Super-Resolution Imaging Informs Paper-Based Electrophoretic Serum Protein Separations

Serum protein electrophoresis (SPEP) is a key diagnostic tool. SPEP uses an electric field to separate proteins based on their mass-to-charge ratios, showing the relative protein fractions to provide diagnostic insights. Paper-based methods are emerging diagnostic tools due to their low cost and ease of use. However, the lack of insight into current protein, paper membrane, and electric field interactions and mechanisms leads to undesired elution peak asymmetry that can obscure diagnostic results. We use super-resolution fluorescence imaging to investigate how protein dynamics on paper membranes influence electrophoretic separation at the single-molecule level. We develop a nano-electromechanical paper-based electrophoresis system to separate serum albumin and IgG proteins under a uniform electric field. Nanoscale imaging is achieved through single-molecule fluorescence microscopy. We track the movement of proteins to observe the speed, isoelectric point, and protein separation mechanisms in a paper membrane. We expect serum albumin will have higher directed diffusion than IgG due to serum albumin's higher mass-to-charge ratio. Understanding the protein, paper membrane, and electric field interactions and mechanisms leads to more effective paper-based SPEP devices. Using this knowledge, we can develop a point-of-care paper-based SPEP device that accurately differentiates between healthy and diseased serum samples.