A microfluidic microbead-based array for screening the binding interactions of biomolecules.

摘要

Platforms which can display cell membrane ligands and receptors as a microarray library of probes for screening against a target are essential tools in drug discovery, biomarker identification, and pathogen detection. The fact that membrane receptors and ligands require their native bilayer environment to retain their selectivity and binding affinity complicates displaying them in a microarray platform. To accommodate this requirement, a design for a microarray of membrane probes is developed in which the probes are first incoporated in supported lipid bilayers formed around micron-sized particles (lipobeads), and the microbeads themselves are then arrayed on a surface by hydrodynamic capture in a microfluidic obstacle course of traps. The traps are "V" shaped, open enclosures, which are arranged in a wide channel of a microfluidic device, and capture the lipobeads (slightly smaller than the channel height) as they are streamed through the course. Screening assays are undertaken directly in the microfluidic device after assembly, by streaming a fluorescently labeled target through the device and detecting the bead fluorescence.;Conditions are first established for which the supported bilayers on the bead surface remain intact during the capture and assay steps, using fluorescent tags in the bilayer to infer bilayer integrity. Numerical calculations of the hydrodynamic drag coefficient on the entrapped beads are calculated and presented in conjunction with the stability experiments to develop a criteria for the bilayer stability as a function of the screening assay perfusion rate. Screening assays are illustrated, assaying fluorescently labeled Neutravidin with biotin, and labeled cholera toxin with its ganglioside binding ligand, GM1. Sequential capturing of sets of lipobeads (one at a time, and with each set bearing a different probe), followed by indexing the bead positions after each set is entrapped, allows for the construction of an indexed array of multiple probes without the need for particle encoding and is illustrated using the Neutravidin-biotin pair. Finally, the microbead platform is used for quantitatively measuring the kinetic rate constants for the binding of a probe (biotin) to a target (Neutravidin) both theoretically and experimentally.