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)