Immobilization and Interaction Strategies in DFS of Biomolecular Partners - Dynamic Force Spectroscopy and Biomolecular Recognition - page 136

in aqueous media (Pagac et al. 1997; Shar et al. 1999; Kim et al. 2003). Moreover,
mechanical compression forces might cause strong adsorption of PEG onto silicon
nitride probe; the resulting physical attachment is capable of withstanding pulling
forces upto hundreds of piconewton (Oesterhelt et al. 1999). Therefore, additional
test experiments are required to determine the nature of measured rupture events
(Averett et al. 2008; Guo et al. 2010b).
Problem of nonspecific interaction of linkers or tethered molecules with the sub-
strate can be solved by using a double-tether approach (illustrated in Figure 4.1d)
where both interacting partners are tethered to surfaces by relatively long linkers
(Kuhner et al. 2004; Ratto et al. 2004; Ray & Akhremitchev, 2005). This double-
tether approach also helps to remove steric hindrance by surfaces and allows to
discriminate (although not perfectly) single-molecule interactions from multiple
interactions (Guo et al. 2008; Guo et al. 2010a; Mayyas et al. 2010). Multiple bond
effects will be considered in Section 4.4 in more detail. Another method to alleviate
nonspecific adsorption problem is to use experimental methodology where the probe
does not apply compressive forces onto the sample surface (Ludwig et al. 1999;
Sekiguchi et al. 2003; Guo et al. 2010b). However, this methodology is not widely
used because it remains challenging to repeatedly approach the substrate without
applying compressive forces to a distance that can be bridged by the linkers.
From a point of view of the weakest bond holding the immobilized biomolecules
on surfaces, attachment methods can be divided in two groups: methods that use
physical adsorption or molecular recognition forces and methods that use cova-
lent attachment. Although physical bonds are generally weaker than covalent bonds,
using such attachment schemes is adequate when physisorption is strong enough for
force spectroscopy methods that utilize low pulling forces (e.g., optical tweezers and
magnetic tweezers techniques) (Averett et al. 2008; Neuman & Nagy, 2008). Below
we describe various immobilization strategies in more detail, indicating the strengths
and weaknesses of different approaches.
4.3 PHYSICAL METHODS OF ATTACHING MOLECULES
Physical methods of attaching biological molecules in AFM experiments were intro-
duced at the early stages of development of force spectroscopy technique (Lee et al.
1994; Florin et al. 1994; Moy et al. 1994; Radmacher et al. 1994; Chilkoti et al. 1995;
Fritz et al. 1998; Willemsen et al. 1998; Baumgartner et al. 2000). In the past decade,
this technique still has been used in AFM-based experiments (Sekiguchi et al. 2003;
Zhang & Moy, 2003; Averett et al. 2008). This approach is often used to attach
cells to the AFM probes (Zhang & Moy, 2003; Zhang et al. 2004; Wojcikiewicz
et al. 2006) and in other force spectroscopy methods like biomembrane force probe,
optical and magnetic tweezers (Evans et al. 2004; Pincet & Husson, 2005; Ferrer
et al. 2008; Todd et al. 2008). In DFS experiments, this approach has been mostly
replaced by chemical methods of attaching molecules that are described in the fol-
lowing subsection. Therefore, here we only briefly describe immobilization strategy
using physical forces and point out advantages and disadvantages of this approach.