Multiphoton lithography with protein photoresists

Abstract

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, respectively. 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 μm³ 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.

Keywords: Extracellular vesicle; Functional photoresist; Multiphoton lithography; Protein printing; Tissue scaffolds.

Graphical abstract

Fig. 1

2D/3D structuring of protein-based photoresists. a) and b) show bright field images of MA-BSA and MA-SA 2D grids, respectively. c) and d) depict confocal fluorescence images of 3D grids in aqueous environment fabricated out of MA-BSA and MA-SA. The panels on the right-side display YZ-plane cross-sections (along the white dashed lines), where three different grid constants are visible. e) and f) atomic force microscopy (AFM) images of MA-BSA and MA-SA lines in aqueous environment (upper panel) and dry state, stored at 25 °C for 5 days, (lower panel) showing achievable lateral and axial dimensions, respectively. g) the cross-section of MA-BSA lines in wet and dry conditions (white dashed line in e). h) the cross-section of MA-SA lines in wet and dry conditions (white dashed line in f). The lateral and axial dimensions of printed lines are represented by height (h) and width at FWHM (w), respectively.

Fig. 2

Mechanical properties of the protein-based resins and writing threshold. a) Young's Modulus of 4 different resin compositions in wet conditions (n_total = 45; 15 points on one line, 3 technical replicas; (4 technical replicas for MA-SA + PEG-DA compositions)) b) writing threshold as a function of writing peak intensity and writing speed for 6 different resin compositions. c) height and Young's moduli of MA-BSA lines as function of the writing peak intensity.

Fig. 3

Mechanical properties of the protein-based resins. a) and b) shows the height and Young's moduli of lines made of four different protein-based resists as a function of the environmental conditions. For MA-SA/PEG-DA in the second cycle only one technical replica was used.

Fig. 4

Dynamics of environmentally induced changes in the size and young's modulus of protein-based resin. The blue solid lines of the fit show a general trend of the curve. The experiments were conducted under standard laboratory conditions (24 °C, 40% humidity).

Fig. 5

Immobilization of EVs. a) schematic of the immobilization procedure. MA-SA/PEG-DA lines are written on a glass substrate and incubated with a biotinylated anti-human CD63 (or CD49) antibody. EVs are captured by the antibodies. The presence of EVs is confirmed via binding of Alexa647-anti-human CD81 antibodies on the EVs. b) and c) shows fluorescent images before and after the incubation with the Alexa647-anti-human CD81 antibody.

Fig. 6

Streptavidin structures for cellular EV uptake a) Schematic representation of the experiment. b) Fluorescence image of eGFP-EVs immobilized on top of MA-SA/PEG-DA lines (optical section along dashed line 1 in a)). c) Fluorescent image of eGFP-EVs taken up by the HeLa cells. (optical section along dashed line 2 in a)). d) Fluorescent image of HeLa cells on a line-free area (negative control). Color scale is the same for b), c), and d) images. White dashed circles in c) and d) show individual cells and average fluorescence intensity in the respective area. The higher background fluorescence (in the cells) in panel c) is due to out-of-focus GFP signal contributions.