Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids.

Single-molecule antigen detection using nanopores offers a promising alternative for accurate virus testing to contain their transmission. However, the selective and efficient identification of small viral proteins directly in human biofluids remains a challenge. Here, we report a nanopore sensing strategy based on a customized DNA molecular probe that combines an aptamer and an antibody to enhance the single-molecule detection of mpox virus (MPXV) A29 protein, a small protein with an M.W. of ca. 14 kDa. The formation of the aptamer–target–antibody sandwich structures enables efficient identification of targets when translocating through the nanopore. This technique can accurately detect A29 protein with a limit of detection of ∼11 fM and can distinguish the MPXV A29 from vaccinia virus A27 protein (a difference of only four amino acids) and Varicella Zoster Virus (VZV) protein directly in biofluids. The simplicity, high selectivity, and sensitivity of this approach have the potential to contribute to the diagnosis of viruses in point-of-care settings.

利用纳米孔进行单分子抗原检测为准确检测病毒以遏制其传播提供了一种有前景的替代方法。然而，直接在人体生物流体中选择性且高效地识别小型病毒蛋白仍然是一个挑战。在此，我们报道了一种基于定制DNA分子探针的纳米孔传感策略，该探针结合了适体和抗体，以增强对猴痘病毒（MPXV）A29蛋白（一种分子量约为14 kDa的小蛋白）的单分子检测。适体 - 靶标 - 抗体夹心结构的形成使得在通过纳米孔转运时能够高效识别靶标。该技术能够以约11飞摩尔的检测限准确检测A29蛋白，并且能够直接在生物流体中区分猴痘病毒A29蛋白与牛痘病毒A27蛋白（仅相差四个氨基酸）以及水痘 - 带状疱疹病毒（VZV）蛋白。这种方法的简便性、高选择性和高灵敏度有可能为即时检测环境中的病毒诊断做出贡献。