DNA origami-based lateral flow biosensors for detection of small molecule, protein, and nucleic acid

Lateral flow assays (LFAs) are widely used in point-of-care diagnostics due to their rapid readout, low cost, and operational simplicity; however, their limited sensitivity hinders the detection of low-abundance biomarkers. Existing signal amplification strategies often fail to precisely modulate signal intensity while maintaining a simple and versatile platform. Here, we introduce a programmable DNA origami-based lateral flow assay (DO-LFA) that employs a triangular DNA origami nanostructure (TDON) as a tunable molecular scaffold. The TDON enables site-specific functionalization with diverse recognition elements (e.g., oligonucleotides, aptamers, and antigens) and reporter labels (Cy5 fluorophores), allowing independent control over the number of signal reporters per binding event. By systematically varying the Cy5 density (6, 12, or 30 labels per TDON), we achieve predictable signal amplification, with the 30-Cy5-TDON delivering a 3.25-fold higher signal than conventional DNA scaffolds. The platform is adapted to both sandwich and competitive LFA formats, demonstrating ultrasensitive detection of nucleic acids (limit of detection, LOD: 70 pM in serum), thrombin (LOD: 50 pM in saliva), and the small molecule digoxigenin (Dig) (LOD: 1.87 pM in saliva), with excellent specificity and performance in complex biological matrices. This work integrates the precision of DNA origami with the practical simplicity of LFAs, offering a generalizable and scalable strategy for next-generation point-of-care diagnostics.