Linking of sensor molecules (e.g., antibodies) to an AFM tip converts it into a biosensor by which single target molecules (e.g., antigens) can be detected and localized on the sample surface. Moreover, the mechanism of interaction can be studied by force spectroscopy if purified target molecules are linked to an ultra-flat surface, such as mica or silicon (nitride). Rapid imaging of the binding sites and force spectroscopy studies are greatly facilitated if 6-10 nm long polyethylene glycol (PEG) chains are used as flexible tethers between the sensor molecule and the tip. Here, we describe a set of methods by which a variety of proteins, oligonucleotides, or small molecules can be tethered to silicon (nitride) tips or to mica. Methods are included which afford site-specific and oriented coupling of the sensor molecules.

Moist mucosal epithelial interfaces that are exposed to external environments are dominated by sugar epitopes, some of which (e.g., sialic acids) are involved in host defense. In this study, we determined the abundance and distribution of two sialic acids to assess differences in their availability to an exogenous probe in isolated mucins and mucous gels. We used atomic force microscopy to obtain force maps of human preocular mucous and purified ocular mucins by probing and locating the interactions between tip-tethered lectins Maackia amurensis and Sambucus nigra and their respective receptors, α-2,3 and α-2,6 N-acetylneuraminic (sialic) acids. The rupture force distributions were not affected by neighboring sugar-bearing molecules. Energy contours for both lectin-sugar bonds were fitted to a two-barrier model, suggesting a conformational change before dissociation. In contrast to data from purified mucin molecules, the preocular gels presented numerous large clusters (19,000 ± 4000 nm2) of α-2,6 sialic acids, but very few small clusters (2000 ± 500 nm2) of α-2,3 epitopes. This indicates that mucins, which are rich in α-2,3 sialic acids, are only partially exposed at the surface of the mucous gel. Microorganisms that recognize α-2,3 sialic acids will encounter only isolated ligands, and the adhesion of other microorganisms will be enhanced by large islands of neighboring α-2,6 sialic acids. We have unveiled an additional level of mucosal surface heterogeneity, specifically in the distribution of pro- and antiadhesive sialic acids that protect underlying epithelia from viruses and bacteria.

The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37°C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 ± 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 ± 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.

The high force sensitivity of the atomic force microscope (AFM) has opened the possibility of measuring interand intramolecular forces of biomolecules on the single molecule level. Covalent binding of bioligands to AFM tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a ~6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly ‘scan’ a large surface area while the tip is at or near the sample surface. Using suchlike modified AFM tips, a recent technology development lead to molecular recognition imaging, where topography and recognition sites are simultaneously localized with nanometer accuracy. Thus, the AFM can identify specific components on complex biological sample and retain its high resolution in imaging.

Two-for-one: The authors discuss a powerful new tool that, in a single dynamic force microscopy scan, maps the topography of a surface and simultaneously recognizes specific target sites (see figure). Topography and recognition images correlated nicely at a lateral resolution of a few nanometers. This clever technique has great potential for a broad range of applications involving the detection of particular binding sites in native biological systems.