High-throughput screening of FRET-based proteins using a hyperspectral microcapillary array

Fluorescence resonance energy transfer (FRET) is a versatile technique used in biology, chemistry, and materials science that involves distance-dependent energy transfer between light-sensitive molecules. Current FRET screening methods are limited in their ability to manage ultra-high-throughput samples. Challenges include low dynamic range, spectral overlap, multiple fluorophores, and high background signals. A high-throughput FRET approach enables rapid, multiplexed screening of interactions, conformations, and dynamics with accurate, real-time measurements. We developed a hyperspectral, high-throughput microcapillary array (HyCAP) that is well-suited for sorting large clone libraries based on fluorescence assays. Our platform provides sensitive, high-resolution hyperspectral imaging with a throughput of approximately 10⁵ per array. We demonstrated the use of FRET to analyze both short polypeptides and longer tandem repeat proteins. HyCAP shows promise in correlating computationally predicted structures with experimentally measured molecular distances from FRET.