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

In the last two decades several groups have investigated the changes of chemical and physical properties of materials with size in nanometric scales. These studies have highlighted a number of possible applications for nanostructures, which are now employed, for example, in biology and medicine for imaging, disease detection, diagnosis, sensing and therapy. Noble metals (especially gold and silver nanoparticles) are particularly versatile for these applications due to the phenomenon known as surface plasmon resonance (SPR), an in-phase oscillations of all the conduction band electrons that resonates with the light wave electric field. The resonance frequency depends on the size, shape, orientation and dielectric constant of the nanoparticle. This coupling of SPR with the electromagnetic field leads to a large enhancement of all the nanoparticle radiative properties, such as absorption and scattering. The extinction cross section of these nanoparticles is 10^5-10^6 larger than that of organic dyes and in contrast to common molecular chromophores they are extremely photostable and, depending on the shape, they convert efficiently light into heat. The SPR, tunable in the visible (for spherical NPs) and near-infrared region (for anisotropic Nps) of the electromagnetic spectrum, can also interact, for gold nanoparticles a few nanometers in size, with the fluorescence emission of dyes and substantially modify their brightness and excited-state lifetime. Depending on the fluorophores-NP distance and the NPs anisotropy one can obtain fluorescence enhancement or quenching. In both cases we expect that any change in the dielectric constant of the NP surface, induced, for example, by a biorecognition process that occurs on the surface itself, can produce a change in the emission properties of the fluorophores. SPR effect becomes also particularly important when combined with two-photon excitation (TPE), which consists in the simultaneous absorption of two photons, each carrying about half the energy necessary to excite the molecule. 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 infrared 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. In fact, the luminescence (TPL) induced by TPE is enhanced (when coupled with an appropriate plasmon resonance) by many orders of magnitude in non spherically symmetric NPs of noble metal with respect to the single photon excitation on smooth noble metal surfaces. These properties promise to improve the usefulness of these nanoparticles for in-vivo imaging in the NIR region of the electromagnetic spectrum. 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. The aim of the first part of this research project 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 occurring through quenching, affects the excited state lifetime that is measured by fluorescence burst analysis in highly diluted suspensions. The binding of protein to the gold NPs through antigen-antibody recognition further modifies the dye excited-state lifetime, which change can then 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); then we have applied the nanoassay in order to recognize the p53 protein, whose detection in the body is highly valuable as marker for early cancer diagnosis and prognosis, both in standard solutions and in total cell extracts. The selectivity of the construct with respect other globular proteins has been also addressed. The data indicate that the FITC excited-state lifetime is a very sensitive parameter in order to detect tiny amounts of protein in solution with an estimated limit of detection of about 5 pM, mostly determined by the statistical accuracy of the lifetime measurement. In the second part of the project we focused on the exploitation of anisotropic gold nanoparticles as probes in cellular imaging. We have then studied their photoluminescence (TPL) properties under two photon excitation. We have focused on gold nanorods that can easily be obtained by synthesis with the standard surfactant CTAB (cetyl trimethylammonium bromide). The synthesis of asymmetric branched gold nanoparticles, obtained using for the first time in the seed growth method approach a zwitterionic surfactant, laurylsulphobetaine (LSB), has been developed in collaboration with the University of Pavia (Prof. P.Pallavicini). We have shown that LSB concentration in the growth solution allows to control the dimension of the NPs and the SPR position, that can be tuned in the 700-1100 nm Near Infrared range. The samples have been analyzed with a number of structural techniques to obtain a complete characterization: absorption spectra in the UV-Visible region, TEM images of the suspensions, Fluorescence Correlation Spectroscopy (FCS) and Dynamic Light Scattering (DLS) experiments in suspensions. From these data we reached information on the nanoparticles shapes, dimensions and aggregation. In particular, three different populations have been found: nanospheres with diameter lower than 20 nm, nanostars characterized by large trapezoidal branches, and asymmetric branched nanoparticles with high aspect ratio (3-4). A full characterization of the NPs TPL was performed for imaging applications by employing two photon excitation (TPE). The dependence of the TPL intensity on the power, wavelength and polarization of the incident light intensity was studied and TPL was exploited to study the cellular uptake of the nanoparticles in different cell lines (macrophages and HEK cells).