Catch strip assay for the relative assessment of two-dimensional protein association kinetics.
Accurate interpretation of recruitment rate measurements of microscale particles, such as cells and microbeads, to biofunctional surfaces is difficult because factors such as uneven ligand distributions, particle collisions, variable particle fluxes, and molecular-scale surface separation distances obfuscate the ability to link the observed particle behavior with the governing nanoscale biophysics. We report the development of a hydrodynamically conditioned micropattern catch strip assay to measure microparticle recruitment kinetics. The assay exploited patterning within microfluidic channels and the mechanostability of selectin bonds to create reaction geometries that confined a microbead flux to within 200 nm of the surface under flow conditions. Systematic control of capillary action enabled the creation of homogeneous or gradient ligand distributions. The method enabled the measurement of particle recruitment rates (keff, s-1) that were primarily determined by the interaction of the biomolecular pair being investigated. The method is therefore well suited for relative measurements of delivery vehicle and cellular recruitment potential as governed by surface-bound molecules.
Schmidt
Brian J
BJ
Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA.
Huang
Peter
P
Breuer
Kenneth S
KS
Lawrence
Michael B
MB
D001665:Binding Sites; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D053758:Nanoparticles / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013499:Surface Properties
D015815:Cell Adhesion Molecules; D008024:Ligands; D011506:Proteins