State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition, Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.
State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition, Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.
J Hazard Mater. 2023 Jun 15;452:131195. doi: 10.1016/j.jhazmat.2023.131195. Epub 2023 Mar 11.
The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.
COVID-19 大流行迫切需要灵敏、便携的 SARS-CoV-2 检测方法。我们设计了一种 SERS 读取、CRISPR/Cas 驱动的纳米生物测定方法，称为 OVER-SARS-CoV-2（针对 SARS-CoV-2 的单管增强 RNA 检测），该方法能够在单个管中以无扩增和抗干扰的方式超灵敏、超快、准确且便携地检测 SARS-CoV-2。SERS 纳米探针通过将金纳米粒子与拉曼报告分子和单链 DNA（ssDNA）探针缀合来构建，其聚集-分散变化可以通过靶标激活的 Cas12a 通过链接 ssDNA 的转切割来精细调节。因此，可以巧妙地将核酸信号转换和放大为 SERS 信号。通过定制巧妙的容器，可以将 RNA 逆转录、Cas12a 转切割和 SERS 纳米探针交联的步骤集成到一个单一且一次性的容器中。事实证明，我们提出的纳米生物测定法无需任何预扩增，即可在 45 分钟内检测到低至 200 拷贝/mL 的 SARS-CoV-2。此外，该纳米生物测定法已通过临床拭子样本得到验证，并用于模拟复杂环境和食物样本中的 SARS-CoV-2 检测。这项工作丰富了基于 CRISPR 的诊断（CRISPR-Dx）的武器库，并为 SARS-CoV-2 的去中心化检测提供了一个新颖而强大的平台，有望在不久的将来付诸实践。
