PROBING CELLULAR ADHESION AT THE SINGLE-MOLECULE LEVEL - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 231

The opposing surface, or substrate, also needs to be functionalized to
link the molecule of interest or suspended cells. Similar strategies to those
described for AFM tips can be used. If the biomolecule of interest is relatively
large (>50 kDa), physisorption can be used to immobilize it to the substrate
by incubation during several hours. 5,6,28-30 For smaller molecules, partial
denaturing due to physisorption could compromise their binding capacity,
thus, an alternative method such as the one described for tip coating is
suggested. Suspended cells can be easily immobilized on Petri dishes coated
with poly-L-lysine at low concentration, allowing them to maintain their
round shape. Monolayers of adherent cells, such as endothelial or epithelial
cells, growing on cell culture dishes or coverslips can be directly used for
AFM force measurements.

31,32

11.3.1.2 Receptor-ligand configuraons

Different conigurations can be adopted to probe cell adhesion at the single-
molecule level. Figure 11.4 shows the four main conigurations commonly
used. Measurements will be similar in all four, although the experimental
conditions will slightly change. The use of puriied proteins allows us to have
a controlled and clean set-up in which only the biomolecules of interest are
present. As mentioned before, molecules can be immobilized on the AFM tip
and on the substrate using various methods ( Fig. 11.3 ) . An advantage of using
puriied protein is that it provides optimal conditions for single-molecule
measurements. Protein can be diluted to very low concentrations and the
relatively small area of contact between the AFM tip and the substrate reduces
the probability of bond formation. Low binding probability (<30%) ensures
that most of the events are mediated by single bonds. AFM cantilever tips
come in different sizes and shapes. Unsharpened pyramidal tips have been
extensively used, because the blunted apex of radius 20-50 nm provides a
relatively bigger area than sharpened tips and wear is less pronounced. 5,25,33-
36

Even if the use of puriied proteins is very convenient and can be used as
a irst approach if the protein is available, puriication of some proteins is
not always possible and, more importantly, puriication may alter the native
conformation and adhesive properties of the protein, as it is known for some
integrins. 37 Thus, when possible, it is recommended to work with proteins
expressed on living cells.
Suspended cells, such as blood cells, can be immobilized on a coated AFM
cantilever to carry out force measurements ( Fig. 11.3 ) . 5,38 For that purpose,
it is recommended to use tipless cantilevers or cantilevers with tips of height
smaller than the cells themselves, to avoid any interaction between the AFM
tip and the substrate that will prevent the cell to contact it (see Fig. 11.4b ) . As