适体
反式激活crRNA
清脆的
分子探针
核酸
荧光团
寡核苷酸
分子信标
DNA
化学
生物传感器
小分子
计算生物学
组合化学
生物物理学
纳米技术
荧光
基因组编辑
生物
材料科学
分子生物学
生物化学
基因
物理
量子力学
作者
Chenqi Niu,Chuyi Wang,Fan Li,Xiang Zheng,Xin‐Hui Xing,Chong Zhang
标识
DOI:10.1016/j.bios.2021.113196
摘要
Molecular diagnostics are vital for the identification, prevention, and treatment of numerous diseases and are of particular demand in point-of-care (POC) settings. Nevertheless, most reported biosensors based on the CRISPR-Cas system have focused on nucleic-acid targets. Here, we report a versatile diagnostic strategy for small molecules called Molecular Radar (Random Molecular Aptamer-Dependent CRISPR-Assist Reporter), The workflow is simple, convenient, and rapid (conducted at 37 °C in under 25 min), indicating the substantial potential of the proposed assay could be adapted into a biosensor for POC settings and on-site molecular diagnostics. This strategy is based on the CRISPR Cas12a-assisted fluorescence reporter system that consists of Cas12a, CRISPR RNA (crRNA), a single-stranded DNA (ssDNA) probe labeled with a fluorophore at the 5′ end and a quencher at the 3’ end (F-Q probe), and a single-stranded DNA aptamer for the target molecule. In the presence of a target molecule, the aptamer binds to this small molecule with high specificity and affinity, resulting in a decrease of aptamer hybridized to the crRNA-Cas12a duplex. This decrease in activated Cas12a leads to a significant reduction in fluorescence signal. In this study, adenosine-5ˊ-triphosphate (ATP) was selected as model target molecule and an ATP detect method was developed with high specificity and sensitivity with a linear range from 25 to 500 μM and a detection limit of 104 nM. Moreover, the particular characteristics of CRISPR-Cas12a that we report here for the first time have enriched our understanding of Cas12a and provided guidance for further research on CRISPR-Cas12a-based biosensors.
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