Biomedical Engineering Reference
In-Depth Information
which molecules interact, and where they are doing it. This is fundamentally different
from bulk techniques, where the molecules of interest are placed in a relatively large
volume, and some signal change observed. In addition, because the molecules are brought
together and pulled apart under the control of the experimentalist, factors such as force of
interaction and the rate of separation can be finely controlled. Combining all this with the
possibility to carry out such reactions in physiological conditions means that AFM-based
biological force measurements represent one of the most powerful techniques available to
make molecular interaction measurements in biology [684].
There are two major ways in which force spectroscopy can be carried out: in one or in
three dimensions. One-dimensional (1-D) force spectroscopy refers to experiments where
the factor of interest is the intermolecular force to be measured, rather than the spatial
distribution of the measured forces. In order to carry out this sort of experiment, the AFM
probe and a flat surface will be modified to bind the two molecules of interest. The flatness
of the substrate will reduce artefacts related to increased adhesion at the edges of features
[159]. For self-interaction, the same molecule could be bound to both the surfaces [144,
685]. There are a number of issues related to the binding strategy used, which were discussed
in Section 3.2.1, but essentially, the molecules must have a resistant yet flexible linker,
and not have their recognition sites blocked [686, 687]. When suitably modified probes and
samples have been generated, experiments are carried out as described in Section 3.2.1, and
any pairs of interacting molecules may be studied. A common way to prove the nature of the
interaction being probed is to add a 'blocking' molecule to the solution [163]. For example,
having established that with molecule A on the tip and molecule B on the surface, a
measurable interaction force is recorded, molecule B is added in excess to the medium.
These excess 'B' molecules bind to 'A' on the tip, and the force spectroscopy experiments
are repeated. If the interaction being probed is really of type 'A-B', the measured force will
be changed (often it will disappear), with the blocking molecules in solution. The main
parameter which is studied is the force or range of forces, at which detachment occurs (hence
force spectroscopy
). However, for molecules bound by a flexible linker, or for macromol-
ecules, the distance of unfolding is also important (see next section). Typically the rate of
pulling (i.e. the speed at which the probe is moved) can be varied, and this can also allow
measurements of the kinetics of the dissociation process [688-690].
Some examples of interactions that have been studied include biotin-avidin binding
[163, 691, 692] (which has become a 'standard measurement' in AFM force spectroscopy,
due to its very high strength and specificity [690, 693]), other antibody-antigen inter-
actions [689, 694, 695], carbohydrate-carbohydrate binding [144, 584], and fibrinogen
binding [685, 688, 696], see the examples in Figure 7.26. AFM can also be used to
measure cell-cell adhesion, or virus-cell adhesion by attachment of a cell or virus to
the probe [697, 698]. This is only a small selection of the interactions that could be studied;
for further details see the reviews [142, 684, 699].
Three-dimensional force spectroscopy or force-mapping is the second major method-
ology of force spectroscopy by AFM. In this sort of experiment, the aim is to use the
specific force of interaction between two molecules to determine the location on the
sample surface of one of the molecules. For instance, this can be used to determine
the location of receptor molecules on a cell surface. Probably the most commonly used
way to do this is by measuring force-distance curves in a grid pattern over the sample
surface. This is sometimes referred to as force volume imaging [700]. In this mode, the
