First implementation of a polymer-polymer aqueous two-phase system to improve lateral-flow immunoassay sensitivity via dextranase integration on paper.

Lateral-flow immunoassays (LFAs) are commonly used to detect proteins in bodily fluids, providing rapid and accessible patient sample testing at the point of care (POC). Although commercially successful, LFAs suffer from low sensitivity. To improve their sensitivity, our group was the first to implement polymer-salt and micellar aqueous two-phase systems (ATPSs) to preconcentrate various targets into a smaller volume prior to LFA application. For small hydrophilic protein targets that distribute fairly evenly in polymer-salt and micellar ATPSs, polymer-polymer ATPSs may be used for preconcentration instead. A poly(ethylene glycol)-dextran ATPS can preconcentrate proteins into the dextran-rich bottom phase, but has not been integrated with LFAs due to poor sample flow caused by the highly viscous dextran-rich phase. To address this limitation, we implemented the enzyme dextranase (DN) to degrade dextran in the bottom phase sample, reducing its viscosity before LFA application. Enzymatic studies were performed to investigate various conditions, which were then further evaluated with imbibition studies to demonstrate improved fluid flow on paper. Subsequently, the ATPS was combined with DN to achieve a tenfold improvement in the limit of detection (LOD) for the protein transferrin spiked in artificial saliva when compared to a conventional LFA setup. To reduce liquid handling steps and thus improve POC accessibility, DN was dehydrated on paper and applied to the LFA to achieve a similar tenfold improvement in sensitivity. By integrating DN, this study represents the first successful use of a polymer-polymer ATPS to improve LFA sensitivity, and introduces the possibility of integrating a different class of ATPSs with the LFA.