Molecular Recognition using BioAFM

PeakForce QNM can be used to map cell stiffness, but it can also provide high-resolution 2D maps of the adhesive interactions between the AFM probe and a surface.  One can obtain a PeakForce QNM adhesion map by attaching a protein or small molecule to the end of the AFM probe which maps a surface containing a complementary binding partner that specifically interacts with the molecule on the tip.  The resulting data represents a map of specific molecular binding interactions – or a molecular recognition map.

To demonstrate this, researchers used PeakForce QNM to conduct molecular recognition mapping of the surface of malaria-infected erythrocytes or IE’s in order to obtain high-resolution images of the distribution of specific molecules expressed on the cell surface.  Due to the resolution limitations in AFM imaging of cells, the presence of these molecules would be quite difficult to detect with traditional topography imaging alone.  Unlike healthy cells, Malaria-Infected erythrocytes exhibit knob-like surface structures.  Infected cells also show cytoadherence which is a tendency to stick to the walls of blood vessels. Not only does this prevent them from being removed from the bloodstream by the spleen, buildup of these cells in the blood vessels can cause other dangerous health issues such as vascular blockages.  Molecules associated with the knob-like structures on the surface of the infected cells are believed to be involved in helping them to adhere to the endothelial cells on blood vessels walls by interacting with endothelium surface receptors.

Schematic of functionalized probe

To examine this further, researchers functionalized AFM probes with the endothelial cell surface receptor CD36, which is believed to be one of the molecules involved in this cytoadherent interaction, and used these probes to visualize the distribution of cytoadherent molecules on the surface of infected erythrocytes.

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Watch a recent webinar with guest speakers David Alsteens & Moritz Pfreundschuh from ETH Zurich

PeakForce-QNM imaging with the CD36-modified probe revealed that areas of high adhesion or high binding affinity (shown as light spots on the adhesion image – in the top right image) all mapped to the knob-like structures in the topography image. The adhesion sites are believed to be specific binding interactions and not just a result of topography variations, as not all raised regions in the topography image correspond to an adhesion or binding site, as indicated by the green arrow. The 3D overlay image on the bottom clearly shows the co-localization of cytoadherent molecules (high adhesion) with the knob structures.

Topography and Adhesion Maps
3D overlay showing colocalization