Biomedical Engineering Reference
In-Depth Information
DFS can be performed by an atomic force microscopy (AFM) equipment, which
is a high-resolution imaging tool based on force measurements, (for a complete
description of AFM, see Chapter 2). Briefly, AFM imaging is obtained by scanning a
very sharp tip, located at the end of a cantilever spring, over the sample that is placed
onto a surface of a piezoelectric scanner, which is able to ensure a three dimensional
positioning with subnanometer resolution (Jena and Horber, 2002). The interaction
force between tip and sample, optically measured from the cantilever deflection, is
used to create a topographical image of the sample when the tip is raster-scanned in
the horizontal x-y plane. In the DFS modality, the interaction forces between two
biomolecules, one anchored to the tip and the other anchored to the substrate, can be
probed during approaching and retraction cycles. From the analysis of the unbinding
force data of biomolecular pairs, the kinetics and thermodynamics properties can be
obtained in the framework of suitable theoretical models. The remarkable force sen-
sitivity (down to pN), coupled with the small probe-sample contact area (as small as
10 nm
2
), involving very few molecules, even down to only one, allows to investigate
the subtle molecular features of biological systems (Janshoff et al., 2000; Zlatanova
et al., 2000; Lee et al., 2007; Bizzarri and Cannistraro, 2009). In this respect, we
remark that DFS requires only a very little amount of interacting species to carry out
reliable experiments. Another interesting advantage of DFS measurements is that
the application of an external force, yielding a reduction of the lifetime of the sys-
tem, makes accessible the study of systems with long lifetimes. Since 1994, DFS
has been applied to investigate a large variety of biomolecular complexes covering a
wide range of different functions and biological processes, such as ligand-receptor
or antibody-antigen pairs, protein unfolding, molecular stretching, conformational
changes, cell deformation, membrane elasticity, cell adhesion, and so on. In this
chapter, the results of some biological complexes investigated by DFS are reviewed
and discussed in connection with the related data in bulk, when available. Selection
of the cited articles has been done with the aim of providing an overview of the actual
capabilities and potentialities of DFS to elucidate the molecular processes underly-
ing some representative interactions of biological and medical interests. Due to the
limited space, only some topics have been selected; therefore, our presentation is far
from being exhaustive. We apologize for the omission of many key references, worth
to be mentioned, that would have certainly enriched the present review.
This chapter is organized as follows: The main aspects involved in a DFS study of
a biomolecular complex are overviewed in Section 6.2. In particular, the fundamental
steps of a DFS experiment (immobilization strategies and data acquisition) are illus-
trated in Subsection 6.2.1, whereas the data analysis and the theoretical background
are summarized in Subsection 6.2.2. A brief description of the computational meth-
ods useful when combined with DFS measurements is given in Subsection 6.2.3.
Section 6.3 is devoted to describe and discuss the results of DFS applied to some rep-
resentative biomolecular complexes. This section has been organized in subsections,
each one of them is focused on a class of complexes playing a specific biological
function; the experimental used setup, the applied data analysis, and the most rele-
vant obtained information being outlined. Applications of DFS to the development
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