Simultaneous measurement of 10,000 protein-ligand affinity constants using microarray-based kinetic constant assays.

Abstract Fluorescence-based endpoint detection of microarrays with 10,000 or more molecular targets is a most useful tool for high-throughput profiling of biomolecular interactions, including screening large molecular libraries for novel protein ligands. However, endpoint fluorescence data such as images of reacted microarrays contain little information on kinetic rate constants, and the reliability of endpoint data as measures of binding affinity depends on reaction conditions and postreaction processing. We here report a simultaneous measurement of binding curves of a protein probe with 10,000 molecular targets in a microarray with an ellipsometry-based (label-free) optical scanner. The reaction rate constants extracted from these curves (kon, koff, and ka=kon/koff) are used to characterize the probe-target interactions instead of the endpoints. This work advances the microarray technology to a new milestone, namely, from an endpoint assay to a kinetic constant assay platform. The throughput of this bin...