Devices and methods for performing assays to determine the presence or quantity of a specific
analyte of interest in a fluid sample. In devices according to this invention two separate flow paths are established sequentially in the device with a single user activation step. The first flow path delivers the
analyte of interest (if present in the sample) and conjugate soluble binding reagents to the
solid phase. If
analyte is present, an analyte:conjugate complex is formed and immobilized. The volume of sample delivered by this first path is determined by the absorbent capacity of the
solid phase, and not by the amount of sample added to the device, relieving the user from the necessity of measuring the sample. The sample / conjugate mixture is prevented from entering the second flow path because the capillarity and the
surface energy of the second flow path prevent it from being wetted by this mixture. The second flow path allows a wash
reagent to remove unbound conjugate and sample from the
solid phase to the absorbant, and optionally to deliver detection reagents. The invention may be adapted to many
assay formats including, sandwich immunoassays,
colloidal gold, or
sol particle assays, heterogeneous generic capture assays and competitive assays. In one embodiment, sandwich assays can be performed by immobilizing an analyte binding
reagent on the solid phase, and
drying a labeled analyte binding
reagent in the first flow path. In a competitive
assay embodiment, the first flow path would contain labeled analyte that is dissolved by the sample, and the analyte binding reagent is immobilized on the solid phase. In each of these embodiments, the
assay can be further modified to run in a "generic capture" format, where the solid phase binding reagent is instead conjugated to a generic ligand such as
biotin, and dried in the first flow path (either together or separately from the other assay reagents), and a generic ligand binding reagent (such as
avidin) is immobilized on the solid phase. Another aspect of the present invention includes a subassembly for the
immunoassay device that is comprised of a plastic housing and a means for delivering fluid and / or wash solution. This subassembly comprises a structure formed from a
hydrophobic polymer selectively treated with a
water insoluble surface
active agent that has been applied as a solution in an
organic solvent rendering portions of the surface hydrophilic. When the surface is contacted with an aqueous liquid, it flows only along the treated areas, creating a defined fluid flow path, thereby delivering sample / conjugate solutions to said solid phase.