• Nessuna parola chiave trovata

This thesis regards the implementation, spectral analysis and optimization of coated
and functionalized metallic nanoparticles, which are made spectrally resolvable also by
exploiting the surface enhanced Raman spectroscopy (SERS) signal of fluorophores appro-
priately bound to their surface; the intended final use of these structures is as smart labels
for diagnostic purposes.
This work is indeed part of an ongoing research in the NEST laboratories, which aims in
developing an inexpensive screening protocol for early diagnosis of some types of cancer,
based on the capture, quantification and characterization of exosomes from biological fluids
using functionalized nanomaterials and their spectroscopic analysis.
Exosomes are natural nanoparticles expelled from cells in an organism, and can bear mark-
ers for several diseases, in particular of tumoral origins; they are present in biological fluids
like urine and plasma, but their extraction, separation and the recognition of the markers
are still too lengthy and with low performances.
There are many biomedical purposes of nanostructured metallic substrates as SERS-
active platforms, e.g. bio-imaging and single molecule tracking, biosensing, diagnostic;
in particular, metallic nanoparticles (Nps) with Raman-active molecules (tipically fluo-
rophores) adsorbed onto them could be used as valid alternatives to fluorophores. SERS
is a powerful vibrational spectroscopy technique that allows for highly sensitive structural
detection of low concentration analytes through the strong enhancement (possible up to
∼ 10
14
) of their Raman signals, partly due to the electromagnetic field amplification gen-
erated by the excitation of localized surface plasmons. The advantages of SERS labels on
the use of fluorophores can arise by: (i) the possibility of multi-labelling with single wave-
length excitation; (ii) the lowest probability of photobleaching when the excitation is in
pre-resonance, i.e. at slightly longer wavelengths than the absorption of the Raman-active
molecule; (iii) the opportunity to exploit other properties of the metal nanoparticles, such
as their dimensions for purification purposes, their multiple functionalizability by using
appropriate coatings, their composition (e.g., they can be magnetic), their strong and typ-
ical light scattering at the wavelength of the surface plasmon resonance, the dependence
of this resonance by the environment and the proximity of other Nps.
Nowadays, metal Nps produced by wet chemistry bottom-up processes are the most used SERS substrates, since the production is inexpensive and easy to reproduce. In the last
decade several experimental conditions were investigated that allow the fine tuning of
size and morphology of Nps in colloidal solutions. These include the choice of suitable
precursors, of stabilizer molecules (surfactants or ligands) and of reducing agents, or the
temperature modulation and adjustment of the relative concentration ratio of the reac-
tants.
In this thesis work I researched the best synthesis protocols for monodisperse and stable
metal Nps of various materials (among which Au, Ag, Pt), shape and size, I verified the
stability of specific multifunctionalizable peptidic coatings for these nanoparticles, and
measured SERS spectra from different nanoparticles functionalized with different optically
active molecules.
The same stabilizing coatings is not necessarily applicable to all types of Nps. We found
that engineered dye-labeled six-aminoacids peptides are able to coat, water-stabilize, and
functionalize Au Nps in a one-step process, albeit the dye produces SERS signals much
less intense than when directly attached to the Nps by a short thiol linker. We verified
that this enhancement loss is in agreement with the expected dependence of the electric
field amplification from the distance between the fluorophore and the metal surface, when
conjugated to the hexapeptide or directly linked to the surface. However, the hexapeptide
coating is more stable than one with the thiolated fluorophore, so it will be necessary to
strike a balance between high SERS intensity and better stability of the colloid, or to
develop novel coatings with fluorophores closer to the metallic surface.
Regarding silver Nps, I found out that amines and thiols have a similar reactivity with
silver; both of them are present on the hexapeptides, the first on the cysteine on the
intended grafting side, and the second also on the lysine on the “bifunctionalization” side.
Therefore, the coating with this hexapeptide of Ag Nps could cause the aggregation of the
colloid itself; anyway, their coating with hexapeptides prefunctionalized with fluorophores
produced SERS signals with intensities of the same order of magnitude as when bound
through the short thiol linker, confirming the fact that the hexapeptide can connect to the
Ag surface with both ends.
In conclusion, this work gave important information on the photophysics and the chem-
istry of metal nanoparticles functionalized with different kind of coating also bearing fluo-
rophores; this is an important step towards the creation of engineered nanosystems, which
will be functionalized in further works with antibodies or other recognition elements for
general or tumoral epitopes on exosomes, and which will then be analyzed with spectro-
scopic methods.