Development and application of
field-flow fractionation (FFF) techniques.
Fundamental research in FFF
concerns:
(i) separation and characterization of macromolecules, colloids
and particulate of biological, environmental and alimentary
field.
(ii) characterization of nano- and microdispersed analytes by
coupling FFF with uncorrelated techniques for morphological
analysis.
(iii) hyphenation of FFF with chemiluminescence (CL) detection for
high sensitivity and high selectivity analysis of dispersed
bioanalytes. Development of flow assisted immunoassays and
biosensors.
(iv) instrumental development of miniaturized, disposable,
biocompatible FFF devices.
Application research topics of
FFF:
i) Invention of cell sorting methods for prokaryotic and
eukaryotic cells;
ii) hyphenation of the developed cell sorting methods with CL,
laser scattering and mass spectrometric detection.
iii) characterization of protein expression of cells and native
protein by coupling hollow fiber field flow fractionation and mass
spectrometry for microorganism protein profiling, study of
functional and allosteric proteins, characterization of recombinant
proteins of pharmaceutical 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.
Cell
sorting: The majority of cell sorting methodologies,
including Magnetic-Activated and Fluorescent-Activated Cell
Sorting, make use of immunological markers. For some applications,
however, specific antigenic markers may be lacking, or the labeling
process itself may affect viability or functionality of the sorted
cells. New techniques for label-free cell sorting would be,
therefore, of great interest particularly for applications to stem
cells. In collaboration with Prof. G.P. Bagnara (Dept. of
Histology, Embriology and Applied Biology, University of Bologna)
GrFFF-related techniques have been developed to sort adult human
stem cells. Such cells can be isolated from different tissues and
used in-vivo to regenerate damaged tissues, or ex-vivo to create
new tissues, with promising applications in regenerative medicine.
A new technique to enrich and sort multipotent human stem cells has
been invented and patented (WO07128737, owned by the University of
Bologna).
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.
Amyloidoses are a class of human diseases that arise from protein
misfolding and subsequent aggregation into amyloid fibrils. In
Alzheimer's disease (AD), a key role is played by a
nucleation-dependent self-aggregation of the
A
β
1-42
peptide. . In cooperation with Prof. E. De Lorenzi (Dept. of
Pharmaceutical Chemistry, University of Pavia), FlFFF-MALS has been
applied to monitor the Aβ1-42 aggregation process
through the separation of transient species and a broad-range,
mass/size characterization (~ 5-1500 nm) of the prefibrillar and
fibrillar Aβ1-42 aggregates. 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).
