Solution NMR Spectroscopy with Isotope-Labeled Cysteine (13C and

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Solution NMR Spectroscopy with Isotope-Labeled Cysteine (13C and 15 N) Reveals the Surface Structure of L‑Cysteine-Coated Ultrasmall Gold Nanoparticles (1.8 nm) Tatjana Ruks,† Christine Beuck,‡ Torsten Schaller,§ Felix Niemeyer,§ Manfred Zähres,∥ Kateryna Loza,† Marc Heggen,⊥ Ulrich Hagemann,# Christian Mayer,∥ Peter Bayer,‡ and Matthias Epple*,†

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Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), ‡Institute of Biology and Center for Medical Biotechnology (ZMB), §Organic Chemistry, ∥Physical Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), and # Interdisciplinary Center for Analytics on the Nanoscale (ICAN) and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, 45117 Essen, Germany ⊥ Ernst Ruska Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, 52428 Jülich, Germany ABSTRACT: Ultrasmall gold nanoparticles with a diameter of 1.8 nm were synthesized by reduction of tetrachloroauric acid with sodium borohydride in the presence of L-cysteine, with natural isotope abundance as well as 13C-labeled and 15Nlabeled. The particle diameter was determined by high-resolution transmission electron microscopy and differential centrifugal sedimentation. X-ray photoelectron spectroscopy confirmed the presence of metallic gold with only a few percent of oxidized Au(+I) species. The surface structure and the coordination environment of the cysteine ligands on the ultrasmall gold nanoparticles were studied by a variety of homo- and heteronuclear NMR spectroscopic techniques including 1H−13Cheteronuclear single-quantum coherence and 13C−13C-INADEQUATE. Further information on the binding situation (including the absence of residual or detached Lcysteine in the solution) and on the nanoparticle diameter (indicating the welldispersed state) was obtained by diffusion-ordered spectroscopy (1H-, 13C-, and 1 H-13C-DOSY). Three coordination environments of L-cysteine on the gold surface were identified that were ascribed to different crystallographic sites, supported by geometric considerations of the nanoparticle ultrastructure. The particle size data and the NMR-spectroscopic analysis gave a particle composition of about Au174(cysteine)67.

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INTRODUCTION Ultrasmall metallic nanoparticles, typically of gold, have gained increasing interest in the last decade because of their small size (between 1 and 3 nm) and their special optical and biomedical properties.1−12 Zarschler et al. have given an extensive review on the biomedical application of ultrasmall inorganic nanoparticles, defined as having a diameter of 1−3 nm, including gold nanoparticles. They are small enough to be rapidly excreted from the body by renal filtration, but can also migrate into the brain across the blood−brain barrier.9 It is also remarkable that a persistent protein corona formation (see Monopoli et al.13 for the formation of short- and long-lived protein coronas around nanoparticles) was not observed on the surface of ultrasmall gold nanoparticles, terminated with zwitterionic molecules such as cysteine14,15 (see Boselli et al. on the formation of a protein corona around ultrasmall nanoparticles).16 The toxic effects and the biodistribution of ultrasmall gold nanoparticles were extensively reviewed by Schmid, Kreyling, and Simon.1 Usually, sulfur-terminated ligands are used for colloidal stabilization. The nature of the gold−ligand surface is essential for optical and biological applications, but typically little is known about the actual surface structure, that is, the bond © XXXX American Chemical Society

between the metal and the ligand. However, in the last decade, a number of atom-precise gold clusters have been crystallized and characterized by single-crystal X-ray diffraction. A landmark was the structural characterization of Au102(pMBA)44 (pMBA = pmercaptobenzoic acid) at high resolution by Kornberg et al. in 2007. The cluster consists of a Au49 core with a Marks decahedron, capped by gold atoms in the periphery. The ligand is bound via sulfur, typically bridging two gold atoms, resulting in a sulfur-to gold ratio at the surface of about 0.7:1.17 The structure of this Au102 cluster was extensively discussed and compared to metalloid aluminum and gallium clusters of similar size by Schnöckel et al. The formal oxidation state of the thiolbound gold atoms can formally be computed as +0.7 with an inner core of “metallic” Au0 atoms.18 Xiong et al. have reported the crystal structure of Au21(SR)15 with a face-centered cubic (fcc) gold atom packing.19 Schnepf et al. have reported the crystal structure of the cluster Au70S20(PPh3)12 with a hydrodynamic diameter (by dynamic light scattering) of 2.5 nm. In the Received: November 14, 2018 Revised: December 18, 2018 Published: December 21, 2018 A

DOI: 10.1021/acs.langmuir.8b03840 Langmuir XXXX, XXX, XXX−XXX

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us to record the corresponding 13C- and 15N-NMR spectra and to unambiguously assign all atoms of surface-bound L-cysteine.

core, 22 Au atoms form a distorted part of an fcc lattice with Au4S4 units as a structural motif of the surrounding shell. The band gap was about 0.6 eV, indicating a molecular structure.20 Wang et al. have recently reported the single crystal structure of the cluster Au144(CCAr)60 with a multishell structure, based on Mackay icosahedra.21 Yan et al. cocrystallized two alloyed clusters, that is, (AuAg) 2 6 7 (SR) 8 0 (1.65 nm) and (AuAg)45(SR)27(PPh3)6 (1.05−1.25 nm) and determined their structure by single-crystal X-ray diffraction. The larger cluster consists of four concentric icosahedral shells and has a fully metallic behavior (no band gap). The smaller cluster is trigonal-prismatic and shows a molecular structure with a computed band gap of 0.71 eV.22 Sakthivel and Dass have recently reviewed the chemistry of aromatic thiolate-protected gold nanoparticles and clusters from 28 to 279 gold atoms, including structural trends with increasing size.23 Xie et al. demonstrated how a Au25 cluster can be enlarged to a Au44 cluster and followed the growth by mass spectrometry.24 Häkkinen et al. analyzed Au68 and Au144 clusters, protected by mMBA (3-mercaptobenzoic acid).25 If there is no crystal structure available due to the lack of single crystals, the characterization of ultrasmall nanoparticles becomes difficult. Azubel et al. have demonstrated how the structure of the cluster Au68(mMBA)ca.32 can be determined by electron microscopy at high resolution.26 This was extended to the structure of the Au144(mMBA)ca.40 cluster by aberrationcorrected electron microscopy at atomic resolution, aggregating the images of about 4300 individual particles.27 In electron microscopy, however, the organic ligands are not visible. Therefore, NMR spectroscopy as a comparatively new tool to study nanoparticles and metallic clusters has been applied to probe the ligand shell with high accuracy.28,29 Unfortunately, the vicinity of the metallic particle usually often results in a substantial line broadening and, in consequence, to a loss of resolution of the NMR spectrum. Especially for larger particles,30−40 the elucidation of the surface structure is hardly possible. If the particles are small enough,28,29,38,41,42 characteristic resonances are observable and distinct ligand species can be identified. Mancin et al. have even proposed to use NMR spectroscopy on metallic nanoparticles for chemosensing of organic molecules.43,44 In the present paper, we continue our earlier studies on ultrasmall thiolate-terminated gold nanoparticles by 1H-NMR spectroscopy,41 and focus on water-dispersed L-cysteinestabilized gold nanoparticles. Cysteine is of special interest: it is the only amino acid containing a thiol group that can bind to gold nanoparticles, permitting to attach peptides and proteins. Surprisingly, only a few studies focused on the structure of cysteine-coated gold nanoparticles. Gullion et al. used solid-state NMR spectroscopy and density functional theory (DFT) calculations and proposed a model for a mono- and a bilayer arrangement of cysteine on the surface of 6.6 nm gold nanoparticles.45−47 In solution, however, the surface structure will differ from the one in the solid state. We have, therefore, carried out a comprehensive study as a step toward an improved understanding of the arrangement of amino acids, peptides, or even proteins on the surface of gold nanoparticles. As expected, 1H-NMR spectroscopy alone is not sufficient to reach this goal because severe line broadening and overlap of signals do not allow conclusive interpretations. A decisive insight was gained with 13C- and 15N-labeled L-cysteine which enabled

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EXPERIMENTAL SECTION

Chemicals. For gold nanoparticle synthesis, we used sodium borohydride (NaBH 4 , Fluka, purity ≥ 96%) and sodium tetrachloridoaurate(III) (NaAuCl4·H2O, Sigma-Aldrich, 99%), both dissolved in ultrapure water. Gold nanoparticles were coated with Lcysteine (Alfa Aesar, purity 98%) and isotope-labeled [13Cβ, purity 99%; 1,2,3-13C, purity 99%; 15N, purity 98%] L-cysteine. All isotopelabeled L-cysteine molecules were obtained from Cambridge Isotope Laboratories. Ultrapure water with a specific resistivity of 18.2 MΩ (Purelab ultra instrument from ELGA) was used in all experiments. Before use, all glassware was cleaned with boiling aqua regia and thoroughly rinsed with ultrapure water. Synthesis of Nanoparticles. For gold nanoparticles coated with Lcysteine, 1 mL of NaAuCl4 (5 mM corresponding to 5 μmol) was dissolved in 20 mL degassed water and 0.1 mL L-cysteine solution (150 mM corresponding to 15 μmol, freshly dissolved in water) was added under stirring. Immediately after that, 0.1 mL of the reducing agent NaBH4 (200 mM corresponding to 20 μmol, freshly dissolved in water at 4 °C) was added to the mixture and stirred for another 10 min. Because of the low pH (between 4 and 5) in the mixture, the carboxy groups of the ligand are protonated, and no electrostatic repulsion is stabilizing the nanoparticles in dispersion. Therefore, the formed nanoparticles agglomerate and can be collected by centrifugation at 2000g (15 min). For purification, the supernatant was removed, and the collected nanoparticles were redispersed in 40 mL water and then centrifuged twice at 2000g and redispersed in water. For NMR characterization, the nanoparticles were dissolved in sodium hydroxide solution (0.2 M NaOD, pH 12). Characterization. The gold content of the nanoparticle solution was determined by atomic absorption spectroscopy (AAS) with a Thermo Electron M-Series spectrometer (a graphite tube furnace according to DIN EN ISO/IEC 17025:2005) after dissolving the particles in aqua regia. A zeta potential titration was performed with a Stabino instrument (Particle Metrix) with a 10 mL measuring cell and a 400 μm pistol. Nanoparticle dispersion (10 mL) at pH 10 was titrated with volumes of 15 μL of 0.1 M HCl at intervals of 10 s. The pH measurements were performed with a pH microelectrode. For pure L-cysteine in H2O, a pH of 4.3−4.5 was determined. For all analyzed samples of cysteine-functionalized ultrasmall gold nanoparticles, a pH of 11.6−12.3 was determined after adding NaOD to a concentration of 0.2 M. Analytical disc centrifugation (differential centrifugal sedimentation; DCS) was performed with a CPS Instruments DC 24000 disc centrifuge (24 000 rpm). Two sucrose solutions (8 and 24 wt %) formed a density gradient which was capped with 0.5 mL dodecane as a stabilizing agent. The calibration standard was a poly(vinyl chloride) latex in water with a particle size of 483 nm provided by CPS Instruments. The calibration was carried out prior to each run. A sample volume of 100 μL was used. High-resolution imaging was performed with an aberrationcorrected FEI Titan transmission electron microscope equipped with a Cs-probe corrector (CEOS Company), operated at 300 kV.48 Imaging was performed by the negative-CS imaging technique (NCSI). Scanning electron microscopy (STEM) was performed with a FEI Titan microscope, equipped with a Cs-probe corrector (CEOS Company) and a high-angle annular dark-field (HAADF) detector, operated at 200 kV. Z-contrast conditions were achieved at a probe semiangle of 25 mrad and an inner collection angle of the detector of 70 mrad. The elemental mapping by energy-dispersive X-ray spectroscopy was conducted on a probe-corrected FEI Titan 80-200 “ChemiSTEM” electron microscope equipped with four symmetrical SDD detectors.49 For NMR spectroscopy, on gold nanoparticles capped with Lcysteine, the collected nanoparticles were dispersed in 400 μL D2O and 100 μL of a 1 M solution of NaOD in D2O. 1D-NMR spectra (1H, 13C, and 15N) and 2D-NMR spectra [1H−1H-correlated spectroscopy B


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Langmuir (COSY), 1H−13C-heteronuclear single-quantum coherence (HSQC), 13 C−13C-INADEQUATE (incredible natural abundance doublequantum transfer experiment)] were recorded with a Bruker-AVANCE III 600 MHz spectrometer equipped with a Prodigy cryo probe head. 1 H-DOSY (diffusion ordered spectroscopy) measurements were performed with a Bruker-AVANCE 500 MHz spectrometer, and 13CDOSY was carried with a Bruker-AVANCE 400 MHz spectrometer. For sufficient resolution, 13C-DOSY was performed in a 5 mm Shigemi tube with 400 μL sample volume and four times higher sample concentration. Additionally, 1H−13C-iDOSY-HSQC measurements were performed on a Bruker-AVANCE 700 MHz spectrometer with a TXI cryo probe head and a 3 mm sample tube with 200 μL sample volume. All 13C-NMR spectra were recorded with 1H-decoupling. The 15 N-NMR spectra were referenced to nitromethane. The diffusion coefficient D from DOSY and the viscosity η of 1.0963 mPa·s for D2O at 25 °C were used for the calculation of the hydrodynamic diameter according to the Stokes−Einstein equation. X-ray photoelectron spectroscopy (XPS) was carried with an ULVAC-PHI VersaProbe II microfocus X-ray photoelectron spectrometer with an aluminum Kα X-ray source (1486.8 eV). As reference for Au(0), a sputtered oxygen-free gold surface was used.

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Figure 1. HRTEM image of cysteine-functionalized ultrasmall gold nanoparticles. The inset shows magnification of one nanoparticle with an fcc polyhedron in the [110] zone axis orientation with {111} and {100} facets.

RESULTS AND DISCUSSION We chose L-cysteine as a typical thiol-terminated biomolecule to elucidate the binding of ligands to ultrasmall gold nanoparticles. The thiol group is coordinated to the gold surface, whereas the deprotonated carboxy group acts as a negatively charged repulsive outer layer. This molecule is well suited for colloidal particle stabilization, but still small enough to allow the assignment of its NMR signals. The ultrasmall nanoparticles are easily dispersable in water at pH > 9. As expected, there were no differences in the colloid-chemical properties and the highresolution transmission electron microscopy (HRTEM) images between nanoparticles stabilized by different kinds of isotopelabeled L-cysteine. Therefore, representative results are given in the following that can be transposed to all L-cysteine species, regardless of the nature of their labeling. However, we give the isotope-labeling pattern in each case and mark labeled atoms with an asterisk in chemical structures. If no labeling is indicated, unlabeled L-cysteine was used. In all cases, the L-isomer of cysteine was used; therefore, the configuration is not added in the following. HRTEM gave the nanoparticle size and morphology (Figure 1). HRTEM showed well separated particles with a high degree of monodispersity with an average size of 1.78 ± 0.34 nm (taken from HAADF-STEM images by counting about 60 nanoparticles). The particles were crystalline with a high degree of order of the constituting atoms, conforming to closed-shell clusters of atoms (see below).1,18,50−52 Formally, gold is oxidized when a thiol group is conjugated. The gold oxidation state in the nanoparticles was determined by XPS. This clearly showed that most of the gold remained in the metallic state as Au(0) (Figure 2). Less than 5% of the gold atoms were present as Au(+I).53 To form a stable colloidal dispersion, the particles have to be dispersed at pH > 9 to avoid agglomeration and subsequent sedimentation (electrostatic stabilization due to the ionized carboxy groups). The particle charge can be assessed by the zeta potential that changes from +79 to −287 mV during titration from pH 2.5 to pH 10. The zwitterionic character of cysteine has to be taken into account. At low pH, the amino group and the carboxy group are both protonated, leading to a positive charge of the nanoparticle. An increase in pH leads to deprotonation of the amino group (first) and of the carboxy group (second).

Figure 2. XPS of cysteine-functionalized ultrasmall gold nanoparticles show the two characteristic asymmetric peaks in the Au 4f region (Au 4f5/2, Au 4f7/2). The binding energy values for both peaks correspond to Au(0) (87.65 eV, 83.95 eV). The deconvolution of the peaks reveals only a small fraction of oxidized gold: more than 95% are present as metallic gold.

Figure 3 shows that a sufficiently negative charge is obtained at a pH above 9. The isoelectric point for cysteine-functionalized

Figure 3. Zeta potential dependence during a pH titration of cysteinefunctionalized ultrasmall gold nanoparticles. The pH-dependent protonation equilibrium of an amino acid is clearly recognizable. C


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amine protons exchange with D2O and are not visible in the 1HNMR spectrum. 1 H-NMR spectroscopy of dispersed nanoparticles gives rise to a poorly resolved spectrum complicating an ad hoc interpretation (Figure 5). The sharp signals of dissolved cysteine become rather broad in the surface-bound state and are paramagnetically shifted downfield. This is similar to our earlier observations for thiocarboxylic acid-stabilized gold nanoparticles41 and the report of Volkert et al. on thioctic acid-functionalized gold nanoparticles.56 Sayevich et al. recorded 1H-NMR spectra (solvent: CDCl3) of butylamine, coordinated to 4.5 nm CdSe quantum dots, and found considerable peak broadening and a downfield shift as well.57 Metallic nanoparticles cause a dipolar magnetic interaction with the proton spins of the adsorbed organic molecules. Such distorting NMR effects are stronger near the gold surface. Interaction parameters are mainly the distance and the angular orientation of the proton toward the particle center and toward the external magnetic field. Consequently, it is partially averaged by conformational and diffusive reorientation.40,58 Because of this strong line broadening and the presumably site-specific shift of the resonances, it is difficult to assess whether the line shape is due to homonuclear coupling or overlap of several signals. In addition, the intensities of the three main signals are not consistent with the assumption of only one cysteine species. The reason for the increased number of signals is unclear at this point, but the signals indicate a complex binding situation on the gold nanoparticle surface as we will outline below. A clear assignment is not possible at this stage. 13 C-NMR spectroscopy with isotope-labeled cysteine gave more insights into the binding situation. The natural abundance of 13C was not sufficient to obtain useful spectra. Therefore, two labeled cysteine species were used: one with a terminal 13Clabeling at the thiol group (denoted as 13Cβ-labeled cysteine in the following) and one that was labeled at all three carbon atoms (denoted as 13C-1,2,3-labeled cysteine in the following). The Cβ carbon which is closest to sulfur and thus to the gold surface gives a single signal at 32 ppm for dissolved 13Cβ-cysteine. On the surface of gold nanoparticles, it splits into three peaks at 39, 40, and 43 ppm (Figure 6). After binding 13C-1,2,3-labeled cysteine to the gold nanoparticle, the signals of Cα and the carboxy carbon differ only marginally from the ones in pure 13C1,2,3-labeled cysteine. The peaks split because of the spin−spin (1J)-coupling of adjacent carbon atoms. As expected, the influence of the gold surface increases with proximity, i.e. the carbon atoms that are closer to the gold surface experience a stronger shift. The fact that Cβ carbon now gives rise to three well-separated peaks indicates that there are (at least) three different chemical environments for cysteine in the sample. A similar picture was found for 15N-labeled cysteine where at least three 15N signals indicate different chemical environments of the cysteine amine group (Figure 7). To assign the proton signals, 1H-COSY and 1H-NOESY/ EXSY spectra were recorded that showed off-diagonal crosspeaks between neighboring protons, i.e. Hα and Hβ. A number of couplings are visible, but the patterns are far more complex than expected and confirm that several signals overlap again similar to the results found earlier for mercaptoacetic acid and similar compounds.41 The assignment of the observed resonances by COSY correlation peaks, however, remained ambiguous. In a number of NOESY/EXSY experiments, the expected timedependent correlation peaks were observed. Unfortunately, because intra- and intermolecular spin diffusion and possible

nanoparticles agrees with free cysteine (pH 5.05), i.e. the Brönsted acid−base properties of cysteine are not influenced by the binding to the gold surface. DCS gave the hydrodynamic diameter of the cysteine-coated nanoparticles with 1.34 ± 0.05 nm (Figure 4). The DCS results

Figure 4. Representative DCS data of cysteine-functionalized ultrasmall gold nanoparticles.

are in good agreement with the HRTEM data, indicating welldispersed (nonagglomerated) nanoparticles. Note that DCS systematically underestimates the particle diameter because of the presence of the ligand shell, leading to an apparently smaller diameter and a lower sedimentation rate.54 L-Cysteine dissolved in pure water has a pH value of about 4.5. This condition is not suitable for cysteine-coated gold nanoparticles because agglomeration and sedimentation occur because of the near-neutral zeta potential. Therefore, a pH value of 12 (adjusted with NaOD) was used for all NMR spectroscopic experiments with dispersed nanoparticles. For comparison, dissolved cysteine was analyzed both at pH 4.5 (D2O) and at pH 12 (D2O with NaOD). It shows a classic ABX spin system in the 1H-NMR spectrum (Figure 5) and the

Figure 5. 1H-NMR spectra of dissolved cysteine (pH 4.5 and pH 12) and of cysteine-functionalized ultrasmall gold nanoparticles (pH 12). Note the downfield shift of the cysteine protons after coupling to the gold nanoparticle. For assignment of peaks, see the text.

expected diamagnetic shift at basic conditions. The α-proton gives a triplet at 3.98 ppm (pH 4.5) or 3.06 ppm (pH 12). The two β-protons are diastereotopic. They give two doublets of doublets because of their magnetic nonequivalency and the chiral nature of α-carbon at 3.1 ppm (β2) and 3.01 ppm (β1) (pH 4.5). At pH 12, the signals of the β-proton split and shift to 2.87 ppm (β2) and 2.43 ppm (β1).55 The carboxy proton and the D


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Figure 6. 13C-NMR spectra of dissolved 13Cβ-labeled cysteine and of ultrasmall gold nanoparticles, functionalized with 13Cβ-labeled cysteine (pH 12) (top). 13C-NMR spectra of dissolved 13C-1,2,3-labeled cysteine (pH 12) and of ultrasmall gold nanoparticles, functionalized with 13C-1,2,3-labeled cysteine (pH 12) (bottom). The carbon atoms labeled with 13C are marked with an asterisk in the molecule structure. Figure 8. 1H−13C-HSQC-NMR spectra of gold nanoparticles, functionalized with 13Cβ-labeled cysteine (top) and 13C-1,2,3-labeled cysteine (bottom) (pH 12).

cysteine not shown). Because the carboxy carbon has no bound proton, it is not visible in the HSQC spectrum. For both 13Cβand 13C-1,2,3-labeled gold-bound cysteine, each of the three Cβ species couples with two distinct diastereotopic Hβ atoms to give signals at 39 ppm/(3.2 and 3.5) ppm, 40 ppm/(3.2 and 3.5 ppm), and 43 ppm/(3.5 and 3.8 ppm). For 13C-1,2,3-labeled cysteine nanoparticles, the Hα/Cα correlation at 3.5 ppm/60 ppm is observed as well. This correlation peak now allows the identification of the Hα proton signals in the 1H-NMR spectrum (see Figure 5) which obviously overlaps with signals of two Hβ protons. Both 1H- and the 13C-NMR spectra of the 13C-1,2,3-labeled cysteine-coated nanoparticles lack spectral resolution, whereas the HSQC spectrum allows the assignment of protons to their respective carbons, line shapes and potential overlaps in the 13CNMR spectrum needs to be clarified. For this purpose, 13C−13CINADEQUATE NMR spectroscopy has been applied: neighboring carbon atoms are detected via their spin−spin (1J) coupling (Figure 9). Here, on the f 2-axis (x-axis), the common one-quantum 13C spectrum is displayed, whereas the f1-axis (y-axis) shows the double-quantum frequency, i.e. the sum of chemical shifts of two correlating carbon atoms. The terminal carboxy carbon (179 ppm) couples with neighboring

Figure 7. 15N-NMR spectra of dissolved 15N-labeled cysteine and of ultrasmall gold nanoparticles, functionalized with 15N-labeled cysteine (pH 12).

site-exchange both result in correlation peaks, the contribution of a single interaction (e.g., the lifetime of a certain chemical state) could not been clearly assigned. 1 H−13C-HSQC-NMR spectra show the connectivity between protons and their bound carbon atoms because of their spin− spin (1J) coupling (Figure 8). For the free 13C-1,2,3-labeled cysteine, the HSQC spectrum shows one signal for the Hα/Cα correlation and two signals with the same carbon chemical shift for the diastereotopic Hβ/Cβ correlation (spectrum of dissolved E


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Figure 9. 13C−13C-INADEQUATE NMR spectrum of ultrasmall gold nanoparticles, functionalized with 13C-1,2,3-labeled cysteine (pH 12). The inset shows a magnification of the Cα/Cβ coupling region. The correlation peak at 60/220 ppm is an experimental artefact of unknown origin as it is not unusual in INADEQUATE NMR spectroscopy.59

Cα carbon (60 ppm, sum of chemical shifts: 239 ppm). The three species of Cβ are coupling with neighboring Cα as indicated by the signals at 39 ppm/59 ppm (sum of frequencies: 98 ppm) and 40 ppm/59 ppm (sum of chemical shifts: 99 ppm). The coupling of Cβ at 43 ppm with corresponding Cα at 60 ppm could not be detected because of its low signal-to-noise ratio (sum of chemical shifts: 103 ppm). Nevertheless, the INADEQUATE spectrum proves the connectivity of the different Cβ carbons with different Cα carbons and their coupling to the carbonyl carbons and clearly shows the integrity of the cysteine molecules on the gold nanoparticle surface. DOSY is a versatile method to determine the hydrodynamic diameter of molecules and particles in dispersion. Furthermore, it is possible to differentiate between species with different diffusion coefficient in one dispersion, for example, dissolved ligand molecules and nanoparticle-bound ligand molecules.29,41,60 1H- and 13C-DOSY NMR spectra as well as an 1 H-13C-iDOSY-HSQC61 experiment were performed. Their corresponding Stejskal−Tanner plots (Figures 10−14) show that free cysteine and nanoparticle-bound cysteine correlate with the same diffusion coefficient both in 1H- and 13C-NMR DOSY spectroscopy. This confirms that the dispersions do not contain a mixture of dissolved cysteine and surface-bound cysteine, and that cysteine does not detach from the surface during storage in dispersion. It also underlines that the three signals of Cβ carbon belong to one particle species with different chemical environments for cysteine on the surface. Table 1 summarizes all DOSY results, i.e. the measured diffusion coefficient and the calculated hydrodynamic particle diameter. The results are in excellent agreement with those from DCS and HRTEM. The quantification of the amount of conjugated cysteine was accomplished by quantitative 13C-NMR spectroscopy with 13Cβlabeled cysteine as the standard. The concentration of cysteine for each obtained peak is computed at 39 ppm to 1.32 mM, at 40

Figure 10. 1H-DOSY NMR spectrum (top) and Stejskal−Tanner plot (bottom) of dissolved cysteine (pH 12). The intensity is averaged from the three proton peaks (α, β1, β2). Error bars represent standard deviations. I signal intensity, I0 signal intensity without gradient, and G gradient strength. The following parameters were constant during the experiment: γ gyromagnetic ratio, δ length of the diffusion gradient pulse, and Δ diffusion time.

ppm to 1.23 mM, and at 43 ppm 1.32 mM. Note that the peak at 39 ppm contained a shoulder. Using a line deconvolution routine, the intensity of the relevant peak was determined. AAS gave a concentration of 2.0 g L−1 gold. With these data, a calculation of the number of cysteine molecules and the number of gold atoms per nanoparticle was carried out as follows VNP =

3 ij 1.78 × 10−9 m yz 4 ji d zy 4 zz ·π ·jj zz = ·3.14·jjj z 3 k2{ 3 2 k {

3

= 2.95 × 10−27 m 3 mNP = VNP·ρAu = 2.95 × 10−27 m 3· 19 302 000

g m3

= 5.69 × 10−20 g

c NP =

2.0 g/L cAu = = 3.51 × 1019 L−1 m(NP) 5.69 × 10−20 g

ccysteine,39 ppm = 1.32 × 10−3

mol ·6.02 × 1023 mol−1 L

= 7.95 × 1020 L−1 F


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Figure 12. 13C-DOSY NMR spectrum (top) and Stejskal−Tanner plot (bottom) of 13Cβ-labeled cysteine with the labeled carbon at 32 ppm. At 43 ppm, minor impurity with a lower diffusion coefficient is observed. This peak was not considered for the calculation of the Stejskal−Tanner plot.

Figure 11. 1H-DOSY NMR spectrum (top) and Stejskal−Tanner plot (bottom) of cysteine-functionalized ultrasmall gold nanoparticles (pH 12). Because of overlapping proton signals between 2.7 and 3.7 ppm, the assignment of the β-protons was unclear. A single integration was carried out over all peaks from 2.7 to 3.7 ppm for the Stejskal−Tanner plot. At 4.8 ppm, the water signal with a higher diffusion coefficient appears. The water peak was not considered for the calculation of the Stejskal−Tanner plot.

ccysteine,40 ppm = 1.23 × 10−3

NAu atoms =

mol ·6.02 × 1023 mol−1 L

Assuming a spherical gold nanoparticle with a diameter of 1.78 nm containing 174 gold atoms per cluster, we calculated that 67 cysteine molecules were bound per gold nanoparticle. From the 13C-NMR results, it can be concluded that there are (at least) three different chemical environments for cysteine on the gold surface strongly indicated by the 13C signals of the Cβ atom (Figure 6) and to a lesser degree by the 15N-signals of the amino group (Figure 7). The integration gave the relative amounts for the 13C−Cβ NMR peaks at 39, 40, and 43 ppm to be 1.08:0.94:1.0, corresponding to 23:21:23 cysteine molecules on each nanoparticle. From the XPS data, we can exclude the presence of Werner-type Au+I cysteine−gold complexes on the nanoparticle surface; therefore, we tentatively assume that the three signals are due to three different crystallographic sites on the surface of the gold cluster. Consequently, we have resorted to modeling cluster structures as proposed in the literature, based on an fcc lattice, including twinned structure models. In all cases, the number of gold surface atoms is sufficient to explain the binding of 67 cysteine molecules, in good agreement with the stoichiometry of published gold nanoclusters (see the Introduction). However, there are differences between the cluster structures in the binding sites for cysteine. For fcc metals, different surface energies exist for different crystal planes. As a

= 7.41 × 1020 L−1 ccysteine,43ppm = 1.32 × 10−3

mol ·6.02 × 1023 mol−1 L

= 7.95 × 1020 L−1

ccysteine,39 ppm c NP ccysteine,40 ppm c NP ccysteine,43 ppm c NP mAu =

=

cysteine 7.95 × 1020 L−1 = 23 NP 3.51 × 1019 L−1

=

cysteine 7.41 × 1020 L−1 = 21 19 −1 NP 3.51 × 10 L

=

cysteine 7.95 × 1020 L−1 = 23 19 −1 NP 3.51 × 10 L

5.69 × 10−20 g mNP = = 174 mAu 3.27 × 10−22 g

197 g/mol MAu = = 3.27 × 10−22 g NA 6.02 × 1023 mol−1 G


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Figure 14. Stejskal−Tanner plots from 1H−13C-iDOSY-HSQC-NMR spectra of dissolved 13C-1,2,3-labeled cysteine (top) and of ultrasmall gold nanoparticles functionalized with 13C-1,2,3-labeled cysteine (bottom), both recorded at pH 12. The intensities represent relative intensities I/I0 of the observed peaks (three and seven, respectively, see Figure 6). Error bars represent standard deviations.

Figure 13. 13C-DOSY NMR spectrum (top) and Stejskal−Tanner plot (bottom) of ultrasmall gold nanoparticles, functionalized with 13Cβlabeled cysteine (pH 12). The intensities represent averaged relative intensities I/I0 of the three peaks between 41 and 46 ppm. Error bars represent standard deviations.

Table 1. Summary of the Diffusion Coefficients D and Hydrodynamic Radii d of all Cysteine-Functionalized Gold Nanoparticles and Their Free Cysteine Counterpartsa

result of the atomic coordination disparity on different surfaces, the low index surface energies follow the general trend σ111 < σ100 < σ110. For isotropic liquids or amorphous particles, the lowest energy configuration is a sphere.62 To further reduce the surface energy, ultrasmall metal particles adopt multiple twinning (formation of icosahedra and decahedra).63,64 Figure 15 shows representative HRTEM images of the major morphologies found for cysteine-coated ultrasmall gold nanoparticles. Inside an fcc lattice, the coordination number of gold is 12 (cuboctahedron). On the surface, the coordination number can decrease to 5. We tentatively assume that the three wellseparated NMR signals for cysteine are due to different chemical environments on the nanoparticle surface. However, it is likely that not all nanoparticles exhibit the same crystal faces as it can also be seen in Figure 16. For a nanoparticle diameter of 1.78 nm as derived from TEM analysis, about 174 gold atoms are expected (see the calculation above). For comparison, the cluster [Au25(SCPh)18]0/1‑ has a core diameter of 1.3 nm,65 the Au55 cluster has a core diameter of 1.4 nm,1 the Au102 cluster has a core diameter of 1.6 nm,17 the Au144 cluster has a core diameter of 1.5−1.6 nm,27 the Au314 cluster has a core diameter of 2.1 nm,66 and the Au887 cluster has a core diameter of about 3 nm in a molecular modeling study.67 We assumed 147 gold atoms to construct several reasonable cluster structures as shown in Figure 16, using DFT-calculated structures from the literature.68 They exhibit different crystal

a

Atoms marked with an asterisk indicate 13C-labeled positions.

faces, and it is assumed that this is responsible for the three different chemical environments of cysteine. The fact that mainly three well-separated NMR lines are observed and not a broad line distribution underlines that we have well-defined chemical environments of cysteine in the sample and not a fully disordered mixture of nanoparticles with many different conjugation sites for cysteine. If we assume that the coordination number of the gold site (5 or 6 for a corner; 7 or 8 for an edge; and 9−11 for a face) H


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Langmuir

A or D. In the following, our results are discussed in the light of the current literature on NMR spectroscopy on gold nanoparticles. The generally observed strong chemical interaction between gold and sulfur is reflected by the two-step reaction where Au+III is first reduced to Au+I by the ligand thiol, followed by reduction to Au0 by NaBH4. If there is no equilibration between the first and the second reduction step for a couple of hours, the nanoparticles become more polydisperse.69 This indicates the presence of soluble Au+I species complexed after reduction with thiol.69 Xie et al. have considered such steps during the seedmediated synthesis in an extensive study by mass spectrometry and UV spectroscopy, leading to Au25 and Au44 nanoparticles.24 Dispersed ultrasmall nanoparticles show much more complex NMR spectra than the dissolved ligands, but at least NMR spectroscopy is possible, in contrast to larger nanoparticles. A paramagnetic NMR shift and a peak broadening/peak splitting have been frequently reported for ligands that are bound to ultrasmall gold nanoparticles. Gobbo and Workentin have prepared water-soluble gold nanoparticles ( m) clusters. Z. Anorg. Allg. Chem. 2011, 637, 15−23. (19) Xiong, L.; Yang, S.; Sun, X.; Chai, J.; Rao, B.; Yi, L.; Zhu, M.; Pei, Y. Structure and electronic structure evolution of thiolate-protected gold nanoclusters containing quasi face-centered-cubic kernels. J. Phys. Chem. C 2018, 122, 14898−14907. (20) Kenzler, S.; Schrenk, C.; Frojd, A. R.; Häkkinen, H.; Clayborne, A. Z.; Schnepf, A. Au70S20(PPh3)12: an intermediate sized metalloid gold cluster stabilized by the Au4S4 ring motif and Au-PPh3 groups. Chem. Commun. 2018, 54, 248−251. (21) Lei, Z.; Li, J.-J.; Wan, X.-K.; Zhang, W.-H.; Wang, Q.-M. Isolation and Total Structure Determination of an All-Alkynyl-Protected Gold Nanocluster Au144. Angew. Chem., Int. Ed. 2018, 57, 8639−8643. (22) Yan, J.; Malola, S.; Hu, C.; Peng, J.; Dittrich, B.; Teo, B. K.; Hakkinen, H.; Zheng, L.; Zheng, N. Co-crystallization of atomically precise metal nanoparticles driven by magic atomic and electronic shells. Nat. Commun. 2018, 9, 3357. (23) Sakthivel, N. A.; Dass, A. Aromatic thiolate-protected series of gold nanomolecules and a contrary structural trend in size evolution. Acc. Chem. Res. 2018, 51, 1774−1783. (24) Yao, Q.; Yuan, X.; Fung, V.; Yu, Y.; Leong, D. T.; Jiang, D.-e.; Xie, J. Understanding seed-mediated growth of gold nanoclusters at molecular level. Nat. Commun. 2017, 8, 927.

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Solution NMR Spectroscopy with Isotope-Labeled Cysteine (13C and

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