Single-Step, Salt-Aging-Free, and Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing CRISPR-Based Diagnostics
Single-Step, Salt-Aging-Free, and Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing CRISPR-Based Diagnostics
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单步、无盐老化、无硫醇冷冻构建基于 AuNP 的生物探针，用于推进基于 CRISPR 的诊断
DOI：
10.1021/jacs.0c00217
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发表时间：
2020-04-22
影响因子：
15
通讯作者：
Zhou, Xiaoming
中科院分区：
文献类型：
--
作者：
Hu, Menglu;Yuan, Chaoqun;Zhou, Xiaoming
最近报道的用于构建DNAAuNP探针的冷冻标记方法快速，但仍需要硫醇修饰。在这里，我们使用基于冷冻的标记方法评估了Poly(A)标记的DNA序列，结果表明序列末端大约有10个A碱基是必不可少的。更详细的观察显示，一些DNA序列倾向于形成二级结构，从而屏蔽暴露的A碱基，导致效率低下或标记失败。然而，通过简单地增加聚(A)碱基数就可以恢复成功的标记。在这些发现的基础上，我们利用冷冻标记的方法开发了三种基于AuNP的生物探针，DNA-AuNP，RNA-AuNP和DNA-酶-AuNP。该方法在一次混合步骤中完成，不需要硫醇修饰，是迄今报道的最方便、成本最低的AuNP生物探针标记技术之一。此外，所产生的AuNP生物探针通过开发用户友好的比色、荧光和侧向流动检测策略，进一步用于推进基于CRISPR的诊断。
The recently reported freezing-based labeling method for constructing DNAAuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling. However, successful labeling was restored by simply increasing the poly(A)-base number. Building on these discoveries, we developed three kinds of AuNP-based bioprobes, DNA-AuNP, RNA-AuNP, and DNA-enzyme-AuNP, using the freezing-based labeling method. This method was completed in a single mixing step with no need for thiol modification, representing one of the most convenient and lowest cost AuNP bioprobe labeling techniques ever reported. In addition, the resulting AuNP bioprobes were further used to advance CRISPR-based diagnostics through the development of user-friendly colorimetric, fluorescence, and lateral flow detection strategies.