Seol Jincheol, Kim Byungju, Yu Eui-Sang, Jeong Cherlhyun, Lee Jong-Bong
Division of Interdisciplinary Bioscience & Bioengineering, Pohang University of Science & Technology (POSTECH), Pohang, 37673, South Korea.
Department of Physics, POSTECH, Pohang, 37673, South Korea.
Small. 2025 Aug;21(31):e2409933. doi: 10.1002/smll.202409933. Epub 2025 Jun 8.
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.
一种名为DNA Hanger的新型单分子免疫分析平台被开发出来，以解决传统基于表面的分析方法的局限性。通过将生物素化的λ噬菌体DNA悬浮在微加工的石英屏障上，该方法能够以最小的非特异性结合实现高特异性蛋白质检测。DNA Hanger显著降低了背景信号，实现了低至每236μm DNA一个蛋白质的非特异性结合率。对mNeonGreen标记的人聚腺苷酸结合蛋白C1（mNG-PABP）的定量以及对人肿瘤坏死因子α（TNF-α）的单分子荧光免疫吸附测定（FLISA）证明了该分析方法在单分子水平上的特异性和灵敏度，在缓冲液中的检测限为0.90 pM，比传统FLISA低38倍，在70%胎牛血清中的检测限为20.6 pM，提高了8倍。DNA Hanger还能够检测和定量人血清中的内源性TNF-α，突出了其临床潜力。DNA Hanger分析方法无需表面封闭，简化了工作流程，可在1小时内完成免疫分析过程。DNA Hanger为生物分子相互作用研究和临床诊断提供了广泛的适用性。
