Stable focusing of proteins within a packed microbead bed by ion concentration polarization in a paper-based analytical device

Paper-based analytical devices (PADs), and particularly lateral flow assays (LFAs), provide many advantages for protein sensing in clinical and resource-limited settings. This class of devices is an important tool for point-of-care (POC) diagnostics, but is limited by sensitivity, which is often inferior to standard benchtop instrumentation. Improvements in sensitivity would increase the range of accessible analytes for biological assays at the POC. Electrokinetic enrichment methods, such as ion concentration polarization focusing (ICPF), have been implemented for target analyte preconcentration. While ICPF has been demonstrated to achieve high enrichment factors (EFs) of proteins in PADs, the focused protein plug experiences instability, leading to bursting (downstream washout) or propagation (gradual upstream displacement) if convective forces are too strong or too weak. Here, we address these challenges by applying a patterned microbead bed to an ICP-PAD. The incorporation of the microbead bed is shown to fix the location of the dye-linked protein enrichment. In contrast, the ICP-PAD without beads exhibits bursting of the focused plug. Besides counteracting bursting, the microbead bed (µb) ICP-PAD increased the average EF by 5-fold and the maximum EF by 4-fold, with a peak EF of over 23-fold. Studies characterizing the effect of bead size were conducted and evaluated using parameters such as EF and the retained fraction of the plug to determine the bead diameters that prevent bursting. To evaluate the stability of the bed, we examined the fluorescence produced by a bed of stained microbeads before and after ICPF, and found less than 1% of fluorescence intensity lost. Finally, we leveraged the microbead bed as a test line for a biotinylated protein in a model LFA. We anticipate that this device architecture is broadly applicable to other real-world systems at the POC. These results establish a platform for the future integration of ICPF with LFAs for stable preconcentration and improved sensitivity of previously inaccessible protein targets.