Guided Electrokinetic Assembly of Functionalized Latex Beads for Fluorescence Signal Enhancement Using AC Electro-Osmosis

Fluorescence-based immunoassays are widely used for sensitive and specific biomarker detection; however, further improvements in sensitivity remain desirable for detecting low-abundance analytes without increasing assay complexity. In this work, we present a proof-of-concept demonstration of guided electrokinetic assembly of functionalized latex beads as a post-assay signal enhancement strategy using alternating-current electro-osmosis (ACEO). Carboxyl-modified 1 μm polystyrene beads conjugated with Alexa Fluor 647-labeled anti-IgG were localized within lithographically defined windows on carbon interdigitated electrode arrays, producing localized fluorescence enhancement through physical bead localization without enzymatic amplification or additional labeling chemistries. Compatibility of the approach with fluorescence-based immunoassays was demonstrated through adaptation of a TNF-α ELISA workflow. Electro-osmotic localization of functionalized bead conjugates was achieved within 120 s, producing an approximately 12-fold increase in corrected total fluorescence relative to the corrected signal of the pre-electro-osmosis condition while demonstrating negligible enrichment of unbound fluorescent protein. Application of the platform to a TNF-α sandwich immunoassay yielded an approximately 5.5-fold enhancement in fluorescence signal, and robust bead localization was maintained across anti-IgG concentrations ranging from 1 to 4 μg/mL. These findings demonstrate that guided electrokinetic bead localization provides an effective signal enhancement strategy for fluorescence-based immunoassays and represents a promising approach for improving the detection of low-abundance analytes.