Analytical Characterization of Size-Dependent Properties of Larger

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Analytical Characterization of Size Dependent Properties of Aqueous Gold Nanoclusters Germán Plascencia-Villa, Borries Demeler, Robert L. Whetten, Wendell P. Griffith, Marcos Alvarez, David M. Black, and Miguel José Yacamán J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b00448 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016

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The Journal of Physical Chemistry

Analytical Characterization of Size Dependent Properties of Aqueous Gold Nanoclusters Germán Plascencia-Villa1*, Borries Demeler2, Robert L. Whetten1, Wendell P. Griffith3, Marcos Alvarez1, David M. Black1, Miguel José-Yacamán1*

1

Department of Physics and Astronomy. The University of Texas at San Antonio (UTSA). San

Antonio, Texas, 78249, USA. 2

Department of Biochemistry. The University of Texas Health Science Center at San Antonio

(UTHSCSA). San Antonio, Texas, 78229, USA. 3

RCMI Protein Biomarkers Core. The University of Texas at San Antonio (UTSA). San

Antonio, Texas, 78249, USA. * Correspondence: [email protected], [email protected] Tel: +1 210-458-5451

ABSTRACT Gold nanoclusters (AuNC) with well-defined structure and arrangement possess particular physical and functional properties. AuNC that differ only by less than 1 nm in diameter corresponding to one atomic layer show different structural, optical and physicochemical properties in a size-dependent mode, making their analytical characterization a challenge. An integrative approach with UV-Vis, dynamic light scattering and zeta-potential in combination with

high-performance

analytical

techniques

such

as

multi-wavelength

analytical

ultracentrifugation and electrospray ionization mass spectrometry were used to separate and determine their specific hydrodynamic diameter, partial abundance, molecular weight and

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mass/charge ratios when present in a complex mixture of AuNC containing Au102 (1.6 nm), Au144 (2 nm) and Au288-328 (2.5 nm). Advanced analytical electron microscopy imaging (spherical aberration corrected BF/HAADF-STEM at low voltages dose) also revealed the structures of discrete arrangements of gold nanocluster populations that were separated by gel electrophoresis.

INTRODUCTION Gold nanoclusters (AuNC) with sizes less than 3 nm in diameter possess unique and interesting structural and functional properties, which have attracted attention in different research areas1-2. The structure of AuNC can be described as superatom complexes with a shell-closing number electronic configuration when the AuNC are monolayer-coordinated with simple ligands2-3. These metallic nanoclusters are thermodynamically and electronically stabilized by the adsorption of high affinity ligands. Thiol-terminated compounds have been the ligands most commonly employed to stabilize the complex geometry of nanoclusters. However, the controlled synthesis, purification and surface modification of AuNC have been a challenging tasks4. Particularly, due to the poor water-solubility of organic thiolated compounds, their biocompatibility, uses and applications in biosciences are limited. Atomically precise nanoclusters such as Au102(SR)44 and Au144(SR)60 are some of the most structurally-stable representative species, with optical and functional properties useful in catalysis, optics and biosciences3. Chemical syntheses of AuNC usually produce a mixture of atomically precise clusters that differ by a few atomic layers5; therefore due to their narrow size distribution, the separation and precise characterization of individual metal nanocluster species


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remains a difficult but crucial task. Partitioning of these ultra-small particles has been attempted with different techniques, including: fractional precipitation, preparative chromatography (size exclusion and ion exchange), high-pressure liquid chromatography (HPLC), differential solvent extraction, electrophoresis (agarose or polyacrylamide), thin-layer chromatography and density gradient ultra-centrifugation5-6. Alternatively, etching of polydisperse metal clusters by adding an excess of coating ligands and incubating at moderate temperatures (60-100 °C) for several days favors obtaining higher geometric and electronically stable species and even a single monodisperse populations7. In the case of water-soluble ultra-small metal particles, the applicability of the aforementioned analytical techniques remains particularly challenging. Mainly due to the large extent of particleparticle interactions, the presence of difficult to remove by-products, interaction with counterions, and nature of the physicochemical environment of the media, which directly impacts the performance and behavior of nanoclusters coated with a monolayer of covalently attached stabilizing ligands. Analytical techniques, such as gel electrophoresis, electrospray ionization mass spectrometry (ESI-MS) and analytical ultra-centrifugation (AUC) have been extensively employed in biotechnology for routine and quantitative analysis of biomolecules, especially proteins and protein complexes, characterizing their size, anisotropy and density distributions8, as well as structure-function relationships9. Advanced instrumentation, sophisticated and optimized methodology can be applied in ESI-MS and AUC to assist with the precise analytical characterization of small metal clusters in the nanometer size range, assuming that each cluster species behaves as an individual molecule or superatom with particular properties, determined by their size, chemistry and number of ligands bound to each particle.


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In this work, we synthesized water-soluble thiolated Au nanoclusters (AuNC) with precise atomic numbers and characterized them using various analytical techniques. Analytical characterization

through

scattering/zeta-potential,

native

polyacrylamide

absorbance

gel

electrophoresis,

spectroscopy,

dynamic

multi-wavelength

light

analytical

ultracentrifugation (MW-AUC) and electrospray ionization mass-spectrometry (ESI-MS) allowed determination of the size-dependent properties of three different nanocluster species that showed good aqueous solubility and colloidal stability. Particularly, this integrative approach of the analytical techniques employed allowed determination of precise hydrodynamic diameters, molecular weights and mass/charges (m/z) of three different Au-pMBA nanoclusters simultaneously produced in aqueous media. Sizes, shapes and nanostructural properties of the purified Au nanoclusters were confirmed with spherical aberration-corrected scanning transmission electron microscopy (Cs-STEM) at low voltage doses and precisely identified as Au102, Au144 and Au288.

EXPERIMENTAL SECTION Synthesis and purification of Au nanoclusters. Au nanoclusters were synthesized using a modification of the two-phase transfer method7, 10-11. First, 4-mercaptobenzoic acid (pMBA) was dissolved in a 300 mM NaOH aqueous solution by stirring for at least 3 h. AuNC were prepared in 25 ml batches in 50 % methanol v/v, by adding HAuCl4 to a final concentration of 3 mM and adding pMBA to 9 mM final concentration. The solution was vigorously stirred overnight until the mixture was colorless. Then, ice-cold aqueous NaBH4 was added to a final concentration of 4.5 mM for reduction of Au(I)-pMBA intermediates with vigorous stirring for 2 h. Consequently, the entire reaction was placed in a 50


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ml conical tube with 1 volume of cold methanol and incubated at 4°C to allow precipitation of nanoparticles. Gold nanoclusters were precipitated by centrifugation (1000 rpm for 15 min), and resuspended in methanol, incubated at -20°C for 15 min and centrifuged again to remove byproducts. Finally, the nanoparticles were air-dried and resuspended in 500 µl of ddH2O for further characterization. The quality and distribution of Au clusters were assessed by polyacrylamide gel electrophoresis (PAGE). Samples were passed through a 10% native gel (19:1) in TBE (Tris-Borate-EDTA) buffer at 100 V for 90 min. PAGE gels were recorded on a white light transilluminator to determine purity and distribution of nanoclusters size. Bands corresponding to AuNC fractions were excised from the PAGE gels and placed into 15 ml conical tubes with ddH2O at 37°C for 1 day to allow diffusion of AuNC from gel matrix into the solution. Finally, AuNC fractions were concentrated in a rotary evaporator. Spectroscopy characterization. Absorption spectroscopy data were acquired using a CARY 100 UV-Vis Spectrometer from 300 to 800 nm using ddH2O or 100 mM NaOH as a blank. Dynamic light scattering (size distribution and zeta-potential) of the AuNC was performed with Zetasizer NanoZS (Malvern) using a He-Ne laser at 633 nm and 5 mW, with a backscattered detector angle at 173° and controlled temperature stage (25°C). Data were recorded and analyzed with Zetasizer Software 7.1. Analytical characterization: Multi-wavelength Analytical Ultracentrifugation (MW-AUC). AuNC were analyzed in four different aqueous solutions: 0, 30, 50 and 100 mM NaOH, at two concentrations: 1:2 and 1:5 dilutions with ddH2O of the AuNC stock. AUC was performed using a Beckman Optima XL equipped with a fiber-based multi-wavelength detector (University of Konstanz, Germany). Samples were sedimented at 20,000 rpm and 20°C for 5 h in Eponcharcoal filled centerpieces (Beckman-Coulter), using an An60Ti rotor. 200 scans were acquired


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for absorbance intensity at wavelengths from 200 to 700 nm, but only wavelengths between 300546 nm were included in the analysis. Data were analyzed with UltraScan-III release 192012-13, using two-dimensional spectrum analysis (2DSA)14-15 on the Stampede and LoneStar clusters at the Texas Advanced Computing Center (TACC) over a sedimentation range from 1–50 s, and a frictional ratio range from 1-3. Time-invariant noise was fitted for each wavelength measurement separately. Then, a meniscus position was fitted for the center wavelength (423 nm) with simultaneous time- and radially-invariant noise correction. The meniscus position was then applied to the experiments from each wavelength. A final 2DSA refinement step including simultaneous time- and radially-invariant noise correction coupled with up to ten iterative refinement steps was performed for each wavelength, using the optimized meniscus position. Finally, a 100-iteration Monte Carlo analysis16 for each wavelength measurement while applying the previously determined noise corrections was performed. Sampling the Monte-Carlo 2DSA signals from all wavelength, a sedimentation-absorbance plot was constructed. Furthermore, a global 2DSA-MC s vs. f/f0 overlay of all wavelengths was constructed, from which the hydrodynamic parameters of each species were integrated. The hydrodynamic radius was derived from the measured diffusion coefficient using the Stokes-Einstein relationship shown in Equation 1:

=

Equation 1

where k is the Boltzmann constant, D the measured diffusion coefficient, T the temperature and η is the viscosity of the solvent. A pseudo-3D movie of the 2DSA showing the sedimentation coefficients vs. frictional ratios with each wavelength representing a different frame is shown in Supporting Information.


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Electrospray-Ionization Mass Spectrometry (ESI-MS). For mass spectrometric analysis, the as-synthesized concentrated Aux(pMBA)y mixture sample was diluted to a working concentration of 0.5 mg ml-1 in an aqueous solution of methanol (50% v/v). The purified AuNC fractions were also diluted to a similar concentration for further analysis. Mass spectrometry data were collected using a micrOTOF II mass spectrometer (Bruker Daltonics) equipped with an electrospray ionization source and operated in the negative ionization mode. Sample solutions were introduced into the ESI source via syringe pump at a flow rate of 5 µl min-1. The instrument parameters were maintained at optimal values for each experiment as follows: capillary voltage, +4000 V; end plate offset, −1000 V; nebulizer gas pressure, 1.4 bar; dry gas flow rate, 10 L min-1; dry gas temperature, 150°C; capillary exit, −35 V; skimmer 1, −33 V; hexapole 1, −21.4 V; hexapole RF, −800 V; skimmer 2, −23 V; lens 1 transfer, 100 µs; and lens 1 pre pulse storage, 50 µs. The calibration was performed externally using cesium-acetate clusters, and mass spectra were recorded in the range 1000 ≤ m/z ≤ 8000. Mass spectra reported were averages of 5 min of scans, each consuming 25 µl of working sample solution, but not exceeding ~10 µg of sample. The data were processed using DataAnalysis Version 3.4 (Bruker Daltonik GmbH). mMass 5.5.0 software17-19 was used to calculate m/z values for the candidate Aux(pMBA)y species and to assign the peaks in the mass spectra. Advanced analytical electron microscopy. AuNC were diluted twenty-fold in ddH2O, 15 µl were loaded onto holey carbon copper grids 300 mesh (Electron Microscopy Sciences). AuNC were characterized by spherical aberrationcorrected atomic resolution scanning transmission electron microscopy (Cs-STEM) (JEOL ARM-200F), operated in STEM mode at an accelerating voltage of 80 kV. Micrographs were recorded using both the Bright Field (BF) and High Angle Annular Dark Field (HAADF)


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detectors, collected with a convergence angle of 26 mrad, collection semiangles of 50-180 mrad, probe size of 0.09 nm and probe current of 22 pA. Micrographs were recorded and analyzed with DigitalMicrograph 1.85 (GATAN).

RESULTS AND DISCUSSION Synthesis and purification of AuNC. To synthesize the AuNC, a modified protocol derived from the Brust-Schiffrin method was employed using water-soluble thiolated pMBA as coating and structure-directing agent7,

10-11

.

The organic phase corresponded to 50 % v/v methanol, which may assist as size-focusing agent during the formation of the AuNC20. After reduction of Au(I)-pMBA precursors with NaBH4, addition of excess cold methanol in combination with incubation at low temperature (4 °C) helped to prevent formation of larger sized Au nanoparticles. Even though it is possible to obtain monodisperse small particles with a narrow size distribution of 2.0 ± 0.5 nm in diameter using this approach7, 20, the final product still possesses nanoclusters with peculiar and significant structural differences. Native (non-denaturing) PAGE allowed resolving discrete subpopulations of AuNC that differed in diameter by only ~0.5 nm, which corresponds to a single atomic layer (Figure 1-A). Electrophoretic techniques have been effectively used for the separation of nanomaterials and bioconjugated nanomaterials, since most metal nanoparticles possess size-dependent characteristic colors, the separation into fractions or sub-populations with differing mobilities can be visually monitored21. To attain good resolution (separation of complex mixtures), it is necessary to optimize conditions of separation, for example by modifying the percentage of polyacrylamide/bis-acrylamide, the electrolyte component of the buffer solution, and the voltage-amperage passed though the gel matrix.


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Rigorous attention to purification-separation techniques as well as adequate and comprehensive analytical characterization of metal nanoclusters is fundamental to achieve controllable applications in nanoscience, nanotechnology and bio-sciences.

Optical properties: absorbance, photon-correlation and zeta-potential. Figure 1-B shows the normalized absorbance spectra of as-produced AuNC in comparison with the three different size-specific fractions recovered from PAGE. The mixture containing all three AuNC presented a slight absorbance peak in the 500-600 nm range. In comparison, the purified AuNC corresponding to the upper PAGE band showed surface plasmon absorption centered at 520 nm, while the other two fractions (middle and lower bands) did not show the presence of surface plasmon resonance effects. These observations correlate with previous reports7,

22-23

,

particularly that AuNC (Au144 and Au102) do not exhibit the characteristic strong surface plasmon resonance absorption peak (at 500-550 nm) that is observed in colloidal gold nanoparticles (1020 nm in diameter). Similar absorption properties were also observed in the absorbance spectra obtained from the AUC analysis (Supporting Information Figure S1). AuNC may present fluorescence in the Vis-NIR region depending on the surface ligands of nanoclusters24. Hydrodynamic diameter and size-dependent physicochemical properties of AuNC were determined by photon-correlation spectroscopy (or dynamic light scattering). Measurements of hydrodynamic diameter of the AuNC separated and recovered from PAGE showed that fractions effectively had different sizes correlated to their different mobilities through the polyacrylamide matrix. Fraction #1 showed a mean hydrodynamic diameter of 5.18 ± 0.81 nm, fraction #2 presented 4.99 ± 0.58 nm and fraction #3 with higher mobility displayed 3.79 ± 0.47 nm. All three nanoclusters showed high monodispersity and narrow size distribution (Figure 2) with no


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perceivable aggregation into higher size populations. Because of limitations in resolution from DLS the crude AuNC sample mixture showed only one population with a significantly broader polydispersity index (data not shown). The hydrodynamic diameter is determined from the translational diffusion coefficient, which correlates with the Brownian motion of the particles in aqueous solution. The Stokes-Einstein relationship (Equation 1) describes the relationship between diffusion and hydrodynamic radius21. Nanoparticle size obtained by DLS reflected not only the dimension of metallic core, but also included the frictional effects of thiolated ligands surrounding their surface, and the hydration layers and ions equally interacting with the nanoclusters. The specific sizes of the metal cores were determined by high-resolution electron microscopy imaging. Determination of physicochemical properties of AuNC, such as zeta-potential (ζ), electrophoretic mobility (µ) and electrical conductivity (EC) showed size- and pH -dependency. In the case of the zeta-potential parameter, all three AuNC showed high colloidal stability with a defined negative charge influenced by free carboxyl radical from pMBA coating agent. The value of ζ showed an influence related to the pH of the solution (by diluting in aqueous NaOH to achieve 30, 50 or 100 mM) (Table 1). An effect of NaOH concentration on the sedimentation behavior was also observed in the AUC experiments, measurements of the ζ-potential, and to some degree on the electrophoretic mobility. Fraction #1 originally displayed a ζ-potential of 16.1 ± 1.70 mV when diluted in ddH2O, indicating a low colloidal stability that could derive into aggregation or precipitation. In comparison, smaller nanoclusters showed good stability in ddH2O with -48.0 ± 0.18 and -44.0 ± 3.80 mV for Au144 (Fraction #2) and Au102 (Fraction #3), respectively. Addition of NaOH resulted in effectively eliminating the correlation between ζpotentials of all three AuNC and their size (Table 1 and Supporting Information-Figure S2).


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ζ-potential of colloidal particles is highly related to the value of electrophoretic mobility (µ), this relationship is expressed by the Smoluchowski mobility formula and the Debye-Hückel parameter, where particle radius and electrolyte concentration are implicit25-26. Determination of µ of AuNC showed a similar tendency as ζ: the largest AuNC had a lower µ of -1.27±0.13 µm cm V-1 s-1 without NaOH than the two smaller Au clusters which displayed µ ranging from -3.76 to -3.45 µm cm V-1 s-1 (Table 1). Addition of NaOH caused a reduction of µ only on clusters from Fraction#1, achieving a similar value as measured for the smaller nanoparticles. Interestingly the values of electrophoretic mobility remained relatively stable for Au144 and Au102 in both scenarios with and without the presence of electrolytes. The Debye-Hückel parameter from µ is applicable when the particle radius is sufficiently large, in the case of ultra-small particles like Au144 and Au102 it has no significant effect on µ, explaining the relative consistency of the values obtained at different ionic strength (0-100 mM NaOH). In contrast, for Au nanoparticles in the range of 15-200 nm, the ζ and µ values are highly influenced by particle size and electrolyte concentration25-27. Finally, EC measurements showed a clear linear dependency on the ionic strength of the solution, with no significant differences among all three AuNC. Gold nanoclusters dissolved in water without any added salts showed a conductivity of 0.263 to 0.405 mS cm-1. The EC increased to 4.14-5.3 and 8.48-9.45 mS cm-1 in the presence of 30 and 50 mM NaOH, respectively. With an excess of 100 mM NaOH the EC reached 20.4-21.8 mS cm-1 (Table 1, Supporting Information Figure S2). EC values obtained for AuNC of 2.5, 2.0 and 1.6 nm at different ionic strengths (0 to 100 mM NaOH) correlate with the electrokinetic expression of Ohshima25, which states that charge density and total charge of gold nanoparticles of different sizes remain apparently constant with fixed electrolyte concentration.


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Measurement of the ζ-potential, µ and EC in solution at different concentrations of counter-ions provided detailed information of the net charge, colloidal stability and mobility properties of water-soluble thiolated AuNC. In all cases, AuNC showed an effective negative electrokinetic surface charge in NaOH buffering solutions. Parameters such as pH, concentration, ionic strength, temperature and surface ligands have a direct impact on physicochemical behavior of the nanoparticles in solution21.

Analytical characterization: Multi-wavelength Analytical Ultracentrifugation (MW-AUC). Characterization of AuNC through MW-AUC and ESI-MS provided quantitative information on the nanoclusters, including hydrodynamic radius, molecular weight, sedimentation and diffusion coefficients, absorbance spectra, and their relative composition. Synthesis resulted in a mixture containing nanomaterials with different size and mass characteristics. Gold nanoclusters synthesized by wet chemistry are usually heterogeneous, requiring additional steps to narrow their size distributions, or to purify particular sub-populations with identical properties. The sedimentation rates of AuNC depend on their size, anisotropy, density and hydration, which in turn depend on their chemical composition and surface chemistry. MW-AUC of AuNC was performed in aqueous solution at 0, 30, 50 and 100 mM NaOH, measuring absorbance in the range of 350-600 nm. Figure 3 shows the sedimentation profile of AuNC in ddH2O. In all cases, the three AuNC were entirely baseline separated by AUC and provided similar, though not identical hydrodynamic parameters and relative composition. Measurements of sedimentation coefficients of AuNC samples in water showed elevated non-ideal solution behavior, such as colloidal instability and overall increased heterogeneity that was attributed to particle-particle interactions through polar carboxyl groups of the pMBA coating agents (Figure 3). With the


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addition of increasing amounts of NaOH as buffering agent and particularly Na+ functioning as counter-ion for the –COO- moiety from pMBA, the sedimentation behavior of AuNC and resolution of MW-AUC to separate and resolve sub-populations of nanoclusters improved markedly. Figure 4 shows the sedimentation profile of AUC of AuNC in 50 mM NaOH. Under basic pH the AuNC showed ideal sedimentation behavior, preventing particle aggregation, improving colloidal stability and reducing heterogeneity. Modeling of sedimentation velocity (SV) data by whole-boundary methods provided detailed measurements for the hydrodynamic diameter of the AuNC species observed through multi-wavelength AUC, as well as absorbance spectrum of each species (Figure 3 and 4). The surface of AuNC was passivated with watersoluble thiolated pMBA. This compound possesses a highly planar and rigid benzoic acid residue with length of approximately 8 Å protruding from the metal core28. Using this size estimate, the pMBA contributed about 1.6 nm to the hydrodynamic diameter of the metal core of the AuNC. SV data was modeled by describing the ideal transport of both sedimentation and diffusion of the particles in a centrifugal force field. This process is described by the Lamm equation29, which is solved by highly efficient adaptive finite element methods30. Hydrodynamic diameters determined experimentally from AUC were: 4.20 ± 0.48 nm, 3.64 ± 0.10 nm and 2.72 ± 0.07 nm; which were in excellent agreement with AuNC mean sizes determined for the three particle populations through Cs-STEM (Table 1), by adding 1.6 nm from pMBA layer obtaining: 4.08 ± 0.25, 3.66 ± 0.27 and 3.22 ± 0.22 nm, respectively. Additionally, from absorbance and SV data we could determine that as-prepared AuNC contained three sub-populations in 1:2.3:3.96 ratios, of the 2.5 (Au288), 2 (Au144) and 1.6 (Au102) nm nanoclusters, respectively. Complementary to PAGE separation of nanoparticles, MW-AUC can achieve high-resolution size distributions, estimate the hydrodynamic diameter of nanoparticle species in solution, and


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provide reliable concentration ratios for all species. AUC is a highly versatile analytical characterization technique complementary to spectroscopy, DLS, electron microscopy with several advantages: non-destructive, no need to create a gradient, small volume of samples, rapid data acquisition and precise analysis, aqueous-base, no need of organic solvents, and high resolution. As demonstrated here for the first time, MW-AUC also permits the direct extraction of wavelength absorbance spectra for all separated species (Figure S1).

Analytical characterization: Electrospray-Ionization Mass Spectrometry (ESI-MS) Electrospray ionization time-of-flight mass spectrometry (ESI-MS) helped to analyze the AuNC based on their mass-to-charge (m/z) ratio. Water-soluble monolayer-protected clusters, including the Au-pMBA clusters of interest here, have presented a formidable challenge to ESI-MS analysis, for reasons that remain unclear. Even the best results published show a charge [z] series of broad, unresolved features. The low information-content of such spectra is in striking contrast to those achieved for non-aqueous (hydrophobic) metal clusters. The mass spectral data are nonetheless consistent with the composition formulations indicated by other analytical techniques, such as X-ray diffraction of single crystals, electron microscopy and electron diffraction of single clusters. Here, we build upon the recent mass spectrometry analysis of Ag44(pMBA)30[4-] system31, which we have repeated and extended to include isotopic exchange (hydrogen-deuterium, or HDX). Various approaches have been attempted including phase transfer and counter-ion exchange, electrospray and API (chemical ionization), but only the one described herein has yielded useful spectra resolved at the limits of the instrument. Briefly, the as-synthesized crude mixture and the three PAGE-separated fractions (Figure 1) were analyzed by direct infusion of the aqueous or methanol-water solution, ionized by electrospray and dried


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by inert gas at 150oC, prior to transfer into the ion-source region of a time-of-flight mass spectrometer, obtaining a resolution exceeding 1x104 but not 2x104, implying that one was unable to achieve isotopic (1 Da) resolution in the relevant mass-range of 25-40 kDa. The MS signal was averaged over typically 5 min, during which time 25 µl solution, containing not more than ~10 µg total cluster material, was consumed. Figure 5 shows ESI-MS for the crude AuNC mixture and for two lower-mass purified fractions. The AuNC mixture containing three major species of clusters was introduced into the mass spectrometer in the negative ion mode, spectra obtained showed 7 distinctive major peaks ranging from 2600 to 4000 m/z evidencing the complexity of Au nanoclusters with different mass and charges (Figure 5-A). ESI-MS confirmed the identity of the two lighter components: Au102pMBA44 and Au144pMBA60. The main peaks centered at 3832 and 4472 m/z were assigned to 7- and 6- charge-states of the Au102pMBA44, respectively. Secondary peak at 5365 possess a close match to 5- charged state, whereas the Au102 4- state at 6707 m/z showed a weak signal intensity. The MS spectra peaks corresponding to the 7- and 6- charged species of monolayerprotected Au102 clusters were prominent and clearly identified, since these gold clusters were preferentially produced corresponding to ~55 % of the clusters mixture (as calculated from AUC). The peak at 5177 m/z with lower intensity was assigned to 7- charge-state of the Au144pMBA60 species, with a molecular weight of 37553 Da and a metal core of 28362 Da7, 32. Additional peaks with low ion count were not assigned to the Au species mentioned and clearly identified by PAGE and MW-AUC. Fraction #1 of AuNC (the heaviest population) when analyzed by MS did not show clear features necessary to determine or even estimate its molecular weight. This was attributed to significantly lower concentration relative to other smaller AuNC species and the possibility that this population contained clusters ranging from


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Au288-Au328 that correlate with particle diameters of ~2.5 nm observed by DLS, AUC and STEM. ESI-MS of AuNC from fraction #2 (Figure 5-B), which was assigned as the elusive Au144pMBA60 based on size, presented two series of major maxima, each composed of a multiplet structure. The series was assigned to the charge-states [z] = [9-] to [12-], with the dominant features occurring at [10-] and [11-]. The sharp onset, or base peak, of each multiplet, occurred at an m/z value corresponding with the compositions of (144,60)[z-] and (142,57)[z-]. The principal spacings within each multiplet were roughly ~60 Da/z, suggesting a distinct sequence of X+ - H+ exchange processes, in addition to the weaker Na-H features at smaller spacings (22 Da/z). Each peak within the multiplet had a breadth (FWHM) of ~10 Da, consistent with the isotopic distribution (cf. 480 C-atoms at natural isotopic abundance). Our interpretation of the main features of the spectrum was that they are consistent with the dominant presence of highpurity (144,60) which partially decays according to: (144,60)[(z+1)-] → (142,57)[z-] + (2,3)[1-] i.e. charge reduction by the expulsion of Au2(pMBA)3 anion, also a decay-mechanism as previously demonstrated for the Ag44(pMBA)30[4-] cluster31.

Evidently this process had an

efficiency increasing with charge, as the (142,57) fragment predominates at [z] = [12-]. Further details presented in Supporting Information (Figure S4 and Table S1. Mass Assignments). Finally, the largest AuNC fraction with the highest mobility and smallest size, assigned as the putative Au102pMBA44, presented a long series of major maxima, each one composed of rich multiplet structure (Figure 5-C). The m/z values of the peaks correlated well with different partially deprotonated states of pure crystallized Au102(pMBA)4433. Structure and arrangement of Au102pMBA44, was resolved at atomic resolution28; the complete thiolated nanocluster has a


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molecular weight of 26829 Da whereas the gold core is 20090 Da. The series was assigned to the charge-states [z] = [7-] to [11-], with the dominant features occurring at [9-] and [10-]. The sharp onset, or base peak, of each multiplet, occurred at a m/z value corresponding, within the limits of instrument resolution and calibration, to the compositions (101,42)[z-], and the spacings within the multiplets were very nearly 22 Da, suggesting a sequence of Na+ — H+ exchange processes. Each peak within the multiplet had a breadth (FWHM) of ~8 Da, consistent with the isotopic distribution (mainly

13

C at natural abundance 1.1%). The dominating features of the spectrum

were consistent with the expected formula (102,44) and the special process: (102,44)[(z+1)-] → (101,42)[z-] + (1,2)[1-] i.e. charge reduction by the expulsion of Au(pMBA)2 anion, the same decay-mechanism as was previously demonstrated for the Ag44(pMBA)30[4-] cluster31. Evidently this process was very efficient, under the conditions applied herein, because only with difficulty could the unfragmented parent ion be resolved, thanks in part to the congestion of the long series of Na-H exchange products. Further details are presented in Supporting Information (Figure S4 and Table S2. Mass Assignments) Because of the complexity of the gold nanoclusters, it has proven quite difficult to obtain higher resolution mass spectra of these larger aqueous polyanionic clusters34,35. Factors include excess solvent retention, presence of counter ions for pMBA monolayer and their tendency to aggregate by cross interaction between carboxyl ligands of clusters36. Despite these challenges, the protocol and instrumentation employed in this paper showed progress in resolving two species of Au nanoclusters with close size and similar surface charge, confirming the purity and identity of Au144 and Au102. The MS results are sufficiently promising to indicate the direction of future analyses of similar water-soluble monolayer-protected metal nanoclusters and their


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bioconjugation products. Clearly, higher resolution instruments will be required to confirm assignments at the isotopic level of resolution. Contrary to mass spectroscopy of biomolecules (particularly proteins), specialized mass spectra databases of metal clusters do not exist, posing additional data processing and interpretation a challenging task.

Advanced analytical STEM imaging. AuNC recovered from PAGE were analyzed by Cs-STEM. Determination of the precise structure-arrangement of water-soluble ultra-small Au nanoparticles has been challenging. Only Au102(SR)44 thiol-protected nanoclusters have been successfully crystallized and structurally resolved at 1.1 Å resolution through X-ray diffraction28, whereas structure and arrangement of Au68 was elucidated at atomic resolution by 3D reconstruction from cryo-electron microscopy imaging6. In this work, we employed spherical aberration-corrected STEM at low voltage doses (80 kV). This advanced analytical imaging technique has the capability to obtain atomic resolution micrographs and direct rapid acquisition of diffraction patterns from ultra-small metal clusters at sub-Angstrom resolution37-38. Under these conditions, radiation damage and ionization of nanoparticles is reduced, contrary to electron microscopy performed at high voltage doses (200-300 kV) where alteration of arrangement and atomic structure of nanomaterials may occur rapidly, caused by the incident-electron energy38-39. Cs-STEM imaging of AuNC with a high angle annular dark field (HAADF) detector revealed high contrast and chemical composition-related information from nanoclusters (Figure 6). AuNC from PAGE fraction #1 showed high monodispersity and a narrow size distribution, with a mean diameter of 2.48 ± 0.25 nm (Figure S3-A). Nanoclusters showed icosahedral arrangement, the metal core of the cluster presented an ordered nanostructure confirming


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crystallinity and organization into a molecule or superatom complex, where the thiolated coating stabilizes the geometry (Figure 6-A-B). FFT and measurement of interplanar distances confirmed nanocluster crystallinity, with spacings of 2.54 Å and 2.38 Å, characteristic of gold. Au nanoclusters corresponding to fraction #2 (middle bands in PAGE) displayed a mean diameter of 2.06 ± 0.27 nm, this nanocluster sub-population showed a narrow distribution containing particles of the same size. HAADF-STEM imaging was able to resolve the icosahedral arrangement and symmetry of these nanoclusters (Figure 6-D-E). The structure of these nanoclusters correlated with the arrangement obtained theoretically by DFT simulations for Au144(SR)60 with an icosahedral metal core, which was also confirmed through X-ray scattering and NMR spectroscopy10, 40. Au-Au bond distances measured were 2.46 Å, which correlated well with DFT predicted inter-atomic distances of 2.45 ± 0.019 nm. A detail of the 5-fold symmetry observed in Au144 is shown in Figure 6-F. Au144(SR)60 is characterized by a compact metallic core, which adopts an arrangement of three concentric layers with 60 symmetry-equivalent atoms surrounded by –SR ligands40. The special atomic and electronic structure adopted by Au144 into a super-atom complex provides high stability, capacitive properties and particular optical properties useful in molecular electronics, bio-applications and electrochemistry. AuNC with the higher electrophoretic mobility in PAGE showed a mean diameter of 1.62 nm (Figure 6-G-H). This sub-population of nanoclusters corresponded to the previously reported Au102(pMBA)44, which is characterized by a central packed decahedral structure surrounded with an additional layer of Au atoms and thiolated ligands28. Au102 represents an example of atomically monodisperse nanoclusters with a closed geometric shell with a magic number configuration of superatom complexes. FFT confirmed the AuNC arrangement and orientation into [111] with an interplanar spacement of 2.34 Å (Figure 6-I). From X-ray atomic structure of


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Au102, it was observed that organic thiols are exchangeable and mobile, a situation that may increase the difficulty of structural analysis, like in the case of atomic resolution electron microscopy imaging28. Cs-STEM imaging under low electron dose (80 kV) helped to reveal the atomic structure of Au102 ultra-small clusters, by reducing the radiation damage which may occur under high-voltage conditions (200-300 kV) typically used for high-resolution electron microscopy imaging.

CONCLUSIONS Advanced

analytical

electron

microscopy

techniques

(spherical

aberration

corrected

BF/HAADF-STEM at low voltages doses) revealed the atomic structure of discrete gold nanocluster populations separated by electrophoresis. The AuNC showed different structural and physicochemical properties in a size-dependent mode as revealed by UV-Vis, dynamic light scattering and zeta-potential. Understanding the particular behavior of AuNC in solution helped to optimize their separation, characterization and determination of hydrodynamic diameter, molecular weight and mass/charge. We showed that three AuNC sizes could be clearly characterized even when present in a complex mixture, and when the AuNC differ only by one single atomic layer (0.5 nm in diameter). Multi-wavelength AUC and ESI-MS were able to efficiently resolve and characterize quantitatively unknown mixtures of ultra-small Au nanoparticles which differ in size, charge and optical properties, determining hydrodynamic diameters that offer excellent correlation with advanced electron microscopy imaging of purified AuNC.


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SUPPORTING INFORMATION Absorbance spectra from AUC, Physicochemical properties of AuNC, Histograms of size distribution, ESI-MS for the assignments of mass, Proposed assignments of peaks in the ESI-MS spectra. Real time sedimentation coefficients vs frictional ratio in MW-AUC.

ACKOWLEDGMENTS This work was supported by The Welch Foundation (AX-1615 and AX-1857), NSF-DAC (1339649) and NSF (TG-MCB-070039). Facilities of NIH RCMI Nanotechnology and Human Health Core (RCMI Grant 5G12RR013646-12) and NIH RCMI Biophotonics (RCMI Grant G12MD007591) at UTSA. We thank Helmut Cölfen and Dirk Haffke from the University of Konstanz (Germany) for access to multi-wavelength analytical ultracentrifugation.

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FIGURES Figure 1.

Size-dependent properties. (A) Polyacrylamide gel electrophoresis, to verify heterogeneity, separate and purify AuNC into fractions or subpopulations of specific size (Lines 1: original sample, Line 2: Fraction #1, Line 3: Fraction #2, Line 4: Fraction #3). (B) Absorbance spectroscopy of gold nanoclusters, asproduced mixture and purified fractions.

Figure 2.

Photon Correlation Spectroscopy of AuNC. Determination of hydrodynamic diameter and size distribution in solution of purified AuNC through photon correlation spectroscopy (dynamic light scattering). Nanoclusters were recovered from PAGE and redissolved in ddH2O. Fraction #1-Black curve: 5.18±0.81 nm. Fraction #2-Dark red curve: 4.99±0.58 nm. Fraction #3-Red curve: 3.79±0.47 nm.


26

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Figure 3.

Multi-wavelength Analytical Ultracentrifugation (MW-AUC) of AuNC in water. 2DSA-Monte Carlo analyses of sedimentation velocity coefficient results from AuNC measurements in water, absorbance measured between 350-600 nm.

Figure 4.

Multi-wavelength Analytical Ultracentrifugation (MW-AUC) of AuNC in 50 mM NaOH. 2DSA-Monte Carlo analyses of sedimentation velocity coefficient results from AuNC measurements in 50 mM NaOH buffer, absorbance measured between 350-600 nm.


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Figure 5.

Page 28 of 30

Electrospray-Ionization Mass Spectrometry (ESI-MS). (A) Spectra of assynthesized sample containing three major AuNC species. (B) Purified AuNC fraction #2, Au144. (C) Purified AuNC fraction #3, Au102. Labels and dashed-line markers for the major features indicate composition-assignments, (x,y)[z] designating z-charged anions AuxLy[z-]. (Details in Supporting Information Figure S4 and Tables S1-S2).


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Figure 6.

HAADF-STEM imaging of AuNC. (A) Au288 low magnification. (B) Au288 high magnification. (C) FFT and interplanar distances. (D) Au144 low magnification. (E) Au144 high magnification. (F) FFT and detail of 5-fold symmetry axis. (G) Au102 low magnification. (H) Au102 high magnification. (I) FFT and detail of interplanar distances of Au102.

Table 1.

Size dependent properties of AuNC. Size determined from STEM or dynamic light scattering (DLS). Zeta-potential (ζ, mV), Electrophoretic mobility (µ, µm cm V-1 s-1) and Electrical Conductivity (EC, mS cm-1) were measured on purified AuNC diluted in ddH2O or Buffer (NaOH 50mM).

AuNC

Size (nm)

Zeta-Potential (ζ ζ)

Electrophoretic Mobility

Electrical Conductivity

(µ)

(EC)

DLS

STEM

H2O

Buffer

H2O

Buffer

H2O

Buffer

AuNC-1

5.18±0.81

2.48±0.25

-16.1±1.70

-45.4±5.75

-1.27±0.13

-3.56±0.45

0.405±0.26

8.48±0.08

AuNC-2

4.99±0.58

2.06±0.27

-48.0±0.18

-44.5±5.42

-3.76±0.01

-3.49±0.42

0.263±0.01

9.45±0.22

AuNC-3

3.79±0.47

1.62±0.22

-44.0±3.80

-43.4±5.78

-3.45±0.29

-3.40±0.45

0.318±0.07

9.33±0.25


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TOC Graphic


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Analytical Characterization of Size-Dependent Properties of Larger

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