Integration of a Genetically Encoded Calcium Molecular Sensor into Photopolymerizable Hydrogels for Micro-Optrode-Based Sensing.

Genetically encoded molecular-protein sensors (GEMS) are engineered to sense and quantify a wide range of biological substances and events in cells, in vitro and even in vivo with high spatial and temporal resolution. Here, we aim to stably incorporate these proteins into a photopatternable matrix, while preserving their functionality, to extend the application of these proteins as spatially addressable optical biosensors. For this reason, we examined the fabrication of 3D hydrogel microtips doped with a genetically encoded fluorescent biosensor, GCaMP3, at the end of an optical fiber. Stable incorporation parameters of GCaMP3 into a photo-cross-linkable monomer matrix were investigated through a series of characterization and optimization experiments. Different precursor-solution formulations and irradiation parameters of in situ photopolymerization were tested to determine the factors affecting protein stability and sensor reproducibility during photoencapsulation. The microstructure and performance of hydrogel microtips were controlled by varying UV irradiation intensity as well as the molecular weight and concentration of the photocurable monomer, PEGDA (polyethylene glycol diacrylate), in precursor solution. Protein-doped hydrogel micro-optrodes (microtip sensors) were fabricated successfully and reproducibly at the distal end of optical fiber. Under optimized conditions, the bioactivity of GCaMP3 within a hydrogel matrix of micro-optrodes remained similar to that of the protein-free matrix in buffer. The limit of detection of protein optrodes for free calcium was also determined to be 4.3 nM. The hydrogel formulation and fabrication process demonstrated here using microtip optrodes can be easily adapted to other conformation-dependent protein biosensors and can be used in sensing applications.