Avidin-Functionalized Gold Nanoparticles Enable Cationic Isotachophoresis for Enhanced Lateral Flow Assays.

Lateral flow assays (LFAs) are widely used for rapid, point-of-care diagnostics but struggle with sensitivity in part due to passive migration and interference from high-abundance serum proteins. Here, we introduce the lateral isotachophoresis-guided migration analysis (LIGMA) assay, a novel diagnostic platform that leverages cationic isotachophoresis (+ITP) to enhance biomolecular separation and detection in LFAs. Using avidin-functionalized gold nanoparticles (AV-S-GNPs) as positively charged nanocarriers, this system enables the direct electrophoretic migration of negatively charged analytes, overcoming a major limitation of conventional cationic electrokinetic focusing methods. The functionalized nanoparticles exhibited superior stability across physiological pH ranges and maintained consistent electrophoretic mobility under + ITP conditions. LIGMA achieved a limit of detection of 1.97 nM and a limit of quantification of 4.12 nM for human immunoglobulin G (IgG), requiring only 1 μL of sample volume and under 2 min of assay time. Compared to standard LFAs, ITP-enhanced migration produced sharper capture line resolution, improved analyte focusing, and minimized background interference from abundant serum proteins. This work establishes the LIGMA assay as a viable electrokinetic strategy for improving paper-based biosensors, providing a foundation for expanded applications in clinical diagnostics, multiplexed detection, and portable field-deployable biosensing platforms.