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Integration of a Glutamate Sensitive Genetically Encoded SensorProtein into Photocrosslinkable Hydrogel Optrodes

Published online by Cambridge University Press:  23 December 2015

Leyla N. Kahyaoglu  and
Jenna L. Rickus
Leyla N. Kahyaoglu*
Affiliation:
Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907-2057, U.S.A. Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907-2057, U.S.A. Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907-2057, U.S.A.
Jenna L. Rickus
Affiliation:
Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907-2057, U.S.A. Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907-2057, U.S.A. Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907-2057, U.S.A. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907-2057, U.S.A.
*
*(Email: lkahyaog@purdue.edu)
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Abstract

Immobilization into 3D matrices stabilizes proteins in comparison to flat planarsurfaces and facilitates the study of the biomolecular interactions as well asintegration into optrodes for cell physiology. Photocrosslinkable hydrogels havereceived significant attention in recent years as they provide not only a highlyhydrophilic 3D environment to promote protein stabilization and its interactionswith analyte molecules, but enable optically addressable patterning for spatialcontrol of protein localization. At the same time, the explosion of newgenetically encoded sensor proteins has greatly expanded the range of opticalmolecular sensors for cell physiology. Here we integrate a genetically encodedglutamate sensor protein into a photocrosslinkable hydrogel via covalentinteraction to create a novel glutamate sensor material. Protein immobilizationcan be achieved through covalent bonds, physical interactions, or physicalentrapment. Although physical entrapment without chemical modifications offers auniversal approach for protein immobilization, leaching of protein through thepores of the hydrogel is a significant challenge. Thus, here an alternativemethod is developed to provide better control of protein localization andimmobilization using naturally existing reactive groups of proteins. To thisend, a genetically encoded FRET based glutamate indicator protein (FLIPE) ismodified with diacrylated poly (ethylene glycol) (PEGDA) by Michael-typeaddition between acrylate groups and the thiol side chains of the cysteineresidues. We optimize the molecular weight of PEGDA (300, 740, and 3400 Da) aswell as concentrations of the photoinitiator (0.1, 0.5 and 1 % (w/w)) andmonomer (10, 20, and 30 % (w/w)) in the precursor solution. Next the precursorsolution is grown at the distal end of an optical fiber to test thespectroscopic properties and characteristic bioactivities of proteins in thehydrogel network. Optimization of the irradiation parameters, light intensityand exposure time, improves the spatial resolution of 3D hydrogel tips. Thisstudy examines the capability of fabricating 3D hydrogel sensors covalentlymodified with a member of recently growing genetically encoded fluorescentbiosensors, which can later be extended to all conformation-dependent proteinbiosensors and be used intracellularly for physiological and biological sensingpurposes.

Keywords

sensoropticalbiological
Type
Articles
Information
MRS Advances , Volume 1 , Issue 8: Biomaterials and Soft Materials , 2016 , pp. 539 - 546
Copyright
Copyright © Materials Research Society 2015 

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