ABSTRACT
Chemistry .........................................................................150 4.5 Multiple Interactions between Immobilized Molecules ...............................151
4.5.1 Probability of Single-Molecule Events ...........................................153 4.5.2 Effects of Multiple Interactions on Measured Data ........................154
4.6 Conclusion ....................................................................................................156 References..............................................................................................................156
In dynamic force spectroscopy (DFS), mechanical forces are applied to intermolecular bond understudy to characterize strength of biomolecular recognition and extract kinetic parameters of bond dissociation. To perform such measurements, interacting partners should be attached to the opposing surfaces of atomic force spectroscopy (AFM) probe and the substrate. Thus, successful immobilization is vital for obtaining
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 interaction 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 careful 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 interpretation 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 spurious (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 simultaneously, and therefore fulfilling the second requirement stated above is not a trivial task. Therefore, advantages and disadvantages of different interaction strategies are considered below.
