Nanoparticles for in-vitro and in-vivo biosensing and imaging

8 Introduction
plasmon resonance) by many orders of magnitude with respect to the single photon excitation
of at surfaces, improving the usefulness of these nanoparticles for in-vivo imaging
in the NIR region of the electromagnetic spectrum.
TPE offers a series of unique features for biological investigation both in vitro and in
vivo. First, the two-photon absorption bands of the dyes commonly used in biological
studies are wider than their one-photon analogous allowing the simultaneous excitation
of multiple fluorophores with a single excitation wavelength. Second, the stimulating
light beam has a high penetration depth because of the long infra-red wavelengths used,
allowing experiments in turbid media. Third, excitation takes place only at the plane of
focus, due to the scaling of the probability of simultaneous photon absorption with the
square of the light intensity. As a consequence TPE avoids the simultaneous absorption
of photons outside the specimen drastically reducing both photo-toxicity and fluorophore
bleaching. These advantages make nowadays TPE a well established tool for scientific
biological and medical research and can be coupled to anisotropic gold nanoparticles.
According to these considerations we have developed our project on two lines related to
the use of gold nanoparticles for sensing and non linear imaging.
In the first part of this work we have reviewed the materials and methods used: in
Chapter 1, the most relevant features of the surface plasmon resonance and the properties
of spherical symmetric and asymmetric nanoparticles are presented. The interaction
between fluorophores and nanoparticles and the basics of fluorescence emission
and two-photon excitation employed in the reported measurements are discussed, while
the physical basis of the Dynamic Light Scattering and the Fluorescence Correlation
Spectroscopy (FCS), the main technique employed in this PhD thesis, are described in
Chapter 3. The instruments employed for the measurements are instead described in
Appendix A.
The aim of the first part of this research project (reported in Chapter 4) is to exploit
changes of the dye excited-state lifetime and brightness induced by its interaction with
the gold surface plasmons for detection of tiny amounts of protein in solution under
physiological conditions. The system we investigated is based on 10 and 5 nm diameter
gold NPs coupled (via a biotin-streptavidin linker) to the FITC dye and to a specific
protein antibody. The interaction of the fluorophore with the gold surface plasmon resonances,
mainly occuring through quenching, affects the excited state lifetime that is
measured by fluorescence burst analysis in standard solutions. The binding of protein
to the gold NPs through antigen-antibody recognition further modifies the dye excitedstate
lifetime. This change can therefore be used to measure the protein concentration.
In particular, we have tested the nanodevice measuring the change of the fluorophore
excited-state lifetime after the binding of the model protein bovine serum albumin (BSA);