CRISPR-Cas12a based aptasensor for sensitive and selective ATP detection
CRISPR-Cas12a based aptasensor for sensitive and selective ATP detection
复制标题
基于 CRISPR-Cas12a 的适体传感器用于灵敏和选择性 ATP 检测
DOI：
10.1016/j.snb.2020.128164
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发表时间：
2020-10-01
影响因子：
8.4
通讯作者：
Ma, Long
中科院分区：
文献类型：
--
作者：
Peng, Lei;Zhou, Jin;Ma, Long
基于CRISPR-CAS的检测平台以其可编程性、序列特异性和对核酸靶标的高碱基分辨率为生物传感开辟了一条新的途径。在本研究中，我们利用靶DNA诱导的CRISPR-Cas12a的非特异性单链DNA切割活性(反式切割)来构建适体介导的荧光生物传感器，用于灵敏和选择性地检测三磷酸腺苷(ATP)。在该传感器中，我们设计了一个ATP结合适配子作为Cas12a的目标单链DNA，因此在有ATP存在的情况下，ATP结合的适体会被ATP占据，而Cas12a可以处理的靶单链DNA较少，这导致了双标记单链DNA报告的反式裂解导致荧光强度随ATP浓度的变化而变化。通过这种方式，我们成功地将ATP信号转化为可以被Cas12a过程放大的核酸信号。动态检测范围为1~200µM，检测下限为400 nm。这种生物传感器从样品到回答的整个时间约为40分钟。总体而言，这种新型生物传感器在灵敏度、特异性、检测时间和易用性之间取得了平衡。我们提出的生物传感器为CRISPR-Cas12a系统检测小分子提供了一个证明的原理，这将释放它的潜力，并进一步发展它在诊断领域的未来应用。
CRISPR-Cas based detection platform opens up a new avenue for biosensing owing to its programmability, sequence specificity and high base resolution upon nucleic acid targets. In current study, we utilised the target DNA-induced non-specific single-stranded DNA cutting activity (trans-cleavage) of CRISPR-Cas12a to fabricate an aptamer mediated fluorescent biosensor for sensitive and selective detection of adenosine triphosphate (ATP). In this sensor, we designed an ATP-binding aptamer as the target ssDNA for Cas12a, thereby in the presence of ATP, the ATP-binding aptamer would be occupied by ATP, and less target ssDNA can be processed by Cas12a, which gave rise to an ATP concentration dependent change in fluorescence resulted from trans-cleavage of doubly labeled ssDNA reporter. In this way, we successfully "translated" ATP signals to nucleic acid signals that can be amplified by Cas12a process. The dynamic detection range was from 1 mu M to 200 mu M and the limit of detection was 400 nM. The entire sample-to-answer time for this biosensor was around 40 min. Overall, this novel biosensor balanced sensitivity, specificity, detection time and ease of use. Our proposed biosensor provided a principle-of-proof for detecting small molecule by using CRISPR-Cas12a system, which would unlock its potential and further its futuristic applications in the field of diagnostics.