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Figure 1. Typical single-molecule force spectroscopy set-up and typical force-distance curves. (A) Typical single-molecule force spectroscopy set-up. The atomic force microscopy tip can be moved relative to the surface with sub-nanometer precision, while forces in the pico-micro Newton range can be determined by the deflection of the cantilever. The molecules are immobilized via a linker on the tip and surface, respectively.
(B) Typical force distance curves (only the retracting part) showing rupture and adhesion events. (1) A typical specific rupture force event of molecules connected via two 30-nm poly(ethylene) glycol linkers. After accounting for the movement of the cantilever due to the bending, the rupture length is 58 nm. (2) A double rupture event. Although this last event might be a single-molecule event, it should not be counted for single-molecule analysis, since the distance-dependent force is not known and cannot be reconstructed. (3) A system with a 30-nm linker on the cantilever and a 2-nm linker on the surface.
(Figure omitted. See article PDF.)
Figure 2. Single-molecule force spectroscopy on antibody-antigen interactions. (A) Experimental set-up of [53]. The antibody fragments are immobilized onto an amino-functionalized glass slide using a heterobifunctional PEG linker. The same procedure is used to immobilize the short antigen peptides on the cantilever. N, M and C correspond to different linker attachment configurations. In N, a cysteine and three glycine residues were attached to the N-terminus of the peptide. In M, alanine on position 8 of the peptide sequence was exchanged for cysteine. In C, three glycine residues followed by a cysteine were attached to the C-terminus of the peptide. While these changes do not affect the equilibrium dissociation rates (i.e., without external force), they change the direction of pulling.
(B) The most probable rupture force plotted (semi-logarithmically) against the corresponding loading rate for all three configurations of N, M and C. By using the standard theory (see theory section) one can gain the corresponding reaction length from the slope of the fitted line and the dissociation rate from the intersection of the fitted line with the y-axis. NHS:
N -hydroxysuccinimide; PEG: Poly(ethylene) glycol. Adapted from [53] with permission from Elsevier © (2008).
(Figure omitted. See article PDF.)
Figure 3. Potential barriers under the influence of external force, as assumed by...