Fluid propagation and protein adsorption patterns in porous nitrocellulose membranes for lateral flow assays

Lateral flow assays (LFAs) have caught new attention in recent years due to extensive use in the containment of the COVID-19 pandemic. Especially the protein and fluid interactions with the nitrocellulose membrane structure are yet to be fully investigated, which affect the fluid and protein distribution of the test and control lines differently due to different adsorptive properties of fluids and proteins. Therefore, the relationship between fluid spread and protein distribution, respectively, and structure needs systematic evaluation. Two procedures were developed based on passive adsorption of complementary fluorescent dyes to investigate these phenomena. These procedures enabled three-dimensional visualization of the membrane structure, fluid as well as the protein spreading, respectively. Confocal laser scanning microscopy was applied after depositing picoliter and nanoliter volumes of the printing buffers containing fluorophore-labeled proteins (immunoglobulin G) and Oregon Green™ 488 onto the membrane using a high precision micro dispenser. The resulting data were correlated with the membrane's tortuosity and permeability. Inverse-proportional dependencies for the lateral spread of the fluid and protein adsorption with the structural parameters were observed. Additionally, surfactants [polysorbate 80 (PS80) and sodium dodecylbenzenesulfonate (SDBS), both at 0.1%] were added individually to the buffers, and the spread of the liquids was evaluated. Both surfactants increase the similarities between fluid and protein shape compared to the reference data. While SDBS increases the general lateral spread, PS80 does increase the penetration depth of the protein into the membrane, which could lead to reduced signal in LFAs.