High-precision two-photon patterning of biomolecules in photo-instructive hydrogels

Photo-responsive hydrogels can be employed to coordinate the organization of proteins in three dimensions (3D) and thus to spatiotemporally control their physiochemical properties by light. However, reversible and user-defined tethering of proteins and protein complexes to biomaterials pose a considerable challenge as this is a cumbersome process, which, in many cases, does not support the precise localization of biomolecules in z-direction. For this reason, we developed a generic approach to micropattern hydrogels with proteins. By exploiting the two-photon excitation of a small lock-and-key interaction pair, we established the protein mounting of hydrogels with micrometer precision. Enhanced by photo-sensitizers, two-photon lithography allowed the user-defined 3D protein immobilization in hydrogels with great flexibility, fidelity, and under mild light doses. Our protein-binding strategy allows the patterning of a wide variety of proteins and offers the possibility to dynamically modify the biofunctional properties of materials in 3D with single-digit micrometer precision.