Development of a direct photochemical method for the generation of biomolecular interfaces for applications in leukocyte biology

Methods for generating surface-immobilized biomolecular patterns and gradients have provided invaluable insight into the molecular basis of numerous complex biological processes. Herein, we describe the development of a general, photochemical method for creating biointerface substrates presenting single- and multi-component biomolecular patterns and gradients. In our approach, the generation of a light density gradient across a photoactive benzophenone monolayer on glass results in covalent attachment of solution-phase biomolecules onto the surface. This simple, direct and molecularly general approach was used to generate surface-immobilized biomolecular gradient substrates for proof-of-principle biomolecular photopatterning demonstrations, as well as investigations of multi-parameter cell-substrate interactions and fundamental leukocyte biology studies. Substrates tailored to present P-selectin or E-selectin, which are proteins expressed on the inflamed endothelium, were applied to flow assays with neutrophils isolated from whole blood for investigating the effects of bromelain, an alternative anti-inflammatory treatment, on neutrophil recruitment in vitro under conditions of physiological shear stress. Beyond applications in leukocyte biology, this methodology for biomolecular substrate generation can serve as an enabling tool for investigating the molecular basis of cell migration and polarization in response to immobilized proteins, and for systematically addressing the combined effects of multiple stimuli, such as immobilized biomolecule density, mechanical cues, and shear stress on cell behavior.