title: Optically diffracting hydrogels for screening kinase activity in vitro and in cell lysate: impact of material and solution properties.
abstract: Optically diffracting films based on hydrogel-encapsulated crystalline colloidal arrays have considerable utility as sensors for detecting enzymaticphosphorylation and, thus, in screening small molecule modulators of kinases. In this work, we have investigated the impact of hydrogel properties, as well as the role of the ionic character of the surrounding environment, on the optical sensitivity of kinase responsive crystalline colloidal array-containing hydrogels. In agreement with a model of hydrogel swelling, the optical sensitivity of such materials increased as the shear modulus and the Flory-Huggins interaction parameter between polymer and solvent decreased. Additionally, elimination of extraneous charges in the polymer backbone by exploiting azide-alkyne click chemistry to functionalize the hydrogels with a peptide substrate for protein kinase A further enhanced the sensitivity of the optically diffracting films. Increasing peptide concentration and, in turn, immobilized charge within the hydrogel network was shown to increase the optical response over a range of ionic strength conditions. Ultimately, we showed that, by tuning the hydrogel and solution properties, as little as 0.1 U/μL protein kinase A could be detected in short reaction times (i.e., 2 h), which is comparable to conventional biochemical kinase assays. We further showed that this approach can be used to detect protein kinase A activity in lysate from HEK293 cells. The sensitivity of the resulting films, coupled with the advantages of photonic crystal based sensors (e.g., label free detection), makes this approach highly attractive for screening enzymatic phosphorylation.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Campus Box 596, Boulder, Colorado 80309, United States.
authors.forename: Kelsey I
authors.initials: KI
authors.lastname: MacConaghy
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Campus Box 596, Boulder, Colorado 80309, United States.
authors.forename: Duncan M
authors.initials: DM
authors.lastname: Chadly
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Campus Box 596, Boulder, Colorado 80309, United States.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Stoykovich
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Campus Box 596, Boulder, Colorado 80309, United States.
authors.forename: Joel L
authors.initials: JL
authors.lastname: Kaar
mesh_terms: D057930:Click Chemistry; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D055620:Optical Phenomena; D009994:Osmolar Concentration; D010766:Phosphorylation / Q000187:drug effects; D012996:Solutions
chemical_list: D020100:Hydrogels; D012996:Solutions; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases