(391e) Femtomolar Detection for Microfluidic Immunoassays Using Controlled Evaporation

Diagnostic protocols based on protein-antigen affinity interactions have become increasingly popular in both clinical and research settings. Heterogeneous affinity assays, in which antigens (or antibodies) immobilized to a surface capture analytes of interest from an aqueous solution, offer many advantages over their traditional homogeneous well-based counterparts, including reductions in footprint size, analysis times, and sample volumes. However, because these methods rely on the reversible protein-antigen affinity interaction, the equilibrium analyte coverage is a function of the association constant for a given system. As a result, affinity interactions possessing poor association kinetics are restricted to limits of detection (LOD) above 1 pM when using standard techniques of detection (e.g. SPR or fluorescence assays).

In this work we present a method that utilizes the effects of evaporation to passively concentrate an analyte solution to lower the LOD for heterogeneous assays. A microfluidic system composed of two reservoirs connected by a single microchannel is reversibly bonded to a substrate such that an outlet reservoir lies over discrete regions of immobilized antibodies. Upon introduction of analyte solution into the inlet reservoir, fluid spontaneously fills microchannel and the corner regions of an outlet reservoir such that the liquid volume present in the outlet reservoir quickly establishes a steady state value on the sub-nL scale and liquid is passively pumped from the inlet to outlet reservoir. With optimized design of the microchannel and outlet reservoir geometries, the ratio of the volumetric flow rate through the channel to the steady state liquid volume can be increased to the point where the liquid solution in the outlet reservoir is concentrated by over three orders of magnitude in less than 30 minutes. Because the liquid volume in the outlet reservoir is steady over time, unwanted deposition of material onto the substrate is avoided.

We show that this passive system can be used to lower the LOD for heterogeneous immunoassays by multiple orders of magnitude while requiring no external pumping mechanism, and eliminates the need for expensive detection methods or complex fabrication strategies.