LATERAL CATIONIC ISOTACHOPHORESIS-GUIDED MIGRATION ANALYSIS (LIGMA) FOR NEXT GENERATION LATERAL FLOW ASSAYS

Devon Michael McCornack

doi:10.7273/000007420

This dissertation presents the development and validation of a novel diagnostic platform integrating cationic isotachophoresis (ITP) with lateral flow assays (LFAs) to overcome the limitations of traditional paper-based diagnostic devices. By enhancing analyte preconcentration and separation, this work addresses the longstanding challenges of sensitivity and specificity in point-of-care (POC) testing for low-abundance biomarkers in complex biological samples. Chapter 1 introduces the constraints of conventional LFAs, including their reliance on passive capillary flow and susceptibility to interference from high-abundance serum pro- teins, which hinder their diagnostic accuracy. It further highlights the untapped potential of cationic ITP, particularly for the analysis of negatively charged proteins, as a transformative approach to improving assay performance. Chapter 2 details the modification of nitrocellulose membranes to enhance biomolecule retention and minimize non-specific interactions, ensuring compatibility with the strong electrophoretic forces inherent to ITP. These modifications laid the groundwork for robust and stable capture-line immobilization under cationic ITP conditions. Chapter 3 provides a systematic guide to designing and optimizing discontinuous electrolyte systems essential for ITP. This chapter evaluates the effects of buffer composition, ionic strength, and pH on analyte stacking and separation, demonstrating how these parameters impact the performance of cationic ITP-enhanced LFAs. Chapter 4 focuses on the synthesis and engineering of avidin-functionalized gold nanoparticles as modular cationic probes. Covalent functionalization strategies were employed to stabilize the bio-nano interface under dynamic ITP conditions, ensuring the reliable migration of target-probe complexes. Zeta potential analyses confirmed the capability of avGNPs to modify the charge of biotinylated protein complexes, rendering them suitable for cationic ITP while maintaining robust signal generation. Chapter 5 validates the platform’s performance in simulated serum samples, demonstrating a limit of detection (LOD) of 1.97 nM and a limit of quantification (LOQ) of 4.12 nM for human IgG. These results represent a 10-fold improvement in sensitivity compared to traditional LFAs, achieved with minimal sample volume (1 μL) and rapid assay times (<2 minutes). The chapter also describes the construction of a portable, battery-operated system for field-deployable applications, emphasizing its potential for resource-limited settings. Finally, Chapter 6 summarizes the findings, underscoring the potential of cationic ITP-enhanced LFAs as a next-generation diagnostic platform. Recommendations for future work include expanding the scope to multiplex detection, higher sample volumes, and diverse analytes such as nucleic acids, extracellular vesicles, and whole cells, as well as integration with complementary sensitivity-enhancing methods. This work demonstrates a significant step forward in the field of paper-based diagnostics, offering a modular, sensitive, and rapid solution for addressing the limitations of conventional LFAs while maintaining their simplicity and affordability.

LATERAL CATIONIC ISOTACHOPHORESIS-GUIDED MIGRATION ANALYSIS (LIGMA) FOR NEXT GENERATION LATERAL FLOW ASSAYS

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