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 μM to 200 μM 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.

基于CRISPR-Cas的检测平台具有可编程性，序列特异性和对核酸靶标的高碱基分辨率，因此为生物传感开辟了一条新途径。在当前研究中，我们利用CRISPR-Cas12a的目标DNA诱导的非特异性单链DNA切割活性（反式切割）来制备适体介导的荧光生物传感器，用于敏感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结合适体作为Cas12a的目标ssDNA，从而在存在ATP的情况下，ATP结合的适体将被ATP占据，而Cas12a可以处理的目标ssDNA较少，这导致了反式导致ATP浓度依赖性的荧光变化-双标记ssDNA报告基因的切割。通过这种方式，我们成功地将ATP信号“翻译”为可以通过Cas12a流程扩增的核酸信号。动态检测范围为1μM至200μM，检测极限为400 nM。该生物传感器的整个从样品到答案的时间约为40分钟。总体而言，这种新颖的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使用CRISPR-Cas12a系统检测小分子提供了证明原理，这将释放其潜力，并进一步将其应用于诊断领域。