Departments of Diagnostic Radiology and Nuclear Medicine and Pediatrics, Center for Blood Oxygen Transport and Hemostasis, University of Maryland Baltimore School of Medicine, Health Sciences Facility III, 670 West Baltimore Street, Baltimore, Maryland 21201, United States.
Bioengineering Department, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
ACS Nano. 2020 Jun 23;14(6):7617-7627. doi: 10.1021/acsnano.0c03822. Epub 2020 May 28.
The current outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) demands its rapid, convenient, and large-scale diagnosis to downregulate its spread within as well as across the communities. But the reliability, reproducibility, and selectivity of majority of such diagnostic tests fail when they are tested either to a viral load at its early representation or to a viral gene mutated during its current spread. In this regard, a selective "naked-eye" detection of SARS-CoV-2 is highly desirable, which can be tested without accessing any advanced instrumental techniques. We herein report the development of a colorimetric assay based on gold nanoparticles (AuNPs), when capped with suitably designed thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2, could be used for diagnosing positive COVID-19 cases within 10 min from the isolated RNA samples. The thiol-modified ASO-capped AuNPs agglomerate selectively in the presence of its target RNA sequence of SARS-CoV-2 and demonstrate a change in its surface plasmon resonance. Further, the addition of RNaseH cleaves the RNA strand from the RNA-DNA hybrid leading to a visually detectable precipitate from the solution mediated by the additional agglomeration among the AuNPs. The selectivity of the assay has been monitored in the presence of MERS-CoV viral RNA with a limit of detection of 0.18 ng/μL of RNA having SARS-CoV-2 viral load. Thus, the current study reports a selective and visual "naked-eye" detection of COVID-19 causative virus, SARS-CoV-2, without the requirement of any sophisticated instrumental techniques.
当前由严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）引起的 2019 年冠状病毒病（COVID-19）大流行要求快速、方便和大规模地进行诊断，以降低其在社区内和社区间的传播。但是，当这些诊断测试针对病毒载量的早期表现或在其当前传播过程中发生突变的病毒基因进行测试时，它们的可靠性、可重复性和选择性都会降低。在这方面，非常需要对 SARS-CoV-2 进行选择性的“肉眼”检测，而无需使用任何先进的仪器技术即可进行检测。在此，我们报告了一种基于金纳米粒子（AuNPs）的比色测定法的开发，当用适当地设计的巯基修饰的针对 SARS-CoV-2 的 N 基因（核衣壳磷蛋白）的反义寡核苷酸（ASO）进行封端时，该方法可以在从分离的 RNA 样本中在 10 分钟内用于诊断阳性 COVID-19 病例。在存在 SARS-CoV-2 的靶 RNA 序列的情况下，巯基修饰的 ASO 封端的 AuNPs 选择性聚集，并表现出表面等离子体共振的变化。此外，添加的核糖核酸酶 H 从 RNA-DNA 杂交体中切割 RNA 链，导致溶液中的可见沉淀，这是由 AuNPs 之间的额外聚集介导的。该测定法的选择性已在存在中东呼吸综合征冠状病毒 RNA 的情况下进行了监测，其检测限为 SARS-CoV-2 病毒载量为 0.18ng/μL 的 RNA。因此，本研究报告了一种选择性和直观的“肉眼”检测 COVID-19 致病病毒 SARS-CoV-2 的方法，而无需使用任何复杂的仪器技术。
