Electrokinetically Driven On-Fiber Active Lateral Flow Assay for MicroRNA Detection

MicroRNAs are promising for early disease detection, particularly in oncology. Their small size ensures stability in biospecimens, but low concentrations require either molecular amplification or large sample volumes, posing challenges toward point-of-care testing (POCT). Here, we present a rapid approach for detecting miRNA-27a, a tumor biomarker, using just 5 μL of sample. Our method integrates electrophoretic separations on low-cost fibers, creating an active lateral flow assay (ALFA), which contrasts with the conventional lateral flow assay (LFA) that relies on passive capillary flow. ALFA separates the target analyte from a complex sample of biomolecules while simultaneously cleaning nonspecific fluorescence probes. Using upconversion nanoparticles that take advantage of near-infrared excitation and anti-Stokes detection, we demonstrate on-fiber electrophoretic separation of unbound probes from those hybridized with target miRNA. This system detects miRNA-27a at concentrations ranging from 1 fM to 10 pM and achieves a detection limit as low as 50 attomolar by optimizing the electrokinetic cleaning time and surface pretreatments. The on-fiber unidirectional ALFA reduces the required sample volume by 10-fold compared to conventional LFAs. This work introduces ALFA as a new candidate for next-generation POCT applications, incorporating sample separation, analyte enrichment, and detection sensitivity allowing large-scale manufacturing and industry adoption using simple and cost-effective materials.