Abstract: Disclosed is a flow-through membrane assay that takes advantage of a high surface area and rapid transport while allowing individual control over flow rates and times for each step of a multi-step assay. A microfluidic card features channels in communication with a porous membrane, channels on either side of membrane to allow transverse flow across the membrane, capturing a labeled target from the sample by flowing the sample across the membrane, or capturing a target from the sample followed by flowing a reagent containing a label that binds to the target. Fluid can be pushed or pulled through the assay membrane by external control. Air near the membrane is managed by diverting air between fluids to a channel upstream of the assay membrane, venting air between fluids through a hydrophobic membrane upstream of the assay membrane, and/or by venting trapped air through a hydrophobic membrane downstream of the assay membrane.