Recently, 2D/3D direct laser writing has attracted increased attention due to its broad applications ranging from
biomedical engineering to aerospace. 3D nanolithography of water-soluble protein-based scaffolds have been
envisioned to provide a variety of tunable properties. In this paper, we present a functional protein-based
photoresist with tunable mechanical properties that is suitable for multiphoton lithography (MPL). Through
the use of methacrylated streptavidin or methacrylated bovine serum albumin in combination with polyethylene
glycol diacrylate or methacrylated hyaluronic acid as crosslinkers and a vitamin-based photoinitiator, we were
able to write two- and three-dimensional structures as small as 200 nm/600 nm lateral/axial features, respec-
tively. We also demonstrated that Young?s modulus can be tuned by the photoresist composition, and we were
able to achieve values as low as 40 kPa. Furthermore, we showed that Young?s modulus can be recovered after
drying and rehydration (i.e. shelf time determination). The retained biological functionality of the streptavidin
scaffolds was demonstrated using fluorescently labelled biotins. Using single-molecule fluorescence microscopy,
we estimated the density of streptavidin in the written features (1.8 ± 0.2 × 105 streptavidins per 1.00 ± 0.05
?m3 of feature volume). Finally, we showed applicability of our 2D scaffold as a support for a fluorescence
absorbance immuno-assay (FLISA), and as a delivery platform of extracellular vesicles to HeLa cells.