Soft Sub-Structured Multi-Material Biosensor Hydrogels with Enzymes Retained by Plant Viral Scaffolds

An all-soft multi-material combination consisting of a hydrogel based on poly(ethylene glycol) (PEG) coated with spatially defined spots of gelatin methacryloyl (GM) containing selectively addressable viral nanorods is presented, and its basic application as a qualitative biosensor with reporter enzymes displayed on the tobacco mosaic virus (TMV) bioscaffolds within the GM is demonstrated. Biologically inert PEG supports are equipped with GM spots serving as biological matrix for enzymes clustered on TMV particles preventing diffusion out of the gel. For this multi-material combination, i) the PEG-based hydrogel surface is modified to achieve a clear boundary between coated and non-coated regions by introducing either isothiouronium or thiol groups. ii) Cross-linking of the GM spots is studied to achieve anchoring to the hydrogel surface. iii) The enzymes horseradish peroxidase or penicillinase (Pen) are conjugated to TMV and integrated into the GM matrix. In contrast to free enzymes, enzyme-decorated TMVs persist in GM spots and show sustained enzyme activity as evidenced by specific color reaction after 7 days of washing, and for Pen after 22 months after dry storage. Therefore, the integration of enzyme-coupled TMV into hydrogel matrices is a promising and versatile approach to obtaining reusable and analyte-specific sensor components. An all-soft biosensor consisting of a poly(ethylene glycol)-based hydrogel coated with spots of gelatin methacryloyl (GM) is generated. The enzymes horseradish peroxidase and penicillinase are conjugated to tobacco mosaic virus (TMV) nanoparticles and integrated into the GM spots. Enzyme-coupled TMV remains captured in GM spots with sustained sensing activity, in contrast to enzymes incorporated without TMV carriers that diffuse out.image