Controlled Nanoconfinement in a Microfluidic Modular Bead Array Device via Elastomeric Diaphragm Collapse for Enhancing Biomolecular Binding Kinetics.

Nanoscale confinement strategies alleviate diffusional transport limitations to enhance target binding kinetics with receptors, motivating their utilization for screening and selecting receptors based on binding affinities with target molecules. Herein, a modular and multiplexed device for creating nanoconfinement is presented through the collapse of an elastomeric diaphragm onto microbead arrays immobilized with biomolecules, followed by repeated diaphragm withdrawal to promote bulk transport, thereby enhancing receptor binding kinetics. To repeatedly create controlled nanoconfinement over large spatial extents on the bead, the diaphragm is integrated on its top side with a strain sensor for modulating vertical displacement, while microfabricated nanoposts (≈500 nm depth) on its bottom side control the lateral extent. The modular platform enables facile assembly of beads, each immobilized with different targets into eight microwells for multiplexed screening of receptors, and facile disassembly for quantifying DNA-binding on each bead by downstream q-PCR. Nanoconfinement enhances biomolecular binding at 1 Hz diaphragm pressurization, as validated by rapid DNA immobilization (time constant of ≈6 min vs >60 min under no confinement) and through saturating the binding of target molecules with optimal aptamer candidates (88% site occupancy vs 5% under no confinement at 10 nm levels), thereby screening candidate receptors based on binding affinity parameters.