Immobilization into 3D matrices stabilizes proteins in comparison to flat planar
surfaces and facilitates the study of the biomolecular interactions as well as
integration into optrodes for cell physiology. Photocrosslinkable hydrogels have
received significant attention in recent years as they provide not only a highly
hydrophilic 3D environment to promote protein stabilization and its interactions
with analyte molecules, but enable optically addressable patterning for spatial
control of protein localization. At the same time, the explosion of new
genetically encoded sensor proteins has greatly expanded the range of optical
molecular sensors for cell physiology. Here we integrate a genetically encoded
glutamate sensor protein into a photocrosslinkable hydrogel via covalent
interaction to create a novel glutamate sensor material. Protein immobilization
can be achieved through covalent bonds, physical interactions, or physical
entrapment. Although physical entrapment without chemical modifications offers a
universal approach for protein immobilization, leaching of protein through the
pores of the hydrogel is a significant challenge. Thus, here an alternative
method is developed to provide better control of protein localization and
immobilization using naturally existing reactive groups of proteins. To this
end, a genetically encoded FRET based glutamate indicator protein (FLIPE) is
modified with diacrylated poly (ethylene glycol) (PEGDA) by Michael-type
addition between acrylate groups and the thiol side chains of the cysteine
residues. We optimize the molecular weight of PEGDA (300, 740, and 3400 Da) as
well as concentrations of the photoinitiator (0.1, 0.5 and 1 % (w/w)) and
monomer (10, 20, and 30 % (w/w)) in the precursor solution. Next the precursor
solution is grown at the distal end of an optical fiber to test the
spectroscopic properties and characteristic bioactivities of proteins in the
hydrogel network. Optimization of the irradiation parameters, light intensity
and exposure time, improves the spatial resolution of 3D hydrogel tips. This
study examines the capability of fabricating 3D hydrogel sensors covalently
modified with a member of recently growing genetically encoded fluorescent
biosensors, which can later be extended to all conformation-dependent protein
biosensors and be used intracellularly for physiological and biological sensing
purposes.