Fig. 1. Laser writing system for microfabrication of proteinaceous structures.(a) Laser writing system is composed of three basic components: a laser, a lens and mirror system, and an objective lens. The beam is directed towards the sample mounted on a scanning stage, optionally observed with a white light camera setup. The system is controlled by a computer. The laser beam typically has a spatially Gaussian profile. In a plane perpendicular to the laser beam axis, the cross-linking occurs only in the beam where the intensity exceeds a threshold that is dependent on wavelength, absorption scheme (single or multiphoton absorption) and the protein precursor. (b) In volume, protein cross-linking occurs in the laser focus along the writing path in the protein precursor material. Diameter and height of volume elements, so-called voxel, in a free space become ellipsoidal shapes due to mismatch between focused spot sizes and Rayleigh length, as indicated by dashed and solid lines. On the substrate, surface bound structures are formed, depending on the position of focus. Determining resolution as the smallest distance between distinguishable structures, well-connected 3D structures can be formed by scanning the focused laser beam with a step smaller than the resolution. The box inset shows that protein molecules (light blue) in the precursor are interacted with photoactivator (orange) to be cross-linked (dark blue). The circular inset shows an SEM image of an icosahedron with submicron feature sizes, scale bar represents 1 μm, reprinted from ref. ³³, with the permission of AIP Publishing, copyright 2015.

Fig. 2. PDB data overview.Properties of 16 different protein structures obtained from selected PDB files: (a) the secondary structures, (b) unique chains, (c) chain lengths, (d) unit weights, and (e)–(f) amino acid distributions. (e) Single letter amino acid codes sorted by classification according to Livingstone et al. from ref. ⁷⁴. (f) The distributions of amino acid occurrence in the PDB FASTA sequence for 16 proteins versus respective database values, calculated from reports by Eitner et al. from ref. ⁷⁵.

Fig. 3. Proposed processes for protein cross-linking and adherence.A schematic illustration depicting the differing degrees of deformation and adhesion that can degenerate protein functionality. Specific surfaces render the functional domain inaccessible or can have little effect on the native protein, leading to retained protein function. Figure reprinted with permission from supplementary material of ref. ⁴³, copyright 2017, American Chemical Society.

Fig. 4. Proposed free radical mechanisms.Three selected protein cross-linking fabrication mechanisms based on radical oxygen formation inside the protein from (a) ref. ²⁶, (b) ref. ³², and (c) ref. ²⁸ are matched the four stages of light-induced radical chain reaction: excitation, radical generation, propagation and termination. Tyr represents tyrosine and M represents DNA-bound metal.

Fig. 5. pH actuation.(a) Conceptual drawing of the fabrication of a device with regions having varying swelling properties in a protein solution in response to the same laser exposure. (b) Lay et al. designed a mesh pattern in which the porosity changes with pH. (c) pH-actuation of a microgripper, in which each pillar is approximately 5 µm wide at the base. (d) Dependence of the focal distance of a pH-tunable lens on pH. (a, b) Reprinted with permission from ref. ⁶², copyright 2015, American Chemical Society. (c) Reprinted with permission from ref. ⁴⁰, copyright 2015, John Wiley and Sons. (d) Reprinted with permission from ref. ⁵⁵, copyright 2015, John Wiley and Sons.

Fig. 6. Soft microoptics.(a) A schematic illustration of the repetitive bending of KPL made of protein formed on a PDMS sheet (upper), and light diffraction results at different positions along the main light axis (lower). (b) A stimulus-responsive whispering-gallery-mode micro-laser fabricated from BSA. This structure requires high accuracy in fabrication and performs in air (shown) and aqueous environments. (a) Adapted by permission from Springer Nature: Light Sci. Appl.⁴¹, copyright 2014. (b) Adapted by permission from Springer Nature: Sci. Rep.⁵³, copyright 2015.

Fig. 7. Cell culture scaffold and in situ guidance.(a) A two-protein microscaffold made with fibronectin and BSA to selectively cultivate cells only on the fibronecting elements. (b) Proteinaceous networks embedded in a mechanically supportive polymer scaffold to provide a microenvironment. (c) In situ observation of a captured bacterial cell with a fluorescent marker. The encapsulated bacterial cell had access to a nutritional medium and multiplied while trapped (scale bar represents 10 μm). (d) In situ guiding of neuronal cells by directly writing free lines (indicated by white arrows) near the cellular extensions (scale bar represents 10 μm). (a) Reprinted with permission from ref. ⁵⁹, copyright 2015, OSA Publishing. (b) Reprinted with permission from ref. ⁴³, copyright 2017, American Chemical Society. (c) Adapted with permission from ref. ⁵⁷, copyright 2012, American Chemical Society. (d) Reprinted with permission from ref. ⁵⁶, copyright 2004, National Academy of Sciences.

Fig. 8. Microfluidic integration.Proteinaceous structures integrated into (a), (b) polydimethylsiloxane (PDMS) and (c) glass microfluidic devices. (a) Live capture of red blood cells (RBCs) based on type A binding to an anti-A antibody protein pad. (b) Enzyme reactors made from trypsin integrated into a PDMS channel. (c) Integration of BSA microstructures (top view during fabrication with 25 μm scale bar and SEM perspective view with 10 μm scale bar) and an EGFP pattern and BSA pattern in a closed glass microfluidic channel with 100 μm scale bars. (a) Reprinted by permission from the authors and SPIE 35, copyright 2015. (b) Adapted by permission from Springer Nature: Microfluids and Nanofluids ³⁶, copyright 2011. (c) Adapted with permission from ref. ³⁷, copyright 2018 MDPI.

Table 1. Protein solution conditions. A summary of previous fabrication conditions, focusing on protein and photosensitizer concentrations. Examples of typical solvents and protein databank samples are included. Photoactivator abbreviations: rose bengal (RB), methylene blue (MB), flavin mononucleotide (FMN), flavin dinucleotide (FAD), rhodamine B (RhB), rhodamine 6 G (R6G), benzophenone dimer (BPD), sodium 4-[2-(4-morpholino)benzoyl-2-dimethylamino]butylbenzenesulfonate (MBS). Solvent abbreviations are: dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

Table 2. Laser fabrication conditions. Fabrication sorted by pulse width: femtosecond (fs), picosecond (ps) and nanosecond (ns). For almost all reports, high-NA lenses were used. BSA was employed for all conditions. Dynamic mirror device (DMD), spatial light modulator (SLM), or galvano mirrors were used in some of these previous works.