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 15N-labeled. 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–13C-heteronuclear
single-quantum coherence and 13C–13C-INADEQUATE.
Further information on the binding situation (including the absence
of residual or detached l-cysteine in the solution) and on
the nanoparticle diameter (indicating the well-dispersed state) was
obtained by diffusion-ordered spectroscopy (1H-, 13C-, and 1H-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.
Ultrasmall gold nanoparticles with a metallic core diameter of 2 nm were surface-conjugated with peptides that selectively target epitopes on the surface of the WW domain of the model protein hPin1 (hPin1-WW). The binding to the gold surface was accomplished via the thiol group of a terminal cysteine. The particles were analyzed by NMR spectroscopy, highresolution transmission electron microscopy, and differential centrifugal sedimentation. The surface loading was determined by conjugating a FAMlabeled peptide, followed by UV−vis spectroscopy, and by quantitative 1 H NMR spectroscopy, showing about 150 peptide molecules conjugated to each nanoparticle. The interaction between the peptide-decorated nanoparticles with hPin1-WW was probed by 1 H− 15 N-HSQC NMR titration, fluorescence polarization spectroscopy (FP), and isothermal titration calorimetry (ITC). The particles showed a similar binding (K D = 10−20 μM) compared to the dissolved peptides (K D = 10−30 μM). Small-angle Xray scattering (SAXS) showed that the particles were well dispersed and did not agglomerate after the addition of hPin1-WW (no cross-linking by the protein). Each nanoparticle was able to bind about 20 hPin1-WW protein molecules. An unspecific interaction with hPin1 was excluded by the attachment of a nonbinding peptide to the nanoparticle surface. The uptake by cells was studied by confocal laser scanning microscopy. The peptide-functionalized nanoparticles penetrated the cell membrane and were located in the cytosol. In contrast, the dissolved peptide did not cross the cell membrane. Peptide-functionalized nanoparticles are promising agents to target proteins inside cells.
The uptake of fluorescently labeled ultrasmall gold nanoparticles (2 nm) by Gram-negative Escherichia coli bacteria occurs within 1-3 hours. This was demonstrated by confocal laser scanning microscopy (CLSM), structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), and flow cytometry. For imaging, eGFP-expressing and DsRed2expressing E. coli strains were used in addition to non-fluorescing E. coli strains. Gold nanoparticles were labeled with fluoresceine (FITC), Cy3, and AF647, respectively. Importantly, gold nanoparticles showed no toxicity to the bacteria, indicating a non-lethal nature of the uptake, that is, not related to cell injury.
The surface of ultrasmall gold nanoparticles with an average diameter of 1.55 nm was conjugated with a 14-3-3 proteinbinding peptide derived from CRaf. Each particle carries 18 CRaf peptides, leading to an overall stoichiometry of Au( 115)Craf( 18). The binding to the protein 14-3-3 was probed by isothermal titration calorimetry (ITC) and fluorescence polarization spectro-scopy (FP). The dissociation constant (K D ) was measured as 5.0 μM by ITC and 0.9 μM by FP, which was close to the affinity of dissolved CRaf to 14-3-3σ. In contrast to dissolved CRaf, which alone did not enter HeLa cells, CRAF-conjugated gold nanoparticles were well taken up by HeLa cells, opening the opportunity to target the protein inside a cell.
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