rico, Mayaguez Campus, PR 00680, United States

)

‡

bS Supporting Information ABSTRACT: Nanoparticles exposed to biofluids become coated with proteins, thus making protein
nanoparticle interactions of particular interest. The consequence on protein conformation and activity depends upon the extent of protein adsorption on the nanoparticle surface. We report the interaction of bovine serum albumin (BSA) with gold nanostructures, particularly gold nanoparticles (GNP) and gold nanorods (GNR). The difference in the geometry and surface properties of nanoparticles is manifested during complexation in terms of different binding modes, structural changes, thermodynamic parameters, and the activity of proteins. BSA is found to retain native-like structure and properties upon enthalpy-driven BSA
GNP complexation. On the contrary, the entropically favored BSA
GNR complexation leads to substantial loss in protein secondary and tertiary structures with the release of a large amount of bound water, as indicated by isothermal calorimetry (ITC), circular dichroism (CD), and Fourier transform infrared (FTIR) and fluorescence spectroscopies. The esterase activity assay demonstrated a greater loss in BSA activity after complexation with GNR, whereas the original activity is retained in the presence of GNP. The formation of large assemblies (aggregates) and reduced average lifetime, as evidenced from dynamic light scattering and fluorescence decay measurements, respectively, suggest that GNR induces protein unfolding at its surface. The effect of temperature on the CD spectra of BSA
GNP was found to be similar to that of pristine BSA, whereas BSA
GNR shows distortion in CD spectra at lower wavelengths, strengthening the perception of protein unfolding. High binding constant and entropy change for BSA
GNR complexation determined by ITC are consistent with large surfacial interaction that may lead to protein unfolding. The present work highlights the differential response of a protein depending on the nature of the nanostructure and its surface chemistry, which need to be modulated for controlling the biological responses of nanostructures for their potential biomedical applications.

’ INTRODUCTION Besides various applications based on physicochemical properties, nanotechnology also offers a great hope for biology and medicine.1 Several engineered nanoparticles are being considered for drug delivery, imaging, diagnostics, and therapeutics.1,2 Their small size enables them to penetrate the cell via different pathways and to interact with various macromolecules of r 2011 American Chemical Society

comparable size. As soon as the particles get into the biological fluid, serum proteins associate with them and form a surrounding protein “corona”.3 This corona defines the biological identity of Received: March 1, 2011 Revised: May 5, 2011 Published: May 17, 2011 7722

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Langmuir the particles within the biological system rather than stand-alone particles, revealing the critical role of proteins in deciding the fate of nanoparticles. Protein adsorption on solid surfaces is of particular interest.3 The large surface to volume ratio of nanoparticles provides a large surface for protein binding even if the nanoparticles are in small amount. The nature of interaction depends upon the physicochemical properties of proteins as well as on the nature of the solid surface such as surface area, curvature, hydrophilicity, and functional groups present on it.4
6 Previous studies on nanoparticle
protein interactions suggest that adsorption of protein on a solid surface is an ensemble of many events such as protein conformational change, the transformation into a molten-globule-like intermediate state of protein, coadsorption of other ions, and the release of existing molecules/ions, which in turn results into entropy gain/free energy change.5
8 Protein stability resulting from the interactions need to be investigated in order to understand the ensuing cellular responses. Lysozyme has been reported to retain a considerable amount of nativelike secondary and tertiary structure when adsorbed on small silica or zinc oxide nanoparticles.5,9 It has been shown that, when using larger nanoparticles (of silica) with greater surface area and lower curvature, there is a higher degree of perturbation on the structure and activity of ribonuclease A.6 It is hard to deliver a generalized statement for protein
nanostructure interactions. Probably it is a result of an ensemble of various parameters that have significant impact on protein
nanoparticle interactions. Essentially, to be useful for biomedical applications, nanoparticles in general should not have any adverse effect on the structure and activity of the biological milieu. Serum albumins have been used as a model protein for carrying out biophysical studies to understand binding to various kinds of ligands. These proteins play an important role in the transport and disposition of a variety of endogenous and exogenous ligands/substances in blood.10 Also, albumins are abundant in the circulatory system of variety of organisms and have important physiological functions, including the control of serum osmotic pressure, pH buffering, and so forth.11 The pharmacological behavior of an administered material in terms of the distribution, availability and metabolism strongly depends upon their interaction with plasma proteins in the bloodstream.10,12 Therefore, albumin is an ideal protein to study protein
nanoparticle interactions. Native bovine serum albumin (BSA) is a globular protein which may undergo structural changes upon interaction with solid surfaces and denaturing agents. It has two tryptophan (Trp) residues located at 134 and 212 positions.13 Trp134 is present in domain IA at a solvent exposed region, whereas Trp212 is located inside the hydrophobic patches of domain IIA. BSA represents a model of serum carrier protein with a sequence identity of 76% with human serum albumin.14 Gold nanoparticles (GNP), due to their unique physicochemical properties, exhibit tremendous potential for biomedical applications from the detection of protein/DNA interactions to drug delivery and cancer therapeutics.2,15 The interest for gold lies in its inert and nontoxic nature, controllable size and ease of functionalization with desired ligands.16 A number of studies on the interaction of GNP with proteins indicated the complexity of protein
nanoparticle interaction.17 Different proteins were observed to have different modes of binding and exhibited different degrees of conformational changes; for example, the complexation of GNP with R-chymotrypsin is enthalpy-driven, whereas

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with histone and cytochrome-c the interaction is entropydriven.4 Protein adsorption on GNP provides stability to the nanoparticles by preventing aggregation and also imparts biocompatible functionality for further biological interactions.18,19 Apart from GNP, gold nanorods (GNR) having large absorption cross section in near-infrared (NIR) frequencies have been demonstrated for use in localized hyperthermia for cancer therapeutics.20 There is a cursory report of the interaction of GNR with BSA.21 Size and shape dependent internalization of gold nanostructures inside the cell is a current hotspot of research.22
24 These may enter the cells via the receptor mediated endocytosis, with the adsorbed BSA on the particle surface dictating these uptake events.22,23 It has been shown that the internalization of spherical GNP in cells is at a much higher rate in comparison to GNR, and the difference in the surface chemistries between the spherical and rod-shaped gold nanoparticles may be one of the reasons for the difference in uptake.22 An understanding of the interaction of BSA with different nanostructures (with the same or different surface coating) would pave way to the elucidation of the mechanism of serum protein mediated internalization process. Here we report a comparative study of the interaction of GNP and GNR of different size, shape, and surface chemistry with BSA under similar experimental conditions at physiological pH, 7.4. The goal was to understand the thermodynamics of interaction of GNP and GNR with BSA to elucidate the resulting conformational or structural changes and impact on protein activity, and to study the effect of these nanostructures on the morphology of the aggregates induced by heat. Any conformational change in protein may cause loss of biological activity or elicit altered immune response and our results would have implications for bioapplications involving nanoparticles.

’ EXPERIMENTAL SECTION Materials. Gold(III) chloride hydrate (99.999%), sodium borohydride (g98%), cetyltrimethylammonium bromide (CTAB, 98% ((C16H33)N(CH3)3Br)), and ascorbic acid were purchased from Sigma-Aldrich and used as received. Bovine serum albumin (BSA) was purchased from Sigma and used without further purification. All other reagents used in these experiments were of analytical grade and purchased from Merck, India; double distilled water was used through out the experiments. Synthesis of GNP and GNR. For the preparation of GNP, 20 mL of aqueous 0.1 mM gold chloride hydrate (HAuCl4 3 xH2O) was reduced by 12 mM ice-cold aqueous sodium borohydride (NaBH4) solution with continuous stirring. GNR were synthesized by seed-mediated growth (using CTAB) of GNP.25 A small amount of silver ion is helpful in increasing the aspect ratio. However, to avoid the incorporation of any Ag on the surface, the use of AgNO3 was substituted by the variation of pH and CTAB concentration.26 The obtained GNR solution was washed properly to remove unbound CTAB. The separation of nanoparticles and nanorods was achieved by centrifugation.27 The concentration of gold nanorods was calculated using the molar extinction coefficient of the longitudinal surface plasmon resonance band (4.2 ( 0.5  109 M
1 cm
1 at 750 nm) of gold nanorods.58 The separated rods when dispersed in 5 mL of aqueous medium gave an approximate concentration of 2.3 nM. This was again centrifuged and redispersed in 23 μL aqueous medium to achieve a stock solution of 100 μM strength. Nanoparticles Characterization. Optical Spectroscopy. The formation of gold nanoparticles and gold nanorods was verified by determining optical density from UV-vis-near IR absorption spectroscopy. 7723

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The characteristic surface plasmon band of colloidal gold nanoparticles is observed in the visible range; however, gold nanorods have a surface plasmon band in the near-infrared region with a coexisting weaker band in the visible region. The optical density spectra were recorded using an Ocean-Optics HR4000 spectrometer equipped with a Toshiba TCD1304AP linear CCD-array detector that enables optical resolution as precise as 0.02 nm (fwhm). The optical response is taken from the 300
1000 nm range in a 10 mm path quartz cuvette. Electron Microscopy. The particle size and dispersity of the prepared nanoparticles were studied using transmission electron microscopy (TEM). TEM grids were prepared by placing 4 μL of the diluted and well sonicated sample solutions on a carbon-coated copper grid and evaporating the solution at room temperature completely. The bright field electron micrographs were acquired with a JEM 2010 electron microscope at the accelerating voltage of 200 kV. Contaminants such as dust particles and not properly washed glass vials were strictly avoided.

Sample Preparation for GNP and GNR Conjugated BSA. BSA protein solution (concentration 5 μM) was exhaustively dialyzed using a dialysis membrane (Spectra biotech membrane, MWCO = 3500, Spectrum Lab, Gardena, CA) against buffer solution at 4 C. A buffer solution, consisting of 0.1 M of sodium phosphate at pH 7.4, was used in all the experiments. To study the interaction between GNP, GNR with BSA, a fixed amount of the GNP/GNR was added to the protein solution, mixed by vortexing, and incubated at room temperature for 2 h. BSA aggregation was studied by incubating the sample in a water bath which maintains a constant temperature of 65 C. The protein/NP (GNP, GNR) ratios in all these samples were 25:1, 50:1, 75:1, 100:1, 150:1, and 500:1, respectively. The incubation was for 4 h. Portions of the incubated sample solutions were removed periodically and placed in Eppendorf tubes and monitored by characterization experiments. For longer storage before measurement, the Eppendorf tubes with protein samples were frozen at
20 C. Both freshly prepared and incubated sample solutions were used in all subsequent characterization experiments Circular Dichroism (CD) Spectropolarimetry. The CD spectra were obtained using a JASCO-810 spectropolarimeter equipped with a thermostatically controlled cell holder. The temperature of the sample was controlled at 25 ( 0.1 C. Protein concentration was taken as 5 μM for all the experiments. The far-UV region was scanned between 200 and 260 nm with an average of three scans and also a bandwidth of 5 nm. The final spectra were obtained by subtracting the buffer contribution from the original protein spectra. The CD results were expressed in terms of mean residual ellipticity (MRE) in deg 3 cm2 3 dmol
1. ½θ ¼ ½θobs =10ncp l

ð1Þ

where [θ]obs is the observed ellipticity in millidegrees, n is the number of amino acid residues (585), cp is the molarity, and l is the path length of the cell (path of light) in centimeters. The temperature dependent CD measurement was carried out with similar parameters Fourier Transform Infrared (FTIR) Spectroscopy. FTIR scanning was preferred in the transmission mode with constant nitrogen purging, and IR grade KBr was used as scanning matrix. The samples of BSA containing GNP and GNR were vacuum-dried, and 1
2 mg of fine sample powder was mixed with 90
100 mg of KBr powder. The mixer was dehumidified completely using a drybox and then transferred to a 13 mm die to make a nearly transparent and homogeneous disk. To characterize aggregates, these were washed with D2O three times and suspended in minimum volume of D2O. Samples were diluted 20 times before the measurement. The final spectra were collected after subtracting the background spectra of D2O. FTIR spectra were recorded on a Perkin-Elmer spectrometer equipped with a DTGS KBr detector and a KBr beam splitter. All spectra were taken at 4 cm
1 resolution, using 20 scans in the range from 1400 to 1800 cm
1

Isothermal Titration Calorimetry (ITC). ITC measurement was performed on a VP-ITC calorimeter (Microcal Inc., Northampton, MA). BSA was dialyzed extensively against 0.1 M sodium phosphate buffer, and the ligand (GNP and GNR) was dissolved in the last dialysate. A typical titration involved 15 injections of the GNP/GNR (titrant) (10 μL aliquot per injection from a 100/50 μM stock solution) at 5 min intervals into the sample cell (volume 1.4359 mL) containing BSA (concentration, 10/5 μM). In order to ensure full occupancy of binding sites in BSA, sequential titrations were carried out by adding ligand to the sample protein solution at 25 C. The titration cell was stirred continuously at 310 rpm. The heat of the ligand dilution in the buffer alone was subtracted from the titration data (both normalized to 0) for each experiment. The data were analyzed to determine the binding stoichiometry (N), affinity constant (Ka), and other thermodynamic parameters28 of the reaction using the Origin software. The reported thermodynamic quantities were the average of two parallel experiments. Esterase Activity. Esterase activity of BSA was determined with the synthetic substrate p-nitrophenyl acetate by following the formation of p-nitrophenol at 400 nm, using a Shimadzu 2401 spectrophotometer. The reaction mixtures contained 50 μM p-nitrophenyl acetate and 20 μM protein in 0.1 M phosphate buffer, pH 7.4 at 37 C. A molar extinction coefficient for p-nitrophenol of ε = 17 700 M
1 3 cm
1 was used for all the calculation. One unit of esterase activity was defined as the amount of enzyme required to liberate 1 μM of p-nitrophenol per minute at 37 C. Dynamic Light Scattering (DLS). The hydrodynamic radii of nanoparticles in solution were determined using DLS with high performance particle sizer (Zetasizer nanoS, Malvern) at 25 C. BSA (concentration 0.005 mM) in phosphate buffer of pH 7.4 was incubated with 0.005 mM of nanoparticles. DLS measurements were carried out using a Zetasizer nanoS instrument (Malvern Instrument, U.K.). Samples were excited with a 633 nm wavelength laser. The scattering intensities were recorded at a 173 angle in kilocounts per second. Fluorescence Spectroscopy. All the fluorescence measurements were carried out using a Hitachi F3000 spectrofluorimeter with 10 μM protein concentration. For fluorescence quenching measurements, GNP was added to the protein from a 0.1 mM stock solution. The excitation wavelength was set as 295 nm to selectively excite tryptophan residues, and the emission scan was monitored in the range of 310
400 nm with the fixed slit width of 5 nm. The fluorescence intensities were determined at the λmax, and inner filter correction and data analysis were done using the Stern
Volmer equation. Fluorescence lifetime of BSA was calculated using a time correlated single photon counter from Edinburgh instrument, U.K. The decays were recorded at excitation of 303 and emission of 340 nm and using an emission polarizer at a magic angle of 54.7. Decay curves were fitted using the following equation GðtÞ ¼

∑i Bi expð
t=τi Þ

where G(t) is the fitted decay curve of the sum of all exponential terms and Bi is the pre-exponential factor for the ith component. The biexponential nature of the fluorescence decay was judged from the reduced χ2 values (1
1.15). Thioflavin T Spectroscopy. A stock solution of thioflavin T was prepared by dissolving the powder in 1 PBS (phosphate buffered saline) and subsequently stored at 4 C and protected from light until use. Protein solution was diluted and mixed with 20 μM thioflavin T. Fluorescence was measured with a Horiba Hitachi spectrofluorometer using a 10 mm quartz cuvette in the range of 460
700 nm, using an excitation wavelength of 450 nm. Emission and excitation slit widths were typically 5 nm. All fluorescence intensity measurements were background-corrected with a 20 μM thioflavin T solution. Scanning Electron Microscopy and X-ray Diffraction. Aggregated suspensions of BSA were applied to rectangular glass slides. 7724

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The samples were washed twice then air-dried and finally fixed at the glass surface. The scanning electron micrograph of the sample was taken using a FEI Quanta 200 instrument operating at an accelerating voltage of 5 kV. Samples were diluted 20- to 200-fold when necessary before deposition on the grids. Microanalysis of the scanning electron microscopy (SEM) samples was also performed to avoid the presence of impurities. X-ray diffraction (XRD) of the BSA aggregate was taken by using a powder X-ray diffractometer (Rigaku Miniflex-II) at room temperature. The samples were scanned between 2 and 30.

’ RESULTS AND DISCUSSION GNP and GNR used by us have different surface modifications. In seed mediated GNR synthesis, CTAB is essential as it forms bilayers over the particles which govern the anisotropic growth of GNRs. Complete removal of CTAB from the GNR surface is very difficult.29 It causes the aggregation of GNR.23,24 Additionally, capping GNR by different ligands makes the surface heterogeneous.22,30 Of the two reduction methods of preparation of GNP, we have used NaBH4 over citrate because the former imparts less electronegative charge on the gold surface,31,19 which makes GNP behave almost like bare particles without having any adsorbed agent. It may be mentioned that we have also used NaBH4 as the reducing agent in the preparation of GNR seeds. Borohydride or borate generally behaves as an inert ion, and it is not known to denature BSA. The effect on BSA structure that we study here would be the result of both the difference in chemistry as well as the shape of the nanostructure. Because of the limitations on the synthesis of GNR, differently capped nanosturctures (spherical and rod) have also been used for cell based experiments.22,23,32 Synthesis of GNP and GNR, Their Physical Characteristics, and Changes Resulting from BSA Binding. GNP has been

prepared by the reduction of gold chloride in aqueous medium using sodium borohydride. This causes Au3þ ions to be reduced into neutral gold atoms which after precipitation form nanoparticles. For the preparation of GNR, small gold particles are used as seeds for the deposition of gold atoms that are reduced from gold salt using a mild reducing agent, ascorbic acid (Supporting Information Figure S1). Cetyltrimethylammonium bromide (CTAB) is used as the structure guiding agent or soft rodlike micellar template for the growth of nanorods, as it allows unidirectional particle growth along the (111) plane. The presence of the positively charged CTAB at the surface stabilizes nanorod size and dispersity, and it itself presents as a surfactant bilayer on the rod surface.33 The suspension was subjected to ultracentrifugation, and excess CTAB was washed out. The collective oscillation of conduction band electrons results in an exceptionally high optical absorption coefficient known as surface plasmon resonance (SPR).34 For spherical GNP, the SPR band appears in the visible region, while for GNR it appears in the infrared region accompanied by a weaker one in the visible region of the electromagnetic spectrum.33,35 Figure 1a shows the SPR bands of GNP and GNR dispersed in aqueous medium. GNP exhibits a sharp SPR band at 522 nm, whereas GNR exhibits a weak transverse band at 517 nm and a sharp and intense longitudinal band at 750 nm, consistent with previous studies.33,35,36 The observed absorption bands suggest the successful formation of spherical and rod-shaped gold nanoparticles. The visual images of GNP and GNR solutions in borosilicate glass tubes are also shown in Figure 1a. High-resolution transmission electron microscopy (HRTEM) showed that the average size

Figure 1. Physical characteristics of gold nanoparticles (GNP) and nanorods (GNR). (a) Representative surface plasmon resonance (SPR) absorption band and (b, c) transmission electron micrographs. Inset of (a) shows the prepared GNP and GNR in aqueous medium.

of GNP is ∼3
4 nm and the average width and length of GNR are 8 ( 1 and 20 ( 1 nm, respectively (an aspect ratio of 1:2.5) (Figure 1b,c and Supporting Information Figure S2). Binding of BSA to GNP and GNR is expected to affect the SPR absorption band as the coating of the gold surface with any type of dielectric material leads to a change in the refractive index and the SPR frequency.36 Figure 2a demonstrates the decrease in absorbance intensity as well as an increase in full width half maxima (fwhm) of the SPR band of GNP after BSA conjugation. The absorbance intensity of the peak at 522 nm diminishes progressively with increasing concentration of BSA (from 0 to 50 μM). The conjugation of BSA to GNR leads to the suppression in transverse SPR peak at 517 nm without any significant shift in peak position (Figure 2b). However, the longitudinal SPR mode of GNR at 750 nm is found to be affected considerably and indicates that the (111) plane is crucial in BSA
GNR complexation. Structure of BSA on Binding to GNP and GNR. Far-UV CD spectroscopy has been used to evaluate the structural changes induced in BSA on complexation with GNP and GNR. The negative peaks appearing at 208 and 222 nm in CD spectra are characteristics of the R helical structure of BSA, and any variation in these bands will indicate conformational change in the native structure.37 The negative ellipticity values of BSA at these wavelengths are decreased slightly in the presence of GNP (Figure 3a), suggesting minor conformational changes in R helical structure. This is in agreement with the previous reports that BSA could retain most of its helical structure on conjugation to gold nanoparticles.18,37 On the other hand, a significant decrease in the ellipticity values is observed for BSA
GNR conjugate showing that the extent of structural perturbation of BSA is more on adsorption on GNR as compared to GNP. These 7725

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Figure 2. Absorbance (SPR) of (a) GNP and (b) GNR solution after the addition of (1) 0, (2) 10, (3) 25, and (4) 50 μM of BSA.

Figure 3. Characteristic spectra of BSA (5 μM in 0.1 M sodium phosphate buffer, pH 7.4) in the absence and presence of gold nanoparticles (GNP) and gold nanorods (GNR). (a) Far-UV CD spectra and (b) FTIR spectra.

observations clearly demonstrate the strong role of the particle size and shape, along with surface chemistry, in inducing conformational change in proteins. We measured the number of protein molecules adsorbed on the nanostructures (Supporting Information Table S1). Given the size difference between the two structures, it is expected that more protein would be adsorbed on GNR than on GNP. Interestingly however, the degree of surface coverage is 3.6% for GNR, as opposed to 12.3% for GNP. It shows that BSA denatured by GNR is less likely to be adsorbed on the surface of the structure. We also measured the temperature-dependent CD data to determine the thermal stability of the conjugates (Supporting Information Figure S3). For BSA alone, a gradual decrease in ellipticity can be seen with increasing temperature, indicating the loss in secondary structure as a result of temperature-induced protein unfolding. The melting temperature (Tm) is found to be 62 C, which changes to 58 C on conjugation with GNP. In contrast, the CD spectra for BSA
GNR are found to be distorted in the lower wavelength region, and provide a Tm value of 45 C. Taken together with the results presented in Figure 3a, it can be concluded that BSA loses its structure substantially on conjugation to GNR, which gets aggregated further on increasing temperature (above 64 C). The secondary structure perturbations are also confirmed by FTIR spectroscopy (Figure 3b). Among the different bands of protein, those corresponding to amide I and amide II provide information on conformational changes. The amide I band (CdO stretch) has correlation with the secondary structure of

protein and appears in the region of 1600
1700 cm
1, whereas the amide II band arising from the coupling of C
N stretch to N
H bending and indicative of the amount of protein adsorbed on surface emerges in the 1500
1600 cm
1 region.38 The amide I band has contributions from different secondary structural elements, such as 1620
1645 cm
1 by β-sheet, 1645
1652 cm
1 by random coil, 1652
1662 cm
1 by R-helix, and 1662
1690 cm
1 by turns.9,39 The peaks around 1650 and 1657 cm
1 observed for free BSA, attributed to coil and helical content, get only a small shift and appear around 1648 and 1656 cm
1 with decreased intensity in GNP-conjugated BSA, indicating the retention of the structure. For BSA
GNR, these peaks are no more distinguishable and only one peak is identified around 1651 cm
1. However, both BSA
GNP and BSA
GNR revealed a slightly shifted and less intense amide II peak around 1536 and 1535 cm
1 compared to 1542 cm
1 for free BSA, suggesting the adsorption of the protein onto the gold surface. However, only in the case of GNR, the protein loses the helical content significantly. Time Resolved Fluorescence Decay. The fluorescence characteristics of protein fluorophore tryptophan (Trp212) in subdomain IIA of BSA provide a convenient way for investigating protein structure and binding.8,18 The average lifetime as well as components of lifetime of Trp are sensitive to the surrounding environment, which significantly affect the population distribution of the exited states of the emitting fluorophores.40 Consequently, time-resolved fluorescence lifetime is an important tool to measure protein conformational change on binding to other 7726

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Table 1. Fluorescence Decay Parameters of BSA on Binding to GNP and GNR fluorescence lifetime component sample

τ1 (ns)

τ2 (ns)

relative abundance of lifetime component τ1

τ2

statistical deviation χ

2

average lifetime τ1 (ns)

BSA

2.9

6.7

16%

84%

1.076

6.1

BSA
GNP

3.2

6.9

16%

84%

1.131

6.30

BSA
GNR

2.21

4.95

36%

64%

1.28

3.92

Figure 4. ITC data from the titration of (a) 10 μM BSA in the presence of 100 μM of GNP and (b) 5 μM BSA in the presence of 50 μM GNR. Heat flow versus time during injection of GNP and GNR at 25 C and heat evolved per mole of added nanoparticles (corrected for the heat of GNP and GNR dilution) against the molar ratio (nanoparticles/BSA) for each injection are shown at the top and bottom, respectively. The data were fitted to a standard model. (The data corresponding to the heat of dilution of nanostructures are shown in Supporting Information Figure S8).

moieties. The fluorescence decay of BSA shows a biexponential curve with relative fluorescence lifetimes of 2.9 ns (16%) and 6.7 ns (84%) and an average lifetime of 6.09 ns (Table 1). The fluorescence decay curve for BSA
GNP remains biexponential with lifetime components, 3.2 ns of relative abundance 16% and 6.9 ns of relative abundance 84%. The average lifetime is observed to be 6.3 ns. The lifetime values for BSA
GNP are quite close to those for BSA, and there is no significant change in the percentage of relative abundance. On the other hand, the fluorescence decay measurement of BSA
GNR shows lifetime components of 2.21 and 4.95 ns of relative abundance 36% and 64%, respectively. Although, the decay for BSA
GNR remains biexponential, there is a noticeable decrease in lifetime components observed with largely varied relative abundances. As a result, the average lifetime is also decreased to a value of 3.9 ns. The decay results show larger conformational changes in protein structure after GNR conjugation leading to a change in lifetime components and their relative population in the excited states. Thermodynamics of Binding. Isothermal titration calorimetry (ITC) offers a powerful tool to investigate the thermodynamics and the stoichiometry of protein
nanoparticle interactions, by quantifying the change in enthalpy, entropy, and Gibbs free energy during the binding process. Depending on the type of the nanostructure, we obtained two different profiles of heat change

for the BSA
GNP and the BSA
GNR conjugation (Figure 4) At the top of both figures, the isothermal titration calorimetric thermograms for raw signals are displayed, while at the bottom the heat flow per mole of the titrant (GNP or GNR) versus mole ratio of protein (GNP or GNR/BSA) is plotted. The derived thermodynamic parameters are presented in Table 2. The binding of GNP with BSA is found to be enthalpically as well as entropically favored (i.e., ΔH < 0, ΔS > 0), with overall favorable negative Gibbs free energy change. The negative value of enthalpy is consistent with the characteristics of weak van der Waals and/or electrostatic interactions, and the positive value of entropy indicates hydrophobic interactions during protein
ligand complex formation.41 The stoichiometry is close to unity, and the binding constant is of order 105, showing moderate binding between GNP and BSA. On the other hand, the complexation of GNR with BSA is found to be endothermic (ΔH > 0) in nature. However, the interaction is favored by a large amount of positive entropy change (TΔS). Hence, an unfavorable enthalpy change is compensated by a favorable entropy change, resulting in an overall negative Gibbs free energy change (ΔG < 0). The binding constant for the interaction between GNR and BSA is about 2 orders of magnitude higher than that between GNP and BSA. Activity of BSA. The retention of the BSA activity is of particular importance for any biological application involving 7727

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nanoparticles. The evaluation was done using the well established esterase activity of BSA using para-nitrophenol. It is observed that BSA retains ∼88% of its original activity in the presence of GNP, while only 5% of the activity is retained with GNR conjugation (Figure 5a). These experiment measurements clearly imply that BSA maintains most of its activity in BSA
GNP conjugates, which is a reflection of the retention of the native structure by BSA. On the contrary, BSA loses most of its activity on BSA
GNR complexation, which can be attributed to the breakdown of the native structure. The loss of BSA activity may be an impediment for the biological application of GNR. Dynamic Light Scattering (DLS) Measurement. The binding of BSA with GNP and GNR has also been monitored by DLS measurements (Figure 5b). DLS measures hydrodynamic radii of particles in solution as opposed to TEM which gives particle size in dried samples. Hence, the size measured using DLS are more significant for samples to be used as biomedicines or drug delivery vehicles. The average size of BSA was observed to be ∼6 nm. After binding to GNP, the size of BSA
GNP conjugates was found to be ∼28 nm. On the other hand, DLS of GNR bound BSA shows peak around 68 nm accompanied with a smaller broad peak at ∼400 nm. The kinetics of BSA
GNP conjugation shows a steady state behavior for the first 40 min (Supporting Information Figure S4), followed by a more than 3-fold increase in the conjugate size for the next 20 min, reaching a plateau after 1 h. Thus, on standing, there is mutual interaction between conjugates resulting in larger Table 2. Thermodynamics of the Binding of GNP and GNR to BSA value ((SD) parameters

BSA
GNP

BSA
GNR

N (nanoparticle/protein

0.65 ((0.04)

0.55 ((0.006)

stoichiometry) Ka (binding constant, M
1)

7.37 ((2.18)

1.17 ((0.17)

 105 ΔH (binding enthalpy, cal/mol)


6.33 ((0.61)  103

ΔS (entropy change, cal/mol 3 K) TΔS (entropy change, cal/mol 3 K)

ΔG (free energy change, kcal/mol)

 107 1.63 ((0.03)  106

5.97

5402

1.8  103

1.64  106


8.13


10

assemblies of BSA
GNP (Supporting Information Figure S5). While our estimate of the size increase of 4 nm GNP is 41 times, an earlier study reported an increase of 33 times.8 It may be mentioned that, in contrast to the aggregation behavior observed in DLS, the UV
visible absorbance kinetics of GNP-BSA conjugation does not indicate any observable scattering (at 340
360 nm) over time due to aggregation. It is possible that the laser used in DLS experiments (633 nm) may initiate the aggregation process, and laser-mediated GNP aggregation is already known.42,43 BSA Aggregation. By using various spectroscopic techniques such as CD, FTIR, and fluorescence lifetime measurements, we have established that BSA is unfolded on binding to GNR. This is further corroborated by results from thermodynamic measurements, which indicate large water release during complexation, and also from the observation on size increment from DLS measurements. It is possible that BSA gets unfolded at the GNR surface and further interacts with native and other unfolded proteins to make larger sized conjugates. Recently, it has been shown that nanoparticles (NP) can significantly increase the rate of protein fibrillation/aggregation.44 The protein aggregation by NP is a two step process; the first step is the protein unfolding on the nanoparticle surface, followed by the hydrophobic patch assembly. In an analogous manner, as GNR induces the unfolding of BSA, we wanted to see if it can further promote the aggregation of the protein. BSA can form two types of aggregates. Generally, at 65 C, it forms regular β-amyloid structure, whereas at higher temperature (85 C) it forms large visible amorphous aggregate.45 With a protein/GNR ratio above 100:1, visible aggregates were formed within 2 min of sample incubation at 65 C (Figure 6a). Visible aggregates are not seen at a lower ratio, as has also been seen involving a hybrid quantum dot.44 We characterized the aggregates by FTIR and found a major peak at 1647 cm
1 which is indicative of the formation of random coil (Figure 6b).9 Further, a hump at 1630 cm
1 is suggestive of β-sheet formation. We further characterized the aggregates using SEM and XRD. SEM clearly revealed that the majority of aggregates are irregular in nature (Figure 6c
f). A small proportion of fibril-like structures was also observed (Figure 6d). Interestingly, in most of our images, we found some smaller spherical beadlike aggregates. These non-amyloid beadlike aggregates are presently getting much attention because it is believed that these states of aggregates have a key role in cell death as well as in protein fibrillation process.46,47 XRD is a very

Figure 5. Effect of GNP and GNR on the activity and hydrodynamic radii of BSA. (a) Relative esterase activity (%) of free BSA and BSA bound to GNP and GNR. (b) DLS measurements of BSA, BSA
GNP, and BSA
GNR, showing the intensity of distribution of their hydrodynamic radii (nm). 7728

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Figure 6. Aggregation of BSA on incubating with GNR at 65 C. (a) Visual image of BSA aggregates: the clear solution of BSA at 65 C (eppendorf on right) gives BSA aggregates in presence of GNR (left). (b) FTIR spectra of BSA aggregates at room temperature. (c
f) SEM images of different types of BSA aggregates formed in presence of GNR: (c) large amorphous aggregates, (d) fibers, and (e, f) small spherical beadlike aggregates.

powerful tool to identify protein aggregates. The XRD profile of protein aggregates shows one strong reflection peak around 22 and a weak peak around 11 with interplanar spacings (dspacings) of 3.94 and 7.3 Å, respectively. Both the characteristic peaks have lesser d-spacing than any conventional β-amyloid (Supporting Information Figure S6). We finally confirmed the presence of nonregular aggregates as the dominant species using thioflavin T fluorescence. Thioflavin T is a fluorescence dye which shows a very weak fluorescence in normal buffer; on binding to protein fibrils, its flouresence intensity increases sharply. Our result clearly indicates that GNP does not alter the rate of BSA fibrillation, in contrast to GNR which promotes the formation of large amorphous aggregates (Supporting Information Figure S7a). Tryptophan fluorescence data showed a large reduction in fluorescence intensity in BSA
GNR when the temperature was raised to 65 C (Supporting Information Figure S7b), indicating a major alteration in protein tertiary structure. Model of Structural Changes in BSA and the Formation of Aggregates Induced by GNR. Solvent reorganization, which is greatly associated with large entropy changes, is an important part of protein
nanoparticle complexation.48 It involves desolvation of protein and nanoparticles and solvation of newly formed protein
nanoparticle complexes that results in the release of some bound water molecules during complexation. Disruption of solvent hydration shells acquires an endothermic process (ΔH > 0 and ΔS > 0).48 Therefore, large positive entropy change observed for BSA
GNR complexation (Table 2) is attributed to the release of a large quantity of water of hydration during reaction as shown in eq 2. BSA 3 xH2 O þ GNR 3 yH2 O T ½BSA
GNR 3 ðx þ y
zÞH2 O ð2Þ þ zH2 O

CTAB is a cationic surfactant which is known to disrupt the structure of BSA on binding, which is mainly hydrophobic in nature.49
51 It is also known to induce aggregation. For BSA
GNR, losing structural content as evident from CD, FTIR, and fluorescence lifetime measurements is indicative of protein unfolding at GNR surfaces. It is believed that BSA gets unfolded at GNR surfaces which further interact with native and other unfolded protein to make a larger sized conjugate (Supporting Information Figure S5). Thus, unfolding resulted in the loss of structure and activity of BSA on GNR conjugation. Putative Binding Site of GNP on BSA. Intrinsic Trp fluorescence quenching is useful for obtaining information on the site of binding in protein
ligand interactions. The change in maximum fluorescence intensity (Imax) of Trp arises from the adsorption of protein onto nanoparticles. The effect of GNP binding on Imax of Trp in BSA is illustrated in Figure 7a
there is quenching that increases progressively with GNP concentration. The binding between BSA and GNP is further investigated using the Stern
Volmer equation (eq 3).52 F0 =Fc ¼ 1 þ KSV ½GNP ¼ 1 þ Kq τ0 ½GNP

ð3Þ

where F0 and Fc denote the steady-state fluorescence intensities in the absence and in the presence of the quencher (GNP), respectively, KSV is the Stern
Volmer quenching constant, [GNP] is the concentration of the quencher, Kq is the bimolecular quenching constant, and τ0 is the lifetime of the fluorophore. The quenching rate constant Kq for BSA
GNP is calculated to be 3.3  1012 M
1 s
1 using τ0 and KSV values of 10
8 s (from lifetime) and 3.3  104 M
1, respectively, in eq 3. The observed value of Kq for BSA
GNP conjugate is greater than the literature value of the maximum scattering quenching constant, that is, 2  1010 M
1 s
1, indicating that the Trp quenching in BSA
GNP is a static rather than a dynamic 7729

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Figure 7. GNP-binding and tryptophan fluorescence in BSA. (a) Quenching of tryptophan (Trp212) fluorescence by varying concentrations of GNP and (b) the Stern
Volmer plot derived from the quenching data. The equation of the fitted line is F0/Fc = 1.0337 þ 0.0323  [GNP] (R2 = 0.94). (c) Displacement of BSA
bound warfarin (5 μM) by GNP using fluorescence spectroscopy. Fluorescence spectrum corresponding to warfarin is shown in blue. (d) Cartoon representation of GNP approaching BSA domain IIA (made using pymol, http://www.pymol.org). Trp212 shown in red with marked domains around it, and GNP is shown in pink color.

event,53 something that can also be concluded from the average lifetime of BSA remaining essentially unaltered in presence of GNP (Table 1). The Stern
Volmer plot showed a straight line with increasing concentration of GNP (Figure 7b), also supporting the existence of a single quenching mechanism. The quenching of Trp212 fluorescence (Figure 7a) which is located at site I of subdomain IIA suggests that GNP is binds to site I of BSA.13 To further investigate the binding site of GNP on BSA, we used warfarin as a model drug with a known binding site. Warfarin (3-(R-acetonylbenzyl)-4-hydroxycoumarin) binds to protein at site I in subdomain IIA. We have observed warfarin displacement from BSA in the presence of GNP (Figure 7c); this indicates that both warfarin and GNP share the same binding site in BSA (Figure 7d). The subdomain IIA has Sudlows’s binding site I characterized by the presence of a central cavity formed from six amphipathic helices arranged in a myoglobin-like fold having Trp212 in the vicinity. The site selective binding studies of a variety of drugs and ligands to BSA have demonstrated that the binding affinity offered by site I is mainly through hydrophobic interactions.54

being caused by the release of large amount of water from the environment of hydrophobic region of CTAB-coated GNR. In line with an earlier study,23 we also find that GNR forms large aggregates on interaction with BSA (Figure 5), which could hamper in the cellular uptake. (2) BSA plays a key role in receptor mediated cell endocytosis, leading to the internalization of nanoparticles.22 In addition to denaturing BSA, GNR has 3.5 times lesser surface protein coverage as compared to GNP (Supporting Information Table S1). The recognition of the GNR
protein conjugates by cell receptors would be less, causing lower internalization of GNR. (3) As CTAB is the material of choice for the anisotropic growth of GNR, the biocompatibility of GNR is likely to be limited. Although GNR is being used for therapy against cancer and other malignant diseases,56,57 it is evident that further study needs to be done with modified gold surfaces under contending conditions to arrive at nanostructures that are safe to be used as nanomedicine.

’ ASSOCIATED CONTENT

bS ’ CONCLUSIONS In this paper, we show how different types of gold nanostructures, GNP and GNR, with different surface modifications, can elicit different effects on the structure and activity of proteins, BSA in particular. The salient features are as follows. (1) While the protein structure is unaltered in the presence of GNP, GNR causes a breakdown. This renders the protein inactive (Figure 5). GNP appears to bind at site I of BSA (Figure 7). The binding of GNP with BSA is exothermic in nature, as is the case with many other nanoparticles;9,55 however, it is uniquely endothermic for GNR (Table 2). For the latter, the interaction is entropy driven,

Supporting Information. Additional experimental details are given in eight figures and two tables. Figure S1 provides a schematic representation of the synthetic methodology of nanostructures; Figure S2 is the distribution of the aspect ratios among the synthesized GNRs; Figure S3 shows the effect of temperature on CD signal of BSA, BSA
GNP ,and GNR; Figure S4 presents the distribution of the hydrodynamic radii of BSA
GNP conjugates at different time intervals; Figure S5 is the cartoon representation of the pathways for the formation of GNP/GNR induced BSA assemblies; Figure S6 gives X-ray diffraction pattern of BSA aggregates induced by GNR; Figure S7 shows time evolution of thioflavin T and Trp fluorescence intensity of BSA alone and in presence of GNP and GNR

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Langmuir at 65 C; Figure S8 is the raw ITC data for the dilution of nanostructues. Table S1 provides the degree of surface coverage of protein over nanostructure; Table S2 explains the data analysis for isothermal titration calorimetry. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (S.P.S.); [email protected] (P.C.). Author Contributions ^

Equal contribution.

’ ACKNOWLEDGMENT P.J. and S.C. acknowledge the Council of Scientific and Industrial Research for research fellowships. P.C. is supported by JC Bose National Fellowship. S.P.S. acknowledges NSF Grant No. HRD 0833112 (CREST program) and IFN start up Grant OIA-0701525. ’ REFERENCES (1) De, M.; Ghosh, P. S.; Rotello, V. M. Adv. Mater. 2008, 20, 4225–4241. (2) Sperling, R. A.; Gil, P. R.; Zhang, F.; Zanella, M.; Parak, W. J. Chem. Soc. Rev. 2008, 37, 1896–1908. (3) I. Lynch, I.; Dawson, K. A. Nano Today 2008, 3, 40–47. (4) De, M.; You, C. C.; Srivastava, S.; Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 10747–10753. (5) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Langmuir 2004, 20, 6800–6807. (6) Shang, W.; Nuffer, J. H.; Dordick, J. S.; Siegel, R. W. Nano Lett. 2007, 7, 1991–1995. (7) Lindman, S.; Lynch, I.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Nano Lett. 2007, 7, 914–920. (8) Lacerda, S. H. D. P.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. ACS Nano 2010, 4, 365–379. (9) Chakraborti, S.; Chatterjee, T.; Joshi, P.; Poddar, A.; Bhattacharyya, B.; Singh, S. P.; Gupta, V.; Chakrabarti, P. Langmuir 2010, 26, 3506–3513. (10) Jisha, V. S.; Arun, K. T.; Hariharan, M.; Ramaiah, D. J. Am. Chem. Soc. 2006, 128, 6024–6025. (11) Uversky, V. N.; Narizhneva, N. V.; Ivanova, T. V.; Tomashevskis, A. Y. Biochemistry 1997, 36, 13638–13645. (12) Banerjee, T.; Singh, S. K.; Kishore, N. J. Phys. Chem. B 2006, 110, 24147–24156. (13) He, X. M.; Carter, D. C. Nature 1992, 358, 209–215. (14) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (15) Sokolov, K.; Follen, M.; Aaron, J.; Pavlova, I.; Malpica, A.; Lotan, R.; Richards-Kortum, R. Cancer Res. 2003, 63, 1999–2004. (16) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Small 2005, 1, 325–327. (17) Gomes, I.; Santos, N. C.; Oliveira, L. M. A.; Quintas, A.; Eaton, P.; Pereira, E.; Franco, R. J. Phys. Chem. C 2008, 112, 16340–16347. (18) Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, 2714–2721. (19) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21, 9303–9307. (20) Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J. X. Adv. Mater. 2007, 19, 3136–3141. (21) Pan, B.; Cui, D.; Xu, P.; Li, Q.; Huang, T.; He, R.; Gao, F. Colloids Surf., A 2007, 295, 217–222. (22) Chithrani., B. D.; Ghazani., A. A.; Chan., W. C. Nano Lett. 2006, 6, 662–668.

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