Injectable Hydrogel Biosensors Based on Fluorogenic DNA and RNA Probes

Development of injectable/implantable sensors that can monitor different biomarkers related to cognitive and physiological performance has gained significant focus in recent years. We report on the initial research related to the development of implantable hydrogel-based sensor for continuous assessment and autonomous monitoring of biomarkers associated with physical/cognitive performance. Several biocompatible hydrogel formulations based on synthetic and natural polymers were selected to investigate the effect of the polymer matrix on the function of the structure switching sensor probes in a hydrogel environment. We optimized the hydrogel formulations to yield biocompatible and transparent network, with optimal mesh size structures and gelation time transitions for sensing. The synthetic hydrogel network was based on hyperbranched polyethylene imine (PEI) polymer network crosslinked with polyethylene glycol (PEG) linkers and natural hydrogel was prepared during enzymatic crosslinking of reconstituted silk fibroin (SF) protein. Both formulations can be processed at physiological conditions in aqueous buffers without the need for organic solvents, and hence can be considered for biocompatible processes. The optimization of sensor function in both hydrogel formulations was done using several fluorogenic DNA and RNA-based sensors including a model fluorogenic DNA aptamer responsive to crystal violet, a spinach RNA sensor for adenosine metabolite and a forced intercalation (FIT) DNA aptamer responsive to dehydroepiandrosterone sulfate (DHEAS) hormone. For all these sensors to be functional and produce a fluorescent signal, the matrix should allow a structure switching mechanism to take place and allow the formation of the aptamer binding pocket. We demonstrate that hydrogels with optimal mesh size network and hydrophilicity can ideally support the diffusion of analytes and the switching of aptamer conformations in DNA-based sensors, leading to generation of the signal within 10-20 min depending on the polymer volume and concentration of sensor probes. While DNA-based sensors were successfully activated in hydrogels, RNA-based sensors function require further optimization. This optimization is necessary to understand the mechanism of sensor activation with long RNA sequences that are composed of several structurally important parts such as ligand-binding pocket, connecting module, stabilizing scaffold and fluorogenic aptamer.