A Quick and Reproducible Silanization Method by Using Plasma Activation for Hydrophobicity-Based Kinesin Single Molecule Fluorescence-Microscopy Assays.

Single-molecule assays often require functionalized surfaces. One approach for microtubule assays renders surfaces hydrophobic and uses amphiphilic blocking agents. However, the optimal hydrophobicity is unclear, protocols take long, produce toxic waste, and are susceptible to failure. Our method uses plasma activation with hydrocarbons for hexamethyldisilazane (HMDS) silanization in the gas phase. We measured the surface hydrophobicity, its effect on how well microtubule filaments were bound to the surface, and the number of nonspecific interactions with kinesin motor proteins. Additionally, we tested and discuss the use of different silanes and activation methods. We found that even weakly hydrophobic surfaces were optimal. Our environmentally friendly method significanty reduced the overall preparation effort and resulted in reproducible, high-quality surfaces with low variability. We expect the method to be applicable to a wide range of other single-molecule assays.

Polycationic gold nanorods as multipurpose in vitro microtubule markers.

Gold nanoparticles are intriguing because of their unique size- and shape-dependent chemical, electronic and optical properties. Gold nanorods (AuNRs) are particularly promising for various sensor applications due to their tip-enhanced plasmonic fields. For biomolecule attachment, AuNRs are often functionalized with proteins. However, by their intrinsic size such molecules block the most sensitive near-field region of the AuNRs. Here, we used short cationic thiols to functionalize AuNRs. We show that the functionalization layer is thin and that these polycationic AuNRs bind in vitro to negatively charged microtubules. Furthermore, we can plasmonically stimulate light emission from single AuNRs in the absence of any fluorophores and, therefore, use them as bleach- and blinkfree microtubule markers. We expect that polycationic AuNRs may be applicable to in vivo systems and other negatively charged molecules like DNA. In the long-term, microtubule-bound AuNRs can be used as ultrasensitive single-molecule sensors for molecular machines that interact with microtubules.