Biomolecular gradients play an essential role in studying various biological phenomena such as development, cancer, inflammation, and wound healing. This paper reports a novel microfluidic device for generating tunable biomolecular gradients over large areas on cell culture surfaces. Laminar streams are stacked above the surface to generate a steady-state gradient via diffusion in the direction orthogonal to the flow and to the surface. Finite-element modelling was used to predict negligible shear forces at the range of gradients possible by tuning flow rates. The surface gradients were characterized with fluorescence microscopy; image analysis verified the presence of a one-dimensional gradient across a 2x2 mm area. Fig. 1 shows a variety of surface gradients obtained simply by changing the inlet configuration and pressure settings. Superimposed onto these images are the linescans taken across 4 different regions of the device which demonstrate lateral uniformity (excluding edge effects near walls <25% of area). Due to the binary branching of the inlets, the perfused area can be arbitrarily wide (here, 2 mm). This enables analyzing responses of large cell populations in parallel and acquiring rich statistics on single-cell variability.View Large Image | View Hi-Res Image | Download PowerPoint Slide