CRISPR/Cas12a和功能性DNA的组合提供了构建用于检测非核酸靶标的生物传感器的可能性。在目前的研究中,CRISPR/Cas12a激活剂中的双链体原型间隔区相邻基序(PAM)用作分子开关,在与CRISPR/Cas12a系统(APF-CRISPR)整合的杂交链反应(HCR)中,使用变构探针缀合的PAM位点形成,构建了灵敏的三磷酸腺苷(ATP)检测生物传感器。如果没有ATP,含有适体的探针(AP)处于茎环结构中,阻止HCR的启动。在ATP存在的情况下,AP的结构在ATP结合时发生变化,导致HCR触发链的释放和具有许多PAM位点的长双链体DNA的产生。由于双链体PAM位点的存在对于触发CRISPR/Cas12a的切割活性至关重要,在HCR产物中PAM位点的ATP依赖性形成可以启动FQ-报告基因裂解,允许通过测量荧光信号进行ATP定量。通过优化序列元素和检测条件,aptasensor表现出卓越的检测性能。测定的检测限(LOD)估计为1.16nM,其中基于六次重复测量计算空白的标准偏差。检测的动态范围为25-750nM,和测定的整个工作流程约为60分钟。此外,通过与市售的用于血清中ATP检测的化学发光试剂盒进行比较,验证了aptasensor的可靠性和实用性。由于其高灵敏度,特异性,和可靠的性能,APF-CRISPR在ATP检测的生物分析研究中具有巨大的潜力。此外,我们为构建基于CRISPR/Cas12a的aptasensor提供了原理证明,其中PAM用于调节Cas12a切割活性。
The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (
ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of
ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of
ATP, the structure of AP is changed upon
ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing
ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for
ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.