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Since its invention in 1986, AFM applications have expanded far beyond simple topographic imaging. AFM tips can be functionalized with a wide range of molecules and used for force-distance measurements to determine binding strengths and single-molecule rupture forces. Image maps can even be generated showing the distribution of interacting molecules on biological surfaces.

Yves Dufrêne and colleagues at the Université Catholique de Louvain now report the use of a kind of AFM called chemical force microscopy (CFM) to map the hydrophobicity of live microbes (Dague et al., 2007). The hydrophobic character of microbial cell surfaces is believed to be important in mediating pathogen-drug and pathogen-host interactions.

To detect hydrophobic interactions, Dufrêne and colleagues functionalized a gold AFM tip with CH3 groups. They obtained nanoscale maps of the hydrophobicity of multiple live pathogens. Whereas the native mycobacteria had homogeneous surface hydrophobicity, treatment of the cells with the antimycobacterial drug isoniazid dramatically decreased the hydrophobicity.

Although AFM generally relies on interactions mediated by the passive physical characteristics of the tip and target, a variant of AFM called Kelvin probe force microscopy (KPFM) requires application of a voltage to the AFM probe. The electrified tip acts as a reference electrode that is scanned over a surface without touching it to measure changes in the surface potential.

Previous work showed that Kelvin probes could be used to detect protein or DNA arrayed on a surface. Ashe Sinensky and Angela Belcher at the Massachusetts Institute of Technology now show that KPFM combined with arrays of protein or DNA could form the basis of a new high-density and label-free class of microarrays (Sinensky & Blecher, 2007). The high spatial resolution of AFM nanoprobes potentially allows detection of spot sizes much smaller than those on existing microarrays. Producing spots small enough to exploit the resolution of KPFM, however, was a challenge.

To create such small spots Sinensky and Belcher used a lithography technique called dip-pen nanolithography (DPN). This allowed them to write sub-micrometer–sized features on a gold substrate by using an AFM tip as a pen, and 'ink' containing protein or ssDNA. Using KPFM they could detect the change in surface potential created by the deposited protein or ssDNA.

For a microarray to be usable though, a user must be able to detect the specific binding of target molecules. Sinensky and Belcher exposed their protein or ssDNA features to protein or ssDNA binding partners, respectively, and showed that KPFM was able to reliably detect the change in potential that occurred. The magnitude of the potential change for the protein-protein interaction was dependent on the ionic properties of the protein, but DNA hybridization gave a reliable twofold signal change.

Sinensky and Belcher showed that the technique is compatible with fast scanning speeds that should allow analyses similar in speed to what is achievable with fluorescent microarray systems but without the requirement for labeling. The limitation at this time is the slow speed of conventional DPN to manufacture the arrays. Once this is overcome, the high-density and label-free nature of KPFM should provide advantages over fluorescence-based arrays.

AFM is expanding quickly into new areas as the nanoscale resolution of the device finds new applications that are quite different from what AFM was developed for. Although it is unlikely that AFM-based devices will ever be as common as light-based microscopes in biology laboratories, the technology seems poised for rapid expansion.