Single-Molecule Binding Assay Using Nanopores and Dimeric NP Conjugates

The ability to measure biomarkers, both specifically and selectively at the single-molecule level in biological fluids, has the potential to transform the diagnosis, monitoring, and therapeutic intervention of diseases. The use of nanopores has been gaining prominence in this area, not only for sequencing but more recently in screening applications. The selectivity of nanopore sensing can be substantially improved with the use of labels, but substantial challenges remain, especially when trying to differentiate between bound from unbound targets. Here highly sensitive and selective molecular probes made from nanoparticles (NPs) that self-assemble and dimerize upon binding to a biological target are designed. It is shown that both single and paired NPs can be successfully resolved and detected at the single-molecule nanopore sensing and can be used for applications such as antigen/antibody detection and microRNA (miRNA) sequence analysis. It is expected that such technology will contribute significantly to developing highly sensitive and selective strategies for the diagnosis and screening of diseases without the need for sample processing or amplification while requiring minimal sample volume.

在生物流体的单分子水平上特异性和选择性地测量生物标志物的能力，有可能改变疾病的诊断、监测和治疗干预。纳米孔的使用在这一领域日益重要，不仅用于测序，而且最近在筛选应用中也是如此。使用标记物可以大幅提高纳米孔传感的选择性，但仍然存在重大挑战，特别是在试图区分结合的和未结合的目标时。在此，设计了由纳米颗粒（NPs）制成的高灵敏度和选择性的分子探针，这些探针在与生物目标结合时会自组装和二聚化。结果表明，单个和成对的纳米颗粒都可以在单分子纳米孔传感中成功地分辨和检测，并且可用于诸如抗原/抗体检测和微小RNA（miRNA）序列分析等应用。预计这种技术将为开发用于疾病诊断和筛选的高灵敏度和选择性策略做出重大贡献，无需样本处理或扩增，同时所需样本量极小。