Biodirected Synthesis and Nanostructural Characterization of

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Bio-directed synthesis and nanostructural characterization of anisotropic gold nanoparticles Germán Plascencia-Villa, Daniel Torrente, Marcelo Marucho, and Miguel José-Yacamán Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00084 • Publication Date (Web): 05 Mar 2015 Downloaded from http://pubs.acs.org on March 11, 2015

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Bio-directed synthesis and nanostructural characterization of anisotropic gold nanoparticles Germán Plascencia-Villa, Daniel Torrente, Marcelo Marucho and Miguel José-Yacamán.

Department of Physics & Astronomy, The University of Texas at San Antonio (UTSA). One UTSA Circle, San Antonio, Texas 78249, USA. [email protected]

ABSTRACT Gold nanoparticles with anisotropic structures have tunable absorption properties and diverse bio-applications as image contrast agents, plasmonics and as therapeutic-diagnostic materials. Amino acids with electrostatically charged side chains possess inner affinity for metal ions. Lysine (Lys) efficiently controlled the growing into star-shape nanoparticles with controlled narrow sizes (30-100 nm) and produced in high yields (85-95%). Anisotropic nanostructures showed tunable absorbance from UV to NIR range, with extraordinary colloidal stability (-26 to 42 mV) and surface-enhanced Raman scattering properties. Advanced electron microscopy characterization through ultra-high resolution SEM, STEM and HR-TEM confirmed the size, nanostructure, crystalline structure and chemical composition. Molecular dynamics simulations revealed that Lys interacted preferentially with Au(I) through the -COOH group instead of their positive side chains with a binding free energy (BFE) of 3.4 kcal mol-1. These highly monodisperse and colloidal stable anisotropic particles prepared with biocompatible compounds may be employed in biomedical applications.

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INTRODUCTION One of the major challenges in nanotechnology is the design of nanoparticles with high affinity, stability and non-toxicity for bio-applications. Recent studies show that the structural and physicochemical properties of nanoparticles such as zeta-potential, surface charge density, shape, size and chemical composition play fundamental roles in the regulation and trigger of specific interactions with biomolecules, bio-distribution and pharmacokinetic properties in living organisms.1, 2 Accordingly, the manipulation of the physicochemical properties of nanoparticles has profound effects on interactions of nanomaterial and cell responses. In this regard, noble metal nanoparticles are of particular importance because it is possible to achieve a precise control of their quality, size, shape, optical properties, stability, aggregation and composition that impact directly on their bio-applications.3,

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Gold (Au) nanoparticles have gained interest in cancer

theranostics, thermal ablation, photoimmunotherapy, as nanosensors, drug/gene delivery systems and contrast agents.5 In particular, anisotropic (nonspherical morphologies) nanoparticles that possess unique structural and size/shape dependent optical properties (tuning surface plasmon resonance and surface-enhanced Raman scattering) make them suitable for bio-applications. Nevertheless, Au nanoparticles have presented cytotoxic effects upon exposition to cells (reduction of viability, programmed cell death and over-production of reactive oxygen species) depending on the coating agents and surfactants used in the synthesis.6 Novel Au nanoparticles coated with biocompatible compounds, particularly proteins, peptides and amino acids have been proposed as an alternative to overcome these limitations when used for biomedical applications7, 8, 9, 10

, producing complex nanostructures with an extraordinary level of control of particle size,

morphology and orientation.11 Peptides and amino acids possess an inner high affinity to metal ions that can be exploited to efficiently synthesize nanoparticles with reduced toxicity and an extraordinary level of control of


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size, morphology and orientation.12,

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In this sense, single amino acids with charged side

chains such as L-Lysine (Lys) are particularly useful functioning as nucleation sites for metal binding and consequently as structure-directing capping agents during synthesis of nanoparticles, with the additional advantage of biocompatibility, efficiently controlling their size, shape and physicochemical properties. Beyond these promising advantages, the bio-directed synthesis of Au nanoparticles with complex-functional nanostructures employing amino acids is still a poorly explored area in contrast with to commonly used coating agents (CTAB, CTAC, hydroxylamine, PVP, among others) that are known to circumvent a variety of cytotoxic effects upon exposure to cells.15, 16 In addition, there is a poor understanding of the molecular mechanisms and interactions between reactant precursors that intervene during the nucleation, growth and stabilization of Au nanoparticles. Thus, the characterization of these parameters is of fundamental importance to comprehend and control the structural organization and functional properties of the Au nanoparticles for bio-applications. In this work, we synthesized anisotropic multibranched Au nanoparticles using Lys as biodirecting agent, achieving a precise control of size-shape of nanostructures, performing a comprehensive characterization of the physicochemical (zeta-potential, electrophoretic mobility, conductivity in solution), optical (absorbance, dynamic light scattering, Raman spectroscopy) and structural (morphology, arrangement, nanostructure, chemical composition) properties of Au nanoparticles

using

integrative

advanced

analytical

electron

microscopy

techniques.

Furthermore, we simulated the Au(I) ion precursors interactions with Lys to provide an understanding of the molecular mechanisms governing the nucleation-growing steps during synthesis of anisotropic Au nanoparticles.


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MATERIALS AND METHODS Two-step synthesis of anisotropic Au nanoparticles. Au seeds were prepared by citrate reduction. In a typical synthesis, 10 ml of ddH2O were added to a scintillation vial, heated to boil under vigorous agitation. When liquid started to boil, 5 µl of HAuCl4 at 0.5 M were added and after 2 min, 20 µl of sodium citrate at 0.5 M were added to the mixture. The solution rapidly turned to red and agitation and heating were stopped after 3 min. Gold seeds (Au-Cit) were stored covered from light, cooled down to room temperature and used after 2 h. Second step of synthesis was on a 10 ml final volume, using Lys buffer (25 mM final concentration), Gly buffer (25 mM final concentration) or ddH2O at 37°C with stirring at 500 rpm. Specific volumes of Au-Cit nanoparticles at 0.25 mM were added to achieve different molar ratios (1:5, 1:10, 1:25, 1:50, 1:66 and 1:100) respect to HAuCl4 , after 5 min 10 µl of NH2OH·HCl (500 mM) were added and mixed for 1 min. Growth of anisotropic nanoparticles started by adding 1 ml of aqueous HAuCl4 (2.5 mM) dropwise at 0.25 ml min-1, the incubation and stirring continued for additional 5 min with no immediate change in color of solution. After 30-60 min reactions started to acquire a pink-purple color and samples were kept at 37°C overnight to achieve final products. Spectroscopy characterization. UV-Vis spectroscopy was performed with a Cary 100 UV-Vis spectrophotometer (Agilent Technologies) from 200-900 nm. Photon correlation spectroscopy (Zetasizer NanoZS, Malvern) was used to obtain size distribution and zeta-potential of nanoparticles in solution, using He-Ne laser 633 nm at 5 mW, backscattered detector angle at 173° and controlled temperature stage (25°C). Raman scattering of nanoparticles coated with fluorescein isothiocyanate (FITC) was performed with HORIBA Jobin Ivon iHR320 imaging spectrometer, operated with a red laser


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(785 nm) or blue laser (488 nm) on samples mounted on ultra-flat silicon wafer. Data was normalized to baseline and analyzed with LabSpec software. Advanced electron microscopy imaging. Au nanoparticles were diluted twenty-fold in ddH2O to load with 5 µl onto holey carbon copper grids 300 mesh (Electron Microscopy Sciences). For SEM imaging, 1 ml of nanoparticles was centrifuged at 3000 rpm for 15 min, supernatant was removed and pellet resuspended in original volume. This process was repeated three times to remove excess organic molecules before loading 10 µl of washed nanoparticles onto an ultraflat silicon wafer chips (TedPella) and dried in a desiccator under vacuum. Au nanoparticles were characterized by ultra-high resolution field emission scanning electron microscopy (UHR FE-SEM HITACHI In-Lens S-5500) coupled with BF/DF Duo-STEM detector and solid-state EDX spectroscopy (Bruker), using an accelerating voltage of 30 kV. Field-Emission High-Resolution Transmission Electron Microscopy (HRTEM) micrographs were obtained with JEOL 2010-F operated at 200 kV. Imaging processing was performed with QUARTZ (PCI) and ImageJ v1.49. Molecular dynamics simulations. We developed an optimized classical mechanics based force field model that reproduced the most relevant solvation and thermodynamics properties of hydrated Au(I) ion. Subsequently, this force field was applied a set of molecular dynamics (MD) simulations to describe the interactions between Au(I) ions and two amino acids (Lys and Gly) surrounded by water molecules. All simulations were performed using NAMD 2.9 package and Charmm22 force field.17 The results were analyzed with VMD.18 The Au(I) ion was considered as a 2 sites (atoms) rigid model to capture the dynamic polarization properties of Au(I). These properties were taken into account by assigning a -1 time charge to the dummy site (atom) and a +2 times charge to the other site (atom), based on a similar reported


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approach used to represent the polarization in the Au atom.19 The bond energy between the two sites was represented by a spring with constant force of 1500 Kcal mol-1 and equilibrium bound length of 0.15 Å. The atomic mass of the dummy atom was set equal to 0.9 u and the other one equal to the atomic mass of Au(I). We employed the Lennard-Jones (LJ) and the Coulomb potentials to model the Van der Waal and electric interactions, respectively. In all simulations, the Au(I) was solvated with the TIP3P water model in a cubic box that extended at least 10 Å from any direction. The MD production was performed in the NPT ensemble at 298 K and 1 atm in a cubic box of 22x22x22 Å under periodic boundary conditions. We used a time step of 1 fs, and the SHAKE algorithm. The long-range electrostatic forces were evaluated by means of the particle-mesh Ewald approach during the 5 ns simulation time. We calculated the height and location of the peaks in the radial distribution function (RDF) between the Au(I) ion and the water molecules to provide useful information on the water layering and coordination number around the Au(I) ion. The coordination number of the first and second shell of water around the Au(I) was estimated from the average number of O atoms within the first and the second minima in the RDF, respectively.20 Additionally, we also calculated the binding free energy (BFE) (difference between the free energy of the bound and unbound states) which represents the mechanical work that must be done against the forces which hold the complex Au(I) ion-H2O molecule together. The BFE was calculated by means of the Potential of Mean Force (PMF) derived from Steer Molecular Dynamic (SMD) simulation. In all SMD simulations we used the same thermodynamics conditions described for the parameterization of the Au(I) with the only difference on the box size (22×22×44 Å). The initial distance between the water molecule and the Au(l) was set equal to 20 Å and the Au(l) was pulled toward the center of mass of the water molecule (or amino acids) along the z direction coordinate. The optimal constant pulling velocity was found to be 0.02 nm ns-1 whereas an appropriate stiff spring approximation was obtained


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using a spring force of 10 kcal mol-1 Å-2.21 The conditions calculated for these simulations were considered reversible due to the same distribution of the reverse versus forward pulling of the Au(I) in water. Nevertheless, we calculated each condition by triplicates to improve the accuracy of our predictions. The binding free energy between the Au(I) and the water molecule or amino acids was determined by the change in the work average for the triplicate simulations. The work was calculated as a function of the time (t):

→ = ′

where v is the constant pulling velocity, is the time step and is the force done by the system. Using the alchemical free energy perturbations (FEP) theory we calculate the overall hydration free energy difference of the Au(l) ion in bulk water and compared our results with the experimental and computational values available.22, 23, 24 In all FEP simulations we use the same thermodynamics conditions described for the parameterization of the Au(l). Briefly, we run a series of FEP simulations (32 windows) equally spaced (λ=0 to λ=1) of a single Au(l) ion placed in the center of a 22×22×22 Å cubic box. In each window, the system was equilibrated and MD production was subsequently started for both the backwards and forwards simulation during 1ns.


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RESULTS AND DISCUSSION Anisotropic nanoparticles were synthesized using Lys as bio-directing agent. Characterization of the optical, physicochemical and nanostructural properties were determined by UV-Vis absorbance, Raman spectroscopy and photon-correlation spectroscopy (dynamic light scattering, DLS). Whereas the structure and shape of the nanoparticles were obtained by ultra-high resolution field-emission scanning electron microscopy (UHR FE-SEM) coupled with energy dispersive X-ray spectroscopy (EDX), and high resolution transmission electron microscopy (HR-TEM). Bio-directed synthesis of anisotropic Au nanoparticles. Small spherical Au nanoparticles were effectively obtained through citrate-reduction using a modified Turkevich method.25 The oxidation of citrate to form acetone dicarboxylic acid facilitated nucleation-reduction of Au ions to obtain biocompatible colloidal nanoparticles. Au nanoparticles capped with citrate (Au-Cit) showed a narrow size distribution and high monodispersity, with a mean diameter of 16.6±2.2 nm (Figure 1-A, Figure S1-Histogram). HRTEM of Au-Cit revealed icosahedral arrangement of particles, Au atoms showed a highly ordered and periodic nanostructure confirming crystallinity (Figure 1-B). Growth into anisotropic nanostructures was achieved through biomineralization at inorganicorganic interfaces. Molarity of Lys (shape-directing), reducing agent (NH2OH) and Au precursors were kept constant in all cases, variation of the amount of reactive nanosurfaces from Au-Cit resulted an effective approach to control specific size range of particles obtained. UHR FE-SEM of reactions at different molar ratios revealed size and morphology of particles (Figure 2). At a low molar ratio (1:5), the nanoparticles started to show enlargement of branches obtaining a mean diameter of 31.7±3.7 nm. Increasing the molar ratio showed a direct effect on the number and length of branches per nanoparticle. Samples produced at 1:10, 1:25, 1:50, 1:66 and 1:100


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showed mean diameters of 41±5.1, 53.8±7.2, 72.4±9.2, 79.5±10.7 and 95.1±12.0 nm respectively (Table 1)(Figure S2-Histograms). Imaging of particles confirmed the efficient formation of multiple branches or peaks around the particle surface, maintaining high level of monodispersion, yield and a narrow size distribution. Control reactions using Glycine or pure ddH2O instead of Lys were performed, obtaining semi-spherical particles with some aggregation instead of starshape morphologies. Similar seed-mediated strategy has been employed by using CTAB, CTAC, PVP, oleylamine and hydroquine as capping agents with variable results in monodispersity and number of branches per particle.15 Unfortunately, all these capping agents are not biocompatible, limiting their uses and applications when direct contact with viable cells is required or causing decrement in viability. In contrast, Lys is highly biocompatible and commonly employed in cell culture and as additive. Au nanorods produced in presence of Lys showed biocompatibility and potential use as contrast agent in tomography acquisition.26 Use of biomolecules as templates to produce inorganic materials can be tailored to achieved different desired nanostructures, like in the case of polylysine that depending on its length resulted in different shapes of silica particles.9 Lysine coating was present even after several rounds of washing. Low voltage BF/DF-STEM revealed presence of an organic continuous layer, whereas EDX microanalysis confirmed chemical composition (Figure S3). Particles obtained at 1:5 showed hexagonal shape with formation of small branches over the edges, BF-STEM imaging revealed the presence of a low contrast layer of ~2 nm surrounding the particles. In comparison, ADF-STEM confirmed that the high contrast core of the particles corresponded to Au. Gold produces high scattering of electrons that is directly proportional to the atomic number (Z-contrast), whereas the organic compounds present no contrast with ADF detector. Similar behavior was observed on particles at molar ratio of 1:100, but with a thinner organic layer. EDX spectroscopy confirmed chemical composition of


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the particles; identifying the distinctive peaks of Au at 2.120 and 9.712 keV (Figure S3). Integration of Au, C, N and O peaks showed that samples contained a relative composition with 52.7 % of Au, 20.4 % of N, 10.6 % of C and 16.4 % of O. The presence of these light elements confirmed that Au nanoparticles were coated with Lys, particularly with the presence of peaks at 0.277 keV (Carbon), 0.392 keV (Nitrogen) and 0.523 keV (Oxygen).

Optical properties: UV-Vis, photon-correlation and Raman scattering. Au nanoparticles have characteristic optical properties that are closely dependent on their size, shape, chemical composition, concentration and agglomeration. The absorption spectra of the Au nanoparticles synthetized at different molar ratios are shown in Figure 3. Noble metal particles exhibit localized surface plasmon resonance (SPR) by a strong optical extinction.27 Au-Cit showed SPR centered at 518 nm. Whereas, anisotropic particles exhibited a red-shift in their SPR peaks correlated with the increase in diameter as well as formation of branches, which correlated with FE-SEM imaging (Figure 2). The red-shift indicated that particles produced at higher molar ratios possessed higher diameters and non-spherical morphology. Lys-Au exhibited well-defined absorption peaks in the Vis and NIR centered at 540, 551, 557, 574, 580 and 645 nm, for particles synthesized at molar ratios of (1:5), (1:10), (1:25), (1:50), (1:66) and (1:100), respectively. Lys-Au particles showed absorbance close to the NIR region; this increment in the longitudinal plasmon resonance of anisotropic particles has been established theorically and experimentally to be proportional to the formation of branches with high aspect ratio.27, 28 This property was more evident for Lys-Au at higher molar ratios, with increment in diameter as well as length of branches (Figure 3). In contrast, anisotropic particles with rod-shape show a transverse SPR around 550 nm plus the longitudinal SPR in the NIR.


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Optical properties of anisotropic particles were also evident by observing the color of the solutions (Figure 3-bottom). Au-Cit had an intense red color characteristic of colloidal gold in this size range, which turned to pink, purple and finally exhibited a bright blue, correlated with the increment in molar ratio of reactants during synthesis using Lys. Since, surface plasmon resonance (SPR) depends sensitively on size and shape, it is extremely important to achieve a controllable synthesis of Au nanoparticles. Anisotropic particles with localized SPR in the NIR window (600-900 nm) have potential applications in photodynamic therapy, photoimmunotherapy, drug and gene delivery, photothermal therapy, radiotherapy, in diagnostic applications and as nanosensors when coupled with specific targeting biomolecules.26, 29, 30

. The particles produced with Lys can be efficiently conjugated through covalent chemistry

or electrostatic interactions with proteins, enzymes, antibodies, DNA, RNA or fluorescent probes through available residues using simple chemistry.3, 4, 14 Size distribution and colloidal stability of nanoparticles were determined by photon-correlation spectroscopy (dynamic light scattering or DLS). The hydrodynamic diameter showed a direct increment related to the increase in the molar ratio of seeds/Au. The initial Au-Cit showed a mean hydrodynamic diameter of 21.8 ± 4.6 nm (Figure 4), displaying a highly monodisperse and narrow distribution confirmed by electron microscopy. DLS allows measuring specific populations of nanoparticles in polydisperse colloids, and effectively determining their size and percentage of the particles.31 Table 1 summarizes DLS data expressed as hydrodynamic diameter and Figure 4 shows the histograms of intensity-hydrodynamic diameter (nm) of as-synthesized Au nanoparticles (without purification or washed). Lys-Au produced at 1:5 presented a mean diameter of 43.0 ± 19.5 nm, this population represented 93 % of particles in solution, the remaining 7 % corresponded to a population of 2-3 nm probably by-products of reaction. Particles produced at 1:10 showed 46.4 ± 12.0 nm with 92.4 %, reactions at higher ratios of 1:25,


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1:50 and 1:66 displayed one population of particles of 58.5 ± 11.7, 87.4 ± 34.7 and 93.8 ± 24.7 nm, respectively with yields ranging from 85 to 94 %. The particles produced at 1:100 showed a mean size of 109 ± 44.9 nm corresponding to 90 % of particles in solution. Noble metal nanoparticles are extensively used, it is important to produce high-quality, high yield, monodisperse narrow size distribution and precise determination of mean particle size.31 Multibranced Au nanoparticles produced with inorganic capping agents show different degrees of dispersity (low, moderate or even high) but most the cases with mean size above 100 nm.15 The two-step reaction with Lys showed to efficiently produce high-yield particles with controlled size ranges, under mild reaction conditions by using biocompatible molecules. The determination of physicochemical parameters of Au particles is of fundamental importance to understand the variables that control colloidal stability and surface functionalities. The ζpotential, electrophoretic mobility (µ) and electrical conductivity (EC) of Au particles were determined by using dynamic light scattering (ZetasizerNano ZS, Malvern). Au-Cit displayed extraordinary colloidal stability and a net negative charge of -42.2 ± 0.57 mV. Lys-Au particles showed good colloidal stability ranging from -36.8 to -40.5 mV, only the particles produced at 1:100 molar ratio showed a reduction of zeta potential to -26.4 ± 2.37 mV that can be related to the higher volume of nanoparticles and limitation of biomolecules to coat all surface (Table 1). The original measurements were obtained in Lys 25 mM that possess a basic pH (9.3). To get an idea of the behavior of the anisotropic particles under physiological conditions, the particles were concentrated by centrifugation and resuspended in PBS buffer (pH 7.2). In general, we observed that the Lys-Au particles suffered a slight reduction in zeta potential ranging to -22.4 to -27.9 mV in PBS buffer (Table 1). Au-Cit and Lys-Au at 1:100 showed a considerable decrement in their colloidal stability to -12 and -13.5 mV, respectively. The behavior of ζ-potentials determined in


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two different buffer of particles of different diameters is presented in Figure S4-A. Lys-coated particles showed higher colloidal stability than Au nanostars produced with HEPES (-26.1 ± 0.49 mV) or particles conjugated with PEG (-23 mV)32, but the values of ζ-potential were very similar when nanoparticles were suspended in PBS buffer. Additionally, electrophoretic mobility (µ) and electrical conductivity (EC) of Au particles were measured in same buffers. Lys-Au nanoparticles displayed similar tendency to ζ-potential, ranging from -2.9 to -3.3 µm cm/Vs, whereas larger particles (~100 nm) displayed -2.0 µm cm/Vs. PBS buffer caused a reduction in µ in all cases, ranging from -2 to -1 µm cm/Vs (Figure S4-B). The mobilities of spherical Au nanoparticles (15 to 200 nm) show that values are independent of electrolyte type but dependent on electrolyte concentration.33 For nonspherical Lys-Au particles we observed variation of µ in PBS, with reduction of mobility for larger particles related to their higher surface area available to interact with the electrolytes in solution. PBS with a higher ionic strength than Lys buffer caused a reduction in µ independently of size and shape of particles. The conductivity of nanoparticles in solution was reproducible independently of the size and shape of particles, samples in Lys buffer showed low conductivity from 0.47-0.55 mS/cm, whereas in PBS buffer nanoparticles showed improved conductive properties achieving 17.2-18.8 mS/cm (Figure S4-C). These observations correlate with electrokinetic formula of Ohsihima that indicates that magnitude of the effective electrokinetic charge density and total charge of particle remained constant with electrolyte concentration.33 Gold particles and in particular those with anisotropic morphologies show localized surface enhanced Raman scattering (SERS) resulting in sharp intense peaks that are characteristic of the sample.4 Figure 5 shows Raman scattering of the different gold nanoparticles synthesized during a noncovalent interaction with FITC as reporter molecule. As expected, small spherical particles


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showed absence of SERS with no increment on signals, whereas particles with anisotropic structures clearly showed an increment in intensity of FITC characteristic peaks. Particularly, SERS effects were present in peaks at 764, 1080, 1171, 1250, 1313, 1334, 1417, 1505, 1562, 1604 and 1634 cm-1, that represent fingerprint of FITC. These distinctive vibrational changes of FITC were assigned to the core xanthene rings at 764 cm-1, ν(C-O-C) asymmetric at 1080 cm-1, -OH phenolic ring at 1171 cm-1, phenoxide ion stretch at 1313 cm-1, vibrations of aromatic rings at 1505 cm-1 and ν(C-C) stretching at 1334, 1562, 1604 and 1634 cm-1.34 SERS occurred preferentially on star-shape particles causing a remarkable augmentation of vibrations and scattering processes observed on FITC fingerprint spectra. Enhancement of Raman peaks was related to growth in nanoparticle diameters and length of branches. SERS enhancement factors are highly dependent or correlated with size and shape of gold nanostars.35 Particles produced at lower molar ratios showed an increase of 3-4 fold the intensity in comparison to small spherical particles; samples produced at molar ratios of 1:25 and 1:50 presented branches of 10-20 nm length that showed 4 fold increase in scattering signals. At higher molar ratios (1:66, 1:100) when nanoparticle branches achieved 20-35 nm length, the enhancement in peak intensities achieved up to 4-5.5 fold (Figure 5). In comparison, anisotropic particles produced using gelatin as coating showed 10-fold SERS enhancement than spherical particles.36 Gold nanoparticles with star-shape have demonstrated their potential in bioapplications, particularly in optical imaging, biosensing, drug delivery, diagnostics and therapy, by their high sensitivity, tunable surface plasmon resonance and SERS properties.35,

36, 37

In

particular, Lysine capped particles showed SERS upon excitation with a 785 nm laser, this property can be exploited to obtain Raman signals directly from cells and tissues in the biological transparency window (650-900 nm) without damaging or heating but with high sensitivity.


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High Resolution-TEM imaging. Figure 6 shows high-resolution TEM of anisotropic nanoparticles. Low magnification micrographs clearly showed a defined hexagonal shape in Lys-Au (1:5) (Figure 6-A) and welldefined star-shape morphology in Lys-Au produced at 1:50 and 1:100 (Figure 6-C-E). Imaging of these samples gave an idea of growth direction and evolution of the crystal from the initial step (1:5), midway (1:50) to final growth step of branches (1:100). Initially, the growth of branches occurred preferentially over the facets of icosahedral particles, by influx of Au ions and catalyzed by soft-reductant hydroxylamine.28,

32, 38

HR-TEM clearly showed the formation of small

branches or projections all over the nanoparticle surface and no perceivable twin boundaries were observed, in contrast to the observations of some twin-boundaries during HEPES-hydroxylamine Au particles.28 Lys-Au at molar ratio of 1:50 gave a perspective of midway step when more Au ions were available allowing growth of longer branches. High-magnification micrographs revealed the crystalline structure and defined lattice planes (Figure 6-D). The branches showed higher length (20-35 nm) and formed over all directions giving nanoparticles of 64 to 80 nm in diameter. Increasing the availability of Lys-Au precursors using a 1:100 seeds/Au molar ratio facilitated the formation of ~100 nm anisotropic nanoparticles; in these conditions the length of branches was superior achieving up to 30-40 nm. Detail of HR-TEM enabled identification of the growth direction of the crystals, branches grew preferentially along (111) face in a regular arrangement along the (110) direction.32,

39

From FFT pattern it was possible to calculate an

interspacing of 2.4 Å characteristic for Au and confirming the crystallinity of as-synthesized nanoparticles. In the case of Au particles synthesized with Lysine but using NaBH4 as strong reducing agent, the (111) plane was more dominant over (100) and (110), favoring the formation of short nanorods.26 In all HR-TEM micrographs it was visible the presence of a cloudy organic layer, corresponding to Lys coating all over the nanoparticle surface. Lys coating was related to


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nanoparticle growth, formation and stabilization into a crystalline shape. Different mechanisms are involved in nanoparticle synthesis and stabilization, particularly for star-shape particles the possibility of formation of planar defects (twins and stacking), introduction of steps and kinks on the surface, and modification of shape by facet truncation to achieve energetically-stable particles.40 Molecular dynamics (MD) simulations. The specific mechanisms involved in the synthesis and control of size/shape of metal nanoparticles have evolved mostly empirically or by trial and error. Additionally, in the case of Au the free surface energies and chemistry of facets are similar making it more challenging.41 Speciation of aqueous HAuCl4 is pH-dependent, influencing the size, structure, morphology and properties of nanoparticles; variation of pH allowed the formation of Au(I)-GSH, Au(I)GSH/Lys-NTA-SH or Au-benzenesulfonate precursors for a controlled synthesis of Au colloids.42, 43, 44 Lysine buffer used in this work showed pH=9.30 at reaction conditions used, promoting liberation of Cl- ions and immediate hydrolysis of AuCl4- ions or Au(III) into Au(I).43 Speciation of Au(III) into Au(I) in Lys 25 mM buffer was confirmed by monitoring reduction of absorption band of Au(III) at 325 nm.45 (Supporting Information-Figure S5). Even though the synthesis of ligand-stabilized Au particles is widely used, the details of mechanisms and interactions between reactants have not been completely elucidated.44 MD simulations helped to understand some of the major steps and specific interactions involved during the synthesis of Au nanoparticles in the presence or mediated by biomolecules. First, we validated Au(I) precursor solvation and thermodynamics properties, these are originated in solution from hydrolysis and partial reduction of Au(III) in high pH solution.42, 43 These set of simulations provided an insight on the molecular mechanisms governing the binding energy of Au(I)-amino acid nucleation complexes. Multiple


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simulations of the system were run for optimization of the proposed Au(I) ion force field. The parametrization of non-bonded Au(I) ion LJ parameters Rmin and ε were adjusted by a trial and error approach, to reproduce results for quantum dynamics (QM), radial distribution functions (RDF), and binding free energy calculations between one water molecule and one Au(I) ion from both, experimental 46, 47 and theoretical studies.24, 48 Rmin represents the Au(l) ion-water molecule separation distance at which the LJ potential reaches its minimum, e.g. -ε. Therefore, obtaining an accurate value for these parameters was of fundamental importance to properly describe the thermodynamic and structural properties of hydrated Au(I) ions. We used the Au atom LJ parameter of the CHARMM-Metal force field as initial approach value for Au(I).49 The best combination of LJ parameters for Au(I) ions was obtained for a Rmin=0.62 Å and ε=2.29 kcal mol-1 (Table 2). These optimized parameters generated a coordination number for the first hydration shell of the Au(I) ion (n(m1)) in good agreement with those predicted by Quantum Mechanics/Molecular Mechanics (QM/MM) and Quantum Mechanics (QM).24 The maxima (rM1) and the minima (rm1) radius of the first solvation shell predicted by our approach showed the same behavior obtained through QM simulations48, therefore providing an accurate description of the number of water molecules bound to the Au(I) ion surface. Additionally, the coordination number predicted for the second solvation shell (n(m2)) of the Au(I) lies in between the QM and QM/MM simulation data (Table 2). As a result, the optimized LJ parameters obtained for Au(I) ions were able to properly capture not only the first, but also the second hydration shells of Au(I) ions, even though electrons and Quantum effects were only considered in our formation implicitly. A binding free energy (BFE) of 34.4 kcal mol-1 was predicted (Figure S6-A). This BFE value correlates with most of the experimental data reported for Au(I)-H2O complexes (34.0, 35.1 and 39.2 kcal mol-1).46, 47 Additionally, we calculated the solvation free energy of the Au(I) in bulk water (134.1 kcal mol-1), this predicted value resulted very similar to those obtained


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experimentally (140.2 kcal mol-1)23 and computationally (139.0 kcal mol-1)24. Overall, the simulations performed in this work demonstrated that the optimized classical mechanics based force field model predicted the thermodynamics and solvation properties of Au(I) ions in good agreement with current available experimental and computational data. The optimized force field parameters obtained for Au(I) ions interacting with water molecules should be transferable to other molecules.50 Therefore, the proposed approach resulted suitable for modeling the interaction between hydrated Au ions and amino acids: Lysine and Glycine. Subsequently, the optimized classical mechanics based force field model developed for Au(I) ion was used to run a set of molecular dynamics (MD) simulations to describe the interactions between Au(I) ion with Lys (positive charge side chain) and Gly (neutral side chain) used for biodirected synthesis of Au nanoparticles. All simulations were performed using the same thermodynamic conditions and configurations described for Au(I) ion in water. The results showed that both amino acids interacted preferentially with Au(I) through the carboxyl terminal group (-COO-) rather than their side chains (Figure 7 and Video S1). Gly was chosen as control because its neutral side chain (-H), as expected Gly showed a low BFE of 1.2 kcal mol-1. In comparison, Lys which has a positive charged side chain with a ε-amino group (NH3+), presented a BFE of 3.4 kcal mol-1. Even though the side chains might not play a direct role in the initial interaction between the Au(I) ion and the free amino acids in solution, the side chains may enable stability of the complex in solution. The fact that the Au(I) preferred an interaction with the -COOH group illustrated the possible active role in the transport of the Au(I) precursors during the growing of the anisotropic Au nanoparticle catalyzed by hydroxylamine.38 Au nanoparticles obtained under same reaction conditions, but in the presence of Gly showed spherical or icosahedral-like morphologies instead of multibranded particles obtained using Lys (Figure S7). This means that positively charged Lys may be seen as Au(I) ion carriers. Since the


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Au(I) interacted favorably with the carboxyl group of the Lys instead of the side chain, this leaves the ε-amino group (NH3+) available to interact with other species. For instance, the –NH3+ terminal might interact with active Au-Cit surfaces with net negative charge (Table 1) meanwhile it carries on the precursor Au(I) bounded to its carboxyl terminal group favoring the growth in a controlled manner (as was observed an slow change in color of the solution over 12 h of reaction). In fact, MD simulations showed a high adsorption energy of positive charged amino acids (His, Lys and Arg) to Au surfaces by the interaction of their side chains, whereas Ala (with a -CH3 side chain) was not absorbed to Au surfaces.19 MD simulations showed that the binding strength of metal binding peptides to Au (100) and Au (111) surfaces is mainly associated with COOH end groups of Asp and Lys.51 The heavy-atom database system (HATODAS II) show that metal binding sites for Au in protein crystals are mainly composed by positively charged residues.52 Thus, we hypothesize that the growth and preferential directionality of Au nanoparticles synthesized in presence of Lys may be due to the high binding affinity between the Au(I) ions and the carboxyl group, as well as the high adsorption energy of positive charged amino acid side chain to Au surfaces. A similar mechanism was proposed for growth of Au nanorods, but in this case the growth of Au seeds is controlled by the flux of CTAB-Au intermediate to the (111) facets of seeds through reduction by ascorbate. Au(I) species formed possess lower tendency for reduction into colloidal gold, usually with a slow gradual change in color.43 This was evident with use of Lys and NH2OH, since samples started coloring after ~1 h of incubation at 37°C and achieved final color after 12 h. This picture may provide insights into the molecular mechanisms involved in the Au nanoparticle formation phenomena observed experimentally, even though the understanding of all variables that provide the selective growing into multibranched Au particles is not completed.


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CONCLUSIONS Anisotropic nanoparticles were efficiently synthesized by using Lys as bio-directing agent, obtaining highly complex nanostructures with reproducibility, high yields and in controlled size ranges between 30 and 100 nm in diameter. The optical, physicochemical and nanostructural properties of these nanoparticles revealed tunable absorbance close to the NIR, extraordinary colloidal stability and surface-enhanced Raman scattering properties. Morphology and nanostructure of the nanoparticles was obtained through advanced electron microscopy techniques, confirmed crystallinity and star-shape formation using Lys. Additionally, the results of MD simulations revealed that Lys interacted preferentially with the Au(I) through the -COOH group instead of their side chains. This suggests that a possible molecular mechanism by which Lys transports Au(I) precursors near the surface of Au-Cit for nucleation. Overall, the results obtained confirmed active role of Lys in bio-directed synthesis of anisotropic nanoparticles, providing a comprehensive understanding on the nucleation, growth, stabilization, structural and physicochemical properties of nanoparticles obtained with Lys.

ACKNOWLEDGEMENTS This work was supported by Welch Foundation (AX-1615), NSF (DMR-1103730), NSF-PREM (DMR-0934218). Facilities of Kleberg Advanced Microscopy Center (KAMiC), NIH RCMI Nanotechnology and Human Health Core (RCMI grant 5G12RR013646-12) and NIH RCMI Biophotonics Core (RCMI grant G12MD007591) at UTSA. Authors thank Drs. Stefano Corni and Liao Chen for their useful comments on the force field optimization used in MD simulations. SUPPORTING INFORMATION AVAILABLE Histograms, STEM-EDX, physicochemical properties, binding affinity and SEM of controls. This material is available free of charge via the Internet at http://pubs.acs.org/.


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force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. Journal of computational chemistry 2010, 31 (4), 671-90. 51. Heinz H, F. B., Pandey RB, Slocik JM, Patnaik SS, Pachter R, Naik RR. Nature of molecular interactions of peptides with gold, palladium, and Pd-Au bimetal surfaces in aqueous solution. J Am Chem Soc 2009, 131, 9704-9714. 52. Sugahara M, A. Y., Shimada H, Taka H, Kunishima N. HATODAS II - heavy-atom database system with potentiality scoring. J Appl Cryst 2009, 42, 540-544.


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Bio-directed synthesis and nanostructural characterization of anisotropic gold nanoparticles. Germán Plascencia-Villa, Daniel Torrente, Marcelo Marucho and Miguel José-Yacamán. Department of Physics & Astronomy, The University of Texas at San Antonio (UTSA). One UTSA Circle, San Antonio, TX 78249, USA. [email protected] TABLES Properties of Lys-Au nanoparticles. Size distribution by electron microscopy and dynamic light scattering (DLS). Zeta-potential measured in Lys 25mM and PBS pH 7.2.

Table 1.

Size Distribution (nm) Electron DLS Microscopy 16.6 ± 2.2 18.1 ± 2.2 31.7 ± 3.7 43.0 ± 19.5 41.0 ± 5.1 46.4 ± 12.0 53.8 ± 7.2 58.5 ± 11.7 72.4 ± 9.2 87.4 ± 34.7 79.5 ± 10.7 93.8 ± 24.7 95.1 ± 12.0 109 ± 44.9

Nanoparticle Au-Cit Lys-Au (1:5) Lys-Au (1:10) Lys-Au (1:25) Lys-Au (1:50) Lys-Au (1:66) Lys-Au (1:100)

-42.2 ± 0.57 -36.9 ± 0.76 -36.8 ± 0.49 -39.6 ± 0.64 -40.2 ± 0.21 -40.5 ± 2.00 -26.4 ± 2.37

-13.5 ± 0.70 -27.9 ± 0.78 -25.6 ± 0.61 -22.9 ± 0.85 -24.2 ± 1.00 -22.4 ± 0.40 -12.0 ± 1.17

Molecular dynamics simulations. Location (in Å) of the maxima rMi and minima rmi in the radial distribution function for Au(l) and Oxygen in aqueous solution. n(mi) is the Oxygen average coordination number integrated up to the minima rmi in the Au(l)-H2O shell. CMD represents the results predicted by our simulations using the optimized LJ potential parameters. QM and QM/MM are the results predicted by other computational approaches.

Table 2.

Method CMD

rM1

rm1

n(m1)

rM2

rm2

n(m2)

2.0

2.9

3.9

4.4

5.6

17.6

2.5

3.8

4.7

4.9

6.5

33.0

QM

a

2.5

3.5

4.1

5.1

6.2

28.0

QM

b

2.0

2.5-3

2.0

3.9

4.6

10.0

QM/MM

a

Zeta Potential (mV) Buffer Lys 25 mM Buffer PBS, pH 7.2

a

QM/MM and QM calculation of RDF values 24. b Values of RDF taken from QM calculation 42.


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Bio-directed synthesis and nanostructural characterization of anisotropic gold nanoparticles. Germán Plascencia-Villa, Daniel Torrente, Marcelo Marucho and Miguel José-Yacamán. Department of Physics & Astronomy, The University of Texas at San Antonio (UTSA). One UTSA Circle, San Antonio, TX 78249, USA. [email protected] FIGURES Figure 1.

Electron microscopy of Au-citrate nanoparticles. (A) Low-magnification HRTEM. (B) High-magnification HR-TEM. Imaging with JEOL 2010F operated at 200 kV.


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Figure 2.

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UHR FE-SEM imaging of Au-Lys anisotropic nanoparticles produced at different reaction conditions. (A) Ratio 1:5, (B) Ratio 1:10, (C) Ratio 1:25, (D) Ratio 1:50, (E) Ratio 1:66, (F) Ratio 1:100. Imaging with UHR-FE-SEM In-lens HITACHI S5500, acquisition with SE detector and operated at 30 kV. Scale bar 300 nm.


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Figure 3.

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Absorbance spectroscopy. Characterization of UV-Vis spectroscopy of initial product Au-citrate (red line) in comparison with Au-Lys produced at different volume ratios. Maximum absorbance of each sample is indicated and used for normalization of data. Bottom image shows microtubes containing the nanoparticles analyzed and indicating the volume ratio used during the synthesis.


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Figure 4.

Size distribution of nanoparticles in solution. Intensity size distribution by Dynamic Light Scattering (DLS) expressed as hydrodynamic diameter (nm) of assynthesized Au nanoparticles (without purification or washed). Mean size of each sample is indicated in Table 1.

Figure 5.

Raman scattering. Surface-enhanced Raman spectroscopy (SERS) of fluorescein isothiocyanete (FITC, black line) and anisotropic Au particles Au-Lys obtained at different volume ratios. The assignments of Raman vibrational bands (cm-1) enhanced for Au-Lys particles coated with FITC conjugates are indicated.


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Figure 6.

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HR-TEM imaging. (A) Low-magnification Au-Lys (1:5). (B) High-magnification Au-Lys (1:5). (C) Low-magnification Au-Lys (1:50). (D) High-magnification AuLys (1:50). (E) Low-magnification Au-Lys (1:100). (F) Low-magnification AuLys (1:100). Imaging with HRTEM JEOL-2010F operated at 200 kV, acquisition with CMOS based TEM camera (TemCam-F416, TVIPS).


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Figure 7.

Langmuir

Molecular dynamics simulations. Graphical representation of the binding site between amino acids and Au(l). Final conformation of interaction positions between (A) Lys-Au(l) and (B) Gly-Au(l). The Au(l) always preferred the binding with the carboxyl group of the two amino acids rather than their side chain or the amine group.


6

Langmuir

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TOC Graphic Bio-directed synthesis and nanostructural characterization of anisotropic gold nanoparticles Germán Plascencia-Villa, Daniel Torrente, Marcelo Marucho and Miguel José-Yacamán. Department of Physics & Astronomy, The University of Texas at San Antonio (UTSA). One UTSA Circle, San Antonio, Texas 78249, USA. [email protected]

Lys-Au

ACS Paragon Plus Environment 1

Biodirected Synthesis and Nanostructural Characterization of

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