Microfluidic, computational, and recombinant protein design for the analysis of the molecular properties of dynamic cell capture systems.

摘要

Interactions between surface-bound proteins play a critical role in many biological processes. Specifically, the interactions between surface-bound selectins and surface-bound glycosylated protein ligands are of considerable interest because they are involved in leukocyte capture from the vasculature to endothelial surfaces in normal trafficking, are altered in inflammatory disease states, and may be an important mediator of disease progression with additional cell types, such as metastatic cancer cells. Despite significant progress characterizing which molecular features facilitate selectin function as assessed by soluble protein binding or cell rolling velocities, determination of the bond formation rates in their surface-attached configuration, and which molecular features enhance bond formation, has proven especially difficult. A hydrodynamically-conditioned micropattern catch strip assay is developed to measure particle recruitment rates that reflect the kinetics of formation for the interaction of the biomolecular pair being investigated. The assay exploits patterning within microfluidic channels and the mechanostability of PSGL-1/P-selectin bonds to create reaction geometries that confine a microbead flux to within 200 nm of the surface under flow conditions. A computational methodology is employed to link hypotheses for how molecules behave at the interface between moving surfaces with experimentally testable motion measurements. The method recreates motion patterns of discrete pauses and skips, as observed in PSGL-1/P-Selectin microbead assays. It is demonstrated that the packaging of adhesion molecules into clusters is critical, whereas an initial increase in bond lifetime with force and the spatial distribution of the reactive density within the volume of contact between surfaces are not important for PSGL-1/P-Selectin dynamic adhesive function. Progress on the development of a covalently oriented PSGL-1 molecular platform that can be employed to experimentally probe the effects of properties such as molecular length and binding pocket chemistry on bond formation is reported. Development of a multifunctional catch strip assay that may be implemented as a multiplexed, internally controlled measurement and screening tool to evaluate the effect of post-translational modifications with the protein scaffold platform technology is reported. In addition to development measuring molecular interactions, preliminary results demonstrating how the catch strips might be developed into cell-based assays for scientific or clinical diagnostic purposes are given.