A Physical Approach to Reduce Nonspecific Adhesion in Molecular Recognition Atomic Force Microscopy

Abstract

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.

Unbinding Molecular Recognition Force Maps of Localized Single Receptor Molecules by Atomic Force Microscopy

“…Their values can be even higher than the avidin–biotin unbinding force (specific adhesion). In order to get rid of this problem, we use the strategy proposed by Willemsen and co‐workers 17. It consists in operating the AFM in the repulsive electrical double layer (REDL) interaction mode.…”

“…This provides a sensor (referring to the whole molecular unit that connects the ligand to the AFM tip) of sufficient length and motility to rapidly lock the ligand into its binding site 6. Furthermore, measurements are obtained in the regime of the repulsive electrical double‐layer interaction to avoid unspecific adhesions, as suggested by Willemsen and co‐workers 17. By operating in this regime it is possible to acquire reliable AFM images of single biomolecules without establishing any mechanical contact (non‐contact NC‐AFM) between the bare tip and the sample 18.…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…When detecting specific forces, non-specific interaction may be a problem, because they make the image quality worse and they may even hide the specific forces. To overcome this problem, Willemsen et al [1202] tested imaging conditions in aqueous solution at low salt concentration, staying within the repulsive part of the DLVO forces and recording only adhesion events when discrete hopping of the tip over this barrier by thermal motion occurs.…”

Measurement of interaction forces between fibrinogen coated probes and mica surface with the atomic force microscope: The pH and ionic strength effect

“…Enhancement of steric repulsion 19,36 at the higher salt concentration should not be expected, since screening of intraand intermolecular repulsions results in a more compact conformation of the protein molecules on the bound layer. 17 Therefore, steric interactions cannot be responsible for the force profile at the 500 mM.…”