Biomedical Engineering Reference
In-Depth Information
meaningful results in DFS experiments. Different attachment strategies can be used
as described in the recently published review articles (Hinterdorfer & Dufrene 2006;
Bizzarri & Cannistraro, 2010). Two basic requirements for such attachments are that
(1) the mechanical strength of attachment significantly exceeds the strength of inter-
action understudy and that (2) various effects arising from attaching biomolecules
do not prohibit the molecular interpretation of the measured biorecognition events.
While the first requirement is rather obvious, the second requirement demands care-
ful consideration of various effects in attaching biomolecules. Ideally, measured
interactions between surfaces that are equipped with recognition partners correspond
to breaking of a single recognition bond under study. Moreover, for accurate inter-
pretation of experimental results, it is important that conditions of measurements
match assumptions of theoretical models that are used for data reduction. Number of
effects that make typical DFS experiments less than ideal are related to attachment
of molecules. Effects detrimental to accuracy of DFS experiments include (1) effects
of surfaces on affinity of recognition between biomolecules, (2) effects of spuri-
ous (nonspecific) interactions, (3) effects of multiple recognition events that might
occur during the measurements, (4) effects of polymeric linkers (which are often
used to attach biomolecules to the surfaces) on extracted parameters of interaction.
The latter two effects arise from effects of attachment density and geometry and from
non-Hookean spring elasticity of polymeric tethers (the non-Hookean spring aspect
is outside the scope of this chapter). Some of these effects might occur simultane-
ously, and therefore fulfilling the second requirement stated above is not a trivial
task. Therefore, advantages and disadvantages of different interaction strategies are
considered below.
The chapter is organized in the following way: First, immobilization strategies
are described and requirements for functional attachment of biomolecules are indi-
cated. Then, we describe physical and chemical methods of attaching biomolecules,
indicating corresponding strengths and weaknesses of different approaches. In the
following section, problem of separating the single rupture events from the multiple
rupture events is described. The chapter concludes with a brief summary.
4.2 IMMOBILIZATION STRATEGIES
Immobilization of biological molecules at interfaces is hardly a new task
(Hermanson, 1996; Wong et al. 2009). However, because of the single-molecule
nature of interactions studied by DFS and because of the mechanical nature of
the measurements, DFS experiments require immobilization approaches that dif-
fer in several aspects from other experimental techniques like chromatography, sur-
face plasmon resonance, and capillary electrophoresis. In DFS, interaction between
recognition partners is measured by analysis of detected rupture events. Registered
rupture events might have spurious nature. For example, false ruptures might be
detected if molecules are not attached strongly enough to surfaces and rupture occurs
at the attachment point or when nonspecific bonds are being ruptured. In addition, if
ruptures of multiple bonds occur simultaneously, then measured distribution of rup-
ture forces might be affected. These effects should be avoided by selecting a proper
immobilization approach.
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