Abstract: A method is provided for performing a gradient-based assay in a microfluidic device. The method includes the steps of passing first and second fluids through a channel in the microfluidic device. The first fluid has a predetermined concentration of particles therein such that the particles in the first fluid diffuse into the second fluid so as to cause a gradient of concentration of particles in the second fluid as the second fluid flows through the channel. The second fluid sequentially intersects a series of targets along the channel wall as the second fluid flows through the channel.