Bianca Buchegger1 Johannes Kreutzer1 Richard Wollhofen1 Jaroslaw Jacak1 2 Thomas Klar1

1, Johannes Kepler Universität Linz, Linz, , Austria
2, Upper Austria University of Applied Sciences, Linz, , Austria

Multiphoton polymerization (MPP) allows fabrication of arbitrary polymer structures in three dimensions with minimum feature sizes down to 100 nm and a lateral resolution around 200 nm. An excitation laser is focused into a photoresist consisting of a highly crosslinking acrylate and a photo-initiator. Adding a second laser beam which induces stimulated emission depletion (STED) of the photo-initiator in the outer rim of the excitation point spread function allows writing of even smaller structures with feature sizes below the diffraction limit.
Including functional groups other than the acrylate groups increases the versatility of the polymer structures, specifically for bio-functionalization. This can be either achieved by mixing of metal-oxo-clusters into the photoresist [1] or by using acrylate monomers with different functional rest groups such as thiols or carboxy groups enabling orthogonal functionalization [2]. The reactivity of the polymer structures was shown by covalent linkage of two different, chemically modified fluorophores. MPP scaffolds with bio-adhesive sites can also be used for 3 dimensional immunoassays [3] and for physiological studies in microfluidic channels.
Using carboxy-acrylate polymer structures in combination with a biotin modified supported lipid bilayer enables orthogonal functionalization with two different fluorescent proteins. One sort of proteins is immobilized on polymer anchors via nickel-nitrilotriacetic acid / histidine interaction. The other one is freely moving within the lipid bilayer surrounding the structures which is enabled using biotin / streptavidin binding [4]. As mobility of proteins and lipids plays a major role in physiological processes, this platform is well suited for modelling of cell interactions with mobilized and immobilized proteins and studying cellular response.

[1] Buchegger, B.; Kreutzer, J.; Plochberger, B.; Wollhofen, R.; Sivun, D.; Jacak, J.; Schütz, G. J.; Schubert, U.; Klar, T. A. ACS Nano 2016, 10 (2), 1954-1959.
[2] Wollhofen, R.; Buchegger, B.; Eder, C.; Jacak, J.; Kreutzer, J.; Klar, T. A., Opt. Mater. Express 2017, 7 (7), 2538-2559
[3] Wollhofen, R.; Axmann, M.; Freudenthaler, P.; Gabriel, C.; Röhrl, C.; Stangl, H.; Klar, T. A.; Jacak, J. ACS Appl. Mater. Inter. 2018, 10 (2), 1474-1479.
[4] Buchegger, B.; Kreutzer, J.; Mayr, S.; Wollhofen, R.; Plochberger, B.; Axmann, M.; Jacak, J.; Klar, T. A, submitted.