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AFM tip functionalization at high ligand density

Development of smart probes (force microscopy tips). The so far weakest point of AFM (atomic force microscope) tip functionalization, i. e. the generation of reactive sites (amino groups) for subsequent attachment of crosslinkers will be replaced by new methods which allow to attach of many ligands per apex, as needed in TREC (topography and recognition imaging) and applications to living cells. This basic work will close the gap between the bare tip surface and the elaborate crosslinker library which already exists at the Institute of Biophysics in Linz. In addition to silicon nitride, silicon, diamond, carbon (EBD), and gold will also considered as tip material. The transfer of chemical protocols to laboratories that are not experts in this field shall be demonstrated.
We aim at maximum dissemination of best practices to the broader scientific community. Innovation and standardization of the primary step of AFM tip functionalization, especially on new materials such as silicon and diamond, potentially also on carbon (EBD) and gold, in addition to silicon nitride, will be pursued. Assessment of surface functionalization by new, fast, sensitive and unbiased fluorescence assay (reversible fluorophore attachment). Adjustment of surface properties between the grafting sites to ensure weak protein adsorption, as needed for fast protein coupling. Standardization of protocols for the conjugation of typical crosslinker molecules (commercial or available in Linz) to the tips. Assessment of the new tip functionalization methods by standardized experiments in single molecule force spectroscopy and dynamic recognition imaging. Evaluation of ease-to-use and performance by Agilent Labs.