Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.
Bibliographical noteFunding Information:
We acknowledge financial support from the National Cancer Institute, National Institutes of Health (R00CA127161 and R01CA182543 to LLP), University of Minnesota (through startup funds) and the Purdue University Center for Cancer Research (through an Innovative Pilot Project Award). We thank Dr. Greg Gillispie (Fluorescence Innovations, Inc.) for his help with instrumentation as well as helpful discussions.