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Tatjana Ruks1 Christine Beuck2 Torsten Schaller3 Felix Niemeyer3 Manfred Zaehres4 Kateryna Loza1 Marc Heggen5 Ulrich Hagemann6 Peter Bayer2 Christian Mayer4 Matthias Epple1

1, Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Essen, , Germany
2, Institute of Biology and Center for Medical Biotechnology (CMB), University of Duisburg-Essen, Essen, , Germany
3, Organic Chemistry, University of Duisburg-Essen, Essen, , Germany
4, Physical Chemistry, University of Duisburg-Essen, Essen, , Germany
5, Ernst Ruska-Center for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, Jülich, , Germany
6, Interdisciplinary Center for Analytics on the Nanoscale (ICAN) and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Duisburg, , Germany

Ultrasmall nanoparticles with a diameter below 2 nm are promising as specialized carriers for targeted drug delivery. Functionalized with specific binding motifs, they open up innovative opportunities in a wide range of applications, e.g. nanomedicine. A possible application is the specific targeting of protein-epitopes.
A versatile approach to the functionalization with specific epitope-binding motifs, i.e. peptides and proteins, is covalent binding of the gold-surface with sulfur-containing molecules. NMR spectroscopy gives valuable insights into the characteristics of the binding situation on the surface.
L-cysteine as a typical sulfur-containing biomolecule was chosen to elucidate the binding of ligands to ultrasmall gold nanoparticles (d < 2 nm). Cysteine is the only thiol-containing amino acid and therefore an ideal model compound for the binding to gold nanoparticles.
The nanoparticle preparation was carried out by reduction of HAuCl4 with NaBH4. L-cysteine was directly attached to the gold nanoparticles via gold-sulfur binding. The purification of the cysteine-capped nanoparticles was performed by multiple centrifugation and washing steps.
To investigate the binding of L-cysteine to the gold nanoparticle surface, isotope-labeled L-cysteine (13C and 15N) was used. 2D 1H-DOSY,13C-DOSY and 3D 1H-13C-HSQC-iDOSY NMR spectroscopy of the gold nanoparticles enabled the determination ofthe hydrodynamic particle diameter in excellent agreement with the metallic core diameter by high-resolution transmission electron microscopy.
The binding of L-cysteine to the gold nanoparticles via the thiol group was confirmed by 1H and 13C NMR spectroscopy and 2D-NMR spectroscopy (1H, 1H-COSY, 1H,13C-HSQC and 13C,13C-INADEQUATE). To exclude the binding of L-cysteine to the gold nanoparticles via the amino group, 15N NMR spectroscopy was carried out.
Quantitative13C NMR spectroscopy and atomic absorption spectroscopy enabled the calculation of a loading of approximately 100 L-cysteine molecules on each gold nanoparticle. By X-ray photoelectron spectroscopy, 95% elemental gold was identified whereas about 5% was oxidized. This confirms the existence of metallic gold nanoparticles, functionalized with L-cysteine as model compound.

We show that NMR spectroscopy is especially well suited to analyze ultrasmall gold nanoparticles due to their small particle size, leading to excellent spectra of dispersed nanoparticles that elucidate the binding situation of L-cysteine on the gold surface.

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