Supported Solid Lipid Bilayers as a Platform for Single-Molecule Force Measurements.

Biocompatible surfaces are important for basic and applied research in life science with experiments ranging from the organismal to the single-molecule level. For the latter, examples include the translocation of kinesin motor proteins along microtubule cytoskeletal filaments or the study of DNA-protein interactions. Such experiments often employ single-molecule fluorescence or force microscopy. In particular for force measurements, a key requirement is to prevent nonspecific interactions of biomolecules and force probes with the surface, while providing specific attachments that can sustain loads. Common approaches to reduce nonspecific interactions include supported lipid bilayers or PEGylated surfaces. However, fluid lipid bilayers do not support loads and PEGylation may require harsh chemical surface treatments and have limited reproducibility. Here, we developed and applied a supported solid lipid bilayer (SSLB) as a platform for specific, load bearing attachments with minimal nonspecific interactions. Apart from single-molecule fluorescence measurements, anchoring molecules to lipids in the solid phase enabled us to perform force measurements of molecular motors and overstretch DNA. Furthermore, using a heating laser, we could switch the SSLB to its fluid state allowing for manipulation of anchoring points. The assay had little nonspecific interactions, was robust, reproducible, and time-efficient, and required less hazardous and toxic chemicals for preparation. In the long term, we expect that SSLBs can be widely employed for single-molecule fluorescence microscopy, force spectroscopy, and cellular assays in mechanobiology.

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.