Topography and recognition imaging of protein-patterned surfaces generated by afm nanolithography

The atomic force microscope (AFM) is a precise, versatile, and powerful tool to pattern biomolecular surfaces and image single molecules. For patterning, the AFM cantilever is used as stylus to scratch nanometer-sized features in soft protein layers via the native-protein nanolithography (NPNL) approach.[ 1, 2] Imaging of individual molecules, on the other hand, exploits the topographic height changes with the cantilever. Detection can also rely on the molecular recognition between surface-bound molecules and complementary receptors tethered to the AFM tip.[3] Recently, both characterization modes have been merged to achieve simultaneous topographic and recognition (TREC) imaging.[4–6] Herein, we combine NPNL and TREC to synergistically use AFM tips to write and image nanoscale protein patterns on a surface. The approach is validated using surface-bound biotinylated bovine serum albumin (BSA) protein and AFM tips carrying streptavidin tethered via a flexible poly(ethylene glycol) (PEG) linker. A detailed biophysical investigation of a key TREC parameter is conducted to optimize the accuracy of the read-out.