DEVELOPMENT OF A NOVEL NANOSENSOR FOR THE STUDY OF BIOMOLECULAR INTERACTIONS

The evaluation and quantification of molecular interactions is of paramount importance in modern biology and molecular medicine. Therefore, there is a continuous exploration for new methodologies capable to detect and to measure binding affinities during reversible molecular interactions. This work is devoted to explore a new tool based on the high sensitivity that the measurement of the scattered light intensity offers when the binding occurs on the surface of index-matched colloids. Static light scattering is not a traditional technique to study molecular association because the binding of insulated ligands and receptors in dilute solutions produce negligible increment of the scattered light, while mesoscopic particles hosting multiple receptors or ligands, including real bacteria, typically scatter too much light compared with the contributions due to molecular adhesion on their surface. This difficulty can be overcome by supporting the receptors on nano-scale latex spheres whose refractive index closely matches the one of water. As \u201cPhantom Nanoparticles\u201d (PNPs), we have used highly hydrophobic monodisperse spherical fluoroelastomer colloids, with radius R = 39 \uc5} 1 nm, and whose refractive index, at our working temperature (30\uc5\ue3C) and wavelength (633 nm), is np0 = 1.3248 (under the same conditions the refractive index of water is nW = 1.3319). Surfactants added to a PNP dispersion readily adsorb on their hydrophobic surfaces, generating a self-assembled monolayer which can be easily equipped with molecular hydrophilic end groups of various kind, including well-known receptors and/or ligands. This label-free method has been assessed through the precise determination of the binding constant of the antibiotic vancomycin with the tripeptide L-Lys-D-Ala-D-Ala and of the vancomycin dimerization constant. We have enlightened the role of bidentate effect and molecular hindrance in the activity of this glycopeptide. After this success first result, an accurate determination of the optimal properties of nanoparticles employed has been performed by comparative experiments and through theoretical evaluation (CHAPETR 3). The effects of size, refractive index, electric charge, and dilution on the reliability and accuracy of the method has been evaluated. Quite surprisingly, perfect index matching and minimal size (i.e., maximum surface), which is almost attained in one of the colloids here employed, do not represent the ideal conditions. Rather, we show that a nanoparticle radius of 100 nm and a refractive index slightly below that of water yields the best signal/background amplitude. We also show that repulsive interactions can lead to artifacts in the adsorption isotherm, thus indicating that electrostatic stabilization should be kept at a minimum. Successively, the particles, already optically phantom, have also been made biologically \u201cinvisible\u201d through PEG coating and decorated by interacting proteins, thus providing a mean to investigate the biological properties of proteins (CHAPETR 4). Avidin decorated Phantom Nanoparticles have been prepared and were employed to detect interactions between different kinds of biotinylated proteins. Using this approach, biotinylated protein A was anchored on the surface of the nanoparticles, and were exploited as a functional probe for the rapid, quantitative, picomolar detection of human IgG antibodies. We used Phantom Nanoparticles to evaluate substrate recognition by Streptomyces PMF Phospholipase D inactivated mutants (CHAPETR 5). The use of this specific technique seems to have some peculiar advantages over other methods in the case of phospholipidsacting enzymes. In fact, the substrate (or a substrate analog) can be organized onto the surface of Phantom Nanoparticles at a desired concentration with optimal display of the polar head group, while the hydrophobic chains result packed into the surfactant monolayer, thus limiting the occurrence of non-specific interactions. The last part of this work is focused on an attempt toward the stable functionalization of Phantom Nanoparticles (CHAPETR 6). Diacetylene surfactants spontaneously adsorb on the nanoparticles and then they are polymerized by exposure to UV light. So far, the stability of the amphiphilic coating around the nanoparticle solely depended on weak hydrophobic interactions. The attachment of the polymer to the particle surface, because of the numerous contact points, is highly stable and can be improved further by crosslinking of the polymer shell. Huns generating a quantifiable number of functional groups suitable for covalent receptor anchorage. All these observations, demonstrate the feasibility of this new technique, which makes it possible to easily generate different synthetic receptors, and highlight this technique as a versatile novel method to study, both qualitatively and quantitatively, of molecular recognition processes. The work described in this thesis is partially published in: Morasso C., Bellini T., Monti D., Bassi M., Prosperi D., Riva S.: Dispersed phantom scatterer technique reveals subtle differences in substrate recognition by phospholipase D inactive mutant; ChemBioChem. Submitted, currently under review Prosperi D., Morasso C., Tortora P., Monti D., Bellini T.: Avidin decorated core-shell nanoparticles for biorecognition studies by elastic light scattering; ChemBioChem. 2007 (8): 1021-1028 (Impact factor: 3,446). Prosperi D., Morasso C., Mantegazza F., Buscaglia M., Houg L., Bellini T.: Phantom nanoparticles as probes of biomolecular interaction; Small. 2006 (8-9):1060-1067 (Impact factor: 6,408)