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. Various microscopy and biomedical applications are based on the particles biocompatibility, surface functionalizability, light absorption, and plasmon resonances. Gold nanorods (AuNRs) are particularly promising for various sensor applications due to their tip-enhanced plasmonic fields. For biomolecule attachment, AuNRs are often stabilized with amphiphilic molecules and functionalized with antibodies or biotin-binding 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 covalently functionalize the gold surface. We show that the functionalization layer is thin and that these polycationic AuNRs bind in vitro to negatively charged microtubule filaments. Furthermore, we can plasmonically stimulate light emission from the AuNRs and, therefore, use them as bleach- and blinkfree microtubule markers. We confirmed colocalization by transmission electron microscopy or the combination of interference reflection and single-molecule fluorescence microscopy of fluorescently-labeled or plasmonic photoluminescent versions of the AuNRs. 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.