Methods and apparatuses are provided for determining presence and concentration of analytes by exploiting molecular binding reactions and differential diffusion rates. Analyte particles and binding particles are allowed to diffuse toward each other, and slowing of the diffusion front is detected when they meet. From the position of the diffusion front, presence and concentration of analyte particles can be determined. One embodiment provides a competitive immunoassay in a microfluidic format. This diffusion immunoassay (DIA) relies on measuring the concentration of labeled antigen along one dimension of a microchannel after allowing it to diffuse for a short time into a region containing specific antibodies. A simple microfluidic device, the T-Sensor, was used to implement a DIA to measure the concentration of phenytoin, a small drug molecule. Concentrations of analyte over the range of 50 to 1600 nM can be measured in less than a minute. The assay is homogeneous, rapid, requires only microliter volumes of reagents and sample, and is applicable to a wide range of analytes, including therapeutic drugs, molecular biological markers, and environmental contaminants. Methods for separating particles of similar size in a diffusion separator are also provided.