A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competi tive assay is based on measuring the distribution of a labeled probe molecu le after it diffuses for a short time from one region into another region c ontaining antigen-specific antibodies. The assay was demonstrated in the T- sensor, a simple microfluidic device that places two fluid streams in conta ct and allows interdiffusion of their components. The model analyte was phe nytoin, a typical small drug molecule. Clinically relevant levels were meas ured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measu rements. Numerical simulations agree well with experimental results and pro vide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 mul of reagents and sample, and is applicable to a wide range of analytes.