DNA Hanger: Surface‐Minimized Single‐Molecule Immunoassay Platform

Abstract A novel single‐molecule immunoassay platform, termed DNA Hanger, is developed to address the limitations of conventional surface‐based assays. By suspending biotinylated λ‐phage DNA across microfabricated quartz barriers, this method enables high‐specificity protein detection with minimal nonspecific binding. DNA Hanger significantly reduces background signals, achieving nonspecific binding rates as low as one protein per 236 µm of DNA. Quantification of mNeonGreen‐tagged human poly(A)‐binding protein C1 (mNG‐PABP) and single‐molecule fluorescence‐linked immunosorbent assay (FLISA) of human tumor necrosis factor α (TNF‐α) demonstrates the assay's specificity and sensitivity at the single‐molecule level, with a detection limit of 0.90 pM in buffer, 38‐fold lower than that of conventional FLISA, and 20.6 pM in 70% fetal bovine serum, an 8‐fold improvement. DNA Hanger also enables the detection and quantification of endogenous TNF‐α in human serum, highlighting its clinical potential. The DNA Hanger assay eliminates the need for surface blocking and simplifies workflow, resulting in completing the immunoassay process within 1 hour. DNA Hanger offers broad applicability for biomolecular interaction studies and clinical diagnostics.