Development and applications of field-flow fractionation (FFF) and
related techniques: a) separation and characterization of
macromolecules, nanoparticles and microparticles of biological,
environmental or food origin; b) hyphenation with uncorrelated
techniques for morphological analysis of nano and microdispersed
analytes; c) miniaturized, disposable, biocompatible devices.
Applications to 1) hyphenation with detection techniques such as
chemiluminescence, laser scattering, and mass spectrometry; 2)
characterization of intact proteins by FFF-MS: protein profiling in
biological fluids and whole cells, characterization of recombinant
protein drugs; 3) fractionation, size analysis and spectroscopical
characterization of nanosystems of nano-biotechnological interest.
Analysis of
nanoparticles: Synthesis and
characterization of nanomaterials are the basis of future
developments and applications of nanotechnologies, from electronics
to biomedical technologies. The ability of hydrodynamic field FFF
(FlFFF) for the analysis of nanodispersed analytes has been
investigated in the fields of material science for nano-biotech
applications. FlFFF has been, for instance, applied to the
fractionation and size analysis of silica and titanium dioxide
synthesized in the presence of fluorescent modifiers. FlFFF
showed able to control the synthesis of such multi-chromophoric
structures, which have been then further characterized by
using spectrometric and microscopic techniques. FlFFF has been also
applied to fractionate functionalized carbon nanotubes (CNT). The
project "LIGHT4HEALTH" (University Strategic Project - Junior,
coordinator N. Zaccheroni) involves application of FlFFF coupled
with multiangle laser scattering (MALS) detection for the analysis
of fluorescent nanoparticles for applications in
medicine.
Analysis of dispersed
microparticles: In the framework
of the project "STARCHitechture" (University Strategic Projects -
Senior, coordinator P. Trost) FlFFF-MALS and gravitational FFF
(GrFFF) are applied to the separation and characterization of
starch granules and enzymatic degradation products from mutant
cereals.
Protein analysis:
In a formal cooperation agreement with Wyatt Technology Europe
(Germany) FlFFF-MALS has been developed from an instrumental and
methodological point of view for the analysis and characterization
of serum lipoproteins. The original
hyphenation of mass spectrometry and hollow-fiber (HF) FlFFF has
been also developed for the analysis of intact proteins and protein
complexes in native form. Functional recombinant protein drugs and
complex protein samples such as whole human blood serum have been
characterized.
In the framework of the locally coordinated project
PRIN 2006 (Integration of novel separation and mass spectrometry
technologies for third-generation proteomics, National coordinator
Prof. G. Marino) , and of the Significant Research Project
within the Bilateral Cooperation Protocol Italy-Korea 2007-2009, HF
FlFFF and FlFFF-MALS are coupled with shot-gun proteomics
techniques such as (nano/chip)LC-ESI(Ion-Trap)-MS/MS for the
identification of native protein complexes in serum and
sub-cellular fractions.
Immunological methods: in cooperation with the Group of Bioanalitical
Chemistry at the Dept. of Pharmaceutical Sciences (Prof. A. Roda)
new analytical formats are developed for flow-through immunometric
methods. Use of flow-through CL detectors online coupled with the
fractionation device (GrFFF-FIA-CL) allows to determine the analyte
concentration by comparing the fractographic peak area with a
proper response curve. Based on the intrinsic advantages of such an
approach, a non-competitive immunoassay has been developed, which
uses GrFFF-FIA-CL for the quantitation of whole cells (pathogenic
bacteria).
