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Electron microscopy of gold nanoparticles at atomic resolution

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Science  22 Aug 2014:
Vol. 345, Issue 6199, pp. 909-912
DOI: 10.1126/science.1251959

Detailed structure of a gold nanoparticle

Adding only a few atoms or changing the capping ligand can dramatically change the structure of individual metal nanoparticles. Azubel et al. used aberration-corrected transmission electron microscopy to derive a three-dimensional reconstruction of water-soluble gold nanoparticles. Small-angle x-ray scattering and other techniques have also corroborated this model. They used this to determine the atomic structure, which compared favorably with density functional theory calculations, without assuming any a priori structural knowledge or the use of model fitting.

Science, this issue p. 909

Abstract

Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102–gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory.

Gold nanoparticles (AuNPs) are of both fundamental and practical interest. Particles on the order of 1 nm in diameter exhibit distinctive physical and chemical properties, with potential applications ranging from quantum electronics to biomedicine (13). The x-ray crystal structure of a 102–gold atom NP (Au102NP), 1.5 nm in diameter, showed the cluster of gold atoms surrounded by 44 thiolate ligands (4). This atomic structure had threefold importance: (i) It identified the Au102NP as a molecule, with a precise composition and distinct arrangement of atoms; (ii) it led to the idea of the gold cluster as a “super atom,” stabilized by the filling of electron shells; and (iii) it revealed a layer of alternating gold and ligand molecules at the interface with solution (5). Subsequent x-ray structures of much smaller organosoluble AuNPs have supported the super-atom idea and have shown a similar gold-thiol surface layer [reviewed in (6)]. Structure determination of other water-soluble AuNPs, and of larger NPs in general, has so far been unsuccessful. Although water-soluble AuNPs ranging from 1 to 3 nm in diameter have been crystallized, x-ray diffraction has not extended beyond ~5 Å resolution. Here, we demonstrate the structure determination of a AuNP by a combination of a low-dose approach and aberration-corrected transmission electron microscopy (TEM), and we report an atomic structure with both similarities and notable differences from the Au102NP.

Whereas the thiolate ligand of the Au102NP was p-mercaptobenzoic acid (p-MBA), we have now performed synthesis with 3-MBA, resulting in a different set of uniform, water-soluble particles. The product of synthesis with a thiol:gold ratio of 2:1 formed a single sharp band upon gel electrophoresis, with a mobility greater than that of the Au102NP, indicative of a smaller size, as confirmed by cryogenic TEM (fig. S1). Electrospray ionization time-of-flight mass spectrometry (MS) revealed four peaks corresponding to various charged forms of a compound of ~18 kD with a gold core of ~14 kD (fig. S2). A stable AuNP with a gold core of similar size was previously reported by Whetten et al. (7). Because the mass difference between three atoms of gold [m = 591 atomic mass units (amu)] and four molecules of 3-MBA (m = 613 amu) could not be resolved, the MS result was consistent with the possible molecular formulas [Au71(3-MBA)27]n, [Au68(3-MBA)31]n, [Au68(3-MBA)32]n, [Au65(3-MBA)35]n, and [Au62(3-MBA)39]n (where n is 5, 6, 7, or 8). We could distinguish among these possibilities by thermogravimetric analysis (TGA) and x-ray photoelectron spectroscopy (XPS). TGA gave a weight loss of 24.4% (fig. S3), compared with values of 26.2% expected for Au68(3-MBA)31 or 26.8% expected for Au68(3-MBA)32, both discrepancies within the error of the method (8). By contrast, expected TGA weight loss values of 29.6% for Au65(3-MBA)35 and 33.0% for Au62(3-MBA)39 represent discrepancies three to five times greater. XPS gave signals corresponding to Au, S, C, and O (fig. S4), and the peak intensities corresponding to Au4f and S2p were integrated to establish the ratio between Au and organic material. The difference between the values of 65.5% Au and 34.5% S measured and those of 68.7% Au and 31.3% S expected for Au68(3-MBA)31 or 68.0% Au and 32.0% S expected for Au68(3-MBA)32 were again within the error of the method (8). Values of 72.4% Au and 27.6% S expected for Au71(3-MBA)27 were substantially different. Therefore, of the five molecular formulas consistent with the results from MS, all but two—Au68(3-MBA)31 and Au68(3-MBA)32—were ruled out by TGA and XPS.

The solubility of the Au68NP and its stability in solution were notable. The Au68NP could be derivatized with proteins and nucleic acids bearing exposed sulfhydryl groups (fig. S5) and is therefore well suited for applications in biological science and biomedicine. The Au68NP could be crystallized, forming hexagonal plates (fig. S6), but x-ray diffraction did not extend beyond 5 Å resolution. Due to the apparent homogeneity of the Au68NP, TEM and single-particle reconstruction (9) could be pursued as an alternative for structure determination. Exposure to an electron dose normally used for data collection in materials science (thousands of electrons per square angstrom) visibly perturbed the Au68 particles. We therefore turned to low-dose procedures, routinely used for biological TEM, which reduce the exposure to the electron beam by several orders of magnitude by focusing on an area adjacent to the one being imaged and then imaging for the minimal time required to record a signal above the noise, followed by improvement of the signal-to-noise ratio by image averaging. The low-dose strategy was successful (Fig. 1A and fig. S7A): Images of 939 particles acquired in this way and processed with the EMAN2 software package (10) (fig. S7) yielded an electron density map with 68 peaks (Fig. 1, B and C). The peaks in the center of the map were of highest intensity, probably because they were most symmetrically arranged and most rigidly fixed. Identification of the peaks with gold atoms is justified by their numbers, the distances between them, and their packing, as well as because their high intensities could be attributed only to heavy atoms. The distances ranged from 2.72 to 3.07 Å (Fig. 1D), in keeping with those for the gold core of the Au102NP, which were from 2.8 to 3.1 Å (4). The packing was face-centered cubic (fcc) or truncated fcc-like, similar to that in a recently reported x-ray crystal structure of the organosoluble Au36(SR)24 (R, 4-tert-butylbenzenethiolate) (11). The arrangement of the 68 gold atoms can be described as a 13-atom cuboctahedron with an atom in the center (Fig. 2, A and B), surrounded by 24 atoms extending the fcc-like framework (Fig. 2C) and an additional 31 atoms deviating from fcc packing (Fig. 2D). A Fourier transform of a section of the electron density map (Fig. 1E) showed spots at 2.4 Å, consistent with this arrangement (atoms 2.7 to 3.0 Å apart with fcc packing have lattice planes with spacings of 2.3 to 2.55 Å) and demonstrating the resolution of the analysis.

Fig. 1 Three-dimensional (3D) reconstruction of Au68NP structure from electron micrographs.

(A) Representative components of the reconstruction. (Left) Back projection from the reconstruction; (middle) corresponding class average of the EM images; (right) EM images. (B to D) Electron density map, blue mesh. Pink stars in (C) and (D) show the position of atomic coordinates for gold atoms. (D) Region of the electron density map surrounding the central atom. Dashed lines show coordination of the central atom; numbers indicate gold-gold distances in angstroms. (E) A cross section of the 3D reconstruction (left) and its Fourier transform (right). The red arrow indicates spots at 2.4 Å resolution.

Fig. 2 Arrangement of gold atoms in Au68NP.

(A to D) Coordinates. (A and B) Two orthogonal views of the central 13 atoms: central atom (yellow) caged in a cuboctahedron (orange). (C) Additional 24 atoms (red) extending the fcc-like framework. (D) All 68 atoms. Atoms with lower or no apparent symmetry are in green. (E) Au68 model from DFT.

Support for the electron microscopy (EM) structure came from small-angle x-ray scattering (SAXS), which gave a radius of gyration (Rg) of 7.6 Å, compared with a Rg calculated for the gold cluster of the EM structure of 5.72 Å. The larger measured Rg could be explained by aggregation of gold particles, which occurs in an ionic strength–dependent manner (12). The SAXS data could be fit by a mixture of monomers and dimers, with χ2 = 1 up to a reciprocal space distance (q value) of 0.44 Å−1 (fig. S8). At higher q values, a small discrepancy was observed, which may arise partially from the organic surface layer, neglected in the calculation of Rg from the EM structure. The center-to-center distance of the dimer calculated from SAXS was 14 Å, consistent with a model of the surface layer (see below), whereas the diameter of the gold cluster from the EM structure, used in the calculation was 11.5 Å.

Sulfur atoms were introduced into the EM structure, with gold-sulfur distances of ~2.3 Å (Fig. 3A). Aside from 15 gold atoms fully coordinated with gold neighbors (the central atom, the 12 atoms on the vertices of the cuboctahedron, and another 2 atoms), all remaining gold atoms are available for Au-S bond formation. The final model, containing 32 atoms of S for consistency with the model from density functional theory (DFT) described below, can be described as an Au15 fcc core decorated with 10 staple motifs (4, 6) and 12 V-shape (or bridge) motifs (13) (Fig. 3A).

Fig. 3 Au68(SH)32 model.

(A) Addition of sulfur atoms (yellow) to the gold framework (orange). (Left) Complete structure. (Right) Gold atoms outside the Au15 fcc core atoms depicted as small spheres to better reveal Au-S motifs. (B) Structure of Au68(SH)32 relaxed by DFT from the atomic positions from EM. Various gold-thiolate motifs (bridging thiolates, short and long staples, and a ringlike structure) are observed. Hydrogen atoms are in white.

Additional support for the EM structure came from DFT for the hypothetical compound Au68(SH)32. When a model of this compound, built from the coordinates of gold atoms in the EM structure, was relaxed to a local energy minimum with DFT, the positions of the gold atoms were largely unaffected (compare Fig. 2, D and E). Most of the gold atoms were shifted by clearly less than 1 Å, with the exception of a few atoms in the surface layer (fig. S9A). The positions of these atoms may be affected by hydrogen bonding between 3-MBA ligands in the surface layer, as observed upon relaxation by DFT of the compound Au68(SH)30(3-MBA)2 (fig. S9B). The DFT model displayed gold-sulfur motifs (Fig. 3B) similar to those produced by manual building of sulfur atoms into the EM structure (Fig. 3A).

The electronic structure of the Au68NP was investigated by infrared (IR) and ultraviolet-visible (UV-vis) spectroscopy and linear-response, time-dependent DFT (LR-TDDFT) calculations. The IR-spectrum showed discrete absorption peaks at ~4200 cm−1 (0.52 eV) and 5250 cm−1 (0.65 eV) and a split transition at 6100 cm−1 (0.76 eV) (Fig. 4). A fourth peak at 2500 cm−1 (0.31 eV) could not be assigned to any known vibrational mode. This peak persisted even after removing the vibrational contribution of the ligand layer (fig. S10), so we attributed the peak to an electronic transition. The optical gap of the Au68NP is therefore at 2500 cm−1 (0.31 eV), which is considerably lower than the gap of 0.45 eV observed for the larger Au102(p-MBA)44 cluster (14). This result is in contrast to the trend toward a smaller optical gap with increasing cluster size, reported for clusters of 25 to 144 gold atoms (1520) and expected to approach zero for bulk metal. The optical gap of the Au68NP is presumably lower because of deviation from spherical symmetry, so the cluster wave functions cannot follow a spherical form as they do in the case of Au102NP. The spectrum of the DFT-relaxed Au68(SH)32 cluster obtained from the LR-TDDFT calculations (Fig. 4) reproduces the experimental spectral features (positions of absorption lines, not line shapes, which depend on the smoothing function applied in the calculations) with notable accuracy. Intensities are in good agreement for all but the first two transitions. In the UV-vis range, both the measured and computational spectra are rather featureless. This is in contrast to the spectra of the smaller organosoluble Au25(SCH2CH2Ph)18 and Au38(SCH2CH2Ph)24 NPs that show notable absorption features in the UV-vis range (1519). Additional LR-TDDFT calculations of possible compounds in the range of Au68(SR)31-34 [including Au68(SR)32 but with different arrangements of gold atoms] did not satisfactorily reproduce the measured IR data in the optical gap region (fig. S11).

Fig. 4 Measured and computed absorption spectra of the Au68NP.

(A) IR region. (B) UV-vis region. Black curves, experimental data; green curves, LR-TDDFT-computed spectra obtained from the individual optical transitions (green vertical lines) with a Gaussian smoothing function [width 0.09 eV in (A) and 0.25 eV in (B)]. The blue curve in (A) denotes a spectrum from which the vibrational contribution has been subtracted by reference to a spectrum of a larger cluster with the same ligand layer (see fig. S10 for details). Both the computed and experimental data indicate that the lowest electronic transition occurs at 2500 cm−1 (0.31 eV).

The notable findings of this work are: (i) the synthesis of a water-soluble AuNP, homogeneous in size, stable in solution, and nevertheless reactive toward sulfhydryl compounds, including proteins; (ii) the successful determination of atomic structure by EM, not previously reported; and (iii) the difference between the structure obtained and the only other structure of a large, water-soluble particle (the Au102NP), solved by x-ray diffraction.

The atomic structure of the Au68NP differs from that of the Au102NP in two important aspects. The symmetry of the Au68NP is lower, and the electronic structure does not indicate a filled electronic shell (5). Whereas the Au102NP exhibits global symmetry (based on a truncated decahedral core), with all remaining atoms following fcc packing rules, the Au68NP is based almost entirely on local fcc packing. The low-symmetry Au68NP structure differs from a proposal from theory for a metal core of higher symmetry for an organosoluble particle of similar size, Au67(SR)35 (21).

Structure determination of water-soluble AuNPs by EM is important because although many can be crystallized, only Au102NP crystals so far diffract to atomic resolution. Aberration-corrected transmission electron microscopes are capable of revealing individual heavy atoms (22). Structures of large metal particles imaged under high-dose conditions have been reported: the results of model fitting to EM data (23, 24); actual reconstructions from electron micrographs, in which the exact positions of individual atoms were not fully resolved (25); and results for bulklike solid-state systems where atoms are arranged in columns (26, 27). Our implementation of low-dose techniques from biological TEM, combined with the power of the aberration-corrected transmission microscope, has revealed the atomic structure of a AuNP without any prior knowledge, model fitting, or assumptions about packing and should be generally applicable. This finding paves the way for structure determination of many more metal NPs and discovery of the general principles of NP organization.

Supplementary Materials

www.sciencemag.org/content/345/6199/909/suppl/DC1

Materials and Methods

Figs. S1 to S11

References (2832)

Coordinates Files

References and Notes

  1. See supplementary materials on Science Online.
  2. Acknowledgments: The work at Stanford University was supported by the Human Frontier Science Program Organization (M.A.) and NIH grant AI-21144 (R.D.K). The work at the University of Jyväskylä was supported by the Academy of Finland (H.H. and M.P.) and the National Graduate School in Computational Chemistry and Spectroscopy LASKEMO (J.K.). G.L.H was supported by U.S. Department of Energy Biological and Environmental Research grant DE-AC02-05CH11231 under Integrated Diffraction Analysis Technologies. We thank P. Robinson for assistance with EM processing and helpful discussions and E. Hulkko and P. A. Clayborne for help with spectroscopic experiments and preliminary computations, respectively.
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