(114d) Development of a Photonic Crystal Biosensor for Assaying Kinase Activity

Protein kinases are a critical family of enzymes that modulate virtually all cellular processes and have been implicated in a myriad of diseases, making kinases among the most important targets for therapeutic molecules. However, kinases are inherently difficult to assay due to the lack of measurable signal and there is a lack of robust, high-throughput screening methods available for kinase inhibitors and activators. To address this problem, we have developed a novel biosensor that is based on a kinase-responsive polymer hydrogel, which enables label-free screening of kinase activity via changes in optical properties.  The hydrogel is specifically designed to swell reversibly upon phosphorylation of a target peptide, triggering a change in optical diffraction from a crystalline colloidal array of particles impregnated into the hydrogel. Changes in optical diffraction upon treatment of the hydrogel with kinase were found to occur on the timescale of minutes to hours and exhibited a dose-dependent response. Such hydrogels may be used as a sensing platform to identify kinase inhibitors or activators of kinase pathways, as well as for assaying kinase selectivity. The lack of exogenous fluorescent reagents or labels enhances the potential utility of the hydrogels for high-throughput screening. We have also demonstrated that the sensor can be used to detect phosphatase activity, involving the removal of the phosphate group from the target peptide. More broadly, such hydrogels may be used to assay the activity of other enzymes that catalyze post-translational modifications that alter substrate charge (e.g., sulfonation or acetylation), thus providing a platform to screen a broad spectrum of protein or biomolecules modifications.