Biomedical Engineering Reference
In-Depth Information
Immobilization approaches used in other techniques are often valued by the sur-
face density of attached molecules (Mallik et al. 2004). In DFS experiments, too
high surface density is detrimental because it hampers analysis of interactions from
a single pair of recognition partners. Another significant difference is that DFS
uses a nanoscale probe that mechanically contacts the substrate. Often, area of the
tip-sample contact significantly exceeds dimensions of molecules. Consequently,
spurious interactions might occur if two surfaces adhere to each other. Figure 4.1
illustrates these effects.
To prevent these and other detrimental effects, polymeric linkers are often used
to tether molecules to surfaces. One advantage in using relatively long linkers is to
remove contribution of nonspecific interactions between the probe and the surface
in analysis of recognition events by using tip-sample separation at rupture as a dis-
criminating parameter (see Figure 4.1c). Such use of the linkers has been proposed
in an early force spectroscopy work by Hinterdorfer et al. (Hinterdorfer et al. 1996)
and this approach became a rather common method to separate rupture forces of
different nature (e.g., specific and nonspecific interactions) (Willemsen et al. 1998;
Hinterdorfer et al. 2002; Kuhner et al. 2004; Ratto et al. 2004; Bonanni et al. 2005;
Kienberger et al. 2005; Ray and Akhremitchev, 2005; Averett et al. 2008; Guo et al.
2008; Guo et al. 2010b; Jiang et al. 2010; Mayyas et al. 2010; Nguyen et al. 2011).
However, nonspecific interaction of tethered molecule or the linker with the substrate
still might contribute to the measured rupture forces (see Figure 4.1c). When using
polymeric linkers, it is important that polymers are water soluble, do not alter recog-
nition of interacting molecules, and do not participate in immobilization reactions
(except in a desired way). The most widely used polymeric linkers are poly(ethylene
glycol) molecules (PEG). Surfaces coated with PEG resist protein adsorption (Ma
et al. 2006). However, PEG itself adsorbs onto hydrophobic and hydrophilic surfaces
(a)
(b)
(c)
(d)
Probe
l
rup
Attached ligands
and receptors
l
rup
Substrate
FIGURE 4.1
Schemes of attachment of recognition partners to the atomic force spectroscopy
probe and substrate. Panels (a) and (b) illustrate attaching molecules without tethers. Panel
(b) illustrates that in such attachment both nonspecific interactions and multiple interactions
are expected. In addition, interactions might be significantly hindered by surfaces. Panel (c)
illustrates attaching one of the molecules by polymeric tether. This reduces probability of
nonspecific interactions and alleviates the steric hindrance. Panel (d) illustrates the double-
tether approach. Here, nonspecific interactions are eliminated from analysis by considering
ruptures occurring at the sum of the tether lengths; the steric hindrance is also greatly reduced.
However, both the single-tether and the double-tether approaches do not prevent multiple bond
ruptures from occurring.
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