Chemical Functionalization and Bioconjugation Strategies for Atomic Force Microscope Cantilevers

Over the last decade, scanning probe microscopy (SPM) techniques, such as atomic force microscopy (AFM), have played an important role in a variety of biophysical research efforts. This straightforward technique has the capability to measure forces down to a few hundred piconewtons, which enables the observation of unique events within or between single molecules. However, in order to successfully carry out these types of biophysical measurements, the anchoring of the biomolecules of interest to the scanning probe cantilever tip needs to be of sufficient strength to avoid rupture prior to the analysis of the specific interaction to be probed. Hence, a covalent linkage of the biomolecule to the SPM probe tip is generally preferred. It is also advantageous to have a long-chain functional linker to separate the biomolecule from the SPM probe tip so as to minimize unwanted interactions between the substrate surface and the tip and to “isolate” the biomolecular forces being probed. The most common materials for SPM cantilevers are silica and silicon nitride, and there are several surface chemistry approaches available to achieve a covalent linkage to such types of materials. In this chapter, we present various strategies and detailed protocols for conducting surface modifications suitable for biomolecular attachment to AFM probe surfaces or other hydroxylated surfaces. The strategies described build upon an initial surface activation treatment using the convenient gas-phase deposition of an organosilane and incorporate various passivation schemes and biomolecular immobilization techniques.