Vincent T. Moy

Sensing specific molecular interactions with the atomic force microscope

One of the unique features of the atomic force microscope (AFM) is its capacity to measure interactions between tip and sample with high sensitivity and unparalleled spatial resolution. Since the development of methods for the functionalization of the tips, the versatility of the AFM has been expanded to experiments where specific molecular interactions are measured. For illustration, we present measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. Here, we compare different techniques that allow for the in situ measurement of the absolute value of the spring constant of cantilevers.

Adhesive forces between ligand and receptor measured by AFM

Abstract Since its invention, the atomic force microscope (AFM) has been used to image a wide range of samples, including soft biological materials. Although various imaging modes have been developed in recent years, the images obtained by these techniques are primarily derived from the mechanical interactions between AFM tip and sample. An imaging mode based on the specific recognition of defined functional groups on the sample by receptor molecules attached to the AFM tip may prove to be beneficial, especially in complex specimens such as the cell membrane. A prerequisite towards this goal is the development of AFM tips functionalized with ligand-specific receptors. Here we report the fabrication of tips functionalized with either avidin or antibodies. These functionalized tips were characterized by force scan measurements on ligands cross-linked to agarose beads. The specificity of the observed adhesion between tip and sample was confirmed by specific blocking of the ligands or the receptors.

Cooperative adhesion of ligand–receptor bonds

Cooperative (simultaneous) breakage of multiple adhesive bonds has been proposed as a mechanism for enhanced binding strength between adhesion molecules on apposing cell surfaces. In this report, we used the atomic force microscopy (AFM) to study how changes in binding affinity and separation rate of force-induced ligand-receptor dissociation affect binding cooperativity. The AFM force measurements were carried out using (strept)avidin-functionalized cantilever tips and biotinylated agarose beads under conditions where multiple (strept)avidin-biotin linkages were formed following surface contact. At slow surface separation of the AFM cantilever from the beads surface, the (strept)avidin-biotin linkages appeared to rupture sequentially. Increasing the separation rate from 210 to 1950 nm/s led to a linear increase in the average rupture force. Moreover, force histograms revealed a quantized force distribution that shifted toward higher values with increasing separation rate. In measurements of streptavidin-iminobiotin adhesion, the force distribution also shifted toward higher values when the buffer was adjusted to a higher pH to raise the binding affinity. Together, these results demonstrate that the cooperativity of ligand-receptor bonds is significantly enhanced by increases in surface separation rate and/or binding affinity.

Nonspecific interactions in AFM force spectroscopy measurements

Sample‐probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand–receptor interaction. A prolonged contact time may be required to initiate ligand–receptor binding as a result of slow on‐rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate‐probe contact, we investigated the interaction of bovine serum albumin (BSA)‐functionalized cantilever with BSA‐coated glass, polyethylene glycol (PEG)‐functionalized glass, Pluronic‐treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA‐functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion. Copyright