An ultrasensitive, two-step immunoassay method was developed for rapid assay of proteins and viruses. In the first step, electrophoresis was used to quickly bring soluble analytes from a flowing solution to a microarray of probe molecules immobilized on a semipermeable membrane. In the second step, the captured analyte was detected by scanning the microarray with functionalized magnetic beads passed over the array surface by shear flow and pressed to the array surface by a magnetic field. We demonstrated that this method routinely detected analytes in concentrations as low as 20 aM (500-1000 molecules, or 1-2 zeptamoles) within 2-3 min in the presence of a similar to 10(11)-fold excess of other protein molecules in the sample.