Seol, Jincheol Kim, Byungju Yu, Eui-Sang Jeong, Cherl hyun Lee, Jong-Bong 2025-06-24T02:00:21Z 2025-06-24T02:00:21Z 2025-06-23 2025-08 1613-6810 https://pubs.kist.re.kr/handle/201004/152674 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. English Wiley - V C H Verlag GmbbH & Co. DNA Hanger: Surface-Minimized Single-Molecule Immunoassay Platform Article 10.1002/smll.202409933 1 Small, v.21, no.31 Small 21 31 Y scie scopus 001503581500001 2-s2.0-105007671733 Chemistry, Multidisciplinary Chemistry, Physical Nanoscience & Nanotechnology Materials Science, Multidisciplinary Physics, Applied Physics, Condensed Matter Chemistry Science & Technology - Other Topics Materials Science Physics Article ADSORPTION DNA skybridge single-molecule immunoassay single-molecule FLISA